Viral and Host Factors Are Associated With Mortality in Hospitalized Patients With COVID-19.

BACKGROUND: Persistent mortality in adults hospitalized due to acute COVID-19 justifies pursuit of disease mechanisms and potential therapies. The aim was to evaluate which virus and host response factors were associated with mortality risk among participants in Therapeutics for Inpatients with COVID-19 (TICO/ACTIV-3) trials. METHODS: A secondary analysis of 2625 adults hospitalized for acute SARS-CoV-2 infection randomized to 1 of 5 antiviral products or matched placebo in 114 centers on 4 continents. Uniform, site-level collection of participant baseline clinical variables was performed. Research laboratories assayed baseline upper respiratory swabs for SARS-CoV-2 viral RNA and plasma for anti-SARS-CoV-2 antibodies, SARS-CoV-2 nucleocapsid antigen (viral Ag), and interleukin-6 (IL-6). Associations between factors and time to mortality by 90 days were assessed using univariate and multivariable Cox proportional hazards models. RESULTS: Viral Ag ≥4500 ng/L (vs <200 ng/L; adjusted hazard ratio [aHR], 2.07; 1.29-3.34), viral RNA (<35 000 copies/mL [aHR, 2.42; 1.09-5.34], ≥35 000 copies/mL [aHR, 2.84; 1.29-6.28], vs below detection), respiratory support (<4 L O2 [aHR, 1.84; 1.06-3.22]; ≥4 L O2 [aHR, 4.41; 2.63-7.39], or noninvasive ventilation/high-flow nasal cannula [aHR, 11.30; 6.46-19.75] vs no oxygen), renal impairment (aHR, 1.77; 1.29-2.42), and IL-6 >5.8 ng/L (aHR, 2.54 [1.74-3.70] vs ≤5.8 ng/L) were significantly associated with mortality risk in final adjusted analyses. Viral Ag, viral RNA, and IL-6 were not measured in real-time. CONCLUSIONS: Baseline virus-specific, clinical, and biological variables are strongly associated with mortality risk within 90 days, revealing potential pathogen and host-response therapeutic targets for acute COVID-19 disease.

Responses to a Neutralizing Monoclonal Antibody for Hospitalized Patients With COVID-19 According to Baseline Antibody and Antigen Levels : A Randomized Controlled Trial.

BACKGROUND: In a randomized, placebo-controlled, clinical trial, bamlanivimab, a SARS-CoV-2-neutralizing monoclonal antibody, given in combination with remdesivir, did not improve outcomes among hospitalized patients with COVID-19 based on an early futility assessment. OBJECTIVE: To evaluate the a priori hypothesis that bamlanivimab has greater benefit in patients without detectable levels of endogenous neutralizing antibody (nAb) at study entry than in those with antibodies, especially if viral levels are high. DESIGN: Randomized, placebo-controlled trial. (ClinicalTrials.gov: NCT04501978). SETTING: Multicenter trial. PATIENTS: Hospitalized patients with COVID-19 without end-organ failure. INTERVENTION: Bamlanivimab (7000 mg) or placebo. MEASUREMENTS: Antibody, antigen, and viral RNA levels were centrally measured on stored specimens collected at baseline. Patients were followed for 90 days for sustained recovery (defined as discharge to home and remaining home for 14 consecutive days) and a composite safety outcome (death, serious adverse events, organ failure, or serious infections). RESULTS: Among 314 participants (163 receiving bamlanivimab and 151 placebo), the median time to sustained recovery was 19 days and did not differ between the bamlanivimab and placebo groups (subhazard ratio [sHR], 0.99 [95% CI, 0.79 to 1.22]; sHR > 1 favors bamlanivimab). At entry, 50% evidenced production of anti-spike nAbs; 50% had SARS-CoV-2 nucleocapsid plasma antigen levels of at least 1000 ng/L. Among those without and with nAbs at study entry, the sHRs were 1.24 (CI, 0.90 to 1.70) and 0.74 (CI, 0.54 to 1.00), respectively (nominal P for interaction = 0.018). The sHR (bamlanivimab vs. placebo) was also more than 1 for those with plasma antigen or nasal viral RNA levels above median level at entry and was greatest for those without antibodies and with elevated levels of antigen (sHR, 1.48 [CI, 0.99 to 2.23]) or viral RNA (sHR, 1.89 [CI, 1.23 to 2.91]). Hazard ratios for the composite safety outcome (<1 favors bamlanivimab) also differed by serostatus at entry: 0.67 (CI, 0.37 to 1.20) for those without and 1.79 (CI, 0.92 to 3.48) for those with nAbs. LIMITATION: Subgroup analysis of a trial prematurely stopped because of futility; small sample size; multiple subgroups analyzed. CONCLUSION: Efficacy and safety of bamlanivimab may differ depending on whether an endogenous nAb response has been mounted. The limited sample size of the study does not allow firm conclusions based on these findings, and further independent trials are required that assess other types of passive immune therapies in the same patient setting. PRIMARY FUNDING SOURCE: U.S. government Operation Warp Speed and National Institute of Allergy and Infectious Diseases.

