Cell-mediated response in cattle experimentally infected with bluetongue virus serotype 2.

Cell-mediated immunity in cattle infected with bluetongue virus serotype 2 was examined using the 3-(4,5, dimethylthiazol- 2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) lymphocyte proliferation assay and the enzyme-linked immunosorbent assay (ELISA) kit for gamma-interferon quantification in serum. Although infection induced the production of neutralising antibodies, no significant statistical differences were observed between the infected and the control animals when tested with the MTT assay. Constant levels of gamma-interferon were detected in the serum infected animals during the trial but again no significant statistical differences were recorded. The results of the study are discussed.

The early growth response gene EGR-1 behaves as a suppressor gene that is down-regulated independent of ARF/Mdm2 but not p53 alterations in fresh human gliomas.

PURPOSE: EGR-1 is an immediate early gene with diverse functions that include the suppression of growth. EGR-1 is down-regulated many cancer cell types, suggesting a tumor suppressor role, and may critically involve the p53 pathway. The aim of this work was to measure the expression of EGR-1 and the p16/INK4a/ARF-Mdm2-p53 pathway status in fresh human gliomas. EXPERIMENTAL DESIGN: Thirty-one human gliomas with different grades of malignancy were investigated for Egr-1 mRNA and the protein expression, frequency, and spectrum of p53 gene mutations, mdm2 gene amplification, and p16/INK4a/ARF allele loss. RESULTS: The amplification of Mdm2 and the deletion of the p16/INK4a gene was found in 3 and 5 cases, respectively, whereas mutations of p53, including two novel mutations, were observed in 10 other cases. The three types of changes occurred strictly mutually exclusively, emphasizing that these genes operate in a common pathway critical to glioma progression. EGR-1 mRNA was significantly down-regulated in astrocytomas (14.7 +/- 5.1%) and in glioblastomas (33.6 +/- 10.0%) versus normal brain. Overall, EGR-1 mRNA was strongly suppressed (average, 15.2 +/- 13.9%) in 27 of 31 cases (87%), independent of changes in p16/INK4a/ARF and Mdm2; whereas 4 of 31 cases with residual EGR-1 expression as well as the highest EGR-1 variance segregated with p53 mutations. Immunohistochemical analyses confirmed the suppression of EGR-1 protein. CONCLUSIONS: These results indicate that EGR-1 is commonly suppressed in gliomas independent of p16/INK4a/ARF and Mdm2 and that suppression is less crucial in tumors bearing p53 mutations, and these results implicate an EGR-1 growth regulatory mechanism as a target of inactivation during tumor progression.

Inhibition of MAPK activity, cell proliferation, and anchorage-independent growth by N-Ras antisense in an N-ras-transformed human cell line.

Mammalian ras genes encode a family of plasma membrane-bound proteins that function as intermediates in signal transduction pathways involved in cell growth and differentiation. Ras oncogene is frequently involved in neoplastic transformation of different cellular histotypes. In this study, we tested the ability of antisense oligodeoxyribonucleotides (AS-ODN) that have mixed phosphodiester/phosphorothioate backbone, targeted against human N-Ras, to inhibit N-ras gene expression and to specifically interfere with the Ras-dependent activity of mitogen-activated protein kinase (MAPK) in two human cell lines carrying an endogenous N-ras mutated allele at codon 61. Three AS-ODN that inhibit basal MAPK activity have been identified. Moreover, AS-ODN treatment resulted in potent antiproliferative effects in cell culture and great inhibition of N-ras mRNA levels in one of two cell lines. These studies suggest that antisense molecules, targeted against N-Ras, could be of considerable value as a tool to study the N-Ras-specific transduction pathway.

CD40 expressed on human melanoma cells mediates T cell co-stimulation and tumor cell growth.

CD40 is a 50 kDa molecule, a member of the tumor necrosis factor/nerve growth factor receptor family. It is expressed on B cells, monocytes, dendritic cells and various malignant cells. While the critical relevance of this molecule in T cell-dependent B cell activation is already established, the biological role of CD40-CD154 interaction in non-hematopoietic cells is still unknown. Here we show that CD40 is functionally expressed on human melanoma-derived cell lines. No correlation between surface CD40 expression and the origin of the cell line, primary versus metastatic, was observed. Melanoma cells were shown to be able to co-stimulate TCR-triggered human T cells; moreover, because they do not express CD80 or CD86 co-stimulatory structures, the involvement of additional pathways have to be postulated. We have identified CD40 as one of the molecules involved in melanoma cell-mediated co-stimulation of anti-CD3-triggered human CD4(+) T lymphocytes. In addition, a CD40-dependent pathway, able to enhance tumor cell proliferation at low serum concentrations, in vitro, has been shown to be functional in human melanoma cell lines.

Increased serum concentrations of soluble HLA-class I antigens in hepatitis C virus related mixed cryoglobulinaemia.

