Study of induction of activation of human peripheral blood mononuclear cells with a non-activating form of anti-CD3 MoAb in autoimmune thyroid disease (AITD).

Anti-CD3 (OKT3) MoAb is a mitogenic agent which activates lymphocytes. We have studied the effects of murine anti-human OKT3 MoAb (IgG1) alone or in combination with IL-2, human thyroglobulin (Tg) and thyroperoxidase (TPO) antigens on the proliferation of whole peripheral blood mononuclear cells (PBMC) (including monocytes) or subtypes (T, CD4+, CD8+, B) as measured by tritiated thymidine (3H-TdR) incorporation. B cell differentiation was studied by measuring numbers of IgG-secreting cells and specific anti-TPO/anti-Tg-secreting cells by SPOT ELISA. PBMC or lymphocyte subtypes, obtained from 45 patients with Hashimoto's thyroiditis (HT), 40 Graves' disease (GD) and 51 normal controls were cultured in 96 microtitre plates for 6 days in the presence of OKT3 MoAb at final concentrations 25-250 ng/ml, IL-2 15 U/ml, Tg and TPO (1 micrograms/ml). Then cultures were pulsed with 0.2 microCi 3H-TdR/well and incorporation was measured after 18 h. IgG and anti-TPO/Tg-secreting cells were detected at 7 days. Higher proliferative responses from whole PBMC preparations in response to any of the combinations including OKT3 MoAb were observed in the HT preparations, while the basal values were the lowest. IL-2 alone increased these responses markedly, but equally in all groups. IL-2 in combination with OKT3 had an additive effect on proliferation, with higher responses in HT. Tg and TPO antigens did not change these responses. Most HT preparations responded with their maximum proliferation to the lowest concentration of OKT3 MoAb (25 ng/ml), whereas in GD and control preparations of PBMC these responses were shifted to higher concentrations (250 ng/ml); even with those, proliferation was not so enhanced in controls when compared with HT and GD preparations. In contrast, the proliferative responses of T cells alone and subpopulations of CD8+ suppressor/cytotoxic cells were decreased in HT preparations compared with controls. Monocytes were necessary for proliferation. In the subpopulation of B cells (> 95% pure) and CD4+ helper/inducer cells, differences did not reach significance. In spite of the effect on proliferation, OKT3 MoAb only mildly but significantly increased the numbers of IgG-secreting cells in HT and GD preparations and did not stimulate synthesis of specific antibodies. Our data suggest that the increased proliferative responses of whole PBMC to OKT3 MoAb in HT preparations might be due to insufficient activation of T suppressor/cytotoxic cells.     

Protection from insulin-dependent diabetes mellitus is linked to a peptide transporter gene

HLA class II association with insulin-dependent diabetes mellitus (IDDM) is well established but is still difficult to map to a particular locus. Polymorphism of the genes coding for transporter associated with antigen processing (TAP1 and TAP2), and located in the HLA class II region, was studied in 167 IDDM patients (116 adult-onset and 51 childhood-onset patients) and 98 normal controls using oligotyping after genomic amplification. A dominant protective effect was observed for theTAP2*0201 allele [relative risk (RR)=0.3, corrected probability (pc) < 0.001]. Conversely, susceptibility to IDDM was associated with apparent homozygosity for the TAP2*0101 allele (RR=3.4, pc < 0.001). Protection was independent from but additive to the protection conferred by the DRB1*02 DQB1*0602 haplotype (RR=0.06, pc<0.05), and antagonistic to the DRB1*03 DQB1*0201 and DRB1*04 DQB 1*0302 haplotypes predisposing effect (RR=1.1, not significant), arguing in favor of an absence of linkage disequilibrium between TAP2 and HLA class II genes. This was assessed by x² analysis. TAP1 allelic distribution was not different among diabetics and controls. A significant association was observed between the presence of TAP2*0101 and that of islet cell antibodies (p < 0.05). These data suggest that the TAP2 gene, which encodes protein required for delivery of antigen peptides to class I molecules in the endoplasmic reticulum, could modulate the autoimmune response leading to β cell destruction. From a practical point of view, they make the combined screening of HLA class II and TAP2 loci a highly valuable tool in IDDM prediction.     

