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2.  Long-lasting oral analgesic effects of N-protected aminophosphinic dual ENKephalinase inhibitors (DENKIs) in peripherally controlled pain 
The peripheral endogenous opioid system is critically involved in neuropathic and inflammatory pain generation as suggested by the modulation of opioid receptors expression and enkephalins (ENKs) release observed in these painful conditions. Accordingly, an innovative approach in the treatment of these nocifensive events is to increase and maintain high local concentrations of extracellular pain-evoked ENKs, by preventing their physiological enzymatic inactivation by two Zn metallopeptidases, the neutral endopeptidase (NEP, neprilysin, EC 3.4.24.11) and the neutral aminopeptidase (APN, EC 3.4.11.2). With this aim, new orally active dual ENKephalinase inhibitors (DENKIs) were designed as soluble prodrugs by introducing a N-terminal cleavable carbamate in the previously described aminophosphinic inhibitors. This induces long-lasting antinociceptive responses after oral administration, in various rodent models of inflammatory and neuropathic pain. These responses are mediated through stimulation of peripheral opioid receptors by DENKIs-protected ENKs as demonstrated by naloxone methiodide reversion. In all tested models, the most efficient prodrug 2a (PL265) was active, at least during 150–180 min, after single oral administration of 25–50 mg/kg in mice and of 100–200 mg/kg in rats. In models of neuropathic pain, both hyperalgesia and allodynia were markedly reduced. Interestingly, combination of inactive doses of 2a (PL265) and of the anti-epileptic drug gabapentin had synergistic effect on neuropathic pain. Pharmacokinetic studies of 2a (PL265) in rats show that the active drug is the only generated metabolite produced. These encouraging results have made 2a (PL265) a suitable candidate for clinical development.
doi:10.1002/prp2.116
PMCID: PMC4324690
Enkephalinase inhibitors; enkephalin-gabapentin analgesic synergy; enkephalins; inflammation; neuropathic pain; peripherally controlled pain; pharmacological assay; physiological analgesia
3.  Use of a Guinea Pig-Specific Transcriptome Array for Evaluation of Protective Immunity against Genital Chlamydial Infection following Intranasal Vaccination in Guinea Pigs 
PLoS ONE  2014;9(12):e114261.
Guinea pigs have been used as a second animal model to validate putative anti-chlamydial vaccine candidates tested in mice. However, the lack of guinea pig-specific reagents has limited the utility of this animal model in Chlamydia sp. vaccine studies. Using a novel guinea pig-specific transcriptome array, we determined correlates of protection in guinea pigs vaccinated with Chlamydia caviae (C. caviae) via the intranasal route, previously reported by us and others to provide robust antigen specific immunity against subsequent intravaginal challenge. C. caviae vaccinated guinea pigs resolved genital infection by day 3 post challenge. In contrast, mock vaccinated animals continued to shed viable Chlamydia up to day 18 post challenge. Importantly, at day 80 post challenge, vaccinated guinea pigs experienced significantly reduced genital pathology - a sequelae of genital chlamydial infections, in comparison to mock vaccinated guinea pigs. Sera from vaccinated guinea pigs displayed antigen specific IgG responses and increased IgG1 and IgG2 titers capable of neutralizing GPIC in vitro. Th1-cellular/inflammatory immune genes and Th2-humoral associated genes were also found to be elevated in vaccinated guinea pigs at day 3 post-challenge and correlated with early clearance of the bacterium. Overall, this study provides the first evidence of guinea pig-specific genes involved in anti-chlamydial vaccination and illustrates the enhancement of the utility of this animal model in chlamydial pathogenesis.
doi:10.1371/journal.pone.0114261
PMCID: PMC4263467  PMID: 25502875
4.  A T Cell Epitope-Based Vaccine Protects Against Chlamydial Infection in HLA-DR4 Transgenic Mice 
Vaccine  2013;31(48):10.1016/j.vaccine.2013.09.036.
