Phenotypic and functional characterization of pulmonary macrophages subpopulations after intratracheal injection of Paracoccidioides brasiliensis cell wall components

A shift in the activation of pulmonary macrophages characterized by an increase of IL-1, INF-alpha and IL-6 production has been induced in mice infected with Paracoccidioides brasiliensis. It is still unclear whether a functional shift in the resident alveolar macrophage population would be responsible for these observations due to the expression of cell surface molecules. We investigated pulmonary macrophages by flow cytometry from mice treated with P. brasiliensis derivatives by intratracheal route. In vivo labeling with the dye PKH26GL was applied to characterize newly recruited pulmonary macrophages from the bloodstream. Pulmonary macrophages from mice inflamed with P. brasiliensis derivatives showed a high expression of the surface antigens CD11b/CD18 and CD23 among several cellular markers. The expression of these markers indicated a pattern of activation of a subpopulation characterized as CD11b(+) or CD23(+), which was modulated in vitro by IFN-gamma and IL-4. Analysis of monocytes labelled with PKH26GL demonstrated that CD11b(+) cells did infiltrate the lung exhibiting a proinflammatoni pattern of activation, whereas CD23(+) cells were considered to be resident in the lung. These findings may contribute to better understand the pathology of lung inflammation caused by P. brasiliensis infection. (C) 2010 Elsevier GmbH. All rights reserved.

Descriptive findings on the morphology of dendritic spines in the rat medial amygdala

The rat posterodorsal medial amygdala (MePD) is a brain area in which gonadal hormones induce notable plastic effects in the density of dendritic spines. Dendritic spines are post-synaptic specializations whose shape and spacing change neuronal excitability. Our aim was to obtain new data on the dendritic spines morphology and density from MePD neurons using the carbocyanine dye Dil under confocal microscopy. In adult male rats, the dendritic spine density of the medial branches of the left MePD (mean +/- SD) was 1.15 +/- 0.67 spines/dendritic mu m. From the total sampled, approximately 53% of the spines were classified as thin, 22.5% as ""mushroom-like"", and 21.5% as stubby/wide. Other spine shapes (3%) included those ramified, with a filopodium-like or a gemule appearance, and others with a protruding spinule. Additional experiment joining Dil and synaptophysin (a pre-synaptic protein) labeling suggested synaptic sites on dendritic shafts and spines. Dendritic spines showed synaptophysin puncta close to their head and neck, although some spines had no evident labeled puncta on them or, conversely, multiple puncta appeared upon one spine. These results advance previous light microscopy results by revealing features and complexities of the dendritic spines at the same time that give new insight on the possible synaptic organization of the adult rat MePD. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

NOVEL 65 kDa RNA-BINDING PROTEIN IN SQUID PRESYNAPTIC TERMINALS

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

In pursuit of prognostic factors in children with pilocytic astrocytomas

This study described a 23-year experience in the treatment of children with pilocytic astrocytomas (piloA) with the aim of identifying putative clinical, histopathological, and/or immunohistochemical features that could be related to the outcome of these patients. Clinical data of 31 patients under 18 years of age with piloA were obtained from 1984 to 2006. The mean age at the time of surgery was 7.8 +/- 4.2 years (1 to 17 years), and the mean follow-up was 5.7 +/- 5.4 years (1 to 20 years). The most common site of tumor formation was the cerebellum (17), followed by brainstem (4), optic chiasmatic hypothalamic region (4), cerebral hemisphere (3), cervical spinal cord (2), and optic nerve (1). Gross total resection (GTR) was achieved in 23 (74.1%), mainly in those with tumors located in the cerebellum and cerebral hemispheres (P = 0.02). The global mortality rate was 6.4%. Nine patients were reoperated. Rosenthal fibers, eosinophilic granular bodies, microvascular proliferation, and lymphocytic infiltration were observed in most cases. The mean Ki-67LI was 4.4 +/- 4.5%. In all cases, Gal-3 expression in tumor cells was observed with variable staining pattern. Aside from GTR, no other clinical, histopathological, or immunohistochemical features were found to be related to the prognosis. We postulate that strict follow-up is recommended if piloA is associated with high mitotic activity/Ki67-LI, or if GTR cannot be achieved at surgery. Tumor recurrence or progression of the residual lesion should be strictly observed. In some aspects, childhood piloA remains an enigmatic tumor.

