[Gene expression in keratoconus. Initial results using DNA microarrays]. / Genexpression bei Keratokonus. Erster Einsatz von DNA-Microarrays.

INTRODUCTION: Keratoconus is a non-inflammatory ocular disease characterised by conical deformation, progressive thinning and scarring of the central cornea. Despite intensive investigations, the exact cause of the disease still remains unclear. Clinical studies provide strong indications of a major genetic role in the aetiology. We set out to examine the involvement in the manifestation of keratoconus of any of the 5,600 gene specificities available on the Affymetrix GeneChip HuGeneFL. METHODS: After examination of two corneas they were stored in RNAlater, RNA was extracted and hybridised on the chips. Using a combination of dyes it was possible to read the chips with laser detection and to visualise the gene expression pattern. RESULTS: We found an upregulation of collagens, versican, metalloproteinases and cell adhesion proteins. A downregulation was observed for TIGR protein, cytokeratins, eyes absent homologue (Eab1) and the proteins for radical treatment selenoprotein P and monooxygenase. CONCLUSIONS: Our results indicate that keratoconus is a process in which repair and scar-formation mechanisms operate at the same time. As candidate genes for this mechanism, collagen IV and related proteoglycans were favoured.

Epidemiology and infection control implications of Acinetobacter spp. in Hong Kong.

In a previous study, we showed that Acinetobacter genomic DNA group 3 was the most common species among blood culture isolates and was commonly found on superficial carriage sites of the healthy and the sick, which are different findings from those reported in Europe and North America. We used amplified ribosomal DNA restriction analysis and pulsed-field gel electrophoresis to study further the molecular epidemiology of acinetobacters in our region. Over a study period of 6 weeks with 136 consecutive routine clinical isolates (1.33% of all specimens), genomic DNA groups 2 (Acinetobacter baumannii), 3, and 13TU were obtained from 59 of 69 positive patients. There is a significant difference in the specimen sources of the three genomic DNA groups, with group 13TU being significantly associated with the respiratory tract (chi-square exact test, P = 0.0064). Settle plates showed a significantly heavier environmental load from the intensive care unit (ICU) than from the four surgical wards examined (22 of 70 versus 76 of 120 plates with <5 colonies; chi-square test, P < 0. 0001). Genomic group 3 accounted for 6 of 12 clusters of possibly related strains among patients, between patients and the ICU environment, and in the ICU environment. Genomic groups 2 and 3 accounted for 21% of the 132 genomically identified isolates recovered from 21 of 41 local vegetables, 53 of 74 fish and meat samples, and 22 of 60 soil samples. Group 13TU was present only in patients' immediate surroundings. The role played by the environment and by human carriage should be evaluated in order to devise a cost-effective infection control program pertinent to our situation of acinetobacter endemicity.

Analysis of aqueous humour proteins of eyes with and without pseudoexfoliation syndrome.

UNLABELLED: Pseudoexfoliation syndrome (PEX) has been suggested to represent a blood-aqueous barrier impairment leading to a higher protein content in aqueous humour of eyes with PEX. However, the nature of a prospective PEX protein has not yet been described. We set out to reevaluate protein content and examine protein composition for prospective PEX protein candidates in aqueous humour of eyes with PEX syndrome. Aqueous humour of 52 patients with PEX and 38 without PEX signs was sampled during cataract or glaucoma surgery. Total aqueous protein concentration in the samples was analysed in 43 PEX specimens and 32 non-PEX specimens according to Bradford. Aqueous protein composition of all samples was determined by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS PAGE) and silver staining. Screening for amyloids was performed in nine PEX samples and six non-PEX samples by Congo Red staining and polarised light microscopy. Aqueous protein concentration was not significantly increased in PEX eyes in comparison with non-PEX eyes. Furthermore, we could not detect any characteristic difference in protein band sizes of the two groups after SDS PAGE. However, we were able to show the presence of amyloid exclusively in aqueous humour of PEX patients. CONCLUSION: our results do not confirm a generally higher protein concentration in pseudoexfoliation syndrome eyes. This does not necessarily contradict a blood-aqueous barrier impairment but illustrates the variance in protein concentration between and within the two groups. No characteristic protein band allocatable to pseudoexfoliation syndrome proteins could be detected in any of the samples. However, our findings support the theory that the pseudoexfoliation syndrome is associated with an amyloid of a serum protein.

17 Alpha-oestradiol and 17 beta-oestradiol do not affect basal and follicle-stimulating hormone-stimulated inhibin B secretion by highly purified rat Sertoli cells.

The effects of 17 alpha-oestradiol and 17 beta-oestradiol on basal and follicle-stimulating hormone (FSH)-stimulated inhibin B secretion by rat Sertoli cells were studied. Sertoli cells were isolated and cultivated from testes of 18-day-old Wistar rats in the presence and absence of FSH and different doses of oestrogens. On day 4 of culture, secreted inhibin was measured by enzyme-linked immunosorbent assay. Neither 17 alpha-oestradiol nor 17 beta-oestradiol had any effect on the secreted inhibin level in either the presence or absence of FSH. It is concluded that these oestradiols do not play an essential role in regulatory processes involving inhibin or FSH.

