GroEL of Lactobacillus johnsonii La1 (NCC 533) is cell surface associated: potential role in interactions with the host and the gastric pathogen Helicobacter pylori

Heat shock proteins of the GroEL or Hsp60 class are highly conserved proteins essential to all living organisms. Even though GroEL proteins are classically considered intracellular proteins, they have been found at the surface of several mucosal pathogens and have been implicated in cell attachment and immune modulation. The purpose of the present study was to investigate the GroEL protein of a gram-positive probiotic bacterium, Lactobacillus johnsonii La1 (NCC 533). Its presence at the bacterial surface was demonstrated using a whole-cell enzyme-linked immunosorbent assay and could be detected in bacterial spent culture medium by immunoblotting. To assess binding of La1 GroEL to mucins and intestinal epithelial cells, the La1 GroEL protein was expressed in Escherichia coli. We report here that La1 recombinant GroEL (rGroEL) binds to mucins and epithelial cells and that this binding is pH dependent. Immunomodulation studies showed that La1 rGroEL stimulates interleukin-8 secretion in macrophages and HT29 cells in a CD14-dependent mechanism. This property is common to rGroEL from other gram-positive bacteria but not to the rGroEL of the gastric pathogen Helicobacter pylori. In addition, La1 rGroEL mediates the aggregation of H. pylori but not that of other intestinal pathogens. Our in vitro results suggest that GroEL proteins from La1 and other lactic acid bacteria might play a role in gastrointestinal homeostasis due to their ability to bind to components of the gastrointestinal mucosa and to aggregate H. pylori.     

Lipoteichoic acids from Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 antagonize the responsiveness of human intestinal epithelial HT29 cells to lipopolysaccharide and gram-negative bacteria

Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines following exposure of the IECs to specific gram-positive bacteria or their lipoteichoic acids (LTAs) in the absence and presence of human milk as a source of sCD14. In contrast to LPS from Escherichia coli or Salmonella enteritidis, neither the gram-positive bacteria Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 nor their LTAs stimulated IECs, even in the presence of sCD14. However, both LTAs inhibited the sCD14-mediated LPS responsiveness of IECs. We have previously hypothesized that sCD14 in human milk is a means by which the neonate gauges the bacterial load in the intestinal lumen and liberates protective proinflammatory cytokines from IECs. The present observations suggest that gram-positive organisms, via their LTAs, temper this response and prevent an exaggerated inflammatory response.     

The cell wall mediates pneumococcal attachment to and cytopathology in human endothelial cells.

Streptococcus pneumoniae interacts with vascular endothelial cells during the course of bacteremia. In this study, we characterized the initial attachment of pneumococci to human endothelial cells (EC) and the response of the endothelium to this interaction. Pneumococci adhered to EC in a dose-dependent fashion. Attachment was rapid, with the majority of bacteria attached by 30 min. No difference was found between the attachment of unencapsulated (R6) and encapsulated (SIII) strains. Purified pneumococcal cell wall components competitively inhibited attachment of R6 by a maximum of 60% in a dose-dependent manner. Following attachment of pneumococci or exposure of EC to pneumococcal cell wall, pronounced changes in EC morphology ensued, resulting in striking separation of the cells of the monolayer and, eventually, destruction of the cells. The cytopathic effects of the cell wall were inhibited by antibodies to interleukin-1 but not to tumor necrosis factor. Both antibodies were required to neutralize the cytopathology caused by intact pneumococci. We conclude that pneumococci attach rapidly to human EC and that the cell wall is important in this interaction. Intact pneumococci and pneumococcal cell wall induce profound morphologic changes in human EC, leading to loss of barrier integrity. These cytopathic effects are likely to be cytokine mediated.     

