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Myocardial infarction (MI) results in aberrant cardiac metabolism, but no therapeutics have been designed to target cardiac metabolism to enhance heart repair. We engineer a humanized monoclonal antibody against the ectonucleotidase ENPP1 (hENPP1mAb) that targets metabolic crosstalk in the infarcted heart. In mice expressing human ENPP1, systemic administration of hENPP1mAb metabolically reprograms myocytes and non-myocytes and leads to a significant rescue of post-MI heart dysfunction. Using metabolomics, single-nuclear transcriptomics, and cellular respiration studies, we show that the administration of the hENPP1mAb induces organ-wide metabolic and transcriptional reprogramming of the heart that enhances myocyte cellular respiration and decreases cell death and fibrosis in the infarcted heart. Biodistribution and safety studies showed specific organ-wide distribution with the antibody being well tolerated. In humanized animals, with drug clearance kinetics similar to humans, we demonstrate that a single "shot" of the hENPP1mAb after MI is sufficient to rescue cardiac dysfunction.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is characterized by rapid functional decline. Current available treatment options aim to delay disease progression or stabilize physical function. To aid in healthcare providers understanding of the symptoms of disease that impact patients experience, this study explored childrens physical functioning, activities of daily living (ADLs), and health-related quality of life (HRQoL) after receiving eteplirsen, a weekly infusion indicated for individuals with DMD with exon 51 skip-amenable mutations. METHODS: Fifteen caregivers of male individuals with DMD participated in a 60-min, semi-structured interview. Open-ended questioning explored changes in the childrens condition or maintenance in abilities since eteplirsen initiation. RESULTS: Children with DMD (age 7-15 years [mean 10.9]; steroid treatment at interview, n = 8; time since eteplirsen initiation 3-24 months [mean 14.9]) were described by caregivers as ambulatory (n = 9) and non-ambulatory (n = 6). Caregivers of ambulatory children reported improvements or maintenance of walking ability (n = 7/9), running (n = 6/9), and using stairs (n = 4/9). Continued decline in using stairs was reported by two caregivers. In upper-limb functioning, improvements or maintenances in fine-motor movements were reported by nearly half of all caregivers (n = 7/15), with one caregiver noting a continued decline. Subsequent improvements or maintenances in ADLs were described. Improvements or maintenances in fatigue (n = 9/15), muscle weakness (n = 7/15), and pain (n = 6/15) were reported, although some caregivers described a continued decline (n = 3/15 fatigue, n = 1/15 muscle weakness, n = 2/15 pain). Importantly, most caregivers who reported maintenances in ability perceived this as a positive outcome (n = 6/9). CONCLUSION: This exploratory study indicated that most caregivers perceived improvements or maintenances in aspects of their childs physical functioning, ADLs, and HRQoL since eteplirsen initiation, which they perceived to be a positive outcome.

Chronic Human Immunodeficiency Virus (HIV) infection remains a significant challenge to global public health. Despite advances in antiretroviral therapy (ART), which has transformed HIV infection from a fatal disease into a manageable chronic condition, a definitive cure remains elusive. One of the key features of HIV infection is chronic immune activation and inflammation, which are strongly associated with, and predictive of, HIV disease progression, even in patients successfully treated with suppressive ART. Chronic inflammation is characterized by persistent inflammation, immune cell metabolic dysregulation, and cellular exhaustion and dysfunction. This review aims to summarize current knowledge of the interplay between chronic inflammation, immune metabolism, and T cell dysfunction in HIV infection, and also discusses the use of humanized mice models to study HIV immune pathogenesis and develop novel therapeutic strategies.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16178. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

PurposeTraditionally, appropriate anchors are used to investigate the amount of change on a clinician-reported outcome assessment that is meaningful to individual patients. However, novel qualitative methods involving input from disease state experts together with patients may better inform the individual improvement threshold for demonstrating the clinical benefit of new treatments. This study aimed to establish a clinically meaningful threshold for treatment success for the clinician-reported Severity of Alopecia Tool (SALT) score for patients with alopecia areata (AA).MethodsA purposive sample of 10 dermatologists expert in AA and 30 adult and adolescent patients with AA and a history of ≥ 50% scalp hair loss were recruited. Semi-structured interview questions explored the outcome that represented treatment success to clinicians and patients. Findings were analyzed using thematic methods to identify treatment success thresholds.ResultsBoth informant groups confirmed scalp hair amount as the outcome of priority. Most expert clinicians considered a static threshold of 80% (n = 5) or 75% (n = 3) of the scalp hair as a treatment success. Most patient responses ranged from 70 to 90% (median: 80% of the scalp hair). Subsequently, queried patients confirmed that achieving SALT score ≤ 20 with treatment would be a success, as reflected in the Alopecia Areata Investigator Global Assessment (AA-IGA™). The novel qualitative processes used to inform this meaningful threshold reflects a clinician-then-patient process for: (a) confirmation of the patient outcome of priority; and (b) clinician input on a preliminary treatment success level for independent understanding among patients.ConclusionThis qualitative investigation of expert clinicians-then-patients with AA confirmed that achieving an amount of 80% or more scalp hair (SALT score ≤ 20) was an appropriate individual treatment success threshold indicating clinically meaningful improvement for patients with ≥ 50% scalp hair loss. A qualitative investigation of a quantifiable treatment success threshold is possible through a well-designed interview process with expert clinicians and the appropriate patient population.

