Non-classic sHLA class I in human oocyte culture medium.

Soluble human leukocyte antigen (sHLA) class I molecules have been described in all human fluids. These molecules play a significant role in immune function. sHLA has been shown to produce tolerance and to induce apoptosis in cytotoxic alloreactive T cells. They are also present in the supernatant of many cultured cells. Similarly, non-classic HLA class I antigens in soluble form are present in human fluids. Among these, HLA-G is the most important because of its location in fetal tissue that suggests maternal immunological tolerance of the fetal semiallograft. In our present study we show that using two monoclonal antibodies, w6/32 and TP25.99, in the enzyme-linked immunosorbent assay allows the detection of non-classic sHLA class I molecules in the medium from human embryo cultures. The sample were collected from oocytes cultures. Oocyte donors were 11 women attending the in vitro fertilization program. The results showed a significant association (chi2 = 9.66, p = 0.002) between sHLA antigens and the oocyte cleavage rate measured 48 h after fertilization.

A method for point mutation analysis that links SSCP and dye primer fluorescent sequencing.

A method is presented for mutation detection directly from single-strand conformational polymorphism (SSCP) variants. This approach is based on: (i) amplification of the exons to be analysed by SSCP using the forward primer with an eight-base tail to form a universal SSCP cassette; (ii) excision from the gel of the shifted silver-stained bands; (iii) reamplification of the eluted DNAs using, as the forward primer, a 26-base universal adaptor primer corresponding to the 18-base-21M13 sequence plus the eight nucleotides of the universal SSCP cassette; and (iv) direct sequencing of the purified products using the standard-21M13 fluorescent primer. This procedure presents several advantages including the avoidance of a cloning step which leads to significant time reduction, while maintaining comparable accuracy at relatively low costs. In conclusion, the presence of the universal SSCP cassette and subsequent reamplification with the same adaptor primer for direct sequencing makes the method universal for scanning and identification of gene alterations.

Immunohistochemical vs molecular biology methods. Complementary techniques for effective screening of p53 alterations in head and neck cancer.

The purpose of this study was to correlate p53 gene alterations and their expression in 85 head and neck squamous cell carcinomas. Genomic p53 was amplified with the polymerase chain reaction (PCR) from formalin-fixed, paraffin-embedded tissues. Exons 5 through 8 were screened for mutations by means of non-isotopic single-strand conformation polymorphism (SSCP) analysis. p53 expression was detected by means of immunohistochemistry (IHC) with the p53 monoclonal antibody DO-7. Twenty-three lesions (27%) showed both SSCP variants and DO-7 immunostaining, whereas 37 (44%) demonstrated both SSCP normal patterns and negative staining. Twenty-five lesions (29%) were discordant, including 12 IHC-positive (14%) and 13 SSCP-positive (15%) lesions. However, discordant IHC-negative, SSCP-positive lesions could have been easily interpreted after sequencing of the abnormal samples. Had these been screened with IHC only, all nonsense or frameshift p53 mutations would have been missed. IHC-positive, SSCP-negative lesions were interpreted in light of current models of p53 immunodetection. Had these been screened with SSCP or sequencing only, a sizeable number of tumors expressing p53 protein abnormalities would have been undetected. Therefore, simultaneous use of both methods increases the number of p53 abnormalities detected, and is warranted.

HLA-B44 subtypes and the chance of finding HLA compatible donor/recipient pairs for bone marrow transplantation: a haplotype study of 303 Italian families.

A total of 1176 HLA-A,B,DR haplotypes were reconstructed by typing 303 unrelated families referred to our laboratory during the last seven years for the search of HLA identical sibs in view of bone marrow transplantation. A total of 614 different three-locus haplotypes were found. Most of them (83.6%) were present only once or twice, whereas 24/614 (3.9%) were found 6-28 times each. HLA-B44 was present in 4 of these most frequent haplotypes. HLA-B44 has been implicated as the molecular target for bone marrow allograft rejection. Therefore, a better knowledge of the HLA-B44 haplotype relationships might prove useful for the programming of registries of unrelated bone marrow donors. Eighty five serologically defined HLA-B44 unrelated subjects, either one or both parents from the above families, were subtyped by a high-resolution sequence-specific oligonucleotide probing approach. Moreover, 34 unrelated potential donors recruited for those patients that did not find a suitable donor among their siblings were subtyped also for HLA-B44. B*4403, which accounted for 47/85 (55.3%) serologically defined B44 alleles, appeared in strong, statistically significant, linkage disequilibrium with HLA-A29, -A23 and -DR7. On the other hand, B*4402, which covered virtually all other B44 alleles, showed prevalent gametic associations with HLA-A2 and HLA-A24. The linkage disequilibrium between HLA alleles is the key for the low frequency of HLA-B44 mismatches in donors selected as HLA-A,B,DRB1 identical to patients waiting for unrelated bone marrow transplantation. If a given patient presents unusual haplotypes, the chance of finding HLA-B44 mismatches may be higher because of the presence of different haplotype relationships in the donors.

