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Exosomal Consignment in Renal Allograft Injury

Abstract

Exosomes are small mobile endocytic vesicles (30-120nm), shredded by every cell to conduct trafficking of cell generated cargo. They are found in almost all body fluids (blood, csf, saliva, urine). These include proteins, lipids, DNA, mi(cro)RNAs etc. In multicellular organisms, they are packaged into numerous vesicles mainly in exosomes to conduct their transport for various cellular activities which can be exploited clinically. Presently the survival of renal allograft is monitored by mostly invasive methods (tissue biopsy, Creatinine, GFR) where curving the injury is quite difficult. Hence potency of molecular markers like proteins and then circulating miRNAs came to picture for early detection of renal injury (Acute Kidney Injury-AKI and Chronic Kidney Disease-CKD). However, due to lack of specificity of circulating miRNAs lose their feasibility and the discovery of these exosomal cargos in cellular communication has become an efficient tool for treatment of various complicated clinical condition including renal allograft injury.

Keywords: micro RNAs,exosome, Renal Allograft Injury

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Introduction

Exosomal world: a prologue

Exosomes are membrane bound mobile vehicles that are found in almost all circulating body fluids like- blood, CSF, saliva, urine, etc. These are responsible for transport of respective cellular cargo to extracellular target sites [1]. Recent studies with exosomes have revealed that exosomal cargo delivery has many important biological, physiological and pathological significance thus, can be an effective diagnostic tool for various diseases [2]. Exosomes are small circulating units of 30-120 nm in diameter, generating from late endosomal compartments of cells by its cell membrane invagination or budding or released as shedding vesicles. Cellular cargos include proteins, lipids, DNAs, mRNAs, miRNAs, etc [1]. The exosomal cell membrane mainly constitute a limiting lipid bilayer, transmembrane proteins and a hydrophilic core containing proteins, mRNAs and microRNAs (miRNAs).

Exsosomes were first discovered by Pan and Johnstone in 1983 [3] when they found that the release of transferrin receptors into extracellular space during sheep reticulocyte maturation was released inside a type of small vesicles. In 1989 Johnstone regarded these mammalian cargo delivering vesicles as exosomes [1-5]. Valadi et al. in 2007 first described about the composition of exosomes that apart from proteins and lipids these also contains DNAs and RNAs [6] which are recorded in ExoCarta database [7,8] . The exosomal cargo delivery requires stimulation of target cell which may be direct by receptor mediated interactions or may aid in transport from cell of origin to various bioactive molecules e.g. membrane receptors, proteins, lipids, mRNAs, miRNAs, etc [7]. When exosomes deliver its contents into the respective target sites the property and behavior of these cells changes to a great extent [8]. It is also understood from various studies done in last couple of years that miRNA composition of parent cell and exosomal components vary a lot [8] and of all the components, miRNAs have drawn the attention due to their regulatory role in gene expression as these are protected against RNAase-dependent degradation [1-8]. Thus exosomal cell-to-cell communication influence both physiological as well as pathological environment of the body. These play important roles in immune reactions, tumorigenesis and in neurodegenerative disorders [1]. e.g. in prostate cancer, ovarian cancers, lymphoma glioblastoma, etc [1].

Biogenesis

Exosomes are formed from late endosomal compartments of cells through endosomal sorting complex required for transport (ESCRT-that recognizes ubiquitylated proteins) to deliver the cargo to target cell or to fuse with lysosomes to degrade the undesired contents [1]. Earlier these exosomes were only considered to be “garbage bags” as their diversified capabilities were unknown then. But now these are the most emerging field of research. The way of formation and secretion of these vesicles from mutlivesicular bodies (MVBs) are of two types [9]:

Microvesicles, which are directly shed from cell membrane.

Exosomes, which are released by exocytosis when MVBs fuse with plasma membrane.

Exosomes can be identified by transmission microscopy as a cup-shaped morphology with negative staining [10-12]. These can be concentrated in 1.10-1.21 g/ml section of a sucrose density gradient [10-12]. Exosomes can be identified by various protein markers e.g. tetraspanin proteins- CD63, CD9, CD81, HSP70 and HSP90, etc [1, 8]. ExoQuick (a one-step precipitation procedure for exosome extraction), Immuno affinity capture, Immunobead (EpCAM), combination of EpCAM and ultracentrifugation, size exclusion chromatography and EpCAM and followed by Quantitative PCR, Microarray techniques for extraction and quantification of exosomes [1,8,13].

Exosomes formation and secretion requires enzymes and ATP. First the cell membrane is internalized to produce endosomes. Subsequently, many small vesicles are formed within endosomes by invagination of its cell membranes [8, 14]. Such endosomes are called MVBs. Finally, the MVBs fuse with endosomal cell membranes to release intraluminal vesicles into extracellular space which become exosomes [14].

The secretion or cell-to-cell communication of exosomes requires certain regulatory factors which were first identified by Ostrowski et al. who observed that Rab27a and Rab27b were associated with exosomal secretion [8]. It was also found that effectors of Rab27- SYTL4 and EXPH5 could also inhibit exosomal secretion in HeLa cells [15]. Also Yu et al. discovered that tumor repressor protein p53 and its downstream effector TSAP6 were required for influencing exosome secretion [16]. Another working group, Baietti et al. observed the importance of syndecan-syntenin which directly interact with ALIX protein via Leu-Tyr-Pro-X(n)-Leu motif in secrection of exosomes by endosomal budding [17]. Koumangoye et al. found that disruption of lipid rafts in exosomal membranes could inhibit its internalization in breast cell carcinoma cell line [18]. Trafficking of exosomes to target sites occurs in following mechanisms:

The transmembrane proteins of exosomes directly interact with signaling receptors of target cell membranes [19].

The exosomal fusion with plasma membrane of recipient cells to deliver the cargo into their cytosol [20].

The exosomes internalization into recipient cells have two fates[21].

in one, some exosomes are engulfed by the cell and may merge with the cell’s endosome and undergo transcytosis

in other case, engulfed exosomes fuse with endosomes and mature into lysosomes for degradation.

As per ExoCarta database records, of all the components proteins and miRNAs have been exploited for various research to correlate some application with different diseased state that could render some remedy. Due to the regulatory role of miRNAs in gene expression these are used as recent area of research as diagnostic tool [8,22]. Goldie et al. demonstrated that among small RNAs, the percentage of miRNAs is higher in exosomes than in parent cells [23]. Studies done with exosomalmiRNAs shows there are specific sorting mechanisms for miRNAs into exosomes. These are:

The neural sphingomyelinase 2 (nSMase-2)-dependent pathway by Kosaka et al. [24].

The miRNA motif and sumoylated heterogeneous nuclear ribonucleoproteins (hnRNPs)-dependent pathway by Villarroya- Beltri et al. [25].

The 3’ end of the miRNA sequence-dependent pathway by Koppers-Lalic et al. [26].

The miRNA induced silencing complex (miRISC)-related pathway and human AGO2 protein [27].

In short there are specific sequence in miRNAs as well as enzymes and proteins that guide them for their sorting into exosomes [8]. Exosomes are shed by cells during both normal as well as pathological conditions. Thus several studies have been made with exosomes in diseased states.

A brief sketch on therapeutic exosomal cargos:

Exosomal miRNA: miRNAs are the recent findings in the field of clinical research which are thought to be crucial in depicting human health and diseases. These biomarkers can also be an indicator for rejection onset of transplanted allograft. miRNAs are a class of small 18-25 nucleotide (nt) long endogenous, noncoding RNAs which play an important role in regulating gene expression [28,29]. A single miRNA has the ability to regulate expression (mostly silencing) of hundreds of mRNAs and have been known to play important role in many cellular functions that include induction of post-translational repression, mRNA degradation/silencing and transcriptional inhibition, cell development, differentiation, proliferation and functional regulation of immune response among others [28-31].

The mystery behind the functional maturation of miRNAs has been solved by research in last couple of years. Similar to mRNAs, miRNAs are also initially transcribed in the nucleus [32]. miRNAs are at first transcribed in nucleus as primary transcript by RNA polymerase II called pri-miRNA [32-35]. This pri-miRNA has a hairpin stem-loop structure (~80nt length) that contains the mature miRNAs [36]. The pri-miRNA processing include recognition of the stem loop followed by its cleavage by DROSHA (a class 2 ribonuclease III) and DGCR8 (a miRNA-processing multiprotein complex) to release pre-miRNA [32-35]. Pre-miRNA is then recognized by Exportin-5 which allows its exports to cytosol for further maturation into 19-25 nucleotide strands by RNA endonuclease III called Dicer [32- 35, 37]. Cleavage of this pre-miRNA by Dicer result in double stranded (ds) RNA molecule of which one of the single strand with more unstable 5’ base pairing is selected and transferred to an Argonaute (AGO) protein and the other strand is degraded [35,38,39]. The selected strand induces silencing of mRNAs through RNA Induced Silencing Complex(RISC) thus affecting various cellular functions like cell differentiation, proliferation as well as development and functional regulation of immune system [32-35,40]. In normal tissues, RISC remain as a low molecular weight entity with reduced regulatory activity while under stressed or replicating conditions these become high molecular weight units with intensified regulatory activity when bound to mRNA [36]. Thus mRNA silencing by miRNAs results in lower protein levels in the body [36,41].

ExosomalProteins: Proteins are the building blocks of life in all living organisms. These are amino acid chains linked by peptide bonds. They are exquisite necessity in every cellular events, may it be the formation of new cells or cell repair. Thus, can be an important biomarker in depicting biological changes. Emerging research have exploited this idea and conducted various proteomic studies. A more burning concept is ofexosomal proteins. The work done and data obtained shows that besides miRNAs another important exosomal load isexosomal proteins. TrairakPisitkun et al had worked on urinary biomarkers and found that urinary exosomal proteins can also be an efficient protein biomarker in reporting renal tubulopathies and other renal disorders [42]. Exosomes normally found in urine accounts for around 3% of the total urinary protein contents and isolation of these exosomes can result in very large enrichment of urinary proteins derived from renal tubular epithelial cells [42]. The exosomal packaging occurs when the apical membrane proteins undergo endocytosis and packaged into MVBs. These MVBs undergo encapsulation of cytosolic proteins into small vesicles. Finally outer membrane of MVBs fuse with apical plasma membrane releasing exosomes containing both membrane and cytosolic proteins [42]. The proteomics study with LC-MS/MS coupled with upstream one dimensional SDS-PAGE separation experiments had disclosed a number of proteins associated with exosome biogenesis like class E vacuolar protein sorting (VPS), ALIX, Aquaporin 1, Aquaporin 2, ESCRT, etc [43]. A total of 295 proteins of urinary exosomeswere found to be associated with renal diseases and hypertension. These have been enlisted in Urinary Exosome Protein Database [42]. In another experiment where polypeptides were considered reflect that β2- microglobulin could be an indicator of damage of renal proximal tubule cells [42,44]. The techniques used to evaluate exosomal protein change is carried out by two dimensional difference in gel electrophoresis and change proteins are identified by mass spectroscopy and validated by Western Blotting [45]. Zhou et al worked with Fetuin-A, a protein of liver as an important exosomal protein that can indicate occurrence of AKI(Acute Kidney Injury) [45].

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Early Molecular Biomarkers for Renal allograft status

Years of research with renal allograft injury for either Acute Kidney Injury (AKI) or Chronic Kidney Disease (CKD) suggest that instead of invasive detection of allograft status there are scopes for early and non-invasive detection of injury through molecular markers. The studies made at the molecular level have disclosed the fact that acute and chronic rejections to a transplanted graft at preliminary stage can be ascertained by alteration in levels as well as expressions of various molecular markers involved in signaling of graft injury. These can be measured from blood/urine samples of patients. In acute rejection the early pathological change is defined by Ischemia-Reperfusion Injury (IRI) where altered expression of various miRNAs [46] is observed 3-7 days post-injury [47]. At later stage when rejection is in progress changes in levels of miR-210,-10a and -10b as well as some proteins (like perforin, granzyme A andB mRNA, FAS Ligand, FOXP3, etc) are observed [48]. Chronic rejection in early graft injury is generally associated with Interstitial Fibrosis and Tubular Atrophy (IF/TA). Pathophysiology of IF/ TA is the deposition of Extracellular matrix (ECM) proteins and Epithelial-Mesenchymal Transition (EMT) which can be stimulated by Transforming Growth Factor beta (TGF-β)/Smad signaling cascades. Ample of literature suggest that TGF-β/ Smad signaling can cause up-regulation and down-regulation of various miRNAs (miR-21,-192 & miR-29 and -200 families under IF/TA conditions) [49,50]. Even though these biomarkers have provided fruitful information but they lack specificity and their cellular source is unknown since they circulate. So to get a much clearer picture of a particular injured cell research at molecular level have unrevealed the next generation biomarker –exosomalmiRNAs for early, specific and non-invasive detection. Moreover their cellular source is also defined so they can deliver exact status of a particular cell [1,8].

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Urinary Exosomal proteins and miRNAs in renal allograft injury as Next Gen Molecular Biomarkers

Studies done with renal diseases is pretty less and still a burning area of research that reveals the fact that urinary exosomal proteins as well as miRNAs can be a potential therapeutic tool for kidney and associated diseases [1,8].

The urinary exosomal proteins can be easily attainable by noninvasive means for diagnosis of ESRD as well as Urinary Tract Infection (UTI) [1]. In 2006 Zhou et al. reported that urinary exosomal protein- fetuin A was found to be increased in AKI (Acute Kidney Injury) patients in ICU than AKI patients in normal care [1,41,45]. In 2008, same group discovered that activating transcription factor-3 (ATF-3) was found in exosomes isolated from AKI patients than CKD patients or control [41,45,51]. Thus suggesting urinary exosomal proteins could be a diagnostic tool. Moreover, a reduced level of urinary exosomal aquaporin-1 has been observed in ischemia-reperfusion injury in rats [7]. ExosomalmiRNAs demonstrate potential diagnostic biomarker for renal fibrosis [8]. MiR-29c and CD2APmRNA [52,53] were observed in urinary exosomes of renal fibrosis patients. The findings by Stefano Gatti1 et al. showed that bone marrow derived Mesenchymal Stem Cells (MSC) Microvesicles (MV) when administered immediately after IR injury can prevent AKI and CKD in rats [8,54] through their paracrine actions. Tara K Sigdel et al have described that in AKI patients with perturbation exosomal proteins like CLCA1, PROS1, KIAA0753 were observed. In addition to that exosomal ApoM is more than soluble ApoM [55]. M.W. Welker group found that in patients with chronic Hepatitis C serum soluble exosomal CD 81, a surface protein marker is elevated [56]..

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Future Prospective and limitations

Lots of work have been done with circulating miRNAs but due to their less specificity with the exact status of injured tissues and accuracy in determining role of a miRNA and its cellular source, still more feasible molecular markers have been searched and scientists have found that the circulating vehicles of cells-circulating exosomes that carry respective cellular cargo to the target sites to conduct cell-to-cell communication can be an option. These can be more proficient in delivering the most specific information on the status of any cell, may it is normal or injured cells. The molecular composition of exosomes that has been found till date is being recorded in the ExoCarta database. By exploiting these data in different pathological diseases scientists have done lots of research with carcinomas. In renal diseases also these exosomal miRNAs are being used to find out a means for noninvasive early detection of graft rejection. The conclusion drawn from these studies that proteins like fetuin-A and activating transcription factor-3 (ATF-3) can be used as marker in acute kidney disease and miR-29c and CD2AP mRNA are identified from urinary exosomes in renal fibrosis patients.

Thus, the various convergent studies made in the field of transplantation have led to the discovery of potential therapeutic targets- non-invasive urinary exosomal miRNAs and proteins which can be used to investigate and confirm the injury of transplanted allograft at an early stage. Though the data obtained define exosomes as an appropriate marker when compared with mRNAs, still it has few limitations:

Diverse isolation procedures that can affect exosomal contents,

Exosomes containing large number of miRNAs that affect the signaling of the cell together but not itself alone and

TDifficultly in measuring the exact quantity of a particular miRNAs in a exosome when miRNA is in low concentration.

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Conclusion

The exosome cell-to-cell communication mechanisms’ experiments are still at its infant stage. There is the need for development of more sophisticated techniques to detect the exact amount of specific functional exosomal proteins and miRNAs and their proper signaling pathways. Thus more investigation are still required to exploit exosomes in clinical fields as therapeutic targets.

