Analysis of chickens for recombination within the MHC (B-complex)

In an attempt to further map the chicken MHC (the B complex), a systematic search for genetic recombinants within the B complex was performed by serotyping the progeny from F2 crosses of chickens by means of specific anti-class I, anti-class II, and anti-class IV alloantisera. Two recombinant B-haplotypes (B21r and B15r) were found by analysing 2,656 F2 chickens representing 5,312 informative typings. In either case, the B-G (class IV) allele was recombined with both the B-F and B-L alleles of the opposite haplotype. MLC typings, tests for direct compatibility by GVH reactions, and absorption analyses confirmed the original serological typing of the two recombinant B haplotypes. No recombination between B-F (class I) and B-L (class II) loci was found. This very low frequency of recombination within the B complex as compared with recombination frequencies found in mammalian MHC's is discussed.     

Peptide-Based OspC Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Lyme Borreliosis

Sera from 210 patients with Lyme borreliosis (LB) were studied by an enzyme-linked immunosorbent assay (ELISA) based on a synthetic peptide (pepC10) comprising the C-terminal 10-amino-acid residues of OspC of Borrelia burgdorferi. We found that 36.3 and 45.0% of the serum samples from patients with erythema migrans (EM) and neuroborreliosis (NB), respectively, displayed immunoglobulin M (IgM) anti-pepC10 reactivities, while these samples rarely (≤8%) displayed IgG antibody reactivities. Sera from patients with acrodermatitis chronica atrophicans did not contain anti-pepC10 antibodies. The diagnostic performance of this newly developed peptide ELISA was compared with those of an ELISA based on the full-length recombinant OspC protein (rOspC) and a commercially available ELISA based on the B. burgdorferi flagellum (Fla). The sensitivity of the IgM pepC10 ELISA was slightly lower (P < 0.04) than that of the rOspC ELISA for EM patients (36.3 versus 43.8%), while there was no difference for NB patients (45.0 versus 48.0%). However, the optical density values obtained by the pepC10 ELISA were generally higher than those obtained by the rOspC ELISA, leading to a significantly better quantitative discrimination between seropositive patients with NB and controls (P < 0.008). The specificity of the pepC10 ELISA was similar to those of the rOspC ELISA and the Fla ELISA for relevant controls including patients with syphilis and mononucleosis. Although the overall diagnostic sensitivity of the Fla ELISA was superior, 8.8 and 12.0% of the EM and NB patients, respectively, were antibody positive only by the pepC10 ELISA. Thus, use of a diagnostic test for LB based on the detection of IgM antibodies to pepC10 and Fla has increased sensitivity for the diagnosis of early LB.     

Genomic approach to identification of Mycobacterium bovis diagnostic antigens in cattle

Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivatives from Mycobacterium bovis and M. avium is the standard for diagnosing bovine tuberculosis. However, improved tests based on defined, specific antigens are urgently needed. In the present study, a combination of bioinformatics, molecular biology, and bovine models of infection were used to screen mycobacterial proteins for their potential as diagnostic reagents which could be used in a whole-blood assay for diagnosis of tuberculosis. Initial screening of 28 proteins selected in silico and expressed as recombinants in Escherichia coli indicated that CFP-10, ESAT-6, TB27.4, TB16.2, TB15.8, and TB10.4 induced strong gamma interferon responses in experimentally infected cattle. A more thorough investigation over time in two groups of animals infected with a high (10(6) CFU) and a low (10(4) CFU) dose of M. bovis revealed that, for both groups, the strength of the in vitro response to individual antigens varied greatly over time. However, combining the results for ESAT-6, CFP-10, and TB27.4, possibly supplemented with TB10.4, gave sensitivities at different infection stages close to those obtained with M. bovis purified protein derivative. Importantly, while responsiveness to ESAT-6 and CFP-10 correlated strongly for individual samples, the same was not the case for ESAT-6 and TB27.4 responsiveness. The results suggest that combinations of specific antigens such as these have great potential in development of optimized diagnostic systems for bovine tuberculosis.     

Inhibition of anti-GD3-ganglioside antibody-induced proliferation of human CD8+ T cells by CD16+ natural killer cells

The ganglioside GD3 has been described as a membrane component of human T cells which is involved in T cell growth. In the present study the activating function of GD3 for human CD4⁺ and CD8⁺ T cells was analyzed by five different monoclonal antibodies (mAb) directed against the GD3 molecule. Three mAb U5, Z21 and R24 induced strong proliferation of peripheral blood mononuclear cells and purified CD8⁺ and CD4⁺ T cells of normal donors containing less than 5% CD16⁺ natural killer (NK) cells. In contrast to CD4⁺ T cells, CD8⁺ T cells proliferated only weakly in the presence of 15% CD16⁺ NK cells. The proliferative response of purified CD4⁺ and CD8⁺ T cells (<5% NK cells) correlated with the antibody-dependent induction of integral and soluble interleukin-2 (IL-2) receptors and was reduced to 20% by an anti-IL-2 receptor antibody. Our results show, that the GD3 molecule represents an activation molecule for both CD4⁺ and CD8⁺ T cells and that CD16⁺ NK cells selectively inhibit anti-GD3 antibody-induced proliferation of CD8⁺ T cells.     

