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BACKGROUND: Antibody-based strategies for COVID-19 have shown promise in prevention and treatment of early disease. COVID-19 convalescent plasma (CCP) has been widely used but results from randomized trials supporting its benefit in hospitalized patients with pneumonia are limited. Here, we assess the efficacy of CCP in severely ill, hospitalized adults with COVID-19 pneumonia. METHODS: We performed a randomized control trial (PennCCP2), with 80 adults hospitalized with COVID-19 pneumonia, comparing up to 2 units of locally sourced CCP plus standard care versus standard care alone. The primary efficacy endpoint was comparison of a clinical severity score. Key secondary outcomes include 14- and 28-day mortality, 14- and 28-day maximum 8-point WHO ordinal score (WHO8) score, duration of supplemental oxygenation or mechanical ventilation, respiratory SARS-CoV-2 RNA, and anti–SARS-CoV-2 antibodies. RESULTS: Eighty hospitalized adults with confirmed COVID-19 pneumonia were enrolled at median day 6 of symptoms and day 1 of hospitalization; 60% were anti–SARS-CoV-2 antibody seronegative. Participants had a median of 3 comorbidities, including risk factors for severe COVID-19 and immunosuppression. CCP treatment was safe and conferred significant benefit by clinical severity score (median [MED] and interquartile range [IQR] 10 [5.5–30] vs. 7 [2.75–12.25], P = 0.037) and 28-day mortality (n = 10, 26% vs. n = 2, 5%; P = 0.013). All other prespecified outcome measures showed weak evidence toward benefit of CCP. CONCLUSION: Two units of locally sourced CCP administered early in hospitalization to majority seronegative participants conferred a significant benefit in clinical severity score and 28-day mortality. Results suggest CCP may benefit select populations, especially those with comorbidities who are treated early. TRIAL REGISTRATION: ClinicalTrials.gov NCT04397757. FUNDING: University of Pennsylvania.

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their antiviral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally derived HIV-1 isolates from the plasma and CD4+ T cells of 26 individuals sampled longitudinally after transmission or after antiretroviral therapy (ART) and analytical treatment interruption. We determined the concentration of IFNα2 and IFNβ that reduced viral replication in vitro by 50% (IC50) and found consistent changes in the sensitivity of HIV-1 to IFN-I inhibition both across individuals and over time. Resistance of HIV-1 isolates to IFN-I was uniformly high during acute infection, decreased in all individuals in the first year after infection, was reacquired concomitant with CD4+ T cell loss, and remained elevated in individuals with accelerated disease. HIV-1 isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just before ART initiation. However, viruses that rebounded after treatment interruption displayed the highest degree of IFNα2 and IFNβ resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control affected by both ART and analytical treatment interruption. Although elevated at transmission, host innate pressures are the highest during viral rebound, limiting the viruses that successfully become reactivated from latency to those that are IFN-I resistant.

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their anti-viral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally-derived HIV-1 isolates from plasma and CD4+ T cells of 26 individuals sampled longitudinally following transmission and/or after antiretroviral therapy (ART) and analytical treatment interruption (ATI). Determining the concentration of IFNα2 and IFNβ that reduced HIV-1 replication by 50% (IC50), we found remarkably consistent changes in the sensitivity of viruses to IFN-I inhibition, both across individuals and over time. IFN-I resistance was uniformly high during acute infection, decreased in all subjects in the first year post-infection, was reacquired concomitant with CD4+ T cell loss, and remained elevated in subjects with accelerated disease. Isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just prior to ART initiation. However, viruses that rebounded following treatment interruption displayed the highest levels of IFNα2 and IFNβ resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate immune responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control impacted by both ART and ATI. Although elevated at transmission, IFN-mediated pressures are the highest during viral rebound, limiting the viruses that successfully reactivate from latency.One Sentence SummaryHIV-1 resistance to IFN-I is highest during acute infection and following analytic treatment interruption, indicating a dynamic interplay between host innate immunity and virus biology.

