The effects of nonsteroidal anti-inflammatory drugs on clinical outcomes, synovial fluid cytokine concentration and signal transduction pathways in knee osteoarthritis. A randomized open label trial.

OBJECTIVE: We investigated the effects of celecoxib, diclofenac, and ibuprofen on the disease-specific quality of life, synovial fluid cytokines and signal transduction pathways in symptomatic knee osteoarthritis (OA). DESIGN: Ninety patients scheduled for a total knee arthroplasty (TKA) were randomized to six groups that were treated with low and high dosages of celecoxib, diclofenac or ibuprofen. At the time of the first admission (T0) and at surgery (T1 = 14 days after beginning of the nonsteroidal anti-inflammatory drugs (NSAIDs)), samples of knee synovial fluid were obtained from each patient for analysis. During the surgery the synovial tissue was harvested from the knee of patients. The Western Ontario and McMaster universities (WOMAC) score was used to evaluate the patient disease-specific quality of life at T0 and T1. Microarray tests performed at T0 and T1 were used to evaluate the effects of NSAIDs on Tumor necrosis factor (TNF)-alpha, Interleukin-6 (IL-6), IL8 and Vascular endothelial growth factor (VEGF) concentration in the synovial fluid. Western blot assays evaluated the effects of NSAIDs on MAP kinase (MAPK) signal transduction pathway in the synovial membrane. RESULTS: NSAID treatment induced a statistically significant improvement in the WOMAC score and a statistically significant decrease in the IL-6, VEGF and TNF-alpha concentration in the synovial fluid. Higher dosages of NSAIDs provided a greater improvement in the disease-specific quality of life of patients and lower concentrations of pro-inflammatory cytokines in the synovial fluid. Inhibition of MAPKs was noted after NSAID treatment. CONCLUSION: Short-term NSAID treatment improves the patient disease-specific quality of life with a parallel decrease in pro-inflammatory synovial fluid cytokine levels in knee OA. Signal transduction pathways may be involved in regulating the anti-inflammatory effects of NSAIDs. ClinicalTrial.gov: NCT01860833.

Postmenopausal women with carotid atherosclerosis: potential role of the serum calcium levels.

BACKGROUND AND AIM: Studies on the association between serum calcium levels and cardiovascular diseases suggested a causative role for hypercalcemia but other studies showed that even serum calcium levels within normal range could be involved in atherosclerosis. However, while dietary calcium intake does not seem to be related to adverse cardiovascular effects, the association between calcium supplementation and the cardiovascular events has not been fully proven. Our aim was to determine the relation between serum calcium levels, within normal range, and the presence of carotid atherosclerosis in a population in whom investigations on this topic are lacking, the postmenopausal women. METHODS AND RESULTS: In this retrospective study, participants were recruited from women aged 49-65 years who underwent an ultrasonography evaluation of the carotid arteries between years 2008-2012. The study included 413 subjects with serum calcium level available, without symptomatic cardiovascular disease. A physical examination, including the evaluation of body mass index, waist and hip circumferences and the blood pressure, as well as, a collection of a venous blood sample was performed. The mean age was 56 ± 7 years. The prevalence of the carotid atherosclerosis was 50.8%. The comparison between women with and without carotid atherosclerosis showed differences for the classical risk factors and for serum calcium levels (p = 0.001). The logistic regression analysis, adjusting for these risk factors, confirmed the association between serum calcium levels and carotid atherosclerosis (p = 0.011). Furthermore, we showed an increasing prevalence of carotid atherosclerosis from lower to higher calcium quartiles (p = 0.016). CONCLUSION: We found a positive relation between serum calcium levels and the carotid atherosclerosis in postmenopausal women. This study may suggest a redetermination of the reference range of calcemia, at least in menopause.

Metformin versus dietary treatment in nonalcoholic hepatic steatosis: a randomized study.

