Genetic analysis of influences on survival following Toxoplasma gondii infection.

Survival of mice during the acute stage of Toxoplasma gondii infection was not influenced by the MHC Class I gene, L(d), but was influenced by the MHC Class II genes, Ia and Ie. As unexplained variability was noted in our initial studies of influence of the L(d) gene on survival, influence of the L(d) gene region on survival in the presence of a number of variables was studied. Although route of administration and dose of parasites, and age and gender of the mice markedly influenced outcome of T. gondii infection, the Class I L(d) gene did not modify survival in any of these circumstances. In separate studies, using mice with a differing genetic background, i.e. H-2(b), C57BL/10 mice, presence of Ia or Ie alone diminished survival even though presence of Ia reduced parasite burden. When neither or both the Ia and Ie genes were present together, survival was greater. In separate analyses of our studies of AxB BxA recombinant inbred mice, similar influences of MHC genes on survival and parasite burden following peroral infection were confirmed. Previously undescribed associations of novel genetic loci and survival and parasite burden also were identified. Genetic loci associated with enhanced survival included D8Mit42, D1Mit3, Iapls1-16, D8Mit14, Hoxb, Mpmv29, Pmv45, and Emv-2; genetic loci associated with reduced parasite burden included H-2, D17Mit62, D17Mit83, D17Mit21, D17Mit34, D17Mit47, D18Mit4, and Gln3-5. These studies demonstrate the importance of MHC region genes (but not L(d)) for survival, and the influence of other novel genes, and endogenous and exogenous variables on survival and parasite burden specified by host genes following T. gondii infection.     

Soil microbial communities and elk foraging intensity: implications for soil biogeochemical cycling in the sagebrush steppe

Foraging intensity of large herbivores may exert an indirect top‐down ecological force on soil microbial communities via changes in plant litter inputs. We investigated the responses of the soil microbial community to elk (Cervus elaphus) winter range occupancy across a long‐term foraging exclusion experiment in the sagebrush steppe of the North American Rocky Mountains, combining phylogenetic analysis of fungi and bacteria with shotgun metagenomics and extracellular enzyme assays. Winter foraging intensity was associated with reduced bacterial richness and increasingly distinct bacterial communities. Although fungal communities did not respond linearly to foraging intensity, a greater β‐diversity response to winter foraging exclusion was observed. Furthermore, winter foraging exclusion increased soil cellulolytic and hemicellulolytic enzyme potential and higher foraging intensity reduced chitinolytic gene abundance. Thus, future changes in winter range occupancy may shape biogeochemical processes via shifts in microbial communities and subsequent changes to their physiological capacities to cycle soil C and N.     

In vitro characterization of four novel non-functional variants of the thiopurine S-methyltransferase

Human thiopurine S-methyltransferase (TPMT) is an enzyme responsible for the detoxification of widely used thiopurine drugs such as azathioprine (Aza). Its activity is inversely related to the risk of developing severe hematopoietic toxicity in certain patients treated with standard doses of thiopurines. DNA samples from four leucopenic patients treated with Aza were screened by PCR-SSCP analysis for mutations in the 10 exons of the TPMT gene. Four missense mutations comprising two novel mutations, A83T (TPMT*13, Glu(28)Val) and C374T (TPMT*12, Ser(125)Leu), and two previously described mutations, G430C (TPMT*10, Gly(144)Arg) and T681G (TPMT*7, His(227)Gln) were identified. Using a recombinant yeast expression system, kinetic parameters (K(m) and V(max)) of 6-thioguanine S-methylation of the four TPMT variants were determined and compared to those obtained with wild-type TPMT. This functional analysis suggests that these rare allelic variants are defective TPMT alleles. The His(227)Gln variant retained only 10% of the intrinsic clearance value (V(max)/K(m) ratio) of the wild-type enzyme. The Ser(125)Leu and Gly(144)Arg variants were associated with a significant decrease in intrinsic clearance values, retaining about 30% of the wild-type enzyme, whereas the Glu(28)Val variant produced a more modest decrease (57% of the wild-type enzyme). The data suggest that the sporadic contribution of the rare Glu(28)Val, Ser(125)Leu, Gly(144)Arg, and His(227)Gln variants may account for the occurrence of altered metabolism of TPMT substrates. These findings improve our knowledge of the genetic basis of interindividual variability in TPMT activity and would enhance the efficiency of genotyping methods to predict patients at risk of inadequate responses to thiopurine therapy.     

In vitro correlates of Ld-restricted resistance to toxoplasmic encephalitis and their critical dependence on parasite strain

Resistance to murine toxoplasmic encephalitis has been precisely and definitively mapped to the L(d) class I gene. Consistent with this, CD8(+) T cells can adoptively transfer resistance to toxoplasmic encephalitis. However, cytotoxic CD8(+) T cells, capable of killing class I-matched, infected target cells, are generated during the course of Toxoplasma gondii infection even in mice lacking the L(d) gene. L(d)-restricted killing could not be demonstrated, and the functional correlate of the L(d) gene has therefore remained elusive. Herein, L(d)-restricted killing of T. gondii-infected target cells is demonstrated for the first time. L(d)-restricted killing is critically dependent on the strain of T. gondii and is observed with all the derivatives of type II strains tested, but not with a type I strain. These results have important implications for vaccine development.     

Toxoplasma gondii tachyzoite-bradyzoite interconversion

During infection in the intermediate host, Toxoplasma gondii undergoes stage conversion between the rapidly dividing tachyzoite that is responsible for acute toxoplasmosis and the slowly replicating, encysted bradyzoite stage. This process of tachyzoite-bradyzoite interconversion is central to the pathogenesis and longevity of infection. Recent research has identified several stage-specific genes and proteins. However, despite recent advances in the understanding of Toxoplasma cell biology, more research is necessary to elucidate the complex events occurring during tachyzoite-bradyzoite interconversion. Here, a brief summary of this process is provided and a new method to characterize gene expression during interconversion is introduced.     

A tracer study with systemically and locally administered dinitrophenylated osteopontin.

Osteopontin (OPN), a major non-collagenous matrix protein of bone, is also found in tissue fluids and in the circulation. It is still not clear whether circulating OPN contributes to bone formation. To elucidate this question, rat OPN was tagged with dinitrophenol groups and administered to rats either intravenously or by infusion with an osmotic minipump through a "surgical window" in the bone of the hemimandible. Dinitrophenylated rat albumin (ALB) was used as a control. The presence and distribution of tagged proteins were revealed by immunogold labeling on sections of tibia and alveolar bone. Tagged molecules of OPN were found in mineralization foci, surfaces and interfaces, and matrix accumulations among calcified collagen fibrils. Even though dinitrophenylated ALB was administered at several-fold higher concentrations, it did not accumulate in these sites. These results show that circulating OPN can be incorporated into specific compartments of forming bone and suggest that such molecules may play a more important role than previously suspected.