Effects of Manduca allatotropin and localization of Manduca allatotropin-immunoreactive cells in earwigs

Manduca sexta allatotropin (Manse-AT) was first isolated on the basis of its ability to stimulate production of juvenile hormone in that insect. We examined whether this neuropeptide affects corpus allatum activity and visceral muscle contraction in adult females of the earwig, Euborellia annulipes. We also assessed the presence of allatotropin-like material in tissues using immunocytochemistry. Manse-AT at 1 nM to 10 muM stimulated juvenile hormone production in vitro by glands of low activity from 2-day virgin females. In glands of high activity from 12-day mated females, 1 and 100 nM allatotropin were effective, but 10 muM was not. Similarly, hindguts of 2-day and 12-day females significantly increased in motility in vitro in response to Manse-AT. A monoclonal antibody to Manse-AT was used to demonstrate allatotropin-like material throughout the nervous system of 2-day, virgin females. Immunoreactivity was most pronounced within varicosities of the corpora cardiaca and perisympathetic organs. No immunofluorescence was observed in gut tissue. Lastly, we showed that extract of retrocerebral complexes also enhanced in vitro hindgut motility from 2-day virgin females, in a dose-dependent manner. These results indicate material similar to M. sexta allatotropin in female earwigs and that such peptides may modulate juvenile hormone biosynthesis and visceral muscle contractions. Sensitivity to the peptides may change with physiological stage.     

A comparison of several serologic tests to detect antibodies to Toxoplasma gondii in naturally exposed bottlenose dolphins (Tursiops truncatus)

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. In a previous study, 138 of 141 (97.8%) bottlenose dolphins (Tursiops truncatus) from the coasts of Florida and California had antibodies to T. gondii by the modified agglutination test (MAT). Although the MAT has been found to be highly sensitive and specific for T. gondii antibodies from several species of terrestrial animals, it has not yet been validated for T. gondii infections in marine mammals. Furthermore, T. gondii has yet not been isolated from dolphins. In the present study, sera from 146 (60 from the 2004 samples and 86 from the 2003 samples) T. truncatus from the coastal areas of South Carolina and Florida were tested for antibodies to T. gondii. Sera from 2004 were tested by the MAT, the indirect fluorescent antibody test (IFAT), the Sabin-Feldman dye test (DT), an indirect hemagglutination test (IHAT), an enzyme-linked immunosorbent assay (ELISA), and Western blot. All 60 dolphins were seropositive, with MAT titers of 1:20 in 3, 1:40 in 19, 1:80 in 29, 1:160 in 2, 1:1,280 in 3, 1:2,560 in 2, and 1:5,120 or higher in 2, and these results were confirmed in another laboratory. The DT titers of these dolphins were <1:10 in 53, 1:800 in 3, 1:1,600 in 2, and 1:3,200 in 2. The IHAT titers were <1:64 in 52, 1:128 in 1, 1:512 in 2, and 1:2,048 in 5. The IFAT titers were <1:20 in 3, 1:20 in 11, 1:40 in 36, 1:80 in 2, 1:160 in 1, and 1:320 or higher in 7. All 7 DT-positive dolphins had high MAT titers, but 2 were negative by the IHAT. Western blot results closely followed MAT results; ELISA results matched MAT results, which were 1:40 or higher. In sera from the 2003 samples, MAT antibodies were found in 86 of 86 dolphins with titers of 1:25 in 29, 1:50 in 23, 1:100 in 27, 1:200 in 3, 1:1,600 in 1, and 1:3,200 in 3; these sera were not tested by other means. Overall, MAT antibodies were found in all 146 dolphin sera tested. Because marine mammals are considered sentinel animals indicative of contamination of the coastal and marine waters by T. gondii oocysts, serologically positive infections need to be validated by the detection of T. gondii organisms in the tissues of seropositive animals.     

Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3⁻ CD56⁺ NK subsets in HIV⁺ and HCV⁺ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56^dim cell fraction compared to CD56^bright cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56^dim NK subset were observed in HIV⁺ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV⁺ and HCV⁺ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections.     

