[Show abstract][Hide abstract] ABSTRACT: Mice were inoculated intranasally with Streptococcus pneumoniae isolates of serotype 14 with different genetic backgrounds (14R, 14DW) and a capsular switch of 14R, strain 9VR (serotype 9V). Inoculation of the mice with 14R and 9VR resulted in 60% mortality. All the mice survived 14DW inoculation. No differences in lungs' bacterial loads were found 3 h following inoculation. Bacterial clearance of 5 logs was observed 48 h after inoculation with 14DW versus within 1 log 48 h after inoculation with 14R and 9VR. No significant differences in bacterial size or the capsular amount could be found between 14R and 14DW. We conclude that factor(s) in addition to the capsule, contribute to disease outcome.
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae infection may result in asymptomatic carriage, mucosal or invasive disease. We hypothesize that self-limiting or fatal disease outcome follows infection with S. pneumoniae differential activation of the host immune response. BALB/c and C57BL/6 mice were inoculated intranasally with S. pneumoniae serotype 3 strain WU2 and serotype 14 strain DW14 and mortality, bacterial load, pathological changes in the lungs and cytokines mRNA levels in the spleen were analysed. No differences between the C57BL/6 and the BALB/c inbred mice were observed except for the severity of their lung pathology and IL-4 expression. Infection of the two mouse strains with S. pneumoniae WU2 resulted in sepsis and death that occurred within 4 days post-inoculation. This death was preceded, in both mouse strains, in an increase over time of the lung bacterial load and bacteraemia. The lung pathology was characterized by diffuse pneumonia with marked congestion of the lungs. Analysis of mRNA expression of cytokines in the spleen revealed no alterations in tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, interleukin (IL)-12 and interferon (IFN)-gamma and induction of IL-10 and IL-4. The two strains of mice survived infection with S. pneumoniae DW14. This was accompanied by a reduction over time of lung bacterial load and bacteraemia. The lung pathology was characterized by focal lymphocyte infiltration and preserved architecture of the organ. Analysis of mRNA expression of cytokines in the spleen revealed a significant decrease in the levels of TNF-alpha, TGF-beta, IL-12 and IFN-gamma mRNA expression, which usually precedes cytokine protein expression. Interestingly, a significant increase in the levels of IL-4 mRNA expression was found in BALB/c mice only. This study suggests that differential activation or evasion of cytokine expression by S. pneumoniae virulent strains determines disease outcome regardless of the host's immunogenetic background.
[Show abstract][Hide abstract] ABSTRACT: Vulnerability to Streptococcus pneumoniae is most pronounced in children. The microbial virulence factors and the features of the host immune response contributing to this phenomenon are not completely understood. In the current study, the humoral immune response to separated Strep. pneumoniae surface proteins and the ability to interfere with Strep. pneumoniae adhesion to cultured epithelial cells were analysed in adults and in children. Sera collected from healthy adults recognized Strep. pneumoniae separated lectin and nonlectin surface proteins in Western blot analysis and inhibited on average 80% of Strep. pneumoniae adhesion to epithelial cells in a concentration-dependent manner. However, sera longitudinally collected from healthy children attending day care centres from 18 months of age and over the course of the following 2 years revealed: (a) development of antibodies to previously unrecognized Strep. pneumoniae surface proteins with age; (b) a quantitative increase in antibody responses, measured by densitometry, towards separated Strep. pneumoniae surface proteins with age; and (c) inhibition of Strep. pneumoniae adhesion to epithelial cells, which was 50% on average at 18 months of age, increased significantly to an average level of 80% inhibition at 42 months of age equalling adult sera inhibitory values. The results obtained in the current study, from the longitudinally collected sera from healthy children with documented repeated Strep. pneumoniae colonization, show that repeated exposures are insufficient to elicit an immune response to Strep. pneumoniae proteins at 18 months of age. This inability to recognize Strep. pneumoniae surface proteins may stem from the inefficiency of T-cell-dependent B-cell responses at this age and/or from the low immunogenicity of the proteins.