OBJECTIVE: We aimed at evaluating whether the addition of low-dose metformin to dietary treatment could be an effective approach in nondiabetic patients with nonalcoholic fatty liver disease (NAFLD). METHODS: We carried out a 6-month prospective study in a series of overweight or obese patients with ultrasonographic diagnosis of hepatic steatosis. In total, 50 patients were enrolled and randomized into two groups: the first group (n=25) was given metformin (1 g per day) plus dietary treatment and the second group (n=25) was given dietary treatment alone. RESULTS: At the end of the study, the proportion of patients with echographic evidence of fatty liver was reduced in both the metformin (P<0.0001) and the diet group (P=0.029). Moreover, patient body mass index and waist circumference significantly decreased in both groups (P<0.001). Fasting glucose, insulin resistance (evaluated as homeostasis model assessment of insulin resistance (HOMA-IR)) and serum adiponectin decreased in both groups, although these changes reached statistical significance only in the metformin group. In this group, HOMA-IR decreased from 3.3+/-1.6 to 2.4+/-1.2 (P=0.003), whereas it decreased from 3.2+/-1.6 to 2.8+/-1.1 (not significant, NS) in the diet group. Similarly, the proportion of patients with impaired fasting glucose declined from 35 to 5% (P=0.04) in the metformin and from 32 to 12% (NS) in the diet group. At baseline, approximately 40% of patients in both groups met the diagnostic criteria of metabolic syndrome. This proportion decreased to 20% in the metformin group (P=0.008) and to 32% in the diet group (NS). CONCLUSIONS: In our 6-month prospective study, both low-dose metformin and dietary treatment alone ameliorated liver steatosis and metabolic derangements in patients with NAFLD. However, metformin was more effective than dietary treatment alone in normalizing several metabolic parameters in these patients.

Inflammatory response to cardiopulmonary bypass with enoximone or steroids in patients undergoing myocardial revascularization: a preliminary report study.

OBJECTIVE: Recent reports have showed an antiinflammatory effect of phosphodiesterase III inhibitors (PDEi) in patients undergoing cardiopulmonary bypass (CPB). We sought to evaluate the immunological and hemodynamic response to enoximone and methylprednisolone in patients undergoing CABG. DESIGN: Prospective, randomized, controlled study. SETTING: Cardiac surgery unit in a university hospital. PATIENTS: 40 patients undergoing CPB-CABG. INTERVENTIONS: Patients receive enoximone (20, Group A) or methylprednisolone (20, Group B). MEASUREMENTS AND MAIN RESULTS: Hemodynamic response was evaluated by Swan-Ganz catheter serial measurements and perioperative Lactate and Troponin I leakage, immunological response was analyzed by IL-2, IL-4, IL-6, TNF-alpah, IFN-gamma, IL-10 before anesthetic induction (T0), at aortic-declamping (T1), at the end of surgery (T2), ITU admission (T3), 24 hs (T4) postoperatively. Morbidity and mortality were comparable between the two groups. Group A demonstrated higher cardiac index at T2 (2.93 l/min m2 vs 2.06, p < 0.001), at T3 (3.01 vs 2.18, p < 0.001), lower indexed systemic vascular resistance at T2 (2,044 dyne s cm-5 m-2 vs 3,132, p < 0.001). Except for higher TNF-alpha in Group B at T2 (15.89 vs 22.68, p = 0.005) proinflammatory cytokines were comparable. IL-10 was higher in Group B at any postoperative time (IL-10: T1 80.74 vs 143.3, p < 0.001, T2 165.7 vs 377.4, p < 0.001, T3 203.4 vs 443.5, p < 0,001, T4 251.8 vs 437.1, p < 0.001), whereas IL-4 and IFN-gamma proved higher in Group A at all time-points (IL-4: T1 45.9 vs 31.2, p = 0.008, T2 67.2 vs 39.7, p < 0.001, T3 77.9 vs 39.2, p < 0.001, T4 102.9 vs 42.2, p < 0.001. IFN-gamma: T1 25.8 vs 15.8, p < 0.001, T2 52.2 vs 30.3, p < 0.001, T3 78.4 vs 40.8, p < 0.001, T4 159.9 vs 67.4, p < 0.001). CONCLUSIONS: Despite comparable major clinical endpoints enoximone showed a different antiinflammatory pattern compared to methylprednisolone, however, the better hemodynamic response in enoximone compared to methylprednisolone suggests enoximone as a potential antiinflammatory tool to improve the outcome in cardiac surgery.

Inflammatory response after cardiopulmonary bypass: a randomized comparison between conventional hemofiltration and steroids.

