A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes

Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P1(Glu)-P1'(Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues. The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system. Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions. Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 +/- 0.3 x 10(6) M-1 s-1, which is in the range for a physiologically significant serpin-proteinase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes. The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B. Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted. Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B. These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation. This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.     

Distribution of insertion sequence IS200 among different clonal lines of the related Salmonella serotypes Livingstone and Eimsbuettel

The copy number and genetic location of IS200 have provided evidence of strain relatedness in many serotypes of Salmonella. In this study, 100 isolates of the related serotypes Livingstone (6,7:d:l,w) and Eimsbuettel (6,7,14:d:l,w), representing 10 ribotype/biotype (RT/BT) groups isolated from human and non-human sources in seven countries over a 26-year period, were examined for their IS200 profiles. The distribution of IS200 in strains of these serotypes was limited, being present in all 53 isolates of ribotype 1 (RT1) and its variant type RT6, in one of five isolates of RT5 but in none of 42 isolates of RTs 2, 3 or 4. Although the seven IS200 profiles identified in RT1 isolates were of little value for further discrimination within different biotype groups, they were extremely valuable for confirming serotype: isolates of RT1/BT8/IS200 profile A (or its variants) and those of RT1/BT3/IS200 profile B (or its variants) were almost invariably associated with serotypes Livingstone and Eimsbuettel, respectively.     

Polarity influences the efficiency of recombinant adenoassociated virus infection in differentiated airway epithelia

To better understand mechanisms that limit rAAV transduction in the lung, we have evaluated several unique features of rAAV infection in polarized primary airway epithelial cultures. rAAV was found to transduce the basolateral surface of airway epithelia 200-fold more efficiently than the apical membrane. These differences in membrane infection correlated with the abundance of apical heparan sulfate proteoglycan (AAV-2 receptor) and virus binding. UV irradiation augmented rAAV transduction greater than 20-fold, only when virus was applied to the apical membrane. Ultrastructural analysis of UV-irradiated primary cultures demonstrated significant changes in microvilli architecture following exposure to 25 J/m2 UV. Although virus binding and the abundance of heparan sulfate proteoglycan were not increased at the apical membrane following UV irradiation, increased receptor-independent endocytosis of fluorescent beads was seen at the apical membrane following UV irradiation. We hypothesize that endocytotic processes associated with apical membrane-specific pathways of viral entry, and/or processing of virus to the nucleus, may be altered following UV irradiation. Interestingly, UV irradiation had an inhibitory effect on rAAV transduction from the basolateral membrane, which correlated with a decrease in the abundance of heparan sulfate proteoglycan at the basal membrane. In summary, these findings suggest that independent pathways of viral transduction may occur in the apical and basolateral compartments of polarized airway epithelia.     

Immunohistochemical diagnosis of typhus rickettsioses using an anti-lipopolysaccharide monoclonal antibody

A monoclonal antibody directed against an epitope on the lipopolysaccharide of typhus-group rickettsiae was developed for the purpose of detecting this heat-stable, proteinase-resistant antigen in formalin-fixed, paraffin-embedded tissues. Rickettsia prowazekii organisms were identified in endothelium and macrophages in sections of the brains of three Egyptian men who died of epidemic louse-borne typhus in Cairo during World War II and in the brain from a recent case of typhus fever acquired in Burundi. R. typhi organisms were identified in endothelial cells from a fatal case of murine typhus and in experimentally infected mice. This approach is applicable not only to the study of archival tissues and experimental animal models but also could be used to establish a timely diagnosis of typhus-group rickettsiosis by immunohistochemical examination of cutaneous biopsies of rash lesions during the acute stage of illness.     

