Biomedical subjects

D W Niesel

Publications and source records attributed to D W Niesel.

At least 37 records · Page 2Linked to original sources

PubMed1993

Tumor necrosis factor (TNF alpha) regulates intestinal mucus production during salmonellosis.

Mucus production by goblet cells in the gastrointestinal tract following Salmonella typhimurium infection using a ligated ileal loop model in mice was investigated. Assessment of the morphology of the loop tissue after Salmonella challenge revealed generalized tissue inflammation, characterized by edema and an inflammatory cell infiltrate. Villi were shortened and blunted, and crypts contained an increased number of cells with mitotic figures. Production of TNF alpha in the loops followed Salmonella challenge and occurred at the same time as the pathological sequelae. A nearly 50% decrease in the number of goblet cells in infected tissue compared to tissue from noninfected controls was observed at these same times. The sulfation of mucins produced by the goblet cells in infected tissues was increased in the villi but was unchanged in the crypts compared to uninfected tissues. Treatment of mice with antibody to TNF alpha before Salmonella challenge abrogated tissue pathology and returned goblet cell numbers and mucin profiles to those observed in noninfected controls. Our results indicate that TNF alpha may mediate changes in goblet cell expression and mucin sulfation in response to Salmonella challenge.

Animals↗

PubMed1993

Immunization of mice with Yersinia enterocolitica leads to the induction of antithyrotropin receptor antibodies.

It is well established that autoimmune Graves' disease, which is characterized by hyperthyroidism, is mediated by autoantibodies to the thyrotropin receptor (TSHr). Although what initially triggers this autoantibody response is not known, a number of studies have suggested that Yersinia enterocolitica, an enterobacteria, could initiate the immune response against the TSHr. In this study, we produced antibodies against purified extracellular domain of human TSHr (ETSHr) and showed that anti-ETSHr antibodies reacted with envelope preparations from Y. enterocolitica. This reactivity was specifically blocked by preincubating sera with purified ETSHr. Moreover, antibodies reactive with ETSHr were induced by immunizing mice with Y. enterocolitica but not with Shigella flexneri SA100, Salmonella typhimurium TML, and Listeria monocytogenes. Anti-Y. enterocolitica antisera specifically reacted with the ETSHr protein and the reactivity could be blocked both by ETSHr and Y. enterocolitica envelope proteins. Our studies provide the first direct evidence that immunization with Y. enterocolitica can lead to the production of antibodies capable of reacting with TSHr and might provide the initial stimulus necessary for breakdown of self-tolerance to TSHr, eventually leading to the development of autoimmunity to TSHr.

Animals↗

PubMed1993

Ity influences the production of IFN-gamma by murine splenocytes stimulated in vitro with Salmonella typhimurium.

The Ity-Lsh-Bcg genetic locus in the mouse has been documented to confer innate resistance to at least three intracellular pathogens: Salmonella typhimurium, Leishmania donovani, and Mycobacterium. Expression of the resistance gene(s) results in a slower net growth of these pathogens in the reticuloendothelial system early postinfection. Although it is clear that the resident macrophages in resistant mice are functionally superior with regard to antimicrobial activity, the exact mechanism(s) underlying the control exerted by this gene is not understood. Using S. typhimurium infection as a model, we have examined the influence of this resistance gene(s) on the production of IFN-gamma, a cytokine known to play an important role in host-defense against several intracellular pathogens. We compared IFN-gamma production by splenocytes from resistant (Ity(r)) and sensitive (Ity(s)) inbred mouse strains after stimulation in vitro with S. typhimurium. Spleen cells from Ity(r) mouse strains produced significantly higher levels of IFN-gamma when compared to spleen cells obtained from Ity(s) mouse strains. Enhanced IFN-gamma production was not a generalized response to bacteria. Listeria monocytogenes induced comparable levels of IFN-gamma production from both Ity(r) (CBA/J) and Ity(s) (C57BL/6) mice. Splenocytes from Ity congenic mouse strains displayed similar differences in the level of IFN-gamma produced after S. typhimurium stimulation, with spleen cells from the Ity(r) strain producing significantly higher levels of IFN-gamma when compared to spleen cells from the Ity(s) strain. A requirement for adherent cells and/or adherent cell-derived factors has been documented for IFN-gamma production by S. typhimurium-stimulated splenocytes. Interestingly, supernatant from adherent cells obtained from Ity(r) mouse strains was found to induce the production of significantly higher levels of IFN-gamma when compared to adherent cell supernatant from Ity(s) strains. Nylon wool nonadherent cells from Ity(s) mouse strains produced high levels of IFN-gamma when exposed to supernatants obtained from adherent cells of Ity(r) mouse strains. In contrast, nylon wool nonadherent cells from Ity(r) mouse strains produced reduced levels of IFN-gamma when exposed to supernatant obtained from adherent cells of Ity(s) mouse strains. Thus, modulation of IFN-gamma production appears to be a function of the Ity(r) gene(s). This study documents for the first time that the Ity locus may play a role in controlling resistance to Salmonella infection by regulating IFN-gamma production by NK cells.

