Physical limitations on Salmonella typhi entry into cultured human intestinal epithelial cells

Kinetic studies of Salmonella typhi invasion of INT407 cells at different multiplicities of infection (MOIs) have revealed a strict physical limitation on S. typhi entry at MOIs of >/=40. Staining of infected monolayers to distinguish intracellular from extracellular bacteria revealed that all monolayer cells are susceptible to infection and that internalized bacteria are typically contained in one to three separate clusters per cell during the first 60 min. Scanning and transmission electron microscopic analyses of time course-infected monolayers showed that at early times postinfection, bacteria bind to shortened, coalesced microvilli in one to three focal aggregate structures per host cell surface. As reported previously for S. typhimurium, focal aggregates progress to conical membrane ruffles that appear to engulf one or a few centrally contained S. typhi cells by a macropinocytic process, which enhanced the entry of simultaneously added Escherichia coli HB101 about 30-fold. Additionally, kinetic studies showed that at an MOI of approximately 400, maximal S. typhi entry is virtually completed within 30 to 35 min. Monolayers pretreated with S. typhi for 30 min to saturate the entry process were severely reduced in the ability to internalize subsequently added kanamycin-resistant strains of S. typhi or S. typhimurium, but E. coli HB101(pRI203) expressing the cloned Yersinia inv gene was not reduced in entry. In invasion inhibition assays, anti-beta1 integrin antibodies markedly reduced E. coli HB101(pRI203) invasion efficiency but did not reduce S. typhi entry. Collectively, these data provide direct physical and visual evidence which indicates that S. typhi organisms are internalized at a limited number (i.e., two to four) of sites on host cells. S. typhi and S. typhimurium likely share INT407 cell entry receptors which do not appear to be members of the beta1 integrin superfamily.     

Interaction of Escherichia coli heat-stable enterotoxin B with cultured human intestinal epithelial cells

Binding of Escherichia coli heat-stable enterotoxin B (STb) to the human intestinal epithelial cell lines T84 and HT29 and to polarized T84 cells was studied to define the initial interaction of this peptide toxin with target cells. Equilibrium and competitive binding isotherms showed that 125I-STb bound specifically to T84 and HT29 cells; however, the toxin-epithelial cell interactions could be characterized by low-affinity binding (< or = 10(5) M(-1)) to a high number of binding sites (> or = 10(6) per cell). STb binding to T84 and HT29 cells as a function of 125I-STb concentration did not approach saturation at levels well above the effective biological concentration of STb for fluid secretion. Treatment of the 125I-STb-bound T84 and HT29 cells with an acidic saline solution to remove surface-bound toxin revealed that only approximately 55% +/- 10% of 125I-STb could be removed by this treatment at 4 degrees C, suggesting that approximately half of the bound STb was stably associated with the plasma membrane and/or internalized into the cytoplasm. Similar results were obtained when binding and internalization experiments were conducted at 22 and 37 degrees C. Immunofluorescence studies demonstrated that the strongest signal for STb appeared in the plasma membrane even after acid treatment. Toxin-treated cells also displayed diffuse cytoplasmic staining, indicating that once cell bound, STb did not appear to preferentially associate with membrane vesicles or cellular organelles. Binding and subsequent internalization of 125I-STb were not affected by treatment of the cells with trypsin, endoglycosidase F/peptide N-glycosidase F, Vibrio cholerae neuraminidase, tunicamycin, or 5 mM sodium chlorate, which blocks sulfation of surface proteoglycans. In addition, the internalization process was not altered by preincubation of the cells with the cytoskeleton inhibitors cytochalasin D and colchicine or cellular perturbants (i.e., 0.45 M sucrose and 5 mM sodium azide), indicating that cell surface proteins or carbohydrates did not function as STb receptors. The binding of 125I-STb to polarized T84 cells was also examined, and the total and nonspecific binding isotherms were found to overlap, indicating that the apical surface of polarized T84 cells did not contain a specific receptor for STb. In comparison to undifferentiated cells, twice the amount of bound STb (approximately 80% +/- 10%) was removable from polarized T84 cells after treatment with acidic solution. The percentage of surface-bound STb to polarized T84 cells did not vary significantly with the transepithelial electrical resistance of the cells or when STb was applied basolaterally. Together, our results indicate that STb binds with relatively low affinity to the plasma membrane of cultured intestinal epithelial cells and polarized T84 cells, probably to membrane lipids, and becomes stably associated with the lipid bilayer. The fact that a significant portion of the bound STb becomes free in the cytoplasm, even at a low temperature, suggests that the bound toxin may directly traverse the membrane bilayer.     

