Modulation of B-1 and B-2 kinin receptors expression levels in the hippocampus of rats after audiogenic kindling and with limbic recruitment, a model of temporal lobe epilepsy

Epileptic seizures are hypersynchronous, paroxystic and abnormal neuronal discharges. Epilepsies are characterized by diverse mechanisms involving alteration of excitatory and inhibitory neurotransmission that result in hyperexcitability of the central nervous system (CNS). Enhanced neuronal excitability can also be achieved by inflammatory processes, including the participation of cytokines, prostaglandins or kinins, molecules known to be involved in either triggering or in the establishment of inflammation. Multiple inductions of audiogenic seizures in the Wistar audiogenic rat (WAR) strain are a model of temporal lobe epilepsy (TLE), due to the recruitment of limbic areas such as hippocampus and amygdata. In this study we investigated the modulation of the B-1 and B-2 kinin receptors expression levels in neonatal WARs as well as in adult WARs subjected to the TLE model. The expression levels of pro-inflammatory (IL-1 beta) and anti-inflammatory (IL-10) cytokines were also evaluated, as well as cyclooxygenase (COX-2). Our results showed that the B-1 and B-2 kinin receptors mRNAs were up-regulated about 7- and 4-fold, respectively, in the hippocampus of kindled WARs. On the other hand, the expressions of the IL-1 beta, IL-10 and COX-2 were not related to the observed increase of expression of kinin receptors. Based on those results we believe that the B, and B2 kinin receptors have a pivotal role in this model of TLE, although their participation is not related to an inflammatory process. We believe that kinin receptors in the CNS may act in seizure mechanisms by participating in a specific kininergic neurochemical pathway. (c) 2007 Elsevier B.V. All rights reserved.

The insulin signaling pathway in honey bee (Apis mellifera) caste development - differential expression of insulin-like peptides and insulin receptors in queen and worker larvae

The insulin/insulin-like signaling (IIS) pathway is an evolutionarily conserved module in the control of body size and correlated organ growth in metazoans. In the highly eusocial bees, the caste phenotypes differ not only in size and several structural features but also in individual fitness and life history. We investigated the developmental expression profiles of genes encoding the two insulin-like peptides (AmILP-1 and AmILP-2) and the two insulin receptors (AmInR-1 and AmInR-2) predicted in the honey bee genome. Quantitative PCR analysis for queen and worker larvae in critical stages of caste development showed that AmILP-2 is the predominantly transcribed ILP in both castes, with higher expression in workers than in queens. Expression of both InR genes sharply declined in fourth instar queen larvae, but showed little modulation in workers. On first sight, these findings are non-intuitive, considering the higher growth rates of queens, but they can be interpreted as possibly antagonistic crosstalk between the IIS module and juvenile hormone. Analyzing AmInR-1 and AmInR-2 expression in ovaries of queen and worker larvae revealed low transcript levels in queens and a sharp drop in AmInR-2 expression in fifth instar worker larvae, indicating relative independence in tissue-specific versus overall IIS pathway activity. (C) 2008 Elsevier Ltd. All rights reserved.

Mars is close to venus - Female reproductive proteins are expressed in the fat body and reproductive tract of honey bee (Apis mellifera L.) drones

Vitellogenin (Vg) and lipophorin (Lp) are lipoproteins which play important roles in female reproductive physiology of insects. Both are actively taken up by growing oocytes and especially Vg and its receptor are considered as female-specifically expressed. The finding that the fat body of in honey bee (Apis mellifera) drones synthesizes Vg and is present in hemolymph has long been viewed as a curiosity. The recent paradigm change concerning the role played by Vg in honey bee life history, especially social division of labor, has now led us to investigate whether a physiological constellation similar to that seen in female reproduction may also be represented in the male sex. By means of Western blot analysis we could show that both Vg and Lp are present in the reproductive tract of adult drones, including the accessory (mucus) glands, but apparently are not secreted. Furthermore, we analyzed the transcript levels of the genes encoding these proteins (vg and lp), as well as their putative receptors (Amvgr and Amlpr) in fat body and accessory glands. Whereas lp, vg and Amlpr transcript levels decreased with age in both tissues. Amvgr mRNA levels increased with age in fat body. To our knowledge this is the first report that vitellogenin and its receptor are co-expressed in the reproductive system of a male insect. We interpret these findings as a cross-sexual transfer of a social physiological trait, associated with the rewiring of the juvenile hormone/vitellogenin circuitry that occurred in the female sex of honey bees. (C) 2010 Elsevier Ltd. All rights reserved.

