Saccharomyces boulardii Protease Inhibits the Effects of Clostridium difficile Toxins A and B in Human Colonic Mucosa

Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis. We have reported that S. boulardii inhibits C. difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225–5232, 1996). The aim of this study was to further evaluate the role of S. boulardii protease in preventing C. difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C. difficile toxins. A polyclonal rabbit antiserum raised against purified S. boulardii serine protease inhibited by 73% the proteolytic activity present in S. boulardii conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of S. boulardii on toxin A-induced intestinal secretion and mucosal permeability to [³H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects of S. boulardii protease on digestion of toxins A and B and on binding of [³H]toxin A and [³H]toxin B to purified human colonic BBM. Purified S. boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S. boulardii protease. We conclude that the protective effects of S. boulardii on C. difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease.     

Lack of Pseudomelanosis coli in colonic adenomas suggests different pathways of apoptotic bodies in normal and neoplastic colonic mucosa

Pseudomelanosis coli is characterized by pigment deposition in the lamina propria and caused by increased epithelial apoptosis. Pseudomelanosis coli is absent in colonic neoplasia. The aim of our studies was to investigate this phenomenon in more detail. Apoptotic fragments of epithelial cells and their distribution, cell proliferation (Ki-67, MIB 1 immunostaining), macrophages (CD68 immunostaining), Bcl-2 expression and apoptosis [terminal-deoxynucleotidyl-transferase mediated dUTP fluorescein nick end labeling (TUNEL) assay] were studied in adenomas arising in normal and melanotic colonic mucosa, in normal colonic mucosa and colonic mucosa with pseudomelanosis alone. In adenomas, we found 7.0 apoptotic bodies per 100 epithelial cells in the epithelial layer and only 0.2 apoptotic bodies per high power field (HPF) in the lamina propria. In contrast, in melanotic mucosa 1.7 apoptotic bodies per 100 epithelial cells in the epithelial layer and 2.5 per HPF in the lamina propria were found. Our results show that apoptotic fragments remain in the neoplastic (adenomatous) epithelium and do not reach (at least in higher amounts) the lamina propria. They can, therefore, not contribute to the development of pseudomelanosis in these lesions. However, macrophages are diminished in adenomas. Proliferation (Ki-67) and also Bcl-2 expression are highly increased in adenomas. The pathway of mucosal macrophages is also discussed. Received: 5 July 1999 / Accepted: 16 December 1999     

Migration of human intestinal lamina propria lymphocytes, macrophages and eosinophils following the loss of surface epithelial cells

Lymphocytes and macrophages are present in the normal intestinal lamina propria, separated from the epithelial monolayer by the basement membrane. There is evidence for movement of mononuclear cells through the lamina propria, entering from the systemic circulation and exiting via lymphatic channels. The goal of our studies was to investigate the capacity of cells to migrate out from the lamina propria into the lumen following the loss of surface epithelial cells. An in vitro model was therefore established in which normal human intestinal mucosal samples, denuded of the surface epithelium, were maintained in culture. Electron microscopy showed that during culture, large numbers (> 2 × 10⁶/g tissue per 24 h) of cells migrated out of the lamina propria via discrete ‘tunnels’ which were in continuity with pores (diameter < 4 μm) in the basement membrane. The emigrating cells were T cells (68.5 ± 5.1%), macrophages (10.5 ± 1.3%) and eosinophils (7.1 ± 1.3%). Our studies have therefore demonstrated, for the first time, the capacity for large numbers of lymphocytes, macrophages and eosinophils to migrate out of the lamina propria, via basement membrane pores. We postulate that such emigration of cells occurs in vivo following the loss of surface epithelial cells due to injury, and could represent an important form of host defence against luminal microorganisms and also facilitate wound repair by enhancing restitution by neighbouring epithelial cells, via peptide factors.     

