Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21(cip1) in vascular remodelling during vein arterialization in the rat

Neo-intima development and atherosclerosis limit long-term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo-intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while alpha-actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs. the media layer (100 vs. 2 times respectively). Cyclin-dependent kinase inhibitors (CDKIs) p27(Kip1) and p16(INKA) remained unchanged, whereas p21(Cip1) was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21(Cip1) is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long-term patency of vein grafts.

Prognostic heterogeneity among patients with chronic stable coronary disease: determinants of long-term mortality after treatment with percutaneous intervention

Aims: To evaluate the risk and predictors of death in a large population of patients with stable coronary disease treated with percutaneous intervention. Methods and results: The study population comprised 1,276 patients with chronic angina or silent ischaemia who underwent elective coronary angioplasty. Baseline and in-hospital mortality data were prospectively collected for all patients during the index hospitalisation. Post-discharge outcome was assessed at out-patient clinic, by review of the patients` records, or direct phone contact. Deaths were classified as cardiac and non-cardiac. Age, peripheral arterial disease, congestive heart failure with NYHA class Ill, triple-vessel disease, and procedural success (i.e. angiographic success for all lesions in the absence of pen-procedural infarction) remained as multivariate independent predictors of death. For the entire population 4-year cumulative all-cause and cardiac mortality were respectively 5.4% and 4.1%. Four-year mortality for patients without any multivariate predictor was 2.4%, while for patients with two or more predictors the death rate was 16.3% after four years. Conclusions: Patients with stable coronary disease undergoing percutaneous treatment have an overall low mortality rate after four years. Nevertheless, stable patients comprise a heterogeneous population in terms of risk profile, ranging from patients at very low risk of late death to individuals with a poor long-term prognosis.

Benefit of antiretroviral therapy on survival of human immunodeficiency virus-infected patients admitted to an intensive care unit

Objective: To evaluate the impact of antiretroviral therapy (ART) and the prognostic factors for in-intensive care unit (ICU) and 6-month mortality in human immunodeficiency virus (HIV)-infected patients. Design: A retrospective cohort study was conducted in patients admitted to the ICU from 1996 through 2006. The follow-up period extended for 6 months after ICU admission. Setting: The ICU of a tertiary-care teaching hospital at the Universidade de Sao Paulo, Brazil. Participants: A total of 278 HIV-infected patients admitted to the ICU were selected. We excluded ICU readmissions (37), ICU admissions who stayed less than 24 hours (44), and patients with unavailable medical charts (36). Outcome Measure: In-ICU and 6-month mortality. Main Results: Multivariate logistic regression analysis and Cox proportional hazards models demonstrated that the variables associated with in-ICU and 6-month mortality were sepsis as the cause of admission (odds ratio [OR] = 3.16 [95% confidence interval [CI] 1.65-6.06]); hazards ratio [HR] = 1.37 [95% Cl 1.01-1.88)), an Acute Physiology and Chronic Health Evaluation 11 score >19 [OR = 2.81 (95% CI 1.57-5.04); HR = 2.18 (95% CI 1.62-2.94)], mechanical ventilation during the first 24 hours [OR = 3.92 (95% CI 2.20-6.96); HR = 2.25 (95% CI 1.65-3.07)], and year of ICU admission [OR = 0.90 (95% CI 0.81-0.99); HR = 0.92 [95% CI 0.87-0.97)]. CD4 T-cell count <50 cells/mm(3) Was only associated with ICU mortality [OR = 2.10 (95% Cl 1.17-3.76)]. The use of ART in the ICU was negatively predictive of 6-month mortality in the Cox model [HR = 0.50 (95% CI 0.35-0.71)], especially if this therapy was introduced during the first 4 days of admission to the ICU [HR = 0.58 (95% CI 0.41-0.83)]. Regarding HIV-infected patients admitted to ICU without using ART, those who have started this treatment during ICU, stay presented a better prognosis when time and potential confounding factors were adjusted for [HR 0.55 (95% CI 0.31-0.98)]. Conclusions: The ICU outcome of HIV-infected patients seems to be dependent not only on acute illness severity, but also on the administration of antiretroviral treatment. (Crit Care Med 2009; 37: 1605-1611)

Expression profiles of the glucose-dependent insulinotropic peptide receptor and LHCGR in sporadic adrenocortical tumors