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Elucidation of Anticancerous Potential of Plant Extracts Against Cervical Cancer Abstract Cancer has been a public health problem that has gained a lot of death. However, inspite of the advances in the diagnosis and treatment of cervical cancer, women follow the struggle versus this disease. Also, those patients suffer from limited efficacy and specificity, undesirable effects, drug resistance, and a high cost of treatments. Early detection and affordable drugs that have clinical efficacy have to go simultaneously in order to seriously address this health challenge. Plant-based drugs with potent anticancer effects should add to the efforts because it will find a cheap drug with limited clinical side effects. So, keeping this in mind, an attempt has been made to explore the potential of plant extracts or constituents known to exhibit anticancerous activity or exert cytotoxic effect in human cervical cells. Alkaloids such as those isolated from C. vincetoxicum and T. Tanakae naulorals A and B, isolated from the roots of N. orientalis, (6aR)-normecambroline isolated from the bark of N. dealbatacan be promising in different human cervical carcinoma cells with the IC50 of 4.0-8μg/mL. However, other compounds such as rhinacanthone and neolignans isolated from different plants are not far behind and kill cervical cancer cells at a very low concentration. Among plant extracts that enhance the effect of known anticancer drugs aloe vera perhaps is the best one. The cytotoxic capability and apoptotic index of certain plant extracts were found to be significant in further enhancing the combination of different human cervical carcinoma cells and therefore they are considered as a promising herbal-based anticancer agent. However, further research needs to be further investigated in various cervical cell lines and most importantly, in in vivo cervical culture for possible use as an alternative and safe anticancer drug. Keywords: Cancer; Carcinoma cells; IC50; Alkaloids; N. dealbata; N. orientalis; Rhinacanthone; Neolignans Go to Introduction A human adult consists of about 1015 cells; which divide and differentiate in order to refurbish organs and tissues [1]. However, if the cells do not stop dividing, they may lead to cancer. Characteristically, cancer is an uncontrolled proliferation of cells which become structurally abnormal and possess the ability to detach them from a tumor and establish a new tumor at a remote site [2]. Every year over 200,000 people are diagnosed with cancer in the United Kingdom only, and approximately 120,000 die as a result of this disease [2]. According to the International Agency for Research on Cancer, in 2002, cancer killed more than 6.7 million people around the world and another 10.9 million new cases were diagnosed [3]. According to World Health Organization, cancer is the second cause of death globally after cardiovascular diseases. An estimated 8.2 million people die from cancer each year, that represents 13% of all deaths worldwide. Cancer basically results from the uncontrolled rapid division of malignant cells that grow beyond their usual limits. Unlike normal cells, cancerous cells do not respond to the controlling signals and consequently, they grow and divide in an uncontrolled manner, infecting normal tissues and organs.There can be many different types of cancers. The type of the cell from where the tumors originate classifies the cancers. Cancers derived from epithelial cells of breast, prostate, lung, pancreas and colon, cause approximately 90% of all human deaths from cancer; lymphomas are cancers of the immune organs such as spleen, white blood cells and lymph glands; leukemias causes the cancers of blood forming bone marrow; while sarcomas are cancers of fibrous connective tissues of bone, cartilage, fat tissue, muscle and neurons; and germ cell tumors are derived from pluripotent stem cells presented in the testicles and ovary. Early detection and effective treatment can help to increase survival rates of cancer patients. So therefore, deliberative plans are needed to improve prevention and treatment of cancer. As we all know cancer rates continue to rise, particularly in the developed world, becoming one of the leading public health problems in many countries [4]. Many of the cancers are associated with longevity, and the possibility of their appearance increases as the life expectancy of individuals increases [5]. Cervical cancer (CC) is a principal cause of death in women in the whole world [6-8]. The research done up to date indicated that this cancer.contributed with approximately 500,000 new cases and produced about 300,000 deaths in 2015. In general, the susceptibility to the pathogens as human papillomaviruses (HPV), lifestyle and cultural factors and inadequate medical system contribute to the development of cervical cancer [9]. Current information suggested that almost 100 serotypes of HPV exist. But, out of these two, 16 and 18 serotypes are important ones and related to the development of cervical cancer. Cervical cancer could produce free radicals that induce damage to the cells, tissues and organs [10]. However, the proper functioning of cells depends on the mitochondria’s ability to regulate metabolic processes and produce molecules, including free radicals as reactive oxygen species (ROS) [11]. ROS controls both physiological as well as pathological process related to cell proliferation, invasion cell, and drug resistance. Several studies have also shown that the cancer risk at the point of specific organs is due to exposure to specific environmental chemicals, biological agents (as Human Papilloma virus, Epstein Barr Virus, HIV1, HCV, Helicobacter pylori) or physical agents (such as ionizing radiation, UV). Chemotherapy and radiotherapy, the conventional cancer treatments used nowadays, are expensive and cause many side effects, including such minor ones like vomiting, diarrhea, constipation, and major ones such as myelosuppression, neurological, cardiac, pulmonary and renal toxicity. All these side effects reduce the quality of life and discourage the patients to follow the medication protocols that further leads to the progression of cancer and future complications. Resection surgery procedures are a major concern because of functional deficiencies or esthetic discomfort. Therefore, there is a need.to produce alternative anticancer drugs, which can be more potent and effective, as well as more selective and less toxic than those of currently in use. In spite of the advances in the diagnosis and treatment of cervical cancer, women follow the struggle related to this disease. Also, the patients suffering from cervical cancer have limited scope and specificity, undesirable effects, drug resistance, and a very high cost of treatments. So therefore, demand of plants and its extracted compounds was increased rapidly. Currently, several studies have demonstrated the efficiency of plant extracts, against cervical cancer cell lines. Certain plant extracts have both antioxidant and pro-oxidant properties. They show high specificity because generally they act as an anti-oxidant and pro-oxidant. Approximately 60% of drugs currently used for cancer treatment have been isolated from natural products and the plant kingdom has been the most significant source. These include Vinca alkaloids, Taxus, diterpenes, Camptotheca alkaloids, Saffron, Bonellia albeflora etc. The pro-oxidant activity is the major reason for obstructing the growth factors related to different signaling pathways that initiates cancer. Although, usually these kind of plant extracts helps for dispatching the apoptosis in cervical cancer cell. Apoptosis describes the programmed cell death. The initial screenings for plants used for cancer treatment are cell-based assays using prepared cell lines, in which the toxic effects of plant extracts or isolated compounds can be measured. According to the National Cancer Institute (NCI), plant extracts and pure compounds with cytotoxic ED50 (Effective Dose 50) values between 4g/l to 30g/l, are generally considered as active Click here to view Large Table 1 Since time immemorial, plants have always been a very good source of drugs and many beneficial uses of medicinal plants are extensively mentioned in the traditional system of medicine of many cultures. Traditional medicines from plants offer great potential for the discovery of novel anti-cervical cancer drugs. There are several sources of anti-cervical cancer drugs: plants, vegetables, herbs and spices used in folk medicine. The combined synergistic effect of the individual active components of these extracts and their molecular mechanisms involved need further investigation in order to evaluate the potential of theseSSSSW compounds as anticancer agents. The protective effects of plantextracts have been related to the presence of phytochemicals, bioactive non-nutrient plant compounds which commonly have complementary and overlapping mechanisms of action, including free radical scavenging, anti-mutagenesis, induction of apoptosis in cancer cell lines. Go to Suggested Mechanism of Action Disruption of cellular homeostasis between cell death and cell proliferation can cause cancer. In general, plant extracts cause cellular toxicity in cervical cancer cells and result in cell death by two primary pathways i.e. cell cycle arrest andapoptosis. Inhibition of cell growth and induction of cell death are two major methods of antitumor growth [12]. Cell cycle progression and apoptosis are two important signaling mechanisms of homeostasis maintenance in healthy tissues and normal cells. The general mechanisms involved include: arrest of cell cycle in G1 phase and induction of apoptosis via the caspasedependent intrinsic pathway. Cell cycle arrest Many anticancerous and DNA-damaging agents arrest the cell cycle at G0/G1, S or G2/M phase and then induce cell apoptosis. Most of the human solid tumors is genetically unstable and have defects in the cell-cycle checkpoint control mechanism. Such tumors frequently contain mutations that disrupt the G1 components of the cell cycle, which affects the abilities of chemotherapeutic drugs to inhibit cell proliferation and induce apoptosis [12]. Since cancer cells usually undergo active cell division (mitosis), a useful approach to finding anticancer drugs is to test whether a compound can selectively kill mitotic cells [13]. Therefore, cell cycle arrest is the special target for many anticancer drugs. Among them, taxols, colchicines and vinca alkaloids are the well-known examples that induce the G2/M phase arrest leading to the subsequent apoptosis [14]. Apoptosis Apoptosis (programmed cell death) plays a crucial role in the homeostasis of organisms under both physiological and pathological conditions. Apoptosis is the most convenient manner of tumor cell elimination, as this type of cell death is a final state which does not cause any possible future danger [15]. The two major pathways leading to apoptosis in cells includes: a) extrinsic pathway which involves the activation of the TNF/Fas death receptor family and the second one b) intrinsic pathway which involves mitochondria [16]. Generally, apoptotic cells are characterized by the reduction of mitochondrial transmembrane potential, intracellular acidification, excessive production of reactive oxygen species (ROS), and externalization of phosphatidylserine residues in membrane bilayers and selective proteolysis of specific proteins and also further degradation of DNA into inter nucleosomal fragments. TRAIL (TNF related apoptosis inducing ligand) is a promising tool for cancer therapeutics due to its ability to selectively induce apoptosis in malignant tumor cells because it causes no toxicity against normal tissue. D fragmentation or degradation is considered as the hallmark of apoptosis. Agents that suppress the proliferation of malignant cells by inducing apoptosis may represent a useful mechanistic approach to both cancer chemoprevention and chemotherapy [17]. Therefore, induction of apoptosis in cancer cells is one of the useful strategies for the development of anticancer drugs [18-28]. Go to Conclusion Although various types of synthetic drugs are being added to this new world of health care, but still no system of medicine in theworld is there that can solve all the health problems. Therefore, the search for new therapeutic compounds from plants is genuine and urgent. Many plant-derived and natural products are being tested for their anti-cancerous activities. The mortality rate due to different types of cancer has been increasing in spite of several treatment strategies for cancer. Plant-derived molecules or drugs could be an effective alternative for the treatment of different types of cancer. This review is just an attempt to understand different types of plants and their different extracts which can be used for the cancer treatment, especially for cervical cancer. As a result of new approaches, the concept of achieving ideal health is changing, and mainly focuses on the importance of a healthy lifestyle highlighting on diet and exercise.Diet as we all know plays a crucial role in the regulation of metabolic pathways both genetically and epigenetically. By the help of modern genetics, and molecular biology, nutrition research will increasingly be able to apply new discoveries to develop designer functional foods by adding specific bioactive properties for preventing and reducing the risk of cancer development. Herbal medicines have been used since ancient times. They are usually a mixture of several compounds, which can affect cells. The information and knowledge collected so far in several civilizations needs to be utilized for the betterment of health issues. Another important problem of the treatment of cervical cancer is the resistance to chemotherapy and radiotherapy. The review focused that several bioactive polyphenols are able to sensitize cervical cancer cells to conventional chemo and radiation therapy. Therefore, this combined approach could improve the efficiency of standard therapies and allow us to decrease the heavy doses of chemotherapy drugs and irradiation leading to reduce the adverse side effects. We summarized in this article some of the polyphenolic compounds that have been studied until now for their possible anti-cancer therapeutic properties. It is crucial to continue these studies for searching therapeutic drugs from natural resource as well as for investigating their mechanism of action in cervical tumor cells.

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Re-Examining the Genetic Bottleneck: Atavistic Regression in Acquired Traits Affects the Outcome for Many Subspecies at the Allelic Level- Juniper Publishers

Preface

Nodular or clear cell hidradenoma denotes an asymptomatic, exceptional, gradually progressive, benign, solid or cystic, intra-dermal adnexal neoplasm of sweat gland origin with eccrine or apocrine differentiation. Initially scripted by Mayer in 1941, the tumefaction describes with a dual subtype as hidradenoma with eccrine or period differentiation and hidradenoma with apocrine or clear cell differentiation [1]. Frequent betwixt fourth and eight-decade, peak incidence of the disorder is cogitated within sixth decade although clear cell hidradenoma can appear in the first decade. Of undetermined genesis, the cutaneous neoplasm arises from eccrine dermal adnexal glands although apocrine derivation is occasionally delineated. Clear cell hidradenoma or the nodular hidradenoma is a frequent histological subtype. The nomenclature includes captions such as eccrine acrospiroma, solid-cystic hidradenoma, clear cell acrospiroma, clear cell myoepithelioma, eccrine sweat gland adenoma, nodular hidradenoma, clear cell hidradenoma, cystic nodule hidradenoma, and eccrine acrospiroma.

Disease Characteristics

Eccrine acrospiroma elucidates as a solitary, firm, nodule with a preferable localization in the head and neck, face and upper extremities although lesions can be cogitated on the chest, shoulder, upper torso, upper or lower extremities. A female to male ratio of 1.7 :1 and a mean age of presentation at 37.2 years is exemplified. An estimated proportion of lesions are cogitated at the head (30.3%), upper limb (25.8%) and trunk (20.2%) [2,3]. Paediatric adnexal tumours demonstrate a predominantly follicular or apocrine/ eccrine genesis. Pilomatrixoma is a frequent and most encountered skin adnexal tumefaction situated in head and neck region. Tumours with sebaceous differentiation are distinctly infrequent. Hidradenoma is generally elucidated betwixt 20 to 50 years of age. Clear cell hidradenoma can occur as a polypoidal umbilical mass, congenital neck swelling or as axillary nodules in infants. Hidradenoma emerge as a gradually progressive, solitary, asymptomatic, firm, mobile tumefaction or nodule [3,4]. A few of the tumour nodules can demonstrate a serous effluvium or may ulcerate. No anatomical site is exempt from the appearance of the tumour. Lesions are disclosed in vulva in females, peri-anal region inmales, scalp, head and neck, face, lower eyelid, external auditory canal, knee and foot. The tumefaction can be associated with hyper-oestrogenaemia as oestrogen and progesterone receptors are discerned on the tumour cells. Hyper-oestrogenaemia can engender multitudinous lesions at various sites [4,5].

Clinical Elucidation

Tumefaction of hidradenoma appears as solitary or multiple lesions. A female preponderance is noted with implication of middle-aged individuals. Adult females can exhibit lesions confined to the vulva. Although the disorder is predominantly cogitated in adults, nodular hidradenoma can arise in children. The nodules are frequently asymptomatic, evolve gradually and rarely demonstrate clinical symptoms such as pain and serous discharge. Solid or cystic, well demarcated, asymptomatic, minimally progressive, endophytic, rarely exophytic nodules of varying magnitude are cogitated usually of dimension betwixt 0.5 centimetres to 3.0 centimetres. The tumefaction enunciates a minimal possibility of malignant transformation and probable ulceration. The generally solitary nodules are skin coloured or crimson and frequently elucidated on the scalp, face, thorax, abdomen and proximal extremities [5,6]. Unconventional clinical appearance is cogently described with tumours exceeding 3 centimetres in diameter, erosion of the superficial surface, a preponderant cystic component and aberrant locations such as the plantar region.

Histological Elucidation

Clear cell hidradenoma is a well circumscribed, un-encapsulated tumour as delineated on gross and morphological examination. Clear cell hidradenoma comprises of lobules of tumour cells situated in the dermis with extensions into subcutaneous fat. The tumefaction is segregated from the epidermis by a dormant Grenz zone. The neoplasm, characteristically confined to the upper dermis, demonstrates a congregation of dual population of miniscule or enlarged, monomorphic or polyhedral epithelial cells with copious clear or eosinophilic cytoplasm and miniature nuclei [6,7]. The dimorphic cell population elucidates polygonal cells with abundant glycogen thereby inducing a clear appearance to thecytoplasm admixed with elongated, darkly stained, miniature cells cogitated at the periphery.

Clear cell metamorphosis and/or squamous metaplasia can be a predominant feature within the tumour cell accumulates. Focal apocrine elements are evidently displayed. The tumour exhibits diverse composites and nodules of benign epithelial cells articulating miniscule ductules and duct lumina generally cogitated within the upper dermis. Duct like configurations recapitulate the eccrine ducts. Additionally, slit like spaces lined by concentric layer of squamous epithelial cells are exemplified. Numerous cystic spaces are elucidated on account of tumour cell degeneration. Apocrine secretion can be delineated within the ductular lumen due to decapitation of cytoplasmic vacuoles [7,8]. Squamous, sebaceous or mucinous epithelial differentiation can be enunciated in specific lesions. The cellular component is variable, although clear cell predominance emerges in proportionately one third instances. Distinctive lesions are enveloped in an intact dermal integument or can demonstrate focal and superficial ulceration with discharge of serous fluid. The tumefaction exhibits singularly solid or cystic elements or an admixture of solid and cystic components in varying proportions or a clear cell predominance. Solid areas enunciate epithelial lobules comprised of preponderant polyhedral cells with a basophilic cytoplasm and clear cells with glycogen containing cells with a clear cytoplasm. Clear cells contain glycogen or a diastase resistant substance which stains with periodic acid Schiff’s (PAS) reagent. Lipid vacuoles are not elucidated.

Clear cells denote a metabolic variant of epidermoid cells, rather than an aberrant category of tumour differentiation. Stroma intervening within the lobules enunciate delicate,vascularised cords of fibrous tissue or dense hyalinised tissue. Myxoid and chondroid stroma are infrequent. Exophytic growth with superficial surface erosion of the tumour is observed [8,9]. As the histological description and annotation is competent, confirmation with immune histochemistry is not a cogent, essential exercise. Malignant transformation is infrequent and is characterized by morphological parameters such as nuclear atypia, necrosis and atypical mitosis. A distinctive histological enunciation may not accurately predict the clinical behaviour of clear cell hidradenoma (Figure 1–14).

Distinguishing Diagnosis

Clear cell hidradenoma necessitates a distinction from a concordant lesion such as haemangioma, glomus tumour, cutaneous lymphoma, dermatofibrosarcoma protuberans, leiomyoma, follicular cyst, trichilemmoma or adjunctive sweat gland or adnexal tumours which are clinically indistinguishable [9,10]. Clear cell neoplasm confined to the dermis necessitate a segregation from metastatic malignancies and primary skin tumours with follicular, sebaceous and sweat gland differentiation. Clear cell hidradenoma in adults require a demarcation from metastatic renal cell carcinoma. However, nodules of hidradenoma are devoid of the classic pulsating vascular prominence as delineated in malignant deposits of renal cell carcinoma [10,11]. Although clear cell hidradenoma, also termed as eccrine acrospiroma, is predominantly a benign neoplasm, the nodules can be subjected to malignant transformation. Engendered malignancy is cogitated as “clear cell hidradenocarcinoma” or “malignant clear cell hidradenoma”. The malignant counterpart is an exceptional neoplasm and is characterized by an infiltrative tumour perimeter, cellular atypia and innumerable atypical mitosis [11,12].

Therapeutic Options

A comprehensive surgical extermination is a pre-requisite in managing clear cell hidradenoma as the nodules are endowed with an enhanced potential of tumour recurrence. Awide tumour perimeter on surgical excision is mandated for a detailed histological confirmation which ensures minimal probable reoccurrence and evaluating prospective malignant transformation. A surgical eradication of the neoplasm with a broad perimeter is the preferred therapy. Malignant transformation is infrequent although neoplasm such as clear cell hidradenocarcinoma tend to reappear. Tumefaction undergoing malignant conversion demonstrate an aggressive clinical course, disseminated disease with adjuvant, enhanced mortality [12,13].

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MazEF homologous Modules System and A Post-segregational killing Mechanism (Bacteriostatic & Bactericidal Mechanism) of Novel Compound Isolated from Spondias monbinon Escherichia Coli and Bacillus Subtilis- Juniper Publishers

Abstract

The basic objective of this research work is to evaluate the mechanism of action of compound Epigallocatechin, Epicatechin and Stigmasterol Phytosterol (Synergy), Aspidofractinine-3-methanol) and Terephthalic acid, dodecyl 2-ethylhexyl ester with Selected clinical isolates by using molecular biomarker MazEF9 TA system. Toxin-Antitoxin (TA) systems are highly conserved in members of the Gram positive +ve and Gram negative –ve bacteria which has been proposed to play an important role in physiology and virulence. Clinical microorganisms were cultured and Sub-culturing in Department of Microbiology and Centre for Biocomputing and Drug Development (CBDD), Adekunle Ajasin University, Akungba Akoko, Ondo-state, Nigeria. A 12 hours old culture of each microorganism was re-suspended in plant extract at 1000 μg mL in a total volume of 500μl for 0, 15, 30, 45, 60, and 180 minutes.

