Impact of mutations in hVISA isolates on decreased susceptibility to vancomycin, through population analyses profile - area under curve (PAP-AUC).

We analyzed sequences of graSR, vraSR, walKR and rpoB genes in hVISA from Brazil. Five isolates showed mutations in at least one gene. rpoB H481N and graS T224I were the most frequent mutations, followed by graR D148Q and walK A468T. Our study reinforces the heterogeneity of genetic patterns among hVISA.

Population studies of the Y-chromosome of loci DYS390, DYS391 and DYS393 in Brazilian subjects and its use in human identification.

Human identification can be accomplished by several technical procedures, especially by the comparative analysis of dental documents. Recent advances in molecular biology have now widened the scope for human identification and defined the protocols for the collection of biological material. Allele patterns of a population are particularly useful and they should be verified since they vary in different populations. We have studied the frequency of the alleles in three STR loci of the Y chromosome (DYS390, DYS391 and DYS393) in a group of Brazilian caucasian subjects. Results presented alleles 21, 22, 23, 24, 25 and 26 in locus DYS390, alleles 8, 9, 10, 11 12 and 13 in STR DYS391 and alleles in STR DYS393 were 11, 12, 13 14 and 15. The highest frequencies were 24 (0.46), 11 (0.37) and 13 (0.45). Each of the three STR systems had a PE (power of exclusion) of 0.6764 (DYS390), 0.5988 (DYS391) and 0.6136 (DYS393), The combination of the three STR systems revealed a PE of 0.9498, suggesting that this data can help in human identification.

[Evaluation of 3 methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA using PCR]. / Avaliação de três métodos de extração de DNA de material parafinado para amplificação de DNA genômico pela técnica da PCR.

There are several protocols reported in the literature for the extraction of genomic DNA from formalin-fixed paraffin-embedded samples. Genomic DNA is utilized in molecular analyses, including PCR. This study compares three different methods for the extraction of genomic DNA from formalin-fixed paraffin-embedded (inflammatory fibrous hyperplasia) and non-formalin-fixed (normal oral mucosa) samples: phenol with enzymatic digestion, and silica with and without enzymatic digestion. The amplification of DNA by means of the PCR technique was carried out with primers for the exon 7 of human keratin type 14. Amplicons were analyzed by means of electrophoresis in an 8% polyacrylamide gel with 5% glycerol, followed by silver-staining visualization. The phenol/enzymatic digestion and the silica/enzymatic digestion methods provided amplicons from both tissue samples. The method described is a potential aid in the establishment of the histopathologic diagnosis and in retrospective studies with archival paraffin-embedded samples.

Capacidade fagocítica de polimorfonucleares (PMN) humanos frente a várias partículas: Candida albicans, Zymosan, Staphylococcus aureus, Micrococcus, Escherichia coli / Phagocytosis capacity of the human neutrophils (PMN) against some particles

O processo de fagocitose envolve a ligação de partículas a receptores de superfície, resultando em indução de sinais intracitoplasmáticos e internalização de partículas, tendo importante papel na evolução das moléstias infecciosas, principalmente das bacterianas. Neste trabalho, nos propusemos a estudar a fagocitose “in vitro”, por meio do isolamento de PMN do sangue periférico de 17 amostras provenientes de doadores do Hemocentro do HURNP colocados em contato com as diversas partículas acima citadas. Com o objetivo de se obter a indicação da partícula mais adequada para uso na rotina laboratorial, as características avaliadas foram a capacidade fagocítica e a facilidade de leitura ao microscópio ótico. Os resultados obtidos indicam maior capacidade fagocítica (em porcentagem de células que apresentaram fagocitose) quando se faz uso da partícula de Zymosan (77 por cento) seguido de C. albicans (70 por cento) e o maior índice de partículas fagocitadas (em porcentagem de células que fagocitaram acima de 4 partículas) quando se fez uso de Zymozan (50 por cento) seguido de S. aureus (37 por cento) permitindo concluir que destas partículas a mais adequada para uso na rotina laboratorial é o Zimosan.