Evaluation of the results of cleft lip and palate surgical treatment: preliminary report

We have analysed randomized controlled studies which reported the incidence of postoperative nausea and vomiting (PONV) after propofol anaesthesia compared with other anaesthetics (control). Cumulative data of early (0-6 h) and late (0-48 h) PONV were recorded as occurrence or non-occurrence of nausea or vomiting. Combined odds ratio and number-needed-to-treat were calculated for propofol as an induction or maintenance regimen, early or late outcomes, and different emetic events. This was performed for all control event rates and within a range of 20-60% control event rates. We analysed 84 studies involving 6069 patients. The effect of propofol on PONV was dependent mainly on the method of administration, time of measurement and range of control event rates. When all studies were included the number-needed-to-treat to prevent PONV with propofol was more than 9 when used for induction of anaesthesia and at best 6 when used for maintenance. Within the 20-60% control event rate range, best results were achieved with propofol maintenance to prevent early PONV: the number-needed-to-treat to prevent early nausea was 4.7 (95% confidence interval 3.8-6.3), vomiting 4.9 (4-6.1) and any emetic event 4.9 (3.7-7.1). Within the 20-60% control event rate, of five patients treated with propofol for maintenance of anaesthesia, one will not vomit or be nauseated in the immediate postoperative period who would otherwise have vomited or been nauseated. This may be clinically relevant. In all other situations the difference between propofol and control may have reached statistical significance but was of doubtful clinical relevance. Treatment efficacy should be established within a defined range of control event rates for meaningful estimates of efficacy and for comparisons.     

Posttraumatic stress disorder symptoms after bone marrow transplantation for breast cancer

Hormonal treatments which have an androgenic effect have the potential to cause vocal changes. The changes in vocal fold structure and voice quality are considered to be irreversible. To date, studies have documented subjective vocal changes or documented single cases without detailed, baseline voice assessments. The impact on laryngeal function of women taking these androgenic treatments requires further detailed, objective assessment. The need for increased awareness of the actions of androgenic hormones on womens' voices, and the benefits of a thorough voice assessment are discussed.     

Environment and health: an analysis of intra-urban differentials in the city of S?o Paulo, Brazil

A field study undertaken in the city of S. Paulo is presented as part of the project Environment and Health in Developing Countries: An Analysis of Intra-Urban Differentials Using Existing Data financed by the Ministry of Foreign Affairs of the United Kingdom with academic support from the London School of Hygiene and Tropical Medicine (LSHTM). The research aim was to fill in the gaps in the information needed to establish associations between mortality, urbanization and the environment. Statistics were produced by means of existing data collected by city departments, research carried out by universities and census data. Data quality was assessed taking into consideration data coverage, accuracy, and sensibility to pinpoint deprived areas in the city of S. Paulo. Socioeconomic data were used to create a composite index for districts and subdistrict in order to form four socioeconomic zones. Mortality differentials between the zones by five broad age groups (0-4, 5-14, 15-44, 45-64 e 65+) and four ICD chapters (circulatory, respiratory, infectious and parasitic and external causes) are presented. The zoning used showed that 43.8% of S. Paulo residents live in areas under the worst socioeconomic conditions. Health data demonstrated that people living in this areas had higher rates of mortality then those living in the areas with the best conditions. Finally, excess mortality data are analyzed and suggested as a good method for allocating health resources.     

Tissue reactions to bacteria-challenged implantable leads with enhanced infection resistance

Tissue reactions to implantable pacemaker leads were investigated in an early infection model in rabbits. Both standard leads and surface-modified leads were used. The surface modification technique was applied to achieve controlled release of the antibiotic gentamicin. The insulating polyurethane tubing material of the leads was provided with an acrylic acid/acrylamide copolymer surface graft and then loaded with gentamicin. Implantation periods varied from day 4, to week 3 1/2, to week 10. We investigated tissue reactions in the absence of an infectious challenge and also the efficacy of surface-modified leads in preventing infection after challenge with Staphylococcus aureus was evaluated. It was demonstrated that the applied surface modification did not induce adverse effects although during early postimplantation an increase in infiltration of granulocytes and macrophages and wound fluid and fibrin deposition were observed. After bacterial challenge, standard leads were heavily infected at each explantation period, denoted by abscesses, cellular debris, and bacterial colonies. In contrast, little or no infection was observed, either macroscopically or by bacterial cultures, with the surface-modified leads. Microscopy showed little evidence of the bacterial challenge, and that primarily at day 4. It was concluded that the applied surface modification demonstrated enhanced infection resistance and thus represents a sound approach to the battle against infectious complications with biomaterials.