Clonotypic IgH Response against Systemic Viral infection in Pronephros and Spleen of a Teleost Fish.

Upon infection, B lymphocytes develop clonal responses. In teleost fish, which lack lymph nodes, the kinetics and location of B cell responses remain poorly characterized. Fish pronephros is the site of B cell differentiation and the main niche for persistence of plasma cells. In this study, we undertook the analysis of the rainbow trout IgHµ repertoire in this critical tissue for humoral adaptive immunity after primary immunization and boost with a rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We used a barcoded 5' RACE-cDNA sequencing approach to characterize modifications of the IgHµ repertoire, including VH usage in expressed V(D)J rearrangements, clonal diversity, and clonotype sharing between individual fish and treatments. In the pronephros, our approach quantified the clonotype frequency across the whole IgH repertoire (i.e., with all VH), measuring the frequency of Ag-responding clonotypes. Viral infection led to extensive modifications of the pronephros B cell repertoire, implicating several VH subgroups after primary infection. In contrast, only modest changes in repertoire persisted 5 mo later, including VHSV-specific public expansions. The IgM public response implicating IgHV1-18 and JH5, previously described in spleen, was confirmed in pronephros in all infected fish, strongly correlated to the response. However, the distribution of top clonotypes showed that pronephros and spleen B cells constitute distinct compartments with different IgH repertoires. Unexpectedly, after boost, the frequency of anti-VHSV clonotypes decreased both in pronephros and spleen, raising questions about B cell circulation. A better monitoring of B cell response kinetics in lymphoid tissues will be an essential step to understand B memory and plasmocyte formation mechanisms in fish.

Cutting Edge: Neutralizing Public Antibody Responses Are an Ancient Form of Defense Conserved in Fish and Mammals.

The repertoire of Abs is generated by genomic rearrangements during B cell differentiation. Although V(D)J rearrangements lead to repertoires mostly different between individuals, recent studies have shown that they contain a substantial fraction of overrepresented and shared "public" clones. We previously reported a strong public IgHµ clonotypic response against the rhabdovirus viral hemorrhagic septicemia virus in a teleost fish. In this study, we identified an IgL chain associated with this public response that allowed us to characterize its functionality. We show that this public Ab response has a potent neutralizing capacity that is typically associated with host protection during rhabdovirus infections. We also demonstrate that the public response is not restricted to a particular trout isogenic line but expressed in multiple genetic backgrounds and may be used as a marker of successful vaccination. Our work reveals that public B cell responses producing generic Abs constitute a mechanism of protection against infection conserved across vertebrates.

Prevalence and risk factors of Strongyloides stercoralis in haemodialysis in Cochabamba, Bolivia: a cross-sectional study.

BACKGROUND: Strongyloidiasis is an infectious disease that can be fatal in immunocompromised patients. Patients with end-stage renal failure who are on dialysis have a considerably weakened immune system, and organ transplantation is a major risk factor for severe strongyloidiasis. Knowledge of the local epidemiology in tropical and subtropical areas is an essential prerequisite for designing an appropriate strategy to prevent this potentially lethal complication. In this study, we aimed to estimate the prevalence and associated risk factors of S. stercoralis infection in patients on dialysis in Cochabamba, Bolivia. METHODS: A cross-sectional study was carried out among patients undergoing haemodialysis in Cochabamba (elevation 2,500 m, temperate climate), collecting information on socio-demographic, lifestyle, and clinical variables, and using one coproparasitological technique (the modified Baermann technique) and one serological (ELISA) test for S.stercoralis diagnosis. RESULTS: In total, 149 patients participated in the study (mean age = 51.4 years, 48.3% male). End-stage renal disease was predominantly (59%) of hypertensive and/or diabetic origin. The positive serological prevalence was 18.8% (95% CI: 13.3%-25.9%). Based on the sensitivity and specificity of the ELISA test, the estimate of the actual prevalence was 15.1% (95% CI: 9.4%-20.7%). Stool samples of 105 patients (70.5%) showed a coproparasitological prevalence of 1.9% (95% CI: 0.52%-6.68%). No potential risk factors were significantly associated with S. stercoralis infection. CONCLUSIONS: We found a high seroprevalence of S. stercoralis in Bolivian patients undergoing haemodialysis in Cochabamba. We recommend presumptive antiparasitic treatment at regular intervals to avoid the potentially fatal complications of severe strongyloidiasis.

