Effect of asiatic and ursolic acids on morphology, hydrophobicity, and adhesion of UPECs to uroepithelial cells

Adhesion of bacteria to epithelial tissue is an essential step in the progression of the urinary tract infections. Reduction of virulence factors responsible for microbial attachment may help to decrease or inhibit colonization of the host organism by pathogens. In the age of increasing bacterial antibiotic resistance, more and more attention is being paid to the use of plants and/or their bioactive components in the prevention and treatment of human infections. Asiatic acid (AA) and ursolic acid (UA), two plant secondary metabolites, were used as potential antibacterial agents. The current study aimed to determine the possible impact of AA and UA on morphology, hydrophobicity, and adhesion of clinical uropathogenic Escherichia coli strains (UPEC) to the uroepithelial cells. Our work describes for the first time the effects exerted by AA and UA on virulence factors of UPECs. The impact of both acids on the cell surface hydrophobicity of the investigated strains was very weak. The results clearly show the influence of AA and UA on the presence of P fimbriae and curli fibers, morphology of the UPECs cells and their adhesion to epithelium; however, some differences between activities of AA and UA were found.     

Clonal growth of normal human uroepithelial cells

Summary We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3×10⁵ lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1±2.5%. A cloning efficiency of 4.9±1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 μg/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9±2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 μg/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7±1.7%. This work was supported by NIH grant 29525 to C. A. R. L. J. L. is a recipient of National Science Foundation predoctoral fellowship.     

Adhesion of Proteus mirabilis and Proteus vulgaris to uroepithelial cells following exposure to various antimicrobial agents.

The effect of subinhibitory concentrations of netilmicin, ceftriaxone, cefotaxime, aztreonam and piperacillin on the adherence of Proteus species to uroepithelial cells was examined. Bacterial adhesion to human uroepithelial cells, measured microscopically, was affected by all five antibiotics but to different extents. The most effective was netilmicin. There was a correlation between the decreased rate of bacterial attachment and morphological changes in the drug-exposed bacteria.     

Monitoring of uropathogens and their susceptibility to antibiotics]

Samples of urine collected from patients with complicated urology infection and hospitalized to the Moscow Region Research Clinical Institute in 1986, 1991, 1995 and 1999 were analysed. Of 11,444 samples examined, bacteriuria was estimated in 7143 samples. 9786 strains (29 genus) of bacteria were isolated--56.9 per cent as mono culture and 43.1 per cent as associations. Susceptibility to 21 antibiotic was determined by disk diffusion method for 1607 strains; beta-lactamase production was determined in 198 strains, MIC was determined for 41 antibiotics. Gram-negative rods relative amount among pathogens decreased substantially (84.7 per cent in 1986 against 61.6 per cent in 1999), particularly Enterobacteriaceae (74.7 per cent in 1986 against 41.4 per cent in 1999). Nonfermenting Gram-negative rods (NFGNR) relative amount increased (10.8 per cent against 19.2 per cent), along with Gram-positive cocci (19.8 per cent against 64.2 per cent), particularly coagulasenegative staphylococci (CNS) (10.8 per cent against 35.9 per cent) and enterococci (5 per cent against 16.5 per cent) and candida and fungi (0.5 per cent in 1986 against 15.9 per cent in 1999). At the period 1986-1999 the main pathogens in urology infection were E. coli, Enterobacter spp., NFGNR (including P. aeruginosa), Staphylococcus, CNS, Enterococcus spp. The problem pathogens for urological department were the following: E. coli, Klebsiella spp., Enterobacter spp., Proteus spp., NFGNR including P. aeruginosa, CNS, Enterococcus spp., candida and fungi. At the period 1991-1997 Gram-negative pathogens susceptibility to amikacin, ofloxacin, ciprofloxacin, imipenem, ceftazidime, cefotaxime was not changed in general, Gram-positive cocci (staphylococci and enterococci) retained the same susceptibility to vancomicin, cefamandol and amoxyclave. Staphylococci were also susceptible to amikacin, imipenem, rifampicin, oxacillin, ciprofloxacin, and ofloxacin. Production of beta-lactamase was registered for 38.7 per cent of CNS, 26.5 per cent of E. coli, 38.5 per cent of K. pneumoniae, 25 per cent of P. mirabilis and 55.6 per cent of P. aeruginosa strains.     

Effect of antibacterials on surface properties and adhesion to uroepithelial cells in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The effect of sub-inhibitory and inhibitory concentrations of antimicrobials including aminoglycosides, third generation cephalosporins and quinolones on the surface properties and adhesion of Klebsiella pneumoniae to uroepithelial cells (UECs) was examined. Antibiotics, ceftazidime and ofloxacin at 1/4, 1/8 × MIC and minimum inhibitory concentration (MIC) induced filament formation in bacteria, however cells treated with amikacin were similar in length to control organisms but showed a rough topology under the scanning electron microscope. An increase in bacterial hydrophobicity and decrease in uronic acid content were noted in the presence of ceftazidime and ofloxacin at MIC and sub-MIC level. However, amikacin at MIC level caused decreased hydrophobicity of the cells and the uronic content remained the same. This study clearly indicates that, although ceftazidime and ofloxacin brought about profound changes in cell surface characteristics, these changes did not result in any advantage to the bacterial cell in terms of adhesion. In contrast, with amikacin, which did not show any appreciable change in cell morphology or surface topology, exposure markedly increased the adherence of bacteria to UECs, indicating that the prophylactic use of this antibiotic not only induces resistance in bacteria but can also promote the colonization of UECs.     

