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1.  SLAP deficiency decreases dsDNA autoantibody production 
Clinical immunology (Orlando, Fla.)  2013;150(2):201-209.
Src-like adaptor protein (SLAP) adapts c-Cbl, an E3 ubiquitin ligase, to activated components of the BCR signaling complex regulating BCR levels and signaling in developing B cells. Based on this function, we asked whether SLAP deficiency could decrease the threshold for tolerance and eliminate development of autoreactive B cells in two models of autoantibody production. First, we sensitized mice with a dsDNA mimetope that causes an anti-dsDNA response. Despite equivalent production of anti-peptide antibodies compared to BALB/c controls, SLAP−/− mice did not produce anti-dsDNA. Second, we used the 56R tolerance model. SLAP−/− 56R mice had decreased levels of dsDNA-reactive antibodies compared to 56R mice due to skewed light chain usage. Thus, SLAP is a critical regulator of B-cell development and function and its deficiency leads to decreased autoreactive B cells that are otherwise maintained by inefficient receptor editing or failed negative selection.
PMCID: PMC4053206  PMID: 24440645
B cells; Autoimmunity; Autoantibodies
2.  Expectations from preceding prosody influence segmentation in online sentence processing 
Psychonomic bulletin & review  2011;18(6):10.3758/s13423-011-0167-9.
Previous work examining prosodic cues in online spoken-word recognition has focused primarily on local cues to word identity. However, recent studies have suggested that utterance-level prosodic patterns can also influence the interpretation of subsequent sequences of lexically ambiguous syllables (Dilley, Mattys, & Vinke, Journal of Memory and Language, 63:274–294, 2010; Dilley & McAuley, Journal of Memory and Language, 59:294–311, 2008). To test the hypothesis that these distal prosody effects are based on expectations about the organization of upcoming material, we conducted a visual-world experiment. We examined fixations to competing alternatives such as pan and panda upon hearing the target word panda in utterances in which the acoustic properties of the preceding sentence material had been manipulated. The proportions of fixations to the monosyllabic competitor were higher beginning 200 ms after target word onset when the preceding prosody supported a prosodic constituent boundary following pan-, rather than following panda. These findings support the hypothesis that expectations based on perceived prosodic patterns in the distal context influence lexical segmentation and word recognition.
PMCID: PMC3811073  PMID: 21968925
Prosody; Expectations; Spoken-word recognition; Lexical competition; Perceptual organization; Visual-world paradigm
3.  Feline models of viral pathogenesis: Opportunity knocks 
PMCID: PMC2988874  PMID: 20716490
4.  A goal-based perspective on eye movements in visual world studies 
Acta psychologica  2010;137(2):172-180.
There is an emerging literature on visual search in natural tasks suggesting that task-relevant goals account for a remarkably high proportion of saccades, including anticipatory eye-movements. Moreover, factors such as “visual saliency” that otherwise affect fixations become less important when they are bound to objects that are not relevant to the task at hand. We briefly review this literature and discuss the implications for task-based variants of the visual world paradigm. We argue that the results and their likely interpretation may profoundly affect the “linking hypothesis” between language processing and the location and timing of fixations in task-based visual world studies. We outline a goal-based linking hypothesis and discuss some of the implications for how we conduct visual world studies, including how we interpret and analyze the data. Finally, we outline some avenues of research, including examples of some classes of experiments that might prove fruitful for evaluating the effects of goals in visual world experiments and the viability of a goal-based linking hypothesis.
PMCID: PMC3109199  PMID: 21067708
5.  Residential Proximity to a Major Roadway Is Associated with Features of Asthma Control in Children 
PLoS ONE  2012;7(5):e37044.
While several studies suggest that traffic-related air pollutants are detrimental for respiratory health, few studies have examined relationships between residential proximity to a major roadway and asthma control in children. Furthermore, a major limitation of existing research is reliance on self-reported outcomes. We therefore determined the spatial relationship between the distance from a major roadway and clinical, physiologic and inflammatory features of asthma in a highly characterized sample of asthmatic children 6–17 years of age across a wide range of severities. We hypothesized that a closer residential proximity to a major roadway would be associated with increased respiratory symptoms, altered pulmonary function and a greater magnitude of airway and systemic inflammation.
