Classification model based on Raman spectra of selected morphological and biochemical tissue constituents for identification of atherosclerosis in human coronary arteries

This study presents the results of Raman spectroscopy applied to the classification of arterial tissue based on a simplified model using basal morphological and biochemical information extracted from the Raman spectra of arteries. The Raman spectrograph uses an 830-nm diode laser, imaging spectrograph, and a CCD camera. A total of 111 Raman spectra from arterial fragments were used to develop the model, and those spectra were compared to the spectra of collagen, fat cells, smooth muscle cells, calcification, and cholesterol in a linear fit model. Non-atherosclerotic (NA), fatty and fibrous-fatty atherosclerotic plaques (A) and calcified (C) arteries exhibited different spectral signatures related to different morphological structures presented in each tissue type. Discriminant analysis based on Mahalanobis distance was employed to classify the tissue type with respect to the relative intensity of each compound. This model was subsequently tested prospectively in a set of 55 spectra. The simplified diagnostic model showed that cholesterol, collagen, and adipocytes were the tissue constituents that gave the best classification capability and that those changes were correlated to histopathology. The simplified model, using spectra obtained from a few tissue morphological and biochemical constituents, showed feasibility by using a small amount of variables, easily extracted from gross samples.

Use of cholesterol-rich nanoparticles that bind to lipoprotein receptors as a vehicle to paclitaxel in the treatment of breast cancer: pharmacokinetics, tumor uptake and a pilot clinical study

Purpose In animal experiments paclitaxel oleate associated with a cholesterol-rich nanoemulsion concentrated in the neoplastic tissues and showed reduced toxicity and increased antitumor activity compared with paclitaxel-Cremophor EL. Here, a clinical study was performed in breast cancer patients to evaluate the tumoral uptake, pharmacokinetics and toxicity of paclitaxel associated to nanoemulsions. Methods Twenty-four hours before mastectomy [(3)H]paclitaxel oleate associated with [(14)C]-cholesteryl oleatenanoemulsion or [(3)H]- paclitaxel in Cremophor EL were injected into five patients for collection of blood samples and fragments of tumor and normal breast tissue. A pilot clinical study of paclitaxel-nanoemulsion administered at 3-week intervals was performed in four breast cancer patients with refractory advanced disease at 175 and 220 mg/m(2) dose levels. Results T(1/2) of paclitaxel oleate associated to the nanoemulsion was greater than that of paclitaxel (t(1/2) = 15.4 +/- 4.7 and 3.5 +/- 0.80 h). Uptake of the [(14)C]-cholesteryl ester nanoemulsion and [(3)H]- paclitaxel oleate by breast malignant tissue was threefold greater than the normal breast tissue and toxicity was minimal at the two dose levels. Conclusions Our results suggest that the paclitaxel-nanoemulsion preparation can be advantageous for use in the treatment of breast cancer because the pharmacokinetic parameters are improved, the drug is concentrated in the neoplastic tissue and the toxicity of paclitaxel is reduced.

Relationship of adhesion molecules expression with epithelial differentiation markers during fetal skin development

Background: Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV). Methods: Using immunohistochemistry expression of E- and P-cadherins, integrins beta-1 and -4, CK10, CK14 and INV was assessed in skin fragments of 10 human fetuses (gestational weeks ranged from 4 to 24, all weighing up to 500 g). Results: At initial phases of development, integrins beta-1 and -4 and E- and P-cadherins were present on epithelial cell membranes in all layers. CK14 and CK10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E-cadherin was detected in all layers, but the cornified stratum and P-cadherin were observed in the lower layers. CK14 was expressed in basal layer, CK10 in suprabasal stratum and INV was observed in cornified layer. Conclusions: Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.

Cellular prion protein (PrP(C)) and superoxide dismutase (SOD) in vascular cells under oxidative stress

The PrP(C) is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP(C) with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP(C) expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrP(C) expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9 +/- 1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP(C) expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP(C) only in cells stimulated for 8 hours with the different stressors. The PrP(C) mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP(C) protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP(C). (C) 2009 Elsevier GmbH. All rights reserved.

