Analysis of microsatellite polymorphism around the HLA-B locus in Iranian patients with Behçet's disease.

We have previously suggested that in a Japanese population the susceptible locus for Behçet's disease (BD) is HLA-B51 itself. To confirm this finding in another population, we performed HLA class I typing using the PCR-SSP method and analyzed eight polymorphic markers distributed within 1100 kb around the HLA-B gene using automated sequencer and subsequent automated fragment detection by fluorescent-based technology with the DNA samples of 84 Iranian patients with BD and 87 healthy ethnically matched controls. As a result, three microsatellite alleles (MICA-A6, MIB-348, C1-4-1-217) and HLA-B51 were found to be strongly associated with BD. Of these alleles HLA-B51 is the most strongly associated allele. There were no alleles that were increased in allele frequency at any microsatellite loci centromeric of MICA or telomeric of HLA-B51. Therefore, HLA-B51 was confirmed to be by far the most strongly associated gene with BD in an Iranian population.

Helicobacter pylori seropositivity in patients with hyperemesis gravidarum.

OBJECTIVES: To test the hypothesis that infection with Helicobacter pylori is associated with hyperemesis gravidarum. METHODS: From November 1999 to February 2001, we enrolled 54 pregnant women with hyperemesis gravidarum and 53 asymptomatic pregnant women in a prospective study. Specific serum immunoglobulin G for Helicobacter pylori was assayed in the sera of the study group and compared with the asymptomatic group. Chi-square and Student's t-test were used accordingly for statistical analysis of the data. RESULTS: Serologically positive Helicobacter pylori infection was detected in 44 out of 54 patients with hyperemesis gravidarum (81.5%) whereas 29 out of 53 asymptomatic gravidas (54.7%) had positive antibody titers for Helicobacter pylori. The ratio of Helicobacter pylori seropositivity in pregnant women with hyperemesis gravidarum was significantly higher than asymptomatic pregnant women (P<0.01). The mean (+/-S.D.) of the IgG titer was 69.7 (+/-77.5) in the hyperemesis gravidarum group and 34.5 (+/-47.8) in the control group (P<0.01). CONCLUSIONS: There is a significant association between Helicobacter pylori infection and hyperemesis gravidarum in our hyperemetic pregnant patients.

HLA class I genotyping including HLA-B*51 allele typing in the Iranian patients with Behçet's disease.

It is well known that Behçet's disease (BD) is strongly associated with human leukocyte antigen (HLA) B51 in many ethnic groups. However, there has been no published report as yet with respect to this association among the Iranian people. Furthermore, since it is now known that the B51 antigen can be encoded by 21 alleles, B*5101-B*5121, we performed HLA-B*51 allele typing as well as HLA class I genotyping of 48 Iranian patients with this disease. As a result, the frequency of the B*51 allele was significantly higher (62.1%) in the patient group as compared with the ethnically matched control group (31.8%) (Pc=0.067, R.R.=3.51). In the genotyping of B*51 alleles, 33 out of the 36 B*51-positive patients possessed B*5101 and the remaining 3 carried B*5108. This study revealed that Iranian patients with BD also had a strong association with HLA-B51. In addition, this significantly high incidence of HLA-B*51 was found to be caused by an increase in both the HLA-B*5101 and HLA-B*5108 alleles. However, there was no significant difference in the HLA-B*51 allelic distribution between the patient and control groups.

Contrasting patterns of polymorphisms at the ABO-secretor gene (FUT2) and plasma alpha(1,3)fucosyltransferase gene (FUT6) in human populations.

The coding sequences ( approximately 1 kb) of FUT2 [ABO-Secretor type alpha(1,2)fucosyltransferase] and of FUT6 [plasma alpha(1,3)fucosyltransferase] were analyzed for allelic polymorphism by direct sequencing in five populations. The nucleotide diversities of FUT2 estimated from pairwise sequence differences were 0.0045, 0.0042, 0.0042, 0.0009, and 0.0008 in Africans, European-Africans, Iranians, Chinese, and Japanese, respectively. The nucleotide diversities of FUT6 were 0.0024, 0.0016, 0.0015, 0.0017, and 0.0020 in Africans, European-Africans, Iranians, Chinese, and Japanese, respectively. At FUT2, excesses in pairwise sequence differences compared to the number of polymorphic sites as indicated by a significantly positive Tajima's D were observed in European-Africans and in Iranians. The data do not fit expectations of the equilibrium neutral model with an infinite number of sites. On the other hand, Tajima's D's at FUT6 in each of the five populations and at FUT2 in Africans, Chinese, and Japanese were not significantly different from zero. F(ST) between the Asians and the others measured at FUT2 was higher than at FUT6. These results suggest that natural selection was responsible for the generation of the FUT2 polymorphism in European-Africans and in Iranians.