Methodology/Principal Findings
224 children 6–17 years with confirmed asthma completed questionnaires and underwent spirometry, plethysmography, exhaled nitric oxide determination, exhaled breath condensate collection and venipuncture. Residential distance from a major roadway was determined by mapping the geographic coordinates of the residential address in Geographic Information System software. The distance between the home address and the nearest major roadway was calculated according to the shortest distance between the two points (i.e., “as the crow flies”). Asthmatic children living in closer proximity to a major roadway had an increased frequency of wheezing associated with increased medication requirements and more hospitalizations even after controlling for potential confounders. These children also had increased airway resistance, increased airway inflammation reflected by a lower breath condensate pH, and higher plasma EGF concentrations.
These findings suggest that closer residential proximity to a major roadway is associated with poorer asthma control in school-age children. Assessment of residential proximity to major roadways may be useful in the clinical evaluation of asthma in children.
PMCID: PMC3355164  PMID: 22615884
6.  Cancer detection and biopsy classification using concurrent histopathological and metabolomic analysis of core biopsies 
Genome Medicine  2012;4(4):33.
Metabolomics, the non-targeted interrogation of small molecules in a biological sample, is an ideal technology for identifying diagnostic biomarkers. Current tissue extraction protocols involve sample destruction, precluding additional uses of the tissue. This is particularly problematic for high value samples with limited availability, such as clinical tumor biopsies that require structural preservation to histologically diagnose and gauge cancer aggressiveness. To overcome this limitation and increase the amount of information obtained from patient biopsies, we developed and characterized a workflow to perform metabolomic analysis and histological evaluation on the same biopsy sample.
Biopsies of ten human tissues (muscle, adrenal gland, colon, lung, pancreas, small intestine, spleen, stomach, prostate, kidney) were placed directly in a methanol solution to recover metabolites, precipitate proteins, and fix tissue. Following incubation, biopsies were removed from the solution and processed for histology. Kidney and prostate cancer tumor and benign biopsies were stained with hemotoxylin and eosin and prostate biopsies were subjected to PIN-4 immunohistochemistry. The methanolic extracts were analyzed for metabolites on GC/MS and LC/MS platforms. Raw mass spectrometry data files were automatically extracted using an informatics system that includes peak identification and metabolite identification software.
Metabolites across all major biochemical classes (amino acids, peptides, carbohydrates, lipids, nucleotides, cofactors, xenobiotics) were measured. The number (ranging from 260 in prostate to 340 in colon) and identity of metabolites were comparable to results obtained with the current method requiring 30 mg ground tissue. Comparing relative levels of metabolites, cancer tumor from benign kidney and prostate biopsies could be distinguished. Successful histopathological analysis of biopsies by chemical staining (hematoxylin, eosin) and antibody binding (PIN-4, in prostate) showed cellular architecture and immunoreactivity were retained.
Concurrent metabolite extraction and histological analysis of intact biopsies is amenable to the clinical workflow. Methanol fixation effectively preserves a wide range of tissues and is compatible with chemical staining and immunohistochemistry. The method offers an opportunity to augment histopathological diagnosis and tumor classification with quantitative measures of biochemicals in the same tissue sample. Since certain biochemicals have been shown to correlate with disease aggressiveness, this method should prove valuable as an adjunct to differentiate cancer aggressiveness.
PMCID: PMC3446261  PMID: 22546470
7.  Association of Rho-associated protein kinase 1 with E-cadherin complexes is mediated by p120-catenin 
Molecular Biology of the Cell  2012;23(1):99-110.
We show that Rho-associated protein kinase 1 coimmunoprecipitates with p120 and colocalizes to adherens junctions. p120 links ROCK1 to E-cadherin, as ROCK1 associates with wild-type, but not p120-uncoupled, E-cadherin. These data suggest that p120 regulates Rho activity at the cadherin complex via interaction with up- and downstream effectors, including ROCK1.
