Diabetes mellitus affects the biomechanical function of the callus and the expression of TGF-beta1 and BMP2 in an early stage of fracture healing

Transforming growth factor beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) are important regulators of bone repair and regeneration. In this study, we examined whether TGF-β1 and BMP-2 expressions were delayed during bone healing in type 1 diabetes mellitus. Tibial fractures were created in 95 diabetic and 95 control adult male Wistar rats of 10 weeks of age. At 1, 2, 3, 4, and 5 weeks after fracture induction, five rats were sacrificed from each group. The expressions of TGF-β1 and BMP2 in the fractured tibias were measured by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction, weekly for the first 5 weeks post-fracture. Mechanical parameters (bending rigidity, torsional rigidity, destruction torque) of the healing bones were also assessed at 3, 4, and 5 weeks post-fracture, after the rats were sacrificed. The bending rigidity, torsional rigidity and destruction torque of the two groups increased continuously during the healing process. The diabetes group had lower mean values for bending rigidity, torsional rigidity and destruction torque compared with the control group (P<0.05). TGF-β1 and BMP-2 expression were significantly lower (P<0.05) in the control group than in the diabetes group at postoperative weeks 1, 2, and 3. Peak levels of TGF-β1 and BMP-2 expression were delayed by 1 week in the diabetes group compared with the control group. Our results demonstrate that there was a delayed recovery in the biomechanical function of the fractured bones in diabetic rats. This delay may be associated with a delayed expression of the growth factors TGF-β1 and BMP-2.

Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts

The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease.

Increased expression of "peripheral-type" benzodiazepine receptors in human temporal lobe epilepsy: implications for PET imaging of hippocampal sclerosis.

Increased binding sites for "peripheral-type" benzodiazepine receptor (PTBR) ligands have been described in a wide range of neurological disorders including both human and experimental epilepsy. This study was undertaken to assess PTBR expression in relation to the presence of hippocampal sclerosis in human temporal lobe epilepsy (TLE). For this purpose, hippocampal CA1 subfields were dissected from surgical samples from patients with therapy-refractive TLE with (n = 5) or without (n = 2) hippocampal sclerosis and from age-matched nonepileptic postmortem controls (n = 5). PTBR expression was assessed by immunohistochemistry and reverse-transcription polymerase chain reaction. Receptor sites were evaluated using an in vitro binding assay and the selective PTBR ligand [3H]PK11195. Epileptic patients with hippocampal sclerosis showed increases in PTBR binding sites, immunoreactivity, and mRNA expression compared to both nonsclerotic TLE patients and postmortem nonepileptic controls. Induction of PTBR expression and binding sites were directly correlated with the presence of hippocampal sclerosis and the accompanying reactive gliosis.

La variante c.-9C>G del promotor de BMP15 está asociadad a la etiología de la falla ovárica prematura no-sindrómica

La falla ovárica prematura es una enfermedad común que conduce a la infertilidad femenina y cuya etiología no es identificable en más del 50% de los casos, lo cual sugiere un origen genético en la patogénesis de esta enfermedad. La función crucial del gen BMP15 en la biología de la reproducción fue propuesta cuando modelos KO de ratón y mutaciones naturales en ovejas revelaron fenotipos ováricos específicos. Aunque la secuenciación de la región codificante de BMP15 en grandes paneles de pacientes afectadas con Falla Ovárica Prematura (FOP) ha identificado algunas mutaciones, estas variaciones explican una baja proporción de casos. Nosotros hipotetizamos que una variante en la secuencia reguladora (promotor BMP15) podrían estar asociada a la etiología de la FOP no-sindrómica. Con la evidencia de los estudios previos que sugirieron la potencial implicación de la variante de secuencia c.-9C>G del promotor de BMP15 en los fenotipos reproductivos incluyendo FOP, evaluamos si este polimorfismo podría modificar las propiedades de transactivación de un factor de transcripción específico. Empleamos aproximaciones in-silico para predecir potenciales sitios de unión a factores de transcripción (TFBS: Transcription Factor Binding Sites) en la región 5´ de BMP15. El ensayo reportero de luciferasa se usó para determinar la modificación de la transacativación del promotor de BMP15 causada por la variante de c-9C>G. Se demostró que aunque los dos constructos del promotor de BMP15 (BMP15- prom-G and BMP15-prom-C) fueron transactivados por el factor de transcripción PITX1, el constructo BMP15- prom-G aumentó 1.6 veces la actividad transcripcional del factor de transcripción de una manera estadísticamente significativa. Por otro lado, se demostró por primera vez que BMP15 y PITX1 son co-expresados en tejido ovárico de humano y de ratón.