Effects of intraduodenal injection of Lactobacillus johnsonii La1 on renal sympathetic nerve activity and blood pressure in urethane-anesthetized rats

Previously, it was shown that milk fermented with lactic acid bacteria lowers blood pressure, suggesting that metabolites or components of the bacteria have hypotensive action. To examine whether one of lactobacilli, Lactobacillus johnsonii La1 (LJLa1), a probiotic strain adhesive onto intestinal epithelial cells, or its metabolite has hypotensive action, and if so the mechanism of action, we determined the effects of intraduodenal injection of LJLa1 on blood pressure (BP) and the activity of autonomic nerves in urethane-anesthetized rats. Intraduodenal injection of LJLa1 reduced renal sympathetic nerve activity (RSNA) and BP and enhanced gastric vagal nerve activity (GVNA). Pre-treatment with thioperamide, a histaminergic H3-receptor antagonist, eliminated the effects of LJLa1 on RSNA, GVNA, and BP. Furthermore, bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN), the master circadian oscillator, abolished the suppression of RSNA and BP and the elevation of GVNA caused by LJLa1. These findings suggest that LJLa1 or its metabolites might lower BP by changing autonomic neurotransmission via the central histaminergic nerves and the suprachiasmatic nucleus in rats.     

The pathogenic A4269G mutation in human mitochondrial tRNA(Ile) alters the T-stem structure and decreases the binding affinity for elongation factor Tu

The A4269G mutation in the human mitochondrial (mt) tRNA(Ile) gene is associated with fatal cardiomyopathy. This mutation completely inhibits protein synthesis in mitochondria, thereby significantly reducing their respiratory activity. The steady-state amount of tRNA(Ile) in cells bearing the A4269G mutation is almost half that of control cells. We previously reported that this mutation causes tRNA(Ile) to be unstable both in vivo and in vitro. To investigate whether the instability of the mutant tRNA(Ile) is due to structural alterations, a nuclease-probing experiment was performed with a mitochondrial enzymatic extract, which showed that the A4269G mutation destabilizes the T-stem of the mutant tRNA(Ile). In addition, measurements of the binding affinity of the aminoacylated mutant tRNA(Ile) for mt elongation factor Tu (EF-Tu) showed that the mutant tRNA(Ile) binds mt EF-Tu less efficiently than the wild-type does. This observation provides insight into the molecular pathology associated with tRNA dysfunction caused by this pathogenic point mutation.     

Lactobacillus johnsonii La1 Attenuates Helicobacter pylori-Associated Gastritis and Reduces Levels of Proinflammatory Chemokines in C57BL/6 Mice

In clinical settings, Lactobacillus johnsonii La1 administration has been reported to have a favorable effect on Helicobacter pylori-associated gastritis, although the mechanism remains unclear. We administered, continuously through the water supply, live La1 to H. pylori-infected C57BL/6 mice and followed colonization, the development of H. pylori-associated gastritis in the lamina propria, and the levels of proinflammatory chemokines macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived cytokine (KC) in the serum and gastric tissue over a period of 3 months. We documented a significant attenuation in both lymphocytic (P = 0.038) and neutrophilic (P = 0.003) inflammatory infiltration in the lamina propria as well as in the circulating levels of anti-H. pylori immunoglobulin G antibodies (P = 0.003), although we did not observe a suppressive effect of La1 on H. pylori colonizing numbers. Other lactobacilli, such as L. amylovorus DCE 471 and L. acidophilus IBB 801, did not attenuate H. pylori-associated gastritis to the same extent. MIP-2 serum levels were distinctly reduced during the early stages of H. pylori infection in the La1-treated animals, as were gastric mucosal levels of MIP-2 and KC. Finally, we also observed a significant reduction (P = 0.046) in H. pylori-induced interleukin-8 secretion by human adenocarcinoma AGS cells in vitro in the presence of neutralized (pH 6.8) La1 spent culture supernatants, without concomitant loss of H. pylori viability. These observations suggest that during the early infection stages, administration of La1 can attenuate H. pylori-induced gastritis in vivo, possibly by reducing proinflammatory chemotactic signals responsible for the recruitment of lymphocytes and neutrophils in the lamina propria.     

