Orexin 2 receptor antagonism sex-dependently improves sleep/wakefulness and cognitive performance in tau transgenic mice.

BACKGROUND AND PURPOSE: Tau pathology contributes to a bidirectional relationship between sleep disruption and neurodegenerative disease. Tau transgenic rTg4510 mice model tauopathy symptoms, including sleep/wake disturbances, which manifest as marked hyperarousal. This phenotype can be prevented by early transgene suppression; however, whether hyperarousal can be rescued after onset is unknown. EXPERIMENTAL APPROACH: Three 8-week experiments were conducted with wild-type and rTg4510 mice after age of onset of hyperarousal (4.5 months): (1) Tau transgene suppression with doxycycline (200 ppm); (2) inactive phase rapid eye movement (REM) sleep enhancement with the dual orexin receptor antagonist suvorexant (50 mg·kg-1 ·day-1 ); or (3) Active phase non-NREM (NREM) and REM sleep enhancement using the selective orexin 2 (OX2 ) receptor antagonist MK-1064 (40 mg·kg-1 ·day-1 ). Sleep was assessed using polysomnography, cognition using the Barnes maze, and tau pathology using immunoblotting and/or immunohistochemistry. KEY RESULTS: Tau transgene suppression improved tauopathy and hippocampal-dependent spatial memory, but did not modify hyperarousal. Pharmacological rescue of REM sleep deficits did not improve spatial memory or tau pathology. In contrast, normalising hyperarousal by increasing both NREM and REM sleep via OX2 receptor antagonism restored spatial memory, independently of tauopathy, but only in male rTg4510 mice. OX2 receptor antagonism induced only short-lived hypnotic responses in female rTg4510 mice and did not improve spatial memory, indicating a tau- and sex-dependent disruption of OX2 receptor signalling. CONCLUSIONS AND IMPLICATIONS: Pharmacologically reducing hyperarousal corrects tau-induced sleep/wake and cognitive deficits. Tauopathy causes sex-dependent disruptions of OX2 receptor signalling/function, which may have implications for choice of hypnotic therapeutics in tauopathies.

Altered EEG power spectrum, but not sleep-wake architecture, in HCN1 knockout mice.

Sleep is a complex biological state characterized by large populations of neurons firing in a rhythmic or synchronized manner. HCN channels play a critical role in generating and sustaining synchronized neuronal firing and are involved in the actions of anaesthetics. However, the role of these channels in sleep-wakefulness per se has yet to be studied. We conducted polysomnographic recordings of Hcn1 constitutive knockout (Hcn1 KO) and wild-type (WT) mice in order to investigate the potential role of HCN1 channels in sleep/wake regulation. EEG and EMG data were analysed using the Somnivore™ machine learning algorithm. Time spent in each vigilance state, bout number and duration, and EEG power spectral activity were compared between genotypes. There were no significant differences in the time spent in wake, rapid eye movement (REM) or non-REM (NREM) sleep between Hcn1 KO and WT mice. Wake bout duration during the inactive phase was significantly shorter in Hcn1 KO mice whilst no other bout parameters were affected by genotype. Hcn1 KO mice showed a reduction in overall EEG power which was particularly prominent in the theta (5-9 Hz) and alpha (9-15 Hz) frequency bands and most evident during NREM sleep. Together these data suggest that HCN1 channels do not play a major role in sleep architecture or modulation of vigilance states. However, loss of these channels significantly alters underlying neuronal activity within these states which may have functional consequences.

Transcriptomic Mapping of Neurotoxicity Pathways in the Rat Brain in Response to Intraventricular Polymyxin B.

