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Notes: HLA class-II allelic diversity is commonly defined using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or the combination of PCR and restriction fragment length polymorphism methods (PCR-RFLP). Nevertheless, the identification of the DRB polymorphism by PCR-SSO is a time-consuming procedure and the PCR-RFLP is cumbersome. A rapid technique which allows a precise and extensive HLA-DRB typing is required, particularly in order to study the role of class-II matching in organ transplantation. A DRB typing method based on the detection and length of PCR products amplified using combination of allele specific primers has been developed. Thirty-four DRB alleles (29 DRB1, 4DRB3, 1DRB4) can be detected using 29 primers distributed into 19 amplification mixtures.

Notes: The role of the MHC class II antigens in the activation of resting human B lymphocytes (B-Go) was examined with respect to both early and late events in the activation process.The (Ca2+)i induced by anti-IgM was enhanced in the presence of, or following pre-incubation with, an anti-MHC class II DR antibody (D1.12). Pre-incubation with a sepharose conjugated antibody (Seph.-D1.12) augmented the proliferation of B-Go in response to a sub-optimal concentration of anti-IgM.The 2D PAGE profile of B-Go differed from that of in vivo activated B lymphocytes. The 2D PAGE profile of B-Go activated by Seph.-D1.12 was not identical to the profile of B-Go activated by either anti-IgM or PMA.These data suggest that the activation of B-Go via the class II antigens shares part of the pathway of anti-IgM induced activation but does not follow an identical pathway.

Notes: Major histocompatibility complex (MHC) class II molecules are responsible for peptide presentation to helper T lymphocytes and as such play an essential role in the immune response. These molecules transmit intracellular signals leading to diverse consequences in B lymphocytes including proliferation and apoptosis. Recent studies have revealed that glycolipid enriched membrane microdomains (GEMs) behave as signalling platforms for a variety of lymphocyte receptors. We have quantified human leucocyte antigen (HLA)-DR molecules localized in GEMs in human B lymphocytes. Use of a model imitating the interaction of HLA-DR with a T-cell receptor (TCR) modified the constituents of the HLA–DR-enriched GEMs. Confocal microscopy demonstrated a recruitment of HLA–DR and the ganglioside GM1 at the site of HLA–DR interaction with the stimulating ligand. Moreover, cholesterol depletion efficiently impaired this recruitment. Co-localizing proteins detected in HLA–DR-enriched GEMs include protein kinase C (PKC)-δ and actin. These data reveal that MHC class II antigens are localized in GEMs in mature human B lymphocytes and indicates that the formation of the immunological synapse regulates the composition of HLA–DR enriched GEMs in the antigen presenting cell (APC).

Notes: Background Nasal polyps are characterized by a proliferation of the epithelial layer of the mucosa. cellular infiltrates and other pathological changes; however the mechanisms involved in polyp pathogenesis remain largely unclear.Objectives We have taken two different approaches to study the cellular events involved in nasal polyposis.Methods First, through use of immunohistochemical methods, we have studied the expression of HLA class II antigens in epithelial cells of nasal polyps and the distribution of lymphocytes in the epithelium and in the subepithelial layer in patients with clinical conditions, such as asthma, atopy, aspirin intolerance or cystic fibrosis, and in subjects with an absence of concomitant diseases. Second, in order to investigate whether HLA class II expression is controlled at the pre- or post-transcriptional level, we studied the effect of interferon gamma (INFγ) on epithelial cells in primary culture, which were derived from HLA class II negative and HLA class II positive nasal polyps. Total RNA was extracted from the cells and reverse-transcribed, and the c-DNA corresponding to DR, DP, DQ loci was amplified by PCRResults Expression of HLA class II antigens by the epithelia of nasal polyps was more common in the presence rather than in the absence of concomitant asthma, atopy or cystic fibrosis (59% versus 409%). HLA-DR was the only HLA class II antigen expressed in the seven polyps taken from cystic fibrosis patients. The number of CD8+ cells was significantly higher in polyps associated with known clinical conditions and HLA class II antigen expression than it was in ‘isolated’ polyps and in HLA class II negative polyps. RNA transcripts for at least one or all three HLA-DR, DP and DQ antigens were detected in 10 cultures of the 11 HLA class II positive polyps. Conversely, 8 of 10 cultures derived from HLA class II negative polyps did not express HLA class II transcripts in the absence of INFγ. Adding INFγ (100U/ml) to the latter cell cultures caused expression of transcripts of one or more HLA class II genes.Conclusions We have shown that HLA class II antigens were more frequently detected in polyps of patients with an identified clinical syndrome than in those of asymptomatic subjects. Our results also suggest that IFN γ regulates expression of HLA class II antigens in airway epithelial cells of the nasal polyps at the transcriptional level, and that cultured cells from nasal polyps represent a suitable modei to investigate immune mechanisms involved in diseases such as atopy, asthma and cystic fibrosis.

Notes: Skin biopsies were taken from 11 patients with morphoea, nine with acrosclerosis and 10 with diffuse systemic sclerosis and processed for immunohistochemical studies using a panel of monoclonal antibodies including antibodies to MHC class II antigens. A significantly higher percentage of HLA-DR positive dermal cells were observed in the reticular dermis in biopsies from patients with morphoea (44·1 ± 16·2%). acrosclerosis (15·9 ± 5·4%) and systemic sclerosis (39·5 ± 2·3%) when compared with the controls (6·6 ± 2%), A smaller percentage of dermal cells also expressed HLA-DP and -DQ. The degree of mononuclear cell infiltrate in the biopsies, however, did not correlate with the percentage of HLA class II positive fibroblasts. In organ culture, the expression of the HLA class II antigens was almost totally lost after 3 days and was no longer detected on fibroblasts after 3 weeks of culture.

Notes: Molecular genotyping of HLA class II genes is commonly carried out using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or a combination of the PCR and restriction fragment length polymorphism methods (PCR-RFLP). However, the identification of the DQB1 type by PCR-SSO and PCR-RFLP is very time-consuming which is disadvantageous for the typing of cadaveric organ donors. We have developed a DQB1 typing method using PCR in combination with allele-specific amplification (PCR-ASA), which allows the identification of the 17 most frequent alleles in one step using seven amplification mixtures. PCR allele-specific amplification HLA-DQB1 typing is easy to perform, and the results are easy to interpret in routine clinical practice. The PCR-ASA method is therefore better suited to DQB1 typing for organ transplantation than other methods.

Notes: The frequencies of HLA-DQA1, DQB1 and DRB1 alleles were compared between 50 Insulin-Dependent Diabetes Melitus (IDDM) patients and 49 healthy controls in the Greek population. Statistically significant difference in the frequencies of HLA-DQA1*0501-DQB1*0201 (P = 10-4), DQA1*0301-DQB1*0201 (P= 0.01) and DQA P0301-DQB 1*0302 (P= 0.001) were observed. The DRB1*0405-DQA1*0301-DQB 1*0201 was the only DR, DQ combination significantly associated with the disease. The unexpected increase of DRB1*0405 observed in the Greek IDDM may suggest as reported in Chinese and Japanese IDDM a contribution of DRβ and DQα in susceptibility. Moreover, in contrast to the Asians, in the Greek, the DRβ, DQα are found with the usual DQβ 57-ve.