Purification and characterization of Clostridium sordellii hemorrhagic toxin and cross-reactivity with Clostridium difficile toxin A (enterotoxin).

Hemorrhagic toxin (toxin HT) was purified from Clostridium sordellii culture filtrate. The purification steps included ultrafiltration through an XM-100 membrane filter and immunoaffinity chromatography, using a monoclonal antibody to toxin A of Clostridium difficile as the ligand. Toxin HT migrated as a major band with a molecular weight of 525,000 and a minor band at 450,000 on nondenaturing gradient polyacrylamide gel electrophoresis. The molecular weight was estimated at 300,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing indicated an apparent pI of 6.1. Toxin HT was cytotoxic for cultured cells and lethal for mice by intraperitoneal injection, and it elicited an accumulation of hemorrhagic fluid in rabbit ileal loops. Immunodiffusion analysis revealed a reaction of partial identity between toxins A and HT. Immunological cross-reactivity between these toxins was further demonstrated by immunoblotting and by neutralization of toxin HT biological activity with antibodies to toxin A. A sensitive indirect enzyme-linked immunosorbent assay was used to examine the affinity involved in homologous and heterologous antigen-antibody interactions. Our findings show that toxin HT has biological activities and immunological properties similar to those of toxin A; however, the toxins are not identical.     

Disease association of antibodies to human and mycobacterial hsp70 and hsp60 stress proteins.

Structural homology between microbial and human stress proteins has been postulated to be a basis for autoimmunization in chronic inflammatory diseases. Therefore, we estimated by ELISA titration the antibody levels to mycobacterial (M) and human (H) recombinant hsp70 and M-hsp65 heat-shock proteins in sera of patients with Crohn's disease (n = 29), ulcerative colitis (n = 20) and nontuberculous mycobacterial disease of the lungs (n = 20). Antibodies to H-hsp60, separated by two-dimensional gel electrophoresis, were tested in six sera of each group of patients. In Crohn's disease, antibody titres to the M-hsp65 antigen without detectable H-hsp60 binding were significantly elevated in 52% of the patients. In contrast titres to both M-hsp70 and H-hsp70 were demonstrable and correlated, but increased over control values only in four (14%) patients. The antibody pattern in ulcerative colitis was found to be quite different: anti-H-hsp60 binding was demonstrable in most patients, although anti-M-hsp65 titres were not elevated. Furthermore, 25% of patients had significantly elevated titres to M-hsp70, but not to H-hsp70. In non-tuberculous mycobacterial pulmonary disease, about 50% of patients had elevated titres to both hsp65 and hsp71 mycobacterial antigens but not to the corresponding human proteins; patients with Mycobacterium xenopi infection had the highest titres in this group. These results demonstrate the existence of distinct disease-associated patterns in the human antibody response to stress protein antigens. However, these data are not sufficient to imply sensitization with mycobacteria in patients with inflammatory bowel diseases, since certain epitopes of heat-shock proteins are shared by several bacterial genera.     

Generation of interleukin-2-dependent T cell lines from synovial fluids in rheumatoid arthritis.

Synovial fluids from rheumatoid arthritis (RA) patients were found to contain activated T lymphocytes that could be maintained as continuous T cell lines (CTCL) in the presence of the T cell growth factor, interleukin (IL)-2. The CTCL predominantly expressed the OKT8 phenotype and were Ia antigen positive. IL-2-dependent RA CTCL could be maintained in an active dividing state by the presence of RA synovial fluids, whereas IL-2-dependent CTCL from mitogen stimulated PBL failed to respond to the fluids, which were shown to contain IL-2. This suggested that RA CTCL exhibit unique properties not possessed by normal PBL CTCL. The CTCL generated from activated synovial T lymphocyte populations in RA may be used to assess the functions of these cells and their responses to regulatory factors.     

