Adherence of human vaginal lactobacilli to vaginal epithelial cells and interaction with uropathogens

Three strains of Lactobacillus, identified as Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus jensenii, were selected from among 70 isolates from the vaginas of healthy premenopausal women for properties relevant to mucosal colonization or antagonism. All three self-aggregated and adhered to epithelial vaginal cells, displacing well-known vaginal pathogens, such as G. vaginalis, and inhibiting the growth in vitro of Escherichia coli and Streptococcus agalactiae. The surface components involved in self-aggregation appeared to be proteins for L. gasseri and lipoproteins for L. acidophilus and L. jensenii, as judged by susceptibility to treatment with appropriate degrading enzymes. The factors responsible for adherence to epithelial vaginal cells seemed to be glycoproteins (L. acidophilus and L. gasseri) and carbohydrate (L. jensenii). The receptors of the vaginal cells were glycolipids, which presumably were the targets of the competition observed between the lactobacilli and the pathogenic microbes.     

An improved method to obtain highly differentiated monolayers of human bronchial epithelial cells

BACKGROUND: Image-guided percutaneous drainage has been shown to be a safe and effective alternative to surgery in the management of psoas abscess in adults and adolescents. There is little information on its use in children. OBJECTIVE: To evaluate the safety and efficacy of US-guided percutaneous needle aspiration and catheter drainage of ilio-psoas abscesses. MATERIALS AND METHODS: A retrospective review of 14 children with 16 ilio-psoas abscesses (10 pyogenic and 4 tuberculous) who were treated by US-guided percutaneous needle aspiration (n = 5) or catheter drainage (n = 9) along with appropriate antimicrobial therapy. RESULTS: Percutaneous treatment was successful in 10 of the 14 patients; all showed clinical improvement within 24-48 h of drainage and subsequent imaging demonstrated resolution of the abscess cavities. Surgery was avoided in all of these ten patients except one, who underwent open surgical drainage of ipsilateral hip joint pus. Of the other four patients, two had to undergo surgical drainage of the ilio-psoas abscesses after failure of percutaneous treatment, one improved with antibiotics after needle aspiration failed to yield any pus, and one died of continuing staphylococcal septicaemia within 24 h of the procedure. There were no procedural complications. CONCLUSIONS: Percutaneous drainage represents an effective alternative to surgical drainage as a supplement to medical therapy in the management of children with ilio-psoas abscesses.     

Adherence to and penetration of human intestinal Caco-2 epithelial cell monolayers by Pseudomonas aeruginosa

Clinical isolates of Pseudomonas aeruginosa from blood adhered to and penetrated intestinal Caco-2 cell monolayers to a greater degree than did isolates from sputum, with a concomitant drastic decrease in transepithelial electrical resistance. PAO-PR1, an avirulent exotoxin A mutant of PAO1, did not cause a decrease in the resistance. The Caco-2 monolayer system may be useful for the evaluation of certain P. aeruginosa virulence factor activities.     

Occurrences of penicillin-binding protein 2B gene in clinically isolated penicillin-resistant Streptococcus pneumoniae

We analyzed 88 strains of Streptococcus pneumoniae (S. pneumoniae) isolated in Showa University Hospital from June 1995 to July 1996. The ratios of antibiotic resistance were 39% to penicillin G, 50% to erythromycin, and 2% to imipenem. No resistant to cefotaxime and ofloxacin was observed. Thirty-four strains (39%) were considered to be penicillin-resistant S. pneumoniae (PRSP) strains (MIC of penicillin G > or = 0.5 microgram/ml), according to the breakpoint determined by the Japanese Working Group for Penicillin-Resistant Streptococcus pneumoniae. The ratio of PRSP was higher in S. pneumoniae isolated from inpatients (25/47) when compared to that from outpatients. By PCR analysis, DNA regions of autolysin were amplified in all the 88 strains, confirming that the isolates were S. pneumoniae. Penicillin-binding protein 2B (PBP2B) class B region was positive in 32 strains, and PBP2B class A was in 2 strains. Twenty eight of 34 strains of PRSP contained the PBP2B class B gene. In the remaining six PRSP strains, neither the PBP2B class A nor B region was amplified. The PBP2B class B region was detected as a 180-kb fragment of SmaI digestion of S. pneumoniae DNA by Southern blot analysis, confirming that the detection of PBP2B class B gene by PCR is reliable. We concluded that the PBP2B class B gene is considered to be a major gene responsible for phenotypic resistance of PRSP. We performed genotyping by SmaI digestion pattern using pulsed field gel electrophoresis. No identical pattern was observed in isolates from inpatients, suggesting that apparent nosocomial infection of S. pneumoniae was negligible.     

