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Biomedical subjects

C Hough

Publications and source records attributed to C Hough.

At least 19 recordsLinked to original sources
PubMed2006

Induction of partial immune tolerance to factor VIII through prior mucosal exposure to the factor VIII C2 domain.

BACKGROUND: The development of anti-factor VIII (FVIII) neutralizing antibodies (inhibitors) is a significant obstacle to FVIII replacement therapy. OBJECTIVE: As mucosal administration of an antigen may induce immune tolerance we have evaluated the efficacy of mucosal antigen exposure to achieve tolerance to FVIII. METHODS: We investigated the effects of oral and nasal administration of the purified FVIII C2 domain (FVIII-C2) to FVIII-deficient BALB/c mice prior to FVIII protein challenge. Mice received oral or nasal doses of FVIII-C2, followed by a subcutaneous challenge of either FVIII-C2 or FVIII. The development of anti-FVIII inhibitors, cytokine production by splenocytes in vitro, and adoptive transfer assays were analyzed. RESULTS AND CONCLUSIONS: Mucosal administration of FVIII-C2 decreases the titer of anti-FVIII-C2 inhibitors after FVIII-C2 challenge, and decreases the percentage of FVIII-C2 specific antibodies after challenge with full-length FVIII. Tolerance induction to FVIII-C2 is associated with increased IL-10 production by splenocytes in vitro, and can be adoptively transferred to naïve mice. This study is the first to demonstrate that tolerance to the FVIII-C2 domain can be induced via the mucosal route. Based on these results, the potential use of FVIII-specific mucosal tolerance induction as an immunotherapy treatment for anti-FVIII inhibitor development warrants further investigation.

Administration, Intranasal↗
PubMed2005

Protracted lithium treatment protects against the ER stress elicited by thapsigargin in rat PC12 cells: roles of intracellular calcium, GRP78 and Bcl-2.

We investigated the cytoprotective effects of lithium, the mood-stabilizer, on thapsigargin-induced stress on the endoplasmic reticulum (ER) in rat PC12 cells. Protracted lithium pretreatment of PC12 cells elicited cytoprotection against thapsigargin-induced cytotoxicity. Lithium protection was concurrent with inhibition of thapsigargin-induced intracellular calcium increase and with elevated expression of the molecular chaperone GRP78. Moreover, lithium pretreatment upregulated the antiapoptotic protein Bcl-2, and blocked Bcl-2 downregulation elicited by thapsigargin. Prior to the induction of GRP78, lithium treatment alone increased the expression of c-Fos whose induction by ER stress is necessary for GRP78 induction. Curcumin, an inhibitor of transcription factor AP-1, blocked lithium cytoprotection against thapsigargin cytotoxicity. Thus, the induction of GRP78 and Bcl-2, and activation of AP-1 likely contribute to lithium-induced protection against cytotoxicity resulting from ER stress. Additionally, thapsigargin-induced cytotoxicity was suppressed by pretreatment with another mood-stabilizer, valproate, indicating that cytoprotection against ER stress is a common action of mood-stabilizing drugs.

Animals↗
PubMed2004

The benefits of radical prostatectomy beyond cancer control in symptomatic men with prostate cancer.

OBJECTIVE: To evaluate lower urinary tract symptoms (LUTS) and the symptom-associated quality of life (QoL) after radical prostatectomy. PATIENTS AND METHODS: We carried out a prospective study (January 1999 to March 2001) of 50 consecutive men initially assessed in the nurse-led prostate assessment clinic for their LUTS and subsequently diagnosed to have localized prostate cancer. They had a radical retropubic prostatectomy and were assessed before, 3 and 6 months after surgery with a self-administered International Prostate Symptom Score (IPSS) and QoL questionnaires, and measurements of urinary flow rate and postvoid residual urinary (PVR) volumes. Finally, each patient completed a self-administered continence questionnaire at 6 weeks, 3 and 6 months after surgery. RESULTS: In men with moderate to severe LUTS before surgery there were significant improvements in total IPSS, symptom-associated QoL, mean peak flow rate and PVR (P < 0.05). However, in eight men with mild LUTS, although there was a 33% improvement in the total symptom score, it was not statistically significant. Thirty-eight patients reported complete continence, nine had infrequent stress leaks only on heavy physical activity, and the remaining three used < 2 pads/day for protection before heavy physical activity at 3 months after surgery. CONCLUSIONS: Radical prostatectomy provides major benefits to men with LUTS besides cancer control. These data are important in counselling patients about the treatment options, especially in view of the current enthusiasm for brachytherapy and conformal radiotherapy, which may worsen LUTS.