[Efficiency of conventional glass wool and SpermFertil columns with respect to ROS-reduction, leukocyte reduction, and CASA-generated sperm counts in semen]. / Effizienz konventioneller Glaswollfiltration und SpermFertil hinsichtlich ROS-Reduktion, Leukozytenreduktion und Erhöhung des Anteils motiler Spermien im Seminalplasma.

OBJECTIVE: Glass wool filtration has proved to be useful to separate motile from defective spermatozoa and leukocytes in semen from patients with oligozoospermia and azoospermia. With regard to that, we set out to investigate the efficiency of two widely used filter systems. MATERIAL AND METHODS: Semen from 10 patients attending the Dept. of Andrology, FSU Jena, were investigated with home-made coarse glass wool filters and SpermFertil columns regarding elimination of reactive oxygen species (ROS) and enrichment of semen with motile and progressive spermatozoa. RESULTS: Filtration with home-made glass wool columns did not affect the percentage of motile spermatozoa but increased that of progressive spermatozoa. The whole cell count was decreased by a third. Leukocytes were reduced by half and ROS by more than half. SpermFertil columns increased the percentage of both motile and progressive spermatozoa and the whole cell count was decreased severely. Leukocytes were eliminated almost completely, as were ROS. CONCLUSIONS: Our results show the higher potential of SpermFertil columns to enrich semen specimens with motile and progressive spermatozoa and to almost completely eliminate leukocytes and ROS in comparison to home-made glass wool columns. However, SpermFertil columns reduce the whole cell count to about 10% of the original cell count.

Isolation of Acinetobacter spp. including A. baumannii from vegetables: implications for hospital-acquired infections.

A. baumannii is rarely recovered from the skin of patients or healthy European subjects as other genospecies predominate, but it isa significant nosocomial pathogen. The natural reservoir of this organism is therefore uncertain. We determined the isolation rates of Acinetobacter spp. from vegetables (as an indicator of the natural environment) using a selective technique and classified the genospecies by amplified ribosomal DNA restriction analysis (ARDRA). Of the 177 samples of vegetables examined, 30 yielded Acinetobacter, with genospecies 2 and 11 being the most common, each with a frequency of 27%. MIC assays showed that strains of genospecies 1, 2, 3, and 13TU (the A. calcoaceticus-A. baumannii complex) were significantly more resistant than other genospecies to ciprofloxacin and gentamicin. Vegetables may therefore be a natural habitat of A. baumannii and provide a route by which these bacteria are introduced into hospitals with obvious implications for infection control.

Distribution of Acinetobacter species on skin of healthy humans.

The distribution of the 19 currently known genospecies of Acinetobacter on human skin, i.e. forehead, forearm and toe webs, was determined. Three selective media were compared for their specificity for all genospecies of Acinetobacter. A minimal-salts agar supplemented with 1% acetate proved to be more efficient than the Leeds medium for the isolation of most genospecies in mixed culture with other bacterial species. Acinetobacter isolates were provisionally identified using biochemical tests and the DNA transformation assay of Juni. Genospecies identification was performed using amplified ribosomal DNA restriction analysis, and duplicate isolates of the same genospecies from individuals were ruled out by random amplified polymorphic DNA analysis. Over 40% of 192 healthy volunteers carried Acinetobacter spp. at one or more body sites, and the frequencies of colonisation were as follows: forearm (51%), forehead (47%) and toe web (34%). Genospecies 8/9 (Acinetobacter lwoffii) was the most common (61%), followed by genospecies 15BJ and 12 (Acinetobacter radioresistens) at 12.5% and 8%, respectively. The Acinetobacter baumannii-Acinetobacter calcoaceticus group (genospecies 1, 2, 3 and 13TU) that predominates in hospital-acquired infections was found in only one individual.

In situ hybridisation and direct fluorescence antibodies for the detection of Chlamydia trachomatis in synovial tissue from patients with reactive arthritis.

BACKGROUND: Chlamydia trachomatis is associated with Reiter's syndrome and reactive arthritis but the form in which the organism survives in synovial cells is unclear. AIM: To compare in situ hybridisation with direct fluorescence in the detection of inapparent chlamydial infection in synovial tissue. METHODS: Synovial tissue from four patients with reactive arthritis patients was examined using biotin labelled probes for chlamydial DNA and fluorescein isothiocyanate (FITC) labelled monoclonal antibodies against the major outer membrane protein. RESULTS: In two of the four patients, evidence of chlamydial infections was detected by in situ hybridisation in parallel sections but not with FITC labelled monoclonal antibodies. CONCLUSIONS: Detection of chlamydial DNA by in situ DNA hybridisation may be a better way to identify chlamydial infection in synovial tissue than phenotype targeting with FITC conjugated antibodies, which is used as a standard procedure for screening clinical specimens for chlamydia.