Adhesion and cytosolic dye transfer between macrophages and intestinal epithelial cells

Activated macrophages (M phi) found in the intestinal lesions of patients with inflammatory bowel disease (IBD) secrete many inflammatory mediators which can regulate intestinal epithelial cell (IEC) function. However, little is known about direct M phi-IEC interactions. Two potential mechanisms by which cells may interact are through specific receptor-ligand binding of adhesion molecules, such as integrins or cadherins, and by exchange of cytoplasmic substances through transmembraneous channels called gap junctions. We investigated whether M phi could adhere to epithelial cells in culture and form transmembrane communication channels as defined by dye transfer. Primary cultures of murine M phi and a M phi cell line, P388D1, adhered to Mode-K and IEC6, but not CMT-93 IEC. Antibody blocking studies determined that P388D1-Mode-K binding was partially dependent on beta 2 integrin (CD18) function, Mode-K constitutively expressed CD106 (VCAM-1) and cell associated fibronectin, while P388D1 expressed low levels of CD49d/CD29 (VLA4) but blocking antibodies to these surface molecules did not inhibit P388D1-Mode-K adherence. Transfer of calcein dye from M phi to IEC was quantitated by flow cytometry and was dependent on M phi-IEC adhesion. Dye transfer was concentration dependent in that the fluorescence intensity of Mode-K was proportional to the number of adherent P388D1 cells as well as the dye load of the M phi. These results indicate that M phi interact with IEC by adhesion and possibly through gap junctions and may thus regulate IEC function by direct cell-cell communication.     

Attachment of Cryptosporidium parvum sporozoites to human intestinal epithelial cells

An enzyme-linked immunosorbent assay-based attachment model using the human intestinal cell line Caco-2A was developed to study attachment of Cryptosporidium parvum sporozoites in vitro and to assess potential inhibitors of sporozoite binding. In this system, attachment was related to sporozoite dose, incubation time, and host cell differentiation status. Polyclonal antibodies to C. parvum as well as glycoprotein inhibitors of a sporozoite lectin reduced attachment. This model will be a valuable tool in elucidating specific molecules and mechanisms involved in sporozoite-host cell attachment.     

Clostridium difficile toxin A binding to human intestinal epithelial cells

Clostridium difficile radiolabelled toxin A ([3H]-toxin A) bound to human duodenal and colonic epithelial cells isolated from endoscopic biopsies. Binding was greater at 4 degrees C than 37 degrees C, consistent with the thermal binding characteristic of toxin A to a carbohydrate moiety. At 37 degrees C colonic cells bound significantly more [3H]-toxin A than duodenal cells. The amount of [3H]-toxin A binding varied considerably between individuals. [3H]-toxin A was displaced by unlabelled toxin A by 50% for duodenal cells and 70% for colonic cells with 94.3 nM unlabelled toxin A. Low non-displacable binding was observed in some samples at 4 degrees C and 37 degrees C, suggesting that these cells came from individuals incapable of specifically binding toxin. Pre-treating cells with alpha- or beta-galactosidases to cleave terminal alpha- and beta-galactose residues reduced [3H]-toxin A binding. There was also a reduction in [3H]-toxin A binding after heat treating cells, which is suggestive of protein binding. The reduction in binding varied between individuals. The reduction of [3H]-toxin A binding, after the removal of beta-linked galactose units, implicates these as components of the receptor and adds credence to the idea that the Lewis X, Y and I antigens may be involved in toxin A binding to human intestinal epithelial cells. However, because the Lewis antigens do not possess terminal alpha-galactose units, the reduction in binding after alpha-galactosidase treatment suggests that other receptors may be involved in toxin A binding to some human intestinal cells. These data are the first demonstration of direct toxin A binding to human intestinal epithelial cells.     