The dynamic functional linkage of cadherins with the underlying actin cytoskeleton is tightly regulated to achieve proper cell–cell adhesion. p120-catenin (p120) regulates both cadherin stability and actin dynamics, but the relationship between these two functions remains unclear. Using a novel proteomic approach called reversible cross-link immunoprecipitation, or ReCLIP, we previously identified a physical interaction between p120 and Rho-associated protein kinase 1 (ROCK1), a major effector of RhoA. In this paper, we show that a discrete fraction of cellular ROCK1 coimmunoprecipitates with p120 and precisely colocalizes to adherens junctions (AJs). Manipulation of AJs using a calcium-switch assay and cadherin-blocking antibodies indicates direct recruitment of ROCK1 to newly forming junctions. Importantly, we find that p120 links ROCK1 to the cadherin complex, as ROCK1 coimmunoprecipitates with wild-type but not p120-uncoupled E-cadherin. Moreover, depletion of ROCK1 using short-hairpin RNA results in dramatic mislocalization of the cadherin complex and junctional actin. These data are consistent with a model in which p120 dynamically regulates Rho-GTPase activity at the cadherin complex through transient interaction with several of its up- and downstream effectors, including ROCK1.
PMCID: PMC3248908  PMID: 22031287
8.  Emerging Viruses in the Felidae: Shifting Paradigms 
Viruses  2012;4(2):236-257.
The domestic cat is afflicted with multiple viruses that serve as powerful models for human disease including cancers, SARS and HIV/AIDS. Cat viruses that cause these diseases have been studied for decades revealing detailed insight concerning transmission, virulence, origins and pathogenesis. Here we review recent genetic advances that have questioned traditional wisdom regarding the origins of virulent Feline infectious peritonitis (FIP) diseases, the pathogenic potential of Feline Immunodeficiency Virus (FIV) in wild non-domestic Felidae species, and the restriction of Feline Leukemia Virus (FeLV) mediated immune impairment to domestic cats rather than other Felidae species. The most recent interpretations indicate important new evolutionary conclusions implicating these deadly infectious agents in domestic and non-domestic felids.
PMCID: PMC3315214  PMID: 22470834
FIV; FCoV; FeLV; Felidae
Journal of wildlife diseases  2008;44(3):537-552.
Feline leukemia virus (FeLV) was not detected in Florida pumas (Puma concolor coryi) in almost 20 yr of surveillance; however, the finding of two FeLV antigen-positive pumas during the 2002–2003 capture season led to an investigation of FeLV in the population. Between January 1990 and April 2007, the proportion of pumas testing FeLV antibody positive increased, with antibody-positive pumas concentrated in the northern portion of puma range. Five of 131 (4%) pumas sampled between July 2000 and April 2007 were viremic, with all cases clustered in Okaloacoochee Slough (OKS). Clinical signs and clinical pathology at capture were absent or included lymphadenopathy, moderate-to-severe anemia, and lymphopenia. All viremic pumas died; causes of death were septicemia (n=2), intraspecific aggression (n=2), and anemia/dehydration (n=1). Outcome after FeLV exposure in pumas was similar to that in domestic cats, with evidence of regressive, latent, and persistent infections. Management of the epizootic included vaccination, and as of April 2007, 52 free-ranging pumas had received one or more inoculations. Vaccinations were concentrated in OKS and in a band between OKS and the remainder of the puma population. There have been no new cases since July 2004; however, the potential for reintroduction of the virus remains.
PMCID: PMC3167064  PMID: 18689639
Feline leukemia virus; Florida panther; infectious disease; Puma concolor coryi; retrovirus; vaccination
10.  Glutathione Oxidation Is Associated With Airway Macrophage Functional Impairment in Children With Severe Asthma 
Pediatric research  2011;69(2):154-159.
