BACKGROUND: Gamma-aminobutyric acid type A receptors (GABAA-Rs) are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including ...benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice. RESULTS: Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10-95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line. CONCLUSION: We conclude that: 1) insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably reduced the amount of γ2, and that 2) attenuated expression of γ2 increased anxiety-like behaviors but did not lead to differences in the hypnotic response to benzodiazepine site ligands. This suggests that reduced synaptic inhibition can lead to a phenotype of increased anxiety-like behavior. In contrast, normal drug effects can be maintained despite a dramatic reduction in GABAA-R targets.
The efficacy of antithymocyte induction therapy in lung transplantation is controversial, and its use varies from center to center. We hypothesized that rabbit antithymocyte globulin (RATG) induction ...therapy would decrease acute rejection after lung transplantation, and we designed a single-center, randomized, prospective study to test this hypothesis.
A total of 44 single or bilateral adult lung transplant recipients were randomly assigned to receive either RATG induction therapy (dosage, 1.5 mg/kg/d for 3 days) at the time of transplantation, along with conventional immunosuppression (cyclosporine, azathioprine, and prednisone), or conventional immunosuppression alone with no induction therapy.
Although a similar number of biopsies were performed in each group, the number of patients experiencing biopsy-proven grade II or greater acute rejection was significantly reduced in the group receiving RATG induction therapy (23% incidence), as compared to the patients treated with conventional immunosuppression alone (55% incidence; p = 0.03). In addition, there was a nonsignificant reduction in the incidence of bronchiolitis obliterans syndrome at the conclusion of the study in patients who received RATG induction (20%), as compared to patients in the control group (38%). The incidence of posttransplant infections and malignancies were similar between the two groups.
Induction therapy with RATG significantly reduces the incidence of acute allograft rejection after lung transplantation.
We studied the cellular and subcellular distribution of GABA(A) receptors in the Bergmann glia and Purkinje cells in the molecular layer of the cerebellum by using electron microscopy postembedding ...immunogold techniques. Gold particles corresponding to alpha2 and gamma1 immunoreactivity were localized in Bergmann glia processes that wrapped Purkinje cell somata, dendritic shafts, and some dendritic spines. The gold particles were mainly located on the glial plasma membrane or intracellularly but near the plasma membrane. The density of gold particles corresponding to alpha2 and gamma1 GABA(A) receptor subunits was 4.3-fold higher in the glial processes wrapping Purkinje cell somata than in the glial processes wrapping Purkinje cell dendritic spines. Moreover, the Bergmann glia GABA(A) receptors were often located in close proximity to the type II GABAergic synapses made by the basket cell axons on Purkinje cell somata. These GABAergic synapses were enriched in neuronal GABA(A) receptors containing alpha1 and beta2/3 subunits. Unexpectedly, 2.8% of the Purkinje cell dendritic spines also showed immunoreactivity for the neuronal alpha1 or beta2/3 subunits, which were located on the spine in type I synapses or extrasynaptically. Double-labeling immunogold experiments showed that approximately 50% of the dendritic spines that were immunolabeled with the neuronal GABA(A) receptors were wrapped by Bergmann glia processes containing glial GABA(A) receptors. These results are consistent with a role of the Bergmann glial GABA(A) receptors in sensing GABAergic synaptic function.
Summary
Due to the poor rate of response to hepatitis C virus (HCV) with pegylated interferon and ribavirin treatment in HCV/HIV coinfected patients, key factors for predicting failure would be ...useful. We performed a retrospective study on 291 patients on HCV treatment, who had early virological response (EVR) data. IL28B and IL28RA polymorphisms were performed using the GoldenGate® assay. Unfavourable genotypes at IL28B (rs12980275 AG/GG and rs8099917 GT/GG) and an unfavourable allele at IL28RA (rs10903035 G) were associated with early treatment failure. However, only the rs12980275 AG/GG genotype and rs10903035 G allele remained independently associated with early failure in the overall population (OR = 4.15 (95% CI = 1.64–10.54) and OR = 2.00 (95% CI = 1.19–3.36), respectively) as well as in GT1/4 patients (OR = 5.07 (95% CI = 1.81–14.22) and OR = 2.03 (95% CI = 1.13–3.66), respectively). Next, a decision tree showed early treatment failure increased from 37.1% to 65.5% when the unfavourable rs12980275 AG/GG and rs10903035 AG/GG genotypes and HCV‐RNA≥ 500.000 IU/mL were taken into account in GT1/4 patients. In contrast, the failure rate decreased from 37.1% to 11.9% when the favourable rs12980275 AA and rs10903035 AA genotypes were detected. The percentage of patients correctly classified was 78.4%, and AUROC was 0.802 ± 0.028. Regarding GT3 patients, the presence of the GCGCA haplotype (all unfavourable alleles) was associated with early treatment failure, while no association was observed for the IL28B polymorphisms. In conclusion, the IL28RA polymorphism was associated with early treatment failure independently of the IL28B SNPs. The combination of IL28B and IL28RA polymorphisms might be a valuable tool for predicting early treatment failure before starting HCV treatment.
We have found that the large intracellular loop of the γ2 GABAA receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. ...In brain extracts, RNF34 co-immunoprecipitates with assembled GABAARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABAAR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2. Mutating several lysines of the γ2IL into arginines makes the γ2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the γ2 subunit when α1 and β3 subunits are co-assembled with γ2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of γ2 GABAAR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased γ2 GABAAR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic γ2-GABAAR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the γ2-GABAAR subunit promoting GABAAR degradation.