Genetic Susceptibility Loci in Rheumatoid Arthritis Establish Transcriptional Regulatory Networks with Other Genes

Linkage studies have identified the human leukocyte antigen (HLA)-DRB1 as a putative rheumatoid arthritis (RA) susceptibility locus (SL). Nevertheless, it was estimated that its contribution was partial, suggesting that other non-HLA genes may play a role in RA susceptibility. To test this hypothesis, we conducted microarray transcription profiling of peripheral blood mononuclear cells in 15 RA patients and analyzed the data, using bioinformatics programs (significance analysis of microarrays method and GeneNetwork), which allowed us to determine the differentially expressed genes and to reconstruct transcriptional networks. The patients were grouped according to disease features or treatment with tumor necrosis factor blocker. Transcriptional networks that were reconstructed allowed us to identify the interactions occurring between RA SL and other genes, for example, HLA-DRB1 interacting with FNDC3A (fibronectin type III domain containing 3A). Given that fibronectin fragments can stimulate mediators of matrix and cartilage destruction in RA, this interaction is of special interest and may contribute to a clearer understanding of the functional role of HLA-DRB1 in RA pathogenesis.

Combining two potential causes of metalloproteinase secretion causes abdominal aortic aneurysms in rats: a new experimental model

P>Progress in understanding the pathophysiology of abdominal aortic aneurysms (AAA) is dependent in part on the development and application of effective animal models that recapitulate key aspects of the disease. The objective was to produce an experimental model of AAA in rats by combining two potential causes of metalloproteinase (MMP) secretion: inflammation and turbulent blood flow. Male Wistar rats were randomly divided in four groups: Injury, Stenosis, Aneurysm and Control (40/group). The Injury group received a traumatic injury to the external aortic wall. The Stenosis group received an extrinsic stenosis at a corresponding location. The Aneurysm group received both the injury and stenosis simultaneously, and the Control group received a sham operation. Animals were euthanized at days 1, 3, 7 and 15. Aorta and/or aneurysms were collected and the fragments were fixed for morphologic, immunohistochemistry and morphometric analyses or frozen for MMP assays. AAAs had developed by day 3 in 60-70% of the animals, reaching an aortic dilatation ratio of more than 300%, exhibiting intense wall remodelling initiated at the adventitia and characterized by an obvious inflammatory infiltrate, mesenchymal proliferation, neoangiogenesis, elastin degradation and collagen deposition. Immunohistochemistry and zymography studies displayed significantly increased expressions of MMP-2 and MMP-9 in aneurysm walls compared to other groups. The haemo-dynamic alterations caused by the stenosis may have provided additional contribution to the MMPs liberation. This new model illustrated that AAA can be multifactorial and confirmed the key roles of MMP-2 and MMP-9 in this dynamic remodelling process.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamus in vitro

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

In vitro susceptibility to antimycotic drug undecanoic acid, a medium-chain fatty acid, is nutrient-dependent in the dermatophyte Trichophyton rubrum

The antimycotic activity of fatty acids has long been known, and their presence in human skin and sweat appears to protect the host against superficial mycoses. Undecanoic acid is a medium-chain fatty acid that has been used in the treatment of dermatophytoses in humans. In this study, we selected one Trichophyton rubrum undecanoic acid-resistant strain that showed a marked reduction in its capacity to grow on human nail fragments, which correlated with the reduced activity of secreted keratinolytic proteases. Moreover, the susceptibility of T. rubrum to undecanoic acid is also dependent on the carbon source utilized by both control and resistant strains. The growth of the control strain was strongly inhibited by undecanoic acid in Sabouraud medium or in cultures supplemented with low-fat milk, whereas it was ineffective when the cultures were supplemented with Tween 20 or keratin as the carbon source, suggesting that nutrient conditions are crucial in establishing a susceptibility to antifungal drugs, which is helpful for the isolation and characterization of resistant strains, and in the screening for new antifungal drugs.

