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PubMed2005

Enhanced survival of Salmonella enterica in vesicles released by a soilborne Tetrahymena species.

Nondestructive ingestion by soilborne protozoa may enhance the environmental resiliency of important bacterial pathogens and may model how such bacteria evade destruction in human macrophages. Here, the interaction of Salmonella enterica serovar Thompson with a soilborne Tetrahymena sp. isolate was examined using serovar Thompson cells labeled with the green fluorescent protein. The bacteria were mixed in solution with cells of Tetrahymena at several ratios. During incubation with serovar Thompson, Tetrahymena cells released a large number of vesicles containing green fluorescent serovar Thompson cells. In comparison, grazing on Listeria monocytogenes cells resulted in their digestion and thus the infrequent release of this pathogen in vesicles. The number of serovar Thompson cells per vesicle increased significantly as the initial ratio of serovar Thompson to Tetrahymena cells increased from 500:1 to 5,000:1. The density of serovar Thompson was as high as 50 cells per vesicle. Staining with propidium iodide revealed that a significantly higher proportion of serovar Thompson cells remained viable when enclosed in vesicles than when free in solution. Enhanced survival rates were observed in vesicles that were secreted by both starved (F = 28.3, P < 0.001) and unstarved (F = 14.09, P < 0.005) Tetrahymena cells. Sequestration in vesicles also provided greater protection from low concentrations of calcium hypochlorite. Thus, the release of this human pathogen from Tetrahymena cells in high-density clusters enclosed in a membrane may have important implications for public health.

Animals↗
PubMed1997

Development of AIDS in a chimpanzee infected with human immunodeficiency virus type 1.

The condition of a chimpanzee (C499) infected with three different isolates of human immunodeficiency virus type 1 (HIV-1) for over 10 years progressed to AIDS. Disease development in this animal was characterized by (i) a decline in CD4+ cells over the last 3 years; (ii) an increase in viral loads in plasma; (iii) the presence of a virus, termed HIV-1JC, which is cytopathic for chimpanzee peripheral blood mononuclear cells; and (iv) the presence of an opportunistic infection and blood dyscrasias. Genetic analysis of the V1-V2 region of the envelope gene of HIV-1JC showed that the virus present in C499 was significantly divergent from all inoculating viruses (> or = 16% divergent at the amino acid level) and was suggestive of a large quasispecies. Blood from C499 transfused into an uninfected chimpanzee (C455) induced a rapid and sustained CD4+-cell decline in the latter animal, concomitant with high plasma viral loads. These results show that HIV-1 can induce AIDS in chimpanzees and suggest that long-term passage of HIV-1 in chimpanzees can result in the development of a more pathogenic virus.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Expanded tissue targets for foamy virus replication with simian immunodeficiency virus-induced immunosuppression.

Foamy viruses (FV) are the oldest known genus of retroviruses and have persisted in nonhuman primates for over 60 million years. FV are efficiently transmitted, leading to a lifelong nonpathogenic infection. Transmission is thought to occur through saliva, but the detailed mechanism is unknown. Interestingly, this persistent infection contrasts with the rapid cytopathicity caused by FV in vitro, suggesting a host defense against FV. To better understand the tissue specificity of FV replication and host immunologic defense against FV cytopathicity, we quantified FV in tissues of healthy rhesus macaques (RM) and those severely immunosuppressed by simian immunodeficiency virus (SIV). Contrary to earlier findings, we find that all immunocompetent animals consistently have high levels of viral RNA in oral tissues but not in other tissues examined, including the small intestine. Strikingly, abundant viral transcripts were detected in the small intestine of all of the SIV-infected RM, which has been shown to be a major site of SIV (and human immunodeficiency virus)-induced CD4+ T-cell depletion. In contrast, there was a trend to lower viral RNA levels in oropharyngeal tissues of SIV-infected animals. The expansion of FV replication to the small intestine but not to other CD4+ T-cell-depleted tissues suggests that factors other than T-cell depletion, such as dysregulation of the jejunal microenvironment after SIV infection, likely account for the expanded tissue tropism of FV replication.