Protective immunosurveillance of the central nervous system by Listeria-specific CD4 and CD8 T cells in systemic listeriosis in the absence of intracerebral Listeria

The invasion of the CNS by pathogens poses a major risk for damage of the highly vulnerable brain. The aim of the present study was to analyze immunological mechanisms that may prevent spread of infections to the CNS. Intraperitoneal application of Listeria monocytogenes to mice induced infection of the spleen, whereas pathogens remained absent from the brain. Interestingly, Listeria-specific CD4 and CD8 T cells homed to the brain and persisted intracerebrally for at least 50 days after both primary and secondary infection. CD4 and CD8 T cells resided in the leptomeninges, in the choroid plexus, and, in low numbers, in the brain parenchyma. CD4 and CD8 T cells isolated from the brain early after infection (day 7) were characterized by an activated phenotype with spontaneous IFN-gamma production, whereas at a later stage of infection (day 28) restimulation with Listeria-specific peptides was required for the induction of IFN-gamma production by CD4 and CD8 T cells. In contrast to splenic T cells, T cells in the brain did not exhibit cytotoxic activity. Adoptively transferred T cells isolated from the brains of Listeria-infected mice reduced the bacterial load in cerebral listeriosis. The frequency of intracerebral Listeria-specific T cells was partially regulated by the time of exposure to Listeria and cross-regulated by CD4 and CD8 T cells. Collectively, these data reveal a novel T cell-mediated pathway of active immunosurveillance of the CNS during bacterial infections.     

Characterization of a novel mammalian phosphatase having sequence similarity to Schizosaccharomyces pombe PHO2 and Saccharomyces cerevisiae PHO13

p34, a specific p-nitrophenyl phosphatase (pNPPase) was identified and purified from the murine cell line EL4 in a screen for the intracellular molecular targets of the antiinflammatory natural product parthenolide. A BLAST search analysis revealed that it has a high degree of sequence similarity to two yeast alkaline phosphatases. We have cloned, sequenced, and expressed p34 as a GST-tagged fusion protein in Escherichia coli and an EE-epitope-tagged fusion protein in mammalian cells. Using p-nitrophenyl phosphate (pNPP) as a substrate, p34 is optimally active at pH 7.6 with a K(m) of 1.36 mM and K(cat) of 0.052 min(-1). Addition of 1 mM Mg(2+) to the reaction mixture increases its activity by 14-fold. Other divalent metal ions such as Co(2+) and Mn(2+) also stimulated the activity of the enzyme, while Zn(2+), Fe(2+), and Cu(2+) had no effect. Furthermore, both NaCl and KCl enhanced the activity of the enzyme, having maximal effect at 50 and 75 mM, respectively. The enzyme is inhibited by sodium orthovanadate but not by sodium fluoride or okadaic acid. Mutational analysis data suggest that p34 belongs to the group of phosphatases characterized by the sequence motif DXDX(T/V).     

Prevalence of antibodies to Neospora caninum and Sarcocystis neurona in sera of domestic cats from Brazil

Parasite Biology, Epidemiology and Systematics Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Building 1001, Beltsville, Maryland 20705-2350 Antibodies to Neospora caninum and Sarcocystis neurona were determined in serum samples of 502 domestic cats from Brazil using direct agglutination tests with the respective antigens. Antibodies to S. neurona were not found in 1:50 dilution of any serum in the S. neurona agglutination test. suggesting that domestic cats from S?o Paulo city were not exposed to S. neurona sporocysts from opossums. Antibodies to N. caninum were found in 60 (11.9%) of 502 cats with titers of 1:40 in 36 cats, 1:80 in 18 cats, 1:160 in 5 cats, and 1:800 in 1 cat using the Neospora agglutination test (NAT). Antibodies to N. caninum were confirmed by Western blotting in the sera of 10 cats with NAT titers of 1:80 to 1:800; this finding suggests that at least 10 cats had N. caninum-specific antibodies confirmed by 2 tests. This is the first documentation of natural exposure of cats to N. caninum.     

Proteome of Oriental ginseng Panax ginseng C. A. Meyer and the potential to use it as an identification tool

Oriental ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius) are two widely used valuable traditional Chinese medicines (TCM). Previously, the identification of ginseng was mainly performed by analyzing the ginsengnosides using high performance liquid chromatography and amplification of polymorphic DNA using polymerase chain reaction. However, these methods cannot be used to distinguish TCM samples which are from different parts (main root, lateral roots, rhizome head and skin) of ginseng and ginseng culture cells from wild-grown ginseng. The present study aimed to identify different species of ginseng, different parts of the same ginseng and cultured cells of ginseng using a proteomic approach. Two-dimensional electrophoresis (2-DE) maps were established from the American ginseng main root, different parts (main root, lateral roots, rhizome head and skins) of Oriental ginseng and Oriental ginseng culture cells. Our results show that the 2-DE maps of different ginseng samples contain sufficient differences to permit easy discrimination. We have also identified common and specific protein spots in the 2-DE maps of different ginseng samples. The use of these "marker proteins" may help to speed up the identification process.     