Airway cellular dysfunction is a differentiating feature of severe asthma in children that may be related to an imbalance of the antioxidant, glutathione (GSH). We hypothesized that oxidation of GSH to glutathione disulfide (GSSG) in the epithelial lining fluid (ELF) of children with severe asthma would contribute to altered airway macrophage (AM) GSH homeostasis and AM cellular dysfunction. Bronchoalveolar lavage (BAL) was performed in 64 asthmatic children (severe asthma, n = 43). GSH, GSSG, markers of lipid peroxidation and DNA oxidation, and IL-8 were quantified in the BAL supernatant. GSH, GSSG, activities of histone deacetylase (HDAC) and histone acetyltransferase, apoptosis, and phagocytosis were assessed in isolated AMs. Children with severe asthma had increased GSSG, lipid peroxidation, byproducts of DNA oxidation, and inflammation in the ELF. This imbalance of GSH homeostasis was also noted intracellularly within the AMs and was associated with decreased HDAC activities, increased apoptosis, and impaired phagocytosis. In vitro GSH supplementation inhibited apoptosis and rescued phagocytosis in children with severe asthma. Severe asthma in children is characterized by altered airway and intracellular AM GSH homeostasis that translates to impaired AM function. Interventions to restore airway GSH homeostasis may be warranted in children with severe asthma.
PMCID: PMC3066054  PMID: 20975618
Feline immunodeficiency virus (FIV), a feline lentivirus related to HIV, causes immune dysfunction in domestic and wild cats. The Pallas’ cat is the only species from Asia known to harbor a species-specific strain of FIV designated FIVOma in natural populations. Here, a 25% seroprevalence of FIV is reported from 28 wild Mongolian Pallas’ cats sampled from 2000-2008. Phylogenetic analysis of proviral RT-Pol from eight FIVOma isolates from Mongolia, Russia, China and Kazakhstan reveals a unique monophyletic lineage of the virus within the Pallas’ cat population, most closely related to the African cheetah and leopard FIV strains. Histopathological examination of lymph node and spleen from infected and uninfected Pallas’ cats suggests that FIVOma causes immune depletion in its’ native host.
PMCID: PMC2822106  PMID: 19926144
FIV; Pallas’ cat; Evolution; Histopathology; Felidae
Experimental cell research  2008;315(1):39-49.
p120-catenin (p120) is required for cadherin stability and is thought to have a central role in modulating cell-cell adhesion. Several lines of evidence suggest that S/T phosphorylation may regulate p120 activity, but the upstream kinases involved have not been established, nor has a discreet measurable function been assigned to an individual site. To approach these issues, we have generated p120 phospho-specific monoclonal antibodies to several individual phosphorylation sites and are using them to pinpoint upstream kinases and signaling pathways that control p120 activity. Protein Kinase C (PKC) has been implicated as a signaling intermediate in several cadherin-associated cellular activities. Signaling events that activate PKC induce rapid phosphorylation at p120 Serine 879 (S879), suggesting that p120 activity is regulated, in part, by one or more PKC isoforms. Here, we find that physiologic activation of a G-protein coupled receptor (i.e., endothelin receptor), as well as several Receptor Tyrosine Kinases, induce rapid and robust p120 phosphorylation at S879, suggesting that these pathways crosstalk to cadherin complexes via p120. Using Va2 cells and PDGF stimulation, we show for the first time that PDGFR-mediated phosphorylation at this site is dependent on PKCα, a conventional PKC isoform implicated previously in disruption of adherens junctions.
PMCID: PMC2925109  PMID: 18950621
Virology  2009;390(1):1-12.