IntroductionWhile antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined.MethodsInterferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD).ResultsIn two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination.DiscussionOverall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

The cellular response to stress is an important determinant of disease pathogenesis. Uncovering the molecular fingerprints of distinct stress responses may identify novel biomarkers and key signaling pathways for different diseases. Emerging evidence shows that transfer RNA-derived small RNAs (tDRs) play pivotal roles in stress responses. However, RNA modifications present on tDRs are barriers to accurately quantifying tDRs using traditional small RNA sequencing. Here, AlkB-facilitated methylation sequencing is used to generate a comprehensive landscape of cellular and extracellular tDR abundances in various cell types during different stress responses. Extracellular tDRs are found to have distinct fragmentation signatures from intracellular tDRs and these tDR signatures are better indicators of different stress responses than miRNAs. These distinct extracellular tDR fragmentation patterns and signatures are also observed in plasma from patients on cardiopulmonary bypass. It is additionally demonstrated that angiogenin and RNASE1 are themselves regulated by stressors and contribute to the stress-modulated abundance of sub-populations of cellular and extracellular tDRs. Finally, a sub-population of extracellular tDRs is identified for which AGO2 appears to be required for their expression. Together, these findings provide a detailed profile of stress-responsive tDRs and provide insight about tDR biogenesis and stability in response to cellular stressors.

BackgroundThe content validity (appropriateness and acceptability) of patient-reported outcome (PRO) measures for scalp hair loss, eyebrow loss, eyelash loss, nail damage and eye irritation has been demonstrated in adults with alopecia areata (AA) but not adolescents.ObjectivesTo explore the content validity of the suite of AA PRO measures and accompanying photoguides in an adolescent sample.MethodsSemi-structured, 90-min, combined concept elicitation and cognitive interviews were conducted face-to-face with adolescents who experienced ≥ 50% AA-related scalp hair loss. Transcripts underwent thematic and framework analysis.ResultsEleven adolescents (aged 12-17 years, 55% female, 45% nonwhite) diagnosed with AA for 5·9 years (mean) participated. Participants had 69·6% scalp hair (mean) and current eyebrow (82%) and/or eyelash loss (82%) and/or nail involvement (36%). Adolescents reported scalp, eyebrow and eyelash hair loss as their top three most bothersome signs/symptoms. Despite mostly accepting their AA, impacts related to visible areas of hair loss were prominent. Participants demonstrated good understanding and appropriate use of the PRO measures, and advocated including hair loss percentages alongside descriptive categories in the Scalp Hair Assessment PRO™. Results confirmed treatment success thresholds established with adults: achievement of ≤ 20% scalp hair loss, no/minimal eyebrow and eyelash loss, no/a little nail damage and eye irritation (PRO measure categories 0 or 1).ConclusionsThe Scalp Hair Assessment PRO™, PRO Measure for Eyebrows™, PRO Measure for Eyelashes™, PRO Measure for Nail Appearance™ and PRO Measure for Eye Irritation™ and accompanying photoguides are fit-for-purpose self-reported measures of AA signs/symptoms that are impactful to adolescents with AA.

Novel therapeutic strategies are needed to attenuate increased systemic and gut inflammation that contribute to morbidity and mortality in chronic HIV infection despite potent antiretroviral therapy (ART). The goal of this study is to use preclinical models of chronic treated HIV to determine whether the antioxidant and anti-inflammatory apoA-I mimetic peptides 6F and 4F attenuate systemic and gut inflammation in chronic HIV. We used two humanized murine models of HIV infection and gut explants from 10 uninfected and 10 HIV infected persons on potent ART, to determine the in vivo and ex vivo impact of apoA-I mimetics on systemic and intestinal inflammation in HIV. When compared to HIV infected humanized mice treated with ART alone, mice on oral apoA-I mimetic peptide 6F with ART had consistently reduced plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) that are products of ADAM17 sheddase activity. Oral 6F attenuated gut protein levels of ADAM17 that were increased in HIV-1 infected mice on potent ART compared to uninfected mice. Adding oxidized lipoproteins and endotoxin (LPS) ex vivo to gut explants from HIV infected persons increased levels of ADAM17 in myeloid and intestinal cells, which increased TNF-α and CX3CL1. Both 4F and 6F attenuated these changes. Our preclinical data suggest that apoA-I mimetic peptides provide a novel therapeutic strategy that can target increased protein levels of ADAM17 and its sheddase activity that contribute to intestinal and systemic inflammation in treated HIV. The large repertoire of inflammatory mediators involved in ADAM17 sheddase activity places it as a pivotal orchestrator of several inflammatory pathways associated with morbidity in chronic treated HIV that make it an attractive therapeutic target.

Cannabis (Cannabis sativa) is a widely used drug in the United States and the frequency of cannabis use is particularly high among people living with HIV (PLWH). One key component of cannabis, the non-psychotropic (-)-cannabidiol (CBD) exerts a wide variety of biological actions, including anticonvulsive, analgesic, and anti-inflammatory effects. However, the exact mechanism of action through which CBD affects the immune cell signaling remains poorly understood. Here we report that CBD modulates type I interferon responses in human macrophages. Transcriptomics analysis shows that CBD treatment significantly attenuates cGAS-STING-mediated activation of type I Interferon response genes (ISGs) in monocytic THP-1 cells. We further showed that CBD treatment effectively attenuates 2'3-cGAMP stimulation of ISGs in both THP-1 cells and primary human macrophages. Interestingly, CBD significantly upregulates expression of autophagy receptor p62/SQSTM1. p62 is critical for autophagy-mediated degradation of stimulated STING. We observed that CBD treated THP-1 cells have elevated autophagy activity. Upon 2'3'-cGAMP stimulation, CBD treated cells have rapid downregulation of phosphorylated-STING, leading to attenuated expression of ISGs. The CBD attenuation of ISGs is reduced in autophagy deficient THP-1 cells, suggesting that the effects of CBD on ISGs is partially mediated by autophagy induction. Lastly, CBD decreases ISGs expression upon HIV infection in THP-1 cells and human primary macrophages, leading to increased HIV RNA expression 24 hours after infection. However, long term culture with CBD in infected primary macrophages reduced HIV viral spread, suggesting potential dichotomous roles of CBD in HIV replication. Our study highlights the immune modulatory effects of CBD and the needs for additional studies on its effect on viral infection and inflammation.