The Association of Baseline Plasma SARS-CoV-2 Nucleocapsid Antigen Level and Outcomes in Patients Hospitalized With COVID-19.

BACKGROUND: Levels of plasma SARS-CoV-2 nucleocapsid (N) antigen may be an important biomarker in patients with COVID-19 and enhance our understanding of the pathogenesis of COVID-19. OBJECTIVE: To evaluate whether levels of plasma antigen can predict short-term clinical outcomes and identify clinical and viral factors associated with plasma antigen levels in hospitalized patients with SARS-CoV-2. DESIGN: Cross-sectional study of baseline plasma antigen level from 2540 participants enrolled in the TICO (Therapeutics for Inpatients With COVID-19) platform trial from August 2020 to November 2021, with additional data on day 5 outcome and time to discharge. SETTING: 114 centers in 10 countries. PARTICIPANTS: Adults hospitalized for acute SARS-CoV-2 infection with 12 days or less of symptoms. MEASUREMENTS: Baseline plasma viral N antigen level was measured at a central laboratory. Delta variant status was determined from baseline nasal swabs using reverse transcriptase polymerase chain reaction. Associations between baseline patient characteristics and viral factors and baseline plasma antigen levels were assessed using both unadjusted and multivariable modeling. Association between elevated baseline antigen level of 1000 ng/L or greater and outcomes, including worsening of ordinal pulmonary scale at day 5 and time to hospital discharge, were evaluated using logistic regression and Fine-Gray regression models, respectively. RESULTS: Plasma antigen was below the level of quantification in 5% of participants at enrollment, and 1000 ng/L or greater in 57%. Baseline pulmonary severity of illness was strongly associated with plasma antigen level, with mean plasma antigen level 3.10-fold higher among those requiring noninvasive ventilation or high-flow nasal cannula compared with room air (95% CI, 2.22 to 4.34). Plasma antigen level was higher in those who lacked antispike antibodies (6.42 fold; CI, 5.37 to 7.66) and in those with the Delta variant (1.73 fold; CI, 1.41 to 2.13). Additional factors associated with higher baseline antigen level included male sex, shorter time since hospital admission, decreased days of remdesivir, and renal impairment. In contrast, race, ethnicity, body mass index, and immunocompromising conditions were not associated with plasma antigen levels. Plasma antigen level of 1000 ng/L or greater was associated with a markedly higher odds of worsened pulmonary status at day 5 (odds ratio, 5.06 [CI, 3.41 to 7.50]) and longer time to hospital discharge (median, 7 vs. 4 days; subhazard ratio, 0.51 [CI, 0.45 to 0.57]), with subhazard ratios similar across all levels of baseline pulmonary severity. LIMITATIONS: Plasma samples were drawn at enrollment, not hospital presentation. No point-of-care test to measure plasma antigen is currently available. CONCLUSION: Elevated plasma antigen is highly associated with both severity of pulmonary illness and clinically important patient outcomes. Multiple clinical and viral factors are associated with plasma antigen level at presentation. These data support a potential role of ongoing viral replication in the pathogenesis of SARS-CoV-2 in hospitalized patients. PRIMARY FUNDING SOURCE: U.S. government Operation Warp Speed and National Institute of Allergy and Infectious Diseases.

Leucine zipper transcription factor-like 1 (LZTFL1), an intraflagellar transporter protein 27 (IFT27) associated protein, is required for normal sperm function and male fertility.

