Mechanistic Understanding of Dexamethasone-Mediated Protection against Remdesivir-Induced Hepatotoxicity.

Remdesivir (RDV), a broad-spectrum antiviral agent, is often used together with dexamethasone (DEX) for hospitalized COVID-19 patients requiring respiratory support. Potential hepatic adverse drug reaction is a safety concern associated with the use of RDV. We previously reported that DEX cotreatment effectively mitigates RDV-induced hepatotoxicity and reduces elevated serum alanine aminotransferase and aspartate aminotransferase levels in cultured human primary hepatocytes (HPH) and hospitalized COVID-19 patients, respectively. Yet, the precise mechanism behind this protective drug-drug interaction remains largely unknown. Here, we show that through the activation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling, RDV induces apoptosis (cleavage of caspases 8, 9, and 3), autophagy (increased autophagosome and LC3-II), and mitochondrial damages (decreased membrane potential, respiration, ATP levels, and increased expression of Bax and the released cytosolic cytochrome C) in HPH. Importantly, cotreatment with DEX partially reversed RDV-induced apoptosis, autophagy, and cell death. Mechanistically, DEX deactivates/dephosphorylates p38, JNK, and ERK1/2 signaling by enhancing the expression of dual specificity protein phosphatase 1 (DUSP1), a mitogen-activated protein kinase (MAPK) phosphatase, in a glucocorticoid receptor (GR)-dependent manner. Knockdown of GR in HPH attenuates DEX-mediated DUSP1 induction, MAPK dephosphorylation, as well as protection against RDV-induced hepatotoxicity. Collectively, our findings suggest a molecular mechanism by which DEX modulates the GR-DUSP1-MAPK regulatory axis to alleviate the adverse actions of RDV in the liver. SIGNIFICANCE STATEMENT: The research uncovers the molecular mechanisms by which dexamethasone safeguards against remdesivir-associated liver damage in the context of COVID-19 treatment.

Stabilization of Cx43 mRNA via RNA-binding protein HuR regulated by polyamines enhances intestinal epithelial barrier function.

Gut barrier dysfunction occurs commonly in patients with critical disorders, leading to the translocation of luminal toxic substances and bacteria to the bloodstream. Connexin 43 (Cx43) acts as a gap junction protein and is crucial for intercellular communication and the diffusion of nutrients. The levels of cellular Cx43 are tightly regulated by multiple factors, including polyamines, but the exact mechanism underlying the control of Cx43 expression remains largely unknown. The RNA-binding protein HuR regulates the stability and translation of target mRNAs and is involved in many aspects of intestinal epithelial pathobiology. Here we show that HuR directly bound to Cx43 mRNA via its 3'-untranslated region in intestinal epithelial cells (IECs) and this interaction enhanced Cx43 expression by stabilizing Cx43 mRNA. Depletion of cellular polyamines inhibited the [HuR/Cx43 mRNA] complex and decreased the level of Cx43 protein by destabilizing its mRNA, but these changes were prevented by ectopic overexpression of HuR. Polyamine depletion caused intestinal epithelial barrier dysfunction, which was reversed by ectopic Cx43 overexpression. Moreover, overexpression of checkpoint kinase 2 in polyamine-deficient cells increased the [HuR/Cx43 mRNA] complex, elevated Cx43 levels, and promoted barrier function. These findings indicate that Cx43 mRNA is a novel target of HuR in IECs and that polyamines regulate Cx43 mRNA stability via HuR, thus playing a critical role in the maintenance of intestinal epithelial barrier function.NEW & NOTEWORTHY The current study shows that polyamines stabilize the Cx43 mRNA via HuR, thus enhancing the function of the Cx43-mediated gap junction. These findings suggest that induced Cx43 by HuR plays a critical role in the process by which polyamines regulate intestinal epithelial barrier.

miR-195 regulates intestinal epithelial restitution after wounding by altering actin-related protein-2 translation.