Apoptosis of human intestinal epithelial cells after bacterial invasion

Epithelial cells that line the human intestinal mucosa are the initial site of host invasion by bacterial pathogens. The studies herein define apoptosis as a new category of intestinal epithelial cell response to bacterial infection. Human colon epithelial cells are shown to undergo apoptosis following infection with invasive enteric pathogens, such as Salmonella or enteroinvasive Escherichia coli. In contrast to the rapid onset of apoptosis seen after bacterial infection of mouse monocyte-macrophage cell lines, the commitment of human intestinal epithelial cell lines to undergo apoptosis is delayed for at least 6 h after bacterial infection, requires bacterial entry and replication, and the ensuing phenotypic expression of apoptosis is delayed for 12-18 h after bacterial entry. TNF-alpha and nitric oxide, which are produced as components of the intestinal epithelial cell proinflammatory program in the early period after bacterial invasion, play an important role in the later induction and regulation of the epithelial cell apoptotic program. Apoptosis in response to bacterial infection may function to delete infected and damaged epithelial cells and restore epithelial cell growth regulation and epithelial integrity that are altered during the course of enteric infection. The delay in onset of epithelial cell apoptosis after bacterial infection may be important both to the host and the invading pathogen since it provides sufficient time for epithelial cells to generate signals important for the activation of mucosal inflammation and concurrently allows invading bacteria time to adapt to the intracellular environment before invading deeper mucosal layers.     

Rotavirus infection of cultured intestinal epithelial cells induces secretion of CXC and CC chemokines

The extracellular glycoproteins fibronectin (FN) and laminin (LMN) are ubiquitously expressed in myocardial tissue. These glycoproteins are important for cellular attachment and differentiation of the cardiac myocytes. Utilizing specific antibodies for the detection of FN and LMN, respectively, the distribution of these extracellular proteins was examined in enzymatically isolated adult cardiac myocytes. Immunofluorescence staining of rod-shaped cardiac myocytes revealed only remnants of immunoreactive FN on the cellular surface and in the transverse tubular membrane system. LMN expression, however, was preserved in a raster-like pattern in the cardiac myocytes. In order to study the distribution of these glycoproteins at high resolution, scanning electron microscopy using the backscattered electron mode was combined with immunogold staining and silver-enhancement. In addition, to confirm the immunofluorescence microscopic observations it was shown that FN labelling was restricted to ill-defined extracellular material and that LMN was absent from the intercalated discs of the cardiac myocytes. The hypercontracted cells were characterized by numerous surface protrusions devoid of immunoreactive LMN. Thus, these results indicate that FN and LMN are differently affected by collagenase treatment, and that these changes of glycoprotein expression may influence the normal function of the cardiac myocytes as well as the membrane stability during the development of irreversible cellular lesions.     

Vectorial release of poliovirus from polarized human intestinal epithelial cells

Polarized epithelial cells represent the primary barrier to virus infection of the host, which must also be traversed prior to virus dissemination from the infected organism. Although there is considerable information available concerning the release of enveloped viruses from such cells, relatively little is known about the processes involved in the dissemination of nonenveloped viruses. We have used two polarized epithelial cell lines, Vero C1008 (African green monkey kidney epithelial cells) and Caco-2 (human intestinal epithelial cells), infected with poliovirus and investigated the process of virus release. Release of poliovirus was observed to occur almost exclusively from the apical cell surface in Caco-2 cells, whereas infected Vero C1008 cells exhibited nondirectional release. Structures consistent with the vectorial transport of virus contained within vesicles or viral aggregates were observed by electron microscopy. Treatment with monensin or ammonium chloride partially inhibited virus release from Caco-2 cells. No significant cell lysis was observed at the times postinfection when extracellular virus was initially detected, and transepithelial resistance and vital dye uptake measurements showed only a moderate decrease. Brefeldin A was found to significantly and specifically inhibit poliovirus biosynthetic processes by an as yet uncharacterized mechanism. The vectorial release of poliovirus from the apical (or luminal) surface of human intestinal epithelial cells has significant implications for viral pathogenesis in the human gut.     