Vascular access dysfunction associated with arteriovenous grafts and fistulas contributes to the morbidity and mortality of chronic kidney disease (CKD) patients receiving hemodialysis. We hypothesized that the uremic conditions associated with CKD promote a pathophysiological vascular smooth muscle cell (VSMC) phenotype that contributes to neointimal hyperplasia. We analyzed the effect of culturing human VSMC with uremic serum. Expression of VSMC contractile marker genes was reduced 50-80% in cells exposed to uremic serum and the decreased expression was accompanied by changes in histone marks. There was an increase in proliferation in cells exposed to uremic conditions, with no change in the levels of apoptosis. Interestingly, we found that uremic serum inhibited PDGF-induced migration of VSMC. Histomorphometric analysis revealed venous neointimal hyperplasia in veins from chronic kidney disease (CKD) patients prior to any surgical manipulation as compared to veins from patients with no kidney disease. We conclude that uremia associated with CKD alters VSMC phenotype in vitro and contributes to neointimal hyperplasia formation in vivo contributing to the pathogenesis of vascular access dysfunction in CKD patients.

We have an operant rat model of upper extremity reaching and grasping in which we examined the impact of performing a high force high repetition (High-ForceHR) versus a low force low repetition (Low-ForceHR) task for 18 weeks on the radius and ulna, compared to age-matched controls. High-ForceHR rats performed at 4 reaches/min and 50% of their maximum voluntary pulling force for 2 h/day, 3 days/week. Low-ForceHR rats performed at 6% maximum voluntary pulling force. High-ForceHR rats showed decreased trabecular bone volume in the distal metaphyseal radius, decreased anabolic indices in this same bone region (e.g., decreased osteoblasts and bone formation rate), and increased catabolic indices (e.g., microcracks, increased osteocyte apoptosis, secreted sclerostin, RANKL, and osteoclast numbers), compared to controls. Distal metaphyseal trabeculae in the ulna of High-ForceHR rats showed a non-significant decrease in bone volume, some catabolic indices (e.g., decreased trabecular numbers) yet also some anabolic indices (e.g., increased osteoblasts and trabecular thickness). In contrast, the mid-diaphyseal region of High-ForceHR rats' radial and ulnar bones showed few to no microarchitecture differences and no changes in apoptosis, sclerostin or RANKL levels, compared to controls. In further contrast, Low-ForceHR rats showed increased trabecular bone volume in the radius in the distal metaphysis and increased cortical bone area its mid-diaphysis. These changes were accompanied by increased anabolic indices, no microcracks or osteocyte apoptosis, and decreased RANKL in each region, compared to controls. Ulnar bones of Low-ForceHR rats also showed increased anabolic indices, although fewer than in the adjacent radius. Thus, prolonged performance of an upper extremity reaching and grasping task is loading-, region-, and bone-dependent, with high force loads at high repetition rates inducing region-specific increases in bone degradative changes that were most prominent in distal radial trabeculae, while low force task loads at high repetition rates induced adaptive bone responses.

The SNF2-related CBP activator protein (SRCAP) serves as a coactivator for several nuclear receptors including the androgen receptor (AR). SRCAP is an ATPase that is the core subunit of a large multiprotein complex and was shown to incorporate the histone variant H2A.Z into nucleosomes. In this report, we demonstrate that SRCAP is expressed in the epithelium of normal prostate and in prostate carcinoma cells, and is associated with AR in the nucleus. Using transient transfection assays we demonstrate that SRCAP activates hormone-dependent transcription of the androgen responsive, prostate specific antigen (PSA)-Luciferase reporter gene in human prostate cells. The in vivo occupancy of SRCAP at the endogenous PSA promoter is demonstrated using chromatin immunoprecipitation assays. ShRNA mediated knockdown of SRCAP resulted in decreased H2A.Z binding at the enhancer region of the PSA promoter and decreased expression of PSA in prostate cancer cells. Furthermore, inhibition of SRCAP expression significantly inhibited androgen dependent prostate cancer cell growth. These data identify SRCAP as a physiologically relevant mediator of PSA expression, and demonstrate that SRCAP plays a role in prostate cancer cell proliferation.

