MIF confers survival advantage to pancreatic CAFs by suppressing interferon pathway-induced p53-dependent apoptosis.

The presence of activated pancreatic stellate cells (PSCs) in the pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a significant role in cancer progression. Macrophage migration inhibitory factor (MIF) is overexpressed in PDAC tissues and expressed by both cancer and stromal cells. The pathophysiological role of MIF in PDAC-associated fibroblasts or PSCs is yet to be elucidated. Here we report that the PSCs of mouse or cancer-associated fibroblast cells (CAFs) of human expresses MIF and its receptors, whose expression gets upregulated upon LPS or TNF-α stimulation. In vitro functional experiments showed that MIF significantly conferred a survival advantage to CAFs/PSCs upon growth factor deprivation. Genetic or pharmacological inhibition of MIF also corroborated these findings. Further, co-injection of mouse pancreatic cancer cells with PSCs isolated from Mif-/- or Mif+/+ mice confirmed the pro-survival effect of MIF in PSCs and also demonstrated the pro-tumorigenic role of MIF expressed by CAFs in vivo. Differential gene expression analysis and in vitro mechanistic studies indicated that MIF expressed by activated CAFs/PSCs confers a survival advantage to these cells by suppression of interferon pathway induced p53 dependent apoptosis.

High PGD2 receptor 2 levels are associated with poor prognosis in colorectal cancer patients and induce VEGF expression in colon cancer cells and migration in a zebrafish xenograft model.

BACKGROUND: Despite intense research, the prognosis for patients with advanced colorectal cancer (CRC) remains poor. The prostaglandin D2 receptors DP1 and DP2 are explored here as potential therapeutic targets for advanced CRC. METHODS: A CRC cohort was analysed to determine whether DP1 and DP2 receptor expression correlates with patient survival. Four colon cancer cell lines and a zebrafish metastasis model were used to explore how DP1/DP2 receptor expression correlates with CRC progression. RESULTS: Analysis of the clinical CRC cohort revealed high DP2 expression in tumour tissue, whereas DP1 expression was low. High DP2 expression negatively correlated with overall survival. Other pathological indicators, such as TNM stage and metastasis, positively correlated with DP2 but not DP1 expression. In accordance, the in vitro results showed high DP2 expression in four CC-cell lines, but only one expressed DP1. DP2 stimulation resulted in increased proliferation, p-ERK1/2 and VEGF expression/secretion. DP2-stimulated cells exhibited increased migration in the zebrafish metastasis model. CONCLUSION: Our results support DP2 receptor expression and signalling as a therapeutic target in CRC progression based on its expression in CRC tissue correlating with poor patient survival and that it triggers proliferation, p-ERK1/2 and VEGF expression and release and increased metastatic activity in CC-cells.

Infection, cases due to SARS-CoV-2 in rural areas during early COVID-19 vaccination: findings from serosurvey study in a rural cohort of eastern India.

COVID-19 serosurvey provides a better estimation of people who have developed antibody against the infection. But limited information on such serosurveys in rural areas poses many hurdles to understand the epidemiology of the virus and to implement proper control strategies. This study was carried out in the rural catchment area of Model Rural Health Research Unit in Odisha, India during March-April 2021, the initial phase of COVID vaccination. A total of 60 village clusters from four study blocks were identified using probability proportionate to size sampling. From each cluster, 60 households and one eligible participant from each household (60 per cluster) were selected for the collection of blood sample and socio-demographic data. The presence of SARS-CoV-2 antibody was tested using the Elecsys Anti-SARS-CoV-2 immunoassay. The overall seroprevalence after adjusting for test performance was 54.21% with an infection to case ratio of 96.89 along with 4.25% partial and 6.79% full immunisation coverage. Highest seroprevalence was observed in the age group of 19-44 years and females had both higher seroprevalence as well as vaccine coverage. People of other backward caste also had higher seropositivity than other caste categories. The study emphasises on continuing surveillance for COVID-19 cases and prioritizing COVID-19 vaccination for susceptible groups for better disease management.