Development of microemulsions to topically deliver 5-aminolevulinic acid in photodynamic therapy

The aim of this study was to obtain and to characterize microemulsions containing 5-aminolevulinic acid (5-ALA) and to investigate the influence of these systems in drug skin permeation for further topical photodynamic therapy (PDT). 5-ALA was incorporated in water-in-oil (W/O), bicontinuous (Bc), and oil-in-water (O/W) microemulsions obtained by the titration of ethyl oleate and PEG-8 caprylic/capric glycerides:polyglyceryl-6 dioleate (3:1) mixtures with water. Selected systems were characterized by conductivity, viscosity, size of the droplets, and drug release. The stability of the drug in the microemulsions was also assessed. Moreover, the in vitro and in vivo skin permeation of 5-ALA was investigated using diffusion cells and confocal scanning laser microscopy (CSLM), respectively. Despite the fact that the O/W microemulsion decreased the 5-ALA diffusion coefficient and retarded the drug release, it also significantly increased the in vitro drug skin permeation when compared to other 5-ALA carriers. It was observed by CSLM that the red fluorescence of the skin increased homogeneously in the deeper skin layers when the 5-ALA microemulsion was applied in vivo, probably due to the formation of the photoactive protoporphyrin IX. The microemulsion developed carried 5-ALA to the deeper skin layers, increasing the red fluorescence of the skin and indicating the potentiality of the system for topical 5-ALA-PDT. (C) 2010 Elsevier B.V. All rights reserved.

Quantitative and qualitative comparison of 3.0 T and 1.5 T MR imaging of the liver in patients with diffuse parenchymal liver disease

Purpose: The purpose of our study was to compare signal characteristics and image qualities of MR imaging at 3.0 T and 1.5 T in patients with diffuse parenchymal liver disease. Materials and methods: 25 consecutive patients with diffuse parenchymal liver disease underwent abdominal MR imaging at both 3.0 T and 1.5 T within a 6-month interval. A retrospective study was conducted to obtain quantitative and qualitative data from both 3.0 T and 1.5 T MRI. Quantitative image analysis was performed by measuring the signal-to-noise ratios (SNRs) and the contrast-to-noise ratios (CNRs) by the Students t-test. Qualitative image analysis was assessed by grading each sequence on a 3- and 4-point scale, regarding the presence of artifacts and image quality, respectively. Statistical analysis consisted of the Wilcoxon signed-rank test. Results: the mean SNRs and CNRs of the liver parenchyma and the portal vein were significantly higher at 3.0 T than at 1.5 T on portal and equilibrium phases of volumetric interpolated breath-hold examination (VIBE) images (P < 0.05). The mean SNRs were significantly higher at 3.0 T than at 1.5 T on T1-weighted spoiled gradient echo (SGE) images (P < 0.05). However, there were no significantly differences on T2-weighted short-inversion-time inversion recovery (STIR) images. Overall image qualities of the 1.5 T noncontrast T1- and T2-weighted sequences were significantly better than 3.0 T (P < 0.01). In contrast, overall image quality of the 3.0 T post-gadolinium VIBE sequence was significantly better than 1.5 T (P< 0.01). Conclusions: MR imaging of post-gadolinium VIBE sequence at 3.0 T has quantitative and qualitative advantages of evaluating for diffuse parenchymal liver disease. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

CXCL12 rs1801157 Polymorphism in Patients with Breast Cancer, Hodgkin`s Lymphoma, and Non-Hodgkin`s Lymphoma

Chemokines and their receptors regulate the trafficking of immune cells during their development, inflammation, and tissue repair. The single-nucleotide polymorphism (SNP) rs1801157 (previously known as CXCL12-A/ stromal cell-derived factor-1 (SDF1)-3`A) in CXCL12/SDF1 gene was assessed in breast cancer, Hodgkin`s lymphoma (HL), and non-Hodgkin`s lymphoma (NHL), since the chemokine CXCL12, previously known as SDF1, and its receptor CXCR4 regulate leukocyte trafficking and many essential biological processes, including tumor growth, angiogenesis, and metastasis of different types of tumors. Genotyping was performed by PCR-RFLP (polymerase chain reaction followed by restriction fragment length polymorphism) using a restriction enzyme Hpall cleavage. No significant difference was observed in genotype distribution between breast cancer patients (GG: 57.3%; GA: 39.8%; AA: 2.9%) and healthy female controls (GG: 62.9%; GA: 33%; AA: 4.1%) nor between HL patients (GG: 61.1%; GA:27.8%; AA: 11.1%) and healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%), whereas a significant difference was observed in genotype distribution between NHL patients (GG: 51.4%; GA: 47.1%; AA: 1.5%) and healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%). Further studies will be necessary to elucidate the cancer chemokine network. However, this study suggests that CXCL12 rs1801157 polymorphism may have important implications in the pathogenesis of NHL. J. Clin. Lab. Anal. 23:387-393, 2009. (C) 2009 Wiley-Liss, Inc.