Arterial disease and thrombosis in the antiphospholipid syndrome (APS): A pathogenic role for endothelin-1 (ET-1)

Objective To explore a possible correlation between endothelin 1 (ET-1), the most potent endothelium-derived contracting factor that modulates vascular smooth muscle tone, and arterial disease in patients with the antiphospholipid syndrome (APS). Methods Plasma levels of ET-1 were measured in APS patients with (n = 16) and without (n = 11) arterial thrombosis and in non-APS patients with arterial thrombosis (n = 9). In addition, steady-state prepro-ET-1 messenger RNA (mRNA) levels were determined in endothelial cells treated with a range of human monoclonal anticardiolipin antibodies (aCL) (as anti-β2-glycoprotein I antibodies) by semiquantitative 32P-dCTP-labeled reverse transcription-polymerase chain reaction. Results Compared with healthy controls, markedly increased plasma levels of ET-1 were found in APS patients with arterial thrombosis (2.00 ± 0.87 versus 0.96 ± 0.37 pg/ml; P = 0.0001) but not in other groups. Three human monoclonal aCL induced prepro-ET-1 mRNA levels significantly more than did control monoclonal antibody lacking aCL activity. Conclusion Plasma ET-1 levels correlated significantly with a history of arterial thrombosis in patients with APS. Prepro-ET-1 mRNA was induced by human monoclonal aCL in the in vitro experimental system. The induction of ET-1 by antiphospholipid antibodies might contribute to increased arterial tone, leading to vasospasm and, ultimately, to arterial occlusion.

Expression and regulation of 80K/MARCKS, a major substrate of protein kinase C, in the developing rat heart

Objective: Protein kinase C (PKC) plays a pivotal role in modulating the growth and differentiation of many cell types including the cardiac myocyte. However, little is known about molecules that act immediately downstream of PKC in the heart. In this study we have investigated the expression of 80K/MARCKS, a major PKC substrate, in whole ventricles and in cardiac myocytes from developing rat hearts. Methods: Poly A+ RNA was prepared from neonatal (2-day) and adult (42-day) cardiac myocytes and whole ventricular tissue and mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) using primers designed to identify a 420 bp fragment in the 80K/MARCKS gene. Protein extracts were prepared from either 2-day and 42-day cardiac myocytes or from whole ventricular tissue at 2, 5–11, 14, 17, 21, 28 and 42 days of age. Protein expression was determined by immunoblotting with an 80K/MARCKS antipeptide antibody and PKC activity was determined by measuring the amount of γ32P-ATP transferred to a specific peptide substrate. Results: RT-PCR analysis of 80K/MARCKS mRNA in neonatal (2-day) and adult (42-day) cardiac myocytes showed the expression of this gene in both cell types. Immunoblotting revealed maximum 80K/MARCKS protein expression in whole ventricular tissue at 5 days (a 75% increase above values at 2 days), followed by a transient decrease in expression during the 6–8-day period (61% of the protein expressed at 2 days for 8-day tissue) with levels returning to 5 day levels by 11 days of age. 80K/MARCKS protein was present in cardiac myocytes at 2 days of age whereas it was not detectable in adult cells. In addition, PKC activity levels increased to 160% of levels present at 2 days in 8-day-old ventricles with PKC activity levels returning to 5-day levels by 9 days of age. This was then followed by a steady decline in both 80K/MARCKS protein expression and PKC activity through to adulthood. Conclusions: Expression of the PKC substrate, 80K/MARCKS, in cardiac myocytes changes significantly during development and the transient loss of immunoreactive protein during the 6–8-day developmental period may reflect 80K/MARCKS phosphorylation and subsequent down-regulation as a result of the concomitant up-regulation of PKC activity at this time.

