Identification of a fibronectin-binding protein from Staphylococcus epidermidis

Staphylococcus epidermidis has been reported to bind to a number of host cell extracellular matrix proteins, including fibronectin. Here we report the identification of a fibronectin-binding protein from S. epidermidis. A phage display library of S. epidermidis genomic DNA was constructed and panned against immobilized fibronectin. A number of phagemid clones containing overlapping inserts were identified, and one of these clones, pSE109FN, contained a 1.4-kb insert. Phage pSE109FN was found to bind to fibronectin but not to collagen, fibrinogen, laminin, or vitronectin. However, pSE109FN also bound to heparin, hyaluronate, and plasminogen, although to a lesser extent than it bound to fibronectin. Analysis of The Institute for Genomic Research S. epidermidis genome sequence database revealed a 1.85-kb region within a putative 30.5-kb open reading frame, to which the overlapping DNA inserts contained within the fibronectin-binding phagemids mapped. We have designated the gene encoding the fibronectin-binding domain embp. A recombinant protein, Embp32, which encompassed the fibronectin-binding domain of Embp, blocked the binding of S. epidermidis, but not the binding of Staphylococcus aureus, to fibronectin. In contrast, a recombinant protein, FnBPB[D1-D4], spanning the fibronectin-binding domain of the S. aureus fibronectin-binding protein FnBPB, blocked binding of S. aureus to fibronectin but had a negligible effect on the binding of S. epidermidis.     

Individual and cultural-diversity competency: focus on the therapist

The Competencies Conference: Future Directions in Education and Credentialing in Professional Psychology was held in Arizona in November 2002. One of the workshops, Individual and Cultural Differences (ICD), focused on racism, homophobia, and ageism. The consensus was that self-awareness and knowledge about the three "isms" are critical components in the education and training of psychologists. This article, authored by four of the workshop attendees, is a review of the current research and theoretical literature. Implications that address both content and context in graduate programs and training sites are presented. This is one of a series of articles published in this issue of the Journal of Clinical Psychology. Several other articles that resulted from the Competencies Conference will appear in Professional Psychology: Research and Practice and The Counseling Psychologist.     

Cytokine induction of neopterin production.

The pteridine neopterin is a marker of immunological activation and has been shown to be a useful marker of graft-versus-host disease (GVHD) in bone marrow transplant patients. High levels of both neopterin and interferon-gamma (IFN-gamma) were produced in vitro during mixed lymphocyte responses, which may be considered to be a model of the primary events leading to GVHD. Neopterin was shown to be produced by monocytes in response to stimulation with IFN-gamma, but not other cytokines. However, the interleukins IL-1 alpha, IL-1 beta, IL-2, and tumour necrosis factor (TNF) alpha and beta, but not IL-6, stimulated neopterin production by unfractionated peripheral blood mononuclear cells (PBMC), and culture supernatants from PBMC stimulated with IL-1 alpha, IL-1 beta, IL-2 and IL-6, but not TNF-alpha or TNF-beta induced neopterin production following transfer to fresh monocyte cultures. It therefore appears that cytokines may generate neopterin by induction of IFN-gamma, by synergy with low levels of induced IFN-gamma, or by non-IFN-gamma-dependent mechanisms.     

The pathology and pathogenesis of chronic lead nephropathy occurring in Queensland.

Many children who suffered acute lead poisoning in Queensland eventually died with contracted kidneys. In most cases the kidneys were granular and showed microscopically fibrosis, hypertensive vascular changes and "alterative glomerulitis". Clinically in these patients, hypertension and chronic renal insufficiency had always preceded death which was usually due to uraemia. In a minority of cases the kidneys showed the changes of benign hypertension but were unusually small; fibrosis and "alterative glomerulitis" were not present. Clinically these patients had had hypertension but minimal renal insufficiency and death was usually due to cerebral haemorrhage. The evidence indicates that lead caused severe damage to the kidney at the time of the lead intoxication by some mechanism other than hypertension. The sequence of events postulated comprises severe renal damage with destruction of glomeruli during childhood lead poisoning, disappearance of the destroyed tissue during childhood and adolescence, onset of hypertension in adolescence or early adult life, gradual onset and progress of chronic uraemia during which fibrosis and granularity developed. In milder cases the sequence is not complete because renal function has remained adequate.     

Vesicular uptake of eosinophil peroxidase by guinea pig basophils and by cloned mouse mast cells and granule-containing lymphoid cells.

Guinea pig basophils, cloned mouse mast cells, and cloned mouse granule-containing lymphoid cells were found to utilize a vesicular transport system to internalize eosinophil peroxidase (EPO) added in vitro. Kinetic analysis indicated that EPO internalization involved the binding of EPO to the plasma membrane, the formation of complex surface invaginations, and the movement of EPO-laden vesicles, tubules, and vacuoles toward the center of the cells. EPO became associated with multivesicular bodies in granule-containing lymphoid cells and mast cells, with immature granules in mast cells, and with mature granules in basophils. In other cells, the endogenous production of granule peroxidases (neutrophils and eosinophils) or the prior uptake of exogenous peroxidatic substances (some basophils) precluded cytochemical analysis of granules for EPO. Vesicular transport of EPO provides a possible explanation for the variable detection of peroxidase activity in mast cells or basophils. It also provides a mechanism for sequestration of this potentially toxic material or for its storage for possible future use.     

