Dunnett's Multiple Comparison Test Research Articles

Effects of Bidens pilosa L. on the molecular targets in the TNBS-model of intestinal inflammation

Inflammatory bowel disease (IBD) comprises two diseases: Crohn's disease and Ulcerative colitis. Its etiology remains unknown and pharmacological treatment is related to several side effects. Several studies indicate ethnopharmacological usage of medicinal plants for the treatment of inflammation disorders. Among these species, Bidens pilosa L. is outstanding. It is rich in polyunsaturated fatty acids, considered key compounds with anti-inflammatory effects. The aim of this investigation was to analyse the preventive effects of a supercritical CO2 extract of B. pilosa L. containing a multi-component standardized mixture of phytol, linolenic, plamitic, linoleic and oleic acids (produced by Chemyunion Quimica Ltda) in the model of TNBS-induced intestinal inflammation. Rats orally received B. pilosa extract in doses of 25, 50 or 100 mg/kg for 5 days before colitis induction. Animals were killed 48h after colitis induction and gene expression evaluation of Hsp70, heparanase, NF-κB, MAPK1, MAPK3, MAPK6 and MAPK9, as well as MUC1, MUC2, MUC3 and MUC4 was made by RT-qPCR using β-actin as internal control. Data were analysed by one-way ANOVA followed by Dunnett's multiple comparison test (p ≤0.05). The results demonstrate that the doses of 25, 50 and 100 mg/kg were able to reduce MAPK3, MUC1 and MUC2 gene expression compared with control colitic animals. Besides that, the dose of 25 mg/kg was able to enhance MUC3 and MUC4 gene expression. Hsp70 expression was reduced by 25 and 100 mg/kg doses and heparanase by 50 and 100 mg/kg doses. In conclusion, this study demonstrates that B. pilosa was able to modulate target genes in the preventive model of intestinal inflammation and provide a basis for the development of the herbal preparation useful to treat human IBD.

English

BACKGROUND: Pre-emptive analgesia is a treatment that is initiated before the surgical procedure in order to reduce sensitization of central and peripheral pain pathways produced by pain signals evoked by tissue damage. Clonidine has demonstrated efficacy in clinical trials as pre-emptive analgesic in postoperative pain management. OBJECTIVE: The present study was conducted to evaluate postoperative analgesic benefit in patients administered clonidine or placebo for below umbilical surgeries to be performed under subarachnoid block (SAB) using 3ml 0.5%bupivacaine & to compare their postoperative efficacy with respect to duration of analgesia, 24hrs postoperative requirements of total analgesics and study side effects. MATERIAL & METHODS: Sixty patients of either sex (30 per group, 20-65yrs, ASA class I-II) received either oral placebo (group PC) or clonidine 150µg (group CL) one hr preoperatively. The postoperative Visual Analogue Scale (VAS) score was assessed for 24hrs every 2hrly. The patients were given iv Diclofenac75mg as rescue analgesic at VAS ≥4.The time at which patient demanded rescue analgesic for first time & total requirement of 24 hrs postoperative analgesics was noted. STATISTICAL ANALYSIS: Software used in the analysis was EPI info software (3.4.3). Data was reported as mean value ± SD, P-value of < 0.05 was considered statistically significant. Unpaired T - test was used to find out significance between two samples. The comparison of normally distributed continuous variables between the groups was performed by means of one-way analysis of variance (ANOVA) and, if appropriate, followed by Dunnett multiple comparison tests. Nominal categorical data among study groups were compared using the chi-square test. Results: Total duration of analgesia in Group-CL was significantly more than Group-PC. (492.66 ±78.29 min. Group-CL, 264.83 ±13.67 min. Group-PC, p=0.000), lower rescue analgesic requirement in Group-CL than in Group-PC (2.20 ±0.61 Group-CL, 4.03 ± 0.66 Group-PC, p=0.000). CONCLUSION: Pre-emptive oral clonidine appears to be effective in prolongation of postoperative analgesia with decreased rescue analgesic requirements. The main side effects observed were hypotension & bradycardia.

Treatment of hypercholesterolemia: screening of Solanum macrocarpon Linn (Solanaceae) as a medicinal plant in Benin.

