Effects of immunosuppressants on hepatocyte cell mitosis during liver regeneration in growing animal models of partial hepatectomy

Objective. We sought to evaluate the effects of immunosuppressant drugs (corticosteroid, cyclosporine [CsA], and tacrolimus [Tac]) on liver regeneration in growing animals submitted to 70% hepatectomy. Materials and Methods. Newborn and weaning rats were submitted to 70% hepatectomy receiving separately methylprednisolone, CsA, or Tac. All animals were sacrificed 24 hours after the procedure. The remnant liver lobes were subjected to histomorphometric analyses with determination of hepatocyte mitotic index. Results., Administration of immunosuppressants did not change the mitotic index of the regenerating liver in newborn animals. In weaning rats, methylprednisolone reduced the mitotic index (P = .01) and Tac caused a greater increase in this rate (P = .001). CsA had no effect on mitotic index. The number of hepatocyte mitoses in newborn animal livers was greater than that in weaning animal livers (P = .001). Conclusion. In situations in which intense, fast processes of liver regeneration are crucial, the advantages of the use of Tac must be considered, such as in pediatric transplant patients.

Is facial pattern a predisposing factor for otitis media with effusion in children?

Abnormalities in craniofacial morphology are associated with Eustachian tube dysfunction and otitis media with effusion (OME). Aim: to evaluate the relationship between facial pattern and craniofacial growth direction, and OME in children with enlarged tonsils and adenoids (ETA). Methods: Clinical prospective survey in 79 children (41 male and 38 female), ranging from 4 to 10 years of age, with tonsil and adenoid enlargement (Brodsky`s grades III and IV). Forty children presented with OME (study group) and 39 did not (control group). Cephalometric analysis was used to determine the facial pattern. Results: There was no correlation observed between facial pattern and OME (c 2 = 0.25 p = 0.88). Facial Axis was larger in the OME group (F(1.75) = 3.68 p = 0.05) and the Lower Anterior Facial height was smaller (F(1. 75) = 3.99 p = 0.05) in children with otitis media with effusion. Conclusions: There was no correlation between OME and facial pattern in children with ETA although a more horizontal facial growth direction, and a smaller lower anterior facial height was observed consistently among subjects in this group. This suggests that abnormal positioning of the eustachian tube influences the development of OME in children with ETA.

Fibroblast Growth Factor 23 in Hemodialysis Patients: Effects of Phosphate Binder, Calcitriol and Calcium Concentration in the Dialysate

Background: Fibroblast growth factor 23 (FGF23) concentrations increase early in chronic kidney disease (CKD), and the influence of current CKD-mineral and bone disorder (MBD) therapies on serum FGF23 levels is still under investigation. Methods: In this post-hoc analysis of a randomized clinical trial, phosphate binders and calcitriol were washed out of 72 hemodialysis patients who were then submitted to bone biopsy, coronary tomography and biochemical measures, including FGF23. They were randomized to receive sevelamer or calcium acetate for 1 year and the prescription of calcitriol and the calcium concentration in the dialysate were adjusted according to serum calcium, phosphate and PTH and bone biopsy diagnosis. Results: At baseline, bone biopsy showed that 58.3% had low-turnover bone disease, whereas 38.9% had high-turnover bone disease, with no significant differences between them with regard to FGF23. Median baseline FGF23 serum levels were elevated and correlated positively with serum phosphate. After 1 year, serum FGF23 decreased significantly. Repeated measures ANOVA analysis showed that the use of a 3.5-mEq/l calcium concentration in the dialysate, as well as the administration of calcitriol and a calcium-based phosphate binder were associated with higher final serum FGF23 levels. Conclusions: Taken together, our results confirm that the current CKD-MBD therapies have an effect on serum levels of FGF23. Since FGF23 is emerging as a potential treatment target, our findings should be taken into account in the decision on how to manage CKD-MBD therapy. Copyright (C) 2010 S. Karger AG, Basel

Expression of insulin-like growth factor-II and its receptor in pediatric and adult adrenocortical tumors

