Arginine deiminase inhibits proliferation of human leukemia cells more potently than asparaginase by inducing cell cycle arrest and apoptosis.

L-Asparaginase is used for the treatment of acute leukemias, but is sometimes ineffective or associated with severe side-effects. We report here that the enzyme arginine deiminase is approximately 100-fold more potent than L-asparaginase in inhibiting the proliferation of cultured human lymphatic leukemia cell lines while it appears to be less effective in leukemia cells of myeloid origin. The inhibition of cell proliferation involves cell growth arrest in the G1- and/or S-phase and eventually apoptotic cell death. Our results suggest the possibility of a future use of arginine deiminase for the therapy of leukemia.

Arginine deiminase inhibits cell proliferation by arresting cell cycle and inducing apoptosis.

We have previously demonstrated that arginine deiminase inhibits the proliferation of vascular endothelial cells, but the mechanisms leading to growth inhibition have remained unclear. We report here that low concentrations of arginine deiminase purified from Mycoplasma arginini inhibit proliferation of various cultured cells by arresting the cell cycle in G(1) and/or S phase with higher arginine deiminase concentrations leading to subsequent apoptosis. Our results demonstrate that arginine deiminase inhibits cell proliferation not only by depletion of arginine, but also by mechanisms involving the cell cycle and death signals.

Haemolytic activity of Helicobacter pylori against human and animal erythrocytes.

Clinical isolates and reference strains of H. pylori (n = 28) were investigated for lytic activity against red blood cells (RBC) of man, sheep, horse, rabbit, hamster and guinea pig. The test medium (HTA) consisted of agar base supplemented with newborn calf serum and washed RBC. In contrast to previous reports, haemolysis of human RBC was detected. Blood group A RBC supported the haemolytic growth of all strains studied. On HTA with blood group 0 RBC, one strain failed to grow and five strains grew nonhaemolytically. The diversity of growth and haemolysis on HTA containing the human and several animal RBC species allowed a differentiation of the strains into eight groups. Using a liquid RBC assay for quantitative measurement of haemolysis, extracts of broth-grown cells of strain ATCC 43504 showed an average haemolysis rate of 75% against sheep RBC and of 83% against human A RBC. Cell-free culture supernatants, however, showed an average haemolysis rate of 57% against sheep RBC and no activity against human A RBC. The data indicate that H. pylori expresses an intracellular haemolytic factor and a cell surface-associated or secreted haemolytic factor which have different RBC specificities. The demonstration of the sensitivity of human RBC supports the hypothesis that the haemolytic property of H. pylori is involved in the pathogenesis of human infection.

Susceptibility of Helicobacter pylori to simethicone and other non-antibiotic drugs.

The antifoaming agent simethicone (Lefax), the protease inhibitor gabexate mesilate (FOY), the antimycotic ketoconazole, and the hydroxyl scavangers dimethylsulphoxide (DMSO) and allopurinol were investigated for growth inhibition of Helicobacter pylori and representative strains of other bacterial species. H. pylori were selectively inhibited by 64-128 mg/L of simethicone, 64-128 mg/L gabexate mesilate, and 16-64 mg/L ketoconazole. Dimethylsulphoxide and allopurinol showed no antibacterial effect at concentrations used therapeutically. It is concluded that gabexate mesilate, ketoconazole and, particularly, simethicone are candidates for treatment of H. pylori infection.

Cat owners' risk of acquiring a Helicobacter pylori infection.

In order to obtain information as to whether or not domestic cats are a significant reservoir for human Helicobacter pylori infection, H. pylori antibodies were determined in 59 members of cat protection clubs and in an age- and sex-matched control group. Using four different assay systems, the prevalence rates of H. pylori antibodies in the cat owner group compared to the control group were 29% and 34%, respectively (Pyloriset Dry), 27%/42% (Pyloriset EIA-G), 14%/29% (Pyloriset EIA-A), and 37%/42% (Cobas Core EIA). The differences between the two groups in the prevalence of H. pylori antibodies were not statistically significant. It is concluded that cat owners, even if they have a very intensive contact with their pets, do not have a higher risk of H. pylori infection than the general population.

Metronidazole susceptibility testing of Helicobacter pylori with the PDM epsilometer test (E test).

