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.     

Differential uptake of systemic fluorochrome Hoechst 33342 in lung and liver metastasis of B16 melanoma

Summary The growth and vascularization patterns of B16 melanoma colonies in the liver and lungs were measured and compared by histological techniques and dye diffusion patterns after injection of the fluorochrome Hoechst 33342. In the liver, the fluorescent pattern of dye diffusion revealed that uninodular tumours measuring up to 146 n in diameter were not functionally vascularized. However, when the nodules fused to give rise to multinodular tumours measuring between 256 and 366 n in diameter, a reticular dye diffusion pattern revealed functional tumour vascularization. In the lungs, subpleural, parenchymal and peritubular (i.e. surrounding blood vessels and airways) tumours were observed. The two former classes were vascularized down to thicknesses and diameters of 49 and 24 m respectively. In contrast, dye diffusion was never seen in peritubular tumour cuffs up to 609 m in thickness. The results indicate differences in vascularization patterns in B16 tumours in the liver and lungs, and differences between tumours growing in different sites within the lungs. If these results are applicable to metastases in these two organs, they indicate potential diffusion-mediated resistance to chemotherapy, and potential hypoxia-mediated resistance to radiotherapy of both metastases and micrometastases.     

A light and electron microscopic study of osteogenesis imperfecta bone samples, with reference to collagen chemistry and clinical phenotype

A detailed morphological study was carried out using light and electron microscopy on 36 bone specimens from patients suffering from osteogenesis imperfecta (OI) and 20 age- and site-matched control bone specimens. The findings were grouped into the clinical types of OI according to the Sillence classification. The morphological and ultrastructural alterations observed in OI bone correlate well with clinical severity. Thus, OI type I, the mildest type, showed the least abnormalities in bone ultrastructure. OI type IV closely resembled type I, with only minor abnormalities in the bone cells and osteoid. OI type III showed abnormalities in the structure and distribution of osteoid collagen fibrils, whilst OI type II, the lethal form, revealed many varied abnormalities such as thin cortical bone, sparse trabecular bone, increased numbers of osteoclasts and osteocytes, thin osteoid with thin collagen fibrils, and patchy mineralization.     

DFNB39, a recessive form of sensorineural hearing impairment, maps to chromosome 7q11.22-q21.12

This article describes the identification of a novel locus (DFNB39) responsible for an autosomal recessive form of hearing loss segregating in a Pakistani consanguineous family. The hearing impaired members of this family present with profound prelingual sensorineural hearing impairment and use sign language for communications. Linkage was established to microsatellite markers located on chromosome 7q with a maximum multipoint lod score of 3.8. The region of homozygosity spans a 19 cM region that is bounded by markers D7S3046 and D7S644.     

Characterization and identification of Oya virus,a Simbu serogroup virus of the genus Bunyavirus,isolated from a pig suspected of Nipah virus infection

Summary.  A virus, named Oya virus, was isolated in Vero cell cultures from the lungs of a pig suspected of Nipah virus infection. The virus was revealed as a spherical enveloped RNA virus with a diameter of 79 nm. For identification of Oya virus, RT-PCR was performed. A common primer set for S-RNA of the Simbu serogroup of the genus Bunyavirus was able to amplify a cDNA from Oya virus RNA. The sequence data of the product revealed that the partial gene of Oya virus S-RNA segment had 65–70% homology with published cDNA sequences of Simbu serogroup viruses. The phylogenetic analysis of the data showed that the Oya virus is grouped in Simbu serogroup, but is genetically distinct from the serogroup viruses that have been analyzed molecularly. Serological surveys revealed that the virus distributed widely and densely in Malaysia. Received January 5, 2002; accepted April 16, 2002 Published online July 19, 2002     

PS2 and HSP70 expression in rectal adenocarcinomas: an immunohistochemical investigation of 45 cases.