Vaccination with recombinant chlamydial protease-like activity factor (rCPAF) has been shown to provide robust protection against genital Chlamydia infection. Adoptive transfer of IFN-γ competent CPAF-specific CD4+ T cells was sufficient to induce early resolution of chlamydial infection and reduction of subsequent pathology in recipient IFN-γ-deficient mice indicating the importance of IFN-γ secreting CD4+ T cells in host defense against Chlamydia. In this study, we identify CD4+ T cell reactive CPAF epitopes and characterize the activation of epitope-specific CD4+ T cells following antigen immunization or Chlamydia challenge. Using the HLA-DR4 (HLA-DRB1*0401) transgenic mouse for screening overlapping peptides that induced T cell IFN-γ production, we identified at least 5 CPAF T cell epitopes presented by the HLA-DR4 complex. Immunization of HLA-DR4 transgenic mice with a rCPAFep fusion protein containing these 5 epitopes induced a robust cell-mediated immune response and significantly accelerated the resolution of genital and pulmonary Chlamydia infection. rCPAFep vaccination induced CPAF-specific CD4+ T cells in the spleen were detected using HLA-DR4/CPAF-epitope tetramers. Additionally, CPAF-specific CD4+ clones could be detected in the mouse spleen following C. muridarum and a human C. trachomatis strain challenge using these novel tetramers. These results provide the first direct evidence that a novel CPAF epitope vaccine can provide protection and that HLA-DR4/CPAF-epitope tetramers can detect CPAF epitope-specific CD4+ T cells in HLA-DR4 mice following C. muridarum or C. trachomatis infection. Such tetramers could be a useful tool for monitoring CD4+ T cells in immunity to Chlamydia infection and in developing epitope-based human vaccines using the murine model.
doi:10.1016/j.vaccine.2013.09.036
PMCID: PMC3843363  PMID: 24096029
5.  IFN-γ Stimulated Human Umbilical-Tissue-Derived Cells Potently Suppress NK Activation and Resist NK-Mediated Cytotoxicity In Vitro 
Stem Cells and Development  2013;22(22):3003-3014.
Umbilical cord tissue represents a unique source of cells with potential for cell therapy applications for multiple diseases. Human umbilical tissue-derived cells (hUTC) are a developmentally early stage, homogenous population of cells that are HLA-ABC dim, HLA-DR negative, and lack expression of co-stimulatory molecules in the unactivated state. The lack of HLA-DR and co-stimulatory molecule expression on unactivated hUTC may account for their reduced immunogenicity, facilitating their use in allogeneic settings. However, such approaches could be confounded by host innate cells such as natural killer (NK) cells. Here, we evaluate in vitro NK cell interactions with hUTC and compare them with human mesenchymal stem cells (MSC). Our investigations show that hUTC suppress NK activation, through prostaglandin-E2 secretion in a contact-independent manner. Prestimulation of hUTC or human MSC with interferon gamma (IFN-γ) induced expression of the tryptophan degrading enzyme indoleamine 2, 3 dioxygenase, facilitating enhanced suppression. However, resting NK cells of different killer immunoglobulin-like receptor haplotypes did not kill hUTC or MSC; only activated NK cells had the ability to kill nonstimulated hUTC and, to a lesser extent, MSC. The cell killing process involved signaling through the NKG2D receptor and the perforin/granzyme pathway; this was supported by CD54 (ICAM-1) expression by hUTC. IFN-γ-stimulated hUTC or hMSC were less susceptible to NK killing; in this case, protection was associated with elevated HLA-ABC expression. These data delineate the different mechanisms in a two-way interaction between NK cells and two distinct cell therapies, hUTC or hMSC, and how these interactions may influence their clinical applications.
doi:10.1089/scd.2013.0028
PMCID: PMC3824722  PMID: 23795941
6.  Live Attenuated Francisella novicida Vaccine Protects against Francisella tularensis Pulmonary Challenge in Rats and Non-human Primates 
PLoS Pathogens  2014;10(10):e1004439.
Francisella tularensis causes the disease tularemia. Human pulmonary exposure to the most virulent form, F. tularensis subsp. tularensis (Ftt), leads to high morbidity and mortality, resulting in this bacterium being classified as a potential biothreat agent. However, a closely-related species, F. novicida, is avirulent in healthy humans. No tularemia vaccine is currently approved for human use. We demonstrate that a single dose vaccine of a live attenuated F. novicida strain (Fn iglD) protects against subsequent pulmonary challenge with Ftt using two different animal models, Fischer 344 rats and cynomolgus macaques (NHP). The Fn iglD vaccine showed protective efficacy in rats, as did a Ftt iglD vaccine, suggesting no disadvantage to utilizing the low human virulent Francisella species to induce protective immunity. Comparison of specific antibody profiles in vaccinated rat and NHP sera by proteome array identified a core set of immunodominant antigens in vaccinated animals. This is the first report of a defined live attenuated vaccine that demonstrates efficacy against pulmonary tularemia in a NHP, and indicates that the low human virulence F. novicida functions as an effective tularemia vaccine platform.