Saliva for diagnosis of SARS-CoV-2: First report from India.

There are very few studies in search of an alternate and convenient diagnostic tool which can substitute nasopharyngeal swab (NPS) specimen for detection of SARS-CoV-2. In the study we analyzed, the comparison and agreement between the feasibility of using the saliva in comparison to NPS for diagnosis of SARS-CoV-2. A total number of 74 patients were enrolled for this study. We analyzed and compared the NPS and saliva specimen collected within 48 h after the symptom onset. We carried out real-time quantitative polymerase chain reaction, gene sequencing for the detection and determination SARS-CoV-2 specific genes. Phylogenetic tree was constructed to establish the isolation of viral RNA from saliva. We used the Bland-Altman model to identify the agreement between two specimens. This study showed a lower cycle threshold (CT ) mean value for the detection of SARS-CoV-2 ORF1 gene (mean, 27.07; 95% confidence interval [CI], 25.62 to 28.52) in saliva methods than that of NPS (mean 28.24; 95% CI, 26.62 to 29.85) specimen although the difference is statistically nonsignificant (p > .05). Bland-Altman analysis produced relatively smaller bias and high agreement between these two clinical specimens. Phylogenetic analysis with the RdRp and S gene confirmed the presence of SARS-CoV-2 in the saliva samples. Saliva represented a promising tool in COVID-19 diagnosis and the collection method would reduce the exposure risk of frontline health workers which is one of the major concerns in primary healthcare settings.

Recombinant outer membrane protein OmpC induces protective immunity against Aeromonashydrophila infection in Labeorohita.

Aeromonashydrophila is an opportunistic pathogen that causes enormous loss to aquaculture industry. The outer membrane proteins of Aeromonas help in bacterium-host interaction, and are considered to be potential vaccine candidates. In the present study, we evaluated immunogenicity and protective efficacy of recombinant OmpC (rOmpC) of A. hydrophila in Indian major carp, Labeorohita. The rOmpC-vaccinated fish produced specific anti-rOmpC antibodies with a significant antibody titer, and the antisera could specifically detect the rOmpC in the cell lysates of Escherichia coli expressing rOmpC and cross-react with different Aeromonas lysates, indicating the suitability of the anti-rOmpC antisera to detect Aeromonas infection. A significant increase was noted in ceruloplasmin level, myeloperoxidase and anti-protease activities in transient and temporal manner the sera of the rOmpC-immunized fish as compared to PBS-control fish. Higher agglutination- and hemolytic activity titers in the anti-rOmpC antisera indicate stimulation of innate immunity. Expression of immune-related genes comprising various acute phase proteins, cytokines and inflammatory response molecules were modulated in the head kidney of rOmpC-immunized L. rohita. While IgM, IL1ß, and TLR-22 were significantly up-regulated at early time points (3 h-72 h), the others showed a transient augmentation at both early and later time points (SOD, lysozymes C and G, NKEF-B, C3, CXCa and TNF-α) in the rOmpC-immunized L. rohita in comparison to PBS-injected controls. These data suggest that the rOmpC-induced immune response is temporally regulated to confer immunity. In vivo challenge of the rOmpC-immunized fish with A. hydrophila showed significantly greater survival when compared to PBS-injected control fish. Thus, our results highlight the immunomodulatory role of rOmpC and demonstrate its protective efficacy in L. rohita, along with the use of anti-rOmpC antisera in detecting Aeromonas infections.

Association of quorum sensing and biofilm formation with Salmonella virulence: story beyond gathering and cross-talk.