Gastrointestinal dysmotility in 5-fluorouracil-induced intestinal mucositis outlasts inflammatory process resolution

Aim To evaluate gastrointestinal motility during 5-fluorouracil (5-FU)-induced intestinal mucositis. Materials and methods Wistar rats received 5-FU (150 mg kg(-1), i.p.) or saline. After the 1st, 3rd, 5th, 15th and 30th day, sections of duodenum, jejunum and ileum were removed for assessment of epithelial damage, apoptotic and mitotic indexes, MPO activity and GSH concentration. In order to study gastrointestinal motility, on the 3rd or 15th day after 5-FU treatment, gastric emptying in vivo was measured by scintilographic method, and stomach or duodenal smooth muscle contractions induced by CCh were evaluated in vitro. Results On the third day of treatment, 5-FU induced a significant villi shortening, an increase in crypt depth and intestinal MPO activity and a decrease in villus/crypt ratio and GSH concentration. On the first day after 5-FU there was an increase in the apoptosis index and a decrease in the mitosis index in all intestinal segments. After the 15th day of 5-FU treatment, a complete reversion of all these parameters was observed. There was a delay in gastric emptying in vivo and a significant increase in gastric fundus and duodenum smooth muscle contraction, after both the 3rd and 15th day. Conclusions 5-FU-induced gastrointestinal dysmotility outlasts intestinal mucositis.

Effect of a thrombin receptor (protease-activated receptor 1, PAR-1) gene polymorphism in chronic hepatitis C liver fibrosis

Background and Aim: Tissue injury leads to activation of coagulation and generation of thrombin. Inhibition of thrombin receptor protease-activated receptor 1 (PAR-1) has been shown to reduce liver fibrosis in animals. This study aimed to evaluate the effect of PAR-1 gene polymorphism on rate of liver fibrosis (RF) in chronic hepatitis C. Methods: Polymorphisms studied: C > T transition 1426 bp upstream of translation start site (-1426C/T), 13 bp repeat of preceding -506 5`-CGGCCGCGGGAAG-3` sequence (-506I/D), and A > T transversion in intervening sequence (IVS) 14 bp upstream of exon-2 start site (IVS-14A/T). A total of 287 European and 90 Brazilian patients were studied. Results: 1426C/T polymorphism: There was a trend to higher RF in patients with the TT genotype (P = 0.06) and an association between genotype CC and slow fibrosis (P = 0.03) in Europeans. In males, RF was significantly higher in those with the TT genotype compared to CT (P = 0.003) and CC (P = 0.007). There was a significant association between TT and fast fibrosis (P = 0.04). This was confirmed in an independent cohort of Brazilians where RF was higher in TT than in CC (P = 0.03). Analysis of -506I/D showed no difference in RF and distribution of slow/fast fibrosis among different genotypes in both populations. Analysis of IVS-14A/T showed no difference between genotypes. Conclusion: In conclusion, these findings suggest that PAR-1 receptor polymorphisms influence the progression of liver fibrosis.

Water excitation MPRAGE: An alternative sequence for postcontrast imaging of the abdomen in noncooperative patients at 1.5 Tesla and 3.0 Tesla MRI

Purpose: To evaluate the diagnostic image quality of post-gadolinium water excitation-magnetization-prepared rapid gradient-echo (WE-MPRAGE) sequence in abdominal examinations of noncooperative patients at 1.5 Tesla (T) and 3.0T MRI. Materials and Methods: Eighty-nine consecutive patients (48 males and 41 females; mean age +/- standard deviation, 54.6 +/- 16.6 years) who had MRI examinations including postgadolinium WE-MPRAGE were included in the study. Of 89 patients, 33 underwent noncooperative protocol at 1.5T. 10 under-went noncooperative protocol at 3.0T, and 46 underwent cooperative protocol at 3.0T. Postgadolinium WE-MPRAGE, MPRAGE, and three-dimensional gradient-echo sequences of these three different groups were qualitatively evaluated for image quality, extent of artifacts, lesion conspicuity, and homogeneity of fat-attenuation by two reviewers retrospectively, independently, and blindly. The results were compared using Wilcoxon signed rank and Mann-Whitney U tests. Kappa statistics were used to measure the extent of agreement between the reviewers. Results: The average scores indicated that the images were diagnostic for WE-MPRAGE at 1.5T and 3.0T in noncooperative patients. WE-MPRAGE achieved homogenous fat-attenuation in 31/33 (94%) of noncooperative patients at 1.5T and 10/10 (100%) of noncooperative patients at 3.0T. WE-MPRAGE at 3.0T had better results for image quality, extent of artifacts, lesion conspicuity and homogeneity of fat-attenuation compared with WE-MPRAGE at 1.5T. in noncooperative patients (P = 0.0008, 0.0006, 0.0024, and 0.0042: respectively). Kappa statistics varied between 0.76 and 1.00, representing good to excellent agreement. Conclusion: WE-MPRAGE may be used as a T1-weighted postgadolinium fat-attenuated sequence in noncooperative patients, particularly at 3.0T MRI.

