CD8+ T cells control SIV infection using both cytolytic effects and non-cytolytic suppression of virus production.

Whether CD8+ T lymphocytes control human immunodeficiency virus infection by cytopathic or non-cytopathic mechanisms is not fully understood. Multiple studies highlighted non-cytopathic effects, but one hypothesis is that cytopathic effects of CD8+ T cells occur before viral production. Here, to examine the role of CD8+ T cells prior to virus production, we treated SIVmac251-infected macaques with an integrase inhibitor combined with a CD8-depleting antibody, or with either reagent alone. We analyzed the ensuing viral dynamics using a mathematical model that included infected cells pre- and post- viral DNA integration to compare different immune effector mechanisms. Macaques receiving the integrase inhibitor alone experienced greater viral load decays, reaching lower nadirs on treatment, than those treated also with the CD8-depleting antibody. Models including CD8+ cell-mediated reduction of viral production (non-cytolytic) were found to best explain the viral profiles across all macaques, in addition an effect in killing infected cells pre-integration (cytolytic) was supported in some of the best models. Our results suggest that CD8+ T cells have both a cytolytic effect on infected cells before viral integration, and a direct, non-cytolytic effect by suppressing viral production.

Heroin-Induced Transverse Myelitis in a Chronic Heroin User: A Case Report.

Transverse myelitis is a rare but documented sequela of heroin use. While the underlying etiology is not clearly elucidated, the prevailing pathophysiologic mechanism amongst existing literature suggests an immune-mediated hypersensitivity reaction due to heroin insufflation following a long period of abstinence. Outcomes vary among the limited reports, but prognosis tends to be poor due to an acute and rapidly progressive disease course. Here, we describe a case of extensive transverse myelitis in a chronic heroin user following heroin insufflation. This report hopes to provide greater insight into the underlying cause of this rare phenomenon due to our patient's discrepancy from the documented norm of heroin abstinence preceding disease onset.

T cell activation is insufficient to drive SIV disease progression.

Resolution of T cell activation and inflammation is a key determinant of the lack of SIV disease progression in African green monkeys (AGMs). Although frequently considered together, T cell activation occurs in response to viral stimulation of acquired immunity, while inflammation reflects innate immune responses to mucosal injury. We dissociated T cell activation from inflammation through regulatory T cell (Treg) depletion with Ontak (interleukin-2 coupled with diphtheria toxin) during early SIV infection of AGMs. This intervention abolished control of T cell immune activation beyond the transition from acute to chronic infection. Ontak had no effect on gut barrier integrity, microbial translocation, inflammation, and hypercoagulation, despite increasing T cell activation. Ontak administration increased macrophage counts yet decreased their activation. Persistent T cell activation influenced SIV pathogenesis, shifting the ramp-up in viral replication to earlier time points, prolonging the high levels of replication, and delaying CD4+ T cell restoration yet without any clinical or biological sign of disease progression in Treg-depleted AGMs. Thus, by inducing T cell activation without damaging mucosal barrier integrity, we showed that systemic T cell activation per se is not sufficient to drive disease progression, which suggests that control of systemic inflammation (likely through maintenance of gut integrity) is the key determinant of lack of disease progression in natural hosts of SIVs.

Prolonged experimental CD4+ T-cell depletion does not cause disease progression in SIV-infected African green monkeys.

CD4+ T-cell depletion is a hallmark of HIV infection, leading to impairment of cellular immunity and opportunistic infections, but its contribution to SIV/HIV-associated gut dysfunction is unknown. Chronically SIV-infected African Green Monkeys (AGMs) partially recover mucosal CD4+ T-cells, maintain gut integrity and do not progress to AIDS. Here we assess the impact of prolonged, antibody-mediated CD4 + T-cell depletion on gut integrity and natural history of SIV infection in AGMs. All circulating CD4+ T-cells and >90% of mucosal CD4+ T-cells are depleted. Plasma viral loads and cell-associated viral RNA in tissues are lower in CD4+-cell-depleted animals. CD4+-cell-depleted AGMs maintain gut integrity, control immune activation and do not progress to AIDS. We thus conclude that CD4+ T-cell depletion is not a determinant of SIV-related gut dysfunction, when gastrointestinal tract epithelial damage and inflammation are absent, suggesting that disease progression and resistance to AIDS are independent of CD4+ T-cell restoration in SIVagm-infected AGMs.