Enteric fever (typhoid and paratyphoid fever) is a public health concern which contributes to mortality and morbidity all around the globe. It is caused mainly due to ingestion of contaminated food and water with a gram negative, rod-shaped, flagellated bacterium known as Salmonella enterica serotype typhi (typhoid fever) or paratyphi (paratyphoid fever). Clinical problems associated with Salmonellosis are mainly bacteraemia, gastroenteritis and enteric fever. The bacteria undergo various mechanisms to escape itself from immune reaction of the host, modulating immune response at the site of infection leading to virulence factor production and anti-microbial resistance. Biofilm is one of the adaptation mechanisms through which Salmonella survives in unfavourable conditions and thus is considered as a major threat to public health. Another property of the bacteria is "Quorum Sensing", which is a cell-cell communication and most of the pathogenic bacteria use it to coordinate the production of several virulence factors and other behaviours such as swarming and biofilm formation. Earlier, quorum sensing was believed to be just a medium for communication but, later on, its role in virulence has been studied. However, there are negligible information relating to interaction between quorum sensing and biofilm formation and how these events play crucial role in Salmonella pathogenesis. The review is a summary of updated information regarding how Salmonella uses these properties to spread more and survive better, making a challenge for clinicians and public health experts. Therefore, this review would help bring an insight regarding how biofilm formation and quorum sensing are inter-related and their role in pathogenesis and virulence of Salmonella.

Tumour-suppressive effect of oestrogen receptor ß in colorectal cancer patients, colon cancer cells, and a zebrafish model.

Oestrogen receptor ß (ERß) has been suggested to have anti-proliferative and anti-tumour effects in breast and prostate cancer cells, but other studies have indicated its tumour-promoting effects. Understanding the complex effects of this receptor in different contexts requires further study. We reported that high ERß expression is independently associated with improved prognosis in female colorectal cancer (CRC) patients. Herein, we investigated the possible anti-tumour effect of ERß and its selective agonist. CRC patients with high ERß expression had significantly higher levels of membrane-associated ß-catenin, cysteinyl leukotriene receptor 2 (CysLT2 R), and 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which have anti-tumour effects, but lower levels of nuclear ß-catenin, cysteinyl leukotriene receptor 1 (CysLT1 R), and cyclooxygenase-2 (COX-2), which have tumour-promoting effects. These interesting findings were further supported by two different publicly available CRC mRNA datasets that showed a significant positive correlation between ERß expression and 15-PGDH and CysLT2 R expression and a negative correlation between ERß expression and ß-catenin, CysLT1 R, and COX-2 expression. We next evaluated ERß expression in three different colon cancer mouse models; ERß expression was negatively correlated with tumourigenesis. Furthermore, treatment with the ERß-agonist ERB-041 reduced CysLT1 R, active ß-catenin, and COX-2 levels but increased phospho-ß-catenin, CysLT2 R, and 15-PGDH levels in HCT-116, Caco-2, and SW-480 colon cancer cells compared to vehicle-treated cells. Interestingly, ERB-041-treated cells showed significantly decreased migration, survival, and colonosphere formation and increased apoptotic activity, as indicated by increased CASPASE-3 and apoptotic blebs. Finally, patients with low ERß expression had significantly more distant metastasis at the time of diagnosis than patients with high ERß expression. Therefore, we studied the effects of ERB-041-treated colon cancer cells in a zebrafish xenograft model. We found significantly less distant metastasis of ERB-041-treated cells compared to vehicle-treated cells. These results further support ERß's anti-tumour role in CRC and the possible use of its agonist in CRC patients. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Lipopolysaccharide (LPS) enhances prostate cancer metastasis potentially through NF-κB activation and recurrent dexamethasone administration fails to suppress it in vivo.

