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1.  Genomic Diversification among Archival Strains of Salmonella enterica Serovar Typhimurium LT7 
Journal of Bacteriology  2003;185(7):2131-2142.
To document genomic changes during long periods of storage, we analyzed Salmonella enterica serovar Typhimurium LT7, a mutator strain that was previously reported to have higher rates of mutations compared to other serovar Typhimurium strains such as LT2. Upon plating directly from sealed agar stabs that had been stocked at room temperature for up to four decades, many auxotrophic mutants derived from LT7 gave rise to colonies of different sizes. Restreaking from single colonies consistently yielded colonies of diverse sizes even when we repeated single-colony isolation nine times. Colonies from the first plating had diverse genomic changes among and even within individual vials, including translocations, inversions, duplications, and point mutations, which were detected by rare-cutting endonuclease analysis with pulsed-field gel electrophoresis. Interestingly, even though the colony size kept diversifying, all descendents of the same single colonies from the first plating had the same sets of detected genomic changes. We did not detect any colony size or genome structure diversification in serovar Typhimurium LT7 stocked at −70°C or in serovar Typhimurium LT2 stocked either at −70°C or at room temperature. These results suggest that, although colony size diversification occurred during rapid growth, all detected genomic changes took place during the storage at room temperature and were carried over to their descendents without further changes during rapid growth in rich medium. We constructed a genomic cleavage map on the LT7 strain that had been stocked at −70°C and located all of the detected genomic changes on the map. We speculated on the significance of mutators for survival and evolution under environmentally stressed conditions.
doi:10.1128/JB.185.7.2131-2142.2003
PMCID: PMC151480  PMID: 12644482
2.  Distinct Regions of the slo Subunit Determine Differential BKCa Channel Responses to Ethanol 
Background:
Ethanol at clinically relevant concentrations increases BKCa channel activity in dorsal root ganglia neurons, GH3 cells, and neurohypophysial terminals, leading to decreases in cell excitability and peptide release. In contrast, ethanol inhibits BKCa channels from aortic myocytes, which likely contributes to alcohol-induced aortic constriction. The mechanisms that determine differential BKCa channel responses to ethanol are unknown. We hypothesized that nonconserved regions in the BKCa channel-forming subunit (slo) are major contributors to the differential alcohol responses of different BKCa channel phenotypes.
Methods:
We constructed chimeras by interchanging the core and the tail domains of two BKCa channel-forming subunits (mslo and bslo) that, after expression, differentially respond to ethanol (activation and inhibition, respectively), and studied ethanol action on these mbslo and bmslo chimeric channels using single-channel, patch-clamp techniques.
Results and Conclusion:
Data from cell-free membranes patches demonstrate that the activity of channels that share a mslo-type core-linker (wt mslo and the mbslo chimera) is consistently and significantly potentiated by acute exposure to ethanol. Thus, a mslo tail is not necessary for ethanol potentiation of slo channels. In contrast, the activity of channels that share a bslo-type core-linker (wt bslo and the bmslo chimera) display heterogenous responses to ethanol: inhibition (in the majority of cases), refractoriness, or activation. Overall, our data indicate that the slo core-linker is a critical region likely contributing to the differential responses of BKCa channels to ethanol.
doi:10.1097/01.ALC.0000094756.41638.5D
PMCID: PMC2494946  PMID: 14574235
Ethanol; Maxi-Potassium Channel; KCNMA1 (slo) Gene; Chimeras; Alcohol
3.  APOE and TGF-ß1 genes are associated with obesity phenotypes 
Long, J | Liu, P | Liu, Y | Lu, Y | Xiong, D | Elze, L | Recker, R | Deng, H
Journal of Medical Genetics  2003;40(12):918-924.
doi:10.1136/jmg.40.12.918
PMCID: PMC1735335  PMID: 14684691
5.  B7DC/PDL2 Promotes Tumor Immunity by a PD-1–independent Mechanism 
The Journal of Experimental Medicine  2003;197(12):1721-1730.
B7H1 (PDL1) and B7DC (PDL2) are two new members of the B7 family that can interact with PD-1, a putative negative regulator for immune function. Recent studies have provided evidence for inhibitory functions of both members via PD-1. Meanwhile, compelling evidence exists for costimulatory function of both members. Here we demonstrate that expression of B7DC on the tumor cells promotes CD8 T cell–mediated rejection of tumor cells, at both the induction and effector phase of antitumor immunity. Moreover, B7DC binds to PD-1(−/−) cells and enhances T cell killing in a PD-1–independent mechanism. Our results demonstrate a novel pathway for B7DC to promote tumor immunity and may reconcile the apparently contradictory findings on the function of B7DC.
doi:10.1084/jem.20022089
PMCID: PMC2193953  PMID: 12810690
tumor immunity; costimulatory molecules; cytolytic T lymphocytes
6.  CII-DC-AdTRAIL cell gene therapy inhibits infiltration of CII-reactive T cells and CII-induced arthritis 
Journal of Clinical Investigation  2003;112(9):1332-1341.
