Cigarette smoke exposure impairs respiratory epithelial ciliogenesis

Background: Cigarette smoke exposure is considered an important negative prognostic factor for chronic rhinosinusitis (CRS) patients. However, there is no clear mechanistic evidence implicating cigarette smoke exposure in the poor clinical evolution of the disease or in the maintenance of the inflammatory state characterizing CRS. This study aimed to evaluate the effects of cigarette smoke exposure on respiratory cilia differentiation. Methods: Monse nasal septal epithelium cultures grown at an air-liquid interface were used as a model of respiratory epithelium. After 5 days of cell growth, cultures were exposed to air on the apical surface. Additionally, cigarette smoke condensate (CSC; the particulate phase of tobacco smoke) or cigarette smoke extract (CSE; the volatile phase) Were diluted in the basolateral compartment in different concentrations. After 15 days of continuous exposure, scanning electron microscopy and immunofluorescence for type IV tubulin were used to determine presence and maturation of cilia. Transepithelial resistance was also recorded to evaluate confluence and physiological barrier integrity. Results: CSC and CSE impair ciliogenesis in a dose-dependent manner with notable effects in concentrations higher than 30 mu g/mL, yielding >70% nonciliation and shorter cilia compared With control. No statistical difference on transepithelial resistance was evident. Conclusion: CSC and CSE exposure negatively impacts ciliogenesis of respiratory cells at concentrations not effecting transepithelial resistance. The impairment on ciliogenesis reduce the mucociliary clearance apparatuts after injury and/or infection and may explain the poor response to therapy for CRS patients exposed to tobacco smoke.

Comparison of clinical features and drug therapies among European and Latin American patients with juvenile dermatomyositis

Objective To compare the demographic features, presenting manifestations, diagnostic investigations, disease course, and drug therapies of children with juvenile dermatomyositis (JDM) followed in Europe and Latin America. Methods Patients were inception cohorts seen between 1980 and 2004 in 27 paediatric rheumatology centres. The following information was collected through the review of patient charts: sex; age at disease onset; date of disease onset and diagnosis; onset type; presenting clinical features; diagnostic investigations; course type; and medications received during disease course. Results Four hundred and ninety patients (65.5% females, mean onset age 7.0 years, mean disease duration 7.7 years) were included. Disease presentation was acute or insidious in 57.1% and 42.9% of the patients, respectively. The course type was monophasic in 41.3% of patients and chronic polycyclic or continuous in 58.6% of patients. The more common presenting manifestations were muscle weakness (84.9%), Gottron`s papules (72.9%), heliotrope rash (62%), and malar rash (56.7%). Overall, the demographic and clinical features of the 2 continental cohorts were comparable. European patients received more frequently high-dose intravenous methylprednisolone, cyclosporine, cyclophosphamide, and azathioprine, while methotrexate and antimalarials medications were used more commonly by Latin American physicians. Conclusion The demographic and clinical characteristics of JDM are similar in European and Latin American patients. We found, however, several differences in the use of medications between European and Latin American paediatric rheumatologists.

Postnatal growth and cardiometabolic profile in young adults born large for gestational age

