Itch inhibits IL-17-mediated colon inflammation and tumorigenesis by ROR-γt ubiquitination.

Dysregulated expression of interleukin 17 (IL-17) in the colonic mucosa is associated with colonic inflammation and cancer. However, the cell-intrinsic molecular mechanisms by which IL-17 expression is regulated remain unclear. We found that deficiency in the ubiquitin ligase Itch led to spontaneous colitis and increased susceptibility to colon cancer. Itch deficiency in the TH17 subset of helper T cells, innate lymphoid cells and γδ T cells resulted in the production of elevated amounts of IL-17 in the colonic mucosa. Mechanistically, Itch bound to the transcription factor ROR-γt and targeted ROR-γt for ubiquitination. Inhibition or genetic inactivation of ROR-γt attenuated IL-17 expression and reduced spontaneous colonic inflammation in Itch(-/-) mice. Thus, we have identified a previously unknown role for Itch in regulating IL-17-mediated colonic inflammation and carcinogenesis.

A novel multi-epitope peptide vaccine candidate targeting hepatitis E virus: An in silico approach.

Hepatitis E virus (HEV) is a foodborne virus transmitted through the faecal-oral route that causes viral hepatitis in humans worldwide. Ever since its discovery as a zoonotic agent, HEV was isolated from several species with an expanding range of hosts. HEV possesses several features of other RNA viruses but also has certain HEV-specific traits that make its viral-host interactions inimitable. HEV leads to severe morbidity and mortality in immunocompromised people and pregnant women across the world. The situation in underdeveloped countries is even more alarming. Even after creating a menace across the world, we still lack an effective vaccine against HEV. Till date, there is only one licensed vaccine for HEV available only in China. The development of an anti-HEV vaccine that can reduce HEV-induced morbidity and mortality is required. Live attenuated and killed vaccines against HEV are not accessible due to the lack of a tolerant cell culture system, slow viral replication kinetics and varying growth conditions. Thus, the main focus for anti-HEV vaccine development is now on the molecular approaches. In the current study, we have designed a multi-epitope vaccine against HEV through a reverse vaccinology approach. For the first time, we have used viral ORF3, capsid protein and polyprotein altogether for epitope prediction. These are crucial for viral replication and persistence and are major vaccine targets against HEV. The proposed in silico vaccine construct comprises of highly immunogenic and antigenic T-cell and B-cell epitopes of HEV proteins. The construct is capable of inducing an effective and long-lasting host immune response as evident from the simulation results. In addition, the construct is stable, non-allergic and antigenic for the host. Altogether, our findings suggest that the in silico vaccine construct may be useful as a vaccine candidate for preventing HEV infections.

Interleukin-17-A multifaceted cytokine in viral infections.

Viral infections are a major threat to the human population due to the lack of selective therapeutic measures. The morbidity and mortality reported worldwide are very alarming against viral pathogens. The proinflammatory environment is required for viral inhibition by initiating the host immune response. The host immune response fights these pathogens by secreting different cytokines. Interleukin-17 (IL-17) a proinflammatory cytokine mainly produced by T helper type 17 cells, plays a vital role in the regulation of host immune response against various pathogens, including viruses. However, dysregulated production of IL-17 induces chronic inflammation, autoimmune disorders, and may lead to cancer. Recent studies suggest that IL-17 is not only involved in the antiviral immune response but also promotes virus-mediated illnesses. In this review, we discuss the protective and pathogenic role of IL-17 against various viral infections. A detailed understanding of IL-17 during viral infections could contribute to improve therapeutic measures and enable the development of an efficient and safe IL-17 based immunotherapy.

Role of interleukin-17 in human papillomavirus infection and associated malignancies.