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella, including mammalian sperm tails. Depletion of IFT27, a component of the IFT complex, in male germ cells results in infertility associated with disrupted sperm flagella structure and motility. Leucine zipper transcription factor-like 1 (LZTFL1) is an IFT27 associated protein. LZTFL1, also known as BBS17, is a Bardet-Biedl syndrome (BBS) associated protein. Patients carrying biallelic variants of LZTFL1 gene exhibit the common BBS phenotypes. The global Lztfl1 knockout mice showed abnormal growth rate and retinal degeneration, typical of BBS phenotype. However, it is not clear if Lztfl1 has a role in male fertility. The LZTFL1 protein is highly and predominantly expressed in mouse testis. During the first wave of spermatogenesis, the protein is only expressed during spermiogenesis phase from the round spermatid stage and displays a cytoplasmic localization with a vesicular distribution pattern. At the elongated spermatid stage, LZTFL1 is present in the developing flagella and appears also close to the manchette. Fertility of Lztfl1 knockout mice was significantly reduced and associated with low sperm motility and a high level of abnormal sperm (astheno-teratozoospermia). In vitro assessment of fertility revealed reduced fertilization and embryonic development when using sperm from homozygous mutant mice. In addition, we observed a significant decrease of the testicular IFT27 protein level in Lztfl1 mutant mice contrasting with a stable expression levels of other IFT proteins, including IFT20, IFT81, IFT88 and IFT140. Overall, our results support strongly the important role of LZTFL1 in mouse spermatogenesis and male fertility.

Administration of interleukin-7 increases CD4 T cells in idiopathic CD4 lymphocytopenia.

Idiopathic CD4 lymphopenia (ICL) is a rare syndrome defined by low CD4 T-cell counts (<300/µL) without evidence of HIV infection or other known cause of immunodeficiency. ICL confers an increased risk of opportunistic infections and has no established treatment. Interleukin-7 (IL-7) is fundamental for thymopoiesis, T-cell homeostasis, and survival of mature T cells, which provides a rationale for its potential use as an immunotherapeutic agent for ICL. We performed an open-label phase 1/2A dose-escalation trial of 3 subcutaneous doses of recombinant human IL-7 (rhIL-7) per week in patients with ICL who were at risk of disease progression. The primary objectives of the study were to assess safety and the immunomodulatory effects of rhIL-7 in ICL patients. Injection site reactions were the most frequently reported adverse events. One patient experienced a hypersensitivity reaction and developed non-neutralizing anti-IL-7 antibodies. Patients with autoimmune diseases that required systemic therapy at screening were excluded from the study; however, 1 participant developed systemic lupus erythematosus while on study and was excluded from further rhIL-7 dosing. Quantitatively, rhIL-7 led to an increase in the number of circulating CD4 and CD8 T cells and tissue-resident CD3 T cells in the gut mucosa and bone marrow. Functionally, these T cells were capable of producing cytokines after mitogenic stimulation. rhIL-7 was well tolerated at biologically active doses and may represent a promising therapeutic intervention in ICL. This trial was registered at www.clinicaltrials.gov as #NCT00839436.

LZTFL1 Upregulated by All-Trans Retinoic Acid during CD4+ T Cell Activation Enhances IL-5 Production.

Retinoic acids, which are metabolites of vitamin A, have been shown to be involved in multiple T cell effector responses through their binding to the retinoic acid receptor, a ligand-activated transcription factor. Because the molecular mechanism of regulation by retinoic acid is still not fully uncovered, we investigated the gene expression profile of all-trans retinoic acid (ATRA)-treated human CD4(+) T cells. Leucine zipper transcription factor-like 1 (LZTFL1) was upregulated by ATRA in a dose- and time-dependent manner. The expression of LZTFL1 depended on both ATRA and TCR signaling. LZTFL1 accumulated in the plasma membrane compartment of human CD4(+) T cells, and, during immunological synapse formation, it transiently redistributed to the T cell and APC contact zone, indicating its role in T cell activation. Live-cell imaging demonstrates that at the initial stage of immunological synapse formation, LZTFL1 is concentrated at the APC contact site, and, during later stages, it relocates to the distal pole. Knockdown of LZTFL1 reduced the basal- and ATRA-induced levels of IL-5 in CD4(+) T cells, and overexpression of LZTFL1 enhanced the TCR-mediated NFAT signaling, suggesting that LZTFL1 is an important regulator of ATRA-induced T cell response. Together, these data indicate that LZTFL1 modulates T cell activation and IL-5 levels.

Repeated Cycles of Recombinant Human Interleukin 7 in HIV-Infected Patients With Low CD4 T-Cell Reconstitution on Antiretroviral Therapy: Results of 2 Phase II Multicenter Studies.

