The arthritis severity quantitative trait locus Cia7 regulates neutrophil migration into inflammatory sites.

Neutrophils are required for the development of arthritis in rodents, and are the predominant cell in the synovial fluid of active rheumatoid arthritis. We hypothesized that neutrophil migration into the inflammed joint is genetically regulated. In addition, this genetic regulation would be accounted for by one of the arthritis loci that we have previously identified in an intercross between arthritis-susceptible DA and arthritis-resistant ACI rats studied for collagen-induced arthritis. We used the synovial-like air pouch model injected with carrageenan, and tested DA, ACI, and four congenic strains. ACI exudates had a significantly lower number of neutrophils compared with DA. Transfer of DA alleles at Cia7 into the ACI background, as in ACI.DA(Cia7) congenics, was enough to increase exudate neutrophil numbers to levels identical to DA, and this locus accounted for the difference between parental strains. None of the other congenic intervals explained the differences in exudate neutrophil counts. In conclusion, we have identified a novel function for Cia7, and determined that it regulates neutrophil migration into a synovial-like inflammatory site. Our data revealed no intrinsic defect in neutrophil responses to chemotactic agents, and suggest that Cia7 regulates an as yet unidentified factor central to neutrophil recruitment into inflammed tissues.

Cia27 is a novel non-MHC arthritis severity locus on rat chromosome 10 syntenic to the rheumatoid arthritis 17q22-q25 locus.

Cia27 on rat chromosome 10 is a collagen-induced arthritis (CIA) severity quantitative trait locus originally identified in a study of (DA x ACI) F2. As an initial step towards the positional cloning of the Cia27 gene, a 17 cM (21 Mb) interval from the DA strain (arthritis-susceptible) containing the two-logarithm of odds support interval comprising Cia27 was introgressed into the ACI (arthritis-resistant) background through genotype-guided congenic breeding. ACI.DA(Cia27) congenics developed a significantly more severe form of arthritis (CIA), with a 5.9-fold increase in median arthritis severity index, a parameter known to correlate with synovial inflammation, and cartilage and bone erosions, compared with ACI (P< or =0.001). The arthritis severity enhancing effect could be detected from day 21 onwards. Rats heterozygous at the congenic interval developed a disease similar to ACI rats, suggesting that DA alleles operate in a recessive manner. Levels of autoantibodies anti-rat type II collagen did not correlate with arthritis severity. Synovial tissue mRNA levels of interleukin-1beta (IL-1beta) were significantly increased in ACI.DA(Cia27) congenics compared with ACI. These results demonstrate that Cia27 harbors a novel arthritis severity regulatory gene. The identification of this gene should facilitate the identification of the rheumatoid arthritis gene mapped to the human syntenic region on chromosome 17q22-q25.

Nitroimidazole and oxygen derived radicals detected by electron spin resonance in hydrogenosomal and cytosolic fractions from Trichomonas vaginalis.

Hydrogenosome-enriched fractions from Trichomonas vaginalis reduce a number of nitroimidazole derivatives to their respective electron spin resonance-detectable nitro-anion radicals. In the presence of of oxygen and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) a superoxide spin trapped adduct of DMPO was formed; the rate-determining step was the prior formation of the nitro-anion radical. Oxygen-derived radicals were detected with cytosolic fractions from a metronidazole-resistant isolate (CDC-85) when incubated with NADH or NADPH as respiratory substrate. The requirement for superoxide dismutase and catalase to completely abolish formation of these signals suggests contributions from both superoxide and peroxide. No oxygen-derived radicals were observed with cytosolic fractions from a metronidazole-susceptible strain (C1-NIH).

Activities of metronidazole and niridazole against Trichomonas vaginalis clinical isolates.

The activities of niridazole and metronidazole against Trichomonas vaginalis C1-NIH and six isolates of clinical origin were compared using the in-vitro assay technique. Under the standard anaerobic assay conditions both metronidazole and niridazole were highly effective at low concentrations against all the strains used. However niridazole, in contrast to metronidazole, was equally effective in the aerobic assay, a feature which may be exploited in the chemotherapy of patients with refractory trichomoniasis.

Reduction of niridazole by metronidazole resistant and susceptible strains of Trichomonas vaginalis.

The inhibitory effect of niridazole on hydrogen production by metronidazole-resistant (CDC-85) and susceptible (C1-NIH) Trichomonas vaginalis strains was investigated. The results show that niridazole is more effective than metronidazole in inhibiting hydrogen production by the resistant isolate. In CDC-85 aerobic inhibition requires a 4-fold increase in metronidazole concentration compared with that required anaerobically, but the corresponding factor for niridazole is only 1.5-fold. Reduction of the drug by a hydrogenosome-enriched preparation gave rise to a multiline electron spin resonance detectable signal, which is due to a nitrogen-centred radical.

Hydrogenosomes in the rumen fungus Neocallimastix patriciarum.

Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed 'hydrogenosomes'. Other typical hydrogenosomal enzymes, namely 'malic' enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.

Ferredoxin-dependent reduction of nitroimidazole derivatives in drug-resistant and susceptible strains of Trichomonas vaginalis.

The inhibitory effect of a range of nitroimidazole-derivatives on H2 production by metronidazole resistant (CDC-85) and susceptible (C1-NIH) Trichomonas vaginalis strains was investigated. The 2-, 4-, and 5-nitro-derivatives used had one-electron reduction potentials within the range -250 to -525 mV. Nitroimidazole concentrations giving 50% inhibition of H2 production (kiH2) for compounds with one-electron reduction potentials in the range -250 to -425 mV were found to be similar for both strains tested. Compounds with one-electron reduction potentials below -425 mV give 10-fold higher KiH2 values for the metronidazole resistant isolate. Both strains showed increased KiH2 for compounds with potentials lower than -500 mV. The addition of 2.1 kPa (0.02 atm) O2 to the gas phase resulted in increasing the kiH2 values for all the compounds tested, but had the greater effect on results obtained with the resistant isolate using nitroimidazoles in the range -425 to -490 mV. The results enable the proposal that the resistant isolate CDC-85 has a ferredoxin with altered redox properties or reduced intracellular levels.

Metronidazole-resistant clinical isolates of Trichomonas vaginalis have lowered oxygen affinities.

Oxygen affinities of metronidazole susceptible and resistant isolates of the parasitic flagellate protozoon Trichomonas vaginalis were determined by mass spectrometric methods. Apparent O2Km values for the respiration of non-proliferating cell suspensions were about 10-fold higher for metronidazole resistant strains than for the susceptible strains C1-NIH or NYH-286. Simultaneous monitoring of hydrogen evolution in the presence of increasing O2 tensions enabled apparent Ki values for H2 to be determined; and this function was independent of metronidazole susceptibility. Apparent O2 affinities of the hydrogenosomal and non-sedimentable fractions were determined for the strains CDC 85 (metronidazole resistant) and C1-NIH, which showed the deficiency in the O2 scavenging capacity by the resistant strain to be associated with the hydrogenosome-containing fraction.