Human papillomavirus infection is among the leading viral infections in the world, causing severe mortality and morbidity. The virus mainly targets the female genital tract-cervix, vulva, anus but it is also reported to infect the lungs and oropharyngeal region of the body. The host immune response plays a vital role in the persistence of viral infection. Interleukin 17 (IL-17) is mainly secreted by Th17 cells and mediates the immune response that enhances the disease severity in HPV infection. IL-17 is reported to promote lesions and tumour progression by creating a hyperinflammatory condition leading to cancer. The current minireview summarizes the pathogenic role of IL-17 in HPV infection and HPV-induced malignancies. Further study on IL-17 associated pathology of HPV infection would be useful in developing therapeutic measures.

Immunological consequences of stress-related proteins--cytosolic tryparedoxin peroxidase and chaperonin TCP20--identified in splenic amastigotes of Leishmania donovani as Th1 stimulatory, in experimental visceral leishmaniasis.

In earlier studies, proteomic characterization of splenic amastigote fractions from clinical isolates of Leishmania donovani, exhibiting significant cellular responses in cured Leishmania subjects, led to the identification of cytosolic tryparedoxin peroxidase (LdcTryP) and chaperonin-TCP20 (LdTCP20) as Th1-stimulatory proteins. Both the proteins, particularly LdTCP20 for the first time, were successfully cloned, overexpressed, purified and were found to be localized in the cytosol of purified splenic amastigotes. When evaluated against lymphocytes of cured Leishmania-infected hamsters, the purified recombinant proteins (rLdcTryP and rLdTCP20) induced their proliferations as well as nitric oxide production. Similarly, these proteins also generated Th1-type cytokines (IFN-γ/IL-12) from stimulated PBMCs of cured/endemic Leishmania patients. Further, vaccination with rLdcTryP elicited noticeable delayed-type hypersensitivity response and offered considerably good prophylactic efficacy (~78% inhibition) against L. donovani challenge in hamsters, which was well supported by the increased mRNA expression of Th1 and Th2 cytokines. However, animals vaccinated with rLdTCP20 exhibited comparatively lesser prophylactic efficacy (~55%) with inferior immunological response. The results indicate the potentiality of rLdcTryP protein, between the two, as a suitable anti-leishmanial vaccine. Since, rLdTCP20 is also an important target, for optimization, further attempts towards determination of immunodominant regions for designing fusion peptides may be taken up.

Efficacy of Leishmania donovani trypanothione reductase, identified as a potent Th1 stimulatory protein, for its immunogenicity and prophylactic potential against experimental visceral leishmaniasis.

In visceral leishmaniasis (VL), Th1-type of immune responses play an important role which correlates with recovery from and resistance to disease resulting in lifelong immunity. Based on this rationale, the soluble leishmanial antigens that elicit cellular responses in peripheral blood mononuclear cells (PBMCs) from cured Leishmania patients were characterized through immunoproteomic approach which led to the identification of trypanothione reductase (TPR) (a cytosolic enzyme explored as a drug target), as one of the potent Th1 stimulatory protein. In this study, the immunogenicity of recombinant Leishmania donovani TPR (rLdTPR) was assessed in PBMCs of cured Leishmania-infected patients/hamsters and further evaluated its prophylactic efficacy against L. donovani challenges in hamsters. Substantial proliferative responses to rLdTPR, as compared to soluble L. donovani antigen, were observed in Leishmania-infected cured patients as well as in hamsters. Moreover, rLdTPR reasonably stimulated PBMCs of cured Leishmania patients to produce IFNγ, IL-12, and TNF-α but not IL-4 or IL-10. On the other hand, the protein downregulated LPS-induced IL-10 as well as soluble L. donovani antigen-induced IL-4 production in PBMCs of Leishmania patients. In case of cured hamsters, rLdTPR generates mixed Th1 and Th2 immune response. Vaccination with rLdTPR along with Bacillus Calmette-Guerin (BCG) was able to provide considerably good prophylactic efficacy (~60%) against L. donovani challenge in hamsters. The efficacy was supported by the increased inducible NO synthase mRNA transcript and Th1-type cytokines IFNγ, IL-12, and TNF-α and downregulation of IL-4, IL-10, and TGF-ß. Since rLdTPR protein is an important target, further attempts towards determination of immunodominant regions for designing fusion peptides may be taken up to optimize its prophylactic efficacy.