Spleen and head kidney differential gene expression patterns in trout infected with Lactococcus garvieae correlate with spleen granulomas.

Lactococcus garvieae is a significant pathogen in aquaculture with a potential zoonotic risk. To begin to characterize the late immune response of trout to lactococcosis, we selected infected individuals showing clinical signs of lactococcosis. At the time lactococcosis clinical signs appeared, infection by L. garvieae induced a robust inflammatory response in the spleen of rainbow trout, which correlated with abundant granulomatous lesions. The response in kidney goes in parallel with that of spleen, and most of the gene regulations are similar in both organs. A correlation existed between the early inflammatory granulomas in spleen (containing macrophages with internalized L. garvieae) and up-regulated gene sets, which defined the presence of macrophages and neutrophils. This is the first analysis of the immune transcriptome of rainbow trout following L. garvieae infection during the initiation of adaptive immune mechanisms and shows a transcriptome induction of antibody response by both IgM (+) and IgT (+) spleen B cells to respond to systemic infection. These results increase our understanding of lactococcosis and pave the way for future research to improve control measures of lactococcosis on fish farms.

The BAFF / APRIL axis plays an important role in virus-induced peritoneal responses in rainbow trout.

IgM+ B cells have been recently demonstrated to be key regulators of peritoneal inflammation in teleost, as a large number of them occupy the peritoneal cavity after 48 h of antigenic stimulation. Despite this, the number of studies addressing the mechanism through which this cell population expands and differentiates in response to stimuli has been scarcely addressed. Because the BAFF/APRIL axis is known to play a major role in B cell survival and differentiation in mammals, we hypothesized that it could be affected in the peritoneal cavity in response to an inflammatory stimulus. To verify this hypothesis, we studied how BAFF, APRIL and the fish-specific related cytokine BALM as well as their putative receptors are regulated in rainbow trout after intraperitoneal (i.p.) injection of viral hemorrhagic septicemia virus (VHSV). When the transcriptional analysis was performed in total cells from the peritoneum, we observed that VHSV provoked an up-regulation of both BAFF and BAFF receptor (BAFF-R) mRNA levels. However, when we examined how isolated peritoneal IgM+ B cells were transcriptionally affected by VHSV i.p. injection, we found that APRIL, BALM and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) were also up-regulated in response to the virus. IgM- cells, on the other hand, only up-regulated BALM transcription in response to VHSV. Finally, to gain further insight on the role that these cytokines play in the peritoneum, we have studied their effect on the survival of peritoneal IgM+ B cells. This work demonstrates a key role for the BAFF/APRIL axis in the peritoneal inflammatory response and contributes to further understanding how IgM+ B cells are regulated at this specific peripheral site.

Identification of Teleost Skin CD8α+ Dendritic-like Cells, Representing a Potential Common Ancestor for Mammalian Cross-Presenting Dendritic Cells.

Although fish constitute the most ancient animal group in which an acquired immune system is present, the presence of dendritic cells (DCs) in teleosts has been addressed only briefly, and the identification of a specific DC subset in teleosts remained elusive because of the lack of specific Abs. In mice, DCs expressing CD8α(+) in lymphoid tissues have the capacity to cross-present extracellular Ags to T cells through MHC I, similarly to tissue-derived CD103(+) DCs and the human CD141(+) DC population. In the current study, we identified a large and highly complex subpopulation of leukocytes coexpressing MHC class II and CD8α. This CD8α(+) MHC II(+) DC-like subpopulation constituted ∼1.2% of the total leukocyte population in the skin, showing phenotypical and functional characteristics of semimature DCs that seem to locally regulate mucosal immunity and tolerance in a species lacking lymph nodes. Furthermore, we identified trout homologs for CD141 and CD103 and demonstrated that, in trout, this skin CD8(+) DC-like subpopulation expresses both markers. To our knowledge, these results provide the first evidence of a specific DC-like subtype in nonimmune tissue in teleosts and support the hypothesis of a common origin for all mammalian cross-presenting DCs.