Hypercholesterolemia is the greatest risk factor for cardiovascular diseases. The present study is conducted to evaluate the lipid lowering activity of leaves and fruits of Solanum macrocarpon, a vegetable, on Wistar rats experimentally rendered hypercholesterolemic by Triton X-100. The leaves and fruits were administered (p.o.) for 7 days to rats at doses of 400 and 800 mg/kg of body weight. Atorvastatin was used as reference treatment drug. The data were analyzed by the Brown-Forsythe ANOVA, Dunnett's T3 multiple comparison test, and Dunnett's t test. All tests were done at the 5% significance level. Administration of S. macrocarpon (fruits as well as leaves) resulted in a statistically significant decrease in total cholesterol, LDL-cholesterol, VLDL-cholesterol, and triglycerides in the treated groups compared with the untreated hypercholesterolemic group, regardless of the administrated doses. A significant increase in HDL-cholesterol was observed in the treated groups. Hepatic disorders due to the Triton have been corrected by S. macrocarpon. This vegetable effectively suppresses experimental hypercholesterolemia in Wistar rats, suggesting a protective role in cardiovascular diseases. Its use by individuals at risk should be promoted.

Glycosylated Haemoglobin (HbA 1 c) - A Marker of Circulating Lipids in Type 2 Diabetic Patients

Diabetic patients with concomitant dyslipidemia are often soft targets for cardiovascular disease and deaths. An early intervention to normalize circulating lipids has been shown to reduce cardiovascular morbidity and mortality. Glycosylated hemoglobin (HbA1c) is routinely used as a marker to indicate long-term glycemic control. Our aim was to test whether HbA1c can serve as a marker of circulating lipids among Type 2 diabetic patients. The sera of 130 Type 2 diabetic patients was analyzed for fasting blood sugar (FBS), HbA1c and lipid profile consisting of total cholesterol (TC), triglycerides (TG), High-density Lipoprotein (HDL) cholesterol and LDL cholesterol. We divided the subjects based on their glycemic index into three groups; HbA1c< 6% as good, HbA1c>6% - <9% as poor and HbA1c>9% as worst glycemic control. One-way analysis of variance (ANOVA) and post-hoc Dunnett's multiple comparison tests was used to examine the significance levels for various biochemical parameters in age-categorized groups. The mean ± SD levels of HbA1c was significantly higher in females (8.598 ± 2.284 %) compared to males (7.323±2.18 %). Older patients had HbA1c, FBS and lipid profile levels similar to younger ones. HbA1c showed direct and significant correlations with cholesterol, TG and LDL. Univariate analysis showed that HbA1c was a good predictor of circulating lipid levels. The study indicates the usefulness of HbA1c as a marker for lipid profile for screening of diabetic patients at high risk of developing cardiovascular diseases.

Does copper enhance the antihypertensive effect of Elaeocarpus ganitrus in experimentally induced hypertensive rats?

Ayurveda, one of the traditional systems of medicine of India, reports that the seeds of Elaeocarpus ganitrus Linn. (Tilaceae) can be used for the treatment of hypertension. The main aim is to evaluate the antihypertensive effect of Elaeocarpus ganitrus (Rudraksha) seeds. Powdered seeds were extracted by maceration, overnight, using water, in copper (E1) and glass vessel (E2) and analyzed for antihypertensive activity in cadmium chloride (1 mg/kg intraperitoneally, for a period of 15 days) induced hypertensive male Wistar rats at three dose levels. E1 was administered at the dose of 5, 10, and 15 mg/kg and E2 at dose of 10, 20, and 30 mg/kg. All the data were analyzed using one way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. E1 and E2 did not show any toxicity at the dose of 5 g/kg in rats. It was found that 15 mg/kg of E1 and 30 mg/kg of E2 decreases the blood pressure by 30.20 mmHg and 28.96 mmHg, respectively, in hypertensive rats. Thus, it can be said that 15 mg/kg of E1 produced similar decrease in blood pressure as was observed with 30 mg/kg of E2. Copper ions in E1 might be additively affecting the reduction in blood pressure with the usage of Elaeocarpus ganitrus extracts.

