Phase I trial of DNA-hsp65 immunotherapy for advanced squamous cell carcinoma of the head and neck

Considering that mycobacterial heat-shock protein 65 (hsp65) gene transfer can elicit a profound antitumoral effect, this study aimed to establish the safety, maximum-tolerated dose (MTD) and preliminary efficacy of DNA-hsp65 immunotherapy in patients with advanced head and neck squamous cell carcinoma (HNSCC). For this purpose, 21 patients with unresectable and recurrent HNSCC were studied. Each patient received three ultrasound-guided injections at 21-day intervals of: 150, 600 or 400 mu g of DNA-hsp65. Toxicity was graded according to CTCAE directions. Tumor volume was measured before and after treatment using computed tomography scan. The evaluation included tumor mass variation, delayed-type hypersensitivity response and spontaneous peripheral blood mononuclear cell proliferation before and after treatment. The MTD was 400 mg per dose. DNA-hsp65 immunotherapy was well tolerated with moderate pain, edema and infections as the most frequent adverse effects. None of the patients showed clinical or laboratory alterations compatible with autoimmune reactions. Partial response was observed in 4 out of 14 patients who completed treatment, 2 of which are still alive more than 3 years after the completion of the trial. Therefore, DNA-hsp65 immunotherapy is a feasible and safe approach at the dose of 400 mg per injection in patients with HNSCC refractory to standard treatment. Further studies in a larger number of patients are needed to confirm the efficacy of this novel strategy.

An 18-Week, Prospective, Randomized, Double-Blind, Multicenter Study of Amlodipine/Ramipril Combination Versus Amlodipine Monotherapy in the Treatment of Hypertension: The Assessment of Combination Therapy of Amlodipine/Ramipril (ATAR) Study

Background: A combination of antihypertensive agents of different drug classes in a fixed-dose combination (FDC) may offer advantages in terms of efficacy, tolerability, and treatment compliance. Combination of a calcium channel blocker with an angiotensin-converting enzyme inhibitor may act synergistically to reduce blood pressure (BP). Objective: The aim of this study was to compare the efficacy and tolerability of an amlodipine/ramipril FDC with those of amlodipine monotherapy. Methods: This 18-week, prospective, randomized, double-blind study was conducted at 8 centers across Brazil. Patients with stage 1 or 2 essential hypertension were enrolled. After a 2-week placebo run-in phase, patients received amlodipine/ramipril 2.5/2.5 mg or amlodipine 2.5 mg, after which the doses were titrated, based on BP, to 515 then 10/10 mg (amlodipme/ramipril) and 5 then 10 mg (amiodipine). The primary end point was BP measured in the intent-to-treat (ITT) population. Hematology and serum biochemistry were assessed at baseline and study end. Tolerability was assessed using patient interview, laboratory analysis, and physical examination, including measurement of ankle circumference to assess peripheral edema. Results: A total of 222 patients completed the study (age range, 40-79 years; FDC group, 117 patients [mean dose, 7.60/7.60 mg]; monotherapy, 105 patients [mean dose, 7.97 mg]). The mean (SD) changes in systolic BP (SBP) and diastolic BP (DBP), as measured using 24-hour ambulatory blood pressure monitoring (ABPM) and in the physician`s office, were significantly greater with combination therapy than monotherapy, with the exception of office DBP (ABPM, -20.76 [1.25] vs -15.80 [1.18] mm Hg and -11.71 [0.78] vs -8.61 [0.74] mm Hg, respectively [both, P = 0.004]; office, -27.51 [1.40] vs -22.84 [1.33] min Hg [P = 0.012] and -16.41 [0.79] vs -14.64 [0.75] mm Hg [P = NS], respectively). In the ITT analysis, the mean changes in ambulatory, but not office-based, BP were statistically significant (ABPM: SBP, -20.21 [1.14] vs -15.31 [1.12] mm Hg and DBP, -11.61 [0.72] vs -8.42 [0.70] mm Hg, respectively [both, P = 0.002]; office: SBP, -26.60 [1.34] vs -22.97 [1.30] mm Hg and DBP, -16.48 [0.78] vs -14.48 [0.75] mm Hg [both, P = NS]). Twenty-nine patients (22.1%) treated with combination therapy and 41 patients (30.6%) treated with monotherapy experienced >= 1 adverse event considered possibly related to study drug. The combmation-therapy group had lower prevalence of edema (7.6% vs 18.7%; P = 0.011) and a similar prevalence of dry cough (3.8% vs 0.8%; P = NS). No clinically significant changes in laboratory values were found in either group. Conclusions: In this population of patients with essential hypertension, the amlodipine/ramipril FDC was associated with significantly reduced ambulatory and office-measured BP compared with amlodipine monotherapy, with the exception of office DBP. Both treatments were well tolerated. (Clin Ther. 2008;30: 1618-1628) (C) 2008 Excerpta Medica Inc.

