35-kDa Antigen Research Articles

Prevalence of tick-transmitted pathogens in cattle reveals that Theileria parva, Babesia bigemina and Anaplasma marginale are endemic in Burundi

BackgroundTick-borne diseases (TBDs) constitute a major constraint for livestock development in sub-Saharan Africa, with East Coast fever (ECF) being the most devastating TBD of cattle. However, in Burundi, detailed information is lacking on the current prevalence of TBDs and on the associated economic losses from mortality and morbidity in cattle as well as the costs associated with TBD control and treatment. The aim of this study was, therefore, to assess the prevalence and spatial distribution of tick-borne pathogens (TBPs) in cattle across the major agro-ecological zones (AEZs) in Burundi.MethodsIn a cross-sectional study conducted in ten communes spanning the five main AEZs in Burundi, blood samples were taken from 828 cattle from 305 farms between October and December 2017. Evidence of Theileria parva infection was assessed by antibody level, measured using a polymorphic immunodominant molecule (PIM) antigen-based enzyme-linked immunosorbent assay (ELISA) and by a T. parva-specific p104 gene-based nested PCR. Antibodies against Theileria mutans infection were detected using the 32-kDa antigen-based indirect ELISA, while the 200-kDa antigen and the major surface protein 5 (MSP5)-based indirect ELISA were used to detect antibodies against Babesia bigemina and Anaplasma marginale, respectively.ResultsThe prevalence of T. parva across the ten communes sampled ranged from 77.5 to 93.1% and from 67.8 to 90.0% based on the ELISA and PCR analysis, respectively. A statistically significant difference in infection was observed between calves and adult cattle; however, T. parva infection levels were not significantly associated with sex and breed. The seroprevalence indicating exposure to T. mutans, B. bigemina and A. marginale ranged from 30 to 92.1%, 33.7 to 90% and 50 to 96.2%, respectively. Mixed infections of TBPs were detected in 82.91% of cattle sampled, with 11 different combinations of pathogen species detected .ConclusionsThe findings indicate that T. parva, A. marginale and B. bigemina infections are endemic in Burundi. Knowledge of the spatial distribution of TBPs will facilitate the design of effective targeted strategies to control these diseases. There is a need for further investigations of the distribution of tick vectors and the population structure of TBPs in order to identify the key epidemiological factors contributing to TBD outbreaks in Burundi.

In Silico Structural Prediction and Production of a Chimeric Recombinant Dickkopf-1 (DKK-1) Antigen.

Dickkopf (DKK) family of proteins are known as antagonists for the Wnt-β-catenin signaling pathway. It is suggested that the Dickkopf-1 (DKK-1) has a role in several diseases such as hepatocellular carcinomas, hepatoblastomas, Wilms' tumors, lung cancer and Myeloma bone disease. The aim of the present study was to produce a chimeric-recombinant DKK-1 protein in order to induce immune response against the antigen. The recombinant Dickkopf-1 (rDKK-1) protein was designed using bioinformatics analysis. The standard methods were used for cloning, expression and purification. The structure of recombinant protein was analyzed by spectroscopy methods. Enzyme-linked immunosorbent assay (ELISA) and Western blotting were performed to confirm the recombinant protein using a commercial anti-DKK-1 (whole protein) polyclonal antibody. The immunogenicityof the recombinant DKK-1 was assessed by immunizing, intraperitoneally, BALB/C mice four times with the 31-kDa and 45-kDa purified rDKK-1 cloned in pET28a and pET32a vectors respectively. The antibody titer was measured in due course of time. Stronger immunogenic parts of the protein were selected based on in-silico predictions and recombinant protein was successfully designed. The chimeric gene was sub-cloned, expressed, purified and refolded. The purified protein was confirmed by Western blotting and ELISA. The three dimensional structural was confirmed by CD spectrum and predicted structures by bioinformatics tools, revealed the stability of helix structures. rDKK-1 protein was capable of inducing immune response with high titer antibody and excessive humoral immune response. No significant difference was observed between immunization by 31-kDa and 45-kDa antigen.