Keywords: Post segregational killing mechanism epigallocatechin; Epicatechin; Stigmasterol phytosterol; Mazef TA System; Bacteriostatic

Introduction

Spondiasmombin L. (Anacardiacaea) also known as hog plum is a plant that grows in almost every part of the world. It is fruit feriousdecidous plant of about 20m high that grows in the rain forest and the coastal area of Africa. It is known locally as “iyeye” by the Yoruba people of Nigeria Ripped fruits are eaten out hand by the old and young and processed into ice-cream, cool beverages, wine, jam and other preservatives. Spondiasmombin alsofound application in folk medicine. Infusion of its leaves has been used for a long time, without any report of collateral effect due to its anti- vitrotic activity against the herpes virus [1]. A tea of the flowers and the leaves is taken to relieve stomachache biliousness, urethritis, cystitis and eye and throat inflammation. Herbalist in South Western Nigeria use the plant in the treatment of typhoid, tuberculosis, diabetics, nervous disorders and psychiatric disorders. Offiah, Anyanwu [2] have reported the abortifacient activity of the aqueous leaf extract of Spondiamombin. In addition, the anthelmintic, molluscicidal, anxiolytic, anti-bacteria, antiviral effect of the plant have been previously described. Idu et al. [3] reported the inhibitory activity of Spondiamombin against Cycasrevoluta induced carcinogenesis. The discovery of toxin-antitoxin gene pairs (also called homologous modules) on extra-chromosomal elements of Escherichia coli, Bacillus subtilis and particularly the discovery of homologous modules on the bacterial chromosome, suggest that a potential for programmed cell death may be inherent in bacterial cultures which is under the scope of this research work, this was reported on the E. coli / Bacillus subtilis mazEF system, a regulatable addiction module located on the bacterial chromosome. MazF is a stable toxin and a labile antitoxin. it show that cell death mediated by the E. coli /Bacillus subtilis mazEF module can be triggered by several medicinal plant extract and antibiotics like Rifampicin, chloramphenicol, and spectinomycinetc, that are general inhibitors of transcription and translation. These medicinal plant extract and antibiotics alike inhibit the continuous expression of the labile antitoxin MazE, and as a result, the stable toxin MazF causes bacteria cell death. The results of this research have implications for the possible mode(s) of action of this group of medicinal plant and antibiotics on selected clinical isolates. The mode of action of the medicinal plants may be either bacteriostatic or bacteriocide [4] but the scope of this research work is based on bacteriostatic mechanism of action of isolated novel compound on selected clinical organisms (E. coli and B. subtilis).

In Escherichia coli/Bacillus subtilis cultures, programmed cell death is mediated through a unique genetic system. This system, called “homologous module,” consists of a pair of genes that specify for two components: a stable toxin and an unstable antitoxin which prevents the lethal action of the toxin. Until recently, such genetic systems for bacterial programmed cell death have been found mainly in E. coli and Bacillus subtilis on low-copy-number plasmids, where they are responsible for what is called the postsegregational killing mechanism. When bacteria lose the plasmid( s) (or other extra-chromosomal elements), the cured cells are selectively killed because the unstable antitoxin is degraded faster than is the more stable toxin [5]. Thus, the cells are “addicted homologous” to the short-lived product, since its de novo synthesis is essential for cell survival [6].Therefore, these homologous modules have been implicated as having a role in maintaining stability in the host of the extrachromosomal elements on which they are bounded, Jensen, Gerdes [7], once this stability is destroyed, this will inevitably leads to the death of the bacterial cell. Pairs of genes homologous to some of these extra-chromosomal addiction modules have been found on the E. coli and Bacillus subtilis chromosome Aizenman et al. [8], Gerdes et al. [9]. The mazEF module consists of two adjacent genes, mazE and mazF, located in the rel operon downstream from the relA gene Metzger et al. [10].

In the study, mazEF was found to have the properties required for an addiction module:

(i) MazF is toxic and MazE is antitoxic

(ii) MazF is long lived, while MazE is a labile protein degraded in vivo by the ATP-dependent ClpPA serine protease

(iii) MazE and MazF interact

(iv) MazE and MazF are coexpressed

Both MazF and MazE are expressed thereby leading overexpressed killing effects of bacteria cell, Moreover, the mazEF system has a unique property: its expression is inhibited by guanosine 3′,5′-bispyrophosphate (ppGpp), which is synthesized under conditions of extreme amino acid starvation by the RelA protein [11] thereby leading to the death of the bacteria cell. Based on these properties of mazEF and on the requirement for the continuous expression of MazE to program cell death, members of our group offered a model for programmed cell death under conditions of nutrient starvation [8]. In this study, MazF (toxic) is used to stimulate the programme cell death of the clinical isolates and evaluates the efficacy of novel compound isolated from Spondiasmombn plant on the selected clinical organisms.

Materials and Method

Microorganism for the research work

Clinical microorganisms were used for this research work, which comprised Escherichia coli ATCC 25922and Bacillus subtilis.

Sources of microorganisms

All typed strains used for this research work were purchased from the University of Pennsylvania, School of Medicine, Philadelphia, United States of America (USA), in an America Type Culture Collection (ATCC) , and the other locally isolated bacteria and fungi were clinical organisms collected from Central Medical Laboratory( CML), Obafemi Awolowo University Teaching, Hospital (OAUTH), Ile Ife, Osun State, and the Institute of Advance Medical Research and Training (IMRAT),University College Hospital, Ibadan, Oyo State Nigeria.

Authentication of test microorganisms

The identity of the test organisms was confirmed using Biomerieux, France, API 20E Kits for bacteria as specified by the manufacturer’s instruction. Analytical Profile Index [12].

Isolation of RNA.

A 12 hours old culture of each microorganism was re-suspended in plant extract at 1000μg mL in a total volume of 500μl for 0, 15, 30, 45, 60, and 180 minutes. The cells were pelleted by centrifugation at 5000g for 5 minutes. The pellets were rinsed twice in phosphate buffer saline (PBS). Then 1/10 volume of 95% ethanol plus 5% saturated phenol were added to the pellets to stabilise cellular RNA. The cells were then re-harvested by centrifugation (8200g, 4°C and 2 minutes). The supernatant was aspirated and pellets re-suspended in 800 μl of lysis buffer (10mMTris, adjusted to pH 8.0 with HCl, 1mM EDTA) and 8.3 U/ml Ready-LyseTM Lysozyme Solution. After the pellets were re-suspended, 80μl of a 10% SDS solution was added, mixed and incubated for 2 minutes at 64°C. Then 88μl of 1 M NaOAc (pH 5.2) was mixed with the lysate followed by an equal volume of water and saturated phenol was added. This was incubated at 64°C for 6 minutes while inverting the tubes every 40 seconds. The aqueous phase was separated following centrifugation at 21,000g for 10 minutes at 4 °C. The RNA was precipitated from the aqueous layer using 1/10 volume of 3M NaOAc (pH 5.2), 1mM EDTA and 2 volumes cold EtOH and centrifugation at 21,000g for 25 minutes at 4°C. Pellets were washed with ice cold 80% EtOH and centrifuged at 21,000g for 5 minutes at 4°C. The ethanol was carefully removed and the pellets were air dried for 20 minutes. The pellets from each split

Solation of RNA from bacterial cell

Figure 1.

PCR protocol

Reverse Transcription–PCR reaction was performed in a 15.0μl final volume. Briefly, 1μl template cDNA (~40 ng) was combined with 1.0μl of forward primer (5nM), 1.0μl of reverse primer(5nM), 4.5 ml nuclease-free water and 7.5 μl of Taq 2X Master Mix. Thermo cycling was performed by 40 cycles at 95°C for 15 seconds, 60 °C for 15 seconds and 72°C for 15 seconds. Analysis of the PCR products was performed using 1.5% agarose gel solution in TBE buffer and visualisation was enabled by soaking gel in ethidium bromide solution for 10 minutes and UV-transilluminator. The data obtained were analyed using Graph pad prism version 6.01 description and frequency. statistic was generated to describe the diameter of inhibition. quantitative phytochemical constituent and toxicological prameter to test for the level of significance [1].

Gel electrophoresis

Assessment of Polymerase Chain Reaction products (amplicons) were electrophoresed in 0.5% of agarose gel using 0.5×TBE buffer (2.6g of Tris base, 5g of Tris boric acid and 2 ml of 0.5M EDTA and adjusted to pH 8.3 with the sodium hydroxide pellet) with 0.5μ lethidum bromide. The expression product was visualized as bands by UV-transilluminator [1] Table 1.

Results

The MazEFhomologous modules system and a Post-segregational killing mechanism (Bacteriostatic & Bactericidal mechanism) of novel compound isolated from Spondiasmonbinon Escherichia coli and Bacillus subtilis were demonstrated in Figure 1–6. The novel compound isolate from Spondiasmombin include the following, (A3-Epigallocatechin, Epicatechin and Stigmasterol phytosterol (synergy), A3-Aspidofractinine-3-methanol) and F3(Terephthalic acid, dodecyl 2-ethylhexyl ester) and the gene expression MazF9was observed in Escherichia coli and bacillus subtilis. Figure 1 shows Bacillus subtilis, mazEF homologous modules system and Post-segregational killing mechanism (Bacteriostatic & Bactericidal mechanism) of compounds A1(Epigallocatechin, Epicatechin and Stigmasterol Phytosterol (Synergy), A3(Aspidofractinine- 3-methanol) and F3 (Terephthalic dodecyl 2-ethylhexyl ester) by MazEF9 (Toxin/ antitoxin sensor. In Figure 2,3 and 4. It was observed that compounds A1, A3 and F3 have a deleterious effect on the cell (via DNA) of the test organism. At 180 minutes, the death phase was ascertained. This is graphically demonstrated in the figure1. Both MazE (toxin) and MazF (antitoxin) were produced, which both were used to activate the death phase of the cell through Post-segregational killing mechanism (Bacteriostatic & Bactericidal mechanism) of MazEFhomologous modules system on E. coli and B. Subtilis. The compounds have bacteriostatic and bactericidal effect on the test organisms (E. coli and B. subtilis). Figure 5 shows E.coli, mazEF homologous modules system and Post-segregational killing mechanism (Bacteriostatic & Bactericidal mechanism)of A1(Epigallocatechin, Epicatechin and Stigmasterol Phytosterol (Synergy),A3 (Aspidofractinine-3-methanol) and F3 (Terephthalic dodecyl 2-ethylhexyl ester)by gene expression MazEF9 (Toxin/ antitoxin sensor ) bacteriostatic and bactericidal effect as the mechanism of action compounds of A1, A3 and F3 as observed in (Figure 6–8) respectively.

Discussion

The purpose of the research work is to determine the Post-segregational killing mechanism (Bacteriostatic & Bactericidal mechanism) of novel compound isolated from Spondiasmonbinon Escherichia coli and Bacillus subtilis by MazEFhomologous modules system. action of compound A1 (Epigallocatechin, Epicatechin and Stigmasterol Phytosterol (Synergy), A3(Aspidofractinine-3-methanol) and F3(Terephthalic acid, dodecyl 2-ethylhexyl ester) with two Selected microorganism (Gram positive +ve), (Gram negative –ve) were demonstrated by toxin-antitoxin gene pairs (also called addiction modules) on extra-chromosomal elements of Escherichia coli, Bacillus subtilis All figs describe the mechanism of action of the isolated compound of ethyl acetate stem bark extract of Spondiasmombin extract. Nine of these TA systems belong to the mazEF family, encoding the intracellular MazF toxin and its antitoxin. Another mechanism of action of compound A1, A3 and F3 on the selected microbe E. coli and Bacillus subtilis is by probing for the toxin-antitoxin Maz F9 bio sensor. It was observed that in Figure 1–3. A1 MazF9, A3Maz F9 and F3 MazF9 stimulate the production of toxin/antitoxin system in the cell of the microorganism, used for this study and a greater effect were found between 0 to 15 minutes and decrease in effect were found at 30 to 180 minutes, this effect of toxin-antitoxin leads to the death of the organism between 0 to 180 minutes, the death ratio is measured and cells were destroyed or damaged by stimulation of toxin-antitoxin system. Toxin-antitoxin system are induced to measure the death phase at 0–180 minutes and this study was conducted to investigate the role of MazF9 to isolated compound. It was also observed that over expression of MazF9 induced a state of irreversible bacteriostasis consistent with the results of the previous study [13- 15]. The main finding of this work is that E. coli mazEF-mediated cell death can be triggered by medicinal plant that are general inhibitors of transcription and/or translation. it was shown that these Spondiasmombin reduce the cellular level of the antitoxic labile protein MazE and seem thereby to permit the lethal action of the toxic protein MazF. The effect of the Spondiasmombin both on cell death and on the reduction in the cellular level of MazE [16].

The isolated compound has a great effect on selected organism. It was observed that the effect of gene MazF9 has more lethal effects in E. coli, taking a cognizant from figure 5–8 represented by A1MazF9, A3MazF9 and F3MazF9. It was observed that the compound complete inhibits the E. coli at 180 minutes i.e. the compound has a better activity on E. coli than B. subtilis. The reasons for this activity must be as a result of their cell wall, the Gram negative (E. coli) has a greater activity because of a thin composition of the peptidoglycan cell wall compares to the gram positive (B. subtilis) which has a large composition of peptidoglycan in the cell wall. The gram negative is easily affected by antibiotic compound and compares the gram positive with very thick cell wall, this account for the activity of MazF9 more on E. coli than B. subtilis but their death rate was constantly measured, and toxin-antitoxin activity were clearly depleted [17].

In analogy to the programmed cell death apparatus in eukaryotic cells [18] reported that the suicide machinery in bacterial cells is always present, it is only requires a trigger to activate it. Moreover, at least for E. coli, the straightforward choice is death caused by a stable intracellular toxin (in this case MazF). The choice of life over the “default” death requires a dynamic antagonistic process manifested either by the continued production of the unstable antitoxin (in this case MazE) or by a process that would prevent the degradation of the unstable antitoxin. Thus, cell death could be caused by anything that would prevent the continuous expression of the antitoxic protein MazE [4]. It should be noted that toxin/ antitoxin system is widespread in bacterial genomes and contribute to prokaryotic stress adaptation and the formation of persister cells biofilms [19]. The mechanism of toxin components is to exert their effects in different ways by targeting essential cellular functions such as DNA replication, protein synthesis, cell division, peptidoglycan biosynthesis (see above) and ribosome assembly.

However, RNA cleavage is the most prevalent mode of action in the pathway [20]. Vasperet al., [21]reported that in E.coli (see above), one of the most well characterized toxin-antoxin gene is MazF9, an operan that encodes that intracellular MazF and its cognate antitoxin MazF9 toxin cleaves MRNA and DNA at 3,5 or 7 base recognition sequences in different bacteria and have been implicated in controlling cellular responses to various adverse conditions encountered by the bacteria in the host. Other mechanism of MazF9 in E. coli is MazF9 protein cleaves both MRNA and 16s ribosomal RNA and proposed to generate a subpopulation of stress ribosomes, thereby enabling the translation of leaderless transcript [22]. MazF9 system in E. coli are activated on exposure to numerous stress condition in an extracellular death factor-dependent manner. Another mechanism of action of compound A1, A3 and F3 on the selected microbes E. coli and Bacillus subtilis was demonstrated by probing for the toxin-antitoxin MazF9 gene. This effect of toxin-antitoxin MazF9 biosensor leads to the death of the organisms between 0 to 180 minutes. The compounds have more lethal effects as they completely inhibited the E. coli at 180 minutes. In addition, the compounds have a better activity on E. coli than B. subtilis. The reasons for this activity must be as a result of differences in their cell wall compositions.

This results in Figure 1–8, showing that the mazEF system is responsible for approximately 90% killing by Spondiasmombin and other medicinal plant and antibiotics alike may illuminate, the until now elusive cause of E. coli killing by medicinal plant. Furthermore, medicinal plant like Spondiasmombin are considered to be bacteriostatic and bactericidal in action [23]. It should be noted that toxin system is widespread in bacterial genomes and contribute to prokaryotic stress adaptation and the formation of persister cells biofilms [19]. The mechanism of toxin components is to exert their effects in different ways by targeting essential cellular functions such as DNA replication, protein synthesis, cell division, peptidoglycan biosynthesis (see above) and ribosome assembly. The mechanism of action of toxin-antitoxin gene MazF9 include RNA cleavage [20]., MRNA and DNA cleavage and protein cleavage of both MRNA and 16s ribosomal RNA [22].

Conclusion

This is better method of measuring the mechanism of action of novel compound by triggering the toxin/antitoxin cell death of inherent nature of bacterial cell.

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The Monoclonal, Massive Globulin- Waldenstrom Macroglobulinaemia- Juniper Publishers

Preface

Wald Enstrom macrogobulinaemia is a disorder designated with a nomenclature of a Swedish physician Jan Gosta Waldenstrom (1906-1996). The exceptional disease was initially scripted in 1944 [1,2]. Waldenstrom macroglobulinaemia may be defined as the appearance of a serum para-protein such as immunoglobulin M (Ig M) in addition to a malignant lymphoplasmacytic infiltrate confined to the bone marrow. Lymphoplasmacytic Lymphoma (LPL) may be cogitated as a neoplasm comprising of miniature B lymphocytes, plasmacytoid lymphocytes and mature plasma cells. The tumefaction generally implicates the bone marrow with an occasional presence in the lymph node and spleen. Lymphoplasmacytic lymphoma is accompanied by Waldenstrom macroglobulinaemia in a majority (95%) of instances [1,2]. The dual conditions may be denominated by an immunoglobulin M (Ig M) monoclonal gammopathy accompanied by an emergence of a lymphoplasmacytoid lymphoma restricted to the bone marrow. Lymphoplasmacytoid lymphoma may concur with an infection of hepatitis C virus (HCV). A familial prevalence may be delineated. An estimated 1.4% of neoplasm of miniature B lymphocytes may be cogitated by lymphoplasmacytoid lymphoma [1,2].