Aged↗
PubMed2004

Factors influencing therapeutic efficacy and the host immune response to helper-dependent adenoviral gene therapy in hemophilia A mice.

BACKGROUND: Adenoviral-based methods of gene therapy have been ineffective at providing sustained factor (F)VIII expression in outbred populations of large animal hemophilic models primarily due to the immunogenicity of these vectors. Improvements have been made in vector design leading to the development of the helper-dependent adenoviral (HD) system. Unfortunately, it remains unclear whether these modifications are sufficient to circumvent the induction of inhibitor formation associated with adenoviral gene transfer. OBJECTIVE: To develop an HD vector capable of mediating sustained FVIII expression and to determine the variables that influence inhibitor development. METHODS: HD vectors were constructed encoding the canine FVIII B-domain deleted transgene under the control of either the cytomegalovirus (CMV) promoter or a tissue-restricted hybrid element consisting of five HNF-1 binding sites, located upstream of the human FVIII proximal promoter. Inbred and outbred populations of hemophilic mice were treated, and monitored for vector-induced toxicity, therapeutic efficacy, and inhibitor formation. RESULTS: When HD vectors utilizing the CMV promoter were administered, all hemophilic mice developed high levels of FVIII inhibitors. In contrast, vectors under the control of the HNF/FVIII element were capable of achieving sustained elevations of FVIII for over 6 months. Strain-specific differences were also observed, with outbred animals showing a greater propensity towards inhibitor development in response to treatment. CONCLUSIONS: HD vectors can be used to provide long-term FVIII expression in hemophilic animals, but treatment outcome and the induction of inhibitors is dependent on a number of variables including the transgene promoter, the vector dose, and the genetic background of the host.

Adenoviridae↗
PubMed2004

A novel type 2A (Group II) von Willebrand disease mutation (L1503Q) associated with loss of the highest molecular weight von Willebrand factor multimers.

Type 2A von Willebrand disease (VWD) is characterized by decreased platelet-dependent function of von Willebrand factor (VWF) associated with an absence of high-molecular-weight multimers. In this study, sequence analysis of the VWF gene from a Type 2A VWD patient showed a novel, heterozygous T-->A transversion at nucleotide 4510, resulting in the non-conservative substitution of L1503Q in the mature VWF subunit. This substitution, which was not found in 55 unrelated normal individuals, was reproduced by in vitro site directed mutagenesis of a full-length VWF cDNA and was subsequently expressed in COS-7 cells. The corresponding recombinant mutant VWF protein was partially retained in COS-7 cells yet the full spectrum of multimers was observed, suggesting that the absence of the highest molecular weight multimers results from increased proteolysis. The recombinant mutant VWF protein was digested with the ADAMTS13 protease from VWF-depleted plasma and the aberrant VWF multimer pattern was observed. These results suggest that the L1503Q substitution induces a conformational change in the VWF protein, which increases the protein's susceptibility to proteolysis. A three-dimensional model of the A2 domain demonstrates that the L1503Q mutation and the physiological proteolytic cleavage site for ADAMTS13 (Y(1605)-M(1606)) are localized close together in two adjacent parallel beta-sheets. The mutation L1503Q does not significantly disrupt the conformation of the protein; thus the subtle loss of multimers in this patient may be due to altered interactions with the ADAMTS13 protease.

ADAM Proteins↗
PubMed2001

Systemic delivery of an adenoviral vector encoding canine factor VIII results in short-term phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs.

Canine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIII-deficient dogs were studied to evaluate an in vivo gene therapy approach using an E1/E2a/E3-deficient adenoviral vector encoding canine FVIII. Results demonstrated a high level of expression of the canine protein and complete phenotypic correction of the coagulation defect in all 4 treated animals. However, FVIII expression was short-term, lasting 5 to 10 days following vector infusion. All 4 dogs displayed a biphasic liver toxicity, a transient drop in platelets, and development of anticanine FVIII antibody. Canine FVIII inhibitor development was transient in 2 of the 4 treated animals. These data demonstrate that systemic delivery of attenuated adenoviral vectors resulted in liver toxicity and hematologic changes. Therefore, the development of further attenuated adenoviral vectors encoding canine FVIII will be required to improve vector safety and reduce the risk of immunologic sequelae, and may allow achievement of sustained phenotypic correction of canine hemophilia A.

Adenoviridae↗
PubMed2000

Ligand-induced dynamic membrane changes and cell deletion conferred by vanilloid receptor 1.

