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Biomedical subjects

U Hadding

Publications and source records attributed to U Hadding.

At least 19 recordsLinked to original sources
PubMed1992

Species differentiation of mycoplasmas by EF-Tu specific monoclonal antibodies.

Ten mouse hybridoma cell lines producing IgG monoclonal antibodies to mycoplasmal elongation factor Tu (EF-Tu) were established. These mAbs showed different degrees of cross-reactivity between mollicutes and even other bacteria. This finding, indicating protein structure diversities of pan bacterial EF-Tu should permit species differentiation of mycoplasmas by epitope pattern analysis of a single protein. Epitope patterns of 23 mollicute type strains and of 40 M. hominis isolates were determined by ELISA. All M. hominis patterns were found to be closely related whereas intrageneric patterns differed in a species specific manner.

Amino Acid Sequence↗
PubMed1992

[A lethal course in pseudomembranous enterocolitis during the parenteral administration of vancomycin and imipenem].

A 48-year-old woman required mechanical ventilation after aortic valve replacement for decompensated aortic valve stenosis when bleeding complications developed and rethoracotomy had to be performed. Acute renal failure necessitated haemodialysis. Septic fever of unknown aetiology failed to respond to oxacillin, cefotaxim and tobramycin. The endotracheal cannula and central venous catheter were changed on the 24th postoperative day and the antibiotic treatment altered to 250 mg imipenem and 125 mg vancomycin three times daily intravenously. The fever soon subsided, but recurred on the 32nd postoperative day, accompanied by increasing leucocytosis. The patient was obstipated but had no intraabdominal signs. Four days later ultrasonography demonstrated thickening of the intestinal wall and coloscopy showed typical pseudomembranous colitis. Intestinal contents were positive for Clostridium difficile toxin. Despite immediate rectal and intragastric administration of 250 mg vancomycin four times daily the patient died of pseudomembranous colitis, confirmed at autopsy. The case demonstrates that vancomycin cannot always prevent the development of pseudomembranous colitis.

Colon↗
PubMed1992

Human glioblastoma cell line 86HG39 activates T cells in an antigen specific major histocompatibility complex class II-dependent manner.

The capacity of three different human glioblastoma cell lines to activate human T cells was analysed by measuring major histocompatibility complex (MHC) antigen expression, monokine secretion and lectin, mAb OKT3 and antigen-driven T cell proliferation. All glioblastoma cells tested were able to induce PHA and concanavalin A (ConA)-driven T cell proliferation in a dose-dependent fashion, while all failed to induce T cell activation with mAb OKT3. In addition, the glioblastoma cell line 86HG39 was able to induce tetanus toxoid and toxoplasma lysate antigen-specific T cell proliferation. The responding T cell lines originated from only one out of five different donors. This foreign antigen-specific T cell proliferation induced by 86HG39 cells could be inhibited with mAb L243 directed against HLA-DR molecules. The study of monokine secretion by 86HG39 cells showed a strong interleukin (IL)-6 secretion after lipopolysaccharide (LPS) treatment, whilst no IL-1 secretion was observed. Furthermore, only 86HG39 cells were positive for HLA-DR molecules, whereas interferon (IFN) gamma treatment of 87HG28 and 87HG31 cells was necessary for the induction of class II antigen expression. Thus, cell line 86HG39 shows many features of an antigen presenting cell and the interaction of these cells with MHC compatible human T cells might be a useful model to study cellular immune reactions within the central nervous system.

Animals↗
PubMed1992

Decreased metabolism and viability of Mycoplasma hominis induced by monoclonal antibody-mediated agglutination.

Monoclonal antibodies (MAbs) were generated against lysates of clinical Mycoplasma hominis isolates. Three of these, designated BG2, BA10, and FE6, recognized an integral membrane protein of M. hominis with an apparent molecular weight of 50,000 (p50). Electron microscopy studies demonstrated that this protein is distributed evenly over the cell surface. These anti-p50 MAbs were species specific for M. hominis; they reacted with 42% of 126 tested clinical M. hominis isolates and showed no reactivity to heterologous mycoplasma species. Immunoblot analysis after limited proteolysis of purified p50 demonstrated that the three MAbs reacted with different epitopes of the protein. Unlike BA10 and FE6, MAb BG2 induced a decrease in arginine metabolism and a reduction of CFU in metabolic inhibition tests. F(ab)2 fragments of MAb BG2 showed the same inhibitory effect as the intact MAb molecule, while Fab and Fc fragments had no influence on vital functions. Preincubation of the mycoplasmas with MAb BG2 followed by trypsin treatment yielded the same amount of CFU as the control without antibodies. In conclusion, the cell aggregates were resolved by the trypsin treatment. These experiments and tests with the antibody fragments led to the conclusion that only the intact MAb structure or the F(ab)2 structure had metabolic inhibition potential and that the observed metabolism inhibition as well as the apparent decrease in viability were a result of agglutination by MAb BG2.

