Low Expression of Human Histocompatibility Soluble Leukocyte Antigen-G (HLA-G5) in Invasive Cervical Cancer With and Without Metastasis, Associated With Papilloma Virus (HPV)

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class lb molecule that acts as a specific immunosuppressor. Some studies have demonstrated that human papillomavirus (HPV) seems to be involved in lower or absent HLA-G expression, particularly in cervical cancer. In this study, we performed a cross-sectional study, systematically comparing the qualitative expression of the HLA-G5 isoform in invasive cervical carcinoma (ICC), stratifying patients according to the presence [ICC with metastasis (ICC(W))) and absence [ICC without metastasis (ICC(WT))] of metastasis, correlating these findings with interference of HPV and demographic and clinical variables. Seventy-nine patients with a diagnosis of ICC were stratified into two groups: ICC(WT) (n=52 patients) and ICC(W) (n=27). Two biopsies were collected from each patient (one from the tumor lesion and one from a lymph node). Immunohistochemistry analyses were performed for the HLA-G5 isoform, for HPV detection, and virus typing. HLA-G5 isoform molecules were detected in 25 cases (31.6%), 17 (32.7%) without metastasis and 8 (29.6%) with metastasis. HPV was detected in the cervical lesions of 74 patients (93.7%), but low expression of the HLA-G5 isoform was observed in all HPV-related cases. These findings are important; however, additional studies are necessary to identify the influence of HPV with HLA-G5 isoform expression on invasive cervical malignancies. (J Histochem Cytochem 58:405-411, 2010)

Evaluation of tests based on the antibody response to keyhole limpet haemocyanin and soluble egg antigen to differentiate acute and chronic human schistosomiasis mansoni

Specific IgG and IgM responses to soluble egg antigen (SEA) and keyhole limpet haemocyanin (KLH) were measured by ELISA in patients with acute and chronic schistosomiasis. The tests based upon IgM and IgG antibodies responses to KLH presented the best diagnostic discrimination, and can be used in conjunction with clinical and epidemiological data to the differential diagnosis of acute schistosomiasis.

A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen.

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8(+) T cell epitope, NY-ESO-1(88-96) (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165) epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1(88-96) is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165). On the other hand, NY-ESO-1(157-165) is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A(26-35); whereas NY-ESO-1(88-96) was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1(88-96) is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18(+) melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1(88-96) from patients, including those who received NY-ESO-1 ISCOMATRIX? vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8(+) T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.

Low expression of human histocompatibility soluble leukocyte antigen-G (HLA-G5) in invasive cervical cancer with and without metastasis, associated with Papilloma Virus (HPV)

Human leukocyte antigen-G (HLA-G) is a non-classical major histocompatibility complex class Ib molecule that acts as a specific immunosuppressor. Some studies have demonstrated that human papillomavirus (HPV) seems to be involved in lower or absent HLA-G expression, particularly in cervical cancer. In this study, we performed a cross-sectional study, systematically comparing the qualitative expression of the HLA-G5 isoform in invasive cervical carcinoma (ICC), stratifying patients according to the presence [ICC with metastasis (ICCW)] and absence [ICC without metastasis (ICCWT)] of metastasis, correlating these findings with interference of HPV and demographic and clinical variables. Seventy-nine patients with a diagnosis of ICC were stratified into two groups: ICCWT (n=52 patients) and ICCW (n=27). Two biopsies were collected from each patient (one from the tumor lesion and one from a lymph node). Immunohistochemistry analyses were performed for the HLA-G5 isoform, for HPV detection, and virus typing. HLA-G5 isoform molecules were detected in 25 cases (31.6%), 17 (32.7%) without metastasis and 8 (29.6%) with metastasis. HPV was detected in the cervical lesions of 74 patients (93.7%), but low expression of the HLA-G5 isoform was observed in all HPV-related cases. These findings are important; however, additional studies are necessary to identify the influence of HPV with HLA-G5 isoform expression on invasive cervical malignancies.

