TRF2 inhibition rather than telomerase disruption drives CD4T cell dysfunction during chronic viral infection.

We investigated the role of telomerase and telomere repeat-binding factor 2 (TRF2 or TERF2) in T-cell dysfunction in chronic viral infection. We found that the expression and activity of telomerase in CD4+ T (CD4T) cells from patients with hepatitis C virus (HCV) infections or people living with HIV (PLWH) were intact, but TRF2 expression was significantly inhibited at the post-transcriptional level, suggesting that TRF2 inhibition is responsible for the CD4T cell dysfunction observed during chronic viral infection. Silencing TRF2 expression in CD4T cells derived from healthy subjects induced telomeric DNA damage and CD4T cell dysfunction without affecting telomerase activity or translocation - similar to what we observed in CD4T cells from HCV patients and PLWH. These findings indicate that premature T-cell aging and dysfunction during chronic HCV or HIV infection are primarily caused by chronic immune stimulation and T-cell overactivation and/or proliferation that induce telomeric DNA damage due to TRF2 inhibition, rather than telomerase disruption. This study suggests that restoring TRF2 presents a novel approach to prevent telomeric DNA damage and premature T-cell aging, thus rejuvenating T-cell functions during chronic viral infection.

Synthetic gRNA/Cas9 ribonucleoprotein targeting HBV DNA inhibits viral replication.

The presence of hepatitis B virus (HBV) covalently closed circular (ccc) DNA (cccDNA), which serves as a template for viral replication and integration of HBV DNA into the host cell genome, sustains liver pathogenesis and constitutes an intractable barrier to the eradication of chronic HBV infection. The current antiviral therapy for HBV infection, using nucleos(t)ide analogues (NAs), can suppress HBV replication but cannot eliminate integrated HBV DNA and episomal cccDNA. Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a powerful genetic tool that can edit integrated HBV DNA and minichromosomal cccDNA for gene therapy, but its expression and delivery require a viral vector, which poses safety concerns for therapeutic applications in humans. In the present study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a nonviral formulation to develop a novel CRISPR/Cas9-mediated gene therapy for eradicating HBV infection. We designed a series of gRNAs targeting multiple specific HBV genes and tested their antiviral efficacy and cytotoxicity in different HBV cellular models. Transfection of stably HBV-infected human hepatoma cell line HepG2.2.15 with HBV-specific gRNA/Cas9 RNPs resulted in a substantial reduction in HBV transcripts. Specifically, gRNA5 and/or gRNA9 RNPs significantly reduced HBV cccDNA, total HBV DNA, pregenomic RNA, and HBV antigen (HBsAg, HBeAg) levels. T7 endonuclease 1 (T7E1) cleavage assay and DNA sequencing confirmed specific HBV gene cleavage and mutations at or around the gRNA target sites. Notably, this gene-editing system did not alter cellular viability or proliferation in the treated cells. Because of their rapid DNA cleavage capability, low off-target effects, low risk of insertional mutagenesis, and readiness for use in clinical application, these results suggest that synthetic gRNA/Cas9 RNP-based gene-editing can be utilized as a promising therapeutic drug for eradicating chronic HBV infection.

Long Noncoding RNA RUNXOR Promotes Myeloid-Derived Suppressor Cell Expansion and Functions via Enhancing Immunosuppressive Molecule Expressions during Latent HIV Infection.