Leptospira interrogans Induces Apoptosis in Macrophages via Caspase-8- and Caspase-3-Dependent Pathways

Apoptosis of host cells plays an important role in modulating the pathogenesis of many infectious diseases. It has been reported that Leptospira interrogans, the causal agent of leptospirosis, induces apoptosis in macrophages and hepatocytes. However, the molecular mechanisms responsible for host cell death remained largely unknown. Here we demonstrate that L. interrogans induced apoptosis in a macrophage-like cell line, J774A.1, and primary murine macrophages in a time- and dose-dependent manner. Apoptosis was associated with the activation of cysteine aspartic acid-specific proteases (caspase-3, caspase-6, and caspase-8), the increased expression of Fas-associated death domain (FADD), and the cleavage of the caspase substrates poly(ADP-ribose) polymerase (PARP) and nuclear lamina protein (lamin A and lamin C). Caspase-9 was activated to a lesser extent, whereas no release of cytochrome c from mitochondria was detectable. Inhibition of caspase-8 impaired L. interrogans-induced caspase-3 and -6 activation, as well as PARP and lamin A/C cleavage and apoptosis, suggesting that apoptosis is initiated via caspase-8 activation. Furthermore, caspase-3 was required for the activation of caspase-6 and seemed to be involved in caspase-9 activation through a feedback amplification loop. These data indicate that L. interrogans-induced apoptosis in macrophages is mediated by caspase-3 and -6 activation through a FADD-caspase-8-dependent pathway, independently of mitochondrial cytochrome c-caspase-9-dependent signaling.     

Antiapoptotic Proteins Bcl-2 and Bcl-XL Inhibit Clostridium difficile Toxin A-Induced Cell Death in Human Epithelial Cells