Animals↗

PubMed1993

Salmonella typhimurium induces IFN-gamma production in murine splenocytes. Role of natural killer cells and macrophages.

IFN-gamma is a cytokine known to play an important role in host defense against Salmonella typhimurium. The lymphoid cells required for in vitro production of IFN-gamma after S. typhimurium stimulation of mouse spleen cells was investigated. Spleen cells depleted of cells bearing NK1.1, asialo GM1, Thy 1.2, or CD5 resulted in a significant reduction in IFN-gamma production after stimulation with S. typhimurium. In contrast, Con A-induced IFN-gamma production was only slightly reduced after depletion of NK1.1- or asialo GM1-bearing cells. Spleen cells from SCID mice produced elevated levels of IFN-gamma after stimulation with S. typhimurium. IFN-gamma production by SCID spleen cells was dependent upon asialo GM1+ T cells, suggesting that NK cells were the cells producing IFN-gamma in response to S. typhimurium. Splenic adherent cells were required for optimal IFN-gamma production. However, direct contact between the adherent and nylon wool nonadherent (NWNA) cell populations was not essential. IFN-gamma production was observed when the adherent and NWNA cell populations were physically separated or when supernatant from S. typhimurium-stimulated adherent cells was added to NWNA cells. Optimal IFN-gamma production was dependent on the presence of TNF-alpha, inasmuch as addition of antibody to TNF-alpha to spleen cell or NWNA cell cultures significantly reduced IFN-gamma production. However, addition of rTNF-alpha did not induce IFN-gamma production by NWNA cells. These findings document the existence of a T-independent mechanism for early IFN-gamma production in response to S. typhimurium, and show that TNF-alpha is necessary but not sufficient for the production of IFN-gamma.

Animals↗

PubMed1993

Tumor necrosis factor-alpha mediates the early pathology in Salmonella infection of the gastrointestinal tract.

Salmonella infection of the intestinal tract results in damage to the gut epithelium. While it is generally believed that bacteria and/or bacterial products account for this pathology, the role of host factors has not been explored. Using a ligated intestinal loop model, we investigated whether tumor necrosis factor-alpha (TNF-alpha) could contribute to the tissue pathology associated with Salmonella infection. Intestinal segments infected with Salmonella typhimurium had high levels of fluid secretion as early as 6 h post-bacterial infection. At this time point, low levels of TNF activity were also present in the fluid obtained from infected segments. At 20 h post-infection, high levels of TNF activity were present in fluids obtained from infected intestinal segments and was characterized as TNF-alpha by neutralization experiments using rabbit antisera to TNF-alpha. TNF-alpha production was further verified by Northern blot analysis using RNA obtained from cells eluted from the infected intestinal segments. In contrast, no TNF activity was found in fluid obtained from intestinal segments challenged with cholera toxin, which induces fluid secretion with little to no inflammatory response. Double labeling by in situ hybridization and immunocytochemistry revealed that macrophages in the lamina propria were producing the TNF-alpha mRNA. To investigate what role TNF-alpha might play in Salmonella-induced inflammation, intestinal segments were injected with recombinant mouse TNF-alpha (rTNF-alpha) or mice were pretreated with antibody to TNF-alpha or a control antibody prior to Salmonella infection. The histological profile of intestinal segments injected with rTNF-alpha appeared identical to segments infected with S. typhimurium. Further, pathology was completely eliminated in infected mice pretreated with antibody to TNF-alpha. These results document the production of TNF-alpha in the intestinal tract following S. typhimurium infection and show that the early pathology induced by Salmonella infection of the gastrointestinal tract is mediated by immune mechanisms.