Previously, we described an APC-adenovirus (APC-Ad) FasL cell gene therapy method which could be used to deplete autoreactive T cells in vivo. FasL was toxic, however, and controlled regulation of FasL was not achieved. Here we describe an improved approach to delivering TNF-related apoptosis-inducing ligand (TRAIL) in vivo in which collagen II–induced (CII-induced) arthritis–susceptible (CIA-susceptible) DBA/1j mice were treated with CII-pulsed DCs that had been transfected with a novel Ad system. The Ad was engineered to exhibit inducible TRAIL under the control of the doxycycline-inducible (DOX-inducible) tetracycline response element (TRE). Four groups of mice were treated with CII-DC-AdTRAIL+DOX, CII-DC-AdTRAIL (no DOX), CII-DC-AdGFP+DOX, or DC-AdTRAIL+DOX (no CII), beginning 2 weeks after priming with CII in CFA. The incidence of arthritis and infiltration of T cells in the joint was significantly decreased in CII-DC-AdTRAIL+DOX–treated mice. The in vitro splenic T cell proliferative response and induction of IFN-γ to bovine CII stimulation were also significantly reduced in mice treated with CII-DC-AdTRAIL+DOX. AdTRAIL+DOX was not toxic to DCs or mice but could induce activated T cells to undergo apoptosis in the spleen. Our results suggest that CII-DC-AdTRAIL+DOX cell gene therapy is a safe and effective method for inhibiting the development of CIA.
doi:10.1172/JCI200319209
PMCID: PMC228459  PMID: 14597760
7.  Comparison of beraprost and ticlopidine in Chinese patients with chronic peripheral arterial occlusion: a multicenter, single-blind, randomized, controlled study 
Background: Chronic peripheral arterial occlusion (CPAO) is a progressive disease that is associated with a variety of symptoms, the 4 most common being a sensation of coolness in the limbs, intermittent claudication (in which pain occurs on walking), limb pain (which occurs spontaneously at rest), and ischemic leg ulcers. Beraprost sodium is an oral prostaglandin I2 analogue that may ameliorate these symptoms.
Objective: The aim of this study was to compare the efficacy and tolerability of beraprost sodium and ticlopidine hydrochloride in the treatment of patients with CPAO in China.
Methods: In this multicenter, single-blind, controlled study, patients with CPAO were randomly assigned to receive beraprost 120-μg tablet TID or ticlopidine 500-mg tablet BID, both administered orally. The clinical efficacy of the drugs was assessed using the 4 main symptoms of CPAO. Ankle-brachial index (ABI) also was measured as a clinical pharmacologic procedure. Adverse events were assessed throughout the study.
Results: A total of 124 patients (96 men, 28 women; mean [SD] age, 65 [12] years) were enrolled in 3 hospitals. Data from 119 patients (93 men, 26 women; mean [SD] age, 65 [12] years) were included in the efficacy analysis (64 and 55 patients in the beraprost and ticlopidine groups, respectively). Although all 4 symptoms of CPAO were ameliorated after 3 and 6 weeks of treatment with both drugs, only the cool sensation was significantly improved with beraprost compared with ticlopidine at 6 weeks (P<0.05). ABI was significantly increased with both beraprost and ticlopidine at 6 weeks versus baseline (P<0.001 and P<0.01, respectively), suggesting that this pharmacologic action may have led to their beneficial effect on various symptoms. The tolerability analysis included 123 patients (65 and 58 patients in the beraprost and ticlopidine groups, respectively). The numbers of patients who (1) experienced adverse events (AEs), (2) experienced adverse drug reactions, and (3) withdrew due to AEs were significantly smaller in the beraprost group than in the ticlopidine group (P<0.001, P<0.05, and P<0.05, respectively).
Conclusions: In this study population of patients with CPAO, beraprost ameliorated cool sensation in the limbs, intermittent claudication, limb pain, and ischemic/leg ulcers. Beraprost was more efficacious in relieving CPAO symptoms and was better tolerated than ticlopidine. Beraprost may be useful for the treatment of patients with CPAO, but more studies are needed.
