Immuno-histochemical and -cytochemical Evidence Suggesting the Presence of Campylobacter jejuni and Campylobacter coli in Cases of Porcine Intestinal Adenomatosis

Antisera against a number of Campylobacter species were used in immuno-histochemical and -cytochemical studies on cases of porcine intestinal adenomatosis. Avidin-biotin-complex (ABC) and streptavidin immunoperoxidase methods were used on formalin-fixed, paraffin-embedded and frozen sections. Protein A gold method was used on formaldehyde fixed and frozen sections for immuno-cytochemistry. The antisera used were raised in rabbits by subcutaneous or intravenous injection of living or formalin treated organisms. Antisera against different serotypes of the thermotolerant, catalase positive Campylobacters, Campylobacter jejuni and Campylobacter coli gave positive reactions in the immuno-histochemical studies. The staining was found in intestinal epithelial cells both in the ileum and in the colon and was restricted to the apical cytoplasm of adenomatous epithelial cells. The staining had a granular pattern, the positive structures sometimes having the shape of Campylobacter. Epithelial cells in areas with normal differentiation of goblet cells did not stain. In contrast, no staining resulted with antisera against Campylobacter sputorum subsp. mucosalis and Campylobacter hyointestinalis. Immuno-cytochemistry, using antisera against Campylobacter jejuni showed that the positive staining in altered epithelial cells were restricted to intracellular organisms having a structure resembling Campylobacter spp.     

Heterogeneity of intercellular adhesion in rat liver cells in culture

The intercellular homotypic adhesive properties of 14 clones derived from a nontumorigenic rat liver epithelial cell line (LEC), derived from neonatal Fischer rats, were examined and compared to those of the hepatoma H4-II-E cell line. Each clone was assayed also for the degree of chromosomal aneuploidy and the ability to grow in soft agar. Over 100-fold differences in adhesive properties were observed among the clones, but no correlation was observed between the degree of aneuploidy in the clones and intercellular adhesive properties. The parent LEC cell line and the clones derived from it were unable to grow in soft agar. The H4-II-E cells showed negligible capacity to reaggregate after dissociation into single cells and these cells readily formed colonies in soft agar. Many of the LEC clones were similar to the H4-II-E cells in their adhesive properties, which suggests that reduced cell-to-cell adhesiveness per se is not a necessary prerequisite of epithelial cells to be able to grow independent of anchorage. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of concanavalin A (Con A)-binding glycoproteins in the "most adhesive" clone 67 and the "least adhesive" clone 201 showed markedly elevated amounts of acidic 105 and 67-kDa glycoproteins in clone 67. Proteins with similar migration patterns in 2D-PAGE have previously been reported to participate in specific homotypic intercellular adhesion of liver cells. The Con A-binding glycoprotein pattern in H4-II-E cells was markedly different from that of LEC cells with a set of six proteins missing and nine proteins appearing new in the H4-II-E cells. It is suggested that, in addition to identifying known epithelial cell polypeptides, systematic screening of cell surface-associated glycoproteins in normal and transformed epithelial cells in vitro and in vivo may lead to identification of novel polypeptides intimately associated with the transformed phenotype.     

Positive correlation between degree of parasitemia, interferon titers, and natural killer cell activity in Plasmodium falciparum-infected children

Natural killer (NK) cell activity and interferon levels have been measured in the peripheral blood of children acutely ill with Plasmodium falciparum infection. The NK cell levels were found to be raised in the malaria-infected children, with a positive correlation between the degree of parasitemia and lytic activity. Comparatively high titers of antiviral activity was discovered in sera from the majority of P. falciparum-infected children, again positively correlating with the degree of parasitemia and NK levels. The characteristics of the antiviral factor indicated alpha-type interferon to be the dominating agent involved. Addition of exogenous interferon in vitro potentiated the NK levels of PBL from normal children while having no significant impact on cells from malaria-infected children.     