[Contamination of flexible fiberoptic bronchoscopes with Mycobacterium chelonae linked to an automated endoscope disinfection machine--on the relationship between the presence of the organism in the intestinal tract and contamination of disinfection machine, and a case of gallbladder and bile duct infection with M. chelonae].

In 1993, thirteen strains (8.7%) of M. chelonae were isolated from bronchoalveolar lavage fluid (BALF) obtained by bronchoscopy of 150 patients in Tachikawa-sogo (T) hospital, where the same automated disinfection machine was commonly used for cleaning, sterilization and disinfection of fiberbronchoscope and fibercolonoscope except 3 bronchoscopes disinfected by gas sterilization. Since January 1994, manual cleaning and sterilization has been applied for bronchoscope, and thereafter no strain of M. chelonae was isolated from BALF of 55 patients in the T hospital. While only one strain (3%) of M. chelonae was isolated from BALF of 33 patients in Ota (O) hospital, but many strains of M. chelonae were isolated from intestinal fluid obtained by fibercolonoscopy of the patients. Manual method of cleaning and disinfection was performed for both bronchoscopes and colonoscopes in the O hospital from the beginning. Based on these results, it was suggested that M. chelonae are commonly present in the colon (intestine) of normal persons. Thus colonoendscope may be often contaminated with the organism and subsequently the automated disinfecting machine may also be contaminated with the organism which is resist and against usual disinfection procedure, and resulted in bronchoscope contamination. If the presence of M. chelonae in intestinal tract is not rare, bile duct may be naturally infected with the organism. A case of cholecystitis and cholangitis caused by M. chelonae, which has not been reported previously, was found in the T hospital.

The functional significance of the second extracellular loop of thyrotropin receptor in thyrotropin- and thyroid stimulating antibody-dependent signal transduction.

In order to determine the functional significance of the extracellular loop of human thyrotropin receptor (hTSHR), two peptides composed of eight amino acids were inserted into hTSHR by ligating synthetic oligonucleotides into +1811 NCol site of hTSHR cDNA. Mutant hTSHR cDNAs which encode a hydrophobic peptide (ATVLVVPM) and a hydrophilic peptide (GTTRTVAM) between +572 Met and +573 Asp were transfected into Chinese hamster ovary (CHO) cells to develop F-cell lines and R-cell lines, respectively. Of the resulting cloned cell lines, F-29 and R-9 were shown to express mutant hTSHs at the protein level by Western blotting and at the mRNA level by reverse transcription-polymerase chain reaction (RT-PCR). We show that neither thyrotropin (TSH) nor IgGs from patients with Graves' disease stimulated cAMP production by F-29 and R-9 cells. 125I-TSH binding study revealed that F-29 and R-9 cells do not bind TSH. Our data demonstrate that the mutations impaired TSH-binding and incapacitated the cells from responding to TSH. The evidence suggests that the second extracellular loop of hTSHR has an important role in TSH and thyroid stimulating antibody (TSAb)-dependent signal transduction.

[Neurocysticercosis with initial clinical picture of acute meningitis]. / Neurocisticercose com quadro clínico inicial de meningite aguda.

Twenty seven cases of neurocysticercosis, with clinical picture of acute meningitis, are described. Twenty (74.1%) patients are male; the age was 4 to 42 years (23.6 +/- 11.7 years). The etiologic diagnosis was defined by the complement fixation test (Weinberg) and/or enzyme-linked immunosorbent assay (ELISA) for cysticercosis in the cerebrospinal fluid (c. s. f.). Six patients that realized cranial computerized tomographic scan resembling neurocysticercosis. Twenty one (77.8%) have predominance of lymphomononuclear cells in the c. s. f. obtained in the admission to the hospital; in 6 (22.2%) there were predominance of polymorphonuclear neutrophils. In this c. s. f. lymphomononuclear pleocytosis and in three that have c. s. f. neutrophil pleocytosis, suggesting the diagnosis of neurocysticercosis. The treatment of acute neurocysticercosis was made with dexamethasone. All the patients survived and were transferred to the ambulatory of Neurology for follow-up and complementary treatment.

Thyrotrophin-dependent desensitization by Chinese hamster ovary cells that express the recombinant human thyrotrophin receptor.