doi:10.1016/S0011-393X(03)00125-5
PMCID: PMC4053019  PMID: 24944399
Beraprost; ticlopidine; prostaglandin I2 analogue; peripheral arterial occlusion
8.  Telithromycin- and Fluoroquinolone-Resistant Streptococcus pneumoniae in Taiwan with High Prevalence of Resistance to Macrolides and β-Lactams: SMART Program 2001 Data 
There is a high prevalence of β-lactam- and macrolide-resistant Streptococcus pneumoniae in Taiwan. To understand the in vitro susceptibilities of recent isolates of S. pneumoniae to fluoroquinolones and telithromycin (which is not available in Taiwan), the MICs of 23 antimicrobial agents for 936 clinical isolates of S. pneumoniae isolated from different parts of Taiwan from 2000 to 2001 were determined by the agar dilution method. Overall, 72% of isolates were not susceptible to penicillin (with 61% being intermediate and 11% being resistant) and 92% were resistant to erythromycin. Telithromycin MICs were ≥1 μg/ml for 16% of the isolates, and for 99% of these isolates the MICs of all macrolides tested were ≥256 μg/ml; all of these isolates had the constitutive macrolide-lincosamide-streptogramin B phenotype. Eighty-eight percent of the isolates were resistant to three or more classes of drugs. The ciprofloxacin MICs were ≥4 μg/ml for six (0.6%) isolates from five patients collected in 2000 and 2001, and the levofloxacin MICs were ≥8 μg/ml for five of these isolates. Seven isolates for which ciprofloxacin MICs were ≥4 μg/ml, including one isolate recovered in 1999, belonged to three serotypes (serotype 19F, five isolates; serotype 23A, one isolate; and serotype 23B, one isolate). The isolates from the six patients for which ciprofloxacin MICs were ≥4 μg/ml had different pulsed-field gel electrophoresis profiles and random amplified polymorphic DNA patterns, indicating that no clonal dissemination occurred over this time period. Despite the increased rate of fluoroquinolone use, the proportion of pneumococcal isolates for which ciprofloxacin MICs were elevated (≥4 μg/ml) remained low. However, the occurrence of telithromycin resistance is impressive and raises concerns for the future.
doi:10.1128/AAC.47.7.2145-2151.2003
PMCID: PMC161856  PMID: 12821460
9.  Telithromycin and Quinupristin-Dalfopristin Resistance in Clinical Isolates of Streptococcus pyogenes: SMART Program 2001 Data 
This study evaluated the current status of antimicrobial resistance in clinical isolates of Streptococcus pyogenes in Taiwan as part of the SMART (Surveillance from Multicenter Antimicrobial Resistance in Taiwan) program. In 2001, 419 different isolates of S. pyogenes, including 275 from respiratory secretions, 87 from wound pus, and 31 from blood, were collected from nine hospitals in different parts of Taiwan. MICs of 23 antimicrobial agents were determined at a central location by the agar dilution method. All of the isolates were susceptible to penicillin (MIC at which 90% of the isolates were inhibited [MIC90], ≤0.03 μg/ml), cefotaxime (MIC90, ≤0.03 μg/ml), cefepime (MIC90, 0.06 μg/ml), meropenem (MIC90, ≤0.03 μg/ml), moxifloxacin (MIC90, 0.25 μg/ml), vancomycin (MIC90, 0.5 μg/ml), and linezolid (MIC90, 1 μg/ml). Overall, 78% of isolates were not susceptible to erythromycin (54% were intermediate, and 24% were resistant), and 5% were not susceptible to clindamycin. Of the 101 erythromycin-resistant isolates, 80.2% exhibited the M phenotype (mefA gene positive), 18.9% exhibited the cMLS (constitutive resistance to macrolides-lincosamides-streptogramin B [MLS]) phenotype (ermB gene positive), and 1% exhibited the iMLS (inducible resistance to MLS) phenotype (ermB gene positive). Fluoroquinolones (sitafloxacin > moxifloxacin > ciprofloxacin = levofloxacin = gatifloxacin > gemifloxacin) demonstrated potent activity against nearly all of the isolates of S. pyogenes tested. Thirty-two isolates (8%) were not susceptible to quinupristin-dalfopristin. Seventeen percent of isolates had telithromycin MICs of ≥1 μg/ml, and all of these isolates exhibited erythromycin MICs of ≥32 μg/ml. The high prevalence of resistance to telithromycin (which is not available in Taiwan) limits its potential use in the treatment of S. pyogenes infections, particularly in areas with high rates of macrolide resistance.
doi:10.1128/AAC.47.7.2152-2157.2003
PMCID: PMC161833  PMID: 12821461
10.  Molecular Epidemiology of Vibrio cholerae O139 in China: Polymorphism of Ribotypes and CTX Elements 
Journal of Clinical Microbiology  2003;41(6):2306-2310.
