Somatic Antigens Research Articles

Isolation, genotyping and antimicrobial resistance of Shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen linked to outbreaks of human gastroenteritis with diverse clinical spectra. In this review, we have examined the currently methodologies and molecular characterization techniques for assessing the phenotypic, genotypic and functional characteristics of STEC O157 and non-O157. In particular, traditional culture and isolation methods, including selective enrichment and differential plating, have enabled the effective recovery of STEC. Following recovery, immunological serotyping of somatic surface antigens (O-antigens) and flagellum (H-antigens) are employed for the classification of the STEC isolates. Molecular genotyping methods, including multiple-locus variable-number tandem repeat analysis, arrays, and whole genome sequencing, can discriminate the isolate virulence profile beyond the serotype level. Virulence profiling is focused on the identification of chromosomal and plasmid genes coding for adhesins, cytotoxins, effectors, and hemolysins to better assess the pathogenic potential of the recovered STEC isolates. Important animal reservoirs are cattle and other small domestic ruminants. STEC can also be recovered from other carriers, such as mammals, birds, fish, amphibians, shellfish and insects. Finally, antimicrobial resistance in STEC is a matter of growing concern, supporting the need to monitor the use of these agents by private, public and agricultural sectors. Certain antimicrobials can induce Shiga toxin production and thus promote the onset of severe disease symptoms in humans. Together, this information will provide a better understanding of risks associated with STEC and will aid in the development of efficient and targeted intervention strategies.

Peptides isolation from crude somatic antigens of Haemonchus contortus through SDS- PAGE

Haemonchus (H.) contortus is a blood feeding gastrointestinal nematode, and is considered one of the major threats for goat health and production globally. Crude somatic antigens (CSA) have been reported as sources of immunogens and provide better level of immunity against gastrointestinal nematodes. The present study aimed to quantify CSA of H. contortus worms (n=25) of both sexes followed by isolation of peptides through Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and their characterization. The triturate contained proteins having concentration of 1.3mg/ml as measured through Bradford assay. Seven different bands were appeared on gel ranging from 35KDa to 170KDa. The future prospects may include identification of the immunogenic peptides which can be used as vaccine candidates against H. contortus.

Phenotypic identification of Escherichia coli O157:H7 isolates from cattle at Suleja Abattoir, Nigeria

Escherichia coli O157:H7 is a well-known pathogen of man and animals and a very low infection dose is needed to propagate the infection and clinical disease. In this study, a total of 515 rectal swab samples were collected from cattle and subjected to conventional biochemical tests. Presumptive identification on Eosine Methylene Blue (EMB) yielded an overall prevalence of 83.1%. Cefixime, Tallurite, Sorbitol MacConkey Agar (CT-SMAC) test yielded 18(4.2) isolates while Indole test, Methyl Red, Voges Proskauer and Citrate utilization test (IMVIC) biochemical test showed prevalence rate of 11(61.1%) and MicrogenTM test performed on the 18 Isolates yielded a prevalence rate of 33.3%. A total of 6 of the isolates were subjected to latex agglutination test, in which 2(0.4%) were confirmed to be E. coli O157. The results of the somatic flagella antigen test performed on the 2 isolates revealed that 1(50%) belonged to E. coli O157:H7. Thus this study is therefore the first research work to confirm the presence of E. coli O157:H7 in cattle presented for slaughter in Suleja abattoir in Nigeria. Humans can contract the infection through exposure, handling and consumption of beef or animal products. Control measures are therefore necessary especially during processing and evisceration of beef, to ensure safety of cattle offal presented to the public for human consumption. Key words: Escherichia coli O157:H7, cattle, Microgen kit, flagella staining, phenotypic identification, cattle, latex agglutination test.