The Protective Effect of CAPE on Hepatic Ischemia/Reperfusion Injury in Rats

Background/Aims. The transcription factor nuclear factor-kappa B (NF-kappa B) exerts a pivotal role in the pathogenesis of hepatic ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent and specific NF-kappa B inhibitor, presents protective effects on I/R injury in some tissues. This study aimed to evaluate the effect of CAPE on hepatic I/R injury in rats. Materials and methods. Wistar rats were submitted to a sham operation, 60 min ischemia, or 60 min ischemia plus saline or CAPE treatment followed by 6 h reperfusion. Liver tissue injury was evaluated by alanine aminotransferase, aspartate aminotransferase, and tissue glutathione measurement, and histological damage score. Apoptotic hepatocytes were determined by the transferase-mediated dUTP-biotin nick-end labeling assay. Hepatic neutrophil accumulation was assessed by the naphthol method. Lipid peroxidation and NF-kappa B activation were evaluated by 4-hydroxynonenal and NF-kappa B p65 immunohistochemistry, respectively. Results. Animals submitted to ischemia showed a marked increase of alanine aminotransferase and aspartate aminotransferase after reperfusion, but with lower levels in CAPE group. Tissue glutathione content declined gradually during ischemia to reperfusion and was partially recovered with CAPE treatment. The histological damage score, apoptosis index, and neutrophil infiltration, as well as 4-hydroxynonenal and NF-kappa B p65 nuclear labeling, were higher in the liver of animals submitted to I/R compared to the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. Conclusions. CAPE was able to protect the liver against normothermic I/R injury in rats. This effect may be associated with the inhibition of the NF-kappa B signaling pathway and decrease of the acute inflammatory response following I/R in the liver. (C) 2008 Elsevier Inc. All rights reserved.

Effect of NF kappa B Inhibition by CAPE on Skeletal Muscle Ischemia-Reperfusion Injury

Background/Aims. Nuclear factor kappa B (NF kappa B) plays important role in the pathogenesis of skeletal muscle ischemia/reperfusion (I/R) injury. Caffeic acid phenyl ester (CAPE), a potent NF kappa B inhibitor, exhibits protective effects on I/R injury in some tissues. In this report, the effect of CAPE on skeletal muscle I/R injury in rats was studied. Methods. Wistar rats were submitted to sham operation, 120-min hindlimb ischemia, or 120-min hindlimb ischemia plus saline or CAPE treatment followed by 4-h reperfusion. Gastrocnemius muscle injury was evaluated by serum aminotransferase levels, muscle edema, tissue glutathione and malondialdehyde measurement, and scoring of histological damage. Apoptotic nuclei were determined by a terminal uridine deoxynucleotidyl transferase dUTP nick end labeling assay. Muscle neutrophil and mast cell accumulation were also assessed. Lipoperoxidation products and NF kappa B were evaluated by 4-hydroxynonenal and NF kappa B p65 immunohistochemistry, respectively. Results. Animals submitted to ischemia showed a marked increase in aminotransferases after reperfusion, but with lower levels in the CAPE group. Tissue glutathione levels declined gradually during ischemia to reperfusion, and were partially recovered with CAPE treatment. The histological damage score, muscle edema percentage, tissue malondialdehyde content, apoptosis index, and neutrophil and mast cell infiltration, as well as 4-hydroxynonenal and NF kappa B p65 labeling, were higher in animals submitted to I/R compared with the ischemia group. However, the CAPE treatment significantly reduced all of these alterations. Conclusions. CAPE was able to protect skeletal muscle against I/R, injury in rats. This effect may be associated with the inhibition of the NF kappa B signaling pathway and decrease of the tissue inflammatory response following skeletal muscle I/R. (C) 2009 Elsevier Inc. All rights reserved.

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis. (C) 2010 Elsevier Ltd. All rights reserved.

