Analysis of trophic responses in lesioned brain: focus on basic fibroblast growth factor mechanisms

The actions of fibroblast growth factors (FGFs), particularly the basic form (bFGF), have been described in a large number of cells and include mitogenicity, angiogenicity and wound repair. The present review discusses the presence of the bFGF protein and messenger RNA as well as the presence of the FGF receptor messenger RNA in the rodent brain by means of semiquantitative radioactive in situ hybridization in combination with immunohistochemistry. Chemical and mechanical injuries to the brain trigger a reduction in neurotransmitter synthesis and neuronal death which are accompanied by astroglial reaction. The altered synthesis of bFGF following brain lesions or stimulation was analyzed. Lesions of the central nervous system trigger bFGF gene expression by neurons and/or activated astrocytes, depending on the type of lesion and time post-manipulation. The changes in bFGF messenger RNA are frequently accompanied by a subsequent increase of bFGF immunoreactivity in astrocytes in the lesioned pathway. The reactive astrocytes and injured neurons synthesize increased amount of bFGF, which may act as a paracrine/autocrine factor, protecting neurons from death and also stimulating neuronal plasticity and tissue repair

p53 as a prognostic factor in adrenocortical tumors of adults and children

Mutations of the tumor suppressor gene p53 have been considered to be important determinants in several kinds of human cancer. Accumulation of p53 protein has been reported to correlate with more aggressive clinical behavior in some neoplasms. The role of p53 expression in adrenal cortical tumors (ACT) has not been elucidated but some studies have suggested its correlation with malignant behavior. Our objective was to determine if there is a correlation between the expression of immunoreactive p53 and the biological behavior of ACT. Fifty-seven ACT (21 from children and 36 from adults) were evaluated for p53 expression by immunohistochemistry in formalin-fixed paraffin-embedded tissue and analyzed in terms of outcome. The p53 parameter was utilized semiquantitatively. Tumors were classified as p53 negative when no positivity was observed, or when only few cells showed weak positivity (0/1+) and scored as p53 positive when there was a diffuse and strong nuclear positivity (2+/3+). In children, p53 positivity was associated with clinically malignant ACT and p53 negativity was associated with clinically benign ACT (P = 0.026). In adults' ACT, p53 positivity had an effect on disease-free survival (P<0.001) and also correlated with Weiss score, with a cutoff = 4 (P = 0.04). p53 expression was related to the clinical behavior of ACT in both children and adults and these findings seem to support a role for p53 in ACT progression.

Changes in endogenous tissue glutathione level in relation to murine ascites tumor growth and the anticancer activity of cisplatin

Changes in glutathione levels were determined in tissues of 11- to 12-week-old Swiss albino mice at different stages of Dalton's lymphoma tumor growth and following cisplatin (8 mg/kg body weight, ip) treatment for 24-96 h, keeping 4-5 animals in each experimental group. Glutathione levels increased in spleen of tumor-bearing compared to normal mice (9.95 ± 0.14 vs 7.86 ± 1.64 µmol/g wet weight, P<=0.05) but decreased in blood (0.64 ± 0.10 vs 0.85 ± 0.09 mg/ml) and testes (9.28 ± 0.15 vs 10.16 ± 0.28 µmol/g wet weight, P<=0.05). Dalton's lymphoma cells showed an increase in glutathione concentration (4.43 ± 0.26 µmol/g wet weight) as compared to splenocytes, their normal counterpart (3.62 ± 0.41 µmol/g wet weight). With the progression of tumor in mice, glutathione levels decreased significantly in testes (~10%) and bone marrow cells (~13%) while they increased in Dalton's lymphoma cells (28-46%) and spleen (15-27%). Glutathione levels in kidney, Dalton's lymphoma cells and bone marrow cells (8.50 ± 1.22, 4.43 ± 0.26 and 3.28 ± 0.17 µmol/g wet weight, respectively) decreased significantly (6.04 ± 0.42, 3.51 ± 0.32 and 2.17 ± 0.14 µmol/g wet weight, P<=0.05) after in vivo cisplatin treatment for 24 h. Along with a decrease in glutathione level, the glutathione-S-transferase (GST) activity also decreased by 60% in tumor cells after cisplatin treatment. The elevated drug uptake by the tumor cells under the conditions of reduced glutathione concentration and GST activity after treatment could be an important contributory factor to cisplatin's anticancer activity leading to tumor regression. Furthermore, lower doses of cisplatin in combination with buthionine sulfoximine (an inhibitor of glutathione synthesis) may be useful in cancer chemotherapy with decreased toxicity in the host.