Disease Characteristics

Lymphoplasmacytic lymphoma may be categorized as a post germinal centre B cell (CD10-, MUM 1+ and BCL6+/-) lymphoma commingled with divergent plasmacytic cellular differentiation. The lymphoma may depict concomitant infection with Hepatitis C Virus (HCV). The bone marrow infiltrate may predominantly be interstitial, nodular or of a diffuse configuration. Bone marrow trephine biopsy and bone marrow aspirate may demonstrate an admixture of miniature lymphocytes, plasmacytoid lymphocytes and mature plasma cells [2,3]. The malignant cellular egress may enunciate a monotypic secretion of serum immunoglobulin M (Ig M) protein which may be elucidated in a majority (> 90%) of instances. Subjects with Waldenstrom macroglobulinaemia frequently depict vascular hyper-viscosity. Thus, Waldenstrom macroglobulinaemia may be cogitated as a non-Hodgkin’s lymphoma concomitant with lymphoplasmacytic lymphoma asmajority (95%) of subjects of lymphoplasmacytic lymphoma elucidate features of Waldenstrom macroglobulinaemia. The indolent lymphoplasmacytic lymphoma and concomitant Waldenstrom macroglobulinaemia may exemplify a disorder of obscure origin [2,3]. Associated aspects of probable disease insurgence may be

1. Male sex

2. Enhancing age of disease emergence with a median age of diagnosis at 65 years,

3. A racial predisposition in Caucasians

4. The concurrence of immunoglobulin M monoclonal gammopathy of undetermined significance (Ig M MGUS).

Waldenstrom macroglobulinaemia may progress to adjunctive B lymphocyte malignancies with an estimated proportion of 10% at 5 years, 18% at 10 years and 24% at 15 years of disease incurrence with an overall ratio of malignant conversion at 1.5 % per year. The neoplasm also displays a familial preponderance and nearly 20% individual’s manifest family members suffering from Waldenstrom macroglobulinaemia and associated B lymphocyte malignancies. Environmental factors such as exposure to radiation or agent orange, hazardous occupation with handling leather, rubber, paints, dyes and solvents, coexistent autoimmune disease and infection with hepatitis C virus (HCV) may be incriminated in the evolution of the malignancy [3,4].

Clinical Elucidation

The circulation of serum monoclonal immunoglobulin M (Ig M) in Waldenstrom macroglobulinaemia may display characteristic constitutional symptoms with concurrent deposition of monoclonal immunoglobulin M (Ig M) protein in several body tissues with a consequent emergence of auto-antibodies. Waldenstrom macroglobulinaemia may manifest systemic symptoms with an estimated serum monoclonal immunoglobulin M (Ig M) protein greater than 3 grams/decilitre and a bone marrow ingress of malignant lymphoplasmacytoid cells greater than 20%. Approximately one fourth (27%) instances of Waldenstrom macroglobulinaemia may be asymptomatic, with anaemia in roughly 38% subjects, the emergence of hyper-viscosity in around 31% individuals, the appearance of B symptoms (fever, weight loss, night sweats) in nearly 23% and neurological symptoms in about 22% of patients [1,2]. Waldenstrom macroglobulinaemia may depict specific complications such as hyper-viscosity, tissue aggregation of immunoglobulin M (Ig M) or autoimmune haemolysis secondary to circulating macro-globulins. Subjects may present with haematemesis, haemorrhage from the nasal cavity and retinal vasculature [5,6]. Anaemia, thrombocytopenia, elevated Erythrocyte Sedimentation Rate (ESR), lymph node enlargement and hepato-splenomegaly may ensue. A bone marrow trephine biopsy may exemplify an abundance of malignant lymphoid cells. Radiographic analysis of the implicated bones may be unremarkable, thereby excluding a multiple myeloma. Serum protein examination may detect the presence of an extremely high molecular weight protein,” a macroglobulin”, cogitated as an excess of immunoglobulin M [5,6].The quantification of monoclonal immunoglobulin M (Ig M) may be concordant with the magnitude of bone marrow infiltration and severity of systemic symptoms. Hyper- viscosity may appear as chronic haemorrhage from the nasal cavity, gingiva or gastrointestinal tract accompanied by headache, dizziness, loss of coordination or balance, impaired hearing with tinnitus with blurring or loss of vision. Retinopathy may ensue on account of distended retinal veins and swelling of the optic disc. Severely affected subjects may display manifestations of heart failure, drowsiness, stupor and coma. Systemic symptoms may be discerned at a quantifiable serum Ig M value greater than 4000 milligrams/decilitre, though immunoglobulin levels may vary. Constitutional or B symptoms such as fever, weight loss greater than 10% of the body weight in preceding six months, drenching night sweats and fatigue may appear [6,7]. Peripheral neuropathy may be a manifestation of the disorder. Cold agglutinin disease may occur on account of elevated circulating antibodies to red blood cells which may aggregate at minimal body temperatures and induce a haemolytic anaemia along with Raynaud’s phenomenon, jaundice and haemoglobinuria. Cryoglobulinemia may be encountered with the precipitation of immunoglobulin M (Ig M) at reduced body temperatures in order to obstruct the miniature blood vessels with emerging consequences such as Raynaud’s phenomenon, thrombocytopenic purpura, haemorrhaging ulcers and gangrene of the fingers, toes, nose and ears [1,2].

Amyloidosis may occur with the configuration of an anomalous “amyloid” protein” which may accumulate in tissues and organs of the body such as gastro-intestinal tract, renal and hepatic tissue or heart and peripheral nerves. Malfunctions such as carpal tunnel syndrome, malabsorption, macroglossia, dermal thickening, swelling of the extremities, congestive heart failure and renal failure may emerge. “BING NEEL” syndrome may be cogitated with a lymphoplasmacytic infiltrate or deposition of immunoglobulin M (Ig M) within the central nervous system (brain or spinal cord). Systemic symptoms such as mental deterioration, confusion, visual disturbance, irritability, altered personality, convulsions and coma may concur. Recurrent sinus and upper respiratory tract infection, pleural effusion, pulmonary infiltrates and occasional rash may be delineated. Tumour cells of lymphoplasmacytic lymphoma may configure nodular aggregates in the skin, extremities, spine, breast and orbital socket [6,7].

Morphological Elucidation

Waldenstrom macroglobulinaemia with coexistent lymphoplasmacytic lymphoma enunciates malignant cells with characteristics of B lymphocytes and plasma cells, denominated as lymphoplasmacytic cells. A diffuse or interfollicular proliferation of malignant lymphoid cells may be cogitated. Cellular aggregates devoid of proliferation centres may be proportionately constituted by miniature B lymphocytes, plasmacytoid lymphocytes and mature plasma cells. A predominant lympho-plasmacytic infiltrate may be situated in the inter-trabecular region of the bone marrow [3]. Peripheral blood picture concordant with acute leukaemia may be demonstrated in an estimated one third (30%) instances. Tumour cells may predominantly omprise solely of miniature lymphocytes or small, mature lymphocytes commingled with plasmacytoid lymphocytes. The bone marrow may be infiltrated by an identical malignant infiltrate. Mature plasma cells, tissue mast cells and histiocytes may be quantifiably augmented. Plasma cells may infrequently be the preponderant cellular component. Serial sections from bone marrow trephine biopsy may delineate a diffuse or a focal lesion.The focal lesions may configure a para trabecular, interstitial or non paratrabecular pattern of tumour incrimination [3]. Expansive marrow replacement by the tumefaction may induce a significant reduction of normal haematopoiesis. Intra-nuclear inclusions termed as “Dutcher’ s bodies” may be articulated within lymphocytes and plasma cells and may be considered diagnostic of lymphoplasmacytic lymphoma. Intra-nuclear inclusions reactive to Periodic Acid Schiff’s (PAS) stain may similarly be configured in plasma cells constituting a multiple myeloma or reactive lymphoid proliferations. Lymphoplasmacytic lymphoma with Waldenstrom macroglobulinaemia may terminate with the evolution of a Richter’s syndrome, thereby recapitulating a Small Lymphocytic Lymphoma (SLL). The malignant egress may lack the presence of monocytoid cells, in contrast to a marginal zone lymphoma. The occurrence of Dutcher ‘s bodies with admixed enlarged, transformed lymphocytes may be characteristic of lymphoplasmacytic lymphoma. Mast cells may be intermingled with epitheloid histiocytes. Intercellular material stained with periodic acid Schiff (PAS+) stain may be exemplified along with scattered amyloid and crystal engulfing histiocytes [7,8].

Immune phenotype

Lymphoplasmacytic lymphoma cells may exemplify a surface immunoglobulin M (Ig M+). Immunoglobulin molecules confined to the cytoplasm may primarily be immunoglobulin M (Ig M) although Ig G or infrequently Ig A may be elucidated. The lymphoma may be immune reactive to CD20+ and associated pan B lymphocyte antigens such as CD19+, CD79a+ and PAX5+. However, a percentage may be non eactive for the aforementioned immune markers. Nonreactive immune molecules may be CD5-, CD10-, CD23- and BCL6- although CD5 may be debatable (-/+) (1,3). Plasmacytic immune markers CD38 and CD138 may be equivocal (+/-) in specific instances.

Molecular Characterization

A frequent genomic abnormality the MYD88L265P mutation may be discerned in a majority (95%) along with chromosomal deletion of del [6] (q21). Chromosomal translocation t (9:14) may be exceptional. The MYD88L265P chromosomal mutation may be universal in Waldenstrom macroglobulinaemia. A whole genome sequencing may depict the mutation in 90% instances. MYD88L265P chromosomal mutation may be infrequent in multiple myeloma, marginal zone lymphoma or immunoglobulin M –monoclonal gammopathy of undetermined significance (Ig M MGUS). Chromosomal mutation CXCR4 may be enunciated which may be identical to the WHIM syndrome (warts, hypogammaglobulinaemia, infections and myelokathexis). Individuals with Waldenstrom macroglobulinaemia devoid of MYD88 or a CXCR4 chromosomal mutation may depict an inferior survival, in contrast to instances delineating the mutations [1,2].

Differential Diagnosis

Lymphoplasmacytic lymphoma may necessitate a distinction from Chronic Lymphocytic Leukaemia (CLL), mantle cell lymphoma and plasmacytoid variants of extra nodal or nodal marginal zone lymphoma. Chronic Lymphocytic Leukaemia (CLL) may exhibit a focal plasmacytic differentiation. The tumour cells may be immune reactive to CD5+, CD23+ and a CD20 dim, in contrast to a lymphoplasmacytic lymphoma. Immunoglobulin M (Ig M) para-protein may be absent or minimal. Splenic marginal zone lymphoma may demonstrate an intra-sinusoidal pattern of marrow incrimination. Plasmacytic differentiation may be reduced or minimal. Immunoglobulin M (Ig M) para-protein may be lacking or be of miniscule quantities [1,2]. Distinction from plasmacytoid lymphoma may be particularly cogitated with demonstration of plasma cells and plasmacytoid lymphocytes, manifesting numerous inclusions confined to the cytoplasm which may react to the Periodic Acid Schiff ‘S (PAS+) stain. The tumour cells may thus recapitulate the appearance of histiocytes. Chromosomal point mutation MYD88 may be elucidated in a majority (90%) of instances of lymphoplasmacytic lymphoma, contrary to an exceptional delineation in multiple myeloma and marginal zone lymphoma [8,9] (Figures 1-14).

Criterion for Discerning Variants of Waldenstrom Macroglobulinaemia

1. A monoclonal gammopathy with Immune Globulin M (IgM) irrespective of the magnitude of M protein and an infiltration of lymphoplasmacytic cells greater than 10% in the bone marrow may be elucidated. Particularly inter-trabecular tumour dissemination may comprise of miniature lymphocytes with a plasmacytoid or plasma cell differentiation and a characteristic immune phenotype of immune reactive surface immune globulin M (Ig M+), CD19+, CD20+ and nonreactivity for CD5-, CD10-and CD23- . The aforementioned evaluation may competently exclude adjunctive lympho proliferative disorders such as Chronic Lymphocytic Leukaemia (CLL) and mantle cell lymphoma [1,2].

2. A monoclonal gammopathy of undetermined significance (Ig M MGUS) may enunciate serum monoclonal immunoglobulin M (Ig M) protein values below 3 grams/ decilitre with a lymphoplasmacytic cellular infiltrate beneath 10% generally confined to the bone marrow. Systemic symptoms of anaemia, hyper-viscosity, lymph node enlargement or hepato-splenomegaly may be absent. Immunoglobulin M monoclonal gammopathy of undetermined significance (Ig M MGUS) may evolve into a florid Waldenstrom macroglobulinaemia or an adjunctive B lymphocyte malignancy. The proportion of malignant transformation may emerge at an estimated 1.5% every year.

3. Smouldering Waldenstrom macroglobulinaemia may be an indolent or asymptomatic disorder. Monoclonal serum protein values for immunoglobulin M (Ig M) exceeding 3 grams/decilitre and/or a lymphoplasmacytic infiltrate restricted to the bone marrow in excess of 10% may be enunciated. End organ damage with coexistent anaemia, hyper-viscosity, lymph node enlargement or hepato-splenomegaly on account of a lymphoplasmacytic proliferation may be absent [1,2].

Investigative Assay

Discernment of Waldenstrom macroglobulinaemia mandates a disease evaluation with complete blood count, serum chemistries such as liver and renal function tests, blood glucose and specific procedures such as bone marrow trephine biopsy and bone marrow aspiration. Serum immunoglobulin assay may depict an overproduction of immunoglobulin M with a decline in the values of immunoglobulin G and A (Ig G and I gA), feature which may enhance the probability of emergent infections. Radiographic investigations may include a plain X-ray, Computerized Tomography (CT) scan, a Magnetic Resonance Imaging (MRI), an ultrasound and Positron Emission Tomography (PET) scan of the lymph node enlargement, enlarged spleen and dermal or tissue infiltrates of lymphoplasmacytic lymphoma cells [9,10]. Ocular examination may incorporate the assessment of retina and ocular fundus.

Therapeutic Options

Commencement of therapeutic intervention may concur with the appearance of B symptoms such as fever, night sweats, weight loss, fatigue, lymph node enlargement or splenomegaly. Haemoglobin declining to below 10 grams/decilitre or a platelet count below 100,000/ microlitre may be cogitated on account of bone marrow infiltration. Additionally, complications such as hyper viscosity syndrome, symptomatic sensory or motor peripheral neuropathy, systemic amyloidosis, renal insufficiency or symptomatic cryoglobulinaemia may require therapy [10,11]. A policy of careful observation may suffice for the indolent disorder. An estimated half (50%) of the subjects with Waldenstrom macroglobulinaemia devoid of constitutional symptoms and a lack of treatment at 3 years following diagnosis may be managed with “active surveillance “. Approximately 10% instances may not require a therapeutic intervention during a 10year duration following detection. It may be crucial to establish the emergence of a hyper-viscosity syndrome prior to initiation of therapy and if plasmapheresis may be necessitated. Waldenstrom macroglobulinemia when associated with hyper-viscosity may display systemic symptoms such as visual deterioration, neurological symptoms and haemorrhage, incurring with immunoglobulin M serum values greater than 4 grams/decilitre and the condition may be benefitted with plasmapheresis [11,12]. Chemotherapeutic agents found efficacious in Waldenstrom macroglobulinaemia include chlorambucil, cyclophosphamide, bendamustine, nucleoside analogues such as fludarabine and cladribine. Corticosteroids prednisone or dexamethasone may be applicable. Biologic therapy may enunciate the utilization of anti monoclonal antibody conjugates such as rituximab, ofatumumab or obinutuzumab. Immune modulators such as thalidomide and lenalidomide may prove to be effective [1,2]. Administration of proteasome inhibitors such as bortezomib, carfilzomib and ixazomib may be advantageous. Targeted therapy implicating the B cell signalling pathway may concur as imbruvica and everolimus [12,13]. Initial or preliminary therapy for previously untreated, symptomatic subjects may employ

a. purine analogues with a combination of fludarabine, cyclophosphamide and rituximab (FCR) or fludarabine and rituximab (FR).

b. Alkylating agents in varying combinations such as rituximab with cyclophosphamide, doxorubicin, vincristine and prednisone (R CHOP), dexamethasone, rituximab and cyclophosphamide (DRC) or rituximab with bendamustine (BR) may be beneficial

c. Bortezomib in diverse combinations such as bortezomib, dexamethasone and rituximab (BDR) may be applicable.

d. Singular antibody conjugate such as rituximab may be utilized for initiation of therapy.

e. Ibrutinib as a Bruton’s tyrosine kinase inhibitor (BTK inhibitor) may be efficacious in instances with MYD88 mutation. Concomitant chemo immunotherapy may be administered

Administration of bendamustine with rituximab may depict a median and inter-quartile range (IQR) of 69.5 months in patients of Waldenstrom macroglobulinaemia [1,2]. The application of R CHOP may display a median and inter-quartile range (IQR) of 28.1 months. Solitary administration of rituximab in Waldenstrom macroglobulinemia may exhibit an objective response rate (ORR) of 52%, a partial response (PR) of 27% and a minor response (MR) of 25%. The median duration of response (DOR) may be elucidated at 27 months. An estimated half (54%) of the individuals may depict an elevated immunoglobulin M (Ig M) “flare” and one fourth (27%) subjects may delineate a persistent elevation of serum immunoglobulin M at 4 months duration following discernment of disease. Administration of ibrutinib may demonstrate an objective response rate (ORR) of 90.5%, a partial response (PR) of 73% and the median time to suitable therapeutic response may appear at 4 weeks. The progression free survival (PFS) at the end of 2 years may be 69.1% and Overall Survival (OS) at 95.2 %. Toxicity levels to the chemotherapeutic agent may be greater than grade 2. Ibrutinib administration may be accompanied by specific toxicities such as thrombocytopenia, neutropenia, stomatitis, atrial fibrillation, diarrhoea, herpes zoster infection, haematoma, secondary hypertension and epistaxis [13].

Contemporary Instances

Contemporary instances of Waldenstrom macroglobulinaemia may necessitate management as described:

1. Monoclonal gammopathy of undetermined significance (Ig M MGUS) with a lymphoplasmacytic infiltrate below 10%, an asymptomatic or smouldering Waldenstrom macroglobulinaemia with haemoglobin greater than 11 grams/decilitre, a platelet count in excess of 120,000/ millilitre may be managed by” watchful waiting”.

2. A symptomatic Waldenstrom macroglobulinaemia with a haemoglobin level below 11 grams/decilitre or a platelet count beneath 120,000/ millilitre.

a. Immunoglobulin M (Ig M) related neuropathy.

b. Haemolytic anaemia secondary to Waldenstrom macroglobulinaemia.

c. Symptomatic cryoglobulinaemia. The aforementioned instances may be managed with a solitary antibody conjugate such as rituximab. A maintenance therapy may not be required. Plasmapheresis may be opted for in instances of hyper-viscosity secondary to chemotherapy [1,2].