The epidemiological aspects of campylobacteriosis in children in arid area]

Specific epidemiological features of Campylobacter infection in children in Samarkand Province during the period of 1987-1990 are discussed. The specific proportion of this infection in the total structure of acute enteric infections was, on the average, 8.5%, and Campylobacter carriership among healthy children, 7.0%. Among children with Campylobacter infection, children aged up to 1 year constituted 71.5%. The results of the study of the biological properties of Campylobacter strains isolated from sick and healthy children and their difference from strains isolated in other regions are discussed.     

Kingella kingae PilC1 and PilC2 are adhesive multifunctional proteins that promote bacterial adherence,twitching motility,DNA transformation,and pilus biogenesis

The gram-negative bacterium Kingella kingae is a leading cause of osteoarticular infections in young children and initiates infection by colonizing the oropharynx. Adherence to respiratory epithelial cells represents an initial step in the process of K. kingae colonization and is mediated in part by type IV pili. In previous work, we observed that elimination of the K. kingae PilC1 and PilC2 pilus-associated proteins resulted in non-piliated organisms that were non-adherent, suggesting that PilC1 and PilC2 have a role in pilus biogenesis. To further define the functions of PilC1 and PilC2, in this study we eliminated the PilT retraction ATPase in the ΔpilC1ΔpilC2 mutant, thereby blocking pilus retraction and restoring piliation. The resulting strain was non-adherent in assays with cultured epithelial cells, supporting the possibility that PilC1 and PilC2 have adhesive activity. Consistent with this conclusion, purified PilC1 and PilC2 were capable of saturable binding to epithelial cells. Additional analysis revealed that PilC1 but not PilC2 also mediated adherence to selected extracellular matrix proteins, underscoring the differential binding specificity of these adhesins. Examination of deletion constructs and purified PilC1 and PilC2 fragments localized adhesive activity to the N-terminal region of both PilC1 and PilC2. The deletion constructs also localized the twitching motility property to the N-terminal region of these proteins. In contrast, the deletion constructs established that the pilus biogenesis function of PilC1 and PilC2 resides in the C-terminal region of these proteins. Taken together, these results provide definitive evidence that PilC1 and PilC2 are adhesins and localize adhesive activity and twitching motility to the N-terminal domain and biogenesis to the C-terminal domain.     

Signal transduction in Campylobacter jejuni-induced cytokine production

Campylobacter jejuni is the leading cause of food-borne illness in the USA and one of the most common causes of diarrhoea worldwide. Central to its pathogenicity is its ability to induce the production of proinflammatory cytokines such as interleukin (IL)-8 in intestinal epithelial cells. Here, we demonstrated that C. jejuni infection of intestinal epithelial cells results in the activation of the ERK and p38 mitogen-activated protein kinases and that the ERK kinase pathway is essential for IL-8 production. We found that MAP kinase stimulation leading to IL-8 secretion requires C. jejuni gene products whose production is stimulated upon contact with epithelial cells. We also found that C. jejuni flagellin is a very poor stimulator of Toll-like receptor (TLR)-5 and therefore does not play a significant role in the stimulation of cytokine production.     

沙眼衣原体感染对大鼠着床期间子宫内膜上皮及其β-连接素表达的影响

目的研究沙眼衣原体(Chlamydiatrachomatis,Ct)感染后大鼠着床期间子宫内膜形态学改变及β-连接素(β-catenin,β-cat)的变化。方法大鼠雌雄合笼建立早孕模型,取正常妊娠和Ct感染后妊娠大鼠子宫,采用扫描电镜(SEM)观察子宫内膜超微结构改变及免疫组化检测β-cat的表达。结果SEM显示对照组子宫内膜腔上皮细胞排列紧密,细胞间隙较窄;实验组腔上皮细胞排列疏松,间隙变宽;免疫组化定性结果示β-cat妊娠4-6d主要定位于内膜上皮细胞膜,妊娠7d在蜕膜细胞膜;半定量结果示在感染组与正常组妊娠第5d其表达量达到高峰,但感染组弱于正常组,且两者之间有显著性差异(P<0·05)。结论Ct感染后可能通过影响子宫内膜黏附分子的表达,破坏着床的微环境;同时影响上皮细胞之间的连接,从而干扰早期妊娠。     