It has been well established that Clostridium difficile toxin A (TcdA) induces cell death in human epithelial cells. However, the mechanism of TcdA-induced cell death remains to be fully characterized. Here, we show that TcdA induces dose-dependent cell death in ovarian carcinoma and colonic carcinoma cell lines. TcdA-mediated cell death, as well as caspase 8 and caspase 3 activation, were specifically abrogated by anti-toxin antibodies. Although caspase 8 and caspase 3 were activated by TcdA in OVCAR3 ovarian carcinoma and T84 colonic cancer cells, pancaspase and caspase 8, 3, and 9 inhibitors did not block TcdA-induced cell death. In contrast, tumor necrosis factor-related apoptosis-inducing ligand-induced cell death was nearly completely blocked by caspase inhibitors in OVCAR3 cells. In these cells, TcdA induces the mitochondrial pathway of apoptosis, as demonstrated by changes in mitochondrial outer membrane permeabilization (MOMP). Furthermore, overexpression of the antiapoptotic proteins Bcl-2 and Bcl-X_L significantly inhibited TcdA-induced cell death, as well as TcdA-induced MOMP. Conversely, small interfering RNA-mediated inhibition of Bcl-X_L in TcdA-resistant SKOV3ip1 cells enhanced TcdA-induced cell death. Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-X_L in T84 cells also inhibited TcdA-induced cell death. Altogether, our data demonstrate that TcdA induces cell death in both ovarian and colonic cancer cells preferentially via the mitochondrial pathway of apoptosis by a death receptor-independent and a caspase-independent mechanism. This process is regulated by antiapoptotic members of the Bcl-2 family.Apoptosis can be mediated by a variety of stimuli, including binding of ligands to death receptors, DNA-damaging agents, and growth factor withdrawal. Depending on the signal, apoptosis is initiated either by the death receptor pathway or by a mitochondrion-dependent pathway (31-33). In both pathways, however, effector caspases (caspases 3, 6, and 7) are activated and cleavage of cellular substrates occurs, leading to the morphological changes observed in apoptosis. In the mitochondrion-dependent pathway of apoptosis, effector caspase activation is triggered by an increase in mitochondrial outer membrane permeabilization (MOMP), resulting in the release of cytochrome c and the formation of the apoptosome (31, 33). Changes in MOMP are regulated by a balance between pro- and antiapoptotic members of the Bcl-2 family (31). The proapoptotic family members Bax and Bak form channels into the outer membrane of the mitochondria that allow the release of cytochrome c and other mitochondrial intermembrane proteins. Insertion of Bax and Bak into the outer mitochondrial membrane is regulated by antiapoptotic members of the Bcl-2 family. Antiapoptotic members, such as Bcl-2 and Bcl-X_L, bind and neutralize Bax and/or Bak. Stimulation of death receptors by death ligands, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), results in activation of initiator caspase 8. Upon binding to TRAIL, activated TRAIL receptors recruit the Fas-associated death domain (3). Via its death effector domain, the Fas-associated death domain recruits caspase 8 and assembles into a death-inducing signaling complex (16, 27). When recruited to the death-inducing signaling complex, pro-caspase 8 is activated and subsequently cleaves downstream effector caspases, leading to apoptosis. This process is efficiently blocked by the inhibition of caspases. An interconnection between cell surface death receptors and mitochondrion-initiated pathways of apoptosis has been found in many cellular systems. In this context, apoptosis can be inhibited by Bcl-X_L or Bcl-2 (2, 10, 13). In contrast to the death receptor pathway, which is highly dependent on caspase activation, the inhibition of caspases fails to prevent apoptosis in caspase-independent cell death (32). Furthermore, as caspase-independent cell death often requires MOMP, this process can be blocked by Bcl-2 overexpression (2, 13).C. difficile is the leading cause of hospital-acquired diarrhea and the etiological agent of pseudomembranous colitis. In humans, the intestinal damage is produced by the actions of toxin A (TcdA) and toxin B (TcdB), which are the major virulence determinants of C. difficile. The emergence in 2000, first in the United States and Canada and more recently in Western Europe, of a hypervirulent strain of C. difficile (NAP1/BI/027) has led to an increase in the incidence and the case-fatality ratio of hospital-acquired diarrhea, resulting, on average, in 10.7 additional days in the hospital (14, 23, 26, 28, 29, 35). This epidemic NAP1/B1/027 strain produces higher levels of TcdA and TcdB (35). TcdA is primarily responsible for the mucosal damage and the inflammatory response in animal models (24). TcdA was shown to induce apoptosis in many human cell types in vitro, including endothelial cells (11), monocytes (34), HeLa cells (30), and intestinal epithelial cells (4, 5, 9). The mechanisms by which TcdA induces apoptosis in the cells remain to be fully characterized. Brito et al. demonstrated that TcdA-induced intestinal cell death involves caspase 8, 3, and 9 activation, but the inhibition of these caspases only partially blocked TcdA-induced DNA laddering (4). Carneiro et al. have shown that TcdA induces caspase 6, 8, 9, and 3 and Bid (a proapoptotic member of the Bcl-2 family) cleavage, resulting in cell death in human intestinal epithelial cells (5). Bid cleavage, however, occurred by a caspase-independent mechanism. Gerhard et al. reported activation of caspases 3, 8, and 9 in colonic crypt cells treated with TcdA (9). Finally, Qa''Dan et al. showed that pancaspase inhibitor slowed but did not inhibit TcdB-induced cell death in HeLa cells, suggesting that cell death occurs by both caspase-dependent and caspase-independent mechanisms (30). In contrast to previous studies, apoptosis was completely blocked by the pancaspase inhibitor z-VAD-fmk. The role of caspase activation in TcdA-induced cell death thus remains controversial. In addition, the degree of mitochondrial contribution to TcdA-induced cell death has not been investigated.In this study, we examined the mechanism of TcdA-induced cell death using two cell culture models involving human colonic carcinoma and ovarian carcinoma cell lines. The advantage of using the ovarian carcinoma cell model was our ability to compare the effect of caspase inhibitors on TcdA-induced cell death to that of a well-established caspase-dependent stimulus (TRAIL), which cannot be assessed in colonic cell lines because of their inherent resistance to TRAIL. In this way, we show that TcdA induces primarily caspase-independent cell death in both ovarian and colonic carcinoma cells. We further demonstrate that TcdA-induced cell death is death receptor pathway independent but strongly dependent on the activation of the mitochondrial pathway. Thus, our findings define a novel mechanism in which TcdA-induced cell death involves a mitochondrion-dependent, caspase- and death receptor-independent signaling pathway that contrasts with previous data.     

Lamina Propria T Cell Subsets in the Small and Large Intestine of Euthymic and Athymic Mice

We investigated lamina propria T cells from the small intestine (jejunum/ileum) and the large intestine (colon) of euthymic (BALB/c, C. B-17, C57BL/6) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/ xid) mice. CD3⁺ T cells represented about 40% of the lamina propria lymphocytes (LPL) from the small or the large intestine of euthymic mice, and 20–30% of the LPL populations from the small or large intestine of athymic mice. In the lamina propria T cell population of the small intestine, 85% were of the αβ lineage in euthymic mice, but only 40% were of the αβ lineage in athymic mice. T cells of the αβ lineage were thus more frequent than T cells of the αβ lineage in the intestinal lamina propria T cells of extrathymic origin. CD4⁺ T cells represented 40% of the lamina propria T cells in the small as well as in the large intestine of euthymic mice, and 20–30% of the T cells in the lamina propria of the nude mouse gut. In euthymic mice, 40% of the T cells in the small intestine lamina propria, and 30% of the T cells in the colonic lamina propria were CD8⁺, In intestinal lamina propria T cell populations of athymic mice, the CD8⁺ T cell population was expanded. Most (60–70%) CD8+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice expressed the homodimeric CD8α⁺β^? form of the CD8 coreceptor. A fraction of 15–20% of all CD3+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice were ‘double negative’ CD4^? CD8^?. A large fraction of the TCRαβ⁺ T cells in the colonic lamina propria (but not in the small intestine lamina propria) of euthymic mice expressed the CD2 and the CD28 costimulator molecules, the adhesion molecule LECAM-1 (CD62 L), and could be activated in vitro by CD3 ligation. These data reveal a considerable heterogeneity in the surface phenotype and the functional phenotype of murine lamina propria T cells.     