Extracellular vesicles (EVs) mediate intercellular signaling by transferring their cargo to recipient cells, but the functional consequences of signaling are not fully appreciated. RBC-derived EVs are abundant in circulation and have been implicated in regulating immune responses. Here, we use a transgenic mouse model for fluorescence-based mapping of RBC-EV recipient cells to assess the role of this intercellular signaling mechanism in heart disease. Using fluorescent-based mapping, we detected an increase in RBC-EV-targeted cardiomyocytes in a murine model of ischemic heart failure. Single cell nuclear RNA sequencing of the heart revealed a complex landscape of cardiac cells targeted by RBC-EVs, with enrichment of genes implicated in cell proliferation and stress signaling pathways compared with non-targeted cells. Correspondingly, cardiomyocytes targeted by RBC-EVs more frequently express cellular markers of DNA synthesis, suggesting the functional significance of EV-mediated signaling. In conclusion, our mouse model for mapping of EV-recipient cells reveals a complex cellular network of RBC-EV-mediated intercellular communication in ischemic heart failure and suggests a functional role for this mode of intercellular signaling.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15539. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Studies of two SARS-CoV-2 mRNA vaccines suggested that they yield ∼95% protection from symptomatic infection at least short-term, but important clinical questions remain. It is unclear how vaccine-induced antibody levels quantitatively compare to the wide spectrum induced by natural SARS-CoV-2 infection. Vaccine response kinetics and magnitudes in persons with prior COVID-19 compared to virus-naı̈ve persons are not well-defined. The relative stability of vaccine-induced versus infection-induced antibody levels is unclear. We addressed these issues with longitudinal assessments of vaccinees with and without prior SARS-CoV-2 infection using quantitative enzyme-linked immunosorbent assay (ELISA) of anti-RBD antibodies. SARS-CoV-2-naı̈ve individuals achieved levels similar to mild natural infection after the first vaccination; a second dose generated levels approaching severe natural infection. In persons with prior COVID-19, one dose boosted levels to the high end of severe natural infection even in those who never had robust responses from infection, increasing no further after the second dose. Antiviral neutralizing assessments using a spike-pseudovirus assay revealed that virus-naı̈ve vaccinees did not develop physiologic neutralizing potency until the second dose, while previously infected persons exhibited maximal neutralization after one dose. Finally, antibodies from vaccination waned similarly to natural infection, resulting in an average of ∼90% loss within 90 days. In summary, our findings suggest that two doses are important for quantity and quality of humoral immunity in SARS-CoV-2-naı̈ve persons, while a single dose has maximal effects in those with past infection. Antibodies from vaccination wane with kinetics very similar to that seen after mild natural infection; booster vaccinations will likely be required.

Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets.

Due to the durability and persistence of reservoirs of HIV-1-infected cells, combination antiretroviral therapy (ART) is insufficient in eradicating infection. Achieving HIV-1 cure or sustained remission without ART treatment will require the enhanced and persistent effective antiviral immune responses. Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy and show promise in treating HIV-1 infection. Persistence, trafficking, and maintenance of function remain to be a challenge in many of these approaches, which are based on peripheral T cell modification. To overcome many of these issues, we have previously demonstrated successful long-term engraftment and production of anti-HIV CAR T cells in modified hematopoietic stem cells (HSCs) in vivo. Here we report the development and in vivo testing of second generation CD4-based CARs (CD4CAR) against HIV-1 infection using a HSCs-based approach. We found that a modified, truncated CD4-based CAR (D1D2CAR) allows better CAR-T cell differentiation from gene modified HSCs, and maintains similar CTL activity as compared to the full length CD4-based CAR. In addition, D1D2CAR does not mediate HIV infection or stimulation mediated by IL-16, suggesting lower risk of off-target effects. Interestingly, stimulatory domains of 4-1BB but not CD28 allowed successful hematopoietic differentiation and improved anti-viral function of CAR T cells from CAR modified HSCs. Addition of 4-1BB to CD4 based CARs led to faster suppression of viremia during early untreated HIV-1 infection. D1D2CAR 4-1BB mice had faster viral suppression in combination with ART and better persistence of CAR T cells during ART. In summary, our data indicate that the D1D2CAR-41BB is a superior CAR, showing better HSC differentiation, viral suppression and persistence, and less deleterious functions compared to the original CD4CAR, and should continue to be pursued as a candidate for clinical study.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) with CCR5- donor cells is the only treatment known to cure HIV-1 in patients with underlying malignancy. This is likely due to a donor cell-mediated graft-versus-host effect targeting HIV reservoirs. Allo-HSCT would not be an acceptable therapy for most people living with HIV due to the transplant-related side effects. Chimeric antigen receptor (CAR) immunotherapies specifically traffic to malignant lymphoid tissues (lymphomas) and, in some settings, are able to replace allo-HSCT. Here, we quantified the engraftment of HSC-derived, virus-directed CAR T cells within HIV reservoirs in a macaque model of HIV infection, using potentially novel IHC assays. HSC-derived CAR cells trafficked to and displayed multilineage engraftment within tissue-associated viral reservoirs, persisting for nearly 2 years in lymphoid germinal centers, the brain, and the gastrointestinal tract. Our findings demonstrate that HSC-derived CAR+ cells reside long-term and proliferate in numerous tissues relevant for HIV infection and cancer.

