Determination of fluoxetine and norfluoxetine enantiomers in human plasma by polypyrrole-coated capillary in-tube solid-phase microextraction coupled with liquid chromatography-fluorescence detection

A sensitive, selective, and reproducible in-tube polypyrrole-coated capillary (PPY) solid-phase microextraction and liquid chromatographic method for fluoxetine and norfluoxetine enantiomers analysis in plasma samples has been developed, validated, and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. Separation of the analytes was achieved with a Chiralcel OD-R column and a mobile phase consisting of potassium hexafluorophosphate 7.5 mM and sodium phosphate 0.25 M solution, pH 3.0, and acetonitrile (75:25, v/v) in the isocratic mode, at a flow rate of 1.0 mL/min. Detection was carried out by fluorescence absorbance at Ex/Em 230/290 nm. The multifunctional porous surface structure of the PPY-coated film provided high precision and accuracy for enantiomers. Compared with other commercial capillaries, PPY-coated capillary showed better extraction efficiency for all the analytes. The quantification limits of the proposed method were 10 ng/mL for R- and S-fluoxetine, and 15 ng/mL for R- and S-norfluoxetine, with a coefficient of variation lower than 13%. The response of the method for enantiomers is linear over a dynamic range, from the limit of quantification to 700ng/mL, with correlation coefficients higher than 0.9940. The in-tube SPME/LC method can therefore be successfully used to analyze plasma samples from ageing patients undergoing therapy with fluoxetine. (C) 2009 Elsevier B.V. All rights reserved.

Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved.

Mapping of suramin binding sites on the group IIA human secreted phospholipase A(2)

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A(2) (hsPLA(2)GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein. namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA(2)GIIA, and indicates that suramin represents a novel class of phosphohpase A(2) inhibitor. (C) 2009 Elsevier Inc. All rights reserved.

Effectiveness of the Combined Recombinant Human Growth Hormone and Gonadotropin-Releasing Hormone Analog Therapy in Pubertal Patients with Short Stature due to SHOX Deficiency

Context: Isolated heterozygous SHOX defects are the most frequent monogenic cause of short stature, and combined therapy with recombinant human GH (rhGH) and GnRH analog (GnRHa) in pubertal patients has been suggested, but there are no data on final height. Objective: The aim of the study was to analyze adult height after rhGH and GnRHa therapy in patients with SHOX haploinsufficiency. Patients: Ten peripubertal patients with isolated SHOX defects participated in the study. Intervention: Five patients were followed without treatment, and five were treated with rhGH (50 mu g/kg/d) and depot leuprolide acetate (3.75 mg/month). Main Outcome Measures: Adult height SD score (SDS) was measured. Results: All patients followed without treatment had marked downward growth shift during puberty (height SDS, -1.2 +/- 0.7 at 11.4 +/- 1.4 yr; adult height SDS, -2.5 +/- 0.5). Conversely, four of five patients treated with rhGH for 2 to 4.9 yr associated to GnRHa for 1.4 to 5.8 yr improved their height SDS from -2.3 +/- 1.3 at 11.8 +/- 2.1 yr to a final height SDS of -1.7 +/- 1.6. The difference between the mean height SDS at the first evaluation and final height SDS was statistically significant in nontreated vs. treated patients (mean height SDS change, -1.2 +/- 0.4 vs. 0.6 +/- 0.4, respectively; P < 0.001). Conclusion: A gain in adult height of patients with isolated SHOX defects treated with combined rhGH and GnRHa therapy was demonstrated for the first time, supporting this treatment for children with SHOX defects who have just started puberty to avoid the loss of growth potential observed in these patients during puberty. (J Clin Endocrinol Metab 95: 328-332, 2010)

In situ immune responses to interstitial pneumonitis in human visceral leishmaniasis

Lung disease during active human visceral leishmaniasis is frequently reported. As such, studies have associated pulmonary symptoms to interstitial pneumonitis with a mononuclear infiltrate. However, the immune response in this condition has never been described before. The aim of this study was to determine the immunophenotypic pattern and cytokine profile of lung involvement (IPL) in human visceral leishmaniasis. Quantitative methods of analysis were performed using immunohistochemistry, and were compared with a control group of normal lung. Interstitial macrophages and cd8 cells were increased in IPL, and IL-4 as well as TNF-alpha displayed increased expression when compared to the control group. This inflammatory process with a Th2 pattern, as suggested by increased IL-4 and low IFN-gamma expression, is consistent with the immune response in other organs of visceral leishmaniasis. The microenvironment of the immune response in this condition is associated with lung disease in patients with interstitial pneumonitis related to visceral leishmaniasis, increasing the chance of bacterial infection.