Performance on the Balance Error Scoring System Decreases After Fatigue

OBJECTIVE: To determine the immediate effects of a whole-body fatigue protocol on performance of the Balance Error Scoring System (BESS), a postural-stability test commonly used as part of a concussion-assessment battery. DESIGN AND SETTING: Subjects were assigned to a fatigue or control group and were assessed before and immediately after a 20-minute fatigue protocol or rest period. SUBJECTS: Fourteen fatigue subjects and 13 control subjects participated in this study. All subjects were male and free of vestibular disorders, and none had suffered a mild head injury or lower extremity injury in the preceding 6 months, as described through self-report. MEASUREMENTS: We measured performance on the BESS for 9 stance-surface conditions and summed each condition to obtain a total score. Using the Borg scale, we also measured ratings of perceived exertion before, during, and after the fatigue protocol or rest period. RESULTS: We found a significant increase in total errors from pretest to posttest in the fatigue group (14.36 +/- 4.73 versus 16.93 +/- 4.32), a significant decrease in errors in the control group (13.32 +/- 3.77 versus 11.08 +/- 3.88), and a significant difference between groups on the posttest. The rating of perceived exertion scores were significantly different between the fatigue and control groups at the middle (13.29 +/- 1.59 versus 6.23 +/- 0.83) and end (15.86 +/- 2.38 versus 6.15 +/- 0.55) of the fatigue or rest period. CONCLUSIONS: The BESS error scores increased immediately after the fatigue protocol, demonstrating that balance ability diminished. Clinicians who use the BESS as part of their sideline assessment for concussion should not administer the test immediately after a concussion due to the effects of fatigue.     

Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases

Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine. Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates. Enzyme activity was associated with the light-chain polypeptide of the toxin. The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain. In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin. A single ADP-ribose group was transferred to each substrate molecule (G-actin). The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine. Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors. The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin. When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins.     

Molecular characterization of the Clostridium difficile toxin A gene

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs had a mol% G + C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Gal alpha 1-3Gal beta 1-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.     

Adhesion of peripheral blood lymphocytes of children with arthritis to human umbilical vein endothelial cells.

To determine whether adhesion of peripheral blood lymphocytes (PBL) of patients with juvenile rheumatoid arthritis (JRA) may be enhanced, adhesion of PBL of children with JRA, children with seronegative spondyloarthropathies (SSA), age-appropriate and adult controls, to human umbilical vein endothelial cells (HUVEC) was assessed in vitro. B and CD4 T lymphocytes in initial, adherent, and non-adherent cell fraction were identified by flow cytometry. B lymphocytes of all the younger subjects combined had a higher adherence to activated HUVEC compared with B lymphocytes of the adult donors. Except for greater adherence of HLA-DR+ CD4 T cells, lymphocytes of children with JRA showed no enhanced adhesion to either unactivated or activated HUVEC. The percentage of B cells adherent to activated HUVEC in each of the subject groups was 1.5-3.6-fold higher than adherent CD4 T lymphocytes. Surface analyses indicated higher percentages of CD49d (alpha 4)+ and CD29 (beta 1)+ CD4 T lymphocytes in adherent cells, but less of a differential in CD49 (alpha 4)+ and no difference in CD29 (beta 1)+ B lymphocytes. There were fewer Leu-8 (L-selectin)+ B and Leu-8+ CD4 T cells among adherent cells. The data suggest a greater adhesive capacity of B lymphocytes compared with CD4 T lymphocytes which is unrelated to disease, and the possibility that B lymphocytes may utilize adhesion molecules distinct from those of CD4 T lymphocytes. Only a small subset of T cells of patients with JRA may have an enhanced capacity for adhesion to endothelium.     

Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A.

The toxin A gene of Clostridium difficile contains a 2.5-kb region encoding a series of contiguous repeating units located at the COOH terminus of the molecule. We previously showed that the monoclonal antibody (MAb) PCG-4, which neutralizes the enterotoxic activity of toxin A, binds to epitopes located within these repeating units. In the present study, we subcloned a series of fragments from this portion of the gene. The recombinant peptides expressed from the gene fragments were examined for reactivity with MAb PCG-4 to identify the epitopes involved in binding. Our results showed that MAb PCG-4 recognizes epitopes in amino acid residues 2097 through 2141 and amino acid residues 2355 through 2398.