Context The association between large for gestational age (LGA) phenotype, postnatal growth and cardiometabolic risk (CMR) in adult life remains unclear. The role of IGF1 genotype on LGA-related outcomes in adult life is unknown. Aim To assess the postnatal growth, IGF-I levels, CMR and the influence of the 737.738 IGF1 in adults born LGA. Subjects Case-control study (n = 515) nested in a population-based prospective cohort (n = 2063); 117 LGA and 398 gender-matched controls appropriate for gestational age (AGA) subjects. Methods Anthropometry was evaluated at birth, at 9-10 and at 23-25 years old. At the age of 23-25 years, blood pressure (BP), glycaemia, insulinaemia, homeostasis model assessment - insulin resistance, lipids, fibrinogen, and plasma IGF-I and 737.738 IGF1 polymorphism were assessed. Results Large for gestational age subjects remained heavier and taller than AGA at 9-10 and 23-25 years (P < 0.05); at 23-25 years, LGA had greater waist circumference (WC; P < 0.05) and higher BP (P < 0.05) than controls. Body proportionality at birth did not predict metabolic outcome. LGA subjects presenting catch-down of weight in childhood had lower body mass index (BMI; P = 0.001), lower WC (P < 0.05) and lower BP (P < 0.05) at 2325 years. 737.738 IGF-I genotype differed between groups (P < 0.001). Homozygosis for polymorphic alleles was associated with increased odds of LGA (OR: 3.2; 95% CI: 1.5-6.9), higher IGF-I (56.9 +/- 16.4 vs 37.7 +/- 16.0 nm; P < 0.01) and lower BP (114/68 vs 121/73 mmHg; P < 0.05). Conclusions Young adults born LGA presented higher BMI, WC and BP and appear to be at higher CMR risk than AGA subjects. The 737.738 IGF1 polymorphism appears to play a role on birth size and LGA-related metabolic outcomes.

Polymorphisms in genes encoding drugs and xenobiotic metabolizing enzymes in a Brazilian population

Polymorphic variations of several genes associated with drugs and xenobiotic metabolism have been linked to the factors that predispose to the carcinogenesis process. As considerable interindividual and interethnic variation in metabolizing enzyme activity has been associated with polymorphic alleles, we evaluated the frequency of the polymorphisms of CYP2D6, EPHX1 and NQO1 genes in 361 Brazilian individuals separated by ethnicity (European and African ancestry), using the polymerase chain reaction-restriction fragment length (PCR-RFLP) method. The allele frequencies of the variants *3 and *4 for the gene CYP2D6 were 0.04 and 0.14 for white subjects and 0.03 and 0.10 for black individuals, respectively. For the both variants of the gene EPHX1, we found higher allele frequencies among white individuals compared with mulatto subjects (0.62 vs 0.54 and 0.18 vs 0.14, respectively); however, these differences were not statistically significant (p = 0.39 and 0.56, respectively). For the NQO1 gene we observed a higher frequency of the homozygous genotype among black individuals (7.9%) compared with white subjects (6.3%) (p = 0.003). The genotype frequencies were within the Hardy-Weinberg equilibrium. We concluded that the allele frequencies of CYP2D6, EPHX1 and NQO1 gene polymorphisms in this Brazilian population showed ethnic variability when compared with those observed in other populations.

Interaction of purinergic and nitrergic mechanisms in the caudal nucleus tractus solitarii of rats

The interaction of purinergic and nitrergic mechanisms was evaluated in the caudal nucleus tractus solitarii (cNTS) using awake animals and brainstem slices. In awake animals, ATP (1.25 nmol/50 nL) was microinjected into the cNTS before and after the microinjection of a selective neuronal nitric oxide synthase (nNOS) inhibitor N-propyl-L-arginine (NPLA, 3 pmoles/50 nL, n=8) or vehicle (saline, n=4), and cardiovascular and ventilatory parameters were recorded. In brainstem slices from a distinct group of rats, the effects of ATP on the NO concentration in the cNTS using the fluorescent dye DAF-2 DA were evaluated. For this purpose brainstem slices (150 pm) containing the cNTS were pre-incubated with ATP (500 mu M; n=8) before and during DAF-2 DA loading. Microinjection of ATP into the cNTS increases the arterial pressure (AP), respiratory frequency (f(R)) and minute ventilation (V(E)), which were significantly reduced by pretreatment with N-PLA, a selective nNOS inhibitor (AP: 39 +/- 3 vs 16 +/- 14 mm Hg; f(R): 75 +/- 14 vs 4 +/- 3 cpm; V(E): 909 159 vs 77 39 mL kg(-1) m(-1)). The effects of ATP in the cNTS were not affected by microinjection of saline. ATP significantly increased the NO fluorescence in the cNTS (62 +/- 7 vs 101 +/- 10 AU). The data show that in the cNTS: a) the NO production is increased by ATP; b) NO formation by nNOS is involved in the cardiovascular and ventilatory responses to microinjection of ATP. Taken together, these data suggest an interaction of purinergic and nitrergic mechanisms in the cNTS. (C) 2009 Elsevier B.V. All rights reserved.