Simplified criteria for the diagnosis of autoimmune hepatitis

Diagnosis of autoimmune hepatitis (AIH) may be challenging. However, early diagnosis is important because immunosuppression is life-saving. Diagnostic criteria of the International Autoimmune Hepatitis Group (IAIHG) were complex and purely meant for scientific purposes. This study of the IAIHG aims to define simplified diagnostic criteria for routine clinical practice. Candidate criteria included sex, age, autoantibodies, immunoglobutins, absence of viral hepatitis, and histology. The training set included 250 AIH patients and 193 controls from 11 centers worldwide. Scores were built from variables showing predictive ability in univariate analysis. Diagnostic value of each score was assessed by the area under the receiver operating characteristic (ROC) curve. The best score was validated using data of an additional 109 AIH patients and 284 controls. This score included autoantibodies, immunoglobulin G, histology, and exclusion of viral hepatitis. The area under the curve for prediction of AIH was 0.946 in the training set and 0.91 in the validation set. Based on the ROC curves, two cutoff points were chosen. The score was found to have 88% sensitivity and 97% specificity (cutoff >= 6) and 81% sensitivity and 99% specificity (cutoff 2:7) in the validation set. Conclusion: A reliable diagnosis of AIH can be made using a very simple diagnostic score. We propose the diagnosis of probable AIH at a cutoff point greater than 6 points and definite AIH 7 points or higher.

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

DETECTION OF CELLULAR-IMMUNITY WITH THE SOLUBLE-ANTIGEN OF THE FUNGUS SPOROTHRIX-SCHENCKII IN THE SYSTEMIC FORM OF THE DISEASE

Sporothrix schenckii is the etiologic agent of sporotrichosis, a mycosis of world-wide distribution more commonly occurring in tropical regions. The immunological mechanisms involved in the prevention and control of sporotrichosis are not fully understood but apparently include both the humoral and cellular responses. In the present investigation, cellular immunity was evaluated by in vivo and in vitro tests in mice infected with yeast-like forms of S. schenckii. The disease developed systemically and cellular immunity was evaluated for a period of 10 weeks. The soluble antigen utilized in the tests was prepared from yeast form of the fungus through the sonication (20 min: 10 sonications at 50 W at 2-min intervals). Delayed hypersensitivity and lymphocyte transformation tests showed that the cellular immune response was depressed between the 4th and 6th week of infection when the animals were challenged with the soluble fungal antigen. This depression frequently indicates worsening of the disease, with greater involvement of the host. This is a promising field of research for a better understanding of the pathogeny of this mycosis.

Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human Schistosomiasis: I. Granuloma formation and modulation around polyacrylamide antigen-conjugated beads

We have developed an in vitro model of granuloma formation for the purpose of studying the immunological components of delayed type hypersensitivity granuloma formation in patients infected with Schistosoma mansoni. Our data show that 1) granulomatous hypersensitivity can be studied by examining the cellular reactivity manifested as multiple cell layers surrounding the antigen conjugated beads; 2) this reactivity is a CD4 cell dependent, macrophage dependent, B cell independent response and 3) the in vitro granuloma response is antigenically specific for parasite egg antigens. Studies designed to investigate the immune regulation of granulomatous hypersensitivity using purified populations of either CD4 or CD8 T cells have demonstrated the complexity of cellular interactions in the suppression of granulomatous hypersensitivity. The anti-S. mansoni egg immune responses of individual patients with chronic intestinal schistosomiasis can be classified either as soluble egg antigen (SEA) hypersensitive with maximal granulomatous hypersensitivity or SEA suppressive with activation of the T cell suppressor pathway with effective SEA granuloma modulation. Our data suggest that T cell network interactions are active in the generation of effective granuloma modulation in chronic intestinal schistosomiasis patients.

The Role of Protein Kinases in Antigen-activation of Peripheral Blood Mononuclear Cells of Schistosoma mansoni Infected Individuals

T cell recognition of antigens displayed on the surface of antigen presenting cell results in rapid activation of protein tyrosine kinases and kinase C. This process leads to second messengers, such as inositol phosphates and diacylgycerol, and phosphorylation of multiple proteins. The role of different protein kinases in the activation of peripheral blood mononuclear cells (PBMC) from Schistosoma mansoni infected individuals was evaluated using genistein and H-7, specific inhibitors of protein tyrosine kinase and kinase C, respectively. Our results showed that proliferation in response to soluble egg antigen or adult worm antigen preparation of S. mansoni was reduced when PBMC were cultured in presence of protein kinase inhibitors. Using these inhibitors on in vitro granuloma reaction, we also observed a marked reduction of granuloma index. Taken together, our results suggest that S. mansoni antigen activation of PBMC involves protein kinases activity