BackgroundValid patient-reported outcome (PRO) measures are required to evaluate alopecia areata (AA) treatments.ObjectivesTo develop a content-valid and clinically meaningful PRO measure to assess AA scalp hair loss with scores comparable with the five-response-level Alopecia Areata Investigator Global Assessment (AA-IGA™).MethodsA draft PRO measure was developed based on input from 10 clinical experts in AA. The PRO measure was cognitively debriefed, modified and finalized through two rounds of qualitative semistructured interviews with patients with AA who had experienced ≥ 50% scalp hair loss. Data were thematically analysed.ResultsAdults (round 1: n = 25; round 2: n = 15) and adolescents aged 15-17 years (round 1: n = 5) in North America participated. All patients named scalp hair loss as a key AA sign or symptom. Patients demonstrated the ability to self-report their current amount of scalp hair using percentages. In round 1 not all patients interpreted the measurement concept consistently; therefore, the PRO was modified to clarify the measurement concept to improve usability. Following modifications, patients in round 2 responded without difficulty to the PRO measure. Patients confirmed that they could use the five-level response scale to rate their scalp hair loss: no missing hair, 0%; limited, 1-20%; moderate, 21-49%; large, 50-94%; nearly all or all, 95-100%. Almost all patients deemed hair regrowth resulting in ≤ 20% scalp hair loss a treatment success.ConclusionsThe Scalp Hair Assessment PRO™ is a content-valid, clinically meaningful assessment of distinct gradations of scalp hair loss for evaluating AA treatment for patients with ≥ 50% hair loss at baseline.

BackgroundContent-valid and clinically meaningful instruments are required to evaluate outcomes of therapeutic interventions in alopecia areata (AA).ObjectivesTo develop an Investigator's Global Assessment (IGA) to interpret treatment response in AA treatment studies.MethodsQualitative interviews were conducted in the USA with expert dermatologists and with patients with AA who had experienced ≥ 50% scalp-hair loss. Thematic data analysis identified critical outcomes and evaluated the content validity of the new IGA.ResultsExpert clinicians (n = 10) judged AA treatment success by the amount of scalp-hair growth (median 80% scalp hair). Adult (n = 25) and adolescent (n = 5) patients participated. Scalp-hair loss was the most bothersome AA sign/symptom for most patients. Perceived treatment success - short of 100% scalp hair - was the presence of ~ 70-90% scalp hair (median 80%). Using additional clinician and patient insights, the Alopecia Areata Investigator Global Assessment (AA-IGA™) was developed. This clinician-reported outcome assessment is an ordinal, static measure comprising five severity categories of scalp-hair loss. Nearly all clinicians and patients in this study agreed that, for patients with ≥ 50% scalp-hair loss, successful treatment would be hair regrowth resulting in ≤ 20% scalp-hair loss.ConclusionsWe recommend using the Severity of Alopecia Tool to assess the extent (0-100%) of scalp-hair loss. The AA-IGA is a robust ordinal measure providing distinct and clinically meaningful gradations of scalp-hair loss that reflects patients' and expert clinicians' perspectives and treatment expectations. What is already known about this topic? The Severity of Alopecia Tool is widely used to assess the extent of scalp-hair loss in patients with alopecia areata. Guidelines define treatment success as a 50% improvement in scalp hair, and clinical trials have used dynamic thresholds of 50% and 90%. However, there is no clinical consensus on these endpoints, and patient perspectives on treatment success are unknown. What does this study add? Through qualitative interviews with 10 expert dermatologists and 30 patients with alopecia areata who had experienced ≥ 50% scalp-hair loss, we developed the Alopecia Areata Investigator Global Assessment (AA-IGA™) to measure five clinically meaningful gradations of alopecia areata scalp-hair loss that reflects patients' and clinicians' perspectives and expectations of treatment success in alopecia areata treatment studies. What are the clinical implications of this work? The AA-IGA is a robust ordinal measure that can inform clinical evaluation of alopecia areata treatment outcomes. The AA-IGA can be used to determine clinically meaningful treatment success for alopecia areata, with success defined by patients and clinicians as reaching ≤ 20% scalp-hair loss. Linked Comment: Blome. Br J Dermatol 2020; 183:609.

BackgroundEyebrow and eyelash hair loss and nail damage-in addition to scalp hair loss-are important signs/symptoms of alopecia areata (AA) to patients and deserve assessment in AA clinical trials.ObjectivesOur objective was to develop clinician-reported outcome (ClinRO) and patient-reported outcome (PRO) measures and accompanying photoguides to aid in the assessment of AA-related eyebrow, eyelash and nail signs/symptoms.MethodsIterative rounds of qualitative, semi-structured interviews were conducted with US expert dermatologists and North American patients with AA. Patients with eyebrow, eyelash and nail involvement were purposefully sampled. Interview transcripts were qualitatively analyzed.ResultsDermatologists (n = 10) described eyebrow and eyelash loss as concerning for affected patients and, along with nail appearance, as deserving assessment. Dermatologist data informed the development of single item, 4-point Likert-type ClinRO and PRO measures of current eyebrow loss, eyelash loss and nail appearance and a PRO measure of eye irritation. Patients (n = 45, age 15-72 years) confirmed the importance and relevance of these signs/symptoms. Interim revision resulted in measures that were understood by and relevant to patients. Dermatologists (n = 5) and patients (n = 10, age 21-54 years) participated in the development of the eyebrow, eyelash and nail photoguides and confirmed that they included photos that appropriately represented different severity levels and were helpful to derive and standardize ratings across raters.ConclusionsThe ClinRO and PRO measures for eyebrow, eyelash and nail appearance, with their accompanying photoguides and the PRO Measure for Eye Irritation provide clear and meaningful assessments of outcomes important to patients with AA.