Current Challenges and Future Perspectives of Diagnosis of Hepatitis B Virus.

It is estimated that approximately 260 million people worldwide are infected with the hepatitis B virus (HBV), which is one of the leading causes of liver disease and liver cancer throughout the world. Compared with developed countries, low-income and middle-income countries have limited access to resources and advanced technologies that require highly specialized staff for HBV diagnosis. In spite of the heavy burden caused by hepatitis B virus, 90% of people are still undiagnosed. The World Health Organization (WHO) goal of eliminating hepatitis B by 2030 seems very difficult to achieve due to the existing diagnostic infrastructure in low-resource regions. The majority of diagnostic laboratories still use hepatitis B surface antigen (HBsAg)-based tests. WHO's elimination plan is at risk of derailment due to phases like the window period, immune control, and occult HBV infection (OBI) not being detected by standard tests. Here, in this article, we are focusing on various diagnostic platforms for the better diagnosis of HBV. The aim of the elimination of HBV can only be achieved by detecting all phases of HBV infection, which can be executed by a combined approach of using new marker assays along with advanced pretesting and testing methods.

Power and vulnerability: managing sensitive language in organizational communication.

Organizational responsibilities can give people power but also expose them to scrutiny. This tension leads to divergent predictions about the use of potentially sensitive language: power might license it, while exposure might inhibit it. Analysis of peoples' language use in a large corpus of organizational emails using standardized Linguistic Inquiry and Word Count (LIWC) measures shows a systematic difference in the use of words with potentially sensitive (ethnic, religious, or political) connotations. People in positions of relative power are ~3 times less likely to use sensitive words than people more junior to them. The tendency to avoid potentially sensitive language appears to be independent of whether other people are using sensitive language in the same email exchanges, and also independent of whether these words are used in a sensitive context. These results challenge a stereotype about language use and the exercise of power. They suggest that, in at least some circumstances, the exposure and accountability associated with organizational responsibilities are a more significant influence on how people communicate than social power.

Identification of novel S-adenosyl-L-homocysteine hydrolase inhibitors through homology-model-based virtual screening, synthesis, and biological evaluation.

The present study describes a successful application of computational approaches to identify novel Leishmania donovani (Ld) AdoHcyase inhibitors utilizing the differences for Ld AdoHcyase NAD(+) binding between human and Ld parasite. The development and validation of the three-dimensional (3D) structures of Ld AdoHcyase using the L. major AdoHcyase as template has been carried out. At the same time, cloning of the Ld AdoHcyase gene from clinical strains, its overexpression and purification have been performed. Further, the model was used in combined docking and molecular dynamics studies to validate the binding site of NAD in Ld. The hierarchical structure based virtual screening followed by the synthesis of five active hits and enzyme inhibition assay has resulted in the identification of novel Ld AdoHcyase inhibitors. The most potent inhibitor, compound 5, may serve as a "lead" for developing more potent Ld AdoHcy hydrolase inhibitors as potential antileishmanial agents.

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications.

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100 nm), zeta (-43.3 ± 2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P ≥ 0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2-8°C for more than 3 months. IC(50) value of Ambisome (0.18 µg/mL) was comparatively similar to F-1a (0.17 µg/mL) and F-2a (0.16 µg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.

Metabolic Pathways of Leishmania Parasite: Source of Pertinent Drug Targets and Potent Drug Candidates.

