Rotavirus-specific cytotoxic T lymphocytes appear at the intestinal mucosal surface after rotavirus infection.

The gastrointestinal tract is constantly exposed to a variety of potentially invasive bacteria and viruses. The first line of defense of the host against these pathogens is the intestinal mucosal surface, which consists of epithelial cells, intraepithelial lymphocytes (IELs), mucus, and secretory immunoglobulins. Little is known about the function, memory, or trafficking of IELs after intestinal infection. We found that IELs obtained 6 days after oral inoculation of mice with the intestinal pathogen rotavirus (simian strain RRV) lysed rotavirus-infected target cells; cytotoxic T lymphocytes (CTLs) were responsible for rotavirus-specific cytotoxic activity. Rotavirus-specific cytotoxic activity by IELs was (i) eliminated by treatment with Thy 1.2-specific immunoglobulin M plus complement, (ii) restricted by proteins encoded at the major histocompatibility complex, and (iii) absent in mock-infected animals. Oral inoculation of mice with RRV also induced rotavirus-specific CTLs in splenic and intestinal lymphocytes (mesenteric lymph nodes, Peyer's patch). Parenteral inoculation induced rotavirus-specific CTLs in splenic, intestinal (IELs, mesenteric lymph nodes, Peyer's patch), and nonintestinal lymphocytes (inguinal nodes). Therefore, presentation of rotavirus to the intestinal mucosal surface was not necessary to induce IELs with virus-specific cytotoxic activity. At 4 weeks after oral or parenteral inoculation of mice with RRV, rotavirus-specific CTL precursors appeared among splenic, Peyer's patch, inguinal, and mesenteric node lymphocytes, but not among IELs. IELs with rotavirus-specific cytotoxic activity may be generated from precursors at a site other than the intestinal mucosal surface. Part of the response of the host to enteric infection may include surveillance and lysis of virus-infected villus epithelial cells by IELs.     

Experimental study of the effect of corticosteroid preparations on the adhesion of Candida genus fungi to the epitheliocytes of mucous membranes

The effect of corticosteroids on the adherence of Candida cells to epithelial cells was studied with the use of the original modification of the in vitro adherence assay. The administration of hydrocortisone acetate to mice resulted in the increased adherence of the pathogenic fungi to epithelial cells: after 10-day hormone treatment the adherence number and the adherence index increased, respectively, to 230% and 360% of control values. After 20-day treatment these characteristics increased, respectively, to 260% and 700%. The adherence of C. albicans to vaginal epithelial cells in female mice receiving corticosteroids increased in comparison with that in the control animals at estrus: the adherence number increased to 220% and the adherence index, to 470% of the level observed in the controls.     

Luminal host-defense mechanisms against invasive amebiasis

Most humans infected with the virulent protozoan parasite Entamoeba histolytica do not develop invasive disease. Available evidence indicates that beneficial bacteria and the mucus gel layer in the colon lumen protect the host mucosa. Glycosidases produced by some normal colonic bacteria and luminal proteases degrade the key adherence lectin on E. histolytica trophozoites and decrease their adherence to epithelial cells. The mucus gel layer prevents those trophozoites that escape the hydrolases from reaching the epithelial cells. Trophozoite mucosal invasion is triggered only when both protective mechanisms are lost, as might occur during an unrelated pathogenic enteric bacterial infection. A newly developed gnotobiotic model of intestinal amebiasis should enable testing of this hypothesis and provide clues to help design practical studies in humans.     

Epithelial cells treated with genistein inhibit adhesion and endocytosis of<Emphasis Type="Italic"> Paracoccidioides brasiliensis</Emphasis>

Paracoccidioidomycosis is caused by Paracoccidioides brasiliensis, which although not formally considered an intracellular pathogen, can be internalized by epithelial cells in vitro and in vivo. The mechanisms used by P. brasiliensis to adhere to and invade non-professional phagocytes have not been identified. The signal-transduction networks, involving protein tyrosine kinase (PTK) and protein phosphatase activities, can modulate crucial events during fungal infections. In this study, the involvement of PTK has been investigated in P. brasiliensis adherence and invasion in mammalian epithelial cells. A significant inhibition of the fungal invasion occurred after the pre-treatment of the epithelial cells with genistein, a specific tyrosine kinase inhibitor, indicating that the tyrosine kinase pathway is involved in P. brasiliensis internalization. In contrast, when the fungus was treated, a slight (not significant) inhibition of PTK was observed, suggesting that PTK might not be the fungus’ transduction signal pathway during the invasion process of epithelial cells. An intense PTK immunofluorescence labeling was observed in the periphery of the P. brasiliensis infected cells, little PTK labeling was found in both uninfected cells and yeast cells, at later infection times (8 and 24 h). Moreover, when the epithelial cells were treated with genistein and infected with P. brasiliensis, no labeling was observed, suggesting the importance of the PTK in the infectious process. These results suggest that PTK pathway participates in the transduction signal during the initial events of the adhesion and invasion processes of P. brasiliensis to mammalian epithelial cells.     