BACKGROUND: Previous studies have shown the effect of bacterial lipopolysaccharide (LPS) on enhanced cancer cells' growth and metastasis. However, the effect of LPS on prostate cancer (PCa) cells metastasis has not been investigated in details. This study aimed to investigate the functional role of LPS on PCa cells metastasis and determine the effect of dexamethasone (DEX) on this event. METHODS: Two different PCa reporter cells lines (DU145-NF-κB-Luc and MAT-LyLu- NF-κB-Luc) were used to assess the direct effect of LPS on NF-κB activation in PCa cells. Plasma collected from LPS-stimulated human and rodent blood were used to check the indirect effect of LPS on NF-κB activation in PCa cells. Trans-well migration assay and two different orthotopic PCa animal models were used to investigate the effect of LPS on DU145 and MAT-LyLu cells migration or metastasis in vitro and in vivo, respectively. In all the studies DEX was used with or without LPS stimulation. RESULTS: LPS and secretory factors present in plasma collected from LPS-stimulated blood, significantly activated NF-κB in DU145, and MAT-LyLu cells and enhanced their migration in vitro. DEX significantly suppressed LPS-mediated activation of cancer and blood cells and abrogated the direct and indirect pro-migratory effect of LPS on PCa cells. Systemic administration of LPS activated NF-κB in DU145 cells in vivo; however, failed to alter the metastatic properties of these cells. On the other hand, systemic administration of LPS to MAT-LyLu tumor bearing animals significantly enhanced the incidence of metastasis without altering the overall growth of primary tumors. Unexpectedly, though DEX significantly suppressed MAT-LyLu primary tumor weights, it aggravated metastasis of cancer cells in presence and absence of LPS. Moreover, consecutive DEX pre-treatment enhanced experimental peritoneal metastasis of MAT-LyLu cells. At the molecular level, LPS, and/or DEX induced overexpression of immunosuppressive molecules in MAT-LyLu tumors. CONCLUSIONS: Overall, our study has shown that LPS and/or LPS induced inflammation can increase PCa metastasis and immunosuppressive dose of DEX might further enhance cancer metastasis.

Experimental models of pancreatic cancer desmoplasia.

Desmoplasia is a fibro-inflammatory process and a well-established feature of pancreatic cancer. A key contributor to pancreatic cancer desmoplasia is the pancreatic stellate cell. Various in vitro and in vivo methods have emerged for the isolation, characterization, and use of pancreatic stellate cells in models of cancer-associated fibrosis. In addition to cell culture models, genetically engineered animal models have been established that spontaneously develop pancreatic cancer with desmoplasia. These animal models are currently being used for the study of pancreatic cancer pathogenesis and for evaluating therapeutics against pancreatic cancer. Here, we review various in vitro and in vivo models that are being used or have the potential to be used to study desmoplasia in pancreatic cancer.

Modulation of immune response and protective efficacy of recombinant outer-membrane protein F (rOmpF) of Aeromonas hydrophila in Labeo rohita.

The outer-membrane proteins (OMPs) of Aeromonas hydrophila, an imperative fish pathogen accountable for massive economic losses to aquaculture industry, are found to be immunogenic and considered as potential vaccine candidates. In spite of development in the formulation of vaccine candidates against Aeromonas infection, no commercial preparation has been done so far; in addition, the molecular mechanisms of immunoprotection induced by various vaccine formulations in Indian major carp, Labeo rohita, are little known. The present study was undertaken to evaluate the modulation of immunity and expression of immune-related genes post-rOmpF (recombinant outer-membrane protein of A. hydrophila, a novel vaccine candidate) immunization and protective efficacy after A. hydrophila challenge. The rOmpF-immunized fish showed a variable expression of the immune-related genes, viz. toll-like receptor 22 (TLR), complement component 3 (C3), chemokine (CXCa), tumor necrosis factor-α (TNFα), interleukin 1ß (IL-1ß), manganese superoxide dismutase (MnSOD) and natural killer enhancing factor (NKEF) in the head kidney tissues, when compared to the control group at different time intervals post-vaccination. A significant increase in serum hemolysin titer, ceruloplasmin level and myeloperoxidase activity was observed on day 140 post immunization. Also, bacterial agglutination titer and antiprotease activity were significantly increased on day 42 post immunization. No significant change was observed in lysozyme activity. Challenge studies with live A. hydrophila on day 140 post-immunization of L. rohita significantly increased the relative percentage survival (∼44%) in the vaccinated group. The results suggest that the rOmpF could be used as a potential vaccine candidate to combat A. hydrophila infection in fish.

Effect of Dactylogyrus catlaius (Jain 1961) infection in Labeo rohita (Hamilton 1822): innate immune responses and expression profile of some immune related genes.