Early activation of teleost B cells in response to rhabdovirus infection.

UNLABELLED: To date, the response of teleost B cells to specific pathogens has been only scarcely addressed. In this work, we have demonstrated that viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, has the capacity to infect rainbow trout spleen IgM-positive (IgM(+)) cells, although the infection is not productive. Consequently, we have studied the effects of VHSV on IgM(+) cell functionality, comparing these effects to those elicited by a Toll-like receptor 3 (TLR3) ligand, poly(I·C). We found that poly(I·C) and VHSV significantly upregulated TLR3 and type I interferon (IFN) transcription in spleen and blood IgM(+) cells. Further effects included the upregulated transcription of the CK5B chemokine. The significant inhibition of some of these effects in the presence of bafilomycin A1 (BAF), an inhibitor of endosomal acidification, suggests the involvement of an intracellular TLR in these responses. In the case of VHSV, these transcriptional effects were dependent on viral entry into B cells and the initiation of viral transcription. VHSV also provoked the activation of NF-κB and the upregulation of major histocompatibility complex class II (MHC-II) cell surface expression on IgM(+) cells, which, along with the increased transcription of the costimulatory molecules CD80/86 and CD83, pointed to VHSV-induced IgM(+) cell activation toward an antigen-presenting profile. Finally, despite the moderate effects of VHSV on IgM(+) cell proliferation, a consistent effect on IgM(+) cell survival was detected. IMPORTANCE: Innate immune responses to pathogens established through their recognition by pattern recognition receptors (PRRs) have been traditionally ascribed to innate cells. However, recent evidence in mammals has revealed that innate pathogen recognition by B lymphocytes is a crucial factor in shaping the type of immune response that is mounted. In teleosts, these immediate effects of viral encounter on B lymphocytes have not been addressed to date. In our study, we have demonstrated that VHSV infection provoked immediate transcriptional effects on B cells, at least partially mediated by intracellular PRR signaling. VHSV also activated NF-κB and increased IgM(+) cell survival. Interestingly, VHSV activated B lymphocytes toward an antigen-presenting profile, suggesting an important role of IgM(+) cells in VHSV presentation. Our results provide a first description of the effects provoked by fish rhabdoviruses through their early interaction with teleost B cells.

CCR7 is mainly expressed in teleost gills, where it defines an IgD+IgM- B lymphocyte subset.

Chemokine receptor CCR7, the receptor for both CCL19 and CCL21 chemokines, regulates the recruitment and clustering of circulating leukocytes to secondary lymphoid tissues, such as lymph nodes and Peyer's patches. Even though teleost fish do not have either of these secondary lymphoid structures, we have recently reported a homolog to CCR7 in rainbow trout (Oncorhynchus mykiss). In the present work, we have studied the distribution of leukocytes bearing extracellular CCR7 in naive adult tissues by flow cytometry, observing that among the different leukocyte populations, the highest numbers of cells with membrane (mem)CCR7 were recorded in the gill (7.5 ± 2% CCR7(+) cells). In comparison, head kidney, spleen, thymus, intestine, and peripheral blood possessed <5% CCR7(+) cells. When CCR7 was studied at early developmental stages, we detected a progressive increase in gene expression and protein CCR7 levels in the gills throughout development. Surprisingly, the majority of the CCR7(+) cells in the gills were not myeloid cells and did not express membrane CD8, IgM, nor IgT, but expressed IgD on the cell surface. In fact, most IgD(+) cells in the gills expressed CCR7. Intriguingly, the IgD(+)CCR7(+) population did not coexpress memIgM. Finally, when trout were bath challenged with viral hemorrhagic septicemia virus, the number of CCR7(+) cells significantly decreased in the gills while significantly increased in head kidney. These results provide evidence of the presence of a novel memIgD(+)memIgM(-) B lymphocyte subset in trout that expresses memCCR7 and responds to viral infections. Similarities with IgD(+)IgM(-) subsets in mammals are discussed.

Teleost fish mount complex clonal IgM and IgT responses in spleen upon systemic viral infection.