Detection of single nucleotide polymorphisms

Single nucleotide polymorphism (SNP) detection technologies are used to scan for new polymorphisms and to determine the allele(s) of a known polymorphism in target sequences. SNP detection technologies have evolved from labor intensive, time consuming, and expensive processes to some of the most highly automated, efficient, and relatively inexpensive methods. Driven by the Human Genome Project, these technologies are now maturing and robust strategies are found in both SNP discovery and genotyping areas. The nearly completed human genome sequence provides the reference against which all other sequencing data can be compared. Global SNP discovery is therefore only limited by the amount of funding available for the activity. Local, target, SNP discovery relies mostly on direct DNA sequencing or on denaturing high performance liquid chromatography (dHPLC). The number of SNP genotyping methods has exploded in recent years and many robust methods are currently available. The demand for SNP genotyping is great, however, and no one method is able to meet the needs of all studies using SNPs. Despite the considerable gains over the last decade, new approaches must be developed to lower the cost and increase the speed of SNP detection.     

Prevalence of Toxoplasma gondii in chickens from an area in southern Brazil highly endemic to humans

The prevalence of Toxoplasma gondii in free-range chickens from Campos dos Goytacazes, Rio de Janeiro State, Brazil, was examined to evaluate environmental contamination by oocysts. Antibodies against T. gondii were assayed by the modified agglutination test (MAT) in sera of chickens. Antibodies against the parasite were found in 129 of 198 chickens with MAT titers > or = 1:25. Brains and hearts of 86 of the 198 chickens were bioassayed in mice for the presence of T. gondii. Viable parasites were isolated from 61 (70.9%) of the 86 chickens. Importantly, viable T. gondii were recovered even from seronegative chickens (MAT titer < or = 1:10). The distribution of parasite-positive chickens by MAT titer was 4 of 17 (titer < or = 1:10), 3 of 4 (titer of 1:20), 2 of 6 (titer of 1:40), and 52 of 59 (titer > or = 1:80). Thus, the high recovery rate of T. gondii observed in mice is indicative of high levels of environmental contamination of free-range chickens by T. gondii oocysts in this area that is endemic to humans.     

Novel immunoglobulin superfamily gene cluster,mapping to a region of human chromosome 17q25, linked to psoriasis susceptibility

Chromosome 17q25 harbors a susceptibility locus for psoriasis ( PSORS2). This locus may overlap with loci for atopic dermatitis and rheumatoid arthritis. To further refine the location of PSORS2, we genotyped 242 primarily nuclear families for 15 polymorphic microsatellites mapping to chromosome 17q23-q25. Non-parametric linkage analysis revealed a linkage peak lying close to a novel cluster of genes from the immunoglobulin (Ig) superfamily. This cluster spans >250 kb and harbors five CMRF35-like genes and a sixth inhibitory receptor ( CMRF35H) with three ITIM motifs that is transcribed in the opposite direction from the rest. The Ig domains encoded by these genes are most similar to those of the TREM (triggering receptor expressed selectively in myeloid cells) molecules, NKp44 and the polymeric immunoglobulin receptor. CMRF35-like genes are only expressed in sub-populations of cells of the myeloid lineage. In order to investigate the association of this region with psoriasis, we genotyped the families for 13 novel microsatellites and 19 SNPs from the region of linkage. A maximum NPL of 1.6 ( P=0.05) was obtained within the interval. Two SNP-based haplotypes revealed some evidence for association with psoriasis. One spanned CMRF35H and includes a non-synonymous polymorphism within CMRF35H (R111Q) (TDT P=0.03). The second was a three-locus haplotype lying within the first intron of CMRF35A2 ( TREM5) (TDT P=0.04). The novel markers described here will facilitate additional linkage and association studies between the CMRF35 family and disease.