AIM: Recent reports have shown anti-inflammatory effects with conventional hemofiltration (CUF) in patients undergoing cardiopulmonary bypass (CPB). The aim of this study was to evaluate the immunological and the hemodynamic response to CUF or metilprednisolone in patients undergoing coronary artery bypass grafting. METHODS: Twenty-four consecutive patients were prospectively randomized to receive CUF (12 patients, Group A) or metilprednisolone (12 patients, Group B). Hemodynamic response was evaluated by Swan-Ganz catheter, immunological response was analyzed by IL-2, IL-4, IL-6, TNF-alpha, IFN-gamma, IL-10 before anesthetic induction (T0), at aortic-declamping (T1), at the end of surgery (T2), ITU admission (T3) and 24 hours (T4). Troponin I was measured at the same time-points. Hematological and coagulative controls were performed. RESULTS: Morbidity and mortality were comparable between the two groups. Group A demonstrated lower cardiac index at T1 (2.1 +/- 0.69 L/min m2 vs. 3.917 +/- 1.28, P = 0.034) without significantly higher indexed-systemic-vascular-resistances at the end of surgery (1 101 +/- 434.3 dyne s cm(-5) m(-2) vs. 797.7 +/- 316.67, P = 0.233). When proinflammatory and anti-inflammatory cytokines were considered, all improved during the postoperative time course, without differences between the 2 Groups (P = NS). Hematological and coagulative data were similar in the two groups, in terms of white blood cells, platelets, prothrombin time, and activated partial thromboplastin time (P = NS). CONCLUSIONS: Anti-inflammatory action of CUF was comparable to steroids, thus determining a similar proinflammatory response to CPB. However, hemodynamics was slightly impaired by CUF. Therefore, there is no reason to prefer CUF to steroids in patients undergoing elective CABG.

Body perfusion during adult cardiopulmonary bypass is improved by pulsatile flow with intra-aortic balloon pump.

PURPOSE: To evaluate if the use of an intra-aortic balloon pump (IABP) during cardioplegic arrest improves body perfusion. METHODS: 158 coronary artery bypass graft (CABG) patients were randomized to linear cardiopulmonary bypass (CPB) (n=71, Group A) or automatic 80 bpm intra-aortic balloon pump (IABP) induced pulsatile CPB (n=87, Group B). We evaluated hemodynamic response by Swan-Ganz catheter, inflammation by cytokines, coagulation and fibrinolysis, transaminase, bilirubin, amylase, lactate and renal function (estimated glomerular filtration rate (eGFR), creatinine, and incidence of renal insufficiency and failure). RESULTS: IABP induced Surplus Hemodynamic Energy was 15.8-/+4.9 mmHg, with higher mean arterial pressure during cross-clamping (p=0.001), and lower indexed systemic vascular resistances during cross-clamping (p=0.001) and CPB discontinuation (p=0.034). IL-2 and IL-6 were lower, while IL-10 proved higher in Group B (p<0.05). Group B showed lower chest drainage (p<0.05), transfusions (p<0.05), INR (p<0.05), and AT-III (p=0.001), together with higher platelets, aPTT (p<0.05), fibrinogen (p<0.05) and D-dimer (p<0.05). Transaminases, bilirubin, amylase, lactate were lower in Group B (p<0.05); eGFR was better in Group B from ITU-arrival to 48 hours, both in preoperative kidney disease Stages 1-2 (p<0.03) and Stage 3 (p<0.05), resulting in lower creatinine from ITU-arrival to 48 hours (p<0.03). Incidence of renal insufficiency (p=0.004) and need for renal replacement therapy (p=0.044) was lower in Group B Stage 3. Group B PaO2/FiO2 and lung compliance improved from aortic declamping to the first day (p<0.003) with shorter intubation time (p=0.01). CONCLUSION: Pulsatile flow by IABP improves whole-body perfusion during CPB.

The insulin receptor: a new anticancer target for peroxisome proliferator-activated receptor-gamma (PPARgamma) and thiazolidinedione-PPARgamma agonists.