BackgroundOne of the objectives of the Health Department of Catalonia (Spain) is the clinical review of medications in chronic complex patients (CCP) and patients with advanced disease (PAD). The ...reconciliation and validation of treatments to detect and prevent drug related problems (DRPs) in a short stay emergency service (SSES) is among the functions of the hospital pharmacist.PurposeTo describe the pharmaceutical interventions (PI) implemented in an SSES and their degree of acceptance by the physician, and to analyse PI according to the type of patient (CCP, PAD and others).Material and methodsA retrospective, descriptive, observational study of data retrieved from January to August 2016 was conducted. Interventions performed by a pharmacist in SSES (20 beds) from Monday to Friday were recorded and communicated to the responsible prescriber. Data collected: class of patient, type of PI and acceptance of the recommendation by the prescriber.ResultsA total of 344 PI in 248 patients were registered: 14 PI in 8 PAD, 95 in 66 CCP and 235 in 174 others. The degree of acceptance of PI were: 79.9% accepted, 6.7% rejected and 13.4% not evaluated due to the discharge of the patient before resolution of the PI. The PI most frequently performed were: 36.6% detection of omission of chronic treatments (65.9% accepted, 7.9% rejected, 26.2% not evaluated), 20.1% therapeutic equivalent switching (98.6% accepted, 1.4% rejected), 16.3% safety monitoring recommendations (96.4% accepted, 3.6% rejected) and 7.3% dosage adjustment (72% accepted, 16% rejected, 12% not evaluated). The most frequent PI in PAD were 35.7% therapeutic equivalent switching and 28.6% detection of omission of chronic treatments, while for CCP the most frequent PI were 47.4% detection of omission of chronic treatments, 14.7% safety monitoring recommendations, 7.4% renal dose adjustment and 7.4% therapeutic equivalent switching. For other patients, 32.8% were detection of omission of chronic treatment, 24.3% therapeutic equivalent switching and 17.4% safety monitoring recommendations.ConclusionDetection of omission of chronic treatments was the most frequent PI recorded. The degree of acceptance of the PI by the prescriber was high. The interventions performed by the pharmacist in the SSES are key to prevent DRPs.References and/or acknowledgementsPrograma de prevenció i atenció a la cronicitat de Catalunya 2011–2014 (PPAC). Health-Department.Government of Catalonia.No conflict of interest
We have found that the γ2 subunit of the GABA(A) receptor (γ2-GABA(A)R) specifically interacts with protocadherin-γC5 (Pcdh-γC5) in the rat brain. The interaction occurs between the large ...intracellular loop of the γ2-GABA(A)R and the cytoplasmic domain of Pcdh-γC5. In brain extracts, Pcdh-γC5 coimmunoprecipitates with GABA(A)Rs. In cotransfected HEK293 cells, Pcdh-γC5 promotes the transfer of γ2-GABA(A)R to the cell surface. We have previously shown that, in cultured hippocampal neurons, endogenous Pcdh-γC5 forms clusters, some of which associate with GABAergic synapses. Overexpression of Pcdh-γC5 in hippocampal neurons increases the density of γ2-GABA(A)R clusters but has no significant effect on the number of GABAergic contacts that these neurons receive, indicating that Pcdh-γC5 is not synaptogenic. Deletion of the cytoplasmic domain of Pcdh-γC5 enhanced its surface expression but decreased the association with both γ2-GABA(A)R clusters and presynaptic GABAergic contacts. Cultured hippocampal neurons from the Pcdh-γ triple C-type isoform knock-out (TCKO) mouse (Pcdhg(tcko/tcko)) showed plenty of GABAergic synaptic contacts, although their density was reduced compared with sister cultures from wild-type and heterozygous mice. Knocking down Pcdh-γC5 expression with shRNA decreased γ2-GABA(A)R cluster density and GABAergic innervation. The results indicate that, although Pcdh-γC5 is not essential for GABAergic synapse formation or GABA(A)R clustering, (1) Pcdh-γC5 regulates the surface expression of GABA(A)Rs via cis-cytoplasmic interaction with γ2-GABA(A)R, and (2) Pcdh-γC5 plays a role in the stabilization and maintenance of some GABAergic synapses.
Summary
Hepatitis C virus (HCV) infection is associated with insulin resistance (IR), although mechanisms leading to IR in these patients are not completely understood. The aim of this study was to ...evaluate the association of interleukin 28B (IL28B) and interleukin 28 receptor alpha (IL28RA) polymorphisms with IR among human immunodeficiency virus (HIV)/HCV‐coinfected patients. We carried out a cross‐sectional study on 203 patients. IL28B (rs8099917) and IL28RA (rs10903035) polymorphisms were genotyped by GoldenGate® assay. IR was defined as homeostatic model assessment (HOMA) values ≥3.00. Univariate and multivariate generalized linear models (GLM) were used to compare HOMA values and the percentage of patients with IR according to IL28B and IL28RA genotypes. In total, 32% (n = 65/203) of the patients had IR. IL28B rs8099917 TT was not significantly associated with HOMA values and IR. In contrast, rs10903035 AA was significantly associated with high HOMA values taking into account all patients (P = 0.024), as well as the subgroups of patients with significant fibrosis (P = 0.047) and infected with HCV genotype 3 (P = 0.024). Additionally, rs10903035 AA was significantly associated with IR (HOMA ≥3.00) in all patients (adjusted odds ratio (aOR) = 2.02; P = 0.034), in patients with significant fibrosis (aOR = 2.86; P = 0.039) and HCV genotype 3 patients (aOR = 4.89; P = 0.031). In conclusions, IL28RA polymorphism (rs10903035) seems to be implicated in the glucose homeostasis because AA genotype increases the likelihood of IR, but this association was different depending on hepatic fibrosis and HCV genotype.
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