3. Waldenstrom macroglobulinaemia elucidating bulky disease (tumour magnitude greater than 10 centimetres) or profound pancytopenia with reduced blood counts such as haemoglobin below 10 grams/decilitre or a platelet count beneath 100,000/ millilitre with the appearance of constitutional symptoms and a lack of features of hyper-viscosity or hyper-viscosity may be managed with plasmapheresis.

The aforementioned subjects may be administered concomitant bendamustine with rituximab with the regimen devoid of maintenance therapy with singular rituximab. Stem cell transplantation may be a pre-requisite. Alternatively, stem cells may be mobilized and cryo-preserved for subjects beneath ≤ 60 years of age or emerge as potential and future candidates of Autologous Stem Cell Transplantation (ASCT). Stem cell transplantation may be suitably employed with subjects of relapsed or refractory Waldenstrom macroglobulinaemia. Autologous stem cell transplant (ASCT) may depict a 5year Progression Free Survival (PFS) of 40% and an overall survival (OS) of 68%. Allogeneic stem cell transplant may exhibit a 5year progression free survival (PFS) of 56% and a 5year overall survival (OS) of 62% [1,2]. Clinical trials with novel agents or drug conjugates may be mandated. Contemporary drugs such as ibrutinib, a Bruton’s tyrosine kinase inhibitor or idelalisib, a PI3kinase inhibitor or everolimus, an m TOR inhibitor may be efficaciously adminstered. Contemporary anti CD20 antibody conjugates such as of tamumab, anti BCL2 agents such as venetoclax, recent histone deacetylase (HDAC) inhibitors such as panobinostat, recent proteasome inhibitor carfilzomib, recent immunomodulatory agents such as pomalidomide may be additionally assessed. Contemporary targeted therapies may include molecules such as ventoclax, acalabrutinib and BGB3111. The aforementioned drugs may be utilized in combination with established agents.

Salvage Therapy

Salvage Therapy may be applicable in specific instances. In subjects where the requirement of subsequent therapy may exceed 4 years, the original therapeutic regimen may be replicated. For therapeutic installation within 4 years, a monotherapy with ibrutinib or combinations such as Dexamathasone, Rituximab and Cyclophosphamide (DRC) may be opted for. Concomitant administration of bendamustine with rituximab (BR), Bortezomib, Dexamethasone and Rituximab (BDR) may be effective.

Supportive Therapy

Supportive Therapy may incorporate modalities such as blood transfusion, administration of growth factors in order to enhance the blood cell counts (white and red blood cells with platelets). Surgical procedures may be specified in particular instances in the form of splenectomy or plasmapheresis in order to reduce the serum immunoglobulin M (Ig M) quantities. Targeted radiation may be employed in order to decimate the magnitude of incriminated lymph nodes [12,13] Table 1.

Table 1: Distinction betwixt Lymphoplasmacytic Lymphoma (LPL) and small cell Plasma Cell Myeloma (PCM) [1,2].

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Recent Trends and Strategies for Targeting M – Cells via Oral Vaccine against Hepatitis B: A Review- Juniper Publishers

Abstract

Background: The presence of a mucus layer that covers the surface of a variety of organs has been capitalized to develop mucoadhesive dosage forms that remain in the administration site for more prolonged times, increasing the local and systemic bioavailability of the administered vaccine. The emergence of micro and nanotechnologies together with the implementation of non‐invasive and painless administration routes has revolutionized the pharmaceutical market and the treatment of disease.

Objectives: To overcome the main drawbacks of the various routes and to maintain patient compliance high, the engineering of innovative drug delivery systems administrable by mucosal routes has come to light and gained the interest of the scientific community due to the possibility to dramatically change the drug pharmacokinetics.

Method: We review herein reported observations on nanoparticle (NP) mediated immunostimulation and immunosuppression, focusing on possible theories regarding how manipulation of particle physicochemical properties can influence their interaction with immune cells to attain desirable immunomodulation and avoid undesirable immunotoxicity.

Result: These results show that both HBV particles and purified HBsAg have an immune modulatory capacity and may directly contribute to the dysfunction of mDC in patients with chronic HBV. The direct immune regulatory effect of HBV and circulating HBsAg particles on the function of DC can be considered as part of the mechanism by which HBV escapes immunity.

Conclusion: NPs are recognized as self or there is an absence of immune recognition, this represents a major area of interest in the field of drug delivery. It is now well accepted due to their huge advantages and properties such as NP size, surface charge, hydrophobicity/hydrophilicity and the steric effects of particle coating can dictate NP compatibility with the immune system.

Keywords: Hepatitis B Virus (HBV); HBV Infection; Vaccines; Nanoparticles; Oral Mucosal Delivery System

Introduction

Hepatitis B virus (HBV) infection is the most common chronic viral infection in the world. An estimated 2 billion people have been infected and more than 350 million are chronic carriers of the virus [1]. In the 2018 Global Burden of Disease study, HBV infection ranked in the top health priorities in the world and the tenth leading cause of death (7,86000 deaths per year). These data have led WHO to include viral hepatitis in its major public health priorities. HBV is transmitted through contact with infected blood or semen [2,3]. Three major modes of transmission prevail. In areas of high endemicity, HBV is transmitted mostly perinatally from infected mothers to neonates, in low endemic areas, sexual transmission is predominant and third major source of infection is unsafe injections, blood transfusions or dialysis. HBV belongs to the Hepadnaviridae family. It is a partly double stranded DNA virus with approximately 3200 base pairs. The transcriptional template of HBV is the cccDNA, which resides inside the hepatocyte nucleus as a mini-chromosome.The maintenance of covalently closed circular DNA (cccDNA) is essential for the persistence of the virus [4,5].

The replication of HBV implicates reverse transcription of the pregenomic RNA intermediate into HBV DNA. Reverse transcriptase is error prone and the mutation rate is high (appendix). The receptor for HBV entry into hepatocytes is sodium taurocholate polypeptide [6]. These mutations abolish or down regulate the production of HBeAg without affecting the replication capacity of the virus and cause HBeAg negative chronic HBV infection. The precore and basal core promoter mutations can occur alone or together. HBV infection can be prevented by avoiding transmission from infected people and by inducing immunity in unexposed people. A safe and effective vaccine has been available since 1982, introduction of HBV vaccine led to a decrease in the incidence of not only HBV infection but also hepatocellular carcinoma [7,8]. Most experience available to date comes from using two recombinant. vaccines like Engerix-B (SmithKline Biologicals, Belgium) and RECOMBIVAX HB-Vax II (Merck & Co., USA). Admministered via different routes such as Pulmonary, Nasal, Intravenous (IV), Intramuscular (IM), Subcutaneous and Oral mucosal (OM) in the form of dried powder, liquid along with nanoparticles (NPs) [9]. Among of all DS NPs offers many potential advantages e.g. large surface area, site-specific delivery of drugs, peptides and genes, improved in vitro, in vivo stability and reduced side effect profile. However, NPs are often first picked up by the phagocytic cells of the immune system (e.g. macrophages), there may be undesirable interactions between NPs and the immune system whenever given via OM. Aim of the present study was to assess advantages of NPs and OM delivery system (DS) in comparison to the other formulation and site of administration [10].

Nps As Potential Delivery System of Vaccine

NPs interact with the immune system and effects on the immune cells may benefit treatment of disorders mediated by unwanted immune responses and enhance immune response to weak antigens [11–13]. On the other hand, undesirable immunostimulation or immunosuppression by NPs may result in safety concerns and should be minimized. One of the few studies on immunosuppression has demonstrated that inhalation of CNTs suppresses B cell function and that the TGF- produced by alveolar macrophages is a key element in the mechanism of the observed immunosuppression. Other studies have shown that NPs can be used to deliver immunosuppressive drugs and prevent immunosuppressive properties of small-molecule drugs. Similarly, allergen-loaded PLGA, chitosan, poly (lactic acid), poly (methyl vinyl ether-co-maleic anhydride) NPs, and dendrosomes have been reported as effective suppressors [14,15]. NPs are also evaluated for their immunostimulatory potential based on their ability to stimulate innate or adaptive immune responses. NP immunogenicity is drawing interest because NPs have been shown to improve antigenicity of conjugated weak antigens and thus serve as adjuvants because some NPs have been shown to be antigenic themselves. The former property has been shown to depend on particle size, surface charge and significantly contribute to the development of improved vaccine formulations [16]. Particle size has been reported as a major factor in determining whether antigens loaded into NPs induce type I (interferon) or type II (IL-4) cytokines, thereby contributing to the type of immune response [17]. A leading hypothesis on why nanotechnology formulations (Polymeric NPs, Nanoliposomes, Solid Lipid NPs (SLNs), Nanoemulsions) are effective in vaccine development is that nonsoluble NPs provide controlled, slow release of antigens, creating a depot at the site of injection and providing protection in the destabilizing in vivo environment [18,19].

M-Cell Targeted Mucosal Vaccine and Transport Mechanism Across the Intestinal Mucosa

Literature surveys were suggested that exploiting the potential of M-cell-specific mechanisms for drug and vaccine. delivery to the mucosal immune system. Many M-cell-targeted molecules have been used for development of mucosal vaccines [20–23].

M-cell-specific molecules in mucosal vaccine development

M cells express a large amount of immune-surveillance receptors on the apical surfaces, contributing to the variety of microbial pathogens and antigens [24]. They are provided with an array of molecules to present luminal antigens to the underlying mucosal lymphoid tissues. Therefore, identifying M-cell-specific targeting molecules has been a focus, by recognizing molecules exploited by pathogens to invade M cells [25–27].

Glycoprotein

GP2 is specially expressed on M cells; this protein is highly expressed on the apical membranes of Peyer’s Patch (PP) M cells, but not highly expressed on other enterocyte populations [28]. Recent studies have revealed that GP2 acts as a transcytotic receptor, bound to FimH+ bacteria such as Escherichia coli and S. Typhmurium, by recognizing FimH, a major component of the type 1 pilus on the outer membrane of a subset of Gramnegative enterobacilli [29,30]. Thus, GP2 on M cells can act as a transcytotic receptor for bacterial antigens, and worthy of note, participate in the mucosal immune responses to these particular bacteria; a subset of commensal and pathogenic enterobacteria (E. coli and S. Typhmurium) [31–33]. Other research has shown that a murine GP2 (mGP2)-specific aptamer, isolated using Systematic evolution of ligands by exponential enrichment (SELEX), with a loop structure and the nucleotide sequence, AAAUA (both important for binding to mGP2), binds to mGP2 expressed on the cell surface, indicating that the aptamer serves as a promising tool for testing M-cell-targeted vaccine delivery in murine model systems [34].

Cellular Prion Protein (PrPC)

PrPC is highly expressed on the luminal side of the apical plasma membrane of murine M cells and co-localized with GP2, suggesting that it is an antigen receptor candidate on M cells [34- 36]. PrPC interacts with heat shock protein (Hsp) of B. abortus, which had been recognized as an immunodominant antigen of many microbes. Accumulated evidence suggests that PrPC on M cells is well placed to contribute to mucosal immunosurveillance by enhancing transcytosis of B. abortus or other exogenous antigens [37–39].

C5a Receptor (C5aR)

The expression and nonredundant role of C5aR in human M-like cells and mouse M cells have been demonstrated, indicating the role of C5aR as a target receptor to induce the immune response [40]. Sae-Hae Kim et al. verified phosphorylation of C5aR in vivo after oral infection of mice by Yersinia enterocolitica. They confirmed the expression of C5aR in the apical area of mouse M cells and human M-like cells by measuring the expression levels of mRNA and protein [41- 43]. Sae-Hae Kim et al. also used the outer membrane protein H (OmpH) ligand of Yersinia enterocolitica, which acts as a targeting ligand to C5aR in M cells, to induce specific mucosal and systemic immunity against envelope domain III (EDIII) of dengue virus (DENV), suggesting OmpH — mediated targeting of antigens to M cells as an efficient oral vaccination against DENV infection [44].

Other specific molecules

There are other M-cell-specific molecules that may specifically bind to components of potential pathogenic organisms [45]. Peptidoglycan recognition protein 1 is an innate recognition protein binding to bacterial peptidoglycan and is also expressed highly in M cells53. Annexin (ANX) A5 expressed by M cells can bind to lipopolysaccharide (LPS) of Gramnegative bacteria and block endotoxin activity, suggesting that ANXA5 on M cells acts as an uptake receptor for Gram-negative bacteria [45]. The discovery of M-cell-targeting receptors using pathogen-exploited molecules could be a promising approach in the development of effective mucosal vaccines. Clusterin, fatty acid binding protein, cathepsin E, secretogranin V and other M-cell-expressed proteins may have potential roles in M cell functions, but these are less clearly understood [46–47]. The increasing evidences have demonstrated that M-cell-specific molecular antibody, which is conjugated with antigen protein or liver vector, can transport the antigen to mucosal tissues, leading to produce efficient immune responses [48]. However, some molecules, selected as M-cell-specific molecules, are not uniquely expressed on M cells, resulting in producing a non-ideal oral delivery system for targeting M cells.With the development research on the mechanism of M cell differentiation, we can regulate the immune processes by means of artificial mediation of the M-cell-specific molecules gene expression [49,50]. For instance, we can increase the efficiency of mucosal vaccination, through booting the expression of certain M-cell-specific molecules. Meanwhile, we can even inhibit the viral infection, by reducing the expression of some molecules, which are necessary for the entry of some virus particles [51].

M cell ligands as novel and effective mucosal vaccine targets

Many researchers have studied M cell ligands, in order to take advantage of the fact that targeting specific receptors on the apical membrane of M cells could specifically increase antigen uptake and presentation, evoking immune responses and providing protection against Infection [52,53].

Co1 ligand

Many studies have investigated the M-cell-targeting ligand, Co1, selected from a phage display library against differentiated M-like cells, and have produced recombinant antigen fused to the selected ligands using the model antigen [58]. Co1 ligand promotes the uptake of fused antigen and enhances the immune response against the fused antigen, indicating that Co1 could be used as an adjuvant for targeted antigen delivery into the mucosal immune system to enhance immune induction [59]. Another promising approach used Co1 ligand to induce specific immune responses against a pathogenic viral antigen, EDIII of DENV. Efficient antigen delivery into PPs was observed and the antibodies induced by the Co1-ligand-conjugated EDIII antigen showed effective virus-neutralizing activity. Taken together, these results reveal that M-cell-targeting ligands with adjuvant activity can be designed to exploit our knowledge of receptors expressed on the apical surface of M cells involved in pathogen invasion [60, 61].

Caveolin-1

Caveolin-1 is the major structural component of caveolae. It was examined its expression in Caco-2-driven M-like cells, and was verified that co-culturing with B lymphocytes, caveolin-1 could increase the susceptibility of M cells to Salmonella infection. Some recent studies have shown that caveolin-1 is not only a good marker of human M cells, but also a potent candidate for understanding M cell transcytosis as a novel target for mucosal immunity [62].

Ulex europaeus agglutinin (UEA)-1

UEA-1 has been confirmed as a specific ligand for α-Lfucose present on the apical membrane of M cells, anchored for selective delivery of antigen to GALT. Some researchers have used NPs coated by UEA-1-conjugated alginate to induce immunological response in BALB/c mice and compared them with aluminum hydroxide gel-based conventional vaccine [63]. The results demonstrated that immunization with the former induced efficient systemic as well as mucosal immune responses against BSA compared to other formulations, which indicated the potential of UEA–alginate-coated NPs as an effective oral delivery system. However, UEA-1 lectin also reacted strongly with other issues, such as goblet cells and the mucus layer covering the intestinal epithelium [64].

Reovirus surface protein α 1 (pα1)

pα1 has the ability to bind M cells, which facilitates reovirus infection via pα1. A genetic fusion between ovalbumin (OVA) and pα1 was applied nasally, to enhance tolerogen uptake [65]. Studies showed that OVA– pα1-mediated tolerance was lost in the absence of interleukin-10, demonstrating that the feasibility of using pα1 as a mucosal delivery platform specifically for low-dose tolerance induction. Another targeted transgene vaccination using pα1 conjugated to polylysine through intranasal immunization, could induce mucosal immunity and enhance cell-mediated immunity, leading to prolong mucosal IgA and produce antigen-specific serum IgG [66].

The number of M cell receptors and their ligands that have been identified so far is limited, and most of them are not just expressed in M cells, but in neighboring enterocytes as well. Tolllike receptor (TLR)-4 and α5β1 integrin, belonging to pathogen recognition receptors (PRRs), are expressed on the surface of human and mouse M cells [67]. Interaction between these innate immune system molecules with pathogen-associated molecular patterns is essential for bacteria translocation across the lumen. Nevertheless, PRRs are also expressed in other enterocytes and not merely in M cells. For example, α5β1 integrin is both dispersed on the lateral and basolateral surfaces of enterocytes and on the apical surface of M cells, which is a challenge in targeting M cells alone [68].

M-cell-targeting ligands can enhance the uptake of oral vaccines by M cells and improve antigen-specific immune responses in both mucosal and systemic immunity. It seems that targeting ligand to antigen is a very promising approach in the development of efficient mucosal vaccine. However, simple targeting of antigen to M cells does not ensure the production of efficient protective immunity. We should pay more attention to the ligand study and find out the “optimal transporter”, presenting antigens to M cells, leading to efficient immune responses [69].

Immune-Olerance

The antibody response to HBV — envelope antigens (HBsAg) is a T-cell dependent process.6 Antibodies to HBsAg serve as neutralising agent. These neutralising antibodies are especially important in the prevention of viral infection, since they could prevent viral attachment and entry into the cells by absorption of the viral particles. Induction of anti-HBs alone during prophylactic vaccination is often sufficient to completely prevent viral infection, irrespective of whether this is the only operative defence mechanism against the viral infection during the course of natural infection [70]. The antibody is detectable in patients who have recovered from acute hepatitis B and in people immunised with HBV vaccine, but it could become undetectable in patients who have recovered fully from infection. Antibody to HBcAg (Hepatitis B core antigen) is detected in virtually all patients who have ever been exposed to HBV. Unlike antibody to HBsAg this antibody is not protective; its presence alone cannot be used to distinguish acute from chronic infection [71].

