Induction of protective immunity after escherichia coli bladder infection in primates. Dependence of the globoside-specific P-fimbrial tip adhesin and its cognate receptor.

Clinical observations suggest that immune mechanisms affect etiology and course of recurrent cystitis. A primate infection model was used to show that primary bladder infection with a uropathogenic P-fimbriated strain (binding to globoside present in the bladder wall) protects against rechallenge with homologous as well as heterologous Escherichia coli strains for up to 5-6 mo. In contrast, mutant derivatives producing P-fimbriae either lacking the tip adhesin protein or carrying an adhesin for which no bladder receptor was present, were unable to induce protection, even though they generated bladder infections of similar duration as the wild type. Therefore, the protective effect mediated by the adhesin seemed to depend upon the presence of its cognate receptor. Since the wild strain also mediated protection against mutants that lacked the adhesin, our data suggest that the globoside-binding PapG adhesin acts as an adjuvant during infection to enhance a specific response against other bacterial antigens. In fact, the globoside-binding strain DS17, but not the mutant DS17-1, unable to bind to membrane-bound globoside, elicited a secretory IgA response to LPS in urine. These in vivo findings suggest that bacterial adhesin-ligand interactions may have signaling functions of importance for the immune response.

Genotypic and phenotypic characterization of two newly established renal cell carcinoma cell lines.

Two new cell lines from human renal cell carcinoma are reported. Primary cell cultures from 75 consecutive cases of nephrectomy and metastatic surgery due to different stages of RCC during 4 years were studied. Two cell cultures could be propagated for more than 50 passages in vitro. HN4 was derived from a grade III clear cell carcinoma. HN51 originated from a metastatic brain lesion of a clear cell carcinoma grade III. Karyotype analysis of HN4 revealed triploidy with a clonal aberration, der(10)t(3;10)(q13;p12). HN51 also had a triploid pattern with different marker chromosomes but without any clonal aberration. Loss of heterozygosity studies revealed no loss of heterozygosity on 3p or other chromosomal markers in HN4 but LOH was found on one 3p marker and one 14q marker in addition to all 17 p and q markers in HN51. In vitro light microscopy showed distinctly different morphology in the two cell lines although they both had a typical epithelial growth pattern. Doubling times in vitro were low but slightly higher for HN51. Repeated tumorigenenic experiments in athymic mice only gave rise to subcutaneous tumors with HN51. On characterization by 2-dimensional gel electrophoresis, the two cell lines exhibited different polypeptide patterns with higher expression of proliferating cell nuclear antigen in HN51 and higher expression of glutathione-S-transferase in HN4 constituting the most prominent differences.

Induction of innate immune responses by Escherichia coli and purified lipopolysaccharide correlate with organ- and cell-specific expression of Toll-like receptors within the human urinary tract.

Mucosal epithelial linings function as physical barriers against microbes. In addition, they participate in the first line of host defence by production of a variety of proinflammatory mediators when exposed to microbes and microbial agents. Here, we use a human urinary tract infection model to demonstrate that organ- and cell-specific innate responses induced by lipopolysaccharides (LPS) present on Gram-negative bacteria correlates with the expression of Toll-like receptor 4 (TLR4). The presence of TLR4 on human bladder epithelial cells enables them to rapidly respond to bacterial infections in vitro and in vivo. In contrast, TLR4 is not expressed on human proximal tubule cells isolated from the renal cortex, which may explain the cortical localization of bacteria in pyelonephritis. TLR4-negative renal epithelial cells, A498, are non-responsive to purified LPS, however, they respond to viable bacteria via a mannose-sensitive attachment-mediated pathway. To identify LPS components recognised by bladder epithelial cells, a bacterial lipid A mutant and LPS of different chemotypes were tested. Full interleukin 8 induction required hexa-acylated lipid A and was decreased by between 50% and 70% in the presence of O-antigen. Taken together, we propose that multiple independent pathways, which are organ- and cell-specifically expressed, mediate bacterial recognition and determine the outcome of innate responses to infection.

Escherichia coli-induced expression of IL-1 alpha, IL-1 beta, IL-6 and IL-8 in normal human renal tubular epithelial cells.

