David Turner

Cytokine gene polymorphism and heart transplant rejection

BACKGROUND Allograft rejection is mediated by cytokines. As polymorphism in cytokine genes can result in interindividual differences in cytokine production, we hypothesize that some patients may have an increased risk of rejection. METHODS We have related polymorphisms that influence cytokine production in the tumor necrosis factor (TNF)-A and interleukin (IL)-10 genes to early graft rejection in 115 heart transplant recipients. RESULTS Certain combinations of TNF-A and IL-10 gene polymorphisms are associated with rejection. Five of 19 patients who had high levels of rejection typed as high TNF-alpha/low IL-10 producers compared with 4 of 96 patients with lower levels of rejection (P<0.005). CONCLUSIONS We have identified a particular cytokine genotype that may confer susceptibility to increased levels of early rejection. Patients with a worse prognosis may be able to be identified pretransplant by DNA analysis of TNF-A, IL-10, and other gene polymorphisms.

IL-10 gene promoter polymorphisms in rheumatoid arthritis

IL-10 is an anti-inflammatory cytokine which may modulate disease expression in RA. Three dimorphic polymorphisms within the IL-10 gene promoter have recently been identified and appear to influence regulation of its expression. The 1082*A allele has been associated with low and the 1082*G allele with high in vitro IL-10 production. We have analysed 117 unrelated Caucasoid RA patients and 119 ethnically matched controls. No significant differences in the allele frequencies of the three polymorphisms were found between controls and RA patients. In contrast, a significant association between the 1082*A allele and the (-1082*A/-819*C/-592*C) haplotype and IgA RF+ve/IgG RF-ve patients was observed. The association of genotypes encoding low IL-10 production with IgA RF in RA is incompatible with its suggested role in antibody isotype switching. IgA RF has been associated with severe RA and may thus be indirectly correlated with a genotype encoding low IL-10 production.

CapA, an Autotransporter Protein of Campylobacter jejuni, Mediates Association with Human Epithelial Cells and Colonization of the Chicken Gut

Two putative autotransporter proteins, CapA and CapB, were identified in silico from the genome sequence of Campylobacter jejuni NCTC11168. The genes encoding each protein contain homopolymeric tracts, suggestive of phase variation mediated by a slipped-strand mispairing mechanism; in each case the gene sequence contained frameshifts at these positions. The C-terminal two-thirds of the two genes, as well as a portion of the predicted signal peptides, were identical; the remaining N-terminal portions were gene specific. Both genes were cloned and expressed; recombinant polypeptides were purified and used to raise rabbit polyclonal monospecific antisera. Using immunoblotting, expression of the ca.116-kDa CapA protein was demonstrated for in vitro-grown cells of strain NCTC11168, for 4 out of 11 recent human fecal isolates, and for 2 out of 8 sequence-typed strains examined. Expression of CapB was not detected for any of the strains tested. Surface localization of CapA was demonstrated by subcellular fractionation and immunogold electron microscopy. Export of CapA was inhibited by globomycin, reinforcing the bioinformatic prediction that the protein is a lipoprotein. A capA insertion mutant had a significantly reduced capacity for association with and invasion of Caco-2 cells and failed to colonize and persist in chickens, indicating that CapA plays a role in host association and colonization by Campylobacter. In view of this demonstrated role, we propose that CapA stands for Campylobacter adhesion protein A.

A Functional Two-Partner Secretion System Contributes to Adhesion of Neisseria meningitidis to Epithelial Cells

Neisseria meningitidis is a frequent commensal of the human nasopharynx causing severe invasive infections in rare cases. A functional two-partner secretion (TPS) system in N. meningitidis, composed of the secreted effector protein HrpA and its cognate transporter HrpB, is identified and characterized in this study. Although all meningococcal strains harbor at least one TPS system, the hrpA genes display significant C-terminal sequence variation. Meningococcal genes encoding the TPS effector proteins and their transporters are closely associated and transcribed into a single mRNA. HrpA proteins are translocated across the meningococcal outer membrane by their cognate transporters HrpB and mainly released into the environment. During this process, HrpA is proteolytically processed to a mature 180-kDa form. In contrast to other known TPS systems, immature HrpA proteins are stable in the absence of HrpB and accumulate within the bacterial cell. A small percentage of mature HrpA remains associated with the bacteria and contributes to the interaction of meningococci with epithelial cells.