eNOS haplotypes associated with gestational hypertension or preeclampsia

Aims: Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene have been inconsistently associated with preeclampsia. We compared genotype and haplotype frequencies of three eNOS gene polymorphisms in normotensive and hypertensive pregnancies. Methods: Genotypes and haplotypes for eNOS polymorphisms (T-786C, Glu298Asp and intron 4 b/a) were determined in 326 pregnant women (1110 healthy pregnancies, 103 gestational hypertensives and 113 preeclamptic). Results: No differences were observed in the frequencies of genotypes and alleles of the three polymorphisms among the groups (all p > 0.05). However, the haplotype `T Glu a` was more common in healthy pregnancies than in gestational hypertensives or preeclamptic (20 vs 6 and 6%, respectively; p < 0.0032). Conversely, the haplotype `C Glu a` was more common in gestational hypertensives and preeclamptic than in healthy pregnancies (117 vs 17 and 5%; p = 0.0061). Conclusion: These findings suggest a contribution of eNOS haplotypes to the development of hypertensive disorders of pregnancy that is obscured when specific eNOS genotypes alone are considered.

Insights on calcium-independent phospholipid membrane damage by Lys49-PLA(2) using tryptophan scanning mutagenesis of bothropstoxin-I from Bothrops jararacussu

Bothropstoxin-I (BthTx-I) is a homodimerie Lys49-PLA(2) from the venom of the snake Bothrops jararacussu, which lacks hydrolytic activity against phospholipid substrates, yet permeabilizes membranes by a Ca2+- independent mechanism. The interaction of the BthTx-I with model membranes has been studied by intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. Nine separate mutants have been created each with a unique tryptophan residue located at a different position in the interfacial recognition site (IRS) of the protein. The rapid and efficient Ca2+-independent membrane damage against unilamellar liposomes composed of DPPC/DMPA in a 9:1 molar ratio was unaffected by these substitutions. Binding studies revealed low protein affinity for these liposomes and no changes were observed in the ITFE properties. In contrast, the binding of all mutants to DPPC/DMPA liposomes in a 1:1 molar ratio was stronger, and was correlated with altered ITFE properties. The blue-shifted emission spectra and increased emission intensity of mutants at positions 31, 67 and 115-117 in the interface recognition surface of the protein suggest these regions are partially inserted into the membrane. These results are consistent with a model for the Ca2+-independent membrane damaging mechanism that involves a transient interaction of the protein with the outer phospholipid leaflet of the target membrane. (C) 2007 Elsevier Masson SAS. All rights reserved.

Genetic and environmental factors affecting ultrasound measures of longissimus muscle area and backfat thickness in Nelore cattle

The aim of the present study was to evaluate the genetic and environmental factors affecting records of longissimus muscle area (LMA) and back fat thickness (BF) obtained between the 12th and 13th ribs, and rump fat thickness (RF) between the hook and pin bones, measured by real-time ultrasound in Nelore cattle. Also, weight records of 22,778 animals born from 1998 to 2003, in ten farms across six Brazilian states were used. Carcass traits as measured by ultrasound of the live animal were recorded from 2002 to 2004 in 2590 males and females with ages varying from 450 to 599 days. Fixed models including farm, year and season of birth, sex and type of feed effects, and the covariates age of dam (AOD) and age of animal at measurement were used to study the effect of environmental factors on these traits. The genetic parameters for LMA, BF and RF were estimated with two and three-trait animal models with 120-day weights using a restricted maximum likelihood method. All environmental effects significantly affected carcass traits, with the exception of year of birth for BF and RF and AOD for LMA. The heritability estimates for LMA, BF and RF were 0.35, 0.51 and 0.39, respectively. Standard errors obtained in one-trait analyses were from 0.07 to 0.09. Genetic correlation estimates between LMA and the two traits of subcutaneous fat were low (close to zero) and 0.74 between BF and RF, indicating that the selection for LMA should not cause antagonism in the genetic improvement of subcutaneous fat measured by real-time ultrasound. (C) 2007 Elsevier B.V. All fights reserved.