Differential binding and internalization of Clostridium difficile toxin A by human peripheral blood monocytes, neutrophils and lymphocytes

Colitis due to Clostridium difficile infection is mediated by secreted toxins A and B and is characterized by infiltration by cells from the systemic circulation. The aim of our study was to investigate interactions between fluorescently labelled toxin A and peripheral blood monocytes, neutrophils and lymphocytes. Purified toxin A was labelled with Alexa Fluor^® 488 (toxin A⁴⁸⁸) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 °C/ice. The ability of trypan blue to quench cell surface–associated (but not cytoplasmic) fluorescence was also investigated. At 37 °C, toxin A⁴⁸⁸‐associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A⁴⁸⁸ in neutrophils was greater on ice than at 37 °C. Studies using trypan blue suggested that over 3 h at 37 °C, most of the toxin A⁴⁸⁸‐associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A⁴⁸⁸‐associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during colonic inflammation in C. difficile infection.     

Melanosis coli. A consequence of anthraquinone-induced apoptosis of colonic epithelial cells.

A condition closely resembling human melanosis coli was induced in the guinea pig large intestine by daily oral administration of the anthraquinone danthron. Each treatment caused a transient, dose-related wave of apoptosis of the colonic surface epithelial cells. Most of the resulting apoptotic bodies were phagocytosed by intraepithelial macrophages and carried by them through fenestrae in the epithelial basement membrane to the lamina propria. Here, the apoptotic bodies were transformed into typical lipofuscin pigment in macrophage heterolysosomes. Continued danthron administration caused progressive accumulation of pigmented macrophages in the bowel wall, whereas ongoing migration of pigmented macrophages to regional lymph nodes resulted, after danthron was ceased, in sequential loss of the pigmented cells from the superficial and deep lamina propria. Examination of colonic biopsies from patients with melanosis coli shows increased numbers of apoptotic bodies in the surface epithelium and lamina propria, suggesting implication of the same cellular processes in the formation of the pigment in man.     

Polyclonal expansion of adoptively transferred CD4+ αβ T cells in the colonic lamina propria of scid mice with colitis

The adoptive transfer of low numbers of peripheral, non-fractionated CD4⁺ αβ T cells into histocompatible, severely immunodeficient (scid) hosts induces a colitis. This disease developed in C.B-17 scid/scid hosts after the injection of 10⁵CD4⁺ T cells purified from different peripheral lymphoid organs of immunocompetent C.B-17 +/+ or BALB/c^dm2 donor mice. Irrespective of their tissue origin, transferred CD4⁺ T cells selectively repopulated the scid host with gut-seeking CD4⁺ T cells. A chronic inflammatory bowel disease (IBD) developed as polyclonal populations of mucosa-seeking memory/effector CD4⁺ T cells accumulated in the gut lamina propria and epithelial layer of the adoptive host. The manifestation of colitis in the scid host correlated with the in situ polyclonal activation and expansion of adoptively transferred CD4⁺ T cells in the colonic lamina propria. Attempts were unsuccessful to select in vivo an oligoclonal CD4⁺ T cell population with an enhanced IBD-inducing potential by repeatedly reinjecting 10⁵ donor-type CD4⁺ T cells from the colonic lamina propria of transplanted scid mice with an early and severe IBD into new scid hosts. The data indicate that the preferential repopulation of gut-associated lymphoid tissues with immunocompetent CD4⁺ T cells, and their polyclonal activation and in situ expansion in the lamina propria of the histocompatible, immunodeficient host are critical events in the pathogenesis of an IBD in this model.     