Background: Adrenocortical tumors are heterogeneous neoplasms with incompletely understood pathogenesis. IGF-II overexpression has been consistently demonstrated in adult adrenocortical carcinomas. Objectives: The objective of the study was to analyze expression of IGF-II and its receptor (IGF-IR) in pediatric and adult adrenocortical tumors and the effects of a selective IGF-IR kinase inhibitor (NVP-AEW541) on adrenocortical tumor cells. Patients: Fifty-seven adrenocortical tumors (37 adenomas and 20 carcinomas) from 23 children and 34 adults were studied. Methods: Gene expression was determined by quantitative real-time PCR. Cell proliferation and apoptosis were analyzed in NCI H295 cells and a new cell line established from a pediatric adrenocortical adenoma. Results: IGF-II transcripts were overexpressed in both pediatric adrenocortical carcinomas and adenomas. Otherwise, IGF-II was mainly overexpressed in adult adrenocortical carcinomas (270.5 +/- 130.2 vs. 16.1 +/- 13.3; P = 0.0001). IGF-IR expression was significantly higher in pediatric adrenocortical carcinomas than adenomas (9.1 +/- 3.1 vs. 2.6 +/- 0.3; P = 0.0001), whereas its expression was similar in adult adrenocortical carcinomas and adenomas. IGF-IR expression was a predictor of metastases in pediatric adrenocortical tumors in univariate analysis (hazard ratio 1.84; 95% confidence interval 1.28 -2.66; P = 0.01). Furthermore, NVP-AEW541 blocked cell proliferation in a dose-and time-dependent manner in both cell lines through a significant increase of apoptosis. Conclusion: IGF-IR overexpression was a biomarker of pediatric adrenocortical carcinomas. Additionally, a selective IGF-IR kinase inhibitor had antitumor effects in adult and pediatric adrenocortical tumor cell lines, suggesting that IGF-IR inhibitors represent a promising therapy for human adrenocortical carcinoma.

Lupus pleuritis: a relevant risk factor for pulmonary tuberculosis

The objective of the study was to evaluate risk factors for pulmonary tuberculosis in systemic lupus erythematosus (SLE). Clinical/laboratorial features of 1283 SLE patients (ACR criteria) followed at the Lupus Clinic were obtained from the electronic register database from 2001 to 2009. Pulmonary tuberculosis was diagnosed in 20 patients (1.6%) (TB+ group). As control group (TB-), 40 patients without tuberculosis matched for age, gender, ethnicity, age at SLE diagnosis, and disease duration were arbitrarily selected. All 20 patients of the TB+ group presented confirmed pulmonary tuberculosis from 1 to 23 years after SLE diagnosis (7.6 +/- 8.1 years). Frequencies of previous SLE involvements (cutaneous, articular, hematological, renal, pericarditis, pneumonitis, and central nervous system) were alike in TB+ and TB- groups (p > 0.05). In contrast, prior pleuritis was more frequent in the TB+ group (40% vs. 5%, p=0.001). In fact, pulmonary tuberculosis was diagnosed in 8/10 patients with previous pleuritis. Immunosuppressive and corticosteroid therapies at the moment of tuberculosis diagnosis were also similar in both groups (p > 0.05). We have identified pleuritis as a relevant risk factor for pulmonary tuberculosis, suggesting that previous pleural injury is a critical part of the complex interplay between altered immune system, socio-economic conditions, and increased susceptibility to this mycobacterial infection. Lupus (2010) 19, 1585-1590.

Vitamin D insufficiency: A risk factor to vertebral fractures in community-dwelling elderly women