Feline immunodeficiency virus (FIV) causes AIDS in the domestic cat (Felis catus) but has not been explicitly associated with AIDS pathology in any of the eight free-ranging species of Felidae that are endemic with circulating FIV strains. African lion (Panthera leo) populations are infected with lion-specific FIV strains (FIVple), yet there remains uncertainty about the degree to which FIV infection impacts their health. Reported CD4+ T-lymphocyte depletion in FIVple infected lions and anecdotal reports of lion morbidity associated with FIV sero-prevalence emphasize the concern as to whether FIVple is innocuous or pathogenic. Here we monitored clinical, biochemical, histological and serological parameters among FIVple-positive (N=47) as compared to FIVple negative (N=17) lions anesthetized and sampled on multiple occasions between 1999 and 2006 in Botswana. Relative to uninfected lions, FIVple infected lions displayed a significant elevation in the prevalence of AIDS defining conditions: lymphandenopathy, gingivitis, tongue papillomas, dehydration, and poor coat condition, as well as displaying abnormal red blood cell parameters and elevated liver enzymes and serum proteins. Spleen and lymph node laparoscopic biopsies from free-ranging FIVple infected lions (N=8) revealed evidence of lymphoid depletion, the hallmark pathology documented in immunodefieciency virus infections of humans (HIV-1), macaques, and domestic cats. We conclude that over time FIVple infections in free-ranging lions can lead to adverse clinical, immunological, and pathological outcomes in some individuals that parallel sequelae caused by lentivirus infection in humans (HIV), Asian macaques (SIV) and domestic cats (FIVfca).
PMCID: PMC2771374  PMID: 19464039
14.  Transforming Growth Factor β Depletion Is the Primary Determinant of Smad Signaling Kinetics▿  
Molecular and Cellular Biology  2009;29(9):2443-2455.
A cell's decision to growth arrest, apoptose, or differentiate in response to transforming growth factor β (TGF-β) superfamily ligands depends on the ligand concentration. How cells sense the concentration of extracellular bioavailable TGF-β remains poorly understood. We therefore undertook a systematic quantitative analysis of how TGF-β ligand concentration is transduced into downstream phospho-Smad2 kinetics, and we found that the rate of TGF-β ligand depletion is the principal determinant of Smad signal duration. TGF-β depletion is caused by two mechanisms: (i) cellular uptake of TGF-β by a TGF-β type II receptor-dependent mechanism and (ii) reversible binding of TGF-β to the cell surface. Our results indicate that cells sense TGF-β dose by depleting TGF-β via constitutive TGF-β type II receptor trafficking processes. Our results also have implications for the role of the TGF-β type II receptor in disease, as tumor cells harboring TGF-β type II receptor mutations exhibit impaired TGF-β depletion, which may contribute to the overproduction of TGF-β and a consequently poor prognosis in cancer.
PMCID: PMC2668365  PMID: 19223462
15.  Genetics and Pathogenesis of Feline Infectious Peritonitis Virus 
Emerging Infectious Diseases  2009;15(9):1445-1452.
Coronavirus sequence analyses demonstrate distinctive circulating strains in natural populations.
Feline coronavirus (FCoV) is endemic in feral cat populations and cat colonies, frequently preceding outbreaks of fatal feline infectious peritonitis (FIP). FCoV exhibits 2 biotypes: the pathogenic disease and a benign infection with feline enteric coronavirus (FECV). Uncertainty remains regarding whether genetically distinctive avirulent and virulent forms coexist or whether an avirulent form mutates in vivo, causing FIP. To resolve these alternative hypotheses, we isolated viral sequences from FCoV-infected clinically healthy and sick cats (8 FIP cases and 48 FECV-asymptomatic animals); 735 sequences from 4 gene segments were generated and subjected to phylogenetic analyses. Viral sequences from healthy cats were distinct from sick cats on the basis of genetic distances observed in the membrane and nonstructural protein 7b genes. These data demonstrate distinctive circulating virulent and avirulent strains in natural populations. In addition, 5 membrane protein amino acid residues with functional potential differentiated healthy cats from cats with FIP. These findings may have potential as diagnostic markers for virulent FIP-associated FCoV.