Genotype-related fingerprints from HLA-DPB1 exon 2 low-stringency PCR.

Techniques based on the formation of heteroduplexes between relevant heterologous amplified HLA loci have proved to be simple and cost-effective screening methods for the detection of DNA sequence diversity. However, the banding patterns produced may not be as complex as required. We used the original procedure of Pena et al. (Proceedings of the National Academy of Sciences USA 91, 1946-1949, 1994) to generate fingerprints from a specific, polymorphic PCR product. HLA-DPB1 exon 2 was amplified, recovered from agarose gel, and used as a template for subsequent low-stringency (30 degrees C) amplification cycles (LS-PCR) in the presence of a single primer. The LS-PCR products were run on 8% PAGE and silver-stained. In total, 22 subjects were characterized by this method. The issues of the reproducibility and specificity of the patterns obtained were addressed by comparing fingerprints from individuals with the same genotype. The results showed that LS-PCR was robust. A further step was the evaluation of the diversity that can be generated, i.e. the sensitivity of the method. Genotype-related fingerprints were produced, and differences as small as a single nucleotide in heterozygous samples could be detected. We then demonstrated the usefulness of LS-PCR in the evaluation of donor/recipient pairs. We believe that LS-PCR may be a valuable adjunct to the battery of tests aimed at the verification of HLA matching before unrelated bone marrow transplantation. We suggest that it could be used to speed up the search process when several candidate donors are retrieved from registries before embarking on SSOP typing or sequencing.

HLA antigens and multiple sclerosis in the Province of Ferrara, northern-Italy: a community-based study.

The association between Multiple Sclerosis (MS) and DR2 HLA antigen is well known in Caucasoids. In the past few years a significant correlation has been found between DQwl and MS in North-East Scotland and in several other countries. In previous HLA studies in Italian MS patients, a lack of any association or an increased frequency of DR2 have been observed; however, the results of most Italian surveys derive from heterogeneous sample of affected individuals. This study was carried out in a homogeneous population of patients living in and originating from the province of Ferrara, Northern-Italy. Among the prevalence cases, 116, indigenous, unrelated patients, were typed for HLA-A-B, -DR and DQ antigens. The comparisons with 185 healthy individuals, originating from the same area, revealed an increased prevalence of DR2 antigen in Ferrara MS patients. This antigen does not appear to be related to the clinical variables of the disease.

Behcet's disease associated with HLA-B51 and DRw52 antigens in Italians.

Thirty-eight Italian patients with Behcet's disease, all with ocular involvement, (28 complete type and ten incomplete) were typed for HLA A,B,DR, and DQ antigens. A significant increase of HLA-B51 (p less than 0.00001) and DRw52 (p = 0.045) with no significant difference between complete and incomplete syndrome was found. The involvement of B51 antigen as the main immunogenetic factor in the disease is suggested by the high value of relative risk (RR = 16.03). However, the association with the II class antigen DRw52 (RR = 2.77) cannot be easily explained as a secondary association due to linkage disequilibria with B51.

Aetiological factors in hypospadias.

Epidemiological and genetic variables in hypospadias were analysed during the years 1978 to 1983 in a case control study of congenital malformations in the Emilia Romagna region of northern Italy. During the observation period, in a sample of 41 078 male newborns, 168 had hypospadias giving a prevalence at birth of 4.1 in 1000 males. Hypospadias was divided into three types: type I or mild (75.0%); type II or moderate (21.4%); and type III or severe (3.6%). Coexisting malformations were found in 8.9% of cases. The heritability coefficient was 0.669. Maternal risk factors correlated with hypospadias were found to be early age at menarche, threatened abortion, and exposure to progestins. Low birth weight and shorter gestation were also correlated with hypospadias.

A monoclonal antibody (GF 22.1) detecting different patterns of HLA class I cross-reactivities at different dilutions.

A murine cytotoxic monoclonal antibody (GF 22.1) was produced by Balb/c immunization with GRA human lymphoblastoid cells. HLA-A, B and C blanketing and SDS-PAGE analysis of the immunoprecipitate demonstrated MHC class I structures as the targets. Cytotoxic assays were performed with peripheral blood lymphocytes from 84 unrelated donors and from members of 15 families at different antibody dilutions. Statistical analysis was performed by Fisher test on each dilution separately and by Mann-Whitney U test on the dilutions taken all together. The data suggest the detection of a cross-reacting group (B15, A32, B17, B40/w41 and B21) with high affinity and the inclusion of other antigens (B12, B35, A2, B13, A11 and A24) with a lower affinity.