Roxithromycin inhibits cytokine production by and neutrophil attachment to human bronchial epithelial cells in vitro

We evaluated the effect of roxithromycin on cytokine production and neutrophil attachment to human airway epithelial cells. Roxithromycin suppressed production of interleukin 8 (IL-8), IL-6, and granulocyte-macrophage colony-stimulating factor. It inhibited neutrophil adhesion to epithelial cells. Roxithromycin modulates local recruitment and activation of inflammatory cells, which may have relevance to its efficacy in airway diseases.     

Antioxidant enzyme regulation and resistance to oxidants of human bronchial epithelial cells cultured under hyperoxic conditions

OBJECTIVE: The measurement of total serum testosterone has an established clinical role in the management of male hypogonadism and female androgen excess disorders. We studied the between-kit variability and precision of six different commercially available testosterone assays and compared them with an established in-house method. DESIGN: Laboratory observational prospective study. SETTING: Tertiary university medical center clinical laboratory. PATIENT(S): Three groups of samples each of men (n = 36) and women (n = 15) who had high, normal, or low levels of sex hormone-binding globulin (SHBG), respectively, were studied. INTERVENTION(S): Individual and pooled (male and female) serum samples were analyzed for total testosterone concentration using six different commercially available assays and one in-house method. MAIN OUTCOME MEASURE(S): The between-kit variability and the effect of the mean (+/- SD) SHBG level were determined, the results obtained with the use of the kits and the in-house method were compared, and the intraassay variability (i.e., precision) was evaluated. RESULT(S): Male samples demonstrated a 26.3%-40.8% variance in the results obtained with different kits, which was greatest for samples with the lowest SHBG levels. For female samples, between-kit variability ranged from 57%-115% (average, 77%). The percent deviation of the results obtained with the use of commercial methods from those obtained with the use of our in-house assay was greater for men (mean variance, 194%) than for women (mean variance, 67%). The female pool intraassay coefficient of variation was 3.8% with the use of the in-house method and ranged from 8.9%-21.2% with the use of the commercial kits. The male pool intraassay coefficient of variation was 3.1% with the use of the in-house method and ranged from 3.3%-5.5% with the use of the commercial kits. CONCLUSION(S): Most commercially available kits for measuring the total serum testosterone level demonstrated significant between-kit variability, which was greatest for female samples. Further, samples with the lowest SHBG levels had the highest between-kit variances. These data strongly suggest that the measurement of total serum testosterone using commercial kits may have limited utility, particularly for the detection of hyperandrogenemia.     

Neutrophil and asbestos fiber-induced cytotoxicity in cultured human mesothelial and bronchial epithelial cells

This study investigates reactive oxygen species generation and oxidant-related cytotoxicity induced by amosite asbestos fibers and polymorphonuclear leucocytes (PMNs) in human mesothelial cells and human bronchial epithelial cells in vitro. Transformed human pleural mesothelial cells (MET 5A) and bronchial epithelial cells (BEAS 2B) were treated with amosite (2 micrograms/cm2) for 48 h. After 24 h of incubation, the cells were exposed for 1 h to nonactivated or amosite (50 micrograms) activated PMNs, washed, and incubated for another 23 h. Reactive oxygen species generation by the PMNs and the target cells was measured by chemiluminescence. Cell injury was assessed by cellular adenine nucleotide depletion, extracellular release of nucleotides, and lactate dehydrogenase (LDH). Amosite-activated (but also to a lesser degree nonactivated) PMNs released substantial amounts of reactive oxygen metabolites, whereas the chemiluminescence of amosite-exposed mesothelial cells and epithelial cells did not differ from the background. Amosite treatment (48 h) of the target cells did not change intracellular adenine nucleotides (ATP, ADP, AMP) or nucleotide catabolite products (xanthine, hypoxanthine, and uric acid). When the target cells were exposed to nonactivated PMNs, significant adenine nucleotide depletion and nucleotide catabolite accumulation was observed in mesothelial cells only. In separate experiments, when the target cells were exposed to amosite-activated PMNs, the target cell injury was further potentiated compared with the amosite treatment alone or exposure to nonactivated PMNs. In conclusion, this study suggests the importance of inflammatory cell-derived free radicals in the development of amosite-induced mesothelial cell injury.     