Intraventricular or intrathecal administration of polymyxins are increasingly used to treat multidrug-resistant (MDR) Gram-negative bacteria caused infections in the central nervous system (CNS). However, our limited knowledge of the mechanisms underpinning polymyxin-induced neurotoxicity significantly hinders the development of safe and efficacious polymyxin dosing regimens. To this end, we conducted transcriptomic analyses of the rat brain and spinal cord 1 h following intracerebroventricular administration of polymyxin B into rat lateral ventricle at a clinically relevant dose (0.5 mg/kg). Following the treatment, 66 differentially expressed genes (DEGs) were identified in the brain transcriptome while none for the spinal cord (FDR ≤ 0.05, fold-change ≥ 1.5). DEGs were enriched in signaling pathways associated with hormones and neurotransmitters, including dopamine and (nor)epinephrine. Notably, the expression levels of Slc6a3 and Gabra6 were decreased by 20-fold and 4.3-fold, respectively, likely resulting in major perturbations of dopamine and γ-aminobutyric acid signaling in the brain. Mass spectrometry imaging of brain sections revealed a distinct pattern of polymyxin B distribution with the majority accumulating in the injection-side lateral ventricle and subsequently into third and fourth ventricles. Polymyxin B was not detectable in the left lateral ventricle or brain tissue. Electrophysiological measurements on primary cultured rat neurons revealed a large inward current and significant membrane leakage following polymyxin B treatment. Our work demonstrates, for the first time, the key CNS signaling pathways associated with polymyxin neurotoxicity. This mechanistic insight combined with pharmacokinetic/pharmacodynamic dosing strategies will help guide the design of safe and effective intraventricular/intrathecal polymyxin treatment regimens for CNS infections caused by MDR Gram-negative pathogens.

Untargeted metabolomics to evaluate polymyxin B toxicodynamics following direct intracerebroventricular administration into the rat brain.

There is a dearth of studies focused on understanding pharmacokinetics, pharmacodynamics and toxicodynamics of polymyxins following direct administration to the central nervous system (CNS). In this study, for the first time, untargeted metabolomics were employed to ascertain the perturbations of brain metabolism in the rat cerebral cortex following direct brain injection of 0.75 mg/kg polymyxin B (1 and 4 h) through the right lateral ventricle. In the right cortex metabolome, ICV polymyxin B induced a greater perturbation at 1 h compared to negligible effect at 4 h. Pathway enrichment analysis showed that sphingolipid, arginine, and histidine metabolism, together with aminoacyl-tRNA biosynthesis were significantly affected in the right cortex metabolome. Furthermore, intracerebroventricular (ICV) polymyxin B dysregulated the two arms (CDP-choline and CDP-ethanolamine) of the Kennedy pathway that governs the de novo biosynthesis of neuronal phospholipids. Importantly, the key intermediates of metabolic pathways that maintain cellular redox balance (e.g., glutathione metabolism) and mitochondrial function (e.g., electron transport chain) were markedly depleted. The abundance of key metabolites (e.g., N-acetyl-l-glutamate) associated with diverse CNS disorders (e.g., neurodegenerative disease) were also significantly perturbed. The biological significance of these metabolic perturbations on the CNS includes impaired oxidant-antioxidant balance, impaired neuronal lipid homeostasis and mitochondrial dysfunction. Furthermore, ICV polymyxin B caused a significant alteration in the abundance of several metabolic biomarkers associated with cerebral ischemia, oxidative stress as well as certain neurological disorders. These findings may facilitate the development of new pharmacokinetic/pharmacodynamic strategies to attenuate polymyxins ICV related CNS toxicities and stimulate the discovery of safer next-generation polymyxin-like lipopeptide antibiotics.

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study.

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous system (CNS) to ameliorate diseases or infections. Drugs are administered directly into the CNS in animals and humans to circumvent the BBB. The present protocol describes a unique way of treating brain infections through intraventricular delivery of antibiotics, i.e., polymyxins, the last-line antibiotics to treat multi-drug resistant Gram-negative bacteria. A straightforward stereotaxic surgery protocol was developed to implant a guide cannula reaching into the lateral ventricle in rats. After a recovery period of 24 h, rats can be injected consciously and repeatedly through a cannula that is fitted to the guide. Injections can be delivered manually as a bolus or infusion using a microinjection pump to obtain a slow and controlled flow rate. The intraventricular injection was successfully confirmed with Evans Blue dye. Cerebrospinal fluid (CSF) can be drained, and the brain and other organs can be collected. This approach is highly amenable for studies involving drug delivery to the CNS and subsequent assessment of pharmacokinetic and pharmacodynamic activity.