Risk of inappropriate exclusion of organ donors by introduction of hepatitis B core antibody testing

BACKGROUND Hepatitis B virus infection originating from hepatitis B surface antigen-negative, hepatitis B core antibody (anti-HBc)-positive organ donors has been documented, and anti-HBc-positive donors have been excluded as liver donors. We assessed the prevalence of anti-HBc in UK organ donors and followed up recipients of organs from anti-HBc-positive donors for serological evidence of posttransplantation hepatitis B virus infection. METHODS Serum samples from 400 hepatitis B surface antigen-negative organ donors were tested for anti-HBc. RESULTS Only five (1.25%) of 20 sera in which anti-HBc was initially detected were confirmed as anti-HBc positive on further testing. Posttransplantation serum samples from four recipients of confirmed anti-HBc-positive organs showed no evidence of de novo hepatitis B infection. CONCLUSIONS The poor specificity of some anti-HBc immunoassays was confirmed and suggests that donor exclusion on the basis of a single anti-HBc-positive result may result in the inappropriate loss of organs.

Heparinised intraocular infusion and bacterial contamination in cataract surgery

BACKGROUND/AIMS Heparin in solution reduces bacterial adhesion to intraocular lenses and a lower incidence of postoperative endophthalmitis has been reported with the use of heparin coated lenses. The safety of adding low molecular weight heparin to the infusion fluid during routine cataract surgery was investigated. Any direct antibacterial effect was looked for by culturing anterior chamber fluid samples taken at the completion of surgery. METHODS A randomised, double blind, controlled study of 111 patients undergoing routine cataract surgery. Low molecular weight heparin at a concentration of 5 IU/ml was added to the infusion fluid in the trial patients. Samples from the anterior chamber taken at completion of surgery were cultured. Twenty nine samples of sterile infusion fluid were also cultured as further controls. RESULTS No complications were found in either group, and no difference in observed postoperative inflammation in each group. In the heparinised group (n=55) bacterial contamination was found in 31% of samples, compared with 27% in the no heparin group (n=56) (no significant difference). CONCLUSIONS There appears to be no direct antibacterial effect of heparin, and other possible mechanisms of action are discussed. Heparin avoids many of the drawbacks of traditional antibiotic prophylaxis and may have the potential to be a safe and effective addition to endophthalmitis prevention.

Functional Characterization of AasP, a Maturation Protease Autotransporter Protein of Actinobacillus pleuropneumoniae

ABSTRACT Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a highly contagious respiratory infection in pigs. AasP, a putative subtilisin-like serine protease autotransporter, has recently been identified in A. pleuropneumoniae. We hypothesized that, similarly to other autotransporters of this type, AasP may undergo autocatalytic cleavage resulting in release of the passenger domain of the protein. Furthermore, AasP may be responsible for cleavage of other A. pleuropneumoniae outer membrane proteins. To address these hypotheses, the aasP gene was cloned and the expressed recombinant AasP protein used to raise monospecific rabbit antiserum. Immunoblot analysis of whole-cell lysates and secreted proteins demonstrated that AasP does not undergo proteolytic cleavage. Immunoblot analysis also confirmed that AasP is universally expressed by A. pleuropneumoniae. Confirmation of the maturation protease function of AasP was obtained through phenotypic analysis of an A. pleuropneumoniae aasP deletion mutant and by functional complementation. Comparison of the secreted proteins of the wild type, an aasP mutant derivative, and an aasP mutant complemented in trans led to the identification of OmlA protein fragments that were present only in the secreted-protein preparations of the wild-type and complemented strains, indicating that AasP is involved in modification of OmlA. This is the first demonstration of a function for any autotransporter protein in Actinobacillus pleuropneumoniae.

Chorioamnionitis due to Pseudomonas aeruginosa: a complication of prolonged antibiotic therapy for premature rupture of membranes

We do not believe that the technique for measurement of SmcO, which we have previously described is invalidated by these observations. We have previously shown that changes in fetal cerebral oxygenation as measured by NIRS correlate well with expected physiological changes in various clinical situationse. This degree of consistency makes it unlikely that the observed changes are due to movement artefact or compartment shifts. In summary, this paper raised some interesting questions on the use of NIRS in the case of a nonviable fetus, but we question its relevance to intrapartum studies of live fetuses.