Effect of various components of the fungal cell wall on adhesion of Candida albicans to the mouth mucosa epithelium in vitro]

An influence of mannan++, its component methyl-D-mannopyranoside+ and N-acetylglucosamine on in vitro adhesion of Candida albicans strains to buccal mucosal epithelium was studied. These substances inhibited adhesion when added to adherence test in a concentration of 10 mg/ml and 25 mg/ml despite whether were added to the test incubation medium or when preincubated with fungi or epithelial cells. Preincubation of fungal cells and epithelial cells with mannan had no influence on attachment; preincubation of epithelial cells with methyl-D-mannopyranoside+ and N-acetylglucosamine decreased adherence significantly. On the other hand preincubation of fungal calls with methyl-D-mannopyranoside+ increased their adhesive properties, having no influence on adherence after preincubation of fungi with N-acetylglucosamine.     

Influence of mucosal cell origin on the in vitro adherence of Candida albicans: Are mucosal cells from different sources equivalent?

The influence of collecting mucosal cells from various anatomical sites, and varying the date of collection and cell donor on adhesion of Candida albicans to human epithelial cells was examined by using an in vitro adherence assay. Examination of buccal mucosal cells from twenty-four donors showed statistically significant differences in the number of attached yeasts between individuals. Sex did not exert a significant influence on adhesion. Examination of buccal mucosal cells from ten donors collected on five different dates revealed that yeast attachment to mucosal epithelial cells varied significantly within subjects across time. Epithelial cells from some donors manifested greater date-to-date variations in yeast adhesion than others. Adherence of Candida to mucosal cells from three anatomical sites (mouth, vagina and urinary tract) collected from ten different donors was also tested. Yeast adherence to buccal cells was highest, lowest using urinary tract cells, while vaginal epithelium was intermediate. Adherence to mucosal cells from three sites was significantly different both within and between individuals although some subjects manifested larger variations than others. These data suggest that the in vitro adherence of Candida albicans is influenced by mucosal cell donor, date of collection and body site of origin. Mucosal cells from different sources do not appear to be equivalent in receptiveness to C. albicans and this might explain some of the discrepancies observed when adhesion studies performed by different investigators are compared. The existing need for a more uniform methodology with which to pursue studies on fungal attachment to mucosal surfaces is emphasized.     

Reconstitution of the gastrointestinal microflora of lactobacillus-free mice.

A colony of mice that do not harbor lactobacilli in their digestive tracts but whose intestinal microflora is otherwise functionally similar to that of conventional animals was derived. Methods used to reconstitute the intestinal microflora of the mice included inoculation of the animals with cultures of specific microbes, noncultivable microbes attached to epithelial cells, and cecal contents from conventional mice treated with chloramphenicol. Twenty-six microflora-associated characteristics were monitored by using relatively simple tests to determine the microflora status of the mice.     

Reconstitution of the gastrointestinal microflora of lactobacillus-free mice

A colony of mice that do not harbor lactobacilli in their digestive tracts but whose intestinal microflora is otherwise functionally similar to that of conventional animals was derived. Methods used to reconstitute the intestinal microflora of the mice included inoculation of the animals with cultures of specific microbes, noncultivable microbes attached to epithelial cells, and cecal contents from conventional mice treated with chloramphenicol. Twenty-six microflora-associated characteristics were monitored by using relatively simple tests to determine the microflora status of the mice.     

Fungal invasion of normally non-phagocytic host cells

Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research.     