Differential sleep/wake response and sex differences following acute suvorexant, MK-1064 and zolpidem administration in the rTg4510 mouse model of tauopathy.

BACKGROUND AND PURPOSE: Transgenic mouse models of tauopathy display prominent sleep/wake disturbances which manifest primarily as a hyperarousal phenotype during the active phase, suggesting that tau pathology contributes to sleep/wake changes. However, no study has yet investigated the effect of sleep-promoting compounds in these models. Such information has implications for the use of hypnotics as potential therapeutic tools in tauopathy-related disorders. EXPERIMENTAL APPROACH: This study examined polysomnographic recordings in 6-6.5-month-old male and female rTg4510 mice following acute administration of suvorexant (50 mg·kg-1 ), MK-1064 (30 mg·kg-1 ) or zolpidem (10 mg·kg-1 ), administered at the commencement of the active phase. KEY RESULTS: Suvorexant, a dual OX receptor antagonist, promoted REM sleep in rTg4510 mice, without affecting wake or NREM sleep. MK-1064, a selective OX2 receptor antagonist, reduced wake and increased NREM and total sleep time. MK-1064 normalised the hyperarousal phenotype of male rTg4510 mice, whereas female rTg4510 mice exhibited a more transient response. Zolpidem, a GABAA receptor positive allosteric modulator, decreased wake and increased NREM sleep in both male and female rTg4510 mice. Of the three compounds, the OX2 receptor antagonist MK-1064 promoted and normalised physiologically normal sleep, especially in male rTg4510 mice. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that hyperphosphorylated tau accumulation and associated hyperarousal does not significantly alter the responses of tauopathy mouse models to hypnotics. However, the sex differences observed in the sleep/wake response of rTg4510 mice to MK-1064, but not suvorexant or zolpidem, raise questions about therapeutic implications for the use of OX2 receptor antagonists in human neurodegenerative disorders.

An Interprofessional Curriculum to Advance Relational Coordination and Professionalism in Early-Career Practitioners.

Introduction: We created a curriculum to help new physicians and nurses develop skills in interprofessional collaboration. This modular, team-based curriculum for early practitioners delivered training in the five following skill areas: listening for meaning, soliciting another's perspective, negotiating a transparent plan of care, attending to nonverbal communication and microaggression, and speaking up the hierarchy. Methods: We brought first-year medical and surgical residents and new nurses together for a 2-hour session monthly for 5 months. Each session began with an interactive large-group presentation, followed by small-group activities covering one of the five skill areas above, which had been identified as critical to interprofessional collaboration by national organizations. We measured relational coordination (RC), a validated measure of how well teams work together, before and after the curriculum was administered. We also obtained qualitative data from participant interviews and end-of-session evaluations. Results: Participants reported that the program helped them gain an understanding of each other's roles and workflow challenges. They felt that the curriculum allowed for the cultivation of professional relationships outside the clinical environment, which improved collegiality via gains in rapport and empathy towards each other. Nurses noted increased approachability of their physician colleagues after participation. RC scores improved for the entire cohort (p = .0232). Nurses had statistically higher RC gains than interns did (p = .0055). Discussion: Curriculum participants demonstrated improved RC scores and reported increased rapport with and empathy for each other. Curriculum development in this area is important because it may lead to better team-based patient care.

Transporters for Antiretroviral Drugs in Colorectal CD4+ T Cells and Circulating α4ß7 Integrin CD4+ T Cells: Implications for HIV Microbicides.