Influence of enzyme inducing antiepileptic drugs on the pharmacokinetics of levetiracetam in patients with epilepsy

To assess whether levetiracetam elimination is influenced by enzyme inducing antiepileptic drugs (EIAEDs), serum levetiracetam levels were determined at frequent intervals after a single oral 1000 mg dose in 15 subjects co-medicated with EIAEDs and 15 matched controls. The EIAED group showed a higher levetiracetam oral clearance (p = 0.01) and a shorter half-life (p = 0.02) than controls. Although the magnitude of interaction is relatively modest, it could have clinical significance for some patients. (C) 2011 Elsevier B.V. All rights reserved.

A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation

Platelet aggregation and acute inflammation are key processes in vertebrate defense to a skin injury. Recent studies uncovered the mediation of 2 serine proteases, cathepsin G and chymase, in both mechanisms. Working with a mouse model of acute inflammation, we revealed that an exogenous salivary protein of Ixodes ricinus, the vector of Lyme disease pathogens in Europe, extensively inhibits edema formation and influx of neutrophils in the inflamed tissue. We named this tick salivary gland secreted effector as I ricinus serpin-2 (IRS-2), and we show that it primarily inhibits cathepsin G and chymase, while in higher molar excess, it affects thrombin activity as well. The inhibitory specificity was explained using the crystal structure, determined at a resolution of 1.8 angstrom. Moreover, we disclosed the ability of IRS-2 to inhibit cathepsin G-induced and thrombin-induced platelet aggregation. For the first time, an ectoparasite protein is shown to exhibit such pharmacological effects and target specificity. The stringent specificity and biological activities of IRS-2 combined with the knowledge of its structure can be the basis for the development of future pharmaceutical applications. (Blood. 2011;117(2):736-744)

Genetic analysis of ticks belonging to the Rhipicephalus sanguineus group in Latin America

Phylogenetic analyses based on mitochondrial 16S rDNA sequences were generated from Rhipicephalus sanguineus group specimens collected in 29 localities among 9 Latin-American countries, plus ticks collected in South Africa, Spain, and Italy. Sequences from Latin America generated six different haplotypes (A, B, C, D, E, and F). Phylogenetic analyses generated trees that segregated our tick sequences into two distinct clades: one is represented by haplotypes A-C, and South African R. sanguineus and Rhipicephalus turanicus ticks; the second clade is represented by haplotypes D-F, and European R. sanguineus and R. turanicus ticks. When haplotypes A-Fare plotted in the Latin America map according to their geographical coordinates, it is clearly seen that haplotypes D-F are restricted to the southern portion of this continent, whereas haplotypes A-C are distributed in areas between northern Mexico and Brazil (except for the extreme south of this last country, where haplotype E was present). Hence, our phylogenetic analyses separated New World specimens of R. sanguineus into two distinct clades, one represented by tropical and subtropical populations (haplotypes A-C), here designated as the `tropical` species. On the other hand, haplotypes D-F are here designated as the `temperate` species because of their distribution in the southern portion of South America. Until recently, it was assumed that the R. sanguineus group was represented by a single species in the New World, namely R. sanguineus. While the present results coupled with recent studies support the presence of at least two species under the taxon R. sanguineus in the New World, they also show that even in the Old World, the taxon R. sanguineus might be represented by more than one species, since our phylogenetic analysis segregated European and South African R. sanguineus ticks into two distinct clades. The same can be applied for Spanish and South African R. turanicus. (C) 2010 Elsevier B.V. All rights reserved.