Immunodiagnostic potency of homologous antigens for natural Paramphistomum epiclitum infection in small ruminants in plate and paper enzyme linked immunosorbent assay

The objective of the present work was to standardize and evaluate indirect plate and dot- enzyme linked immunosorbent assay (ELISA) using purified Paramphistomum epiclitum homologous antigens in the small ruminants. Electrophoretic separation of somatic antigen (PeSAg) in reducing condition on 15% polyacrylamide gel resolved into 16 proteins of the molecular weight ranging from 14 -100 kDa. Two step ethanolic precipitation of supernatant of in-vitro culture of the fluke yielded P. epiclitum excretory-secretory antigen (PeESAg) of molecular weight 28 kDa. The animals (Goats=123; Sheep=91) were broadly kept into post-mortem and faecal examined groups. At many occasion the PeSAg found to cross reacts with other helminths parasites thus minimizing the specificity of the tests and antigens. There was no any direct correlation between the parasites load and ELISA reactivity pattern. The noted prevalence rate after combining the results of post-mortem examination and PeESAg based ELISA (plate and paper/ dot) was 30.08% (37/123) in goats and 28.57% (26/91) in sheep. While using PeESAg, the calculated overall sensitivity% was 92.86 (goats)/ 100 (sheep) in both plate and dot-ELISA, specificity% was 91.58 (goats)/ 91.55 (sheep) in plate ELISA while 88.42 (goats)/ 92.96 (sheep) in dot-ELISA, positive predictive value% was 76.47 (goats)/ 76.92 (sheep) in plate ELISA while 70.27 (goats)/ 80 (sheep) in dot-ELISA and negative predictive value% was 97.75 (goats)/ 100 (sheep) in plate ELISA while 97.67 (goats)/ 100 (sheep) in dot-ELISA, these values were optimum for the field sera sample so the tests and PeESAg can be recommended for the detection P. epiclitum infection in the small ruminants.

Efficacy of glutathione-S-transferase purified antigen of the gastro-intestinal nematode Haemonchus contortus in diagnosis of sheep haemonchosis.

Haemonchus contortus is one of the most pathogenic and economically important parasites of sheep. Different H. contortus antigens; crude somatic antigen (CSA), excretory/secretory antigen (ESA), crude larval antigen (CLA), glutathione-S-transferase antigen (GST) and recombinant protein (rhcp 26/23) were prepared and characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The antigens were immunologically evaluated through indirect enzyme linked immunosorbent assay (ELISA) for diagnosis of haemonchosis in experimentally and naturally infected sheep. Analysis of the resultant bands of SDS-PAGE demonstrated that 13, 6, 11, 2 and 1 protein bands from CSA, ESA, CLA, GST and rhcp 26/23, respectively and analysis of the resultant bands of western blot showed that 13, 6, 4 and 1 reactive bands detected from CSA, ESA, CLA and GST, respectively. The results of ELISA of different antigens revealed that sero-prevelance of CSA, ESA, CLA, GST and rhcp 26/23 were 78.51, 82.34, 85.319, 45.319 and 90.8% respectively, sensitivity were 100, 90, 100, 96.66 and 90%, respectively and specificity were 0, 70, 10, 70 and 6.66%, respectively with diagnostic potency were 50, 80, 55, 83.33 and 48.33%, respectively. Statistical analysis using Chi square test found that GST is the best one that can be used. The cross reactivity of GST antigen, crude Fasciola antigen and crude Moniezia antigen tested versus their homologous hyper immune sera at different dilutions using ELISA. The current study reported that GST antigen could be considered as a promising antigen for diagnosis of haemonchosis.