Objective:To determine the risk factors for the presence of moderate/severe vertebral fracture, specifically 25-hydroxyvitamin D (25-OHD). Study design: Cross-sectional study conducted for 2 years in the city of Sao Paulo, Brazil including community-dwelling elderly women. Methods: Bone mineral density (BMD), serum 25-OHD, intact parathyroid hormone (iPTH), calcium and estimated glomerular filtration rate (eGFR) were examined in 226 women without vertebral fractures (NO FRACTURE group) and 189 women with at least one moderate/severe vertebral fracture (FRACTURE group). Vertebral fracture assessment (VFA) was evaluated using both the Genant semiquantitative (SQ) approach and morphometry. Results: Patients in the NO FRACTURE group had lower age, increased height, higher calcium intake, and higher BMD compared to those patients in the FRACTURE group (p < 0.05). Of interest, serum levels of 25-OHD in the NO FRACTURE group were higher than those observed in the FRACTURE group (51.73 nmol/L vs. 42.31 nmol/L, p < 0.001). Reinforcing this finding, vitamin D insufficiency (25-OHD < 75 nmol/L) was observed less in the NO FRACTURE group (82.3% vs. 93.65%, p = 0.001). After adjustment for significant variables within the patient population (age, height, race, calcium intake, 25-OHD, eGFR and sites BMD), the logistic-regression analyses revealed that age (OR = 1.09, 95% Cl 1.04-1.14, p < 0.001) femoral neck BMD (OR = 0.7, 95% CI 0.6-0.82, p < 0.001) and 25-OHD <75 nmol/L (OR = 2.38, 95% CI 1.17-4.8, p = 0.016) remains a significant factor for vertebral fracture. Conclusion: Vitamin D insufficiency is a contributing factor for moderate/severe vertebral fractures. This result emphasizes the importance of including this modifiable risk factor in the evaluation of elderly women. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Intramyocardial transplantation of fibroblasts expressing vascular endothelial growth factor attenuates cardiac dysfunction

In this study, we analyzed whether transplantation of cardiac fibroblasts (CFs) expressing vascular endothelial growth factor (VEGF) mitigates cardiac dysfunction after myocardial infarction (MI) in rats. First, we observed that the transgene expression lasts longer (45 vs 7 days) when fibroblasts are used as vectors compared with myoblasts. In a preventive protocol, induction of cardiac neovascularization accompanied by reduction in myocardial scar area was observed when cell transplantation was performed 1 week before ischemia/reperfusion and the animals analyzed 3 weeks later. Finally, the therapeutic efficacy of this approach was tested injecting cells in a fibrin biopolymer, to increase cardiac retention, 24 h post-MI. After 4 weeks, an increase in neovascularization and a decrease in myocardial collagen were observed only in rats that received cells expressing VEGF. Basal indirect or direct hemodynamic measurements showed no differences among the groups whereas under pharmacological stress, only the group that received cells expressing VEGF showed a significant reduction in end-diastolic pressure and improvement in stroke volume and cardiac work. These results indicate that transplantation of CFs expressing VEGF using fibrin biopolymer induces neovascularization and attenuates left ventricle fibrosis and cardiac dysfunction in ischemic heart. Gene Therapy (2010) 17, 305-314; doi:10.1038/gt.2009.146; published online 10 December 2009

NFAT1 transcription factor is central in the regulation of tissue microenvironment for tumor metastasis

Members of the nuclear factor of activated T cell (NFAT) family of transcription factors were originally described in T lymphocytes but later shown to be expressed in several immune and non-immune cell types. NFAT proteins can modulate cellular transformation intrinsically, and NFAT-deficient (NFAT1-/-) mice are indeed more susceptible to transformation than wild-type counterparts. However, the contribution of an NFAT1-/- microenvironment to tumor progression has not been studied. We have addressed this question by inoculating NFAT1-/- mice with B16F10 melanoma cells intravenously, an established model of tumor homing and growth. Surprisingly, NFAT1-/- animals sustained less tumor growth in the lungs after melanoma inoculation than wild-type counterparts. Even though melanoma cells equally colonize NFAT1-/- and wild-type lungs, tumors do not progress in the absence of NFAT1 expression. A massive mononuclear perivascular infiltrate and reduced expression of TGF-beta in the absence of NFAT1 suggested a role for tumor-infiltrating immune cells and the cytokine milieu. However, these processes are independent of an IL-4-induced regulatory tumor microenvironment, since lack of this cytokine does not alter the phenotype in NFAT1-/- animals. Bone marrow chimera experiments meant to differentiate the contributions of stromal and infiltrating cells to tumor progression demonstrated that NFAT1-induced susceptibility to pulmonary tumor growth depends on NFAT1-expressing parenchyma rather than on bone marrow-derived cells. These results suggest an important role for NFAT1 in radio-resistant tumor-associated parenchyma, which is independent of the anti-tumor immune response and Th1 versus Th2 cytokine milieu established by the cancer cells, but able to promote site-specific tumor growth.