Changes to the Simian Immunodeficiency Virus (SIV) Reservoir and Enhanced SIV-Specific Responses in a Rhesus Macaque Model of Functional Cure after Serial Rounds of Romidepsin Administrations.

HIV persistence requires lifelong antiretroviral therapy (ART), calling for a cure. The histone deacetylase inhibitor, romidepsin, is used in the "shock and kill" approach with the goal of reactivating virus and subsequently clearing infected cells through cell-mediated immune responses. We tested serial and double infusions of romidepsin in a rhesus macaque (RM) model of SIV functional cure, which controls virus without ART. Off ART, romidepsin reactivated SIV in all RMs. Subsequent infusions resulted in diminished reactivation, and two RMs did not reactivate the virus after the second or third infusions. Therefore, those two RMs received CD8-depleting antibody to assess the replication competence of the residual reservoir. The remaining RMs received double infusions, i.e., two doses separated by 48-h. Double infusions were well tolerated, induced immune activation, and effectively reactivated SIV. Although reactivation was gradually diminished, cell-associated viral DNA was minimally changed, and viral outgrowth occurred in 4/5 RMs. In the RM which did not reactivate after CD8 depletion, viral outgrowth was not detected in peripheral blood mononuclear cells (PBMC)-derived CD4+ cells. The frequency of SIV-specific CD8+ T cells increased after romidepsin administration, and the increased SIV-specific immune responses were associated, although not statistically, with the diminished reactivation. Thus, our data showing sequential decreases in viral reactivation with repeated romidepsin administrations with all RMs and absence of viral reactivation after CD8+ T-cell depletion in one animal suggest that, in the context of healthy immune responses, romidepsin affected the inducible viral reservoir and gradually increased immune-mediated viral control. Given the disparities between the results of romidepsin administration to ART-suppressed SIVmac239-infected RMs and HIV-infected normal progressors compared to our immune-healthy model, our data suggest that improving immune function for greater SIV-specific responses should be the starting point of HIV cure strategies. IMPORTANCE HIV cure is sought after due to the prevalence of comorbidities that occur in persons with HIV. One of the most investigated HIV cure strategies is the "shock and kill" approach. Our study investigated the use of romidepsin, a histone deacetylase (HDAC) inhibitor, in our rhesus macaque model of functional cure, which allows for better resolution of viral reactivation due to the lack of antiretroviral therapy. We found that repeated rounds of romidepsin resulted in gradually diminished viral reactivation. One animal inevitably lacked replication-competent virus in the blood. With the accompanying enhancement of the SIV-specific immune response, our data suggest that there is a reduction of the viral reservoir in one animal by the cell-mediated immune response. With the differences observed between our model and persons living with HIV (PWH) treated with romidepsin, specifically in the context of a healthy immune system in our model, our data thereby indicate the importance of restoring the immune system for cure strategies.

Lack of Specific Regulatory T Cell Depletion and Cytoreduction Associated with Extensive Toxicity After Administration of Low and High Doses of Cyclophosphamide.

Up to 93% of the human immunodeficiency virus (HIV) latent reservoir comprised defective proviruses, suggesting that a functional cure is possible through the elimination of a small population of cells containing intact virus, instead of the entire reservoir. Cyclophosphamide (Cy) is an established chemotherapeutic agent for immune cell cancers. In high doses, Cy is a nonselective cytoreductor, used in allogeneic stem-cell transplantation, while in a low dose, metronomic schedule, Cy selectively depletes regulatory T cells (Tregs). We administered low and high doses to simian immunodeficiency virus (SIV)-infected rhesus macaques (RM) to assess their effects on the SIV reservoirs. As a Treg-depleting agent, Cy unselectively depleted Treg and total lymphocytes, resulting in minimal immune activation and no viral reactivation. As a cytoreductive agent, Cy induced massive viral reactivation in elite controller RMs without ART. However, when administered with antiretroviral therapy (ART), Cy had substantial adverse effects, including mortality. Our study thus dissuades further investigation of Cy as an HIV cure agent.

Neutrophil and Eosinophil Extracellular Traps in Hodgkin Lymphoma.