Early gut epithelial restitution reseals superficial wounds after acute injury, but the exact mechanism underlying this rapid mucosal repair remains largely unknown. MicroRNA-195 (miR-195) is highly expressed in the gut epithelium and involved in many aspects of mucosal pathobiology. Actin-related proteins (ARPs) are key components essential for stimulation of actin polymerization and regulate cell motility. Here, we reported that miR-195 modulates early intestinal epithelial restitution by altering ARP-2 expression at the translation level. miR-195 directly interacted with the ARP-2 mRNA, and ectopically expressed miR-195 decreased ARP-2 protein without effect on its mRNA content. In contrast, miR-195 silencing by transfection with anti-miR-195 oligo increased ARP-2 expression. Decreased ARP-2 levels by miR-195 overexpression were associated with an inhibition of early epithelial restitution, as indicated by a decrease in cell migration over the wounded area. Elevation of cellular ARP-2 levels by transfection with its transgene restored cell migration after wounding in cells overexpressing miR-195. Polyamines were found to decrease miR-195 abundance and enhanced ARP-2 translation, thus promoting epithelial restitution after wounding. Moreover, increasing the levels of miR-195 disrupted F-actin cytoskeleton organization, which was prevented by ARP2 overexpression. These results indicate that miR-195 inhibits early epithelial restitution by decreasing ARP-2 translation and that miR-195 expression is negatively regulated by cellular polyamines.

Off-the-shelf, steroid-resistant, IL13Rα2-specific CAR T cells for treatment of glioblastoma.

BACKGROUND: Wide-spread application of chimeric antigen receptor (CAR) T cell therapy for cancer is limited by the current use of autologous CAR T cells necessitating the manufacture of individualized therapeutic products for each patient. To address this challenge, we have generated an off-the-shelf, allogeneic CAR T cell product for the treatment of glioblastoma (GBM), and present here the feasibility, safety, and therapeutic potential of this approach. METHODS: We generated for clinical use a healthy-donor derived IL13Rα2-targeted CAR+ (IL13-zetakine+) cytolytic T-lymphocyte (CTL) product genetically engineered using zinc finger nucleases (ZFNs) to permanently disrupt the glucocorticoid receptor (GR) (GRm13Z40-2) and endow resistance to glucocorticoid treatment. In a phase I safety and feasibility trial we evaluated these allogeneic GRm13Z40-2 T cells in combination with intracranial administration of recombinant human IL-2 (rhIL-2; aldesleukin) in six patients with unresectable recurrent GBM that were maintained on systemic dexamethasone (4-12 mg/day). RESULTS: The GRm13Z40-2 product displayed dexamethasone-resistant effector activity without evidence for in vitro alloreactivity. Intracranial administration of GRm13Z40-2 in four doses of 108 cells over a two-week period with aldesleukin (9 infusions ranging from 2500-5000 IU) was well tolerated, with indications of transient tumor reduction and/or tumor necrosis at the site of T cell infusion in four of the six treated research subjects. Antibody reactivity against GRm13Z40-2 cells was detected in the serum of only one of the four tested subjects. CONCLUSIONS: This first-in-human experience establishes a foundation for future adoptive therapy studies using off-the-shelf, zinc-finger modified, and/or glucocorticoid resistant CAR T cells.

Real price of health-experiences of out-of-pocket costs in Australia: protocol for a systematic review.

INTRODUCTION: Australians have substantial out-of-pocket (OOP) health costs compared with other developed nations, even with universal health insurance coverage. This can significantly affect access to care and subsequent well-being, especially for priority populations including those on lower incomes or with multimorbidity and chronic illness. While it is known that high OOP healthcare costs may contribute to poorer health outcomes, it is not clear exactly how these expenses are experienced by people with chronic illnesses. Understanding this may provide critical insights into the burden of OOP costs among this population group and may highlight policy gaps. METHOD AND ANALYSIS: A systematic review of qualitative studies will be conducted using Pubmed, CINAHL Complete (EBSCO), Cochrane Library, PsycINFO (Ovid) and EconLit from date of inception to June 2022. Primary outcomes will include people's experiences of OOP costs such as their preferences, priorities, trade-offs and other decision-making considerations. Study selection will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and methodological appraisal of included studies will be assessed using the Critical Appraisal Skills Programme. A narrative synthesis will be conducted for all included studies. ETHICS AND DISSEMINATION: Ethics approval was not required given this is a systematic review that does not include human recruitment or participation. The study's findings will be disseminated through conferences and symposia and shared with consumers, policymakers and service providers, and published in a peer-reviewed journal. PROSPERO REGISTRATION NUMBER: CRD42022337538.