The HIV reservoir remains to be a difficult barrier to overcome in order to achieve a therapeutic cure for HIV. Several strategies have been developed to purge the reservoir, including the "kick and kill" approach, which is based on the notion that reactivating the latent reservoir will allow subsequent elimination by the host anti-HIV immune cells. However, clinical trials testing certain classes of latency reactivating agents (LRAs) have so far revealed the minimal impact on reducing the viral reservoir. A robust immune response to reactivated HIV expressing cells is critical for this strategy to work. A current focus to enhance anti-HIV immunity is through the use of chimeric antigen receptors (CARs). Currently, HIV-specific CARs are being applied to peripheral T cells, NK cells, and stem cells to boost recognition and killing of HIV infected cells. In this review, we summarize current developments in engineering HIV directed CAR-expressing cells to facilitate HIV elimination. We also summarize current LRAs that enhance the "kick" strategy and how new generation and combinations of LRAs with HIV specific CAR T cell therapies could provide an optimal strategy to target the viral reservoir and achieve HIV clearance from the body.

Invariant natural killer T (iNKT) cells are potent immune cells for targeting cancer; however, their clinical application has been hindered by their low numbers in cancer patients. Here, we developed a proof-of-concept for hematopoietic stem cell-engineered iNKT (HSC-iNKT) cell therapy with the potential to provide therapeutic levels of iNKT cells for a patient's lifetime. Using a human HSC engrafted mouse model and a human iNKT TCR gene engineering approach, we demonstrated the efficient and long-term generation of HSC-iNKT cells in vivo. These HSC-iNKT cells closely resembled endogenous human iNKT cells, could deploy multiple mechanisms to attack tumor cells, and effectively suppressed tumor growth in vivo in multiple human tumor xenograft mouse models. Preclinical safety studies showed no toxicity or tumorigenicity of the HSC-iNKT cell therapy. Collectively, these results demonstrated the feasibility, safety, and cancer therapy potential of the proposed HSC-iNKT cell therapy and laid a foundation for future clinical development.

HIV-1-infected individuals are treated with lifelong antiretroviral drugs to control the infection. A means to strengthen the antiviral T cell response might allow them to control viral loads without antiretroviral drugs. We report the development of a lentiviral vector-based dendritic cell (DC) vaccine in which HIV-1 antigen is co-expressed with CD40 ligand (CD40L) and a soluble, high-affinity programmed cell death 1 (PD-1) dimer. CD40L activates the DCs, whereas PD-1 binds programmed death ligand 1 (PD-L1) to prevent checkpoint activation and strengthen the cytotoxic T lymphocyte (CTL) response. The injection of humanized mice with DCs transduced with vector expressing CD40L and the HIV-1 SL9 epitope induced antigen-specific T cell proliferation and memory differentiation. Upon HIV-1 challenge of vaccinated mice, viral load was suppressed by 2 logs for 6 weeks. Introduction of the soluble PD-1 dimer into a vector that expressed full-length HIV-1 proteins accelerated the antiviral response. The results support development of this approach as a therapeutic vaccine that might allow HIV-1-infected individuals to control virus replication without antiretroviral therapy.

To develop a map of cell-cell communication mediated by extracellular RNA (exRNA), the NIH Extracellular RNA Communication Consortium created the exRNA Atlas resource (https://exrna-atlas.org). The Atlas version 4P1 hosts 5,309 exRNA-seq and exRNA qPCR profiles from 19 studies and a suite of analysis and visualization tools. To analyze variation between profiles, we apply computational deconvolution. The analysis leads to a model with six exRNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4), each detectable in multiple biofluids (serum, plasma, CSF, saliva, urine). Five of the cargo types associate with known vesicular and non-vesicular (lipoprotein and ribonucleoprotein) exRNA carriers. To validate utility of this model, we re-analyze an exercise response study by deconvolution to identify physiologically relevant response pathways that were not detected previously. To enable wide application of this model, as part of the exRNA Atlas resource, we provide tools for deconvolution and analysis of user-provided case-control studies.

The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.

Genes implicated in neuropsychiatric disorders are active in human fetal brain, yet difficult to study in a longitudinal fashion. We demonstrate that organoids from human pluripotent cells model cerebral cortical development on the molecular level before 16 weeks postconception. A multiomics analysis revealed differentially active genes and enhancers, with the greatest changes occurring at the transition from stem cells to progenitors. Networks of converging gene and enhancer modules were assembled into six and four global patterns of expression and activity across time. A pattern with progressive down-regulation was enriched with human-gained enhancers, suggesting their importance in early human brain development. A few convergent gene and enhancer modules were enriched in autism-associated genes and genomic variants in autistic children. The organoid model helps identify functional elements that may drive disease onset.

To broaden our understanding of human neurodevelopment, we profiled transcriptomic and epigenomic landscapes across brain regions and/or cell types for the entire span of prenatal and postnatal development. Integrative analysis revealed temporal, regional, sex, and cell type-specific dynamics. We observed a global transcriptomic cup-shaped pattern, characterized by a late fetal transition associated with sharply decreased regional differences and changes in cellular composition and maturation, followed by a reversal in childhood-adolescence, and accompanied by epigenomic reorganizations. Analysis of gene coexpression modules revealed relationships with epigenomic regulation and neurodevelopmental processes. Genes with genetic associations to brain-based traits and neuropsychiatric disorders (including MEF2C, SATB2, SOX5, TCF4, and TSHZ3) converged in a small number of modules and distinct cell types, revealing insights into neurodevelopment and the genomic basis of neuropsychiatric risks.