Animals↗

PubMed1993

Shigella flexneri invasion of HeLa cells induces NF-kappa B DNA-binding activity.

Although information about the genetic basis and mechanisms of Shigella flexneri cellular invasion is accumulating, little is known about changes in cell signaling and their consequences following bacterium-host cell interactions. A general result of signal transduction is alterations in the levels and/or activities of transcription factors. Alterations in transcription factor binding activities were observed following challenge with S. flexneri. Changes in the DNA-binding activities of cellular transcription factors to AP1, AP2, cyclic AMP response element, CTF1/NF1, NF-kappa B/Rel, OCT1, and SP1 DNA-binding sites were investigated by electrophoretic mobility shift assays. NF-kappa B/Rel DNA-binding activity was enhanced more than 11-fold by cellular invasion; noninvasive S. flexneri strains induced low levels of kappa B DNA binding. Both subunits of the NF-kappa B transcription factor, p50 and p65, but not c-Rel (p85), are components of the kappa B DNA-binding activity. These data suggest that changes in cellular transcription factor binding activity are a consequence of S. flexneri invasion, and these changes could play a role in the initial host response or in the pathogenesis of the disease.

Base Sequence↗

PubMed1993

Tumor necrosis factor alpha binding to bacteria: evidence for a high-affinity receptor and alteration of bacterial virulence properties.

Human and murine receptors for tumor necrosis factor alpha (TNF-alpha) are present on most somatic cells and have been characterized and cloned. In contrast, very little is currently known about whether TNF-alpha can bind to pathogens and whether such binding results in important biological consequences for the infected host. We now report that a number of gram-negative bacteria have receptors for TNF-alpha. Using 125I-labeled TNF-alpha, we show that Shigella flexneri has 276 receptors for TNF-alpha, with a Kd of 2.5 nM. The binding of labeled TNF-alpha to these bacterial receptors can be inhibited by cold TNF-alpha but not by cold TNF-beta. Binding of 125I-TNF-alpha to S. flexneri was inhibited by trypsin treatment of bacterial cells or incubation at 52 degrees C for 3 min. Monoclonal antibody to either the 55-kDa or the 75-kDa TNF-alpha receptor, which are present on different eukaryotic cells, had no effect on 125I-TNF-alpha binding to bacteria. A number of gram-negative bacteria were capable of binding 125I-TNF-alpha. Gram-positive bacteria bound significantly less 125I-TNF-alpha than gram-negative bacteria. Pretreatment of S. flexneri with TNF-alpha resulted in enhanced bacterial invasion of HeLa cells and enhanced uptake by human and murine macrophages. Pretreatment of HeLa cells with antibody to the 55-kDa TNF-alpha receptor abrogated enhanced invasion of HeLa cells by TNF-alpha-bacterium complexes. These results suggest that TNF-alpha-bacterium complexes can interact with TNF-alpha receptors present on eukaryotic cells. This report shows that gram-negative bacteria have receptors for TNF-alpha and that a virulence property of a bacterium is altered as a consequence of cytokine binding.