RUNX1 overlapping RNA (RUNXOR) is a long noncoding RNA and a key regulator of myeloid-derived suppressor cells (MDSCs) via targeting runt-related transcription factor 1 (RUNX1). We and others have previously reported MDSC expansion and inhibition of host immune responses during viral infections; however, the mechanisms regulating MDSC differentiation and suppressive functions, especially the role of RUNXOR-RUNX1 in the regulation of MDSCs in people living with HIV (PLHIV), remain unknown. In this study, we demonstrate that RUNXOR and RUNX1 expressions are upregulated in MDSCs that expand and accumulate in human PBMCs derived from PLHIV. We found that the upregulation of RUNXOR and RUNX1 is associated with the expressions of several key immunosuppressive molecules, including arginase 1, inducible NO synthase, STAT3, IL-6, and reactive oxygen species. RUNXOR and RUNX1 could positively regulate each other's expression and control the expressions of these suppressive mediators. Specifically, silencing RUNXOR or RUNX1 expression in MDSCs from PLHIV attenuated MDSC expansion and immunosuppressive mediator expressions, whereas overexpressing RUNXOR in CD33+ myeloid precursors from healthy subjects promoted their differentiation into MDSCs and enhanced the expression of these mediators. Moreover, loss of RUNXOR-RUNX1 function in MDSCs improved IFN-γ production from cocultured autologous CD4 T cells derived from PLHIV. These results suggest that the RUNXOR-RUNX1 axis promotes the differentiation and suppressive functions of MDSCs via regulating multiple immunosuppressive signaling molecules and may represent a potential target for immunotherapy in conjunction with antiviral therapy in PLHIV.

Immune Activation Induces Telomeric DNA Damage and Promotes Short-Lived Effector T Cell Differentiation in Chronic HCV Infection.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) leads to a high rate of chronic infection and T cell dysfunction. Although it is well known that chronic antigenic stimulation is a driving force for impaired T cell functions, the precise mechanisms underlying immune activation-induced T cell dysfunctions during HCV infection remain elusive. APPROACH AND RESULTS: Here, we demonstrated that circulating CD4+ T cells from patients who are chronically HCV-infected exhibit an immune activation status, as evidenced by the overexpression of cell activation markers human leukocyte antigen-antigen D-related, glucose transporter 1, granzyme B, and the short-lived effector marker CD127- killer cell lectin-like receptor G1+ . In contrast, the expression of stem cell-like transcription factor T cell factor 1 and telomeric repeat-binding factor 2 (TRF2) are significantly reduced in CD4+ T cells from patients who are chronically HCV-infected compared with healthy participants (HP). Mechanistic studies revealed that CD4+ T cells from participants with HCV exhibit phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signaling hyperactivation on T cell receptor stimulation, promoting proinflammatory effector cell differentiation, telomeric DNA damage, and cellular apoptosis. Inhibition of Akt signaling during T cell activation preserved the precursor memory cell population and prevented inflammatory effector cell expansion, DNA damage, and apoptotic death. Moreover, knockdown of TRF2 reduced HP T cell stemness and triggered telomeric DNA damage and cellular apoptosis, whereas overexpression of TRF2 in CD4 T cells prevented telomeric DNA damage. CONCLUSIONS: These results suggest that modulation of immune activation through inhibiting Akt signaling and protecting telomeres through enhancing TRF2 expression may open therapeutic strategies to fine tune the adaptive immune responses in the setting of persistent immune activation and inflammation during chronic HCV infection.

Telomere and ATM Dynamics in CD4 T-Cell Depletion in Active and Virus-Suppressed HIV Infections.

CD4 T-cell depletion is a hallmark of HIV/AIDS, but the underlying mechanism is still unclear. We have recently shown that ataxia-telangiectasia-mutated (ATM) deficiency in CD4 T cells accelerates DNA damage, telomere erosion, and cell apoptosis in HIV-infected individuals on antiretroviral therapy (ART). Whether these alterations in ART-treated HIV subjects occur in vitro in HIV-infected CD4 T cells remains unknown. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the telomeric DNA damage response (DDR) and cellular apoptosis in highly permissive SupT1 cells, followed by the validation of our observations in primary CD4 T cells with active or drug-suppressed HIV infection. Specifically, we established an in vitro HIV T-cell culture system with viral replication and raltegravir (RAL; an integrase inhibitor) suppression, mimicking active and ART-controlled HIV infection in vivo We demonstrated that HIV-induced, telomeric DDR plays a pivotal role in triggering telomere erosion, premature T-cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This in vitro model provides a new tool to investigate HIV pathogenesis, and our results shed new light on the molecular mechanisms of telomeric DDR and CD4 T-cell homeostasis during HIV infection.IMPORTANCE The hallmark of HIV infection is a gradual depletion of CD4 T cells, with a progressive decline of host immunity. How CD4 T cells are depleted in individuals with active and virus-suppressed HIV infection remains unclear. In this study, we employed a cellular model of HIV infection to characterize the mechanisms underlying CD4 T-cell destruction by analyzing the chromosome end (telomere) DNA damage response (DDR) and cellular apoptosis in a T-cell line (highly permissive SupT1 cells), as well as in primary CD4 T cells with active or drug-suppressed HIV infection. We demonstrated that HIV-induced telomeric DDR plays a critical role in inducing telomere loss, premature cell aging, and CD4 T-cell apoptosis or depletion via dysregulation of the PI3K/ATM pathways. This study sheds new light on the molecular mechanisms of telomeric DDR and its role in CD4 T-cell homeostasis during HIV infection.