The monogenean ectoparasite, Dactylogyrus sp. is a major pathogen in freshwater aquaculture. The immune responses in parasitized fish were analyzed by quantitation of innate immune factors (natural agglutinin level, haemolysin titre, antiprotease, lysozyme and myeloperoxidase activities) in serum and immune-relevant gene expression in gill and anterior kidney. The antiprotease activity and natural agglutinin level were found to be significantly higher and lysozyme activity was significantly lower in parasitized fish. Most of the genes viz., beta2-microglobulin (beta2M), major histocompatibility complex I (MHCI), MHCII, tumor necrosis factor alpha (TNFalpha) and toll-like receptor 22 (TLR22) in gill samples were significantly down-regulated in the experimental group. In the anterior kidney, the expression of superoxide dismutase and interleukin 1beta (IL1beta) were significantly up-regulated whereas a significant down regulation of MHCII and TNFalpha was also observed. The down-regulation of most of the genes viz, MHCI, beta2M, MHCII, TLR22 and TNFalpha in infected gills indicated a well evolved mechanism in this parasite to escape the host immune response. The modulation of innate and adaptive immunity by this parasite can be further explored to understand host susceptibility.

Antimicrobial Resistance and Biofilm Formation of Staphylococcus aureus Isolates from Febrile Cases: Findings from a Rural Cohort of Odisha, India.

This study investigated the major pathogens in fever patients' blood in a rural cohort and characterized its virulence. A total of 718 blood samples received from IPD/OPD (inpatient department/outpatient department) patients with H/O (history of) fever were cultured, and 73 out of 83 culture-positive samples were identified as Staphylococcus aureus. The isolates showed higher resistance to penicillin, most being multidrug resistant. They formed biofilm in vitro, and 27.4% of the isolates were strong biofilm producers. They were sensitive towards linezolid, gentamicin, and tetracycline. The findings emphasize the necessity of preventing and managing staphylococcal infection and regular antimicrobial surveillance in rural areas.

Factors Associated with Chronic Kidney Disease of Unknown Etiology (CKDu): A Systematic Review.

(1) Background: Despite ample research, the factors, specific causes, and pathways associated with chronic kidney disease of unknown etiology (CKDu) remain elusive. Therefore, we performed a systematic review to explore the potential etiologies for the development of CKDu globally. (2) Methods: A systematic literature review was conducted using databases CINAHL, Cochrane Library, Embase, Google Scholar, MEDLINE, and PsycINFO on the specific causes and pathophysiology related to CKDu from inception until April 2021. Study selection, data extraction of included articles, and quality appraisal were assessed. The narrative approach was used to summarize and comprehend the findings. (3) Results: Our study included 25 studies, considering 38,351 participants. Twelve studies were case-control, ten were cross-sectional, and three were cohort designs. All articles were from low-and middle-income countries (LMICs). The findings suggest 12 factors are associated with CKDu. Most studies (n = 8) identified farming and water sources as the factors related to CKDu, with heavy metal toxicity coming in second (n = 7). (4) Conclusion: The systematic review reported various factors associated with CKDu, from which most studies reported farming, water sources, and heavy metal poisoning. Considering the findings, the study recommends future strategies and public health initiatives to prevent the epidemiological/environmental factors contributing to CKDu.

Prevalence and pattern of multimorbidity among chronic kidney disease patients: a community study in chronic kidney disease hotspot area of Eastern India.