TRPC1-mediated Ca2+ signaling enhances intestinal epithelial restitution by increasing α4 association with PP2Ac after wounding.

Gut epithelial restitution after superficial wounding is an important repair modality regulated by numerous factors including Ca2+ signaling and cellular polyamines. Transient receptor potential canonical-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs) and its activation increases epithelial restitution by inducing Ca2+ influx after acute injury. α4 is a multiple functional protein and implicated in many aspects of cell functions by modulating protein phosphatase 2A (PP2A) stability and activity. Here we show that the clonal populations of IECs stably expressing TRPC1 (IEC-TRPC1) exhibited increased levels of α4 and PP2A catalytic subunit (PP2Ac) and that TRPC1 promoted intestinal epithelial restitution by increasing α4/PP2Ac association. The levels of α4 and PP2Ac proteins increased significantly in stable IEC-TRPC1 cells and this induction in α4/PP2Ac complexes was accompanied by an increase in IEC migration after wounding. α4 silencing by transfection with siRNA targeting α4 (siα4) or PP2Ac silencing destabilized α4/PP2Ac complexes in stable IEC-TRPC1 cells and repressed cell migration over the wounded area. Increasing the levels of cellular polyamines by stable transfection with the Odc gene stimulated α4 and PP2Ac expression and enhanced their association, thus also promoting epithelial restitution after wounding. In contrast, depletion of cellular polyamines by treatment with α-difluoromethylornithine reduced α4/PP2Ac complexes and repressed cell migration. Ectopic overexpression of α4 partially rescued rapid epithelial repair in polyamine-deficient cells. These results indicate that activation of TRPC1-mediated Ca2+ signaling enhances cell migration primarily by increasing α4/PP2Ac associations after wounding and this pathway is tightly regulated by cellular polyamines.

MicroRNA-195 regulates Tuft cell function in the intestinal epithelium by altering translation of DCLK1.

Intestinal Tuft cells sense luminal contents to influence the mucosal immune response against eukaryotic infection. Paneth cells secrete antimicrobial proteins as part of the mucosal protective barrier. Defects in Tuft and Paneth cells occur commonly in various gut mucosal disorders. MicroRNA-195 (miR-195) regulates the stability and translation of target mRNAs and is involved in many aspects of cell processes and pathologies. Here, we reported the posttranscriptional mechanisms by which miR-195 regulates Tuft and Paneth cell function in the small intestinal epithelium. Mucosal tissues from intestinal epithelial tissue-specific miR-195 transgenic (miR195-Tg) mice had reduced numbers of double cortin-like kinase 1 (DCLK1)-positive (Tuft) and lysozyme-positive (Paneth) cells, compared with tissues from control mice, but there were no effects on Goblet cells and enterocytes. Intestinal organoids expressing higher miR-195 levels from miR195-Tg mice also exhibited fewer Tuft and Paneth cells. Transgenic expression of miR-195 in mice failed to alter growth of the small intestinal mucosa but increased vulnerability of the gut barrier in response to lipopolysaccharide (LPS). Studies aimed at investigating the mechanism underlying regulation of Tuft cells revealed that miR-195 directly interacted with the Dclk1 mRNA via its 3'-untranslated region and inhibited DCLK1 translation. Interestingly, the RNA-binding protein HuR competed with miR-195 for binding Dclk1 mRNA and increased DCLK1 expression. These results indicate that miR-195 suppresses the function of Tuft and Paneth cells in the small intestinal epithelium and further demonstrate that increased miR-195 disrupts Tuft cell function by inhibiting DCLK1 translation via interaction with HuR.