Molecular and Structural Evaluation of Dentin Caries-Like Lesions Produced by Different Artificial Models

This study evaluated structural and molecular issues of dentin caries-like lesions produced by different artificial models (ACL) compared with natural caries lesions (NCL). One hundred twenty-four sound occlusal dentin blocks and 47 carious blocks were obtained and surface hardness was analyzed (SH1). They were assigned to groups according to ACL: GB: Biological; GC: Chemical; GIS: In situ; GNC: natural caries (control). Blocks from groups 1, 2 and 3 were submitted to caries lesion induction. NCL and ACL blocks were submitted to surface hardness (SH 2), FT-Raman and µEDXRF analysis. All blocks were longitudinally sectioned and one of the halves was submitted to cross-sectional hardness (CSH) and the other to SEM analysis. SH1 and SH2 data were submitted to t test (unpaired and paired, respectively), CSH and SEM data to two-way and one-way ANOVA respectively, and Tukey and t tests, respectively (p<0.05). Data from FT-Raman/µEDXRF were submitted to one-way ANOVA and Dunnett multiple-comparisons test (a=0.05). GB and GNC showed lowest SH2 values that were significantly different from GC and GIS. Regarding CSH, GB and GNC showed no significant difference between them. SEM showed similar caries lesion depth for GB and GNC, being significantly higher than for GC and GIS. µEDXRF showed similar values of calcium and phosphate for GB and GNC; GNC values were significantly different from GIS. No significant difference was found among the groups concerning phosphate, carbonate and CH bonds values. For collagen type I, GC values were significantly different compared to other groups. It may be concluded that caries-like lesions produced by GB were the closest model to NCL.

Monitoring Novel Anticoagulants Dabigatran, Rivaroxaban and Apixaban By Thrombelastography. Proof Of Concept

Background The novel anticoagulants dabigatran, rivaroxaban and apixaban have entered the clinical arena for treatment of atrial fibrillation and thromboebolism. Although these agents were designed not to require monitoring of the anticoagulation effect, there are certain clinical situations where such measurement would be useful including bleeding situations, trauma, and thromboembolic event while on treatment with the anticoagulant agent. At this point monitoring of these agents is under investigation and not fully understood and standardized. Thrombelastography (TEG) evaluates the viscoelastic properties of blood during coagulation. The clinical application of TEG in monitoring novel anticoagulant treatment is not yet well defined. The purpose of this in vivo study was to systematically evaluate the anticoagulant effect of the novel agents dabigatran, rivaroxaban and apixaban with TEG. Methods Healthy male volunteers (n = 8) were given oral dabigatran 150 mg once, or rivaroxaban 20 mg, or apixaban 10 mg once. TEG parameters Reaction time R, angle of Alfa and Maximum Amplitude (MA) were measured at times 0, 2, 4, and 24 hours after intake of the anticoagulant agent. We applied different TEG assays including, Koalin, Funtional Fibrinogen, Rapid TEG and Platelet mapping. The measurement of each parameter was compared to baseline measurement at time 0. Repeated measures ANOVA and Dunnett's multiple Comparison test was performed. Results All following results are with the Koalin assay. The other TEG assays did not demonstrate clinically relevant responsiveness after the intake of anticoagulant. For dabigatran, Reaction time R was increased by 73% as compared to baseline (p &lt; 0.008) by 2 hours after intake. Alfa was decreased by 8% at 2 hours (p &lt; 0.008). see Figure 1. MA was not significantly altered. For rivaroxaban, R was increased by 75% as compared to baseline at 2 hours after intake (p = 0.03). Alfa was decreased by 14% and MA was decreased by 6% at 2 hours after intake (p = 0.03 for both). For apixaban R was increased by 18% as compared to baseline (p &lt; 0.05) at 4 hours after intake. The MA was decreased by 5% (p= 0.016) and alfa was not significantly altered. Conclusion We have for the first time demonstrated that Thromelastograph has the potential of monitoring the novel anticogulant's effect on the hemostatis system. This technology is relatively easy to use on whole blood without need for specialized laboratory expertise. The reaction time R with Koalin assay is the most suitable TEG parameters for evaluation of the antithrombotic effect of the novel anticoagulant. The reaction time R is most responsive to the effect of rivaroxaban and least responsive to the effect of apixaban. This finding demonstrates different effects on the hemostatic system by 2 agents in the class of factor Xa inhibitors. Efforts to find the appropriate TEG assay for optimization of R responsiveness to apixaban will likely improve the clinical usefulness of this technology. Larger clinical trials are needed to correlate R with specific agent drug level and clinical outcomes. Disclosures: No relevant conflicts of interest to declare.