High Frequency of Lamivudine Resistance Mutations in Brazilian Patients Co-Infected With HIV and Hepatitis B

This study analyzed the genotype distribution and frequency of lamivudine (LAM) and tenofovir (TDF) resistance mutations in a group of patients co-infected with HIV and hepatitis B virus (HBV). A cross-sectional study of 847 patients with HIV was conducted. Patients provided blood samples for HBsAg detection. The load of HBV was determined using an ""in-house"" real-time polymerase chain reaction. HBV genotypes/subgenotypes, antiviral resistance, basal core promoter (BCP), and precore mutations were detected by DNA sequencing. Twenty-eight patients with co-infection were identified. The distribution of HBV genotypes among these patients was A (n = 9; 50%), D (n = 4; 22.2%), G (n = 3; 16.7%), and F (n = 2; 11.1%). Eighteen patients were treated with LAM and six patients were treated with LAM plus TDF. The length of exposure to LAM and TDF varied from 4 to 216 months. LAM resistance substitutions (rtL180M + rtM204V) were detected in 10 (50%) of the 20 patients with viremia. This pattern and an accompanying rtV173L mutation was found in four patients. Three patients with the triple polymerase substitution pattern (rtV173L+ rtL180M + rtM204V) had associated changes in the envelope gene (sE164D + sl195M). Mutations in the BCP region (A1762T, G1764A) and in the precore region (G1896A, G1899A) were also found. No putative TDF resistance substitution was detected. The data suggest that prolonged LAM use is associated with the emergence of particular changes in the HBV genome, including substitutions that may elicit a vaccine escape phenotype. No putative TDF resistance change was detected after prolonged use of TDF. J. Med. Virol. 82:1481-1488, 2010. (C) 2010 Wiley-Liss, Inc.

Reconstruction of large cranial defects in nonimmunosuppressed experimental design with human dental pulp stem cells

The main aim of this study is to evaluate the capacity of human dental pulp stem cells (hDPSC), isolated from deciduous teeth, to reconstruct large-sized cranial bone defects in nonimmunosuppressed (NIS) rats. To our knowledge, these cells were not used before in similar experiments. We performed two symmetric full-thickness cranial defects (5 x 8 mm) on each parietal region of eight NIS rats. In six of them, the left side was supplied with collagen membrane only and the right side (RS) with collagen membrane and hDPSC. In two rats, the RS had collagen membrane only and nothing was added at the left side (controls). Cells were used after in vitro characterization as mesenchymal cells. Animals were euthanized at 7, 20, 30, 60, and 120 days postoperatively and cranial tissue samples were taken from the defects for histologic analysis. Analysis of the presence of human cells in the new bone was confirmed by molecular analysis. The hDPSC lineage was positive for the four mesenchymal cell markers tested and showed osteogenic, adipogenic, and myogenic in vitro differentiation. We observed bone formation 1 month after surgery in both sides, but a more mature bone was present in the RS. Human DNA was polymerase chain reaction-amplified only at the RS, indicating that this new bone had human cells. The us e of hDPSC in NIS rats did not cause any graft. rejection. Our findings suggest that hDPSC is an additional cell resource for correcting large cranial defects in rats and constitutes a promising model for reconstruction of human large cranial defects in craniofacial surgery.

Is procalcitonin useful to differentiate rejection from bacterial infection in the early post-operative period of liver transplantation in children?