Functional hemispherectomy: can preoperative imaging predict outcome?

OBJECTIVE: Although hemispherectomy is an effective treatment for children with intractable hemispheric epilepsy syndromes, as many as 40% of patients eventually develop seizure recurrence. The causes of seizure recurrence in these patients are incompletely understood. The authors sought to evaluate the efficacy of hemispherectomy at their center and determine whether contralateral MRI abnormalities can predict seizure recurrence. METHODS: A retrospective review of consecutive hemispherectomies performed at Miami Children's Hospital between January 2000 and June 2014 was performed. Time-to-event analysis was performed. The "event" was defined as any seizures following resective epilepsy surgery (not including seizures in the first postoperative week and auras). Several preoperative variables were analyzed to determine their suitability to predict seizure recurrence following surgery. RESULTS: Sixty-nine patients (44 boys) with a mean age of 8.2 ± 5.9 years (range 0.1-20.8 years) underwent 72 hemispherectomies; 67 of these were functional hemispherectomies, while another 5 were completion of a previous functional hemispherectomy (2 completions of functional hemispherectomies, 3 anatomical hemispherectomies). The duration of epilepsy was 5.8 ± 5.5 years with 66 cases (91.7%) having daily seizures. Etiology included stroke (n = 28), malformation of cortical development (n = 11), hemimegalencephaly (n = 11), encephalitis (n = 13), and other (n = 7). Engel class I outcome was achieved in 59 (86%) and 56 (81%) patients at 1 and 2 years of follow-up, respectively. The mean time to seizure recurrence was 33.5 ± 31.1 months. In univariate analyses, the absence of contralateral abnormalities on MRI (HR 4.09, 95% CI 1.41-11.89, p = 0.009) was associated with a longer duration of seizure freedom. The presence of contralateral MRI abnormalities was associated with contralateral ictal seizures on preoperative scalp EEG (p = 0.002). Fifteen patients experienced 20 complications (20/72, 27.8%), including the development of hydrocephalus necessitating CSF diversion in 9 cases (13%), hygroma in 1, hemispheric edema in 1, aseptic meningitis in 2, postoperative hemorrhage in 2, infection in 2, ischemic stroke in 2, and blood transfusion-contracted hepatitis C in 1 case. CONCLUSIONS: Patients with bihemispheric abnormalities, as evidenced by contralateral MRI abnormalities, have a higher risk of earlier seizure recurrence following functional hemispherectomy. ABBREVIATIONS: EVD = external ventricular drain; MCD = malformation of cortical development; MEG = magnetoencephalography; PVWM = periventricular white matter; TTE = time-to-event; VPS = ventriculoperitoneal shunt.

miR-222 represses expression of zipcode binding protein-1 and phospholipase C-γ1 in intestinal epithelial cells.

Both zipcode binding protein-1 (ZBP1) and phospholipase C-γ1 (PLCγ1) are intimately involved in many aspects of early intestinal mucosal repair after acute injury, but the exact mechanisms that control their cellular abundances remain largely unknown. The present study shows that microRNA-222 (miR-222) interacts with the mRNAs encoding ZBP1 and PLCγ1 and regulates ZBP1 and PLCγ1 expression in intestinal epithelial cells (IECs). The biotinylated miR-222 bound specifically to the ZBP1 and PLCγ1 mRNAs in IECs. Ectopically expressed miR-222 precursor destabilized the ZBP1 and PLCγ1 mRNAs and consequently lowered the levels of cellular ZBP1 and PLCγ1 proteins. Conversely, decreasing the levels of cellular miR-222 by transfection with its antagonism increased the stability of the ZBP1 and PLCγ1 mRNAs and increased the levels of ZBP1 and PLCγ1 proteins. Overexpression of miR-222 also inhibited cell migration over the wounded area, which was partially abolished by overexpressing ZBP1 and PLCγ1. Furthermore, prevention of the increased levels of ZBP1 and PLCγ1 in the miR-222-silenced cells by transfection with specific small interfering RNAs targeting ZBP1 or PLCγ1 mRNA inhibited cell migration after wounding. These findings indicate that induced miR-222 represses expression of ZBP1 and PLCγ1 at the posttranscriptional level, thus inhibiting IEC migration during intestinal epithelial restitution after wounding.