Purification of bovine conglutinin using pepsin digestion

This paper describes a new method for the purification of bovine conglutinin based on the relative resistance of this protein to pepsin digestion. First, conglutinin is purified by absorption on yeast, then the preparation is treated with 2% pepsin (w/w) at 4°C for 18 hr, and finally gel filtrated on agarose A5m. The yield is 60–75% and conglutinin thus prepared appears physically, immunochemically and functionally intact. This procedure allows for a rapid production of sufficient amounts of conglutinin for immune complex detection or purification methods.     

Biological properties associated with the enhanced lung-colonizing potential in a B16 murine melanoma line grown in a medium conditioned by syngeneic Corynebacterium parvum-elicited macrophages

A previous study by our laboratory showed that the peritoneal murine Corynebacterium parvum-elicited macrophages released into their growth medium an activity which enhanced the ability of B16-F10 melanoma cells to form experimental metastases in the lung of syngeneic mice. In the present study, we used a clone of B16-F10 line (F10-M3 cells) to investigate whether the increase in lung-colonizing potential due to the pro-clonogenic activity released by C. parvum-elicited macrophages was associated with biological properties characteristic of a metastatic phenotype. We have found that the pulmonary retention, growth rate in lung parenchyma, invasiveness through Matrigel, adhesiveness to IL-1-activated endothelium and MHC class I expression were increased in F10-M3 cells stimulated by the macrophage pro-clonogenic activity. By using an in vitro experimental protocol, the enhancement of lung-colonizing potential in the stimulated melanoma cells turned out to be a transient phenomenon as was the increase of invasiveness through Matrigel and the higher expression of MHC class I antigens. In conclusion, the melanoma cells stimulated by the pro-clonogenic activity released by C. parvum-elicited macrophages showed changes in biological parameters which are relevant to metastatic diffusion. These changes appeared as a temporary phenomenon which sustains the view that the metastatic phenotype represents a transient biological character influenced by host factors. This revised version was published online in July 2006 with corrections to the Cover Date.     

The expression of γΔ T cell receptor and the prevalence of primed,activated and IgA-bound T cells in Behçet's syndrome

Mucosal ulceration of the oral, and to a lesser extent genital tissues is an essential feature of Behçet''s syndrome and is associated with changes in the IgA class of immune responses. Indeed, a significant increase in the proportion of cytophilic IgA1 was found in circulating CD8 and CD4 cells (P less than 0.01), with a corresponding decrease in IgA-Fc receptors on these T cells. Furthermore, 30-40% of the cytophilic IgA1 on T cells may have been of the polymeric secretory type and the rest of the monomeric variety. IgA isotype of B cells was also significantly increased (P less than 0.001), without an overall change in circulating B cells. However, a surprising finding was the significant up-regulation of gamma delta T cell receptor in the CD8 (P less than 0.01) in the absence of a change in the proportion of alpha beta T cell receptor. The results suggest that some common microbial antigen might initiate at the mucosal surface an immune defence reaction characterized by T cells with gamma delta receptors and IgA-specific B cells. However, IgA1 bound to circulating T cells may down-regulate the central T cell function.     

