A polarized Th1 type immune response to Cowdria ruminantium infection is detected in immune DBA/2 mice

Immune responses to Cowdria ruminantium, an intracellular organism that causes heartwater in domestic ruminants, were characterized in a DBA/2 mouse model. Immunity induced by infection and treatment was adoptively transferable by splenocytes and could be abrogated by in vivo depletion of T cells but not by inhibition of nitric oxide synthase using NG-monomethyl-L-arginine. IgG2a and IgG2b C. ruminantium-specific responses were detected in immune mice. Culture supernatants of splenocytes from immune DBA/2 mice, which were stimulated with crude C. ruminantium antigens or recombinant major antigenic proteins 1 or 2, contained significant levels of interferon (IFN)-gamma and interleukin (IL)-6, but insignificant levels of IL-1alpha, IL-2, IL-4, IL-5, IL-10, IL-12, tumor necrosis factor-alpha (TNF), and nitric oxide. A similar response was detected during primary infection, although IFN-gamma levels decreased significantly during clinical illness and then increased following natural or antibiotic-aided recovery. These data support the conclusion that protective immunity to C. ruminantium in DBA/2 mice is mediated by T cells and is associated with a polarized T helper 1 type of immune response. This murine model could be utilized to screen for protective C. ruminantium antigens that provoke Th1 type immune responses and for evaluation of these antigens in recombinant vaccines against heartwater.     

Vaccine strategies for Cowdria ruminantium infections and their application to other ehrlichial infections.

Suman Mahan and co-authors review the strategies applied to develop improved vaccines for Cowdria ruminantium infections (heartwater). Inactivated vaccines using cell-cultured C. ruminantium organisms combined with an adjuvant are capable of protecting goats, sheep and cattle against lethal C. ruminantium challenge. Immune responses induced with this vaccine, or after recovery from infection, target outer membrane proteins of C. ruminantium, in particular the major antigenic protein 1 (MAP-1). Genetic immunizations with the gene encoding MAP-1 induce protective T helper cell type 1 responses against lethal challenge in a mouse model. Similarly, homologues of MAP-1 in other phylogenetically and antigenically related ehrlichial agents such as Anaplasma marginale and Ehrlichia chaffeensis are also targets of protective responses. Given the antigenic similarities between the related ehrlichial agents, common strategies of vaccine development could be applied against these agents that cause infections of importance in animals and humans.     

The map1 gene of Cowdria ruminantium is a member of a multigene family containing both conserved and variable genes

Heartwater is an economically important disease of ruminants caused by the tick-transmitted rickettsia Cowdria ruminantium. The disease is present in Africa and the Caribbean and there is a risk of spread to the Americas, particularly because of a clinically asymptomatic carrier state in infected livestock and imported wild animals. The causative agent is closely related taxonomically to the human and animal pathogens Ehrlichia chaffeensis and Ehrlichia canis. A dominant immune response of infected animals or people is directed against variable outer membrane proteins of these agents known, in E. chaffeensis and E. canis, to be encoded by polymorphic multigene families. We demonstrate, by sequence analysis, that map1 encoding the major outer membrane protein of C. ruminantium is also encoded by a polymorphic multigene family. Two members of the gene family are located in tandem in the genome. The upstream member, orf2, is conserved, encoding only 2 amino acid substitutions among six different rickettsial strains from diverse locations in Africa and the Caribbean. In contrast, the downstream member, map1, contains variable and conserved regions between strains. Interestingly, orf2 is more closely related in sequence to omp1b of E. chaffeensis than to map1 of C. ruminantium. The regions that differ among orf2, map1, and omp1b correspond to previously identified variable sequences in outer membrane protein genes of E. chaffeensis and E. canis. These data suggest that diversity in these outer membrane proteins may arise by recombination among gene family members and offer a potential mechanism for persistence of infection in carrier animals.     

Susceptibility and carrier status of impala, sable, and tsessebe for Cowdria ruminantium infection (heartwater).

Three species of wild African ruminants, impala (Aepyceros melampus), sable (Hippotragus equinus), and tsessebe (Damaliscus lunatus), were experimentally inoculated with in vitro culture-derived Cowdria ruminantium organisms, the tick-borne causative agent of heartwater in domestic ruminants, to determine their susceptibility to infection. No clinical disease was observed in any of the ruminants. However, C. ruminantium was detected in the sable by the transmission of heartwater to susceptible sheep, through the tick vector Amblyomma hebraeum, at 10 and 37 days postinfection (PI). Attempts to detect infection in the impala and tsessebe by tick transmission at 54 days PI failed. The impala and tsessebe were reinoculated with C. ruminantium organisms at 146 days after the first inoculation; however, a tick transmission attempt at 66 days after the reinoculation also failed. Seroconversion, as detected by immunoblotting, was demonstrated in the sable and the tsessebe but not in the impala. The results demonstrate that sable can be carriers of C. ruminantium. The susceptibility of tsessebe and impala, however, remains undetermined.     