A liquid bead array for the identification and characterization of fljB-positive and fljB-negative monophasic variants of Salmonella Typhimurium

Salmonella1,4,[5],12:i:- accounts currently for one of the most common serotypes observed worldwide. These isolates do not express the FljB flagellin and mostly derive from Salmonella Typhimurium. They are therefore termed Salmonella Typhimurium monophasic variants (STMV) and are considered of comparable public health risk. Since serological identification of the somatic and flagellar antigens of STMV is not sufficient to demonstrate relatedness with Salmonella Typhimurium, additional assays detecting genetic markers unique to Salmonella Typhimurium are required. In addition, identification of the mutations affecting expression of the flagellar gene fljB can be useful to support the monophasic character observed phenotypically. Finally, genetic subtyping of the various mono- and biphasic Salmonella Typhimurium clonal groups can facilitate their epidemiological follow-up. Here, we present a home-made liquid bead array able to fulfill these requirements. This array confirmed the monophasic character of 240 STMV isolates collected in Belgium during 2014–2015 and identified 10 genetic subtypes. Microevolution in and around the fljB locus linked to IS26 insertions is probably one of the driven force accounting for STMV population diversity. Thanks to its open design, other genetic signatures could later be merged to the assay to subtype additional STMV clonal groups and to detect rare mutations.

Enzyme-linked immunosorbent assay for diagnosis of Amphimerus spp. liver fluke infection in Humans.

BACKGROUNDAmphimerus spp. is a liver fluke that infects humans and domestic animals. It is highly prevalent in some Ecuadorian communities. Currently, diagnosis is based on the microscopic observation of eggs in faeces, but this has variable sensitivity. More sensitive methods are needed for diagnostic testing.OBJECTIVE The main objective of this work was to develop an enzyme-linked immunosorbent assay (ELISA) using crude antigens from Amphimerus spp. adult worms to detect anti-Amphimerus IgG in human sera.METHODS Crude somatic antigens were obtained from adult Amphimerus spp. worms. Human sera from 119 patients were tested: 48 from individuals with a confirmed Amphimerus spp. infection, 78 from non-infected Ecuadorians living in the endemic region, 60 from persons living in non-endemic areas (20 Ecuadorians, 20 Europeans, and 20 Africans), and 33 who had other parasitic and non-parasitic infections.PRINCIPAL FINDINGS Results were analysed using the receiver-operator characteristic (ROC) curve analysis with an area under curve (AUC) value of 0.967. The accuracy of the ELISA was high. The sensitivity was 85.0% [95% confidence interval (CI): 80.3-89.7%] and the specificity was 71.0% (95% CI: 65.2-76.8%). Some cross reactivity was detected against Paragonimus mexicanus, Fasciola hepatica, Schistosomiasis, Taenia solium, Strongyloides stercoralis, Mansonella spp., and Vampirolepis nana.MAIN CONCLUSIONS We have developed the first ELISA technique that detects anti-Amphimerus IgG in human sera with good sensitivity, repeatability and reproducibility. However, more specific antigens are needed to further enhance performance of this assay. Regardless, this ELISA test could be useful for early diagnosis and prompt treatment of human Amphimerus spp. infections.

Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis

BackgroundSalmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O9 antigen (O9 MAb) and O9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition.ResultsA total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD50) of the rfbG deletion mutant strain was 106.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally.ConclusionsThese data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

Serological diagnosis of cystic echinococcosis in cattle.

An IgM murine monoclonal antibody (MAb) was obtained against the excretory-secretory antigen (ES-Ag) of in vitro reared protoscoleces of Echinococcus granulosus (Batsch, 1786). Western blotting revealed that the MAb recognised a 20.6 kDa protein of this ES-Ag. The MAb was used in sandwich enzyme-linked immunosorbent assay (s-ELISA) for selective sensitisation of the solid phase with the protoscolex-specific protein from its ES-Ag and somatic antigen (S-Ag) to examine serum samples of 108 cows from a cystic echinococcosis (CE) endemic area for specific antibodies and to compare the results with those from necropsies and an indirect ELISA (i-ELISA). The sensitivity of s-ELISA/ES-Ag, s-ELISA/S-Ag and i-ELISA/S-Ag was 48%, 52% and 62%, respectively. The low sensitivity of the ELISA was probably caused by the fact that 13 cows (62%) were infected with sterile cysts (acephalocysts and/or calcified foci) only. A relatively high specificity (80%) of s-ELISA/ES-Ag was observed in cows with fertile cysts. It also detected antibodies in the serum of two cows that had recovered from the disease according to the necropsy. The i-ELISA/S-Ag gave false results in testing sera from a healthy animal and from a cow with tubercular foci. Further analysis will be necessary to define more precisely the value of this study, because the duration of antibody elimination from the bloodstream of recovered cattle remains unknown. The solution of this problem will increase the specificity of the proposed test in monitoring herbivorous animals for CE.