α4 Coordinates Small Intestinal Epithelium Homeostasis by Regulating Stability of HuR.

The mammalian intestinal epithelium is a rapidly self-renewing tissue in the body, and its homeostasis depends on a dynamic balance among proliferation, migration, apoptosis, and differentiation of intestinal epithelial cells (IECs). The protein phosphatase 2A (PP2A)-associated protein α4 controls the activity and specificity of serine/threonine phosphatases and is thus implicated in many cellular processes. Here, using a genetic approach, we investigated the mechanisms whereby α4 controls the homeostasis of the intestinal epithelium. In mice with ablated α4, the small intestinal mucosa exhibited crypt hyperplasia, villus shrinkage, defective differentiation of Paneth cells, and reduced IEC migration along the crypt-villus axis. The α4-deficient intestinal epithelium also displayed decreased expression of different intercellular junction proteins and abnormal epithelial permeability. In addition, α4 deficiency decreased the levels of the RNA-binding protein HuR in the mucosal tissue. In cultured IECs, ectopic overexpression of HuR in α4-deficient cells rescued the production of these intercellular junction proteins and restored the epithelial barrier function to a nearly normal level. Mechanistically, α4 silencing destabilized HuR through a process involving HuR phosphorylation by IκB kinase α, leading to ubiquitin-mediated proteolysis of HuR. These findings indicate that the critical impact of α4 upon the barrier function and homeostasis of the intestinal epithelium depends largely on its ability to regulate the stability of HuR.

ß-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding.

Early gut mucosal restitution is a process by which intestinal epithelial cells (IECs) migrate over the wounded area, and its defective regulation occurs commonly in various critical pathological conditions. This rapid reepithelialization is mediated by different activating small GTP-binding proteins, but the exact mechanism underlying this process remains largely unknown. Recently, it has been reported that interaction between p21-activated kinase-interacting exchange factor (ß-PIX) and G protein-coupled receptor kinase-interacting protein 1 (GIT1) activates small GTPases and plays an important role in the regulation of cell motility. Here, we show that induced association of ß-PIX with GIT1 is essential for the stimulation of IEC migration after wounding by activating Rac1. Levels of ß-PIX and GIT1 proteins and their association in differentiated IECs (line of IEC-Cdx2L1) were much higher than those observed in undifferentiated IECs (line of IEC-6), which was associated with an increase in IEC migration after wounding. Decreased levels of endogenous ß-PIX by its gene-silencing destabilized ß-PIX/GIT1 complexes, repressed Rac1 activity and inhibited cell migration over the wounded area. In contrast, ectopic overexpression of ß-PIX increased the levels of ß-PIX/GIT1 complexes, stimulated Rac1 activity, and enhanced intestinal epithelial restitution. Increased levels of cellular polyamines also stimulated ß-PIX/GIT1 association, increased Rac1 activity, and promoted the epithelial restitution. Moreover, polyamine depletion decreased cellular abundances of ß-PIX/GIT1 complex and repressed IEC migration after wounding, which was rescued by ectopic overexpression of ß-PIX or GIT1. These results indicate that ß-PIX/GIT1/Rac1 association is necessary for stimulation of IEC migration after wounding and that this signaling pathway is tightly regulated by cellular polyamines. NEW & NOTEWORTHY Our current study demonstrates that induced association of ß-PIX with GIT1 is essential for the stimulation of intestinal epithelial restitution by activating Rac1, and this signaling pathway is tightly regulated by cellular polyamines.