Dexmedetomidine Dose-Dependently Enhances Local Anesthetic Action of Lidocaine

The combination of α2-adrenoceptor agonists, such as dexmedetomidine (DEX) and clonidine, with local anesthetics has been found to extend the duration of peripheral nerve blocks, probably owing to the resultant local vasoconstriction in the peripheral nerves. However, because the clear elucidation of the effect of DEX requires examination of the local anesthetic effect with DEX alone and the combination of various concentrations of DEX with local anesthetics, we evaluated the local anesthetic effect of various concentrations of DEX alone and with a local anesthetic. The present study assessed the tail-flick (TF) latencies after injection of the appropriate drug in male Sprague-Dawley rats, using an epidural model that allowed constant pain stimulation intensity, dispersion of the anesthetic, and a precise injection site and dose. Lidocaine alone, lidocaine with 2.5-ppm DEX, lidocaine with 5.0-ppm DEX, lidocaine with 7.5-ppm DEX, and DEX alone were administered at the predetermined dose. The TF latency changes over time were compared using repeated measures analysis of variance (ANOVA). Comparisons among the groups were analyzed using ANOVA followed by a post hoc Dunnett's multiple comparison test or Tukey's multiple comparison test. The addition of DEX to lidocaine increased the TF latency and dose-dependently prolonged its duration as follows: 0-ppm DEX, 20 minutes; 2.5-ppm, 40 minutes; 5.0-ppm, 40 minutes; and 7.5-ppm, 50 minutes. DEX alone did not change the TF latency. Our results have demonstrated that DEX dose-dependently enhances the local anesthetic action of lidocaine in a rat TF model.

Agonist-biased Trafficking of Somatostatin Receptor 2A in Enteric Neurons

Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.

Hypoxia increases the release of salmon cardiac peptide (sCP) from the heart of rainbow trout (Oncorhynchus mykiss) under constant mechanical load in vitro

Our aim was to study the effects of hypoxia on the release of salmon cardiac peptide (sCP) from an isolated heart ventricle of trout during a constant mechanical load. Trout heart ventricles were studied in vitro. The ventricle was placed in an organ bath at 12 °C in which a constant mechanical load could be imposed on the ventricle while buffer solution was circulating. Ventricles were field-stimulated with a supramaximal voltage pulse at a rate of about 0.3 s⁻¹. Samples of 1 ml were collected at an interval of 10 min for 200 min from the organ bath and assessed with a radioimmunoassay for sCP. After a control period of 20 min, ventricles were exposed to hypoxia produced with N₂ gassing (n = 9) or to hypoxia with 20 mM BDM, a nonselective myosin ATPase inhibitor locking cross-bridges in a pre-power-stroke state inhibiting force production with normal electrical activity (n = 10). In this model and setup, hypoxia stimulated the release of sCP, but the interindividual variation in the response was large. At the end of hypoxia exposure, the concentration of sCP in the organ bath was about sixfold higher than at the start of the exposure (P < 0.05, one-way ANOVA for repeated measurements, followed by Dunnett's multiple comparison test). When BDM was introduced into the bath, the ventricle still secreted sCP but the hypoxic response was smaller than in the experiments without BDM. In the trout heart ventricle, there is a hypoxia-sensitive component in the release mechanism of sCP which is independent of contraction.

Oligodendrocyte Progenitor Cell Susceptibility to Injury in Multiple Sclerosis

Remyelination in multiple sclerosis (MS) is often incomplete. In experimental models, oligodendrocyte progenitor cells (OPCs) rather than previously myelinating oligodendrocytes (OLs) are responsible for remyelination. This study compares the relative susceptibility of adult human OPCs and mature OLs to injury in actively demyelinating MS lesions and under invitro stress conditions. In all lesions (n = 20), the number of OLs (Olig2 weak/NogoA positive) was reduced compared to control white matter (mean 38± 4% of control value). In 11 cases, OPC numbers (Olig2 strong; NogoA negative) were also decreased; in eight of these, the reduction was greater for OPCs than for OLs. In the other nine samples, OPC numbers were greater than control white matter, indicating ongoing OPC migration and/or proliferation. Analysis of co-cultures with rat dorsal root ganglia neurons confirmed that OPCs were more capable of contacting and ensheathing axons than OLs. In isolated culture under stress conditions (withdrawal of serum/glucose and/or antioxidants), OPCs showed increased cell death and reduced process extension compared to OLs. Under all culture conditions, OPCs up-regulated expression of genes in the extrinsic proapoptotic pathway, and had increased susceptibility to tumor necrosis factor-induced cell death as compared to OLs. Our data suggest that susceptibility of OPCs to injury within the MS lesion environment contributes to the limited remyelination in MS.