Effects of Toxin A from Clostridium difficile on Mast Cell Activation and Survival

Toxins A and B from Clostridium difficile are the main cause of antibiotic-associated diarrhea and pseudomembranous colitis. They cause fluid accumulation, necrosis, and a strong inflammatory response when inoculated in intestinal loops. Since mast cells are a rich source of inflammatory mediators, abundant in the gut, and known to be involved in C. difficile-induced enteritis, we studied the in vitro effect of toxin A on isolated mast cells. Normal rats sensitized by infection with Nippostrongilus brasiliensis were used to isolate peritoneal mast cells (PMC). PMC from naive rats were stimulated with calcium ionophore A23187 as a model of antigen-independent activation, and PMC from sensitized rats were stimulated with N. brasiliensis antigens to study immunoglobulin E-dependent mast cell activation. After 4 h, toxin A did not induce release of nitric oxide or histamine in naive PMC. However, 10 ng of toxin per ml caused a significant release of tumor necrosis factor alpha (TNF-α). In contrast, 1 μg of toxin per ml inhibited antigen or A23187-induced histamine release by PMC. Toxin A at 1 μg/ml for 4 h caused disruption of actin which aggregated in the cytoplasm and around the nucleus. After 24 h, chromatin condensation, cytoplasmic blebbing, and apoptotic-like vesicles were observed; DNA fragmentation was documented also. These results suggest that mast cells may participate in the initial inflammatory response to C. difficile infection by releasing TNF-α upon interaction with toxin A. However, longer exposure to toxin A affects the release of inflammatory mediators, perhaps because of the alteration of the cytoskeleton and induction of apoptosis. The impaired functions and survival of mast cells by C. difficile toxin A could hamper the capacity of these cells to counteract the infection, thus prolonging the pathogenic effects of C. difficile toxins.

Clostridium difficile is the etiologic agent of antibiotic-associated diarrhea and pseudomembranous colitis (1). Antibiotics and cytotoxic drugs disturb colonic flora, allowing overgrowth of C. difficile and production of toxins A and B. Toxin A elicits an acute inflammatory response, congestion, and necrosis when inoculated in the gut (1, 14, 36, 44). It is chemotactic and induces the release of inflammatory mediators by macrophages and neutrophils (9, 24, 31). Some studies suggested that mast cells also play an important role in the pathophysiology of toxin A (25). Thus, toxin A administered into ileal loops of rats elicited the release of inflammatory mediators such as leukotriene B4, platelet-activating factor, and rat mucosal mast cell protease II (RMCPII) (6, 26, 35). Moreover, treatment of animals with the antiallergy and antiinflammatory agent ketotifen, with the H1 histamine antagonist iodoxamide, or with histaminase reduced the inflammation and secretory responses caused by toxin A (12, 25, 34). It has been proposed that toxin A induces the secretion of inflammatory mediators from mast cells either directly, or indirectly through the release of substance P, a known activator of mucosal mast cells (7, 19, 29).Mast cells are widely distributed in the intestinal mucosa, in skin and around blood and lymphatic vessels, and in many other tissues and organs. They can be activated to release inflammatory mediators via immunoglobulin E (IgE)-dependent and IgE-independent mechanisms (16). In IgE-independent mechanisms, mast cells can be activated by substances such as calcium ionophore, compound 48/80, substance P, and microbial products (11, 16). They can release potent mediators of inflammation and recently have been shown to play a pivotal role in host defense against bacterial infection (11, 28). The defenses in sepsis are dependent on mast cells that produce tumor necrosis factor alpha (TNF-α), which in turn attracts and activates neutrophils to the site of infection (28). However, in all these studies, direct evidence of C. difficile toxin A effect on mast cells has not been described.Thus, to investigate whether toxin A has direct effects on mast cells, we analyzed the influence of toxin A on the secretion of histamine, TNF-α, and nitric oxide (NO) in vitro. We found that toxin A did not induce the release of histamine and NO, although it induced the release of small amounts of TNF-α. Moreover, exposure to large doses of toxin A inhibited mast cell activation induced by IgE-dependent and IgE-independent mechanisms and also altered the mast cell cytoskeleton and induced cell death by apoptosis.     

