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1.  Falling up the stairs: the equivalent of ‘bashing it with a bible’ for an ACL ganglion cyst of the knee 
BMJ Case Reports  2012;2012:bcr0120125591.
Intra-articular anterior cruciate ligament (ACL) cysts are rare, the pathogenesis remains unknown, with trauma often implicated. Often asymptomatic, incidental MRI findings, 11% produce symptoms such as pain, locking or instability. Treatment of intra-articular ganglia differs from the traditional ‘bash it with a bible’ mantra for ganglia elsewhere with surgical debridement generally indicated for symptomatic cases. This case report describes a 43-year-old male car mechanic who presented with a symptomatic ACL cyst diagnosed on MRI. While waiting for surgery the patient fell up his stairs at home, causing forced hyperflexion of his knee. After an initial sharp pain, within 24 h the patient experienced complete resolution of symptoms. Postfall MRI showed no evidence of the initial lesion, leading to our conclusion that for this patient, a fall up the stairs was the equivalent of ‘bashing it with a bible’ for an ACL ganglion cyst of the knee.
PMCID: PMC3316796  PMID: 22605799
2.  The Role of Fear of Movement in Subacute Whiplash-Associated Disorders Grades I and II 
Pain  2012;154(3):393-401.
Fear and avoidance of activity may play a role in fostering disability in whiplash-associated disorders (WAD). This study examined the role of fear following WAD and assessed the effectiveness of three treatments targeting fear. People still symptomatic from WAD Grade I–II injuries approximately 3 months previously (n = 191) completed questionnaires (e.g., Neck Disability Index [NDI]) and were randomized to one of the treatments: (1) informational booklet (IB) describing WAD and the importance of resuming activities; (2) IB + didactic discussions (DD) with clinicians reinforcing the booklet; and (3) IB + imaginal and direct exposure desensitization (ET) to feared activities. DD and ET participants received three 2-hour treatment sessions. Absolute improvements in NDI were in predicted direction (ET = 14.7, DD = 11.9, IB = 9.9). ETs reported significantly less post-treatment pain severity, compared to the IB (M = 1.5 vs. 2.3, p < 0.001, d = 0.6) and DD (M = 1.5 vs. 2.0, p = 0.039, d = 0.6) groups. Reduction in fear was the most important predictor of improvement in NDI (β = 0.30, p < 0.001), followed by reductions in pain (β = 0.20, p = 0.003) and depression (β = 0.18, p = 0.004). The mediational analysis confirmed that fear reduction significantly mediated the effect of treatment group on outcome. Results highlight the importance of fear in individuals with subacute WAD, and suggest the importance of addressing fear, via exposure therapy and/or educational interventions, to improve function.
PMCID: PMC3582794  PMID: 23318127
3.  Topological Analysis of HIV-1 Glycoproteins Expressed In Situ on Virus Surfaces Reveals Tighter Packing but Greater Conformational Flexibility than for Soluble gp120 
Journal of Virology  2013;87(16):9233-9249.
In natural infection, antibodies interact with HIV-1 primarily through nonfunctional forms of envelope glycoproteins (Env), including uncleaved (UNC) gp160 and gp41 stumps. These antigens are important to fully characterize, as they may be decoys that promote nonneutralizing responses and may also be targets for nonneutralizing effector responses. In this study, we compared the antigenic properties of Env expressed in situ on pseudovirion virus-like particle (VLP) surfaces and soluble gp120 using harmonized enzyme-linked immunosorbent assays (ELISAs) and a panel of 51 monoclonal antibodies (MAbs). Only 32 of 46 soluble gp120-reactive MAbs recognized the primary UNC gp160 antigen of VLPs. Indeed, many epitopes were poorly exposed (C1, V2, C1-C4, C4, C4-V3, CD4 induced [CD4i], and PGT group 3) or obscured (C2, C5, and C1-C5) on VLPs. In further studies, VLP Env exhibited an increased degree of inter-MAb competition, the epicenter of which was the base of the V3 loop, where PGT, 2G12, V3, and CD4 binding site specificities competed. UNC gp160 also underwent more drastic soluble CD4 (sCD4)-induced conformational changes than soluble gp120, exposing CD4i, C1-C4, and V2 epitopes. A greater propensity of UNC gp160 to undergo conformational changes was also suggested by the induction of CD4i MAb binding to VLPs by a V3 MAb as well as by soluble CD4. The same effect was not observed for soluble gp120. Taken together, our data suggest that membrane-expressed UNC gp160 exists in a less “triggered” conformational state than soluble gp120 and that MAb binding to UNC gp160 tends to have greater conformational consequences.
PMCID: PMC3754051  PMID: 23740975
4.  Report from the Immunogenomic Data Analysis Working Group (IDAWG) 16th International HLA and Immunogenetics Workshop (IHIW) Project: Immunogenomic Data-Management Methods 
The goal of the IDAWG is to facilitate the consistent analysis of HLA and KIR data, and the sharing of those data among the immunogenomic and larger genomic communities. However, the data-management approaches currently applied by immunogenomic researchers are not widely discussed or reported in the literature, and the effect of different approaches on data-analyses is not known.