Topological DNA damage, telomere attrition and T cell senescence during chronic viral infections.

BACKGROUND: T cells play a key role in controlling viral infections; however, the underlying mechanisms regulating their functions during human viral infections remain incompletely understood. Here, we used CD4 T cells derived from individuals with chronic viral infections or healthy T cells treated with camptothecin (CPT) - a topoisomerase I (Top 1) inhibitor - as a model to investigate the role of DNA topology in reprogramming telomeric DNA damage responses (DDR) and remodeling T cell functions. RESULTS: We demonstrated that Top 1 protein expression and enzyme activity were significantly inhibited, while the Top 1 cleavage complex (TOP1cc) was trapped in genomic DNA, in T cells derived from individuals with chronic viral (HCV, HBV, or HIV) infections. Top 1 inhibition by CPT treatment of healthy CD4 T cells caused topological DNA damage, telomere attrition, and T cell apoptosis or dysfunction via inducing Top1cc accumulation, PARP1 cleavage, and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. Moreover, T cells from virally infected subjects with inhibited Top 1 activity were more vulnerable to CPT-induced topological DNA damage and cell apoptosis, indicating an important role for Top 1 in securing DNA integrity and cell survival. CONCLUSION: These findings provide novel insights into the molecular mechanisms for immunomodulation by chronic viral infections via disrupting DNA topology to induce telomeric DNA damage, T cell senescence, apoptosis and dysfunction. As such, restoring the impaired DNA topologic machinery may offer a new strategy for maintaining T cell function against human viral diseases.

Hepatitis C virus-induced myeloid-derived suppressor cells regulate T-cell differentiation and function via the signal transducer and activator of transcription 3 pathway.

T cells play a pivotal role in controlling viral infection; however, the precise mechanisms responsible for regulating T-cell differentiation and function during infections are incompletely understood. In this study, we demonstrated an expansion of myeloid-derived suppressor cells (MDSCs), in particular the monocytic MDSCs (M-MDSCs; CD14(+) CD33(+) CD11b(+) HLA-DR(-/low) ), in patients with chronic hepatitis C virus (HCV) infection. Notably, HCV-induced M-MDSCs express high levels of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and interleukin-10 (IL-10) compared with healthy subjects. Blocking STAT3 signalling reduced HCV-mediated M-MDSC expansion and decreased IL-10 expression. Importantly, we observed a significant increase in the numbers of CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells following incubation of healthy peripheral blood mononuclear cells (PBMCs) with MDSCs derived from HCV-infected patients or treated with HCV core protein. In addition, depletion of MDSCs from PBMCs led to a significant reduction of Foxp3(+) Treg cells developed during chronic HCV infection. Moreover, depletion of MDSCs from PBMCs significantly increased interferon-γ production by CD4(+) T effector (Teff) cells derived from HCV patients. These results suggest that HCV-induced MDSCs promote Treg cell development and inhibit Teff cell function, suggesting a novel mechanism for T-cell regulation and a new strategy for immunotherapy against human viral diseases.

KLRG1 impairs CD4+ T cell responses via p16ink4a and p27kip1 pathways: role in hepatitis B vaccine failure in individuals with hepatitis C virus infection.

