Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections

The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.     

Alternative splicing of IL-24 in melanocytes by deletion of exons 3 and 5

Two novel interleukin-24 (IL-24) splice variants were identified in normal human melanocytes by sequencing cloned polymerase chain reaction (PCR) products that are not expressed in metastatic melanoma. These gene products have been generated by differential skipping of exons 3 (IL-24 delE3) and 5 (IL-24 delE5). IL-24 delE3 has limited sequence identity to the IL-24-interacting protein mda-7s, and IL-24 delE5 is homologous to IL-24.     

Use of the omp50 gene for identification of Campylobacter species by PCR

We studied the prevalence of the omp50 gene and the Omp50 protein in Campylobacter strains. Immunodetection assays and DNA-DNA hybridizations showed that most C. coli strains tested were negative and most C. jejuni and C. lari strains tested were positive. A PCR assay was developed, using the omp50 gene as a species-specific target. We propose a combination of a hippurate test and an omp50 assay to perform identification of Campylobacter species.     

Evidence of two genetic entities inBothriocephalus funiculus (Cestoda) detected by arbitrary-primer polymerase chain reaction random amplified polymorphic DNA fingerprinting

The genetic diversity of two samples of Cestoda (Bothriocephalus funiculus, Renaud and Gabrion, 1984) parasitizing two sympatric teleostean species was assessed using random amplified polymorphic DNA (RAPD). A total of 72Bothriocephalus were analyzed individually, and electrophoretic analysis of the amplification products of 65 primers among the 68 tested revealed monomorphic patterns, reflecting the close genetic relatedness within and between the parasites of the two samples. However, 3 primers showed polymorphic patterns at 6 RAPD sites. Analysis of the distribution of these genomic fragments, assuming random mating, showed strong linkage disequilibria (only 8 genetic combinations were observed among the 32 expected). Two genetic entities displaying a high degree of host specificity were evidence within our two samples ofB. funiculus. This powerful molecular technique can be used as a diagnostic tool in studies concerning the biodiversity of related genetic entities and could have broad applications in parasitology.     

Expression of growth associated protein 43 and neural cell adhesion molecule in congenital fibre type disproportion with interstitial myositis

We report on the expression of growth associated protein (GAP)43 and neural cell adhesion molecule (NCAM) in congenital fibre type disproportion (CFTD) with myopathological additional signs of interstitial myositis. We assume that sarcolemmal GAP43 in developmental disordered myocytes plays a role in maintenance of growth morphology. In muscular dystrophy light microscopical evaluation reveals no GAP43 immunoreactivity in regenerating fibres. The expression of GAP43 seems to be a characteristic feature of CFTD. The expression of NCAM, particularly in the sarcolemma of small muscle fibres of CFTD, indicates a functional state of permanent partial denervation. Whether the steroid-responsive interstitial myositis is pathogenetically related to CFTD or a coincidental inflammation is not known. Because of the clinical and myopathological data the differential diagnosis of Emery-Dreifuss muscular dystrophy is considered.     

Heterogeneity of histamine H2-receptor mediated responses in the rabbit aorta

Distribution and properties of histamine H₂-receptor mediated responses in segments of rabbit aorta was studied with histamine and H₂-receptor stimulating drugs, dimaprit and impromidine. All agonists produced concentration-dependent tonus decreased in precontracted vascular strips, which were antagonised by selective H₂-receptor antagonists, cimetidine and oxmetidine. Activities of the agonists were segment-dependent, and increased caudally along the aorta. A nonspecific smooth muscle relaxant, papaverine had homogeneous activity along the vessel, suggesting receptor specific nature of the observed heterogeneity.     

Class II (DR) antigen expression on CD8+ lymphocyte subsets in acquired immune deficiency syndrome (AIDS)

In a selected group of human immunodeficiency virus (HIV)-infected patients we confirm the expansion of a CD8⁺ T-lymphocyte subset, i.e., the CD8⁺/Leu7⁺ cells, which account for 30% of the lymphocytes, compared to 3% in the control donors. In addition, a CD8⁺ T-lymphocyte subset that coexpresses class II (DR) antigens, i.e., CD8⁺/DR⁺ cells, is also increased from 1.5% in controls to 27% in the HIV-infected patients. Using three-color immunofluorescence and flow cytometry we can demonstrate that the CD8⁺/Leu7⁺ and the CD8⁺/class II⁺ cells are not distinct but overlapping subsets. In the HIV-infected patients 42% of the CD8⁺/Leu7⁺ cells were strongly positive for class II and these CD8⁺/Leu7⁺/class II⁺ cells accounted for 13% of all lymphocytes. These findings indicate that the expanded CD8⁺/Leu7⁺ cells are activated and hence might be actively involved in immune defense in acquired immune deficiency syndrome (AIDS).     