CD4+ T lymphocytes in the colorectal mucosa are key in HIV-1 transmission and dissemination. As such they are also the primary target for antiretroviral (ARV)-based rectal microbicides for pre-exposure prophylaxis. Drug transporters expressed in mucosal CD4+ T cells determine ARV distribution across the cell membrane and, most likely, efficacy of microbicides. We describe transporters for antiretroviral drugs in colorectal mucosal CD4+ T lymphocytes and compare gene expression with circulating α4ß7+CD4+ T cells, which traffic to the intestine and have been shown to be preferentially infected by HIV-1. Purified total CD4+ T cells were obtained from colorectal tissue and blood samples by magnetic separation. CD4+ T cells expressing α4ß7 integrin were isolated by fluorescence-activated cell sorting from peripheral blood mononuclear cells of healthy volunteers. Expressions of 15 efflux and uptake drug transporter genes were quantified using Taqman qPCR assays. Expression of efflux transporters MRP3, MRP5, and BCRP and uptake transporter CNT2 were significantly higher in colorectal CD4+ T cells compared to circulating CD4+ T cells (p = 0.01-0.03). Conversely, circulating α4ß7+CD4+ T cells demonstrated significantly higher expression of OATPD compared to colorectal CD4+ T cells (p = 0.001). To the best of our knowledge this is the first report of drug transporter gene expression in colorectal CD4+ and peripheral α4ß7+CD4+ T cells. The qualitative and quantitative differences in drug transporter gene expression profiles between α4ß7+CD4+ T cells and total mucosal CD4+ T cells may have significant implications for the efficacy of rectally delivered ARV-microbicides. Most notably, we have identified efflux drug transporters that could be targeted by selective inhibitors or beneficial drug-drug interactions to enhance intracellular accumulation of antiretroviral drugs.

Older Patients Are Immunocompromised by Cytokine Depletion and Loss of Innate Immune Function After HIP Fracture Surgery.

PURPOSE/INTRODUCTION: We have examined the immune status of elderly patients who underwent surgery for a hip fracture, an injury associated with poor postoperative outcomes, to identify specific immune defects. METHODS: In a cohort observational study, 16 patients undergoing surgery for hip fractures had immune function evaluation prior to surgery, and then at 3 and 7 days postoperatively, using flow cytometry for phenotype and for monocyte and granulocyte phagocytic function and respiratory burst. Serum samples were stored and batch analyzed using a human cytokine 25-plex panel. RESULTS: We report significant loss of innate immune function, related specifically to reduced granulocyte numbers by day 7 (P < .0001, flow cytometry; P < .05 white blood cells), and although granulocyte ability to take up opsonized Escherichia coli was increased (P < .05), the ability of those cells to generate a respiratory burst was reduced at days 3 and 7 (P < .05). Monocyte respiratory burst was also significantly reduced (P < .05). Serum cytokine levels indicated very poor T-cell function. CONCLUSION: We have demonstrated that the antimicrobial immune response is profoundly reduced after surgery in elderly patients with hip fractures. The effect was sustained up to 7 days postoperatively, identifying these patients as particularly vulnerable to bacterial infections.

Regulation of T-lymphocyte CCL3 and CCL4 production by retinal pigment epithelial cells.

PURPOSE: Retinal pigment epithelial (RPE) cells have an important role in the immune suppression associated with the immune privilege of the eye. Some aspects of this remain unclear and this study aimed to determine how RPE cells could influence the production of chemokines by T lymphocytes. METHODS: T lymphocytes, separated from peripheral blood of normal volunteers, and RPE cells, cultured from donor eyes, were cultured separately and together, either in contact or in transwells. Supernatants were analyzed for CCL3, CCL4, and soluble CD54 (sCD54) by ELISA. Blocking agents were used to determine which soluble mediators were involved. RESULTS: Coculture of RPE cells with activated lymphocytes resulted in a reduction in CCL3 and CCL4 production by lymphocytes, primarily by soluble mediators. Soluble CD54 was markedly increased on coculture of lymphocytes with RPE cells. Soluble CD54 reduced CCL3 and CCL4 production by RPE cells, and inhibition of CCL3 and CCL4 on coculture with RPE cells was reduced by anti-CD54. Blocking prostaglandin E2 (PGE2) abrogated the inhibition of CCL4, but not CCL3, by RPE cells. Blocking TGFß and nitric oxide production had no effect. CONCLUSIONS: RPE cells are able to down-regulate high levels of CCL3 and CCL4 production by T lymphocytes by using the soluble mediators sCD54 and PGE2. Reducing this production of CCL3 and CCL4 will dampen down the cascade effect and recruitment of more inflammatory cells, protecting the retina from an excessive immune response.