Vaccination with DNA encoding ES 43-kDa /45-kDa antigens significantly reduces Trichinella spiralis infection in mice

Trichinella spiralis is an intestinal nematode parasite that can cause trichinellosis in humans and animals worldwide. The most important known hosts of T. spiralis are pigs, horses, dogs and cats. Pork and its products are the main sources of infection in human trichinellosis. Vaccines against these infections are urgently needed. In this study, the genes encoding the 43-kDa or 45-kDa glycoprotein present in the excretory-secretory (ES) products from T. spiralis muscle larvae (ML) were cloned into the eukaryotic expression vector pVAX1, resulting plasmids pVAX1-Ts43 and pVAX1-Ts45, respectively. Then BALB/c mice were intramuscularly immunized with the DNA vaccine pVAX1-Ts43, pVAX1-Ts45, or both to evaluate their immunogenicity and host protective potential. After the third immunization, mice of each group were challenged with 300 T. spiralis ML. The results showed that the mice immunized with the DNA vaccine pVAX1-Ts43 or pVAX1-Ts45 developed significant numbers of FAS+PNA+ B220+ B cells indicating the formation of the germinal centers (GCs), IFN-γ-secreting (mesenteric lymph nodes, MLN) cells, and IL-4-, and IL-10-secreting splenocytes. Mice immunized with the pVAX1-Ts43 or pVAX1-Ts45 vaccine elicited partial protective immunity against challenge infections with T. spiralis as shown by significant reduction in ML. Most notably, the combined immunity of pVAX1-Ts43 and pVAX1-Ts45 induced better immune responses than either of the DNA vaccines given alone and provided as high as 75.9% reductions in muscle larval burden. These results suggest that the plasmid DNA encoding the 43-kDa or 45-kDa glycoprotein could be considered as a potential vaccine candidate against T. Spiraling infection.

Immune response elicited by two rBCG strains devoid of genes involved in c-di-GMP metabolism affect protection versus challenge with M. tuberculosis strains of different virulence

Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection, and as a source of antigens for humoral response in infected guinea pigs. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used the live vaccine Mycobacterium bovis BCG to determine whether deletion of genes involved in c-di-GMP metabolism would affect interaction with macrophages, capacity to induce immune response in a murine cell line and mice, and how the protein profile was modified when grown as surface pellicles. We found that deletion of the BCG1419c (Delta c-di-GMP phosphodiesterase, ΔPDE) gene, or deletion of the BCG1416c (Delta c-di-GMP diguanylate cyclase, ΔDGC) gene, altered production of TNF-α, IL-6, and IL-1β, in murine macrophages, and resulted in attenuation in intra-macrophage replication. Moreover, in addition to the improved immunogenicity of the BCGΔBCG1419c mutant already reported, deletion of the BCG1416c gene leads to increased T CD4+ and T CD8+ activation. This correlated with protection versus lethality in mice infected with the highly virulent M. tuberculosis 5186 afforded by vaccination with all the tested BCG strains, and controlled the growth of the mildly virulent M. tuberculosis H37Rv in lungs by vaccination with BCGΔBCG1419c during chronic late infection from 4 to 6 months after challenge. Furthermore, when grown as surface pellicles, a condition used to manufacture BCG vaccine, in comparison to BCG wild type, both rBCGs changed expression of antigenic proteins such as DnaK, HbhA, PstS2, 35KDa antigen, GroEL2, as well as AcpM, a protein involved in synthesis of mycolic acids, molecules relevant to modulate inflammatory responses.

Association of Helicobacter pylori with hepatobiliary stone disease, a prospective case control study.