Anopheles funestus s.s. is a primary vector of malaria in sub-Saharan Africa. Despite its important role in human Plasmodium transmission, evolutionary history, genetic diversity, and population structure of An. funestus in southern and central Africa remains understudied. We deep sequenced, assembled, and annotated the complete mitochondrial genome of An. funestus s.s. for the first time, providing a foundation for further genetic research of this important malaria vector species. We further analyzed the complete mitochondrial genomes of 43 An. funestus s.s. from three sites in Zambia, Democratic Republic of the Congo, and Tanzania. From these 43 mitogenomes we identified 41 unique haplotypes that comprised 567 polymorphic sites. Bayesian phylogenetic reconstruction confirmed the co-existence of two highly divergent An. funestus maternal lineages, herein defined as lineages I and II, in Zambia and Tanzania. The estimated coalescence time of these two mitochondrial lineages is ~500,000 years ago (95% HPD 426,000-594,000 years ago) with subsequent independent diversification. Haplotype network and phylogenetic analysis revealed two major clusters within lineage I, and genetic relatedness of samples with deep branching in lineage II. At this time, data suggest that the lineages are partially sympatric. This study illustrates that accurate retrieval of full mitogenomes of Anopheles vectors enables fine-resolution studies of intraspecies genetic relationships, population differentiation, and demographic history. Further investigations on whether An. funestus mitochondrial lineages represent biologically meaningful populations and their potential implications for malaria vector control are warranted.

MotivationAnalysis of RNA sequencing (RNA-Seq) data in human saliva is challenging. Lack of standardization and unification of the bioinformatic procedures undermines saliva's diagnostic potential. Thus, it motivated us to perform this study.ResultsWe applied principal pipelines for bioinformatic analysis of small RNA-Seq data of saliva of 98 healthy Korean volunteers including either direct or indirect mapping of the reads to the human genome using Bowtie1. Analysis of alignments to exogenous genomes by another pipeline revealed that almost all of the reads map to bacterial genomes. Thus, salivary exRNA has fundamental properties that warrant the design of unique additional steps while performing the bioinformatic analysis. Our pipelines can serve as potential guidelines for processing of RNA-Seq data of human saliva.Availability and implementationProcessing and analysis results of the experimental data generated by the exceRpt (v4.6.3) small RNA-seq pipeline (github.gersteinlab.org/exceRpt) are available from exRNA atlas (exrna-atlas.org). Alignment to exogenous genomes and their quantification results were used in this paper for the analyses of small RNAs of exogenous origin.Contactdtww@ucla.edu.

To better understand the molecular and cellular differences in brain organization between human and nonhuman primates, we performed transcriptome sequencing of 16 regions of adult human, chimpanzee, and macaque brains. Integration with human single-cell transcriptomic data revealed global, regional, and cell-type-specific species expression differences in genes representing distinct functional categories. We validated and further characterized the human specificity of genes enriched in distinct cell types through histological and functional analyses, including rare subpallial-derived interneurons expressing dopamine biosynthesis genes enriched in the human striatum and absent in the nonhuman African ape neocortex. Our integrated analysis of the generated data revealed diverse molecular and cellular features of the phylogenetic reorganization of the human brain across multiple levels, with relevance for brain function and disease.

Despite the success of combination antiretroviral therapy (cART) for suppressing HIV and improving patients' quality of life, HIV persists in cART-treated patients and remains an incurable disease. Financial burdens and health consequences of lifelong cART treatment call for novel HIV therapies that result in a permanent cure. Cellular immunity is central in controlling HIV replication. However, HIV adopts numerous strategies to evade immune surveillance. Engineered immunity via genetic manipulation could offer a functional cure by generating cells that have enhanced antiviral activity and are resistant to HIV infection. Recently, encouraging reports from several human clinical trials using an anti-CD19 chimeric antigen receptor (CAR) modified T-cell therapy for treating B-cell malignancies have provided valuable insights and generated remarkable enthusiasm in engineered T-cell therapy. In this review, we discuss the development of HIV-specific chimeric antigen receptors and the use of stem cell based therapies to generate lifelong anti-HIV immunity.

Chronic immune activation, immunosuppression, and T cell exhaustion are hallmarks of HIV infection, yet the mechanisms driving these processes are unclear. Chronic activation can be a driving force in immune exhaustion, and type I interferons (IFN-I) are emerging as critical components underlying ongoing activation in HIV infection. Here, we have tested the effect of blocking IFN-I signaling on T cell responses and virus replication in a murine model of chronic HIV infection. Using HIV-infected humanized mice, we demonstrated that in vivo blockade of IFN-I signaling during chronic HIV infection diminished HIV-driven immune activation, decreased T cell exhaustion marker expression, restored HIV-specific CD8 T cell function, and led to decreased viral replication. Antiretroviral therapy (ART) in combination with IFN-I blockade accelerated viral suppression, further decreased viral loads, and reduced the persistently infected HIV reservoir compared with ART treatment alone. Our data suggest that blocking IFN-I signaling in conjunction with ART treatment can restore immune function and may reduce viral reservoirs during chronic HIV infection, providing validation for IFN-I blockade as a potential therapy for HIV infection.

The humanized bone marrow-liver-thymus (BLT) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34+ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain human leukocyte antigen (HLA) haplotypes or monogene deficiencies. To circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rγ-/-) recipient mice, we were able to expand one primary BLT mouse into a colony of 4-5 proBLT (propagated BLT) mice in 6-8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to those of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2-restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable preclinical research tool.