Lactobacillus johnsonii La1 attenuates Helicobacter pylori-associated gastritis and reduces levels of proinflammatory chemokines in C57BL/6 mice

In clinical settings, Lactobacillus johnsonii La1 administration has been reported to have a favorable effect on Helicobacter pylori-associated gastritis, although the mechanism remains unclear. We administered, continuously through the water supply, live La1 to H. pylori-infected C57BL/6 mice and followed colonization, the development of H. pylori-associated gastritis in the lamina propria, and the levels of proinflammatory chemokines macrophage inflammatory protein 2 (MIP-2) and keratinocyte-derived cytokine (KC) in the serum and gastric tissue over a period of 3 months. We documented a significant attenuation in both lymphocytic (P=0.038) and neutrophilic (P=0.003) inflammatory infiltration in the lamina propria as well as in the circulating levels of anti-H. pylori immunoglobulin G antibodies (P=0.003), although we did not observe a suppressive effect of La1 on H. pylori colonizing numbers. Other lactobacilli, such as L. amylovorus DCE 471 and L. acidophilus IBB 801, did not attenuate H. pylori-associated gastritis to the same extent. MIP-2 serum levels were distinctly reduced during the early stages of H. pylori infection in the La1-treated animals, as were gastric mucosal levels of MIP-2 and KC. Finally, we also observed a significant reduction (P=0.046) in H. pylori-induced interleukin-8 secretion by human adenocarcinoma AGS cells in vitro in the presence of neutralized (pH 6.8) La1 spent culture supernatants, without concomitant loss of H. pylori viability. These observations suggest that during the early infection stages, administration of La1 can attenuate H. pylori-induced gastritis in vivo, possibly by reducing proinflammatory chemotactic signals responsible for the recruitment of lymphocytes and neutrophils in the lamina propria.     

Cloning of a human antibody directed against human neuroblastoma cells and specific for human translation elongation factor 1alpha

Human sera have shown antitumor effects mediated by tumor-specific immunoglobulin M (IgM) antibodies. Most people who have cytotoxic serum are in good health and show no evidence of exposure to tumor antigens. We characterized the serum of a healthy female adult that was highly lytic to a neuroblastoma cell line via IgM-activated complement (>60% of malignant cells were killed during the 60-min assay). Complement-dependent lysis was not mediated by other classes of serum antibodies (data not shown) which is consistent with the findings of Ollert et al. To identify the target antigen on neuroblastoma cells, we fractionated neuroblastoma cell lysates by ion-exchange chromatography. In the fraction that showed maximal IgM binding, the dominant protein was identified as the 47-kDa translational elongation factor 1alpha (eEF1alpha). We used the donor's B-cells to create hybridomas producing the antibody (B12.6.22) that bound to neuroblastoma cells and mediated cytotoxicity. This antibody recognized eEF1alpha in a specific manner. Sequence analysis of the heavy chain of B12.6.22 showed usage of VH3-23 and JH6 gene segments, with no somatic mutation. The structural similarity of B12.6.22 to antibodies of the innate immune system supports the assumption that natural antibodies are a potential source of therapeutic antibodies.     

Bovine herpesvirus type 1 gp87 mediates both attachment of virions to susceptible cells and hemagglutination

Summary The 87,000-dalton glycoprotein (gp87) of bovine herpesvirus type 1 (BHV-1) was found to selectively attach to susceptible cells. The attachment of gp87 to the cells was markedly decreased by the prior adsorption of intact virions. Anti-gp87 (site Ia) monoclonal antibody, which inhibited BHV-1 adsorption to the cells and neutralized the virus without complement [Okazaki et al., Virology 250: 260–264], was effective in inhibiting the adsorption of gp87. Only the same antibody was able to inhibit the hemagglutination activity of BHV-1. Other monoclonal antibodies to the glycoproteins of BHV-1, including antibodies directed to sites Ib and Ic on gp87, were ineffective in inhibiting either virus adsorption or hemagglutination. The results of this study indicate that site Ia of gp87 molecule is the critical site of virus attachment for initiation of infection as well as the hemagglutination of BHV-1.     