The aim of the present study was to investigate whether the IL-1 family cytokines, in addition to IL-6 and IL-8, could be induced in normal human cortical epithelial cells in response to bacterial stimuli. Human renal tissue was obtained from 9 patients undergoing elective tumour nephrectomy. Renal cortical epithelial cells of tubular origin were prepared from the unaffected tissue. The proximal tubular cells were stimulated for 2, 6 and 24 h with a heat-inactivated pyelonephritogenic Escherichia coli strain DS-17. Cultured unstimulated tubular cells served as controls. IL-1 alpha, IL-1 beta, IL-1 receptor antagonist, IL-6, IL-8, IL-10, TNF-alpha, G-CSF and GM-CSF were analysed using immunohistochemistry at the single cell level. The nonstimulated cells were found to express low levels of IL-6 and IL-8 (mean value < 3% of total cells). In contrast, E. coli exposure resulted in significantly increased incidences of IL-6 and IL-8 expressing cells (mean values approximately 18% of total cells) peaking within two hours of stimulation (P < 0.008 and P < 0.02 versus non-stimulated cells, respectively). A gradual decrease was thereafter observed at 6 and 24 h, respectively, although persistently higher compared to controls. A different kinetic response was found for IL-1 alpha, IL-1 beta and IL-1 receptor antagonist-expressing cells, which peaked 24 h after E. coli stimulation (mean values 3--10%) (P < 0.008, P < 0.02, P < 0.02 versus non-stimulated cells, respectively). Low levels of TNF-alpha and GM-CSF were found in 3 of the 9 donated epithelial cells, peaking at 2 h, and IL-10 and G-CSF producing cells in 1 patient each. In conclusion we found that heat-inactivated pyelonephritic E. coli induced a proinflammatory cytokine response in the normal human proximal tubular cells including the IL-1 family, IL-6 and IL-8.

Molecular evidence for pap-G specific adhesion of Escherichia coli to human renal cells.

PURPOSE: To study the interaction between class II G-adhesin of Escherichia coli and human urogenital cells. MATERIAL AND METHODS: The adherence of two wild type P-fimbriated E. coli strains, both carrying a class II G-adhesion, and two constructed mutants (one class II G-adhesion knock-out mutant and one class switch mutant in which the papG gene was exchanged with a prsJ96 allele which is a representative of the class III G-adhesin) to human urogenital cells were examined by light microscopy and flow cytometry. RESULTS: The wild type E. coli strains adhered avidly to proximal tubular cells, but the isogenic mutant strains did only adhere in one of the experiments. A soluble receptor analogue inhibited bacterial attachment. CONCLUSIONS: These experiments strongly suggest that the papG class II tip adhesin of P-fimbriae is essential in the pathogenesis of human kidney infection.

The role of nitric oxide in bacillus Calmette-Guérin mediated anti-tumour effects in human bladder cancer.

Bacillus Calmette-Guérin (BCG) has been used for many years to treat cancer of the urinary bladder. It constitutes effective intravesical therapy of carcinoma in situ and recurrent superficial bladder cancer. Although the mechanism of action is unknown, most evidence suggests an immune-mediated mechanism. BCG treatment is known to increase cytokine production in the urinary bladder. As cytokines may induce nitric oxide synthase (NOS) activity and as nitric oxide (NO) exerts cytotoxic effects on tumour cells, we investigated the role of NO in BCG-mediated anti-tumour activity. Here we demonstrate a marked induction of both calcium-dependent and calcium-independent NOS activity in the human urinary bladder after BCG treatment. The presence of NOS in the urothelial cells was also demonstrated by the use of immunohistochemistry. Furthermore, patients treated with BCG showed a 30 times higher production of gaseous NO as measured in the urinary bladder by chemiluminescence. Finally, NO donors exerted cytotoxic effects on bladder cancer cell lines. These findings suggest that NO synthesis may be an important mechanism in BCG-mediated anti-tumour therapy.

Vaccination with FimH adhesin protects cynomolgus monkeys from colonization and infection by uropathogenic Escherichia coli.

Escherichia coli FimH adhesin mediates binding to the bladder mucosa. In mice, a FimH vaccine protects against bacterial challenge. In this study, 4 monkeys were inoculated with 100 microgram of FimCH adhesin-chaperone complex mixed with MF59 adjuvant, and 4 monkeys were given adjuvant only intramuscularly. After 2 doses (day 0 and week 4), a booster at 48 weeks elicited a strong IgG antibody response to FimH in the vaccinated monkeys. All 8 monkeys were challenged with 1 mL of 108 E. coli cystitis isolate NU14. Three of the 4 vaccinated monkeys were protected from bacteruria and pyuria; all control monkeys were infected. These findings suggest that a vaccine based on the FimH adhesin of E. coli type 1 pili may have utility in preventing cystitis in humans.