CD4+ T lymphocytes injected into severe combined immunodeficient (SCID) mice lead to an inflammatory and lethal bowel disease

Transfer of 2 × 10⁵ congenic or semiallogenic purified TCRαβ⁺ CD4⁺ T cells to SCID mice leads to an infiltration of the recipient gut lamina propria and epithelium with a donor-derived CD4⁺ T cell subset which induces a lethal inflammatory bowel disease (IBD) in the recipients. In contrast, IBD was not observed in SCID mice transplanted with unfractionated splenic cells. The earliest detectable pathological changes after CD4⁺ T cell transfer were proliferation and hypertrophy of the entire colonic epithelial layer, including increased mitotic activity, increased expression of epithelial nuclear proliferation antigen, and elongation of the crypts. Later on, massive mononuclear cell infiltration, hypertrophy of all layers of the colon and occasional epithelial ulcerations were observed. At this stage, accumulations of IgA, IgM and small numbers of IgG1-, IgG2-and IgG3-secreting plasma cells were present in the lamina propria of both the small and large intestine. We conclude that low numbers of intraveneously transferred CD4⁺ T cells induce IBD in SCID mice. In the late stages of CD4⁺ T cell-induced IBD, the colonic lamina propria becomes infiltrated with macrophages, neutrophils and plasma cells secreting IgA, IgM, and to a lesser degree IgG antibodies which might play an accessory role in the pathogenesis of IBD.     

A C-Type Lectin MGL1/CD301a Plays an Anti-Inflammatory Role in Murine Experimental Colitis

Inflammatory bowel disease is caused by abnormal inflammatory and immune responses to harmless substances, such as commensal bacteria, in the large bowel. Such responses appear to be suppressed under healthy conditions, although the mechanism of such suppression is currently unclear. The present study aimed to reveal whether the recognition of bacterial surface carbohydrates by the macrophage galactose-type C-type lectin-1, MGL1/CD301a, induces both the production and secretion of interleukin (IL)-10. Dextran sulfate sodium salt (DSS) was orally administrated to mice that lacked MGL1/CD301a (Mgl1^−/− mice) and their wild-type littermates. Mgl1^−/− mice showed significantly more severe inflammation than wild-type mice after administration of DSS. MGL1-positive cells in the colonic lamina propria corresponded to macrophage-like cells with F4/80-high, CD11b-positive, and CD11c-intermediate expression. These cells in Mgl1^−/− mice produced a lower level of IL-10 mRNA compared with wild-type mice after the administration of DSS for 2 days. Recombinant MGL1 was found to bind both Streptococcus sp. and Lactobacillus sp. among commensal bacteria isolated from mesenteric lymph nodes of DSS-treated mice. Heat-killed Streptococcus sp. induced an increase in IL-10 secretion by MGL1-positive colonic lamina propria macrophages, but not the macrophage population from Mgl1^−/− mice. These results strongly suggest that MGL1/CD301a plays a protective role against colitis by effectively inducing IL-10 production by colonic lamina propria macrophages in response to invading commensal bacteria.     

Cytotoxic T cells in AIDS colonic cryptosporidiosis

BACKGROUND/AIMS: It is not known how enteric cryptosporidiosis induces severe intestinal impairment despite minimal invasion by the parasite. The aim of this study was to analyse the histological features and locally implicated immune cells in colonic biopsies of AIDS related cryptosporidiosis. PATIENTS/METHODS: Colonic biopsies from patients with AIDS related cryptosporidiosis (n = 10, group I), patients with AIDS but without intestinal infection (n = 9, group II), and human seronegative controls (n = 9, group III) were studied. Using immunohistochemistry the infiltrating mononuclear cells were analysed in both the epithelium and lamina propria for the expression of CD3, CD8, TiA1, granzyme B, and CD68 and for glandular expression of human major histocompatibility complex DR antigen (HLA-DR). RESULTS: Severe histological changes, resulting in abundant crypt epithelial apoptosis and inflammatory infiltrate in the lamina propria, were seen in all biopsies from group I. A significant increase of CD8+, TiA1+, and granzyme B+ T cells in the lamina propria and HLA-DR glandular expression was noted in group I compared with groups II and III. However, the number of intraepithelial lymphocytes, lamina propria CD3+ T cells, and macrophages was not significantly increased in cryptosporidiosis specimens compared with controls. CONCLUSION: Epithelial apoptosis mediated by granzyme B+ cytotoxic host T cells might play a major role in the development of colonic lesions in AIDS related cryptosporidiosis.     