High-Density SNP genotyping defines 17 distinct haplotypes of the TNF block in the Caucasian population: implications for haplotype tagging

The region spanning the tumor necrosis factor (TNF) cluster in the human major histocompatibility complex (MHC) has been implicated in susceptibility to numerous immunopathological diseases, including type 1 diabetes mellitus and rheumatoid arthritis. However, strong linkage disequilibrium across the MHC has hampered the identification of the precise genes involved. In addition, the observation of "blocks" of DNA in the MHC within which recombination is very rare, limits the resolution that may be obtained by genotyping individual SNPs. Hence a greater understanding of the haplotypes of the block spanning the TNF cluster is necessary. To this end, we genotyped 32 human leukocyte antigen (HLA)-homozygous workshop cell lines and 300 healthy control samples for 19 coding and promoter region SNPs spanning 45 kb in the central MHC near the TNF genes. The workshop cell lines defined 11 SNP haplotypes that account for approximately 80% of the haplotypes observed in the 300 control individuals. Using the control individuals, we defined a further six haplotypes that account for an additional 10% of donors. We show that the 17 haplotypes of the "TNF block" can be identified using 15 SNPs.     

Properdin factor B and complement factor C4 allotypes in rheumatoid arthritis: results of a follow-up study.

The association between allotypes of properdin factor B (Bf), the fourth component of complement (C4A and C4B), and rheumatoid arthritis (RA), was investigated in a well-characterized cohort of RA patients who were followed from an early phase of the disease for a mean duration of 6 years. The frequencies of probable heterozygous C4AQ0 and of C4A3 were lower in RA patients compared to controls, irrespective of the presence of DR4 [relative risk (RR): 0.52 and 0.49, respectively, 95% confidence intervals (95% CI): 0.34-0.80 and 0.29-0.82]. The frequency of C4A4 was higher in RA patients compared to controls (RR: 1.86, 95% CI: 1.03-3.35), especially in DR4 positive RA patients compared to DR4 positive controls (RR: 2.58, 95% CI: 1.07-6.25), indicating a positive association of this allotype with RA additional to DR4. Bf and C4B allotypes were comparable in RA patients and controls. We did not find significant differences in Bf and C4 allotype frequencies in RA patients subdivided according to severity of the disease into a mild group and a progressive group. Because of inconsistent results in all studies on Bf and C4 allotypes, we conclude that C4 and Bf allotypes do not seem to have an important independent effect on determining disease susceptibility.     

Hydrolytic Polycondensation of Bisphenol‐A‐Bischloroformate Catalyzed by Phase‐Transfer Catalysts

The hydrolytic interfacial polycondensation of bisphenol‐A‐bischloroformate was performed with four different phase‐transfer (PT) catalysts: N‐butylpyridinium bromide, triethylbenzylammonium (TEBA) chloride, tetrabutylammonium hydrogen sulfate, and tetraphenylphosphonium bromide. These polycondensations were conducted at 5 or 35 °C initial reaction temperature. The resulting polycarbonates were characterized by viscosity and SEC measurements and by MALDI‐TOF mass spectrometry. The four PT catalysts gave quite different results with respect to molecular weight and formation of cyclic polycarbonates. The highest molecular weights (number average, and weight average, ) were obtained with TEBA‐Cl. Lower temperatures and high feed ratios of TEBA‐Cl proved to be favorable for both high molecular weights and high fractions of cycles. Cyclic polycarbonates were detectable in the mass spectra up to 14 kDa (technical limit of the measurements). Low molecular weights in combination with unreacted chloroformate groups proved that the other PT‐catalysts were less efficient under the given reaction conditions.

MALDI‐TOF mass spectrum of the polycarbonate No. 3b .     

Pharmacokinetics of tolfenamic acid in patients with cirrhosis of the liver

Summary Six patients with alcoholic cirrhosis of the liver received 100 mg tolfenamic acid p.o. and i.v. The disposition of tolfenamic acid could be described by a two-compartment open body model, with a mean central compartment volume of 8.71, and a -phase volume of 251. The elimination rate constant k_e averaged 1.13 h^–1 and the half-life of the -phase was 1.73 h; the mean total plasma clearance was 159 ml/min. These pharmacokinetic parameters differed only slightly from those in two groups of healthy volunteers studied previously; k_e was significantly reduced by about 30% in the patients but none of the other parameters differed significantly. There was good correlation between individual elimination rate constants or plasma clearances with the liver function tests, serum albumin and P-coagulation factors. Oral absorption was good and bioavailability of about 100% was shown by comparison of the areas under the plasma concentration — time curves after i.v. and p.o. administration. Metabolism was qualitatively and quantitatively very similar to previous observations in healthy volunteers. There seems no reason to reduce the dose of tolfenamic acid in patients with compensated alcoholic cirrhosis.     

Polyneuropathy in paraproteinaemia

Summary Paraproteinaemias are frequently associated with peripheral neuropathies. Benign paraproteinaemia, myeloma and Waldenströms macroglobulinaemia may present clinically as polyneuropathy. Therefore immunoelectrophoresis is strongly recommended in the routine diagnosis of polyneuropathies of unknown origin. Peripheral neuropathies associated with paraproteinaemia are clinically, electrophysiologically, pathologically and probably also pathogenetically heterogeneous. There are subgroups such as demyelinating neuropathy associated with IgM paraproteinaemia, which show quite distinctive features. This survey describes the different types of paraproteinaemia and their associated peripheral neuropathies. The incidence, pathogenesis and therapy of peripheral neuropathy associated with monoclonal gammopathies are discussed.