OBJECTIVE: To investigate whether quantitative alterations of both beta(2)microglobulin (beta(2)micro) associated HLA class I heavy chains (sHLA-I) and beta(2) micro free class I heavy chains (sHLA-FHC) in sera of patients with hepatitis C virus (HCV) infection occur and whether they distinguish patients with mixed cryoglobulinaemia (MC). METHODS: 83 HCV infected patients were studied and divided into three groups: (A) without cryoglobulinaemia (n=21), (B) with polyclonal MC (n=20), (C) with monoclonal MC (n=42). Serum sHLA-I and sHLA-FHC were measured by double determinant radioimmunoassay using monoclonal antibodies: TP25.99 as catching antibody, and NAMB-1 and HC-10 as revealing antibodies. Western blot identified HLA-I isoforms. RESULTS: The serum concentrations of sHLA-I and of sHLA-FHC in HCV infected patients versus controls were respectively 1.3(0.5) microg/ml (mean (SD)) versus 0.8 (0.3) (p<0. 001) and 13.9 (7.1) ng/ml versus 9.2 (5) (p<0.001). sHLA-I were 1.01 (0.4) microg/ml in group A, 1.04 (0.4) microg/ml in group B, and 1. 47 (0.4) microg/ml in group C (p=0.001). Statistical analysis showed a significant difference versus controls for groups B (p<0.02) and C (p<0.001). sHLA-FHC were 12.8 (8.3) ng/ml in group A, 17.2 (7.1) ng/ml in group B, and 12.9 (6.2) ng/ml in group C (p<0.02). A significant difference versus controls for each group was found (p<0. 02, p<0.001, and p<0.02, respectively). Different patterns of sHLA-I isoforms were observed. CONCLUSIONS: Increased serum concentrations of sHLA-I and sHLA-FHC characterise HCV infected patients. The highest sHLA-I concentrations seem to distinguish patients with monoclonal MC. In this last condition sHLA could play a part in the HCV escape and in B cell proliferation. The significance of sHLA-FHC is still undefined.

Common human leukocyte antigen alleles in pemphigus vulgaris and pemphigus foliaceus Italian patients.

Pemphigus refers to a group of autoimmune blistering skin diseases, mainly identified as pemphigus vulgaris and pemphigus foliaceus, both characterized by the presence of autoantibodies against keratinocyte adhesion molecules, leading to loss of cell-cell adhesion with consequent blister formation. Pemphigus vulgaris is reported to be associated with human leukocyte antigen DR4 and/or DR6 whereas no data are available on pemphigus foliaceus, except for the endemic Brazilian form (fogo selvagem), which is reported to be associated with DR1 and DR4. We here report human leukocyte antigen molecular typing on a total of 87 patients, 61 with pemphigus vulgaris and 26 with pemphigus foliaceus, versus 128 healthy matched controls. Generic typing showed an increase of DRB1*04 and DRB1*14 and a decrease of DRB1*07 in both pemphigus vulgaris and pemphigus foliaceus patients. Molecular subtyping of DR4+ and DR14+ subjects showed a highly significant association between the DRB1*1401 and both pemphigus vulgaris (p < 0.0001) and pemphigus foliaceus patients (p < 0.0001) together with a significant increase of the linked DQB1*0503 (pemphigus vulgaris p < 0.0001; pemphigus foliaceus p < 0.0001). Moreover, whereas the association between DRB1*0402 and pemphigus vulgaris (p < 0.0001) has been confirmed, no significant association between a specific allele of the DR4 group and pemphigus foliaceus, has been found. Therefore, at least in Italian patients, pemphigus vulgaris and pemphigus foliaceus share DRB1*1401 and DQB1*0503, as susceptible human leukocyte antigen alleles, whereas DRB1*0402 is only found associated with pemphigus vulgaris. The observation that both diseases, pemphigus vulgaris and pemphigus foliaceus, carry the same susceptible human leukocyte antigen alleles has been interpreted as a common genetic background predisposing to pemphigus as, like in other autoimmune disorders, it is not sufficient to explain the onset of the disease on the basis of the sole aforementioned alleles. Other linked genes and/or environmental factors should play a facilitating role in the outbreak of pemphigus, either pemphigus vulgaris or pemphigus foliaceus.

Molecular analysis of HLA DRB1 and DQB1 polymorphism in Italian melanoma patients.

Several studies have reported association between a variety of malignancies and human leukocyte antigens (HLA) genes. However, conflicting data have been reported on HLA association and melanoma. We report here serologic and molecular analysis by polymerase chain reaction sequence-specific primers (PCR-SSP) and PCR sequence-specific oligonucleotides (PCR-SSO) of HLA class II DRB1 and DQB1 loci in 132 patients with melanoma and 102 ethnically matched controls. Molecular typing of DQB1 polymorphism showed a significant increase of DQB1 *0501 (25.0% versus 14.7%; p = 0.038). Moreover, an increase of DQB1*0301, which was present in 62.8% of patients and 54.9% of controls (p = 0.136), was noted. Because DQB1*0501 and DQB1*0301 are strongly linked to DRB1*01 and DRB1*11, respectively, both found increased in patients with melanoma, to look for a more stringent association with a particular allele specificity of the DR locus, we performed PCR-SSP high-resolution typing of DR1 and DR11 positive subjects. Results showed no significant difference between the frequencies of the alleles found in patients with respect to controls. Analysis of the distribution of DQB1*0501 and DQB1*0301 according to the AJCC clinical stage of the disease showed no significant difference in the frequency of these alleles between the localized and the metastatic form of the disease. However, none of the HLA class II alleles showed significant association after correction of the p value. These results indicate that HLA class II alleles may not contribute to a strong susceptibility to melanoma, at least in Italian patients.