Abatacept in children with juvenile idiopathic arthritis: a randomised, double-blind, placebo-controlled withdrawal trial

Background Some children with juvenile idiopathic arthritis either do not respond, or are intolerant to, treatment with disease-modifying antirheumatic drugs, including anti-tumour necrosis factor (TNF) drugs. We aimed to assess the safety and efficacy of abatacept, a selective T-cell costimulation modulator, in children with juvenile idiopathic arthritis who had failed previous treatments. Methods We did a double-blind, randomised controlled withdrawal trial between February, 2004, and June, 2006. We enrolled 190 patients aged 6-17 years, from 45 centres, who had a history of active juvenile idiopathic arthritis; at least five active joints; and an inadequate response to, or intolerance to, at least one disease-modifying antirheumatic drug. All 190 patients were given 10 mg/kg of abatacept intravenously in the open-label period of 4 months. Of the 170 patients who completed this lead-in course, 47 did not respond to the treatment according to predefined American College of Rheumatology (ACR) paediatric criteria and were excluded. Of the patients who did respond to abatacept, arthritis, and 62 were randomly assigned to receive placebo at the same dose and timing. The primary endpoint was time to flare of arthritis. Flare was defined as worsening of 30% or more in at least three of six core variables, with at least 30% improvement in no more than one variable. We analysed all patients who were treated as per protocol. This trial is registered, number NCT00095173. Findings Flares of arthritis occurred in 33 of 62 (53%) patients who were given placebo and 12 of 60 (20%) abatacept patients during the double-blind treatment (p=0.0003). Median time to flare of arthritis was 6 months for patients given placebo (insufficient events to calculate IQR); insufficient events had occurred in the abatacept group for median time to flare to be assessed (p=0.0002). The risk of flare in patients who contined abatacept was less than a third of that for controls during that double-blind period (hazard ratio 0.31, 95% CI 0.16-0.95). During the double-blind period, the frequency of adverse events did not differ in the two treatment groups, Adverse events were recorded in 37 abatacept recipients (62%) and 34 (55%) placebo recipients (p=0.47); only two serious adverse events were reported, bouth in controls (p=0.50). Interpretation Selective modulation of T-cell costimulation with abatacept is a rational alternative treatment for children with juvenile idiopathic arthritis. Funding Bristol-Myers Squibb.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor. (c) 2006 Elsevier Inc. All rights reserved.

Immunohistochemical analysis of p53, APE1, hMSH2 and ERCC1 proteins in actinic cheilitis and lip squamous cell carcinoma

Aims: This study has compared the tissue expression of the p53 tumour suppressor protein and DNA repair proteins APE1, hMSH2 and ERCC1 in normal, dysplastic and malignant lip epithelium. Methods and results: Morphological analysis and immunohistochemistry were performed on archived specimens of normal lip mucosa (n = 15), actinic cheilitis (AC) (n = 30), and lip squamous cell carcinoma (LSCC) (n = 27). AC samples were classified morphologically according to the severity of epithelial dysplasia and risk of malignant transformation. LSCC samples were morphologically staged according to WHO and invasive front grading (IFG) criteria. Differences between groups and morphological stages were determined by bivariate statistical analysis. Progressive increases in the percentage of epithelial cells expressing p53 and APE1 were associated with increases in morphological malignancy from normal lip mucosa to LSCC. There was also a significant reduction in epithelial cells expressing hMSH2 and ERCC1 proteins in the AC and LSCC groups. A higher percentage of malignant cells expressing APE1 was found in samples with an aggressive morphological IFG grade. Conclusions: Our data showed that epithelial cells from premalignant to malignant lip disease exhibited changes in the expression of p53, APE1, hMSH2 and ERCC1 proteins; these molecular change might contribute to lip carcinogenesis.