The bacteriostatic activity of metronidazole against Helicobacter pylori was determined with the PDM epsilometer test (E test) and with an agar dilution test. Both methods correlated in the minimum inhibitory concentration (MIC) ranges (< or = 0.5- > 32 mg/l) and in the MIC 50 (E test: 1 mg/l; agar dilution test: 2 mg/l) and MIC 90 (> 32 mg/l) values. However, comparison of test results of single strains revealed that 14 out of 105 strains (13.3%) were classified as resistant by one method but classified susceptible by the other. The correlation coefficient of 0.71 also indicated a low congruence of test results. It is concluded that both methods should be used in order to ascertain all strains resistant to metronidazole.

No cultural detection of Helicobacter pylori in dental plaque.

Helicobacter pylori causes human type B gastritis and is involved in the etiology of peptic ulcer disease. The routes of transmission of H. pylori are still unclear. The microorganism may be transmitted orally, since H. pylori has been detected in dental plaques. To confirm the hypothesis that dental plaques are a reservoir of H. pylori, 100 dental plaque specimens from 55 dental surgery patients were incubated on one nonselective and up to four selective agar media for the detection of H. pylori. In addition, urease activity of the plaque material was tested, and the gingival status of the patients was assessed. H. pylori was not cultivated from any of the specimens investigated. Plaque material from 12 patients with moderate and severe gingivitis showed urease activity. The results do not confirm the hypothesis that dental plaques are a relevant reservoir of viable H. pylori cells. However, non-cultivatable forms of H. pylori may survive in dental plaques. Urea cleaving activity of dental plaque may be a marker of gingival inflammation.

Activity of antibiotics and azole antimycotics against Helicobacter pylori.

The bacteristatic and bactericidal activities of six antibiotics from different substance classes against Helicobacter pylori were determined. Ampicillin, imipenem, tetracycline, and amikacin inhibit the growth of all isolates at concentrations achievable in serum. Cefpirome and ofloxacin are ineffective against three and two of 41 strains, respectively. However, the minimum bactericidal concentrations (MBC) of the substances are two- to sixteen-fold higher than the minimum inhibitory concentrations (MIC). There is sufficient bactericidal activity of ampicillin and imipenem against all strains, but amikacin, ofloxacin, tetracycline, and cefpirome are unable to kill 2, 8, 12, and 18 of 25 strains, respectively, at concentrations achievable in serum. Differences between MIC and MBC of antibiotics may contribute to the explanation of therapy failures. In addition, the inhibitory activity of seven nitroimidazole antimycotics and the triazole fluconazole was evaluated. The nitroimidazole MICs range from 2 to 64 mg/l, with tioconazole, miconazole, bifonazole, and ketoconazole as the most active substances. Fluconazole, however, was ineffective at concentrations < or = 128 mg/l. The efficacy of the nitroimidazole antimycotics against H. pylori in vivo should be tested in a clinical trial.

Cell-associated haemolytic activity of Helicobacter pylori.

Helicobacter pylori cells cultured on solid medium were quantitatively tested for haemolytic activity against erythrocytes of man, sheep, the guinea pig and rabbit. Using 4-day and 8-day cultures of two standard strains (ATCC 43504, IMMi 676), human erythrocytes were not lysed by 10% bacterial suspensions. Rabbit erythrocytes were the most sensitive to 8-day cultures. Hot-cold incubation yielded the highest haemolysis titres. The extent of haemolysis strongly correlated with the number of bacterial cells. Supplementation of the test medium (PBS, pH 7.4) with L-cysteine, dithiothreitol, MgCl2, EDTA, cholesterol, lecithin or sphingomyelin did not influence the haemolysis titres. They were significantly reduced in the presence of pronase E, human serum, bovine serum albumin or CaCl2, and by heat treatment of the bacteria. Supplementation of the test medium with cardiolipin strongly increased the haemolysis titres. Comparing the cell-associated haemolytic activity of 18 strains, the titres ranged from < 2 to 64, with a median titre of 16. No correlation was found between the haemolytic activity and phospholipase C activity of the cell suspensions. It was concluded that the formation of lysophosphatides and non-enzymatic factors rather than a sulphydryl-activated cytolysin or phospholipase C are responsible for the cell-associated haemolytic activity. This property may be involved in the pathogenicity and virulence of Helicobacter pylori.

Cholesterol binding of Helicobacter pylori.

