Detection of serum antibodies against Leishmania 94 kDa antigen in visceral and cutaneous leishmaniosis due to Leishmania infantum.

Leishmania promastigotes polypeptides are analyzed by immunoblotting with sera from patients infected with different Leishmania species and presenting visceral or cutaneous infections. These sera recognize Leishmania polypeptides in several molecular masses. The major findings of this study are as follow. 1) The Leishmania 94 kDa antigen, which is specifically recognized by all sera from L. infantum-infected patients with visceral infection, is recognized by some sera from L. infantum-infected patients presenting cutaneous infection. 2) All patients with cutaneous infections due to L. tropica, L. amazonensis, or L. guyanensis do not develop anti-94 kDa antibodies, whatever the Leishmania species used as antigens. 3) Difference in electrophoretic mobilities is seen between the 94 kDa antigen identified by sera from Leishmania infantum-infected patients, and the antigen both recognized by the Concavalin A lectin and a rabbit antiserum raised against deglycosylated Promastigote Surface Protease.

CD8(+) T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1.

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.

Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD.

During the selection of monoclonal antibodies (MAb) raised against purified carcinoembryonic antigen (CEA), two MAbs were identified which immunoprecipitated a glycoprotein of 95 kD present both in perchloric acid extracts of normal lung and on the surface of normal granulocytes. This antigen was distinct from the previously reported normal glycoprotein crossreacting with CEA (NCA) which had a molecular weight of 55 kD. The difference between the smaller and the larger crossreacting antigens termed NCA-55 and NCA-95, respectively, was demonstrated by SDS-polyacrylamide gel electrophoresis, by elution from Sephadex-G200 and by selective binding to a series of anti-CEA MAb. Out of six MAb which all bound CEA purified from colon carcinoma, three did not react with these two crossreacting antigens, one bound only NCA-95, one reacted only with NCA-55 and one reacted with both NCA-55 and NCA-95. Immunoadsorbent purified preparations of 125I labelled NCA-95 and NCA-55 were found useful for the screening of new anti-CEA MAb. In addition, when tested on frozen sections of colon carcinoma, normal spleen, normal lung and pancreas, each type of MAb gave a clearly different pattern of reactivity. The three anti-CEA MAb which did not bind any of the crossreacting antigens stained only the colon carcinoma cells; the MAb binding to either one of the two types of NCA gave a similar pattern of reactivity both on colon carcinoma cells and on granulocytes. However, on normal lung and pancreas, the MAb binding NCA-55 stained granulocytes as well as bronchiolar and alveolar epithelial cells in lung and inter- and intra-lobular duct epithelial cells in pancreas, whereas the MAb binding only NCA-95 stained only the granulocytes. Thus, the newly identified NCA-95 appears to differ from NCA-55 not only in terms of molecular size and antigenicity but also by the fact that in normal lung and pancreas it is found in granulocytes but not in epithelial cells.

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.

Evaluation of a new serological test for the detection of anti-Coxiella and anti-Rickettsia antibodies.

Coxiella burnetii and members of the genus Rickettsia are obligate intracellular bacteria. Since cultivation of these organisms requires dedicated techniques, their diagnosis usually relies on serological or molecular biology methods. Immunofluorescence is considered the gold standard to detect antibody-reactivity towards these organisms. Here, we assessed the performance of a new automated epifluorescence immunoassay (InoDiag) to detect IgM and IgG against C. burnetii, Rickettsia typhi and Rickettsia conorii. Samples were tested with the InoDiag assay. A total of 213 sera were tested, of which 63 samples from Q fever, 20 from spotted fever rickettsiosis, 6 from murine typhus and 124 controls. InoDiag results were compared to micro-immunofluorescence. For acute Q fever, the sensitivity of phase 2 IgG was only of 30% with a cutoff of 1 arbitrary unit (AU). In patients with acute Q fever with positive IF IgM, sensitivity reached 83% with the same cutoff. Sensitivity for chronic Q fever was 100% whereas sensitivity for past Q fever was 65%. Sensitivity for spotted Mediterranean fever and murine typhus were 91% and 100%, respectively. Both assays exhibited a good specificity in control groups, ranging from 79% in sera from patients with unrelated diseases or EBV positivity to 100% in sera from healthy patients. In conclusion, the InoDiag assay exhibits an excellent performance for the diagnosis of chronic Q fever but a very low IgG sensitivity for acute Q fever likely due to low reactivity of phase 2 antigens present on the glass slide. This defect is partially compensated by the detection of IgM. Because it exhibits a good negative predictive value, the InoDiag assay is valuable to rule out a chronic Q fever. For the diagnosis of rickettsial diseases, the sensitivity of the InoDiag method is similar to conventional immunofluorescence.

