An isoform of GTPase regulator DOCK4 localizes to the stereocilia in the inner ear and binds to harmonin (USH1C).

The driving forces for the regulation of cell morphology are the Rho family GTPases that coordinate the assembly of the actin cytoskeleton. This dynamic feature is a result of tight coupling between the cytoskeleton and signal transduction and is facilitated by actin-binding proteins (ABPs). Mutations in the actin bundling and PDZ domain-containing protein harmonin are the causes of Usher syndrome type 1C (USH1C), a syndrome of congenital deafness and progressive blindness, as well as certain forms of non-syndromic deafness. Here, we have used the yeast two-hybrid assay to isolate molecular partners of harmonin and identified DOCK4, an unconventional guanine exchange factor for the Rho family of guanosine triphosphatases (Rho GEF GTPases), as a protein interacting with harmonin. Detailed molecular analysis revealed that a novel DOCK4 isoform (DOCK4-Ex49) is expressed in the brain, eye and inner ear tissues. We have further provided evidence that the DOCK4-Ex49 binds to nucleotide free Rac as effectively as DOCK2 and DOCK4 and it is a potent Rac activator. By immunostaining using a peptide antibody specific to DOCK4-Ex49, we showed its localization in the inner ear within the hair bundles along the stereocilia (SC). Together, our data indicate a possible Rac-DOCK4-ABP harmonin-activated signaling pathway in regulating actin cytoskeleton organization in stereocilia.

The NF-kappa B and Sp1 motifs of the human immunodeficiency virus type 1 long terminal repeat function as novel thyroid hormone response elements.

We report that thyroid hormone (T3) receptor (T3R) can activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Purified chick T3R-alpha 1 (cT3R-alpha 1) binds as monomers and homodimers to a region in the LTR (nucleotides -104 to -75 [-104/-75]) which contains two tandem NF-kappa B binding sites and to a region (-80/-45) which contains three Sp1 binding sites. In contrast, human retinoic acid receptor alpha (RAR-alpha) and mouse retinoid X receptor beta (RXR-beta) do not bind to these elements. However, RXR-beta binds to these elements as heterodimers with cT3R-alpha 1 and to a lesser extent with RAR-alpha. Gel mobility shift assays also revealed that purified NF-kappa B p50/65 or p50/50 can bind to one but not both NF-kappa B sites simultaneously. Although the binding sites for p50/65, p50/50, and T3R, or Sp1 and T3R, overlap, their binding is mutually exclusive, and with the inclusion of RXR-beta, the major complex is the RXR-beta-cT3R-alpha 1 heterodimer. The NF-kappa B region of the LTR and the NF-kappa B elements from the kappa light chain enhancer both function as T3 response elements (TREs) when linked to a heterologous promoter. The TREs in the HIV-1 NF-kappa B sites appear to be organized as a direct repeat with an 8- or 10-bp gap between the half-sites. Mutations within the NF-kappa B motifs which eliminate binding of cT3R-alpha 1 also abolish stimulation by T3, indicating that cT3R-alpha 1 binding to the Sp1 region does not independently mediate activation by T3. The Sp1 region, however, is converted to a functionally strong TRE by the viral tat factor. These studies indicate that the HIV-1 LTR contains both tat-dependent and tat-independent TREs and reveal the potential for T3R to modulate other genes containing NF-kappa B- and Sp1-like elements. Furthermore, they indicate the importance of other transcription factors in determining whether certain T3R DNA binding sequences can function as an active TRE.

Regulation of the mdm2 oncogene by thyroid hormone receptor.