Functional reactivation of the deafferented hippocampus by embryonic septal grafts as assessed by measurements of local glucose utilization

Summary Transection of the septo-hippocampal connections through fimbria-fornix damage in the rat results in profound hippocampal cholinergic deafferentation, and, when applied bilaterally, leads to severe and long-lasting impairments in learning and memory. Previous studies have shown that intrahippocampal septal grafts can reestablish a new cholinergic innervation in the inititally denervated hippocampal formation and at least partly compensate for the lesion-induced learning impairments in fimbria-fornix lesioned rats. The purpose of the present study was to determine the magnitude of lesion-induced alterations in cerebral function as reflected in local glucose use measured by (¹⁴C)-2-deoxyglucose (2-DG) autoradiography, and the degree to which this index of functional activity could be normalized following reinnervation from transplants of fetal cerebral tissue from the primordial septal region. Six months after unilateral fimbriafornix transection the rate of glucose utilization was reduced markedly throughout the ipsilateral hippocampus when compared to the intact contralateral side, while in the neocortex only the cingulate cortex showed long-lasting reductions in glucose use. Rats that received a transplant of fetal septal-diagonal band tissue at the time of fimbria-fornix transection, and were sacrificed 6 months later, displayed significantly greater glucose utilization in the ipsilateral hippocampus and cingulate cortex than was measured in these areas in rats with lesion alone. The recovery in glucose use was paralleled by a significant increase in acetylcholinesterase (AChE) staining in several areas of the ipsilateral hippocampal formation and cingulate cortex. This index of graft-induced cholinergic reinnervation was, moreover, significantly correlated with the rate of glucose use. Thus, in the fimbria-fornix transected animals the magnitude of glucose depression correlated with the extent of reduction in AChE staining, and in the grafted animals the degree of normalization of glucose use was correlated with the graft-induced increase in AChE-staining density. These results thus indicate that the 2-DG autoradiographic technique can provide a unique opportunity to map both altered functional activity in localized areas of the brain following specific lesions and the extent to which transplant-derived reinnervation of the host may induce a return to normal functional levels in the target site.ETP and Royal Society (London) visiting fellow     

Assignment of gene coding for cell surface glycoprotein with a molecular weight of 75,000 to human chromosome 11

The gene for a cell surface glycoprotein recognized by a mouse monoclonal antibody (Mab 4), has been assigned to human chromosome 11 by the study of mouse-human lymphocyte hybrids. The antigen is present on all human peripheral blood leukocytes, on human fibroblasts, and on human lymphoid and erythroid cell lines, but not on erythrocytes. Immunoprecipitation and polyacrylamide slab gel electrophoresis of both human cells and mouse-human hybrid clones carrying human chromosome 11 show that the apparent molecular weight of this glycoprotein is 75,000.     

Increase in vascular permeability induced by leukotriene b4 and the role of polymorphonuclear leukocytes

Leukotriene B₄ (LTB₄), a metabolite of arachidonic acid, is known to be a potent chemotactic and chemokinetic substance. We have used the hamster cheek pouch microcirculation model to study the effect of LTB₄ on vascular permeability and the involvement of neutrophil granulocytes in this response. Intravascular fluorescein-labeled dextran (mol wt 150,000) was used as a tracer of macromolecular permeability. Topical application of LTB₄ (150 nM-5 M) to the hamster cheek pouch resulted in an immediate increase in adhering leukocytes in postcapillary venules and later larger venules. Leukocyte accumulation was reversible, but continued longer the higher the dose of LTB₄ used. Subsequently, a dose-dependent increase in vascular permeability was seen at postcapillary and larger venules, with a maximum 10–20 min after application; the maximum occurred later the higher the dose of LTB₄. Depletion of neutrophil granulocytes by pretreatment of the animals with antineutrophil serum obtained from immunized rabbits significantly decreased the permeability response to LTB₄, whereas the response to histamine was unaffected. These results suggest that neutrophil granulocytes play a role in LTB₄-mediated permeability increase. LTB₄ may be of importance both for the leukocyte accumulation and for the edema formation seen in inflammatory reactions.