Glycosylation of random IgG distinguishes seropositive and seronegative rheumatoid arthritis.

The N-glycosylation of human immunoglobulins, especially IgGs, plays a critical role in determining affinity of IgGs towards their effector (pro- and anti-inflammatory) receptors. However, it is still not clear whether altered glycosylation is involved in only antibody-dependent disorders like seropositive rheumatoid arthritis (RA) or also in pathologies with similar clinical manifestations, but no specific autoantibodies like seronegative RA. The clarification of that uncertainty was the aim of the current study. Another study aim was the detection of specific glycan forms responsible for altered exposure of native glycoepitopes. We studied sera from seropositive RA (n = 15) and seronegative RA (n = 12) patients for exposure of glycans in native IgG molecules, followed by determination of specific glycans by capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). Aged-matched groups of normal healthy donors (NHD) and samples of intravenous immunoglobulin IgG preparations (IVIG) served as controls. There was significantly stronger binding of Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) lectins towards IgG from seropositive RA compared to seronegative RA or NHD. CE-LIF analysis revealed statistically significant increases in bisecting glycans FA2BG2 (p = .006) and FABG2S1 (p = .005) seropositive RA, accompanied by decrease of bisecting monogalactosylated glycan FA2(6)G1 (p = .074) and non-bisecting monosialylated glycan FA2(3)G1S1 (p = .055). The results suggest that seropositive RA is distinct from seronegative RA in terms of IgG glycan moieties, attributable to specific immunoglobulin molecules present in seropositive disease. These glycans were determined to be bisecting GlcNAc-bearing forms FA2BG2 and FABG2S1, and their appearance increased the availability of LCA and AAL lectin-binding sites in native IgG glycoepitopes.

Restriction enzyme analysis of ribosomal DNA shows that Candida inconspicua clinical isolates can be misidentified as Candida norvegensis with traditional diagnostic procedures.

We identified 29 yeast isolates from 22 patients using the API ID32C panel. Twenty-eight of these isolates were Candida norvegensis and one was C. inconspicua. Although C. norvegensis is considered a pseudohypha-producing species, only one isolate produced pseudohyphae. Restriction enzyme analysis of PCR-amplified ribosomal DNA with four different enzymes proved that all isolates were C. inconspicua.

Genetic polymorphism of the human tumor necrosis factor region in insulin-dependent diabetes mellitus. Linkage disequilibrium of TNFab microsatellite alleles with HLA haplotypes.

The TNF region within the MHC includes a number of immunologically important genes. Microsatellites TNFa and TNFb adjacent to TNF exhibit extensive polymorphism. Employing a PCR-based technique, we identified TNFab haplotypes and defined their distribution in 97 controls and 48 diabetics of Caucasoid origin in a search for other genes within the MHC potentially associated with IDDM. Twenty-five different TNFab haplotypes were identified. A significant difference (p < 0.0005) in frequency between patients and controls was found for TNFa1b5 (relative risk 53). However, no other TNFab microsatellites demonstrated significantly different frequencies. Among diabetics TNFa1b5 was found to be in linkage disequilibrium with HLA-DR3-B18, a haplotype known to be associated with IDDM. Thus the increased frequency of TNFa1b5 among diabetics could reflect a linkage disequilibrium with a gene within the TNF region or with other genes, including the HLAs, which characterize this haplotype. In both controls and diabetics TNFa2b3 and TNFa7b4 were in linkage disequilibrium with DR3-B8 and DR7, respectively. Among diabetics, TNFa2b1 and TNFa6b5 were in linkage disequilibrium with DR4-B62 and DR4-B44, respectively. It is intriguing that TNFab haplotypes, represented by a short piece of about 200 nucleotides in the untranslated region upstream of TNF beta gene, maintain strong linkage disequilibria with different HLA haplotypes extending over 1 million base pairs. The identification of TNFab microsatellites exhibiting a high polymorphic index in a region lacking known polymorphic markers may provide potentially important information regarding the association of HLA haplotypes with autoimmune diseases, as they are in close proximity to other genes of immunologic importance.

Potentiation of HIV envelope glycoprotein and other immunogens by endotoxin (ET) and its molecular fragments.

The structural requirements for the immunopotentiating (adjuvant) effect of endotoxin (ET) were investigated. Mild hydrolysis (0.2 N acetic acid at 95 degrees C) was applied to various ET preparations and the lipid rich (Lipid A) and polysaccharide-rich (PS) preparations obtained were tested as adjuvants on three immunogens: sheep red blood cells (SRBC), L-glutamine: L-lysine: L-alanine containing random synthetic polypeptide (GLA-40), and recombinant HIV viral envelope polypeptide (CBre3). It was found that not only the Lipid A precipitates, but under certain hydrolytic conditions the non-toxic PS preparations were also potent adjuvants. The exact conditions of hydrolysis which led to the isolation of immune adjuvant bacterial products were established. These materials were also tested for endotoxicity (Limulus lysate clotting, chick embryo lethality and local Shwartzman skin reactivity), as well as for TNF generating activities. It was found that TNF generation runs parallel with toxicity of the samples, but it does not follow the adjuvant activity of the isolates. Chemical analysis of the preparations indicated that they did not contain residual ET or Lipid A, however, they did not exclude that deacylated and dephosphorylated skeletal remains of ET are among those components in these preparations which have immunomodulatory activity.

Molecular requirements of endotoxin (ET) actions: changes in the immune adjuvant, TNF liberating and toxic properties of endotoxin during alkaline hydrolysis.

The effect of alkaline pH and alkaline hydrolysis on the physico-chemical and biological properties of endotoxin (ET) isolated from Serratia marcescens ATCC 13477 by the Biovin procedure was studied. Major emphasis was put on the ion exchange column chromatography and immune adjuvant activity (ADA) of the alkali treated samples. To measure changes in some endotoxicity parameters, Limulus lysate clotting (LAL), chick embryo lethality, Shwartzman skin reactivity and in vitro TNF release were measured. The toxic properties of ET, with the unique exception of the Shwartzman skin reactivity, rapidly diminished during alkaline treatment. As immunogen CBre3, a recombinant HIV glycoprotein which spans the C terminus of gp 120 and the N terminus of gp 41, was used in CD-1 mice, alkali treated and immediately neutralized ET samples (zero time) were inactive as adjuvants, in some cases immunosuppression could be clearly seen. But if the alkaline hydrolysis was continued for 6 h, the ADA became higher than it had been for the starting ET sample. Further alkaline hydrolysis eliminated the ADA of the samples. Both NaOH and propylamine acted similarly on the ET preparation. Reaction kinetic studies of the NaOH detoxification indicated the cleavage of ester bound acyl groups with low binding energy. Chemical analyses of the samples revealed that changes occurred in the fatty acid composition, characterized by a loss of approximately half of the 3-OH myristic acid content.