Local cooperation dominates over competition between CD4+ T cells of different antigen/MHC specificity

Interactions between CD4(+) T cells in vivo are controlled by a balance between cooperation and competition. In this study the interaction between two populations of CD4(+) T cells of different MHC/peptide specificity was probed at different precursor frequencies, delivering one or both Ags to APC using particle-mediated DNA delivery. Expansion of clonal populations of Ag (OVA and pigeon cytochrome c-specific) CD4(+) T cells was limited at higher precursor frequencies, presumably reflecting intraclonal competition. In contrast, a strong enhancement of the number of cells expressing IFN-gamma, IL-4, and IL-2 was observed in populations of cells at low precursor frequency in the presence of a high frequency of activated cells of a different Ag specificity. The helper effect was most potent when both Ags were delivered to the same dendritic cell (i.e., linked). This reflects the requirement of epicrine or paracrine help for optimal activation of T cell clones at low frequency. A measure of help was also delivered in an endocrine manner (unlinked), especially for Th1 responses, suggesting that there is also limited diffusion of cytokines between dendritic cell clusters. The dominant effects of cooperation over competition between CD4(+) T cells responding to different Ags may have important implications in terms of the efficacy of multivalent vaccines.     

Deficient CD4+ T cell priming and regression of CD8+ T cell functionality in virus-infected mice lacking a normal B cell compartment

In this study, we investigate the state of T cell-mediated immunity in B cell-deficient (B(-/-)) mice infected with two strains of lymphocytic choriomeningitis virus known to differ markedly in their capacity to persist. In B(-/-) C57BL mice infected with the more persisting virus, virus-specific CD8(+) T cells are initially generated that are qualitatively similar to those in wild-type mice. However, although cell numbers are well sustained over time, the capacity to produce cytokines is rapidly impaired. In similarly infected B(-/-) BALB/c mice, virus-specific CD8(+) T cells are completely deleted, indicating that host genotype influences the severity of the T cell defect. In B(-/-) C57BL mice infected with the less persisting virus, CD8(+) T cell dysfunction was not as pronounced, although it was clearly present. Most importantly, the appearance of dysfunctional CD8(+) T cells clearly precedes recrudescence of detectable virus, indicating that the T cell defect is not simply a secondary event due to virus buildup resulting from the failure of B(-/-) mice to produce neutralizing Abs. In contrast with CD8(+) T cells, which initially respond almost as in wild-type mice, the priming of virus-specific CD4(+) T cells was markedly impaired in B(-/-) mice infected with either virus strain. Thus, our results indicate that B cells play an important role in antiviral immunity not only as Ab producers, but also in promoting an optimal and sustained T cell response. The T cell defects are likely to contribute to the chronic course of viral infection in B(-/-) mice.     

Mapping of QTL for Body Conformation and Behavior in Cattle

Genome scans for quantitative trait loci (QTL) in farm animals have concentrated on primary production and health traits, and information on QTL for other important traits is rare. We performed a whole genome scan in a granddaughter design to detect QTL affecting body conformation and behavior in dairy cattle. The analysis included 16 paternal half-sib families of the Holstein breed with 872 sons and 264 genetic markers. The markers were distributed across all 29 autosomes and the pseudoautosomal region of the sex chromosomes with average intervals of 13.9 cM and covering an estimated 3155.5 cM. All families were analyzed jointly for 22 traits using multimarker regression and significance thresholds determined empirically by permutation. QTL that exceeded the experiment-wise significance threshold (5% level) were detected on chromosome 6 for foot angle, teat placement, and udder depth, and on chromosome 29 for temperament. QTL approaching experiment-wise significance (10% level) were located on chromosome 6 for general quality of feet and legs and general quality of udder, on chromosome 13 for teat length, on chromosome 23 for general quality of feet and legs, and on chromosome 29 for milking speed. An additional 51 QTL significant at the 5% chromosome-wise level were distributed over 21 chromosomes. This study provides the first evidence for QTL involved in behavior of dairy cattle and identifies QTL for udder conformation on chromosome 6 that could form the basis of recently reported QTL for clinical mastitis.     