Pentoxifylline decreases tumor necrosis factor and interleukin-1 during high tidal volume

Tumor necrosis factor-alpha (TNF-alpha) is one of the most important proinflammatory cytokines which plays a central role in host defense and in the acute inflammatory response related to tissue injury. The major source of TNF-alpha are immune cells such as neutrophils and macrophages. We tested the hypothesis that pentoxifylline, a methylxanthine derivative, down-regulates proinflammatory cytokine expression during acute lung injury in rats. Male Wistar rats weighing 250 to 450 g were anesthetized ip with 50 mg/kg sodium thiopental and randomly divided into three groups: group 1 (N = 7): tidal volume (V T) = 7 ml/kg, respiratory rate (RR) = 50 breaths/min and normal saline infusion; group 2 (N = 7): V T = 42 ml/kg, RR = 9 breaths/min and normal saline infusion; group 3 (N = 7): V T = 42 ml/kg, RR = 9 breaths/min and pentoxifylline infusion. The animals were ventilated with an inspired oxygen fraction of 1.0, a positive end-expiratory pressure of 3 cmH2O, and normal saline or pentoxifylline injected into the left femoral vein. The mRNA of TNF-alpha rapidly increased in the lung tissue within 180 min of ventilation with a higher V T with normal saline infusion. The concentrations of inflammatory mediators were decreased in plasma and bronchoalveolar lavage (BAL) in the presence of higher V T with pentoxifylline infusion (TNF-alpha: plasma, 102.2 ± 90.9 and BAL, 118.2 ± 82.1; IL-1ß: plasma, 45.2 ± 42.7 and BAL, 50.2 ± 34.9, P < 0.05). We conclude that TNF-alpha produced by neutrophil influx may function as an alert signal in host defense to induce production of other inflammatory mediators.

Modulation of the expression of the transcription factor Max in rat retinal ganglion cells by a recombinant adeno-associated viral vector

Exclusion of the transcription factor Max from the nucleus of retinal ganglion cells is an early, caspase-independent event of programmed cell death following damage to the optic axons. To test whether the loss of nuclear Max leads to a reduction in neuroprotection, we developed a procedure to overexpress Max protein in rat retinal tissue in vivo. A recombinant adeno-associated viral vector (rAAV) containing the max gene was constructed, and its efficiency was confirmed by transduction of HEK-293 cells. Retinal ganglion cells were accessed in vivo through intravitreal injections of the vector in rats. Overexpression of Max in ganglion cells was detected by immunohistochemistry at 2 weeks following rAAV injection. In retinal explants, the preparation of which causes damage to the optic axons, Max immunoreactivity was increased after 30 h in vitro, and correlated with the preservation of a healthy morphology in ganglion cells. The data show that the rAAV vector efficiently expresses Max in mammalian retinal ganglion cells, and support the hypothesis that the Max protein plays a protective role for retinal neurons.

Bradysia hygida (Diptera, Sciaridae) presents two eukaryotic Elongation Factor 1A gene homologues: partial characterization of the eukaryotic Elongation Factor 1A-F1 gene

Elongation factor 1A is a highly conserved protein that participates in translation. We report the occurrence of two genes homologous to the eukaryotic Elongation Factor 1A in Bradysia hygida and describe the partial cloning and characterization of the B. hygida eukaryotic Elongation Factor 1A-F1 (BheEF1A-F1) gene. The pattern of BheEF1A-F1 expression in the salivary gland at the end of the fourth larval instar was investigated using real-time PCR. The results showed that BheEF1A-F1 expression levels are relatively constant at the time when rapid changes in protein synthesis occur in this tissue. In situ hybridization experiments coupled to Southern blot analyses showed that the BheEF1A-F1 gene is located at position 3d of the A chromosome and a second gene homologous to eEF1A is located at position 6a of the X chromosome. Southern blot analyses showed that both the BheEF1A-F1 gene and the second gene homologous to eEF1A constitute non-amplified genes. The present results contribute to the molecular characterization of a sciarid eEF1A gene.

Interaction between growth differentiation factor 9, insulin-like growth factor I and growth hormone on the in vitro development and survival of goat preantral follicles