Author Summary
Francisella tularensis is a bacterium that causes the infectious disease tularemia. F. tularensis has been developed as a biothreat agent, because it causes high morbidity and mortality when spread by aerosol. There is currently no approved vaccine for human use, making mankind vulnerable to the illicit use of this organism. F. tularensis contains a cluster of genes in the Francisella Pathogenicity Island (FPI) that are required for replication inside host macrophages and virulence. In the current study we created a live vaccine strain by inactivating an FPI gene, iglD, in a closely-related species that does not cause disease in humans, F. novicida (Fn iglD). We demonstrate that vaccination with Fn iglD protects against exposure to airborne F. tularensis. Fn iglD vaccination induces antibody and cellular immune responses and protects two different animals, rats and non-human primates, against lethal pulmonary tularemia challenges. These two animal models reflect human sensitivity to F. tularensis. Our results suggest that a vaccine made from the low virulence F. novicida will protect humans against aerosol exposure to this dangerous pathogen.
doi:10.1371/journal.ppat.1004439
PMCID: PMC4207810  PMID: 25340543
7.  Cocaine Dysregulates Opioid Gating of GABA Neurotransmission in the Ventral Pallidum 
The Journal of Neuroscience  2014;34(3):1057-1066.
The ventral pallidum (VP) is a target of dense nucleus accumbens projections. Many of these projections coexpress GABA and the neuropeptide enkephalin, a δ and μ opioid receptor (MOR) ligand. Of these two, the MOR in the VP is known to be involved in reward-related behaviors, such as hedonic responses to palatable food, alcohol intake, and reinstatement of cocaine seeking. Stimulating MORs in the VP decreases extracellular GABA, indicating that the effects of MORs in the VP on cocaine seeking are via modulating GABA neurotransmission. Here, we use whole-cell patch-clamp on a rat model of withdrawal from cocaine self-administration to test the hypothesis that MORs presynaptically regulate GABA transmission in the VP and that cocaine withdrawal changes the interaction between MORs and GABA. We found that in cocaine-extinguished rats pharmacological activation of MORs no longer presynaptically inhibited GABA release, whereas blocking the MORs disinhibited GABA release. Moreover, MOR-dependent long-term depression of GABA neurotransmission in the VP was lost in cocaine-extinguished rats. Last, GABA neurotransmission was found to be tonically suppressed in cocaine-extinguished rats. These substantial synaptic changes indicated that cocaine was increasing tone on MOR receptors. Accordingly, increasing endogenous tone by blocking the enzymatic degradation of enkephalin inhibited GABA neurotransmission in yoked saline rats but not in cocaine-extinguished rats. In conclusion, our results indicate that following withdrawal from cocaine self-administration enkephalin levels in the VP are elevated and the opioid modulation of GABA neurotransmission is impaired. This may contribute to the difficulties withdrawn addicts experience when trying to resist relapse.
doi:10.1523/JNEUROSCI.4336-13.2014
PMCID: PMC3891949  PMID: 24431463
addiction; cocaine; electrophysiology; enkephalin; μ opioid receptor; ventral pallidum
8.  Mast cells: multitalented facilitators of protection against bacterial pathogens 
Mast cells are crucial effector cells evoking immune responses against bacterial pathogens. The positioning of mast cells at the host–environment interface, and the multitude of pathogen-recognition receptors and preformed mediator granules make these cells potentially the earliest to respond to an invading pathogen. In this review, the authors summarize the receptors used by mast cells to recognize invading bacteria and discuss the function of immune mediators released by mast cells in control of bacterial infection. The interaction of mast cells with other immune cells, including macrophages, dendritic cells and T cells, to induce protective immunity is highlighted. The authors also discuss mast cell-based vaccine strategies and the potential application in control of bacterial disease.
doi:10.1586/eci.12.95
PMCID: PMC4070289  PMID: 23390944
infection; mast cells; vaccine adjuvant
9.  Anaphylaxis and mortality induced by treatment of mice with anti-VLA-4 antibody and pertussis toxin 
Antibody-mediated blockade of the adhesion molecule very late antigen-4 (VLA-4) has been shown to ameliorate disease in human multiple sclerosis (MS) patients and experimental autoimmune encephalomyelitis (EAE) animal models. We wanted to determine whether anti-VLA-4 antibody treatment affected the function and persistence of autoreactive T cells in mice with EAE. Unexpectedly, we observed a high level of mortality in anti-VLA-4 mAb (PS/2) treated mice with actively induced EAE despite decreased disease severity. Investigation of the underlying mechanism showed that injection of PS/2 mAb in combination with pertussis toxin (PTX) resulted in anaphylaxis and mortality. Furthermore, the data showed that CD4+ T cells were required for this effect and suggested a role for IL-1β and TNF-α in the underlying pathology. The results reveal a previously not appreciated deleterious effect of anti-VLA-4 antibody treatment in combination with exposure to PTX.