Immunolocalisation of secreted-excreted products of Meloidogyne spp. using polyclonal and monoclonal antibodies

Molecules expressed at the surface cuticle (SC) of plant parasitic nematodes represent the primary plant-nematode interface, and together with secreted-excreted (S-E) products are probably the first signals perceived by the host. These molecules, which are released into plant tissue, probably play important roles in the host-parasite interactions. Characterisation of these antigens will help in the identification of nematode targets useful for novel control strategies, which interfere with the nematode infection of plants. Three monoclonal (MAbs) and three polyclonal (PAbs) antibodies produced to S-E products of Meloidogyne spp. and Heterodera avenae were used to examine their reactivity towards M. incognita and/or M. arenaria second stage juveniles and adult females. The three PAbs showed cross-reactivity with M. incognita and M. arenaria. Antibody Roth-PC 373 strongly recognised molecules present in the SC, amphids and intestine, antibody Roth-PC 389 recognised the nematode amphids and metacorpus, while antibody Roth-PC 419 bound to molecules present in the subventral glands. Reactivity of the MAbs was only tested against M. arenaria. Monoclonal antibody Roth-MAb T116C1.1 showed intense reactivity with molecules present in the amphidial and phasmidial glands. Monoclonal antibodies Roth-MAb T46.2 and T42D.2 labeled the nematode amphids and molecules present in the nematode oesophagus (metacorpus), respectively.

Development of IgY antibodies in chickens and IgG in rabbits immunized against proteins of Pythium insidiosum isolated from horses in the state of Rio de Janeiro

Pythiosis is caused by Pythium insidiosum and the occurrence of disease in horses was described in the North and Northwest State of Rio de Janeiro, Brazil. The disease was described in cattle, sheep, humans, and horses in different states and regions across the country. This paper describes the development of IgY and IgG polyclonal antibodies, in chicken and rabbits, respectively against proteins extracted from kunkers and hyphae of P. insidiosum from affected horses. The proteins were recognized by chicken, rabbit and horse antibodies by immunodiffusion and Western blot against majority bands of 27 and 43 KDa, and titrated by ELISA. The antibodies IgY developed by the first time against Brazilian strains of P. insidiosum may represent a valuable tool in the detection of antigens of the pathogen and contribute to further studies aimed at immunotherapy and knowledge about this disease in endemic areas in Rio de Janeiro and in Brazil.

Anti-Rickettsia spp. antibodies in free-ranging and captive capybaras from southern Brazil

Capybaras (Hydrochaeris hydrochaeris) are among the main hosts of Amblyomma spp. ticks, which is able to transmit Rickettsia species to human beings and animals. Since they are often infested with potential vector ticks, capybaras may be used as sentinels for rickettsiosis, such as the Brazilian Spotted Fever. The aim of the present study was to determine the prevalence of antibodies against Rickettsia spp. using the indirect immunofluorescence assay (IFA) in 21 free-ranging and 10 captive animals from the Zoological Park of the 'Bela Vista Biological Sanctuary' (BVBS), Itaipu Binational, Foz do Iguaçu, Southern Brazil. Antigens of six rickettsial species already identified in Brazil (Rickettsia rickettsii, R. parkeri, R. bellii, R. rhipicephali, R. amblyommii and R. felis) were used for IFA. Ticks from each capybara were collected for posterior taxonomic identification. A total of 19 (61.3%) samples reacted to at least one of tested species. Seropositivity was found in 14 (45.2%), 12 (38.7%), 5 (16.1%), 4 (12.9%), 3 (9.7%) and 3 (9.7%) animals for R. rickettsii, R. bellii, R. parkeri, R. amblyommii, R. felis and R. rhipicephali, respectively. Two captive capybaras presented suggestive titers of R. rickettsii infection and one sample showed homologous reaction to R. parkeri. Only one free-ranging capybara presented evidence R. bellii infection. Ticks collected on capybaras were identified as Amblyomma dubitatum e Amblyomma sp. Results evidenced the rickettsial circulation in the area, suggesting a potential role of capybaras on bacterial life cycle.

Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV) were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs) (Corapi WV, Donis RO and Dubovi EJ (1990) American Journal of Veterinary Research, 55: 1388-1394). Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3%) reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R) was calculated, 49 of 66 comparisons (74.24%) between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

Prevalence of antibodies to human herpesvirus-8 in populations with and without risk for infection in São Paulo State

Human herpesvirus 8 (HHV-8) is a newly described herpesvirus that is etiologically associated with all forms of Kaposi's sarcoma (KS). Seroepidemiological studies have shown high prevalence rates of HHV-8 antibodies among men who have sex with men (MSM) and AIDS patients, African children, Brazilian Amerindians, and elderly individuals in certain regions of Europe. The aim of the present study was to determine the prevalence of HHV-8 antibodies in healthy children and young adults from different cities in São Paulo State, and in a population at high risk for HHV-8 infection: HIV-negative MSM, and AIDS patients with and without KS. Antibodies to HHV-8 latency-associated nuclear antigen and lytic-phase antigens were detected by immunofluorescence assays. In 643 healthy children and young adults from the general population attending a vaccination program for yellow fever in ten different cities in São Paulo State, the prevalence of HHV-8 antibodies detected by the presence of latent or lytic antigens ranged from 1.0 to 4.1% in the different age groups (mean = 2.5%). In the MSM group, the prevalence was 31/95 (32.6%). In the group of patients with AIDS, the prevalence was 39.2% (51/130) for non-KS patients and 98.7% (77/78) for AIDS patients with the diagnosis of KS confirmed by histopathological examination. We conclude that HHV-8 has a restricted circulation among healthy children and young adults in the general population of São Paulo State and a high prevalence among MSM and AIDS patients.

Production and characterization of monoclonal antibodies to a Brazilian bovine herpesvirus type 5

Antigens of a bovine herpesvirus type 5 (BHV-5), isolated from a cow with a neurological infection in Rio Grande do Sul State, Brazil, were used to immunize BALB/c mice to produce monoclonal antibodies (mAbs). Eleven hybridomas secreting mAbs directed at BHV-5 antigens were obtained after two fusions and screening of 356 hypoxanthine-aminopterin-thymidine-resistant clones. The mAbs reacted at dilutions up to 1:500 (hybridoma culture supernatant) and up to >1:10,000 (ascitic fluid) in an indirect fluorescent antibody assay (IFA) and in immunoperoxidase staining of BHV-5-infected cells. Four mAbs (1D12, 2E2, 2G10 and 4E4) showed virus-neutralizing activity against the parental BHV-5 isolate. Five mAbs (1F3, 2A6, 2F9, 2G10 and HB24L) reacted in Western immunoblotting with a protein of approximately 90 kDa. Three other mAbs (2E2, 3D6 and 4E4) reacted in IFA with antigens of a BHV-1 mutant glycoprotein C- negative strain, demonstrating that they are directed at a viral antigen other than glycoprotein C. The eleven mAbs tested reacted with 20 BHV-5 field isolates and nine mAbs reacted with 10 BHV-1 isolates. Two mAbs (1F3 and 2F9) failed to react with BHV-1 field isolates, although they displayed a weak and nonreproducible reaction with the BHV-1 reference strain Los Angeles. These mAbs may be very useful in distinguishing between BHV-1 and BHV-5 infections since most of the traditional reagents and techniques are unable to do so. One mAb (2F9) was shown to bind to viral antigens by immunohistochemistry of histological sections of the brain of a BHV-5-infected calf. These results demonstrate that the mAbs produced here are suitable for use in a variety of immunological techniques and therefore may be useful for diagnostic and research purposes.