Transient disruption of liver gap junctional intercellular communication and induction of apoptosis after administration of 1,4-bis[2-(3,5 dichloropyridyloxy)]benzene in mice

Gap junctional intercellular communication (GJIC) and connexin expression (Cx26 and Cx32) in mouse liver were studied after administration of 4-bis[2-(3,5 dichloropyridyloxy)]benzene (TCPOBOP), a phenobarbital-like enzyme inducer. Female C57BI/6 mice were administered TCPOBOP (5.8 mg/kg BW) and euthanized 0, 24, 48 and 72 hours later. Liver samples were snap frozen, or fixed in formalin, or submitted to GJIC analysis. The proliferating cell nuclear antigen (PCNA) immunohistochemistry and the Western blotting for Cx26 and Cx32 were performed. After 48 and 72 h of drug administration the liver-to-body weight ratio was increased 70% and 117% (p < 0.0001), respectively. There were temporal-dependent alterations in liver histopathology and a significant increase in cell proliferation was noted after 48h and sustained after 72h, though to a lesser extent (p < 0.0001). In addition. TCPOBOP administration induced apoptosis, which appeared to be time-dependent showing statistical significance only after 72h (p < 0.0001). Interestingly, a transient disruption by nearly 50% of GJIC capacity was detected after 48 h of drug ingestion, which recovered after 72 h (p = 0.003). These GJIC changes were due to altered levels of Cx26 and Cx32 in the livers of TCPOBOP-treated mice. We concluded that a single administration of TCPOBOP transiently disrupted the levels of GJIC due to decreased expression of connexins and increased apoptotic cell death in mouse liver. (C) 2009 Elsevier GmbH. All rights reserved.

NONINVASIVE MONITORING OF ANDROGENS IN MALE AMAZONIAN MANATEE (TRICHECHUS INUNGUIS): BIOLOGIC VALIDATION

The Amazonian manatee (Trichechus inunguis) is endemic in the Amazonian basin and is the only exclusively fresh water sirenian. Historically hunted on a large scale, this species is now considered endangered, and Studies on the reproductive physiology are critical for the improvement of reproductive management of captive and wild Populations of manatees. The aim of this Study was to verify the viability of androgen measurement in saliva, lacrimal, urine, and fecal samples of the Amazonian manatee by conducting a hormone challenge. Two adult male manatees (A-1 and A-2) were Submitted to an experimentation protocol of 12 day (D1 to D10). On D0, the animals received an intramuscular injection of gonadotropin-releasing hormone (GnRH)-analogue. Salivary, lacrimal, urinary, and fecal samples were collected daily (between 0800 hours and 0900 hours) and frozen at -20 degrees C until assayed. Fecal samples were lyophilized, extracted with 80% methanol, and diluted in buffer before the radioimmunoassay (RIA). Urine samples underwent acid hydrolysis and were diluted in depleted bovine serum. Salivary and lacrimal samples were assayed without the extraction step. Hormonal assays were conducted with a commercial testosterone RIA kit. An androgen peak (>median + 2 interquartile range [IQR]) was observed in all matrices of both animals, although it was less prominent in the lacrimal samples of A-2. However, the fecal androgen peak (A-1 peak = 293.78 ng/g dry feces, median [IQR] = 143.58 [32.38] ng/g dry feces; A-2 peak = 686.72 ng/g dry feces, median [IQR] = 243.82 [193.16] ng/g dry feces) occurred later than urinary (A-1 peak = 648.16 ng/mg creatinine [Cr], median [IQR] = 23.88 [30.44] ng/mg Cr; A-2 peak = 370.44 ng/mg Cr, median [IQR] = 113.87 [117.73] ng/mg Cr) and salivary (A-1 peak = 678.89 pg/ml, median [IQR] = 103.69 [119.86] pg/ml; A-2 peak = 733.71 pg/ml, median [IQR] = 262.92 [211.44] pg/ml) androgen peaks. These intervals appear to be correlated with the long digesta passage time in this species. The salivary and urinary peaks were closely associated. These results demonstrate that androgen concentrations in saliva, urine, or feces samples reflect reliably physiologic events and are a powerful tool for noninvasive reproductive monitoring of Amazonian manatees.