Leishmaniasis is a tropical disease caused by a protozoan parasite Leishmania that is transmitted via infected female sandflies. At present, leishmaniasis treatment mainly counts on chemotherapy. The currently available drugs against leishmaniasis are costly, toxic, with multiple side effects, and limitations in the administration route. The rapid emergence of drug resistance has severely reduced the potency of anti-leishmanial drugs. As a result, there is a pressing need for the development of novel anti-leishmanial drugs with high potency, low cost, acceptable toxicity, and good pharmacokinetics features. Due to the availability of preclinical data, drug repurposing is a valuable approach for speeding up the development of effective anti-leishmanial through pointing to new drug targets in less time, having low costs and risk. Metabolic pathways of this parasite play a crucial role in the growth and proliferation of Leishmania species during the various stages of their life cycle. Based on available genomics/proteomics information, known pathways-based (sterol biosynthetic pathway, purine salvage pathway, glycolysis, GPI biosynthesis, hypusine, polyamine biosynthesis) Leishmania-specific proteins could be targeted with known drugs that were used in other diseases, resulting in finding new promising anti-leishmanial therapeutics. The present review discusses various metabolic pathways of the Leishmania parasite and some drug candidates targeting these pathways effectively that could be potent drugs against leishmaniasis in the future.

Designing of multi-epitope chimeric vaccine using immunoinformatic platform by targeting oncogenic strain HPV 16 and 18 against cervical cancer.

Cervical cancer is the most common gynaecological cancer and reaches an alarming stage. HPVs are considered the main causative agents for cervical cancer and other sexually transmitted infections across the globe. Currently, three prophylactic vaccines are available against HPV infections with no therapeutic values. Due to a lack of effective therapeutic and prophylactic measures, the HPV infection is spreading in an uncontrolled manner. Next-generation of vaccine is needed to have both prophylactic and therapeutic values against HPV. Here first time we have designed a multi-epitope chimeric vaccine using the most oncogenic strain HPV 16 and HPV 18 through an immunoinformatic approach. In this study, we have used the L1, E5, E6 and E7 oncoproteins from both HPV 16 and HPV 18 strains for epitope prediction. Our recombinant chimeric vaccine construct consists, selected helper and cytotoxic T cell epitopes. Our computational analysis suggests that this chimeric construct is highly stable, non-toxic and also capable of inducing both cell-mediated and humoral immune responses. Furthermore, in silico cloning of the multi-epitope chimeric vaccine construct was done and the stabilization of the vaccine construct is validated with molecular dynamics simulation studies. Finally, our results indicated that our construct could be used for an effective prophylactic and therapeutic vaccine against HPV.

Immunization with the DNA-encoding N-terminal domain of proteophosphoglycan of Leishmania donovani generates Th1-type immunoprotective response against experimental visceral leishmaniasis.

Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) which exist as secretory as well as surface-bound forms in both promastigotes and amastigotes. The structure and function of PPGs have been reported to be species and stage specific as in the case of Leishmania major and Leishmania mexicana; there has been no such information available for Leishmania donovani. We have recently demonstrated that PPG is differentially expressed in sodium stibogluconate-sensitive and -resistant clinical isolates of L. donovani. To further elucidate the structure and function of the ppg gene of L. donovani, a partial sequence of its N-terminal domain of 1.6 kb containing the majority of antigenic determinants, was successfully cloned and expressed in prokaryotic as well as mammalian cells. We further evaluated the DNA-encoding N-terminal domain of the ppg gene as a vaccine in golden hamsters (Mesocricetus auratus) against the L. donovani challenge. The prophylactic efficacy to the tune of approximately 80% was observed in vaccinated hamsters and all of them could survive beyond 6 mo after challenge. The efficacy was supported by a surge in inducible NO synthase, IFN-gamma, TNF-alpha, and IL-12 mRNA levels along with extreme down-regulation of TGF-beta, IL-4, and IL-10. A rise in the level of Leishmania-specific IgG2 was also observed which was indicative of enhanced cellular immune response. The results suggest the N-terminal domain of L. donovani ppg as a potential DNA vaccine against visceral leishmaniasis.

Mechanism involved in the pathogenesis and immune response against SARS-CoV-2 infection.

SARS CoV-2, a causative agent of human respiratory tract infection, was first identified in late 2019. It is a newly emerging viral disease with unsatisfactory treatments. The virus is highly contagious and has caused pandemic globally. The number of deaths is increasing exponentially, which is an alarming situation for mankind. The detailed mechanism of the pathogenesis and host immune responses to this virus are not fully known. Here we discuss an overview of SARS CoV-2 pathogenicity, its entry and replication mechanism, and host immune response against this deadly pathogen. Understanding these processes will help to lead the development and identification of drug targets and effective therapies.