BACKGROUND: Phase I/II studies in human immunodeficiency virus (HIV)-infected patients receiving antiretroviral therapy have shown that a single cycle of 3 weekly subcutaneous (s/c) injections of recombinant human interleukin 7 (r-hIL-7) is safe and improves immune CD4 T-cell restoration. Herein, we report data from 2 phase II trials evaluating the effect of repeated cycles of r-hIL-7 (20 µg/kg) with the objective of restoring a sustained CD4 T-cell count >500 cells/µL. METHODS: INSPIRE 2 was a single-arm trial conducted in the United States and Canada. INSPIRE 3 was a 2 arm trial with 3:1 randomization to r-hIL-7 versus control conducted in Europe and South Africa. Participants with plasma HIV RNA levels <50 copies/mL during antiretroviral therapy and with CD4 T-cell counts between 101 and 400 cells/µL were eligible. A repeat cycle was administered when CD4 T-cell counts fell to <550 cells/µL. RESULTS: A total of 107 patients were treated and received 1 (n = 107), 2 (n = 74), 3 (n = 14), or 4 (n = 1) r-hIL-7 cycles during a median follow-up of 23 months. r-hIL-7 was well tolerated. Four grade 4 events were observed, including 1 case of asymptomatic alanine aminotransferase elevation. After the second cycle, anti-r-hIL-7 binding antibodies developed in 82% and 77% of patients in INSPIRE 2 and 3, respectively (neutralizing antibodies in 38% and 37%), without impact on the CD4 T-cell response. Half of the patients spent >63% of their follow-up time with a CD4 T-cell count >500 cells/µL. CONCLUSIONS: Repeated cycles of r-hIL-7 were well tolerated and achieved sustained CD4 T-cell restoration to >500 cells/µL in the majority of study participants. CLINICAL TRIALS REGISTRATION: INSPIRE II: clinicaltrials.gov (NCT01190111) and INSPIRE III: EudraCT (No. 2010-019773-15) and clinicaltrials.gov (NCT01241643).

Decreases in colonic and systemic inflammation in chronic HIV infection after IL-7 administration.

Despite antiretroviral therapy (ART), some HIV-infected persons maintain lower than normal CD4(+) T-cell counts in peripheral blood and in the gut mucosa. This incomplete immune restoration is associated with higher levels of immune activation manifested by high systemic levels of biomarkers, including sCD14 and D-dimer, that are independent predictors of morbidity and mortality in HIV infection. In this 12-week, single-arm, open-label study, we tested the efficacy of IL-7 adjunctive therapy on T-cell reconstitution in peripheral blood and gut mucosa in 23 ART suppressed HIV-infected patients with incomplete CD4(+) T-cell recovery, using one cycle (consisting of three subcutaneous injections) of recombinant human IL-7 (r-hIL-7) at 20 µg/kg. IL-7 administration led to increases of both CD4(+) and CD8(+) T-cells in peripheral blood, and importantly an expansion of T-cells expressing the gut homing integrin α4ß7. Participants who underwent rectosigmoid biopsies at study baseline and after treatment had T-cell increases in the gut mucosa measured by both flow cytometry and immunohistochemistry. IL-7 therapy also resulted in apparent improvement in gut barrier integrity as measured by decreased neutrophil infiltration in the rectosigmoid lamina propria 12 weeks after IL-7 administration. This was also accompanied by decreased TNF and increased FOXP3 expression in the lamina propria. Plasma levels of sCD14 and D-dimer, indicative of systemic inflammation, decreased after r-hIL-7. Increases of colonic mucosal T-cells correlated strongly with the decreased systemic levels of sCD14, the LPS coreceptor - a marker of monocyte activation. Furthermore, the proportion of inflammatory monocytes expressing CCR2 was decreased, as was the basal IL-1ß production of peripheral blood monocytes. These data suggest that administration of r-hIL-7 improves the gut mucosal abnormalities of chronic HIV infection and attenuates the systemic inflammatory and coagulation abnormalities that have been linked to it.

Voriconazole metabolism, toxicity, and the effect of cytochrome P450 2C19 genotype.

BACKGROUND: Prospective evaluation of the antifungal drug, voriconazole, is needed to determine whether drug toxicity correlates with CYP2C19 genotype or serum concentrations of voriconazole or its metabolites. METHODS: We conducted a prospective study of 95 patients to determine voriconazole toxicity and its relationship to genotype and serum levels of voriconazole and its two metabolites. Efficacy was not evaluated because, in most cases, the drug was given for empirical or prophylactic therapy. RESULTS: Hallucinations occurred in 16 patients (16.8%), visual changes in 17 (17.9%), photosensitivity in 10 (10.5%), and hepatotoxicity in 6 (6.3%). There was no correlation between photosensitivity or hepatotoxicity and levels of voriconazole or metabolites. Patients with hallucinations had higher average voriconazole levels (4.5 vs 2.5 µg/mL) but with extensive overlap. The recommended oral dose of 200 mg did not provide consistently detectable serum voriconazole levels in adults. CYP2C19 and CYP2C9 genotypes had a minor influence over levels, though the 4 patients homozygous for the 2C19*2 genotype had higher average levels for voriconazole (4.3 vs 2.5 µg/mL) and lower N-oxide levels (1.6 vs 2.5 µg/mL). CONCLUSIONS: CYP2C19 and 2C9 genotypes were not major determinants of voriconazole metabolism. No toxic serum level of voriconazole or its metabolites could be identified.