Upon infection, B-lymphocytes expressing antibodies specific for the intruding pathogen develop clonal responses triggered by pathogen recognition via the B-cell receptor. The constant region of antibodies produced by such responding clones dictates their functional properties. In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT remain unknown. The expression of IgM and IgT are mutually exclusive, leading to the existence of two B-cell subsets expressing either both IgM and IgD or only IgT. Here, we undertook a comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout (Onchorhynchus mykiss) before, and after challenge with a rhabdovirus, the Viral Hemorrhagic Septicemia Virus (VHSV), using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, we observed distinct repertoires for IgM, IgD and IgT, respectively, with a few amplified µ and τ junctions, suggesting the presence of IgM- and IgT-secreting cells in the spleen. In infected animals, we detected complex and highly diverse IgM responses involving all VH subgroups, and dominated by a few large public and private clones. A lower number of robust clonal responses involving only a few VH were detected for the mucosal IgT, indicating that both IgM(+) and IgT(+) spleen B cells responded to systemic infection but at different degrees. In contrast, the IgD response to the infection was faint. Although fish IgD and IgT present different structural features and evolutionary origin compared to mammalian IgD and IgA, respectively, their implication in the B-cell response evokes these mouse and human counterparts. Thus, it appears that the general properties of antibody responses were already in place in common ancestors of fish and mammals, and were globally conserved during evolution with possible functional convergences.

Evaluation of Microscopic Observation Drug Susceptibility (MODS) and the string test for rapid diagnosis of pulmonary tuberculosis in HIV/AIDS patients in Bolivia.

BACKGROUND: Tuberculosis (TB) is the most common opportunistic infection and the leading cause of death in HIV-positive people worldwide. Diagnosing TB is difficult, and is more challenging in resource-scarce settings where culture-based diagnostic methods rely on poorly sensitive smear microscopy by Ziehl-Neelsen stain (ZN). METHODS: We performed a cross-sectional study examining the diagnostic utility of Microscopic Observation Drug Susceptibility liquid culture (MODS) versus traditional Ziehl-Neelsen staining (ZN) and Lowenstein Jensen culture (LJ) of pulmonary tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB) in HIV-infected patients in Bolivia. For sputum scarce individuals we assessed the value of the string test and induced sputum for TB diagnosis. The presence of Mycobacterium tuberculosis (Mtb) in the sputum of 107 HIV-positive patients was evaluated by ZN, LJ, and MODS. Gastric secretion samples obtained by the string test were evaluated by MODS in 102 patients. RESULTS: The TB-HIV co-infection rate of HIV patients with respiratory symptoms by sputum sample was 45 % (48/107); 46/48 (96 %) were positive by MODS, 38/48 (79 %) by LJ, and 30/48 (63 %) by ZN. The rate of MDRTB was 9 % (4/48). Median time to positive culture was 10 days by MODS versus 34 days by LJ (p < 0.0001). In smear-negative patients, MODS detected TB in 17/18 patients, compared to 11/18 by LJ (94.4 % vs 61.0 %, p = 0.03 %). In patients unable to produce a sputum sample without induction, the string test cultured by MODS yielded Mtb in of 9/11 (82 %) TB positive patients compared to 11/11 (100 %) with induced sputum. Of the 10 patients unable to produce a sputum sample, 4 were TB-positive by string test. CONCLUSION: MODS was faster and had a higher Mtb detection yield compared to LJ, with a greater difference in yield between the two in smear-negative patients. The string test is a valuable diagnostic technique for HIV sputum-scarce or sputum-absent patients, and should be considered as an alternative test to induced sputum to obtain sample for Mtb in resource-limited settings. Nine percent of our TB+ patients had MDRTB, which reinforces the need for rapid detection with direct drug susceptibility testing in HIV patients in Bolivia.

Effect of CaS Nanostructures in the Proliferation of Human Breast Cancer and Benign Cells In Vitro.