Cecocolitis in immunodeficient mice associated with an enteroinvasive lactose negative E. coli

Infection with an atypical (lactose-negative) E. coli was associated with increased mortality rates in a colony of triple immune deficient N:NIH(S) III mice. Affected mice were lethargic and exhibited perianal fecal staining. Slight-to-moderate thickening of the wall of the cecum and colon was found on necropsy examination. Microscopic examination revealed segmental hyperplasia of the cecal and colonic mucosa with clusters of gram negative bacteria on the surface and within the cytoplasm of mucosal epithelial cells. Scattered foci of epithelial invasion and hyperplasia were observed in the colons of C57B1/6N-nunu mice after per os inoculation with the atypical E. coli. Immunocompetant mice housed in the same room as the N:NIH(S) III's remained healthy and exhibited no gross or microscopic lesions in spite of infection.     

Interaction of Eimeria tenella with intestinal mucin in vitro

The mucus gel layer overlying the gastrointestinal epithelium plays an important role in host-pathogen interactions. The initial interaction between the coccidian parasite Eimeria tenella and host cells of the intestinal epithelium must occur across this mucus interface. In this study, we examined the relationship between E. tenella and avian mucin, in particular the effect of purified intestinal regional mucin on parasite adherence and invasion in vitro. Secreted mucin from the chicken duodenum and cecum was purified by density gradient centrifugation and gel chromatography. Parasite invasion studies were performed in the Madin-Darby bovine kidney cell model. Eimeria tenella adherence to chicken duodenal mucin was detected, whereas adherence to cecal or bovine mucin was not shown. Parasite invasion into epithelial cells was not influenced by bovine mucin, whereas chicken mucin purified from the duodenum and cecum significantly inhibited invasion. Inhibition of E. tenella invasion into cells by mucin from the duodenum was marginally greater than that of the cecum, but this was not significant. This study demonstrated E. tenella interaction with native chicken intestinal mucin, which in turn inhibited parasite invasion into epithelial cells in vitro.     

实验性痴呆动物的肠道菌群和粘附性研究

用ＡＦ６４Ａ复制实验性痴呆动物模型,分析该动物的肠道菌群,并以双歧杆菌和大肠杆菌作为肠道菌的代表,初步探讨它们对实验性痴呆动物肠道粘膜上皮细胞表面的粘附特性。结果表明,实验性痴呆动物的肠道菌群是紊乱的,二种试验菌均能粘附到正常小鼠肠上皮细胞上,双歧杆菌的粘附率明显高于大肠杆菌,而双歧杆菌对实验性痴呆小鼠肠上皮细胞的粘附率明显低于对照组小鼠,大肠杆菌则相反。     

Peptide fragments of AMP-18, a novel secreted gastric antrum mucosal protein,are mitogenic and motogenic

Antrum mucosal protein (AMP)-18 is a novel 18-kDa protein synthesized by cells of the gastric antrum mucosa. The protein is present in secretion granules of murine gastric antrum epithelial cells and is a component of canine antrum mucus, suggesting that it is secreted into the viscoelastic gel layer on the mucosal surface. Release of the protein appears to be regulated because forskolin decreased the amount of immunoreactive AMP-18 in primary cultures of canine antrum mucosal epithelial cells, and indomethacin gavaged into the stomach of mice reduced AMP-18 content in antrum mucosal tissue before inducing histological injury. A functional domain of the protein was identified by preparing peptides derived from the center of human AMP-18. A 21-mer peptide stimulated growth of gastric and intestinal epithelial cells, but not fibroblasts, and increased restitution of scrape-wounded gastric epithelial monolayers. These functions of AMP-18 suggest that its release onto the apical cell surface is regulated and that the protein and/or peptide fragments may protect the antral mucosa and promote healing by facilitating restitution and proliferation after injury.     

Neisseria gonorrhoeae-induced transactivation of EGFR enhances gonococcal invasion

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhoea, adheres to and invades into genital epithelial cells. Here, we investigate host components that are used by the bacteria for their entry into epithelial cells. We found that gonococcal microcolony formation on the surface of HEC-1-B cells disrupted the polarized, basolateral distribution of both epidermal growth factor receptor (EGFR) and ErbB2, a related family member, and induced their accumulation under the microcolonies at the apical membrane. Gonococcal infection increased EGFR and ErbB2 phosphorylation. The EGFR kinase inhibitor, AG1478, reduced gonococcal invasion by 80%, but had no effect on adherence or the recruitment of EGFR and ErbB2 to the microcolonies. Gonococcal inoculation upregulated the mRNA levels of several ligands of EGFR. Prevention of EGFR ligand shedding by blocking matrix metalloproteinase activation reduced gonococcal invasion without altering their adherence, while the addition of the EGFR ligand, HB-EGF, was able to restore invasion to 66% of control levels. These data indicate that N. gonorrhoeae modulates the activity and cellular distribution of host EGFR, facilitating their invasion. EGFR activation does not appear to be due to direct gonococcal binding to EGFR, but instead by its transactivation by gonococcal induced increases in EGFR ligands.     

