NADPH-oxidase-driven oxygen radical production determines chondrocyte death and partly regulates metalloproteinase-mediated cartilage matrix degradation during interferon-γ-stimulated immune complex arthritis

In previous studies we have found that FcγRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during IFN-γ-regulated immune complex arthritis (ICA). Binding of immune complexes (ICs) to FcγRI leads to the prominent production of oxygen radicals. In the present study we investigated the contribution of NADPH-oxidase-driven oxygen radicals to cartilage destruction by using p47phox^-/- mice lacking a functional NADPH oxidase complex. Induction of a passive ICA in the knee joints of p47phox^-/- mice resulted in a significant elevation of joint inflammation at day 3 when compared with wild-type (WT) controls as studied by histology. However, when IFN-γ was overexpressed by injection of adenoviral IFN-γ in the knee joint before ICA induction, a similar influx of inflammatory cells was found at days 3 and 7, comprising mainly macrophages in both mouse strains. Proteoglycan depletion from the cartilage layers of the knee joints in both groups was similar at days 3 and 7. Aggrecan breakdown in cartilage caused by MMPs was further studied by immunolocalisation of MMP-mediated neoepitopes (VDIPEN). VDIPEN expression in the cartilage layers of arthritic knee joints was markedly lower (between 30 and 60%) in IFN-γ-stimulated arthritic p47phox^-/- mice at day 7 than in WT controls, despite significant upregulation of mRNA levels of various MMPs such as MMP-3, MMP-9, MMP-12 and MMP-13 in synovia and MMP-13 in cartilage layers as measured with quantitative RT-PCR. The latter observation suggests that oxygen radicals are involved in the activation of latent MMPs. Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, ranged between 20 and 60% at day 3 and between 30 and 80% at day 7 in WT mice, and was completely blocked in p47phox^-/- mice at both time points. FcγRI mRNA expression was significantly lower, and FcγRII and FcγRIII were higher, in p47phox^-/- mice than in controls. NADPH-oxidase-driven oxygen radical production determines chondrocyte death and aggravates MMP-mediated cartilage destruction during IFN-γ-stimulated IC-mediated arthritis. Upregulation of FcγRI by oxygen radicals may contribute to cartilage destruction.     

Cellular responses to <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> erythrocyte membrane protein-1: use of relatively conserved synthetic peptide pools to determine CD4 T cell responses in malaria-exposed individuals in Benin,West Africa

Background

Plasmodium falciparum erythrocyte membrane protein-1, a variant antigen of the malaria parasite, is potentially a target for the immune response. It would be important to determine whether there are CD4 T cells that recognise conserved regions. However, within the relatively conserved region, there is variation. It is not possible to test T cell responses from small field samples with all possible peptides.

Methods

We have aligned sequences that are relatively conserved between several PfEMP1 molecules, and chosen a representative sequence similar to most of the PfEMP1 variants. Using these peptides as pools representing CIDRα, CIDRβ and DBLβ-δ domains, DBLα domain, and EXON 2 domain of PfEMP1, we measured the CD4 T cell responses of malaria-exposed donors from Benin, West Africa by a FACS based assay.

Results

All the three peptide pools elicited a CD4 T cell response in a proportion of malaria-exposed and non-exposed donors. CD4 T cell proliferation occurs at a relatively higher magnitude to peptide pools from the DBLα and EXON 2 in the malaria-exposed donors living in Benin than in the UK malaria-unexposed donors.

Conclusions

These findings suggest that an immunological recall response to conserved peptides of a variant antigen can be measured. Further testing of individual peptides in a positive pool will allow us to determine those conserved sequences recognised by many individuals. These types of assays may provide information on conserved peptides of PfEMP1 which could be useful for stimulating T cells to provide help to P. falciparum specific B cells.

     

Algorithms for the search of amino acid patterns in nucleic acid sequences.

Some algorithms are described for the search of regions in a nucleic acid sequence that, when translated into amino acids, are homologous to a given amino acid pattern. All algorithms are modifications of the dynamic programming method for sequence comparison such that the translation of codons is taken into account. One of the algorithms has been implemented as a FORTRAN 77 program. The program operates on files that follow the format of the EMBL Nucleotide Sequence Data Library.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

The Mitochondrial Toxin Produced by Streptomyces griseus Strains Isolated from an Indoor Environment Is Valinomycin