With ASHI’s support, the IDAWG developed a forty-five question survey on HLA and KIR data-generation, data-management, and data-analysis practices. Survey questions detailed the loci genotyped, typing systems used, nomenclature versions reported, computer operating systems and software used to manage and transmit data, the approaches applied to resolve HLA ambiguity, and the methods used for basic population-level analyses. Respondents were invited to demonstrate their HLA ambiguity resolution approaches in simulated data sets.By May 2012, 156 respondents from 35 nations had completed the survey . These survey respondents represent a broad sampling of the Immunogenomic community; 52% were European, 30% North American, 10% Asian, 4% South American, and 4% from the Pacific.
The project will continue in conjunction with the 17th Workshop, with the aim of developing community data-sharing standards, ambiguity resolution documentation formats, single-task data-Management tools, and, novel data-analysis methods and applications. While additional project details and plans for the 17th IHIW will be forthcoming, we welcome the input and participation in these projects from the histocompatibility and immunogenetics community.
PMCID: PMC3789600  PMID: 23280068
Meta-analysis; Statistics; HLA; Genetics
5.  Mutations causing medullary cystic kidney disease type 1 (MCKD1) lie in a large VNTR in MUC1 missed by massively parallel sequencing 
Nature genetics  2013;45(3):299-303.
While genetic lesions responsible for some Mendelian disorders can be rapidly discovered through massively parallel sequencing (MPS) of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple Mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing, and de novo assembly, we found that each of six MCKD1 families harbors an equivalent, but apparently independently arising, mutation in sequence dramatically underrepresented in MPS data: the insertion of a single C in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5-5 kb), GC-rich (>80%), coding VNTR in the mucin 1 gene. The results provide a cautionary tale about the challenges in identifying genes responsible for Mendelian, let alone more complex, disorders through MPS.
PMCID: PMC3901305  PMID: 23396133
6.  Increased plasma levels of soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1) in women by moderate exercise and increased plasma levels of VEGF in overweight/obese women 
The incidence of breast cancer is increasing worldwide, and this seems to be related to an increase in lifestyle risk factors, including physical inactivity, and overweight/obesity. We previously reported that exercise induced a circulating angiostatic phenotype characterized by increased sFlt-1 and endostatin and decreased unbound-VEGF in men. However, there is no data on women. The present study determines the following: 1) whether moderate exercise increased sFlt-1 and endostatin and decreased unbound-VEGF in the circulation of adult female volunteers; 2) whether overweight/obese women have a higher plasma level of unbound-VEGF than lean women. 72 African American and Caucasian adult women volunteers aged from 18–44 were enrolled into the exercise study. All the participants walked on a treadmill for 30 minutes at a moderate intensity (55–59% heart rate reserve), and oxygen consumption (VO2) was quantified by utilizing a metabolic cart. We had the blood samples before and immediately after exercise from 63 participants. ELISA assays (R&D Systems) showed that plasma levels of sFlt-1 were 67.8±3.7 pg/ml immediately after exercise (30 minutes), significantly higher than basal levels, 54.5±3.3 pg/ml, before exercise (P < 0.01; n=63). There was no significant difference in the % increase of sFlt-1 levels after exercise between African American and Caucasian (P=0.533) or between lean and overweight/obese women (P=0.892). There was no significant difference in plasma levels of unbound VEGF (35.28±5.47 vs. 35.23±4.96 pg/ml; P=0.99) or endostatin (111.12±5.48 vs. 115.45±7.15 ng/ml; P=0.63) before and after exercise. Basal plasma levels of unbound-VEGF in overweight/obese women were 52.26±9.6 pg/ml, significantly higher than basal levels of unbound-VEGF in lean women, 27.34±4.99 pg/ml (P < 0.05). The results support our hypothesis that exercise-induced plasma levels of sFlt-1 could be an important clinical biomarker to explore the mechanisms of exercise training in reducing breast cancer progression and that VEGF is an important biomarker in obesity and obesity-related cancer progression.
PMCID: PMC3449013  PMID: 22609636
Exercise; Young adult women; Overweight/obese; sFlt-1; Endostatin; VEGF
7.  A high throughput Chromatin ImmunoPrecipitation approach reveals principles of dynamic gene regulation in mammals 
Molecular cell  2012;47(5):10.1016/j.molcel.2012.07.030.
Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in-vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.
PMCID: PMC3873101  PMID: 22940246
8.  Variation in Hospital Costs, Payments, and Profitabilty for Cardiac Valve Replacement Surgery 
Health Services Research  2011;46(6 Pt 1):1928-1945.
Examine the variation for Medicare and privately insured patients in hospital costs, payments, and contribution margins and their association with characteristics of the patients, hospitals, and hospital markets.
