Virulent Toxoplasma gondii strain RH promotes T-cell-independent overproduction of proinflammatory cytokines IL12 and gamma-interferon.

The aim of this study was the analysis of the cytokine response in BALB/c mice infected with the highly virulent RH or the weakly virulent Beverley strains of Toxoplasma gondii. Analysis of cytokine messages showed increased expression of IL12, IFN-gamma and TNF-alpha, but not IL4 mRNAs in spleen cells after infection with the T. gondii strains RH and Beverley. High levels of circulating IL12 and IFN-gamma were detected in the serum of mice infected with strain RH, although TNF-alpha levels remained low. In contrast, the same cytokines were detected at only low levels in the serum of mice infected with the Beverley strain. Administration of antibody against IL12 or IFN-gamma significantly delayed time to death of mice infected with strain RH compared to controls. T-Cell-deficient as well as normal mice were equally infected by strain RH, suggesting that T lymphocytes do not contribute to the response. Depletion of natural killer cells from the splenocyte population abolished the in vitro production of IFN-gamma. Together, our data suggest that the virulent strain RH induces in BALB/c mice a type 1 cytokine pattern with T-cell-independent overproduction of IL12 and IFN-gamma that may be involved in the pathogenesis of this micro-organism.

P2Y(6) nucleotide receptor mediates monocyte interleukin-8 production in response to UDP or lipopolysaccharide.

Extracellular nucleotides are autocrine and paracrine cellular mediators that signal through P2 nucleotide receptors. Monocytic cells express several P2Y receptors but the role of these G protein-coupled receptors in monocytes is not known. Here, we present evidence that P2Y(6) regulates chemokine production and release in monocytes. We find that UDP, a selective P2Y(6) agonist, stimulates interleukin (IL)-8 release in human THP-1 monocytic cells whereas other nucleotides are relatively inactive. P2 receptor antagonists or P2Y(6) antisense oligonucleotides inhibit IL-8 release induced by UDP. Furthermore, UDP specifically activated IL-8 production in astrocytoma 1321N1 cells transfected with human P2Y(6). Since lipopolysaccharide has been suggested to activate P2 receptors via nucleotide release, we tested whether IL-8 production stimulated by lipopolysaccharide might result from P2Y(6) activation. P2 antagonists or apyrase, an enzyme which hydrolyzes nucleotides including UDP, inhibit IL-8 production induced by lipopolysaccharide but not by other stimuli. Furthermore, IL-8 gene expression activated by lipopolysaccharide is enhanced by P2Y(6) overexpression and inhibited by P2Y(6) antisense oligonucleotides. Thus, UDP activates IL-8 production via P2Y(6) in monocytic cells. Furthermore, lipopolysaccharide mediates IL-8 production at least in part by autocrine P2Y(6) activation. These findings indicate a novel role for P2Y(6) in innate immune defenses.

Saccharomyces boulardii stimulates intestinal immunoglobulin A immune response to Clostridium difficile toxin A in mice.

Saccharomyces boulardii is a nonpathogenic yeast that protects against antibiotic-associated diarrhea and recurrent Clostridium difficile colitis. The administration of C. difficile toxoid A by gavage to S. boulardii-fed BALB/c mice caused a 1.8-fold increase in total small intestinal immunoglobulin A levels (P = 0.003) and a 4.4-fold increase in specific intestinal anti-toxin A levels (P < 0.001). Enhancing host intestinal immune responses may be an important mechanism for S. boulardii-mediated protection against diarrheal illnesses.

Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea.

BACKGROUND: We have reported that symptom-free carriers of Clostridium difficile have a systemic anamnestic immune response to toxin A. The aim of this study was to determine whether an acquired immune response to toxin A, during an episode of C. difficile diarrhoea, influences risk of recurrence. METHODS: We prospectively studied 63 patients with nosocomial C. difficile diarrhoea. Serial serum IgA, IgG, and IgM concentrations against C. difficile toxin A, toxin B, or non-toxin antigens were measured by ELISA. Individuals were followed for 60 days. FINDINGS: 19 patients died (30%). Of the 44 who survived, 22 had recurrent C. difficile diarrhoea. Patients with a single episode of C. difficile diarrhoea (n=22) had higher concentrations of serum IgM against toxin A on day 3 of their first episode of diarrhoea than those with recurrent diarrhoea (n=22, p=0.004). On day 12, serum IgG values against toxin A were higher in patients who had a single episode of diarrhoea (n=7) than in those who subsequently had recurrent diarrhoea (n=9, p=0.009). The odds ratio for recurrence associated with a low concentration of serum IgG against toxin A, measured 12 days after onset of C. difficile diarrhoea, was 48.0 (95% CI 3.5-663). INTERPRETATION: A serum antibody response to toxin A, during an initial episode of C. difficile diarrhoea, is associated with protection against recurrence.