Coinfection of hepatitis B virus (HBV) with hepatitis C virus (HCV) is quite common, leading to an increase in morbidity and mortality. As such, HBV vaccination is recommended in HCV-infected individuals. However, HBV vaccine responses in HCV-infected individuals are often blunted compared with uninfected populations. The mechanism for this failure of vaccine response in HCV-infected subjects remains unclear. In this study, we investigated the expression and function of an inhibitory receptor, killer cell lectin-like receptor subfamily G member 1 (KLRG1), in the regulation of CD4(+) T cells and HBV vaccine responses during HCV infection. We demonstrated that KLRG1 was overexpressed on CD4(+) T cells from HCV-infected, HBV vaccine nonresponders compared with HBV vaccine responders. The capacity of CD4(+) T cells to proliferate and secrete IL-2 cytokine was inversely associated with the level of KLRG1 expression. Importantly, blocking KLRG1 signaling resulted in a significant improvement in CD4(+) T cell proliferation and IL-2 production in HCV-infected, HBV vaccine nonresponders in response to TCR stimulation. Moreover, blockade of KLRG1 increased the phosphorylation of Akt (Ser(473)) and decreased the expression of cell cycle inhibitors p16(ink4a) and p27(kip1), which subsequently enhanced the expression of cyclin-dependent kinase 2 and cyclin E. These results suggest that the KLRG1 pathway impairs CD4(+) T cell responses to neoantigen and induces a state of immune senescence in individuals with HCV infection, raising the possibility that blocking this negative-signaling pathway might improve HBV vaccine responses in the setting of chronic viral infection.

HCV-infected hepatocytes drive CD4+ CD25+ Foxp3+ regulatory T-cell development through the Tim-3/Gal-9 pathway.

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T-cell Ig and mucin domain protein-3 (Tim-3) and galectin-9 (Gal-9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim-3/Gal-9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim-3/Gal-9 interactions regulate HCV-mediated Treg-cell development, here we provide pilot data showing that HCV-infected human hepatocytes express higher levels of Gal-9 and TGF-ß, and upregulate Tim-3 expression and regulatory cytokines TGF-ß/IL-10 in co-cultured human CD4(+) T cells, driving conventional CD4(+) T cells into CD25(+) Foxp3(+) Treg cells. Additionally, recombinant Gal-9 protein can transform TCR-activated CD4(+) T cells into Foxp3(+) Treg cells in a dose-dependent manner. Importantly, blocking Tim-3/Gal-9 ligations abrogates HCV-mediated Treg-cell induction by HCV-infected hepatocytes, suggesting that Tim-3/Gal-9 interactions may regulate human Foxp3(+) Treg-cell development and function during HCV infection.

KLRG1 negatively regulates natural killer cell functions through the Akt pathway in individuals with chronic hepatitis C virus infection.

In this study, we demonstrate that killer cell lectin-like receptor subfamily G member 1 (KLRG1), a transmembrane protein preferentially expressed on T cells, is highly expressed on CD56(+) NK cells, which are significantly reduced in their numbers and functions in the peripheral blood of patients with chronic hepatitis C virus (HCV) infection compared to subjects without infection. KLRG1 expression is also upregulated on healthy NK cells exposed to Huh-7 hepatocytes infected with HCV in vitro. Importantly, the expression levels of KLRG1 are inversely associated with the capacity of NK cells to proliferate and to produce gamma interferon (IFN-γ) but positively associated with apoptosis of NK cells in response to inflammatory cytokine stimulation. KLRG1(+) NK cells, including CD56(bright) and CD56(dim) subsets, exhibit impaired cell activation and IFN-γ production but increased apoptosis compared to KLRG1(-) NK cells, particularly in HCV-infected individuals. Importantly, blockade of KLRG1 signaling significantly recovered the impaired IFN-γ production by NK cells from HCV-infected subjects. Blockade of KLRG1 also enhanced the impaired phosphorylation of Akt (Ser473) in NK cells from HCV-infected subjects. Taken together, these results indicate that KLRG1 negatively regulates NK cell numbers and functions via the Akt pathway, thus providing a novel marker and therapeutic target for HCV infection.