Lymphatic Vessel Memory Stimulated by Recurrent Inflammation

Inflammation stimulates new lymphatic vessel growth (inflammatory lymphangiogenesis). One key question is how recurrent inflammation, a common clinical condition, regulates lymphatic vessel remodeling. We show here that recurrent inflammation accelerated the development a functional lymphatic vessel network. This observation suggests a novel program of lymphangiogenesis and identifies a property of lymphatic vessel memory in response to recurrent inflammation. A brief episode of initial inflammation regressed lymphatic vessels, and a significant increase in CD11b(+) macrophages were associated with the development of lymphatic vessel memory. These vessels had major differences in the structure and the spatial distribution of specialized lymphatic vessel features. Surprisingly, we found that the lymphatic vessel memory response did not depend on the vascular endothelial growth factor C or A pathway, indicating that different molecular pathways regulate inflammatory lymphangiogenesis and lymphatic vessel memory. These findings uncover a priming mechanism to facilitate a rapid lymphatic vessel memory response: a potential important component of peripheral host defense.

Minocycline Blocks Asthma-associated Inflammation in Part by Interfering with the T Cell Receptor-Nuclear Factor κB-GATA-3-IL-4 Axis without a Prominent Effect on Poly(ADP-ribose) Polymerase

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

Behavioural responses of lambs to plastic clips as an alternative procedure to mulesing

Compare the effects on the behaviour of lambs of applying occlusive plastic clips, as an alternative procedure to surgical mulesing, with tail docking, surgical mulesing and a control treatment. We allocated 48 6-7-week-old Merino lambs to four treatment groups: plastic clips (Clip); surgical mulesing (Mules); tail docking with a rubber ring (Tail ring); no treatment (Control). For each posture and behaviour observed on each of the 4 days post-treatment, a Dunnett's multiple comparison test was used to simultaneously compare the Clip treatment with each of the comparator treatments (Control, Tail ring and Mules treatments). Most of the significant differences (P < 0.05) detected between the comparator treatments occurred on day 1. For four of these measurements, the Clip treatment differed (P < 0.01) from the Mules treatment, but from not the Control and Tail ring treatments: the Clip lambs spent less time standing immobile not interacting with ground, hay or feeder, less time standing immobile head down not interacting with ground, hay or feeder, more time walking and more time interacting with ground, hay or feeder. These behavioural results, together with previous behavioural and physiological research, indicate that the effect on lamb welfare of applying occlusive clips is less than that of surgical mulesing.

The effects of doxycycline and micronized purified flavonoid fraction on human vein wall remodeling are not hypoxia-inducible factor pathway-dependent

Doxycycline and micronized purified flavonoid fraction (MPFF) modulate vein wall remodeling that may be associated with hypoxia in varicose veins (VVs), vein graft stenosis, and deep venous thrombosis. We recently reported that in vitro exposure of non-VV (NVVs) and VVs to hypoxic conditions activates the hypoxia-inducible factor (HIF) pathway. This study investigated the in vitro effects of doxycycline and MPFF on the HIF pathway in hypoxic NVVs and VVs. Six NVVs and six VVs obtained from surgery were used to prepare vein organ cultures, which were exposed to hypoxia (1% O(2)), with and without MPFF (10(-5) mol/L) or doxycycline (5 μg/mL) for 16 hours. The veins were analyzed for HIF-1α, HIF-2α, and their target gene expression, with real-time polymerase chain reaction and Western blot. The differences between gene expressions were tested with one-way analysis of variance with repeated measures, followed by the Dunnett test for multiple comparisons. P < .05 was considered significant. Treatment of NVV organ cultures exposed to hypoxia with doxycycline or MPFF did not significantly alter the expression of HIF-1α and HIF-2α messenger (m)RNA and protein compared with untreated. Doxycycline also did not significantly affect the expression of HIF-1α and HIF-2α mRNA and protein in VVs exposed to hypoxia compared with untreated VVs. However, MPFF significantly reduced the expression of HIF-1α but not HIF-2α mRNA in VVs exposed to hypoxia compared with untreated VVs. Interestingly, the reduction of the expression of HIF-1α mRNA in VVs by MPFF was not reflected at the protein level. The mRNA expression of HIF target genes, namely glucose transporter-1, carbonic anhydrase-9, vascular endothelial growth factor, B-cell lymphoma 2/adenovirus E1B 19-kDa protein-interacting protein 3, prolyl hydroxylase domain-2, and prolyl hydroxylase domain-3, was not significantly altered in NVVs and VVs exposed to hypoxia and treated with doxycycline or MPFF compared with those untreated. Doxycycline and MPFF at a concentration corresponding to a therapeutic dose do not alter the activation of the HIF pathway in NVV and VV organ cultures exposed to hypoxia. Our findings suggest vein wall remodeling actions in NVVs and VVs are likely not HIF-dependent.

