Ashok Khatri

Vpr Is Preferentially Targeted by CTL During HIV-1 Infection

The HIV-1 accessory proteins Vpr, Vpu, and Vif are essential for viral replication, and their cytoplasmic production suggests that they should be processed for recognition by CTLs. However, the extent to which these proteins are targeted in natural infection, as well as precise CTL epitopes within them, remains to be defined. In this study, CTL responses against HIV-1 Vpr, Vpu, and Vif were analyzed in 60 HIV-1-infected individuals and 10 HIV-1-negative controls using overlapping peptides spanning the entire proteins. Peptide-specific IFN-γ production was measured by ELISPOT assay and flow-based intracellular cytokine quantification. HLA class I restriction and cytotoxic activity were confirmed after isolation of peptide-specific CD8+ T cell lines. CD8+ T cell responses against Vpr, Vpu, and Vif were found in 45%, 2%, and 33% of HIV-1-infected individuals, respectively. Multiple CTL epitopes were identified in functionally important regions of HIV-1 Vpr and Vif. Moreover, in infected individuals in whom the breadth of HIV-1-specific responses was assessed comprehensively, Vpr and p17 were the most preferentially targeted proteins per unit length by CD8+ T cells. These data indicate that despite the small size of these proteins Vif and Vpr are frequently targeted by CTL in natural HIV-1 infection and contribute importantly to the total HIV-1-specific CD8+ T cell responses. These findings will be important in evaluating the specificity and breadth of immune responses during acute and chronic infection, and in the design and testing of candidate HIV vaccines.

Extensive HLA class I allele promiscuity among viral CTL epitopes

Promiscuous binding of T helper epitopes to MHC class II molecules has been well established, but few examples of promiscuous class I‐restricted epitopes exist. To address the extent of promiscuity of HLA class I peptides, responses to 242 well‐defined viral epitopes were tested in 100 subjects regardless of the individuals’ HLA type. Surprisingly, half of all detected responses were seen in the absence of the originally reported restricting HLA class I allele, and only 3% of epitopes were recognized exclusively in the presence of their original allele. Functional assays confirmed the frequent recognition of HLA class I‐restricted T cell epitopes on several alternative alleles across HLA class I supertypes and encoded on different class I loci. These data have significant implications for the understanding of MHC class I‐restricted antigen presentation and vaccine development.

CTL Responses of High Functional Avidity and Broad Variant Cross-Reactivity Are Associated with HIV Control

Cytotoxic T lymphocyte (CTL) responses targeting specific HIV proteins, in particular Gag, have been associated with relative control of viral replication in vivo. However, Gag-specific CTL can also be detected in individuals who do not control the virus and it remains thus unclear how Gag-specific CTL may mediate the beneficial effects in some individuals but not in others. Here, we used a 10mer peptide set spanning HIV Gag-p24 to determine immunogen-specific T-cell responses and to assess functional properties including functional avidity and cross-reactivity in 25 HIV-1 controllers and 25 non-controllers without protective HLA class I alleles. Our data challenge the common belief that Gag-specific T cell responses dominate the virus-specific immunity exclusively in HIV-1 controllers as both groups mounted responses of comparable breadths and magnitudes against the p24 sequence. However, responses in controllers reacted to lower antigen concentrations and recognized more epitope variants than responses in non-controllers. These cross-sectional data, largely independent of particular HLA genetics and generated using direct ex-vivo samples thus identify T cell responses of high functional avidity and with broad variant reactivity as potential functional immune correlates of relative HIV control.

GLP-1 (9–36) Amide, Cleavage Product of GLP-1 (7–36) Amide, Is a Glucoregulatory Peptide

Objective: Glucagon‐like peptide‐1 (GLP‐1) (7–36) amide is a glucoregulatory hormone with insulinotropic and insulinomimetic actions. We determined whether the insulinomimetic effects of GLP‐1 are mediated through its principal metabolite, GLP‐1 (9–36) amide (GLP‐1m).