We report on the effect of naked CaS nanostructures on the proliferation of carcinoma cancer cells and normal fibroblasts in vitro. The CaS nanostructures were prepared via the microwave-mediated decomposition of dimethyl sulfoxide (DMSO) in the presence of calcium acetate Ca ( CH 3 CO 2 ) 2 . Light scattering measurements revealed that dispersions contain CaS nanostructures in the size range of a few Å to about 1 nanometer, and are formed when DMSO is decomposed in the presence of Ca ( CH 3 CO 2 ) 2 . Theoretical calculations at the DFT/B3LYP/DGDZVP level of theory on ( C a S ) n clusters ( n = 1 , 2 , 3 , and 4) are consistent with clusters in this size range. The absorption spectra of the CaS nanostructures are dominated by strong bands in the UV, as well as weaker absorption bands in the visible. We found that a single dose of CaS nanoclusters smaller than 0.8 nm in diameter does not affect the survival and growth rate of normal fibroblasts and inhibits the proliferation rate of carcinoma cells in vitro. Larger CaS nanostructures, approximately (1.1 ± 0.2) nm in diameter, have a similar effect on carcinoma cell proliferation and survival rate. The CaS nanoclusters have little effect on the normal fibroblast cell cycle. Human carcinoma cells treated with CaS nanocluster dispersion exhibited a decreased ability to properly enter the cell cycle, marked by a decrease in cell concentration in the G0/G1 phase in the first 24 h and an increase in cells held in the SubG1 and G0/G1 phases up to 72 h post-treatment. Apoptosis and necrotic channels were found to play significant roles in the death of human carcinoma exposed to the CaS nanoclusters. In contrast, any effect on normal fibroblasts appeared to be short-lived and non-detrimental. The interaction of CaS with several functional groups was further investigated using theoretical calculations. CaS is predicted to interact with thiol ( R-SH ), hydroxide ( R - OH ), amino ( R - NH 2 ), carboxylic acid ( R - COOH ), ammonium ( R-NH 3 + ), and carboxylate ( R-COO - ) functional groups. None of these interactions are predicted to result in the dissociation of CaS. Thermodynamic considerations, on the other hand, are consistent with the dissociation of CaS into Ca 2 + ions and H 2 S in acidic media, both of which are known to cause apoptosis or cell death. Passive uptake and extracellular pH values of carcinoma cells are proposed to result in the observed selectivity of CaS to inhibit cancer cell proliferation with no significant effect on normal fibroblast cells. The results encourage further research with other cell lines in vitro as well as in vivo to translate this nanotechnology into clinical use.

Cortisol modulates the induction of inflammatory gene expression in a rainbow trout macrophage cell line.

Glucocorticoid actions on the immune system are diverse and cell type dependent, and little is known about cell type-specific interactions and cross-talk between hormones and cytokines. In this study we have analyzed the gene expression patterns of the rainbow trout macrophage cell line RTS-11 by quantitative PCR, after exposure to combinations of cortisol plus a pro-inflammatory cytokine (e.g. recombinant trout IL-1ß, IFN-γ), type I IFN or a PAMP (LPS or poly I:C). Several key genes of the inflammatory process were targetted to assess whether any modulation of their expression occurred due to the addition of cortisol to this cell line. Incubation of macrophages for 3 or 6 h with a physiological concentration of cortisol caused a decrease in expression of IL-6 and IL-8, but no significant changes were observed for the other genes examined. Co-stimulation of cortisol with the inflammatory agents resulted in a general suppression of genes related to the inflammatory response. Cortisol inhibited the up-regulation of IL-8 by all the stimulants after 3 h of co-incubation. Suppression of the up-regulation of IL-6 by rIL-1ß, rIFN-γ and poly I:C, of γIP by rIFN-γ or poly I:C, and of Cox-2 by rIL-1ß was seen after 6 h. In contrast, cortisol in combination with the pro-inflammatory agents has a synergistic effect on IL-10 expression, an anti-inflammatory molecule, suggesting that the activation of certain macrophage functions that lead to the resolution of inflammation occurs in fish macrophages in response to cortisol treatment.

HIV and Chagas Disease: An Evaluation of the Use of Real-Time Quantitative Polymerase Chain Reaction to Measure Levels of Trypanosoma cruzi Parasitemia in HIV Patients in Cochabamba, Bolivia.