Role of Cytokines in Experimental and Human Visceral Leishmaniasis.

Visceral Leishmaniasis (VL) is the most fatal form of disease leishmaniasis. To date, there are no effective prophylactic measures and therapeutics available against VL. Recently, new immunotherapy-based approaches have been established for the management of VL. Cytokines, which are predominantly produced by helper T cells (Th) and macrophages, have received great attention that could be an effective immunotherapeutic approach for the treatment of human VL. Cytokines play a key role in forming the host immune response and in managing the formation of protective and non-protective immunities during infection. Furthermore, immune response mediated through different cytokines varies from different host or animal models. Various cytokines viz. IFN-γ, IL-2, IL-12, and TNF-α play an important role during protection, while some other cytokines viz. IL-10, IL-6, IL-17, TGF-ß, and others are associated with disease progression. Therefore, comprehensive knowledge of cytokine response and their interaction with various immune cells is very crucial to determine appropriate immunotherapies for VL. Here, we have discussed the role of cytokines involved in VL disease progression or host protection in different animal models and humans that will determine the clinical outcome of VL and open the path for the development of rapid and accurate diagnostic tools as well as therapeutic interventions against VL.

Laboratory diagnosis of COVID-19: current status and challenges.

The magnitude and pace of global affliction caused by Coronavirus Disease-19 (COVID-19) is unprecedented in the recent past. From starting in a busy seafood market in the Chinese city of Wuhan, the virus has spread across the globe in less than a year, infecting over 76 million people and causing death of close to 1.7 million individuals worldwide. As no specific antiviral treatment is currently available, the major strategy in containing the pandemic is focused on early diagnosis and prompt isolation of the infected individuals. Several diagnostic modalities have emerged within a relatively short period, which can be broadly classified into molecular and immunological assays. While the former category is centered around real-time PCR, which is currently considered the gold standard of diagnosis, the latter aims to detect viral antigens or antibodies specific to the viral antigens and is yet to be recommended as a stand-alone diagnostic tool. This review aims to provide an update on the different diagnostic modalities that are currently being used in diagnostic laboratories across the world as well as the upcoming methods and challenges associated with each of them. In a rapidly evolving diagnostic landscape with several testing platforms going through various phases of development and/or regulatory clearance, it is prudent that the clinical community familiarizes itself with the nuances of different testing modalities currently being employed for this condition.

Photodynamic vaccination of hamsters with inducible suicidal mutants of Leishmania amazonensis elicits immunity against visceral leishmaniasis.

Leishmania, naturally residing in the phagolysosomes of macrophages, is a suitable carrier for vaccine delivery. Genetic complementation of these trypanosomatid protozoa to partially rectify their defective heme-biosynthesis renders them inducible with delta-aminolevulinate to develop porphyria for selective photolysis, leaving infected host cells unscathed. Delivery of released "vaccines" to antigen-presenting cells is thus expected to enhance immune response, while their self-destruction presents added advantages of safety. Such suicidal L. amazonensis was found to confer immunoprophylaxis and immunotherapy on hamsters against L. donovani. Neither heat-killed nor live parasites without suicidal induction were effective. Photodynamic vaccination of hamsters with the suicidal mutants reduced the parasite loads by 99% and suppressed the development of disease. These suppressions were accompanied by an increase in Leishmania-specific delayed-type hypersensitivity and lymphoproliferation as well as in the levels of splenic iNOS, IFN-gamma, and IL-12 expressions and of Leishmania-specific IgG2 in the serum. Moreover, a single intravenous administration of T cells from vaccinated hamsters was shown to confer on naïve animals an effective cellular immunity against L. donovani challenges. The absence of lesion development at vaccination sites and parasites in the draining lymphnodes, spleen and liver further indicates that the suicidal mutants provide a safe platform for vaccine delivery against experimental visceral leishmaniasis.