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.     

Inhibition of Toxoplasma gondii replication in IFN-gamma-activated human intestinal epithelial cells

A therapeutic trial, involving 130 Schistosoma mansoni-infected children, with no previous history of antischistosomal treatment, was carried out to evaluate the efficacy of two different dose regimens of praziquantel. The study was carried out because low cure rates were described in this recently established (1990) S. mansoni focus in northern Senegal, following treatment with a standard dosage of 40 mg/kg. The subjects were randomly allocated into two groups: one group (1) received 40 mg/kg in one oral dose, the other group (2) was treated with two oral doses of 30 mg/kg at a 6-hr interval. Parasitologic examination and circulating anodic antigen (CAA) detection were performed before, 10 days, three, six, and 21 weeks after chemotherapy. No significant differences in cure rates were found between the two groups. Six weeks after treatment, 34% and 44% of the individuals were found to be stool negative in group 1 and group 2, respectively. However, only 10-15% became completely negative according to the serum CAA antigen assay. Mean egg counts were reduced by 99% in both groups. Antigen detection confirmed the parasitologic results. Fewer side effects were observed in the group treated with 2 x 30 mg/kg, which may be explained by split dosage administration. Our study shows that the low cure rates observed in this area could not be improved by using a higher dosage of praziquantel.     

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.     

Transport of quercetin and its glucosides across human intestinal epithelial Caco-2 cells

There is mounting evidence from human epidemiological, animal in vivo, and in vitro studies to suggest beneficial effects related to the consumption of quercetin and its glucosides. However, there is limited knowledge on the oral bioavailability of these natural products. This study examined the intestinal epithelial membrane transport of quercetin, quercetin 4'-glucoside, and quercetin 3,4'-diglucoside, using the Caco-2 human colonic cell line, a model of human intestinal absorption. The apparent permeability (Papp) of each agent was measured in both apical to basal and basal to apical directions. The apical to basolateral flux of quercetin, Papp 5.8 +/- 1.1 x 10(-6) cm x sec(-1) (mean +/- SEM), was more than 10-fold higher than for the paracellular transport marker mannitol, 0.48 +/- 0.09 x 10(-6) cm x sec(-1) (P < 0.01). Under identical conditions, the Papp for the transcellular marker propranolol was about 5-fold higher than for quercetin (P < 0.001). Interestingly, the reverse, basolateral to apical, flux of quercetin (Papp 11.1 +/- 1.2 x 10(-6) cm x sec(-1)) was almost 2-fold higher than the apical to basolateral flux (P < 0.001). In similar experiments, quercetin 4'-glucoside demonstrated no absorption, Papp < 0.02 x 10(-6) cm x sec(-1) in the apical to basal direction, but did demonstrate basal to apical flux, Papp 1.6 +/- 0.2 x 10(-6) cm x sec(-1). Quercetin 3,4'-diglucoside showed a low apical to basolateral transport (Papp 0.09 +/- 0.03 x 10(-6) cm x sec(-1)); its reverse, basolateral to apical, transport was, however, 4-fold higher (P < 0.05). In these cells, glucose was actively transported with an apical to basolateral Papp of 36.8 +/- 1.1 x 10(-6) cm x sec(-1). These observations suggest facile absorption of quercetin through the human intestinal epithelium, but contrary to a previous proposal, they do not support an active transport process for quercetin glucosides.     

TGF-beta1 effects on proliferation of rat intestinal epithelial cells are due to inhibition of cyclin D1 expression