The mdm2 gene is positively regulated by p53 through a p53-responsive DNA element in the first intron of the mdm2 gene. mdm2 binds p53, thereby abrogating the ability of p53 to activate the mdm2 gene, and thus forming an autoregulatory loop of mdm2 gene regulation. Although the mdm2 gene is thought to act as an oncogene by blocking the activity of p53, recent studies indicate that mdm2 can act independently of p53 and block the G1 cell cycle arrest mediated by members of the retinoblastoma gene family and can activate E2F1/DP1 and the cyclin A gene promoter. In addition, factors other than p53 have recently been shown to regulate the mdm2 gene. In this article, we report that thyroid hormone (T3) receptors (T3Rs), but not the closely related members of the nuclear thyroid hormone/retinoid receptor gene family (retinoic acid receptor, vitamin D receptor, peroxisome proliferation activation receptor, or retinoid X receptor), regulate mdm2 through the same intron sequences that are modulated by p53. Chicken ovalbumin upstream promoter transcription factor I, an orphan nuclear receptor which normally acts as a transcriptional repressor, also activates mdm2 through the same intron region of the mdm2 gene. Two T3R-responsive DNA elements were identified and further mapped to sequences within each of the p53 binding sites of the mdm2 intron. A 10-amino-acid sequence in the N-terminal region of T3Ralpha that is important for transactivation and interaction with TFIIB was also found to be important for activation of the mdm2 gene response element. T3 was found to stimulate the endogenous mdm2 gene in GH4C1 cells. These cells are known to express T3Rs, and T3 is known to stimulate replication of these cells via an effect in the G1 phase of the cell cycle. Our findings, which indicate that T3Rs can regulate the mdm2 gene independently of p53, provide an explanation for certain known effects of T3 and T3Rs on cell proliferation. In addition, these findings provide further evidence for p53-independent regulation of mdm2 which could lead to the development of tumors from cells that express low levels of p53 or that express p53 mutants defective in binding to and activating the mdm2 gene.

Constitutive activation of gene expression by thyroid hormone receptor results from reversal of p53-mediated repression.

Thyroid hormone receptor (T3R) is a member of the steroid hormone receptor gene family of nuclear hormone receptors. In most cells T3R activates gene expression only in the presence of its ligand, L-triiodothyronine (T3). However, in certain cell types (e.g., GH4C1 cells) expression of T3R leads to hormone-independent constitutive activation. This activation by unliganded T3R occurs with a variety of gene promoters and appears to be independent of the binding of T3R to specific thyroid hormone response elements (TREs). Previous studies indicate that this constitutive activation results from the titration of an inhibitor of transcription. Since the tumor suppresser p53 is capable of repressing a wide variety of gene promoters, we considered the possibility that the inhibitor is p53. Evidence to support this comes from studies indicating that expression of p53 blocks T3R-mediated constitutive activation in GH4C1 cells. In contrast with hormone-independent activation by T3R, p53 had little or no effect on T3-dependent stimulation which requires TREs. In addition, p53 mutants which oligomerize with wild-type p53 and interfere with its function also increase promoter activity. This enhancement is of similar magnitude to but is not additive with the stimulation mediated by unliganded T3R, suggesting that they target the same factor. Since p53 mutants are known to target wild-type p53 in the cell, this suggests that T3R also interacts with p53 in vivo and that endogenous levels of p53 act to suppress promoter activity. Evidence supporting both functional and physical interactions of T3R and p53 in the cell is presented. The DNA binding domain (DBD) of T3R is important in mediating constitutive activation, and the receptor DBD appears to functionally interact with the N terminus of p53 in the cell. In vitro binding studies indicate that the T3R DBD is important for interaction of T3R with p53 and that this interaction is reduced by T3. These findings are consistent with the in vivo studies indicating that p53 blocks constitutive activation but not ligand-dependent stimulation. These studies provide insight into mechanisms by which unliganded nuclear hormone receptors can modulate gene expression and may provide an explanation for the mechanism of action of the v-erbA oncoprotein, a retroviral homolog of chicken T3R alpha.

The ligand-binding domains of the thyroid hormone/retinoid receptor gene subfamily function in vivo to mediate heterodimerization, gene silencing, and transactivation.