H. pylori cells grown on cholesterol-free medium adsorb cholesterol from serum, egg yolk, and VDRL reagent. The binding of cholesterol does not influence the hydrophobicity of the cells. The haemagglutinating activity is slightly diminished. The cell-bound haemolytic activity is completely inhibited. The affinity of H. pylori for cholesterol probably acts as factor of chemotaxis and adherence.

Flocculation of venereal disease research laboratory reagent by Helicobacter pylori.

Helicobacter pylori strains flocculated with Venereal Disease Research Laboratory (VDRL) reagent in a glass slide test. Other pathogenic bacterial and fungal strains were nonreactive. The specific VDRL reaction property of Helicobacter pylori indicates an affinity of the cells for lipoidal substances, and can be used as a diagnostic aid for species identification.

Evaluation of techniques for isolation, subcultivation, and preservation of Helicobacter pylori.

With sheep blood agar (SBA), Belo Horizonte medium, and Brussels campylobacter charcoal agar, 104 strains of Helicobacter pylori were detected in 309 gastric biopsies. Each medium revealed only 69 to 71% of the strains. Ten strains grew solely on SBA, and four strains each grew on Belo Horizonte medium and Brussels campylobacter charcoal agar. Subculturing of 50 fresh H. pylori isolates on SBA revealed a progressive reduction in growth with increasing passage. Thirty strains stopped growing between passages two and seven. Four strains survived more than 20 passages. The preservation of fresh H. pylori isolates at -193 and -70 degrees C and by lyophilization was compared by use of 10% porcine mucin solution, fetal calf serum, and a commercial cryopreservative fluid. Of 30 strains, 77 to 90% could be recultivated on SBA after preservation at -70 degrees C in all three storage media. The data indicate that for the primary isolation of H. pylori, not only one selective medium but several selective media with different antibiotic supplements plus at least one nonselective medium should be used to yield the highest culture rates. Frequent subculturing of H. pylori on SBA selects strains which may not be representative of clinical isolates. Storage of fresh H. pylori isolates at -70 degrees C in 10% mucin solution is a simple and effective preservation procedure.

[Use of microbiologic-serologic studies for Chlamydia and Mycoplasma in differential diagnosis of inflammatory rheumatic diseases]. / Einsatz mikrobiologisch-serologischer Untersuchungen auf Chlamydien und Mykoplasmen für die Differentialdiagnostik entzündlich-rheumatischer Erkrankungen.

Chlamydiae are obligate intracellular bacteria that lack ATP-synthesis; mycoplasmas are free-living bacteria without cell walls. Chlamydia trachomatis and mycoplasmas may be associated with reactive rheumatic diseases. Local chlamydial infections are identified by isolating the bacteria in cell cultures or by detecting bacterial antigens in specimens containing epithelial cells. Disseminated chlamydial infection can be detected by specific serum antibodies. Mycoplasma infections of the genitourinary tract are detected by quantitative isolation methods. Diagnosis of Mycoplasma pneumoniae infections requires demonstration of a rise in specific serum antibodies using complement fixation or indirect hemagglutination tests. For the diagnosis of reactive rheumatic diseases, the microbiological and serological findings have to be connected synoptically with other laboratory data and with the clinical symptoms.

Bacteriophages in Helicobacter (Campylobacter) pylori.

Bacteriophages in different stages of maturation were found in thin sections of a clinical isolate of Helicobacter (Campylobacter) pylori. Mature phage heads measured 70 x 60 nm and the tail at least 120 nm. Lysogeny was maintained during subculture on blood agar for more than 3 months after isolation from a gastric biopsy.

Inhibitory and bactericidal activity of cefpirome and cefotaxime against blood culture isolates.

Using a broth microtiter dilution method, minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) were determined for the aminothiazolyl cephalosporins cefpirome and cefotaxime against 436 blood culture isolates. At concentrations of less than or equal to 16 mg/l, cefpirome inhibited 82.3% of the isolates and cefotaxime 66.5%. At the same concentrations, cefpirome killed 60.0% of the isolates and cefotaxime 46.7%. Whereas the MIC values indicated a far better activity of cefpirome than cefotaxime against oxacillin-resistant Staphylococcus aureus, Enterococci and Pseudomonas aeruginosa, the MBC values showed a poor activity of both drugs against these strains. By comparison, cefpirome was the more active agent, covering a similar spectrum to that of cefotaxime with a slight additional activity against oxacillin-susceptible S. aureus and Staphylococcus epidermidis.