To determine whether thyrotrophin (TSH)-induced desensitization requires a thyroid-specific factor(s), the human TSH (hTSH) receptor was expressed in Chinese hamster ovary cells. The first incubation of the cells with TSH decreased the subsequent response of adenosine 3',5'-cyclic monophosphate to freshly added TSH in the second incubation. This homologous desensitization was observed as early as after 3 h of the first incubation. The lowest dose of TSH that elicited desensitization was 0.1 nmol/l. The desensitization was not overcome by adding higher doses of TSH in the second incubation. A 125I-labelled TSH-binding study revealed a decrease in the number of high-affinity binding sites but not in that of low-affinity binding sites. The data suggest that TSH-induced desensitization in hTSH receptor-transfected cells is caused, at least in part, by a decrease in the number of TSH receptors on the cell surface. The evidence demonstrates, contrary to an earlier report, that a thyroid-specific factor(s) is not required for hTSH receptor desensitization.

Detection of verocytotoxin from stool and serological testing of patients with diarrhea caused by Escherichia coli O157 : H7.

The detection of verocytotoxin (VT) in stool and measurement of antibodies against VT and three antigens (unheated-antigen, LPS, and flagellin) of Escherichia coli O157 : H7 in the serum of patients with diarrhea were examined. Five of 14 inpatients during an outbreak had fecal VT2 in stool taken within 5 days of onset to hospitalization. Among these 5, 3 of them also had fecal VT-producing E. coli (VTEC) serotype O157 : H7, whereas the other 2 did not. In the passive hemagglutination (PHA) test with formalinized sheep red blood cells sensitized with three VTEC O157 : H7 antigens, 49 (74.2%) of 66 outbreak patients and 3 of 3 sporadic cases had antibodies against both or one of unheated-antigen and LPS of E. coli O157, but none had antibody against flagellin. In addition, anti-VT2 antibody was demonstrated in serum samples from 15 (94%) of 16 inpatients and 2 (4%) of 50 outpatients in an outbreak by a VT-enzyme-linked immunosorbent assay (VT-ELISA). These results showed that serological assay particularly for antibodies against VT and unheated-antigen or LPS of VTEC O157 may provide a useful tool for diagnosis of infection with VTEC O157.

Single subunit structure of the human thyrotropin receptor.

We have produced rabbit antibody against a synthetic peptide corresponding to N-terminal region of the extracellular domain of human thyrotropin receptor (hTSH-R) (N peptide, aminoacid residues 29-57). Western blot analysis revealed that N-peptide antibody recognized recombinant hTSH-R stably expressing in CHO-K1 cells as a mol. wt. about 104 kDa regardless in the presence or absence of disulfide-reducing agent. The band was not detected in untransfected CHO-K1 cells and no band was also stained by the antibody absorbed with N-peptide. In a reducing condition, the antibody also bound the rat receptor from FRTL5 cells as the same molecular size (104 kDa). These results clearly indicate that TSH-R is composed of a single subunit and that two subunit model for the TSH-R may reflect artifactual proteolytic cleavage of the receptor during membrane preparation.

Heterogeneous responses of recombinant human thyrotropin receptor to immunoglobulins from patients with Graves' disease.

Non-thyroid mammalian cells, CHO-K1 cells, stably expressing human thyrotropin receptor (CHO-TSH-R cells) were used for the assay of thyroid stimulating antibody (TSAb) activities of IgGs from 24 patients with Graves' disease and we compared them with the values obtained in porcine thyroid cells. A significant positive correlation was observed between the results given by CHO-TSH-R cells (hTSAb) and porcine thyrocytes (pTSAb) (r = 0.94, p less than 0.001). However, we found that hTSAb values of IgGs from 5 patients were extremely different from their hTSAb values. Four out of these 5 IgGs showed strong pTSAb activity but exhibited a weak or negative hTSAb activity. Conversely, one out of 5 autoantibodies was very strong for hTSAb but its pTSAb was low. These heterogeneous responses of recombinant hTSH-R to Graves' IgGs suggest that there exist different types of TSAb and also that the epitope(s) for TSAb may be different from case to case.

[Studies on epidemiology of nontuberculous mycobacteriosis--on the regional difference of the incidence of pulmonary diseases due to M. kansasii and M. avium complex in Tokyo area].