[Show abstract][Hide abstract] ABSTRACT: We determined apoptosis in whole rat colonic tissue and in isolated colonocytes from the various rat crypt regions in preneoplastic stages up to frank neoplasia following administration of the procarcinogen, dimethylhydrazine (DMH). Apoptotic cells were determined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-method, by evaluating sections stained with hematoxylin and eosin, and caspase-1 immunostaining. Apoptotic cells in whole colonic tissue from untreated rats were confined to the upper crypt while, in DMH-treated rats apoptotic and caspase-1 positive cells were located in the crypt proliferative regions. Numerous apoptotic and caspase-1-positive cells were found in sections from early tumors while in the delayed tumors, apoptotic-positive cells were absent and number of caspase-1-positive cells was negligible. A marked reduction in the apoptotic index along the crypt was observed in isolated transformed colonic cells, this was not the case for caspase-1-positive cells. We conclude that: (i) in colorectal tumors at progressive stage apoptosis is altered, (ii) the mechanistic alteration in apoptosis may be located between caspase-1-protease activity and the fragmentation process of DNA.
Cancer Letters 03/2001; 163(2):229-38. DOI:10.1016/S0304-3835(00)00624-8 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Streptococcus pneumoniae (Pnc) is one of the leading pathogens in the world. Attachment to respiratory mucosal and lung surfaces is presumed to be involved in carriage, in disease and in the interaction with macrophages initiating innate immune responses. We hypothesized that bacterial adhesins mediate Pnc adhesion and host cell invasiveness. Initial studies have focused on the purification of cell wall and membrane proteins using fetuin affinity chromatography, SDS PAGE and western blot analysis probed with pooled healthy human sera. Using a Pnc clinical isolate, and a gpt mutant we have detected 10-lectin proteins isolated from the cell wall and adherent to the affinity column and 15 lectins isolated from membrane extracts. The fetuin-captured lectins agglutinated rabbit erythrocytes. 15 proteins in the cell wall and 18 proteins in the membrane that failed to bind to the fetuin column did not agglutinate rabbit erythrocytes. Further purification of the cell wall and membrane fetuin-separated fractions was achieved via anion exchange FPLC, was verified by SDS PAGE. These proteins maintained their agglutinating activity, and were subsequently tested for their ability to interfere with Pnc adhesion and invasion of epithelial cells in culture. Additional biochemical, immunological and molecular techniques are being used in attempt to identify relevant proteins.
Advances in Experimental Medicine and Biology 02/2000; 479:61-71. DOI:10.1007/0-306-46831-X_6 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apoptosis in cells of different lineages is restrained by survival signals which depend upon cell-to-cell communication. The aim of this study was to determine whether colonic cells deprived of crypt ambient are doomed to die prior to their normal chronological demise. Apoptosis was studied in rat whole colonic tissue, in isolated intact crypts, and in colonic cell populations collected from the crypt axis at different stages of proliferation and differentiation. In a number of experiments, cell harvest was performed in the presence of either a tetrapeptide (YVAD-CMK) inhibitor of interleukin-1beta-converting enzyme (ICE), or tyrphostin A25, a protein tyrosine kinase inhibitor, or sodium-orthovanadate, a phosphatase inhibitor. DNA fragmentation was assessed by electrophoretic and nonisotopic-labeling procedures. The ultrastructure of colonic tissue specimens and isolated cells was examined by transmission electron microscopy. Apoptosis in whole colonic tissue and in isolated crypts was confined predominantly to cells resident in the upper crypt regions. In contrast, extensive apoptotic death was observed in isolated colonic cells, irrespective of their developmental stage and positional hierarchy within the crypt continuum at harvest time. An apoptotic gradient, however, was evident. Exposure to YVAD-CMK resulted in a marked decrease in the number of apoptotic cells. Treatment with tyrphostin A25 caused a sharp rise in the apoptotic index; conversely, vanadate significantly impeded apoptosis. Cumulatively, these results indicate that disordered intercellular communication provokes unscheduled ICE-mediated apoptosis of colonocytes, and that local signals along the crypt continuum control both the reprieve from death and the timely demise of distinct colonic cell populations. Attenuation of tyrosine phosphorylation may be a contributory event in the acquisition of the apoptotic phenotype.