Differential regulation of interleukin-12 (IL-12)/IL-23 by Tim-3 drives T(H)17 cell development during hepatitis C virus infection.

Cytokine production by innate immunity is critical for shaping the adaptive immunity through regulation of T cell differentiation. In this report, we studied T cell immunoglobulin mucin domain protein 3 (Tim-3) expression on monocytes and its regulatory effect on interleukin-12 (IL-12)/IL-23 production by CD14(+) monocytes, as well as IL-17 production by CD4(+) T cells in individuals with chronic hepatitis C virus (HCV) infection. We found that Tim-3 and IL-23p19 are highly expressed and that IL-12p35 is inhibited in human CD14(+) monocytes, while IL-17 expression is upregulated in CD4(+) T cells, in chronically HCV-infected individuals compared to healthy subjects. Interestingly, Tim-3 expression is closely associated with the differential regulation of IL-12/IL-23 expression in CD14(+) monocytes and correlated to IL-17 production by CD4(+) T cells. These Tim-3-associated IL-12/IL-23/IL-17 dysregulations in HCV-infected individuals are also recapitulated in vitro by incubating healthy monocytes or peripheral blood mononuclear cells with Huh-7 hepatoma cells transfected with HCV RNA. Importantly, blocking Tim-3 signaling on monocytes restores the balance of IL-12/IL-23 through the intracellular STAT3 signaling, which in turn reverses the upregulated IL-17 expression both ex vivo and in vitro. Our findings suggest that Tim-3-mediated differential regulation of IL-12/IL-23 drives T(H)17 cell development, a milieu favoring viral persistence and autoimmune phenomenon during HCV infection.

Tim-3 pathway controls regulatory and effector T cell balance during hepatitis C virus infection.

Hepatitis C virus (HCV) is remarkable at disrupting human immunity to establish chronic infection. Upregulation of inhibitory signaling pathways (such as T cell Ig and mucin domain protein-3 [Tim-3]) and accumulation of regulatory T cells (Tregs) play pivotal roles in suppressing antiviral effector T cell (Teff) responses that are essential for viral clearance. Although the Tim-3 pathway has been shown to negatively regulate Teffs, its role in regulating Foxp3(+) Tregs is poorly explored. In this study, we investigated whether and how the Tim-3 pathway alters Foxp3(+) Treg development and function in patients with chronic HCV infection. We found that Tim-3 was upregulated, not only on IL-2-producing CD4(+)CD25(+)Foxp3(-) Teffs, but also on CD4(+)CD25(+)Foxp3(+) Tregs, which accumulate in the peripheral blood of chronically HCV-infected individuals when compared with healthy subjects. Tim-3 expression on Foxp3(+) Tregs positively correlated with expression of the proliferation marker Ki67 on Tregs, but it was inversely associated with proliferation of IL-2-producing Teffs. Moreover, Foxp3(+) Tregs were found to be more resistant to, and Foxp3(-) Teffs more sensitive to, TCR activation-induced cell apoptosis, which was reversible by blocking Tim-3 signaling. Consistent with its role in T cell proliferation and apoptosis, blockade of Tim-3 on CD4(+)CD25(+) T cells promoted expansion of Teffs more substantially than Tregs through improving STAT-5 signaling, thus correcting the imbalance of Foxp3(+) Tregs/Foxp3(-) Teffs that was induced by HCV infection. Taken together, the Tim-3 pathway appears to control Treg and Teff balance through altering cell proliferation and apoptosis during HCV infection.

Pulsed 808-nm infrared laser stimulation of the auditory nerve in guinea pig cochlea.