Data Sources
Administrative records for 1,858 patients undergoing cardiac valve replacement surgery were obtained from 37 hospitals in 7 states for 2008.
Study Design
Bivariate and multivariate statistical analyses of costs, payments, and profitability (contribution margin) for Medicare and privately insured patients, adjusting for patient, hospital, and market characteristics.
Data Collection
Integrated Health Care Association, Aspen Health Metrics, American Hospital Association Annual Survey of Hospitals.
Principal Findings
Cardiac valve replacement surgery is an expensive but profitable procedure, with average cost and contribution margin per case of U.S.$38,667 and U.S.$21,967, respectively. Average costs per case for Medicare patients are 16.1 percent higher in concentrated than in competitive local markets after adjusting for patient comorbidities, complications, and other relevant factors (p<.01). Payments per case were 33.2 percent (p<.01) lower from Medicare than from private insurers. The average contribution margin earned by hospitals from Medicare was U.S.$30,986 lower than the margin earned from private insurers (p<.01), after adjusting for patient, hospital, and market characteristics.
Hospitals charge significantly higher prices and earn significantly higher contribution margins from private insurers than from Medicare for patients undergoing cardiac valve replacement.
PMCID: PMC3393019  PMID: 21762141
Hospital costs and prices; Medicare cost shifting; health care reform; prospective payment; insurance
9.  Compression of Structured High-Throughput Sequencing Data 
PLoS ONE  2013;8(11):e79871.
Large biological datasets are being produced at a rapid pace and create substantial storage challenges, particularly in the domain of high-throughput sequencing (HTS). Most approaches currently used to store HTS data are either unable to quickly adapt to the requirements of new sequencing or analysis methods (because they do not support schema evolution), or fail to provide state of the art compression of the datasets. We have devised new approaches to store HTS data that support seamless data schema evolution and compress datasets substantially better than existing approaches. Building on these new approaches, we discuss and demonstrate how a multi-tier data organization can dramatically reduce the storage, computational and network burden of collecting, analyzing, and archiving large sequencing datasets. For instance, we show that spliced RNA-Seq alignments can be stored in less than 4% the size of a BAM file with perfect data fidelity. Compared to the previous compression state of the art, these methods reduce dataset size more than 40% when storing exome, gene expression or DNA methylation datasets. The approaches have been integrated in a comprehensive suite of software tools ( that support common analyses for a range of high-throughput sequencing assays.
PMCID: PMC3832420  PMID: 24260313
10.  Alcohol decreases RhoA activity through a Nitric Oxide (NO)/Cyclic GMP(cGMP)/Protein Kinase G (PKG)-dependent pathway in the airway epithelium 
Alcoholism, clinical and experimental research  2011;35(7):10.1111/j.1530-0277.2011.01463.x.
Alcohol has been shown to have a number of harmful effects on the lung, including increasing the risk of pneumonia and bronchitis. How alcohol increases the risk of these diseases is poorly defined. RhoA is a small guanosine triphosphate (GTP)ase that plays an integral role in many basic functions of airway epithelial cells. It is not known how alcohol affects RhoA activity in the airway epithelium. We hypothesized that brief alcohol exposure modulates RhoA activity in the airway epithelium through a nitric oxide (NO)/Cyclic GMP (cGMP)/Protein Kinase G (PKG) dependent pathway.
Primary airway epithelial cells were cultured and exposed to ethanol at various concentrations and times. The cell layers were harvested and RhoA activity was measured.
Alcohol induced a time- and concentration-dependent decrease in RhoA activity in airway epithelial cells. We were able to block this decrease in activity using Nω-Nitro-l-arginine methyl ester hydrochloride (L-NAME), a nitric oxide synthase (NOS) inhibitor. Likewise, we were able to demonstrate the same decrease in RhoA activation using 0.1µM sodium nitroprusside (SNP), an NO donor. To determine the role of cGMP/PKG, we pretreated the cells with a cGMP antagonist analogue, Rp-8Br-cGMPS. This blocked the decrease in RhoA activity caused by alcohol, suggesting that alcohol exerts its effect on RhoA activity through cGMP/PKG.
Alcohol decreases airway epithelial RhoA activity through an NO/cGMP/PKG- dependent pathway. RhoA activity controls many aspects of basic cellular function, including cell morphology, tight junction formation, and cell cycle progression and gene regulation. Dysregulation of RhoA activity can potentially have several consequences, including dysregulation of inflammation. This may partially explain how alcohol increases the risk of pneumonia and bronchitis.
PMCID: PMC3821532  PMID: 21410486
ethanol; nitric oxide; PKG; cGMP; airway epithelium
11.  Heterogeneity in Neutralization Sensitivities of Viruses Comprising the Simian Immunodeficiency Virus SIVsmE660 Isolate and Vaccine Challenge Stock 
Journal of Virology  2013;87(10):5477-5492.