Expression of Th17-related transcription factor ROR.GAMMA.t mRNA in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

Objective To investigate the action mechanism of Th17 cells in immunoinflammatory response in systemic lupus erythematosus (SLE).Methods Reverse-transcription PCR was performed to measure the mRNA expre ssion of the retinoic acid receptor-related orphan nuclear receptor RORγt in peripheral blood mononuclear cells (PBMCs) from 12 patients with active SLE,9 patients with inactive SLE and 12 normal human controls.Data were statistically analyzed by approximate F test (Welch test) and Dunnett's T3 multiple comparison test (corrected).Results In the case of RORγt mRNA expression in PBMCs,significant differences existed among the 3 groups (F =23.286,P ＜ 0.01 ); in detail,the patients with active SLE were significantly higher than patients with inactive SLE and normal controls ( 1.06 ± 0.44 vs.0.65 ± 0.25,F =2.453,P ＜ 0.05;1.06 ± 0.44 vs.0.22 ± 0.08,F =6.504,P ＜ 0.05),and the patients with inactive SLE were significantly increased compared with the normal controls (F =3.343,P ＜ 0.05).The expression level of RORγt mRN A was significantly positively correlated with SLE disease activity index (rp =0.623,P ＜ 0.01 ).Conclusions There is a polarization of Th17 cells in patients with SLE.To antagonize the transcription factor RORγt,which plays an essential role in the regulation of Th17 cell differentiation,may facilitate the control of SLE via attenuating the immunoinflammatory response.

Tumor necrosis factor α release in peripheral blood mononuclear cells of cutaneous lupus and dermatomyositis patients

IntroductionSeveral studies have reported that TNFα is substantially increased within skin lesions of patients with discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE) and dermatomyositis (DM) compared to controls. Elevated TNFα has been reported in the sera of some patients with systemic lupus erythematosus, DLE and SCLE, but not in the sera of patients with DM. Because of the key pathogenic role of autoimmunity in these diseases, in this study we sought to evaluate TNFα production by a readily available source of immune cells (namely, peripheral blood mononuclear cells (PBMCs)) taken from controls and from patients with cutaneous lupus or DM.MethodsFreshly isolated PBMCs were cultured overnight, and TNFα protein accumulation in conditioned medium was determined. In addition, flow cytometry using cell-type-specific markers was performed to determine the sources of TNFα. One-way analysis of variance and Dunnett's multiple comparisons test were performed for statistical comparisons.ResultsAccumulation of TNFα protein in conditioned medium containing PBMCs from DLE patients, but not from SCLE, TLE or DM patients, was significantly greater (19-fold) than that from controls (P < 0.001). In DLE PBMCs, increased TNFα was produced by circulating monocytes and myeloid dendritic cells (mDCs). The mean TNFα fluorescence intensity, but not the total number, of both monocytes and mDCs (P < 0.01) from DLE patients was significantly greater (2.3-fold) than that of controls. There were significantly more (13.3-fold) mDCs with intracellular TNFα in blood from DLE patients (P < 0.001) and DM patients (P < 0.001) compared to controls. Most importantly, a positive correlation was seen in DLE patients between their disease activity measured using the Cutaneous Lupus Erythematosus Disease Area and Severity Index and TNFα protein secretion (r = 0.61, P < 0.08).ConclusionsTNFα protein production by PBMCs is greater in DLE patients than in patients with other cutaneous forms of lupus and DM or in controls. Flow cytometric studies demonstrated that circulating monocytes and mDCs contributed to this increased TNFα production. Monocytes and mDCs are present in lesional skin, and the increased TNFα production by these cells and other PBMCs likely increase the number of inflammatory cells seen in DLE skin relative to other subsets of cutaneous lupus erythematosus and DM. These results provide a possible biological explanation for the denser infiltrate seen in DLE relative to DM.