The priority test request form: a method for improving communication between the physician and the Emergency Clinical Biochemistry Laboratory.

Two forms--a priority test request form and a telephone results form--provide improved communication between the physician and the emergency laboratory of the Clinical Biochemistry Department. The priority test request form contains a list of available tests and it allows the physician to tell the laboratory exactly when emergency test results are required. The telephone results form ensures that the physician will receive a report, by telephone, of a test result although it may not be an urgent test. This allows a greater control of work flow, both routine and emergency, through the laboratory.     

Recombinant Actinobacillus actinomycetemcomitans cytolethal distending toxin proteins are required to interact to inhibit human cell cycle progression and to stimulate human leukocyte cytokine synthesis

It has recently been discovered that Actinobacillus actinomycetemcomitans, an oral bacterium causing periodontitis, produces cytolethal distending toxin (CDT), a cell cycle-modulating toxin that has three protein subunits: CdtA, CdtB, and CdtC. In this study, we have cloned and expressed each toxin gene from A. actinomycetemcomitans in Escherichia coli and purified the recombinant Cdt proteins to homogeneity. Individual Cdt proteins failed to induce cell cycle arrest of the human epithelial cell line HEp-2. The only combinations of toxin proteins causing cell cycle arrest were the presence of all three Cdt proteins and the combination of CdtB and CdtC. A similar experimental protocol was used to determine if recombinant Cdt proteins were able to induce human peripheral blood mononuclear cells (PBMCs) to produce cytokines. The individual Cdt proteins were able to induce the synthesis by PBMCs of interleukin-1beta (IL-1beta), IL-6, and IL-8 but not of tumor necrosis factor alpha, IL-12, or granulocyte-macrophage colony-stimulating factor, with CdtC being the most potent and CdtB being the least potent cytokine inducer. There was evidence of synergism between these Cdt proteins in the stimulation of cytokine production, most markedly with gamma interferon, which required the minimum interaction of CdtB and -C to stimulate production.     

COMT and DRD3 polymorphisms, environmental exposures, and personality traits related to common mental disorders

In a community sample of 2,327 Caucasians, we tested the hypotheses that polymorphisms in the COMT and DRD3 genes are associated with personality traits conferring vulnerability to anxiety, depression, or alcohol misuse, or with current symptoms of these; and that the association is stronger in persons who also have been exposed to stressor experiences. To conserve resources and to allow replication, the genetic analysis was undertaken in two stages. For the COMT polymorphism, no statistically significant associations were found in the first sample of 862 persons. The remainder of the sample was therefore not analysed for that gene. For the DRD3 polymorphism, those in the first sample with at least one of the Ser(9) alleles had significantly higher scores in neuroticism (p=0.006) and behavioral inhibition (p=0.003). There was a trend, failing to meet the 1% significance criterion, for those with this genotype also to have higher depression and anxiety. The groups did not differ in alcohol use. In persons with the Ser(9) allele who were also exposed to stressors, there was a higher level of depression at the 5% level; and the depression level was higher in homozygotes. But when the remainder of the sample (1,465) was analysed, none of the associations reached statistical significance. We conclude that neither the COMT nor DRD3 polymorphisms are associated with anxiety, depression, or alcohol abuse. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:102-107, 2000 Copyright 2000 Wiley-Liss, Inc.     

Preferential apoptosis of CD56dim natural killer cell subset in patients with cancer

Natural killer (NK) cells (CD56(+)/CD3(-)) in the circulation of cancer patients were reported to have low NK activity and undergo spontaneous apoptosis. A possible relationship between apoptosis and impaired NK activity was studied by Annexin V-binding and NK-cell assays performed with peripheral blood mononuclear cells of patients with head and neck cancer (HNC), breast cancer (BC) and normal controls (NC). Cells stained with Annexin V (Anx) and antibodies to CD56, CD3, CD95, CD25, CD122 or CD132 were examined by flow cytometry. NK activity was tested against K562 targets in 4-h (51)Cr-release assays. The ratio of CD56(dim)/CD56(bright) NK cells was significantly different in patients vs. controls (10 vs. 16; p<0.01). A significantly greater percentage of CD56(dim) NK cells bound Anx in HNC patients (27+/-17%, median +/- SD) or BC (46+/-18%) than in NC (15+/-18%, p<0.04 and p<0.0002, respectively). CD56(dim) NK cells were preferentially targeted for apoptosis. NK activity was significantly lower in patients with HNC and BC than in NC (p<0.009). An inverse correlation between NK activity and the percent of Anx(+)CD56(dim) NK cells was observed in cancer patients (p =0.002) but not in NC. In patients, circulating CD56(dim) NK cells were targeted for apoptosis, leading to low levels of NK activity.