Pulsed near-infrared radiation has been proposed as an alternative stimulus for auditory nerve stimulation and could be potentially used in the design of cochlear implant. Although the infrared with high absorption coefficient of water (i.e., wavelength ranged from 1.8 to 2.2 µm) has been widely investigated, the lymph in the cochlea absorbs most of the infrared energies, and only a small part can arrive at the target auditory nerves. The present study is aimed to test whether the short-wavelength near-infrared irradiation with lower absorption coefficients can penetrate the lymph fluid to stimulate the auditory nerves. An 808-nm near-infrared laser was chosen to stimulate the auditory nerve in the guinea pig cochlea. The infrared pulse was delivered by an optical fiber that was surgically inserted near the round window membrane and oriented toward the spiral ganglion cells in the basal turn of the cochlea. The 2-Hz infrared pulses were used to stimulate the cochlea before and after the deafness with different pulse durations (100-1,000 µs). Optically evoked compound action potentials (oCAPs) were recorded during the infrared radiation. We successfully recorded oCAPs from both normal hearing animals and deafened animals. The oCAP amplitude increased with the infrared radiation energy. The preliminary experiment suggests that the near-infrared with lower absorption coefficients can effectively pass through the lymph filled in the cochlea and stimulate the auditory nerve. Further studies will optimize the deafness animal model and determine the optimal stimulation parameters.

Circulating GDF-15: a biomarker for metabolic dysregulation and aging in people living with HIV.

Despite effective control of HIV replication by antiretroviral therapy (ART), a significant number of people living with HIV (PLWH) fail to achieve complete immune reconstitution and thus are deemed immune non-responders (INRs). Compared with immune responders (IRs) who have restored their CD4 T cell numbers and functions, CD4 T cells from these INRs exhibit prominent mitochondrial dysfunction and premature aging, which play a major role in increasing the incidence of non-AIDS, non-communicable diseases (NCDs). To date, there are no reliable biomarkers that can be used to typify and manage PLWH, especially INRs with non-AIDS NCDs. Growth differential factor-15 (GDF-15) is a transforming growth factor-ß (TGF-ß) family member known to regulate several biological processes involved in cell aging and stress responses. Since PLWH exhibit premature aging and metabolic dysregulation, here we measured the plasma levels of GDF-15 by ELISA and metabolic proteins by proteomic array and correlated the results with clinical parameters in ART-controlled PLWH (including INRs and IRs) and healthy subjects (HS). We found that GDF-15 levels were significantly elevated in PLWH compared to HS. GDF-15 levels were positively correlated with age and negatively associated with body mass and LDL cholesterol levels in the study subjects. Also, elevated GDF-15 levels were correlated with differential dysregulation of multiple metabolic proteins in PLWH. These results suggest that GDF-15 protein may serve as a biomarker of metabolic dysregulation and aging, and this biomarker will be useful in clinical trials targeting aging and metabolic disorders in ART-treated PLWH.

Cross-talk between programmed death-1 and suppressor of cytokine signaling-1 in inhibition of IL-12 production by monocytes/macrophages in hepatitis C virus infection.

Hepatitis C virus (HCV) dysregulates innate immune responses and induces persistent viral infection. We previously demonstrated that HCV core protein impairs IL-12 expression by monocytes/macrophages (M/M(Φ)s) through interaction with a complement receptor gC1qR. Because HCV core-mediated lymphocyte dysregulation occurs through the negative immunomodulators programmed death-1 (PD-1) and suppressor of cytokine signaling-1 (SOCS-1), the aim of this study was to examine their role in HCV core-mediated IL-12 suppression in M/M(Φ)s. We analyzed TLR-stimulated, primary CD14(+) M/M(Φ)s from chronically HCV-infected and healthy subjects or the THP-1 cell line for PD-1, SOCS-1, and IL-12 expression following HCV core treatment. M/M(Φ)s from HCV-infected subjects at baseline exhibited comparatively increased PD-1 expression that significantly correlated with the degree of IL-12 inhibition. M/M(Φ)s isolated from healthy and HCV-infected individuals and treated with HCV core protein displayed increased PD-1 and SOCS-1 expression and decreased IL-12 expression, an effect that was also observed in cells treated with gC1qR's ligand, C1q. Blocking gC1qR rescued HCV core-induced PD-1 upregulation and IL-12 suppression, whereas blocking PD-1 signaling enhanced IL-12 production and decreased the expression of SOCS-1 induced by HCV core. Conversely, silencing SOCS-1 expression using small interfering RNAs increased IL-12 expression and inhibited PD-1 upregulation. PD-1 and SOCS-1 were found to associate by coimmunoprecipitation studies, and blocking PD-1 or silencing SOCS-1 in M/M(Φ) led to activation of STAT-1 during TLR-stimulated IL-12 production. These data suggested that HCV core/gC1qR engagement on M/M(Φ)s triggers the expression of PD-1 and SOCS-1, which can associate to deliver negative signaling to TLR-mediated pathways controlling expression of IL-12, a key cytokine linking innate and adaptive immunity.