The objective of this study was to determine the effects of GDF-9, IGF-I, and GH alone or combined on preantral follicle survival, activation and development after 1 and 7 days of in vitro culture. Either fresh (non-cultured) or cultured ovarian tissue was processed for histological and fluorescence analysis. For all media tested, the percent of normal follicles was greater when compared to minimum essential medium supplemented (MEM+) alone, except when ovarian tissue was cultured with GDF-9/IGF-I or GDF-9/GH (P < 0.05). Fluorescence analysis showed that the percent of viable follicles after 7 days of culture was similar for non-cultured tissue and for all treatments tested. The percent of primordial follicles was reduced (P < 0.05) and there was a significant and concomitant increase in the percent of intermediate and primary follicles in all treatments tested after 7 days of culture when compared to non-cultured tissue. After 7 days of culture, the highest percent of intermediate follicles was observed with IGF-I/GH (61.3%), and the highest percent of primary follicles was achieved with IGF-I (57.7%). After 7 days of culture in MEM+ containing GDF-9, IGF-I and GH alone or in all associations, a significant increase in follicular diameter was observed when compared to MEM+ alone and non-cultured tissue. In conclusion, GDF-9, IGF-I and GH alone or in combination maintain preantral follicle survival and promote primordial follicle activation. Nevertheless, the data showed that IGF-I/GH and IGF-I alone are efficient in promoting the transition from primordial to intermediate follicles and from intermediate to primary follicles, respectively.

Plasma concentrations and placental immunostaining of interleukin-10 and tumornecrosis factor-α as predictors of alterations in the embryo-fetal organism and the placental development of diabetic rats

Interleukin-10 (IL-10) appears to be the key cytokine for the maintenance of pregnancy and inhibits the secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α). However, there are no studies evaluating the profile of these cytokines in diabetic rat models. Thus, our aim was to analyze IL-10 and TNF-α immunostaining in placental tissue and their respective concentrations in maternal plasma during pregnancy in diabetic rats in order to determine whether these cytokines can be used as predictors of alterations in the embryo-fetal organism and in placental development. These parameters were evaluated in non-diabetic (control; N = 15) and Wistar rats with streptozotocin (STZ)-induced diabetes (N = 15). At term, the dams (100 days of life) were killed under anesthesia and plasma and placental samples were collected for IL-10 and TNF-α determinations by ELISA and immunohistochemistry, respectively. The reproductive performance was analyzed. Plasma IL-10 concentrations were reduced in STZ rats compared to controls (7.6 ± 4.5 vs 20.9 ± 8.1 pg/mL). The placental scores of immunostaining intensity did not differ between groups (P > 0.05). Prevalence analysis showed that the IL-10 expression followed TNF-α expression, showing a balance between them. STZ rats also presented impaired reproductive performance and reduced plasma IL-10 levels related to damage during early embryonic development. However, the increased placental IL-10 as a compensatory mechanism for the deficit of maternal regulation permitted embryo development. Therefore, the data suggest that IL-10 can be used as a predictor of changes in the embryo-fetal organism and in placental development in pregnant diabetic rats.

Effect of neurotrophic factor, MDP, on rats’ nerve regeneration

Our objective was to determine the immune-modulating effects of the neurotrophic factor N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) on median nerve regeneration in rats. We used male Wistar rats (120-140 days of age, weighing 250-332 g) and compared the results of three different techniques of nerve repair: 1) epineural neurorrhaphy using sutures alone (group S - 10 rats), 2) epineural neurorrhaphy using sutures plus fibrin tissue adhesive (FTA; group SF - 20 rats), and 3) sutures plus FTA, with MDP added to the FTA (group SFM - 20 rats). Functional assessments using the grasp test were performed weekly for 12 weeks to identify recovery of flexor muscle function in the fingers secondary to median nerve regeneration. Histological analysis was also utilized. The total number and diameter of myelinated fibers were determined in each proximal and distal nerve segment. Two indices, reported as percentage, were calculated from these parameters, namely, the regeneration index and the diameter change index. By the 8th week, superiority of group SFM over group S became apparent in the grasping test (P = 0.005). By the 12th week, rats that had received MDP were superior in the grasping test compared to both group S (P < 0.001) and group SF (P = 0.001). Moreover, group SF was better in the grasping test than group S (P = 0.014). However, no significant differences between groups were identified by histological analysis. In the present study, rats that had received MDP obtained better function, in the absence of any significant histological differences.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

Plasma von Willebrand factor as a predictor of survival in pulmonary arterial hypertension associated with congenital heart disease