The role of p38 mitogen-activated protein kinase in serum-induced leukemia inhibitory factor secretion by bone marrow stromal cells from pediatric myelodysplastic syndromes

Stromal cells from pediatric myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) associated with MDS(MDS-AML) present high expression of leukemia inhibitor factor (LIF). We demonstrated using mitogen-activated protein kinase ( MAPK) inhibitors that in stromal cells from pediatric MDS and MDS-AML, p38MAPK was critical in serum-induced secretion of LIF. The serum induction of phosphorylated p38MAPK form was observed only in stromal cells from healthy children, whereas in MDS and MDS-AML basal levels were maintained suggesting constitutive p38MAPK activation. Our study suggested the possible importance in pediatric MDS of p38MAPK signaling pathway which may be a future therapeutic target. (C) 2009 Elsevier Ltd. All rights reserved.

Insulin-like growth factor-1 and 17 beta-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells

The PKC apoptosis WTI regulator gene, also named prostate apoptosis response-4 (PAR-4), encodes a pro-apoptotic protein that sensitizes cells to numerous apoptotic stimuli. Insulin-like growth factor-1 (IGF-1) and 17 beta-estradiol (E2), two important factors for breast cancer development and progression, have been shown to down-regulate PAR-4 expression and inhibit apoptosis induced by PAR-4 in neuronal cells. In this study, we sought to investigate the mechanisms of regulation of PAR-4 gene expression in MCF-7 cells treated with E2 or IGF-1. E2 (10 nM) and IGF-1 (12.5 nM) each down-regulated PAR-4 expression in MCF-7 cells after 24 h of treatment. The effect of E2 was dependent on ER activation, as demonstrated by an increase in PAR-4 expression when cells were pretreated for 1 h with 1 mu M ICI-182,780 (ICI) before receiving E2 plus ICI. The effect of IGF-1 was abolished by pre-treatment for 1 h with 30 mu M LY294002 (a specific PI3-K inhibitor), and significantly inhibited by 30 mu M SB202190 (a specific p38MAPK inhibitor). We also demonstrated that E2 acts synergistically with IGF-1, resulting in greater down-regulation of PAR-4 mRNA expression compared with E2 or IGF-1 alone. Our results show for the first time that E2 and IGF-1 inhibit PAR-4 gene expression in MCF-7 cells, suggesting that this down-regulation may provide a selective advantage for breast cancer cell survival.

Concomitant expression of epithelial-mesenchymal transition biomarkers in breast ductal carcinoma: Association with progression

Epithelial to mesenchymal transition (EMT) is a process implicated in cancer progression in which the underlying cellular changes have been identified mainly using in vitro models. We determined the expression of some putative EMT biomarkers including E-cadherin, beta-catenin, zinc finger factor Snail (Snail), transforming growth factor beta 1 (TGF beta 1), TGF beta type II receptor (TBRII) and the HGF receptor (c-met) and their possible correlation to progression and overall survival in a series of breast ductal carcinoma in situ (DCIS) and invasive ductal carcinomas (IDC). Biomarkers were immunohistochemically determined in 55 IDC specimens from which 21 had lymph node metastases and in 95 DCIS specimens, 46 of these cases associated to invasive carcinoma, in a tissue microarray (TMA). Positive cytoplasmic staining of TGF beta 1 (78.2%), c-met (43.6%), Snail (34.5%), TBRII (100%), membranous E-cadherin (74.5%) and membranous/cytoplasmic beta-catenin (71%) were detected in the IDC samples. Metastatic lymph node samples displayed similar frequencies. A significant increase of c-met and TGF beta 1 positivity along DCIS to IDC progression was noted but only TGF beta 1 positivity was associated with presence of lymph node metastases and advanced stages in IDC. The evaluation of the other EMT markers in DCIS did not show differences in positivity rate as compared to invasive carcinomas. DCIS either pure or associated to IDC showed similar expression of the analyzed biomarkers. All the carcinomas exhibited positive expression of TBRII. Associations between the markers, determined by Spearman`s correlation coefficient, showed a significant association between TGF beta 1 and respectively E-cadherin, beta-catenin and cmet in DCIS cases, but in invasive carcinomas only cadherin and catenin were positively correlated. Kaplan-Meier survival curves revealed that none of the EMT biomarkers analyzed were correlated with survival, which was significantly determined only by clinical and hormone receptor parameters.