Tadalafil and fluoxetine in premature ejaculation: Prospective, randomized, double-blind, placebo-controlled study

Introduction: Premature ejaculation ( PE) is a common male sexual disorder. An ideal, reliable and effective treatment is desired by many men and couples affected by this condition. Aim: Evaluate if the association of a phosphodiesterase- 5 inhibitor, tadalafil, and a selective serotonin reuptake inhibitor, fluoxetine, can prolong the intravaginal ejaculatory latency time ( IELT) in men with lifelong premature ejaculation. Methods: Sixty patients with lifelong premature ejaculation and without erectile dysfunction ( ED) with IELT less than 90 s were enrolled in the protocol and randomized into 4 groups to use a combination of medications: ( 1) tadalafil 20 mg plus fluoxetine 90 mg, ( 2) fluoxetine 90 mg plus placebo, ( 3) tadalafil 20 mg plus placebo, and ( 4) two different placebo capsules ( control). Before starting the medications, each man timed his IELT with a stopwatch, and likewise during the treatment period. Fluoxetine 90 mg or placebo was taken once a week plus tadalafil 20 mg or placebo within a 36- hour frame of intended sexual intercourse with a steady partner. Patients were prospectively followed for 12 weeks. One- way ANOVA was used for statistical comparisons of IELT results in each group. Results: Mean IELT before starting treatment was 51.3 +/- 23 s. With one- way ANOVA, a statistically significant difference in post- treatment IELT was seen with combination treatment compared to placebo ( p < 0.001). There were increases in IELT from baseline in patients using fluoxetine plus tadalafil ( 49.57 +/- 25.87 to 336.13 +/- 224.77) (p < 0.001), fluoxetine (56.55 +/- 18.55 to 233.62 +/- 105.08) ( p < 0.001) and tadalafil (49.26 +/- 19.43 to 186.53 +/- 159.05) (p = 0.001). The increases in each group were statistically significant compared to the placebo (49.86 +/- 19.43 to 67.82 +/- 46.18) ( p = 0.042). Conclusion: Fluoxetine plus tadalafil significantly increased the IELT from baseline in men with lifelong premature ejaculation when compared to placebo, tadalafil or fluoxetine. Copyright (C) 2008 S. Karger AG, Basel.

A tale of two terminators: Crystal structures sharpen the debate on DNA replication fork arrest mechanisms

The structure of the Tus-Ter DNA replication fork arrest complex of Escherichia coli reveals a novel architecture for the bound Tus protein and a new type of DNA-binding motif, The structure of the complex may explain how Tus can block movement of a replication fork approaching from one direction and not the other.

Reorganization of terminator DNA upon binding replication terminator protein: Implications for the functional replication fork arrest complex

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B. subtilis DNA terminator, Terl, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of Terl causes the DNA to become slightly unwound and bent by similar to 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to similar to 60 degrees. We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner, in the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry at the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

Creatine supplementation does not impair kidney function in type 2 diabetic patients: a randomized, double-blind, placebo-controlled, clinical trial

Creatine supplementation may have a therapeutic role in diabetes, but it is uncertain whether this supplement is safe for kidney function. The aim of this study was to investigate the effects of creatine supplementation on kidney function in type 2 diabetic patients. A randomized, double-blind, placebo-controlled trial was performed. The patients were randomly allocated to receive either creatine or placebo for 12 weeks. All the patients underwent exercise training throughout the trial. Subjects were assessed at baseline and after the intervention. Blood samples and 24-h urine samples were obtained for kidney function assessments. Additionally, (51)Cr-EDTA clearance was performed. To ensure the compliance with creatine intake, we also assessed muscle phosphorylcreatine content. The creatine group presented higher muscle phosphorylcreatine content when compared to placebo group (CR Pre 44 +/- A 10, Post 70 +/- A 18 mmol/kg/wt; PL Pre 52 +/- A 13, Post 46 +/- A 13 mmol/kg/wt; p = 0.03; estimated difference between means 23.6; 95% confidence interval 1.42-45.8). No significant differences were observed for (51)Cr-EDTA clearance (CR Pre 90.4 +/- A 16.9, Post 96.1 +/- A 15.0 mL/min/1.73 m(2); PL Pre 97.9 +/- A 21.6, Post 96.4 +/- A 26.8 mL/min/1.73 m(2); p = 0.58; estimated difference between means -0.3; 95% confidence interval -24.9 to 24.2). Creatinine clearance, serum and urinary urea, electrolytes, proteinuria, and albuminuria were unchanged. CR supplementation does not affect kidney function in type 2 diabetic patients, opening a window of opportunities to explore its promising therapeutic role in this population. ClinicalTrials.gov registration number: NCT00992043.