PCT is a protein that is recognized as an acute marker of inflammation. Previous studies performed in adults who underwent liver or heart transplantation indicated that PCT plasmatic levels help to differentiate between rejection and infection. The objective of this study was to evaluate whether PCT has the same role in liver-transplanted children. Thirty-six patients were studied between the first and the thirtieth post-operative days, and PCT determinations were prospectively performed according to the clinical status of the patient. In the non-complicated patients, PCT measurements performed on the first and second post-operative days revealed a median value of 1.60 ng/mL (mean 5.68 +/- 7.05; range 0.69-18.30). After the fourth day of transplantation, PCT plasma concentrations decreased to a median value of 0.21 ng/mL (mean 0.47 +/- 0.59; range 0.05-2.00; normal values are less than 0.5 ng/mL). In infected patients, PCT plasma levels demonstrated a significant increase, differing from the patients with acute liver rejection whose levels were similar to those of non-complicated patients. In conclusion, we could demonstrate that in the early post-operative period of liver transplantation in children, measuring PCT plasmatic levels might be a useful tool for differentiation between bacterial infection and acute liver rejection.

Serum from children with polyarticular juvenile idiopathic arthritis (pJIA) inhibits differentiation, mineralization and may increase apoptosis of human osteoblasts ""in vitro""

We examined the effects of polyarticular juvenile idiopathic arthritis (pJIA) serum on proliferation, differentiation, mineralization, and apoptosis of human osteoblast cells (hOb) in culture. The hOb were cultured with 10% serum from active pJIA and healthy controls (CT) and were tested for DNA synthesis, alkaline phosphatase (AP) activity, osteocalcin (OC) secretion, calcium levels, caspase 3 activity, and DNA fragmentation. None of the patients had used glucocorticoids for at least 1 month before the study, or any other drug that can affect bone mineral metabolism. Human inflammatory cytokine levels (IL-6, IL-8, IL-10, IL-1 beta, TNF-alpha, and IL-12p70) were measured in pJIA and CT sera. Low levels of AP activity was observed in pJIA cultures compared with CT cultures (67.16 +/- 53.35 vs 100.11 +/- 50.64 mu mol p-nitrophenol/h(-1) mg(-1) protein, P=0.008). There was also a significant decrease in OC secretion (9.23 +/- 5.63 vs 12.82 +/- 7.02 ng/mg protein, P=0.012) and calcium levels (0.475 +/- 0.197 vs 0.717 +/- 0.366 mmol/l, P=0.05) in pJIA hOb cultures. No difference was observed in cell proliferation (323.56 +/- 108.23 vs 328.91 +/- 88.03 dpm/mg protein, P=0.788). Osteoblasts cultured with JIA sera showed lower levels of DNA and increased fragmentation than osteoblasts cultured with CT sera. pJIA sera showed higher IL-6 values than CT (21.44 +/- 9.31 vs 3.58 +/- 2.38 pg/ml, P<0.001), but no difference was observed related to IL-8, IL-10, IL-1 beta, TNF-alpha, and IL-12p70 between pJIA and controls. This study suggests that serum from children with pJIA inhibits differentiation, mineralization and may increase apoptosis of hOb cultures, and inflammatory cytokines such as IL-6 might be a mechanism in this find. These results may represent an alternative therapeutic target for prevention and treatment of bone loss in JIA.

HUMAN CHOLESTERYL ESTER TRANSFER PROTEIN EXPRESSION ENHANCES THE MOUSE SURVIVAL RATE IN AN EXPERIMENTAL SYSTEMIC INFLAMMATION MODEL: A NOVEL ROLE FOR CETP

Mice expressing human cholesteryl ester transfer protein (huCETP) are more resistant to Escherichia coli bacterial wall LIPS because death rates 5 days after intraperitoneal inoculation of LIPS were higher in wild-type than in huCETP(+/-) mice, whereas all huCETP(+/+) mice remained alive. After LIPS inoculation, plasma concentrations of TNF-alpha and IL-6 increased less in huCETP(+/+) than in wild-type mice. LPS in vitro elicited lower TNF-alpha production by CETP expressing than by wild-type macrophages. In addition, TNF-alpha production by RAW 264.7 murine macrophages increased on incubation with LPS but decreased in a dose-dependent manner when human CETP was added to the medium. Human CETP in vitro enhanced the LIPS binding to plasma high-density lipoprotein/low-density lipoprotein. The liver uptake of intravenous infused C-14-LPS from Salmonella typhimurium was greater in huCETP(+/+) than in wild-type mice. Present data indicate for the first time that CETP is an endogenous component involved in the first line of defense against an exacerbated production of proinflammatory mediators.