Clostridium difficile toxin A causes early damage to mitochondria in cultured cells.

BACKGROUND & AIMS: The mechanism by which Clostridium difficile toxin A causes actin depolymerization and cell rounding involves toxin internalization and subsequent monoglucosylation of the Rho family of proteins. This study explored toxin internalization and effects on mitochondrial function before cell rounding. METHODS: Chinese hamster ovary (CHO) cells were exposed to toxin A, and mitochondrial localization was assayed by confocal microscopy. Mitochondrial function was measured by adenosine triphosphate (ATP) concentration, mitochondrial permeability, and leakage of cytochrome c. RESULTS: Confocal microscopy showed toxin A colocalization with the mitochondrial protein GRP 75 at 5 minutes after toxin exposure. Between 5 and 15 minutes, toxin A caused an 80% diminution in cellular ATP levels; cell rounding and Rho glucosylation commenced between 15 and 30 minutes. Toxin A also resulted in reduction of mitochondrial membrane potential and a 2-3-fold increase in reactive oxygen radicals. Preincubation of CHO cells with the antioxidants butylated hydroxyanisole or butylated hydroxytoluene blocked the toxin A-induced increase in oxygen radicals and diminished cell rounding. Western blot analysis of toxin A-exposed isolated mitochondria showed a direct effect of toxin A on leakage of cytochrome c. CONCLUSIONS: The results show that extensive mitochondrial damage occurs within 15 minutes in CHO cells exposed to toxin A. Diminished ATP concentrations and increased oxygen radicals are likely to contribute to cytotoxicity from this bacterial toxin.

p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis.

Clostridium difficile toxin A causes acute neutrophil infiltration and intestinal mucosal injury. In cultured cells, toxin A inactivates Rho proteins by monoglucosylation. In monocytes, toxin A induces IL-8 production and necrosis by unknown mechanisms. We investigated the role of mitogen-activated protein (MAP) kinases in these events. In THP-1 monocytic cells, toxin A activated the 3 main MAP kinase cascades within 1 to 2 minutes. Activation of p38 was sustained, whereas stimulation of extracellular signal-regulated kinases and c-Jun NH(2)-terminal kinase was transient. Rho glucosylation became evident after 15 minutes. IL-8 gene expression was reduced by 70% by the MEK inhibitor PD98059 and abrogated by the p38 inhibitor SB203580 or by overexpression of dominant-negative mutants of the p38-activating kinases MKK3 and MKK6. SB203580 also blocked monocyte necrosis and IL-1beta release caused by toxin A but not by other toxins. Finally, in mouse ileum, SB203580 prevented toxin A-induced neutrophil recruitment by 92% and villous destruction by 90%. Thus, in monocytes exposed to toxin A, MAP kinase activation appears to precede Rho glucosylation and is required for IL-8 transcription and cell necrosis. p38 MAP kinase also mediates intestinal inflammation and mucosal damage induced by toxin A.

Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A.

BACKGROUND: Clostridium difficile infection can result in asymptomatic carriage, mild diarrhea, or fulminant pseudomembranous colitis. We studied whether antibody responses to C. difficile toxins affect the risks of colonization, diarrhea, and asymptomatic carriage. METHODS: We prospectively studied C. difficile infections in hospitalized patients who were receiving antibiotics. Serial stool samples were tested for C. difficile colonization by cytotoxin assay and culture. Serum antibody (IgA, IgG, and IgM) levels and fecal antibody (IgA and IgG) levels against C. difficile toxin A, toxin B, and nontoxin antigens were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Of 271 patients, 37 (14 percent) were colonized with C. difficile at the time of admission, 18 of whom were asymptomatic carriers. An additional 47 patients (17 percent) became infected in the hospital, 19 of whom remained asymptomatic. The baseline antibody levels were similar in the patients who later became colonized and those who did not. After colonization, those who became asymptomatic carriers had significantly greater increases in serum levels of IgG antibody against toxin A than did the patients in whom C. difficile diarrhea developed (P<0.001). The adjusted odds ratio for diarrhea was 48.0 (95 percent confidence interval, 3.4 to 678) among patients with colonization who had a serum level of IgG antibody against toxin A of 3.00 ELISA units or less, as compared with patients with colonization who had a level of more than 3.00 ELISA units. CONCLUSIONS: We find no evidence of immune protection against colonization by C. difficile. However, after colonization there is an association between a systemic anamnestic response to toxin A, as evidenced by increased serum levels of IgG antibody against toxin A, and asymptomatic carriage of C. difficile.