Hepatobiliary stone disease is one of the most common surgical conditions worldwide. There are multiple causative agents responsible for the formation of hepatobiliary stones, and bacterial infection is one of them. The presence of Helicobacter DNA species has been investigated in the biliary epithelium of patients with biliary diseases. However, conflicting results have been observed that may have been due to the small number of subjects studied, difficulty in obtaining a healthy control group, absence of controlling for confounding factors, or ethical and regional differences among populations. We investigated the presence of Helicobacter pylori species by PCR of 26-kDa surface antigen specific to H. pylori in bile samples from 50 cases with hepatobiliary stones and 25 controls without hepatobiliary stones. The control group comprised of 20 patients of hydatid cyst disease of liver and 5 patients of right colonic growth. H. pylori was present in 20 bile samples among cases and was absent in 30 bile samples among cases. Among controls, H. pylori could not be detected. A significant association of the presence of H. pylori with hepatobiliary stone disease was seen (p < 0.001). There is an association between bile infection with H. pylori and hepatobiliary stone disease.

Brazilian donkeys (Equus asinus) have a low exposure to Neospora spp.

Donkeys (Equus asinus) are closely related to horses and are known to be infected by several equine pathogens. Neospora caninum and Neospora hughesi are protozoan parasites that infect horses, but they were not confirmed in donkeys up to this date. The aim of this study was to evaluate the exposure of donkeys (Equus asinus) to Neospora spp. using tachyzoites of N. caninum as antigen and employing two common serologic methods, IFAT and immunoblot. Sera from 500 donkeys were obtained from 30 municipalities in Bahia state and tested by IFAT. Two of 500 sera were positive for Neospora spp. by IFAT with antibody titers of 100, and recognized a 37kDa antigen in immunoblot. Approximately 22% of the samples showed strong apical reactions and/or incomplete fluorescence, what may cause confusion in the interpretation of IFAT. We concluded that Neospora spp. are possibly of minor importance for Brazilian donkeys. Future studies are necessary to prove that Neospora spp. can naturally infect donkeys.

The changes in antigenic components of Vibrio cholerae strains isolated in Vietnam

Whole cells of Vibrio cholerare serotype Inaba and serotype Ogawa (strains I389 and O395) were injected into rabbits to obtain antiserum. The antiserums were used for immune reaction with antigenic components of 25 strains of V. cholerae isolated from five provinces of Vietnam and the two standard strains I389 and O395 by Western-blot technique. Analysis of immune hybrid results showed that there were 11 antigenic components with molecular weights approximately 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa and 20kDa. In which the antigens of 45kDa, 42kDa, 31kDa and 20kDa were similar to OmpT, OmpS, Omp-31kDa and TcpA that have been considered as vaccine-candidate antigens. Among 25 V. cholerae strains, there were 6 antigenic components in common including 79kDa, 62kDa, 45kDa, 35kDa, 31kDa and 20kDa. 23/25 strains contained 42kDa antigen; 5/25 strains contained 38kDa and 23kDa antigens; 11/25 had 26kDa antigen. In addition, 7/25 strains contained antigens identical to V. cholerae I389 serotype Inaba; 6/25 strains contained antigens of I389 and O395; 12/25 strains had changes of antigenic components. These changes were actually the lack of antigens, not appearing new antigens. These results are considered as basis for researches about immune response and prevention of cholera disease.&#x0D; Toàn bộ tế bào của các chủng Vibrio cholerare typ huyết thanh Inaba và typ huyết thanh Ogawa (chủng I389 và O395) được sử dụng để gây miễn dịch trên thỏ để thu kháng huyết thanh. Các kháng huyết thanh được dùng để thực hiện phản ứng miễn dịch với các thành phần kháng nguyên của 25 chủng V. cholerae phân lập từ 5 tỉnh thành của Việt Nam và hai chủng chuẩn I389 và O395 bằng kỹ thuật Western-blot. Phân tích kết quả lai miễn dịch cho thấy, có tổng số 11 thành phần kháng nguyên có kích thước khoảng 79kDa, 62kDa, 52kDa, 45kDa, 42kDa, 38kDa, 35kDa, 31kDa, 26kDa, 23kDa và 20kDa. Các kháng nguyên này chủ yếu là các protein màng ngoài (Omp) và kháng nguyên lông (TcpA). Trong đó các kháng nguyên 45kDa, 42kDa, 31kDa và 20kDa trùng với các kháng nguyên OmpS, OmpT, Omp-31kDa và TcpA được xem là những kháng nguyên dự tuyển vacxin tả. Có 6 kháng nguyên chung giữa 25 chủng với kích thước 79kDa, 62kDa, 45kDa, 35kDa, 31kDa và 20kDa. 7/25 chủng có các kháng nguyên giống với kháng nguyên của chủng V. cholerae I389 typ huyết thanh Inaba; 6/25 chủng có các kháng nguyên giống với kháng nguyên của cả hai chủng V. cholerae I389 và O395; 12/25 chủng có sự biến đổi thành phần kháng nguyên. Tuy nhiên, sự biến đổi này thực chất là sự thiếu hụt chứ không phải là sự xuất hiện các thành phần kháng nguyên mới. Các kết quả nghiên cứu này có thể được xem là nền tảng ban đầu cho các nghiên cứu về miễn dịch và dự phòng bệnh tả.