Bacterial Proteins↗

PubMed1992

Listeria monocytogenes activation of human peripheral blood lymphocytes: induction of non-major histocompatibility complex-restricted cytotoxic activity and cytokine production.

Gram-negative bacteria have been shown to activate human natural killer (NK) cells. In this report, we show that the gram-positive bacterium Listeria monocytogenes can also activate human NK cells with regard to non-major histocompatibility complex (MHC)-restricted killing and the production of cytokines. Overnight incubation of peripheral blood mononuclear (PBM) cells or enriched NK cell populations with live or Formalin-fixed L. monocytogenes resulted in high levels of non-MHC-restricted cytotoxic activity. Listeria-stimulated non-MHC-restricted cytotoxic activity could be achieved with pathogenic as well as nonpathogenic Listeria strains. PBM cells also produced tumor necrosis factor alpha and different interferons (IFNs) after incubation with Listeria strains. Optimal cytokine production appeared to be dependent on nylon wool- and plastic-adherent cells. Different IFNs were produced by Listeria-stimulated PBM cells obtained from different donors. IFN-gamma was always produced but was sometimes associated with IFN-alpha and/or IFN-beta. Interleukin-2 (IL-2) activity was never detected in culture supernatants obtained from Listeria-stimulated PBM cell cultures. However, IL-2 appeared to be produced by Listeria-stimulated PBM cells, since antibody to IL-2 inhibited Listeria-stimulated NK cell cytotoxic activity. Listeria activation of NK cell cytotoxic activity was also dependent on tumor necrosis factor alpha production. Antibody to IFN-gamma, IFN-beta, or IFN-alpha had no effect on Listeria-stimulated NK cell cytotoxic activity. These results demonstrate that NK cells can be activated by Listeria strains and add further evidence that NK cells may play an important role in host defense against bacterial infections.

Cytokines↗

PubMed1991

The interferons. Mechanisms of action and clinical applications.

The interferons (IFN) are one of the body's natural defensive responses to such foreign components as microbes, tumors, and antigens. The IFN response begins with the production of the IFN proteins (alpha, beta, and gamma), which then induce the antiviral, antimicrobial, antitumor, and immunomodulatory actions of IFN. Recent advances have led to Food and Drug Administration approval of five clinical indications for IFN. Interferon alfa is approved for hairy-cell leukemia, condyloma acuminatum, Kaposi's sarcoma in the acquired immunodeficiency syndrome, and non-A, non-B (type C) viral hepatitis. Interferon gamma has properties distinctive from those of IFNs alpha and beta and is approved as an immunomodulatory treatment for chronic granulomatous disease. Promising clinical results with IFNs have also been reported for basal cell carcinoma, chronic myelogenous leukemia, cutaneous squamous cell carcinoma, early human immunodeficiency virus infection, hepatitis B, and laryngeal papillomatosis. Future clinical uses of IFNs may emphasize combination therapy with other cytokines, chemotherapy, radiation, surgery, hyperthermia, or hormones.

Humans↗

PubMed1991

Interferon gamma (IFN-gamma) production by gut-associated lymphoid tissue and spleen following oral Salmonella typhimurium challenge.

Although IFN-gamma has been shown to play an important role in protection against a systemic S. typhimurium challenge, the in vivo and in vitro production of this cytokine following S. typhimurium infection of the gastrointestinal tract has not been investigated. In this study, IFN-gamma production by gut-associated lymphoid tissue and spleen was investigated in mice following oral challenge with S. typhimurium. Cells obtained from the Peyer's patches (PP), mesenteric lymph nodes (MLN) and spleen (Sp) of mice orally challenged with S. typhimurium were assessed for levels of IFN-gamma mRNA after varying times following in vivo infection. RNA obtained from the above tissues was subjected to reverse transcription followed by PCR amplification using primers specific for murine IFN-gamma. Elevated levels of IFN-gamma mRNA were first detected in the PP at 6 h post-challenge. Elevated levels of IFN-gamma mRNA were then detected in the MLN at 24 h and in the spleen at 4 days post-challenge. These in vivo results were in agreement with the ability of these lymphoid tissues to produce IFN-gamma upon in vitro stimulation with killed S. typhimurium. Neutralization of endogenously produced IFN-gamma by administration of mAb to IFN-gamma completely abrogated resistance to an oral challenge of S. typhimurium. A significant difference in the percent mortality was observed between the antibody-treated and control groups. Evaluation of bacterial spread in the antibody treated group versus the control group at 4 days following oral challenge revealed higher numbers of bacteria in the spleen and liver of antibody treated mice. These results clearly show that IFN-gamma is rapidly produced by gut-associated lymphoid tissue and spleen following oral S. typhimurium infection, and that endogenous production of IFN-gamma is essential in host resistance to S. typhimurium.