Erythromycin modulates IL-8 expression in normal and inflamed human bronchial epithelial cells

Previous studies have shown that blood plasma levels of 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) and 17alpha, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S) increase in striped bass (Morone saxatilis) undergoing final oocyte maturation (FOM). Both hormones are produced by ovarian fragments undergoing hCG-induced germinal vesicle breakdown (GVBD) in vitro. In the present study, we investigated binding of DHP and 20beta-S to ovarian membranes from striped bass undergoing FOM. Saturable binding sites for DHP were not detected. Saturation of 20beta-S binding sites with 5 nM [3H]20beta-S occurred within 40 min at 0 degrees C (at 3 min, half of the maximum specific binding of steroid was calculated to have occurred), and the binding was pH-dependent. Scatchard analyses revealed the presence of a single class of high-affinity (dissociation constant [Kd] = 1.4 +/- 0.2 nM), limited-capacity (estimated concentration [Bmax] = 2.7 +/- 0.3 pmol/g ovary) 20beta-S binding sites on membranes from striped bass ovaries undergoing FOM. In contrast, only low levels of specific binding (Bmax < 0.04 pmol/g tissue) were detected on membranes from testes, liver, brain, and muscle. Ovarian membranes prepared from vitellogenic females also had low levels (Bmax < 0.1 pmol/g ovary) of specific 20beta-S binding, less than 5% of that found during FOM. Results of competition assays showed that DHP was approximately 250 times less effective than 20beta-S for displacing 20beta-S from ovarian membranes. In contrast, 20beta, 21-dihydroxy-4-pregnen-3-one was a very effective competitor, although it is only a weak inducer of oocyte GVBD in vitro. Of several other steroids tested, only progesterone showed affinity for the 20beta-S binding site within a physiological range of concentrations. Taken together with previous studies of striped bass FOM, these findings indicate that 20beta-S is the oocyte maturation-inducing steroid hormone in striped bass.     

Increased interleukin-8 release by beta-adrenoceptor activation in human transformed bronchial epithelial cells

1. The effect of beta-adrenoceptor activation on release of the neutrophil chemoattractant, interleukin-8 (IL-8), was examined in human transformed bronchial epithelial cells (16HBE cells). 2. The combined beta 1- and beta 2-adrenoceptor agonist, isoprenaline, time- (100 nM, 2-18 h) and concentration- (1-30 nM) dependently increased IL-8 protein content in the cell culture supernatant as measured by an enzyme immunosorbent assay standardized for DNA by fluoro-colorimetry. 3. Isoprenaline (1-100 nM, 15 min) increased cyclic AMP concentration-dependently. 4. The effect of isoprenaline (100 nM) was inhibited by the beta-adrenoceptor blocker propranolol (10 microM). The maximum magnitude of IL-8 increase caused by beta-adrenoceptor activation was 40% of that caused by the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha 100 ng ml-1). 5. The selective beta 2-adrenoceptor agonist salbutamol (1 microM), increased IL-8 protein similarly to isoprenaline and the cyclic AMP analogue, dibutyryl cyclic AMP (1 mM) produced a corresponding effect. 6. Pretreatment with isoprenaline (100 nM) followed by TNF-alpha (20 ng ml-1) increased IL-8 additively. 7. In conclusion, beta-adrenoceptor stimulation increased the release of the neutrophil chemoattractant, IL-8 in 16HBE cells, via an increase in intracellular cyclic AMP. beta-adrenoceptor stimulation adds to the IL-8 increase caused by the pro-inflammatory cytokine TNF-alpha. If this mechanism exists in vivo, beta-adrenoceptor activation may increase neutrophil chemotaxis into the airways.     

Localization of histidyl-tRNA synthetase (Jo-1) in human laryngeal epithelial carcinoma cell line (HEp-2 cells)

The present study was designed to determine the subcellular localization of histidyl-tRNA synthetase (Jo-1) in human laryngeal epithelial carcinoma cell line (HEp-2 cells). Indirect immunofluorescence using commercial HEp-2 cells with human serum and human-affinity-purified anti-Jo-1 antibodies was performed using confocal microscopy. Anti-histidyl-tRNA-synthetase-positive sera showed distinct nuclear and cytoplasmic granular staining in HEp-2 cells. Affinity purified anti-Jo-1 produced an identical pattern to the whole serum, whereas the serum fraction that did not bind to the affinity column was negative by immunofluorescence on HEp-2 cells. Two commercial human anti-Jo-1-positive control sera and seven anti-Jo-1-positive sera from patients with myositis reproduced the nuclear and cytoplasmic granular pattern. We conclude that Jo-1 is present in cytoplasm and in intact nuclei from HEp-2 cells. The presence of tRNA synthetases in intact nuclei suggests that they have an unsuspected function in the nucleus.     