HIV remains a highly important public health and clinical issue despite many recent advances in attempting to develop a cure, which has remained elusive for most people infected with HIV. HIV disease can be controlled with pharmacologic therapies; however, these treatments are expensive, may have severe side effects, and are not curative. Consequently, an improved means to control or eliminate HIV replication is needed. Cytotoxic T lymphocytes (CTLs) play a critical role in controlling viral replication and are an important part in the ability of the immune response to eradicate most viral infections. There are considerable efforts to enhance CTL responses in HIV-infected individuals in hopes of providing the immune response with armaments to more effectively control viral replication. In this review, we discuss some of these efforts and focus on the development of a gene therapy-based approach to engineer hematopoietic stem cells with an HIV-1-specific chimeric antigen receptor, which seeks to provide an inexhaustible source of HIV-1-specific immune cells that are MHC unrestricted and superior to natural antiviral T cell responses. These efforts provide the basis for further development of T cell functional enhancement to target and treat chronic HIV infection in hopes of eradicating the virus from the body.

BackgroundTo address the lack of standard terminology to describe extracellular RNA (exRNA) data/metadata, we have launched an inter-community effort to extend the Gene Ontology (GO) with subcellular structure concepts relevant to the exRNA domain. By extending GO in this manner, the exRNA data/metadata will be more easily annotated and queried because it will be based on a shared set of terms and relationships relevant to extracellular research.MethodsBy following a consensus-building process, we have worked with several academic societies/consortia, including ERCC, ISEV, and ASEMV, to identify and approve a set of exRNA and extracellular vesicle-related terms and relationships that have been incorporated into GO. In addition, we have initiated an ongoing process of extractions of gene product annotations associated with these terms from Vesiclepedia and ExoCarta, conversion of the extracted annotations to Gene Association File (GAF) format for batch submission to GO, and curation of the submitted annotations by the GO Consortium. As a use case, we have incorporated some of the GO terms into annotations of samples from the exRNA Atlas and implemented a faceted search interface based on such annotations.ResultsWe have added 7 new terms and modified 9 existing terms (along with their synonyms and relationships) to GO. Additionally, 18,695 unique coding gene products (mRNAs and proteins) and 963 unique non-coding gene products (ncRNAs) which are associated with the terms: "extracellular vesicle", "extracellular exosome", "apoptotic body", and "microvesicle" were extracted from ExoCarta and Vesiclepedia. These annotations are currently being processed for submission to GO.ConclusionsAs an inter-community effort, we have made a substantial update to GO in the exRNA context. We have also demonstrated the utility of some of the new GO terms for sample annotation and metadata search.

UnlabelledAlthough the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log10 units) to transduced CD8(+) T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1.ImportanceWhile chimeric antigen receptors (CARs) using single-chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were done for HIV-1 infection. However, those trials failed, and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on the use of CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (BNAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single-chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse BNAb types and show that all these CARs are functional against HIV-1.

The number of humanized mouse models for the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and other infectious diseases has expanded rapidly over the past 8 years. Highly immunodeficient mouse strains, such as NOD/SCID/gamma chain(null) (NSG, NOG), support better human hematopoietic cell engraftment. Another improvement is the derivation of highly immunodeficient mice, transgenic with human leukocyte antigens (HLAs) and cytokines that supported development of HLA-restricted human T cells and heightened human myeloid cell engraftment. Humanized mice are also used to study the HIV reservoir using new imaging techniques. Despite these advances, there are still limitations in HIV immune responses and deficits in lymphoid structures in these models in addition to xenogeneic graft-versus-host responses. To understand and disseminate the improvements and limitations of humanized mouse models to the scientific community, the NIH sponsored and convened a meeting on April 15, 2015 to discuss the state of knowledge concerning these questions and best practices for selecting a humanized mouse model for a particular scientific investigation. This report summarizes the findings of the NIH meeting.

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (cγ(-/-))-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

Recent research on disparate psychiatric disorders has implicated rare variants in genes involved in global gene regulation and chromatin modification, as well as many common variants located primarily in regulatory regions of the genome. Understanding precisely how these variants contribute to disease will require a deeper appreciation for the mechanisms of gene regulation in the developing and adult human brain. The PsychENCODE project aims to produce a public resource of multidimensional genomic data using tissue- and cell type–specific samples from approximately 1,000 phenotypically well-characterized, high-quality healthy and disease-affected human post-mortem brains, as well as functionally characterize disease-associated regulatory elements and variants in model systems. We are beginning with a focus on autism spectrum disorder, bipolar disorder and schizophrenia, and expect that this knowledge will apply to a wide variety of psychiatric disorders. This paper outlines the motivation and design of PsychENCODE.

Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.

UnlabelledA unique aspect of human monocytes, compared to monocytes from many other species, is that they express the CD4 molecule. However, the role of the CD4 molecule in human monocyte development and function is not known. We determined that the activation of CD4 via interaction with major histocompatibility complex class II (MHC-II) triggers cytokine expression and the differentiation of human monocytes into functional mature macrophages. Importantly, we determined that CD4 activation induces intracellular signaling in monocytes and that inhibition of the MAPK and Src family kinase pathways blocked the ability of CD4 ligation to trigger macrophage differentiation. We observed that ligation of CD4 by MHC-II on activated endothelial cells induced CD4-mediated macrophage differentiation of blood monocytes. Finally, CD4 ligation by MHC-II increases the susceptibility of blood-derived monocytes to HIV binding and subsequent infection. Altogether, our studies have identified a novel function for the CD4 molecule on peripheral monocytes and suggest that a unique set of events that lead to innate immune activation differ between humans and mice. Further, these events can have effects on HIV infection and persistence in the macrophage compartment.ImportanceThe CD4 molecule, as the primary receptor for HIV, plays an important role in HIV pathogenesis. There are many cell types that express CD4 other than the primary HIV target, the CD4(+) T cell. Other than allowing HIV infection, the role of the CD4 molecule on human monocytes or macrophages is not known. We were interested in determining the role of CD4 in human monocyte/macrophage development and function and the potential effects of this on HIV infection. We identified a role for the CD4 molecule in triggering the activation and development of a monocyte into a macrophage following its ligation. Activation of the monocyte through the CD4 molecule in this manner increases the ability of monocytes to bind to and become infected with HIV. Our studies have identified a novel function for the CD4 molecule on peripheral monocytes in triggering macrophage development that has direct consequences for HIV infection.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.