Peptides containing T cell epitopes, derived from Sm14, but not from paramyosin, induce a Th1 type of immune response, reduction in liver pathology and partial protection against Schistosoma mansoni infection in mice

Sm14 and paramyosin are two major Schistosoma mansoni vaccine candidate antigens. Recently, we have identified Sm14 and paramyosin epitopes that are recognized by T cells of resistant individuals living in endemic areas for schistosomiasis. Herein, mice were immunized with these peptides separately or in association in order to evaluate their vaccine potential. Immunization of mice with Sm14 peptides alone or mixed with paramyosin peptides was able to induce 26%-36.7% or 28%-29.2% of worm burden reduction, 67% or 46% of intestinal eggs reduction and also 54%-61% or 43%-52% of liver pathology reduction, respectively. Protection was associated with a Th1 type of immune response induced by Sm14 peptide immunization. In contrast, paramyosin peptide vaccination did not engender protective immunity or liver pathology reduction and immunization was associated with a Th2 type of immune response. (C) 2008 Elsevier B.V. All rights reserved.

CD4+T cells from HIV-1-infected patients recognize wild-type and mutant human immunodeficiency virus-1 protease epitopes

P>Human immunodeficiency virus (HIV)-1 protease is a known target of CD8+ T cell responses, but it is the only HIV-1 protein in which no fully characterized HIV-1 protease CD4 epitopes have been identified to date. We investigated the recognition of HIV-1 protease by CD4+ T cells from 75 HIV-1-infected, protease inhibitor (PI)-treated patients, using the 5,6-carboxyfluorescein diacetate succinimidyl ester-based proliferation assay. In order to identify putative promiscuous CD4+ T cell epitopes, we used the TEPITOPE algorithm to scan the sequence of the HXB2 HIV-1 protease. Protease regions 4-23, 45-64 and 73-95 were identified; 32 sequence variants of the mentioned regions, encoding frequent PI-induced mutations and polymorphisms, were also tested. On average, each peptide bound to five of 15 tested common human leucocyte antigen D-related (HLA-DR) molecules. More than 80% of the patients displayed CD4+ as well as CD8+ T cell recognition of at least one of the protease peptides. All 35 peptides were recognized. The response was not associated with particular HLA-DR or -DQ alleles. Our results thus indicate that protease is a frequent target of CD4+ along with CD8+ proliferative T cell responses by the majority of HIV-1-infected patients under PI therapy. The frequent finding of matching CD4+ and CD8+ T cell responses to the same peptides may indicate that CD4+ T cells provide cognate T cell help for the maintenance of long-living protease-specific functional CD8+ T cells.

Subclinic atherosclerosis and cardiovascular risk factors in HIV-infected children: PERI study

Objectives To compare carotid intima-media thickness (cIMT) of children and adolescents with and without HIV infection and to determine associations among independent socio-demographic, clinical or cardiovascular variables and cIMT in HIV-infected children and adolescents. Patients and methods This is a matched case-control study comparing 83 HIV-infected and 83 healthy children and adolescents. Clinical and laboratorial parameters, cIMT and echocardiogram were measured. Results The cIMT was higher in HIV-infected individuals (median 480 mu m; interquartile range 463-518 mu m) compared with controls (426 mu m; range 415-453 mu m, P < 0.001). In addition, the HIV-infected group showed higher levels of high-sensitive C-reactive protein (medians 1.0 mg/l vs. 0.4 mg/l, P < 0.001), glycated hemoglobin (6.1 +/- 0.9 vs. 5.7 +/- 0.8%, P= 0.028) and triglycerides (medians 0.9 vs. 0.8 mmol/l, P= 0.031). Finally, this group showed lower levels of total and high-density lipoprotein-cholesterol. After multivariate analysis, increased cIMT was positively associated with stavudine use [odds ratio (OR): 18.9, P=0.005], left atrial/aorta index (OR: 15.6, P=0.019), suprailiac skinfold (OR: 7.9, P=0.019), tachypnea (OR: 5.9, P=0.031), CD8 lymphocyte count (OR: 5.7, P=0.033) and CD4 T-lymphocyte count (OR: 5.5, P=0.025). cIMT increment was negatively associated with total cholesterol (OR: 0.2, P=0.025) and with CD8 zenith (OR: 0.1, P=0.007). Conclusion In this sample of children and adolescents, having HIV infection was associated with increased cIMT and elevated prevalence of cardiovascular risk factors. These findings suggest that this group should be included in cardiovascular prevention programs.