The ligand-binding domains (LBDs) of the thyroid/retinoid receptor gene subfamily contain a series of heptad motifs important for dimeric interactions. This subfamily includes thyroid hormone receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), 9-cis RA receptors (RARs and retinoid X receptors [RXRs]), the 1,25-dihydroxyvitamin D3 receptor (VDR), and the receptors that modulate the peroxisomal beta-oxidation pathway (PPARs). These receptors bind to their DNA response elements in vitro as heterodimers with the RXRs. Unliganded receptors in vivo, in particular the T3Rs, can mediate gene silencing and ligand converts these receptors into a transcriptionally active form. The in vivo interactions of these receptors with RXR were studied by using a GAL4-RXR chimera containing the yeast GAL4 DNA-binding domain and the LBD of RXR beta. GAL4-RXR activates transcription from GAL4 response elements in the presence of 9-cis RA. Unliganded T3R, which does not bind or activate GAL4 elements, represses the activation of GAL4-RXR by 9-cis RA in HeLa cells. However, addition of T3 alone leads to transcriptional activation. These findings suggest that T3R can repress or activate transcription while tethered to the LBD of GAL4-RXR and that heterodimerization can occur in vivo without stabilization by hormone response elements. Similar ligand-dependent activation was observed in HeLa cells expressing RAR, VDR, or PPAR and in GH4C1 cells from endogenous receptors. Replacement of the last 17 amino acids of the LBD of RXRbeta with the 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein leads to a GAL4 constitutive activator that is repressed by wild-type T3R but not by a ninth heptad mutant that does not form heterodimers. This finding suggests that the ninth heptad or T3R is important for gene silencing and that the LBD of RXR does not exhibit silencing activity. This conclusion was verified with GAL4-LBD chimeras and with wild-type receptors in assays using appropriate response elements. These studies indicate that the LBD has diverse functional roles in gene regulation.

NRIF3 is a novel coactivator mediating functional specificity of nuclear hormone receptors.

Many nuclear receptors are capable of recognizing similar DNA elements. The molecular event(s) underlying the functional specificities of these receptors (in regulating the expression of their native target genes) is a very important issue that remains poorly understood. Here we report the cloning and analysis of a novel nuclear receptor coactivator (designated NRIF3) that exhibits a distinct receptor specificity. Fluorescence microscopy shows that NRIF3 localizes to the cell nucleus. The yeast two-hybrid and/or in vitro binding assays indicated that NRIF3 specifically interacts with the thyroid hormone receptor (TR) and retinoid X receptor (RXR) in a ligand-dependent fashion but does not bind to the retinoic acid receptor, vitamin D receptor, progesterone receptor, glucocorticoid receptor, or estrogen receptor. Functional experiments showed that NRIF3 significantly potentiates TR- and RXR-mediated transactivation in vivo but has little effect on other examined nuclear receptors. Domain and mutagenesis analyses indicated that a novel C-terminal domain in NRIF3 plays an essential role in its specific interaction with liganded TR and RXR while the N-terminal LXXLL motif plays a minor role in allowing optimum interaction. Computer modeling and subsequent experimental analysis suggested that the C-terminal domain of NRIF3 directly mediates interaction with liganded receptors through an LXXIL (a variant of the canonical LXXLL) module while the other part of the NRIF3 protein may still play a role in conferring its receptor specificity. Identification of a coactivator with such a unique receptor specificity may provide new insight into the molecular mechanism(s) of receptor-mediated transcriptional activation as well as the functional specificities of nuclear receptors.

A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor alpha is essential for transcriptional activation and interaction with the general transcription factor TFIIB.

The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription.

Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor.

The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor suppresses transactivation by the T3Rs and RARs while these receptors are bound to DNA and that ligands act, in part, by inactivating or promoting dissociation of a receptor-inhibitor complex.

The impact of co-existing immune-mediated diseases on phenotype and outcomes in inflammatory bowel diseases.