A monoclonal antibody to human transitional-cell carcinoma of the bladder cross-reacting with a differentiation antigen of neutrophilic lineage.

A monoclonal antibody (MAb) to human transitional-cell carcinoma of the bladder (TCCB) was obtained by immunization of a BALB/c mouse with formalin-fixed TCCB cells and subsequent fusion of the spleen cells with SP2-OAg14 myeloma line. GF 26.7.3 MAb was selected by indirect immunofluorescence (IIF) as reacting agent with target cells and negative with autologous lymphocytes and Epstein-Barr virus (EBV)-transformed lymphoblastoid cell-line. GF 26.7.3 reacts with a high percentage of bladder and colon carcinomas when examined by IIF and immunoperoxidase techniques and cross-reacts with a determinant expressed on neutrophilic cell lineage. The IIF analysis performed on bone marrow and peripheral blood (PB) from healthy subjects and leukemic patients and on leukemic cell lines showed that the expression of the structure detected by GF 26.7.3 is restricted to the neutrophilic cell lineage and first expressed at the promyelocytic level. Immunoprecipitation and SDS-polyacrylamide gel electrophoresis (PAGE) of 125 I-labelled membrane proteins from target cells were performed, but no bands were detected by autoradiography. In addition, pronase insensitivity and periodate sensitivity suggest the possible involvement of a carbohydrate determinant.

Impaired HLA capping capacity of peripheral blood lymphocytes in Duchenne muscular dystrophy.

The cap capacity in nine Duchenne muscular dystrophy (DMD) patients and in 23 healthy male subjects (14 adults and nine neonates) has been investigated by inducing capping of HLA molecules. The evaluation of capping percentages ranged in healthy controls from 44 to 61 with a mean value of 53.39 +/- 4.89, while DMD patients displayed cap capacity of percentages varying from 15 to 38 with a mean value of 31.5 +/- 7.42. Statistical significance of the differences between the two groups, assessed by the Mann-Whitney U test, was p less than 0.00003. A correlation was found in DMD patients between cap capacity and age (tau = +0.657, p = 0.012). The results confirm previous reports of Ig capping impairment noted in B cells of the whole lymphocyte population, supporting the hypothesis of a systemic cellular defect in DMD patients. The data obtained suggest that HLA capping could overcome some of the technical difficulties of Ig capping and could be used as a diagnostic aid in antenatal detection of DMD.

[Occurrence of HLA B27 antigen in ankylosing spondylitis in Italy]. / Contribution à l'étude de la fréquence de l"antigène HLA B27 dans la spondylite ankylosante en Italie.

39 individuals from Middle and South Italy were selected for being affected by ankylosing spondylitis (AS) (N = 22) or by sacroiliitis (SI) (N = 17) and typed for the HLA system antigens. 320 healthy individuals were typed as control population. The following phenotypic frequencies of HLA-B27 were found; 68% in AS patients, 71% in SI patients and 5% in controls. The frequency of B27 observed among our AS patients is lower than those observed in other caucasian patients. Our data may be explained either by a genetic hypothesis or by an environmental hypothesis; data reported by other authors let we believe that the last one is more reliable.

Maternal lymphocytotoxic antibodies and bilirubin levels in the postpartum offspring.

HLA antigens are present on immature red cells (Harris & Zervas 1969), but disappear from their surface when they become mature and enter into the circulation (Silvestre et al. 1970). Nevertheless some remnants of HLA substance may survive this antigenic switch and possibly persist on adult red cells (Morton et al. 1969, 1971, Doughty et al. 1973). A shortened survival of HLA incompatible reticulocytes has been reported when they are transfused into recipients with circulating anti-HLA antibodies (Zervas et al. 1972). As to anti-HLA antibodies possibly reacting with erythrocytes in cases of fetomaternal immunization, Moulinier (1970) claimed that they would potentiate any hemolytic process in newborns of Rh-immunized women, but successive investigations failed to confirm this (Ahrons & Glavind-Kristensen 1971, Nymand 1975). Also, Nymand (1975) found no association between lymphocytotoxic antibodies in mothers and high bilirubin levels in the umbilical cord blood collected at delivery. On the contrary, when the onset of neonatal jaundice in the postpartum offspring was monitored, newborns of mothers with lymphocytotoxins appeared to become jaundiced more frequently than expected (Reekers et al. 1975). The present study has been undertaken to investigate the relationships between complement dependent lymphocytotoxins (CdL) in the mothers and the bilirubin levels in the newborns during the first days of life.