Inhaled Hydrogen Sulfide Prevents Endotoxin-Induced Systemic Inflammation and Improves Survival by Altering Sulfide Metabolism in Mice

AIMS The role of hydrogen sulfide (H(2)S) in endotoxin (lipopolysaccharide [LPS])-induced inflammation is incompletely understood. We examined the impact of H(2)S breathing on LPS-induced changes in sulfide metabolism, systemic inflammation, and survival in mice. RESULTS Mice that breathed air alone exhibited decreased plasma sulfide levels and poor survival rate at 72 h after LPS challenge. Endotoxemia markedly increased alanine aminotransferase (ALT) activity and nitrite/nitrate (NOx) levels in plasma and lung myeloperoxidase (MPO) activity in mice that breathed air. In contrast, breathing air supplemented with 80 ppm of H(2)S for 6 h after LPS challenge markedly improved survival rate compared to mice that breathed air alone (p<0.05). H(2)S breathing attenuated LPS-induced increase of plasma ALT activity and NOx levels and lung MPO activity. Inhaled H(2)S suppressed LPS-induced upregulation of inflammatory cytokines, while it markedly induced anti-inflammatory interleukin (IL)-10 in the liver. Beneficial effects of H(2)S inhalation after LPS challenge were associated with restored sulfide levels and markedly increased thiosulfate levels in plasma. Increased thiosulfate levels after LPS challenge were associated with upregulation of rhodanese, but not cystathionine-γ-lyase (CSE), in the liver. Administration of sodium thiosulfate dose-dependently improved survival after LPS challenge in mice. INNOVATION By measuring changes in plasma levels of sulfide and sulfide metabolites using an advanced analytical method, this study revealed a critical role of thiosulfate in the protective effects of H(2)S breathing during endotoxemia. CONCLUSION These observations suggest that H(2)S breathing prevents inflammation and improves survival after LPS challenge by altering sulfide metabolism in mice.

A novel hydrogen sulfide-releasing N-methyl-D-aspartate receptor antagonist prevents ischemic neuronal death

Background: Hydrogen sulfide (H2S) exerts neuroprotective effects, whereas H2S may cause neurotoxicity via N-methyl-d-aspartate receptor (NMDAR) activation. Results: A newly-synthesized H2S-releasing NMDAR antagonist S-memantine exerted lower neurotoxicity and prevented ischemic neuronal death more markedly than conventional H2S-releasing compounds or memantine alone. Conclusion: S-memantine prevents ischemic brain injury without neurotoxicity. Significance: H2S-releasing NMDAR antagonists may prevent neurodegeneration of various causes. Physiological levels of H2S exert neuroprotective effects, whereas high concentrations of H2S may cause neurotoxicity in part via activation of NMDAR. To characterize the neuroprotective effects of combination of exogenous H2S and NMDAR antagonism, we synthesized a novel H2S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide (S-memantine) and examined its effects in vitro and in vivo. S-memantine was synthesized by chemically combining a slow releasing H2S donor 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48) with a NMDAR antagonist memantine. S-memantine increased intracellular sulfide levels in human neuroblastoma cells (SH-SY5Y) 10-fold as high as that was achieved by ACS48. Incubation with S-memantine after reoxygenation following oxygen and glucose deprivation (OGD) protected SH-SY5Y cells and murine primary cortical neurons more markedly than did ACS48 or memantine. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by sodium sulfide (Na2S) or ACS48, but suppressed by memantine and S-memantine. S-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y cells more markedly than did Na2S or ACS48. Administration of S-memantine after global cerebral ischemia and reperfusion more robustly decreased cerebral infarct volume and improved survival and neurological function of mice than did ACS48 or memantine. These results suggest that an H2S-releasing NMDAR antagonist derivative S-memantine prevents ischemic neuronal death, providing a novel therapeutic strategy for ischemic brain injury.

Endosomal GPCR signaling turned off by negative feedback actions of PKA and v-ATPase

The PTH receptor is one of the first GPCR found to sustain cAMP signaling after internalization of the ligand–receptor complex in endosomes. This unexpected model is adding a new dimension on how we think about GPCR signaling, but its mechanism is incompletely understood. We report here that endosomal acidification mediated by the PKA action on the v-ATPase provides a negative feedback mechanism by which endosomal receptor signaling is turned-off.

Molecular Imaging of Fibrin Deposition in Deep Vein Thrombosis Using Fibrin-Targeted Near-Infrared Fluorescence