Introduction: Chronic kidney disease (CKD) is mostly asymptomatic until reaching an advanced stage. Although conditions such as hypertension and diabetes can cause it, CKD can itself lead to secondary hypertension and cardiovascular disease (CVD). Understanding the types and prevalence of associated chronic conditions among CKD patient could help improve screening for early detection and case management. Methods: A cross sectional study of 252 CKD patients in Cuttack, Odisha (from the last 4 years CKD data base) was telephonically carried out using a validated Multimorbidity Assessment Questionnaire for Primary Care (MAQ-PC) tool with the help of an android Open Data Kit (ODK). Univariate descriptive analysis was done to determine the socio-demographic distribution of CKD patients. A Cramer's heat map was generated for showing Cramer's coefficient value of association of each diseases. Results: The mean age of participants was 54.11 (±11.5) years and 83.7% were male. Among the participants, 92.9% had chronic conditions (24.2% with one, 26.2% with two and 42.5% with three or more chronic conditions). Most prevalent chronic conditions were hypertension (48.4%), peptic ulcer disease (29.4%), osteoarthritis (27.8%) and diabetes (13.1%). Hypertension and osteoarthritis were found to be most commonly associated (Cramer's V coefficient = 0.3). Conclusion: Increased vulnerability to chronic conditions among CKD patients make them at higher risk for mortality and compromised quality of life. Regular screening of CKD patient for other chronic conditions (hypertension, diabetes, peptic ulcer disease, osteoarthritis and heart diseases) would help in detecting them early and undertake prompt management. The existing national program could be leveraged to achieve this.

Metabolomics of Multimorbidity: Could It Be the Quo Vadis?

Multimorbidity, the simultaneous presence of two or more chronic diseases, affects the health care to a great extent. Its association with health care cost, more disability, and poor quality of life makes it a major public health risk. The matter of worry is that management of a multimorbid condition is complicated by the fact that multiple types of treatment may be required to treat different diseases at a time, and the interaction between some of the therapies can be detrimental. Understanding the causal factors of simultaneously occurring disease conditions and investigating the connected pathways involved in the whole process may resolve the complication. When different disease conditions present in an individual share common responsible factors, treatment strategies targeting at those common causes will certainly reduce the chance of development of multimorbidity occurring because of those factors. Metabolomics that can dig out the underlying metabolites/molecules of a medical condition is believed to be an effective technique for identification of biomarkers and intervention of effective treatment strategies for multiple diseases. We hypothesize that understanding the metabolic profile may shed light on targeting the common culprit for different/similar chronic diseases ultimately making the treatment strategy more effective with a combinatorial effect.

Sequence analysis of Indian SARS-CoV-2 isolates shows a stronger interaction of mutant receptor-binding domain with ACE2.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected the whole world, including Odisha, a state in eastern India. Many people have migrated to the state from different countries as well as other states during this SARS-CoV-2 pandemic. The aim of this study was to analyse the receptor-binding domain (RBD) sequence of the spike protein from isolates collected from throat swab samples of COVID-19-positive patients and further to assess the RBD affinity for angiotensin-converting enzyme 2 (ACE2) of different species, including humans. METHODS: Whole-genome sequencing for 35 clinical SARS-CoV-2 isolates from COVID-19-positive patients was performed by ARTIC amplicon-based sequencing. Sequence analysis and phylogenetic analysis were performed for the spike region and the RBD region of all isolates. The interaction between the RBD and ACE2 of five different species was also analysed. RESULTS: The spike region of 32 isolates showed one or multiple alterations in nucleotide bases in comparison with the Wuhan reference strain. One of the identified mutations, at position 1204 (Ref A, RMRC 22 C), in the RBD coding region of the spike protein showed stronger binding affinity for human ACE2. Furthermore, RBDs of all the Indian isolates showed binding affinity for ACE2 of different species. CONCLUSION: As mutant RBD showed stronger interaction with human ACE2, it could potentially result in higher infectivity. The binding affinity of the RBDs for ACE2 of all five species studied suggests that the virus can infect a wide variety of animals, which could also act as natural reservoir for SARS-CoV-2.