Seroepidemiological survey of rubella immunity among three populations in Shiraz, Islamic Republic of Iran.

We used indirect ELISA assay to test 1193 sera for rubella IgG and IgM antibodies in a seroepidemiological survey of three age- and gender-differentiated sample populations in Shiraz: 203 children aged 2-7 years, 255 paired mothers and neonates (cord blood) and 480 women aged 14-70 years. Seropositivity among women aged 14-70 years was 96.2%. No IgM positive case was found among the 255 tested cord blood samples. Seropositivity among the 203 children was 97.0% (much higher than previously reported). This may be due to rubella epidemics, which tend to occur every 6-10 years. The impact of introducing rubella vaccination is discussed.

Seroepidemiological survey of rubella immunity among three populations in Shiraz, Islamic Republic of Iran

We used indirect ELISA assay to test 1193 sera for rubella IgG and IgM antibodies in a seroepidemiological survey of three age- and gender-differentiated sample populations in Shiraz: 203 children aged 2-7 years, 255 paired mothers and neonates [cord blood] and 480 women aged 14-70 years. Seropositivity among women aged 14-70 years was 96.2%. No IgM positive case was found among the 255 tested cord blood samples. Seropositivity among the 203 children was 97.0% [much higher than previously reported]. This may be due to rubella epidemics, which tend to occur every 6-10 years. The impact of introducing rubella vaccination is discussed

IS6110-RFLP and spoligotyping of Mycobacterium tuberculosis isolates in Iran.

To evaluate and compare the usefulness of IS6110-restriction fragment length polymorphism (RFLP) and spoligotyping in the epidemiology of tuberculosis in Iran, Mycobacterium tuberculosis complex strains, isolated in 2 different areas of Iran, were subjected to RFLP and spoligotyping. The average number of IS6110 copies per strain was 11 and ranged from 5 to 18 among the M. tuberculosis strains. In total, among the 62 isolates, 56 different patterns were observed. 50 strains had unique RFLP patterns (89%) and 12 (11%) revealed patterns that were found among at least 1 other isolate. Spoligotyping of 97 isolates resulted in 42 different patterns, of which 72% were found in 15 clusters. 14 (29%) out of 48 investigated isolates were resistant to 1 or more antituberculosis drugs and 57% of the resistant isolates were isolated from Afghan immigrants. Ten percent of the isolates represented the Beijing genotype, including 4 of the 14 (36%) resistant strains. Three of these resistant Beijing strains were isolated from Afghan patients. IS6110-RFLP typing could be useful for studying the epidemiology of tuberculosis in Iran. IS6110 patterns were polymorphic and the average IS6110 copy number was high.

Cross-linking of CD38 molecules induced expression of interleukin-2 receptor (IL-2R) and HLA-DR on human thymocytes.

CD38 is a 45 KD glycoprotein which is mainly expressed on early lymphoid progenitors and plasma cells. Role and function of CD38 on thymocytes and plasma cells has not yet been elucidated. By cross-linking of CD38 molecules using a specific monoclonal antibody on thymocytes and a neoplastic T cell line (Jurkat), it was shown that percentage of HLA-DR and IL-2R molecules is significantly upregulated. Moreover, CD11a and CD18 were among two adhesion molecules which showed a sharp increase in expression, whereas no changes on expression of CD11b, CD44 and CD54 were detected. Spontaneous proliferation of Jurkat cell line after addition of different concentrations of CD38 monoclonal antibody was unchanged.

Cross-linking of a sequential epitope within the beta-chain of HLA-DR/DP molecules suppressing B lymphocyte growth and inducing homotypic cell aggregation.