OBJECTIVES The goal of this study was to develop and validate a new fibrin-targeted imaging agent that enables high-resolution near-infrared fluorescence (NIRF) imaging of deep vein thrombosis (DVT). BACKGROUND NIRF imaging of fibrin could enable highly sensitive and noninvasive molecular imaging of thrombosis syndromes in vivo. METHODS A fibrin-targeted peptide was conjugated to a near-infrared fluorophore Cy7, termed FTP11-Cy7. The NIRF peptide is based on a fibrin-specific imaging agent that has completed Phase II clinical magnetic resonance imaging trials. In vitro binding of FTP11-Cy7 to human plasma clots was assessed by using fluorescence reflectance imaging. Next, FTP11-Cy7 was intravenously injected in mice with femoral DVT induced by topical 7.5% ferric chloride treatment. Intravital fluorescence microscopy and noninvasive fluorescence molecular tomography-computed tomography were performed in 32 mice with DVT, followed by histological analyses. RESULTS In vitro human clot-binding analyses showed a 6-fold higher NIRF clot target-to-background ratio (TBR) of FTP11-Cy7 than free Cy7 (6.3 ± 0.34 vs. 1.2 ± 0.03; p < 0.0001). The thrombus TBR of acute and subacute femoral DVT with FTP11-Cy7 obtained by using intravital fluorescence microscopy was >400% higher than control free Cy7. Binding of FTP11-Cy7 to thrombi was blocked by a 100-fold excess of unlabeled competitor peptide both in vitro and in vivo (p < 0.001 for each). Histological analyses confirmed that FTP11-Cy7 specifically accumulated in thrombi. Noninvasive fluorescence molecular tomography-computed tomography imaging of fibrin in jugular DVT demonstrated strong NIRF signal in thrombi compared with sham-operated jugular veins (mean TBR 3.5 ± 0.7 vs. 1.5 ± 0.3; p < 0.05). CONCLUSIONS The fibrin-targeted NIRF agent FTP11-Cy7 was shown to avidly and specifically bind human and murine thrombi, and enable sensitive, multimodal intravital and noninvasive NIRF molecular imaging detection of acute and subacute murine DVT in vivo.

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH

Agonist-induced phosphorylation of the parathyroid hormone (PTH) receptor 1 (PTHR1) regulates receptor signaling in vitro, but the role of this phosphorylation in vivo is uncertain. We investigated this role by injecting “knock-in” mice expressing a phosphorylation-deficient (PD) PTHR1 with PTH ligands and assessing acute biologic responses. Following injection with PTH (1–34), or with a unique, long-acting PTH analog, PD mice, compared with WT mice, exhibited enhanced increases in cAMP levels in the blood, as well as enhanced cAMP production and gene expression responses in bone and kidney tissue. Surprisingly, however, the hallmark hypercalcemic and hypophosphatemic responses were markedly absent in the PD mice, such that paradoxical hypocalcemic and hyperphosphatemic responses were observed, quite strikingly with the long-acting PTH analog. Spot urine analyses revealed a marked defect in the capacity of the PD mice to excrete phosphate, as well as cAMP, into the urine in response to PTH injection. This defect in renal excretion was associated with a severe, PTH-induced impairment in glomerular filtration, as assessed by the rate of FITC-inulin clearance from the blood, which, in turn, was explainable by an overly exuberant systemic hypotensive response. The overall findings demonstrate the importance in vivo of PTH-induced phosphorylation of the PTHR1 in regulating acute ligand responses, and they serve to focus attention on mechanisms that underlie the acute calcemic response to PTH and factors, such as blood phosphate levels, that influence it.

A novel hydrogen sulfide-releasing NMDA receptor antagonist prevents ischemic neuronal death

Background: Hydrogen sulfide (H2S) exerts neuroprotective effects, whereas H2S may cause neurotoxicity via N-methyl-d-aspartate receptor (NMDAR) activation. Results: A newly-synthesized H2S-releasing NMDAR antagonist S-memantine exerted lower neurotoxicity and prevented ischemic neuronal death more markedly than conventional H2S-releasing compounds or memantine alone. Conclusion: S-memantine prevents ischemic brain injury without neurotoxicity. Significance: H2S-releasing NMDAR antagonists may prevent neurodegeneration of various causes. Physiological levels of H2S exert neuroprotective effects, whereas high concentrations of H2S may cause neurotoxicity in part via activation of NMDAR. To characterize the neuroprotective effects of combination of exogenous H2S and NMDAR antagonism, we synthesized a novel H2S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide (S-memantine) and examined its effects in vitro and in vivo. S-memantine was synthesized by chemically combining a slow releasing H2S donor 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48) with a NMDAR antagonist memantine. S-memantine increased intracellular sulfide levels in human neuroblastoma cells (SH-SY5Y) 10-fold as high as that was achieved by ACS48. Incubation with S-memantine after reoxygenation following oxygen and glucose deprivation (OGD) protected SH-SY5Y cells and murine primary cortical neurons more markedly than did ACS48 or memantine. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by sodium sulfide (Na2S) or ACS48, but suppressed by memantine and S-memantine. S-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y cells more markedly than did Na2S or ACS48. Administration of S-memantine after global cerebral ischemia and reperfusion more robustly decreased cerebral infarct volume and improved survival and neurological function of mice than did ACS48 or memantine. These results suggest that an H2S-releasing NMDAR antagonist derivative S-memantine prevents ischemic neuronal death, providing a novel therapeutic strategy for ischemic brain injury.