Antibodies to Recombinant Clostridium difficile Toxins A and B Are an Effective Treatment and Prevent Relapse of C. difficile-Associated Disease in a Hamster Model of Infection

Clostridium difficile causes antibiotic-associated diarrhea and colitis in humans through the actions of toxin A and toxin B on the colonic mucosa. At present, broad-spectrum antibiotic drugs are used to treat this disease, and patients suffer from high relapse rates after termination of treatment. This study examined the role of both toxins in pathogenesis and the ability of orally administered avian antibodies against recombinant epitopes of toxin A and toxin B to treat C. difficile-associated disease (CDAD). DNA fragments representing the entire gene of each toxin were cloned, expressed, and affinity purified. Hens were immunized with these purified recombinant-protein fragments of toxin A and toxin B. Toxin-neutralizing antibodies fractionated from egg yolks were evaluated by a toxin neutralization assay in Syrian hamsters. The carboxy-terminal region of each toxin was most effective in generating toxin-neutralizing antibodies. With a hamster infection model, antibodies to both toxins A and B (CDAD antitoxin) were required to prevent morbidity and mortality from infection. In contrast to vancomycin, CDAD antitoxin prevented relapse and subsequent C. difficile reinfection in the hamsters. These results indicate that CDAD antitoxin may be effective in the treatment and management of CDAD in humans.     

Ultrastructural evaluation of apoptosis induced by Helicobacter pylori infection in human gastric mucosa: novel remarks on lamina propria mucosae

It has been considered that Helicobacter pylori (H. pylori) infection is a major cause of human gastritis and gastroduodenal ulcers (G-DU). Many investigations of the relationship between H. pylori and apoptosis have been reported recently. However, these studies focused mostly on epithelium, using the TUNEL method. In the present study, we evaluated by electron microscopy the occurrence of apoptosis in the mesenchymal cells of lamina propria mucosae infected with H. pylori. Gastric biopsy specimens from 37 H. pylori-infected G-DU patients and 8 noninfected volunteers were examined with both light and electron microscopy and analyzed by the TUNEL method. The TUNEL method showed no significant difference between H. pylori-infected and noninfected cases. In contrast, electron microscopy revealed significant numbers of apototic fibroblasts and smooth muscle cells in H. pylori-infected lamina propria mucosae, with a diminished number of collagen fibers in surrounding areas. These areas showed edematous changes histopathologically. These results indicated that H. pylori infection induces apoptosis of fibroblasts and smooth muscle cells in lamina propria, with decrease in the numbers of collagen fibers, suggesting that these alterations may be affected by exaggerate acid secretion, decrease mucus protecting factors, and result in ulcer formation.     

Caspase Activation as a Versatile Assay Platform for Detection of Cytotoxic Bacterial Toxins

Pathogenic bacteria produce several virulence factors that help them establish infection in permissive hosts. Bacterial toxins are a major class of virulence factors and hence are attractive therapeutic targets for vaccine development. Here, we describe the development of a rapid, sensitive, and high-throughput assay that can be used as a versatile platform to measure the activities of bacterial toxins. We have exploited the ability of these toxins to cause cell death via apoptosis of sensitive cultured cell lines as a readout for measuring toxin activity. Caspases (cysteine-aspartic proteases) are induced early in the apoptotic pathway, and so we used their induction to measure the activities of Clostridium difficile toxins A (TcdA) and B (TcdB) and binary toxin (CDTa-CDTb), Corynebacterium diphtheriae toxin (DT), and Pseudomonas aeruginosa exotoxin A (PEA). Caspase induction in the cell lines, upon exposure to toxins, was optimized by toxin concentration and intoxication time, and the specificity of caspase activity was established using a genetically mutated toxin and a pan-caspase inhibitor. In addition, we demonstrate the utility of the caspase assay for measuring toxin potency, as well as neutralizing antibody (NAb) activity against C. difficile toxins. Furthermore, the caspase assay showed excellent correlation with the filamentous actin (F-actin) polymerization assay for measuring TcdA and TcdB neutralization titers upon vaccination of hamsters. These results demonstrate that the detection of caspase induction due to toxin exposure using a chemiluminescence readout can support potency and clinical immunogenicity testing for bacterial toxin vaccine candidates in development.     