Biomarkers have been identified for pulmonary arterial hypertension, but are less well defined for specific etiologies such as congenital heart disease-associated pulmonary arterial hypertension (CHDPAH). We measured plasma levels of eight microvascular dysfunction markers in CHDPAH, and tested for associations with survival. A cohort of 46 inoperable CHDPAH patients (age 15.0 to 60.2 years, median 33.5 years, female:male 29:17) was prospectively followed for 0.7 to 4.0 years (median 3.6 years). Plasma levels of von Willebrand factor antigen (VWF:Ag), tissue plasminogen activator (t-PA) and its inhibitor (PAI-1), P-selectin, reactive C-protein, tumor necrosis factor alpha, and interleukin-6 and -10 were measured at baseline, and at 30, 90, and 180 days in all subjects. Levels of six of the eight proteins were significantly increased in patients versus controls (13 to 106% increase, P < 0.003). Interleukin-10 level was 2.06 times normal (P = 0.0003; Th2 cytokine response). Increased levels of four proteins (t-PA, PAI-1, P-selectin, and interleukin-6) correlated with disease severity indices (P < 0.05). Seven patients died during follow-up. An average VWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was independently associated with a high risk of death (hazard ratio = 6.56, 95%CI = 1.46 to 29.4, P = 0.014). Thus, in CHDPAH, microvascular dysfunction appears to involve Th2 inflammatory response. Of the biomarkers studied, plasma vWF:Ag was independently associated with survival.

Physical training prevents body weight gain but does not modify adipose tissue gene expression

The relationship of body weight (BW) with white adipose tissue (WAT) mass and WAT gene expression pattern was investigated in mice submitted to physical training (PT). Adult male C57BL/6 mice were submitted to two 1.5-h daily swimming sessions (T, N = 18), 5 days/week for 4 weeks or maintained sedentary (S, N = 15). Citrate synthase activity increased significantly in the T group (P < 0.05). S mice had a substantial weight gain compared to T mice (4.06 ± 0.43 vs 0.38 ± 0.28 g, P < 0.01). WAT mass, adipocyte size, and the weights of gastrocnemius and soleus muscles, lung, kidney, and adrenal gland were not different. Liver and heart were larger and the spleen was smaller in T compared to S mice (P < 0.05). Food intake was higher in T than S mice (4.7 ± 0.2 vs 4.0 ± 0.3 g/animal, P < 0.05) but oxygen consumption at rest did not differ between groups. T animals showed higher serum leptin concentration compared to S animals (6.37 ± 0.5 vs 3.11 ± 0.12 ng/mL). WAT gene expression pattern obtained by transcription factor adipocyte determination and differentiation-dependent factor 1, fatty acid synthase, malic enzyme, hormone-sensitive lipase, adipocyte lipid binding protein, leptin, and adiponectin did not differ significantly between groups. Collectively, our results showed that PT prevents BW gain and maintains WAT mass due to an increase in food intake and unchanged resting metabolic rate. These responses are closely related to unchanged WAT gene expression patterns.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

Adipose tissue-derived stem cells promote pancreatic cancer cell proliferation and invasion

To explore the effects of adipose tissue-derived stem cells (ADSCs) on the proliferation and invasion of pancreatic cancer cells in vitroand the possible mechanism involved, ADSCs were cocultured with pancreatic cancer cells, and a cell counting kit (CCK-8) was used to detect the proliferation of pancreatic cancer cells. ELISA was used to determine the concentration of stromal cell-derived factor-1 (SDF-1) in the supernatants. RT-PCR was performed to detect the expression of the chemokine receptor CXCR4 in pancreatic cancer cells and ADSCs. An in vitro invasion assay was used to measure invasion of pancreatic cancer cells. SDF-1 was detected in the supernatants of ADSCs, but not in pancreatic cancer cells. Higher CXCR4 mRNA levels were detected in the pancreatic cancer cell lines compared with ADSCs (109.3±10.7 and 97.6±7.6 vs 18.3±1.7, respectively; P<0.01). In addition, conditioned medium from ADSCs promoted the proliferation and invasion of pancreatic cancer cells, and AMD3100, a CXCR4 antagonist, significantly downregulated these growth-promoting effects. We conclude that ADSCs can promote the proliferation and invasion of pancreatic cancer cells, which may involve the SDF-1/CXCR4 axis.

New concepts in white adipose tissue physiology

Numerous studies address the physiology of adipose tissue (AT). The interest surrounding the physiology of AT is primarily the result of the epidemic outburst of obesity in various contemporary societies. Briefly, the two primary metabolic activities of white AT include lipogenesis and lipolysis. Throughout the last two decades, a new model of AT physiology has emerged. Although AT was considered to be primarily an abundant energy source, it is currently considered to be a prolific producer of biologically active substances, and, consequently, is now recognized as an endocrine organ. In addition to leptin, other biologically active substances secreted by AT, generally classified as cytokines, include adiponectin, interleukin-6, tumor necrosis factor-alpha, resistin, vaspin, visfatin, and many others now collectively referred to as adipokines. The secretion of such biologically active substances by AT indicates its importance as a metabolic regulator. Cell turnover of AT has also recently been investigated in terms of its biological role in adipogenesis. Consequently, the objective of this review is to provide a comprehensive critical review of the current literature concerning the metabolic (lipolysis, lipogenesis) and endocrine actions of AT.