Major operative injury initiates immunologic changes that may result in a systemic inflammatory response, leading to organ dysfunction/sepsis. Catecholamines seem to be key mediators. The effects of beta(2) receptor blockade in vitro and in vivo before and after operative injury were studied to clarify their role.In vitro studies were done in RAW 264.7 cells using epinephrine (50 micromol/L) with or without alpha(2)- and beta(2)-receptor blockade. Comparative gene expression analysis on the Toll-like receptor (TLR)-4 receptor signaling pathway was performed between RAW cells pretreated with epinephrine with subsequent lipopolysaccharide (LPS) stimulation versus LPS alone. Confirmatory studies were performed by real-time reverse transcription polymerase chain reaction (RT-PCR). Tumor necrosis factor (TNF)-alpha gene and protein expression were determined via real time RT-PCR and enzyme-linked immunosorbent assay, respectively. In vivo, Balb/C mice received no treatment nor injury (group I). Group II received operative injury and vehicle injection, group III received ICI 118,551 (beta(2)-receptor antagonist) 30 minutes before or in separate studies after operative injury. At 7 days, splenic macrophages were harvested and cytokine production was measured with or without LPS. In separate experiments, cecal ligation and puncture (CLP) was done 7 days after operation and survival determined.In vitro studies demonstrated that epinephrine pretreatment significantly increased TNF-alpha production with LPS stimulation (P.05). beta(2)-Receptor blockade significantly attenuated (P.05) LPS stimulated TNF-alpha production. MD-2, an essential coactivator of TLR-4 signaling, gene expression was significantly elevated when cells were pretreated with epinephrine before LPS exposure (P.001). In vivo studies demonstrated a significant decrease (P.05) in TNF-alpha and interleukin-6 production in the ICI 118,551 group. Similar findings were demonstrated measuring monocyte chemoattractant protein-1 and interferon-gamma cytokine levels (P.05) versus no treatment. ICI 118,551 treatment 30 minutes before operation demonstrated a 31% reduction in mortality after CLP (P.05).This study demonstrates that beta(2)-receptor blockade reduces macrophage cytokine production and improves survival showing the critical importance of catecholamines to the immunologic response in surgery.

Prostaglandin metabolite 15-Deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPARgamma. We investigated the ability of 15d-PGJ2 to inhibit TNF-alpha gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 microM) inhibited LPS-stimulated TNF-alpha mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPARgamma ligand, GW1929, failed to inhibit LPS-induced TNF-alpha mRNA expression nor did a PPARgamma antagonist, GW9662, alter the repression of TNF-alpha mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPARgamma-independent inhibition of TNF-alpha mRNA in THP-1 cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-alpha promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-alpha promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-alpha promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-alpha promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-alpha promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-alpha transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter.

We have identified and mapped a gene in Toxoplasma gondii that encodes a homologue of SRCAP (Snf2-related CBP activator protein), a member of the SNF/SWI family of chromatin remodeling factors. The genomic locus (TgSRCAP) is present as a single copy and contains 16 introns. The predicted cDNA contains an open reading frame of 8,775 bp and encodes a protein of 2,924 amino acids. We have identified additional SRCAP-like sequences in Apicomplexa for comparison by screening genomic databases. An analysis of SRCAP homologues between species reveals signature features that may be indicative of SRCAP members. Expression of mRNA encoding TgSRCAP is upregulated when tachyzoite (invasive form) parasites are induced to differentiate into bradyzoites (encysted form) in vitro. Recombinant TgSRCAP protein is functionally equivalent to the human homologue, being capable of increasing transcription mediated by CREB.

Objective— The role of receptors for endogenous metabolic danger signals–associated molecular patterns has been characterized recently as bridging innate immune sensory systems for danger signals–associated molecular patterns to initiation of inflammation in bone marrow–derived cells, such as macrophages. However, it remains unknown whether endothelial cells (ECs), the cell type with the largest numbers and the first vessel cell type exposed to circulating danger signals–associated molecular patterns in the blood, can sense hyperlipidemia. This report determined whether caspase-1 plays a role in ECs in sensing hyperlipidemia and promoting EC activation. Approach and Results— Using biochemical, immunologic, pathological, and bone marrow transplantation methods together with the generation of new apoplipoprotein E (ApoE) −/− /caspase-1 −/− double knockout mice, we made the following observations: (1) early hyperlipidemia induced caspase-1 activation in ApoE −/− mouse aorta; (2) caspase-1 −/− /ApoE −/− mice attenuated early atherosclerosis; (3) caspase-1 −/− /ApoE −/− mice had decreased aortic expression of proinflammatory cytokines and attenuated aortic monocyte recruitment; and (4) caspase-1 −/− /ApoE −/− mice had decreased EC activation, including reduced adhesion molecule expression and cytokine secretion. Mechanistically, oxidized lipids activated caspase-1 and promoted pyroptosis in ECs by a reactive oxygen species mechanism. Caspase-1 inhibition resulted in accumulation of sirtuin 1 in the ApoE −/− aorta, and sirtuin 1 inhibited caspase-1 upregulated genes via activator protein-1 pathway. Conclusions— Our results demonstrate for the first time that early hyperlipidemia promotes EC activation before monocyte recruitment via a caspase-1–sirtuin 1–activator protein-1 pathway, which provides an important insight into the development of novel therapeutics for blocking caspase-1 activation as early intervention of metabolic cardiovascular diseases and inflammations.