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.

Monocytic cell necrosis is mediated by potassium depletion and caspase-like proteases.

Apoptosis is a physiological cell death that culminates in mitochondrial permeability transition and the activation of caspases, a family of cysteine proteases. Necrosis, in contrast, is a pathological cell death characterized by swelling of the cytoplasm and mitochondria and rapid plasma membrane disruption. Necrotic cell death has long been opposed to apoptosis, but it now appears that both pathways involve mitochondrial permeability transition, raising the question of what mediates necrotic cell death. In this study, we investigated mechanisms that promote necrosis induced by various stimuli (Clostridium difficile toxins, Staphylococcus aureus alpha toxin, ouabain, nigericin) in THP-1 cells, a human monocytic cell line, and in monocytes. All stimuli induced typical features of necrosis and triggered protease-mediated release of interleukin-1beta (IL-1beta) and CD14 in both cell types. K+ depletion was actively implicated in necrosis because substituting K+ for Na+ in the extracellular medium prevented morphological features of necrosis and IL-1beta release. N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum caspase inhibitor, prevented morphological features of necrosis, plasma membrane destruction, loss of mitochondrial membrane potential, IL-1beta release, and CD14 shedding induced by all stimuli. Thus, in monocytic cells, necrosis is a cell death pathway mediated by passive K+ efflux and activation of caspase-like proteases.

Bovine immunoglobulin concentrate-clostridium difficile retains C difficile toxin neutralising activity after passage through the human stomach and small intestine.

BACKGROUND: Bovine immunoglobulin concentrate (BIC)-Clostridium difficile is prepared from the colostrum of cows immunised against C difficile toxins and contains high concentrations of neutralising IgG antitoxin. AIMS: To determine the proportion of BIC-C difficile which survives passage through the human stomach and small intestine. METHODS: Six volunteers with an end ileostomy took 5 g of BIC-C difficile containing 2.1 g of bovine IgG on four occasions: alone, with an antacid, during treatment with omeprazole, and within enteric coated capsules. RESULTS: When BIC-C difficile was taken alone, a mean (SEM) of 1033 (232) mg of bovine IgG was recovered in the ileal fluid representing 49% of the total ingested dose. Bovine IgG recovery was not significantly increased by antacid (636 (129) mg) or omeprazole (1052 (268) mg). The enteric capsules frequently remained intact or only partially opened in the ileal effluent and free bovine IgG levels were low in this treatment group (89 (101) mg). Bovine IgG recovery was higher in volunteers with shorter (less than two hours) mouth to ileum transit times (68% versus 36%, p<0. 05). Specific bovine IgG against C difficile toxin A was detected in ileal fluid following oral BIC. Toxin neutralising activity was also present and correlated closely with bovine IgG levels (r=0.95, p<0. 001). CONCLUSION: BIC-C difficile resists digestion in the human upper gastrointestinal tract and specific anti-C difficile toxin A binding and neutralising activity was retained. Passive oral immunotherapy with anti-C difficile BIC may be a useful non-antibiotic approach to the prevention and treatment of C difficile antibiotic associated diarrhoea and colitis.

Intravenous immunoglobulin therapy for severe Clostridium difficile colitis.