doi:10.4049/jimmunol.1000907
PMCID: PMC4064569  PMID: 21270409
Pertussis toxin; anti-VLA-4; mice; EAE
10.  Mutational and Structural Analysis of KIR3DL1 Reveals a Lineage-Defining Allotypic Dimorphism That Impacts Both HLA and Peptide Sensitivity 
Killer Ig-like receptors (KIRs) control the activation of human NK cells via interactions with peptide-laden HLAs. KIR3DL1 is a highly polymorphic inhibitory receptor that recognizes a diverse array of HLA molecules expressing the Bw4 epitope, a group with multiple polymorphisms incorporating variants within the Bw4 motif. Genetic studies suggest that KIR3DL1 variation has functional significance in several disease states, including HIV infection. However, owing to differences across KIR3DL1 allotypes, HLA-Bw4, and associated peptides, the mechanistic link with biological outcome remains unclear. In this study, we elucidated the impact of KIR3DL1 polymorphism on peptide-laden HLA recognition. Mutational analysis revealed that KIR residues involved in water-mediated contacts with the HLA-presented peptide influence peptide binding specificity. In particular, residue 282 (glutamate) in the D2 domain underpins the lack of tolerance of negatively charged C-terminal peptide residues. Allotypic KIR3DL1 variants, defined by neighboring residue 283, displayed differential sensitivities to HLA-bound peptide, including the variable HLA-B*57:01–restricted HIV-1 Gag-derived epitope TW10. Residue 283, which has undergone positive selection during the evolution of human KIRs, also played a central role in Bw4 subtype recognition by KIR3DL1. Collectively, our findings uncover a common molecular regulator that controls HLA and peptide discrimination without participating directly in peptide-laden HLA interactions. Furthermore, they provide insight into the mechanics of interaction and generate simple, easily assessed criteria for the definition of KIR3DL1 functional groupings that will be relevant in many clinical applications, including bone marrow transplantation.
doi:10.4049/jimmunol.1303142
PMCID: PMC3948114  PMID: 24563253
11.  Genome Sequences of Four Acinetobacter baumannii-A. calcoaceticus Complex Isolates from Combat-Related Infections Sustained in the Middle East 
Genome Announcements  2014;2(1):e00026-14.
Acinetobacter baumannii is among the most prevalent bacterial causes of combat-related infections on the battlefield. Antibiotic resistance and a poor understanding of the protective host immune responses make treatment difficult. Here, we report the genome sequences of four clinical Acinetobacter baumannii-A. calcoaceticus complex isolates exhibiting significant differences in virulence in a mouse sepsis model.
doi:10.1128/genomeA.00026-14
PMCID: PMC3916481  PMID: 24503987
12.  Mesenchymal Stem Cell Osteodifferentiation in Response to Alternating Electric Current 
Tissue Engineering. Part A  2012;19(3-4):467-474.
The present study addressed adult human mesenchymal stem cell (MSC) differentiation toward the osteoblastic lineage in response to alternating electric current, a biophysical stimulus. For this purpose, MSCs (chosen because of their proven capability for osteodifferentiation in the presence of select bone morphogenetic proteins) were dispersed and cultured within electric-conducting type I collagen hydrogels, in the absence of supplemented exogenous dexamethasone and/or growth factors, and were exposed to either 10 or 40 μA alternating electric current for 6 h per day. Under these conditions, MSCs expressed both early- (such as Runx-2 and osterix) and late- (specifically, osteopontin and osteocalcin) osteogenic genes as a function of level, and duration of exposure to alternating electric current. Compared to results obtained after 7 days, gene expression of osteopontin and osteocalcin (late-osteogenic genes) increased at day 14. In contrast, expression of these osteogenic markers from MSCs cultured under similar conditions and time periods, but not exposed to alternating electric current, did not increase as a function of time. Most importantly, expression of genes pertinent to the either adipogenic (specifically, Fatty Acid Binding Protein-4) or chondrogenic (specifically, type II collagen) pathways was not detected when MSCs were exposed to the aforementioned alternating electric-current conditions tested in the present study. The present research study was the first to provide evidence that alternating electric current promoted the differentiation of adult human MSCs toward the osteogenic pathway. Such an approach has the yet untapped potential to provide critically needed differentiated cell supplies for cell-based assays and/or therapies for various biomedical applications.
doi:10.1089/ten.tea.2012.0091
PMCID: PMC3542886  PMID: 23083071
13.  Assessment of left ventricular volumes and primary mitral regurgitation severity by 2D echocardiography and cardiovascular magnetic resonance 
Background
Two-dimensional transthoracic echocardiography (2DTTE) remains the first-line diagnostic imaging tool to assess primary mitral regurgitation although cardiovascular magnetic resonance (CMR) has proven to establish left ventricular function more accurately and might evaluate mitral regurgitation severity more reliably. We sought to compare routine evaluation of left ventricular function and mitral regurgitation severity by 2DTTE with assessment by CMR in moderate to severe primary mitral regurgitation without overt left ventricular dysfunction.