Irradiation of 850-nm laser light changes the neural activities in rat primary visual cortex.

Although infrared laser was proven to be an alternative approach for neural stimulation, there is very little known about the neural response to infrared laser irradiation in visual cortex. This study is to investigate the effect of near-infrared laser irradiation on neural activities at the cortex level. A 850-nm pigtailed diode laser was applied to stimulate the rat primary visual cortex while the horizontal black and white stripe pattern was used as standard visual stimulation to evoke visual-evoked potential (VEP). Both amplitude and latency of VEP P100 was measured with or without infrared pulse stimulation applied in rat primary visual cortex. Paired t test and one-way analysis of variance were used to evaluate the impact of infrared irradiation and its pulse width on the amplitudes and latencies of P100, respectively. The results from our preliminary study revealed that, the pulsed near-infrared laser depressed the VEP amplitude and shortened the latency of P100; with the increment of pulse width of infrared irradiation, further decline of VEP amplitude and much shortened latency of P100 were observed. The present work suggests that near-infrared laser irradiation can alter the neural activities in primary visual cortex transiently, and could provide a novel contactless artificial neural stimulus to brain cortex with high spatial selectivity.

Beta Changes Induced by Acute Hand Loss Model during NMES.

Neuromuscular electrical stimulation (NMES) has been demonstrated to effectively modulate cortical activities by evoking muscle contraction in upper limb and generating joint movements, which showed an excellent performance in motor rehabilitation. However, due to hand loss and cortical function reorganization induced by hand amputation, how neural activities in sensorimotor cortex response to NMES-evoked muscle contraction in the end of an amputation stump is not clear. In this paper, Ischemic nerve block (INB) technique was used to build an acute hand loss model, and 64-channel EEG signals were recorded from 11 healthy subjects to perform a 2×2 factorial design protocol, with the INB state and the current intensity as factors. The changes of NMES-evoked sensorimotor cortical activities were quantified by computing Beta-band event-related desynchronization (Beta ERD) patterns and the time-varying functional connectivity using adaptive directed transfer function (ADTF) before and during INB. The acute hand "loss" resulted in ipsilateral dominance of Beta ERD induced by NMES with two current intensities in the topographic maps, that is, ipsilateral Beta ERD was significantly higher than that the contralateral one (p<0.05). However, before INB, Beta ERD in the contralateral sensorimotor cortex induced by NMES above motor threshold was significantly higher than that in the ipsilateral area (p< 0.01). Meanwhile, whatever before or during INB, clustering coefficients of the ADTF network in sensorimotor cortex showed temporal dynamics during two NMES tasks. During INB, NMES above motor threshold-evoked lower clustering coefficients of the time-varying network in sensorimotor cortex than that before INB (p<0.05). The present results suggest that the loss of the hand proprioception will degrade cortical activities in the contralateral area, and increase cortical activities in the ipsilateral area compensatively responding to NMES. This finding may be particularly important to improve the reconstruction of the proprioception function of hand prosthesis.

The Kinematic-Muscle Synergies during Infant Crawling: A Pilot Study.

