Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells.

Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis is likely important in the establishment of a primary infection in the lung. M. tuberculosis binds to a variety of phagocyte receptors, of which the mannose receptor and complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a beta2 integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tuberculosis H37Rv to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesized complement, which are inherent in studies with mononuclear phagocytes. The surface expression of CD11b and CD18 was assessed by immunofluorescence, immunobead binding, flow cytometry, and immunoprecipitation with anti-CD11b and anti-CD18 monoclonal antibodies (MAbs). The functional activity of the surface-expressed CD11b/CD18 (CR3) heterodimer was confirmed by rosetting with C3bi-coated microspheres. We found that M. tuberculosis bound four- to fivefold more avidly to CR3-expressing CHO cells than to wild-type cells and, importantly, that this binding was at similar levels in the presence of fresh or heat-inactivated human or bovine serum or no serum. In contrast, Mycobacterium smegmatis bound poorly to CR3-expressing CHO cells in the absence of serum, but after opsonization in serum, binding was comparable to that of M. tuberculosis. The binding of M. tuberculosis to the transfected CHO cells was CR3 specific, as it was inhibited by anti-CR3 MAbs, particularly the anti-CD11b MAbs LM2/1 (I domain epitope) and OKM1 (C-terminal epitope), neither of which inhibit C3bi binding. MAb 2LPM19c, which recognizes the C3bi-binding site on CD11b, had little or no effect on M. tuberculosis binding. The converse was found for the binding of opsonized M. smegmatis, which was strongly inhibited by 2LPM19c but unaffected by LM2/1 or OKM1. CR3-specific binding was also evidenced by the failure of M. tuberculosis to bind to CHO cells transfected with an irrelevant surface protein (angiotensin-converting enzyme) in the presence or absence of serum. We conclude that the binding of M. tuberculosis H37Rv to CR3 expressed in CHO cells is predominantly nonopsonic and that the organism likely expresses a ligand that binds directly to CR3.     

Outer membrane protein YadA of enteropathogenic yersiniae mediates specific binding to cellular but not plasma fibronectin.

The binding of bacteria or bacterial products to host proteins of tissue extracellular matrix may be a mechanism of tissue adherence. We investigated interactions of the plasmid-encoded outer membrane protein YadA, which confers pathogenic functions on enteropathogenic yersiniae, with fibronectin. Attachment of YadA-positive and YadA-negative recombinant Yersinia enterocolitica strains to cartilage-derived human cellular fibronectin and human plasma fibronectin in the solid phase revealed that YadA mediates binding of yersiniae to cellular fibronectin in a saturable, concentration-dependent manner. The interaction could be inhibited by an anti-YadA-specific anti-serum. An anti-beta 1-integrin antibody and the synthetic peptide G-R-G-D-S-P, representing the binding site for alpha 5 beta 1-integrin on fibronectin, did not block attachment of YadA-positive yersiniae to cellular fibronectin, indicating a binding site for YadA on cellular fibronectin independent of the R-G-D-S-containing site. By contrast, YadA failed to mediate binding to plasma fibronectin immobilized on nitrocellulose or plastic surfaces. These observations provide evidence for the hypothesis that the binding region for YadA in cellular fibronectin is not present in plasma fibronectin. This study is the first report on differential binding of bacteria to splicing variants of fibronectin. Further experiments might answer the question whether binding of YadA to cellular fibronectin contributes to the pathogenesis of yersiniae, both to the initial adhesion of the bacteria to the matrices of the host and to the arthritogenic potential of enteropathogenic yersiniae.     

Life expectancy in British Marfan syndrome populations

A total of 206 patients with Marfan syndrome were ascertained throughout genetic clinics in Wales and Scotland during the period 1970–1990. There were 45 deaths representing 22% of the cohort. Mean age at death was 45.3 ± 16.5 years. 50% median cumulative survival in the total cohort (n = 206) was 53 years for males and 72 years for females. Multivariate analysis confirmed severity as the best independent indicator of survival. These findings and survival curves will assist in the counselling of British families and individuals with Marfan syndrome.     

The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.      

Total serum IgD is increased in atopic subjects

We have developed a sandwich-type ELISA system for measuring total IgD levels in the serum of atopics and non-atopic controls. In this ELISA system, affinity purified goat anti-human IgD was used for capture. Results were superior to those obtained with monoclonal anti-human IgD antibody. No cross-reactivity could be demonstrated to IgG, IgM, IgA or IgE. The assay showed minimal non-specific binding even with initial serum dilutions of 1:2. The results obtained were reproducible among replicates (Mean CV +/- SEM = 0.03 +/- 0.002; n = 251), between dilutions (CV = 0.08 +/- 0.006; n = 108), and between assays (CV = 0.05 +/- 0.12; n = 5). We used routine radioimmunoassay for measuring total serum IgE. Using these assays total serum IgD and IgE levels were measured in 75 atopic patients and 33 normal subjects. None of the atopics had recent immunotherapy. As expected, the geometric mean serum IgE in atopics (373 ng/ml) was significantly higher than that in normal subjects (49 ng/ml) (P less than 0.01). However, geometric mean serum IgD was also significantly higher in atopics (20.3 micrograms/ml) than that in normal subjects (8.4 micrograms/ml) (P less than 0.02). In both atopic and normal groups, mean serum IgD level did not differ significantly on the bases of age, sex or asthmatic status. Furthermore, total serum IgD was not significantly correlated with total serum IgE (r = 0.14; P = 0.14; n = 108), indicating that immunoregulatory control of the basal levels of the two isotypes is not linked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reliable detection of macrolide-resistant Helicobacter pylori via fluorescence in situ hybridization in formalin-fixed tissue.

Macrolide-resistant Helicobacter (H.) pylori represent an increasing therapeutic problem. Macrolide resistance is usually determined phenotypically in vitro with methods such as E-test or agar dilution test. A prerequisite for those tests, however, is bacterial culture that is not routinely set up in the course of gastroscopy. In contrast, formalin-fixed, paraffin-embedded biopsies are regularly available from patients who have undergone gastroscopy. In such biopsies macrolide-resistant H. pylori can be detected by the genotype-based technique of fluorescence in situ hybridization (FISH). Experience gained by this new method, however, is still extremely limited, especially in formalin-fixed tissue. Therefore, we retrospectively investigated formalin-fixed, paraffin-embedded biopsy specimens by FISH in 104 patients suffering from therapy-resistant H. pylori gastritis. To test the accuracy of FISH, we initially examined specimens from 53 patients for whom results of the E-test were available. Next we analyzed biopsies from another 51 patients that had been selected since phenotypical resistance testing had failed despite documented culturing attempts. In all 104 patients, H. pylori was detected by FISH and could thus be investigated for macrolide resistance. Overall, macrolide-resistant bacteria were found in 71 patients (68.3%). In 49 of 53 patients (92.4%), FISH and E-test returned identical results (no significant discordance according to McNemar's chi(2)-test). Taken together, our study demonstrates that FISH is a highly sensitive and reliable method for detecting macrolide-resistant H. pylori in formalin-fixed, paraffin-embedded biopsy specimens, which represents the routine method of processing tissue obtained upon gastroscopy.