The effect of gangliosides on the adhesive interaction of Neisseria meningitidis with human cells

The influence of a number of gangliosides and sialic acid on the adhesive interaction of meningococci and human cells have been studied. Sialic acid has been found to produce no influence on adhesion, and the preliminary treatment of meningococci with gangliosides or their preparations suppresses the capacity of meningococci for attachment to epithelial cells and erythrocytes. At the same time the degree of the inhibition of adhesion depends on the kind and concentration of gangliosides. On the contrary, after the treatment of target cells with gangliosides (1.25 micrograms/ml) the adhesion indices of meningococci with respect to these cells increase 5- to 8-fold. These data are indicative of the participation of gangliosides in the adhesive interaction of meningococci and human cells.     

口腔细菌对铜绿假单胞菌黏附及侵入肺上皮细胞能力的影响

目的 通过比较铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入细胞的能力,探讨细菌间相互作用在呼吸道感染的最初阶段的作用机制.方法 应用培养法和抗生素保护法检测铜绿假单胞菌和口腔细菌单独或共同作用于肺上皮细胞时,细菌黏附和侵入肺上皮细胞的能力.结果 铜绿假单胞菌与口腔细菌共同作用于肺上皮细胞,牙龈卟啉单胞菌和伴放线放线杆菌降低了铜绿假单胞菌的黏附能力,却增强了其侵入能力;而铜绿假单胞菌能够影响口腔细菌对肺上皮细胞的黏附,同时增强口腔细菌侵入肺上皮细胞的能力.结论 口腔细菌,尤其是牙周可疑致病菌主要通过增强铜绿假单胞菌对肺上皮细胞的侵入而影响呼吸道感染过程.     

The adhesive activity of diphtheritic strains in relation to the characteristics of the infectious process they induce]

The degree of adhesiveness of 602 C. diphtheriae strains isolated from patients with different forms of diphtheria was studied on trypsinized sheep red blood cells (SRBC) used as an experimental model. The titer of bacterial suspension, i.e. its highest dilution ensuring the agglutination of 50% of SRBC, was assumed to be the index of adhesive activity. The toxigenic strains were more homogeneous with respect to the degree of their activity and proved to be moderately and highly adhesive, while among the nontoxigenic strains faintly and moderately adhesive ones prevailed. The degree of adhesiveness was not linked with the cultural biological strains variants, but depended on the form of C. diphtheriae infection. The toxigenic strains isolated from diphtheria patients were essentially more active than those isolated from carriers. Both toxigenic and nontoxigenic strains isolated in cases of prolonged carrier state (more than 4 weeks) did not differ in the degree of their adhesiveness and were essentially more active than the strains isolated from short-term carriers. The strains circulating in 10 closed groups with a high proportion of pronounced cases of carrier state (70.6% to 86.7%) were essentially more active than those circulating in 10 similar groups, but having a low proportion of pronounced cases of carrier state (6.7% to 23.8%). The conclusion was made that the degree of adhesiveness proved to be an important factor of C. diphtheriae pathogenicity, responsible for the formation of carrier state. Along with pathogenicity, this factor should be taken into consideration in the evaluation of the epidemiological importance of different sources of infection.     