The real time dynamics of vanilloid-induced cytotoxicity and the specific deletion of nociceptive neurons expressing the wild-type vanilloid receptor (VR1) were investigated. VR1 was C-terminally tagged with either the 27-kDa enhanced green fluorescent protein (eGFP) or a 12-amino acid epsilon-epitope. Upon exposure to resiniferatoxin, VR1eGFP- or VR1epsilon-expressing cells exhibited pharmacological responses similar to those of cells expressing the untagged VR1. Within seconds of vanilloid exposure, the intracellular free calcium ([Ca(2+)](i)) was elevated in cells expressing VR1. A functional pool of VR1 also was localized to the endoplasmic reticulum that, in the absence of extracellular calcium, also was capable of releasing calcium upon agonist treatment. Confocal imaging disclosed that resiniferatoxin treatment induced vesiculation of the mitochondria and the endoplasmic reticulum ( approximately 1 min), nuclear membrane disruption (5-10 min), and cell lysis (1-2 h). Nociceptive primary sensory neurons endogenously express VR1, and resiniferatoxin treatment induced a sudden increase in [Ca(2+)](i) and mitochondrial disruption which was cell-selective, as glia and non-VR1-expressing neurons were unaffected. Early hallmarks of cytotoxicity were followed by specific deletion of VR1-expressing cells. These data demonstrate that vanilloids disrupt vital organelles within the cell body and, if administered to sensory ganglia, may be employed to rapidly and selectively delete nociceptive neurons.

Animals↗
PubMed2000

Rubella virus capsid protein induces apoptosis in transfected RK13 cells.

Rubella virus is an enveloped positive-strand RNA virus that can cause mild to severe birth defects or death in an infected fetus. RV induction of programmed cell death, demonstrated in cell culture, has been implicated in the pathogenesis. The timing of apoptosis, 48 h p.i., suggested that accumulation of RV structural proteins might induce cell death in infected cells. Expression of RV structural proteins, capsid, envelope glycoproteins E1 and E2, in transiently transfected RK13 cells was as potent an inducer of cell death as RV infection. Immunofluorescence microscopy revealed that RV structural protein transfected cells exhibited the condensed nuclei typical of apoptotic cell death. Transfection with the capsid protein construct, but not E2 and E1, resulted in as much cell death as joint expression of all three RV structural proteins. Capsid required a membrane-anchoring domain to induce cell death, but a heterologous polypeptide fused to the capsid membrane anchor did not cause apoptosis. Deletion mutants demonstrated that the apoptosis-inducing activity resides in the N-terminal 170 amino acids of capsid. Though apoptosis-inducing capsid constructs appear to have an ER sub-cellular localization, disruption of the ER calcium storage capacity does not correlate with cell death. Mechanisms consistent with these results are discussed.

Animals↗
PubMed1999

In vivo evaluation of an adenoviral vector encoding canine factor VIII: high-level, sustained expression in hemophiliac mice.

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.

Adenoviridae↗
PubMed1999

Elevated basal and thapsigargin-stimulated intracellular calcium of platelets and lymphocytes from bipolar affective disorder patients measured by a fluorometric microassay.

BACKGROUND: A number of investigators have reported finding elevated basal and stimulated intracellular calcium levels in the platelets or lymphocytes of bipolar disorder patients. METHODS: Intracellular calcium was measured by a micro fura-2 fluorometric method in the platelets and lymphocytes of 30 affective disorder patients and 14 control subjects. RESULTS: We observed significantly elevated basal calcium concentrations in bipolar patient platelets and lymphocytes compared to control subjects. Bipolar patient platelet calcium responses to thrombin, serotonin, and thapsigargin were also significantly greater than control subjects. The peak calcium levels of lymphocytes of bipolar patients were greater than control subjects only when stimulated by thapsigargin. There were significant differences between bipolar and unipolar patients in basal and thapsigargin-stimulated calcium measures but not between bipolar I and bipolar II patients. Unmedicated versus medicated calcium measures were not significantly different. We also found little correlation between calcium measures and the severity of mood rating. CONCLUSIONS: Using this method, we were able to confirm and extend the work of others, indicating altered intracellular calcium homeostasis in the blood cells of bipolar disorder patients. In addition, our data suggest that storage operated calcium channels may be the source of the elevated intracellular calcium in platelets and lymphocytes of bipolar patients.

Adult↗
PubMed1998

3'-Azido-3'-deoxythymidine (AZT) mediates cross-resistance to nucleoside analogs in the case of AZT-resistant human immunodeficiency virus type 1 variants.