Animals↗
PubMed2004

Comparative analysis of the spatial distribution and severity of cerebral microbleeds and old lacunes.

BACKGROUND: Cerebral microbleeds, which result from microangiopathic changes following chronic hypertension, may reflect bleeding-prone microangiopathy. However, the distribution of these lesions has not been compared with that of lacunes, which represent occlusive type microangiopathy. OBJECTIVES: To compare the cerebral distribution of microbleeds and lacunes and correlate their severity. METHODS: The study population comprised 129 hypertensive patients who underwent brain magnetic resonance imaging (MRI), including gradient echo (GE) sequences. Cerebral microbleeds were counted using GE-MRI data, and lacunes were also counted by comparing T1 and T2 weighted MRI. To investigate the distributions, the number of patients with each type of lesion was compared, and the occurrence index (the total number of the specific lesions divided by the total number of patients) was examined statistically. Correlation analyses were done on the relations between the different grades of microbleeds, lacunes, and leukoaraiosis. RESULTS: Cerebral microbleeds and lacunes were found at various foci in the brain, with a preference for the cortico-subcortical region and the deep grey matter. The occurrence index of microbleeds, but not of lacunes, was significantly higher in the cortico-subcortical region than in the deep grey matter. The severity of the microbleeds was positively correlated with the severity of lacunes, and both types of lesion were closely correlated with the degree of leukoaraiosis. CONCLUSIONS: These data suggest that microbleeds and lacunes tend to occur to a similar extent in long standing hypertension, but not necessarily in the same locations.

Aged↗
PubMed1996

Virus-induced immunosuppression is linked to rapidly fatal disease in infant rhesus macaques infected with simian immunodeficiency virus.

Six newborn rhesus macaques were experimentally infected with pathogenic Simian immunodeficiency virus of macaques (SIVmac251), and three newborn macaques were infected with avirulent SIVmac1A11. The former developed rapidly fatal simian AIDS and died within 26 wk of age, whereas the latter remained clinically normal. Infant monkeys that developed rapidly progressive disease had rapid declines in CD4+ cells and were unable to mount IgG and IgA antibody responses to SIV or to an unrelated antigen, tetanus toxoid. IgM antibody responses were near normal to both SIV-specific and nonspecific antigens. Cytotoxic T lymphocyte (CTL) responses to SIV envelope were observed in animals infected with either virulent or avirulent SIV. These studies demonstrated that virulent SIVmac infection induced a rapid immunosuppression that was both SIV-specific and nonspecific in nature. The observation that virulent strains of SIV can rapidly induce a global immunosuppression provides one explanation for the rapid disease course in some HIV-infected children and supports the strategy of early and vigorous antiviral drug therapy to alter the disease course even if this does not prevent infection.

Amino Acid Sequence↗
PubMed2004

Selection in context: patterns of natural selection in the glycoprotein 120 region of human immunodeficiency virus 1 within infected individuals.

Evolution of the HIV-1 V3 loop was monitored in 15 subjects over a period of 5 years at approximately 6-month intervals. Putative recombination was detected in many of the sequences. Evolutionary trees were estimated from the nonrecombinant viral sequences found in each individual. Selection and altered demographic regimes were detected with logit and other contingency analyses in a highly context-dependent fashion. Mutations leading to amino acid substitutions are subject to positive selection over a broad range of clinical conditions in the nonsyncytium-inducing (NSI) form, and the growth rates of the NSI strains and their level of genetic subdivision change little in going from a healthy immune system to a severely compromised immune system. In contrast, the SI form has a significant increase in growth rate as the immune system goes from healthy to compromised, particularly in those subjects who did not receive any antiviral drug therapy. This increase in SI growth rate results in a significant growth advantage of SI over NSI when the immune system is compromised. The SI strains also show more demographic subdivision when the immune system is healthy than when the immune system is compromised, and the SI form has greater demographic subdivision than NSI in subjects with healthy immune systems who also are not receiving antiviral drug therapy. Positive selection on amino-acid-changing mutations weakens and then intensifies again in the SI strains in going from healthy to compromised immune systems. These patterns are consistent with other studies that suggest that NSI strains inhibit replication of SI strains, that the V3 loop is more hidden from the immune system in the NSI form, that evolution in the V3 loop influences cell tropism and coreceptor usage, that substrate for replication of SI forms increases as the disease progresses, and that death of CD8 cells is influenced by the type of coreceptor usage typically found in SI but not in NSI strains. Finally, the transition between NSI and SI forms is associated with a burst of evolutionary change due to strong positive selection at sites other than those that define the NSI/SI phenotypes.