A monoclonal antibody (SU2) directed against HLA class II antigens has been produced by immunizing BALB/c mice with a lymphoblastoid cell line, RPMI-8866 cells. The specificity of this mAb has been determined using a panel of HLA class II transfectants. SU2 stained all HLA-DR and HLA-DP transfectants tested, but reacted with only one HLA-DQ (HLA-DQw2). From comparison of the available data sequences of known HLA class II molecules, it appeared that the epitope recognized by the SU2 mAb contains a sequence of 6 amino acids (DSDVGE) at position 41-46 of the beta-chain of HLA-DR/DP. Functional studies indicated that SU2 mAb strongly induced cell aggregation and large clumping in resting tonsillar B cells. SU2 mAb inhibited the spontaneous growth and proliferation of B lymphoblastoid cell lines and drastically inhibited [3H]thymidine uptake by phorbol 12-myristate 13-acetate (PMA)-activated resting B cell. Results are discussed in relation to the dual recognition of HLA-DR/DP by SU2 mAb.

Epitope mapping of the site(s) of binding of Fc epsilon RII/CD23 within human IgE. Determination of the B lymphocyte-binding sites by use of synthetic peptides and anti-peptide antibodies representative of linear Fc sequences.

The work undertaken has investigated the structure-function relationship between IgE and its low affinity receptor on B lymphocytes. To identify sites on the IgE molecule which interact with the low affinity receptor (Fc epsilon RII/CD23), 10 different peptide sequences within the CH2, CH3 and CH4 domains of human IgE were selected according to charge, overall hydrophobicity and possible accessibility on native IgE sequences. Peptides representative of these were synthesized by the solid phase procedure; and their cytophilic activities were examined by determining their capacity to inhibit the binding of radiolabelled or erythrocyte-bound IgE to a Fc epsilon RII/CD23 positive B cell line (RPMI-8866). Moreover, these linear sequences were rendered immunogenic by conjugation to a protein carrier (KLH) and used to produced polyclonal antibodies in rabbits. The reactivity of the anti-peptide antibodies with both free peptides and native IgE bound to a solid phase, as well as their capacity to inhibit binding of IgE to a Fc epsilon RII/CD23 positive cell line, were investigated. Results from such use of peptides and anti-peptide antibodies indicate that two sequences, representative of residues 364-383 and 401-415, could be involved in the binding of IgE to both membrane-bound and soluble form Fc epsilon RII/CD23; indicating that the B lymphocyte-binding site on human IgE may be restricted to the CH3 domain.

Production and characterization of two murine monoclonal antibodies directed against epitopes exclusive to soluble fragments of Fc epsilon RII/CD23.

Two murine monoclonal antibodies (mAb) designated as SU1 and SU3 directed against soluble Fc epsilon RII/CD23 have been generated by fusing X.63.AG.8653 (a mouse myeloma cell line) with spleen cells from mice immunized with an Epstein Barr virus (EBV)-transformed B cell line (RPMI-8866). The antibodies have been shown to be capable of detecting affinity purified soluble Fc epsilon RII/CD23 in an enzyme-linked immunosorbent assay. Indirect immunofluorescence has shown that the SU1 and SU3 mAb do not stain RPMI-8866, a Fc epsilon RII/CD23+ B cell line. By studying the migration profiles of affinity purified SU1- and SU3-reactive molecules on sodium dodecylsulfate-polyacrylamide gel electrophoresis it has been shown that SU1 mAb immunoprecipitates 33- and 12-kDa components, while the SU3 mAb recognized 25- and 45-kDa proteins from culture supernatants of RPMI-8866 cells. Moreover, affinity purified SU1- and SU3-reactive proteins have been shown to be recognized by human IgE but not by the human IgG molecule. These results provide evidence that SU1 and SU3 mAb may recognize some putative post-cleavage epitopes on the N-terminal end of the low affinity receptor which appear, perhaps, following the process of fragmentation. In addition, the effect of these antibodies on continuous growth of a panel of lymphoblastoid cell lines indicates that SU1 mAb was found incapable of influencing the spontaneous proliferation of EBV-immortalized B cell lines; whereas SU3 mAb completely blocked the spontaneous growth and proliferation of all B cell lines tested. The results are discussed in relation to the appearance of a functional post-cleavage epitope on soluble Fc epsilon RII/CD23.

Affinity-purified soluble Fc epsilon RII/CD23 derived from a culture supernatant of an EBV-immortalized B-cell line induced a monophasic fever in rabbits.

A soluble form of Fc epsilon RII/CD23 is spontaneously released from most lymphoblastoid cell lines established by the Epstein-Barr virus (EBV). Such a product was purified on an IgE-Sepharose column and its pyrogenic effect was investigated in rabbits. This preparation induced a monophasic fever in rabbits, with a peak response appearing 75 min after injection. Since IgE was found to be capable of abrogating such an effect, it is suggested that IgE might be involved in the control of the effectiveness of this soluble protein.