Isolation and characterization of flagellar filament from zoospores of Dermatophilus congolensis

Highly motile zoospores from Dermatophilus congolensis bovine isolates from clinical dermatophilosis in Japan were obtained by culturing at 27°C in an ambient atmosphere on heart infusion agar supplemented with 5% defibrinated sheep blood for 72h or in heart infusion broth for 48h with gentle shaking. After vigorous mechanical agitation of the zoospore suspension, the flagellar filaments detached from motile zoospores and were isolated in the clear gelatinous part of the final pellet by differential centrifugation. Typical morphology of a flagellar filament, with a width of approximately 15nm, was observed in the isolated flagellar filament by electron microscopy. A single major protein (flagellin) band with an apparent molecular mass of 35kDa was detected in the flagellar filament of D. congolensis strain AM-1 and that of 33kDa was detected in strain IT-2 by SDS-PAGE. In immunoblot analysis of whole-cell proteins from seven isolates of D. congolensis, antiserum to strain AM-1 zoospores reacted with the 35-kDa antigen band of strain AM-1, but not with any antigen band of other strains in a similar molecular mass range. In contrast, antiserum to strain IT-2 zoospores reacted with antigen bands at 33kDa from six strains, except strain AM-1. Similar strain-specific reactions of these anti-zoospore sera with isolated flagellar filaments from strains AM-1 and IT-2 were confirmed by immunoblot, indicating the presence of antigenic variations of flagellins of D. congolensis zoospores.

Evaluation of Antigen Detection Test (Chromatographic Immunoassay): Potential to Replace the Antibody Assay Using Purified 45‐kDa Protein for Rapid Diagnosis of Tuberculosis

The current strategy for combating tuberculosis (TB) is based on the early detection and treatment of patients to halt transmission. The present study was conducted to evaluate the diagnostic potential of three Mycobacterium tuberculosis antigens, 45-kDa, A60, and sonicated MTB antigen (SmTB-Ag), as antibody/antigen detection methods for the rapid and accurate diagnosis of TB. The SmTB-Ag and 45-kDa antigens were purified and A60 antigen was supplied by Anda-Biologicals, France. The 45-kDa and A60 antigens (for antibody detection procedures) and SmTB-Ag (for antigen detection test) were tested in the same study subjects. ELISA and immunochromatographic (rapid) test were performed on 201 sputum and serum samples. Ninety-eight samples from TB patients and 103 samples from control individuals were studied. The mean absorbance value of antibodies against 45-kDa antigen in the TB patients were (1.17 ± 0.44, CI 1.09-1.26), significantly higher than in the non-TB group, (0.8± 0.28, CI 0.74-0.85, P< 0.05). The sensitivities of tests using two antigens, 84% for the 45-kDa antigen and 65% for the A60 antigen, were lower than SmTB-Ag(93%). The rapid test yielded 93% sensitivity and 92% specificity. Findings highlighted the importance of antigen detection as a diagnostic tool. The rapid test evaluated in this study may be useful for diagnosis of TB.

