Prognostic index expression of cyclin-D1, cerbB-2 and VEGF: metastases vs corresponding primary cancers and metastatic vs non-metastatic adenocarcinomas

The prognostic relevance of different molecular markers in lung cancer is a crucial issue still worth investigating, and the specimens collected and analyzed represent a valuable source of material. Cyclin-D1, c-erbB-2 and vascular endothelial growth factor (VEGF) have shown to be promising as prognosticators in human cancer. In this study, we sought to examine the importance of Cyclin-D1, c-erbB-2 and VEGF, and to study the quantitative relationship among these factors and disease progression in metastases vs corresponding primary cancer, and metastatic vs non metastatic cancers. Material and Methods: We used immunohistochemistry and morphometric analysis to evaluate the amount of tumour staining for Cyclin-D1, c-erbB-2 and VEGF in 52 patients with surgically excised ademocarcinoma of the lung, and the outcome for our study was survival time until death from hematogenic metastases. Results: Metastasis presented lower c-erbB-2 expression than corresponding primary cancers (p=0.02). Cyclin-D1 and VEGF expression were also lower in metastases than in corresponding primary cancers, but this difference did not achieve statistical significance. Non-metastatic cancers also presented significantly lower Cyclin-D1 and c-erbB-2 expression than metastatic cancers (p<0.01 and p<0.01, respectively). Equally significant was the difference between higher c-erbB-2 expression by metastatic cancers compared to non-metastatic cancers (p=0.02). Considering survival in Kaplan-Maier analysis, Cyclin-D1 (p=0.04), c-erbB-2 (p=0.04) and VEGF (p<0.01) were important predictors of survival in metastatic cancers.

Association between intratumoral lymphatic microvessel density (LMVD) and clinicopathologic features in endometrial cancer: a retrospective cohort study

Background: Lymph node metastasis in endometrial cancer significantly decreases survival rate. Few data on the influence of intratumoral lymphatic microvessel density (LMVD) on survival in endometrial cancer are available. Our aim was to assess the intratumoral LMVD of endometrial carcinomas and to investigate its association with classical pathological factors, lymph node metastasis and survival. Methods: Fifty-seven patients with endometrial carcinoma diagnosed between 2000 and 2008 underwent complete surgical staging and evaluation of intratumoral LMVD and other histologic variables. Lymphatic microvessels were identified by immunohistochemical staining using monoclonal antibody against human podoplanin (clone D2-40) and evaluated by counting the number of immunostained lymphatic vessels in 10 hot spot areas at 400x magnification. The LMVD was expressed by the mean number of vessels in these 10 hot spot microscopic fields. We next investigated the association of LMVD with the clinicopathologic findings and prognosis. Results: The mean number of lymphatic vessels counted in all cases ranged between 0 and 4.7. The median value of mean LMVD was 0.5, and defined the cut-off for low and high LMVD. We identified low intratumoral LMVD in 27 (47.4%) patients and high LMVD in 30 (52.6%) patients. High intratumoral LMVD was associated with lesser miometrial and adnaexal infiltration, lesser cervical and peritoneal involvement, and fewer fatal cases. Although there was lower lymph node involvement among cases with high LMVD, the difference did not reach significance. No association was seen between LMVD and FIGO staging, histological type, or vascular invasion. On the other hand, low intratumoral LMVD was associated with poor outcome. Seventy-five percent of deaths occurred in patients with low intratumoral LMVD. Conclusion: Our results show association of high intratumoral LMVD with features related to more localized disease and better outcome. We discuss the role of lymphangiogenesis as an early event in the endometrial carcinogenesis.

Effect of Laser (670 nm) on Healing of Wounds Covered with Occlusive Dressing: A Histologic and Biomechanical Analysis

Objectives: To analyze the effects of low-level laser therapy (LLLT), 670 nm, with doses of 4 and 7 J/cm(2), on the repair of surgical wounds covered by occlusive dressings. Background Data: The effect of LLLT on the healing process of covered wounds is not well defined. Materials and Methods: For the histologic analysis with HE staining, 50 male Wistar rats were submitted to surgical incisions and divided into 10 groups (n=5): control; stimulated with 4 and 7 J/cm(2) daily, for 7 and 14 days, with or without occlusion. Reepithelization and the number of leukocytes, fibroblasts, and fibrocytes were obtained with an image processor. For the biomechanical analysis, 25 rats were submitted to a surgical incision and divided into five groups (n=5): treated for 14 days with and without occlusive dressing, and the sham group. Samples of the lesions were collected and submitted to the tensile test. One-way analysis of variance was performed, followed by post hoc analysis. A Tukey test was used on the biomechanical data, and the Tamhane test on the histologic data. A significance level of 5% was chosen (p <= 0.05). Results: The 4 and 7J/cm(2) laser with and without occlusive dressing did not alter significantly the reepithelization rate of the wounds. The 7 J/cm(2) laser reduced the number of leukocytes significantly. The number of fibroblasts was higher in the groups treated with laser for 7 days, and was significant in the covered 4 J/cm(2) laser group. Conclusions: Greater interference of the laser-treatment procedure was noted with 7 days of stimulation, and the occlusive dressing did not alter its biostimulatory effects.