Acquired Immunodeficiency Syndrome↗
PubMed2002

A novel approach to the analysis of specificity, clonality, and frequency of HIV-specific T cell responses reveals a potential mechanism for control of viral escape.

Escape from the CD8(+) T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8(+) T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes, thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8(+) T cell response to six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally, multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8(+) T cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of the response to recognize virus escape mutants.

Adult↗
PubMed1993

Bovine leukocyte adhesion deficiency in Holstein cattle.

Two Holstein heifers with persistent and recurrent infections including ulcerative gingivitis, periodontitis, pneumonia, loss of teeth and stunted growth associated with marked neutrophilia were evaluated clinically and for neutrophil function, CD18 expression on neutrophils and CD18 genotype analysis by DNA-polymerase chain reaction (PCR) test. Adherence to nylon fibers and phagocytic activity of neutrophils from affected animals were significantly (p < 0.05) impaired as compared with those of controls. Neutrophils from affected heifers had decreased chemiluminescent (CL) responses when stimulated with opsonized zymosan, compared with those of controls. In contrast, neutrophils from affected heifers produced increased CL responses when stimulated with latex beads and phorbol myristate acetate compared with those of controls. The clinical findings, functional leukocyte abnormalities, deficiency in expression of CD18 on neutrophils, and the D128G mutation detected by DNA-PCR testing of affected heifers demonstrated that these heifers have bovine leukocyte adhesion deficiency (BLAD). Although both animals were confirmed to be homozygotes for BLAD by DNA-PCR test, they had differences in clinical, hematological and neutrophil functional characteristics.

Animals↗
PubMed1993

Detection of HIV-specific cell-mediated cytotoxicity in the peripheral blood from infected children.

Cytotoxic T lymphocytes may play a significant role in containing the spread of HIV in infected individuals. Although HIV-infection is associated with immune suppression, a vigorous T lymphocyte response has been detected in infected adults. HIV can be transmitted from mother to child, either during pregnancy, when differentiation of the T lymphoid compartment is ongoing, or at birth when the neonate immune system is partially competent. The shorter asymptomatic period of pediatric infection could be related to differences in the host immune control of viral replication. HIV-specific cell-mediated cytotoxicity (CMC) from fresh and in vitro stimulated PBMC of HIV-infected children was measured. CD8+CD3+ T lymphocytes were found to be the major effector population. The vast majority of children examined had detectable HIV-specific CMC. A cross-sectional analysis of CMC responses as a function of clinical status revealed that 71% of asymptomatic children (CDC stage P1) recognized the Env protein, 14% the Gag protein, but none of them recognized the Pol protein. Cytolytic activities directed against these three proteins were detected in two thirds of paucisymptomatic children (P2A). In contrast, symptomatic children (P2B-F) did not show cytolytic activities toward the Gag and Pol proteins, and only 20% recognized the Env protein. In contrast in vitro generated secondary CTL were consistently detected at all stages of disease, even in children with low CD4+ cells counts.

Amino Acid Sequence↗
PubMed1996

Clonal populations of CD4+ and CD8+ T cells in patients with multiple myeloma and paraproteinemia.