A major role for intestinal epithelial nucleotide oligomerization domain 1 (NOD1) in eliciting host bactericidal immune responses to Campylobacter jejuni

Campylobacter jejuni is the foremost cause of bacterial-induced diarrhoeal disease worldwide. Although it is well established that C. jejuni infection of intestinal epithelia triggers host innate immune responses, the mechanism(s) involved remain poorly defined. Innate immunity can be initiated by families of structurally related pattern-recognition receptors (PRRs) that recognize specific microbial signature motifs. Here, we demonstrated maximal induction of epithelial innate responses during infection with live C. jejuni cells. In contrast when intestinal epithelial cells (IECs) were exposed to paraformaldehyde-fixed bacteria, host responses were minimal and a marked reduction in the number of intracellular bacteria was noted in parallel. These findings suggested a role for intracellular host-C. jejuni interactions in eliciting early innate immunity. We therefore investigated the potential involvement of a family of intracellular, cytoplasmic PRRs, the nucleotide-binding oligomerization domain (NOD) proteins in C. jejuni recognition. We identified NOD1, but not NOD2, as a major PRR for C. jejuni in IEC. We also found that targeting intestinal epithelial NOD1 with small interfering RNA resulted in an increase in number of intracellular C. jejuni, thus highlighting a critical role for NOD1-mediated antimicrobial defence mechanism(s) in combating this infection at the gastrointestinal mucosal surface.     

Architecture and adhesive activity of the Haemophilus influenzae Hsf adhesin

Haemophilus influenzae type b is an important cause of meningitis and other serious invasive diseases and initiates infection by colonizing the upper respiratory tract. Among the major adhesins in H. influenzae type b is a nonpilus protein called Hsf, a large protein that forms fiber-like structures on the bacterial surface and shares significant sequence similarity with the nontypeable H. influenzae Hia autotransporter. In the present study, we characterized the structure and adhesive activity of Hsf. Analysis of the predicted amino acid sequence of Hsf revealed three regions with high-level homology to the HiaBD1 and HiaBD2 binding domains in Hia. Based on examination of glutathione S-transferase fusion proteins corresponding to these regions, two of the three had adhesive activity and one was nonadhesive in assays with cultured epithelial cells. Structural modeling demonstrated that only the two regions with adhesive activity harbored an acidic binding pocket like the binding pocket identified in the crystal structure of HiaBD1. Consistent with these results, disruption of the acidic binding pockets in the adhesive regions eliminated adhesive activity. These studies advance our understanding of the architecture of Hsf and the family of trimeric autotransporters and provide insight into the structural determinants of H. influenzae type b adherence.     

Proliferative activity of gastric epithelial cells in Helicobacter pylori infected children

Helicobacter pylori infection has been associated with gastric carcinogenesis. Gastric epithelial cells proliferative rate is accelerated in H. pylori infected adult patients. Our study was performed to evaluate proliferative cell activity in gastric epithelium in the course of H. pylori infection in the early stage of its natural history. Gastric antral biopsy specimens were obtained from thirteen H. pylori positive and seven negative children. To assess replication rates we used nucleolar organiser regions staining with colloidal silver nitrate technique (AgNOR). The number of AgNORs per nucleus, area of single AgNOR, and the quotient of these two parameters (AgNOR content) were analysed. The mean area of AgNOR was lower in H. pylori positive than in negative children. Conversely, both the mean number of AgNOR per nucleus and AgNOR content were higher in infected than non infected subjects. These results show accelerated proliferation of gastric antral epithelial cells in the course of H. pylori infection in children. Such alteration of cell replication occurring in an initial phase of natural history of long lasting infection provides an explanation for the association between acquisition of H. pylori infection in the first years of life and the development of gastric cancer.     