Deregulation of LOXL1 and HTRA1 Gene Expression in Endometriosis

Endometriosis is a gynecologic disease characterized by the presence of endometrial tissue outside the uterine cavity. Although 15% of the female population in reproductive age is affected by endometriosis, its pathogenesis remains unclear. According to the most accepted pathogenesis hypothesis, endometrial fragments from the menstrual phase are transported through the uterine tubes to the peritoneal cavity, where they undergo implantation and growth, invading adjacent tissues. However, the establishment of the disease requires that endometrial cells present molecular characteristics favoring the onset and progression of ectopic implantation. In this investigation, we analyzed the differential gene expression profiles of peritoneal and ovarian endometriotic lesions compared to the endometrial tissue of nonaffected women using rapid subtraction hybridization (RaSH). In our study, this method was applied to samples of endometriotic lesions from affected women and to biopsies of endometrium of healthy women without endometriosis, where we could identify 126 deregulated genes. To evaluate the expression of genes found by RaSH method, we measured LOXL1, HTRA1, and SPARC genes by real-time polymerase chain reaction. Significant different expression was obtained for HTRA1 and LOXL1, upregulated in the ectopic endometrium, suggesting that these genes are involved in the physiopathology of endometriosis and may favor the viability of endometrial cells at ectopic sites.

Morphological comparison of different protocols of skeletal muscle remobilization in rats after hindlimb suspension

The aim of this study was to evaluate and compare the efficacy of different remobilization protocols in different skeletal muscles considering the changes induced by hindlimb suspension of the tail. Thirty-six female Wistar rats were divided into six groups: control I, control II, suspended, suspended free, suspended trained on a declined treadmill and suspended trained on a flat treadmill. Fragments of soleus and tibialis anterior (TA) muscle were frozen and processed by different histochemical methods. The suspended soleus showed a significant increase in the proportional number of intermediate/hybrid fibers and a decrease in the number of type I fibers. Some of these changes proved to be reversible after remobilization. The three remobilization programs led to the recovery of both the proportional number of fibers and their size. The TA muscle presented a significant increase in the number and size of type I fibers and a cell size reduction of type IIB fibers, which were recovered after training on a declined treadmill and free movement. Especially regarding the soleus, the present findings indicate that, among the protocols, training on a declined treadmill was found to induce changes of a more regenerative nature, seemingly indicating a better tissue restructuring after the suspension procedure.

Effects of eccentric and concentric training on capillarization and myosin heavy chain contents in rat skeletal muscles after hindlimb suspension

We studied the effects of different protocols of post-disuse rehabilitation on angiogenesis and myosin heavy chain (MHC) content in rat hindlimb muscles after caudal suspension. Thirty female Wistar rats were divided into five groups: (1) Control I, (2) Control II, (3) Suspended, (4) Suspended trained on declined treadmill, and (5) Suspended trained on flat treadmill. Fragments of the soleus and tibialis anterior (TA) muscles were frozen and processed by electrophoresis and immunohistochemistry (CD31 antibody). Hindlimb suspension caused reduction of capillary/fiber (C/F) ratios and contents of MHC type I (MHCI) in the soleus in parallel to increased capillary density. Flat treadmill protocols increased the content of the MHCI isoform. The C/F ratio was increased by concentric training after hypokinesis, but was not modified by eccentric training, which caused a greater reduction of capillary density compared to the other protocols. In the TA muscle, hindlimb suspension caused a non-significant increase in capillary density and C/F ratio with limited changes in MHC. The present data demonstrate that the different training protocols adopted and the functional performance of the muscles analyzed caused specific changes in capillarization and in the content of the various MHC types. (C) 2010 Published by Elsevier GmbH.

Occurrence of TRGV-BJ hybrid gene in SV40-transformed fibroblast cell lines

Illegitimate V(D)J-recombination in lymphoid malignancies involves rearrangements in immunoglobulin or T-cell receptor genes, and these rearrangements may play a role in oncogenic events. High frequencies of TRGV-BJ hybrid gene (rearrangement between the TRB and TRG loci at 7q35 and 7p14-15, respectively) have been detected in lymphocytes from patients with ataxia telangiectasia (AT), and also in patients with lymphoid malignancies. Although the TRGV-BJ gene has been described only in T-lymphocytes, we previously detected the presence of TRGV-BJ hybrid gene in the genomic DNA extracted from SV40-transformed AT5BIVA fibroblasts from an AT patient. Aiming to determine whether the AT phenotype or the SV40 transformation could be responsible for the production of the hybrid gene by illegitimate V(D)J-recombination, DNA samples were extracted from primary and SV40-transformed (normal and AT) cell lines, following Nested-PCR with TRGV- and TRBJ-specific primers. The hybrid gene was only detected in SV40-transformed fibroblasts (AT-5BIVA and MRC-5). Sequence alignment of the cloned PCR products using the BLAST program confirmed that the fragments corresponded to the TRGV-BJ hybrid gene. The present results indicate that the rearrangement can be produced in nonlymphoid cells, probably as a consequence of the genomic instability caused by the SV40-transformation, and independently of ATM gene mutation.