Tamoxifen inhibits transforming growth factor-alpha gene expression in human breast carcinoma samples treated with triiodothyronine

Objectives: To examine the effects of triiodothyronine (T(3)), 17 beta-estradiol (E(2)), and tamoxifen (TAM) on transforming growth factor (TGF)-alpha gene expression in primary breast cancer cell cultures and interactions between the different treatments. Methods and results: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3-mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T(3); dish 3: T(3)+TAM; dish 4: TAM; dish 5: E(2); dish 6: E(2)+TAM. TGF-alpha mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T(3) for 48 h significantly increased TGF-alpha mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-alpha mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. Conclusion: We demonstrate that TGF-alpha mRNA expression is more efficiently upregulated by T(3) than E(2). Concomitant treatment with TAM had a mitigating effect on the T(3) effect, while E(2) induced TGF-alpha upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-alpha, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER alpha and beta; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E(2).. Endocrinol. Invest. 31: 1047-1051, 2008) (c) 2008, Editrice Kurtis

The influence of the amount of sunscreen applied and its sun protection factor (SPF): evaluation of two sunscreens including the same ingredients at different concentrations

Background To estimate labeled sun protection factor (SPF) for sunscreen, the amount of product applied on volunteers, according to food and drug administration (FDA) and International protocols, is 2 mg/cm(2). However, different studies have shown that consumers actually apply much less product when exposed to the sun. Previous studies have reported contradictory findings in an attempt to correlate the amount applied in relation to SPF. The objective of the present study was to estimate the influence of the quantity of sunscreen applied in the determination of SPF, according to the FDA methodology. Subjects and methods Forty volunteers were included in two groups (SPF 15 and 30). The selected sunscreen was then applied in four different quantities (2, 1.5, 1.0 and 0.5 mg/cm(2)). All areas were irradiated with a solar simulator. After 24 h, the minimal erythemal dose (MED) and SPF were determined. Results In both groups, we observed that the SPF decreased when the amount of sunscreen applied was decreased. The differences between the 2 mg/cm(2) area and the others were significant in both groups (P < 0.001). The correlation between specified SPF and applied amount grew exponentially. Conclusion The protection provided by sunscreen is related to the amount of product applied. It is essential to educate consumers to apply larger amounts of sunscreen for adequate photoprotection.

Experimental models of sepsis and their clinical relevance

Sepsis remains a major cause of morbidity and mortality mainly because of sepsis-induced multiple organ dysfunction. In contrast to preclinical studies, most clinical trials of promising new treatment strategies for sepsis have failed to demonstrate efficacy. Although many reasons could account for this discrepancy, the misinterpretation of preclinical data obtained from experimental studies and especially the use of animal models that do not adequately mimic human sepsis may have been contributing factors. In this review, the potentials and limitations of various animal models of sepsis are discussed to clarify to which extent these findings are relevant to human sepsis. Such models include intravascular infusion of endotoxin or live bacteria, bacterial peritonitis, cecal ligation and perforation, soft tissue infection, pneumonia or meningitis models using different animal species including rats, mice, rabbits, dogs, pigs, sheep, and nonhuman primates. Despite several limitations, animal models remain essential in the development of all new therapies for sepsis and septic shock because they provide fundamental information about the pharmacokinetics, toxicity, and mechanism of drug action that cannot be replaced by other methods. New therapeutic agents should be studied in infection models, even after the initiation of the septic process. Furthermore, debility conditions need to be reproduced to avoid the exclusive use of healthy animals, which often do not represent the human septic patient.

Establishment and characterization of androgen-independent human prostate cancer cell lines, PcBra1, PcBra2, and PcBra3

One of the main obstacles for understanding biological events involved in cancer is the lack of experimental models for in vitro studies especially for prostate cancer (PC).There are a limited number of PC cell lines being the majority originated from metastatic tumors mostly acquired from American Tissue Cell Culture which demands importation an expensive and bureaucratic process. Also it is well known that there are ethnic differences between populations concerning the behavior of tumors and the research based on cell lines derived from Brazilians should be interesting. Our aim was to develop tumor cell lines from primary PC.