Effects of Nd:YAG Laser Irradiation on the Hybrid Layer of Different Adhesive Systems

Purpose: The aim of this in vitro study was to evaluate the microtensile bond strength (mu TBS) and hybrid layer morphology of different adhesive systems, either followed by treatment with Nd:YAG laser irradiation or not. Previous studies have shown the effects of Nd:YAG laser irradiation on the dentin surface at restoration margins, but there are few reports about the significance of the irradiation on the hybrid layer. Materials and Methods: The flattened coronal and root dentin samples of 24 bovine teeth were randomly divided into 8 groups, according to the adhesive system used - Scotchbond Multi Purpose (SBMP) or Clearfil SE Bond (CSEB) - and were either irradiated with Nd:YAG or not, with different parameters: 0.8 W/10 Hz, 0.8 W/20 Hz, 1.2 W/10 Hz, 1.2 W/20 Hz. The left sides of specimens were the control groups, and right sides were irradiated. A composite crown was built over bonded surfaces and stored in water (24 h at 37 degrees C). Specimens were sectioned vertically into slabs that were subjected to mu TBS testing and observed by SEM. Results: Control groups (27.81 +/- 1.38) showed statistically higher values than lased groups (21.37 +/- 0.99), and CSEB control group values (31.26 +/- 15.71) were statistically higher than those of SBMP (24.3 +/- 10.66). There were no significant differences between CSEB (20.34 +/- 10.01) and SBMP (22.43 +/- 9.82) lased groups. Among parameters tested, 0.8 W/10 Hz showed the highest value (25.54 +/- 11.74). Nd:YAG laser irradiation caused dentin to melt under the adhesive layer of both adhesive systems tested. Conclusion: With the parameters used in this study, Nd:YAG laser irradiation of the hybrid layer promoted morphological changes in dentin and negatively influenced the bond strength of both adhesive systems.

Micro-shear bond strength of Er : YAG-laser-treated dentin

This study tested if dentin adhesion is affected by Er:YAG laser. Ninety dentin disks were divided in groups (n=10): G1, control; G2, Er:YAG laser 150 mJ, 90 degrees contact, 38.8 J/cm(2); G3, Er:YAG laser 70 mJ, 90 degrees contact, 18.1 J/cm(2); G4, Er:YAG laser 150 mJ, 90 degrees non-contact, 1.44 J/cm(2); G5, Er:YAG laser 70 mJ, 90 degrees non-contact, 0.67 J/cm(2); G6, Er:YAG laser 150 mJ, 45 degrees contact, 37.5 J/cm(2); G7, Er:YAG laser 70 mJ, 45 degrees contact, 17.5 J/cm(2); G8, Er:YAG laser 150 mJ, 45 degrees non-contact, 1.55 J/cm(2); and G9, Er:YAG laser 70 mJ, 45 degrees non-contact, 0.72 J/cm(2). Bonding procedures were carried out and the micro-shear-bond strength (MSBS) test was performed. The adhesive surfaces were analyzed under SEM. Two-way ANOVA and multiple comparison tests revealed that MSBS was significantly influenced by the laser irradiation (p < 0.05). Mean values (MPa) of the MSBS test were: G1 (44.97 +/- 6.36), G2 (23.83 +/- 2.46), G3 (30.26 +/- 2.57), G4 (35.29 +/- 3.74), G5 (41.90 +/- 4.95), G6 (27.48 +/- 2.11), G7 (34.61 +/- 2.91), G8 (37.16 +/- 1.96), and G9 (41.74 +/- 1.60). It was concluded that the Er:YAG laser can constitute an alternative tool for dentin treatment before bonding procedures.