Comparative neuroanatomical and temporal characterization of FluoroJade-positive neurodegeneration after status epilepticus induced by systemic and intrahippocampal pilocarpine in Wistar rats

The aims of this study were to characterize the spatial distribution of neurodegeneration after status epilepticus (SE) induced by either systemic (S) or intrahippocampal (H) injection of pilocarpine (PILO), two models of temporal lobe epilepsy (TLE), using FluoroJade (FJ) histochemistry, and to evaluate the kinetics of FJ staining in the H-PILO model. Therefore, we measured the severity of behavioral seizures during both types of SE and also evaluated the FJ staining pattern at 12, 24, and 168 h (7 days) after the H-PILO insult. We found that the amount of FJ-positive (FJ+) area was greater in SE induced by S-PILO as compared to SE induced by H-PILO. After SE induced by H-PILO, we found more FJ+ cells in the hilus of the dentate gyrus (DG) at 12 h, in CA3 at 24 h, and in CA1 at 168 h. We found also no correlation between seizure severity and the number of FJ+ cells in the hippocampus. Co-localization studies of FJ+ cells with either neuronal-specific nuclear protein (NeuN) or glial fibrillary acidic protein (GFAP) labeling 24 h after H-PILO demonstrated spatially selective neurodegeneration. Double labeling with FJ and parvalbumin (PV) showed both FJ+/PV+ and FJ+/PV- cells in hippocampus and entorhinal cortex, among other areas. The current data indicate that FJ+ areas are differentially distributed in the two TLE models and that these areas are greater in the S-PILO than in the H-PILO model. There is also a selective kinetics of FJ+ cells in the hippocampus after SE induced by H-PILO, with no association with the severity of seizures, probably as a consequence of the extra-hippocampal damage. These data point to SE induced by H-PILO as a low-mortality model of TLE, with regional spatial and temporal patterns of FJ staining. (C) 2010 Elsevier B.V. All rights reserved.

Reconstruction of mandibular segmental defects using the guided-bone regeneration technique with polylactide membranes and/or autogenous bone graft: A preliminary study on the influence of membrane permeability

Purpose: Bone maintenance after mandibular reconstruction with autogenous iliac crest may be disappointing due to extensive resorption in the long term. The potential of the guided-bone regeneration (GBR) technique to enhance the healing process in segmental defects lacks comprehensive scientific documentation. This study aimed to investigate the influence of polylactide membrane permeability on the fate of iliac bone graft (BG) used to treat mandibular segmental defects. Materials and Methods: Unilateral 10-mm-wide segmental defects were created through the mandibles of 34 mongrel dogs. All defects were mechanically stabilized, and the animals were divided into 6 treatment groups: control, BG alone, microporous membrane (poly L/DL-lactide 80/20%) (Mi); Mi plus BG; microporous laser-perforated (15 cm(2) ratio) membrane (Mip), and Mip plus BG. Calcein fluorochrome was injected intravenously at 3 months, and animal euthanasia was carried out at 6 months postoperatively. Results: Histomorphometry showed that BG protected by Mip was consistently related to larger amounts of bone compared with other groups (P <= .0001). No difference was found between defects treated with Mip alone and BG alone. Mi alone rendered the least bone area and reduced the amount of grafted bone to control levels. Data from bone labeling indicated that the bone formation process was incipient in the BG group at 3 months postoperatively regardless of whether or not it was covered by membrane. In contrast, GBR with Mip tended to enhance bone formation activity at 3 months. Conclusions: The use of Mip alone could be a useful alternative to BG. The combination of Mip membrane and BG efficiently delivered increased bone amounts in segmental defects compared with other treatment modalities. (C) 2008 American Association of Oral and Maxillofacial Surgeons.

Slight activation of nuclear factor kappa-B is associated with increased hepatic stellate cell apoptosis in human schistosomal fibrosis