BACKGROUND: Many individuals have serum antibodies against Clostridium difficile toxins. Those with an impaired antitoxin response may be susceptible to recurrent, prolonged, or severe C difficile diarrhoea and colitis. AIMS: To examine whether treatment with intravenous immunoglobulin might be effective in patients with severe pseudomembranous colitis unresponsive to standard antimicrobial therapy. PATIENTS: Two patients with pseudomembranous colitis not responding to metronidazole and vancomycin were given normal pooled human immunoglobulin intravenously (200-300 mg/kg). METHODS: Antibodies against C difficile toxins were measured in nine immunoglobulin preparations by ELISA and by cytotoxin neutralisation assay. RESULTS: Both patients responded quickly as shown by resolution of diarrhoea, abdominal tenderness, and distension. All immunoglobulin preparations tested contained IgG against C difficile toxins A and B by ELISA and neutralised the cytotoxic activity of C difficile toxins in vitro at IgG concentrations of 0.4-1.6 mg/ml. CONCLUSION: Passive immunotherapy with intravenous immunoglobulin may be a useful addition to antibiotic therapy for severe, refractory C difficile colitis. IgG antitoxin is present in standard immunoglobulin preparations and C difficile toxin neutralising activity is evident at IgG concentrations which are readily achieved in the serum by intravenous immunoglobulin administration.

IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes.

Neutrophil infiltration is central to the pathogenesis of Clostridium difficile toxin A-induced enterocolitis. This study examines whether monocyte activation by C. difficile toxins is instrumental in initiating neutrophil activation and recruitment. Human monocytes were exposed to low concentrations of highly purified C. difficile toxins, and the conditioned media were harvested for cytokine and functional assays. Monocytes exposed to C. difficile toxin A (10(-10) M) or toxin B (10(-12) M) released 100 and 20 times basal levels, respectively, of the neutrophil chemoattractant interleukin-8 (IL-8). Reverse transcriptase-polymerase chain reaction demonstrated a marked increase in IL-8 mRNA expression by monocytes 3 h after toxin exposure. Conditioned media from toxin A- and toxin B-treated monocytes stimulated neutrophil migration (324 and 245% of control, respectively). This effect was completely blocked by IL-8 antiserum. These media also upregulated neutrophil CD11b/CD18 and endothelial cell intercellular adhesion molecule-1 expression. C. difficile toxins, at low concentrations, potently activate monocytes to release factors, including IL-8, that facilitate neutrophil extravasation and tissue infiltration. Our findings indicate a major role for toxin-mediated monocyte and macrophage activation in C. difficile colitis.

Effect of Clostridium difficile toxin A on CD11/CD18 expression in vitro.

Clostridium difficile toxin A is chemotactic for neutrophils and induces their emigration into the colonic mucosae of rodents. We found that toxin A did not upregulate neutrophil beta 2 integrins on isolated human neutrophils. These data support the hypothesis that in C. difficile colitis, these adhesion molecules are upregulated by endogenous mediators.

The anti-Hu syndrome: a clinical and immunological study of 7 cases.

The authors describe the clinical and biological data of seven patients with anti-Hu antibodies. Six of them displayed a small cell lung carcinoma (SCLC), but no cancer was detected in the 7th patient in spite of an extensive workup. The clinical heterogeneity of the anti-Hu syndrome is emphasized. The major symptoms were linked to a severe sensory neuropathy in three cases, to cerebellitis in two cases, to dysautonomia in one case, and to gastro-intestinal pseudo-obstruction in one case. One patient also displayed EMG abnormalities characteristic of the Lambert-Eaton myasthenic syndrome. Two patients developed opsoclonus or ocular flutter associated with severe confusion in the late stage of their disease. In four patients, the neurological signs and symptoms preceded the discovery of the SCLC, and in two cases the initial detection of anti-Hu antibodies prompted the successful search for this tumor. Immunopathological events injuring the peripheral and central nervous system are briefly discussed.

Detection of inner ear disease autoantibodies by immunoblotting.