Local and systemic induction of CD4+CD25+ regulatory T-cell population by non-Hodgkin lymphoma.

Regulatory T (Treg) cells contribute to immune evasion by malignancies. To investigate their importance in non-Hodgkin lymphoma (NHL), we enumerated Treg cells in peripheral blood mononuclear cells (PBMCs) and involved tissues from 30 patients. CD25(+)FoxP3(+)CD127(low)CD4(+) Treg cells were increased markedly in PBMCs (median = 20.4% CD4 T cells, n = 20) versus healthy controls (median = 3.2%, n = 13, P < .001) regardless of lymphoma subtype, and correlated with disease stage and serum lactate dehydrogenase (R(s) = 0.79, P < .001). T-cell hyporesponsiveness was reversed by depleting CD25(+) cells, or by adding anti-CTLA-4, supporting the view that Treg cells explain the systemic immunosuppression seen in NHL. A high proportion of Treg cells was also present in involved tissues (median = 38.8% CD4 T cells, n = 15) versus reactive nodes (median = 11.6%, n = 2, P = .02). When autologous CD25(-) PBMC fractions were incubated with tumor cells from patients (n = 6) in vitro, there was consistent strong induction and then expansion of cells with the CD4(+)CD25(+)FoxP3(+) phenotype of classic "natural" Treg cells. This population was confirmed to be suppressive in function. Direct cell-cell interaction of tumor cells with CD25(-) PBMCs was important in Treg induction, although there was heterogeneity in the mechanisms responsible. We conclude that NHL cells are powerful inducers of Treg cells, which may represent a new therapeutic target.

Cross presentation of antigen on MHC class II via the draining lymph node after corneal transplantation in mice.

We investigated Ag trafficking from the cornea and T effector cell activation in secondary lymphoid tissue after corneal transplantation. In preliminary experiments, the central cornea was shown to contain a population of CD45(+), CD11b(+), CD11c- cells, with a few MHC class II(+) cells, and F4/80(+) cells. However, MHC class II(+) passenger leukocytes in donor cornea after allografting did not traffic to the draining lymph node. Instead, Ag (plasmid) delivered to the eye via the donor cornea during allograft was detected in host CD11c(+) and F4/80(+) APC in the draining lymph nodes and spleen. The earliest detection of APC-associated Ag was at 6 h in the draining lymph node and 24 h in the spleen. After 48 h Ag was not detected in the draining lymph node but was still present in the spleen. Ag applied to the donor corneal epithelium before allografting induced Ag-specific T cell activation and expansion in the draining lymph node with a peak response at 4-6 days, indicating that cross-presentation of Ag had occurred. We conclude therefore, that Ag is transported from the donor cornea within host APC and that this event occurs within hours after grafting. Ag is cross-presented to host CD4(+) T cells on MHC class II and leads to the activation of Ag-specific effector T cells and clonal expansion in the draining lymph node.

Skin integrity in critically ill and injured children.