Resistance to antibiotics of Streptococcus pneumoniae strains

Streptococcus pneumoniae strains are exhibiting increasing rates of antibiotics resistance. A rapid increase of resistance was seen not only to penicillin but also other antimicrobial agents and therefore this paper describes the study of resistance and multiresistance of pneumococci to 7 antibiotics: penicillin (P), erythromycin (E), clindamycin (CC), tetracycline (T), co-trimoxazole (SXT), cefotaxime (CTX) and vancomycin (Va), using the disk-diffusion technique according to NCCLS procedure. We tested a total of 218 S. pneumoniae strains isolated from various materials: from sputum (54), noses (117), throats (28) and different swabs specimens (19). The overall percentage of resistant isolates to penicillin was 3.7%, to erythromycin--4.1%, to clindamycin--10.6%, to tetracycline--17.4%, to co-trimoxazole--15.6%, to cefotaxime--2.3%. In the sputum was most the monoresistant strains (66.7%). The multiresistance was highest in the penicillin resistant pneumococci. With the exception of vancomycin, the number of resistant strains to non-beta-lactam antibiotics (erythromycin, clindamycin, tetracycline, co-trimoxazole) was higher in penicillin-resistant strains compared with penicillin susceptible isolates. All isolates were susceptible to vancomycin.     

Induction of lymphocytes cytotoxic to oral epithelial cells by Streptococcus mitis superantigen

The preparation of a superantigenic fraction F-2 from the culture supernatant of Streptococcus mitis 108, a fresh isolate from human tooth surfaces, was reported previously. Now, to determine the possible pathogenic role of the superantigen in oral mucosal diseases, we examined the cytotoxic effects of human peripheral blood T-cells activated with F-2 on human oral epithelial cells. T-cells activated with F-2 were cytotoxic to the human squamous carcinoma HO-1-N-1 cells derived from the oral mucosa, similar to those activated with Staphylococcus aureus enterotoxin B (SEB). This cytotoxic effect was increased in a dose-dependent manner by the addition of the respective stimulant, F-2 or SEB, to the cytotoxic assay system. F-2 endowed mainly CD8+ T-cells with cytotoxic activity. Pretreatment with human interferon gamma increased the sensitivity of the HO-1-N-1 cells to the cytotoxic effects of F-2-activated T-cells. The F-2-activated T-cells were also cytotoxic to human keratinocytes derived from gingiva. There was no correlation between the degree of cytotoxicity and the levels of tumor necrosis factor alpha in co-cultures of F-2-activated T-cells and HO-1-N-1 cells. A double-chamber plate experiment revealed no cytotoxic effects when the F-2-activated T-cells were separated from the HO-1-N-1 cells. Supernatants of the co-cultures of target and effector cells were not cytotoxic to HO-1-N-1 cells. These findings suggest that the cytotoxic effects of the F-2-activated T-cells on HO-1-N-1 cells were mediated not by soluble factors but by the direct interaction between the activated T-cells and the target cells. The cytotoxicity of the F-2-activated T-cells against HO-1-N-1 cells was markedly inhibited by monoclonal antibodies (MAbs) against CD11a and CD54, but was only slightly inhibited by MAbs against human leukocyte antigen (HLA)-DR and CD2. Thus, the interaction between lymphocyte-function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) was crucial for the F-2-dependent T-cell-mediated cytotoxicity against oral epithelial cells, while HLA-DR and CD2 molecules are not necessarily involved in the cytotoxicity observed.     