Comparative Study of In Vitro Ocular Surface Cytotoxicity of a Fixed Combination of 0.5% Timolol/1% Dorzolamide Eyedrop and Its Components with 0.005% Benzalkonium Chloride

We evaluated the cytotoxicity of antiglaucoma ophthalmic solutions preserved with the same concentration of benzalkonium chloride (BAK) in four cultured corneal and conjunctival cell lines. The viability of cell cultures was determined following the exposure of cells to timolol maleate, dorzolamide, and their fixed combination, Kosoputo(®) (MSD, a Japanese formulation of Cosopt(®) (Merck)), and two commercially available eyedrop solutions, 0.5% Timpotol(®) (containing 0.5% timolol maleate, MSD) and 1% Trusopt(®) (containing 1% dorzolamide, MSD) for varying exposure times and at various dilutions using the MTT and neutral red assays. All the three commercially available eyedrop solutions tested in this study were preserved with 0.005% BAK. The toxicity of each solution was compared using the % cell viability score (CVS) . Cell viability was also subjected to statistical analysis using ANOVA, Dunnett's multiple comparison tests and a chi-square test. %CVS50/%CVS40/80s for the tested solutions were 53/-13 for 0.5% Timoptol(®), 100/88 for preservative-free 0.5% timolol maleate, 50/ -10 for 1% Trusopt(®), 72/100 for preservative-free 1% dorzolamide, and 44/-17 for Kosoputo(®). The results of statistical analysis were consistent to them. In conclusion, Kosoputo(®) had greater cytotoxicity than each component; however, in actual use it may have the advantages of reduced toxicity (side effect) due to reduced instillation frequency, and better patient adherence to the treatment regimen as well as a comparable pressure reduction effect.

Determination of dose dependence in repeated dose toxicity studies when mid-dose alone is insignificant

Repeated dose toxicity studies with rodents are regulatory requirements for registering chemical substances like drugs and pesticides with the government regulatory agencies. Usually 4 groups of animals, including a control group, are used in repeated dose toxicity studies. Williams' test, Dunnett's test and Jonckheere's trend test are generally used to evaluate the data obtained from these studies. Selection of a statistical tool is relatively easy, when the data obtained from the groups of animals show a dose-dependency. But, occasionally a significance difference, compared to control, is not seen in the mid-dose group alone, thus losing the dose-dependency. We attempted to find the appropriate statistical tool for analyzing the quantitative data obtained from repeated dose toxicity studies, when the data of the mid-dose group alone do not show a significant difference, compared to control. The commonly used Williams' test to analyse such data has a disadvantage as it assigns an estimated mean value for the mid-dose group, rather than the original mean value, for the analysis. Hence, it is likely that Williams' test may misjudge in establishing a dose dependency, when in reality it does not exists. Therefore, to analyse such data we suggest the use of Dunnett's multiple comparison test, to compare each dose group with the control, followed by Jonckheere's trend test for examining dose dependency.

Nephroprotective effect of Kabab chini (Piper cubeba) in gentamycin-induced nephrotoxicity

Kabab chini (KC) (Piper cubeba) is an important drug in Unani Medicine, widely described to be effective in renal diseases, and physicians are using it as a protective and curative agent in various renal disorders from ancient times. The present study was designed to evaluate the nephroprotective effect of KC against gentamycin-induced nephrotoxicity in Wistar rats. This was studied in two different sets of tests, in which both the protective as well as the curative effects were evaluated in groups of albino rats. The powder of the test drug was administered orally in a dose of 810 mg/kg and 1220 mg/kg, in suspension form, in the pre- and post-treated models. The nephroprotective effect was assessed on the basis of biochemical estimation of serum urea and creatinine levels and histopathological examination of the treated kidney. The effect observed in the pre-treated and post-treated groups was compared with plain as well as negative control groups using one-way ANOVA with Dunnett's multiple pair comparison test. The findings of the two tests demonstrated that KC produced a significant nephroprotective effect in both pre-treated and post-treated groups. The results of our study indicate that KC possesses significant benefit against gentamycin-induced nephrotoxicity.