HIV-1 with gag processing defects activates cGAS sensing.

BACKGROUND: Detection of viruses by host pattern recognition receptors induces the expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), which suppress viral replication. Numerous studies have described HIV-1 as a poor activator of innate immunity in vitro. The exact role that the viral capsid plays in this immune evasion is not fully understood. RESULTS: To better understand the role of the HIV-1 capsid in sensing we tested the effect of making HIV-1 by co-expressing a truncated Gag that encodes the first 107 amino acids of capsid fused with luciferase or GFP, alongside wild type Gag-pol. We found that unlike wild type HIV-1, viral particles produced with a mixture of wild type and truncated Gag fused to luciferase or GFP induced a potent IFN response in THP-1 cells and macrophages. Innate immune activation by Gag-fusion HIV-1 was dependent on reverse transcription and DNA sensor cGAS, suggesting activation of an IFN response by viral DNA. Further investigation revealed incorporation of the Gag-luciferase/GFP fusion proteins into viral particles that correlated with subtle defects in wild type Gag cleavage and a diminished capacity to saturate restriction factor TRIM5α, likely due to aberrant particle formation. We propose that expression of the Gag fusion protein disturbs the correct cleavage and maturation of wild type Gag, yielding viral particles that are unable to effectively shield viral DNA from detection by innate sensors including cGAS. CONCLUSIONS: These data highlight the crucial role of capsid in innate evasion and support growing literature that disruption of Gag cleavage and capsid formation induces a viral DNA- and cGAS-dependent innate immune response. Together these data demonstrate a protective role for capsid and suggest that antiviral activity of capsid-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.

Lytic efficiency of immunosuppressive drug-resistant armoured T cells against circulating HBV-related HCC in whole blood.

Recurrence of hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) after liver transplant (LT) is mediated by circulating tumour cells (CTCs) and exacerbated by the immunosuppressants required to prevent graft rejection. To circumvent the effects of immunosuppressants, we developed immunosuppressive drug-resistant armoured HBV-specific T-cell receptor-redirected T cells (IDRA HBV-TCR). However, their ability to eliminate HBV-HCC circulating in the whole blood has never been tested, and whether their lytic efficacy is compatible with the number of adoptively transferred T cells in vivo has never been measured. Hence, we developed a microscopy-based assay to quantify CTCs in whole blood. The assay was then used to quantify the efficacy of IDRA HBV-TCRs to lyse free-floating HBV-HCC cells in the presence of Tacrolimus and Mycophenolate Mofetil (MMF). We demonstrated that a panel of antibodies (AFP, GPC3, Vimentin, pan-Cytokeratin, and CD45) specific for HCC tumour antigens and immune cells can effectively differentiate HCC-CTCs in whole blood. Through dose-titration experiments, we observed that in the presence of immunosuppressive drugs, a minimum of 20 000 IDRA HBV-TCR T cells/ml of whole blood is necessary to lyse ~63.5% of free-floating HBV-HCC cells within 16 hours. In conclusion, IDRA HBV-TCR T cells can lyse free-floating HBV-HCC cells in whole blood in the presence of Tacrolimus and MMF. The quantity of IDRA-HBV TCR T cells required can be achieved by the adoptive transfer of 5 × 106 IDRA-HBV TCR-T cells/kg, supporting the utilisation of IDRA HBV-TCR T cells to eliminate CTCs as prophylaxis against recurrence after LT.

Neuromagnetic evidence of abnormal automatic inhibitory function in subjective memory complaint.