Classic Hodgkin lymphoma (cHL), nodular sclerosis (NS) subtype, is characterized by the presence of Hodgkin/Reed-Sternberg (HRS) cells in an inflammatory background containing neutrophils and/or eosinophils. Both types of granulocytes release extracellular traps (ETs), web-like DNA structures decorated with histones, enzymes, and coagulation factors that promote inflammation, thrombosis, and tumor growth. We investigated whether ETs from neutrophils (NETs) or eosinophils (EETs) are detected in cHL, and evaluated their association with fibrosis. We also studied expression of protease-activated receptor-2 (PAR-2) and phospho-extracellular signal-related kinase (p-ERK), potential targets/effectors of ETs-associated elastase, in HRS cells. Expression of tissue factor (TF) was evaluated, given the procoagulant properties of ETs. We analyzed 32 HL cases, subclassified as 12 NS, 5 mixed-cellularity, 5 lymphocyte-rich, 1 lymphocyte-depleted, 4 nodular lymphocyte-predominant HL (NLPHL), and 5 reactive nodes. Notably, a majority of NS cHL cases exhibited NET formation by immunohistochemistry for citrullinated histones, with 1 case revealing abundant EETs. All other cHL subtypes as well as NLPHL were negative. Immunofluorescence microscopy confirmed NETs with filamentous/delobulated morphology. Moreover, ETs formation correlates with concurrent fibrosis (r = 0.7999; 95% CI, 0.6192-0.9002; P ≤ 0.0001). Results also showed that HRS cells in NS cHL expressed PAR-2 with nuclear p-ERK staining, indicating a neoplastic or inflammatory phenotype. Remarkably, TF was consistently detected in the endothelium of NS cHL cases compared with other subtypes, in keeping with a procoagulant status. A picture emerges whereby the release of ETs and resultant immunothrombosis contribute to the inflammatory tumor microenvironment of NS cHL. This is the first description of NETs in cHL.

Pharmacokinetics and Immunological Effects of Romidepsin in Rhesus Macaques.

HIV/SIV persistence in latent reservoirs requires lifelong antiretroviral treatment and calls for effective cure strategies. Romidepsin (RMD), a histone deacetylase inhibitor, was reported to reactivate HIV/SIV from reservoirs in virus-suppressed individuals. We characterized in detail the pharmacokinetics and safety profile of RMD in three SIV-naïve rhesus macaques which received two rounds of treatment. In plasma, RMD mean terminal half-life was 15.3 h. In comparison, RMD mean terminal half-life was much longer in tissues: 110 h in the lymph nodes (LNs) and 28 h in gastrointestinal tract. RMD administration was accompanied by transient liver and systemic toxicity. Isoflurane anesthesia induced near-immediate transient lymphopenia, which was further exacerbated and extended with the extensive immune modifications by RMD. The effect of RMD on circulating immune cells was complex: (i) slight increase in lymphocyte death rates; (ii) transient, robust increase in neutrophils; (iii) massive downregulation of lymphocyte surface markers; (iv) important migration of CD3+ T cells to the gut and LNs; and (v) hindrance to CD8+ T cell functionality, yet without reaching significance. Our results show that, in contrast to transient plasma concentrations, RMD has a long-term presence in tissues, with multiple immunomodulatory effects and minimal to moderate kidney, liver, and lymphocyte toxicities. As such, we concluded that RMD can be used for "shock and kill" approaches, preferentially in combination with other latency reversal agents or cytotoxic T lymphocyte boosting strategies with consideration taken for adverse effects.

Nonhuman Primate Testing of the Impact of Different Regulatory T Cell Depletion Strategies on Reactivation and Clearance of Latent Simian Immunodeficiency Virus.

Regulatory T cells (Tregs) may be key contributors to the HIV/SIV latent reservoir, since they harbor high levels of HIV/SIV; reverse CD4+ T cell immune activation status, increasing the pool of resting CD4+ T cells; and impair CD8+ T cell function, favoring HIV persistence. We tested the hypothesis that Treg depletion is a valid intervention toward an HIV cure by depleted Tregs in 14 rhesus macaque (RM) controllers infected with SIVsab, the virus that naturally infects sabaeus monkeys, through different strategies: administration of an anti-CCR4 immunotoxin, two doses of an anti-CD25 immunotoxin (interleukin-2 with diphtheria toxin [IL-2-DT]), or two combinations of both. All of these treatments resulted in significant depletion of the circulating Tregs (>70%) and their partial depletion in the gut (25%) and lymph nodes (>50%). The fractions of CD4+ T cells expressing Ki -67 increased up to 80% in experiments containing IL-2-DT and only 30% in anti-CCR4-treated RMs, paralleled by increases in the inflammatory cytokines. In the absence of ART, plasma virus rebounded to 103 vRNA copies/ml by day 10 after IL-2-DT administration. A large but transient boost of the SIV-specific CD8+ T cell responses occurred in IL-2-DT-treated RMs. Such increases were minimal in the RMs receiving anti-CCR4-based regimens. Five RMs received IL-2-DT on ART, but treatment was discontinued because of high toxicity and lymphopenia. As such, while all treatments depleted a significant proportion of Tregs, the side effects in the presence of ART prevent their clinical use and call for different Treg depletion approaches. Thus, based on our data, Treg targeting as a strategy for HIV cure cannot be discarded.IMPORTANCE Regulatory T cells (Tregs) can decisively contribute to the establishment and persistence of the HIV reservoir, since they harbor high levels of HIV/SIV, increase the pool of resting CD4+ T cells by reversing their immune activation status, and impair CD8+ T cell function, favoring HIV persistence. We tested multiple Treg depletion strategies and showed that all of them are at least partially successful in depleting Tregs. As such, Treg depletion appears to be a valid intervention toward an HIV cure, reducing the size of the reservoir, reactivating the virus, and boosting cell-mediated immune responses. Yet, when Treg depletion was attempted in ART-suppressed animals, the treatment had to be discontinued due to high toxicity and lymphopenia. Therefore, while Treg targeting as a strategy for HIV cure cannot be discarded, the methodology for Treg depletion has to be revisited.