HMGB1-derived peptide acts as adjuvant inducing immune responses to peptide and protein antigen

There is a need for new adjuvants that will induce immune responses to subunit vaccines. We show that a short peptide, named Hp91, whose sequence corresponds to an area within the endogenous molecule high mobility group box (HMGB1) protein 1 potentiates cellular immune responses to peptide antigen and cellular and humoral immune responses to protein antigen in vivo. Hp91 promoted the in vivo production of the immunomodulatory cytokines, IFN-gamma, TNF-alpha, IL-6, and IL-12 (p70), as well as antigen-specific activation of CD8+ T cells. These results demonstrate the ability of a short immunostimulatory peptide to serve as an adjuvant for subunit vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

Diagnostic value of interleukin-6 and C-reactive protein on early onset bacterial infection in preterm neonates with respiratory distress

Aims: To evaluate the C-reactive protein (CRP) and interleukin-6 (IL-6) as diagnostic tools for early onset infection in preterm infants with early respiratory distress (RD). Methods: CRP and IL-6 were quantified at identification of RD and 24 h after in 186 newborns. Effects of maternal hypertension, mode of delivery, Apgar score, birth weight, gestational age, mechanical ventilation, being small for gestational age (SGA), and the presence of infection were analyzed. Results: Forty-four infants were classified as infected, 42 as possibly infected, and 100 as uninfected. Serum levels of IL-6 (0 h), CRP (0 h), and CRP (24 h), but not IL-6 (24 h) were significantly higher in infected infants compared to the remaining groups. The best test for identification of infection was the combination of IL-6 (0 h) 36 pg/dL and/or CRP (24 h) 0.6 mg/dL, which yielded 93% sensitivity and 37% specificity. The presence of infection and vaginal delivery independently increased IL-6 (0 h), CRP (0 h) and CRP (24 h) levels. Being SGA also increased the CRP (24 h) levels. IL-6 (24 h) was independently increased by mechanical ventilation. Conclusions: The combination of IL-6 (0 h) and/or CRP (24 h) is helpful for excluding early onset infection in preterm infants with RD but the poor specificity limits its potential benefit as a diagnostic tool.

Crucial Role of TNF Receptors 1 and 2 in the Control of Polymicrobial Sepsis

Sepsis is still a major cause of mortality in the intensive critical care unit and results from an overwhelming immune response to the infection. TNF signaling pathway plays a central role in the activation of innate immunity in response to pathogens. Using a model of polymicrobial sepsis by i.p. injection of cecal microflora, we demonstrate a critical role of TNFR1 and R2 activation in the deregulated immune responses and death associated with sepsis. A large and persistent production of TNF was found in wild-type (B6) mice. TNFR1/R2-deficient mice, compared with B6 mice, survive lethal polymicrobial infection with enhanced neutrophil recruitment and bacterial clearance in the peritoneal cavity. Absence of TNFR signaling leads to a decreased local and systemic inflammatory response with diminished organ injury. Furthermore, using TNFR1/R2-deficient mice, TNF was found to be responsible for a decrease in CXCR2 expression, explaining reduced neutrophil extravasation and migration to the infectious site, and in neutrophil apoptosis. In line with the clinical experience, administration of Enbrel, a TNF-neutralizing protein, induced however only a partial protection in B6 mice, with no improvement of clinical settings, suggesting that future TNF immunomodulatory strategies should target TNFR1 and R2. In conclusion, the present data suggest that the endogenous TNFR1/R2 signaling pathway in polymicrobial sepsis reduces neutrophil recruitment contributing to mortality and as opposed to pan-TNF blockade is an important therapeutic target for the treatment of polymicrobial sepsis. The Journal of Immunology, 2009, 182: 7855-7864.