Influence of Laser Photobiomodulation on Collagen IV During Skeletal Muscle Tissue Remodeling After Injury in Rats

Objective: The aim of the present study was to determine the effect of GaAlAs low-level laser therapy (LLLT) on collagen IV remodeling of the tibialis anterior (TA) muscle in rats after cryolesion. Background: Considerable interest exists in skeletal muscle regeneration in situations such as repair after exercise-induced muscle injury, after muscle transplantation, in muscular dystrophy, exercise-induced muscle injury, and the recovery of strength after atrophy due to disuse. A number of studies have demonstrated the potential of LLLT in facilitating the muscle-healing process; however, no consensus is found in the literature regarding the best laser-irradiation parameters. Methods: Adult male Wistar rats (n = 45) were used and randomly divided into three groups: control (n = 5); nontreated cryolesioned group (n = 20), and LLLT-cryolesioned group (n = 20). The cryolesioned groups were analyzed at 1, 7, 14, and 21 days after the injury procedure. Laser irradiation was performed 3 times per week on the injured region by using the GaAlAs laser (660 nm; beam spot of 0.04 cm(2), output power of 20 mW, power density of 500 mW/cm(2), and energy density of 5 J/cm(2), for 10 sec). The muscles were removed, frozen, cryosectioned, and then stained with hematoxylin-eosin for the visualization of general morphology or used for immunohistochemical analysis of collagen IV. Results: It was demonstrated that LLLT promotes an increase in collagen IV immunolabeling in skeletal muscle in the first 7 days after acute trauma caused by cryoinjury, but does not modify the duration of the tissue-repair process. Even with LLLT, the injured muscle tissue needs similar to 21 days to achieve the same state of organization as that in the noninjured muscle. Conclusion: The collagen IV content is modulated in regenerating skeletal muscle under LLLT, which might be associated with better tissue outcome, although the histologic analysis did not detect tissue improvement in the LLLT group.

Glut1 and Glut3 as Potential Prognostic Markers for Oral Squamous Cell Carcinoma

We associated clinical-pathological features of 142 OSCC with the expression pattern of GLUT1 and GLUT3 in order to estimate their prognostic value. Methods: Clinical-pathological features and overall survival data of 142 patients with Oral Squamous Cell Carcinoma (OSCC) were retrospectively reviewed from A. C. Camargo hospital records. A tissue microarray (TMA) was built for the immunohistochemical (IHC) analysis of GLUT 1 and GLUT 3. IHC results were evaluated according to the staining pattern and number of positive cells. Results: GLUT 1 was over expressed in 50.3% of OSSC cases showing membrane staining pattern. However, nuclear expression was observed in 49.7% of the analyzed cases. GLUT 3 over expression was detected in 21.1% of OSCC cases. The pattern of GLUT 1 expression showed significant association with alcohol consumption (p = 0.004). Positive cell membrane GLUT 3 protein expression was associated with advanced clinic-staging of tumours (p = 0.005) as well as with vascular embolization (p = 0.005). Positive expression of GLUT 3 was associated with unfavorable free-disease survival (p = 0.021). Conclusion: GLUT1 and GLUT3 protein expression evaluated by immunohistochemistry are, significantly, indicators of poor prognosis outcome in oral squamous cell carcinoma, probably due to the enhanced glycolytic metabolism of more aggressive neoplastic cells.