Administration, Oral↗

PubMed1990

Bacteria-infected fibroblasts have enhanced susceptibility to the cytotoxic action of tumor necrosis factor.

The susceptibility of bacteria-infected fibroblasts to the cytotoxic action of tumor necrosis factor was investigated. L cells infected with Shigella flexneri, Salmonella typhimurium, or Listeria monocytogenes, had an enhanced susceptibility to the cytotoxic activity of TNF-alpha. This enhanced susceptibility was dependent upon the challenge dose of bacteria, the concentration of TNF, and upon the exposure time of bacteria-infected cells to TNF. L cells infected with S. flexneri were susceptible to the cytotoxic action of TNF at 2 to 6 h after bacterial infection. In contrast, L cells infected with S. typhimurium or L. monocytogenes did not show enhanced susceptibility to TNF until 14 h postbacterial infection and exposure to TNF. Enhanced susceptibility to TNF was dependent on bacterial invasion because fibroblasts pretreated with a noninvasive isogenic variant of S. flexneri, UV-treated invasive bacteria, bacterial cultural supernatant, or bacteria LPS were no more susceptible to TNF than untreated cells. Enhanced susceptibility to TNF by bacteria-infected cells was not unique to L cells. Mouse embryo fibroblasts and HeLa cells also showed similar reactivities after bacteria infection. Bacteria-infected cells were greatly suppressed in host cell protein synthesis that may play an important role in their enhanced susceptibility to TNF. These results suggest that an important role of TNF in host defense against bacterial infections is its cytotoxic activity against bacteria-infected cells.

Animals↗

PubMed1990

Interferon production by Shigella flexneri-infected fibroblasts depends upon intracellular bacterial metabolism.

The role of bacterial invasion and subsequent intracellular metabolism or replication, or both, in the induction of interferon (IFN) production in primary cultures of murine embryo fibroblasts (MEFs) was examined. IFN production appeared to be dependent upon bacterial invasion. MEFs that were challenged with Shigella flexneri cultured at 30 degrees C to inhibit the temperature-dependent virulence gene expression that is essential for invasion failed to produce IFN. Furthermore, inhibition of S. flexneri invasion by pretreatment of MEFs with cytochalasin B resulted in a reduction in IFN production. Intracellular bacterial residence alone, however, was not sufficient for the induction of IFN production since an avirulent isogenic variant of S. flexneri which invades but fails to grow intracellularly did not induce IFN production. In fact, the blocking of bacterial RNA synthesis immediately after cellular uptake of S. flexneri by rifampin inhibited IFN production by MEFs. Transfer of the invasion-encoding plasmid to a noninvasive Escherichia coli strain conferred upon the bacteria the ability to invade MEFs and induce IFN production. These results suggest that the induction of IFN production in S. flexneri-infected fibroblasts requires bacterial invasion and intracellular bacterial metabolism or replication, or both.

Animals↗

PubMed1989

The induction of interferon production in fibroblasts by invasive bacteria: a comparison of Salmonella and Shigella species.