Creatine in Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial

GUALANO, B., V. DE. SALLES PAINNELI, H. ROSCHEL, G. G. ARTIOLI, M. NEVES JR, A. L. DE SA PINTO, M. E. DA SILVA, M. R. CUNHA, M. C. G. OTADUY, C. DA COSTA LEITE, J. C. FERREIRA, R. M. PEREIRA, P. C. BRUM, E. BONFA, and A. H. LANCHA JR. Creatine in Type 2 Diabetes: A Randomized, Double-Blind, Placebo-Controlled Trial. Med. Sci. Sports Exerc., Vol. 43, No. 5, pp. 770-778, 2011. Creatine supplementation improves glucose tolerance in healthy subjects. Purposes: The aim was to investigate whether creatine supplementation has a beneficial effect on glycemic control of type 2 diabetic patients undergoing exercise training. Methods: A 12-wk randomized, double-blind, placebo-controlled trial was performed. The patients were allocated to receive either creatine (CR) (5 g.d(-1)) or placebo (PL) and were enrolled in an exercise training program. The primary outcome was glycosylated hemoglobin (Hb(A1c)). Secondary outcomes included the area under the curve of glucose, insulin, and C-peptide and insulin sensitivity indexes. Physical capacity, lipid profile, and GLUT-4 protein expression and translocation were also assessed. Results: Twenty-five subjects were analyzed (CR: n = 13; PL: n = 12). Hb(A1c) was significantly reduced in the creatine group when compared with the placebo group (CR: PRE = 7.4 +/- 0.7, POST = 6.4 +/- 0.4; PL: PRE = 7.5 +/- 0.6, POST = 7.6 +/- 0.7; P = 0.004; difference = -1.1%, 95% confidence interval = -1.9% to -0.4%). The delta area under the curve of glucose concentration was significantly lower in the CR group than in the PL group (CR = -7790 +/- 4600, PL = 2008 +/- 7614; P = 0.05). The CR group also presented decreased glycemia at times 0, 30, and 60 min during a meal tolerance test and increased GLUT-4 translocation. Insulin and C-peptide concentrations, surrogates of insulin sensitivity, physical capacity, lipid profile, and adverse effects were comparable between the groups. Conclusions: Creatine supplementation combined with an exercise program improves glycemic control in type 2 diabetic patients. The underlying mechanism seems to be related to an increase in GLUT-4 recruitment to the sarcolemma.

Cutaneous vasculitis in systemic lupus erythematosus: association with anti-ribosomal P protein antibody and Raynaud phenomenon

Ninety-one consecutive systemic lupus erythematosus (SLE) patients (American College of Rheumatology criteria) with a history of cutaneous vasculitis were compared to 163 SLE controls without this clinical manifestation from July to December 2007 in order to determine the possible clinical and serological association of this manifestation. Data were obtained in an ongoing electronic database protocol and autoantibodies to anti-double-stranded DNA, anti-Sm, anti-RNP, anti-Ro/SS-A, anti-La/SS-B, and anticardiolipin and ribosomal P protein antibody (anti-P) were detected by standard techniques. Exclusion criteria were the presence of anti-phospholipid syndrome or antibodies, Sjogren syndrome, and a history of thrombosis. The mean age (38.5 +/- 11.5 vs. 37.8 +/- 11.6 years, p = 0.635), disease duration (12.5 +/- 7.8 vs. 11.8 +/- 7.9 years, p = 0.501), and frequency of white race (71.4% vs. 70.5%, p = 0.872) and female sex (96.8% vs. 93.7%, p = 0.272) were comparable in both groups. The vasculitis group had a higher frequency of malar rash (97.9% vs. 87.4%, p = 0.004), photosensitivity (91.4% vs. 81.6%, p = 0.030), and Raynaud phenomenon (RP; 27.7% vs. 7.5%, p < 0.001), whereas all other clinical manifestation including renal and central nervous system involvements were similar to the control group. Laboratorial data revealed that only anti-P (35.1% vs. 12.1%, p < 0.001) was more frequent in patients with vasculitis. In a multivariate logistic regression model, cutaneous vasculitis was associated to the presence of RP (OR = 3.70; 95% confidence interval [CI] = 1.73-8.00) and anti-P (OR = 3.42; 95% CI = 1.76-6.66). In summary, SLE cutaneous vasculitis characterizes a subgroup of patients with more RP and anti-P antibodies but not accompanied by a higher frequency of renal and central nervous system involvements.