This cross-sectional study evaluated epidemiologic characteristics of persons living with HIV (PWH) coinfected with Trypanosoma cruzi in Cochabamba, Bolivia, and estimated T. cruzi parasitemia by real-time quantitative polymerase chain reaction (qPCR) in patients with and without evidence of reactivation by direct microscopy. Thirty-two of the 116 HIV patients evaluated had positive serology for T. cruzi indicative of chronic Chagas disease (27.6%). Sixteen of the 32 (50%) patients with positive serology were positive by quantitative polymerase chain reaction (qPCR), and four of the 32 (12.5%) were positive by direct microscopy. The median parasite load by qPCR in those with CD4+ < 200 was 168 parasites/mL (73-9951) compared with 28.5 parasites/mL (15-1,528) in those with CD4+ ≥ 200 (P = 0.89). There was a significant inverse relationship between the degree of parasitemia estimated by qPCR from blood clot and CD4+ count on the logarithmic scale (rsBC= -0.70, P = 0.007). The correlation between T. cruzi estimated by qPCR+ blood clot and HIV viral load was statistically significant with rsBC = 0.61, P = 0.047. Given the significant mortality of PWH and Chagas reactivation and that 57% of our patients with CD4+ counts < 200 cells/mm3 showed evidence of reactivation, we propose that screening for chronic Chagas disease be considered in PWH in regions endemic for Chagas disease and in the immigrant populations in nonendemic regions. Additionally, our study showed that PWH with advancing immunosuppression have higher levels of estimated parasitemia measured by qPCR and suggests a role for active surveillance for Chagas reactivation with consideration of treatment with antitrypanosomal therapy until immune reconstitution can be achieved.

Establishment of an IFN-gamma specific reporter cell line in fish.

An interferon (IFN)-gamma responsive stable cell line RTG-3F7 has been developed for rainbow trout by modifying the RTG-2 cell line through transfection with a plasmid construct (pGL4.14[luc2/hygro]-PrTAP2) containing a promoter element from the IFN-gamma responsive gene TAP2 linked to a luciferase reporter gene and a hygromycin resistance gene. Following transfection single clones were selected in 96 well plates using hygromycin B, and those showing specific activation after rIFN-gamma stimulation were maintained. Five clones that showed the highest reporter activity to rIFN-gamma were incubated with different stimuli to examine specificity. No significant induction of luciferase was observed following exposure to recombinant type I IFN, LPS, PHA or poly I:C. The cell line was responsive to rIFN-gamma at concentrations between 150 pg and 20 ng ml(-1). Supernatants of primary cultures of head kidney leucocytes stimulated with PHA, known to induce IFN-gamma gene expression, were also used to assess the reporter activity of the stable cell line. A dose-dependent induction of the promoter activity was observed with these supernatants indicating the presence of IFN-gamma. These results indicate that the stable cell line RTG-3F7 is an excellent tool for monitoring the presence of trout IFN-gamma in biological samples, and in addition, enables the study of intracellular signalling pathways of IFNs, their receptor interactions, and other closely related signalling networks.

Effect of jasmonic acid on major terpenes and density of glandular trichomes in Lippia graveolens kunth (Verbenaceae).

The effect of exogenous application of jasmonic acid (JA) on the concentration of main terpenes and density of glandular trichomes was investigated in the Mexican oregano, propagated from seeds from 3 localities. JA 1 mM was applied locally and to the whole plant. JA locally applied increased the number of trichomes, with a mean of 20 trichomes more with respect to the controls in plants from Tecomavaca and Zapotitlán Salinas, and significantly increased the thymol concentration by 185% systemically and 255% locally, compared to the control. JA applied to the whole plant decreased the number of trichomes and increased the concentration of caryophyllene from 0.79 to 1.7 mg g-1, and α-caryophyllene from 0.3 to 0.8 mg g-1 in plants from San Rafael with reference to water control. The results suggest a plasticity of morphologic and phytochemical responses, and a potential use of JA to improve phenolic monoterpenes and sesquiterpenes production.

Isolation and molecular cloning of a fish myeloperoxidase.