Pemphigoïde anti-p200 : rémission sous mycophénolate mofétil (Cellcept®)

Anti-p200 pemphigoid is a recently described autoimmune subepidermal bullous dermatosis characterized by its target antigen and the associated anatomoclinical picture. The treatment is not as yet well defined. A 73-year-old man consulted for a pruritic bullous eruption with buccal involvement. Direct immunofluorescence revealed linear deposits of IgG and C3 at the dermal-epidermal junction. Elisa screening for circulating anti-BP180 and anti-BP230 antibodies was negative. A diagnosis of bullous pemphigoid was suspected. After an unfavourable clinical outcome under clobetasol and then prednisolone and methotrexate, other immunological tests were performed. Indirect immunofluorescence on NaCl-cleaved skin revealed a deposit of IgG4 antibodies on the dermal side. Immunoblotting showed antibodies directed against a 200-kDa antigen on a dermal extract. A diagnosis of anti-p200 pemphigoid was made. The patient was treated with dapsone combined with prednisolone. Seventy-two hours later, treatment was stopped due to hepatic cytolysis related to immunoallergic hepatitis. Treatment with mycophenolate mofetil was then initiated and resulted in complete remission, which persisted at seven months. The diagnosis of anti-p200 pemphigoid was made on the basis of a set of clinical and immunological factors. Anti-p200 pemphigoid differs from standard bullous pemphigoid in terms of more frequent cephalic, acral and mucous membrane involvement, as well as a greater degree of miliary scarring. There was no eosinophilia. Elisa screening for anti-BP180 and anti-BP230 antibodies was negative. Immunoblotting showed antibodies directed against a 200kDa protein on dermal extract. The treatment is not well defined, even if dapsone appears to be the most effective therapy. To our knowledge, our patient is the first to be successfully treated with mycophenolate mofetil. Treatment of anti-p200 pemphigoid is difficult. In our case, treatment by mycophenolate mofetil was effective and could offer an alternative to dapsone.

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.

Anti-p200 pemphigoid responding to dapsone

Abstract Anti-p200 pemphigoid is a rare autoimmune subepidermal blistering disease. Clinical presentation is similar to standard bullous pemphigoid (BP) but mucous membranes and cephalic lesions are more frequent. Histology and direct immunofluorescence (IF) are identical to BP but indirect IF discloses linear deposits of immunoglobulin G (IgG) on the dermal side of artificial salt-split skin. Specific diagnosis is based on western immunoblotting that shows circulating IgG recognizing a 200-kDa protein localized on the dermal extract. The 200-kDa antigen was recently identified as laminin γ1. Anti-p200 pemphigoid should be considered before all atypical or topical steroid-resistant bullous disease, as well as mucous membranes pemphigoid or epidermolysis bullosa acquisita. Dapsone appears to be the most effective treatment and should be used as the first option in combination with topical steroids. In this report, we describe the case of a patient with a typical clinical and immunopathological anti-p200 pemphigoid, responding to a combination of topical steroids and dapsone.