Methods
We prospectively included 38 patients (79% of male, age 57 ± 14 years) with at least moderate primary mitral regurgitation, a left ventricular ejection fraction ≥60% and a left ventricular end-systolic diameter ≤45 mm. Patients with evidence of coronary artery disease, arrhythmias or significant concomitant valvular disease were excluded. All patients were scheduled for 2DTTE and CMR.
Results
Left ventricular end-diastolic and end-systolic volumes were significantly underestimated by 2DTTE in comparison with CMR, although there was a strong correlation (Pearson r = 0.81, p < 0.00001 and r = 0.7, p < 0.00001, respectively). Measurement of the regurgitant orifice was similar between 2DTTE PISA method and planimetry by CMR (47 ± 24 vs. 42 ± 16 mm2, p = 0.12) with a strong correlation between both imaging techniques (Pearson r = 0.76, p < 0.0001). By contrast, assessment of the regurgitant volume by 2DTTE and by phase contrast velocity mapping by CMR showed poor agreement.
Conclusions
In moderate to severe primary mitral regurgitation without overt left ventricular dysfunction, 2DTTE significantly underestimates left ventricular remodelling in comparison to CMR. Measurement of the regurgitant orifice with planimetry by CMR shows good agreement with the PISA method by 2DTTE and thus may be a valuable alternative to assess mitral regurgitation severity.
doi:10.1186/1476-7120-11-46
PMCID: PMC3880971  PMID: 24373138
Valve; Mitral regurgitation; Left ventricular function; Echocardiography; Cardiac magnetic resonance
14.  Looking Like a Leader–Facial Shape Predicts Perceived Height and Leadership Ability 
PLoS ONE  2013;8(12):e80957.
Judgments of leadership ability from face images predict the outcomes of actual political elections and are correlated with leadership success in the corporate world. The specific facial cues that people use to judge leadership remain unclear, however. Physical height is also associated with political and organizational success, raising the possibility that facial cues of height contribute to leadership perceptions. Consequently, we assessed whether cues to height exist in the face and, if so, whether they are associated with perception of leadership ability. We found that facial cues to perceived height had a strong relationship with perceived leadership ability. Furthermore, when allowed to manually manipulate faces, participants increased facial cues associated with perceived height in order to maximize leadership perception. A morphometric analysis of face shape revealed that structural facial masculinity was not responsible for the relationship between perceived height and perceived leadership ability. Given the prominence of facial appearance in making social judgments, facial cues to perceived height may have a significant influence on leadership selection.
doi:10.1371/journal.pone.0080957
PMCID: PMC3851990  PMID: 24324651
15.  Degenerate recognition of MHC class I molecules with Bw4 and Bw6 motifs by a KIR3DL expressed by macaque natural killer cells 
The killer cell immunoglobulin-like receptors (KIRs) expressed on the surface of natural killer (NK) cells recognize specific major histocompatibility complex class I (MHC-I) molecules and regulate NK cell activities against pathogen-infected cells and neoplasia. In human immunodeficiency virus (HIV) infection, survival is linked to host KIR and MHC-I genotypes. In the simian immunodeficiency virus (SIV) macaque model, however, the role of NK cells is unclear due to the lack of information on KIR-MHC interactions. Here, we describe the first characterization of a KIR-MHC interaction in pig-tailed macaques (Macaca nemestrina). Initially, we identified three distinct subsets of macaque NK cells that stained ex vivo with macaque MHC-I tetramers loaded with SIV peptides. We then cloned cDNAs corresponding to 15 distinct KIR3D alleles. One of these, KIR049-4, was an inhibitory KIR3DL that bound MHC-I tetramers and prevented activation, degranulation and cytokine production by macaque NK cells after engagement with specific MHC-I molecules on the surface of target cells. Furthermore, KIR049-4 recognized a broad range of MHC-I molecules carrying not only the Bw4 motif but also Bw6 and non-Bw4/Bw6 motifs. This degenerate, yet peptide-dependent, MHC reactivity differs markedly from the fine specificity of human KIRs.
doi:10.4049/jimmunol.1201360
PMCID: PMC3507536  PMID: 23041569
Natural Killer cells; comparative immunology/evolution; macaque; MHC; NK cell receptor
16.  Local and systemic innate immune response to secondary human peritonitis 
Critical Care  2013;17(5):R201.