Glucose-dependent insulinotropic peptide receptor (GIPR) and LHCGR are G-protein-coupled receptors with a wide tissue expression pattern. Aberrant expression of these receptors has rarely been demonstrated in adult sporadic adrenocortical tumors with a lack of data on pediatric tumors. We quantified the GIPR and LHCGR expression in a large cohort of 55 patients (25 children and 30 adults) with functioning and non-functioning sporadic adrenocortical tumors. Thirty-eight tumors were classified as adenomas whereas 17 were carcinomas. GIPR, and LHCGR expression were analyzed by real-time PCR and normal human pancreatic and testicular tissue samples were used as positive controls. Mean expression values were determined by fold increase in comparison with a normal adrenal pool. GIPR mRNA levels were significantly higher in adrenocortical carcinomas than in adenomas from both pediatric and adult groups. LHCGR expression was similar in both carcinomas and adenomas from the pediatric group but significantly lower in carcinomas than in adenomas from the adult group (median 0.06 and 2.3 respectively, P<0.001). GIPR was detected by immunohistochemistry in both pediatric and adult tumors. Staining and real-time PCR results correlated positively only when GIPR in RN A levels were increased at least two-fold in comparison with normal adrenal expression levels. In Conclusion, GIPR overexpression was observed in pediatric and adult adrenocortical tumors and very low levels of LHCGR expression were found in all adult adrenocortical carcinomas.

Talc pleurodesis: Evidence of systemic Inflammatory response to small size talc particles

The mechanisms of the systemic response associated with talc-induced pleurodesis are poorly understood. The aim of this study was to assess the acute inflammatory response and migration of talc of small. size particles injected in the pleural space. Rabbits were injected intrapleurally with talc solution containing small. or mixed particles and blood and pleural fluid samples were collected after 6, 24 or 48 h and assayed for leukocytes, neutrophils, lactate dehydrogenase, IL-8, VEGF, and TGF-beta. The lungs, spleen, liver and kidneys were assessed to study deposit of talc particles. Both types of talc produced an acute serum inflammatory response, more pronounced in the small particles group. Pleural fluid IL-8 and VEGF levels were higher in the small particle talc group. Correlation between pleural VEFG and TGF-beta levels was observed for both groups. Although talc particles were demonstrated in the organs of both groups, they were more pronounced in the small talc group. In conclusion, intrapleural injection of talc of small size particles produced a more pronounced acute systemic response and a greater deposition in organs than talc of mixed particles. (C) 2008 Elsevier Ltd. All rights reserved.

Factors determining normal adult height in girls with gonadotropin-dependent precocious puberty treated with depot gonadotropin-releasing hormone analogs

Context: Several factors can affect adult height (AH) of patients with gonadotropin-dependent precocious puberty (GDPP) treated with depot GnRH analogs. Objective: Our objective was to determine factors influencing AH in patients with GDPP treated with depot GnRH analogs. Patients: A total of 54 patients (45 girls) with GDPP treated with depot GnRH analog who reached AH was included in the study. Design: Univariate and multivariate analyses of the factors potentially associated with AH were performed in all girls with GDPP. In addition, clinical features of the girls who attained target height (TH) range were compared with those who did not. Predicted height using Bayley and Pinneau tables was compared with attained AH. Results: In girls the mean AH was 155.3 +/- 6.9 cm (-1.2 +/- 1 SD) with TH range achieved by 81% of this group. Multiple regression analysis revealed that the interval between chronological age at onset of puberty and at the start of GnRH analog therapy, height SD scores (SDSs) at the start and end of therapy, and TH explained 74% of AH variance. The predicted height at interruption of GnRH therapy, obtained from Bayley and Pinneau tables for average bone age, was more accurate than for advanced bone age in both sexes. In boys the mean AH was 170.6 +/- 9.2 cm (-1 +/- 1.3 SDS), whereas TH was achieved by 89% of this group. Conclusions: The major factors determining normal AH in girls with GDPP treated with depot GnRH analogs were shorter interval between the onset of puberty and start of therapy, higher height SDS at the start and end of therapy, and TH. Therefore, prompt depot GnRH analog therapy in properly selected patients with GDPP is critical to obtain normal AH.

High frequency of submicroscopic chromosomal imbalances in patients with syndromic craniosynostosis detected by a combined approach of microsatellite segregation analysis, multiplex ligation-dependent probe amplification and array-based comparative genome hybridisation

We present the first comprehensive study, to our knowledge, on genomic chromosomal analysis in syndromic craniosynostosis. In total, 45 patients with craniosynostotic disorders were screened with a variety of methods including conventional karyotype, microsatellite segregation analysis, subtelomeric multiplex ligation-dependent probe amplification) and whole-genome array-based comparative genome hybridisation. Causative abnormalities were present in 42.2% (19/45) of the samples, and 27.8% (10/36) of the patients with normal conventional karyotype carried submicroscopic imbalances. Our results include a wide variety of imbalances and point to novel chromosomal regions associated with craniosynostosis. The high incidence of pure duplications or trisomies suggests that these are important mechanisms in craniosynostosis, particularly in cases involving the metopic suture.

