IntroductionClostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis in humans (1). Pathogenic strains of C. difficile release 2 large exotoxins: toxin A (308 kDa) and toxin B (269 kDa). In animal models, only toxin A is enterotoxic and causes fluid secretion, mucosa edema, and villous disruption by inducing massive acute inflammation with neutrophil infiltration. We have shown that blocking neutrophil extravasation using an anti-CD18 antibody prevented toxin A-induced enteritis and mucosal damage (2).Toxin A and toxin B show 63% homology (3) and share similar domains. Their COOH-terminal portions carry repeating sequences that may be involved in receptor binding. The NH 2 -terminal portions carry an enzymatic domain, which hydrolyzes UDP-glucose and transfers the glucose moiety to a conserved threonine residue of the small GTP-binding proteins Rho, Cdc42, and Rac (4-6). The small GTP-binding protein Rap is glucosylated only by toxin A (7). These covalent modifications inactivate Rho proteins, which in turn induce cytoskeleton disaggregation and cell rounding. Whether Rho glucosylation is involved in toxin A enterotoxicity and in vitro cytokine production is not known. Toxin A or toxin B were shown to block Rho-regulated signaling pathways, including basophilic cell activation (8), receptor signaling to phospholipase D (9), NF-κB activation by bradykinin (10), and mitogen-activated protein-kinase (MAP kinase) activation (11,12). Blockage of the above signal transduction pathways by C. difficile toxins is consistent with the role of Rho proteins in signaling to MAP kinases and NF-κB (13-15). However, it appears at variance with the known in vivo and in vitro inflammatory actions of toxin A.In cells of the monocyte lineage, C. difficile toxins stimulate inflammatory cytokine release, including TNF-α, IL-1β, . In addition, we have reported recently that both toxins induce necrosis in human monocytes and in THP-1 human monocytic cells. This cell-death pathway is associated with potassium depletion, caspase activation, and maturation and release of preformed IL-1β (19). The mechanisms whereby these toxins activate and kill monocytes are not known.In this study, we investigated the role of MAP kinases in IL-8 production, IL-1β release, and necrosis induced by toxin A in monocytic cells. MAP kinases regulate cell responses to growth factors and stress stimuli and transmit signals from the cell surface to the nucleus via 3 distinct but related pathways. These culminate in the selective activation of extracellular signal-related kinases (ERK), p38, and c-Jun NH 2 -terminal kinase (JNK) 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 cascad...

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.

JAK2 V617F mutation recently was identified as a pathogenic factor in typical chronic myeloproliferative diseases (CMPD). Some forms of myelodysplastic syndromes (MDS) show a significant overlap with CMPD (classified as MDS/MPD), but the diagnostic assignment may be challenging. We studied blood or bone marrow from 270 patients with MDS, MDS/MPD, and CMPD for the presence of JAK2 V617F mutation using polymerase chain reaction, sequencing, and melting curve analysis. The detection rate of JAK2 V617F mutants for polycythemia vera, chronic idiopathic myelofibrosis, and essential thrombocythemia (n ‫؍‬ 103) was similar to the previously reported results. In typical forms of MDS (n ‫؍‬ 89) JAK2 V617F mutation was very rare (n ‫؍‬ 2). However, a higher prevalence of this mutation was found in patients with MDS/MPD-U (9 of 35). Within this group, most of the patients harboring JAK2 V617F mutation showed features consistent with the provisional MDS/MPD-U entity refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T). Among 9 RARS-T patients, 6 showed the presence of JAK2 V617F mutation, and in 1 patient without mutation, aberrant, positive phospho-STAT5 staining was seen that is typically present in association with JAK2 V617F mutation. In summary, we found that RARS-T reveals a high frequency of JAK2 V617F mutation and likely constitutes another JAK2 mutation-associated form of

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.