Comprehensive analysis of unique cases with extraordinary control over HIV replication.

True long-term nonprogressors (LTNPs)/elite controllers (ECs) maintain durable control over HIV replication without antiretroviral therapy. Herein we describe 4 unique persons who were distinct from conventional LTNPs/ECs in that they had extraordinarily low HIV burdens and comparatively weak immune responses. As a group, typical LTNPs/ECs have unequivocally reactive HIV-1 Western blots, viral loads below the lower threshold of clinical assays, low levels of persistent viral reservoirs, an over-representation of protective HLA alleles, and robust HIV-specific CD8(+) T-cell responses. The 4 unique cases were distinguished from typical LTNPs/ECs based on weakly reactive Western blots, undetectable plasma viremia by a single copy assay, extremely low to undetectable HIV DNA levels, and difficult to isolate replication-competent virus. All 4 had at least one protective HLA allele and CD8(+) T-cell responses that were disproportionately high for the low antigen levels but comparatively lower than those of typical LTNPs/ECs. These unique persons exhibit extraordinary suppression over HIV replication, therefore, higher-level control than has been demonstrated in previous studies of LTNPs/ECs. Additional insight into the full spectrum of immune-mediated suppression over HIV replication may enhance our understanding of the associated mechanisms, which should inform the design of efficacious HIV vaccines and immunotherapies.

Long-term persistence of transcriptionally active 'defective' HIV-1 proviruses: implications for persistent immune activation during antiretroviral therapy.

OBJECTIVES: People with HIV-1 (PWH) on effective antiretroviral therapy (ART) continue to exhibit chronic systemic inflammation, immune activation, and persistent elevations in markers of HIV-1 infection [including HIV-DNA, cell-associated HIV-RNA (CA HIV-RNA), and antibodies to HIV-1 proteins] despite prolonged suppression of plasma HIV-RNA levels less than 50 copies/ml. Here, we investigated the hypothesis that nonreplicating but transcriptionally and translationally competent 'defective' HIV-1 proviruses may be one of drivers of these phenomena. DESIGN: A combined cohort of 23 viremic and virologically suppressed individuals on ART were studied. METHODS: HIV-DNA, CA HIV-RNA, western blot score (measure of anti-HIV-1 antibodies as a surrogate for viral protein expression in vivo ), and key biomarkers of inflammation and coagulation (IL-6, hsCRP, TNF-alpha, tissue factor, and D-dimer) were measured in peripheral blood and analyzed using a combined cross-sectional and longitudinal approaches. Sequences of HIV-DNA and CA HIV-RNA obtained via 5'-LTR-to-3'-LTR PCR and single-genome sequencing were also analyzed. RESULTS: We observed similar long-term persistence of multiple, unique, transcriptionally active 'defective' HIV-1 provirus clones (average: 11 years., range: 4-20 years) and antibody responses against HIV-1 viral proteins among all ART-treated participants evaluated. A direct correlation was observed between the magnitude of HIV-1 western blot score and the levels of transcription of 'defective' HIV-1 proviruses ( r  = 0.73, P  < 0.01). Additional correlations were noted between total CD8 + T-cell counts and HIV-DNA ( r  = 0.52, P  = 0.01) or CA HIV-RNA ( r  = 0.65, P  < 0.01). CONCLUSION: These findings suggest a novel interplay between transcription and translation of 'defective' HIV-1 proviruses and the persistent immune activation seen in the setting of treated chronic HIV-1 infection.

Retinoic acid and liver X receptor agonist synergistically inhibit HIV infection in CD4+ T cells by up-regulating ABCA1-mediated cholesterol efflux.