Characterization of Salmonella Gallinarum from an outbreak in Raigarh, Chhattisgarh.

Aim:The present investigation was conducted to isolate and characterize Salmonella Gallinarum from an outbreak of fowl typhoid in layer birds.Materials and Methods:Clinically ill and dead layer birds from an outbreak were investigated. History, clinical signs, and postmortem lesions were suggestive of fowl typhoid. Postmortem samples including heart blood, intestinal contents, pieces of ovary, and liver were collected and processed immediately for bacterial culture, serotyping and antibiotic sensitivity tests. Isolates were further screened for the presence of extended spectrum beta lactamase (ESBL) (blaTEM) gene by polymerase chain reaction.Results:On the basis of cultural, staining and biochemical characteristics; three bacterial isolates were confirmed as S. Gallinarum. On serotyping, somatic antigen O: 9 and 12 with nonflagellated antigen were detected in all three isolates. Isolates were intermediate sensitive to amoxycillin, amoxyclav, gentamicin and ciprofloxacin and resistant to most of the antibiotics including chloramphenicol, ampicillin, ceftazidime, cefexime, cefepime, azithromycin, nalidixin, tetracycline, oxytetracycline, and streptomycin. Two isolates were found to harbor ESBL (blaTEM) gene.Conclusion:Beta lactamase producer S. Gallinarum was confirmed as cause of increased mortality in layer birds during present investigation. Existence of multi drug resistant Salmonella poses serious threat to poultry industry in Chhattisgarh.

Molecular characterization and antimicrobial resistance of Salmonella enterica from swine slaughtered in two different types of Philippine abattoir

Salmonella enterica is a well-known pathogen commonly acquired from the consumption of contaminated food. It has been estimated to affect millions of humans and cause hundreds of thousands of deaths per year globally. Pork, one of the most commonly consumed meats worldwide, has been identified as one of the main sources of human salmonellosis. In this study, we aimed to detect and characterize S. enterica from slaughtered swine and generate antimicrobial resistance profiles of select isolates. Tonsils and jejunum with mesenteric lymph nodes (MLN) were collected from a total of 240 swine from eight abattoirs (five accredited and three locally registered abattoirs) across Metro Manila. S. enterica were isolated using conventional culture methods and confirmed by PCR amplification of the invA gene. Isolates were further characterized based on somatic antigen by multiplex PCR. We report that there is no significant difference (P = 0.42) between the incidences of S. enterica in swine slaughtered in accredited (44.0%) and in locally registered abattoirs (46.7%). Most samples were contaminated with S. enterica under serogroup O:3,10. Antimicrobial susceptibility testing of 183 isolates using the VITEK® 2 system revealed high resistance to ampicillin (67.8%) and trimethoprim/sulfamethoxazole (80.3%). Multidrug-resistance was found in 124 (67.8%) isolates.