OBJECTIVE: To evaluate the expression of HSP70 and pS2 and to determine whether it may be an additional prognostic variable in the prediction of recurrence and survival in rectal adenocarcinomas. METHODS: The paraffin sections of 45 patients with rectal carcinoma who were treated with surgical resection were stained with HSP70 and pS2 antibodies by using the standard biotin immunoperoxidase method. Cytoplasmic staining for both antibodies was scored semiquantitatively. RESULTS: Only 16 (35.6%) tumors showed a positive cytoplasmic reaction with HSP70 antibody, while pS2 expression was observed in 26 (57.8%) tumors. There was an association between HSP70 and pS2 expression (P=0.002). No correlations were found between HSP70 and pS2 expression and tumor recurrence or overall survival and other prognostic factors. However, the type of surgical resection was significantly associated with pS2 expression status (P=0.013). Significant correlations were detected between tumor recurrence and other clinicopathologic parameters, such as clinical stage, lymph node involvement, and resection type (P=0.015, P=0.015, and P=0.03, respectively). Resection type was significantly associated with clinical outcome, recurrence, and metastasis (P=0.009, P=0.03, P<0.01, respectively). In addition, there was a statistically significant relationship between clinical stage and final outcome (P=0.005). CONCLUSIONS: The strong correlation between pS2 expression and incomplete surgical resection suggests that pS2 may be related to invasive tumor behavior and may also play a role in tumor recurrence, although this latter association did not reach statistical significance in this study. HSP70 expression does not appear to be related to tumor invasiveness or tumor recurrence.     

Participation of serum and membrane lectins on the oxidative burst regulation in Macrobrachium rosenbergii hemocytes

Using a spectrophotometric NBT reduction assay and phagocytosis, we identified that production of superoxide anions and phagocytic activity of hemocytes from Macrobrachium rosenbergii were significantly higher in the presence of rat, rabbit, and chicken erythrocytes than with human, pig, or horse erythrocytes. Hemocytes stimulated with MrL, MrLMab, or PMA increased 4.7, 5.1, and 6.1 fold, respectively, the oxidative response as compared to non-stimulated hemocytes. MrLMab together with MrL increased 5.7 fold the oxidative capacity of hemocytes as compared to non-stimulated cells. These effects were inhibited with 100 mM GalNAc, GlcNAc, or Neu5Ac and 0.2 microM of sialylated submaxillary gland mucin and fetuin. Piroxicam inhibited (P < 0.05) the production of O(2)(-) induced by MrL, whereas iodoacetamide inhibited the effect of MrLMAb (P < 0.05) in a dose-dependent manner. Our results suggest that MrLMab might activate the oxidative burst through the metabolism of glucose as opposed to MrL which utilizes NADPH-independent mechanisms, very probably through pro-inflammatory metabolites.     

Epidemic typhoid in vietnam: molecular typing of multiple-antibiotic-resistant Salmonella enterica serotype typhi from four outbreaks

Multidrug-resistant Salmonella enterica serotype Typhi isolates from four outbreaks of typhoid fever in southern Vietnam between 1993 and 1997 were compared. Pulsed-field gel electrophoresis, bacteriophage and plasmid typing, and antibiotic susceptibilities showed that independent outbreaks of multidrug-resistant typhoid fever in southern Vietnam are caused by single bacterial strains. However, different outbreaks do not derive from the clonal expansion of a single multidrug-resistant serotype Typhi strain.     

The effect of foster feeding and bottle feeding expressed breast-milk on the susceptibility of guinea-pig infants to influenza virus

Infant guinea-pigs born to mothers immunized against influenza virus by infection during pregnancy were reared from birth by non-immune foster mothers. As a control for the effects of fostering, a similar group were fostered to immune mothers. Fostering, regardless of the immune state of the foster-mother, increased the susceptibility of the infant to upper respiratory tract infection. Increased susceptibility was associated with ablation of the infants IgM and IgA antibody responses and reduced secretion of transplacentally acquired IgG antibody in nasal secretions. In the reciprocal experiment, infants of non-immune mothers fostered to immune mothers cleared virus more rapidly than their peers who were fed by their own mothers. This protective effect was associated with an enhanced nasal IgM and IgA antibody response. Infants of immune mothers separated from their mothers at birth and hand-reared on a cow's-milk-based formula feed suffered an increased susceptibility to the virus similar to that seen in fostered infants. Addition of a pool of expressed milk from a group of immune mothers, including their own, to the feed of hand-reared infants did not reduce their susceptibility. However, a further group of infants fed a non-cellular whey fraction of the same milk pool secreted significantly lower titres of virus. This increased protection was associated with elevated levels of IgG antibody secretion into nasal washes early in infection.