BACKGROUND: Skin breakdown increases the cost of care, may lead to increased morbidity, and has negative psychosocial implications because of secondary scarring or alopecia. The scope of this problem has not been widely studied in critically ill and injured children. OBJECTIVES: To determine the incidence of skin breakdown in critically ill and injured children and to compare the characteristics of patients who experience skin breakdown with those of patients who do not. METHODS: Admission and follow-up data for a 15-week period were collected retrospectively on children admitted to a large pediatric intensive care unit. The incidence of skin breakdown was calculated. The risk for skin breakdown associated with potential risk factors (relative risk) and 95% confidence intervals were determined. RESULTS: The sample consisted of 401 distinct stays in the intensive care unit for 373 patients. During the 401 stays, skin breakdown occurred in 34 (8.5%), redness in 25 (6.2%), and breakdown and redness in 13 (3.2%); the overall incidence was 18%. Patients who had skin breakdown or redness were younger, had longer stays, and were more likely to have respiratory illnesses and require mechanical ventilatory support than those who did not. Patients who had skin breakdown or redness had a higher risk of mortality than those who did not. CONCLUSIONS: Risk factors for skin breakdown were similar to those previously reported. Compared with children of other ages, children 2 years or younger are at higher risk for skin breakdown.

Infusion nursing: aspects of practice liability.

As the healthcare arena continues to evolve with the advent of novel clinical practices and technologies, the practice of infusion nursing is no exception to this phenomenon. Given this situation, it is imperative that nurses practicing infusion therapy be aware of the Infusion Nursing Standards of Practice to optimize patient care and safety and avoid potential litigious situations. Aspects of practice liability specific to the delivery of nursing care, product liability concepts, and a case study including the role of the nurse expert witness provide a basis for ascertaining the importance of adherence to these standards.

CD4+CD25+ T regulatory cells induced by LPS-activated bone marrow dendritic cells suppress experimental autoimmune uveoretinitis in vivo.

BACKGROUND: Tolerance-inducing DC are considered to be less mature than immunogenic DC, but the conditions promoting a less mature DC phenotype are not clear. We have previously shown that lipopolysaccharide (LPS) can have differential effects on DC function depending on the timing of DC exposure to LPS. Here, we show that early LPS-activated bone marrow derived DC (early DC, eDC), when administered subcutaneously to mice in vivo, promote tolerance to EAU induced via immunisation with interphotoreceptor retinol binding protein (IRBP) peptide 161-180. The effect correlates with the failure of eDC to secrete IL-12, and appears to be mediated in part via expansion of naturally occurring CD4+ CD25+ T regulatory cells (Tregs), which also mediate suppression of EAU on adoptive transfer to naive mice followed by immunization with autoantigen. METHODS: Immature DC were prepared from BMDC cultures. Early DC (eDC) and late DC (lDC) for tolerance experiments were obtained by differential timing of LPS addition and their cytokine secretion profile was analyzed. eDC and lDC were subcutaneously injected into mice. From the dLN CD4+ CD25+ GITR+ T regulatory cells found to express FoxP3 were isolated and transferred into mice prior to immunisation with IRBP. The immune response was scored by histopathology. Tregs were characterized in vitro by intracellular staining, cytokine secretion assay and transwell experiments. RESULTS: eDC secrete IL-10 but no IL-12 or IFNgamma. When injected subcutaneously into naive mice, they expand the population of CD4+ CD25(+high) GITR+ T cells expressing FoxP3 in the dLN, thus increasing the total number of IL-10 producing cells. eDC induced Tregs inhibit CD4+ CD25- T effector cell proliferation by a contact dependent process, and both eDC and Tregs suppress retinal damage when adoptively transferred. CONCLUSIONS: We suggest that DC maturation may be necessary for both tolerance and immunity, but differential levels of activation and/or cytokine production direct the outcome of DC-T cell interaction and this is determined by IL-12 production. T regulatory cells induced in vivo by contact with eDC are able to suppress disease in the EAU model by adoptive transfer.

Interferon-alpha: a key factor in autoimmune disease?