EFFECT OF SOMATIC ANISAKIS SIMPLEX EXTRACT TO DEVELOPMENT CHICK EMBRYOS

Helminthes have embryotoxic and teratogenic effects to host’s cells and tissues.Investigations were carried out mainly on embryo and fetus in mammals.Blaszkowska, J. (1998); Kadlubowski R. (2000) conducted studies of in chickembryos. Dose-dependent effect is also studied and found that small doses causeembryotoxic effect, while higher doses - teratogenic. Anisakis simplex antigenshave also embryotoxic effect has been found. The aim of our research was toexamine the effect of A. simplex somatic antigen to the development of chickembryos in its experimental introduction in various ways and at different stages ofembryogenesis. For the study chicken embryos were used at different stages ofembryogenesis: 7.5, 10.5 and 12.5 days. Methods for introducing antigen chickenembryos: in the allantois cavity, on-chorion allantois membrane and yolk sac at 0.2ml. The autopsy was performed on embryos 2 and 8 days. The eggs and embryoswere weighed and evaluated the embryonic development. As a result of the firstexperience the greatest change observed by mass, and when administered in thedevelopment of antigen in both yolk sac on day 2 (1.68 ± 0.06, unlike control 2.25± 0.20), and at 8 days ( 9.97 ± 6.21 in the experiment versus 14.89 ± 0.78 in thecontrol), respectively. In the second experiment eggs were used at 1 and 5 dayincubation embryos. The antigen was administered in both cases also in the yolksac at a dose of 0.2 ml. In the second experiment there was a delay in thedevelopment of both cases at autopsy after 2 days in non-incubated eggs missingthe development of, unlike the control, the development of which 48 hourscorresponded; including 5 daily observed decrease in weight 0.88 ± 0.22, against1.05 ± 0.05 and 8 days in a similar way. As a result of experiments on the effect ofthe Anisakis simplex somatic antigen on the development of chick embryosembryotoxic action installed. Thus antigen has the greatest effect on developmentof early embryos and putting it into the yolk sac.

Clonal Expansion and Long-Term Persistence of Rhesus Macaque NK Cells with an Adaptive Phenotype

Natural killer (NK) cells are defined as lymphocytes capable of target cell killing and cytokine production independent of somatic antigen receptor recombination. Challenging the paradigm of continuous production of non-specific cytotoxic NK cells from undifferentiated precursors, a subset of murine NK cells sharing many properties with cytotoxic effector T cells was shown to specifically respond to a viral infection, resulting in expansion and protective immunity upon adoptive transfer, uncovering unexpected "adaptive" capabilities for NK cells. In humans, CD56dimCD16+ NK cells expressing NKG2C and variegated patterns of KIR are associated with Cytomegalovirus (CMV) infection, and may represent human analogs of murine adaptive NK cells. In contrast to CD56bright and canonical CD56dim NK cells, we recently showed that human adaptive NK cells lack expression of specific signaling proteins and transcription factors, and epigenetically resemble CD8+ effector T cells, suggesting shared pathogen-driven developmental pathways (Schulms et al, 2015). NK cell heterogeneity, ontogeny, and mechanisms sustaining NK cell memory are poorly understood, particularly in humans, because in contrast to adaptive T and B cells, clonal tracking of NK cells based on receptor gene structure is not possible. We applied autologous transplantation of genetically barcoded CD34+ rhesus macaque hematopoietic stem and progenitor cells (HSPC) to interrogate the landscape of NK cell production and expansion at a clonal level. We followed 3 monkeys for up to 4 years post-transplantation, and observed a distinct clonal ontogeny for CD56-CD16+ (corresponding to human CD56dimCD16+ NK) versus CD56+CD16- rhesus NK cells. CD56-CD16+ NK were oligoclonal, and dominated by small numbers of highly biased clones, a pattern showing little overlap with highly polyclonal multipotent contributions to T, B, myeloid and CD56+CD16- NK cell lineages. There were clear expansions and contractions of groups of these highly biased CD56-CD16+ NK cell clones over time, with individual clones contributing up to 20% to this lineage, similar to patterns of waxing and waning T cell clones, suggesting responses of both cell types to extrinsic cues such as viruses. In contrast, all other lineages, including CD56+CD16- NK cells, were characterized by very stable contributions from long-term engrafting multipotent HSPC clones over time. The highly biased, greatly expanded, and waxing and waning CD56-CD16+ NK cell clones were specifically enriched in a subpopulation of CD56+CD16- NK with high KIR and low SYK expression,and decreased methylation at IFNG the CNS1 locus, analogous to human CMV-associated adaptive NK cells. This population of cells was increased in CMV+ compared to CMV- macaques; our transplanted macaques were CMV+. Canonical CD56+CD16- NK cells, lacking an adaptive phenotype, had clonal patterns similar to CD56+CD16- NK cells. We profoundly depleted CD56-CD16+ NK cells with an anti-CD16 antibody, and observed rapid regeneration of the same CD56-CD16+ NK clonal pattern, without recruitment of contributions from multipotent HSPC via CD56+CD16- putative precursor NK cells. There was a marked increase in cycling of CD56-CD16dim NK cells just prior to reappearance of CD56-CD16bright NK cells, and no change in cycling of CD56+CD16- cells. Thus, adaptive NK cell clones, both at steady state and under regenerative stress do not appear to require ongoing production from HSPC via CD56+CD16- precursors. These results suggest that adaptive NK cells or their immediate precursors have acquired self-renewal properties and high proliferative potential, with clonal restriction potentially based selection and expansion of cells with specific, epigenetically-controlled receptor expression able to interact with external stimuli such as viruses. In conclusion, via tracking of individual NK cell clonal histories in rhesus macaques, we provide the first direct evidence for persistence of adaptive NK cell clones in any species, offering potential insights into the acquisition of NK cell memory. DisclosuresDunbar:GSK/Novartis: Research Funding.