Transforming growth factor-beta 1 (TGF-beta1) arrests intestinal epithelial cells (RIE-1 and IEC-6) in the G1 phase of the cell cycle and inhibits cyclin D1 expression. This report describes experiments designed to elucidate the mechanism of cyclin D1 inhibition and to determine whether inhibition of cyclin D1 expression is the cause, rather than the result, of TGF-beta1-mediated cell cycle arrest. TGF-beta1 inhibition of IEC-6 cell proliferation was associated with a decrease in the abundance of cyclin D1/Cdk4 complexes and a corresponding decrease in Cdk4-dependent phosphorylation of the retinoblastoma protein. Metabolic labeling studies indicated that TGF-beta1 inhibited cyclin D1 synthesis without altering the rate of cyclin D1 protein degradation. Cyclin D1 antisense oligonucleotides blocked serum-stimulated induction of cyclin D1 and DNA synthesis, whereas cyclin D1 sense oligonucleotides had no effect. RIE-1 cells were engineered to overexpress human cyclin D1 under the control of a tetracycline-repressible promoter. These cells entered S phase in the presence of TGF-beta1 only when human cyclin D1 was derepressed by the withdrawal of tetracycline. These data indicate that TGF-beta1 inhibits the synthesis of cyclin D1 in gut epithelial cells and that this inhibition is the cause, rather than the result, of TGF-beta1-mediated arrest of intestinal epithelial cell proliferation.     

The regulation and functional consequence of proinflammatory cytokine binding on human intestinal epithelial cells

Products of an activated immune system may affect cells within the immune system as well as nonlymphoid cells in the local environment. Given the immunologically activated state of the intestinal tract, it is conceivable that locally produced cytokines could regulate epithelial cell function. To assess whether epithelial cells are targets for particular cytokines, we initiated studies on the binding of a panel of proinflammatory cytokines in freshly isolated epithelial cells from normal and inflammatory bowel disease (IBD) patients as well as in cell lines. Isolated intestinal epithelial cells (IEC) were stained with phycoerythrin-conjugated or biotinylated cytokines to determine the expression and density of receptors for IL-1beta, IL-6, granulocyte-macrophage CSF (GM-CSF), and TNF-alpha. Receptors for IL-1beta, IL-6, and GM-CSF were readily detectable in all epithelial cell preparations at levels equal to (GM-CSFR) or lower than those seen on monocytes. However TNFalpha-R were not detectable on freshly isolated IECs. Receptor density was greater in surface vs crypt epithelial cells, but no significant differences were seen between normal and IBD epithelial cells. Expression of IL-1R and IL-6R was enhanced by LPS and IFN-gamma. Functionally, IL-1beta enhanced proliferation of the IEC cell line, DLD1, whereas GM-CSF treatment of de-differentiated crypt-like DLD1 and HT29 cells resulted in enhanced expression of ICAM-1. Furthermore, TNF-alpha treatment enhanced the secretion of IL-8 and GRO-alpha in HT29 cells, but not in freshly isolated IEC cultures. The differential binding and function of proinflammatory cytokines on IEC support the hypothesis that these cytokines may be involved in normal physiological processes as well as in regulating mucosal immune responses.     

Eosinophil adhesion to cultured airway epithelial cells from guinea pig lungs

We examined the effects of histamine, tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), indomethacin and dexamethasone on the adherence of eosinophils to airway epithelial cells. Epithelial cells were isolated from guinea pig trachea through enzymatic digestion, and cultured until they showed confluence. Eosinophils were taken by peritoneal lavage from guinea pigs sensitized with horse serum and purified. The adhesion of eosinophils to cultured epithelial cells was examined using 51Cr. The percentage adhesion was obtained by dividing counts per minute of lysed eosinophils adhered to epithelial cells by suspended eosinophils. Histamine significantly increased the adhesion of eosinophils to epithelial cells, while TNF alpha or IFN gamma did not significantly alter the adhesion. Indomethacin significantly inhibited the adhesion of eosinophils to epithelial cells in both nontreated and histamine-induced conditions. Dexamethasone also reduced adhesion, although it did not reach statistical significance. These findings indicate that histamine induced eosinophil adherence to airway epithelial cells, suggesting a role of endogenous prostaglandins in adherence.     