PURPOSE: Interferon (IFN)-alpha is an effective drug for treatment of uveitis in Behçet's disease. This study was undertaken to investigate the mechanism of action of IFN-alpha in the treatment of various types of noninfectious sight-threatening uveitis. METHODS: Eleven patients with refractory uveitis, and 13 healthy individuals were enrolled. The number of circulating plasmacytoid dendritic cells (pDCs) and their capacity to produce IFN-alpha in culture on stimulation with synthetic oligodinucleotides containing the CpG-motif were studied. Peripheral blood CD4+ T-cell phenotype and activation status were evaluated by flow cytometry at 0, 2, and 8 weeks after treatment for expression of CD69, CD62L, chemokine receptors (CCR4, CXCR3, and CCR5), and intracellular cytokines (TNF-alpha, IFN-gamma, and IL-10). RESULTS: All patients experienced a positive clinical response to IFN-alpha treatment. There was no significant difference between patients and control subjects in the number of circulating pDCs, but there was a significant decrease in the capability of patients' pDCs to produce IFN-alpha in response to CpG (P < 0.001). Peripheral blood CD4+ T cells expressed reduced levels of surface CD62L (P < 0.005) as a measure of activation and higher levels of chemokine receptors CXCR3, CCR4, and CCR5 (P < 0.005, P < 0.05, and P < 0.05, respectively); in addition, intracellular T-cell IL-10 levels were increased once the treatment was initiated (P < 0.01). CONCLUSIONS: The data suggest that IFN-alpha may control uveitis by promoting induction of IL-10-producing T-cells, possibly T-regulatory cells. Dysregulation of the T-cell population in patients with uveitis may be associated with a defect in the pDCs' ability to produce IFN-alpha, which can be circumvented with administration of exogenous IFN-alpha.

Cyclosporine vs tacrolimus therapy for posterior and intermediate uveitis.

OBJECTIVES: To compare the efficacy and tolerability of tacrolimus and cyclosporine therapy for noninfectious posterior segment intraocular inflammation and to evaluate their effect on peripheral blood CD4(+) T-cell phenotype and activation status. METHODS: Thirty-seven patients who required second-line immunosuppression for posterior segment intraocular inflammation were enrolled in this prospective randomized trial of tacrolimus vs cyclosporine therapy. The main outcome measures were visual acuity, binocular indirect ophthalmoscopy score, adverse effects, and quality of life. In addition, peripheral blood CD4(+) T-cell phenotype and activation status were evaluated by flow cytometry before treatment and at 2, 4, and 12 weeks using CD69, chemokine receptor (CCR4, CCR5, and CXCR3), and intracellular cytokine (tumor necrosis factor alpha, interferon-gamma, and interleukin 10) expression. RESULTS: Thirteen patients (68%) taking tacrolimus and 12 patients (67%) taking cyclosporine responded to treatment. Cyclosporine therapy was associated with a higher incidence of reported adverse effects. Mean arterial pressure and serum cholesterol level were significantly higher at 3 months in the cyclosporine group than the tacrolimus group. No significant difference was detected with regard to effect on quality of life or CD4(+) T-cell phenotype. CONCLUSIONS: Tacrolimus and cyclosporine were similar with regard to efficacy for posterior segment intraocular inflammation, but the results suggested a more favorable safety profile for tacrolimus therapy.

Generation of a monoclonal human single chain antibody fragment to hepatic stellate cells--a potential mechanism for targeting liver anti-fibrotic therapeutics.

BACKGROUND/AIMS: Hepatic stellate cells are pivotal to fibrogenesis in the liver and many potential anti-fibrotic therapeutics are required to act on targets within hepatic stellate cells. The aim of this study was to generate a human antibody fragment to hepatic stellate cells. METHODS: Phage display was used to generate a human monoclonal antibody fragment to a peptide sequence present on an extracellular domain of synaptophysin, a protein expressed on the surface of hepatic stellate cells. RESULTS: An antibody fragment was isolated (termed C1-3), expressed in bacteria and purified. Fluorescently-labelled C1-3 antibody associated with human hepatic stellate cells but not hepatocytes in culture. Binding of fluorescently labelled C1-3 to hepatic stellate cells was blocked by the extracellular synaptophysin peptide sequence and uptake of the antibody intracellularly was inhibited by monensin. The toxin tributyl tin-when conjugated to C1-3-retained the ability to kill hepatic stellate cells confirming that C1-3 is sequestered intracellularly. CONCLUSIONS: This antibody fragment may be an effective means to target therapeutics to human hepatic stellate cells.