As the role of interferon (IFN) in host defense against facultative intracellular bacterial infections continues to expand, it has become increasingly important to understand what cell types can produce IFN following infection and/or interaction with these invasive bacteria. We have demonstrated previously that Shigella flexneri was able to induce high levels of IFN in primary cultures of human and murine fibroblasts following bacterial invasion. In this study, we examined the ability of Salmonella typhimurium to induce IFN production in different cell lines. S. typhimurium-infected primary cell cultures of mouse embryo-fibroblasts (MEF) were shown to produce high levels of IFN following bacterial challenge. In contrast to Shigella, Salmonella required a much lower multiplicity of infection for optimal IFN induction. Examination at the RNA level of IFN production by MEF following challenge with either bacteria revealed that the IFN produced was a mixture of IFN alpha and IFN beta (IFN alpha/beta), with IFN beta 1 as the predominant species. As previously demonstrated for Shigella, bacterial invasion of cells appeared to be required for the induction of IFN production by S. typhimurium. Salmonella rendered non-invasive by UV-treatment failed to induce IFN production in MEF. Furthermore, Salmonella LPS, when tested over a wide range of concentrations, was unable to induce IFN production in these cells. In contrast to MEF, human and murine continuous cell lines did not produce IFN following Salmonella challenge. These results taken together suggest that IFN may be a common factor involved in Salmonella and Shigella infections. Furthermore, IFN may play an important role in the front line host defense against these types of infections.

Animals↗

PubMed1988

Natural killer cell activation and interferon production by peripheral blood lymphocytes after exposure to bacteria.

We have previously shown that peripheral blood natural killer (NK) cells have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. In this report, we show that NK cell activity against K562 tumor cells and Shigella flexneri-infected HeLa cells can be greatly enhanced by preincubating peripheral blood lymphocytes (PBL) for 18 h with kanamycin-treated Shigella flexneri or Salmonella typhimurium. Cell-free supernatants obtained from PBL-bacteria cultures contained high levels of interferon (IFN) activity, which was characterized as a mixture of IFN-gamma and IFN-alpha. Cytotoxic activity associated with PBL precultured with shigellae was associated with predominantly CD16+ (Leu-11+) and CD2+ (OKT-11+) cells. Further, IFN production was dependent upon the presence of CD16+ and CD2+ cells at culture initiation. Enhancement of cytotoxic activity associated with PBL-bacteria cultures did not, however, appear to be dependent upon IFN production, since low numbers of bacteria which failed to stimulate IFN production induced high levels of NK cell activity. Lipopolysaccharide appeared not to be involved in bacteria-induced IFN production or enhanced NK cell activity, since Salmonella lipopolysaccharide failed to induce IFN production or enhance NK cell activity. These results suggest that IFN production by NK cells and the killing of bacteria-infected cells play an important role in host defense against facultative intracellular bacterial infections.

Adjuvants, Immunologic↗

PubMed1987

Bacterial invasion of fibroblasts induces interferon production.

Although bacteria and/or bacterial products have been shown to induce interferon (IFN) production in cells of the immune system, the ability of non-lymphoid cells to produce IFN in response to bacterial products and/or bacterial invasion is currently unknown. In this study we examined murine and human fibroblasts for their ability to produce IFN in response to challenge with the invasive bacteria Shigella flexneri and enteroinvasive Escherichia coli. Both human and murine primary cell cultures produced high levels (500 to 1000 U/ml) of IFN-beta within 12 hr after Shigella invasion. The production of IFN appeared to be dependent upon bacterial invasion because no IFN was produced by cells pretreated with a non-invasive isogenic variant of S. flexneri. Furthermore, UV treated invasive S. flexneri, which lose the ability to invade cells, failed to induce IFN production in fibroblasts after bacterial challenge. In contrast to primary cells, most human and murine continuous cell lines did not produce IFN in response to Shigella invasion. The above results demonstrate that bacterial invasion of primary cultures of fibroblasts can induce IFN production. Taken together with previous findings, these results suggest that IFN may play an important front line host defense against invasive bacterial infections.