BACKGROUND: Retinoic acids regulate the reverse cholesterol transport by inducing the ATP binding cassette transporter A1 (ABCA1) dependent cholesterol efflux in macrophages, neuronal as well as intestine cells. In the present study, we aim to test the effect of all trans retinoic acid (ATRA) on ABCA1 expression in human CD4+ T cells and the involvement of cholesterol in ATRA mediated anti-HIV effect. RESULTS: Treatment with ATRA dramatically up-regulated ABCA1 expression in CD4+ T cells in a time and dose dependent manner. The expression of ABCA1 paralleled with increased ABCA1-dependent cholesterol efflux. This induction was dependent on T cell receptor (TCR) signaling and ATRA failed to induce ABCA1 expression in resting T cells. Moreover, ATRA and liver X receptor (LXR) agonist-TO-901317 together had synergistic effect on ABCA1 expression as well as cholesterol efflux. Increased ABCA1 expression was associated with lower cellular cholesterol staining. Cells treated with either ATRA or TO-901317 were less vulnerable to HIV-1 infection. Combination of retinoic acid and TO-901317 further inhibited HIV-1 entry and their inhibitory effects could be reversed by cholesterol replenishment. METHODS: ABCA1 RNA and protein were determined by real-time PCR and immuno blot methods in cells treated with ATRA. Cholesterol efflux rate was measured in cells treated with ATRA and TO-901317. CONCLUSIONS: ATRA up-regulates ABCA1 expression and cholesterol efflux in CD4+ T cells and combination of ATRA and liver X receptor (LXR) agonist further enhanced these effects. Increased cholesterol efflux contributed to reduced HIV-1 entry, suggesting that anti-HIV effect of ATRA is mediated through ABCA1.

LIM kinase 1 - dependent cofilin 1 pathway and actin dynamics mediate nuclear retinoid receptor function in T lymphocytes.

BACKGROUND: It is known that retinoid receptor function is attenuated during T cell activation, a phenomenon that involves actin remodeling, suggesting that actin modification may play a role in such inhibition. Here we have investigated the role of actin dynamics and the effect of actin cytoskeleton modifying agents on retinoid receptor-mediated transactivation. RESULTS: Agents that disturb the F-actin assembly or disassembly attenuated receptor-mediated transcription indicating that actin cytoskeletal homeostasis is important for retinoid receptor function. Overexpression or siRNA-induced knockdown of cofilin-1 (CFL1), a key regulator of F-actin assembly, induced the loss of receptor function. In addition, expression of either constitutively active or inactive/dominant-negative mutants of CFL1or CFL1 kinase LIMK1 induced loss of receptor function suggesting a critical role of the LIMK1-mediated CFL1 pathway in receptor-dependent transcription. Further evidence of the role of LMK1/CFL1-mediated actin dynamics, was provided by studying the effect of Nef, an actin modifying HIV-1 protein, on receptor function. Expression of Nef induced phosphorylation of CFL1 at serine 3 and LIMK1 at threonine 508, inhibited retinoid-receptor mediated reporter activity, and the expression of a number of genes that contain retinoid receptor binding sites in their promoters. The results suggest that the Nef-mediated inhibition of receptor function encompasses deregulation of actin filament dynamics by LIMK1 activation and phosphorylation of CFL1. CONCLUSION: We have identified a critical role of LIMK1-mediated CFL1 pathway and actin dynamics in modulating retinoid receptor mediated function and shown that LIMK1-mediated phosphocycling of CFL1 plays a crucial role in maintaining actin homeostasis and receptor activity. We suggest that T cell activation-induced repression of nuclear receptor-dependent transactivation is in part through the modification of actin dynamics.

Prolonged Posttreatment Virologic Control and Complete Seroreversion After Advanced Human Immunodeficiency Virus-1 Infection.

BACKGROUND: Possible human immunodeficiency virus (HIV)-1 clearance has rarely been reported. In this study, we describe a unique case of an HIV-positive, combination antiretroviral therapy (cART)-experienced woman with prior acquired immunodeficiency syndrome (AIDS) who has not experienced viral rebound for over 12 years since discontinuing cART. METHODS: Leukapheresis, colonoscopy, and lymph node excision were performed for detailed examination of virologic (including HIV reservoir) and immunologic features. Comparisons were made with chronically infected patients and healthy controls. RESULTS: No HIV-specific antibodies were detected in serum. Plasma HIV ribonucleic acid (RNA) levels were <0.2 copies/mL, and, except for low-frequency HIV deoxyribonucleic acid (DNA)+ cells in lymph node tissue (1 copy/3 × 106 cells), HIV antigen could not be detected by quantitative virus outgrowth (<0.0025 infectious units/106 CD4+ T cells) or by most measurements of HIV RNA or DNA in blood, lymph node, or gut-associated mononuclear cells. Human immunodeficiency virus-specific T-cell responses were detectable but low. Brain imaging revealed a prior biopsy site and persistent white matter disease since 1996. Human immunodeficiency virus DNA+ cells in the 1996 brain biopsy specimen confirmed her identity and initial HIV diagnosis. CONCLUSIONS: This represents the first report of complete seroreversion, prolonged posttreatment virus suppression, a profoundly small HIV reservoir, and persistent HIV-specific T cells in an adult with prior AIDS.