BACKGROUND: Inflammatory bowel diseases lead to progressive bowel damage and need for surgery. While the increase in prevalence of other immune-mediated diseases in IBD is well recognised, the impact of this on the natural history of IBD is unknown. AIM: To determine the impact of concomitant immune-mediated diseases on phenotypes and outcomes in IBD. METHODS: Patients with IBD enrolled in a prospective registry were queried about the presence of other immune-mediated diseases, defined as those where immune dysregulation plays a role in pathogenesis. Demographics and disease-related information were obtained. Subjects also completed measures of quality of life. Multivariable regression models compared disease phenotype and outcomes of IBD patients with and without other immune-mediated diseases. RESULTS: The cohort included 2145 IBD patients among whom 458 (21%) had another immune-mediated disease. There was no difference in CD phenotype between the two groups. UC patients were more likely to have pancolitis in the presence of another immune-mediated disease (62%) compared to those without (52%, P = 0.02). IBD patients with another immune-mediated disease had higher rates of needing anti-TNF biologics [Odds ratio (OR) 1.31, 95% CI 1.05-1.63] and surgery (OR 1.26, 95% CI 0.99-1.61). The presence of another immune-mediated disease was also associated with lower disease-specific and general physical quality of life. CONCLUSIONS: The presence of another immune-mediated disease in IBD patients was associated with higher likelihood of pancolonic involvement in UC, and a modest increase in need for IBD-related surgery and anti-TNF biological therapy. Such patients also experienced worse quality of life.

Adjuvant use of antibiotics with corticosteroids in inflammatory bowel disease exacerbations requiring hospitalisation: a retrospective cohort study and meta-analysis.

BACKGROUND: Patients hospitalised with an exacerbation of inflammatory bowel disease (IBD) often receive antibiotics in addition to intravenous steroids. However, their efficacy in this setting is unclear. AIM: To ascertain if the addition of antibiotics to intravenous steroids modifies short and long-term clinical outcomes. METHODS: Our study included IBD patients hospitalised between 2009 and 2014 who received intravenous (IV) steroids with or without adjuvant antibiotics. Outcomes of interest included length of stay (LOS), need for medical and surgical rescue therapy during the hospitalisation, and at 90 and 365 days. A meta-analysis of previously published randomised trials was additionally performed. RESULTS: A total of 354 patients were included [145 ulcerative colitis (UC); 209 Crohn's disease (CD)]. In CD, combination of IV steroids and antibiotics did not change need for in-hospital medical rescue therapy, surgery or hospitalisations at 1 year but was associated with greater LOS (6.1 vs. 4.6 days, P = 0.02). In UC, patients receiving antibiotics were less likely to require in-hospital medical rescue therapy [odds ratio (OR): 0.42, 95% confidence interval (CI): 0.19-0.93] but experienced no statistically significant differences in LOS, in-hospital surgery, re-hospitalisations or surgery by 1 year. A meta-analysis of three relevant randomised trials demonstrated no difference in clinical improvement with antibiotics over placebo (OR: 1.08, 95% CI: 0.50-2.32). CONCLUSIONS: The addition of antibiotics to intravenous steroids for treatment of IBD exacerbations was associated with a reduced need for in-hospital medical rescue therapy in ulcerative colitis without significant long-term benefit, and did not affect short- or long-term outcomes in Crohn's disease.

Genetic basis of TNF-α antagonist associated psoriasis in inflammatory bowel diseases: a genotype-phenotype analysis.