c-Jun enhances intestinal epithelial restitution after wounding by increasing phospholipase C-γ1 transcription.

c-Jun is an activating protein 1 (AP-1) transcription factor and implicated in many aspects of cellular functions, but its exact role in the regulation of early intestinal epithelial restitution after injury remains largely unknown. Phospholipase C-γ1 (PLCγ1) catalyzes hydrolysis of phosphatidylinositol 4,5 biphosphate into the second messenger diacylglycerol and inositol 1,4,5 triphosphate, coordinates Ca2+ store mobilization, and regulates cell migration and proliferation in response to stress. Here we reported that c-Jun upregulates PLCγ1 expression and enhances PLCγ1-induced Ca2+ signaling, thus promoting intestinal epithelial restitution after wounding. Ectopically expressed c-Jun increased PLCγ1 expression at the transcription level, and this stimulation is mediated by directly interacting with AP-1 and CCAAT-enhancer-binding protein (C/EBP) binding sites that are located at the proximal region of the rat PLCγ1 promoter. Increased levels of PLCγ1 by c-Jun elevated cytosolic free Ca2+ concentration and stimulated intestinal epithelial cell migration over the denuded area after wounding. The c-Jun-mediated PLCγ1/Ca2+ signal also plays an important role in polyamine-induced cell migration after wounding because increased c-Jun rescued Ca2+ influx and cell migration in polyamine-deficient cells. These findings indicate that c-Jun induces PLCγ1 expression transcriptionally and enhances rapid epithelial restitution after injury by activating Ca2+ signal.

RhoA enhances store-operated Ca2+ entry and intestinal epithelial restitution by interacting with TRPC1 after wounding.

Early mucosal restitution occurs as a consequence of epithelial cell migration to resealing of superficial wounds after injury. Our previous studies show that canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca(2+) channel (SOC) in intestinal epithelial cells (IECs) and plays an important role in early epithelial restitution by increasing Ca(2+) influx. Here we further reported that RhoA, a small GTP-binding protein, interacts with and regulates TRPC1, thus enhancing SOC-mediated Ca(2+) entry (SOCE) and epithelial restitution after wounding. RhoA physically associated with TRPC1 and formed the RhoA/TRPC1 complexes, and this interaction increased in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1). Inactivation of RhoA by treating IEC-TRPC1 cells with exoenzyme C3 transferase (C3) or ectopic expression of dominant negative RhoA (DNMRhoA) reduced RhoA/TRPC1 complexes and inhibited Ca(2+) influx after store depletion, which was paralleled by an inhibition of cell migration over the wounded area. In contrast, ectopic expression of wild-type (WT)-RhoA increased the levels of RhoA/TRPC1 complexes, induced Ca(2+) influx through activation of SOCE, and promoted cell migration after wounding. TRPC1 silencing by transfecting stable WT RhoA-transfected cells with siRNA targeting TRPC1 (siTRPC1) reduced SOCE and repressed epithelial restitution. Moreover, ectopic overexpression of WT-RhoA in polyamine-deficient cells rescued the inhibition of Ca(2+) influx and cell migration induced by polyamine depletion. These findings indicate that RhoA interacts with and activates TRPC1 and thus stimulates rapid epithelial restitution after injury by inducing Ca(2+) signaling.

Caveolin-1 enhances rapid mucosal restitution by activating TRPC1-mediated Ca2+ signaling.