Cardiac Na+/H+ exchanger during post-natal development in the rat: Changes in mRNA expression and sarcolemmal activity

We examined Na+–H+exchanger isoform 1 (NHE-1) mRNA expression in ventricular myocardium and its correlation with sarcolemmal NHE activity in isolated ventricular myocytes, during postnatal development in the rat. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA did not change in ventricular myocardium between 2 and 42 days after birth. Therefore, at seven time points within that age range, GAPDH expression was used to normalize NHE-1 mRNA levels, as determined by reverse transcription polymerase chain reaction analysis. There was a progressive five-fold reduction in NHE-1 mRNA expression in ventricular myocardium from 2 days to 42 days of age. As an index of NHE activity, acid efflux rates (JH) were determined in single neonatal (2–4-day-old) and adult (42-day-old) ventricular myocytes (n=16/group) loaded with the pH fluoroprobe carboxy-seminaphthorhodafluor-1. In HEPES-buffered medium, basal intracellular pH (pHi) was similar at 7.28±0.02 in neonatal and 7.31±0.02 in adult myocytes, but intrinsic buffering power was lower in the former age group. The rate at which pHirecovered from a similar acid load was significantly greater in neonatal than in adult myocytes (0.36±0.07v0.16±0.02 pH units/min at pHi=6.8). This was reflected by a significantly greaterJH(22±4v9±1 pmol/cm2/s at pHi=6.8), indicating greater sarcolemmal NHE activity in neonatal myocytes. The concomitant reductions in tissue NHE-1 mRNA expression and sarcolemmal NHE activity suggest that myocardial NHE-1 is subject to regulation at the mRNA level during postnatal development.

Presence and bronchomotor activity of protease-activated receptor-2 in guinea pig airways

The protease activated receptor-2 (PAR-2) belongs to a family of G-protein-coupled receptors that are activated by proteolysis. Trypsin cleaves PAR-2, exposing an N-terminal tethered ligand (SLIGRL) that activates the receptor. Messenger RNA (mRNA) for PAR-2 was found in guinea pig airway tissue by reverse transcription-polymerase chain reaction, and PAR-2 was found by immunohistochemistry in airway epithelial and smooth-muscle cells. In anesthetized guinea pigs, trypsin and SLIGRL-NH(2) (given intratracheally or intravenously) caused a bronchoconstriction that was inhibited by the combination of tachykinin-NK(1) and -NK(2) receptor antagonists and was potentiated by inhibition of nitric oxide synthase (NOS). Trypsin and SLIGRL-NH(2) relaxed isolated trachea and main bronchi, and contracted intrapulmonary bronchi. Relaxation of main bronchi was abolished or reversed to contraction by removal of epithelium, administration of indomethacin, and NOS inhibition. PAR-1, PAR-3, and PAR-4 were not involved in the bronchomotor action of either trypsin or SLIGRL-NH(2), because ligands of these receptors were inactive either in vitro or in vivo, and because thrombin (a PAR-1 and PAR-3 agonist) did not show cross-desensitization with PAR-2 agonists in vivo. Thus, we have localized PAR-2 to the guinea-pig airways, and have shown that activation of PAR-2 causes multiple motor effects in these airways, including in vivo bronchoconstriction, which is in part mediated by a neural mechanism.