PMCID: PMC2819880  PMID: 19788813
Coronavirus; infectious peritonitis; feline; virulence; genetic; marker; viruses; Maryland; USA; research
16.  An essential role for p120-catenin in Src- and Rac1-mediated anchorage-independent cell growth 
The Journal of Cell Biology  2009;184(3):437-450.
p120-catenin regulates epithelial cadherin stability and has been suggested to function as a tumor suppressor. In this study, we used anchorage-independent growth (AIG), a classical in vitro tumorigenicity assay, to examine the role of p120 in a different context, namely oncogene-mediated tumorigenesis. Surprisingly, p120 ablation by short hairpin RNA completely blocked AIG induced by both Rac1 and Src. This role for p120 was traced to its activity in suppression of the RhoA–ROCK pathway, which appears to be essential for AIG. Remarkably, the AIG block associated with p120 ablation was completely reversed by inhibition of the downstream RhoA effector ROCK. Harvey-Ras (H-Ras)–induced AIG was also dependent on suppression of the ROCK cascade but was p120 independent because its action on the pathway occurred downstream of p120. The data suggest that p120 modulates oncogenic signaling pathways important for AIG. Although H-Ras bypasses p120, a unifying theme for all three oncogenes is the requirement to suppress ROCK, which may act as a gatekeeper for the transition to anchorage independence.
PMCID: PMC2646551  PMID: 19188496
17.  Genetic Characterization of Feline Leukemia Virus from Florida Panthers 
Emerging Infectious Diseases  2008;14(2):252-259.
The emergent strain of FeLV, a novel subgroup A, was probably transmitted to panthers by a domestic cat.
From 2002 through 2005, an outbreak of feline leukemia virus (FeLV) occurred in Florida panthers (Puma concolor coryi). Clinical signs included lymphadenopathy, anemia, septicemia, and weight loss; 5 panthers died. Not associated with FeLV outcome were the genetic heritage of the panthers (pure Florida vs. Texas/Florida crosses) and co-infection with feline immunodeficiency virus. Genetic analysis of panther FeLV, designated FeLV-Pco, determined that the outbreak likely came from 1 cross-species transmission from a domestic cat. The FeLV-Pco virus was closely related to the domestic cat exogenous FeLV-A subgroup in lacking recombinant segments derived from endogenous FeLV. FeLV-Pco sequences were most similar to the well-characterized FeLV-945 strain, which is highly virulent and strongly pathogenic in domestic cats because of unique long terminal repeat and envelope sequences. These unique features may also account for the severity of the outbreak after cross-species transmission to the panther.
PMCID: PMC2600209  PMID: 18258118
Communicable diseases; emerging; leukemia virus; feline; molecular biology; immunodeficiency virus; research
18.  Seroprevalence and Genomic Divergence of Circulating Strains of Feline Immunodeficiency Virus among Felidae and Hyaenidae Species†  
Journal of Virology  2005;79(13):8282-8294.
Feline immunodeficiency virus (FIV) infects numerous wild and domestic feline species and is closely related to human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Species-specific strains of FIV have been described for domestic cat (Felis catus), puma (Puma concolor), lion (Panthera leo), leopard (Panthera pardus), and Pallas' cat (Otocolobus manul). Here, we employ a three-antigen Western blot screening (domestic cat, puma, and lion FIV antigens) and PCR analysis to survey worldwide prevalence, distribution, and genomic differentiation of FIV based on 3,055 specimens from 35 Felidae and 3 Hyaenidae species. Although FIV infects a wide variety of host species, it is confirmed to be endemic in free-ranging populations of nine Felidae and one Hyaenidae species. These include the large African carnivores (lion, leopard, cheetah, and spotted hyena), where FIV is widely distributed in multiple populations; most of the South American felids (puma, jaguar, ocelot, margay, Geoffroy's cat, and tigrina), which maintain a lower FIV-positive level throughout their range; and two Asian species, the Pallas' cat, which has a species-specific strain of FIV, and the leopard cat, which has a domestic cat FIV strain in one population. Phylogenetic analysis of FIV proviral sequence demonstrates that most species for which FIV is endemic harbor monophyletic, genetically distinct species-specific FIV strains, suggesting that FIV transfer between cat species has occurred in the past but is quite infrequent today.
PMCID: PMC1143723  PMID: 15956574

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