Cellular immune response during uncomplicated genital infection with Chlamydia trachomatis in humans.

Extraction of staphylococcal abscesses by the Folch procedure revealed that all of the staphylocidal activity was present in the lipid fraction. Further separation of the lipids indicated that the bactericidal activity resided in the free fatty acid pool. Lipids similarly extracted from mesenteric or epididymal fat tissue, either before of after activation, did not possess comparable activity. Myristic, palmitic, palmitoleic, linoleic, and oleic acids, as well as lysolecithin, also failed to exhibit the properties of the fatty acid fraction obtained from abscess homogenates. These findings suggest the staphylocidal fatty acid is not a common host lipid.     

Behavior Problems in Children With Diabetes: Disentangling Possible Scoring Confounds on the Child Behavior Checklist

Objective: The Child Behavior Checklist (CBCL; T. M. Achenbach,1991), when used to assess the behavior of children with diabetes,may contain confounds because some behavioral items can havea physiologic etiology, and may skew reports of behavioral disturbance. Methods: Two techniques were used to disentangle possible scoringconfounds in the behavioral ratings of children with and withoutdiabetes: (1) the Somatic Complaints scale was deleted, or (2)Diabetes Items, identified a priori with 89% agreement by ninemedical personnel, were deleted. Results: As expected, with traditionally scored protocols, childrenwith diabetes obtained higher Internalizing and Total BehaviorProblem scores than controls. This group difference persistedwhether the Somatic Complaints scale or the Diabetes Items weredeleted. Conclusions: Compared to controls, children with diabetes obtainedmildly elevated scores on six of the eight CBCL scales, regardlessof scoring method, suggesting that their mildly elevated behavioralprofile is not confounded by physiologic symptomatology.     

Inhibition of endoplasmic reticulum-associated degradation in CHO cells resistant to cholera toxin,Pseudomonas aeruginosa exotoxin A,and ricin

Many plant and bacterial toxins act upon cytosolic targets and must therefore penetrate a membrane barrier to function. One such class of toxins enters the cytosol after delivery to the endoplasmic reticulum (ER). These proteins, which include cholera toxin (CT), Pseudomonas aeruginosa exotoxin A (ETA), and ricin, move from the plasma membrane to the endosomes, pass through the Golgi apparatus, and travel to the ER. Translocation from the ER to the cytosol is hypothesized to involve the ER-associated degradation (ERAD) pathway. We developed a genetic strategy to assess the role of mammalian ERAD in toxin translocation. Populations of CHO cells were mutagenized and grown in the presence of two lethal toxins, ETA and ricin. Since these toxins bind to different surface receptors and attack distinct cytoplasmic targets, simultaneous acquisition of resistance to both would likely result from the disruption of a shared trafficking or translocation mechanism. Ten ETA- and ricin-resistant cell lines that displayed unselected resistance to CT and continued sensitivity to diphtheria toxin, which enters the cytosol directly from acidified endosomes, were screened for abnormalities in the processing of a known ERAD substrate, the Z form of alpha1-antitrypsin (alpha1AT-Z). Compared to the parental CHO cells, the rate of alpha1AT-Z degradation was decreased in two independent mutant cell lines. Both of these cell lines also exhibited, in comparison to the parental cells, decreased translocation and degradation of a recombinant CTA1 polypeptide. These findings demonstrated that decreased ERAD function was associated with increased cellular resistance to ER-translocating protein toxins in two independently derived mutant CHO cell lines.     

Modified oxidation-fermentation medium for detection of acid production from carbohydrates by Neisseria spp. and Branhamella catarrhalis

A modified oxidation-fermentation medium was developed as a practical medium for highly sensitive and specific detection of acid production from carbohydrates by Neisseria spp. and Branhamella catarrhalis. A total of 756 strains representing 17 Neisseria spp. and Branhamella catarrhalis were tested in this medium, in which the protein concentration was reduced relative to the carbohydrate concentration, phenol red was substituted for bromthymol blue at a low concentration, and the initial pH was adjusted to 7.2. Sugar utilization patterns were consistent with published results and with other cultural and biochemical characteristics for these species. The reactions obtained using this medium were qualitatively better and more reproducible than those obtained in cystine-Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) medium.     

Iron-dependent regulation of diphtheria toxin and siderophore expression by the cloned Corynebacterium diphtheriae repressor gene dtxR in C. diphtheriae C7 strains.

A regulatory gene (dtxR) responsible for iron-dependent repression of the toxin (tox) and siderophore genes in Corynebacterium diphtheriae was cloned and characterized. A DNA fragment carrying dtxR repressed expression of a tox-lacZ gene fusion in Escherichia coli DH5 alpha in a high-iron environment but not under low-iron conditions. A protein with mobility corresponding to approximately 28 to 29 kDa was identified as the product of the dtxR gene by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A shuttle vector designated pCM2.6 was constructed which carries the origin of replication from C. diphtheriae plasmid pNG2 and confers resistance to chloramphenicol in E. coli and C. diphtheriae. DNA fragments carrying dtxR were cloned into pCM2.6, and the hybrid shuttle plasmids were transformed by electroporation into wild-type C. diphtheriae C7(beta) and the regulatory mutant C7(beta)hm723, which produces toxin and siderophore constitutively under high-iron conditions. Expression of the cloned dtxR determinant did not affect the phenotype of C. diphtheriae C7(beta). In C. diphtheriae C7(beta)hm723, expression of cloned dtxR restored full repression of siderophore production and partial repression of diphtheria toxin production during growth in a high-iron environment.     