Animals↗

PubMed1986

Natural cytotoxic effector cell activity against Shigella flexneri-infected HeLa cells.

Virus and facultative intracellular bacteria both replicate within a host cell. The recognition and killing of virus-infected cells by natural killer (NK) cells is thought to be an important host immune function. However, little is known about immune recognition of bacteria-infected cells. In this report, we show for the first time that human peripheral blood lymphocytes (PBL) and large granular lymphocytes (LGL) purified from PBL have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. This cytotoxic activity was dependent on bacterial invasion of the HeLa cells, because HeLa cells pretreated with a noninvasive isogenic variant of S. flexneri or soluble bacterial products were not killed. Pretreatment of PBL with interleukin 2 (IL 2) or interferon-alpha greatly enhanced the cytotoxic activity of PBL against Shigella-infected HeLa cells. Cytotoxic activity present in PBL or in PBL pretreated with IL 2 was shown to be associated with both Leu-11+ and Leu-11- cell populations. These results suggest that NK cell killing of bacteria-infected cells may play an important role in host defense against facultative intracellular bacterial infections.

Antigens, Surface↗

PubMed1986

Natural and recombinant interferons inhibit epithelial cell invasion by Shigella spp.

The effect of natural and recombinant interferons (IFNs) on the abilities of Shigella flexneri, S. sonnei, and Salmonella typhimurium to invade different human and murine cells was examined. Pretreatment of cell monolayers with natural and recombinant IFNs reduced the number of Shigella-infected cells in a dose-dependent manner. Establishment of an anti-invasive cellular state was time dependent, requiring 10 h for 50% inhibition of bacterial invasion. The inhibitory effect of IFN was species specific, with human or murine IFN effective against homologous but not heterologous cells. Gamma IFN was slightly more potent than alpha IFN at inhibiting bacterial invasion. Inhibition of Shigella invasion was dependent on the challenge dose of bacteria. Little inhibition of invasion was seen when cells were pretreated with low concentrations of IFN and challenged with high multiplicities of infection of Shigella sp. In contrast to Shigella invasion, the maximum inhibitory effect of IFN on Salmonella invasion of cells was observed at low levels (5 to 50 U) of IFN. These results suggest that Shigella and Salmonella invasions occur at unique sites on eucaryotic cells or by different penetration mechanisms. More importantly, these data suggest that IFN may play a significant role in host defense against Shigella and Salmonella infections.

Animals↗

PubMed1985

Quantitation of HeLa cell monolayer invasion by Shigella and Salmonella species.

A major determinant in the virulence of Salmonella and Shigella spp. is the ability of these organisms to invade epithelial cells of the gastrointestinal mucosa and multiply intracellularly. The invasion of cell culture monolayers is a convenient experimental system to evaluate eucaryotic cell penetration and is correlated with the potential of a strain to cause human disease. We have developed an agarose-L agar overlay technique which allows for the convenient quantitation of the number of infected tissue culture cells in a monolayer. Bacterial strains were introduced onto antibiotic-free HeLa cell monolayers. Infected monolayers were washed, and noninternalized bacteria were counterselected with kanamycin (50 micrograms/ml). The number of infected HeLa cells present was determined by overlaying the monolayer with distilled water-agarose (0.5 to 1.5%) followed by an equal volume of 2X L agar. Bacterial colonies formed over infected cells in 24 h at 37 degrees C, and wells were counted with a dissecting microscope under X2 power. Bacterial colonies were not observed with noninvasive variants of Shigella spp. To obtain countable wells (20 to 200 CFU) the multiplicity of infection or invasion times were adjusted. With a 90-min invasion time, the invasive potential of a strain was reflected by the multiplicity of infection needed to produce countable wells. The quantitation of bacterium-invaded cells by using standard bacteriological methods is a convenient and rapid method to evaluate the invasive potential of bacterial strains. Additionally, parameters essential for the invasive process can easily be investigated.

Agar↗