Schwann cells can be reprogrammed to multipotency by culture

Adult neural crest related-stem cells persist in adulthood, making them an ideal and easily accessible source of multipotent cells for potential clinical use. Recently, we reported the presence of neural crest-related stem cells within adult palatal ridges, thus raising the question of their localization in their endogenous niche. Using immunocytochemistry, reverse transcription-polymerase chain reaction, and correlative fluorescence and transmission electron microscopy, we identified myelinating Schwann cells within palatal ridges as a putative neural crest stem cell source. Palatal Schwann cells expressed nestin, p75(NTR), and S100. Correlative fluorescence and transmission electron microscopy revealed the exclusive nestin expression within myelinating Schwann cells. Palatal neural crest stem cells and nestin-positive Schwann cells isolated from adult sciatic nerves were able to grow under serum-free conditions as neurospheres in presence of FGF-2 and EGF. Spheres of palatal and sciatic origin showed overlapping expression pattern of neural crest stem cell and Schwann cell markers. Expression of the pluripotency factors Sox2, Klf4, c-Myc, Oct4, the NF-κB subunits p65, p50, and the NF-κB-inhibitor IκB-β were up-regulated in conventionally cultivated sciatic nerve Schwann cells and in neurosphere cultures. Finally, neurospheres of palatal and sciatic origin were able to differentiate into ectodermal, mesodermal, and endodermal cell types emphasizing their multipotency. Taken together, we show that nestin-positive myelinating Schwann cells can be reprogrammed into multipotent adult neural crest stem cells under appropriate culture conditions.

Sole infection by human metapneumovirus among children with radiographically diagnosed community-acquired pneumonia in a tropical region

Background Limited information is available on the role of human metapneumovirus (HMPV) as the unique pathogen among children hospitalized for community-acquired pneumonia (CAP) in a tropical region. Objective We aimed to describe HMPV infection among children with CAP investigating bacterial and viral co-infections. Patients and methods A prospective study was carried out in Salvador, North-East Brazil. Overall, 268 children aged <5 years hospitalized for CAP were enrolled. Human metapneumovirus RNA was detected in nasopharyngeal aspirates (NPA) by reverse transcription polymerase chain reaction. Sixteen other bacterial and viral pathogens were investigated by an expanded panel of laboratory methods. Chest X-ray taken on admission was read by an independent paediatric radiologist unaware of clinical information or the established aetiology. Results Human metapneumovirus RNA was detected in NPAs of 11 (4.1%) children, of which 4 (36%) had sole HMPV infection. The disease was significantly shorter among patients with sole HMPV infection in comparison with patients with mixed infection (4 +/- 1 versus 7 +/- 2 days, P = 0.03). Three of those four patients had alveolar infiltrates. Conclusion Sole HMPV infection was detected in children with CAP in Salvador, North-East Brazil. HMPV may play a role in the childhood CAP burden.

Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.

Short-Term Modulation of Extracellular Signal-Regulated Kinase 1/2 and Stress-Activated Protein Kinase/c-Jun NH2-Terminal Kinase in Pancreatic Islets by Glucose and Palmitate Possible Involvement of Ceramide

Objectives: The effect of glucose and palmitate on the phosphorylation of proteins associated with cell growth and survival (extracellular signal-regulated kinase 1/2 [ERK1/2] and stress-activated protein kinase/c-Jun NH2-terminal kinase [SAPK/JNK]) and on the expression of immediate early genes was investigated. Methods: Groups of freshly isolated rat pancreatic islets were incubated in 10-mmol/L glucose with palmitate, LY294002, or fumonisin B1 for the measurement of the phosphorylation and the content of ERK1/2, JNK/SAPK, and v-akt murine thymoma viral oncongene (AKT) (serine 473) by immunoblotting. The expressions of the immediate early genes, c-fos and c-jun, were evaluated by reverse transcription-polymerase chain reaction. Results: Glucose at 10 mmol/L induced ERK1/2 and AKT phosphorylations and decreased SAPK/JNK phosphorylation. Palmitate (0.1 mmol/L) abolished the glucose effect on ERK1/2, AKT, and SAPK/JNK phosphorylations. LY294002 caused a similar effect. The inhibitory effect of palmitate on glucose-induced ERK1/2 and AKT phosphorylation changes was not observed in the presence of fumonisin B1. Glucose increased c-fos and decreased c-jun expressions. Palmitate and LY294002 abolished these latter glucose effects. The presence of fumonisin B1 abolished the effect induced by palmitate on c-jun expression. Conclusions: Our results suggest that short-term changes of mitogen-activated protein kinase and AKT signaling pathways and c-fos and c-jun expressions caused by glucose are abolished by palmitate through phosphatidylinositol 3-kinase inhibition via ceramide synthesis.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Hospital-based Surveillance to Evaluate the Impact of Rotavirus Vaccination in Sao Paulo, Brazil