IgA Anti-p200 Pemphigoid

Anti-p200 pemphigoid is a rare autoimmune subepidermal blistering disorder. Clinically, it may resemble bullous pemphigoid, linear IgA bullous dermatosis, or dermatitis herpetiformis. Immunologically, anti-p200 pemphigoid is characterized by the development of IgG antibodies directed against a basement membrane zone protein with a molecular weight of 200 kDa. We report the first case, to our knowledge, of anti-p200 pemphigoid associated with IgA antibodies and having clinical features resembling pemphigus herpetiformis or dermatitis herpetiformis localized on traumatized areas. Histopathological examination of lesional skin showed dermal-epidermal separation and microabscesses composed of neutrophils in the dermal papillae. Direct immunofluorescence disclosed the presence exclusively of linear in vivo-bound IgA along the basement membrane zone. With the use of laser scanning confocal microscopy, in vivo-bound IgA was localized above collagen type IV and colocalized with laminin 332. Indirect immunofluorescence showed circulating IgA antibodies against basement membrane zone at a titer of 1:160 that reacted with the floor of an artificial blister of salt-split skin. Western immunoblot analysis using dermal extract confirmed the reactivity of circulating IgA antibodies with the 200-kDa antigen corresponding to laminin γ1; however, immunoblotting using recombinant protein of 107 amino acid C-terminus of laminin γ1 was negative for circulating IgA antibodies. Immunoelectron microscopy disclosed the reactivity of circulating IgA autoantibodies within the lower lamina lucida. To the best of our knowledge, this is the first case fulfilling the immunopathological criteria for anti-p200 pemphigoid associated with IgA antibodies and having unusual clinical features.

A 35 kDa antigenic protein from Shigella flexneri: In silico structural and functional studies

Shigella flexneri serotype 2a is a major public health concern in the developing and under-developed countries which contributes to shigellosis endemic and mortality. Thus, there is an urgent need for a rapid diagnostic test for effective therapy and disease management. Previous study showed that a ∼35kDa antigenic protein from S. flexneri is a potential biomarker. We therefore modelled the three-dimensional structure of the antigen to probe its functionality which could aid in the development of an antigen-based diagnostic. Results showed that the antigen is a transmembrane protein consists of OmpA and OmpA-like domains. The OmpA domain is a beta-barrel embedded in the outer membrane with four surface-exposed extracellular loops. The OmpA-like domain is linked to the OmpA domain with a 17 amino acids linker and located in the periplasmic. Docking of peptidoglycan into the groove of OmpA-like domain might help in catalyzing the bacterial cell wall formation. Both domains are expected to be involved in the virulence, structural stability, pathogenesis and survival of Shigella thus made the 35kDa protein a suitable shigellosis diagnostic biomarker. This structural elucidation will also enable a better identification of the epitope regions for the development of specific binders to the 35kDa antigen.

Exposure to Extremely Low Frequency Magnetic Fields Induces Fos-Related Antigen-Immunoreactivity Via Activation of Dopaminergic D1 Receptor

We previously demonstrated that repeated exposure to extremely low frequency magnetic fields (ELF-MF) increases locomotor activity via stimulation of dopaminergic D1 receptor (J. Pharmacol. Sci., 2007;105:367-371). Since it has been demonstrated that activator protein-1 (AP-1) transcription factors, especially 35-kDa fos-related antigen (FRA), play a key role in the neuronal and behavioral adaptation in response to various stimuli, we examined whether repeated ELF-MF exposure induces FRA-immunoreactivity (FRA-IR) in the striatum and nucleus accumbens (striatal complex) of the mice. Repeated exposure to ELF-MF (0.3 or 2.4 mT, 1 h/day, for consecutive fourteen days) significantly induced hyperlocomotor activity and FRA-IR in the striatal complex in a field intensity-dependent manner. ELF-MF-induced FRA-IR lasted for at least 1 year, while locomotor activity returned near control level 3 months after the final exposure to ELF-MF. Pretreatment with SCH23390, a dopaminergic D1 receptor antagonist, but not with sulpiride, a dopaminergic D2 receptor antagonist, significantly attenuated hyperlocomotor activity and FRA-IR induced by ELF-MF. Our results suggest that repeated exposure to ELF-MF leads to prolonged locomotor stimulation and long-term expression of FRA in the striatal complex of the mice via stimulation of dopaminergic D1 receptor.

The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein.

Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF) sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.