Effects of perinatal protein deprivation and recovery on esophageal myenteric plexus

AIM: To evaluate effects of pre- and postnatal protein deprivation and postnatal recovery on the myenteric plexus of the rat esophagus. METHODS: Three groups of young Wistar rats (aged 42 d) were studied: normal-fed (N42), protein-deprived (D42), and protein-recovered (R42). The myenteric neurons of their esophagi were evaluated by histochemical reactions for nicotinamide adenine dinucleotide (NADH), nitrergic neurons (NADPH)-diaphorase and acetylcholinesterase (AChE), immunohistochemical reaction for vasoactive intestinal polypeptide (VIP), and ultrastructural analysis by transmission electron microscopy. RESULTS: The cytoplasms of large and medium neurons from the N42 and R42 groups were intensely reactive for NADH. Only a few large neurons from the D42 group exhibited this aspect. NADPH detected in the D42 group exhibited low reactivity. The AChE reactivity was diffuse in neurons from the D42 and R42 groups. The density of large and small varicosities detected by immunohistochemical staining of VIP was low in ganglia from the D42 group. In many neurons from the D42 group, the double membrane of the nuclear envelope and the perinuclear cisterna were not detectable. NADH and NADPH histochemistry revealed no group differences in the profile of nerve cell perikarya (ranging from 200 to 400 mu m(2)). CONCLUSION: Protein deprivation causes a delay in neuronal maturation but postnatal recovery can almost completely restore the normal morphology of myenteric neurons. (C) 2010 Baishideng. All rights reserved.

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background: Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination. Methods: Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR. Results: In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus. Conclusion: These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Background: Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption. Methodology/Principal Findings: In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues. Conclusions/Significance: These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.

Low-Level Laser Irradiation (InGaAlP-660 nm) Increases Fibroblast Cell Proliferation and Reduces Cell Death in a Dose-Dependent Manner

Background and Objective: Impaired cell metabolism and increased cell death in fibroblast cells are physiological features of chronic tendinopathy. Although several studies have shown that low-level laser therapy (LLLT) at certain parameters has a biostimulatory effect on fibroblast cells, it remains uncertain if LLLT effects depend on the physiological state. Study Design/Material and Methods: High-metabolic immortal cell culture and primary human keloid fibroblast cell culture were used in this study. Trypan blue exclusion and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test were used to determine cell viability and proliferation. Propidium iodide stain was used for cell-cycle analysis by flow cytometry. Laser irradiation was performed daily on three consecutive days with a GaAlAs 660-nm laser (mean output: 50 mW, spot size 2 mm(2), power density = 2.5 W/cm(2)) and a typical LLLT dose and a high LLLT dose (irradiation times: 60 or 420 s; fluences: 150 or 1050 J/cm(2); energy delivered: 3 or 21 J). Results: Primary fibroblast cell culture from human keloids irradiated with 3 J showed significant proliferation by the trypan blue exclusion test (p < 0.05), whereas the 3T3 cell culture showed no difference using this method. Propidium iodide staining flow cytometry data showed a significant decrease in the percentage of cells being in proliferative phases of the cell cycle (S/g(2)/M) when irradiated with 21 J in both cell types (hypodiploid cells increased). Conclusions: Our data support the hypothesis that the physiological state of the cells affects the LLLT results, and that high-metabolic rate and short-cell-cycle 3T3 cells are not responsive to LLLT. In conclusion, LLLT with a dose of 3 J reduced cell death significantly, but did not stimulate cell cycle. A LLLT dose of 21 J had negative effects on the cells, as it increased cell death and inhibited cell proliferation.

Cellular Renewal and Improvement of Local Cell Effector Activity in Peritoneal Cavity in Response to Infectious Stimuli

The peritoneal cavity (PerC) is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p.) inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (Mempty set) subsets, namely small peritoneal Mempty set (SPM) and large peritoneal Mempty set (LPM), in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for beta-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO) and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-gamma stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal Mempty set subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

Induction of Heme Oxygenase-1 Can Halt and Even Reverse Renal Tubule-Interstitial Fibrosis

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-beta protein production was significantly lower in Hemin-treated animals. Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.

Effects of Aflatoxin B(1) and Fumonisin B(1) on the Viability and Induction of Apoptosis in Rat Primary Hepatocytes

The present study evaluated the effect of aflatoxin B(1) (AFB(1)) and fumonisin B(1) (FB(1)) either alone, or in association, on rat primary hepatocyte cultures. Cell viability was assessed by flow cytometry after propidium iodine intercalation. DNA fragmentation and apoptosis were assessed by agarose gel electrophoresis and acridine orange and ethidium bromide staining. At the concentrations of AFB(1) and FB(1) used, the toxins did not decrease cell viability, but did induce apoptosis in a concentration and time-dependent manner.