The sooty mangabey-derived simian immunodeficiency virus (SIV) strain E660 (SIVsmE660) is a genetically heterogeneous, pathogenic isolate that is commonly used as a vaccine challenge strain in the nonhuman primate (NHP) model of human immunodeficiency virus type 1 (HIV-1) infection. Though it is often employed to assess antibody-based vaccine strategies, its sensitivity to antibody-mediated neutralization has not been well characterized. Here, we utilize single-genome sequencing and infectivity assays to analyze the neutralization sensitivity of the uncloned SIVsmE660 isolate, individual viruses comprising the isolate, and transmitted/founder (T/F) viruses arising from low-dose mucosal inoculation of macaques with the isolate. We found that the SIVsmE660 isolate overall was highly sensitive to neutralization by SIV-infected macaque plasma samples (50% inhibitory concentration [IC50] < 10−5) and monoclonal antibodies targeting V3 (IC50 < 0.01 μg/ml), CD4-induced (IC50 < 0.1 μg/ml), CD4 binding site (IC50 ∼ 1 μg/ml), and V4 (IC50, ∼5 μg/ml) epitopes. In comparison, SIVmac251 and SIVmac239 were highly resistant to neutralization by these same antibodies. Differences in neutralization sensitivity between SIVsmE660 and SIVmac251/239 were not dependent on the cell type in which virus was produced or tested. These findings indicate that in comparison to SIVmac251/239 and primary HIV-1 viruses, SIVsmE660 generally exhibits substantially less masking of antigenically conserved Env epitopes. Interestingly, we identified a minor population of viruses (∼10%) in both the SIVsmE660 isolate and T/F viruses arising from it that were substantially more resistant (>1,000-fold) to antibody neutralization and another fraction (∼20%) that was intermediate in neutralization resistance. These findings may explain the variable natural history and variable protection afforded by heterologous Env-based vaccines in rhesus macaques challenged by high-dose versus low-dose SIVsmE660 inoculation regimens.
PMCID: PMC3648171  PMID: 23468494
12.  Akt Signaling Accelerates Tumor Recurrence Following Ras Inhibition in the Context of Ink4a/Arf Loss 
Genes & Cancer  2013;4(11-12):476-485.
Aberrant activation of the RAS signaling pathway contributes to nearly all human cancers, including gliomas. To determine the dependence of high-grade gliomas on this signaling pathway, we developed a doxycycline-regulated KRas glioma mouse model. Using this model we previously demonstrated that inhibition of KRas expression in gliomas induced by activated KRas and Akt results in complete tumor regression. We have also shown that, in the context of Ink4a/Arf loss, abrogation of KRas signaling is sufficient to decrease tumor burden but resistance ensues. In this study, we sought to determine the effect of activated Akt signaling in combination with activated KRas and loss of Ink4a/Arf on the growth and recurrence of brain tumors following suppression of KRas expression. We observed significant tumor formation in Ink4a/Arflox/lox mice injected with retroviruses containing tetracycline responsive element (TRE)-KRas, Tet-off, Akt, and Cre. Abrogation of KRas signaling resulted in significant tumor regression; however, resistance developed after a relatively short latency. Tumor recurrence occurred more rapidly and the tumors were more aggressive in the presence of activated Akt signaling compared with loss of Ink4a/Arf alone suggesting that this pathway contributes to tumor progression in this context.
PMCID: PMC3877666  PMID: 24386508
Akt; Ras; Ink4a/Arf; glioma; mouse
13.  Metabolic and transcriptional response to a high-fat diet in Drosophila melanogaster★ 
Molecular Metabolism  2013;3(1):42-54.
Obesity has dramatically increased in prevalence, making it essential to understand its accompanying metabolic changes. Modeling diet-induced obesity in Drosophila melanogaster (fruit flies), we elucidated transcriptional and metabolic changes in w1118 flies on a high-fat diet (HFD). Mass spectrometry-based metabolomics revealed altered fatty acid, amino acid, and carbohydrate metabolism with HFD. Microarray analysis uncovered transcriptional changes in nitrogen metabolism, including CG9510, homolog of human argininosuccinate lyase (ASL). CG9510 knockdown in flies phenocopied traits observed with HFD, namely increased triglyceride levels and decreased cold tolerance. Restoration of CG9510 expression ameliorated observed negative consequences of HFD. Metabolomic analysis of CG9510 knockdown flies confirmed functional similarity to ASL, regulating the balance of carbon and nitrogen metabolism. In summary, we found that HFD suppresses CG9510 expression, a gene required for proper triglyceride storage and stress tolerance. These results draw an important link between regulation of amino acid metabolism and the response to diet-induced obesity.