On 397 patients with pulmonary disease due to nontuberculous mycobacteria (NTM) [102 due to M. kansasii (MK) and 295 due to M. avium complex (MAC)] observed at National Tokyo Chest Hospital, 191 patients with pulmonary disease (59 due to MK and 132 due to MAC) observed at Fukujuji Hospital of Antituberculosis Association and 257 patients from whose sputum MK (36) or MAC (221) were isolated in Byotai-Seiri Clinical Laboratory, the distribution of these patients by domicile in Tokyo area was analysed. The percentage of patients with MK disease among the whole patients with MK disease and MAC disease (MK ratio) in each community area was investigated. MK ratio was 30.9% in the 23 wards and 16.4% in Tama section of Tokyo in the patients observed at National Tokyo Chest Hospital. It was 37.0% in the 23 wards and 32.1% in Tama section in the patients observed at Fukujuji Hospital and was 17.7% in the 23 wards and 8.1% in Tama section in the patients observed at Byotai-Seiri Clinical Laboratory. MK ratio in Tokyo was considerably higher in the 23 wards that were densely populated industrial and commercial areas than in Tama section, a comparatively sparsely populated suburb. MK ratio in patients of Ota Hospital located in the 23 wards was higher (36.4%) than that of Tachikawa-Sogo Hospital located in Tama area (10.0%). Regional differences in MK ratio were remarkable in Tokyo area. A high MK ratio appeared to correlate with a high incidence rate of tuberculosis. From the results mentioned above, it was suggested that M.K. disease may be transmitted from person to person.

[123I-iodoamphetamine single photon emission computed tomography in three patients with amyotrophic lateral sclerosis].

We examined the 123I-iodoamphetamine SPECT for 3 patients with ALS, who were clinically diagnosed. Patient 1 was a 31-years-old man, who had bilateral muscle weakness of his upper extremities, and spasticity in lower extremities. Patient 2 was a 51-years-old woman, who had marked weakness of her upper extremities and bulbar sign. Patient 3 was a 68-years-old man, who had severe degree of marked weakness of his upper extremities and mild bulbar signs. Cerebral cognitive function were all normal in three patients. Computed tomographic and magnetic resonance imagings showed moderate degree of cortical atrophy in patient 1, but no abnormalities in patients 2 and 3. In 123I-IMP SPECT, however, hypoperfusion were recognized on the bilateral fronto-parietal border zone areas in these three patients with ALS. It was suggested that patients with ALS showed varying degrees of impaired perfusion in the fronto-parietal border zone areas in spite of normal cognitive functions.

[Basic and clinical studies on the effect of cefmetazole on infection in gyneco-obstetrics].

Cefmetazole (CMZ) was administered to 102 infectious cases in the gynecoobstetric patients, and the basic and clinical studies have been performed. The main findings obtained in the present study are: Amont 78 cases of CMZ administration immediately after the initial infection (A group), 42 remarkably effective (53.8%), 36 effective (46.2%) cass were observed. When other drugs were not effective after the initial infection, CMZ was administered (B group) to 24 cases, and 15 remarkably effective (62.5%), 9 effective (37.5%) cases were observed. overall effectiveness in the A and B groups was 57 remarkably effective (55.9%) and 45 effective (44.1%) cases, which are very excellent clinical effects. Among 102 cases, the pathogenic bacteria were found in 55 cases, and 29 cases out of the 55 (52.7%) showed infections with E. coli and with other bacteria having mixed infections. The effect of CMZ to E. coli as judged by MIC was excellent, providing the excellent clinical results. E. coli and other Gram-negative pathogenic bacteria in the B group showed resistance to ABPC, CEX, CEZ and CET, and after administering CMZ, all cases showed disappearance of these bacteria, but the increase in the resistant bacteria for CEX, CET and CEZ was obviously shown. Subjective and objective adverse effects and clinical laboratory analysis showed no abnormal effect and values due to CMZ, and it was true of the of the case received 140 g in total of CMZ over 35 days, 4 g/day. It may be conducted from the above findings that CMZ is very effective and safe antibiotic agent in the infections in the gynecoobsterics .