Difficulties in deciphering the mechanisms of 3'-azido-3'-deoxythymidine (AZT)-resistance by human immunodeficiency virus type 1 (HIV-1) variants are due in part to an inability to reconstitute resistance in vitro using AZT-resistant reverse transcriptases. We decided to characterize mechanisms of AZT resistance in tissue culture infections by studying the ability of drug-resistant viruses to synthesize viral DNA in the presence or absence of drug. Through use of PCR amplifications, we discovered an AZT-mediated stimulation of reverse transcription by AZT-resistant viruses carrying the M41L and T215Y mutations that can apparently override the inhibitory effects of AZT-5'-triphosphate. In addition, the presence of AZT also causes viruses containing the M41L and T215Y substitutions to have diminished sensitivity to other nucleoside analogs (i.e., ddC, ddI, and d4T). This AZT-mediated cross-resistance may help to explain the virological failure of treatment regimens that included ddI plus AZT or ddC plus AZT in situations in which the T215Y and/or M41L mutations were present (F. Brun-Vézinet, C. Boucher, C. Loveday, D. Descamps, V. Fauveau, J. Izopet, D. Jeffries, S. Kaye, C. Krzyanowski, A. Nunn, R. Schuurman, J. M. Seigneurin, C. Tamalet, R. Tedder, J. Weber, and G. J. Weverling, Lancet 350:983-990, 1997). Our results suggest that the use of AZT may be contraindicated in those patients for whom resistance to this compound (M41L and/or T215Y) has been demonstrated.

Anti-HIV Agents↗
PubMed1998

The canine factor VIII cDNA and 5' flanking sequence.

Factor VIII is a trace plasma glycoprotein involved as a cofactor in the activation of factor X by factor IXa. Inherited deficiency of factor VIII results in the X-linked bleeding disorder hemophilia A which has been documented in humans, horses, sheep and dogs. In this report, the putative proximal promoter, 5' untranslated region, complete coding sequence and 3' untranslated region of the canine factor VIII gene have been characterized. When compared to the human gene, the 5' flanking region shows conservation of transcription factor binding sites in the 5' untranslated region. Alignment of the amino acid sequence with that of the previously reported human, mouse and pig proteins demonstrates sequence identity of between 77 and 92% for the A1, A2, A3, C1 and C2 domains but an identity of only between 44 and 62% for the central B domain. The three thrombin cleavage sites are conserved in the canine sequence as are the protein C cleavage sites and the von Willebrand factor binding region. In addition, all six tyrosine residues that are known to undergo sulfation in the human protein are conserved in the dog. The 3' untranslated region of the canine gene extends 1.5 kilobases. The initial 700 basepairs of this sequence are highly GC rich and the sequence terminates with 2 alternative potential polyadenylation sites. The knowledge of this sequence, in combination with a well described canine model of hemophilia A, provides the necessary starting point for studies addressing the long-term evaluation of factor VIII gene therapy using a homologous transgene.

Amino Acid Sequence↗
PubMed1997

The K channel blocker, tetraethylammonium, displaces beta-endorphin and naloxone from T-cell binding sites.

Beta-endorphin and naloxone bind to Jurkat cell membrane preparations and can mutually displace each other from membrane binding sites. Tetraethylammonium ion, a potassium channel blocker, competitively displaces beta-endorphin and naloxone from membrane binding sites. Mitogen stimulated calcium ion flux is inhibited by tetraethyl ammonium and this inhibition is relieved by naloxone. With data derived from whole cell calcium ion flux studies, we accurately calculated the competitive displacement of beta-endorphin and naloxone from membrane preparations by tetraethylammonium thus showing that the action of these agents on potassium channels does not require second messengers. Using the resuspension induced ion flux technique, we find that beta-endorphin competes against naloxone for binding to Jurkat cells and naloxone competes against charybdotoxin, a potassium channel inhibitor, which like tetraethylammonium, is known to bind to the outer vestibule of the channel. Patch clamp electrophysiological studies show that beta-endorphin and naloxone exert complex actions on potassium channels in the presence or absence of mitogens. We conclude that one molecule of beta-endorphin or naloxone, but not both at the same time, bind to an area near the charybdotoxin/tetraethylammonium binding locus of Jurkat potassium channels.

Binding Sites↗
PubMed1997

Effects of chronic ethanol on the mobilization of arachidonate and docosahexaenoate stimulated by the type 2A serotonin receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in C6 glioma cells.