The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear hormone receptor superfamily. Ligand activation of PPARgamma is associated with differentiation and inhibition of proliferation in the normal and malignant cells. Herein, we studied the effects of PPARgamma and the PPARgamma agonists thiazolidinediones (TZDs) on the insulin receptor (IR), a cell membrane tyrosine kinase receptor protein, whose role is of paramount importance in mediating the metabolic and growth-promoting effects of the peptide hormone insulin. Overexpression of the PPARgamma1 in human hepatocellular (HepG2) cells was associated with decreased IR gene transcription and protein expression levels, and these reductions were more evident in the presence of TZDs. Since no PPARgamma response elements were identified on the IR promoter, we postulated that PPARgamma adversely affects the IR gene transcription by perturbing the assembly and stability of the transcriptionally active multiprotein-DNA complex identified previously, which includes the high-mobility group A1 protein, the ubiquitously expressed transcription factor (Sp1), the CAAT enhancer-binding protein (C/EBPbeta), and, in some cell lines, the developmentally regulated activator protein-2 (AP-2) transcription factor. Using glutathione S-transferase pull-down assays combined with electrophoretic mobility shift assay and chromatin immunoprecipitation, we demonstrated that by interacting with Sp1, C/EBPbeta, and AP-2, PPARgamma can prevent Sp1/AP-2 protein-protein association and inhibit binding of Sp1 and C/EBPbeta to DNA, thus reducing IR gene transcription. Our results demonstrate that IR is a new target gene of PPARgamma, and support a potential use of TZDs as anti-proliferative agents in selected neoplastic tissues overexpressing IRs.

The pleiotropic ts15 mutation of E. coli is an IS1 insertion in the rho structural gene.

Rho protein regulates transcription termination in E. coli. Some of the temperature-sensitive mutants defective in Rho protein, e.g., ts15, show remarkable pleiotropic phenotypes. The ts mutations map between the ilv and cya loci on the E. coli chromosome. We have cloned the gene that restores the wild-type phenotypes of these mutants. Genetic and biochemical characterizations have shown that the cloned DNA segment carries the structural gene for the Rho polypeptide. Analysis of the rhots15 mutation has revealed the presence of an IS1 insertion in the carboxy terminal segment of the rho cistron, thereby truncating the 52-kilodalton (kd) Rho polypeptide to a 50-kd size and also making it thermolabile. This provides an example of how an IS1 insertion mutation can cause a TS phenotype. We have also shown that the multiple phenotypes of the mutant cell, including the temperature sensitivity, are caused by a single mutation (rhots15::IS1) in the rho structural gene. How a rho structural gene mutation may cause such pleiotropy is discussed.

Relationship between angiotensin-converting enzyme gene polymorphism and insulin resistance in never-treated hypertensive patients.

The association between angiotensin-converting enzyme (ACE) gene polymorphism and insulin resistance (IR) in hypertensive subjects remains controversial. Thus, we evaluated the possible association between IR and ACE gene polymorphism in a group of hypertensive, never-treated patients compared with that in a normotensive control group. We enrolled 200 (114 men and 86 women; age, 45.5 +/- 4.7 yr) hypertensive patients and 96 (54 men and 42 women; age, 44.0 +/- 4.7 yr) normotensive subjects. A double PCR assay was used to identify ACE genotypes. We determined fasting glucose and insulin by the glucose oxidase method and using a standard RIA technique. IR was estimated using the homeostasis model assessment (HOMA(IR)). Both fasting glucose (5.0 +/- 0.3 vs. 4.7 +/- 0.3 mmol/L; P < 0.0001), insulin levels (12.3 +/- 4.7 vs. 4.9 +/- 1.5 muU/mL; P < 0.0001), and HOMA(IR) (2.7 +/- 1.1 vs. 1.1 +/- 0.3; P < 0.0001) were significantly higher in hypertensive patients than in the normotensive control group. When we subdivided hypertensive patients according to ACE genotype, we observed that fasting insulin and HOMA(IR) were 16.3 +/- 3.3 and 3.6 +/- 0.8 in the DD genotype, 9.4 +/- 3.1 and 2.1 +/- 0.7 in the ID genotype, and 8.3 +/- 2.8 and 1.9 +/- 0.7 muU/mL in the II group (P < 0.0001, by ANOVA). No significant differences were observed in the normotensive control group. In conclusion, we extended previous data regarding the relationship of hypertension and IR by demonstrating a dependence of this relationship upon the ACE gene polymorphism.

Recolonization of the subgingival microflora after scaling and root planing in human periodontitis.