Introduction
Our aim was to describe inflammatory cytokines response in the peritoneum and plasma of patients with peritonitis. We also tested the hypothesis that scenarios associated with worse outcome would result in different cytokine release patterns. Therefore, we compared cytokine responses according to the occurrence of septic shock, mortality, type of peritonitis and peritoneal microbiology.
Methods
Peritoneal and plasma cytokines (interleukin (IL) 1, tumor necrosis factor α (TNFα), IL-6, IL-10, and interferon γ (IFNγ)) were measured in 66 patients with secondary peritonitis.
Results
The concentration ratio between peritoneal fluid and plasma cytokines varied from 5 (2 to 21) (IFNγ) to 1310 (145 to 3888) (IL-1). There was no correlation between plasma and peritoneal fluid concentration of any cytokine. In the plasma, TNFα, IL-6, IFNγ and IL-10 were higher in patients with shock versus no shock and in nonsurvivors versus survivors (P ≤0.03). There was no differential plasma release for any cytokine between community-acquired and postoperative peritonitis. The presence of anaerobes or Enterococcus species in peritoneal fluid was associated with higher plasma TNFα: 50 (37 to 106) versus 38 (29 to 66) and 45 (36 to 87) versus 39 (27 to 67) pg/ml, respectively (P = 0.02). In the peritoneal compartment, occurrence of shock did not result in any difference in peritoneal cytokines. Peritoneal IL-10 was higher in patients who survived (1505 (450 to 3130) versus 102 (9 to 710) pg/ml; P = 0.04). The presence of anaerobes and Enterococcus species was associated with higher peritoneal IFNγ: 2 (1 to 6) versus 10 (5 to 28) and 7 (2 to 39) versus 2 (1 to 6), P = 0.01 and 0.05, respectively).
Conclusions
Peritonitis triggers an acute systemic and peritoneal innate immune response with a simultaneous release of pro and anti-inflammatory cytokines. Higher levels of all cytokines were observed in the plasma of patients with the most severe conditions (shock, non-survivors), but this difference was not reflected in their peritoneal fluid. There was always a large gradient in cytokine concentration between peritoneal and plasma compartments highlighting the importance of compartmentalization of innate immune response in peritonitis.
doi:10.1186/cc12895
PMCID: PMC4057228  PMID: 24028733
18.  Preclinical Characterization of GS-9669, a Thumb Site II Inhibitor of the Hepatitis C Virus NS5B Polymerase 
GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC50) of ≤11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.)
doi:10.1128/AAC.02052-12
PMCID: PMC3553741  PMID: 23183437
19.  Killer Immunoglobulin Receptor 3DL1-mediated recognition of Human Leukocyte Antigen B 
Nature  2011;479(7373):401-405.
Members of the Killer Immunoglobulin-Like Receptor (KIR) family, a large group of polymorphic receptors expressed on Natural Killer (NK) cells, recognise particular peptide-laden Human Leukocyte Antigen (pHLA) class I molecules and play a pivotal role in innate immune responses1. Allelic variation and extensive polymorphism within the three-domain KIR family (KIR3D, domains D0–D1–D2) affects pHLA binding specificity and is linked to the control of viral replication and the treatment outcome of certain haematological malignancies1–3. We describe the structure of the KIR3DL1 receptor, bound to HLA-B*5701 complexed with a self-peptide. KIR3DL1 clamped around the C-terminal end of the HLA-B*5701 antigen (Ag)-binding cleft, resulting in two discontinuous footprints on the pHLA. Firstly, the D0 domain, a distinguishing feature of the KIR3D family, extended towards β2-microglobulin and abutted a region of the HLA molecule that exhibited limited polymorphism, thereby acting as an “innate HLA sensor” domain. Secondly, while the D2-HLA-B*5701 interface exhibited a high degree of complementarity, the D1-pHLA-B*5701 contacts were sub-optimal and accommodated a degree of sequence variation both within the peptide and the polymorphic region of the HLA molecule. While the two-domain KIR (KIR2D) and KIR3DL1 docked similarly onto HLA-C4,5 and HLA-B respectively, the corresponding D1-mediated interactions differed markedly, thereby providing insight into the specificity of KIR3DL1 for discrete HLA-A and HLA-B allotypes. Collectively, in association with extensive mutagenesis studies at the KIR3DL1-pHLA B*5701 interface, we provide a framework for understanding the intricate interplay between peptide variability, KIR3D and HLA polymorphism in determining the specificity requirements of this essential innate interaction that is conserved across primate species.
