Identification of Aeromonas strains to the genospecies level in the clinical laboratory.

One hundred thirty-three strains of Aeromonas (human, n = 102; animal, n = 16; environmental, n = 15) previously identified to the DNA group level by molecular methods were biochemically analyzed for 58 properties. On the basis of the use of between 9 and 16 selected tests, 132 of the 133 strains (99%) could be assigned to their correct hybridization group using this biochemical scheme. The results suggest a feasible approach for identifying aeromonads to genospecies level under appropriate conditions.     

Bcl-2 and p53 protein expression, apoptosis, and p53 mutation in human epithelial ovarian cancers

Bcl-2 and p53 gene products have been both linked to cell death by apoptosis. In the present study, we examined the relationship of Bcl-2 and p53 protein expression, p53 mutation and apoptosis in normal human ovaries and different types of human ovarian epithelial tumors by immunohistochemical localization, in situ terminal transferase-mediated dUTP nick end labeling and polymerase chain reaction-single strand conformation polymorphism. It was found that Bcl-2 expressed strongly in the surface epithelium of normal ovaries and benign and borderline ovarian tumors but weakly in the malignant tumors. On the contrary, strong protein expression of p53 was found in 54% (25/46) of the malignant epithelial tumors examined but similar expression of p53 was not observed in borderline and benign tumors and normal ovarian surface epithelium. A significant inverse correlation between Bcl-2 and p53 expression was found in the malignant ovarian tumors examined. p53 gene mutation at exons 5-11 was however not a pre-requisite for p53 expression in both borderline and malignant tumors. Apoptotic activities, as reflected by apoptotic indices, were low in normal ovarian surface epithelium and benign tumors but were increased in borderline and malignant tumors, with the highest average apoptotic index found in grade III malignant tumors. Statistical analyses showed a positive correlation between apoptosis and p53 expression, but similar correlation was not found between apoptosis and Bcl-2 expression. Our results also indicate that although expression of Bcl-2 is important during ovarian carcinogenesis, the Bcl-2 protein may have other roles to play apart from being a modulator of apoptosis in human ovarian epithelial cancers.     

Staphylococcus aureus induces platelet aggregation via a fibrinogen-dependent mechanism which is independent of principal platelet glycoprotein IIb/IIIa fibrinogen-binding domains.

Platelet aggregation by bacteria is felt to play an important role in the pathogenesis of infective endocarditis. However, the mechanisms involved in bacterium-induced platelet aggregation are not well-defined. In the present study, we examined the mechanisms by which Staphylococcus aureus causes rabbit platelet aggregation in vitro. In normal plasma, the kinetics of S. aureus-induced platelet aggregation were rapid and biphasic. The onset and magnitude of aggregation phase 1 varied with the bacterium-platelet ratio, with maximal aggregation observed at a ratio of 5:1. The onset of aggregation phase 2 was delayed in the presence of apyrase (an ADP hydrolase), suggesting that this later aggregation phase may be triggered by secreted ADP. The onset of aggregation phase 2 was delayed in the presence of prostaglandin I2-treated platelets, and this phase was absent when paraformaldehyde-fixed platelets were used, implicating platelet activation in this process. Platelet aggregation phase 2 was dependent on S. aureus viability and an intact bacterial cell wall, and it was mitigated by antibody directed against staphylococcal clumping factor (a fibrinogen-binding protein) and by the cyclooxygenase inhibitor indomethacin. Similarly, aggregation phase 2 was either delayed or absent in three distinct transposon-induced S. aureus mutants with reduced capacities to bind fibrinogen in vitro. In addition, a synthetic pentadecapeptide, corresponding to the staphylococcal binding domain in the C terminus of the fibrinogen delta-chain, blocked aggregation phase 2. However, phase 2 of aggregation was not inhibited by two synthetic peptides (alone or in combination) analogous to the two principal fibrinogen-binding domains on the platelet glycoprotein (GP) IIb/IIIa integrin receptor: (i) a recognition site on the IIIa molecule for the Arg-Gly-Asp (RGD) sequence of the fibrinogen alpha-chain and (ii) a recognition site on the IIb molecule for a dodecapeptide sequence of the fibrinogen delta-chain. This differs from ADP-induced platelet aggregation, which relies on an intact platelet GP IIb/IIIa receptor with an accessible RGD sequence and dodecapeptide recognition site for fibrinogen. Furthermore, a monoclonal antibody directed against the RGD recognition site on rabbit platelet GP IIb/IIIa receptors failed to inhibit rabbit platelet aggregation by S. aureus. Collectively, these data suggest that S. aureus-induced platelet aggregation requires bacterial binding to fibrinogen but is not principally dependent upon the two major fibrinogen-binding domains on the platelet GP IIb/IIIa integrin receptor, the RGD and dodecapeptide recognition sites.     

Use of PCR to study epidemiology of Serratia marcescens isolates in nosocomial infection.

A method to characterize strains of Serratia marcescens based on the PCR amplification of enterobacterial repetitive intergenic consensus sequences has been developed. The PCR fingerprints were generated from boiled supernatants prepared directly from bacterial colonies without the need for DNA extraction. The technique was applied to isolates obtained during an outbreak of pneumonia from seven mechanically ventilated patients, and its result indicated that the outbreak was due to the spread of two epidemic strains. This technique was validated by comparison with rRNA gene restriction analysis. There was complete concordance between these two techniques in discriminating the outbreak-related strains from epidemiologically unrelated isolates. Typing with both biochemical profile and antibiogram profile, though simple, was found to be less reliable than genotyping. The results show that this enterobacterial repetitive intergenic consensus PCR provides a rapid and simple means of typing S. marcescens isolates for epidemiologic studies.     

Assessment of cell proliferation in hydatidiform mole using monoclonal antibody MIB1 to Ki-67 antigen.