Isolation and partial characterization of excretory/secretory antigens of Gastrothylax crumenifer

The present study was carried out to identify the excretory/secretory (E/S) antigens of the rumen infecting digenetic trematode Gastrothylax crumenifer that may be useful for the immunodiagnosis of rumen amphistomosis particularly during the pre-monsoon season during which this rumen parasite stops shedding eggs. The in vitro released E/S proteins were purified on a Sephadex G-200 column. The gel filtration profile revealed three distinct fractions F1–F3 where F1 and F3 appeared as sharp peaks while the F2 fraction was dispersed. The antibody titre against each of the purified E/S fractions was determined by ELISA using anti-whole E/S polyclonal antibodies raised in rabbit. Among the three fractions, the antibody titre against F1 was highest (1:12,800) whereas IgG titre was very low (1:50) for fraction F2 and F3 (1:100). Of the total polypeptides resolved on gradient SDS–PAGE, only a few antigenic polypeptides were detected in each fraction with hyperimmune anti-serum as revealed by Western Blot analysis. However, a 33kDa antigen detected in each fraction appeared to be immunodominant which could be exploited for the diagnosis of the pouched amphistome.

Identification and characterization of myeloma-associated antigens in Trichinella spiralis

To investigate the presence of myeloma-associated antigens in Trichinella spiralis and their anti-tumor effect, cross-immune responses between antigens of the myeloma cell SP2/0 versus positive sera to T. spiralis, and antigens of T. spiralis versus positive sera to myeloma cell SP2/0 were determined using T. spiralis and myeloma specific enzyme-linked immunosorbent assays (ELISA). The myeloma-associated antigens in T. spiralis were separated by ultrafiltration and 2-D electrophoresis, and the amino acid sequences and molecular weights were determined by spectrometry. An obvious reaction was found between a 33kDa antigen and positive sera, and the major component of the antigen was tropomyosin (TM), which is an surface acidic protein with 284 amino acids. Mice were immunized with TM to determine the anti-tumor effect in vivo. The results showed that CD4+, CD8+ T lymphocyte, and CD19+ B lymphocyte were significantly increased (P<0.05). The anti-tumor effects were significantly different between mice immunized with the antigens or adjuvant alone (P<0.05), while the difference between mice immunized with antigens and whole T. spiralis was not significant (P>0.05). The results indicated that TM identified in this study may play a role in eliciting cross-protective immunity.

Cell Wall Anchoring of the 37-Kilodalton Oncofetal Antigen by Lactobacillus plantarum for Mucosal Cancer Vaccine Delivery

The 37-kDa oncofetal antigen (OFA), a tumor immunogen expressed on all mammalian cancers examined to date, was secreted and anchored to the cell wall of Lactobacillus plantarum using homologous signal peptides and LPxTG anchors. Orally administered L. plantarum expressing anchored OFA induced a specific immune response against OFA in mice.

Immunocytochemical Localization of a 58kDa Glycoprotein of Epididymal Fluid of Rhesus Monkey (Macaca mulatta)

Jamal, F. and Srivastav, A. 2010. Immunocytochemical localization of a 58kDa glycoprotein of epididymal fluid of rhesus monkey (Macaca mulatta). J. Appl. Anim. Res., 37: 177–183. A 58kDa glycoprotein of epididymal fluid of rhesus monkey, using lectins Lens culinaris agglutinin (LCA) and Ricinus communis agglutinin (RCA), was studied raising polyclonal antisera against this protein to study cross-reactivity with the epididymal fluids of mice, rat, hamster, guinea pig, rabbit and Triton X-100 human sperm extract. The present study aimed at delineating the distribution of this 58kDa antigen in testis, epididymis, vas deferens, seminal vesicle and prostate tissues of rhesus monkey using immunological probes, depicting their ontogeny and its association with maturing spermatozoa. The epitopes cross-reacting with 58kDa antisera were predominantly distributed in the stroma region and luminal spermatozoa throughout the epididymis, vas deferens and prostate tissues. The epitopes against this 58kDa antisera have been conserved and could be explored for immuno-contraception in humans.