Release of TcdA and TcdB from Clostridium difficile cdi 630 is not affected by functional inactivation of the tcdE gene

The small open reading frame tcdE is located between the genes tcdA and tcdB which encode toxin A (TcdA) and B (TcdB), respectively, within the pathogenicity locus of Clostridium difficile. Sequence and structure similarities to bacteriophage-encoded holins have led to the assumption that TcdE mediates the release of the toxins from C. difficile into the extracellular environment. A TcdE-deficient C. difficile 630 strain was generated by insertional inactivation of the tcdE gene. Data revealed that TcdE does not regulate or affect growth or sporogenesis. TcdE-deficiency was accompanied by a moderately increased accumulation of TcdA and TcdB prior to sporulation in this microorganism. Interestingly, this observation did not correlate with a delayed or inhibited toxin release: inactivation of TcdE neither significantly altered kinetics of release nor the absolute level of secreted TcdA and TcdB, indicating that TcdE does not account for the pathogenicity of C. difficile strain 630. Furthermore, mass spectrometry analysis could not reveal differences in the secretome of wild type and TcdE-deficient C. difficile, indicating that TcdE did not function as a secretion system for protein release. TcdE was expressed as a 19 kDa protein in C. difficile, whereas TcdE expressed in Escherichia coli appeared as a 19 and 16 kDa protein. Expression of the short 16 kDa TcdE correlated with bacterial cell death. We conclude that TcdE does not exhibit pore-forming function in C. difficile since in these cells only the non-lytic full length 19 kDa protein is expressed.     

Characterization of monoclonal antibodies specific for monocytes, macrophages and granulocytes from porcine peripheral blood and mucosal tissues

A panel of four monoclonal antibodies produced in our laboratory, MIL1, MIL2, MIL3, MIL4, and the type-specific monocyte/granulocyte marker 74-22-15 were used to isolate and to discriminate between monocytes, macrophages and granulocytes derived from porcine peripheral blood, lung and gut lamina propria.

Two-colour flow cytometry and cell sorting showed that while no monoclonal antibody was specific for just a single cell population, each cell type had a unique and characteristic combination of surface antigens. These differences could be used to identify and purify monocytes, macrophages, neutrophils, eosinophils and basophils from the three different sites.

The study also demonstrated similarities and differences within cell types from the same site and from different sites: polymorphonuclear neutrophils (PMN) from peripheral blood were subdivided into two subpopulations by the presence or absence of the surface antigen recognized by MIL4, while PMN from alveolar lavage did not express this antigen. Peripheral blood eosinophils were also divided into subpopulations by the presence or absence of the same surface antigen. Lamina propria eosinophils strongly expressed the MIL4 marker and differed morphologically from blood eosinophils. Peripheral blood basophils and lamina propria mast cells were morphologically similar and expressed similar antigens. Monocytes and alveolar macrophages also expressed the same surface antigens.     

Apoptosis and reduced microvascular density of the lamina propria during tooth eruption in rats

During tooth eruption, structural and functional changes must occur in the lamina propria to establish the eruptive pathway. In this study, we evaluate the structural changes that occur during lamina propria degradation and focus these efforts on apoptosis and microvascular density. Fragments of maxilla containing the first molars from 9‐, 11‐, 13‐ and 16‐day‐old rats were fixed, decalcified and embedded in paraffin. The immunohistochemical detection of vascular endothelial growth factor (VEGF), caspase‐3 and MAC387 (macrophage marker), and the TUNEL method were applied to the histological molar sections. The numerical density of TUNEL‐positive cells and VEGF‐positive blood vessel profiles were also obtained. Data were statistically evaluated using a one‐way anova with the post‐hoc Kruskal–Wallis or Tukey test and a significance level of P ≤ 0.05. Fragments of maxilla were embedded in Araldite for analysis under transmission electron microscopy (TEM). TUNEL‐positive structures, fibroblasts with strongly basophilic nuclei and macrophages were observed in the lamina propria at all ages. Using TEM, we identified processes of fibroblasts or macrophages surrounding partially apoptotic cells. We found a high number of apoptotic cells in 11‐, 13‐ and 16‐day‐old rats. We observed VEGF‐positive blood vessel profiles at all ages, but a significant decrease in the numerical density was found in 13‐ and 16‐day‐old rats compared with 9‐day‐old rats. Therefore, the establishment of the eruptive pathway during the mucosal penetration stage depends on cell death by apoptosis, the phagocytic activity of fibroblasts and macrophages, and a decrease in the microvasculature due to vascular cell death. These data point to the importance of vascular rearrangement and vascular neoformation during tooth eruption and the development of oral mucosa.