The zinc-finger factor Insm1 (IA-1) is essential for the development of pancreatic beta cells and intestinal endocrine cells

The pancreatic and intestinal primordia contain epithelial progenitor cells that generate many cell types. During development, specific programs of gene expression restrict the developmental potential of such progenitors and promote their differentiation. The Insm1 (insulinoma-associated 1, IA-1) gene encodes a Zinc-finger factor that was discovered in an insulinoma cDNA library. We show that pancreatic and intestinal endocrine cells express Insm1 and require Insm1 for their development. In the pancreas of Insm1 mutant mice, endocrine precursors are formed, but only few insulin-positive beta cells are generated. Instead, endocrine precursor cells accumulate that express none of the pancreatic hormones. A similar change is observed in the development of intestine, where endocrine precursor cells are formed but do not differentiate correctly. A hallmark of endocrine cell differentiation is the accumulation of proteins that participate in secretion and vesicle transport, and we find many of the corresponding genes to be down-regulated in Insm1 mutant mice. Insm1 thus controls a gene expression program that comprises hormones and proteins of the secretory machinery. Our genetic analysis has revealed a key role of Insm1 in differentiation of pancreatic and intestinal endocrine cells.     

Antibody to ICAM-1 mediates enhancement of HIV-1 infection of human endothelial cells

Summary Human umbilical vein endothelial cells (HUVEC) can be abortively infected with HIV-1, but virus production is rescued by the addition of T cells. Monoclonal antibody (Mab) to ICAM-1, but not its Fab' fragment or MAbs to LFA-1 and PECAM-1, increases HIV-1 infection of HUVEC by enhancing HIV-1 absorption. Enhancement by anti ICAM-1 is probably due to a bridging effect different from the ADE mediated by anti-gp120 that involves FcR or CR-mediated capture of the virus-antibody complex. Since antibodies to cell membrane molecules are present in HIV-1 infected patients, the ADE mediated by such a mechanism can be important in AIDS pathogenesis.     

Genetic analysis of Pseudomonas aeruginosa adherence: distinct genetic loci control attachment to epithelial cells and mucins.

Infection of mucosal tissues by the opportunistic pathogen Pseudomonas aeruginosa is initiated by attachment of the bacterium to host tissues. To gain a better understanding of this interaction, we used two methods to isolate mutants of P. aeruginosa with altered adherence to cultured A549 cells and to mucins. First, from a population of nonpiliated mutants of P. aeruginosa mutagenized with transposon Tn5G, we have isolated variants that are defective in binding to both A549 cells and respiratory mucins. Using a cloned transposon plus flanking DNA from one such mutant as a DNA probe, we have isolated plasmids from a cosmid bank, which, upon reintroduction to the original mutants, restored adhesion to both A549 cells and mucin. The second strategy to identify genes involved in adhesion used mutagenesis of P. aeruginosa N1G, an rpoN mutant which is unable to bind to either A549 cells or mucin, with transposon Tn5 containing an outward-directed promoter. From this bank of mutagenized P. aeruginosa N1G, two classes of adhesion variants were isolated; one class attached to A549 cells and to mucin, and the other class restored binding of the rpoN mutant to mucin but not to A549 cells. These findings suggest that P. aeruginosa can express at least two adhesins distinct from pili, one recognizing receptors shared by epithelial cells and mucins and the other recognizing mucins alone.     

Cytokine-stimulated release of decay-accelerating factor (DAF; CD55) from HT-29 human intestinal epithelial cells

Expression of DAF (CD55) is enhanced on colonic epithelial cells of patients with ulcerative colitis (UC), and stool DAF concentrations are increased in patients with active disease. Cytokines are known to modulate DAF expression in various human cells, and lesions of UC reveal altered profiles of cytokine production. In this study, we evaluate the effects of various cytokines, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, and interferon-gamma (IFN-γ), on the synthesis and kinetics of DAF protein in HT-29 human intestinal epithelial cells. Using flow cytometry and an ELISA, we found that HT-29 cells constitutively express DAF on the cell surface and spontaneously release DAF into the culture supernatant under standard culture conditions. When the culture supernatant was centrifuged at 100 000 g , nearly a half of DAF was precipitated, indicating that one half of the released DAF was present as a membrane-bound form and the other half as a soluble form. Analysis of the culture supernatant of biotin surface-labelled HT-29 cells suggested that the soluble form DAF was derived by secretion from within the cell or by cleavage from the cell surface. Among the cytokines, IL-4 markedly, and IL-1β moderately, enhanced the expression and the release of DAF. Actinomycin D, cycloheximide, and brefeldin A inhibited the increase in DAF release induced by IL-4 and IL-1β stimulation. These results suggest that DAF is released from intestinal epithelial cells in response to cytokine stimulation and that IL-4 and IL-1β are possible cytokines involved in DAF release into the colonic lumen of patients with UC.     