Neutrophil extracellular trap production contributes to pathogenesis in SIV-infected nonhuman primates.

Neutrophil extracellular traps (NETs) are involved in the pathogenesis of many infectious diseases, yet their dynamics and impact on HIV/SIV infection have not yet been assessed. We hypothesized that SIV infection and the related microbial translocation trigger NET activation and release (NETosis), and we investigated the interactions between NETs and immune cell populations and platelets. We compared and contrasted the levels of NETs between SIV-uninfected, SIV-infected, and SIV-infected antiretroviral-treated nonhuman primates. We also cocultured neutrophils from these animals with either peripheral blood mononuclear cells or platelets. Increased NET production was observed throughout SIV infection. In chronically infected animals, NETs were found in the gut, lung, and liver, and in the blood vessels of kidney and heart. Antiretroviral therapy (ART) decreased NETosis, albeit above preinfection levels. NETs captured CD4+ and CD8+ T cells, B cells, and monocytes, irrespective of their infection status, potentially contributing to the indiscriminate generalized immune cell loss characteristic to HIV/SIV infection, and limiting the CD4+ T cell recovery under ART. By capturing and facilitating aggregation of platelets, and through expression of increased tissue factor levels, NETs may also enhance HIV/SIV-related coagulopathy and promote cardiovascular comorbidities.

Regulatory T Cells As Potential Targets for HIV Cure Research.

T regulatory cells (Tregs) are a key component of the immune system, which maintain a delicate balance between overactive responses and immunosuppression. As such, Treg deficiencies are linked to autoimmune disorders and alter the immune control of pathogens. In HIV infection, Tregs play major roles, both beneficial and detrimental. They regulate the immune system such that inflammation and spread of virus through activated T cells is suppressed. However, suppression of immune activation also limits viral clearance and promotes reservoir formation. Tregs can be directly targeted by HIV, thereby harboring a fraction of the viral reservoir. The vital role of Tregs in the pathogenesis and control of HIV makes them a subject of interest for manipulation in the search of an HIV cure. Here, we discuss the origin and generation, homeostasis, and functions of Tregs, particularly their roles and effects in HIV infection. We also present various Treg manipulation strategies, including Treg depletion techniques and interventions that alter Treg function, which may be used in different cure strategies, to simultaneously boost HIV-specific immune responses and induce reactivation of the latent virus.

Cutting Edge: T Regulatory Cell Depletion Reactivates Latent Simian Immunodeficiency Virus (SIV) in Controller Macaques While Boosting SIV-Specific T Lymphocytes.

T regulatory cells (Tregs) are critical in shaping the latent HIV/SIV reservoir, as they are preferentially infected, reverse CD4+ T cell activation status, and suppress CTL responses. To reactivate latent virus and boost cell-mediated immune responses, we performed in vivo Treg depletion with Ontak (denileukin diftitox) in two SIVsab-infected controller macaques. Ontak induced significant (>75%) Treg depletion and major CD4+ T cell activation, and only minimally depleted CD8+ T cells. The overall ability of Tregs to control immune responses was significantly impaired despite their incomplete depletion, resulting in both reactivation of latent virus (virus rebound to 103 viral RNA copies/ml plasma in the absence of antiretroviral therapy) and a significant boost of SIV-specific CD8+ T cell frequency, with rapid clearance of reactivated virus. As none of the latency-reversing agents in development have such dual activity, our strategy holds great promise for cure research.