Subjective memory complaint (SMC), a self-perceived worsening in memory capacity concurrent with normal performance on standardized cognitive assessments, is considered a risk factor for the development of Alzheimer's disease (AD). Deficient sensory gating (SG), referring to the lack of automatic inhibition of neural responses to the second identical stimulus, has been documented in prodromal and incident AD patients. However, it remains unknown whether the cognitively normal elderly with SMC demonstrate alterations of SG function compared with those without SMC. A total of 19 healthy controls (HC) and 16 SMC subjects were included in the present study. Neural responses to the auditory paired-stimulus paradigm were recorded by the magnetoencephalography and analyzed by the distributed source imaging method of minimum norm estimate. The SG of M50 and M100 components were measured using the amplitude ratio of the second response over the first response at the cortical level. Compared to HC, subjects with SMC showed significantly increased M50 SG ratios in the inferior parietal lobule (IPL). Furthermore, M50 SG ratios in the right IPL yielded an acceptable discriminative ability to distinguish SMC from HC. However, we did not find a significant association between SG ratios and cognitive function requiring inhibitory control either in the HC or SMC group. In conclusion, although SMC subjects have intact cognitive functioning revealed by objective neuropsychological tests, their deficits in automatic inhibitory function could be detected through neurophysiological recordings. Our results suggest that altered brain function occurs in SMC prior to the obvious decline of cognitive performance.

Immunosuppressive Drug-Resistant Armored T-Cell Receptor T Cells for Immune Therapy of HCC in Liver Transplant Patients.

BACKGROUND AND AIMS: HBV-specific T-cell receptor (HBV-TCR) engineered T cells have the potential for treating HCC relapses after liver transplantation, but their efficacy can be hampered by the concomitant immunosuppressive treatment required to prevent graft rejection. Our aim is to molecularly engineer TCR-T cells that could retain their polyfunctionality in such patients while minimizing the associated risk of organ rejection. APPROACH AND RESULTS: We first analyzed how immunosuppressive drugs can interfere with the in vivo function of TCR-T cells in liver transplanted patients with HBV-HCC recurrence receiving HBV-TCR T cells and in vitro in the presence of clinically relevant concentrations of immunosuppressive tacrolimus (TAC) and mycophenolate mofetil (MMF). Immunosuppressive Drug Resistant Armored TCR-T cells of desired specificity (HBV or Epstein-Barr virus) were then engineered by concomitantly electroporating mRNA encoding specific TCRs and mutated variants of calcineurin B (CnB) and inosine-5'-monophosphate dehydrogenase (IMPDH), and their function was assessed through intracellular cytokine staining and cytotoxicity assays in the presence of TAC and MMF. Liver transplanted HBV-HCC patients receiving different immunosuppressant drugs exhibited varying levels of activated (CD39+ Ki67+ ) peripheral blood mononuclear cells after HBV-TCR T-cell infusions that positively correlate with clinical efficacy. In vitro experiments with TAC and MMF showed a potent inhibition of TCR-T cell polyfunctionality. This inhibition can be effectively negated by the transient overexpression of mutated variants of CnB and IMPDH. Importantly, the resistance only lasted for 3-5 days, after which sensitivity was restored. CONCLUSIONS: We engineered TCR-T cells of desired specificities that transiently escape the immunosuppressive effects of TAC and MMF. This finding has important clinical applications for the treatment of HBV-HCC relapses and other pathologies occurring in organ transplanted patients.

Effect of Age in Auditory Go/No-Go Tasks: A Magnetoencephalographic Study.

Response inhibition is frequently examined using visual go/no-go tasks. Recently, the auditory go/no-go paradigm has been also applied to several clinical and aging populations. However, age-related changes in the neural underpinnings of auditory go/no-go tasks are yet to be elucidated. We used magnetoencephalography combined with distributed source imaging methods to examine age-associated changes in neural responses to auditory no-go stimuli. Additionally, we compared the performance of high- and low-performing older adults to explore differences in cortical activation. Behavioral performance in terms of response inhibition was similar in younger and older adult groups. Relative to the younger adults, the older adults exhibited reduced cortical activation in the superior and middle temporal gyrus. However, we did not find any significant differences in cortical activation between the high- and low-performing older adults. Our results therefore support the hypothesis that inhibition is reduced during aging. The variation in cognitive performance among older adults confirms the need for further study on the underlying mechanisms of inhibition.

SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.

