Defensin deficiency, intestinal microbes, and the clinical phenotypes of Crohn's disease

Crohn's disease is a chronic, inflammatory disease of the intestinal mucosa. Although intestinal bacteria are implicated in disease pathogenesis, the etiology is still unclear. The main location of disease is the small intestine (ileum) and the colon. Ileal disease has been linked to a mutation in the NOD2 gene. Defensins are antimicrobial peptides and in the ileum, are mainly expressed in Paneth cells, epithelial cells that also express NOD2. In the colon, defensins are expressed by enterocytes or metaplastic Paneth cells. Crohn's disease patients with ileal involvement, compared with controls or Crohn's patients without ileal involvement, have diminished expression of ileal Paneth cell defensins. This decrease is even more pronounced in Crohn's patients displaying a NOD2 mutation. In contrast, Crohn's disease of the colon is characterized by an impaired induction of beta-defensins in enterocytes. The colonic expression of the constitutive beta-defensin 1 is also decreased in the inflamed colonic mucosa, but this decrease is less specific to Crohn's disease, as it can also be found in ulcerative colitis patients. In conclusion, the regional localizations of Crohn's disease, ileal or colonic disease, can be linked to different defensin profiles. Crohn's disease of the ileum is associated with diminished defensin expression in Paneth cells. Crohn's disease of the colon is associated with diminished beta-defensin expression in enterocytes. Thus, it can be speculated that decreased defensin levels lead to a weakened intestinal barrier function to intestinal microbes and might be crucial in the pathophysiology of Crohn's disease.     

Proteasomal degradation of cytotoxic necrotizing factor 1-activated rac

The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli has been shown to activate members of the Rho family by deamidation of glutamine 63. This amino acid is essential for hydrolysis of GTP, and any substitution results in a constitutively active Rho. Activation of Rho induces the formation of stress fibers, filopodia, and membrane ruffles due to activation of RhoA, Cdc42, and Rac, respectively. Here we show that the level of endogenous Rac decreased in CNF1-treated HEK293 and HeLa cells. The amount of mRNA remained unaffected, leaving the possibility that Rac is subject to proteolytic degradation. Treatment of cells with lactacystin, an inhibitor of the 26S proteasome, protected Rac from degradation. We have previously shown that CNF1 activates the c-Jun N-terminal kinase (JNK) only transiently in HeLa cells (M. Lerm, J. Selzer, A. Hoffmeyer, U. R. Rapp, K. Aktories, and G. Schmidt, Infect. Immun. 67:496-503, 1998). Here we show that CNF1-induced JNK activation is stabilized in the presence of lactacystin. The data indicate that Rac is degraded by a proteasome-dependent pathway in CNF1-treated cells.     

Chimeric Clostridial Cytotoxins: Identification of the N-Terminal Region Involved in Protein Substrate Recognition

Clostridium sordellii lethal toxin is a member of the family of large clostridial cytotoxins that glucosylate small GTPases. In contrast to Clostridium difficile toxins A and B, which exclusively modify Rho subfamily proteins, C. sordellii lethal toxin also glucosylates Ras subfamily proteins. By deletion analysis and construction of chimeric fusion proteins of C. sordellii lethal toxin and C. difficile toxin B, we localized the enzyme activity of the lethal toxin to the N terminus of the holotoxin and identified the region involved in protein substrate specificity. The toxin fragment of the N-terminal 546 amino acid residues of C. sordellii lethal toxin glucosylated Rho and Ras subfamily proteins, as the holotoxin did. Deletion of a further 30 amino acid residues from the C terminus of this active fragment drastically reduced glucotransferase activity and blocked glucohydrolase activity. Exchange of amino acid residues 364 through 516 of lethal toxin for those in the active toxin B fragment (1 to 546) allowed glucosylation of Ras subfamily proteins. In contrast, the chimera with amino acids 1 to 364 from toxin B, 365 to 468 from lethal toxin, and 469 to 546 from toxin B exhibited markedly reduced modification of Ras subfamily proteins, whereas modification of Rac and Cdc42 was hardly changed. The data indicate that the region of amino acid residues 364 through 516 primarily defines the substrate specificity of C. sordellii lethal toxin.     

Evaluation of Mannitol Salt Agar for Detection of Oxacillin Resistance in Staphylococcus aureus by Disk Diffusion and Agar Screening

Mannitol salt agar was evaluated for detection of oxacillin resistance in 136 Staphylococcus aureus isolates. All mecA-positive isolates (n = 54) were correctly categorized as oxacillin resistant by the disk diffusion test (1-μg disk; zone diameter, <16 mm); the specificity was 97.6%. Agar screening (2 μg of oxacillin per ml) revealed a sensitivity of 98.1% and a specificity of 95.1%.     

The Dominant Epitope of Borrelia garinii Outer Surface Protein C Recognized by Sera from Patients with Neuroborreliosis Has a Surface-Exposed Conserved Structural Motif

Epitope mapping of outer surface protein C (OspC) by using sera from patients with neuroborreliosis led to the identification of one single major immunodominant epitope within the C-terminal 10 amino acid residues. Peptide binding studies and alanine replacement scanning of the C-terminal decapeptide, PVVAESPKKP, revealed a critical role for the PKKP sequence and its terminal carboxyl group for the binding of immunoglobulin M (IgM) antibodies from patients with Lyme borreliosis. Electron microscopy of antibody-labeled spirochetes indicated that the C-terminal region is exposed on the surface of the spirochete. Based on homology to proteins of known function, this region most probably adopts a polyproline II-like helix, which is found in surface-exposed structures involved in protein-protein interactions. This structural motif is highly conserved in Borrelia species causing Lyme borreliosis and subjected to purifying selection. We suggest that the abundance of the C-terminal region of OspC on the surface of B. burgdorferi allows a multimeric high-avidity interaction between the spirochete and surface Igs on B cells. The resulting cross-linking of surface Igs on B cells may induce a T-cell-independent B-cell activation without IgM-to-IgG switching, thus explaining the lack of IgG antibodies to OspC in neuroborreliosis.     