Effect of neuraminidase treatment on serum reactivity to autologous leukemic blast cells

Summary The sera of 35 patients with acute lymphoblastic leukemia (ALL) and acute non-lymphoblastic leukemia (ANLL) were tested for reactivity against cell surface antigens of autologous leukemic blast cells by protein A assay (PA), immune adherence assay (IA), and anti-C3 mixed hemadsorption assay (C3-MHA). Autologous serum reactivity was detectable by PA in four cases and by LA and C3-MHA in about half the patients. Autologous serum reactivity occurred more often in ALL than in ANLL. Absorption studies revealed that in one patient only the autologous reactivity was directed against a restricted antigen, which could be detected only on the individual T-ALL blast cells. All other autologous antibodies detected unspecific antigens. Neuraminidase treatment had two effects: first, it increased antibody attachment to antigens which are also present on untreated cells; secondly, after neuraminidase treatment an antigen was detectable on the cell surface which could also be demonstrated on neuraminidase-treated non-leukemic cells (e.g., erythrocytes). Neither of these two effects of neuraminidase treatment seems to be tumor-specific. Possible therapeutic effects of neuraminidase are probably caused by unspecific adjuvant effects of the enzyme.     

Pathogenesis of urinary tract infection in the elderly: the role of bacterial adherence to uroepithelial cells

The in vitro adherence of nine strains of Gram-negative bacteria to uroepithelial cells from 24 women patients (>65 years) was significantly higher than to cells from 24 premenopausal women (18–40 years). Uroepithelial cells from patients with a history of previous urinary tract infection (UTI) were marginally more receptive to attachment of uropathogens than cells from women without a history of UTI, but this was not statistically significant. Serum from four elderly women with asymptomatic bacteriuria was used to stabilize samples for electron-microscopic examination, which showed the presence of fibrous glycocalyx material surrounding the bacteria and attached to the uroepithelial cells. Eighty uropathogenic isolates from elderly and premenopausal women were found to express adhesins, to produce urease and hemolysins, and to ferment sucrose, salicin, and dulcitol. These results suggest that the increased receptivity of uroepithelial cells to bacterial attachment may be a predisposing factor in the onset of UTI in the hospitalized and domiciliary elderly population.     

Activities of MAP-kinase pathways in normal uroepithelial cells and urothelial carcinoma cell lines

It is often assumed that MAPK pathways drive proliferation of normal uroepithelial (UEC) and urothelial carcinoma (TCC) cells. To check this assumption, activities and inducibilities of promoters containing serum-response elements (SRE) or AP-1 binding sites were investigated in cultured UEC and seven TCC lines. Reporter plasmids dependent on SRE or AP-1 sites were highly active in UEC, but significantly less so in TCC lines. Reporter activity in TCC lines could be induced by constitutively active MEKK4 or TPA. Accordingly, phosphorylation of the MAPK pathway components MEK, ERK, and ELK1 was most pronounced in UEC and lower in TCC lines. MAPK-dependent promoter activities and bromodeoxyuridine incorporation decreased in UEC upon withdrawal of growth factors, but less so in TCC lines, in which serum diminution increased apoptosis. Likewise, E2F-dependent promoters responded to growth factors in UEC, but were more serum-independent in the TCC lines, which lack either RB1 or p16(INK4A). MEK inhibitors inhibited BrdU incorporation in UEC more strongly than in TCC lines. Thus, proliferation of normal uroepithelial cells is indeed associated with activation of MAPK pathways. However, autonomous proliferation of TCC lines--unexpectedly--appears much less dependent on MAPK activation and may rather be promoted by defects in cell cycle regulation.     

Effect of Bacillus Callmette Guerin (BCG) on the receptivity of nude mice to Schistosoma mansoni]

"Nude" mice are injected intravenously with 1 mg live BCG each. Pulmonary schistosomules, counted 5 days after infestation with Schistosoma mansoni cercaria are observed to be fewer in injected mice than in non-injected controls. This show that BCG immunostamulation can be obtained when mature T lymphocytes are absent.     