Treatment with trastuzumab for 1 year after adjuvant chemotherapy in patients with HER2-positive early breast cancer: a 4-year follow-up of a randomised controlled trial

Background Treatment with adjuvant trastuzumab for 1 year improves disease-free survival and overall survival in patients with human epidermal growth factor receptor 2 (HER2)-positive early breast cancer. We aimed to assess disease-free survival and overall survival after a median follow-up of 4 years for patients enrolled on the Herceptin Adjuvant (HERA) trial. Methods The HERA trial is an international, multicentre, randomised, open-label, phase 3 trial comparing treatment with trastuzumab for 1 and 2 years with observation after standard neoadjuvant, adjuvant chemotherapy, or both in patients with HER2-positive early breast cancer. The primary endpoint was disease-free survival. After a positive first interim analysis at a median follow-up of 1 year for the comparison of treatment with trastuzumab for 1 year with observation, event-free patients in the observation group were allowed to cross over to receive trastuzumab. We report trial outcomes for the 1-year trastuzumab and observation groups at a median follow-up of 48.4 months (IQR 42.0-56.5) and assess the effect of the extensive crossover to trastuzumab. Our analysis was by intention-to-treat. The HERA trial is registered with the European Clinical Trials Database, number 2005-002385-11. Findings The HERA trial population comprised 1698 patients randomly assigned to the observation group and 1703 to the 1-year trastuzumab group. Intention-to-treat analysis of disease-free survival showed a significant benefit in favour of patients in the 1-year trastuzumab group (4-year disease-free survival 78.6%) compared with the observation group (4-year disease-free survival 72.2%; hazard ratio [HR] 0.76; 95% CI 0.66-0.87; p<0.0001). Intention-to-treat analysis of overall survival showed no significant difference in the risk of death (4-year overall survival 89.3% vs 87.7%, respectively; HR 0.85; 95% CI 0.70-1.04; p=0.11). Overall, 885 patients (52%) of the 1698 patients in the observation group crossed over to receive trastuzumab, and began treatment at median 22.8 months (range 4.5-52.7) from randomisation. In a non-randomised comparison, patients in the selective-crossover cohort had fewer disease-free survival events than patients remaining in the observation group (adjusted HR 0.68; 95% CI 0.51-0.90; p=0.0077). Higher incidences of grade 3-4 and fatal adverse events were noted on 1-year trastuzumab than in the observation group. The most common grade 3 or 4 adverse events, each in less than 1% of patients, were congestive cardiac failure, hypertension, arthralgia, back pain, central-line infection, hot flush, headache, and diarrhoea. Interpretation Treatment with adjuvant trastuzumab for 1 year after chemotherapy is associated with significant clinical benefit at 4-year median follow-up. The substantial selective crossover of patients in the observation group to trastuzumab was associated with improved outcomes for this cohort.

White matter microstructure underlying default mode network connectivity in the human brain

Resting state functional magnetic resonance imaging (fMRI) reveals a distinct network of correlated brain function representing a default mode state of the human brain The underlying structural basis of this functional connectivity pattern is still widely unexplored We combined fractional anisotropy measures of fiber tract integrity derived from diffusion tensor imaging (DTI) and resting state fMRI data obtained at 3 Tesla from 20 healthy elderly subjects (56 to 83 years of age) to determine white matter microstructure e 7 underlying default mode connectivity We hypothesized that the functional connectivity between the posterior cingulate and hippocampus from resting state fMRI data Would be associated with the white matter microstructure in the cingulate bundle and fiber tracts connecting posterior cingulate gyrus With lateral temporal lobes, medial temporal lobes, and precuneus This was demonstrated at the p<0001 level using a voxel-based multivariate analysis of covariance (MANCOVA) approach In addition, we used a data-driven technique of joint independent component analysis (ICA) that uncovers spatial pattern that are linked across modalities. It revealed a pattern of white matter tracts including cingulate bundle and associated fiber tracts resembling the findings from the hypothesis-driven analysis and was linked to the pattern of default mode network (DMN) connectivity in the resting state fMRI data Out findings support the notion that the functional connectivity between the posterior cingulate and hippocampus and the functional connectivity across the entire DMN is based oil distinct pattern of anatomical connectivity within the cerebral white matter (C) 2009 Elsevier Inc All rights reserved