Impact of Antifibrotic Treatment on the Course of Schistosoma mansoni Infection in Murine Model

Administration of an antifibrotic agent as an adjunct to antihelmintic treatment with the objective of morbidity reduction was investigated in the murine schistosomiasis mansoni model. Antifibrotic, ß-aminopropionitrile treatment has a profound effect on the cellular matrix composition of the liver granuloma of Schistosoma mansoni infected mice when given alone, resulting in increase macrophage infiltration. These macrophages, in response to stimulation with soluble egg antigen or lipopolysaccharide produced elevated levels of nitric oxide but low levels of tumor necrosis factor alpha compared to untreated infected mice. This also correlated with reduced liver granuloma size. In spite of low numbers of eggs in the liver, mice receiving a combine treatment had a high level of resistance to a challenge infection compared with mice receiving only praziquantel. Those mice also exhibited a reduced lymphocyte proliferative response, similar to that of infected untreated mice. Antifibrotic treatment has an impact on the dynamic of the cellular nature of granulomas and impacts on the host immunity to infection

Inhibition of antigen-induced T-cell clone proliferation by antigen-specific antibodies.

Attempts to inhibit the recognition of soluble antigens by T lymphocytes using antibodies specific for the antigen in question have been uniformally unsuccessful, in contrast to the observed specific inhibition of antibody generation by B cells. One exception is the unique situation whereby anti-hapten antisera inhibit the T-cell proliferative responses observed when hapten-specific T lymphocytes or clones are cultured with hapten-derivatized cells or proteins. The inability to inhibit T-cell functions by antigen-specific antibodies has been interpreted in several ways: (1) T cells possess a different repertoire from B cells; (2) the antibodies tested recognize epitopes present on the native antigen, whereas T cells recognize non-native (processed) structures; (3) the antigenic determinant(s) recognized by T cells on the surface of antigen presenting cells are either not accessible to antibodies, or are present in low amounts. The development of antigen-specific T-cell clones and monoclonal antibodies both specific for the same antigenic determinants now allows this question to be investigated definitively. Here, we report for the first time the specific inhibition of antigen-induced T-cell clone proliferation by a monoclonal antibody directed against the relevant soluble protein antigen.

Immunization with PIII, a fraction of Schistosoma mansoni soluble adult worm antigenic preparation, affects nitric oxide production by murine spleen cells

Nitric oxide (NO) is an important effector molecule involved in immune regulation and defense. NO produced by cytokine-activated macrophages was reported to be cytotoxic against the helminth Schistosoma mansoni. Identification and characterization of S. mansoni antigens that can provide protective immunity is crucial for understanding the complex immunoregulatory events that modulate the immune response in schistosomiasis. It is, then, essential to have available defined, purified parasite antigens. Previous work by our laboratory identified a fraction of S. mansoni soluble adult worm antigenic preparation (SWAP), named PIII, able to elicit significant in vitro cell proliferation and at the same time lower in vitro and in vivo granuloma formation when compared either to SEA (soluble egg antigen) or to SWAP. In the present work we report the effect of different in vivo trials with mice on their spleen cells ability to produce NO. We demonstrate that PIII-immunization is able to significantly increase NO production by spleen cells after in vitro stimulation with LPS. These data suggest a possible role for NO on the protective immunity induced by PIII.

Malnutrition and hepatic fibrosis in murine schistosomiasis

In this paper, four different approaches attempting to reproduce the schistosomal liver fibrosis in undernourished mice are reported: shifting from a deficient to a balanced diet and vice-versa, repeated infections, influence of the genetic background, and immunological response. Infections were performed with 30 cercariae of Schistosoma mansoni and lasted at least four months. Undernourished mice were unable to reproduce the picture of "pipestem" fibrosis, except the C57 BL/10 inbred strain, four out of 21 mice developing the liver lesion. A link of this histological finding to the type of parasite strain can not be discarded at the moment. Repeated infections increased collagen deposition mainly in well nourished animals (seven out of 16 Swiss mice developed "pipestem"-like fibrosis). In undernourished infected Swiss mice the serum levels of soluble egg antigen specific antibodies IgG1, IgG2a, IgG2b, and IgG3 were two to four times lower than those detected for well nourished controls. The decreased humoral immune response coupled to the morphological, morphometric, and biochemical results reinforce the influence of the host nutritional status on the connective tissue changes of hepatic schistosomiasis.