Glycosylation of Candida albicans Cell Wall Proteins Is Critical for Induction of Innate Immune Responses and Apoptosis of Epithelial Cells

C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.     

Adherence of Lactobacillus to Intestinal 407 Cells in Culture Correlates with Fibronectin Binding

Lactobacilli are members of the normal mucosal microflora of most animals. Many isolates of Lactobacillus spp. are adherent to epithelial cells. In this study, using Lactobacillus acidophilus and L. agilis, we detected adherence in a pattern that suggested that the bacteria were binding to extracellular matrix proteins. Fluorescent microscopy, by using anti-fibronectin antibody, demonstrated that the isolates localize in those areas where fibronectin was detected. In addition, fibronectin pretreatment of the bacterial cells decreased adherence to Intestinal 407 epithelial cell monolayers. Cellular binding to fibronectin was detected by enzyme-linked immunosorbent assay and affinity binding to radio-labeled fibronectin. Fibronectin may be one of the eukaryotic receptors mediating attachment of Lactobacillus to mucosal surfaces. Received: 19 January 2000 / Accepted: 2 March 2000     

Behavior of in vitro grown normal human mucosal epithelial cells and tumorigenic rat cells inoculated into nude mice

The present study describes the behavior of in vitro grown normal human oral mucosal epithelial cells and that of a tumorigenic epithelial cell line following subcutaneous inoculation into nude mice. A successful recovery of viable human epithelial cell inocula was seen in 25-90% of mice and there was no improvement in recovery rates after addition of fibroblasts. These inocula resulted in cyst formation lined by a 2-6 cell layer unkeratinized squamous epithelium without rete ridges. There was no increase in recovery rate or size of cysts when coinoculated with fibroblasts. The tumorigenic cell inocula were successfully recovered in all cases. Tumors established from these inocula had a low grade of differentiation and were without signs of metastasis. Inocula of tumorigenic cells showed an increased size after addition of fibroblasts to the inocula. The model may be useful in studies of interactions between inoculations of heterologous normal and pathologic cells as well as in studies of differentiation of carcinogen-treated epithelial cells.     

Blockade of Salmonella enteritidis passage across the basolateral barriers of human intestinal epithelial cells by specific antibody

Antibodies specific to Salmonella enteritidis (S.E.) were obtained from immunized egg yolk, and their protective effects against S.E. were studied by using monolayer-cultured human intestinal epithelial cells, Caco-2 and T84. The Salmonella adherence and entry to the cells were partially inhibited by the antibodies. The antibodies inhibited the decrease in transepithelial electrical resistance (TEER) of the intestinal epithelial monolayers and IL-8 secretion of the cells induced by S.E. invasion. Also, the antibodies blocked the penetration of bacteria through the cell layer although they did not inhibit the growth of bacteria in the cells. Confocal microscopic photographs revealed the bacteria in the infected monolayer cells were bound to antibodies. These results indicate that anti-S.E. antibodies may protect the cells from destruction induced by S.E. invasion in intestinal epithelial cells in addition to the partial inhibition of adhesion and invasion of S.E. at the cell surface. Passive antibodies against invasive bacteria would be useful to prevent the migration of S.E. to blood not only at the cell surface but also inside of intestinal epithelial cells.     

Virulence Factors and Antifungal Susceptibility of Candida albicans Isolates from Oral Candidosis Patients and Control Individuals

Sixty isolates of Candida albicans, 30 obtained from the oral cavity of denture wearers presenting signs of candidosis and 30 obtained from the oral cavity of denture wearers with normal palatal mucosa were assayed for phospholipase and proteinase production, as well as for adherence to buccal epithelial cells. Likewise, susceptibility of the isolates to antifungals was determined by the NCCLS reference method and the E-test method. Proteinase activity was increased among the strains obtained from oral candidosis patients. In contrast, no significant differences between the two groups of isolates were observed in their adherence ability in vitro, in phospholipase production, and susceptibility to antifungal drugs.     

Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.