Phylogenetic position of Cowdria ruminantium (Rickettsiales) determined by analysis of amplified 16S ribosomal DNA sequences.

The 16S ribosomal DNA sequence of Cowdria ruminantium, the causative agent of heartwater disease in ruminants, was determined. An analysis of this sequence showed that C. ruminantium forms a tight phylogenetic cluster with the canine pathogen Ehrlichia canis and the human pathogen Ehrlichia chaffeensis. Although a close relationship between the genus Cowdria and several members of the tribe Ehrlichieae has been suspected previously, the tight phylogenetic cluster with E. canis and E. chaffeensis is surprising in view of known differences in host preference and target cells.     

Evidence of Cowdria ruminantium infection (heartwater) in Amblyomma sparsum ticks found on tortoises imported into Florida

Amblyomma marmoreum and A. sparsum ticks were collected from tortoises imported into Florida from Africa and were tested for Cowdria ruminantium infection using a C. ruminantium-specific pCS20 polymerase chain reaction assay. In I shipment imported from Zambia, 15 of the 38 A. sparsum male ticks collected from the leopard tortoises (Geochelone pardalis) were found to be positive for infection with C. ruminantium. In contrast, all 148 A. marmoreum tested were negative for C. ruminantium infection. This is the first reported evidence of the introduction of heartwater-infected ticks into the United States, but there were no opportunities to confirm isolation of C. ruminantium from the ticks by either culture or transmission studies.     

Correlation between antibodies to Cowdria ruminantium (Rickettsiales) in cattle and the distribution of Amblyomma vector ticks in Zimbabwe

Cowdriosis, caused by Cowdria ruminantium, is transmitted by Amblyomma ticks, which are widely distributed in Zimbabwe. To assess the distribution of this disease in Zimbabwe, cattle either exposed to Amblyomma ticks or maintained in areas free from these ticks were tested for antibodies to Cowdria. A total of 324 sera were tested using competitive ELISA and the indirect fluorescent antibody test (IFAT). At diptanks in Amblyomma-infested areas 52% (n=95) and 26% (n=47) of sera were positive by cELISA and IFAT, respectively. At diptanks in Amblyomma-free areas 11% (n=125) and 10% (n=134) of sera were positive by cELISA and IFAT, respectively. The results were significantly different between Amblyomma-infested and tick-free areas (²=24.73, P0.005 for IFAT and ²=57.53, P0.005 for cELISA). High background readings in field sera, possibly due to cross-reactive antibodies to Ehrlichia spp., complicated the determination of a realistic cut-off point, especially in cELISA. On the basis of the distribution of Amblyomma ticks, currently a large part of Zimbabwe can be considered endemic for the disease.     

Identification of sequences important for recognition of vnf genes by the VnfA transcriptional activator in Azotobacter vinelandii

Abstract To analyze regulation of the vanadium-dependent nitrogenase of Azotobacter vinelandii , plasmids carrying vnfE-, vnfH- , or vnfD-lacZ fusions were transferred to Escherichia coli . These genes were expressed only if VnfA was present. Deletions of the vnfE upstream region were constructed and comparison of a region necessary for expression with sequences upstream of other vnf genes indicated a substantially conserved motif, GTAC-N6-GTAC, hypothesized to be the binding site for VnfA. This motif was duplicated with 17 or 18 bases lying between each in the vnfH and vnfD promoters. Deletion analysis of the vnfH promoter indicated that both motifs were necessary for full expression. In footprinting experiments, VnfA significantly protected from methylation the guanine residues within or immediately adjacent to the proposed VnfA recognition motifs. The active form of VnfA is probably interacting dimers, a tetramer, or a higher order oligomer since two regions of dyad symmetry are required for its interaction with the DNA.     

Testing gene set enrichment for subset of genes: Sub-GSE

Background  

Many methods have been developed to test the enrichment of genes related to certain phenotypes or cell states in gene sets. These approaches usually combine gene expression data with functionally related gene sets as defined in databases such as GeneOntology (GO), KEGG, or BioCarta. The results based on gene set analysis are generally more biologically interpretable, accurate and robust than the results based on individual gene analysis. However, while most available methods for gene set enrichment analysis test the enrichment of the entire gene set, it is more likely that only a subset of the genes in the gene set may be related to the phenotypes of interest.     