BACKGROUND: Anti-tumour necrosis factor (anti-TNF) biologic associated psoriasis has been reported in inflammatory bowel disease (IBD) patients. However, little is known regarding its pathogenesis. AIM: To identify potential genetic predispositions to anti-TNF associated psoriasis in IBD patients. METHODS: This retrospective chart review included IBD patients enrolled in a prospective registry. Cases of anti-TNF associated psoriasis and idiopathic psoriasis unrelated to anti-TNF exposure were confirmed by an expert dermatologist. All patients were genotyped on the Illumina Immunochip. A weighted genetic risk score ascertaining genetic pre-disposition towards psoriasis was calculated and overall genetic pre-disposition as well as differential distribution of individual polymorphisms was compared across the three groups. RESULTS: Our study included 724 IBD patients who initiated anti-TNF therapy and did not develop psoriasis, 35 patients with anti-TNF associated psoriasis, and 38 patients with idiopathic psoriasis. Anti-TNF users who developed psoriasis had a modest but statistically significantly greater psoriasis genetic risk score than anti-TNF controls (mean 0.64 vs. 0.61, P = 0.04), and had a similar genetic risk score as those with idiopathic psoriasis (0.64 vs. 0.62, P = 0.22). Two loci associated with NOS2 and ETS1 genes achieved P < 0.05 when comparing anti-TNF associated psoriasis to anti-TNF controls. Three loci were significantly different between anti-TNF associated psoriasis and idiopathic psoriasis including a polymorphism near NOS2 encoding for inducible nitric oxide synthase that is produced by dendritic cells in skin lesions in psoriasis. CONCLUSION: Patients with anti-TNF associated psoriasis had a modestly greater genetic pre-disposition towards psoriasis but no single causative polymorphism was identified.

The cloning of Grb10 reveals a new family of SH2 domain proteins.

SH2 domains function to bind proteins containing phosphotyrosine and are components of proteins that are important signal transducers for tyrosine kinases. We have cloned SH2 domain proteins by screening bacterial expression libraries with the tyrosine phosphorylated carboxyterminus of the epidermal growth factor (EGF) receptor. Here we report the identification of a new SH2 domain protein, Grb10. Grb10 is highly related to Grb7, an SH2 domain protein that we have previously identified. In addition to an SH2 domain, Grb7 and Grb10 have a central domain with similarity to a putative C. elegans gene likely to be involved in neuronal migration. At least three forms of Grb10 exist in fibroblasts apparently due to alternate translational start sites. Grb10 undergoes serine but not tyrosine phosphorylation after EGF treatment resulting in a shift mobility in a large fraction of Grb10 molecules. However Grb10 appears to bind poorly to EGF-Receptor and the true binding partner for the Grb10 SH2 domain is unclear. Grb10 maps to mouse chromosome 11 very close to the EGF-Receptor which is remarkably similar to Grb7 that maps near the EGF-Receptor related HER2 receptor. The finding of multiple family members with evolutionarily conserved domains indicates that these SH2 domain proteins are likely to have an important, although as of yet, unidentified function.

New onset idiosyncratic liver enzyme elevations with biological therapy in inflammatory bowel disease.

BACKGROUND: Anti-tumour necrosis factor α (anti-TNF) agents have been implicated in drug-induced liver injury. There is minimal data on this occurrence in inflammatory bowel disease (IBD) patients. AIM: To identify the characteristics of liver enzyme elevations following anti-TNF therapy initiation in IBD. METHODS: A retrospective cohort of patients initiating anti-TNF therapy were analysed for new onset alanine transaminase (ALT) elevation (≥60 U/L). We collected data on natural history, outcomes and patient characteristics compared with controls with persistent normal liver enzymes. Likelihood of causal association was assessed using the RUCAM score. RESULTS: From 1753 patients initiating an anti-TNF (1170 infliximab, 575 adalimumab, 8 certolizumab), 102 (6%) developed new onset ALT elevation. In 54 (53%), this could be linked to an alternate aetiology. Among those with idiopathic ALT elevations, the median time to ALT elevation from anti-TNF initiation was 18 weeks and median peak ALT was 96 U/L. Six underwent liver biopsy, all demonstrating hepatitis with autoimmune features. Compared to controls, cases were on a lower dose of infliximab (5.7 vs. 6.7 mg/kg, P = 0.02) but were otherwise similar in body mass index, sex and age. On follow-up, 34 continued the anti-TNF, 14 stopped therapy and 4 initiated steroids. Most (85%) normalised their LFTs after a median of 17 weeks including 28 (82%) of those who continued anti-TNF therapy. Ten patients were transitioned to a second anti-TNF without recurrence. CONCLUSIONS: ALT elevations occurred in 6% of IBD patients initiating anti-TNF therapy. Most idiopathic elevations were mild, transient and resolved despite therapy continuation.