AIMS--To assess the role of Ki67 immunoreactivity in predicting the clinical progress of hydatidiform mole. METHODS--Tissue from 87 hydatidiform moles, 11 normal first trimester placentas, 11 normal term placentas and 17 spontaneous abortions were examined for expression of Ki67 antigen, using the monoclonal antibody MIB1. RESULTS--Ki67 immunoreactivity was significantly higher in the tissue from normal first trimester placentas than in that from normal term placentas and spontaneous abortions. Among the 87 patients with hydatidiform moles studied, 20 developed persistent gestational trophoblastic disease and required subsequent treatment. There was no statistically significant difference in the Ki67 index between the 20 patients who developed persistent disease and those who did not. CONCLUSION--Hydatidiform moles which give rise to persistent trophoblastic disease do not have a higher proliferative rate than those which do not. The Ki67 index is not useful for predicting the prognosis of molar pregnancies.     

Carbon plasma immersion ion implantation of nickel-titanium shape memory alloys

Nickel-titanium (NiTi) shape memory alloys possess super-elasticity in addition to the well-known shape memory effect and are potentially suitable for orthopedic implants. However, a critical concern is the release of harmful Ni ions from the implants into the living tissues. We propose to enhance the corrosion resistance and other surface and biological properties of NiTi using carbon plasma immersion ion implantation and deposition (PIII&D). Our corrosion and simulated body fluid tests indicate that either an ion-mixed amorphous carbon coating fabricated by PIII&D or direct carbon PIII can drastically improve the corrosion resistance and block the out-diffusion of Ni from the materials. Our tribological tests show that the treated surfaces are mechanically more superior and cytotoxicity tests reveal that both sets of plasma-treated samples favor adhesion and proliferation of osteoblasts.     

In vitro method to study antifungal perfusion in Candida biofilms

Antimycotic perfusion through Candida biofilms was demonstrated by a modification of a simple in vitro diffusion cell bioassay system. Using this model, the perfusion of three commonly used antifungal agents, amphotericin B, fluconazole, and flucytosine, was investigated in biofilms of three different Candida species (i.e., Candida albicans, Candida parapsilosis, and Candida krusei) that were developed on microporous filters. Scanning electron microscopy revealed that C. albicans formed a contiguous biofilm with tightly packed blastospores and occasional hyphae compared with C. parapsilosis and C. krusei, which developed confluent biofilms displaying structural heterogeneity and a lesser cell density, after 48 h of incubation on nutrient agar. Minor structural changes were also perceptible on the superficial layers of the biofilm after antifungal perfusion. The transport of antifungals to the distal biofilm-substratum interface was most impeded by C. albicans biofilms in comparison to C. parapsilosis and C. krusei. Fluconazole and flucytosine demonstrated similar levels of perfusion, while amphotericin B was the least penetrant through all three biofilms, although the latter appeared to cause the most structural damage to the superficial cells of the biofilm compared with the other antifungals. These results suggest that the antifungal perfusion through biofilm mode of growth in Candida is dependent both on the antimycotic and the Candida species in question, and in clinical terms, these phenomena could contribute to the failure of Candida biofilm-associated infections. Finally, the in vitro model we have described should serve as a useful system to investigate the complex interactions that appear to operate in vivo within the biofilm-antifungal interphase.     

Expression of c-myc and c-fms oncogenes in trophoblastic cells in hydatidiform mole and normal human placenta.

AIMS: To compare the expression of c-myc and c-fms proto-oncogenes in the placenta and hydatidiform mole. METHODS: Twelve hydatidiform moles and six induced abortion cases were collected. c-myc and c-fms proto-oncogene expression was analysed by northern blot hybridisation and immunohistochemical staining. RESULTS: The results of northern blot hybridisation analysis showed that c-fms was expressed more strongly in hydatidiform moles compared with normal placenta of similar gestational age. Moreover, c-fms mRNA concentrations increased with more advanced gestational age in moles but not in normal placentas. c-myc expression was very low in hydatidiform moles and normal placentas. Both oncogenes, however, had no direct correlation with the clinical course of the molar pregnancies. CONCLUSION: The difference in c-fms expression between hydatidiform moles and normal placentas suggests that c-fms may have a role in the development of molar pregnancies.     

Protective efficacy of protein A-specific antibody against bacteremic infection due to Staphylococcus aureus in an infant rat model.

Staphylococcal protein A (SpA) is a potent antiphagocytic component of the cell wall of most pathogenic Staphylococcus aureus strains. We studied the in vitro opsonophagocytic and in vivo protective activities of rabbit immunoglobulin G (IgG) antibody to purified SpA obtained from two unencapsulated S. aureus strains (Cowan I and 17A). Postimmune serum contained high titers of specific IgG to SpA, as measured by a modified enzyme-linked immunosorbent assay that blocked nonspecific binding of IgG to SpA. In vitro, both S. aureus strains were efficiently phagocytosed and killed by polymorphonuclear leukocytes in the presence of nonimmune sera and complement. With one strain (Cowan I), opsonophagocytosis was significantly enhanced in the presence of SpA antibody, but with the other strain (17A), killing was significantly decreased with immune serum. We then evaluated the potential protective benefit of SpA antibody in preventing S. aureus bacteremia in infant rats. Two-day-old rats received saline or various doses of SpA antiserum and were challenged subcutaneously 1 day later, but even the highest levels of antibody did not significantly reduce mortality, bacteremia or metastatic infection to lungs or liver (frequency or magnitude). This lack of protective efficacy was not related to a failure of SpA F(ab')2 to bind to cell surface-exposed epitopes, since F(ab')2 fragments prepared from hyperimmune serum bound avidly to the whole organism in an enzyme-linked immunosorbent assay.