Myeloperoxidase (MPO) is a conspicuous enzyme in neutrophils of many fish species. Although the MPO gene has been identified in some fish species, the structure and functions of the protein remain to be determined in these vertebrates. In the present study, we isolated turbot neutrophil MPO from kidney cells by affinity chromatography, with Ulva rigida acidic sulphated polysaccharides (ASP), some of which resemble glycosaminoglycans, and Sepharose. The product obtained, of approximately 150kDa molecular weight and with peroxidase activity, was examined by SDS-page electrophoresis under reduced conditions and immunoblotting, and a single band of about 75kDa was observed. The results obtained suggest that turbot MPO is a dimer and that the band of 75kDa probably corresponds to a monomer generated by treatment of the samples with the reducing agent. The band was analysed by electromatrix-assisted laser desorption ionization-time-of flight-mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization-ion trap mass spectrometry, dynamic exclusion mode (LC-ESI-IT DE), to determine the amino acid composition of some peptides. The peptides obtained were very similar to myeloperoxidases of other organisms, including other fish and mammals, and were used to design the primers for cDNA amplification. A 567bp product was amplified and the deduced amino acid sequence, which contains several putative N-glycosylation and O-glycosylation sites, was compared with other myeloperoxidases. As expected, turbot MPO was more similar to MPO from other fish species (67-86% identity), where the phylogenetic tree obtained agrees with the taxonomic hierarchy, than to MPO from mammals (55-57% identity) and other groups. The results obtained in the present study will also allow functional studies to be carried out with turbot neutrophil MPO enzyme, as well as analysis of MPO gene expression under different stimuli.

Characterisation of gamma-interferon responsive promoters in fish.

Reporter constructs of three interferon (IFN)-gamma-induced rainbow trout genes were generated to examine specificity to type I or type II IFN. Constructs included gammaIP-10, LMP2 and TAP2 and were used to transfect trout fibroblast cells (RTG-2) which were then exposed to rainbow trout rIFNs. The gammaIP-10 construct showed high reporter activity even in the absence of rIFNs. The LMP2 promoter contained one GAS element and two double ISRE elements, of four constructs made, only those with ISRE elements showed significant reporter activity following rIFN-gamma stimulation. The TAP2 regulatory region contained two GAS, two ISRE and one C/EBP element from which four constructs were made. Reporter expression for the construct containing all five elements showed an 11- and 2-fold increase in response to rIFN-gamma and type I rIFN, respectively. Constructs containing only the GAS elements did not respond to rIFNs. The TAP2 construct with two ISRE and the C/EBP gave the greatest dose-dependent reporter response to rIFN-gamma, with no significant response to type I rIFN. These data suggest that the ISRE elements, or elements nearby, are essential for the induction of type II IFN responsive genes in trout. The TAP2 construct is a candidate to develop a IFN-gamma reporter stable cell line.

Standardized IMGT® Nomenclature of Salmonidae IGH Genes, the Paradigm of Atlantic Salmon and Rainbow Trout: From Genomics to Repertoires.

In teleost fish as in mammals, humoral adaptive immunity is based on B lymphocytes expressing highly diverse immunoglobulins (IG). During B cell differentiation, IG loci are subjected to genomic rearrangements of V, D, and J genes, producing a unique antigen receptor expressed on the surface of each lymphocyte. During the course of an immune response to infections or immunizations, B cell clones specific of epitopes from the immunogen are expanded and activated, leading to production of specific antibodies. Among teleost fish, salmonids comprise key species for aquaculture. Rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) are especially important from a commercial point of view and have emerged as critical models for fish immunology. The growing interest to capture accurate and comprehensive antibody responses against common pathogens and vaccines has resulted in recent efforts to sequence the IG repertoire in these species. In this context, a unified and standardized nomenclature of salmonid IG heavy chain (IGH) genes is urgently required, to improve accuracy of annotation of adaptive immune receptor repertoire dataset generated by high-throughput sequencing (AIRRseq) and facilitate comparisons between studies and species. Interestingly, the assembly of salmonids IGH genomic sequences is challenging due to the presence of two large size duplicated IGH loci and high numbers of IG genes and pseudogenes. We used data available for Atlantic salmon to establish an IMGT standardized nomenclature of IGH genes in this species and then applied the IMGT rules to the rainbow trout IGH loci to set up a nomenclature, which takes into account the specificities of Salmonid loci. This unique, consistent nomenclature for Salmonid IGH genes was then used to construct IMGT sequence reference directories allowing accurate annotation of AIRRseq data. The complex issues raised by the genetic diversity of salmon and trout strains are discussed in the context of IG repertoire annotation.