Invasion of human epithelial cells by Campylobacter upsaliensis

Few data exist on the interaction of Campylobacter upsaliensis with host cells, and the potential for this emerging enteropathogen to invade epithelial cells has not been explored. We have characterized the ability of C. upsaliensis to invade both cultured epithelial cell lines and primary human small intestinal cells. Epithelial cell lines of intestinal origin appeared to be more susceptible to invasion than non-intestinal-derived cells. Of three bacterial isolates studied, a human clinical isolate, CU1887, entered cells most efficiently. Although there was a trend towards more efficient invasion of Caco-2 cells by C. upsaliensis CU1887 at lower initial inocula, actual numbers of intracellular organisms increased with increasing multiplicity of infection and with prolonged incubation period. Confocal microscopy revealed C. upsaliensis within primary human small intestinal cells. Both Caco-2 and primary cells in non-confluent areas of the infected monolayers were substantially more susceptible to infection than confluent cells. The specific cytoskeletal inhibitors cytochalasin B, cytochalasin D and vinblastine attenuated invasion of Caco-2 cells in a concentration-dependent manner, providing evidence for both microtubule- and microfilament-dependent uptake of C. upsaliensis. Electron microscopy revealed the presence of organisms within Caco-2 cell cytoplasmic vacuoles. C. upsaliensis is capable of invading epithelial cells and appears to interact with host cell cytoskeletal structures in order to gain entry to the intracellular environment. Entry into cultured primary intestinal cells ex vivo provides strong support for the role of host cell invasion during human enteric C. upsaliensis infection.     

Selective association and transport of Campylobacter jejuni through M cells of rabbit Peyer's patches

M cells in the Peyer's patches may facilitate transport of pathogens such as Campylobacter jejuni from the intestine. We evaluated this hypothesis by using electron microscopy to examine Peyer's patches in ligated adult rabbit ileal loops inoculated with 5-mL suspensions of 10(9) cfu/mL of Campylobacter jejuni. Peyer's patches taken at intervals from 15 min to 2 h after inoculation of loops in anaesthetized rabbits provided evidence that Campylobacter jejuni selectively adhered to M cells as opposed to absorptive epithelial cells and was transported, apparently intact, into the M cell follicle. Although intercellular organisms were seen within the follicle, many others were phagocytosed by lymphoid cells. The proximity of the lymphatic and blood circulatory systems to the M cell follicle makes this a probable route for systemic spread of Campylobacter jejuni.     

Helicobacter pylori infection activates NF-kappaB signaling pathway to induce iNOS and protect human gastric epithelial cells from apoptosis

Helicobacter pylori infection induces apoptosis and inducible nitric oxide synthase (iNOS) expression in gastric epithelial cells. In this study, we investigated the effects of NF-kappaB activation and iNOS expression on apoptosis in H. pylori-infected gastric epithelial cells. The suppression of NF-kappaB significantly increased caspase-3 activity and apoptosis in H. pylori-infected MKN-45 and Hs746T gastric epithelial cell lines as well as primary gastric epithelial cells. An NF-kappaB signaling pathway via NF-kappaB-inducing kinase and IkappaB kinase-beta activation was found to be involved in the inhibition of apoptosis in H. pylori-infected gastric epithelial cells. In gastric epithelial cells transfected with retrovirus containing IkappaBalpha superrepressor, iNOS mRNA and protein levels were reduced, indicating that H. pylori infection induced the expression of iNOS by activating NF-kappaB. Moreover, a NO donor, S-nitroso-N-acetylpenicillamine (100 microM), decreased caspase-3 activity and apoptosis in NF-kappaB-suppressed cells infected with H. pylori. These results suggest that NF-kappaB activation may play a role in protecting gastric epithelial cells from H. pylori-induced apoptosis by upregulating endogenous iNOS.     

Infection with Campylobacter jejuni induces tyrosine-phosphorylated proteins into INT-407 cells

The mechanisms used by Campylobacter jejuni to induce internalization into host intestinal epithelial cells have not been defined. In this study, we obtained evidence that exposure of INT-407 cells to protein kinase inhibitors results in decreased invasion of these cells by C. jejuni in a dose dependent manner. Preincubation of INT-407 cells in the presence of staurosporine, tyrphostin 46 and genistein decreased invasion of these cells by C. jejuni significantly. Moreover, C. jejuni infection of INT-407 cells induced tyrosine phosphorylation of several Triton X-100 soluble proteins with approximate molecular weights of 170, 145, 90, 60 and 55 kDa that were absent or reduced in the presence of genistein in cells after 1 hr of pretreatment. These data suggest that tyrosine protein kinase-linked pathways strongly regulate the internalization of C. jejuni into intestinal epithelial cells.     