Despite the enormous success of combined anti-retroviral therapy, HIV infection is still a lifelong disease and continues to spread rapidly worldwide. There is a pressing need to develop a treatment that will cure HIV infection. Recent progress in stem cell manipulation and advancements in humanized mouse models have allowed rapid developments of gene therapy for HIV treatment. In this review, we will discuss two aspects of HIV gene therapy using human hematopoietic stem cells. The first is to generate immune systems resistant to HIV infection while the second strategy involves enhancing anti-HIV immunity to eliminate HIV infected cells.

Despite the enormous success of combined anti-retroviral therapy, HIV infection is still a lifelong disease and continues to spread rapidly worldwide. There is a pressing need to develop a treatment that will cure HIV infection. Recent progress in stem cell manipulation and advancements in humanized mouse models have allowed rapid developments of gene therapy for HIV treatment. In this review, we will discuss two aspects of HIV gene therapy using human hematopoietic stem cells. The first is to generate immune systems resistant to HIV infection while the second strategy involves enhancing anti-HIV immunity to eliminate HIV infected cells.

Currently, few rodent models of AIDS-associated non-Hodgkin's lymphoma (AIDS-NHL) exist. In these studies, a novel mouse/human xenograft model of AIDS-associated Burkitt lymphoma (AIDS-BL) was created by injecting cells of the human AIDS-BL cell line, 2F7, intraperitoneally into NOD-SCID mice. Mice developed tumors in the peritoneal cavity, with metastases to the spleen, thymus, and mesenteric lymph nodes. Expression of the chemokine receptor, CXCR5, was greatly elevated in vivo on BL tumor cells in this model, as shown by flow cytometry. CXCL13 is the ligand for CXCR5, and serum and ascites levels of murine, but not human, CXCL13 showed a striking elevation in tumor-bearing mice, with levels as high as 200,000 pg/ml in ascites, as measured by ELISA. As shown by immunohistochemistry, murine CXCL13 was associated with macrophage-like tumor-infiltrating cells that appeared to be histiocytes. Blocking CXCR5 on 2F7 cells with neutralizing antibodies prior to injection into the mice substantially delayed tumor formation. The marked elevations in tumor cell CXCR5 expression and in murine CXCL13 levels seen in the model may potentially identify an important link between tumor-interacting histiocytes and tumor cells in AIDS-BL. These results also identify CXCL13 as a potential biomarker for this disease, which is consistent with previous studies showing that serum levels of CXCL13 were elevated in human subjects who developed AIDS-lymphoma. This mouse model may be useful for future studies on the interactions of the innate immune system and AIDS-BL tumor cells, as well as for the assessment of potential tumor biomarkers for this disease.

Recent research in autism spectrum disorder (ASD) has aroused interest in anterior cingulate cortex and in the neurometabolite glutamate. We report two studies of pregenual anterior cingulate cortex (pACC) in pediatric ASD. First, we acquired in vivo single-voxel proton magnetic resonance spectroscopy ((1)H MRS) in 8 children with ASD and 10 typically developing controls who were well matched for age, but with fewer males and higher IQ. In the ASD group in midline pACC, we found mean 17.7% elevation of glutamate + glutamine (Glx) (p

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.

The rates of immunologic and clinical progression are lower in patients with drug-resistant HIV compared to wild-type HIV. This difference is not fully explained by viral load. It has been argued that reductions in T cell activation and/or viral fitness might result in preserved target cells and an altered relationship between the level of viremia and the rate of CD4+ T cell loss. We tested this hypothesis over time in a cohort of patients with highly resistant HIV. Fifty-four antiretroviral-treated patients with multi-drug resistant HIV and detectable plasma HIV RNA were followed longitudinally. CD4+ T cell counts and HIV RNA levels were measured every 4 weeks and T cell activation (CD38/HLA-DR) was measured every 16 weeks. We found that the levels of CD4+ T cell activation over time were a strong independent predictor of CD4+ T cell counts while CD8+ T cell activation was more strongly associated with viremia. Using spectral analysis, we found strong evidence for oscillatory (or cyclic) behavior in CD4+ T cell counts, HIV RNA levels, and T cell activation. Each of the cell populations exhibited an oscillatory behavior with similar frequencies. Collectively, these data suggest that there may be a mechanistic link between T cell activation, CD4+ T cell counts, and viremia and lends support for the hypothesis of altered predator-prey dynamics as a possible explanation of the stability of CD4+ T cell counts in the presence of sustained multi-drug resistant viremia.

There is a desperate need for effective therapies to fight chronic viral infections. The immune response is normally fastidious at controlling the majority of viral infections and a therapeutic strategy aimed at reestablishing immune control represents a potentially powerful approach towards treating persistent viral infections. We examined the potential of genetically programming human hematopoietic stem cells to generate mature CD8+ cytotoxic T lymphocytes that express a molecularly cloned, "transgenic" human anti-HIV T cell receptor (TCR). Anti-HIV TCR transduction of human hematopoietic stem cells directed the maturation of a large population of polyfunctional, HIV-specific CD8+ cells capable of recognizing and killing viral antigen-presenting cells. Thus, through this proof-of-concept we propose that genetic engineering of human hematopoietic stem cells will allow the tailoring of effector T cell responses to fight HIV infection or other diseases that are characterized by the loss of immune control.