To investigate the relationship between NF-kappa B activation and hepatic stellate cell (HSC) apoptosis in hepatosplenic schistosomiasis, hepatic biopsies from patients with Schistosoma mansoni-induced periportal fibrosis, hepatitis C virus-induced cirrhosis, and normal liver were submitted to alpha-smooth muscle actin (alpha-SMA) and NF-kappa B p65 immunohistochemistry, as well as to NF-kappa B Southwestern histochemistry and TUNEL assay. The numbers of alpha-SMA-positive cells and NF-kappa B- and NF-kappa B p65-positive HSC nuclei were reduced in schistosomal fibrosis relative to liver cirrhosis. In addition, increased HSC NF-kappa B p65 and TUNEL labeling was observed in schistosomiasis when compared to cirrhosis. These results suggest a possible relationship between the slight activation of the NF-kappa B complex and the increase of apoptotic HSC number in schistosome-induced fibrosis, taking place to a reduced HSC number in schistosomiasis in relation to liver cirrhosis. Therefore, the NF-kappa B pathway may constitute an important down-regulatory mechanism in the pathogenesis of human schistosomiasis mansoni, although further studies are needed to refine the understanding of this process. (C) 2009 Elsevier B.V. All rights reserved.

Neuronal maturation in an experimental model of brain tissue heterotopia in the lung

Neural maturation involves diverse interaction and signaling mechanisms that are essential to the development of the nervous system. However, little is known about the development of neurons in heterotopic brain tissue in the lung, a rare abnormality observed in malformed babies and fetuses. The aim of this study was to identify the neurons and to investigate their maturation in experimental brain tissue heterotopia during fetal and neonatal periods. The fetuses from 24 pregnant female Swiss mice were used to induce brain tissue heterotopia on the 15th gestational day. Briefly, the brain of one fetus of each dam was extracted, disaggregated, and injected into the right hemithorax of siblings. Six of these fetuses with pulmonary brain tissue implantation were collected on the 18th gestational day (group E18), and six others were collected on the 8th postnatal day (group P8). The brain of each fetus from dams not submitted to any experimental procedure was collected on the 18th gestational day (group CE18) and on the 8th postnatal day (group CP8) to serve as a control for neuronal quantitation and maturation. Immunohistochemical staining of NeuN was used to assess neuron quantity and maturation. The NeuN labeling index was greater in the postnatal period than in the fetal period for the experimental and control groups (138 > E18 and CP8 > CE18), although there were fewer neurons in experimental than in control groups (P8 < CP8 and El 8 < CE1 8) (P < 0.005). These results indicate that fetal neuroblasts/neurons not only survive a dramatic event such as mechanical disaggregation, in the same way as it happens in human cases, but also they retain their development in heterotopia, irrespective of local tissue influences.

Exercise reduces inflammation and cell proliferation in rat colon carcinogenesis

Purposes: There is evidence that the risk of colon cancer is reduced by appropriate levels of physical exercise. Nevertheless, the mechanisms involved in this protective effect of exercise remain largely unknown. Inflammation is emerging as a unifying link between a range of environment exposures and neoplastic risk. The carcinogen dimethyl-hydrazine (DMH) induces an increase in epithelial cell proliferation and in the expression of the inflammation-related enzyme cyclooxigenase-2 (COX-2) in the colon of rats. Our aim was to verify whether these events could be attenuated by exercise. Methods: Four groups of eight Wistar rats were used in the experiment. The groups G1 and G3 were sedentary (controls), and the groups G2 and G4 were submitted to 8 wk of swimming training, 5 d wk(-1). The groups G3 and G4 were given subcutaneous injections of DMH immediately after the exercise protocols. Fifteen days after the neoplasic induction, the rats were sacrificed and the colon was processed for histological examination and immunohistochemistry staining of proliferating cell nuclear antigen (PCNA) and COX-2. Results: We found a significant increase in the PCNA-labeling index in both DMH-treated groups of rats. However, this increase was significantly attenuated in the training group G4 (P < 0.01). Similar results were observed in relation to the COX-2 expression. Conclusions: From our findings, we conclude that exercise training exerts remarkable antiproliferative and antiinflammatory effects in the rat colonic mucosa, suggesting that this may be an important mechanism to explain how exercise protects against colonic cancer.