Agglutination↗
PubMed1992

Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays.

Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized. Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule. No MAb was able to neutralize cytotoxic activity significantly. The two IgG1 MAbs were purified and applied to various immunodiagnostic assays. MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay. An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml.

Animals↗
PubMed1991

Sequence and expression in Escherichia coli of a Mycoplasma hominis gene encoding elongation factor Tu.

We describe the molecular cloning and the complete nucleotide (nt) sequence of a Mycoplasma hominis gene common to a broad range of Mycoplasma species, as defined by hybridization analysis with the cloned gene. Production of M. hominis protein in Escherichia coli was assayed by use of a monoclonal antibody. The nt sequence analysis revealed a 1194-bp open reading frame that could encode a 43 516-Da protein. Computer-aided sequence comparison indicated that the gene codes for elongation factor Tu.

Amino Acid Sequence↗
PubMed1991

Granulocyte colony-stimulating factor (G-CSF) treatment in a neutropenic leukemia patient with diffuse interstitial pulmonary infiltrates.

Adult respiratory distress syndrome (ARDS) in patients suffering from acute leukemia usually occurs during chemotherapy-induced neutropenia. In addition, intensified chemotherapy with high-dose cytosine arabinoside and mediastinal irradiation may contribute to the development of ARDS. This complication is usually refractory to conservative treatment with antibiotics, steroids, and mechanical ventilation. In this report, we describe a 25-year-old patient with acute lymphoblastic leukemia who developed ARDS during the phase of chemotherapy-induced neutropenia. Subcutaneous administration of granulocyte colony-stimulating factor (G-CSF) at doses of 300-600 micrograms/day led to a prompt increase of peripheral granulocyte counts. With resolution of neutropenia, respiratory function gradually improved, and mechanical ventilatory support was stopped after 2 weeks. From this observation we surmise that the application of G-CSF may be an effective therapeutic approach for preventing the fatal outcome of ARDS in leukemia patients with bone marrow aplasia.

Adult↗
PubMed1991

Cytokine response by human monocytes to Clostridium difficile toxin A and toxin B.

Clostridium difficile toxins A and B isolated from strain VPI 10463 were tested for induction of cytokine release by human monocytes. Toxin B at 10(-12) M activated human monocytes as measured by release of interleukin-1 (IL-1), tumor necrosis factor (TNF), or IL-6. These effects of toxin B were heat labile (51 degrees C, 30 min). Toxin B was as effective as bacterial lipopolysaccharides in inducing IL-1 beta but less effective in inducing TNF or IL-6. Toxin B and lipopolysaccharides were synergistic in induction of IL-1 beta, TNF, and IL-6. The toxin A preparation used was 1,000-fold less active than toxin B. Apart from the difference in activity, the two toxins showed identical patterns of reaction and there was no synergism between them. A short pulse with toxin B was sufficient to trigger IL-1 release. Toxin B was also extremely toxic for monocytes. The toxicity and the induced proinflammatory monokines (IL-1 and TNF) may contribute to the pathogenic mechanisms of C. difficile infection and pseudomembranous colitis.

Bacterial Proteins↗
PubMed1990

Fast and simple procedure for the detection of cell culture mycoplasmas using a single monoclonal antibody.

The detection of mycoplasmas in cell cultures is still a problem, especially in those laboratories in which the detection and identification of microorganisms is not established as a routine procedure. In our laboratory, monoclonal antibodies (mAbs) have been prepared to Acholeplasma laidlawii, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini and Mycoplasma salivarium. 30 mAbs were obtained and one of these, designated CCM-2, was shown to bind to all five mycoplasma species. It also bound to Mycoplasma fermentans, Mycoplasma hominis and to all wild types (n = 54), isolated from cell cultures submitted to our laboratory. The mAb was used in a immunofluorescent assay (IF) and the method correlated with the microbiological assay. Using this mAb immunofluorescent staining of cells is a fast and simple procedure for mycoplasma detection in cell cultures.

Animals↗
PubMed1990

Septicemia due to Streptococcus mitis in neutropenic patients with acute leukemia.

Eight neutropenic patients with acute lymphocytic or nonlymphocytic leukemia had septicemia due to different strains of Streptococcus mitis (St. mitis), a microorganism not commonly recognized as a special pathogen in leukemic patients. Four of the patients had been treated with high-dose cytosine arabinoside as part of the cytostatic regimen, six had a central venous line and four patients had oral lesions prior to the infection. Selective gut decontamination consisted of co-trimoxazole/colistin in five patients and quinolones in three patients. The first three patients died, either due to interstitial pneumonia with the adult respiratory distress syndrome (ARDS), or due to infection-triggered disseminated intravascular coagulation despite prompt empiric antibiotic therapy including vancomycin. The other patients improved after empiric supplementation of penicillin G (30 Mega/day) to the antibiotic regimen. Beginning ARDS in two of these patients dramatically responded to high-dose steroids. We conclude that St. mitis is a major pathogen in neutropenic leukemic patients. Infection appears to occur independently of acute leukemic cell type, regimen of selective gut decontamination, venous access, visible oral lesions or treatment with high-dose cytosine arabinoside. The clinical course of our patients raises questions about the value of commonly recommended empiric antibiotic regimens, which were clearly ineffective to control infections with St. mitis in this patient group. Our data indicate that immediate antibiotic therapy with penicillin G is indicated and may be life-saving for suspected St. mitis infections in neutropenic leukemic patients.