PMCID: PMC3929909  PMID: 24567903
AcCoA, acetyl-coenzyme A; arm-GAL4, armadillo-GAL4; ASL, argininosuccinate lyase; BCAA, branch chain amino acid; CAFE, capillary feeder; da-GAL4, daughterless-Gal4; EASE, Expression Analysis Systematic Explorer (DAVID analysis); FAME, fatty acid methyl ester; Fdr, false discovery rate; GC/MS, gas chromatography/mass spectrometry; HFD, high-fat Diet; MeOH, methanol; PCR, polymerase chain reaction; RT-PCR, reverse-transcriptase PCR; TBDMS, tert-butyldimethylsilyl; TCA, tricarboxylic acid; TG, triglyceride; TMS, trimethylsilyl; VDRC, Vienna Drosophila RNAi Center; w1118, white-1118; Argininosuccinate lyase; Lifespan; Metabolism; Obesity; Triglyceride
14.  Designing CXCL8-based decoy proteins with strong anti-inflammatory activity in vivo 
Bioscience Reports  2013;33(5):e00068.
IL (interleukin)-8 [CXCL8 (CXC chemokine ligand 8)] exerts its role in inflammation by triggering neutrophils via its specific GPCRs (G-protein-coupled receptors), CXCR1 (CXC chemokine receptor 1) and CXCR2, for which additional binding to endothelial HS-GAGs (heparan sulphate-glycosaminoglycans) is required. We present here a novel approach for blocking the CXCL8-related inflammatory cascade by generating dominant-negative CXCL8 mutants with improved GAG-binding affinity and knocked-out CXCR1/CXCR2 activity. These non-signalling CXCL8 decoy proteins are able to displace WT (wild-type) CXCL8 and to prevent CXCR1/CXCR2 signalling thereby interfering with the inflammatory response. We have designed 14 CXCL8 mutants that we subdivided into three classes according to number and site of mutations. The decoys were characterized by IFTs (isothermal fluorescence titrations) and SPR (surface plasmon resonance) to determine GAG affinity. Protein stability and structural changes were evaluated by far-UV CD spectroscopy and knocked-out GPCR response was shown by Boyden chamber and Ca2+ release assays. From these experiments, CXCL8(Δ6F17KF21KE70KN71K) emerged with the most promising in vitro characteristics. This mutant was therefore further investigated in a murine model of mBSA (methylated BSA)-induced arthritis in mice where it showed strong anti-inflammatory activity. Based on these results, we propose that dominant-negative CXCL8 decoy proteins are a promising class of novel biopharmaceuticals with high therapeutic potential in inflammatory diseases.
PMCID: PMC3775513  PMID: 23919527
anti-inflammatory; chemokine decoys; competition; GAG affinity; heparan sulphate; AIA, antigen induced arthritis; CCL, CC chemokine ligand; CS, chondroitin sulphate; CXCL, CXC chemokine ligand; CXCR, CXC chemokine receptor; GAG, glycosaminoglycan; GPCR, G-protein-coupled receptor; HBSS, Hanks balanced salt solution; HS, heparan sulphate; IFT, isothermal fluorescence titration; IL, interleukin; Kd, dissociation constant; KC, keratinocyte chemoattractant; LMWH, low molecular weight heparin; mBSA, methylated BSA; MIP, macrophage inflammatory protein; MPO, myeloperoxidase; RA, rheumatoid arthritis; SP, sulphopropyl; SPR, surface plasmon resonance; TFA, trifluoroacetic acid; TMB, tetramethylbenzidine; UFMG, Universidade Federal de Minas Gerais; WT, wild-type
16.  Interspecific synchrony of seabird population growth rate and breeding success 
Ecology and Evolution  2013;3(7):2013-2019.
Environmental variability can destabilize communities by causing correlated interspecific fluctuations that weaken the portfolio effect, yet evidence of such a mechanism is rare in natural systems. Here, we ask whether the population dynamics of similar sympatric species of a seabird breeding community are synchronized, and if these species have similar exceptional responses to environmental variation. We used a 24-year time series of the breeding success and population growth rate of a marine top predator species group to assess the degree of synchrony between species demography. We then developed a novel method to examine the species group – all species combined – response to environmental variability, in particular, whether multiple species experience similar, pronounced fluctuations in their demography. Multiple species were positively correlated in breeding success and growth rate. Evidence of “exceptional” years was found, where the species group experienced pronounced fluctuations in their demography. The synchronous response of the species group was negatively correlated with winter sea surface temperature of the preceding year for both growth rate and breeding success. We present evidence for synchronous, exceptional responses of a species group that are driven by environmental variation. Such species covariation destabilizes communities by reducing the portfolio effect, and such exceptional responses may increase the risk of a state change in this community. Our understanding of the future responses to environmental change requires an increased focus on the short-term fluctuations in demography that are driven by extreme environmental variability.
PMCID: PMC3728942  PMID: 23919147
Community variability; environmental forcing; extreme events; positive correlation; stability
17.  Impact of Fluoroquinolone Resistance Mutations on Gonococcal Fitness and In Vivo Selection for Compensatory Mutations 
The Journal of Infectious Diseases  2012;205(12):1821-1829.