Vibrio cholerae O139, the second etiological serogroup of cholera, triggered the first outbreak of O139 cholera in China in 1993. To analyze the clone polymorphism of O139 isolates in China, 117 strains of V. cholerae O139, isolated from different areas in China between 1993 and 1999, were selected to characterize the phylogenetic relationships by molecular techniques. Analysis of restriction fragment length polymorphism in the conserved 16S rRNA gene revealed seven different ribotypes within the 117 strains. Among these strains, there were eight that lacked the cholera toxin gene (ctxAB), zot, and the repetitive sequence (RS); these eight strains belonged to three individual ribotypes. Our results suggested that V. cholerae O139 strains in China had clone diversity in phylogeny. The results of our hybridization patterns for CTX genetic elements (ctxAB, zot, and RS) showed that CTXΦ genomes in most V. cholerae O139 strains had two or more copies and had extensive restriction patterns even for the strains which belong to the same ribotype. For 22 (20.1%) strains, the copies of ctxAB were different from those of zot, suggesting that a ctxAB-negative CTXΦ genome may exist in O139 strains. This ctxAB-negative CTXΦ genome may coexist with the intact CTXΦ genome in a strain. In addition, the dendrogram for I-CeuI-generated pulsed-field gel electrophoresis patterns showed that V. cholerae serogroup O139 has a closer relationship with one strain of serogroup O22 than with the strains of serogroup O1. The results of this study showed the clonal diversity and the distribution of O139 strains in China, suggesting multiple origins of the O139 cholera epidemic or sporadic events.
doi:10.1128/JCM.41.6.2306-2310.2003
PMCID: PMC156495  PMID: 12791841
11.  Antigenic drift as a mechanism for tumor evasion of destruction by cytolytic T lymphocytes 
Journal of Clinical Investigation  2003;111(10):1487-1496.
It is established that mutations in viral antigenic epitopes, or antigenic drifts, allow viruses to escape recognition by both Ab’s and T lymphocytes. It is unclear, however, whether tumor cells can escape immune recognition via antigenic drift. Here we show that adoptive therapy with both monoclonal and polyclonal transgenic CTLs, specific for a natural tumor antigen, P1A, selects for multiple mutations in the P1A antigenic epitope. These mutations severely diminish T cell recognition of the tumor antigen by a variety of mechanisms, including modulation of MHC:peptide interaction and TCR binding to MHC:peptide complex. These results provide the first evidence for tumor evasion of T cell recognition by antigenic drift, and thus have important implications for the strategy of tumor immunotherapy.
doi:10.1172/JCI200317656
PMCID: PMC155049  PMID: 12750398
12.  Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer 
Journal of Clinical Investigation  2003;111(9):1347-1356.
Gene replacement therapy is an attractive approach for treatment of genetic disease, but may be complicated by the risk of a neutralizing immune response to the therapeutic gene product. There are examples of humoral and cellular immune responses against the transgene product as well as absence of such responses, depending on vector design and the underlying mutation in the dysfunctional gene. It has been unclear, however, whether transgene expression can induce tolerance to the therapeutic antigen. Here, we demonstrate induction of immune tolerance to a secreted human coagulation factor IX (hF.IX) antigen by adeno-associated viral gene transfer to the liver. Tolerized mice showed absence of anti-hF.IX and substantially reduced in vitro T cell responses after immunization with hF.IX in adjuvant. Tolerance induction was antigen specific, affected a broad range of Th cell subsets, and was favored by higher levels of transgene expression as determined by promoter strength, vector dose, and mouse strain. Hepatocyte-derived hF.IX expression induced regulatory CD4+ T cells that can suppress anti-hF.IX formation after adoptive transfer. With a strain-dependent rate of success, tolerance to murine F.IX was induced in mice with a large F.IX gene deletion, supporting the relevance of these data for treatment of hemophilia B and other genetic diseases.
doi:10.1172/JCI200316887
PMCID: PMC154443  PMID: 12727926
13.  GGAPs, a New Family of Bifunctional GTP-Binding and GTPase-Activating Proteins 
Molecular and Cellular Biology  2003;23(7):2476-2488.
G proteins are molecular switches that control a wide variety of physiological functions, including neurotransmission, transcriptional activation, cell migration, cell growth. and proliferation. The ability of GTPases to participate in signaling events is determined by the ratio of GTP-bound to GDP-bound forms in the cell. All known GTPases exist in an inactive (GDP-bound) and an active (GTP-bound) conformation, which are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), respectively. In this study, we identified and characterized a new family of bifunctional GTP-binding and GTPase-activating proteins, named GGAP. GGAPs contain an N-terminal Ras homology domain, called the G domain, followed by a pleckstrin homology (PH) domain, a C-terminal GAP domain, and a tandem ankyrin (ANK) repeat domain. Expression analysis indicates that this new family of proteins has distinct cell localization, tissue distribution, and even message sizes. GTPase assays demonstrate that GGAPs have high GTPase activity through direct intramolecular interaction of the N-terminal G domain and the C-terminal GAP domain. In the absence of the GAP domain, the N-terminal G domain has very low activity, suggesting a new model of GGAP protein regulation via intramolecular interaction like the multidomain protein kinases. Overexpression of GGAPs leads to changes in cell morphology and activation of gene transcription.
doi:10.1128/MCB.23.7.2476-2488.2003
PMCID: PMC150724  PMID: 12640130
14.  Human DF3/MUC1 carcinoma-associated protein functions as an oncogene 
Oncogene  2003;22(38):6107-6110.