First Report of the Occurrence of Trichinella-Specific Antibodies in Domestic Pigs in Central and Eastern Uganda.

Previous research on trichinellosis in Africa focused on isolating Trichinella from wildlife while the role of domestic pigs has remained highly under-researched. Pig keeping in Uganda is historically recent, and evidence on zoonotic pig diseases, including infection with Trichinella species, is scarce. A cross-sectional survey on Trichinella seroprevalence in pigs was conducted in three districts in Central and Eastern Uganda from April 2013 to January 2015. Serum from a random sample of 1125 pigs from 22 villages in Eastern and Central Uganda was examined to detect immunoglobulin G (IgG) against any Trichinella spp. using a commercially available ELISA based on excretory-secretory antigen. ELISA positive samples were confirmed using Western Blot based on somatic antigen of Trichinella spiralis as recommended in previous validation studies. Diaphragm pillar muscle samples (at least 5 g each) of 499 pigs from areas with high ELISA positivity were examined using the artificial digestion method. Overall, 78 of all 1125 animals (6.9%, 95% CI: 5.6–8.6%) tested positive for antibodies against Trichinella spp. in the ELISA at significantly higher levels in Kamuli district compared to Masaka and Mukono districts. Thirty-one percent of the ELISA positive samples were confirmed IgG positive by the Western Blot leading to an overall seroprevalence of 2.1% (95% CI: 1.4–3.2%). The large proportion of ELISA positive samples that could not be confirmed using Western blot may be the result of cross-reactivity with other gastrointestinal helminth infections or unknown host-specific immune response mechanisms in local pig breeds in Uganda. Attempts to isolate muscle larvae for species determination using the artificial digestion method were unsuccessful. Due to the large number of muscle samples examined we are confident that even if pigs are infected, the larval burden in pork is too low to pose a major risk to consumers of developing trichinellosis. This was the first large systematic field investigation of Trichinella infection in domestic pigs in Uganda and its results imply that further studies are needed to identify the Trichinella species involved, and to identify potential sources of infection for humans.