Background. Quinolone-resistant Neisseria gonorrhoeae (QRNG) arise from mutations in gyrA (intermediate resistance) or gyrA and parC (resistance). Here we tested the consequence of commonly isolated gyrA91/95 and parC86 mutations on gonococcal fitness.
Methods. Mutant gyrA91/95 and parC86 alleles were introduced into wild-type gonococci or an isogenic mutant that is resistant to macrolides due to an mtrR−79 mutation. Wild-type and mutant bacteria were compared for growth in vitro and in competitive murine infection.
Results. In vitro growth was reduced with increasing numbers of mutations. Interestingly, the gyrA91/95 mutation conferred an in vivo fitness benefit to wild-type and mtrR−79 mutant gonococci. The gyrA91/95, parC86 mutant, in contrast, showed a slight fitness defect in vivo, and the gyrA91/95, parC86, mtrR−79 mutant was markedly less fit relative to the parent strains. A ciprofloxacin-resistant (CipR) mutant was selected during infection with the gyrA91/95, parC86, mtrR−79 mutant in which the mtrR−79 mutation was repaired and the gyrA91 mutation was altered. This in vivo–selected mutant grew as well as the wild-type strain in vitro.
Conclusions. gyrA91/95 mutations may contribute to the spread of QRNG. Further acquisition of a parC86 mutation abrogates this fitness advantage; however, compensatory mutations can occur that restore in vivo fitness and maintain CipR.
PMCID: PMC3415892  PMID: 22492860
18.  Shaping T Cell – B Cell Collaboration in the Response to Human Immunodeficiency Virus Type 1 Envelope Glycoprotein gp120 by Peptide Priming 
PLoS ONE  2013;8(6):e65748.
Prime-boost vaccination regimes have shown promise for obtaining protective immunity to HIV. Poorly understood mechanisms of cellular immunity could be responsible for improved humoral responses. Although CD4+ T-cell help promotes B-cell development, the relationship of CD4+ T-cell specificity to antibody specificity has not been systematically investigated. Here, protein and peptide-specific immune responses to HIV-1 gp120 were characterized in groups of ten mucosally immunized BALB/c mice. Protein and peptide reactivity of serum antibody was tested for correlation with cytokine secretion by splenocytes restimulated with individual gp120 peptides. Antibody titer for gp120 correlated poorly with the peptide-stimulated T-cell response. In contrast, titers for conformational epitopes, measured as crossreactivity or CD4-blocking, correlated with average interleukin-2 and interleukin-5 production in response to gp120 peptides. Antibodies specific for conformational epitopes and individual gp120 peptides typically correlated with T-cell responses to several peptides. In order to modify the specificity of immune responses, animals were primed with a gp120 peptide prior to immunization with protein. Priming induced distinct peptide-specific correlations of antibodies and T-cells. The majority of correlated antibodies were specific for the primed peptides or other peptides nearby in the gp120 sequence. These studies suggest that the dominant B-cell subsets recruit the dominant T-cell subsets and that T-B collaborations can be shaped by epitope-specific priming.
PMCID: PMC3679139  PMID: 23776539
19.  Epitope Mapping of Broadly Neutralizing HIV-2 Human Monoclonal Antibodies 
Journal of Virology  2012;86(22):12115-12128.
Recent studies have shown that natural infection by HIV-2 leads to the elicitation of high titers of broadly neutralizing antibodies (NAbs) against primary HIV-2 strains (T. I. de Silva, et al., J. Virol. 86:930–946, 2012; R. Kong, et al., J. Virol. 86:947–960, 2012; G. Ozkaya Sahin, et al., J. Virol. 86:961–971, 2012). Here, we describe the envelope (Env) binding and neutralization properties of 15 anti-HIV-2 human monoclonal antibodies (MAbs), 14 of which were newly generated from 9 chronically infected subjects. All 15 MAbs bound specifically to HIV-2 gp120 monomers and neutralized heterologous primary virus strains HIV-27312A and HIV-2ST. Ten of 15 MAbs neutralized a third heterologous primary virus strain, HIV-2UC1. The median 50% inhibitory concentrations (IC50s) for these MAbs were surprisingly low, ranging from 0.007 to 0.028 μg/ml. Competitive Env binding studies revealed three MAb competition groups: CG-I, CG-II, and CG-III. Using peptide scanning, site-directed mutagenesis, chimeric Env constructions, and single-cycle virus neutralization assays, we mapped the epitope of CG-I antibodies to a linear region in variable loop 3 (V3), the epitope of CG-II antibodies to a conformational region centered on the carboxy terminus of V4, and the epitope(s) of CG-III antibodies to conformational regions associated with CD4- and coreceptor-binding sites. HIV-2 Env is thus highly immunogenic in vivo and elicits antibodies having diverse epitope specificities, high potency, and wide breadth. In contrast to the HIV-1 Env trimer, which is generally well shielded from antibody binding and neutralization, HIV-2 is surprisingly vulnerable to broadly reactive NAbs. The availability of 15 human MAbs targeting diverse HIV-2 Env epitopes can facilitate comparative studies of HIV/SIV Env structure, function, antigenicity, and immunogenicity.
