Endopeptidase activities of selected Porphyromonas spp., Prevotella spp. and Fusobacterium spp. of oral and non-oral origin

The ability of three Porphyromonas spp., seven Prevotella spp., seven Fusobacterium spp. and two related Bacteroides spp. (B. levii and B. macacae) to degrade an extensive range of synthetic endo-, amino- and diamino peptidase substrates linked to the fluorescent leaving group 7-amido-4-methylcoumarin (NHMec) was investigated. Many more species than was previously recognized exhibited peptidase activities, albeit at lower levels than those already described for Porphyromonas gingivalis. Detection of chymotrypsin-like activity was dependent on which of three NHMec-linked substrates was used, but all species exhibited degradative activity with at least one of these substrates. Elastase-like activity was detected in all species though not all species reacted with each of the elastase substrates. Glycylprolyl peptidase activity was detected in all of the species tested with the exception of F. mortiferum, F. gonidiaformans, F. naviforme and F. necrophorum. While the detection of peptidase activities does not appear to be useful for the differentiation of species within the genera Bacteroides and Prevotella, its ability to differentiate species of the genus Porphyromonas or Fosobacterium further investigation.     

VS38: a new monoclonal antibody for detecting plasma cell differentiation in routine sections.

AIMS--To characterise a new mouse monoclonal antibody, VS38, which recognises an intracytoplasmic antigen of 64 kilodaltons present in normal and neoplastic plasma cells; and to establish its value as a diagnostic reagent for routine pathological practice. METHODS--A range of normal and neoplastic tissue sections, both frozen and routinely fixed, were immunostained, using the microwave method of antigen retrieval for routinely fixed specimens. The antibody was also tested on blood and bone marrow specimens and a range of human cell lines. The molecular weight of the antigen recognised by the antibody was obtained by western blot analysis. FACS analysis was used to demonstrate the cellular location of the antigen and its presence on tonsil cell suspensions and myeloma cases. RESULTS--VS38 recognised normal and neoplastic plasma cells in all of the tissues, including all routinely fixed plasma cell neoplasms tested. The antibody also weakly stained epithelial elements within the tissue but was absent from haemopoietic cells of other lineages. CONCLUSION--Antibody VS38 is of potential value in identifying myeloma or plasmacytoma in bone marrow or other tissues. It differentiates lymphoplasmacytoid lymphoma from lymphocytic and follicular lymphoma. It also subdivides large cell lymphomas into two groups which may be a more reliable method of separating these tumours than morphology alone.     

Influence of age on the reactivity of the BANA test among Brazilian children.

Three hundred and twenty samples of subgingival plaque were obtained from 80 caucasian girls, ranging from 10 to 13 years of age. The samples were analyzed to verify the influence of age upon colonization of the gingival sulcus by microorganisms potentially pathogenic to the periodontal tissues. The gingival and plaque status were evaluated through the gingival index (GI) and plaque index (PlI) and the microflora was assessed by the enzymatic method benzoyl-arginine-naphthylamide (BANA). The results of the BANA test were positive for 62.50% of the tested individuals and 40% of the examined sites. The influence of age was statistically significant on BANA reactivity, and the number of positive sites was greater at 11 (57.5%) than at 12 years (28.8%).     

Evidence for the presence of a type III secretion system in diffusely adhering Escherichia coli (DAEC).

Diffusely adhering Escherichia coli (E. coli) strains (DAEC) represent a potential cause of diarrhoea in infants, and the detection of type three secretion system (TTSS) genes in DAEC would substantiate their pathogenic nature. In this work, four isolates of DAEC, recovered from stools of diarrhoeic children, were analysed by PCR, in order to detect the presence of TTSS genes. Primers targeted to the escC, escJ, escN and escV, some of the most conserved TTSS genes in enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC), were used in order to verify the occurrence of homologous genes in our DAEC isolates. By this approach, we were able to characterise DNA fragments corresponding to putative escJ and escN genes in all DAEC isolates. Furthermore, DNA fragments homologous to the escC and escV genes were also amplified from all isolates. Besides the similarity found among the DAEC esc homologues with EPEC and EHEC esc genes, the nucleotide sequence analysis of the flanking regions of the amplified DNA fragments suggests that the putative DAEC esc genes are organised in the same manner as observed in EPEC and in EHEC strains. The results described here provide strong evidence for the presence of a TTSS in the DAEC strains analysed, implicating a pathogenic nature of these isolates.     