Influences of pre- and postnatal testosterone treatment on defeminization of sexual receptivity in pigs

Sexual receptivity was evaluated in female and male pigs that had experienced varying periods of exposure to testosterone pre- and postnatally. For prenatal exposure, pregnant sows were treated with testosterone propionate (TP) from Day 29-35 or Day 39-45 of gestation at a dosage that caused virilization of the external genitalia of their female offspring. Eighty-three percent of the females that received TP prenatally had regular estrous cycles, but reached puberty later than control females. Only 26% of the females that received TP both pre- and postnatally (4-6 mo of age) were observed in estrus by 10 mo of age. After ovariectomy and acute treatment with estradiol benzoate (EB), the proportion of females that showed the immobilization response (receptivity) was similar for all groups of females independent of pre- or postnatal TP treatment. Females treated prenatally from Day 39-45 showed the immobilization response for fewer days after treatment with a high dosage of EB than did controls. On the basis of these observations, we conclude that receptivity in female pigs is not affected greatly by testosterone treatment at the stages of development that were investigated. Males castrated at birth and treated with a single injection of EB after 9.5 mo showed the immobilization response. In contrast, few males castrated at 8 mo or castrated at birth and treated with TP from 3 to 6 mo showed the immobilization response after EB treatment. These observations provide direct evidence for a postnatal component of testosterone-dependent defeminization of receptivity in male pigs.     

Effect of nitrogen supplementation on the longevity of antibiotic production by immobilized cells of Penicillium urticae

Summary Conidia of Penicillium urticae were immobilized in Kappa-Carrageenan beads (2–3 mm) by a previously described procedure to yield an in situ grown immobilized cell population which could be induced to produce the antibiotic and mycotoxin, patulin. When repeatedly transferred into a nitrogen-free production medium every 2 days, the patulin productivity of these cells gradually decreased to 50% within 14 days while the total cell protein remained constant. This decline was due to the gradual loss of the cells' catalytic capacity for converting glucose to 6-methylsalicylic acid (6-MSA), the first metabolite of the patulin pathway, as well as for converting 6-MSA to patulin. When these 14 day-old cells were incubated in a nutrient rich growth medium for 2 days their patulin producing activity increased from 50% to 130%. On the other hand the addition of a protein synthesis inhibitor, cycloheximide, to the N-free production medium drastically reduced the patulin producing activity of the immobilized cells; in particular, their capacity for converting 6-MSA to patulin. The cells' patulin producing activity was maintained at >100% for longer than 15 days when the cells were repeatedly transferred into a yeast extract supplemented production medium or when they were occasionally transferred into 10 or 20% strength growth medium. Repeated transfers to a 10% strength growth medium appeared to stabilize the cells' capacity for converting 6-MSA to patulin.     

Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium

Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum-free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 microgram/ml hydrocortisone, 5 micrograms/ml transferrin, 10 micrograms/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 2.7 mg/ml D-glucose, 10(-4) M ethanolamine or 10(-4) M phosphoethanolamine, and 5 X 10(-8) M selenium. HUC grown in F12 on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3-5 times during log phase of growth (20-25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serum-free F12 on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12 did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12 was mitogenic to HUC in a dose-dependent manner in the concentration range 0.01-1.00% (4-400 micrograms/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS-F12 decreased to 21 hours, and the potential population doublings in vitro increased to 31-36. Small amounts (140 micrograms/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum-containing or serum-free medium. Higher calcium concentrations (0.30-0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum-free F12 (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12 medium did not confer a long-term growth advantage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of aeration on antibiotic production byStreptomyces clavuligerus

Summary During the rapid growth phase ofStreptomyces clavuligerus in a 10 litre fermentor, the level of dissolved oxygen (DO) was found to drop to almost zero for a period of approximately 10 h, delaying the appearance of and lowering the production of the antibiotic cephamycin C. Controlling the DO at either 50% or 100% throughout the fermentation did not significantly alter the specific growth rate of the culture, but did elevate final antibiotic levels two- and three-fold respectively. The improved oxygen availability affected antibiotic production both by increasing the rate of specific cephamycin C bisosynthesis and by maintaining this higher rate throughout the production period. These results demonstrate that controlling dissolved oxygen levels close to saturation during periods of rapid growth markedly improves the efficiency and duration of cephamycin C biosynthesis inS. clavuligerus.     

Effect of terrilytin on antifungal antibiotic activity]

The antifungal activity of terrilitine, an enzymatic preparation of microbial origin and its effect on the activity of antifungal polyenic antibiotics and griseofulvine were studied in vitro. It was found with the method of serial dilutions in Sabourand's liquid medium that terrilitine was active against C. albicans and certain dermatophytes. In combination with amphotericin B, amphoglucamine, mycoheptine, levorin, nystatin or griseofulvin it increased the activity of these antibiotics 2-16 times.     

Effect of anti-TNF-α treatment in an antibiotic treated murine model of shock due to Streptococcus pyogenes

Abstract Experimentally, Gram-negative septic shock can be prevented by the prophylactic use of an anti-TNF-α monoclonal antibody. The clinical similarity between Gram-negative and Gram-positive septic shock suggested that anti-TNF-α therapy might have a wide application. Increased levels of TNF-α were seen in a murine model of septic shock due to Streptococcus pyogenes but administration of an anti-TNF-α monoclonal antibody had no beneficial effect on the outcome.