The purpose of this study was to evaluate the recolonization patterns of the subgingival microflora of adult periodontitis patients after a single session of scaling and root planing. In each of eight patients, three clinically diseased sites were investigated microbiologically by darkfield microscopy and cultural analysis. After initial clinical and microbiological parameters were determined, each subject received a single session of scaling and root planing but no oral hygiene instructions. Clinical indices were measured and microbial parameters were reassessed 7, 21, and 60 days after treatment in a manner such that each of the test sites was sampled only once after treatment. Recolonization was evaluated by matching any single site with its own preoperative site. A significant improvement in probing depth was noted for up to 60 days after treatment, while the gingival index did not change markedly during the course of the study. The microbial composition of treated sites 7 days after scaling and root planing, as determined by both cultural and darkfield data, was similar to that of periodontally healthy sites. Differences between cultural and darkfield data became apparent at the 21 day sampling point. The darkfield data showed that the sites consisted of cocci with few spirochetes. Cultural data demonstrated that the majority of the cocci were anaerobic, namely Streptococcus intermedius, Veillonella parvula, and Peptostreptococcus micros. At 60 days, there was no significant variation in any of the parameters from pretreatment levels. The most prevalent anaerobic rods prior to and 60 days after therapy were Fusobacterium nucleatum, Bacteroides gingivalis, and B. intermedius.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of Chlamydia trachomatis 26K protein expressed in Escherichia coli.

Molecular genetics appears to be the most promising approach to understanding the biology and pathology of Chlamydia. This report focuses on the cloning and the protein expression of a DNA fragment from Chlamydia trachomatis DK20 chromosome. Results of hybridization experiments suggest that this sequence is specifically present within chlamydial DNA. The coding capacity of this DNA fragment is supported by the expression of a 26,000 m.w. peptide, in an Escherichia coli maxicell system.

High Th1-type cytokine serum levels in patients with infectious mononucleosis.

Most adults are asymptomatically infected with Epstein-Barr virus (EBV). Primary EBV infection is commonly associated with acute infectious mononucleosis (IM). T cell immune activation plays an important role in EBV-associated diseases. IM shows a mainly Th1-type profile, so Th1-type cytokines such as interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and lymphotoxin (LT) are moderately enhanced. We measured IL-2 and IFN-gamma in serum during acute phase of the disease and during convalescence. Sera were collected from 23 IM patients, 13 patients with similar clinical manifestations but without IM, and 10 healthy donors. The levels of IL-2, IFN-gamma and IL-12 were significantly higher in patients with acute IM than in healthy individuals. IL-2, IFN-gamma and IL-12 decreased during convalescence. These three cytokines may be useful as sensitive markers of IM during severe illness and its later phases.

Cloning of the RHO gene of Escherichia coli.

The map position of the rho gene on E coil chromosome, as determined by phage P1 generalized transduction, was determined. The gene order is ilv-rho-cya. By Hind III endonuclease shotgun technique, the rho+ gene was cloned into a lambda-Charon 25 vector. The sizing of the restriction endonuclease generated DNA fragments by agarose gel electrophoresis, the heteroduplex analysis of cloned DNA molecules by electron microscope and the demonstration of the synthesis of rho protein by the cloned DNA shows that a functional piece of DNA, containing the entire rho gene of E coli has been cloned.

Cloning and expression of the Escherichia coli rho gene in a plasmid vector.

In order to further elucidate the role of Rho protein on transcription termination and cells growth control, we have subcloned by two steps the rho+ structural gene of Escherichia coli from Lambda rho+524 into a plasmid vector. The resulting plasmid pEG25 contains a 2.9 kbp insert which is able to complement several different rho mutations and to express a functional Rho protein in U.V. irradiated maxicells.

Rapid diagnosis by immunofluorescence of Chlamydia trachomatis cervical infections.

In this study the MicroTrak (Syva Co.) system has been evaluated in comparison with the isolation of Chlamydia trachomatis onto McCoy cell monolayer, testing 120 endocervical samples taken from an unselected women population. The overall results show a higher incidence of positivity in women under 30 years of age; a sensitivity of the immunofluorescence of 76.3% and a specificity of 89.8%. The agreement between the two methods is significative when we test symptomatic patients.

Comparison of two systems for identification of anaerobic bacteria.

The RapID Ana and the API 20A systems for identification of anaerobic bacteria were compared for accuracy on 108 recent isolates of gram-negative and gram-positive anaerobic bacteria. No additional tests or gas-liquid chromatography were used. RapID Ana identified 91.7% of the isolated strains to species level and 6.5% to genus level but failed to identify 1.8% of the total strains. API 20A identified 85.2% of strains to species level, 3.7% to genus level but failed to identify 11.1% of the total strains. Although the difference between the identification rates of the two systems was not significant, it was concluded that RapID Ana was easier to perform and interpret.