Actinomycete isolates from indoor air and dust in water-damaged schools and children’s day care centers were tested for toxicity by using boar spermatozoa as an indicator. Toxicity was detected in extracts of four strains which caused a loss of sperm motility, and the 50% effective concentrations (EC₅₀) were 10 to 63 ng (dry weight) ml of extended boar semen⁻¹. The four strains were identified as Streptomyces griseus strains by 16S ribosomal DNA and chemotaxonomic methods. The four S. griseus strains had similar effects on sperm cells, including loss of motility and swelling of mitochondria, but we observed no loss of plasma membrane integrity or depletion of cellular ATP. None of the effects was observed with sperm cells exposed to extracts of other indoor actinomycete isolates at concentrations of ≥5,000 to 72,000 ng ml⁻¹. The toxin was purified from all four strains and was identified as a dodecadepsipeptide, and the fragmentation pattern obtained by tandem mass spectrometry was identical to that of valinomycin. Commercial valinomycin had effects in sperm cells that were identical to the effects of the four indoor isolates of S. griseus. The EC₅₀ of purified toxin from the S. griseus strains were 1 to 3 ng ml of extended boar semen⁻¹, and the EC₅₀ of commercial valinomycin was 2 ng ml of extended boar semen⁻¹. To our knowledge, this is the first report of the presence of ionophoric toxin producers in an indoor environment and the first report of valinomycin-producing strains identified as S. griseus.     

Effect of copper on membrane lipids and on methane monooxygenase activity of Methylococcus capsulatus (Bath)

The effect of copper supplementation on growth, methane monooxygenase activity and lipid composition of Methylococcus capsulatus (Bath) was studied. Copper increased biomass yield, methane monooxygenase activity and phospholipid content from 7.7 to 10.2% of dry weight. Cells from copper-deficient and copper supplemented cultures contained the same major fatty acids but in the presence of copper only the contents of C16:0 and the three C16:1 isomers were increased while the contents of C14:0 and cyclic C17:0 remained unchanged. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol and cardiolipin were analysed amongst the polar lipids. PE was the main component (about 60 mol-%) but the most notable copper-induced increment occurred in the proportion of PC, from about 10 to 16 mol-%. Concomitantly with this increment the fatty acids of PC were changed so that the mol-% of C16: 1 isomers were increased at the expense of other acids. Similar trends were seen also in the fatty acid compositions of other polar lipid fractions. It is therefore concluded that phosphatidylcholine would be one of the key factors when the role of membranous lipids in methane monooxygenase activity is to be considered.     

Seasonal occurrence and ecological aspects of Afrodiplozoon polycotyleus (Monogenea: Diplozoidae) from the gills of three cyprinids from the Nwanedi-Luphephe dams,South Africa

The effect of seasonal changes and ecological aspects of Afrodiplozoon polycotyleus (Paperna, 1973) collected on Labeobarbus marequensis (Smith, 1841), Enteromius trimaculatus (Peters, 1852) and Enteromius radiatus (Peters, 1853) was investigated from January to October 2008. Fish were collected at the Nwanedi-Luphephe dams, Limpopo River System, South Africa using gill, cast and seine nets and electrofishing gear. Enteromius radiatus and E. trimaculatus were the most infested compared with L. marequensis. Seasonal changes had an influence on the intensity of A. polycotyleus, with infestation rates higher in spring, summer and autumn. The adults predominantly infected the medial region of the second gill arch, whereas diporpae were mainly found on the first gill. Neither sex nor water quality changes had an influence on the intensity of the parasite. The health condition of the hosts was not affected by the parasite. The different infestation rates of the parasite between the species could be attributed to host characteristics, behaviour and habitat preferences. The selection of a particular gill/region could be attributed to space for attachment organs and food supply, as well as flow and velocity of the water over the gills. The occurrence of A. polycotyleus on E. radiatus constitutes a new host record.     

Fluidized-bed denitrification for mine waters. Part I: low pH and temperature operation

Mining often leads to nitrate and metal contamination of groundwater and water bodies. Denitrification of acidic water was investigated in two up-flow fluidized-bed reactors (FBR) and using batch assays. Bacterial communities were enriched on ethanol plus nitrate in the FBRs. Initially, the effects of temperature, low-pH and ethanol/nitrate on denitrification were revealed. Batch assays showed that pH 4.8 was inhibitory to denitrification, whereas FBR characteristics permitted denitrification even at feed pH of 2.5 and at 7–8 °C. Nitrate and ethanol were removed and the feed pH was neutralized, provided that ethanol was supplied in excess to nitrate. Subsequently, Fe(II) and Cu impact on denitrification was investigated within batch tests at pH 7. Iron supplementation up to 100 mg/L resulted in iron oxidation and soluble concentrations ranging from 0.4 to 1.6 mg/L that stimulated denitrification. On the contrary, 0.7 mg/L of soluble Cu significantly slowed denitrification down resulting in about 45 % of inhibition in the first 8 h. Polymerase chain reaction—denaturant gradient gel electrophoresis demonstrated the co-existence of different denitrifying microbial consortia in FBRs. Dechloromonas denitrificans and Hydrogenophaga caeni were present in both FBRs and mainly responsible for nitrate reduction.