Early rapid mucosal restitution occurs as a consequence of epithelial cell migration to reseal superficial wounds, a process independent of cell proliferation. Our previous studies revealed that the canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca(2+) channel (SOCs) in intestinal epithelial cells (IECs) and regulates epithelial restitution after wounding, but the exact mechanism underlying TRPC1 activation remains elusive. Caveolin-1 (Cav1) is a major component protein that is associated with caveolar lipid rafts in the plasma membrane and was recently identified as a regulator of store-operated Ca(2+) entry (SOCE). Here, we showed that Cav1 plays an important role in the regulation of mucosal restitution by activating TRPC1-mediated Ca(2+) signaling. Target deletion of Cav1 delayed gastric mucosal repair after exposure to hypertonic NaCl in mice, although it did not affect total levels of TRPC1 protein. In cultured IECs, Cav1 directly interacted with TRPC1 and formed Cav1/TRPC1 complex as measured by immunoprecipitation assays. Cav1 silencing in stable TRPC1-transfected cells by transfection with siCav1 reduced SOCE without effect on the level of resting [Ca(2+)]cyt. Inhibition of Cav1 expression by siCav1 and subsequent decrease in Ca(2+) influx repressed epithelial restitution, as indicated by a decrease in cell migration over the wounded area, whereas stable ectopic overexpression of Cav1 increased Cav1/TRPC1 complex, induced SOCE, and enhanced cell migration after wounding. These results indicate that Cav1 physically interacts with and activates TRPC1, thus stimulating TRPC1-mediated Ca(2+) signaling and rapid mucosal restitution after injury.

Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.

BACKGROUND: CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe. METHODS: We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. RESULTS: One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CONCLUSIONS: CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).

Differentiated intestinal epithelial cells express high levels of TGF-ß receptors and exhibit increased sensitivity to growth inhibition.

BACKGROUND: Intestinal epithelial cells (IECs) within crypts continuously divide and differentiate as they migrate up towards the luminal surface of the mucosa. With the onset of differentiation, IECs lose their proliferative potential, but the exact mechanism remains unknown. This current study examined the involvement of the TGF-ß signaling pathway in this process. METHODS: Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cell differentiation was induced by forced expression of the Cdx2 gene, a transcription factor responsible for controlling intestinal epithelial cell differentiation. RESULTS: Forced expression of the Cdx2 gene in stable Cdx2-transfected IEC-6 cells resulted in a differentiated phenotype as indicated by morphological features and increased expression of sucrase-isomaltase. Levels of TGF-ß type I receptor (TGFß-RI) and TGF-ß type II receptor (TGFß-RII) increased in these differentiated epithelial cells. The induced TGFß-RI and TGFß-RII expression in Cdx2-transfected IEC-6 cells was associated with increased sensitivity to TGF-ß-induced growth inhibition. Depletion of cellular polyamines further increased TGF-ß receptor expression and additionally enhanced the response to TGF-ß-induced growth inhibition. Increased TGFß-RI and RII in polyamine-deficient cells were also associated with an induction in JunD/AP-1 activity. CONCLUSIONS: These results indicate that the loss of the proliferative potential in differentiated IECs results partially from the increased expression of TGF-ß receptors.

Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection.

UNLABELLED: Oxidative stress has been identified as a key mechanism of hepatitis C virus (HCV)-induced pathogenesis. Studies have suggested that HCV increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage, but the source of reactive oxygen species (ROS) remains incompletely characterized. We hypothesized that HCV increases the generation of superoxide and hydrogen peroxide close to the hepatocyte nucleus and that this source of ROS is reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase 4 (Nox4). Huh7 human hepatoma cells and telomerase-reconstituted primary human hepatocytes, transfected or infected with virus-producing HCV strains of genotypes 2a and 1b, were examined for messenger RNA (mRNA), protein, and subcellular localization of Nox proteins along with the human liver. We found that genotype 2a HCV induced persistent elevations of Nox1 and Nox4 mRNA and proteins in Huh7 cells. HCV genotype 1b likewise elevated the levels of Nox1 and Nox4 in telomerase-reconstituted primary human hepatocytes. Furthermore, Nox1 and Nox4 proteins were increased in HCV-infected human liver versus uninfected liver samples. Unlike Nox1, Nox4 was prominent in the nuclear compartment of these cells as well as the human liver, particularly in the presence of HCV. HCV-induced ROS and nuclear nitrotyrosine could be decreased with small interfering RNAs to Nox1 and Nox4. Finally, HCV increased the level of transforming growth factor beta 1 (TGFbeta1). TGFbeta1 could elevate Nox4 expression in the presence of infectious HCV, and HCV increased Nox4 at least in part through TGFbeta1. CONCLUSION: HCV induced a persistent elevation of Nox1 and Nox4 and increased nuclear localization of Nox4 in hepatocytes in vitro and in the human liver. Hepatocyte Nox proteins are likely to act as a persistent, endogenous source of ROS during HCV-induced pathogenesis.