Differential expression of complement regulatory proteins decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 during human spermatogenesis.

We have examined the distribution of the complement (C) regulatory proteins CD59, membrane cofactor protein (MCP) and decay-accelerating factor (DAF) on mature sperm and compared expression of these proteins in parallel both during spermatogenesis and in the prostate. Enhanced immunoperoxidase staining and radioimmunoassay confirmed that C regulators are differentially expressed on sperm; CD59 was strongly expressed on the surface of acrosome intact sperm while MCP and DAF appear to be located primarily on the inner acrosomal membrane. While the MW of CD59 on sperm is typical of other systems, we confirm that in addition to a novel 40,000-46,000 MW MCP protein, sperm also express a novel 55,000 MW DAF product. Examination of normal testis by immunostaining revealed that although C regulators are differentially expressed within the germinal epithelium, all three proteins were present on the acrosomal region of condensing spermatids. We show that novel, low MW forms of MCP and DAF are expressed in normal testis membranes but are absent from testis membranes obtained from patients undergoing gender reassignment surgery in whom the germinal epithelium is diminished. Novel MW C3 convertase regulators are therefore associated with differentiating germinal epithelium. Typical CD59 components were also present on normal testis membranes confirming that CD59 is acquired during spermatogenesis. We demonstrate that the prostatic epithelium, in addition to MCP, expresses CD59 but not DAF. By comparison with CD59, therefore, our studies suggest that DAF may be acquired only in the testis. Overall, our data suggest that, on leaving the testis, sperm express the repertoire of C regulators required for protection from C during their transit through the male and female reproductive tracts.     

DNA-synthesizing cells in the blood in coeliac disease and inflammatory bowel disease.

The level of 3H-labelled thymidine ([3H]TdR) incorporation of blood cells of patients with coeliac disease was shown in two separate studies to be significantly lower than that of a normal control group. In the first study the 'background' DNA synthesis in unstimulated cultures containing a standard number of blood lymphocytes was measured on days 4, 5 and 6. In the second study a standard volume of freshly drawn whole blood was added to culture medium and the [3H]TdR incorporation measured over the first 24 hr and from 48 to 72 hr. In all cases the [3H]TdR incorporation of cells of coeliac patients on a normal or a gluten-free diet was lower than that of the control group. It is suggested that sequestration of DNA-synthesizing cells in the mucosa of the small bowel may partly explain these results. In whole-blood cultures from patients with inflammatory bowel disease in remission [3H]TdR incorporation over the first 24 hr was raised in Crohn's disease but normal in ulcerative colitis.     

Lineage extinction and replacement in dengue type 1 virus populations are due to stochastic events rather than to natural selection

Between 1996 and 1998, two clades (B and C; genotype I) of dengue virus type 1 (DENV-1) appeared in Myanmar (Burma) that were new to that location. Between 1998 and 2000, a third clade (A; genotype III) of DENV-1, which had been circulating at that locality for at least 25 years, became extinct. These changes preceded the largest outbreak of dengue recorded in Myanmar, in 2001, in which more than 95% of viruses recovered from patients were DENV-1, but where the incidence of severe disease was much less than in previous years. Phylogenetic analyses of viral genomes indicated that the two new clades of DENV-1 did not arise from the, now extinct, clade A viruses nor was the extinction of this clade due to differences in the fitness of the viral populations. Since the extinction occurred during an inter-epidemic period, we suggest that it was due to a stochastic event attributable to the low rate of virus transmission in this interval.     

Staphylococcus aureus isolates carrying Panton-Valentine leucocidin genes in England and Wales: frequency, characterization, and association with clinical disease

Staphylococcus aureus isolates carrying the genes that encode for Panton-Valentine leucocidin (PVL), a highly potent toxin, have been responsible for recent outbreaks of severe invasive disease in previously healthy children and adults in the United States of America and Europe. To determine the frequency of PVL-positive isolates sent to the Staphylococcus Reference Unit (United Kingdom) for epidemiological purposes, we tested 515 isolates of S. aureus, and 8 (1.6%) were positive for the PVL locus. A further 470 isolates were selected to explore the association of PVL-positive S. aureus with clinical disease. Of these, 23 (4.9%) were PVL positive and most were associated with skin and soft tissue infections (especially abscesses). The PVL genes were also detected in isolates responsible for community-acquired pneumonia, burn infections, bacteremia, and scalded skin syndrome. Genotyping by pulsed-field gel electrophoresis and multilocus sequence typing revealed that the PVL-positive isolates were from diverse genetic backgrounds, although one prevalent clone of 12 geographically dispersed methicillin-resistant S. aureus (MRSA) isolates was identified (ST80). All 12 isolates were stapylococcal cassette chromosome mec type IVc, had an agr3 allele, and shared a common toxin gene profile (sea-see, seg-sej, eta, etb, and tst negative but etd positive). ST80 strains with similar genetic characteristics have been responsible for community-acquired infections in France and Switzerland. The remaining PVL-positive isolates were mostly methicillin-sensitive S. aureus and belonged to 12 different sequence types, including ST22 and ST30, which are closely related to the most prevalent MRSA clones in United Kingdom hospitals, EMRSA-15 and EMRSA-16, respectively.