Patients with paraproteinemia have abnormalities in their T-cell subsets including inversion of the CD4:CD8 ratio and increased expression of activation markers. Recently, distortions in T-cell receptor (TCR) TCRAV and TCRBV gene segment expression have been reported, although the significance of these observations is unclear given the finding of clonal populations of CD8+ T cells in healthy elderly individuals. We have used an extensive range of TCR V-region-specific monoclonal antibodies to assess TCRAV and TCRBV expression in patients with myeloma and paraproteinemia. TCR sequence analysis was used to assess the clonality of expansions and 3-color fluorescence-activated cell sorting analysis determined the phenotype of the expanded populations. The patients show novel oligoclonal expansions within the CD4+ subset and show an increased frequency of CD8+ expansions. Oligoclonal CD4+ T cells belong to the rare CD4+CD28- T-cell subset, a phenotype associated with granular morphology. CD45RA and CD11b are expressed on many of the CD8 T-cell expansions. Comparison of T-cell receptor sequences from two T-cell clones in one patient suggests a possible role for a common peptide antigen in the generation of the expansions. Further work is needed to identify the relevance of such T cells to the B-cell proliferation.

Aged↗
PubMed1998

Human immunodeficiency virus in semen arises from a genetically distinct virus reservoir.

The reservoir of human immunodeficiency virus (HIV) in semen is unknown. Several lines of evidence suggest that semen HIV may not arise from the same reservoir of infection as peripheral blood. If true, the viral burden in the two compartments could be qualitatively and quantitatively different, a scenario of potentially profound significance for the design of effective strategies of treatment, disease monitoring, and infection containment. We report here that the ratio of infected to uninfected leukocytes in ejaculated semen specimens is highly discordant with paired blood samples, demonstrating that they derive from distinct populations of infected cells. In addition, infectious HIV was isolated from semen cells, but not from blood cells, of an individual on triple antiretroviral therapy; the absence of major resistance-conferring mutations in the semen virus indicates that it was replicating in isolation from the antiviral agents. The compartmentalization of blood and semen infection was further supported by genetic analysis of several infectious HIV clones isolated from semen cells and peripheral blood cells of another donor not on antiretroviral therapy. Protease gene sequence analyses revealed significant divergence of the two viral populations. These findings confirm the distinct compartmentalization of HIV in the semen of this study cohort, and support the concept that semen HIV arises from an isolated reservoir of infection that may function independently in the pathobiology of HIV disease.

Amino Acid Sequence↗
PubMed2005

Not so different after all: a comparison of methods for detecting amino acid sites under selection.

We consider three approaches for estimating the rates of nonsynonymous and synonymous changes at each site in a sequence alignment in order to identify sites under positive or negative selection: (1) a suite of fast likelihood-based "counting methods" that employ either a single most likely ancestral reconstruction, weighting across all possible ancestral reconstructions, or sampling from ancestral reconstructions; (2) a random effects likelihood (REL) approach, which models variation in nonsynonymous and synonymous rates across sites according to a predefined distribution, with the selection pressure at an individual site inferred using an empirical Bayes approach; and (3) a fixed effects likelihood (FEL) method that directly estimates nonsynonymous and synonymous substitution rates at each site. All three methods incorporate flexible models of nucleotide substitution bias and variation in both nonsynonymous and synonymous substitution rates across sites, facilitating the comparison between the methods. We demonstrate that the results obtained using these approaches show broad agreement in levels of Type I and Type II error and in estimates of substitution rates. Counting methods are well suited for large alignments, for which there is high power to detect positive and negative selection, but appear to underestimate the substitution rate. A REL approach, which is more computationally intensive than counting methods, has higher power than counting methods to detect selection in data sets of intermediate size but may suffer from higher rates of false positives for small data sets. A FEL approach appears to capture the pattern of rate variation better than counting methods or random effects models, does not suffer from as many false positives as random effects models for data sets comprising few sequences, and can be efficiently parallelized. Our results suggest that previously reported differences between results obtained by counting methods and random effects models arise due to a combination of the conservative nature of counting-based methods, the failure of current random effects models to allow for variation in synonymous substitution rates, and the naive application of random effects models to extremely sparse data sets. We demonstrate our methods on sequence data from the human immunodeficiency virus type 1 env and pol genes and simulated alignments.