Density of immunogenic antigens does not explain the presence or absence of the T-cell–inflamed tumor microenvironment in melanoma

Melanoma metastases can be categorized by gene expression for the presence of a T-cell-inflamed tumor microenvironment, which correlates with clinical efficacy of immunotherapies. T cells frequently recognize mutational antigens corresponding to nonsynonymous somatic mutations (NSSMs), and in some cases shared differentiation or cancer-testis antigens. Therapies are being pursued to trigger immune infiltration into non-T-cell-inflamed tumors in the hope of rendering them immunotherapy responsive. However, whether those tumors express antigens capable of T-cell recognition has not been explored. To address this question, 266 melanomas from The Cancer Genome Atlas (TCGA) were categorized by the presence or absence of a T-cell-inflamed gene signature. These two subsets were interrogated for cancer-testis, differentiation, and somatic mutational antigens. No statistically significant differences were observed, including density of NSSMs. Focusing on hypothetical HLA-A2+ binding scores, 707 peptides were synthesized, corresponding to all identified candidate neoepitopes. No differences were observed in measured HLA-A2 binding between inflamed and noninflamed cohorts. Twenty peptides were randomly selected from each cohort to evaluate priming and recognition by human CD8+ T cells in vitro with 25% of peptides confirmed to be immunogenic in both. A similar gene expression profile applied to all solid tumors of TCGA revealed no association between T-cell signature and NSSMs. Our results indicate that lack of spontaneous immune infiltration in solid tumors is unlikely due to lack of antigens. Strategies that improve T-cell infiltration into tumors may therefore be able to facilitate clinical response to immunotherapy once antigens become recognized.

腸内細菌とくに大腸菌属による生態の感作とその意義に関する研究 : 第8報 培養白血球反応を指標としての大腸菌及び赤痢菌菌体物質による遅延型アレルギー反応の証明

腸内細菌とくに大腸菌属による生態の感作とその意義に関する研究 : 第8報 培養白血球反応を指標としての大腸菌及び赤痢菌菌体物質による遅延型アレルギー反応の証明

CpG oligodeoxynucleotides with crude parasite antigens reduce worm recovery in Opisthorchis viverrini infected hamsters

Opisthorchis viverrini, a human liver fluke, is still an endemic parasitic infection in Thailand and nearly all countries in Southeast Asia. O. viverrini induces a chronic stage of infection in hamsters. During the first 2 weeks of infection, Th1 inducing cytokine, IL-12, increased but was down regulated in chronic infection. In this study it was found that unmethylated-CpG ODN (oligodeoxynucleotides) 1826 increased hamster mononuclear cell proliferation and stimulated IFN-γ production in vitro. The IFN-γ levels in hamster sera were significantly increased in hamsters injected with CpG ODN 1826 alone or plus crude somatic antigens (CSAg). Further investigation using the flow cytometer found that CD4+T cells and IFN-γ+ CD4+T cells (Th1-like cells) in the hamster blood were significantly increased. The role of these cells in the protective responses in hamsters was evaluated by challenging with 25 metacercaria and observation for 3 months. The number of worms recovered was significantly reduced in the hamsters injected with CpG ODN 1826 with CSAg, but not in CpG ODN 1826 alone groups when compared to PBS control. The percent of reduction in hamsters against this parasite were 32.95% and 21.49% in the CpG ODN 1826 with CSAg and CpG ODN 1826 alone. This study indicates that CpG ODN 1826 plus parasite antigens elicit a Th1-like response that leads to the enhancement of worm reduction.

Immunodiagnostic potency of homologous antigens for natural Haemonchus contortus infection in small ruminants in plate and paperenzyme linked immunosorbent assay