We studied the effects of chronic ethanol exposure on the mobilization of polyunsaturated fatty acids stimulated by activation of the type 2A serotonin receptor in C6 glioma cells. In our in vitro model, we prelabeled cells with [3H]arachidonate and [14C]docosahexaenonate and subsequently stimulated with the type 2A serotonin receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride. In as early as 10 days of exposure to 20 or 50 mM ethanol, the (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride-simulated mobilization of [3H]arachidonic acid [[3H]AA) and [14C]docosahexaenoic acid ([14C]DHA) was significantly inhibited, and this inhibition was accompanied by decreased mobilization of intracellular [Ca2+]i. Exposure to ethanol did not alter significantly the release of [3H]AA and [14C]DHA stimulated by the calcium ionophore A23187 nor the incorporation of [3H]AA and [14C]DHA into cellular lipids. Decreased mobilization of polyunsaturated fatty acids and calcium in astroglia may contribute to neurotoxicity caused by chronic ethanol exposure.

Amphetamines↗
PubMed1996

Regulation of synapse structure and function by the Drosophila tumor suppressor gene dlg.

Mutations of the tumor suppressor gene discs-large (dlg) lead to postsynaptic structural defects. Here, we report that mutations in dlg also result in larger synaptic currents at fly neuromuscular junctions. By selectively targeting DLG protein to either muscles or motorneurons using Gal-4 enhancer trap lines, we were able to rescue substantially the reduced postsynaptic structure in mutants. Rescue of the physiological defect was accomplished by presynaptic, but not postsynaptic targeting, consistent with our finding that miniature excitatory junctional currents were not changed in dlg mutants. These results suggest that DLG functions in the regulation of neurotransmitter release and postsynaptic structure. We propose that DLG is an integral part of a mechanism by which changes in both neurotransmitter release and synapse structure are accomplished during development and plasticity.

Animals↗
PubMed1996

Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia.

The Discs large (Dlg) protein of Drosophila is the prototypic member of a growing family of proteins termed membrane-associated guanylate kinase homologs (MAGUKs). The MAGUKs are composed of a series of peptide domains that include one or three DHR/PDZs, an SH3, and a region homologous to guanylate kinase (GUK). We have previously shown that the product of this gene, the Dlg protein, is localized at the septate junctions between epithelial cells, and that mutations in the gene cause neoplastic overgrowth of the imaginal discs. The dlg locus is therefore defined as a tumor suppressor gene. In this paper, we show that the Dlg protein is localized on the cytoplasmic face of the septate junction and is required for the maintenance of this structure. It is also required for proper organization of the cytoskeleton, for the differential localization of membrane proteins, and for apicobasal polarity of epithelial cells. However, these other functions can be uncoupled from Dlg's role as a tumor suppressor since mutations in two domains of the protein, the SH3 and GUK, cause loss of normal cell proliferation control without affecting the other functions of the protein. These results suggest that, besides regulating cellular proliferation, the Dlg protein is a critical component of the septate junctions and is required for maintaining apicobasal polarity in Drosophila epithelium.

Alleles↗
PubMed1996

Modular organization of the PDZ domains in the human discs-large protein suggests a mechanism for coupling PDZ domain-binding proteins to ATP and the membrane cytoskeleton.

The human homologue (hDIg) of the Drosophila discs-large tumor suppressor (DIg) is a multidomain protein consisting of a carboxyl-terminal guanylate kinase-like domain, an SH3 domain, and three slightly divergent copies of the PDZ (DHR/GLGF) domain. Here have examined the structural organization of the three PDZ domains of hDIg using a combination of protease digestion and in vitro binding measurements. Our results show that the PDZ domains are organized into two conformationally stable modules one (PDZ, consisting of PDZ domains 1 and 2, and the other (PDZ) corresponding to the third PDZ domain. Using amino acid sequencing and mass spectrometry, we determined the boundaries of the PDZ domains after digestion with endoproteinase Asp-N, trypsin, and alpha-chymotrypsin. The purified PDZ1+2, but not the PDZ3 domain, contains a high affinity binding site for the cytoplasmic domain of Shaker-type K+ channels. Similarly, we demonstrate that the PDZ1+2 domain can also specifically bind to ATP. Furthermore, we provide evidence for an in vivo interaction between hDIg and protein 4.1 and show that the hDIg protein contains a single high affinity protein 4.1-binding site that is not located within the PDZ domains. The results suggest a mechanism by which PDZ domain-binding proteins may be coupled to ATP and the membrane cytoskeleton via hDlg.

Adenosine Triphosphate↗