The HBV-specific T-cell and B-cell responses are generally undetectable. The exact mechanism by which HBV escapes immunity is still not known. Dendritic Cells (DC) play an important role in antiviral immunity and have the unique capacity to activate naive T cells and stimulate B and natural killer cells. Both circulating and tissue-resident immature DC sample the environment for the presence of foreign antigens and upon activation, DC migrate to lymphoid tissues to initiate immune responses [72]. Depending on their maturation status, represented by the expression level of costimulatory and Human Leucocyte Antigen (HLA) molecules and the capacity to produce proinflammatory cytokines, DC can induce either immunity or tolerance. Immature and semi-mature DC are associated with tolerogenic responses, so in the context of HBV a defect in the maturation process of DC may lead to tolerogenic T-cell responses and HBV persistence [73].

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Nasopharyngeal Lymphoma in a developing Community- juniper Publishers

Abstract

The nasopharyngeal lymphoma has been reported from countries as far apart as China, Hong Kong, India, Korea, Morocco, Tunisia, and USA. Therefore, this report comes from Nigeria with special reference to the Ibo ethnic group. It was found that mostly young patients of either sex were involved. Follow-up data are not available.

Keywords: Nasopharynx; Lymphoma; Age; Nigeria; Ibos

Introduction

Squamous cell carcinoma is defined as “a malignant epithelial neoplasm exhibiting squamous differentiation as characterized by the formation of keratin and/or the presence of intercellular bridges” [1]. This was illustrated in a case report from India [1] as well as from Saudi Arabia [2] and from the UK [3]. Therefore, this paper presents a series from the Ibo ethnic group’s developing community [4], this being done on account of the author’s headship of a histopathology data pool such as the one fostered by a Birmingham (UK) group [5]. To facilitate matters, the tabular form is used as shown hereunder.

Investigation

As the pioneer pathologist in charge of the Regional Pathology Laboratory established in Enugu by the Regional Government, I was able to provide Histology Request Forms which stipulated the required epidemiological data. Moreover, as I kept personal copies of all the results, their analysis was facilitated as in this study.

Results

The tabular form is deemed to be practicable Table 1

Discussion

The cohort consisted of 5 patients. It is clear that relatively young patients were involved. In this context, as the US authors generalized concerning nasopharyngeal malignancies in children, “They are, almost without exception, either lymphomas, rhabdomyosarcomas or nasopharyngeal carcinomas” [14]. Curiously, of the single case reports from India [6], and Korea [8], both were old. Indeed, in the publications dealing with numerous cases, the mean age was 46 years in China [3], 52.7 years in another Chinese report [9], and 59.3 years in USA [11]. In contrast, the local mean age is only 27 years. As for sex, our cohort showed male preponderance of 5 out of the 8 cases. The ratio was 84 males to 28 females in China [3].

Incidentally, unlike these series in which the treatment was at issue, as it was in China [1–4], the present paper differs. Thus, the practice here is to make the diagnosis available to the clinicians who provided the biopsy itself. The doctors were encouraged to even hazard a provisional diagnosis. Apparently, although all were impressed by the malignant disposition, only three correct diagnoses were made in terms of lymphoma.

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Lymphoma of the vulva in Nigeria: Case Report- Juniper Publishers

Abstract

Lymphoma of the vulva is acknowledged to occur rarely. Thus, only single case reports came from 9 countries worldwide. Therefore, a typical case is reported here from Nigeria, the patient hailing from the Ibo ethnic group. It is concluded that the series collected from USA provided a good platform for appreciating the epidemiology of this rare tumor including the ranges in age.

Keywords:  Vulva; Lymphoma; Single cases; Collected cases, Epidemiology, Nigeria

Introduction

Vulva lymphoma is acclaimed to be rare. This is attested to by the single case reports emanating from countries as far apart as Italy [1,2], Japan [3], Korea [4], Spain [5], Switzerland [6], and Turkey [7]. Therefore, this single such report comes from Nigeria, especially as it is from the Ibo ethnic group [8].

Case Report

OB, a 47-year-old woman, attended our Institution where she first saw Dr Arthur C. Ikeme in Ward 10 with a large fungating single mass involving the right part of the mons and right labium majus. Biopsied five irregular pale pieces were submitted to the corresponding author. The largest showed hairy skin 4cm across. Microscopy revealed ulcerated skin undermined by sheets of plump, round, hyperchromatic, tumor cells. They showed no differentiation. Therefore, Non-Hodgkin’s lymphoma was diagnosed.

Discussion

Oddly enough, the USA contribution concerned 6 cases [9]. These patients were aged from 43 to 71 years (mean 60 years). In this range is the local case as well as those from Italy [2], Japan [3], Korea [4] and Spain [5]. Outside the range are the patients from Switzerland [6] and Turkey [7]. What of the exactitude of the sites of origins? The Italian had “a non-tender mass in the upper part of the left labium major” [2]. The Japanese was described as suffering from “vulvar swelling” [3]. The Korean was having “a mass in the right upper labium” [4]. The Spaniard exhibited “vulva lymphoma predominantly involving the clitoris” [5]. Finally, the Swiss “patient underwent biopsy of the vulvar nodule” [6].

As regards treatment, the Japanese patient failed to respond [3]; while, in a Spanish woman, the “response to chemotherapy was good and the patient remains asymptomatic after three years of follow-up.” Concerning the group of 6 USA patients, who were clinically followed up, deaths occurred among 4 of them [9]. Incidentally, our local patient did not have any follow up. This was because she emerged strictly from a histopathology data pool as was advocated for easily facilitating epidemiological analysis by a Birmingham (UK) group [10]. Thus, it is important that what eventually emerged included the age patterns.

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Undifferentiated Carcinoma of the Larynx in a Developing Community- Juniper Publishers

Abstract

A rare form of laryngeal carcinoma is the undifferentiated form. There are but a few cases in the literature. Therefore, two cases from among the Ibos of South Eastern Nigeria are deemed to be worthy of documentation.

Keywords: Larynx; Undifferentiated carcinoma; Ibos; Males; Rarity

Introduction

A search of the Internet revealed but a few reports on the larynx exhibiting undifferentiated carcinoma. From Turkey [1], a 7-year-old male patient was reported to have “Undifferentiated laryngeal carcinoma with pagetoid spread.” From both France [2] and India [3], the authors went as far as to differentiate undifferentiated carcinoma of “nasopharyingeal type.” In this context, another differentiation was mooted in USA as the “small cell” type [4]. Therefore, the author presents two cases on the strength of carcinomas which were “undifferentiated,” the patients being of the Ibo ethnic group [5].

Case Report

1. NA, a 78-year-old Ibo man, presented to Dr. Udeh at the University of Nigeria Teaching Hospital, Enugu, with the history of cough, hoarseness and weight loss that had lasted for a year. Tracheostomy tube was inserted. Direct laryngoscopy was used to take a biopsy. Thereafter, the author received pale and dark fragments. On microscopy, much cartilage was seen with malignant epithelial cells whose differentiation was not discerned. Accordingly, it was diagnosed as undifferentiated carcinoma.

2. NF, an 82-year-old Ibo man, attended the Balsam Clinic at Enugu under Dr. Ezeanolue. Hoarseness was of a year’s duration. Difficulty in swallowing and stridor were lasting for 3 months. A few small, pale fragments were biopsied. The corresponding author received them. On processing, malignant epithelial cells were seen, no differentiation being apparent. Accordingly, undifferentiated carcinoma was diagnosed.

Discussion

In pursuit of these researches, the useful pool had come from Birmingham (UK). The group there had hypothesized that the fruit is “epidemiological analysis” whereas planting the tree constituted “the histopathology data pool” [6]. So far, apart from another “undifferentiated” carcinoma [7], the past examples in my collection are the following: clear cell [8], papillary [9], adenoid cystic [10], inflammatory [11], and mucoepidermoid [12]. Curiously, the connection between the “undifferentiated” carcinoma and the “differentiated” type emerged in Japan [13]. There, a 78-year-old man presented with undifferentiated carcinoma of the anal canal. However, at necropsy, it was found that he had “metastatic squamous cell carcinoma to inguinal nodes.”

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Distinct Pathways of Regulation Seed Dormancy and Germination by Abscisic Acid- Juniper Publishers

Abscisic Acid (ABA) is the phytohormone inducing seed dormancy, whereas seed germination occurs at low ABA concentration. Two pathways of their regulation are considered. One represents ABA reception and its signal transduction to ABA-responsive genes to induce dormancy genes, a modification of this way leading to germination in the absence of ABA. Another way suggests direct ABA inhibition of plasmamembrane H+-ATPase in dormant seeds that prevents germination and its activation in the absence of ABA, thus promoting seed germination.

Keywords: Seed dormancy; Germination; Transcription of dormancy genes; Activation of plasma membrane H+-ATPase.

Introduction

After the discovery of ABA receptors belonging to PYR/PYL/ RCAR protein family [1,2], and mechanism of their functioning [3], the components of signal system transducing the ABA signal to nuclear ABA-sensitive genes were identified [4,5]. According to the modern point of view, this regulatory pathway is common for the induction of primary seed dormancy and its maintenance. However, dormancy release includes the ABA degradation [6], and non-dormant seeds contain low ABA concentrations, which do not prevent seed germination [7,8]. In the absence of ABA, this signal system is modified to make the germination possible [9,10]. Figure 1 schematically describes such model of ABA regulation established on Arabidopsis seeds. ABA signaling system in seeds consists of three components, namely protein phosphatases, protein kinases and transcription factor ABI5.

Protein phosphatases PP2C, known also as ABI1 and ABI2 factors, negatively regulate ABA responses. Arabidopsis seeds have six genes of PP2Cs, the expression of which results in the production of these proteins taking part in the formation of the ternary complex with ABA and its receptor and in modulating the phosphorylation activity of protein kinases. Three protein kinases SnRK2 take part in ABA signaling. They mediate phosphorylation and activation of transcription factor ABI5- the step critical in the dormancy and inhibition of germination.

ABI5 protein always has a phosphorylation site to be activated by SnRK2 protein kinases. This ABA-responsive activation is accompanied by its stabilization during dormancy induction and maintenance. The participation of ABI5 permits the expression of ABA-responsive dormancy–specific genes. The degradation of ABI5 is necessary for the initiation of germination. It begins by ABI5 dephosphorylation by another family of serine/threonine protein phosphatases, namely FYPP/PP6 phosphoprotein phosphatases, followed by its breakdown through the 26S proteasome-dependent pathway. ABI5 dephosphorylation is an essential prerequisite for eliminating the inhibitory effect of ABA on germination. The abolishment of ABI5 transcription factor causing subsequent changes in gene expression is a key event for seed germination.

The successful functioning of ABA reception and subsequent transduction of the signal to the corresponding nuclear genes determines the so-called ABA sensitivity; any disturbance of receptor — signaling system, for example in mutants, results in a loss of ABA sensitivity.. The list of genes involved in dormancy regulation was presented by Graeber et al. [11]. In dormant Arabidopsis seeds gene expression is enriched for proteins involved in ABA synthesis, gibberellin catabolism, protein kinases, protein phosphatases, numerous transcription factors, and typical maturation proteins, namely small heat shock proteins and Late Embryogenesis Abundant (LEA) proteins [12,13]. Special attention was paid to DOG1 protein regulating seed dormancy induction and delaying germination by interfering with PP2C protein phosphatase activity [14]. The translation of proteins occurs slowly in dormant seeds.

The set of germination proteins radically differs from the proteins translated during dormancy. In imbibing and germinating seeds, translational machinery operates much more rapidly than in dormant seeds [15]. Protein composition depends first of all on mRNAs stored during maturation [16,17]. At radicle emergence state and after germination, de novo transcription of other genes starts. As a result, germinating seeds contain the complete set of “house-keeping” enzymes providing glycolysis, TCA cycle, gluconeogenesis, glyoxalate cycle, amino-acid metabolism, respiration, pentose phosphate cycle, ribosome functioning and other processes constituting main metabolic and energy utilization systems. They contain also a set of enzymes participating in the mobilization of the reserves (proteins, starch and lipids) as well as aquaporins and gibberellin synthesizing enzymes. Numerous transcription factors are produced while the components of ABA signal system are downregulated. Some maturation proteins still are produced (LEA and heat shock proteins), but stress-proteins and cell cycle proteins appeared. The list of proteins identified in germinating rice embryos is presented in [18].

We have considered the pathway of seed dormancy and germination regulation based either on inhibitory action of ABA on growth initiation resulting in dormancy or on the absence of such ABA action leading to germination commencement (Figure1). This pathway operates through the receptor — signaling–transcription system. However, quite ananother regulatory pathway can function in parallel, namely via inactivation (in the case of dormancy) or activation (in the case of germination) of the enzyme H+-ATPase responsible for H+ ions transfer from cytoplasm through the plasma membrane to cell wall. H+ efflux is accompanied by K+ infux into cytoplasm. The acidified cell walls acquire the ability to activate the enzymes modifying the structure of hemicellulose polymers and of expansin proteins facilitating the displacement of polymer chains relative each other. As a result, activated enzyme increases the extensibility of cell wall, i.e. the prerequisite for growth initiation. The cell extension itself begins under the pressure of water entering cell. In addition, K+ ions delivered to cytoplasm support the osmotic potential and favor water uptake. In the presence of inhibitory ABA concentrations, no activation of plasma membrane H+-ATPase occurs.

Plasma membrane H+-ATPase (proton ATPase) is a transmembrane protein, the most part of the molecule is situated in cytoplasm while five loops are localized outside the membrane [19]. In cytoplasm, both hydrophilic N and C termini as well as three domains are located, namely for binding nucleotides, for ATP hydrolysis and for activity regulation. Two activity states of enzyme are known: an autoinhibited state, when ATP hydrolysis is only loosely coupled to H+ transport, and an upregulated state with tight coupling between ATP hydrolysis and H+ pumping. In autoinhibited state, eight times more ATP is required to pump the same number of H+ ions as in upregulated state. In autoinhibited state, the regulatory C domain is pressed into the intramolecular binding site of protein [20]. Both N and C termini together take part in its detachment, apparently due to a structural rearrangement making the penultimate threonine residue accessible for protein kinase-mediated phosphorylation and subsequent activation of the enzyme [21].

The activation occurs inside the terminal region of regulatory domain at the level of penultimate threonine, the phosphorylation of which creates a binding site for 1–3–3 protein, which stabilizes the upregulated state of the enzyme, i.e. stabilizes its conformation [19]. Fusicoccin, a specific activator of plasma membrane H+-ATPase [22], binds to the enzyme simultaneously with 14–3–3 protein, its molecule being situated within the cavity in the interaction surface between H+-ATPase and 14–3–3 protein to tightly couple both partners [19]. In the upregulated state, the enzyme actively extrudes the H+ ions to cell walls [23]. The activity of plasma membrane H+-ATPase is measured either by rate of ATP hydrolysis, usually by the release of inorganic phosphate, or by H+ ion extrusion, estimated by acidification of ambient solution or by pH shift recorded by indicator color. In the experiments, fusicoccin is applied as a specific activator, whereas vanadate and diethylstilbestrol are applied as the inhibitors of ATP hydrolysis.

The capacity of plasma membrane H+-ATPase to acidify cell walls and promote cell elongation is well-known as an “acid growth” (acid-induced growth) exhibited in coleoptiles and hypocotyls, the growth of which starts after radicle protrusion [24,25]. However, no data on the participation of plasma membrane H+-ATPase in seed germination is available in literature. We have observed the acidification of cell walls in broad bean, wheat and horse chestnut seeds prior to radicle protrusion that is prior to growth beginning in the embryos of germinating seeds [26,27]. Such acidification is similar to the effect of acid buffer, it is stimulated by fusicoccin that confirms the participation of this enzyme. Analogous activation of ATP hydrolysis occurs just before growth initiation, which is blocked by vanadate. These data indicate a direct participation of the enzyme in seed germination. The experiments with seed incubation in the solutions of fusicoccin stimulating radicle emergence or in diethylstilbestrol and vanadate inhibiting it have confirmed the conclusion.

In seeds, proteomic analysis failed to record the presence of plasma membrane H+-ATPase, because the method does not permit to see the proteins of high molecular weight. The mol. wt. of this enzyme is about 100 kDa. The enzyme can be identified by western blotting and application of specific antibodies. It was shown in broad bean embryo axes in the course of imbibition, radicle emergence and growth [27]. Similarly, in non-dormant horse chestnut seeds, the enzyme protein was identified in imbibing, protruding and growing embryo axes. In all samples, 14–3–3 protein was also present [27]. The plasma membrane H+-ATPase protein was also shown in the embryos of dry maize seeds, they exhibit a weak activity after short 5h-imbibition, not accompanied by protein synthesis [28,29]. The presence of the enzyme in dry seeds shows that it was synthesized and accumulated earlier, apparently during seed maturation. In seed embryo axes, the delay of imbibition by PEG-6000, an osmoticum, retards the acidification, which needs about 68- 70% water content for enzyme activation. Therefore, during imbibition the transition of autoinhibited form of H+-ATPase to an active one occurs. We followed this transition in horse chestnut seeds, which experience the state of deep physiological dormancy and then dormancy release to acquire the capacity of rapid germination after four months of cold wet stratification [29]. The long period of dormancy and dormancy release culminated in the enzyme activation and germination indicates rather prolonged time of the transition from autoinhibited form to an active one.

In dormant seeds, the rate of cell wall acidification in embryo axes is rather low at the initial stage of embryo dormancy, but twofold increased after 6th week of stratification up to the level typical of emerged embryos, when seeds experience only coatimposed dormancy [29]. During embryo dormancy, fusicoccin does not exert any stimulating effect either on H+ ion extrusion into cell walls or on the size of embryos themselves. It means that during embryo dormancy plasma membrane H+-ATPase is still autoinhibited. Its activity and responsiveness to fusicoccin significantly rose after embryo dormancy, when water content in embryo increased and cell preparation for germination occurred. Such activation of the enzyme is not related to indolyl-acetic acid as it was shown for coleoptiles and hypocotyls [24,25], i.e. for postgerminative growth, because the dormancy release and germination of horse chestnut seeds do not respond to the treatments by indolyl-acetic acid or gibberellin.