HCV in peripheral blood mononuclear cells are predominantly carried on the surface of cells in HIV/HCV co-infected individuals.

HCV replication in extra-hepatic reservoirs has been suggested to occur in many tissues including PBMCs. A recent study showed evidence for compartmentalization and evolution of HCV in PBMCs. However, the cells that support HCV replication in PBMCs have not been identified. In this study we have fractionated the PBMC from HIV/HCV co-infected patients into T, monocytes, B and NK cells, and most of the HCV was located in CD3-cell fractions. Protease treatment of PBMCs to remove cell surface receptors resulted in the loss of HCV RNA suggesting that most of the HCV is present on the cell surface. PBMCs were treated by freeze-thaw nuclease method that would protect the HCV RNA in the virus but not the intracellular viral RNA. Data from this analysis support the conclusion that most of HCV is present on the cell surface. Even though the presence of minus strand RNA in PBMCs suggests that a low level HCV replication takes place within the PBMCs of HIV/HCV co-infected individuals, HCV in PBMC is present mainly on the surface of non-T cells, mostly on NK, monocytes and B cells. These results suggest a unique pathogenic role of NK, monocyte and B cells as carriers of HCV.

RNA-activated protein kinase differentially modulates innate immune response mediated by supraphysiological concentrations of thyroid hormone.

Nuclear hormone receptor ligands are known to modulate innate immunity by dampening the immune response induced by pathogens. Here, we report that unlike other ligands, 3,3',5-triiodo-l-thyronine (T3) induced the type 1 IFN response and expression of IFN-stimulated genes (ISGs). T3 action was found to be significantly amplified at supraphysiological concentrations (SPC) and in combination with double-stranded RNA mimic polyinosinic-polycytidylic acid. Induction by T3 was due to non-genomic mechanisms involving integrin binding, calcium mobilization, and phosphatidyl-inositol 3-kinase-AKT pathways, but was independent of TLR3, RIG-I, and IFN-ß1 pathways. Whereas siRNA-induced knockdown of RNA-activated protein kinase (PKR) was found to abrogate the T3-induced expression of select ISGs, expression of other T3-induced ISGs was strongly induced by PKR knockdown, indicating the differential role of PKR in modulating T3 action. Together, we describe a novel role of T3 in modulating the innate immune response and identify the importance of PKR in regulating T3-induced immune activation. These findings have important implications in the basic understanding of the mechanisms of T3 function at SPCs and crosstalk involved in the thyroid hormone function and the innate immune response.

Leucine zipper transcription factor-like 1 binds adaptor protein complex-1 and 2 and participates in trafficking of transferrin receptor 1.

LZTFL1 participates in immune synapse formation, ciliogenesis, and the localization of ciliary proteins, and knockout of LZTFL1 induces abnormal distribution of heterotetrameric adaptor protein complex-1 (AP-1) in the Lztfl1-knockout mouse photoreceptor cells, suggesting that LZTFL1 is involved in intracellular transport. Here, we demonstrate that in vitro LZTFL1 directly binds to AP-1 and AP-2 and coimmunoprecipitates AP-1 and AP-2 from cell lysates. DxxFxxLxxxR motif of LZTFL1 is essential for these bindings, suggesting LZTFL1 has roles in AP-1 and AP-2-mediated protein trafficking. Since AP-1 and AP-2 are known to be involved in transferrin receptor 1 (TfR1) trafficking, the effect of LZTFL1 on TfR1 recycling was analyzed. TfR1, AP-1 and LZTFL1 from cell lysates could be coimmunoprecipitated. However, pull-down results indicate there is no direct interaction between TfR1 and LZTFL1, suggesting that LZTFL1 interaction with TfR1 is indirect through AP-1. We report the colocalization of LZTFL1 and AP-1, AP-1 and TfR1 as well as LZTFL1 and TfR1 in the perinuclear region (PNR) and the cytoplasm, suggesting a potential complex between LZTFL1, AP-1 and TfR1. The results from the disruption of adaptin recruitment with brefeldin A treatment suggested ADP-ribosylation factor-dependent localization of LZFL1 and AP-1 in the PNR. Knockdown of AP-1 reduces the level of LZTFL1 in the PNR, suggesting that AP-1 plays a role in LZTFL1 trafficking. Knockout of LZTFL1 reduces the cell surface level and the rate of internalization of TfR1, leading to a decrease of transferrin uptake, efflux, and internalization. However, knockout of LZTFL1 did not affect the cell surface levels of epidermal growth factor receptor and cation-independent mannose 6-phosphate receptor, indicating that LZTFL1 specifically regulates the cell surface level of TfR1. These data support a novel role of LZTFL1 in regulating the cell surface TfR1 level by interacting with AP-1 and AP-2.