Monoclonal antibodies specific for elongation factor Tu and complete nucleotide sequence of the tuf gene in Mycobacterium tuberculosis.

Monoclonal antibodies against mycobacterial antigens were produced by immunizing LOU/C rats with live Mycobacterium bovis BCG. The antibodies were characterized by an enzyme-linked immunosorbent assay and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting (immunoblotting). One antibody, MAMB 2, reactive with a 47-kDa protein was used to screen a lambda gt11 M. tuberculosis gene library (R. A. Young, B. R. Bloom, C. M. Grosskinsky, J. Ivanji, D. Thomas, and R. W. Davis, Proc. Natl. Acad. Sci. USA 82:2583-2587, 1985). Three recombinant phages reactive with MAMB 2 in plaque lysates were isolated, and part of the insert was sequenced. The mycobacterial inserts were all expressed as proteins fused with beta-galactosidase when the phages were induced as lysogens in Escherichia coli. The entire M. tuberculosis tuf gene was obtained by screening the lambda gt11 library with a DNA probe specific for the primary clones. A phage isolated from this screening was able to express the native protein in E. coli when introduced as a lysogen. A comparison of the entire gene sequence and the deduced protein sequence with the EMBL DNA and Swiss-Prot protein data libraries revealed strong homologies with elongation factors of bacteria, yeast mitochondria, and a plant chloroplast.     

Interferon regulatory factor 6 (IRF6) and fibroblast growth factor receptor 1 (FGFR1) contribute to human tooth agenesis

Phenotypic characteristics expressed in syndromes give clues to the factors involved in the cause of isolated forms of the same defects. We investigated two genes responsible for craniofacial syndromes, FGFR1 and IRF6, in a collection of families with isolated tooth agenesis. Cheek swab samples were obtained for DNA analysis from 116 case/parent trios. Probands had at least one developmentally missing tooth, excluding third molars. In addition, we studied 89 cases and 50 controls from Ohio to replicate any positive findings. Genotyping was performed by kinetic polymerase chain-reaction or TaqMan assays. Linkage disequilibrium analysis and transmission distortion of the marker alleles were performed. The same variants in the IRF6 gene that are associated with isolated orofacial clefts are also associated with human tooth agenesis (rs861019, P = 0.058; rs17015215-V274I, P = 0.0006; rs7802, P = 0.004). Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes. The craniofacial phenotypic characteristics of these syndromes include oral clefts and preferential tooth agenesis of incisors and premolars, besides pits on the lower lips. Also it appears that preferential premolar agenesis is associated with FGFR1 (P = 0.014) and IRF6 (P = 0.002) markers. There were statistically significant data suggesting that IRF6 interacts not only with MSX1 (P = 0.001), but also with TGFA (P = 0.03).     

Cell attachment of human gingival fibroblasts in vitro to porous-surfaced titanium alloy discs coated with collagen and platelet-derived growth factor

The influence of biological coating, with or without the incorporation of growth factor, on the migration, attachment and orientation of human gingival fibroblasts in relation to porous-surfaced titanium alloy (Ti6AI4V) discs, was measured. Comparison was made between coating the discs with collagen and with collagen incorporating platelet-derived growth factor (PDGF); controls comprised porous-surfaced discs coated with agar or collagen containing bovine serum albumin (used as a carrier for the PDGF), uncoated porous-surfaced Ti6AI4V discs (with or without additional protein additives) exhibited significantly higher attachment indices (AI) and orientation indices (OI) compared with naked control discs (p less than 0.01); OI was also significantly higher than that of surface-demineralized root slices (p less than 0.001) on days 1, 2 and 3. Addition of PDGF to the collagen resulted in a further enhancement in OI on days 1 and 2 (p less than 0.01) over that shown by discs coated with collagen incorporating the bovine serum albumin vehicle. There was no cell attachment and consequently, no cell orientation, in relation to Ti alloy discs that had been coated with agar. These data suggest that attachment and orientation of cells following migration in relation to porous-surfaced Ti6AI4V discs can be modified by the application of biological molecules to the surface of the disc. This may have a useful application in clinical implantology.