A Morphological and Histological Characterization of Bisexual and Male Flower Types in Pomegranate

Pomegranate [Punica granatum (Punicaceae)] is characterized by having two types of flowers on the same tree: hermaphroditic bisexual flowers and functionally male flowers. This condition, defined as functional andromonoecy, can result in decreased yields resulting from the inability of male flowers to set fruit. Morphological and histological analyses of bisexual and male flowers were conducted using light and scanning electron microscopy (SEM) to characterize the different flower types observed in pomegranate plants and to better understand their developmental differences. Bisexual flowers had a discoid stigma covered with copious exudate, elongated stigmatic papillae, a single elongate style, and numerous stamens inserted on the inner wall of the calyx tube. Using fluorescence staining, high numbers of pollen tubes were observed growing through a central stylar canal. Ovules were numerous, elliptical, and anatropous. In contrast, male flowers had reduced female parts and exhibited shortened pistils of variable heights. Stigmatic papillae of male flowers had little exudate yet supported pollen germination. However, pollen tubes were rarely observed in styles. Ovules in male flowers were rudimentary and exhibited various stages of degeneration. Pollen from both types of flowers was of similar size, approximate to 20 mu m, and exhibited similar percent germination using in vitro germination assays. Pollen germination was strongly influenced by temperature. Maximal germination (greater than 74%) was obtained at 25 and 35 degrees C; pollen germination was significantly lower at 15 degrees C (58%) and 5 degrees C (10%).

A comparison between the concentration of mast cells in squamous cell carcinomas of the skin and oral cavity

FUNDAMENTS: The lethality of squamous cell carcinomas (SCC) of the skin is considered low. SCC in the mouth is usually associated with poor prognosis. Current evidence suggests that mast cells in the normal tissue contribute to the tumorigenesis of SCC, probably by promoting angiogenesis. OBJECTIVE: The aim of this study was to compare the concentration of mast cells in SCC of the mouth and skin and evaluate whether there is a correlation with the degree of differentiation of these tumors. MATERIAL AND METHODS: Thirty cases of SCC of the skin and 34 of the mouth were investigated. Toluidine blue staining was used to identify mast cells in blocks containing the central portion of the neoplasm. RESULTS: A concentration of between 0 and 10 mast cells was found in one single case of SCC of the skin and there were no cases of SCC of the mouth with concentrations of mast cells in the tumor >201. In the majority of cases of SCC of the mouth (47%; n=16), mast cell concentration was between 0 and 10, with a concentration >51 mast cells in 80% of cases of SCC of the skin. All the cases of SCC of the mouth with a concentration of mast cells between 100 and 200 and 80% of those with a concentration of 51-99 were located on the lip. The concentration of mast cells was unrelated to the degree of differentiation of the tumor. CONCLUSION: The concentration of mast cells is lower in SCC of the mouth except when the tumor is located on the lip. This may reflect a lower need for cell activation in the microenvironment to improve vascularization in oral cancer.

Evaluation of aluminium tolerance in grapevine rootstocks

Aluminum (Al) toxicity is a major worldwide agricultural problem. At low pH, Al speciates into the soluble and phyto-toxic form Al(3+), inhibiting the root growth and affecting plant development. In Brazil, agriculture in acidic soils with elevated concentration of Al has significantly increased in the last decades. Therefore, in order to achieve efficient agriculture practices, the selection of plant cultivars with improved Al resistance has become crucial in this type of soils. In this work we have evaluated the Al resistance of six genotypes of grapevine rootstocks. The grapevine hardwood cuttings were grown in nutrient solution in the absence and presence of 250 and 500 mu M Al at pH 4.2. The phenotypic indexes of relative root growth, fresh and dry root weight, root area, hematoxylin staining profile, and Al content were evaluated for all six genotypes. These phenotypic indexes allowed us to identify the `Kober 5BB`, `Gravesac`, `Paulsen 1103`, and `IAC 766` grapevine rootstocks genotypes as the ones with the highest resistance to Al. Likewise, `IAC 572` and `R110` genotypes were the most Al-sensitive cultivars. We evaluated the root organic acid exudation profile in the most Al-resistant (`Kober 5BB`) and most Al-sensitive (`R110`) in plantlets cultivated in vitro in the absence and presence of 100, 200, and 400 mu M of Al. Among several compounds detected, citrate was the only organic acid related to the Al resistance phenotype observed in the `Kober 5BB` genotype. The high constitutive citrate exudation observed in `Kober 5BB` strongly suggests that exudation of this particular organic acid may impart Al-resistance/a melioration in grapevine.