Comparison of efficacy of American and African Amblyomma ticks as vectors of heartwater (Cowdria ruminantium) infection by molecular analyses and transmission trials

The ability of Amblyomma americanum, Amblyomma cajennense, Amblyomma maculatum, and Amblyomma variegatum to acquire and transmit Cowdria ruminantium infection was investigated. Uninfected nymphs were fed on clinically reacting C. ruminantium-infected sheep and then analyzed for infection by specific DNA detection assays and by tick transmission trials. By polymerase chain reaction (PCR), the mean infection prevalence of A. maculatum ticks (50.7%) was similar to that of A. variegatum, Elevage strain (43.5%; P = 0.83) and Petit Bourg strain (45.9%; P = 0.26) ticks. Though Amblyomma hebraeum were not tested by PCR, by DNA probe their infection prevalence was 94%. In contrast, A. americanum and A. cajennense ticks demonstrated very low susceptibility to C. ruminantium, and the prevalence of infection by PCR was approximately 1%. The higher susceptibility of A. maculatum and A. variegatum to C. ruminantium correlated with superior vector efficiency, depicted by similar prepatent periods and severity of disease transmissions to sheep. Amblyomma americanum and A. cajennense failed to transmit infection, confirming that low susceptibility to C. ruminantium correlates with the poor vector status of these species. These results highlight the importance of A. maculatum as a potential vector that is likely to play a major role in the establishment and maintenance of heartwater, if the disease were to be introduced to the U.S.A., Central, and South America.     

Structural basis for preferential recognition of diaminopimelic acid-type peptidoglycan by a subset of peptidoglycan recognition proteins

Drosophila peptidoglycan recognition protein (PGRP)-LCx and -LCa are receptors that preferentially recognize meso-diaminopimelic acid (DAP)-type peptidoglycan (PGN) present in Gram-negative bacteria over lysine-type PGN of gram-positive bacteria and initiate the IMD signaling pathway, whereas PGRP-LE plays a synergistic role in this process of innate immune defense. How these receptors can distinguish the two types of PGN remains unclear. Here the structure of the PGRP domain of Drosophila PGRP-LE in complex with tracheal cytotoxin (TCT), the monomeric DAP-type PGN, reveals a buried ionic interaction between the unique carboxyl group of DAP and a previously unrecognized arginine residue. This arginine is conserved in the known DAP-type PGN-interacting PGRPs and contributes significantly to the affinity of the protein for the ligand. Unexpectedly, TCT induces infinite head-to-tail dimerization of PGRP-LE, in which the disaccharide moiety, but not the peptide stem, of TCT is positioned at the dimer interface. A sequence comparison suggests that TCT induces heterodimerization of the ectodomains of PGRP-LCx and -LCa in a closely analogous manner to prime the IMD signaling pathway, except that the heterodimer formation is nonperpetuating.     

A novel device for islet transplantation providing immune protection and oxygen supply

Islet transplantation as a biological β-cell replacement therapy has emerged as a promising option for achieving restoration of metabolic control in type 1 diabetes patients. However, partial or complete loss of islet graft function occurs in relatively short time (months to few years) after implantation. The high rate of early transplant dysfunction has been attributed to poorly viable and/or functional islets and is mediated by innate inflammatory response at the intravascular (hepatic) transplant site and critical lack of initial nutrient/oxygen supply prior to islet engraftment. In addition, the diabetogenic effect of mandatory immunosuppressive agents, limited control of alloimmunity, and the recurrence of autoimmunity limit the long-term success of islet transplantation. In order to abrogate instant blood-mediated inflammatory reaction and to provide oxygen supply for the islet graft, we have developed an extravascular (subcutaneous) transplant macrochamber (the 'βAir' device). This device contains islets immobilized in alginate, protected from the immune system by a thin hydrophilized teflon membrane impregnated with alginate and supplied with oxygen by daily refueling with oxygen-CO (2) mixture. We have demonstrated successful utilization of the oxygen-refueling macrochamber for sustained islet viability and function as well as immunoprotection after allogeneic subcutaneous transplantation in healthy minipigs. Considering the current limitations of intraportal islet engraftment and the restricted indication for islet transplantation mainly due to necessary immunosuppressive therapy, this work could very likely lead to remarkable improvements in the procedure and moreover opens up further strategies for porcine islet cell xenotransplantation.     

CHX10 targets a subset of photoreceptor genes

The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10 we sought to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, we observed interactions between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 bound sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. Our data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells.     

A model for simulating cognate recognition and response in the immune system.

We have constructed a model of the immune system that focuses on the clonotypic cell types and their interactions with other cells, and with antigens and antibodies. We carry out simulations of the humoral immune system based on a generalized cellular automaton implementation of the model. We propose using computer simulation as a tool for doing experiments in machine, in the computer, as an adjunct to the usual in vivo and in vitro techniques. These experiments would not be intended to replace the usual biological experiments since, in the foreseeable future, a complete enough computer model capable of reliably simulating the whole immune would not be possible. However a model simulating areas of interest could be used for extensively testing ideas to help in the design of the critical biological experiments. Our present model concentrates on the cellular interactions and is quite adept at testing the importance and effects of cellular interactions with other cells, antigens and antibodies. The implementation is quite general and unrestricted allowing most other immune system components to be added with relative ease when desired.