The difference in plasma membrane H+-ATPase behavior over the whole period of dormancy and dormancy release is apparently due to the dynamics of ABA in embryo axis tissues. The content of endogenous ABA manifold increased during the period of embryo dormancy and then declined to very low level by the germination commencement [30]. ABA accumulation correlates in time with the autoinhibited state of the enzyme. The treatment of horse chestnut embryos with 10–7 M ABA did not inhibit the acidification over this time interval, 10–6 M influenced weakly, while 10–5 M ABA actively inhibited the H+ extrusion, apparently because the concentrations of endogenous and exogenous ABA summated. Therefore, the target of ABA inhibitory action on dormant seeds is plasma membrane H+- ATPase resulted in the cessation of H+ ion delivery to cell walls, the prerequisite of cell growth initiation. Decline of endogenous ABA content leads to the activation of the enzyme and commencement of germination. In non-dormant seeds lacking ABA, the transition of the enzyme to active state is related to an increase in water content.

The described pathway of ABA action on seed germination differs from that described above, namely from the receptor — signaling — transcription system, in two aspects: this pathway is shorter (no ABA–receptor activation) and it does not directly depend on the transcription of new mRNA in imbibing seeds, as follows from the experiments with α-amanitin , the inhibitor of the new mRNAs transcription. After radicle protrusion, when the growth of seedling organs occurs, a postgerminative program includes active operation of plasma membrane H+-ATPase [19,31]; the inhibitory action of ABA on their cell elongation is explained by ABA interaction with the receptor, ABA inhibition of protein phosphatase and ABA activation of protein kinase leading to the phosphorylation of an unknown amino acid residue in regulatory domain of the enzyme. This chain of events can result in the inhibition of enzyme, that in its turn causes K+ efflux from cytoplasm and its acidification due to the damage of H+ ion transport by the enzyme [31]. These observations might give insight into the transition from germination program of seeds to postgerminative program of seedling development.

Acknowledgements

In our patient the rare combination of High-risk HPV, Lymphoma and High grade squamous intraepithelial lesion was quite interesting. But the efficient interdisciplinary discussions gave her a wonderful outcome.

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High Risk HPV, HSIL and Primary Diffuse Large B Cell Lymphoma of Cervix: An Unsual Case- Juniper Publishers

Abstract

Primary malignant non-Hodgkin’s lymphomas in the female genital tract are rare, accounting for less than 1% of all extranodal non-Hodgkin’s lymphomas. HPV infection has well been documented as the causative factor for Carcinoma cervix; not malignant lymphoma of cervix. Here we report an interesting case of primary non-Hodgkin’s lymphomas of uterine cervix with proven HPV 16 infection and High grade squamous intraepithelial lesion with co-existing diffuse large B-cell lymphomas (DLBCL) on histopathological examination and immunohistochemical study. The patient was successfully treated with chemotherapy after Cervical Conisation for HSIL and is now in complete remission with follow up period of 11 months. Gynecological lymphomas can be a diagnostic challenge due to nonspecific symptoms. However, when diagnosed and treated they have a good prognosis. Clinicians should therefore include lymphoma in their differential diagnosis when investigating gynecological symptoms.

Case report

28-year-old lady went for a routine gynecological checkup where a Pap smear was taken. The report came as Low grade squamous intra-epithelial lesion (LSIL). HPV test was done; it came as positive for genotype 16 which is consistent with high risk HPV, which is a proven carcinogenic virus for cervical cancer. Colposcopic biopsy soon followed and it showed (Figure 1) High grade squamous intraepithelial lesion (HSIL) (CIN III). Cervical LEEP excision was done to cure the disease. The specimen measured 4.5x3x1cm and was oriented at 12 o’ clock. It was inked and serially sliced. The sections showed foci of epithelial dysplasia involving the entire thickness with loss of maturation and presence of hyperchromatic pleomorphic nuclei with involvement of the endocervical glands. The features were in keeping with High grade squamous intraepithelial lesion (HSIL) (CIN III) (Figure 2a). No histological evidence of stromal invasion was seen.

The sub epithelial region showed (Figure 2b) sheets of neoplastic lymphoid cells with irregular nuclear contours, vesicular nuclei, occasional prominent nucleoli and moderate amount of eosinophilic cytoplasm. Mitosis was frequent. Spindling of tumour cells was evident in areas. Immunohistochemical work up showed the tumour cells were positive for CD20 (Figure 2c) while negative for CD3, CD10, Bcl-6 and Cyclin D1. The neoplasm extended up to the deep resection margin. The final diagnosis was given as High grade squamous intraepithelial lesion (HSIL) (Cervical intraepithelial Neoplasia III/ CIN III) with Non-Hodgkin’s Lymphoma B-cell type; Diffuse Large B Cell (DLBCL) Further staging work up and imaging was done (Figure 3) which showed extension of neoplasm to the vaginal fornices, the anterior vaginal wall and the parametrium with involvement of the left ureteric wall and subsequent mild hydronephrosis.

CT neck, chest and abdomen showed no evidence of metastasis. In chest and abdomen; no significant abnormality noted. No significant lymphadenopathy or metastasis in chest. In abdomen, liver was displaying multiple hepatic focal lesions with progressive fill in the contrast likely hemangiomatous left hydroureteronephrosis, left ovarian cyst, retained fluid within the uterine cavity. PET CT scan impression came as hypermetabolic lesions involving the cervix with vaginal and parametrial involvement in keeping with biopsy proven metabolically active lymphoma. The remainder of the study shows no evidence of FDG avid lymphoma. Mild left hydronephrosis was noted. Since Lymphoma of cervix is a very uncommon lymphoma type and surgery has removed most of the disease, in the tumour board it was planned to consider chemotherapy followed by radiotherapy based on available data; measures that could be used in treating lymphoma of cervix. Different options of treatment was discussed and finally planned to give her 6 Cycles of R-CHOP followed by PET- CT and involved field Radiotherapy.

Since the patient was just engaged only and with no children, fertility issue had been discussed with her. Patient decided not to do any fertility preservation procedure. So prophylactic Goserelin injections to increase the chance of being fertile was also planned; once every 4th week up to 6 months after last chemotherapy. After 6 cycles of R- CHOP the patient was sent for PET- CT and impression showed a complete response to therapy. Hence a follow up PET- CT was planned after 3 months. This also showed complete response. The case was rediscussed in the tumour board and since the patient being young, unmarried and with no children and had a complete metabolic response to treatment, it was decided not to give her any Radiotherapy; but to follow up with 6 monthly PET- CT and bloods. To date 13 months after the diagnosis, she is healthy and disease free.

Discussion

Primary cervical lymphoma is rare and involvement of the cervix by a lymphoproliferative disorder is more commonly seen in the setting of systemic disease [1]. It affects adult women over a wide age range. Vaginal bleeding is the most common symptom. Lymphomas are often bulky tumours, sometimes with circumferential enlargement of the cervix (“barrel-shaped” cervix) [1,2]. Cervical diffuse large B-cell lymphomas, which is the predominant type are are often associated with prominent sclerosis and may be associated with a cord-like arrangement or spindle-shaped tumor cells (“spindle cell variant”) [3]. Rare cervical marginal zone lymphomas (MALT lymphomas), Burkitt lymphomas [4] and extranodal NK/Tcell lymphomas, nasaltype, have been described. Differential diagnoses of cervical lymphomas include sarcoma, poorly differentiated carcinoma, neuroendocrine tumours, Malignant Mixed Mullerian tumour, Melanoma, extraosseous Ewing’s sarcoma, and chronic cervicitis. Most cases of NHL involving uterine cervix presents at stage I or II. The optimal treatment of such tumours is not clear. These tumours have been managed with chemotherapy, [5] radiotherapy, and surgery, alone or in combination [6]. Heredia et al. demonstrate the use of combination of CHOP × 3 plus involved field radiotherapy as therapy for this malignancy [7]. The association of HPV infection with cervical cancer is well known; though literature on HPV association to cervical lymphoma is scanty. A Danish study [8] noted that HPV infection is associated with an increased risk of Lymphoma. This association may be attributable to a chronic immune activation induced by persistent HPV infection and/or failure of the immune system both to clear HPV infection and to control lymphoma development.

Conclusion

In our patient the rare combination of High-risk HPV, Lymphoma and High grade squamous intraepithelial lesion was quite interesting. But the efficient interdisciplinary discussions gave her a wonderful outcome.

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Squamous Cell Carcinoma of The Tongue in A Developing Community- Juniper publishers

Abstract

Squamous cell carcinoma of the tongue is characterized by malignant cells which show squamous differentiation in the direction of “formation of keratin and/or the presence of intercellular bridges.” As single case reports have appeared in the literature recently, this paper presents a good number of them traced among the Ibo ethnic group in Nigeria, this being facilitated by the author who heads an institution that provides a histopathology data pool.

Keywords: Tongue; Malignancy; Squamous cell carcinoma; Epidemiology; Developing community

Introduction

Squamous cell carcinoma is defined as “a malignant epithelial neoplasm exhibiting squamous differentiation as characterized by the formation of keratin and/or the presence of intercellular bridges” [1]. This was illustrated in a case report from India [1] as well as from Saudi Arabia [2] and from the UK [3]. Therefore, this paper presents a series from the Ibo ethnic group’s developing community [4], this being done on account of the author’s headship of a histopathology data pool such as the one fostered by a Birmingham (UK) group [5]. To facilitate matters, the tabular form is used as shown hereunder.

Results

Table 1

Table 2

In contrast, only few of the published cases designated the sites, including

1) Side

2) Lateral border

3) Margin.

Discussion

Wide interest in squamous cell carcinomas of the tongue is shown in the publications which have appeared recently in countries as far apart as China [6], France [7], UK [8] and USA [9,10]. Therefore, the local cohort ought to be compared with them. In this context, the mean age was given as 60 years from India [1]. Age stands out. The Chinese group was aged 28 to 88 years, but the mean age was not apparent [6]. The local cohort was aged 34 years to 79 years (mean 56 years). As to sex, the local cohort was almost equal. It was disparate in China to the tune of 102 males to 83 females [6]. What stood out was a rare histological variant. It was reported from Spain [11]. It turned out to be the infrequent and aggressive “spindle cell type” that featured in a 11-year-old boy.

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Basosquamous Carcinoma of Skin in A Developing Community- Juniper Publishers

Abstract

Recently, single case reports were published from India, Romania, UK, and USA. Its rarity was stressed as well as the histological combination of both basal cell carcinoma and squamous cell carcinoma. The ages ranged from 71 to 86 years and most were females. Therefore, this paper reports the experience from a developing community in Nigeria with special reference to members of the Ibo ethnic group in whom the site of preponderance was the head and the sex was the female.

Keywords: Skin; Cancer; Basosquamous carcinoma; Head; Sex; Developing community

Introduction

Recent single case reports appeared in the literature from India [1], Romania [2], UK [3], and USA [4]. Not only was its rarity stressed but also its combining of the histology of basal cell carcinoma and squamous cell carcinoma. Among the findings was the age group of 61 years to 86 years as well as the preponderance of female sufferers. Therefore, this paper sets out to present the findings among the Ibo ethnic group, of whom a British anthropologist wrote about copiously [5]. Moreover, there was the advantage that a Birmingham (UK) group proposed that the establishment of a histopathology data pool facilitates epidemiological analysis [6]. In this context, the author became the pioneer of such a pool established by the Government of the Eastern Region of Nigeria at Enugu. Moreover, he trained at the prestigious Glasgow Western Infirmary [7]. Therefore, having insisted on biopsies being sent with epidemiological data, and carefully keeping personal duplicates, the results in respect of basosquamous carcinoma of skin can be presented below in tabular form.

Discussion

Australian authors [8] defined basosquamous carcinoma as “a basal cell carcinoma (BCC) differentiating into squamous cell carcinoma (SCC).” I am persuaded that both features were present in the local cases. The local cohort consisted of 7 females out of a total of 9 cases. A Swiss paper bothered not with the sex incidence but with recurrence rate which was reported to be “highly aggressive” [9]. A clear point is the frequency of attack of the head and neck region in Germany [10], Italy [11], and UK [12]. Incidentally, the head alone was the preponderant site among the Ibos.

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Impact of Heat Shock Proteins in Hepatocellular Carcinoma- Juniper Publishers

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Abstract

Heat shock proteins are highly conserved proteins, expressed at low levels under normal conditions. Heat shock proteins significantly induced in response to cellular stresses and lead to heat shock response, which could help cancer cells to adapt to stress conditions. As molecular chaperones, Heat shock proteins play critical roles in protein homeostasis, apoptosis, invasion and cellular signaling transduction. A heat shock protein over expression is widely reported in many human cancers due to constant stress condition in tumor microenvironment. Heat shock proteins often associated with poor prognosis in many types of human cancers. Up regulation of Heat shock proteins may serve as diagnostic and prognostic markers in hepatocellular carcinoma. Targeting Heat shock proteins with specific inhibitor alone or in combination with chemotherapy regimens holds promise for the improvement of outcomes for hepatocellular carcinoma patients. In addition, our study suggests progression and challenges in targeting these Heat shock proteins as novel therapeutic strategies in hepatocellular carcinoma.

Keywords: Heat shock proteins; Hepatocellular carcinoma; Heat shock response

Abbrevations: HSPs: Heat Shock Proteins; HCC: Hepatocellular Carcinoma; HSF1: Heat Shock Factor

Introduction

Heat shock proteins (HSPs) are ubiquitous proteins found in the cells of all studied organisms, which are expressed at low levels under normal conditions. Many types of stress, including heat, induce expression of a family of genes known as the heat shock protein genes. Heat shock proteins originally were discovered when it was observed that heat shock produced chromosomal puffs in the salivary glands of fruit flies (Drosophilia) [1–2]. The DNA sequence that makes up this family of genes is highly conserved across species. This family of genes originally was named because of their expression after exposure to heat. However, the genes are now known to be induced by a wide variety of environmental or metabolic stresses that include the following: anoxia (hypoxia), ischemia, heavy metal ions, ethanol, nicotine, surgical stress, viral agents, genotoxic agents, nutrient starvation and overexpression of oncoproteins [3].

Thus, the term “heat shock protein” is a misnomer because many agents other than heat induce the expression of the heat shock protein gene. Consequently, “stress protein” is the preferred term. Stress proteins are critically important because they appear to be necessary in the critical step of three-dimensional folding of some newly formed proteins within the cell. In fact, they ensure that newly formed polypeptides proceed correctly through folding and unfolding to eventually achieve a functional shape (Figure 1). Stress proteins also assist in the repair of denatured proteins or promote their degradation after stress or injury. They have been referred to as “molecular chaperones” because of this function [1,4].

Upregulation of HSPs is a critical part of heat shock response, which could help cells to cope with the stress condition. Many members of HSPs perform their functions as molecular chaperons by stabilizing proteins to ensure their correct folding, inhibiting stress-induced protein aggregation or regulating cellular signalling and transcriptional networks. The increased expression of HSPs under stress condition is often transcriptionally regulated by heat shock factor 1 (HSF1). In response to stress conditions, HSF1 is phosphorylated and forms homotrimers, then binds to heat shock elements (HSEs) located upstream of HSPs genes and activates the transcription of heat shock genes [5–6]. Stress proteins belong to a multigene family and often named according to their molecular weight from 8 to 150kd. In this review, we will discuss three major members of the HSP family, which are closely related to human malignancies, namely HSP27, HSP70 and HSP90. It is thought that stress proteins are produced in response to nonlethal stress to protect organisms from subsequent severe stress that would otherwise be lethal. In the case of exposure to heat, this phenomenon has been called “thermotolerance” and has launched many experiments in which an association has been found between the heat shock response and protection against other stresses, such as hypoxia or ischemia. The addition of one type of stress may provide protection against other types of insults, which results in cross-tolerance. As examples, stress protein induction by hyperthermia may provide protection during a subsequent arterial injury or exposure to a heavy metal may provide subsequent protection against heat or ischemic injury. This thermotolerance treatment strategy has proved successful in experimental models of cardiac ischemia, arterial injury, endotoxic shock, renal and hepatic ischemia, ethanol-induced gastric ulcerations, and skeletal muscle ischemia-reperfusion [7]. HSP27 overexpression is reported in a wide range of human cancers. Report shows that HSP27 performs crucial roles in cell cycle modulation, apoptosis inhibition, cytoskeleton organization, regulation of translation initiation, DNA repair, RNA splicing and degradation of oxidized proteins via the ubiquitin-proteasome pathway.

Stress proteins play a critical role in the maintenance of normal cellular homeostasis. These proteins almost certainly have a pivotal role in cell cycle progression and cell death (apoptosis) and are involved in many disease processes, including cardiovascular disease. Currently, the manipulation of stress proteins remains cumbersome because hyperthermia and pharmacologic manipulations are relatively nonspecific. Eventually, as we gain more insight into the exact role and function of these fascinating molecules, the clinical manipulation of the stress proteins will almost certainly prove beneficial [8]. Liver cancer is one of the most frequent malignancies worldwide [9]. An estimated 782,500 new liver cancer cases and 745,500 deaths occurred worldwide during 2012 [10]. About half of the new cases and related deaths of liver cancer occurred in China. Hepatocellular carcinoma (HCC) accounts for more than 80% of primary liver cancers. Although treatment techniques for HCC have experienced great progress, prognosis is still poor for HCC patients. Only 30–40% of HCC patients are suitable for curative treatment at the time of diagnosis. Long-term survival following radical surgical resection remains unsatisfactory because of high rates of recurrence and metastasis [11]. A better understanding of molecular mechanisms involved in tumorigenesis and metastasis of HCC will provide novel and potential therapeutic implications in HCC treatment. HSPs have become attractive therapeutic targets in HCC. Novel therapeutic strategies that target HSPs alone or combined with other anticancer agents are widely investigated [12].

Conclusion and Future Directions

As stress induction of HSPs plays a crucial role in tumorigenesis, metastasis and therapeutic resistance, the clinical efficacies of HSPs as biomarkers for the diagnosis and prognosis of HCC patients warrant further testing in clinical settings. Targeting HSPs with their specific inhibitors holds attractive therapeutic promise for the improvement of outcomes for HCC patients. Despite promising preclinical data, there are still some challenges to target HSPs in HCC. First, little is known about the therapeutic responses of HSPs inhibitors in HCC patients. Therefore, more clinical trials about the antitumor activity of HSPs inhibitors in HCC patients are urgently required. Second, the future of HSPs inhibitors in the treatment of cancer individuals may lie in combining them with cytotoxic chemotherapy or other targeted therapies. Although some preliminary studies have shown enhanced efficacy of HSPs inhibition combined with targeted drugs such as rapamycin and sorafenib, the best strategy for combination treatment still needs to be validated in further studies. Third, we can focus our attention on HSPs inhibitors directly. Intrinsic or acquired resistance often limits the efficiencies of HSPs inhibitors CRISPR/Cas9- or shRNAbased loss-of-function genetic screens can help us to identify mechanisms of the resistance and find potential combined drug targets whose inactivation is effective to improve efficiencies of HSPs inhibitors.123–125 Fourth, HSPs constitute 1–2% of total proteins in most celltypes.126 HSPs are essential for both cancer and normal cells, and play important roles in a wide range of cellular processes such as maintaining protein homeostasis, intracellular trafficking, signal transduction and regulating innate immune responses. HSPs targeting may have unacceptable deleterious effects on non-malignant cells and normal organs in clinical trials. As a consequence, novel HSPs inhibitors with high selectivity and potency for tumor cells are eagerly to be developed. Finally, molecular mechanisms of HSPs in cytoprotective effects and tumor metastasis are still not fully understood. Answers to key issues of these basic mechanisms will significantly accelerate the applications of HSPs inhibitors in HCC treatment.