Significance of membrane attack complex inhibitory factor expression in cultured human glomerular epithelial cells

We report five cases of pancreatitis in systemic lupus erythematosus. Three patients died. The cause of death was directly related to pancreatitis in two cases. Several causes of pancreatitis may be suspected: vasculitis, thrombosis when associated to antiphospholipid syndrome, biliary calculi, infection, metabolic abnormalities or adverse effects of therapy. About seventy cases of pancreatitis have been reported in systemic lupus erythematosus in the literature Pancreatitis was the inaugural sign of lupus in six cases. In 12 cases, besides lupus, no other cause of pancreatitis was found. However, pancreatic vasculitis or thrombosis was rarely demonstrated even in post-mortem examinations. The role of corticosteroids in pancreatitis in lupus is controversial since the evolution of pancreatitis in lupus is generally good with corticosteroids. We think that diagnosis of pancreatitis in lupus should not implicate discontinuation or decrease of corticosteroids dosage, unless their responsibility is absolutely demonstrated.     

Comparison of the toxic effects of hydrogen peroxide and ozone on cultured human bronchial epithelial cells

In this study, we compared the cytotoxic and genotoxic effects of hydrogen peroxide and ozone on cultured human airway epithelial cells in primary culture. Both agents caused a dose-dependent loss in the replicative ability of epithelial cells and at higher levels of exposure caused acute cytotoxicity as measured by release of lactate dehydrogenase. Differences were seen, however, between the agents' effects with regard to induction of DNA single strand breaks as measured by alkaline elution:; whereas single-strand breaks were detected in significant amounts at concentration of hydrogen peroxide that cause acute cytotoxicity, none were detected at any of the levels of ozone exposure examined. A difference was also seen in the ability of the iron chelator deferoxamine to protect cells from the effect of the two oxidants. Preincubation of cultures with deferoxamine appreciably attenuated the toxicity of hydrogen peroxide but not of ozone. These data suggest that ozone has significant toxic effects on bronchial epithelial cells not mediated through the generation of hydrogen peroxide or hydroxyl radical. Furthermore, the data indicate that the inhibiting action of ozone on cell replicative ability is not mediated through a mechanism related to DNA single strand breaks.     

Differential early interactions between Salmonella enterica serovar Typhi and two other pathogenic Salmonella serovars with intestinal epithelial cells

Salmonella enterica serovar Typhi (hereafter referred to as S. typhi) is a host-restricted pathogen that adheres to and invades the distal ileum and subsequently disseminates to cause typhoid fever in humans. However, S. typhi appears to be avirulent in small animals. In contrast, other pathogenic salmonellae, such as S. enterica serovars Typhimurium and Dublin (S. typhimurium and S. dublin, respectively), typically cause localized gastroenteritis in humans but have been used as models for typhoid fever because these organisms cause a disease in susceptible rodents that resembles human typhoid. In vivo, S. typhi has been demonstrated to attach to and invade murine M cells but is rapidly cleared from the Peyer's patches without destruction of the M cells. In contrast, invasion of M cells by S. typhimurium is accompanied by destruction of these M cells and subsequently sloughing of the epithelium. These data have furthered our view that the early steps in the pathogenesis of typhoidal and nontyphoidal Salmonella serovars are distinct. To extend this concept, we have utilized an in vitro model to evaluate three parameters of initial host-pathogen interactions: adherence of three Salmonella serovars to human and murine small intestinal epithelial cell (IEC) lines, the capacity of these salmonellae to invade IECs, and the ability of the bacteria to induce interleukin-6 (IL-6) in these cell lines as a measure of host cell activation and the host acute-phase response. The results demonstrate that S. typhi adheres to and invades human small IECs better than either S. typhimurium or S. dublin. Interestingly, invA and invE null mutants of S. typhi are able neither to adhere to nor to invade IECs, unlike S. typhimurium invA and invE mutants, which adhere to but cannot invade IECs. S. typhi also induces significantly greater quantities of IL-6 in human small IEC lines than either of the other two Salmonella serovars. These findings suggest that differential host cytokine responses to bacterial pathogens may play an important role in the pathological sequelae that follow infection. Importantly, S. typhimurium did not induce IL-6 in murine IECs. Since S. typhimurium infection in mice is often used as a model of typhoid fever, these findings suggest that, at least in this case, the mouse model does not reflect the human disease. Taken together, our studies indicate that (i) marked differences occur in the initial steps of S. typhi, S. typhimurium, and S. dublin pathogenesis, and (ii) conclusions about S. typhi pathogenesis that have been drawn from the mouse model of typhoid fever should be interpreted conservatively.