Many feature extraction algorithms have been separately used for kinematic or muscle synergy analysis during human movement. However, very few studies focus on the co-extraction of kinematic and muscle synergies. Therefore, the aim of this study was to propose a novel and efficient approach for extracting the kinematic-muscle synergies during infant crawling. Surface electromyography signals and three-dimensional joint trajectories were collected from 20 typically developing infants during self-paced hands-and-knees crawling. Angular accelerations of shoulder, elbow, hip and knee flexion/extension computing from those joint trajectories were divided into two independent directional positive degrees-of-freedom. The kinematic-muscle synergies and corresponding activation coefficients were extracted using the non-negative matrix factorization algorithm based on two selection criteria of synergy number (i.e., criterion 1: the total constraint, criterion 2: a combination of the total constraint and a local constraint for each joint/muscle). Then, the data of each joint/muscle were reconstructed by those synergies and corresponding activation coefficients. Our results indicated that the minimum number of kinematic-muscle synergies based on criterion 1 is less than that based on criterion 2. The data reconstruction of joint flexion/extension based on criterion 2 is better than that based on criterion 1, whereas the data reconstruction of muscles is similar between criterion 1 and 2. These promising results show the feasibility of applying the proposed approach to clinical assessments of motor function for infants.Clinical Relevance- Extracting kinematic-muscle synergies during infant crawling has the potential for professional therapists or rehabilitation physicians to conduct the early assessment and rehabilitation treatment of infants with the central nervous system disorders.

ROS-Induced Mitochondrial Dysfunction in CD4 T Cells from ART-Controlled People Living with HIV.

We have previously demonstrated mitochondrial dysfunction in aging CD4 T cells from antiretroviral therapy (ART)-controlled people living with HIV (PLWH). However, the underlying mechanisms by which CD4 T cells develop mitochondrial dysfunction in PLWH remain unclear. In this study, we sought to elucidate the mechanism(s) of CD4 T cell mitochondrial compromise in ART-controlled PLWH. We first assessed the levels of reactive oxygen species (ROS), and we observed significantly increased cellular and mitochondrial ROS levels in CD4 T cells from PLWH compared to healthy subjects (HS). Furthermore, we observed a significant reduction in the levels of proteins responsible for antioxidant defense (superoxide dismutase 1, SOD1) and ROS-mediated DNA damage repair (apurinic/apyrimidinic endonuclease 1, APE1) in CD4 T cells from PLWH. Importantly, CRISPR/Cas9-mediated knockdown of SOD1 or APE1 in CD4 T cells from HS confirmed their roles in maintaining normal mitochondrial respiration via a p53-mediated pathway. Reconstitution of SOD1 or APE1 in CD4 T cells from PLWH successfully rescued mitochondrial function as evidenced by Seahorse analysis. These results indicate that ROS induces mitochondrial dysfunction, leading to premature T cell aging via dysregulation of SOD1 and APE1 during latent HIV infection.

Impacts of Motor Developmental Delay on the Inter-Joint Coordination Using Kinematic Synergies of Joint Angles During Infant Crawling.

Motor developmental delay (MDD) usually affects the inter-joint coordination for limb movement. However, the mechanism between the abnormal inter-joint coordination and MDD is still unclear, which poses a challenge for clinical diagnosis and motor rehabilitation of MDD in infant's early life. This study aimed to explore whether the joint activities of limbs during infant crawling are represented with kinematic synergies of joint angles, and evaluate the impacts of MDD on the inter-joint coordination using those synergies. 20 typically developing infants, 16 infants at risk of developmental delay, 11 infants at high risk of developmental delay and 13 infants with confirmed developmental delay were recruited for self-paced crawling on hands and knees. A motion capture system was employed to trace infants' limbs in space, and angles of shoulder, elbow, hip and knee over time were computed. Kinematic synergies were derived from joint angles using principal component analysis. Sample entropy and Spearman's rank correlation coefficients were calculated among those synergies to evaluate the crawling complexity and the symmetry of bilateral limbs, respectively. We found that the first two synergies with different contributions to the crawling movements sufficiently represented the joint angular profiles of limbs. MDD further delayed the development of motor function for lower limbs and mainly increased the crawling complexity of joint flexion/extension to some extent, but did not obviously change the symmetry of bilateral limbs. These results suggest that the time-varying kinematic synergy of joint angles is a potential index for objectively evaluating the abnormal inter-joint coordination affected by MDD.