The effect of thyroid hormone treatment on the gene expression and enzyme activity of rat liver sodium-potassium dependent adenosine triphosphatase.

The effects of thyroid hormone (T3) treatment on liver Na,K-adenosine triphosphatase (Na,K-ATPase) at the levels of subunit messenger RNA (mRNA), enzymatic activity, and enzyme content were studied in euthyroid rats injected for 5 consecutive days with T3. Northern and slot blot analyses of polyadenylated mRNA revealed that T3 treatment coordinately increases the level of mRNA encoding the alpha 1- and beta 1-subunits, approximately 4- and 3-fold, respectively, above basal levels. To determine whether this increase in the subunit mRNA consequently results in an increase in the synthesis of the enzyme, a modified liver cell fractionation procedure was developed, and the subcellular fractions from control and T3-treated livers were examined biochemically. Western blot analysis and Na,K-ATPase assay demonstrated that T3 treatment resulted in a 2-fold increase in both the amount and activity of the enzyme. Furthermore, the Western blot analysis of endoglycosidase-H-treated membrane fractions revealed an increase in the amount of the precursor beta-subunit in the T3-treated liver rough microsomal fraction, suggesting that an increase in subunit synthesis contributes at least partially to the increase in the rat liver Na,K-ATPase by T3 treatment.

Penbutolol in hypertension: a pilot study with single daily doses.

A pilot single-blind placebo controlled crossover within-patient study was undertaken in essential hypertension. In ten patients single daily doses of 25 mg and 50 mg and in two patients 25 mg, 50 mg and 100 mg were used. Satisfactory reductions in both systolic and diastolic blood pressure in the supine and erect postures were observed. Reduction in heart rate was of the order of 6-32%, there being no correlation between reductions in blood pressure and decrements in heart rate. Three patients were dropped from the final analyses. Seventy-eight per cent (7/9) of patients had a final diastolic pressure (lying) of 90 mm Hg or less. Single doses of penbutolol controlled blood pressure for at least twenty-four hours. At the end of two weeks on placebo medication, following nine weeks of active drug medication, blood pressure had reverted to near pre-treatment levels. Penbutolol was well tolerated.

Evaluation of amlodipine in mild to moderate hypertension--a clinical report.

Twenty two patients having mild to moderate hypertension were treated with a single daily dose of amlodipine for 4 weeks. Satisfactory response defined as final diastolic blood pressure < 90 mm of Hg and a reduction from baseline values > 10 mm of Hg could be achieved in 81.8% of patients in supine position and 70% of patients in standing position. Thirteen patients responded to 5 mg dose and 9 patients required 10 mg. Postural hypotension and reflex tachycardia were absent. Three patients has mild leg cramps and constipation. No deleterious effects were observed on liver, kidney and hemopoetic function, or on E.C.G. Changes. Amlodipine given once daily is effective and safe, and is a useful addition to the existing armamentarium of antihypertensive drugs.

Effects of purified insulins on insulin requirement and metabolic control.

Seventeen diabetics requiring high insulin doses were transferred from conventional insulin to purified chromatographed porcine insulins (Actrapid and Lentard-Novo, Denmark). At the end of 8 to 12 weeks, there was a 46% reduction in insulin dosage while metabolic control improved. Some of these patients when transferred again to conventional insulins demonstrated poor metabolic control and an increase in insulin requirements. Use of purified insulin is beneficial as insulin requirement is reduced with improved metabolic control.

Ramipril vs captopril in mild to moderate hypertension.