Background: Brazil implemented routine immunization with the human rotavirus vaccine, Rotarix, in 2006 and vaccination coverage reached 81% in 2008 in Sao Paulo. Our aim was to assess the impact of immunization on the incidence of severe rotavirus acute gastroenteritis (AGE). Methods: We performed a 5-year (2004-2008) prospective surveillance at a sentinel hospital in Sao Paulo, with routine testing for rotavirus in all children less than 5 years of age hospitalized with AGE. Genotypes of positive samples were determined by reverse transcription polymerase chain reaction. Results: During the study, 655 children hospitalized with AGE were enrolled; of whom 169 (25.8%) were positive for rotavirus. In the post-vaccine period, a 59% reduction in the number of hospitalizations of rotavirus AGE and a 42.2% (95% confidence interval [CI], 18.6%-59.0%; P = 0.001) reduction in the proportion of rotavirus-positive results among children younger than 5 years were observed, with the greatest decline among infants (69.2%; 95% CI, 24.7%-87.4%; P = 0.004). Furthermore, the number of all-cause hospitalizations for AGE was reduced by 29% among children aged <5 years. The onset and peak incidences of rotavirus AGE occurred 3 months later in the 2007 and 2008 seasons compared with previous years. Genotype G2 accounted for 15%, 70%, and 100% of all cases identified, respectively, in 2006, 2007, and 2008. Conclusions: After vaccine implementation, a marked decline in rotavirus AGE hospitalizations was demonstrated among children younger than 5 years of age, with the greatest reduction in the age groups targeted for vaccination. The predominance of genotype G2P[4] highlights the need of continued postlicensure surveillance studies.

Genetic diversity and toxic activity of Aggregatibacter actinomycetemcomitans isolates

Introduction: Very little is known of the diversity and expression of virulence factors of serotypes of Aggregatibacter actinomycetemcomitans. Toxic activity on Chinese hamster ovary (CHO) cells and cdt and ltx genotyping were evaluated in A. actinomycetemcomitans serotypes. Methods: Forty-one A. actinomycetemcomitans isolates were analysed for CHO cell growth inhibition. Genotyping was performed by polymerase chain reactions specific to the ltx promoter region, serotype-specific and cdt region and by sequencing of cdtB. Results: cdtABC was detected in 40 strains. Analysis of the cdtA upstream region revealed 10 cdt genotypes. Toxicity to CHO cells was detected for 92.7% of the isolates; however, no correlation between the toxic activity and the cdt genotype was detected. Serotype c was more prevalent among Brazilian samples (68.0%). Four serotype b isolates from subjects with aggressive periodontitis were associated with high leukotoxin production and exhibited moderate to strong toxic activity in CHO cells, but were classified in different cdt genotypes. High levels of toxicity in CHO cells were not associated with a particular serotype; 57.1% of serotype a isolates presented low toxicity to CHO cells whereas the highly toxic strains belonged to serotypes b and c. Sequencing of cdtB revealed a single nucleotide polymorphism of amino acid 281 but this was not related to the toxic activity in CHO cells. Conclusion: Differences in prevalence of the low and highly cytotoxic strains among serotypes reinforce the hypothesis that serotype b and c isolates of A. actinomycetemcomitans are more virulent than serotype a strains.