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Translational Cell Carcinoma of the Nose in a Developing Community- Juniper Publishers

Abstract

Transitional cell carcinoma of the nasal cavity is rare. Single case reports have appeared in the recent literature. Accordingly, we report a case from a developing community in Nigeria. It is deemed to be worthy of documentation.

Keywords: Nose; Transitional cell carcinoma; Different countries; Developing community; Nigeria

Introduction

The transitional cell carcinoma of the nose is a rarity. Single cases of it have been reported recently from India [1–3] as well as Australia [4]. Therefore, we report a case from Nigeria with special reference to the Ibo ethnic group [5]. It is deemed to be worthy of documentation, especially as it followed the recommendation of a Birmingham (UK) group concerning epidemiological analysis being the result of the establishment of a histopathology data pool [6].

Case Report

ON, a 70-year-old man of the Ibo ethnic group, attended the Balsam Clinic, Enugu, Nigeria, where Dr Basil Ezeanolue, the junior co-author, attended to him. He complained of left nasal mass associated with bleeding for 3 months. There was a fleshy mass obstructing the left nasal cavity, arising from the ethmoid air cell system. Inverted papilloma was suspected, and biopsy undertaken. Numerous, irregularly surfaced soft, pale masses measuring up to 3.5cm across were obtained. On microscopy by the corresponding author, there are benign looking papillary structures as well as mitotically active tumor cells diagnostic of transitional cell carcinoma.

Discussion

Indian associate mentioned that transitional cell carcinoma is “also known as non-keratinizing carcinoma of sinonasal tract” [2]. They went further to add that “According to the World Health Organization (WHO) classification, it has many synonyms, including, Schneiderian carcinoma, transitional cell carcinoma, cylindrical cell carcinoma, Ringertz carcinoma, and respiratory epithelial carcinoma.” Another group included only three of these, namely, “Respiratory epithelial carcinoma, Ringertz carcinoma, Cylindrical carcinoma” [1].The age range is said to be “most commonly seen in 5th to 7th decade” [3]. Our 70-year-old patient is in this range.

Conclusion

Good news followed a review of the relevant literature which ended nicely thus: “A dramatic and complete tumor response to chemotherapy with cis-platinum, methotrexate, and bleomycin is described in a patient with advanced proptosis due to extensive local recurrence of transitional cell carcinoma” [7]. Of course, this is a fan cry in a developing community such as ours. Indeed, as regards treatment, our patient was lost to follow up.

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In vitro Assessment of Biofield Energy Treated DMEM on Thermogenesis Using Myoblasts Cell Line (C2C12)- Juniper Publishers

Abstract

Mitochondrial dysfunction lead to various serious disorders, which are considered as one of the important components related with the aging, such as type-2 diabetes and Alzheimer’s disease. The aim of the present study was to examine the effect of Consciousness Energy Healing based DMEM medium on murine myoblasts (C2C12) cells to evaluate the mitochondrial mass content using 10-N-nonyl acridine orange (NAO) dye assay. The test item (DMEM medium) was divided into two parts, one part received Consciousness Energy Healing Treatment by a renowned Biofield Energy Healer, Dahryn Trivedi and was labeled as the Biofield Energy Treated DMEM group, while the other part did not receive any kind of Treatment and denoted as the untreated DMEM group. The level of mitochondrial mass content was assessed using 10-N-nonyl acridine orange (NAO) dye method. Cell viability of the test items using MTT assay showed 72.32% and 125.32% viable cells in the untreated DMEM and Biofield Energy Treated DMEM groups, respectively suggested a safe and nontoxic profile of the test items. Besides, the mitochondrial mass content in terms of Fluorescence Unit (FU) was significantly (p≤0.05) increased by 81.78% in the Biofield Energy Treated DMEM group compared to the untreated DMEM group. Overall, the experimental data suggested that the Consciousness Energy Healing Based DMEM showed a significant improvement of mitochondrial mass content and results in better thermogenesis with respect to naive DMEM. Thus, an increased level of NAO dye accumulation in muscle cells indicated increased mitochondrial mass content and hence, better thermogenesis. In the present study, results demonstrated that an increased mitochondrial mass content in the cells when treated with The Trivedi Effect®. This indicates that the test sample has the potential to improve thermogenesis, which can be used against various metabolic diseases, such as insulin resistance, type-2 diabetes, and cardiovascular diseases, etc.

Keywords: Biofield energy; The Trivedi Effect®; Thermogenesis; Mitochondrial biogenesis; Metabolic disorders; Murine myoblast cell; DMEM

Abbreviations: CAM: Complementary and Alternative Medicine, NCCAM: National Center for Complementary and Alternative Medicine; DMEM: Dulbecco’s Modified Eagle’s Medium; FBS: Fetal Bovine Serum

Introduction

Mitochondria (also known as power generator of the cell) produce most of the vital energy required for the cellular function through oxidative phosphorylation involved in electron transport and ATP synthesis. They produce ATP through the process of cellular respiration mainly aerobic respiration, which requires oxygen. Number and amount of mitochondria in a cell be governed by the energy requirement of the cell [1]. For example, the muscle cells have found comparatively more number of mitochondria since, they need to produce energy to move the body. On the other hand, red blood cells carry oxygen to other cells, do not need to produce energy as compared with the muscle cells. Mitochondria are the powerhouse in the cell, which produce energy from basic components. They undergo fusion, fission, transport, and degradation, all of the process is vital to maintain a healthy mitochondrial population [1]. However, the mitochondrial biogenesis process is involved an increased and controlled mitochondrial mass with number that helped to produce greater production of ATP as a response to greater energy expenditure [2]. Physiologic, metabolic, and pathologic changes along with morphological and functional adaptability are the vital factor to regulate the process of mitochondrial biogenesis. In addition, proteins and transcription factors, upstream regulatory proteins and secondary mechanisms are also involved in the biogenesis process, which also stabilizes the new mitochondrial DNA [3].

Mitochondrial biogenesis regulates and control various therapeutic interference in wide number of diseases such as metabolic syndrome, neurodegenerative disorders, sarcopenia, cardiac pathophysiology and physiological processes like aging [4]. Nonyl-acridine orange (NAO) is a non-fluorescent dye that converts into fluorescent dye in the presence of oxidative species [5]. NAO assay is one of the gold standard assays to detect the mitochondrial mass alteration, which is a metachromatic dye that binds to cardiolipin, an inner mitochondrial membrane lipid, regardless of the energetic state of the cell. Therefore, mitochondrial mass of the cells could be estimated by studying accumulation of the fluorescent dye in the mitochondria. Furthermore, an alternative therapies such as nuclear gene was reported to regulate total mitochondrial mass in response to mitochondrial dysfunction [6]. In order to improve the mitochondrial mass content via thermogenesis process, some alternative treatment approach without any associated side-effect is needed.

Biofield Energy Healing is a categorized as one of the Complementary and Alternative Medicine (CAM) accepted worldwide for the various treatment. Biofield Energy Therapy was accepted by National Center for Complementary and Alternative Medicine (NCCAM). Biofield Energy Healing is one of the emerging frontier in medicine, which has been increased in order to promote wellness by uncovering the root cause of diseases with universal solutions. CAM therapies have shown various significant clinical benefits. Over the past few decades, many energy healing practices have been reported a significant outcomes in various clinical and non-clinical fields. The effects of the CAM therapies have great potential, which include external qigong, Johrei, Reiki, therapeutic touch, yoga, Qi Gong, polarity therapy, Tai Chi, pranic healing, deep breathing, chiropractic/osteopathic manipulation, guided imagery, meditation, massage, homeopathy, hypnotherapy, progressive relaxation, acupressure, acupuncture, special diets, relaxation techniques, Rolfing structural integration, healing touch, movement therapy, pilates, mindfulness, Ayurvedic medicine, traditional Chinese herbs and medicines in biological systems both In vitro and in vivo [7]. Every living organisms possess some kind of unique energy known as Biofield Energy, which is infinite, para-dimensional and electromagnetic field surrounding the human body. Biofield (Putative Energy Fields) based Energy Healing Therapies have been reported to have significant outcomes against various disease conditions. Biofield Energy Healing Treatment (The Trivedi Effect®) contain a putative bioenergy, which is channeled by a renowned practitioner from a distance. Biofield Energy Healing as a CAM showed a significant results in biological studies [8]. However, the National Center for Complementary and Alternative Medicine (NCCAM), well-defined Biofield Therapies in the subcategory of Energy Therapies [9]. The Trivedi Effect®- Consciousness Energy Healing Treatment has been reported with significant revolution in the physicochemical properties of metals, chemicals, ceramics and polymers [10- 12], improved agricultural crop yield, productivity, and quality [13,14], transformed antimicrobial characteristics [15–17], biotechnology [18,19], improved bioavailability [20–22], skin health [23, 24], nutraceuticals [25,26], cancer research [27,28], bone health [29–31], human health and wellness. On the basis of outstanding benefits of Biofield Energy Treatment, the present study was aimed to evaluate the impact of the Biofield Energy Treatment (The Trivedi Effect®) on DMEM as test sample to alter the mitochondrial mass content on thermogenesis using NAO dye staining using standard in vitro assay in murine myoblasts (C2C12) cells.

Material and Methods

Chemicals and reagents

Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Life Technology, USA. Antibiotics solution (penicillin-streptomycin) were procured from HiMedia, India, and ethylenediaminetetraacetic acid (EDTA) was purchased from Sigma, USA. All the other chemicals used in this experiment were analytical grade procured from India.

Cell culture

C2C12 (murine myoblasts) was used as a test system in the present study. The C2C12 cell line was maintained in DMEM growth medium for routine culture supplemented with 10% FBS. Growth conditions were maintained at 37°C, 5% CO2, and 95% humidity and subcultured by trypsinisation followed by splitting the cell suspension into fresh flasks and supplementing with fresh cell growth medium. Before initiation of the experiment, cells were incubated in DMEM+2% horse serum (HS) for 3 days to allow the cells to differentiate into myotubes.

Experimental design

The experimental groups consisted of group 1 (G-I) with untreated DMEM and group 2 (G-II) included the Biofield Energy Treated DMEM.

Consciousness energy healing treatment strategies

The test item, DMEM was divided into two parts. One part of the test item was treated with the Biofield Energy by a renowned Biofield Energy Healer, Dahryn Trivedi remotely for ~5 minutes and coded as the Biofield Energy Treated DMEM, while the second part did not receive any sort of treatment and denoted as the untreated DMEM group. Biofield Energy Healer was located in the USA, while the test items were located in the research laboratory of Dabur Research Foundation, New Delhi, India. This Biofield Energy Treatment was administered through Healer’s unique Energy Transmission process to the test sample under laboratory conditions. Dahryn Trivedi in this study never visited the laboratory in person, nor had any contact with the test item (DMEM). Further, the untreated DMEM group was treated with a “sham” healer for comparative purposes. The “sham” healer did not have any knowledge about the Biofield Energy Treatment. After that, the Biofield Energy Treated and untreated samples were kept in similar sealed conditions for experimental study.

Assessment of cell viability using MTT assay

The cell viability was performed by MTT assay in C2C12 cell line. The cells were counted and plated in 96-well plates at the density corresponding to 10 X 103 cells/well/180μL of cell growth medium (DMEM+2% HS). The above cells were incubated overnight under growth conditions and allowed the cell recovery and exponential growth, which were subjected to serum stripping or starvation. The cells were treated with the Untreated and Biofield Energy Treated test item. The cells in the above plate(s) were incubated for 24 to 72 hours in a CO2 incubator at 37°C, 5% CO2, and 95% humidity. Following incubation, the plates were taken out and 20μL of 5 mg/mL of MTT solution were added to all the wells followed by additional incubation for 3 hours at 37°C. The supernatant was aspirated and 150μL of DMSO was added to each well to dissolve formazan crystals. The absorbance of each well was read at 540nm using Synergy HT microplate reader, Bio Tek, USA [29]. The percentage cytotoxicity at each tested concentrations of the test items were calculated using the following equation (1):

%cytotoxicity = (1− X / R)*100………..(1)

Where, X = Absorbance of the Biofield Treated cells; R = Absorbance of untreated cells

The percentage cell viability corresponding to each treatment was obtained using the following equation (2):

% Cell Viability = (100- % Cytotoxicity)………(2)

The concentrations exhibiting ≥70% cell viability was considered as non-cytotoxic.

Assessment of mitochondrial content

For the assessment of mitochondrial mass, the cells were counted using an hemocytometer and plated at 4500 cells/well in dark walled 96-well plates in DMEM supplemented with 2% HS. The cells were incubated overnight under standard growth conditions to allow the cell recovery and exponential growth, which were treated by the test items in different groups followed by incubation with the test items for 72 hours. After incubation with the test items, mitochondrial content was determined by 10-N-nonyl acridine orange (NAO) dye. 50nM dye was added to each well and the cells were incubated for 30 minutes at 37°C and 5%CO2. After 30 minutes of incubation, media was discarded and cells were washed with phosphate buffer saline (PBS). 150μL of PBS was added to each well and fluorescence was read at 485/20 excitation, 528/20 emission filter using synergy HT microplate reader. The percentage increase in mitochondrial content was calculated using following equation

Where, FU denotes Fluorescence unit

Statistical analysis

All the values were represented as Mean ± SEM (standard error of mean) of three independent experiments. The statistical analysis was performed using Sigma Plot statistical software (v11.0). For two groups comparison student’s t-test was used. Statistically significant values were set at the level of p≤0.05.

Results and Discussion

Cell viability using MTT assay

Cell viability data of the untreated and Biofield Energy Treated DMEM groups in C2C12 cells using MTT assay is shown in Figure 1. The percentage of cell viability in the untreated DMEM group was 72.32%, while it was 125.32% in the Biofield Energy Treated DMEM group (Figure 1). Overall, data suggest that all the test samples were found safe against the tested C2C12 cells, which were used for the estimation of mitochondrial mass content, which indicate extend of thermogenesis.

Extend of mitochondrial mass content in C2C12 cells

Mitochondrial mass content or biogenesis showed a significant effects against various metabolic diseases. The increased cell capacity to control and maintain the cell metabolism, signal transduction, and regulation of mitochondrial ROS production [2]. Alteration or decrease in the mitochondrial biogenesis are related with the mitochondrial dysfunction and mitochondrial oxidative stress, which leads to various diseases [32]. Mitochondrial mass content results in the improved production of ATP as a response to greater energy expenditure [33]. Various factors such as physical exercise, nutritional factors, etc. reported to have an improved mitochondrial mass content, which results in greater glucose uptake by muscles, along with an increased metabolic enzymes level for glycolysis, oxidative phosphorylation and ultimately a greater mitochondrial metabolic capacity [34]. Aging process leads to decrease level of mitochondrial mass content and results in various diseases such as enhanced aging, insulin resistance, type-2 diabetes, cardiovascular diseases, obesity, etc. [35]. The experiment was conducted to study the influence of Biofield Energy Healing Treatment on mitochondrial content in C2C12 cells using NAO dye assay. The results of mitochondrial mass content in terms of increase number of fluorescence unit (FU) among different groups in C2C12 cells using NAO dye assay are presented in Figure 2. The untreated DMEM group showed 272.3 ± 12.14 FU. Besides, the Biofield Energy Treated DMEM group showed 81.78% increase the level of mitochondrial mass in terms of FU, compared to the untreated DMEM group (Figure 2).

Thus, the data suggested that the Consciousness Energy Blessed DMEM showed a significant improvement of thermogenesis, which results in mitochondrial mass content. This phenomenon can be significantly used against various metabolic diseases, such as insulin resistance, type 2 diabetes, and cardiovascular diseases. Overall, the Biofield Energy Healing Treatment (The Trivedi Effect®) has the significant capacity to improve the overall Quality of life with an improved thermogenesis and mitochondrial content.

Conclusion

MTT data showed a significant cell viability with ranges from 72.32% to 125.32% in different test item groups, which indicated that the test items were safe and non-toxic in nature. Correspondingly, The Trivedi Effect® showed a significant change in mitochondrial mass among different groups. The Biofield Energy Treated DMEM group demonstrated significant increase in the mitochondrial mass by 81.78% as compared to the untreated DMEM group. Thus, Biofield Energy Healing based DMEM can be significantly used to improve the energy level, endurance, body energy, which can be utilized against many diseases such aging, diabetes, cancer, depression, hypertension, cardiovascular disease, aging, and physical strength. The Biofield Energy Treated (The Trivedi Effect®) DMEM were found to have a significant impact on thermogenesis, which might significantly improve the mitochondrial content in muscle cells. Therefore, the Consciousness Energy Healing based DMEM might be a suitable alternative media for cell growth. It can be useful for the management of various disorders such as Lupus, Systemic Lupus Erythematosus, Fibromyalgia, Addison Disease, Hashimoto Thyroiditis, Celiac Disease (gluten-sensitive enteropathy), Multiple Sclerosis, Dermatomyositis, Graves’ Disease, Myasthenia Gravis, Pernicious Anemia, Aplastic Anemia, Scleroderma, Psoriasis, Rheumatoid Arthritis, Reactive Arthritis, Diabetes, Sjogren Syndrome, Crohn’s Disease, Vasculitis, Vitiligo, Chronic Fatigue Syndrome and Alopecia Areata, as well as inflammatory disorders such as Irritable Bowel Syndrome (IBS), Asthma, Ulcerative Colitis, Alzheimer’s Disease, Parkinson’s Disease, Atherosclerosis, Dermatitis, Hepatitis, and Diverticulitis. Further, the Biofield Energy Healing Treatment can also be used in the prevention of immune-mediated tissue damage in cases of organ transplants (for example heart transplants, kidney transplants and liver transplants), for anti-aging, stress prevention and management, and in the improvement of overall health and Quality of Life.

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