Comparative study of five different DNA fingerprint techniques for molecular typing of Streptococcus pneumoniae strains.

The aim of this study was to identify the strengths and weaknesses of five DNA fingerprint methods for epidemiological typing of Streptococcus pneumoniae. We investigated the usefulness of (i) ribotyping, (ii) BOX fingerprinting with the BOX repetitive sequence of S. pneumoniae as a DNA probe, (iii) PCR fingerprinting with a primer homologous to the enterobacterial repetitive intergenic consensus sequence, (iv) pulsed-field gel electrophoresis of large DNA fragments, and (v) restriction fragment end labeling to detect restriction fragment length polymorphism of small DNA fragments. Twenty-eight S. pneumoniae strains isolated from the blood and/or cerebrospinal fluid of 21 patients were analyzed. Genetic clustering among the 28 strains was independent of the DNA fingerprint technique used. However, the discriminatory power and the similarity values differed significantly among the individual techniques. BOX fingerprinting, pulsed-field gel electrophoresis, and restriction fragment end labeling provided the highest degree of discriminatory power. Furthermore, the ease with which computerized fingerprint analysis could be conducted also varied significantly among the techniques. Ribotyping, BOX fingerprinting, and restriction fragment end labeling were very suitable techniques for accurate computerized data analysis. Because of their high discriminatory potential and ease of accurate analysis, we conclude that BOX fingerprinting and restriction fragment end labeling are the most suitable techniques to type pneumococcal strains.     

Guillain-Barré syndrome in South-West Stockholm, 1973–1991, 3. Clinicoepidemiological subgroups

Using hierarchical cluster analysis, applied to 47 cases of Guillain-Barre Syndrome (GBS) incident in South-West Stockholm (SWS) during the period from January 1973 to June 1992, we identified three major clinicoepidemiological subgroups. The first subgroup, 25.5% of the cases (26.7 ± 6.7 years), recorded a peak incidence at ages 20–29 years and presented significant differences from other subgroups, a high proportion of cases with onset at low age preceded by respiratory infection (83.3%) and with normal motor conduction velocity (50.0%). Also found, were less affected biological parameters, a rapidly progressive course and independence in gait at one month after onset. A second subgroup, 27.7% of cases, was severely affected, clinically and functionally. It consisted predominantly of young individuals (22.7 ± 11.1 years), with a high incidence (69.2% of cases) in autumn. A third subgroup, comprising 40.47; of cases, was older (61.1 ± 11.0 years) and, in general, also severely affected. The incidence of this form appeared to be invariant with time.     

A chemically defined medium for the culture of mature oligodendrocytes

A new chemically defined medium consisting of equal parts of Dulbecco modified Eagle's and Ham's F-12 media supplemented with insulin, sodium selenite, putrescine, and D+ galactose, which allows the long-term survival of mature oligodendrocyte pure cultures, is described. Immunohistochemical staining has shown that over 90% of the cells become positive for myelin proteins shortly following subculture. Contaminating astrocytes (2%) do not survive in this medium. Biochemical data have indicated that these purified oligodendrocytes express 2'3'-cyclic nucleotide 3'-phosphohydrolase and UDP-galactose ceramide galactosyltransferase activities. Electron microscopical examination revealed that the oligodendrocytes were mostly of medium-dark type and appeared to be identical to cells cultured in serum-containing medium. The ability to maintain pure oligodendrocyte cultures in such a defined medium will allow investigations concerning exogenous and endogenous factors involved in oligodendrocyte metabolism.     

Vitrectomy for idiopathic epiretinal membranes causing macular pucker.

We used vitreous surgery to remove idiopathic epiretinal membranes from the macular area in 70 consecutive cases. The abnormal tissue was successfully removed in each case. Vision improved postoperatively in 61 eyes (87%), remained unchanged in six eyes (9%), and worsened in three eyes (4%). However, at the time of final examination vision was improved in only 47 eyes (67%), primarily because of the occurrence or progression of nuclear sclerosis, which occurred in 38 of 60 phakic eyes (63%). Four preoperative factors were associated with final visual acuity of 20/60 or better: (1) initial vision of 20/100 or better, (2) shorter preoperative duration of blurred vision, (3) thin epiretinal membrane, and (4) absence of traction retinal detachment.