The human DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed by most carcinomas of the breast and other epithelia. The contribution of MUC1 overexpression to the malignant phenotype is, however, not known. In the present studies, we have stably expressed MUC1 in rat 3Y1 fibroblasts. MUC1-positive cells were selected from independent transfections. The results demonstrate that, as found in human carcinomas, MUC1 is expressed on the cell surface and as a complex with α-catenin in the nucleus of the transfectants. Colony formation in soft agar demonstrates that cells expressing MUC1, but not the empty vector, exhibit anchorageindependent growth. The results also show that MUC1 expression confers tumor formation in nude mice. These findings provide the first evidence that MUC1 induces cellular transformation.
doi:10.1038/sj.onc.1206732
PMCID: PMC4209839  PMID: 12955090
DF3/MUC1; 3Y1 cells; anchorage-independent growth; cellular transformation
15.  c-Myc Transformation Domain Recruits the Human STAGA Complex and Requires TRRAP and GCN5 Acetylase Activity for Transcription Activation* 
The Journal of biological chemistry  2003;278(22):20405-20412.
Deregulation of the c-Myc oncoprotein (Myc) is implicated in many types of cancer. Myc is a sequence-specific transcription factor that regulates transcription of genes involved in the control of cell proliferation and apoptosis via mechanisms that are still poorly understood. Cell transformation by Myc involves its association with the transformation-transactivation domain-associated protein (TRRAP) and the human histone acetyltransferase (HAT) GCN5. TRRAP and GCN5 are components of a variety of shared and distinct multiprotein HAT complexes with diverse functions. Myc induces TRRAP recruitment and histone hyperacetylation at specific Myc-activated genes in vivo. However, the identity of the HAT complexes recruited by Myc and the roles of TRRAP and GCN5 in Myc function are still unclear. Here we show that Myc co-recruits TRRAP and GCN5 via direct physical interactions of its N-terminal activation/transformation domain with the human STAGA (SPT3-TAF-GCN5 acetylase) coactivator complex. We demonstrate that GCN5 and TRRAP cooperate to enhance transcription activation by the N-terminal activation domain of Myc in vivo and that this synergy requires both the SPT3/GCN5 interaction domain of TRRAP and the HAT activity of GCN5. Thus, TRRAP might function as an adaptor within the STAGA complex, which helps recruit GCN5 HAT activity to Myc during transcription activation.
doi:10.1074/jbc.M211795200
PMCID: PMC4031917  PMID: 12660246
16.  Malnutrition at Age 3 Years and Lower Cognitive Ability at Age 11 Years 
Background
Early malnutrition is linked to poor cognition, but long-term effects have not been extensively examined and psychosocial confounds have not always been controlled.
Objective
To test the hypothesis that malnutrition at age 3 years will be associated with poorer cognitive ability at age 11 years independent of psychosocial confounds.
Design
A prospective, longitudinal study of a birth cohort of 1559 children originally assessed at age 3 years for malnutrition (low hemoglobin level, angular stomatitis, kwashiorkor, and sparse, thin hair) and followed up to age 11 years.
Setting and Participants
A community sample of 1559 children (51.4% boys and 48.6% girls) born between September 1, 1969, and August 31, 1970, in 2 towns in the island of Mauritius, with 68.7% Indians and 25.7% Creoles (African origin).
Main Outcome Measures
Verbal and spatial ability measured at ages 3 and 11 years and reading, scholastic ability, and neuropsychologic performance measured at age 11 years.
Results
Malnourished children had poorer cognition at both ages. Deficits were stable across time, applied to all sex and ethnic groups, and remained after controlling for multiple measures of psychosocial adversity. Children with 3 indicators of malnutrition had a 15.3-point deficit in IQ at age 11 years.
Conclusions
Malnutrition at age 3 years is associated with poor cognition at age 11 years independent of psychosocial adversity. Promoting early childhood nutrition could enhance long-term cognitive development and school performance, especially in children with multiple nutritional deficits.