To define further the character of autoantibodies against the inner ear in patients with inner ear disease, Autoantibodies in sera from 82 patients with inner ear disease were investigated by immunoblotting. The inner ear antigens were extracted from Hartley guinea pigs. Brain, kidney, lung, heart and liver extracts were also prepared. Antibodies against the inner ear were found in 32 of 82 (39%) patients with inner ear disease. These sera reacted with the 30 and 58 kDa bands of the inner ear extracts. The 30 kDa band was detected in sera from patients with various inner ear diseases, while the 58 kDa band reacted with sera of patients with idiopathic progressive sensorineural hearing loss. Only two of the 52 normal control sera had a very faint band at 30 kDa. Sixteen of 32 positive sera were then used to probe Western blots of the brain, kidney, lung heart and liver extracts. The 58 kDa band was also found in the protein extracts of the brain, the lung, and the liver. On the other hand, preliminary purification of the 30 and 58 kDa proteins from the inner ear extracts were achieved by anion exchange chromatography. These results show that antibodies in sera from patients with inner ear disease reacted with at least two polypeptide bands (30 and 58 kDa) of guinea pig inner ear extracts, and the 58 kDa antigenic epitope was not cochlea specific.

Clostridium difficile colitis: recent therapeutical and immunological considerations.

Clostridium difficile is the main etiological agent of antibiotic associated diarrhoea and pseudomembranous colitis (PMC). It is considered as the most frequent agent of infectious diarrhoea occurring in hospitalized patients, in whom it is responsible for a high morbidity and occasional mortality even when the diagnosis and the treatment are pursued aggressively (1). The pathology is due to the production of at least two toxins: toxin A is an enterotoxin which induces intestinal tissue damage and a fluid response and toxin B is a cytotoxin which lacks any enterotoxic activity but is believed to exert an additive effect in vivo (2).

Gamma globulin administration in relapsing Clostridium difficile-induced pseudomembranous colitis with a defective antibody response to toxin A.

A 53-year-old woman suffered six episodes of Clostridium difficile-pseudomembranous colitis. The serological follow-up demonstrated the absence of a rise in IgG and IgA to toxin A. Human pooled gamma globulin was administered during the fifth relapse and raised IgG levels to toxin A. Normal stools reappeared a week later. The role of the antibodies to toxin A and gamma globulin in C. difficile colitis are discussed.

Human antibody response to Clostridium difficile toxin A in relation to clinical course of infection.

This study investigated whether differences in fecal and serum antitoxin A antibody levels may account for the duration of Clostridium difficile-associated diarrhea (CDAD) and the occurrence of relapses. By an enzyme linked-immunosorbent assay, we tested 40 patients with CDAD including 25 patients without immunodeficiency and 15 patients receiving antineoplastic drugs. Two hundred eighty serum samples and 80 normal stool samples were investigated as controls. In nonimmunocompromised patients, serum immunoglobulin (IgG) and fecal IgA antitoxin A antibody titers were significantly higher in patients who suffered a single episode (n = 21) than in those with relapsing CDAD (n = 4) whose titers were at control levels. Of these 25 patients, eight suffered from diarrhea which lasted for more than 2 weeks. These patients had significantly lower serum- and feces-specific antibody levels than the others who presented symptoms of shorter duration. In cytostatic-treated patients, antitoxin A antibody levels were similar to controls, but relapses occurred in a single case. These data suggest an association between a defective humoral response to toxin A and a more severe form of C. difficile infection. They also indicate that other host-related factors control the severity of CDAD and remain to be elucidated.

Anti-neutrophil antibodies in chronic hepatitis and the effect of alpha-interferon therapy.

In 34 patients with non-A non-B, 28 with type B and 11 with autoimmune chronic hepatitis, anti-neutrophil antibodies were investigated using indirect immunofluorescence and anti-myeloperoxidase antibodies by enzyme-linked immunosorbent assay. Granulocyte-specific antinuclear antibodies, were detected in 14 patients with advanced stages of non-A, non-B hepatitis (41%). Their presence correlated with histological features of disease activity but not with response to interferon therapy. Within 24 h after the first dose of interferon, 9 of these became negative and 3 more became negative after 1, 3 and 5 months. Myeloperoxidase-positive perinuclear neutrophil cytoplasmic antibodies were detected in a single patient and increased reaching a peak level after 8 weeks of interferon, decreasing thereafter. In type B, all were negative before and during the 6 months of therapy. In 6 patients with autoimmune hepatitis (55%), myeloperoxidase-negative perinuclear neutrophil cytoplasmic antibodies were detected in high titers. The association of granulocyte-specific anti-nuclear antibodies with non-A, non-B hepatitis support the hypothesis that hepatitis C virus infection might trigger humoral autoimmune response. In chronic autoimmune hepatitis, perinuclear neutrophil cytoplasmic antibodies appear as another marker of autoimmunity.