PMCID: PMC3486499  PMID: 22933274
20.  Viral Escape from Neutralizing Antibodies in Early Subtype A HIV-1 Infection Drives an Increase in Autologous Neutralization Breadth 
PLoS Pathogens  2013;9(2):e1003173.
Antibodies that neutralize (nAbs) genetically diverse HIV-1 strains have been recovered from a subset of HIV-1 infected subjects during chronic infection. Exact mechanisms that expand the otherwise narrow neutralization capacity observed during early infection are, however, currently undefined. Here we characterized the earliest nAb responses in a subtype A HIV-1 infected Rwandan seroconverter who later developed moderate cross-clade nAb breadth, using (i) envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants, (ii) longitudinal autologous plasma, and (iii) autologous monoclonal antibodies (mAbs). Initially, nAbs targeted a single region of gp120, which flanked the V3 domain and involved the alpha2 helix. A single amino acid change at one of three positions in this region conferred early escape. One immunoglobulin heavy chain and two light chains recovered from autologous B cells comprised two mAbs, 19.3H-L1 and 19.3H-L3, which neutralized the founder Env along with one or three of the early escape variants carrying these mutations, respectively. Neither mAb neutralized later nAb escape or heterologous Envs. Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions. Resistance to mAb neutralization arose in later Envs through alteration of two glycans spatially adjacent to the initial escape signatures. The cross-neutralizing nAbs that ultimately developed failed to target any of the defined V3-proximal changes generated during the first year of infection in this subject. Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env. This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.
Author Summary
Since cases were first recognized in the United States in 1981, human immunodeficiency virus (HIV-1) has infected over one million Americans. Globally, this scale reaches into the tens of millions, but no effective vaccine exists. Of those infected, approximately 20–30% of patients will develop broadly neutralizing antibodies. The reasons for maturation of these potentially protective responses are presently unknown, but being able to elicit such antibodies via vaccination could curb the pandemic. Here, we defined the earliest neutralizing antibody targets and the consequent routes of viral escape in one subtype A HIV-1 infected subject who developed modest breadth. We also determined the genetic and structural characteristics of early neutralizing monoclonal antibodies circulating in this subject and found that subtle light chain alteration enhanced target contact and neutralization. Overall, our data support the idea that exposure to a specific sequence of viral variants, which have escaped from immune pressure, could program long-term potential for antibody breadth.
PMCID: PMC3585129  PMID: 23468623
21.  Antiviral Resistance and Correlates of Virologic Failure in the first Cohort of HIV-Infected Children Gaining Access to Structured Antiretroviral Therapy in Lima, Peru: A Cross-Sectional Analysis 
The impact of extended use of ART in developing countries has been enormous. A thorough understanding of all factors contributing to the success of antiretroviral therapy is required. The current study aims to investigate the value of cross-sectional drug resistance monitoring using DNA and RNA oligonucleotide ligation assays (OLA) in treatment cohorts in low-resource settings. The study was conducted in the first cohort of children gaining access to structured ART in Peru.
Between 2002–5, 46 eligible children started the standard regimen of AZT, 3TC and NFV Patients had a median age of 5.6 years (range: 0.7-14y), a median viral load of 1.7·105 RNA/ml (range: 2.1·103 – 1.2·106), and a median CD4-count of 232 cells/μL (range: 1–1591). Of these, 20 patients were classified as CDC clinical category C and 31/46 as CDC immune category 3. At the time of cross-sectional analysis in 2005, adherence questionnaires were administered. DNA OLAs and RNA OLAs were performed from frozen PBMC and plasma, RNA genotyping from dried blood spots.
During the first year of ART, 44% of children experienced virologic failure, with an additional 9% failing by the end of the second year. Virologic failure was significantly associated with the number of resistance mutations detected by DNA-OLA (p < 0.001) during cross-sectional analysis, but also with low immunologic CDC-scores at baseline (p < 0.001). Children who had been exposed to unsupervised short-term antiretrovirals before starting structured ART showed significantly higher numbers of resistance mutations by DNA-OLA (p = 0.01). Detection of M184V (3TC resistance) by RNA-OLA and DNA-OLA demonstrated a sensitivity of 0.93 and 0.86 and specificity of 0.67 and 0.7, respectively, for the identification of virologic failure. The RT mutations N88D and L90M (NFV resistance) detected by DNA-OLA correlated with virologic failure, whereas mutations at RT position 215 (AZT resistance) were not associated with virologic failure.