Identification of an obeche (Triplochiton scleroxylon) wood allergen as a class I chitinase

BACKGROUND: Wood dust is known to cause allergic occupational asthma and obeche (Triplochiton scleroxylon) is a prominent exponent in this field. However, the knowledge about wood allergens is still limited. The aim of this study was to identify and characterize obeche wood allergens. METHODS: Obeche extracts were prepared from freshly ground in comparison to 7 years stored wood dust and investigated by Sodium dodecyl sulfate-polyacrylamid gel electrophoresis, enzyme-linked allergosorbent test and immunoglobulin (Ig)E-immunoblot. Allergens were detected by specific IgE of seven obeche allergic patients' sera and protein analysis was performed by mass spectrometry. Cross-reactivity was demonstrated by ImmunoCAP-inhibition with sera of seven obeche and four latex-allergic patients. RESULTS: Obeche extracts showed different protein pattern and IgE-binding capacities depend on the age of the wood dust. A 38 kDa protein was identified as major obeche wood allergen, detected by six of seven (85%) obeche allergic patients' sera and was entitled as Trip s 1. Trip s 1 is homologous to plant class I chitinases and exhibited enzyme activity demonstrated by chitinolysis. Co-recognition or cross-reactivity of Trip s 1 according to structural similarity was seen in sera of latex allergic patients. IgE inhibition studies with obeche as solid phase and Trip s 1 and latex hevein as inhibitor demonstrated that Trip s 1 was a more effective inhibitor in obeche as well as in latex allergic patients' sera. CONCLUSIONS: Trip s 1 is a new obeche wood allergen of the plant class I chitinase family. This finding may explain the dominant role of obeche in sensitization against wood dust.     

A promiscuous T cell hybridoma restricted to various I-A molecules

In a previous study, we identified T cell receptor and major histocompatibility complex (MHC) contact sites on the pigeon cytochrome c p43-58 peptide. Positions 46 and 54 of p43-58 were shown to be the MHC-binding sites. Specific amino acids were identified on the MHC-binding sites which bound to the relevant I-A molecule. In the present study, using NOD (I-A^g7) mice, we established a T cell hybridoma, NOE33-1-2, specific for a p43-58 analog 46R50E54A with arginine (R) and alanine (A) at positions 46 and 54, respectively. Interestingly, NOE 33-1-2 recognized 46R50E54A in the presence of not only I-A^g7, but also I-A^{d, s, u and v}. In contrast to previous reports that promiscuous T cells were able to recognize peptide antigens with various HLA-DR or I-E molecules consist of monomorphic α and polymorphic β chains, the promiscuous T cell clone NOE33-1-2 recognized peptides with various I-A molecules lacking the monomorphic chain.     

Molecular defects in the beta-globin gene identified in different ethnic groups/populations during prenatal diagnosis for beta-thalassemia: a Malaysian experience

Abstract   β-thalassemia is the most-common genetic disorder of hemoglobin synthesis in Malaysia, and about 4.5% of the population are heterozygous carriers of the disorder. Prenatal diagnosis was performed for 96 couples using the Amplification Refractory Mutation System and Gap-Polymerase Chain Reaction. We identified 17 β-globin defects-initiation codon for translation (T-G), -29 (A-G), -28 (A-G), CAP +1 (A-C), CD 8/9 (+G), CD 15 (G-A), CD 17 (A-T), CD 19 (A-G), Hb E (G-A), IVS1-1 (G-T), IVS1-5 (G-C), CD 41/42 (-CTTT), CD 71–72 (+A), IVS2-654 (CT), poly A(A-G), 100-kb ^Gγ(^Aγδβ)° and 45-kb Filipino deletions. The 192 β-alleles studied comprised Chinese (151 patients), Malay (21), Orang Asli from East Malaysia (15), Filipino (1), Indian (1), Indonesian Chinese (2), and Thai (1). In the Chinese, 2 β-globin defects at CD 41/42 and IVS2-654 were responsible for 74% of β-thalassemia. β-mutations at CD 19, IVS1-1 (G-T), IVS1-5, poly A, and hemoglobin E caused 76% of the hemoglobin disorders in the Malays. The Filipino 45-kb deletion caused 73.3% of bthalassemia in the Orang Asli. Using genomic sequencing, the rare Chinese β-mutation at CD 43 (G-T) was confirmed in 2 Chinese, and the Mediterranean mutation IVS1-1 (G-A) was observed in a Malay β-thalassemia carrier. The β-globin mutations confirmed in this prenatal diagnosis study were heterogenous and 65 (68%) couples showed a different globin defect from each other. The use of specific molecular protocols has allowed rapid and successful prenatal diagnosis of β-thalassemia in Malaysia.     