Memory T cells induced by previous pathogens can shape susceptibility to, and the clinical severity of, subsequent infections1. Little is known about the presence in humans of pre-existing memory T cells that have the potential to recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we studied T cell responses against the structural (nucleocapsid (N) protein) and non-structural (NSP7 and NSP13 of ORF1) regions of SARS-CoV-2 in individuals convalescing from coronavirus disease 2019 (COVID-19) (n = 36). In all of these individuals, we found CD4 and CD8 T cells that recognized multiple regions of the N protein. Next, we showed that patients (n = 23) who recovered from SARS (the disease associated with SARS-CoV infection) possess long-lasting memory T cells that are reactive to the N protein of SARS-CoV 17 years after the outbreak of SARS in 2003; these T cells displayed robust cross-reactivity to the N protein of SARS-CoV-2. We also detected SARS-CoV-2-specific T cells in individuals with no history of SARS, COVID-19 or contact with individuals who had SARS and/or COVID-19 (n = 37). SARS-CoV-2-specific T cells in uninfected donors exhibited a different pattern of immunodominance, and frequently targeted NSP7 and NSP13 as well as the N protein. Epitope characterization of NSP7-specific T cells showed the recognition of protein fragments that are conserved among animal betacoronaviruses but have low homology to 'common cold' human-associated coronaviruses. Thus, infection with betacoronaviruses induces multi-specific and long-lasting T cell immunity against the structural N protein. Understanding how pre-existing N- and ORF1-specific T cells that are present in the general population affect the susceptibility to and pathogenesis of SARS-CoV-2 infection is important for the management of the current COVID-19 pandemic.

Discovering Putative Protein Targets of Small Molecules: A Study of the p53 Activator Nutlin.

Small molecule drugs bind to a pocket in disease causing target proteins based on complementarity in shape and physicochemical properties. There is a likelihood that other proteins could have binding sites that are structurally similar to the target protein. Binding to these other proteins could alter their activities leading to off target effects of the drug. One such small molecule drug Nutlin binds the protein MDM2, which is upregulated in several types of cancer and is a negative regulator of the tumor suppressor protein p53. To investigate the off target effects of Nutlin, we present here a shape-based data mining effort. We extracted the binding pocket of Nutlin from the crystal structure of Nutlin bound MDM2. We next mined the protein structural database (PDB) for putative binding pockets in other human protein structures that were similar in shape to the Nutlin pocket in MDM2 using our topology-independent structural superimposition tool CLICK. We detected 49 proteins which have binding pockets that were structurally similar to the Nutlin binding site of MDM2. All of the potential complexes were evaluated using molecular mechanics and AutoDock Vina docking scores. Further, molecular dynamics simulations were carried out on four of the predicted Nutlin-protein complexes. The binding of Nutlin to one of these proteins, gamma glutamyl hydrolase, was also experimentally validated by a thermal shift assay. These findings provide a platform for identifying potential off-target effects of existing/new drugs and also opens the possibilities for repurposing drugs/ligands.

Age Effects on Spatiotemporal Dynamics of Response Inhibition: An MEG Study.

Inhibition, the ability to suppress irrelevant information, thoughts or movements, is crucial for humans to perform context-appropriate behaviors. It was suggested that declined cognitive performance in older adults might be attributed to inhibitory deficiencies. Although previous studies have shown an age-associated reduction in inhibitory ability, the understanding regarding its cortical spatiotemporal maps remained limited. Thus, we used a whole-head magnetoencephalography (MEG) to elucidate the age effects on response inhibition, and to explore the brain activation differences in high- and low-performing seniors. We recruited 22 younger and 22 older adults to participate in the visual Go/No-go task. Both behavioral performance and neuromagnetic responses to No-go stimuli were analyzed. The behavioral results showed that the older adults made more false alarm (FA) errors than the younger adults did. The MEG results showed that the seniors exhibited declined cortical activities in middle temporal gyrus (MTG) and delayed activation in MTG, prefrontal cortex (PFC) and pre-supplementary motor area (pre-SMA). Furthermore, among the older adults, more recruitment of the left PFC was found in the high-performers than in the lower-performers. In conclusion, age-related deficiencies in response inhibition were observed in both behavioral performance and neurophysiological measurement. Our results also suggested that frontal recruitment plays a compensatory role in successful inhibition.

Age Effect on Automatic Inhibitory Function of the Somatosensory and Motor Cortex: An MEG Study.