Ramipril 5 mg once daily was compared to Captopril 50 mg twice daily in a randomised, double-blind, parallel group study in 60 patients with a diastolic blood pressure between 95 to 120 mmHg over a period of 2 months. Both drugs in the dose regimen used in this study exerted a similar anti-hypertensive effect at the end of 2 months of treatment resulting in a fall of supine diastolic blood pressure with Ramipril = 19.27 +/- 3.34 mmHg and Captopril = 19.15 +/- 2.63, in patients receiving the drugs without the diuretic. The mean fall in supine diastolic blood pressure 4 hours after the first dose of Ramipril was 6.5 mmHg and Captopril 8 mmHg. None of the patients developed first dose hypotension or orthostatic hypotension and there was no significant alteration of the heart rate in either group. The serum K+ levels remained unchanged in both groups of patients. Both drugs were well tolerated and there were no adverse effects observed on the liver, kidney, blood sugar or haemopoietic system. Based on the results of this study, it can be concluded that the antihypertensive efficacy of 5 mg ramipril in a once daily dose is equivalent to 50 mg captopril given twice daily. However an appreciably greater number of patients reported improvement in the "quality of life' parameters with ramipril as compared to captopril. Thus for the routine treatment of mild to moderate arterial hypertension, ramipril offers reliable antihypertensive efficacy in a once daily dose, thereby helping to improve patient compliance and making the treatment more economical.

An open clinical trial of benazepril--a new ACE inhibitor in mild-moderate hypertension.

Benazepril hydrochloride, a new non-sulfhydryl ACE inhibitor (ACEI) was studied in a titrated dose of 10 mg-20 mg once a day for 6 weeks in 42 mild to moderate adult hypertensive patients with sitting diastolic blood pressure (SDBP) 95-114 mm Hg. The pre-drug SDBP(mean +/- SE) of 102.5 +/- 0.8 mm Hg showed a significant reduction to 87.5 +/- 0.93 mm Hg at the end of treatment. BP was controlled (SDBP < or = 90 mm Hg) in 34 (81%) patients and a drop of at least 10 mm Hg from the pre-treatment SDBP value was noted in 34 (81%) patients. Common adverse reaction was cough in 8(19%) patients. Clinically significant changes in laboratory evaluations were not seen in any patient. Study showed that benazepril in a dose range of 10 to 20 mg per day is an effective agent for treatment of mild to moderate hypertension.

Genetic risk factors for Clostridium difficile infection in ulcerative colitis.

BACKGROUND: Patients with inflammatory bowel disease (IBD) are at higher risk for Clostridium difficile infection (CDI). Disruption of gut microbiome and interaction with the intestinal immune system are essential mechanisms for pathogenesis of both CDI and IBD. Whether genetic polymorphisms associated with susceptibility to IBD are also associated with risk of CDI is unknown. AIMS: To use a well-characterised and genotyped cohort of patients with UC to (i) identify clinical risk factors for CDI; (ii) examine if any of the IBD genetic risk loci were associated with CDI; and (iii) to compare the performance of predictive models using clinical and genetic risk factors in determining risk of CDI. METHODS: We used a prospective registry of patients from a tertiary referral hospital. Medical record review was performed to identify all ulcerative colitis (UC) patients within the registry with a history of CDI. All patients were genotyped on the Immunochip. We examined the association between the 163 risk loci for IBD and risk of CDI using a dominant genetic model. Model performance was examined using receiver operating characteristics curves. RESULTS: The study included 319 patients of whom 29 developed CDI (9%). Female gender and pancolitis were associated with increased risk, while use of anti-TNF was protective against CDI. Six genetic polymorphisms including those at TNFRSF14 [Odds ratio (OR) 6.0, P-value 0.01] were associated with increased risk while 2 loci were inversely associated. On multivariate analysis, none of the clinical parameters retained significance after adjusting for genetics. Presence of at least one high-risk locus was associated with an increase in risk for CDI (20% vs. 1%) (P = 6 × 10⁻9). Compared to 11% for a clinical model, the genetic loci explained 28% of the variance in CDI risk and had a greater AUROC. CONCLUSION: Host genetics may influence susceptibility to Clostridium difficile infection in patients with ulcerative colitis.