Current approaches for target-specific drug discovery using natural compounds against SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) recently caused a pandemic outbreak called coronavirus disease 2019 (COVID-19). This disease has initially been reported in China and also now it is expeditiously spreading around the globe directly among individuals through coughing and sneezing. Since it is a newly emerging viral disease and obviously there is a lack of anti-SARS-CoV-2 therapeutic agents, it is urgently required to develop an effective anti-SARS-CoV-2-agent.Through recent advancements in computational biology and biological assays, several natural compounds and their derivatives have been reported to confirm their target specific antiviral potential against Middle East respiratory syndrome coronavirus (MERS-CoV) or Severe Acute Respiratory Syndrome(SARS-CoV).These targets including an important host cell receptor, i.e., angiotensin-converting enzyme ACE2 and several viral proteins e.g. spike glycoprotein (S) containing S1 and S2 domains, SARS CoV Chymotrypsin-like cysteine protease (3CLpro), papain-like cysteine protease (PLpro), helicases and RNA-dependent RNA polymerase (RdRp). Due to physical, chemical, and some genetic similarities of SARS CoV-2 with SARS-COV and MERS-COV, repurposing various anti-SARS-COV or anti-MERS-COV natural therapeutic agents could be helpful for the development of anti-COVID-19 herbal medicine. Here we have summarized various drug targets in SARS-COV and MERS-COV using several natural products and their derivatives, which could guide researchers to design and develop a safe and cost-effective anti-SARS-COV-2 drugs.

Pro-apoptotic effect of the landrace Bangla Mahoba of Piper betle on Leishmania donovani may be due to the high content of eugenol.

In the absence of effective and safe treatment for visceral leishmaniasis or Kala-azar - a devastating parasitic disease caused by Leishmania donovani - the search for anti-leishmanial agents from natural resources in common use is imperative. Recently, the comparative in vitro anti-leishmanial activity of methanolic extracts from two landraces of Piper betle - P. betle landrace Bangla Mahoba (PB-BM) and P. betle landrace Kapoori Vellaikodi (PB-KV) - has been reported. Here, the putative pathway responsible for death induced by the effective extract of PB-BM methanolic extract in promastigotes, as well as the intracellular amastigote form of L. donovani, was assessed using various biochemical approaches. It was found that PB-BM was capable of selectively inhibiting both stages of Leishmania parasites by accelerating apoptotic events by generation of reactive oxygen species targeting the mitochondria without any cytotoxicity towards macrophages. The study was extended to determine the presence or absence of activity of the methanolic extract of PB-BM and PB-KV on the basis of differences in essential oil composition present in the extract assessed by GC and MS. The essential oil from PB-BM was found to be rich in eugenol compared with that from PB-KV. The anti-leishmanial efficacy of PB-BM methanolic extract mediated through apoptosis is probably due to the higher content of eugenol in the active landrace. This observation emphasizes the need to extend studies related to traditional medicines from bioactive plants below the species level to the gender/landrace level for better efficacy and reproducibility.

SUMOylation of ROR-γt inhibits IL-17 expression and inflammation via HDAC2.

Dysregulated ROR-γt-mediated IL-17 transcription is central to the pathogenesis of several inflammatory disorders, yet the molecular mechanisms that govern the transcription factor activity of ROR-γt in the regulation of IL-17 are not fully defined. Here we show that SUMO-conjugating enzyme Ubc9 interacts with a conserved GKAE motif in ROR-γt to induce SUMOylation of ROR-γt and suppress IL-17 expression. Th17 cells expressing SUMOylation-defective ROR-γt are highly colitogenic upon transfer to Rag1-/- mice. Mechanistically, SUMOylation of ROR-γt facilitates the binding of HDAC2 to the IL-17 promoter and represses IL-17 transcription. Mice with conditional deletion of HDAC2 in CD4+ T cells have elevated IL-17 expression and severe colitis. The identification of the Ubc9/ROR-γt/HDAC2 axis that governs IL-17 expression may open new venues for the development of therapeutic measures for inflammatory disorders.