Genetic analysis of forest species Eugenia uniflora L. through of newly developed SSR markers

Nine microsatellite loci for genetic analysis of three populations of the tropical tree Eugenia uniflora L. (pitanga or Brazilian cherry) from fragments of semideciduous forest were developed. We used the technique of building a (GA)(n) and (CA)(n) microsatellite-enriched library by capture with streptavidin-coated magnetic beads. We assessed the polymorphism of seven microsatellites in 84 mature trees found in three areas (Ribeir (a) over tildeo Preto, Tambau and S (a) over tildeo Jose do Rio Pardo), highly impacted by the agricultural practices, in a large region among Pardo river and Mogi-Guacu river basins, in state of S (a) over tildeo Paulo, Brazil. All loci were polymorphic, and the number of alleles was high, ranging from 6 to 24, with a mean of 14.4. All stands showed the same high level of genetic diversity (mean H(E) = 0.83) and a low genetic differentiation (mean F(ST) = 0.031), indicating that genetic diversity was higher within rather than among populations. Seven of the nine loci were highly variable, and sufficiently informative for E. uniflora. It was concluded that these new SSR markers can be efficiently used for gene flow studies.

A quantitative analysis of the nerve fibres of the acetabular periosteum of dogs

There are many techniques for the treatment of hip dysplasia, and novel research is currently being undertaken in the hope of obtaining more efficient and less traumatic techniques. The denervation of the hip joint capsule is a simple and effective technique that allows recovery of the functional activity of the affected limbs in significantly less time than other techniques. This surgical procedure consists of removing the acetabular periosteum, thus eliminating the nerve fibres with consequent analgesia. The aim of this investigation was to quantify the number of nerve fibres present in different regions of the acetabular periosteum. The knowledge of regional differences is potentially valuable for the refining of the denervation technique of the hip joint capsule. Thirty canine acetabular fragments were used to compare the nerve fibre density of the periosteum. The results showed a significant difference between the mean density of nerve fibres at the cranial and dorsal-lateral portion (approximately 75 fibres/mm(2)) and caudal lateral portion (approximately 60 fibres/mm(2)) of the acetabulum. Those fibres at the pedosteum are almost positioned in a sagittal plane, pointing towards the joint capsule, suggesting the some density in the latter region. These results indicate a new approach to the articular denervation technique, thus obtanining even better results for the treatment of hip dysplasia in dogs.

Ticks (Acari : ixodidae) infesting wild birds in an Atlantic forest area in the state of Sao Paulo, Brazil, with isolation of Rickettsia from the tick Amblyomma longirostre

During field work in Nazare Paulista, state of Sao Paulo, Brazil, we found 13 (56.5%) of 23 birds (mostly Passeriformes) to be infested by 28 larvae and I nymph of Amblyomma spp. Two larvae were reared to the adult stage, being taxonomically identified as Amblyomma parkeri Fonseca and Aragao, whereas five larvae and one nymph were identified as Amblyomma longirostre Koch. All six A. longirostre specimens were shown to be infected by rickettsia, as demonstrated by polymerase chain reaction (PCR) targeting two rickettsial genes (gltA and ompA) or isolation of rickettsia in cell culture from one of the ticks. This isolate was designated as strain AL, which was established in Vero cell culture and was molecularly characterized by DNA sequencing fragments of the rickettsial genes gltA, htrA, ompA, and ompB. Phylogenetic analyses inferred from ompA and ompB partial sequences showed a high degree of similarity of strain AL with Rickettsia sp. strain ARANHA, previously detected by PCR in A. longirostre ticks from Rondonia, northern Brazil. We conclude that strain AL is a new rickettsia genotype belonging to the same species of strain ARANHA, which are closely related to Candidatus `R. amblyomniii`. Further studies should elucidate if strains AL and ARANHA are different strains of Candidatus `R. amblyommii` or are a new species.