Macrophage migration inhibitory factor of Syrian golden hamster shares structural and functional similarity with human counterpart and promotes pancreatic cancer.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases. Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions, the information obtained from these models is biased towards a specific species. In experimental science, results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans. Syrian golden hamster (Mesocricetus auratus), is a clinically relevant animal model for multiple human diseases. Hence, the major objectives of this study were to characterize the structure and function of Mesocricetus auratus MIF (MaMIF) and finally evaluate its effect on pancreatic tumor growth in vivo. Initially, the recombinant MaMIF was cloned, expressed and purified in a bacterial expression system. The MaMIF primary sequence, biochemical properties, and crystal structure analysis showed greater similarity with human MIF. The crystal structure of MaMIF illustrates that it forms a homotrimer as known in human and mouse. However, MaMIF exhibits some minor structural variations when compared to human and mouse MIF. The in vitro functional studies show that MaMIF has tautomerase activity and enhances activation and migration of hamster peripheral blood mononuclear cells (PBMCs). Interestingly, injection of MaMIF into HapT1 pancreatic tumor-bearing hamsters significantly enhanced the tumor growth and tumor-associated angiogenesis. Together, the current study shows a structural and functional similarity between the hamster and human MIF. Moreover, it has demonstrated that a high level of circulating MIF originating from non-tumor cells might also promote pancreatic tumor growth in vivo.

Th2-biased immune response and agglutinating antibodies generation by a chimeric protein comprising OmpC epitope (323-336) of Aeromonas hydrophila and LTB.

Aeromonas hydrophila is responsible for causing fatal infections in freshwater fishes. Besides chemical/antibiotic treatment and whole-cell vaccine, no subunit vaccine is currently available for A. hydrophila. Outer membrane proteins of gram-negative bacteria have been reported as effective vaccine candidates. Peptide antigens elicit focused immune responses against immunodominant stretches of the antigen. We have attempted to characterize the immunogenicity of linear B-cell epitopes of outer membrane protein (OmpC) of A. hydrophila identified using in silico tools, in conjugation with heat-labile enterotoxin B (LTB) subunit of Escherichia coli as a carrier protein. Antisera against the fusion protein harboring 323-336 residues of the AhOmpC (raised in mice) showed maximum cross-reactivity with the parent protein OmpC and LTB. The fusion protein displayed efficient GM1 ganglioside receptor binding, retaining the adjuvanicity of LTB. Antibody isotype profile and in vitro T-cell response analysis, cytokine ELISA, and array analysis collectively revealed a Th2-biased mixed T-helper cell response. Agglutination assay and flow cytometry analysis validated the ability of anti-fusion protein antisera to recognize the surface exposed epitopes on Aeromonas cells, demonstrating its neutralization potential. Oral immunization studies in Labeo rohita resulted in the generation of long-lasting humoral immune response, and the antisera could cross-react with the fusion protein as well as both the fusion partners. Considering significant similarity among OmpC of different enteric bacteria, the use of A. hydrophila OmpC epitope323-336 in fusion with LTB could have a broader scope in vaccine design.

Structural and Functional Characterization of Recombinant Interleukin-10 from Indian Major Carp Labeo rohita.

Interleukin-10, an important regulator of both the innate and adaptive immune systems, is a multifunctional major cytokine. Though it is one of the major cytokines, IL-10 from the Indian major carp, Labeo rohita, has not yet been characterized. In the present study, we report large scale production and purification of biologically active recombinant IL-10 of L. rohita (rLrIL-10) using a heterologous expression system and its biophysical and functional characterization. High yield (~70 mg/L) of soluble rLrIL-10 was obtained at shake flask level. The rLrIL-10 was found to exist as a dimer. Far-UV CD spectroscopy showed presence of predominantly alpha helices. The tertiary structure of the purified rLrIL-10 was verified by fluorescence spectroscopy. Two-dimensional gel analysis revealed the presence of six isoforms of the rLrIL-10. The rLrIL-10 was biologically active and its administration significantly reduced serum proinflammatory cytokines, namely, interleukin 1ß, TNFα, and IL-8, and augmented the NKEF transcript levels in spleen of L. rohita. Anti-inflammatory role of the rLrIL-10 was further established by inhibition of phagocytosis using NBT reduction assay in vitro. The data indicate that the dimeric alpha helical structure and function of IL-10 of L. rohita as a key regulator of anti-inflammatory response have remained conserved during evolution.