Humoral response to low molecular weight antigens of Mycobacterium tuberculosis by tuberculosis patients and contacts

Much effort has been devoted to the identification of immunologically important antigens of Mycobacterium tuberculosis and to the combination of target antigens to which antibodies from serum of tuberculous patients could react specifically. We searched for IgG antibodies specific for antigens of 45 to 6 kDa obtained after sonication of the well-characterized wild M. tuberculosis strain in order to detect differences in the antibody response to low molecular weight antigens from M. tuberculosis between patients with pulmonary tuberculosis and contacts. Specific IgG antibodies for these antigens were detected by Western blot analysis of 153 serum samples collected from 51 patients with confirmed pulmonary tuberculosis. Three samples were collected from each patient: before therapy, and after 2 and 6 months of treatment. We also analyzed 25 samples obtained from contacts, as well as 30 samples from healthy individuals with known tuberculin status, 50 samples from patients with other lung diseases and 200 samples from healthy blood donors. The positive predictive value for associated IgG reactivity against the 6-kDa and 16-kDa antigens, 6 and 38 kDa, and 16 and 38 kDa was 100% since simultaneous reactivity for these antigens was absent in healthy individuals and individuals with other lung diseases. This association was observed in 67% of the patients, but in only 8% of the contacts. The humoral response against antigens of 16 and 6 kDa seems to be important for the detection of latent tuberculosis since the associated reactivity to these antigens is mainly present in individuals with active disease.

A Comparative Study of the Frequency of Antibody and Titers Against Human Herpesvirus 8 Latent and Lytic Antigens in ""At-Risk"" Individuals and Among Patients With Kaposi`s Sarcoma

Differences in the prevalence of human herpesvirus 8 (HHV-8) and Kaposi`s sarcoma (KS) have been described, depending on the study population and their geographic origin. A cross-sectional study aimed at detecting the frequency and titers of antibodies against HHV-8 latent and lytic antigens in serum samples from individuals with different risk-factors for HHV-8 infection, as well as predictive marker identification in patients with KS, was conducted. Serum samples were collected from seven groups of individuals: 75 patients with AIDS-KS, 5 with classic KS, 16 with African KS, 495 with HIV/AIDS, 805 patients with chronic kidney disease, 683 handicapped individuals, and 757 health care workers. Samples were evaluated for the presence and titers of HHV-8-specific antibodies to latent and lytic antigens using ""in house"" immunofluorescence assays. The results were analyzed by the Chi-square, Fisher`s exact test, Kruskal-Wallis and/or Mann-Whitney U-tests. The frequencies of HHV-8 antibodies were as follows: 87.5-100% in patients with KS, 20.4% in patients with HIV/AIDS, 18% in patients with chronic kidney disease, 1.6% in handicapped individuals, and 1.1% in health care workers. A greater number of samples were antibody positive to lytic antigens. Elevated titers of antibodies to latent and lytic antigens, mostly among patients with KS, were detected. Using established serological assays, different ""at-risk"" populations for HHV-8 infection/disease were detected in this geographic area, confirming HIV/AIDS and identifying patients with chronic kidney disease as high-risk groups. It is suggested that a longitudinal evaluation of antibody titers in patients with chronic kidney disease be undertaken to confirm their predictive value in the development of KS. J. Med. Virol. 81: 1292-1297, 2009. (C) 2009 Wiley-Liss, Inc.

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin beta(13). It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V(H) CDR3 peptide from mAb A4 and V(L) CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.

Mycoplasmal lipid-associated membrane proteins and Mycoplasma arthritidis mitogen recognition by serum antibodies from patients with rheumatoid arthritis

Mycoplasmal lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. The objective of this work was to detect antibodies to MAM and LAMPs of Mycoplasma hominis and M. fermentans in the sera of patients affected by rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) to identify mycoplasmal products that can be involved in the etiopathogenesis of these autoimmune diseases. Serum samples from female RA and SLE patients and controls, recombinant MAM, and LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blot assays. A similar frequency of sera from patients and controls reactive to MAM was detected. A larger number of M. hominis and M. fermentans LAMPs were recognized by sera from RA patients than controls, but no differences were detected between sera from SLE patients and controls. Among the LAMPs recognized by IgG antibodies from RA patients, proteins of molecular masses in a range of < 49 and a parts per thousand yen20 KDa (M. hominis) and < 102 and a parts per thousand yen58 KDa (M. fermentans) were the most reactive. These preliminary results demonstrate the strong reactivity of antibodies of RA patients with some M. hominis and M. fermentans LAMPs. These LAMPs could be investigated as mycoplasmal antigens that can take part in the induction or amplification of human autoimmune responses.