Structural analysis of alkene/maleic anhydride copolymers

Structural investigations on maleic anhydride (MAn) copolymers with ethene, propene and styrene, their products of hydrolysis, and their methyl half-esters by means of ¹³C NMR spectroscopy are presented. The spectra of 2,3-diethylsuccinic acid and its anhydrides in the erythro- and threo-configuration and of butylsuccinic acid and its anhydride were obtained and compared with the spectra of the copolymers. In each case the results show the formation of both threo(trans)- and erythro(cis)-structures. At a polymerization temperature of 60°C the proportions were 88% threo to 12% erythro for ethene/MAn copolymers and 80% threo to 20% erythro for propene/MAn copolymers, a ratio which was confirmed also by the hydrolyzed forms. Copolymerization at 150°C leads, in the case of propene/MAn, to 67% threo and 33% erythro. The production of ethene/MA copolymer via the copolymerization of ethene and fumaric acid half-ester and its saponification to ethene/fumaric acid leads to a ratio of 38% threo to 62% erythro. These results are in accordance with the thermal stabilities of the configurations. In the case of styrene/MAn copolymer it is not possible to obtain a unique interpretation of the configurations from the spectra. For the conversion of propene/MAn and styrene/MAn copolymers by means of methanol to half-esters it can be derived from the ¹³C NMR spectra how many of the half-ester moieties are obtained in the neighbourhood of the methyl and phenyl groups, respectively, of the olefin.     

Detection of cytomegaloviral DNA in human milk cells and cell free milk whey by nested PCR

Human cytomegalovirus (HCMV) DNA can be detected in different compartments of human milk. A protocol for the preparation of milk whey free of fat and cells for the detection of human cytomegalovirus (HCMV) by nested PCR is presented. This is based upon the experience of the separation of more than 200 milk specimens of healthy seropositive breast feeding mothers. HCMV DNA could be detected in freshly centrifuged and filtrated milk whey specimens without contamination by cellular DNA. In limiting dilution experiments using HCMV plasmid DNA, the effect of different DNA extraction procedures from native milk and milk whey on the detection limit of cytomegaloviral DNA was demonstrated. About 200 viral genome equivalents/ml in milk whey or native milk were detectable by classical organic phenol/chloroform extraction or a spin column method, respectively. The detection of viral DNA in milk cells depended on a minimum number of milk cells (10⁵–2×10⁵) available for DNA extraction. In contrast to the findings of cytomegaloviral DNA in native sera or plasma of immunosuppressed patients we failed to amplify low level viral DNA from native breast milk by nested PCR due to an inhibition of Taq polymerase by lipid components. Finally, the course of cell associated and cell free DNAlactia was monitored. Analyzing sequential milk specimens, in some cases the presence of HCMV DNA in colostrum could be demonstrated. DNAlactia of milk cells and whey was partially discordant. Onset (week 1–4 after delivery) and duration (2 weeks up to more than 3 months) of DNAlactia showed distinct individual patterns. The methods described, allow further analysis of the mechanisms involved in the postnatal HCMV transmission by breast feeding seropositive mothers.     

Fusion with mouse thymocytes leads to expression of p30 antigen in fibroblasts of BALB/Mo mice

Embryonic fibroblasts of BALB/Mo mice carrying the endogenous genome of Moloney murine leukemia virus (M-MuLV) in addition to murine type C viruses were fused with Swiss mouse thymocytes, B lymphocytes, macrophages, or embryonic fibroblasts. We wanted to determine whether these cells contained physiological factors involved in induction of the integrated viral genome(s). Fusion with thymocytes and, to a lesser extent, fusion with B lymphocytes induced viral genome expression as demonstrated by the appearance of viral structural protein p30 detected by immunofluorescence. Maximal expression of p30 was observed about 36 hr after the fusion event, using thymocytes from 1- to 2-week-old Swiss mice. This temporary expression of p30 was not accompanied by release of infectious virus. Fusion of BALB/Mo fibroblasts with Swiss mouse macrophages or embryonic fibroblasts led to neither the production of p30 nor the release of detectable infectious virus. These results suggest the existence of thymocyte-specific and possibly B lymphocyte-specific factor(s) involved in the control of expression of integrated viral genome(s).     

Efficacy of Amodiaquine in uncomplicated falciparum malaria in Nigeria in an area with high-level resistance to Chloroquine and Sulphadoxine/Pyrimethamine

Falciparum Malaria is hyperendemic in southern Nigeria and chloroquine resistance is an increasing problem. Therefore, the parasitological and haematological response to treatment with amodiaquine was studied in children under 5 years during a 14-day follow-up. Of 105 children who accomplished the study (out of 114 who were enrolled), 95.3% were parasite-negative on thick blood film on day 7, which decreased to 89.5% on day 14. The haemoglobin levels increased on average by 1.3% on day 14 (±1.9) and more pronounced in children with anaemia < 10 g/dl on enrolment. The number of patients with adverse events (mainly pruritus and nausea) was few. This study shows that amodiaquine is effective, safe and affordable in an area with high resistance to chloroquine.