Neutralizing tumor necrosis factor activity leads to remission in patients with refractory noninfectious posterior uveitis.

OBJECTIVE: To evaluate the efficacy and safety of tumor necrosis factor (TNF) inhibition with the p55 TNF receptor fusion protein (TNFr-Ig) for severe sight-threatening noninfectious posterior segment intraocular inflammation. METHODS: Seventeen patients with refractory noninfectious posterior segment intraocular inflammation received TNFr-Ig by intravenous infusion in this nonrandomized, open-label, pilot study. The primary outcome measure was logMAR visual acuity. Secondary outcome measures were binocular indirect ophthalmoscopy score, cystoid macular edema, adverse effects, and vision-related (visual core module 1) and health-related (36-Item Short-Form Health Survey) quality of life. RESULTS: Within 1 month of TNFr-Ig therapy, 9 patients (53%) achieved at least a 2-line improvement in visual acuity, 8 (57%) of 14 patients with vitreous haze before treatment achieved an improvement in binocular indirect ophthalmoscopy score to 0, and macular edema resolved in 5 (56%) of 9 affected patients. Twelve (71%) of the patients achieved complete cessation of intraocular inflammation following TNFr-Ig therapy. A reduction in concomitant immunosuppression was possible for 11 patients (65%) following TNFr-Ig therapy. However, all but 1 patient required continuing adjuvant therapy during the response to TNFr-Ig, which had a median duration of 3 months. Adverse effects included mild infusion reactions in 3 patients and transient lymphocytopenia in 2 patients. CONCLUSION: Therapy with TNFr-Ig was safe and effective for treating patients with sight-threatening noninfectious posterior segment intraocular inflammation resistant to conventional immunotherapy, but adjuvant immunosuppression and repeat infusions would be required to maintain long-term remission.

Anti-TNFalpha therapy modulates the phenotype of peripheral blood CD4+ T cells in patients with posterior segment intraocular inflammation.

PURPOSE: Posterior segment intraocular inflammation (PSII) is a putative Th1 CD4+ cell mediated autoimmune disorder. In experimental autoimmune uveoretinitis, neutralization of tumor necrosis factor (TNF)-alpha induces suppression of Th1 cells, macrophage activation, and target organ damage. Previous studies implicated an efficacy of anti-TNFalpha therapies in patients with PSII. This study investigated the immunomodulatory effect of anti-TNFalpha therapy to find predictors of effective immunosuppression in PSII. METHODS: Fifteen patients with PSII refractory to conventional immunosuppressive therapy received a single infusion of a recombinant protein generated by fusing the p55 TNFalpha receptor with human IgG1. During 17 treatment periods, visual acuity (logarithm of the minimum angle of resolution [logMAR]) was monitored as a response criterion. Phenotype markers of CD4+ T cells were analyzed before and 2, 4, and 12 weeks after therapy. Expression of intracellular cytokines (interferon [IFN]-gamma, interleukin [IL]-10, and TNFalpha) and Th1/Th2-specific chemokine receptors (CXCR3, CCR4, and CCR5) on peripheral blood CD4+ T cells was determined using flow cytometry. RESULTS: The fraction of IL-10-expressing CD4+ T cells was increased during 12 of 17 treatment periods within 2 weeks after treatment. During eight treatment periods, this increase was associated with an improvement of visual acuity of at least 0.2 logMAR within 4 weeks (P = 0.029). The ratio between IL-10- and IFNgamma-expressing CD4+ T cells was significantly increased 2 weeks after therapy (P = 0.015). There was no significant change of CXCR3, CCR4, or CCR5 expression. CONCLUSIONS: Neutralizing TNFalpha activity in PSII increases the fraction of peripheral blood CD4+ T cells expressing IL-10, which correlates with a recovery of visual function.