Background: Vascular access dysfunction associated with arteriovenous grafts and fistulas contributes significantly to the morbidity and mortality of end-stage renal disease (ESRD) patients receiving hemodialysis. The mechanism involved in the development of peri-anastomotic intimal hyperplasia, a major cause of the arteriovenous access dysfunction, is poorly elucidated. We hypothesized that the uremic condition associated with ESRD promotes a pathophysiological vascular smooth muscle cell (VSMC) phenotype that contributes to accelerated development of intimal hyperplasia in blood vessels, both de novo and following the arteriovenous anastomosis. Methods and Results: We examined the effect of culturing human VSMC with human uremic serum on its phenotype. We utilized quantitative real-time PCR to measure the expression of the VSMC-specific contractile marker genes, and found that these genes were reduced 50-80% in VSMC exposed to serum from hemodialysis patients as compared to serum from patients with normal renal function. Chromatin immunoprecipitation assays demonstrated that there were also significant changes in epigenetic markers associated with contractile gene promoters in VSMC cultured in uremic conditions. Moreover, there was a 60% increase in proliferation rate in cells exposed to uremic conditions, with no change in the levels of apoptosis as compared to cells exposed to non-uremic conditions. Interestingly, uremic serum from ESRD patients inhibited PDGF-induced migration of VSMC. We also examined the histopathology of veins from patients with normal kidney function and chronic kidney disease (CKD). Indeed, we found significant de novo venous intimal hyperplasia exists in CKD and ESRD patients prior to any surgical manipulation as compared to veins from patients with normal kidney function. Conclusion: CKD alters VSMC phenotype in vitro and contributes to intimal hyperplasia formation in vivo resulting in the pathogenesis of vascular access dysfunction in ESRD patients.

The SNF2-related CBP activator protein, SrCap (pronounced “sir cap”), shares homology with the SNF2/SWI2 protein family. SrCap was cloned through its ability to bind CBP. SrCap can function as a CBP coactivator and can activate transcription in a reporter assay when expressed as a Gal-SrCap fusion protein. A monoclonal antibody raised against the carboxyl terminus of SrCap coimmunoprecipitates CBP/p300, supporting the model that SrCap is a CBP binding protein and that these proteins can be found together in a cellular protein complex. In addition, several cellular proteins are coimmunoprecipitated by the SrCap-specific antibody. Since adenovirus E1A proteins interact with CBP/p300 proteins, we examined what proteins could be copurified in a SrCap-specific coimmunoprecipitation assay from lysates of adenovirus-infected cells. While E1A proteins were not detected in this complex, to our surprise, we observed the presence of an infected-cell-specific band of 72 kDa, which we suspected might be the adenovirus DNA binding protein, DBP. The adenovirus DBP is a multifunctional protein involved in several aspects of the adenovirus life cycle, including an ability to modulate transcription. The identity of DBP was confirmed by DBP-specific Western blot analysis and by reimmunoprecipitating DBP from denatured SrCap-specific protein complexes. Using in vitro-translated DBP and SrCap proteins, we demonstrated that these proteins interact. To determine whether this interaction could affect SrCap-mediated transcription, we tested whether increasing amounts of DBP could modulate the Gal-SrCap transcription activity. We observed that DBP inhibited Gal-SrCap transcription activity in a dose-dependent manner. These data suggest a novel mechanism of adenovirus host cell control by which DBP binds to and inactivates SrCap, a member of the SNF2 chromatin-remodeling protein family.