Polyamines are required for phospholipase C-gamma1 expression promoting intestinal epithelial restitution after wounding.

Intestinal mucosal restitution occurs by epithelial cell migration, rather than by proliferation, to reseal superficial wounds after injury. Polyamines are essential for the stimulation of intestinal epithelial cell (IEC) migration during restitution in association with their ability to regulate Ca2+ homeostasis, but the exact mechanism by which polyamines induce cytosolic free Ca2+ concentration ([Ca2+]cyt) remains unclear. Phospholipase C (PLC)-gamma1 catalyzes the formation of inositol (1,4,5)-trisphosphate (IP3), which is implicated in the regulation of [Ca2+]cyt by modulating Ca2+ store mobilization and Ca2+ influx. The present study tested the hypothesis that polyamines are involved in PLC-gamma1 activity, regulating [Ca2+]cyt and cell migration after wounding. Depletion of cellular polyamines by alpha-difluoromethylornithine inhibited PLC-gamma1 expression in differentiated IECs (stable Cdx2-transfected IEC-6 cells), as indicated by substantial decreases in levels of PLC-gamma1 mRNA and protein and its enzyme product IP3. Polyamine-deficient cells also displayed decreased [Ca2+]cyt and inhibited cell migration. Decreased levels of PLC-gamma1 by treatment with U-73122 or transfection with short interfering RNA specifically targeting PLC-gamma1 also decreased IP3, reduced resting [Ca2+]cyt and Ca2+ influx after store depletion, and suppressed cell migration in control cells. In contrast, stimulation of PLC-gamma1 by 2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)-benzenesulfonamide induced IP3, increased [Ca2+]cyt, and promoted cell migration in polyamine-deficient cells. These results indicate that polyamines are absolutely required for PLC-gamma1 expression in IECs and that polyamine-mediated PLC-gamma1 signaling stimulates cell migration during restitution as a result of increased [Ca2+]cyt.

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-kappaB signaling in intestinal epithelial cells.

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-kappaB signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-kappaB activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF-alpha plus cycloheximide (TNF-alpha/CHX). Specific inhibition of NF-kappaB by the recombinant adenoviral vector containing the IkappaBalpha superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-kappaB activity and increased the sensitivity to TNF-alpha/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-kappaB activity and resulted in increased resistance to TNF-alpha/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-alpha/CHX-induced apoptosis, at least partially, through the activation of NF-kappaB signaling in IECs.

Pain and fatigue in community-dwelling adults.

BACKGROUND: Pain and fatigue are common debilitating symptoms reported by both patient and community-dwelling populations. However, population-based studies typically focus on psychosocial variables as correlates of fatigue, with little effort toward assessing pain as a risk factor. This study examines the specific relationship between pain and fatigue and the importance of mood and sleep as covariates of fatigue in a community-dwelling sample of adults. We also assessed the prevalence of mild, moderate, and severe fatigue for this sample. METHODS: Data were collected using self-administered questionnaires assessing demographic characteristics and symptoms, including pain, fatigue, mood, and sleep disturbance, in a sample (N=274) of community-dwelling adults. RESULTS: Regression analyses showed pain, mood, and sleep as significantly associated with fatigue, with pain being the most highly correlated. However, while pain accounted for the largest proportion of the variability in fatigue, mood modified the relationship between pain and fatigue. We also found that 9% of the sample reported no fatigue, 41% reported mild fatigue, 42% reported moderate fatigue, and 8% reported severe fatigue. CONCLUSIONS: Our findings provide empirical support of the importance of assessing pain as an important risk factor of fatigue and examining mood as a covariate in population-based studies of fatigue.