Magnetic resonance imaging quantification of regional cerebral blood flow and cerebrovascular reactivity to carbon dioxide in normotensive and hypertensive rats

Hypertension afflicts 25% of the general population and over 50% of the elderly. In the present work, arterial spin labeling MRI was used to non-invasively quantify regional cerebral blood flow (CBE), cerebrovascular resistance and CO(2) reactivity in spontaneously hypertensive rats (SHR) and in normotensive Wistar Kyoto rats (WKY), at two different ages (3 months and 10 months) and under the effects of two anesthetics, alpha-chloralose and 2% isoflurane (1.5 MAC). Repeated CBE measurements were highly consistent, differing by less than 10% and 18% within and across animals, respectively. Under alpha-chloralose, whole brain CBE at normocapnia did not differ between groups (young WKY: 61 3 ml/100 g/min; adult WKY: 62 +/- 4 ml/100 g/min; young SHR: 70 +/- 9 ml/100 g/min: adult SHR: 69 8 ml/100 g/min), indicating normal cerebral autoregulation in SHR. At hypercapnia, CBE values increased significantly, and a linear relationship between CBE and PaCO(2) levels was observed. In contrast, 2% isoflurane impaired cerebral autoregulation. Whole brain CBE in SHR was significantly higher than in WKY rats at normocapnia (young SHR: 139 +/- 25 ml/100 g/min; adult SHR: 104 +/- 23 ml/100 g/min; young WKY: 55 +/- 9 ml/100 g/min; adult WKY: 71 +/- 19 ml/100 g/min). CBE values increased significantly with increasing CO(2): however, there was a clear saturation of CBF at PaCO(2) levels greater than 70 mm Hg in both young and adult rats, regardless of absolute CBE values, suggesting that isoflurane interferes with the vasoclilatory mechanisms of CO(2). This behavior was observed for both cortical and subcortical structures. Under either anesthetic, CO(2) reactivity values in adult SHR were decreased, confirming that hypertension, when combined with age, increases cerebrovascular resistance and reduces cerebrovascular compliance. Published by Elsevier Inc.

LOW-INTENSITY ULTRASOUND APPLICATION IN DISTAL RADIUS METAPHYSEAL BONE DEFECTS OF DOGS

We assessed the repair of transverse, 3-mm wide bone gaps created at the distal radius in 28 dogs that were randomly divided into two 14-animal groups; one was the control group and the other received a daily, 20-min application of low-intensity pulsed ultrasound for 100 days. Sequential radiographs, histomorphometrics, bone fluorescent histology and bone vascularity assessments found that all animals from both groups obtained a stage of hypertrophic-type nonunion with fibrocartilage tissue formation throughout the region of osteotomy. However, treated animals exhibited areas of endochondral ossification within the fibrocartilage region. There was no difference in type of vascularity or the newly formed bone process obtained by tetracycline labeling. Application of low-intensity ultrasound was not capable of significantly changing the reparative process and it may not be sufficiently powerful to overcome a combination of local deleterious effects on bone healing, created by gapping, excessive motion and periosteal resection. (E-mail: jbvolpon@fmrp.usp.br) (C) 2010 World Federation for Ultrasound in Medicine & Biology.

Intramammary infusion of leptin decreases proliferation of mammary epithelial cells in prepubertal heifers

High energy intake and excessive body fatness impair mammogenesis in prepubertal ruminants. High energy intake and excessive fatness also increase serum leptin. Our objective was to determine if an infusion of leptin decreases proliferation of mammary epithelial cells of prepubertal heifers in vivo. Ovine leptin at 100 mu g/quarter per d with or without 10 mu g of insulin-like growth factor (IGF)-I was infused via the teat canal into mammary glands of prepubertal dairy heifers; contralateral quarters were used as controls. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered similar to 2 h later. Tissue from 3 regions of the mammary parenchyma was collected and immunostained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (Ki-67), and caspase-3. Leptin decreased the number of mammary epithelial cells in the S-phase of the cell cycle by 48% in IGF-I-treated quarters and by 19% in saline-treated quarters. Leptin did not alter the number of mammary epithelial cells within the cell cycle, as indicated by Ki-67 labeling. Caspase-3 immunostaining within the mammary parenchyma was very low in these heifers, but leptin significantly increased labeling in saline-treated quarters. Leptin enhanced SOCS-3 expression in IGF-I-treated quarters but did not alter SOCS-1 or SOCS-5 expression. We conclude that a high concentration of leptin in the bovine mammary gland reduces proliferation of mammary epithelial cells. The reduced proliferation is accompanied by an increase in SOCS-3 expression, suggesting a possible mechanism for leptin inhibition of IGF-I action. Whether leptin might be a physiological regulator of mammogenesis remains to be determined.