THE MEDIAL AMYGDALOID NUCLEUS MODULATES CARDIOVASCULAR RESPONSES TO ACUTE RESTRAINT IN RATS

The medial amygdaloid nucleus (MeA) modulates several physiological and behavioral processes and among them, the cardiovascular correlates of behavioral responses to stressful stimuli. Acute restraint evokes cardiovascular responses, which are characterized by both elevated blood pressure (BP) and intense heart rate (HR) increase. We presently report effects of MeA pharmacological manipulations on BP and HR responses evoked by acute restraint in rats. Bilateral microinjection of 100 nL of the unspecific synaptic blocker COCl(2) (1 mM) into the MeA increased HR response to acute restraint, without significant effect on the BP response. This result indicates an inhibitory influence of MeA on restraint-evoked HR changes. Injections of the non-selective muscarinic receptor antagonist atropine (3 nmol); the inhibitor of choline uptake hemicholinium (2 nmol) or the selective M(1)-receptor antagonist pirenzepine (6 nmol) caused effects that were similar to those caused by cobalt. These results suggest that local cholinergic neurotransmission and M(1)-receptors mediate the MeA inhibitory influence on restraint-related HR responses. Pretreatment with the M3 receptor antagonist 4-DAMP (4-Diphenylacetoxy-N-methylpiperidine methiodide-2 nmol) did not affect restraint-related cardiovascular responses, reinforcing the idea that M(1)-receptors mediate MeA-related inhibitory influence on restraint-evoked HR increase. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Targeting endothelin ET(A) and ET(B) receptors inhibits antigen-induced neutrophil migration and mechanical hypernociception in mice

Endothelin may contribute to the development of inflammatory events such as leukocyte recruitment and nociception. Herein, we investigated whether endothelin-mediated mechanical hypernociception (decreased nociceptive threshold, evaluated by electronic pressure-meter) and neutrophil migration (myeloperoxidase activity) are inter-dependent in antigen challenge-induced Th1-driven hind-paw inflammation. In antigen challenge-induced inflammation, endothelin (ET) ET(A) and ET(B) receptor antagonism inhibited both hypernociception and neutrophil migration. Interestingly, ET-1 peptide-induced hypernociception was not altered by inhibiting neutrophil migration or endothelin ET(B) receptor antagonism, but rather by endothelin ET(A) receptor antagonism. Furthermore, endothelin ET(A), but not ET(B), receptor antagonism inhibited antigen-induced PGE(2) production, whereas either selective or combined blockade of endothelin ET(A) and/or ET(B) receptors reduced hypernociception and neutrophil recruitment caused by antigen challenge. Concluding, this study advances knowledge into the role for endothelin in inflammatory mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation.

Mechanisms involved in the pressor response to noradrenaline microinjection into the supraoptic nucleus of unanesthetized rats

We report on the cardiovascular effects of noradrenaline (NA) microinjection into the hypothalamic supraoptic nucleus (SON) as well as the central and peripheral mechanisms involved in their mediation. Microinjections of NA 1, 3, 10, 30 or 45 nmol/100 nL into the SON caused dose-related pressor and bradycardiac response in unanesthetized rats. The response to NA 10 nmol was blocked by SON pretreatment with 15 nmol of the alpha(2)-adrenoceptor antagonist RX821002 and not affected by pretreatment with equimolar dose of the selective alpha(1)-adrenoceptor antagonist WB4101, suggesting that local alpha(2)adrenoceptors mediate these responses. Pretreatment of the SON with the nonselective beta-adrenoceptor antagonist propranolol 15 nmol did not affect the pressor response to NA microinjection of into the SON. Moreover, the microinjection of the 100 nmol of the selective alpha(1)-adrenoceptor agonist methoxamine (MET) into the SON did not cause cardiovascular response while the microinjection of the selective alpha(2)adrenoceptor agonists BHT920 (BHT, 100 nmol) or clonidine (CLO, 5 nmol) caused pressor and bradycardiac responses, similar to that observed after the microinjection of NA. The pressor response to NA was potentiated by intravenous pretreatment with the ganglion blocker pentolinium and was blocked by intravenous pretreatment with the V(1)-vasopressin receptor antagonist dTyr(CH2)5(Me)AVP, suggesting an involvement of circulating vasopressin in this response. In conclusion, our results suggest that pressor responses caused by microinjections of NA into the SON involve activation of local alpha(2)-adrenoceptor receptors and are mediated by vasopressin release into circulation. (c) 2008 Published by Elsevier B.V.