Epidemiology of Strongyloides stercoralis infection in Bolivian patients at high risk of complications.

BACKGROUND: Strongyloidiasis can be fatal in immunocompromised patients, but few epidemiological studies investigated the burden of this neglected tropical disease among these populations, particularly in low- and middle-income countries such as Bolivia. This study aimed to fill in this gap by estimating prevalence rate and risk factors associated with strongyloidiasis among patients at high risk of complications. METHODS: A cross-sectional study was carried out in Santa Cruz (elevation 400 meters, tropical climate) and Cochabamba (elevation 2,500 meters, temperate climate), among patients with cancer, HIV infection and rheumatic or hematologic disease, using four coproparasitological techniques and one serological (ELISA) test. RESULTS: In total, 1,151 patients participated in this study, including individuals who were HIV-positive (30%) or with rheumatic (29%), oncologic (32%) or hematologic (9%) diseases. The serological and coproparasitological prevalence was 23.0% (95% confidence interval [CI], 20.7-25.5; n = 265/1151) and 7.6% (95% CI, 6.2-9.3; n = 88/1151), respectively, with an estimated actual prevalence of 20.2% (95% CI, 17.9-22.5). Positive serology and positive coproparasitology were associated with younger age and lower education levels. There was no significant difference in prevalence between Cochabamba and Santa Cruz as defined by coproparasitology (6.4% vs. 8.9%; p = 0.11) or serology (24.0% vs. 22.0%; p = 0.4). Among 64 patients in Cochabamba who had never travelled to the tropical lowlands, 5 (7.8%) had a positive coproparasitology. CONCLUSIONS: Strongyloidiasis is widely prevalent in Bolivia among vulnerable patients at increased risk of life-threatening complications. Transmission of the parasite occurs both in tropical lowlands and temperate elevation (≥ 2,500 m). Control strategies to prevent transmission and complications of this serious parasitic disease should be urgently reinforced.

Micropapillary Cervical Adenocarcinoma: A Clinicopathologic Study of 44 Cases.

Micropapillary adenocarcinoma has been reported as an aggressive variant of adenocarcinoma in several organs, where it is associated with poor clinical outcome. This study reports the clinicopathologic features and outcomes of cervical adenocarcinomas with a micropapillary component (micropapillary cervical adenocarcinomas); this represents the largest reported study of these neoplasms. The study comprised 44 cervical adenocarcinomas of usual (human papillomavirus-related)-type (84%), mucinous, not otherwise specified (4.5%), gastric-type (4.5%), endometrioid (4.5%), and adenosquamous carcinoma (2%). The micropapillary component comprised >50% of the neoplasm in 34 cases (77%) (group 1), and 10% to 50% in 10 cases (23%) (group 2). Lymph node metastasis was present in 41 of 44 (93%) cases and typically the nodal tumor retained a prominent micropapillary morphology. Follow-up ranged from 7 to 123 months (mean, 65.9 mo). Seventeen of 44 (38.6%) patients had no evidence of disease on follow-up, 6/44 (13.6%) were alive with disease, and 21/44 (47.7%) died of disease. There were no survival differences between group 1 and group 2. On univariate analysis, lymph node metastasis (P=0.0015), lymphovascular space invasion (P=0.002), parametrial involvement (P=0.03), and depth of stromal invasion (P=0.045) were related to tumor recurrence. On multivariate analysis, lymph node metastasis (P=0.001), and extent of lymphovascular space invasion (P=0.027) were significant independent predictors of tumor recurrence. Our study shows that a micropapillary component in cervical adenocarcinoma may be associated with aggressive behavior and that a micropapillary architecture may occur within a variety of types of cervical adenocarcinoma.