Multi-dose Romidepsin Reactivates Replication Competent SIV in Post-antiretroviral Rhesus Macaque Controllers.

Viruses that persist despite seemingly effective antiretroviral treatment (ART) and can reinitiate infection if treatment is stopped preclude definitive treatment of HIV-1 infected individuals, requiring lifelong ART. Among strategies proposed for targeting these viral reservoirs, the premise of the "shock and kill" strategy is to induce expression of latent proviruses [for example with histone deacetylase inhibitors (HDACis)] resulting in elimination of the affected cells through viral cytolysis or immune clearance mechanisms. Yet, ex vivo studies reported that HDACis have variable efficacy for reactivating latent proviruses, and hinder immune functions. We developed a nonhuman primate model of post-treatment control of SIV through early and prolonged administration of ART and performed in vivo reactivation experiments in controller RMs, evaluating the ability of the HDACi romidepsin (RMD) to reactivate SIV and the impact of RMD treatment on SIV-specific T cell responses. Ten RMs were IV-infected with a SIVsmmFTq transmitted-founder infectious molecular clone. Four RMs received conventional ART for >9 months, starting from 65 days post-infection. SIVsmmFTq plasma viremia was robustly controlled to <10 SIV RNA copies/mL with ART, without viral blips. At ART cessation, initial rebound viremia to ~106 copies/mL was followed by a decline to < 10 copies/mL, suggesting effective immune control. Three post-treatment controller RMs received three doses of RMD every 35-50 days, followed by in vivo experimental depletion of CD8+ cells using monoclonal antibody M-T807R1. RMD was well-tolerated and resulted in a rapid and massive surge in T cell activation, as well as significant virus rebounds (~104 copies/ml) peaking at 5-12 days post-treatment. CD8+ cell depletion resulted in a more robust viral rebound (107 copies/ml) that was controlled upon CD8+ T cell recovery. Our results show that RMD can reactivate SIV in vivo in the setting of post-ART viral control. Comparison of the patterns of virus rebound after RMD administration and CD8+ cell depletion suggested that RMD impact on T cells is only transient and does not irreversibly alter the ability of SIV-specific T cells to control the reactivated virus.

Nutritional status of children aged 3-6 years in a rural area of tamilnadu.

BACKGROUND: A child's entire life is determined in large measures by the food given to him during his first five years. Since childhood is the most vulnerable phase in the life of human being, nutritional inadequacies will result in the hampering of the development of the body. Future of the country is determined by the growing generation of the country. OBJECTIVES: a) To assess the nutritional status of children aged 3-6 y in a rural area of TamilNadu. b) To identify the factors associated with the nutritional status of the above study population. SETTINGS AND DESIGN: Kuthambakkam village in Tamilnadu, Cross sectional study. MATERIALS AND METHODS: Total number of children aged 3-6 y is 172 in Kuthambakkam village. The entire children aged 3-6 y was included in the study. Mothers of the children were interviewed using an interview schedule to collect information regarding the sociodemographic profile, feeding practices, and immunization status. Socio-economic status was assessed using modified BG Prasad's classification. Weight of the children was measured using a portable weighing machine. Nutritional status among 3-6 y old children was assessed by computing weight for age (standard used - National Centre For Health Statistics (NCHS) standards for weight for age) and grading of nutritional status of the children was done using the Indian Academy of Paediatrics (IAP) classification. Grade I to Grade IV nutritional grade is taken as undernourished. Statistical Analytical: Prevalence will be expressed in percentage and Chi-square test will be used to find association with factors. RESULTS: The prevalence of under-nutrition (≤ 80 percentage of standard weight for age) was 66.5%. The prevalence of grade 1 malnourishment was 46.2%.The prevalence of under nourishment increased with increasing age and the difference was found to be statistically significant (p< 0.05).Prevalence of under nourishment was higher among male children (76.9%)than female children (56.3%) and was statistically significant. As the socioeconomic status increased the prevalence of undernourishment decreased and the difference was found to be statistically significant(p<0.05). Duration of exclusive breast feeding had influence on the nutritional status. CONCLUSION: Community based preventive measures should be taken to allievate malnutrition. Health education to the mothers on dietary practices like feeding their children with locally available low cost but healthy food should be given. Nutritional rehabilitation centres should be established. Improving the socioeconomic standards is mandatory.