Amino Acids↗
PubMed2004

Prokaryote phylogeny without sequence alignment: from avoidance signature to composition distance.

This is a review of a new and essentially simple method of inferring phylogenetic relationships from complete genome data without using sequence alignment. The method is based on counting the appearance frequency of oligopeptides of a fixed length (up to K = 6) in the collection of protein sequences of a species. It is a method without fine adjustment and choice of genes. Applied to prokaryotic genomes it has led to results comparable with the bacteriologists' systematics as reflected in the latest 2002 outline of the Bergey's Manual of Systematic Bacteriology. The method has also been used to compare chloroplast genomes and to the phylogeny of Coronaviruses including human SARS-CoV. A key point in our approach is subtraction of a random background from the original counts by using a Markov model of order K-2 in order to highlight the shaping role of natural selection. The implications of the subtraction procedure is specially analyzed and further development of the new approach is indicated.

Algorithms↗
PubMed1994

Molecular evidence on primate phylogeny from DNA sequences.

Evidence from DNA sequences on the phylogenetic systematics of primates is congruent with the evidence from morphology in grouping Cercopithecoidea (Old World monkeys) and Hominoidea (apes and humans) into Catarrhini, Catarrhini and Platyrrhini (ceboids or New World monkeys) into Anthropoidea, Lemuriformes and Lorisiformes into Strepsirhini, and Anthropoidea, Tarsioidea, and Strepsirhini into Primates. With regard to the problematic relationships of Tarsioidea, DNA sequences group it with Anthropoidea into Haplorhini. In addition, the DNA evidence favors retaining Cheirogaleidae within Lemuriformes in contrast to some morphological studies that favor placing Cheirogaleids in Lorisiformes. While parsimony analysis of the present DNA sequence data provides only modest support for Haplorhini as a monophyletic taxon, it provides very strong support for Hominoidea, Catarrhini, Anthropoidea, and Strepsirhini as monophyletic taxa. The parsimony DNA evidence also rejects the hypothesis that megabats are the sister group of either Primates or Dermoptera (flying lemur) or a Primate-Dermoptera clade and instead strongly supports the monophyly of Chiroptera, with megabats grouping with microbats at considerable distance from Primates. In contrast to the confused morphological picture of sister group relationships within Hominoidea, orthologous noncoding DNA sequences (spanning alignments involving as many as 20,000 base positions) now provide by the parsimony criterion highly significant evidence for the sister group relationships defined by a cladistic classification that groups the lineages to all extant hominoids into family Hominidae, divides this ape family into subfamilies Hylobatinae (gibbons) and Homininae, divides Homininae into tribes Pongini (orangutans) and Hominini, and divides Hominini into subtribes Gorillina (gorillas) and Hominina (humans and chimpanzees). A likelihood analysis of the largest body of these noncoding orthologues and counts of putative synapomorphies using the full range of sequence data from mitochondrial and nuclear genomes also find that humans and chimpanzees share the longest common ancestry.

Animals↗
PubMed2005

Identification of a dysfunctional missense single nucleotide variant of human adenylyl cyclase VI.