The objective of this study was to characterize Haemonchus contortus antigens, and to standardize and evaluate indirect plate and dot-ELISA using homologous antigens in the small ruminants. Electrophoretic separation of somatic antigen in reducing condition on 15% polyacrylamide gel resolved into 16 proteins of molecular weight ranging from 14-100 kDa. Two step ethanolic extraction of the supernatant of in-vitro culture of H. contortus yielded excretory-secretory (E-S) antigen/ cathepsin L cysteine proteinase of molecular weight 28 kDa. The animals (Goats=103; Sheep=91) were broadly kept into post-mortem (PM) and faecal examined groups and further sub-grouped based on mono or multiply helminths infection. At many occasion, the somatic antigen found to cross reacts with other helminths parasites thus minimizing the specificity of the selected tests and antigens. There was no any direct correlation between the parasites load and ELISA reactivity pattern. The prevalence rate of haemonchosis was 55.7 (34/61) in goats/ 47.6 (40/84) % in sheep as per PM examination while it was 45.63 (47/103) in goats/ 41.76% (38/91) in sheep and 36.89 (38/103) in goats/ 35.16% (32/91) in sheep using E-S antigen based plate and dot-ELISA, respectively. With E-S antigen, the overall % sensitivity, specificity, positive and negative predictive values of plate-ELISA was 89.74 (goats)/ 80.95 (sheep), 81.25 (goats)/ 91.84 (sheep), 74.47 (goats)/ 89.47 (sheep), 92.86 (goats)/ 84.91 (sheep), respectively while for dot-ELISA it was 66.67 (goats)/ 61.9 (sheep), 81.25 (goats)/ 87.76 (sheep), 68.42 (goats)/ 81.25 (sheep), 80 (goats)/ 72.88 (sheep), respectively, so the tests and E-S antigen can be recommended for the detection haemonchosis in the small ruminants.

Induction of regulatory T cells by Opisthorchis viverrini.

Opisthorchis viverrini causes public health problems in South-East Asia. Recently, TGF-β and IL-10 have been reported to increase in O. viverrini-infected hamsters but the sources of these cytokines are still unknown. In this study, the CD4+ T cells in infected hamsters were investigated. It was demonstrated that IL-4+ CD4+ T cells were significantly increased in hamster spleens and mesenteric lymph nodes (MLNs) during chronic infection. Interestingly, IL-10+ CD4+ T cells were also discovered at a significant level while Treg (T regulatory)-like TGF- β+ CD4+ T cells were in MLNs of infected hamsters. Moreover, the CD4+ CD25+ Foxp3+ Treg cell response was significantly found both in spleens and MLNs in infected hamsters. The findings were then confirmed by development of T-cell clones against crude somatic antigens (CSAg) in immunized BALB/c mice. Five clones named TCC21, TCC23, TCC35, TCC41 and TCC108 were established. The TCC21 was found to be the TGF-β+ CD4+ while TCC35, TCC41 and TCC108 were IL-4+ CD4+ and TCC23 was IFN-γ+ CD4+ . This TGF-β+ CD4+ T clone showed an inhibitory function in vitro in mononuclear cell proliferation via TGF-β-mediated mechanisms. This study indicated that O. viverrini-infected hamsters could induce TGF-β+ CD4+ Treg-like cells. The CSAg-specific Tregs secreted high TGF-β, and limited immune cell proliferation.

Humoral and cellular immune responses in mice against secreted and somatic antigens from a Corynebacterium pseudotuberculosis attenuated strain: Immune response against a C. pseudotuberculosis strain.

BackgroundCorynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CL), a chronic disease that affects goats and sheep. CL is characterized by the formation of granulomas in lymph nodes and other organs, such as the lungs and liver. Current knowledge of CL pathogenesis indicates that the induction of humoral and cellular immune responses are fundamental to disease control. The aim of this study was to evaluate the humoral and cellular immune responses in BALB/c mice inoculated with a C. pseudotuberculosis strain isolated in the state of Bahia, Brazil.ResultsThe lymphocyte proliferation and in vitro production of IFN-γ, IL-4, IL-10, IL-12 and nitric oxide by spleen cells stimulated with secreted and somatic antigens from the studied strain were evaluated. IgG subclasses were also analyzed. Results showed a significant increase of Th1-profile cytokines after 60 days post-inoculation, as well as an important humoral response, represented by high levels of IgG2a and IgG1 against C. pseudotuberculosis.ConclusionThe T1 strain of C. pseudotuberculosis was shown to induce humoral and cellular immune responses in BALB/c mice, but, even at a dosage of 1x107 CFU, no signs of the disease were observed.