Advanced immunosuppression at baseline and previous exposures to unsupervised brief cycles of ART significantly impaired treatment outcomes at a time when structured ART was finally introduced in his cohort. Brief maternal exposures to with AZT +/− NVP for the prevention of mother-to-child transmission did not affect treatment outcomes in this group of children. DNA-OLA from frozen PBMC provided a highly specific tool to detect archived drug resistance. RNA consensus genotyping from dried blood spots and RNA-OLA from plasma consistently detected drug resistance mutations, but merely in association with virologic failure.
PMCID: PMC3782360  PMID: 23280237
22.  Ink4a/Arf loss promotes tumor recurrence following Ras inhibition 
Neuro-Oncology  2011;14(1):34-42.
Aberrant activation of rat sarcoma (Ras) signaling contributes to the development of a variety of human cancers, including gliomas. To determine the dependence of high-grade gliomas on continued Ras signaling, we developed a doxycycline-regulated Kirsten Ras (KRas) glioma mouse model. We previously demonstrated that KRas is required for the maintenance of glioblastoma multiforme tumors arising in the context of activated Akt signaling in vivo; inhibition of KRas expression resulted in apoptotic tumor regression and significantly increased survival. We utilized a well-established glioma mouse model to determine the reliance of gliomas on continued KRas signaling in the context of Ink4a/Arf deficiency, a common occurrence in human gliomas. Despite the dependency of primary gliomas on continued KRas signaling, a significant percentage of tumors progressed to a KRas-independent state in the absence of Ink4a/Arf expression, demonstrating that these tumor suppressors play a critical role in the suppression of glioma recurrence. While even advanced stages of gliomas may remain dependent upon KRas signaling for maintenance and growth, our findings demonstrate that loss of Ink4a/Arf facilitates the acquisition of oncogene independence and tumor recurrence. Furthermore, reactivation of the Ras mitogen-activated protein kinase pathway in the absence of virally delivered KRas expression is a common mechanism of recurrence in this context.
PMCID: PMC3245997  PMID: 22015595
glioma; Ink4a/Arf; mouse; Ras; tumor maintenance
23.  Neutralization Resistance of Virological Synapse-Mediated HIV-1 Infection Is Regulated by the gp41 Cytoplasmic Tail 
Journal of Virology  2012;86(14):7484-7495.
Human immunodeficiency virus type 1 (HIV-1) infection can spread efficiently from infected to uninfected T cells through adhesive contacts called virological synapses (VSs). In this process, cell-surface envelope glycoprotein (Env) initiates adhesion and viral transfer into an uninfected recipient cell. Previous studies have found some HIV-1-neutralizing patient sera to be less effective at blocking VS-mediated infection than infection with cell-free virus. Here we employ sensitive flow cytometry-based infection assays to measure the inhibitory potency of HIV-1-neutralizing monoclonal antibodies (MAb) and HIV-1-neutralizing patient sera against cell-free and VS-mediated infection. To various degrees, anti-Env MAbs exhibited significantly higher 50% inhibitory concentration (IC50s) against VS-mediated infection than cell-free infection. Notably, the MAb 17b, which binds a CD4-induced (CD4i) epitope on gp120, displayed a 72-fold reduced efficacy against VS-mediated inocula compared to cell-free inocula. A mutant with truncation mutation in the gp41 cytoplasmic tail (CT) which is unable to modulate Env fusogenicity in response to virus particle maturation but which can still engage in cell-to-cell infection was tested for the ability to resist neutralizing antibodies. The ΔCT mutation increased cell surface staining by neutralizing antibodies, significantly enhanced neutralization of VS-mediated infection, and had reduced or no effect on cell-free infection, depending upon the antibody. Our results suggest that the gp41 CT regulates the exposure of key neutralizing epitopes during cell-to-cell infection and plays an important role in immune evasion. Vaccine strategies should consider immunogens that reflect Env conformations exposed on the infected cell surface to enhance protection against VS-mediated HIV-1 spread.
PMCID: PMC3416307  PMID: 22553332
24.  Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop 
Journal of Virology  2013;87(1):52-66.
There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.
PMCID: PMC3536401  PMID: 23077306
25.  IPD—the Immuno Polymorphism Database 
Nucleic Acids Research  2012;41(D1):D1234-D1240.
The Immuno Polymorphism Database (IPD), is a set of specialist databases related to the study of polymorphic genes in the immune system. The IPD project works with specialist groups or nomenclature committees who provide and curate individual sections before they are submitted to IPD for online publication. The IPD project stores all the data in a set of related databases. IPD currently consists of four databases: IPD-KIR, contains the allelic sequences of killer-cell immunoglobulin-like receptors, IPD-MHC, a database of sequences of the major histocompatibility complex of different species; IPD-HPA, alloantigens expressed only on platelets; and IPD-ESTDAB, which provides access to the European Searchable Tumour Cell-Line Database, a cell bank of immunologically characterized melanoma cell lines. The data is currently available online from the website and FTP directory. This article describes the latest updates and additional tools added to the IPD project.
PMCID: PMC3531162  PMID: 23180793

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