Affinity-purified soluble Fc epsilon RII/CD23 derived from RPMI-8866 cells induces histamine release from human nasal polyp mast cells through a non-IgE-mediated mechanism.

Soluble Fc epsilon RII/CD23 (IgE-binding factor) is released spontaneously from activated B cells and most EBV-immortalised B cell lines. We have purified soluble Fc epsilon RII/CD23 from culture supernatants of RPMI-8866 cells on an IgE Sepharose column, and studied its ability to release histamine from human nasal polyp mast cells. Soluble Fc epsilon RII/CD23 induces release of a significant amount of histamine from nasal polyp mast cells in a dose-dependent manner. IgE, and a monoclonal antibody specific for the soluble form of this receptor, were shown to neutralise this effect. It was found that soluble Fc epsilon RII/CD23 was still capable of triggering histamine release from nasal polyp mast cells from which IgE had been eluted by incubation in a low pH buffer, suggesting that a non-IgE mediated mechanism was responsible for this effect.

Functional implication for the topographical relationship between MHC class II and the low-affinity IgE receptor: occupancy of CD23 prevents B lymphocytes from stimulating allogeneic mixed lymphocyte responses.

Following the observation of Bonnefoy et al. (J. Exp. Med. 1988. 167:57), that the low-affinity IgE receptor (CD23) on B lymphocytes can be coupled (with the use of chemical cross-linking reagents) to major histocompatibility complex (MHC) class II DR molecules, we now report that ligands binding within the lectin-homology region of CD23 prevent B cells from stimulating allogeneic mixed lymphocyte responses. Ligands capable of blocking mixed lymphocyte responses include the anti-CD23 antibodies MHM6 and EBVCS 4 but not EBVCS 1 and 5. IgE itself, and small peptides representing sequences within the CH3 domain of IgE. The detailed topographical relationship between CD23 and MHC class II on the B lymphocyte surface was examined using dual immuno-fluorescence labeling of cells and direct visualization of the staining by confocal laser scanning microscopy. On transformed B lymphoblasts, the two antigens were seen to co-localize in discrete patches; on normal B cells which had been cultured for 2 days with interleukin 4, CD23 and MHC class II converged at a single pole which exhibited a tendency to pseudopod formation and provided a focus for homotypic cell-cell interactions. The possibility that CD23 could serve as a co-stimulatory-adhesion molecule in antigen presentation by B lymphocytes is discussed with special reference to a potential role in the regulation of IgE synthesis.

Stimulation of B-chronic lymphocytic leukemia populations by recombinant interleukin-4 and other defined growth-promoting agents.

Monoclonal populations from 10 cases of phenotypically well-characterized B-chronic lymphocytic leukemia (B-CLL) and from a single case of hairy cell leukemia were assessed for their ability to respond by mitogenic stimulation to a number of agents described as growth-promoting for normal B cells. These included the recombinant factors interleukin-1 (IL1), IL2, IL4, IL5, and gamma-interferon, partially purified B cell growth factor (BCGF), B cell stimulatory factor 2 (BSF2), and a CDw40 antibody to the Bp50 antigen. With only few exceptions, no factor or combination of factors stimulated B-CLL populations directly to DNA synthesis. By marked contrast, the hairy cells were responsive to IL4, BCGF, and the CDw40 antibody. B-CLL cells could become responsive with the inclusion of the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) as co-stimulant such that half of the populations were now activated by IL4, particularly when BCGF was also present. Populations refractory to IL4 were, nonetheless, still responsive to BCGF. In only three cases was a significant effect seen with IL2. gamma-interferon could be either inhibitory or stimulatory and, in a few cases, modulated specifically the effects of IL4. In contrast to normal B cell activations, neither the CDw40 antibody nor a calcium ionophore synergized with TPA for stimulating the majority of B-CLL populations. BSF2 was stimulatory in the two cases examined while both IL1 and IL5 were ineffective where studied. No simple correlation was observed between the patterns of responsiveness and the expression of a panel of CD markers assayed on cells both freshly isolated and after TPA stimulation. The data demonstrate a functional heterogeneity not disclosed by simple phenotypic analysis and also indicate the range of activities which can impinge on the growth regulation of monoclonal B cell populations.