Acinetobacter baumannii is an important nosocomial pathogen in civilian intensive care units. Recently the incidence has increased in wounded military personnel. Morphine is documented in numerous animal studies to be immunosuppressive and to sensitize to infection. The hypotheses were tested that morphine, administered for analgesia in the battlefield, predisposes to Acinetobacter infection, and that the opioid may have an additive or synergistic effect with trauma. To test these hypotheses, an intraperitoneal infection model was established in mice using several Acinetobacter strains. Morphine administered for 48 h by implantation of a slow-release morphine pellet increased mortality compared to animals receiving a placebo pellet, an effect that was blocked by the mu-opioid receptor antagonist, naltrexone. Acinetobacter burdens in the blood, spleens, livers, and lungs of morphine-treated mice, were significantly higher than those in placebo-treated animals, confirming that mortality was due to potentiated growth of the bacteria. There were also elevated levels of pro-inflammatory cytokines in morphine-treated versus placebo-treated mice. Morphine caused a reduction in the total number of cells in the peritoneal cavity, a decrease in the percentage and total numbers of neutrophils, and a decrease in the total number of macrophages. Morphine treatment also suppressed levels of the neutrophil-inducing molecules, IL-17A and KC/CXCL1. However, IL-17A(-/-) mice given morphine were not sensitized to Acintobacter infection to a greater degree than similarly treated wild-type mice. Trauma alone did not sensitize to Acinetobacter infection, and there was no additive effect between morphine and trauma. These results support the hypothesis that morphine potentiates Acinetobacter infection.

Osteoactivin (OA) is required for the differentiation of osteoblast cells. OA expression is stimulated by bone morphogenetic protein-2 (BMP-2). BMP-2 recruits homeodomain transcription factors Dlx3, Dlx5, and Msx2 to selectively activate or repress transcription of osteogenic genes and hence tightly regulate their transcription during osteoblast differentiation. Considering the key roles of Dlx3, Dlx5, and Msx2 in osteoblast differentiation, here we hypothesize that homeodomain proteins regulate BMP-2-induced OA transcription during osteoblast differentiation. Four classical homeodomain binding sites were identified in the proximal 0.96 kb region of rat OA promoter. Deletions and mutagenesis studies of the OA promoter region indicated that all four homeodomain binding sites are crucial for BMP-2-induced OA promoter activity. Simultaneous disruption of homeodomain binding sites at -852 and -843 of the transcription start site of OA gene significantly decreased the BMP-2-induced OA transcription and inhibited binding of Dlx3, Dlx5, and Msx2 proteins to the OA promoter. Dlx3 and Dlx5 proteins were found to activate the OA transcription, whereas, Msx2 suppressed BMP-2-induced OA transcription. Using chromatin immunoprecipitation assays, we demonstrated that the OA promoter is predominantly occupied by Dlx3 and Dlx5 during the proliferation and matrix maturation stages of osteoblast differentiation, respectively. During the matrix mineralization stage, BMP-2 robustly enhanced the recruitment of Dlx5 and to a lesser extent of Dlx3 and Msx2 to the OA promoter region. Collectively, our results show that the BMP-2-induced OA transcription is differentially regulated by Dlx3, Dlx5, and Msx2 during osteoblast differentiation.

Background Ovarian cancer is the most common cause of cancer related death from gynecologic tumors in the United States. The insidious nature of the disease precludes early diagnosis, therefore surgical debulking and chemotherapy are considered as standard treatment modalities for advanced stages. We investigated the effect of the LXR agonist, T0901317, on ovarian cancer cell proliferation and apoptosis as a potential therapeutic agent. Results T0901317 treatment resulted in a significant (P <0.001) inhibition of cell proliferation in a time- and dose-dependent manner in CaOV3, SKOV3 and A2780 cells. Western blot analysis demonstrated an induction of p21 and p27 with a concominant reduction in phospho-RB protein levels. Cell cycle analysis demonstrated a significant (P <0.001) arrest in the G1 cell cycle phase. Significant induction of Caspase-3 and BAX gene expression occurred with treatment. Induction of apoptosis was confirmed by significant (P < 0.001) elevation of caspase activity on FACS analysis, caspase-glo assay, BAX protein induction and decreased caspase 3 precursor protein expression on Western blot analysis. LXR α/β knockdown experiments did not reverse the anti-proliferative and cytotoxic effects of T0901317. Conclusions The LXR agonist, T0901317, significantly suppresses cell proliferation and induces programmed cell death in a dose- and time-dependent manner. Our results indicate that T0901317 induces its anti-proliferative and cytotoxic effects via an LXR-independent mechanism.