doi:10.1038/nature10517
PMCID: PMC3723390  PMID: 22020283
20.  Mast cell toll-like receptor 2 signaling is crucial for effective killing of Francisella tularensis1 
Toll-like receptor (TLR) signaling is critical for early host defense against pathogens, but the contribution of mast cell TLR-mediated mechanisms and subsequent effector functions during pulmonary infection is largely unknown. We have previously demonstrated that mast cells, through the production of IL-4, effectively control Francisella tularensis replication. In this study, the highly human virulent strain of F. tularensis SCHU S4 and the Live Vaccine Strain (LVS) were utilized to investigate the contribution of mast cell-TLR regulation of Francisella. Mast cells required TLR2 for effective bacterial killing, regulation of the hydrolytic enzyme cathepsin L, and for coordination and trafficking of MHCII and lysosomal associated membrane protein 2 (LAMP2). Infected TLR2−/− mast cells, in contrast to WT and TLR4−/−, lacked detectable IL-4 and displayed increased cell death with a 2–3 log increase of F. tularensis replication, but could be rescued with recombinant IL-4 treatment. Importantly, MHCII and LAMP2 localization with labeled F. tularensis in the lungs was greater in WT than in TLR2−/− mice. These results provide evidence for the important effector contribution of mast cells and TLR2-mediated signaling on early innate processes in the lung following pulmonary F. tularensis infection and provide additional insight into possible mechanisms by which intracellular pathogens modulate respiratory immune defenses.
doi:10.4049/jimmunol.1200039
PMCID: PMC3358431  PMID: 22529298
21.  Development of Neurological Disease Is Associated with Increased Immune Activation in Simian Immunodeficiency Virus-Infected Macaques 
Journal of Virology  2012;86(24):13795-13799.
Simian immunodeficiency virus (SIV) infection of macaques can result in central nervous system disorders, such as meningitis and encephalitis. We studied 10 animals inoculated with brain-derived virus from animals with SIV encephalitis. Over half of the macaques developed SIV-induced neurologic disease. Elevated levels of systemic immune activation were observed to correlate with viral RNA in the cerebral spinal fluid but not with plasma viral load, consistent with a role for SIV in the pathogenesis of neurologic disease.
doi:10.1128/JVI.02174-12
PMCID: PMC3503031  PMID: 23035225
22.  Identification of the peptide-binding motif recognized by the pigtail macaque class I MHC molecule Mane-A1*082:01 (Mane A*0301) 
Immunogenetics  2012;64(6):461-468.
Rhesus and pigtail macaques have proven to be valuable animal models for several important human diseases, including HIV, where they exhibit similar pathology and disease progression. Because rhesus macaques have been extensively characterized in terms of their major histocompatibility complex (MHC) class I alleles, their demand has soared, making them increasingly difficult to obtain for research purposes. This problem has been exacerbated by a continued export ban in place since 1978. Pigtail macaques represent a potential alternative animal model. However, because their MHC class I alleles have not been characterized in detail, their use has been hindered. To address this, in the present study, we have characterized the peptide binding specificity of the pigtail macaque class I allele Mane-A1*082:01 (formerly known as Mane A*0301), representative of the second most common MHC class I antigen detected across several cohorts. The motif was defined on the basis of binding studies utilizing purified MHC protein and panels of single amino acid substitution analog peptides, as well as sequences of peptide ligands eluted from Mane- A1*082:01. Based on these analyses, Mane-A1*082:01 was found to recognize a motif with H in position 2 and the aromatic residues F and Y, or the hydrophobic/aliphatic residue M, at the C-terminus. Finally, analysis of the binding of a combinatorial peptide library allowed the generation of a detailed quantitative motif that proved effective in the prediction of a set of high-affinity binders derived from chimeric SIV/HIV, an important model virus for studying HIV infection in humans.
doi:10.1007/s00251-012-0600-x
PMCID: PMC3626442  PMID: 22278177
MHC; Macaques; Peptide binding motif; T cell epitope; Class I
23.  Intranasal Vaccination with Chlamydia pneumoniae Induces Cross-Species Immunity against Genital Chlamydia muridarum Challenge in Mice 
PLoS ONE  2013;8(5):e64917.