GADD34 attenuates HIV-1 replication by viral 5'-UTR TAR RNA-mediated translational inhibition.

Role of GADD34, a protein that is induced following cellular stress, in HIV-1 replication was investigated. GADD34 was induced during the late phase of HIV-1 infection. siRNA-knockdown of GADD34 stimulated whereas overexpression of GADD34 inhibited HIV-1 replication. GADD34 N-terminal ER-binding-helix amino acid region 1-192 alone was found to be sufficient for the inhibition of HIV-1 replication whereas protein-phosphatase -1-binding domain and eIF-2α-phosphatase activity of GADD34 were not crucial for anti-HIV-1 activity. GADD34 did not alter the HIV-1 RNA levels but reduced the viral protein expression suggesting that GADD34 interferes in HIV protein synthesis. Studies on the effect of HIV-1-5'-UTR and its mutants on a human promoter-driven luciferase expression indicated that GADD34-inhibition was mediated by 5'-UTR/TAR RNA, probably by modulating TAR RNA structure. In summary, our data support a novel function of GADD34 as a putative anti-HIV-1 restriction factor.

Adoptive lymphocyte transfer to an HIV-infected progressor from an elite controller.

BACKGROUNDHIV-infected patients with poor virologic control and multidrug-resistant virus have limited therapeutic options. The current study was undertaken to evaluate the safety, immunologic effects, and antiviral activity of peripheral lymphocytes transferred from an elite controller, whose immune system is able to control viral replication without antiretroviral medications, to an HLA-B*2705-matched progressor.METHODSApproximately 22 billion cells were collected from an elite controller by lymphapheresis and infused within 6 hours into a recipient with a preinfusion CD4+ T cell count of 10 cells/µL (1%) and HIV plasma viral load of 114,993 copies/mL.RESULTSDonor cells were cleared from the recipient's peripheral blood by day 8. A transient decrease in viral load to 58,421 (day 3) was followed by a rebound to 702,972 (day 6) before returning to baseline values by day 8. The decreased viral load was temporally associated with peak levels of donor T cells, including CD8+ T cells that had high levels of expression of Ki67, perforin, and granzyme B. Notably, recipient CD8+ T cells also showed increased expression of these markers, especially in HIV-specific tetramer-positive cells.CONCLUSIONThese results suggest that the adoptive transfer of lymphocytes from an HIV-infected elite controller to an HIV-infected patient with progressive disease may be able to perturb the immune system of the recipient in both positive and negative ways.TRIAL REGISTRATIONClinicalTrials.gov NCT00559416.FUNDINGIntramural Research Programs of the US NIH Clinical Center and the National Institute of Allergy and Infectious Diseases (NIAID); the National Cancer Institute.

In vivo expansion of CD4CD45RO-CD25 T cells expressing foxP3 in IL-2-treated HIV-infected patients.

Administration of IL-2 to HIV-infected patients leads to expansion of a unique subset of CD4CD45ROCD25 cells. In this study, the origin, clonality, and function of these cells were investigated. Analysis of TCR excision circles revealed that the CD4CD45ROCD25 cells were the product of peripheral expansion but remained polyclonal as determined by TCR repertoire analysis. Phenotypically, these cells were distinct from naturally occurring Tregs; they exhibited intermediate features, between those of memory and naive cells, and had lower susceptibility to apoptosis than CD45ROCD25 or memory T cells. Studies of intracellular cytokine production and proliferation revealed that cytokine-expanded naive CD25 cells had low IL-2 production and required costimulation for proliferation. Despite elevated expression of forkhead transcription factor P3 (foxP3), they exerted only weak suppression compared with CD45ROCD25 cells (Tregs). In summary, in vivo IL-2 administration to HIV-infected patients leads to peripheral expansion of a population of long-lived CD4CD45ROCD25 cells that express high levels of foxP3 but exert weak suppressive function. These CD4CD25 cytokine-expanded naive cells, distinct from antigen-triggered cells and Tregs, play a role in the maintenance of a state of low turnover and sustained expansion of the CD4 T cell pool.