Genetic variants have been described for a range of G protein-coupled receptors (as well as for G proteins) linked to adenylyl cyclase. Furthermore, expression of these variants resulted in alterations in receptor-mediated activation of adenylyl cyclase, as well as alterations in more "downstream" effector pathways mediated by cyclic adenosine monophosphate. However, the identification of dysfunctional variants of adenylyl cyclase has been far more limited. Screening a region of the molecule that we recently demonstrated to be critical in regulation of enzyme activity, we have identified a missense single-nucleotide variant at amino acid 674 of human adenylyl cyclase isoform VI. In a population of 286 healthy white subjects, this variant has an allelic frequency of 3.1% (although 0/90 nonwhite subjects had this variant). Expression of this variant of adenylyl cyclase VI (whether expressed as the S674 human adenylyl cyclase VI [ADCY6] or the S686 ADCY6 rat analog) is characterized by a significant decrease in stimulated adenylyl cyclase activity (forskolin-stimulated activity of the S674 human ADCY6 variant was decreased to 56% +/- 6% of the activity of the A674 variant [mean +/- SEM]; n = 9; P = .004). Furthermore, subjects with the S674 variant demonstrated a significantly higher lymphocyte count (2.68 +/- 4.13 x 10(3)/mm3 versus 1.90 +/- 0.72 x 10(3)/mm3, P = .019). Paralleling this phenotype, expression of the variant was associated with attenuation of the forskolin-mediated reduction in cell growth rate to 64% +/- 5% of the effect seen with expression of the wild-type ADCY6 (n = 4; P = .001). In summary, these data demonstrate an unappreciated variant of adenylyl cyclase isoform VI that has a functional impact on both enzyme activity and cyclic adenosine monophosphate-mediated regulation of cell growth.

Adenylyl Cyclases↗
PubMed1994

Alphanumeric sequencing: a report on a brief measure of information processing used among persons with multiple sclerosis.

Alphanumeric Sequencing involves the alternating recitation of counting and the alphabet. We report data on the use of this measure with two clinical samples of persons with multiple sclerosis, having either the chronic progressive (n = 23) or relapsing-remitting form (n = 52) of the disease. Patients were administered Alphanumeric Sequencing and several other tests of information-processing speed/capacity and short-term memory. Chronic progressive MS patients performed worse than 23 healthy controls on both the speed and error components of the test, while relapsing-remitting patients were worse than 35 controls only on the total time to complete the task. The time score was correlated with several measures of information processing and short-term memory.

Adult↗
PubMed1995

Antithrombotic effect of a recombinant von Willebrand factor, VCL, on nitrogen laser-induced thrombus formation in guinea pig mesenteric arteries.

To assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded. In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i.v. injection inhibits risocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1999

PCR-based identification of individuals of Schistosoma japonicum representing different subpopulations using a genetic marker in mitochondrial DNA.

A mitochondrial NADH dehydrogenase I gene fragment (NDI) was sequenced for three laboratory maintained isolates of Schistosoma japonicum. Comparison of sequences representing the isolates (originally obtained from the Anhui and Zhejiang provinces of the People's Republic of China, and from the Philippines) revealed inter-isolate sequence variations of 0.2-0.6% and no intra-isolate variation was found. The sequences also indicated that while the amplification products of the Zhejiang and Philippine isolates contained a recognition site for the endonuclease RsaI, there was no such site in the Anhui isolate. This was tested by digesting amplification products from a number of individual worms with RsaI. Then an infection experiment was designed to test the value of this genetic marker for studies of the population biology of S. japonicum in the final host. For this, the two Chinese isolates were used. Three groups of mice (A-C) were exposed firstly to a primary infection and then challenge-infected at weeks 4 and 7 of the experiment. In group A the first infection was done with the Anhui isolate, and the two others with the Zhejiang isolate, thereby providing a specific, detectable cohort. In groups B and C the Anhui isolate was used for the second and third infection. All mice were perfused 5 weeks after the last challenge infection, and the NDI was subsequently amplified from DNA of the perfused worms and digested with RsaI. The digestion revealed that while infection groups A and B contained mixed populations of the Anhui and Zhejiang isolates, only Zhejiang worms were present in group C. We concluded that the absence/presence of the RsaI site in the NDI provides a useful marker for the delineation of cohorts of S. japonicum.

Amino Acid Sequence↗