Age-related deficiency in the top-down modulation of cognitive inhibition has been extensively documented, whereas the effects of age on a bottom-up or automatic operation of inhibitory function were less investigated. It is unknown that whether the older adults (OA)' reduced behavioral performance and neural responses are due to the insufficient bottom-up processes. Compared to behavioral assessments which have been widely used to examine the top-down control of response inhibition, electrophysiological recordings are more suitable to probe the early-stage processes of automatic inhibitory function. Sensory gating (SG), a phenomenon of attenuated neural response to the second identical stimulus in a paired-pulse paradigm, is an indicator to assess automatic inhibitory function of the sensory cortex. On the other hand, electricity-induced beta rebound oscillation in a single-pulse paradigm reflects cortical inhibition of the motor cortex. From the neurophysiological perspective, SG and beta rebound oscillation are replicable indicators to examine the automatic inhibitory function of human sensorimotor cortices. Thus, the present study aimed to use a whole-head magnetoencephalography (MEG) to investigate the age-related alterations of SG function in the primary somatosensory cortex (SI) and of beta rebound oscillation in the primary motor cortex (MI) in 17 healthy younger and 15 older adults. The Stimulus 2/Stimulus 1 (S2/S1) amplitude ratio in response to the paired-pulse electrical stimulation to the left median nerve was used to evaluate the automatic inhibitory function of SI, and the beta rebound response in the single-pulse paradigm was used to evaluate the automatic inhibitory function of MI. Although there were no significant age-related differences found in the SI SG ratios, the MI beta rebound power was reduced and peak latency was prolonged in the OA. Furthermore, significant association between the SI SG ratio and the MI beta rebound power, which was seen in the younger adults (YA), was absent in the OA. In conclusion, our data suggested an age-related defect of association between sensorimotor cortices regarding automatic inhibitory function.

Prevention and Reversal of Diabetes by All-Trans Retinoid Acid and Exendin-4 in NOD Mice.

It has been shown that all-trans retinoid acid (ATRA) hinders the development of autoimmune diabetes by inducing immune tolerance status. Meanwhile, exendin-4 increases beta-cell function and mass. Thus, we hypothesized that ATRA and exendin-4 combination therapy would prevent and reverse autoimmune diabetes. NOD/scid mice were intravenously transferred with splenocytes isolated from 12-week-old female NOD mice. After adoptive transfer, mice were treated with vehicle, ATRA (0.5 mg/mouse intraperitoneally every other day), exendin-4 (3 µ g/kg subcutaneously twice daily), or combination for 6 weeks. Compared with vehicle, ATRA (P = 0.022) and ATRA plus exendin-4 (P = 0.013) treatment delayed the onset of diabetes. The pancreatic insulin content in mice treated with ATRA (P = 0.013) and exendin-4 (P < 0.02) was significantly higher than that of control mice. All but one spontaneous diabetic NOD mouse treated with ATRA and/or exendin-4 remained persistent hyperglycemic. ATRA and/or exendin-4 treatment did not alter their blood glucose levels and survival. Our results indicate that, before the onset of autoimmune diabetes, ATRA and exendin-4 treatment alone preserves pancreatic beta cells; ATRA and ATRA plus exendin-4 treatment delays the onset of autoimmune diabetes. However, after the onset of autoimmune diabetes, ATRA and/or exendin-4 treatment is unable to reverse hyperglycemia or improve survival.

Quantitative NanoProteomics approach for protein complex (QNanoPX) using gold nanoparticle-based DNA probe.

Affinity purification by pulldown methods using target-bound gel beads provides a powerful approach for purifying endogenous protein complexes. Such methods can be improved by using nanoparticle-based probe, coupled with immunoblot analysis or quantitative proteomics method using stable isotope labeling via liquid chromatography-mass spectrometry (LC-MS). Here, we describe sample preparation and a pulldown method using gold nanoparticle-based DNA probe for characterizing the transcriptional complex of estrogen response element (ERE). The described protocol includes the fabrication of gold nanoparticle-based probe, nuclear extract preparation, and affinity purification for the analysis by immunoblotting, as well as the subsequent trypsin digestion and stable isotope dimethyl labeling for the analysis by LC-MS.