Interaction between glucocorticoids and beta2-agonists: alpha and beta glucocorticoid-receptor mRNA expression in human bronchial epithelial cells

Disabled-2 (Dab2), a mammalian structural homolog of Drosophila Disabled (Dab), is a mitogen-responsive phosphoprotein. It has been speculated to be a negative regulator of growth since its expression is lost in ovarian carcinomas. Dab2 contains a C-terminal proline-rich domain with sequences similar to those found in Sos, a guanine nucleotide exchange factor for Ras. The proline-rich sequences of Sos mediate the interaction of Sos with Grb2, an adaptor protein which coupled tyrosine kinase receptors to Sos. Herein, we have investigated the possibility that Dab2 interacts with Grb2. In experiments of co-immunoprecipitation from BAC1.2F5 macrophage cell lysates, significant quantities of Grb2 were associated with both Sos and Dab2, although Dab2 and Sos were not present in the same complex. Transfection of Dab2 into a Dab2-negative cell line (293 cells) decreased the amount of Grb2 associated with Sos, suggesting that Dab2 competes with Sos for binding to Grb2. Proline-rich peptides corresponding to Dab2 (#661-669) and to Sos (#1146-1161) inhibited the binding of Dab2 to Grb2, but were less effective in disrupting the Grb2-Sos complex. The expressed proline-rich domain of Dab2 (#600-730) bound Grb2, but other regions of Dab2 failed to bind Grb2. Both of the individual SH3 domains of Grb2 bound to Sos (N-terminal SH3 domain > C-terminal SH3 domain), but binding to Dab2 required the intact Grb2, suggesting cooperative binding using both SH3 domains of Grb2. These data indicate that Dab2 binds to the SH3 domains of Grb2 via its C-terminal proline-rich sequences. Dab2 may modulate growth factor/Ras pathways by competing with Sos for binding to Grb2.     

Polypeptide pattern of human breast epithelial cells following human chorionic gonadotropin (hCG) treatment

Numerous attempts have made to describe the particular protein pattern of malignant cells by using high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The placental hormone human chorionic gonadotropin (hCG) inhibits tumor initiation and progression in experimental animals and has an inhibitory effect on the proliferation of human breast epithelial cells (HBEC) in vitro. The inhibitory effect on the immortalized HBEC MCF-10F is accompanied by the immunocytochemical expression of inhibin alpha and beta subunits by treated cells. With the purpose of clarifying the molecular mechanisms involved in this effect, the pattern of protein synthesis and mRNA were studied by 2-D PAGE in the immortalized HBEC MCF-10F cells treated in vitro 1001U for 24 h. The effect of hCG treatment on the synthesis of MCF-10F cells was monitored by labeling both control and treated cells with [S35]methionine and separation by 2-D PAGE. At least 11 proteins were preferentially synthesized and five specific polypeptides were decreased in hCG treated cells in comparison with controls. The hCG induced at least four new mRNAs which encoded protein in the molecular mass range of 24-72 kDa. It also increased the expression of at least six mRNAs and reduced the expression of least four mRNAs in comparison with control cells. The hCG-treated cells actively synthesized a 33-kDa polypeptide which was not present in control cells. The nature of this hCG-inducible 33 kDa protein elucidated by immunoprecipating [S35]methionine-labeled proteins with antisera directed against rat inhibin subunit alpha and beta b.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the human junctional epithelial cells directly attached to the tooth (DAT cells) in periodontal disease

This study examined the directly attached cells (DAT cells) of the human junctional epithelium from teeth extracted for advanced periodontal disease. The monolayer nature of the DAT cells remaining on the tooth surface after extraction offers a unique opportunity to study their morphology and activity in situ. We collected DAT cells and examined them by transmission electron microscopy and by autoradiography of cells labeled with 3H-thymidine. Our morphological results suggest that degenerative changes associated with pocket formation affect individual cells rather than regions of the DAT cell population at one time. The epithelial attachment apparatus (EAA) appeared to be the most resistant structure persisting on the tooth surface during the degeneration of the individual DAT cells. The new technique developed for two-dimensional observation of the sheet of DAT cells labeled in situ on the tooth surface in culture showed that the attached cells, even in periodontal disease, exhibit proliferative ability, suggesting a regenerative role for the DAT cells.     

Management of infections due to antibiotic-resistant Streptococcus pneumoniae

Antibiotic-resistant strains of Streptococcus pneumoniae are becoming more prevalent throughout the world; this has resulted in modifications of treatment approaches. Management of bacterial meningitis has the greatest consensus. Strategies for treating other systemic infections such as pneumonia, bacteremia, and musculoskeletal infections are evolving, in part related to the availability of new antibiotics which are active in vitro against isolates resistant to penicillin and the extended-spectrum cephalosporins. However, there are currently very limited data related to the clinical efficacy of these new agents. The studies upon which current recommendations are based are reviewed. Otitis media represents the single most common infection due to S. pneumoniae. Recommendations for treatment of acute otitis media due to drug-resistant strains and the rationale for these recommendations are discussed.