In macrophages, peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to be important for differentiation, and it serves as a negative regulator of activation. Major trauma/injury causes a dramatic host response that disrupts cellular immune homeostasis and initiates an inflammatory cascade that predisposes the injured host to subsequent infections. In prior studies using a murine trauma model consisting of femur fracture and hemorrhage, splenic macrophages from traumatized mice had significantly enhanced LPS-induced cyclooxygenase enzyme (subtype 2) and iNOS production as well as elevated levels of inflammatory cytokines at 1 week after injury compared with uninjured controls. These up-regulated cellular responses corresponded to increased mortality when animals were challenged with LPS or Candida. In the current study, we used the injury model to determine the effect of treatment of injured mice with the endogenous PPARgamma ligand 15-deoxy-Delta(12-, 14)-PGJ2 (15d-PGJ2). It was found that in vivo 15d-PGJ2 treatment significantly reduced the levels of inflammatory mediators produced by splenic macrophages 7 days after injury. The mechanism of inhibition is dependent on PPARgamma because concomitant treatment of animals with the PPARgamma antagonist GW9662 reversed the inhibitory effect of 15d-PGJ2. Endogenous PPARgamma modulated activation of LPS-induced p38 mitogen-activated protein kinase. Furthermore, treatment of injured mice with 15d-PGJ2 conferred a significant survival advantage after infectious challenge induced by cecal ligation and puncture. Thus, this PPARgamma ligands significantly attenuate the postinjury inflammatory response and improve survival after infectious challenge.

SRCAP (SNF2-related CBP activator protein) is a 350-kDa protein that shares homology with the SNF2 family of proteins whose members function in various aspects of transcriptional regulation. In various cell types, SRCAP is found in distinct multiprotein complexes that include proteins found in SWI/SNF chromatin remodeling complexes. SRCAP was identified by its ability to bind to CBP and was found to potentiate the ability of CBP to activate transcription. Studies in our laboratory have demonstrated that SRCAP functions as a coactivator for CREB-mediated transcription of a number of promoters, including that of the phosphoenolpyruvate carboxykinase gene. Our current studies demonstrate that SRCAP enhances phosphoenolpyruvate carboxykinase promoter transcription induced by glucocorticoids. SRCAP also enhances glucocorticoid receptor-mediated transcription of a simple promoter containing only two glucocorticoid response elements, indicating that SRCAP functions as a glucocorticoid receptor coactivator. In similar studies, SRCAP was also found to serve as a coactivator for the androgen receptor. SRCAP exhibits synergistic activation with nuclear receptor coactivators and functionally interacts in vivo with glucocorticoid receptor-interacting protein-1 and coactivator-associated arginine methyltransferase-1. We propose that SRCAP, by virtue of its ability to interact with CBP, functions as a coactivator to regulate transcription initiated by several signaling pathways.

Introduction: Significant immune changes occur after trauma. Prostaglandin-E2 (PGE2) and nitric oxide (NO ) are key inflammatory mediators involved in trauma-related immune dysfunction. The relative importance of the early response genes nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) after trauma is controversial. This study investigated macrophage cytokine production after trauma and followed survival from subsequent sepsis in COX-2 and iNOS knockout (−/−) and wild-type mice. Methods: Female C57/Bl6 COX-2 and iNOS KO and WT mice were randomized to control or trauma (femur fracture plus 40% blood volume hemorrhage) groups. Expt. 1. Seven days after injury, splenic macrophages were isolated, stimulated with LPS (10ng/mL) for 24 hours and pro- (IL-6 and TNFa) and anti-inflammatory (IL-10 and IL-12) cytokine levels measured by cytometric bead array. Expt. 2. At 7 days after trauma the mice were subjected to cecal ligation and puncture and followed for survival. Results: Macrophages produced significantly decreased TNFa levels in iNOS (−/−) mice compared with WT mice after trauma. iNOS (−/−) mice has a significant survival advantage compared with WT mice following sepsis (10/13 vs 4/12 p < 0.005). IL-10 levels were significantly elevated in COX-2 (−/−) but IL-6, IL-10 and IL-12 levels were unchanged compared with WT mice. Survival was similar between the COX-2 (−/−) and WT mice following septic challenge. Conclusions: These data suggest that inhibition of iNOS is more important than COX-2 in protecting the host from sepsis after trauma. Significantly decreased TNFa production in iNOS (−/−) mice correlated with improved outcome suggesting that NO regulates TNFa production in trauma.