Electron and video-light microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia lamblia

Electron and video-light microscopy analysis of the in vitro effects of pyrantel pamoate on Giardia lamblia. Experimental Parasitology 97, 9-14. Giardia infection is predominant in the small intestine of vertebrates, where the trophozoites attach to epithelial cells and adversely affect the microvilli and other epithelial cell structures. Giardiasis, the disease caused by this protozoan, is very common in developing countries and mainly affects children. Drugs currently used to treat Giardia infection, such as some benzimidazole derivatives, were originally designed to treat helminthic infections. Many of the drugs are known to cause severe side effects and disturbances to the patient. Using transmission electron microscopy and video-light microscopy, we studied the effects of pyrantel pamoate, a drug commonly used in the treatment of helminthic infections in horses and ruminants, on Giardia lamblia trophozoites. Pyrantel pamoate was administered to Giardia cells in four different concentrations. Using video-light microscopy, we observed the decrease in flagella beating frequency and severe changes in the lateral flange and in the general aspect of the cell. Using transmission electron microscopy, we observed changes in the cytoplasm and peripheral vesicles. The flagella and adhesive disk structure were not affected. Apparently, the effects of pyrantel pamoate are irreversible.     

Ultrastructure of cyclic changes in the murine uterus,cervix, and vagina

The regional and cyclic changes in the murine genital epithelium were studied by transmission and scanning electron microscopy to provide a morphological standard to serve as a basis for investigation of host-parasite relationships in genital infections. Thus, we examined not only mucosal epithelial cell changes, but also surface mucus, normal flora and inflammatory cells. Ultrastructurally, at proestrus/estrus, we found uterine and most cervical epithelial cells covered with microvilli overlaid with mucus-like secretions and evidence of internal secretory activity. There was little normal flora anywhere in the tract. At early metestrus, we found squamous cervicovaginal epithelial cells with low discontinuous microrugae, extensive normal flora and many neutrophils beginning to migrate through the epithelium. The flora and neutrophils could explain the relative lack of susceptibility to infection at that time. At diestrus the appearance of a newly regenerated epithelium and lack of normal flora suggested that initiation of infection could occur at this stage; however, the presence of large numbers of neutrophils ready to phagocytize invading bacteria indicated a deterrent to infection. This study of cyclic changes in flora, mucus, neutrophils and epithelial cells provided ultrastructural evidence to support an earlier hypothesis that the greatest susceptibility to gonococcal infection in mice occurred at proestrus/estrus.     

Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157

A strain of Escherichia coli serotype O157 was grown in steady state chemostat culture under aerobic, oxygen-limited and anaerobic conditions. The growth and metabolic efficiency of oxygen-limited and anaerobic cultures was impaired, with biomass yield and the molar growth yield for glucose, Yglucose, reduced markedly in comparison with aerobic cultures. Steady state cells were typically short rods 2-3 microns long, and were encapsulated by a layer of extracellular material. The majority of cells were non-flagellated and fimbriae were not observed. Chemostat-grown cells were significantly more adhesive for HEp-2 monolayers than cells grown in aerobic batch culture. Furthermore, oxygen-limited and anaerobic cultures were significantly more adhesive for Hep-2 cells when compared with cells grown in aerobic chemostat culture, possibly reflecting increased pathogenicity associated with the induction of novel adhesins. Type 1 pili were not responsible for increased adherence. Verocytotoxins, VT1 and VT2, were expressed constitutively and were not influenced by oxygen availability. This study demonstrates that E. coli O157 is a versatile micro-organism, which responds to environmental conditions likely to be encountered during infection by inducing a phenotype which is more adhesive for human epithelial cells.