Amino acid changes within conserved region III of the herpes simplex virus and human cytomegalovirus DNA polymerases confer resistance to 4-oxo-dihydroquinolines,a novel class of herpesvirus antiviral agents

The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.     

PCR-RFLP is used to investigate relations among species in the entomopathogenic genera Eryniopsis and Entomophaga

The shape and nucleation of primary conidia are important characters in the classification of the Entomophthoraceae (Zygomycetes). The five species in the genus Eryniopsis vary in the shapes of primary conidia, although within most genera in the order Entomophthorales species have the same shapes of primary conidia. Using PCR-RFLP, we investigated two species in Eryniopsis, Ery. caroliniana with oblong-ovoid primary conidia and Ery. ptychopterae with pear-shaped primary conidia, with five species of Entomophaga, all having pear-shaped conidia. Molecular results merged with morphological data indicate that Ery. ptychopterae belongs in the genus Entomophaga while Ery. caroliniana clearly differs from Entomophaga. Ery. ptychopterae and Ery. transitans are transferred to the genus Entomophaga. Our results support the idea that morphology of primary conidia is of major importance in defining entomophthoralean genera. These results also show that such studies can be conducted with species that have not been isolated, if fungal-filled cadavers can be obtained.     

Cardiovascular and neuroendocrine responses to left lateral position in non-obese young males

Previous results from our laboratory indicate that the heart is distended by the left lateral position (LAT) compared to horizontal supine (SUP). We therefore tested the hypothesis that cardiac output is increased by LAT and that mean arterial pressure is maintained unchanged or even decreased through peripheral vasodilatation induced by cardiopulmonary low-pressure receptor stimulation. Twelve non-obese young males were investigated. The location of the mid-aorta between the aortic valves was used as the hydrostatic reference point for the arterial pressure measurements. It was determined by magnetic resonance (n=6) to be 7.0 +/- 0.2 cm below the sternum in SUP (1/3 of anteroposterior chest diameter below the sternum) and 2.5 +/- 0.2 cm below the midsternal level in LAT. Brachial mean (auscultation) and finger mean arterial pressures (infrared photoplethysmography), cardiac output (foreign gas rebreathing), heart rate, and plasma concentrations (n=6) of vasoactive hormones were unchanged by LAT. In conclusion, cardiac output, mean arterial pressures, and vasoactive hormone releases were unaffected by 30 min of LAT. Furthermore, the hydrostatic reference points for arterial pressure measurements is located one third of the antero-posterior chest diameter below the sternum in SUP and 2.5 cm below the midsternal level in LAT in non-obese young males.     

Consensus and comprehensive linkage maps of bovine chromosome 24

This study describes development of a consensus genetic linkage map of bovine chromosome 24 (BTA24). Eight participating laboratories contributed data for 58 unique markers including a total of 25 409 meioses. Eighteen markers, which were typed in more than one reference population, were used as potential anchors to generate a consensus framework map. The framework map contained 16 loci ordered with odds greater than 1000:1 and spanned 79.3 cM. Remaining markers were included in a comprehensive map relative to these anchors. The resulting BTA24 comprehensive map was 98.3 cM in length. Average marker intervals were 6.1 and 2.5 cM for framework and comprehensive maps, respectively. Marker order was generally consistent with previously reported BTA24 linkage maps. Only one discrepancy was found when comparing the comprehensive map with the published USDA-MARC linkage map. Integration of genetic information from different maps provides a high-resolution BTA24 linkage map.     

Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.     

Chromosome location, genomic organization of the porcine COL10A1 gene and model structure of the NC1 domain

The porcine COL10A1 gene, encoding the alpha1(X) chain of type X collagen, has been sequenced. The gene structure is evolutionarily conserved, consisting of three exons and two introns spanning 7100 bp. Linkage mapping localized the gene to chromosome 1, which is in agreement with human-pig homology maps. Furthermore, protein structure comparison of the functionally important carboxyl domain between species revealed that amino acid changes were few and mainly situated in loop regions.