Adult↗
PubMed1989

Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile.

Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits. To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C. difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum. Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C. difficile genome. All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector. Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30-170 kDa) in Escherichia coli strains harbouring recombinant plasmids. As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300-600 kDa protein. This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A. No additional gene(s) with homology to the cloned toxin A gene was detected.

Bacterial Toxins↗
PubMed1988

A safe and efficient method for elimination of cell culture mycoplasmas using ciprofloxacin.

The antibacterial activity of ciprofloxacin, a 4-fluoroquinolone antibiotic, in the control of mycoplasma contamination in experimentally infected cell lines has been investigated. Seven mycoplasma species, including M. hyorhinis, M. gallisepticum, M. orale, M. salivarium, M. hominis, M. fermentans, and M. arginini, which had chronically infected the murine plasmocytoma line X63-Ag8 653, were eradicated with 10 micrograms/ml ciprofloxacin. Wild type laboratory infections of two human cell lines, HL-60 and U-937, were eliminated by 12 days of such treatment. Mycoplasma decontamination of cell cultures was monitored by the cultivation method 4 weeks after treatment. No side effects were seen in cell cultures and complex proliferation assays with cells of human and murine origin, using ciprofloxacin in doses up to 2.5 times the usual bactericidal concentration.

Animals↗
PubMed1988

[The complement system: a regulator of infection defense mechanisms and immune regulation].

Complement is a physiological constituent of bloodplasma. In its activated state complement is able to stimulate various cells for special performance, e.g., monocytes, macrophages, granulocytes, platelets, but also T- and B-lymphocytes. The biological functions of the complement system cover the following areas: induction of inflammation, defence against microbial infections, handling of immune complexes, and modulation of the immune response.

Communicable Diseases↗
PubMed1988

Rosmarinic acid: a new inhibitor of complement C3-convertase with anti-inflammatory activity.

Rosmarinic acid (RA) is a naturally occurring compound, isolated from Rosmarinus officinalis or Melissa officinalis which inhibits the in vitro immunohaemolysis of antibody-coated sheep erythrocytes by guinea pig serum. In further experiments this reduced immunohaemolysis was found to be due to inhibition of the C3-convertase of the classical complement pathway. The threshold concentration for inhibition of C3-convertase was 10(-6) mol/l. RA with an optimal inhibitory concentration between 5 and 10 mumol/l., resulting in about 70% inhibition of haemolysis. However, higher concentrations of RA were less effective at inhibiting C3-convertase. The inhibition may not be specific for C3-convertase, since another serine protease, elastase, was also weakly inhibited by RA in vitro. RA also exhibited inhibitory activity in three in vivo models in which complement activation plays a role. Thus, RA (0.316-3.16 mg/kg i.m.) reduced paw oedema induced by cobra venom factor (CVF) in the rat, and at 1-100 mg/kg p.o. inhibited passive cutaneous anaphylaxis in the rat. In addition, at 10 mg/kg i.m. RA impaired in vivo activation by heat-killed Corynebacterium parvum (i.p.) of mouse macrophages, as measured by the decreased capacity of the activated macrophages to undergo the oxidative burst. RA (0.1-10 mg/kg i.m.) did not inhibit t-butyl hydroperoxide-induced paw oedema in the rat, indicating selectivity for complement-dependent processes.

Animals↗
PubMed1988

Clostridium difficile toxins A and B inhibit human immune response in vitro.

Two Clostridium difficile toxins isolated from strain VPI 10463 were tested for their effect on different human T-cell proliferation systems. In mitogen- and antigen-driven T-cell proliferation systems, toxins inhibited the proliferative response in a dose-dependent fashion. In interleukin-2-driven culture systems, no effect of toxins could be found on preactivated T cells. We suspected that monocytes were the influenced cells, since in antigen- and mitogen-driven systems monocytes were necessary for the proliferative response, whereas the interleukin-2-driven system was independent of monocytes. To prove this concept, purified monocytes were treated with toxins. The treatment was found to markedly reduce the capacity of monocytes to stimulate T-cell proliferation. No inhibition of the proliferative response was measured when, instead of monocytes, resting or preactivated T cells were treated with toxins. These experiments clearly show that C. difficile toxins interact with monocytes and not with T cells. The effect of toxins on cells of the immune system might be one factor in the development of pseudomembranous colitis.

Bacterial Proteins↗