doi:10.1001/archpedi.157.6.593
PMCID: PMC3975917  PMID: 12796242
17.  Interleukin-1 Mediates Pathological Effects of Microglia on Tau Phosphorylation and on Synaptophysin Synthesis in Cortical Neurons through a p38-MAPK Pathway 
The presence of tangles of abnormally phosphorylated tau is a characteristic of Alzheimer's disease (AD), and the loss of synapses correlates with the degree of dementia. In addition, the overexpression of interleukin-1 (IL-1) has been implicated in tangle formation in AD. As a direct test of the requirement for IL-1 in tau phosphorylation and synaptophysin expression, IL-1 actions in neuron–microglia cocultures were manipulated. Activation of microglia with secreted β-amyloid precursor protein or lipopolysaccharide elevated their expression of IL-1α, IL-1β, and tumor necrosis factor α (TNFα) mRNA. When such activated microglia were placed in coculture with primary neocortical neurons, a significant increase in the phosphorylation of neuronal tau was accompanied by a decline in synaptophysin levels. Similar effects were evoked by treatment of neurons with recombinant IL-1β. IL-1 receptor antagonist (IL-1ra) as well as anti-IL-1β antibody attenuated the influence of activated microglia on neuronal tau and synaptophysin, but anti-TNFα antibody was ineffective. Some effects of microglial activation on neurons appear to be mediated by activation of p38 mitogen-activated protein kinase (p38-MAPK), because activated microglia stimulated p38-MAPK phosphorylation in neurons, and an inhibitor of p38-MAPK reversed the influence of IL-1 β on tau phosphorylation and synaptophysin levels. Our results, together with previous observations, suggest that activated microglia may contribute to neurofibrillary pathology in AD through their production of IL-1, activation of neuronal p38-MAPK, and resultant changes in neuronal cytoskeletal and synaptic elements.
PMCID: PMC3833596  PMID: 12629164
Alzheimer's disease; β-amyloid precursor protein; cortical neuron; interleukin-1; microglia; mitogen-activated protein kinase; synaptophysin; phosphorylated tau
18.  Caveolin-1 Contributes to Assembly of Store-operated Ca2+ Influx Channels by Regulating Plasma Membrane Localization of TRPC1 
The Journal of biological chemistry  2003;278(29):27208-27215.
TRPC1, a component of store-operated Ca2+ entry (SOCE) channels, is assembled in a complex with caveolin- 1 (Cav1) and key Ca2+ signaling proteins. This study examines the role of Cav1 in the function of TRPC1. TRPC1 and Cav1 were colocalized in the plasma membrane region of human submandibular gland and Madin-Darby canine kidney cells. Full-length Cav1 bound to both the N and C termini of TRPC1. Amino acids 271–349, which includes a Cav1 binding motif (amino acids 322–349), was identified as the Cav1 binding domain in the TRPC1 N terminus. Deletion of amino acids 271–349 or 322–349 prevented plasma membrane localization of TRPC1. Importantly, TRPC1Δ271–349 induced a dominant suppression of SOCE and was associated with wild-type TRPC1. Although the role of the C-terminal Cav1 binding domain is not known, its deletion did not affect localization of TRPC1 (Singh, B. B., Liu, X., and Ambudkar, I. S. (2000) J. Biol. Chem. 275, 36483–36486). Further, expression of a truncated Cav1 (Cav1Δ51–169), but not full-length Cav1, similarly disrupted plasma membrane localization of endogenously and exogenously expressed TRPC1 in human Submandibular gland and Madin-Darby canine kidney cells. Cav1Δ51–169 also suppressed thapsigargin- and carbachol-stimulated Ca2+ influx and increased the detergent solubility of TRPC1, although plasma membrane lipid raft domains were not disrupted. These data demonstrate that plasma membrane localization of TRPC1 depends on an interaction between its N terminus and Cav1. Thus, our data suggest that Cav1 has an important role in the assembly of SOCE channel(s).
doi:10.1074/jbc.M301118200
PMCID: PMC3621139  PMID: 12732636
19.  Ac-SDKP Reverses Inflammation and Fibrosis in Rats With Heart Failure After Myocardial Infarction 
Hypertension  2003;43(2):229-236.
Inflammation may play an important role in the pathogenesis of cardiac fibrosis in heart failure (HF) after myocardial infarction (MI). N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring antifibrotic peptide whose plasma concentration is increased 4- to 5-fold by angiotensin-converting enzyme inhibitors. We tested the hypothesis that in rats with HF after MI, Ac-SDKP acts as an anti-inflammatory cytokine, preventing and also reversing cardiac fibrosis in the noninfarcted area (reactive fibrosis), and thus affording functional improvement. We found that Ac-SDKP significantly decreased total collagen content in the prevention group from 23.7±0.9 to 15.0±0.7 μg/mg and in the reversal group from 22.6±2.2 to 14.4±1.6 (P<0.01). Interstitial collagen volume fraction and perivascular collagen were likewise significantly reduced. We also found that infiltrating macrophages were reduced from 264.7±8.1 to 170.2±9.2/mm2, P<0.001 (prevention), and from 257.5±9.1 to 153.1±8.5 mm2, P<0.001 (reversal), while transforming growth factor (TGF)-β-positive cells were decreased from 195.6±8.4 to 129.6±5.7/mm2, P<0.01 (prevention), and from 195.6±8.4 to 130.7±10.8/mm2, P<0.01 (reversal). Ac-SDKP did not alter either blood pressure or left ventricular hypertrophy (LVH); however, it depressed systolic cardiac function in the prevention study while having no significant effect in the reversal group. We concluded that Ac-SDKP has an anti-inflammatory effect in HF that may contribute to its antifibrotic effect; however, this decrease in fibrosis without changes in LVH was not accompanied by an improvement in cardiac function.