Inhibition of nitric oxide synthase delays the development of tolerance to LPS in rats

The role of nitric oxide (NO) was investigated in endotoxin (lipopolysaccharide, LPS) tolerance in freely moving biotelemetered rats. We monitored changes in febrile response and feeding behavior (food intake, water intake) during the development of tolerance to repeated intraperitoneal injections of LPS (50 microg/kg) along with injections of N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg/kg), an inhibitor of NO synthase. Rats were treated with LPS and L-NAME for three consecutive days. On the fourth day, all rats were injected with LPS alone. Control rats were injected with saline along with saline or with L-NAME for four consecutive days. Rats repeatedly injected with LPS became tolerant to pyrogenic and hypophagic/cachexic effects of LPS as early as on the second day of experiment. The treatment with L-NAME prevented the attenuation of febrile response following the second LPS injection. Moreover, the depressive effects of LPS on body weight as well as on water and food intake were prolonged in rats treated with a combination of L-NAME and LPS. Injection of LPS caused a 3.5-fold increase in plasma nitrite within 3 h and nitrite levels remained significantly elevated 6 and 24 h after LPS. Rats injected secondly with LPS did have still 2.5- to 3-fold increase in plasma nitrite levels 3 and 6 h, but not 24 h, after injection. Third injection of LPS did not elevate nitrite level in plasma. Taken together, presented data provide clear evidence that NO formation is involved in mechanisms responsible for development of early-stage tolerance to endotoxin.     

DQCAR 113 and DQCAR 115 in combination with HLA-DRB1 alleles are significant markers of susceptibility to rheumatoid arthritis in the Korean population

We have investigated HLA region microsatellite polymorphisms in rheumatoid arthritis (RA) which are known to be associated with HLA class II alleles in the Korean population. Ninety patients with RA and 106 controls were employed for this study, in which TAP1CA, DQCAR, D6S273, HLA-DRB1, -DQA1 and -DQB1 allele typing were performed. DQCAR 113 (RR = 3.2, P<0.0002), DQCAR 115 (RR = 3.6, P<0.0001) and heterozygous DQCAR 113/115 (RR = 11.2, P<0.0001) frequencies were significantly increased in the RA group compared with the control group. The HLA-DRB1 genotypes of patients who had DQCAR 113/115 alleles were defined as DRB1*04 and/or DRB1*09. There was no significant difference between RA and controls in D6S273 and TAP1CA allele frequencies. We demonstrated that HLA-DRB1*0405 (RR = 6.6, P<10(-6)), DQA1*03 (RR = 5.2, P<10(-6)), DQB1*04 (RR = 3.5, P<0.002) alleles were useful markers of susceptibility to RA in Koreans. The frequency of HLA-DRB1*0405 was higher in DQCAR 113 allele-positive RA (68.1%) than in DQCAR 113 allele-negative (16.3%) and total RA (43.3%) groups, and the susceptibility risk of DQCAR 113 allele to RA was more increased in the DRB1*0405-positive group (RR = 5.5, P<0.04). On the other hand, DQCAR 115 allele was more significantly associated with susceptibility to RA in HLA-DRB1*0405-negative patients (RR = 5.1, P<0.0005), and the association between RA and HLA-DRB1*0405 was also significantly associated with DQCAR 115 allele-negative patients (RR = 13.2, P<0.00001) as compared with DQCAR 115 allele-negative control groups. HLA-DRB1*0405-DQA1*03-DQCAR113-DQB1*03 haplotype showed high relative risk value (RR= 17.7, P<0.0002). In conclusion, the DQCAR allele in combination with HLA class II, especially DR, is probably a useful risk marker for RA susceptibility in the Korean population.