Late response to anti-TNF-alpha therapy in refractory mucocutaneous lesions of Beh double dagger et`s disease

Beh double dagger et`s disease (BD) is a multisystem chronic inflammatory disorder characterized by oral and genital ulceration and ocular involvement. Recurrent oral and genital ulcers are the most common symptoms of BD and occur in more than 80% of patients. The treatments of these disease manifestations include colchicine, corticosteroids and immunosuppressive drugs in severe cases. Anti-TNF-alpha therapy may be useful in refractory severe BD, particularly for ocular, central nervous system, gastrointestinal and refractory mucocutaneous lesions. During a 2-year period, 280 patients suffering from rheumatic diseases received anti-TNF-alpha agents at the infusion center of our University Hospital. Two patients (0.7%) presented BD; one of them had celiac disease as well, with recalcitrant mucocutaneous lesions that were not responsive to immunosuppressive drugs. We reported those patients who were successfully treated with infliximab and adalimumab, despite their late response.

A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (formerly Pseudomonas solanacearum)

Six Burkholderia solanacearum (formerly Pseudomonas solanacearum) genomic DNA fragments were isolated, using RAPD techniques and cloning, from the three genetically diverse strains: ACH092 (Biovar 4), ACH0158 (Biovar 2) and ACH0171 (Biovar 3) (1). One of these cloned fragments was selected because it was present constantly in all bacterial strains analysed. The remaining five clones were selected because Southern hybridisation revealed that each showed partial or complete specificity towards the strain of origin. A seventh genomic fragment showing a strain-specific distribution in Southern hybridisations was obtained by differential restriction, hybridisation and cloning of genomic DNA. Each of these clones was sequenced and primers to amplify the insert were designed. When DNA from the strain of origin was used as template, PCR amplification for each of these fragments yielded a single band on gel analysis. One pair of primers amplified the species-constant fragment of 281 bp from DNA of all B. solanacearum strains investigated, from DNA of the closely related bacterium which causes ''blood disease'' of banana (BDB) and in P. syzigii. The sensitivity of detection of B. solanacearum using these ubiquitous primers was between 1.3 and 20 bacterial cells. The feasibility and reliability of a PCR approach to detection and identification of B. solanacearum was tested in diverse strains of the bacterium in several countries and laboratories.

Quantum dynamics of an atomic Bose-Einstein condensate in a double-well potential

We consider the quantum dynamics of a neutral atom Bose-Einstein condensate in a double-well potential, including many-body hard-sphere interactions. Using a mean-field factorization we show that the coherent oscillations due to tunneling are suppressed when the number of atoms exceeds a critical value. An exact quantum solution, in a two-mode approximation, shows that the mean-field solution is modulated by a quantum collapse and revival sequence.

AP-1 and Egr DNA-binding activities are increased in rat brain during ethanol withdrawal

The DNA-binding activities of AP-1 and Egr proteins were investigated in nuclear extracts of rat brain regions during ethanol withdrawal. Both DNA-binding activities were transiently elevated in the hippocampus and cerebellum 16 h after withdrawal. In the cerebral cortex, AP-1 and Egr DNA-binding activities increased at 16 h and persisted until 32 and 72 h, respectively. The AP-1 DNA-binding activities in all regions at all times after withdrawal were composed of FosB, c-Jun, JunB, and JunD. c-Fos was detected at all times in the cerebral cortex, at 16 h only in the hippocampus, and from 16 to 72 h in the cerebellum. Withdrawal severity did not affect the composition of the AP-1 DNA-binding activities. Two Egr DNA-binding activities were present in the cortex and hippocampus. The faster-migrating complex predominated in hippocampus, and only the slower-migrating complex (identified as Egr-1) was present in the cerebellum. The increase in DNA-binding activity of immediate early gene-encoded transcription factors supports their proposed role in initiating a cascade of altered gene expression underlying the long-term neuronal response to ethanol withdrawal.