doi:10.1161/01.HYP.0000107777.91185.89
PMCID: PMC3259854  PMID: 14691195
rat; myocardial infarction; cardiac function; collagen; macrophages; transforming growth factor-β
20.  Mechanisms of Ganglioside Inhibition of APC Function1 
Gangliosides shed by tumor cells exert potent inhibitory effects on cellular immune responses. Here we have studied ganglioside inhibition of APC function. When human monocytes were preincubated in 50 µM highly purified ganglioside GD1a, pulsed with tetanus toxoid (TT), and washed, the expected Ag-induced proliferative response of autologous normal T cells added to these monocytes was inhibited by 81%. Strikingly, there was also almost complete (92%) and selective inhibition of the up-regulation of the monocyte costimulatory molecule CD80, while I-CAM-1, LFA-3, HLA-DR, and CD86 expression were unaffected. Purified LPS-stimulated monocytes that had been preincubated in GD1a likewise showed inhibition of CD80 up-regulation (59%) as well as down-regulation of CD40 (54%) and impaired release of IL-12 and TNF-α (reduced by 59 and 51%). GD1a-preincubated human dendritic cells (DC) were also affected. They had reduced constitutive expression of CD40 (33%) and CD80 (61%), but not CD86, and marked inhibition of release of IL-6 (72%), IL-12 (70%), and TNF-α (46%). Even when pulsed with TT, these ganglioside-preincubated DC remained deficient in costimulatory molecule expression and cytokine secretion and were unable to induce a normal T cell proliferative response to TT. Finally, significant inhibition of nuclear localization of NF-κB proteins in activated DC suggests that disruption of NF-κB activation may be one mechanism contributing to ganglioside interference with APC expression of costimulatory molecules and cytokine secretion, which, in turn, may diminish antitumor immune responses.
PMCID: PMC2849639  PMID: 12902465
21.  C-Reactive Protein and Ischemia in Users and Nonusers of β-Blockers and Statins: Data From the Heart and Soul Study 
Circulation  2003;107(2):245-250.
Background
Elevated levels of C-reactive protein (CRP) are associated with an increased risk of coronary events, but whether inflammation is associated with inducible ischemia in patients with stable coronary disease is unknown.
Methods and Results
We recruited patients with known coronary disease from 2 VA Medical Centers and 1 University-based medical center for the Heart and Soul Study. We measured CRP levels in 118 participants who had exercise-induced ischemia and in 111 who did not have inducible ischemia, as determined by stress echocardiography. We used logistic regression to examine the risk of exercise-induced ischemia associated with elevated CRP. We found that 75% (39/52) of participants in the highest CRP category (>0.38 mg/dL) had inducible ischemia, compared with 45% (79/177) in the lower 4 categories combined (adjusted odds ratio 4.2; 95% confidence interval 1.6 to 11; P=0.004). However, this association differed in users and nonusers of β-blockers and statins. Among 89 participants who did not use β-blockers, 93% in the highest CRP category had exercise-induced ischemia, compared with 42% in the lower 4 categories (P=0.03). Among 67 participants who did not use statins, 94% in the highest CRP category had exercise-induced ischemia, compared with 44% in the lower 4 categories (P=0.009). We did not observe a significant association between CRP and ischemia among participants who were treated with either of these medications.
Conclusion
Elevated CRP levels are associated with inducible ischemia in patients with stable coronary disease, particularly among those not treated with β-blockers or statins.
PMCID: PMC2776690  PMID: 12538423
coronary disease; ischemia; risk factors; inflammation; epidemiology
22.  Depressive Symptoms and Health-Related Quality of Life: The Heart and Soul Study 
Context
Little is known regarding the extent to which patient-reported health status, including symptom burden, physical limitation, and quality of life, is determined by psychosocial vs physiological factors among patients with chronic disease.
Objective
To compare the contributions of depressive symptoms and measures of cardiac function to the health status of patients with coronary artery disease.
Design, Setting, and Participants
Cross-sectional study of 1024 adults with stable coronary artery disease recruited from outpatient clinics in the San Francisco Bay Area between September 2000 and December 2002.
Main Measures
Measurement of depressive symptoms using the Patient Health Questionnaire (PHQ); assessment of cardiac function by measuring left ventricular ejection fraction on echocardiography, exercise capacity on treadmill testing, and ischemia on stress echocardiography; and measurement of a range of health status outcomes, including symptom burden, physical limitation, and quality of life, using the Seattle Angina Questionnaire. Participants were also asked to rate their overall health as excellent, very good, good, fair, or poor.