Chlamydia trachomatis is the most common bacterial sexually transmitted disease in the world and specifically in the United States, with the highest incidence in age-groups 14–19 years. In a subset of females, the C. trachomatis genital infection leads to serious pathological sequelae including pelvic inflammatory disease, ectopic pregnancy, and infertility. Chlamydia pneumoniae, another member of the same genus, is a common cause of community acquired respiratory infection with significant number of children aged 5–14 yr displaying sero-conversion. Since these bacteriae share several antigenic determinants, we evaluated whether intranasal immunization with live C. pneumoniae (1×106 inclusion forming units; IFU) in 5 week old female C57BL/6 mice would induce cross-species protection against subsequent intravaginal challenge with Chlamydia muridarum (5×104 IFU), which causes a similar genital infection and pathology in mice as C. trachomatis in humans. Mice vaccinated intranasally with live C. pneumoniae, but not mock (PBS) immunized animals, displayed high levels of splenic cellular antigen-specific IFN-γ production and serum antibody response against C. muridarum and C. trachomatis. Mice vaccinated with C. pneumoniae displayed a significant reduction in the vaginal C. muridarum shedding as early as day 12 after secondary i.vag. challenge compared to PBS (mock) immunized mice. At day 19 after C. muridarum challenge, 100% of C. pneumoniae vaccinated mice had cleared the infection compared to none (0%) of the mock immunized mice, which cleared the infection by day 27. At day 80 after C. muridarum challenge, C. pneumoniae vaccinated mice displayed a significant reduction in the incidence (50%) and degree of hydrosalpinx compared to mock immunized animals (100%). These results suggest that respiratory C. pneumoniae infection induces accelerated chlamydial clearance and reduction of oviduct pathology following genital C. muridarum challenge, and may have important implications to the C. trachomatis-induced reproductive disease in humans.
doi:10.1371/journal.pone.0064917
PMCID: PMC3669087  PMID: 23741420
24.  Dried Plum’s Unique Capacity to Reverse Bone Loss and Alter Bone Metabolism in Postmenopausal Osteoporosis Model 
PLoS ONE  2013;8(3):e60569.
Interest in dried plum has increased over the past decade due to its promise in restoring bone and preventing bone loss in animal models of osteoporosis. This study compared the effects of dried plum on bone to other dried fruits and further explored the potential mechanisms of action through which dried plum may exert its osteoprotective effects. Adult osteopenic ovariectomized (OVX) C57BL/6 mice were fed either a control diet or a diet supplemented with 25% (w/w) dried plum, apple, apricot, grape or mango for 8 weeks. Whole body and spine bone mineral density improved in mice consuming the dried plum, apricot and grape diets compared to the OVX control mice, but dried plum was the only fruit to have an anabolic effect on trabecular bone in the vertebra and prevent bone loss in the tibia. Restoration of biomechanical properties occurred in conjunction with the changes in trabecular bone in the spine. Compared to other dried fruits in this study, dried plum was unique in its ability to down-regulate osteoclast differentiation coincident with up-regulating osteoblast and glutathione (GPx) activity. These alterations in bone metabolism and antioxidant status compared to other dried fruits provide insight into dried plum’s unique effects on bone.
doi:10.1371/journal.pone.0060569
PMCID: PMC3612052  PMID: 23555991
25.  Australian Diabetes Foot Network: practical guideline on the provision of footwear for people with diabetes 
Trauma, in the form of pressure and/or friction from footwear, is a common cause of foot ulceration in people with diabetes. These practical recommendations regarding the provision of footwear for people with diabetes were agreed upon following review of existing position statements and clinical guidelines. The aim of this process was not to re-invent existing guidelines but to provide practical guidance for health professionals on how they can best deliver these recommendations within the Australian health system. Where information was lacking or inconsistent, a consensus was reached following discussion by all authors. Appropriately prescribed footwear, used alone or in conjunction with custom-made foot orthoses, can reduce pedal pressures and reduce the risk of foot ulceration. It is important for all health professionals involved in the care of people with diabetes to both assess and make recommendations on the footwear needs of their clients or to refer to health professionals with such skills and knowledge. Individuals with more complex footwear needs (for example those who require custom-made medical grade footwear and orthoses) should be referred to health professionals with experience in the prescription of these modalities and who are able to provide appropriate and timely follow-up. Where financial disadvantage is a barrier to individuals acquiring appropriate footwear, health care professionals should be aware of state and territory based equipment funding schemes that can provide financial assistance. Aboriginal and Torres Strait Islanders and people living in rural and remote areas are likely to have limited access to a broad range of footwear. Provision of appropriate footwear to people with diabetes in these communities needs be addressed as part of a comprehensive national strategy to reduce the burden of diabetes and its complications on the health system.
doi:10.1186/1757-1146-6-6
PMCID: PMC3599221  PMID: 23442978
Footwear; Diabetes; Foot; Guideline; Australia

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