Results
Of the 1024 participants, 201 (20%) had depressive symptoms (PHQ score ≥10). Participants with depressive symptoms were more likely than those without depressive symptoms to report at least mild symptom burden (60% vs 33%; P<.001), mild physical limitation (73% vs 40%; P<.001), mildly diminished quality of life (67% vs 31%; P<.001), and fair or poor overall health (66% vs 30%; P<.001). In multivariate analyses adjusting for measures of cardiac function and other patient characteristics, depressive symptoms were strongly associated with greater symptom burden (odds ratio [OR], 1.8; 95% confidence interval [CI], 1.3-2.7; P=.002), greater physical limitation (OR, 3.1; 95% CI, 2.1-4.6; P<.001), worse quality of life (OR, 3.1; 95% CI, 2.2-4.6; P<.001), and worse overall health (OR, 2.0; 95% CI, 1.3-2.9; P<.001). Although decreased exercise capacity was associated with worse health status, left ventricular ejection fraction and ischemia were not.
Conclusions
Among patients with coronary disease, depressive symptoms are strongly associated with patient-reported health status, including symptom burden, physical limitation, quality of life, and overall health. Conversely, 2 traditional measures of cardiac function—ejection fraction and ischemia—are not. Efforts to improve health status should include assessment and treatment of depressive symptoms.
doi:10.1001/jama.290.2.215
PMCID: PMC2776689  PMID: 12851276
23.  Relation of Self-Reported Angina Pectoris to Inducible Myocardial Ischemia in Patients With Known Coronary Artery Disease: The Heart and Soul Study 
To determine whether self-reported angina pectoris is associated with objective evidence of myocardial ischemia, we assessed angina symptoms, using the Seattle Angina Questionnaire, and measured ischemia using stress echocardiography in 933 patients with known coronary artery disease. We observed no association between self-reported angina pectoris and objective evidence of inducible ischemia.
PMCID: PMC2776686  PMID: 12972112
24.  Association between Renal Insufficiency and Inducible Ischemia in Patients with Coronary Artery Disease: The Heart and Soul Study 
Chronic renal insufficiency (CRI) is a predictor of stroke, cardiovascular, and all-cause mortality, but the mechanisms responsible for these associations are unclear. Whether CRI was associated with severity of coronary artery disease (CAD) as measured by exercise stress echocardiography among outpatients with stable CAD was evaluated. This study is a cross-sectional analysis of the Heart and Soul study, a prospective cohort of patients with known CAD. Renal function was assessed by 24-h urine collection, and CRI was defined as measured creatinine clearance ≤60 ml/min. Exercise stress echocardiography was used to identify inducible ischemia, defined as any wall motion abnormality seen at stress but not at rest. Logistic regression was used to evaluate the association of CRI with exercise-induced ischemia after adjustment for cardiovascular risk factors. Participants with CRI composed 97 (23%) of the 431 participants and were characterized by older age, worse CAD, lower ejection fraction, greater left ventricular mass and higher C-reactive protein values. The prevalence of exercise-induced ischemia was also substantially greater in the participants with CRI (42% versus 23%; odds ratio [OR], 2.3; 95% confidence interval [CI], 1.4 to 3.8; P < 0.001). This association was minimally changed by adjustment for traditional cardiovascular risk factors and coronary disease history (OR, 2.0; 95% CI, 1.3 to 3.3; P < 0.01) and remained strong even after adjustment for C-reactive protein (OR, 2.3; 95% CI, 1.0 to 5.1; P < 0.04). CRI is strongly associated with exercise-induced ischemia in patients with CAD. The greater severity of atherosclerotic disease observed in patients with CRI may in part explain the association of CRI with increased cardiovascular risk among individuals with CAD.
PMCID: PMC2770344  PMID: 14638921
25.  Methods for Estimating and Interpreting Provider-Specific Standardized Mortality Ratios 
Standardized Mortality Ratios (SMRs) are widely used as a measurement of quality of care for profiling and otherwise comparing medical care providers. Invalid estimation or inappropriate interpretation may have serious local and national consequences. Estimating an SMR entails producing provider-specific expected deaths via a statistical model and then computing the “observed/expected” ratio. Appropriate comparison of estimated SMRs requires considering both estimated values and statistical uncertainty. With statistical uncertainty that varies over providers, hypothesis testing to identify poor performers unfairly penalizes large providers; use of direct estimates unfairly penalizes small providers. Since neither approach suffices, we report on a suite of comparisons, each addressing an important aspect of the comparison. Our approach is based on a hierarchical statistical model. Goals include estimating and ranking (percentiling) provider-specific SMRs and calculating the probability that a provider's true SMR percentile falls within a specified percentile range. We present the issues and related statistical models for comparing SMRs and apply our approaches to the 1998 United States Renal Data System (USRDS) dialysis provider data.
doi:10.1023/B:HSOR.0000031400.77979.b6
PMCID: PMC2709867  PMID: 19606272
Standardized Mortality Ratio (SMR); Bayesian hierarchical models; percentile estimates; End-Stage Renal Disease (ESRD); United States Renal Data System (USRDS)

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