Evaluation of rhBMP-2 and Natural Latex as Potential Osteogenic Proteins in Critical Size Defects by Histomorphometric Methods

This in vivo study evaluated the osteogenic potential of two proteins, recombinant human bone morphogenetic protein-2 (rhBMP-2) and a protein extracted from natural latex (Hevea brasiliensis, P-1), and compared their effects on bone defects when combined with a carrier or a collagen gelatin. Eighty-four (84) Wistar rats were divided into two groups, with and without the use of collagen gelatin, and each of these were divided into six treatment groups of seven animals each. The treatment groups were: (1) 5 mu g of pure rhBMP-2; (2) 5 mu g of rhBMP-2/monoolein gel; (3) pure monoolein gel; (4) 5 mu g of pure P-1; (5) 5 mu g of P-1/monoolein gel; (6) critical bone defect control. The animals were anesthetized and a 6 mm diameter critical bone defect was made in the left posterior region of the parietal bone. Animals were submitted to intracardiac perfusion after 4 weeks and the calvaria tissue was removed for histomorphometric analysis. In this experimental study, it was concluded that rhBMP-2 allowed greater new bone formation than P-1 protein and this process was more effective when the bone defect was covered with collagen gelatin (P < 0.05). Anat Rec, 293:794-801, 2010. (C) 2010 Wiley-Liss, Inc.

Apoptosis induced by Oropouche virus infection in HeLa cells is dependent on virus protein expression

Oropouche (OROV) is a single-stranded RNA arbovirus of the family Bunyaviridae, genus Orthobunyavirus, which has caused over half a million cases of febrile illness in Brazil in the past 30 years. OROV fever has been registered almost exclusively in the Amazon region, but global warming, deforestation and redistribution of vectors and animal reservoirs increases the risk of Oropouche virus emergence in other areas. OROV causes a cytolytical infection in cultured cells with characteristic cytopathic effect 48 h post-infection. We have studied the mechanisms of apoptosis induced by OROV in HeLa cells and found that OROV causes DNA fragmentation detectable by gel electrophoresis and by flow cytometric analysis of the Sub-G1 population at 36 h post-infection. Mitochondrial release of cytochrome C and activation of caspases 9 and 3 were also detected by western blot analysis. Lack of apoptosis induced by UV-inactivated OROV reveals that virus-receptor binding is not sufficient to induce cell death. Results obtained in cells treated with chloroquine and cycloheximide indicated that viral uncoating and replication are required for apoptosis induction by OROV. Furthermore, treatment of the cells with pan-caspase inhibitor prevented OROV-induced apoptosis without affecting virus progeny production. The results show that OROV infection in vitro causes apoptosis by an intracellular pathway involving mitochondria, and activated by a mechanism dependent on viral replication and protein synthesis. (C) 2010 Elsevier B.V. All rights reserved.

Pore Formation Triggered by Legionella spp. Is an Nlrc4 Inflammasome-Dependent Host Cell Response That Precedes Pyroptosis

Legionella pneumophila, the etiological agent of Legionnaires disease, is known to trigger pore formation in bone marrow-derived macrophages (BMMs) by mechanisms dependent on the type IVB secretion system known as Dot/Icm. Here, we used several mutants of L. pneumophila in combination with knockout mice to assess the host and bacterial factors involved in pore formation in BMMs. We found that regardless of Dot/Icm activity, pore formation does not occur in BMMs deficient in caspase-1 and Nlrc4/Ipaf. Pore formation was temporally associated with interleukin-1 beta secretion and preceded host cell lysis and pyroptosis. Pore-forming ability was dependent on bacterial Dot/Icm but independent of several effector proteins, multiplication, and de novo protein synthesis. Flagellin, which is known to trigger the Nlrc4 inflammasome, was required for pore formation as flaA mutant bacteria failed to induce cell permeabilization. Accordingly, transfection of purified flagellin was sufficient to trigger pore formation independent of infection. By using 11 different Legionella species, we found robust pore formation in response to L. micdadei, L. bozemanii, L. gratiana, L. jordanis, and L. rubrilucens, and this trait correlated with flagellin expression by these species. Together, the results suggest that pore formation is neither L. pneumophila specific nor the result of membrane damage induced by Dot/Icm activity; instead, it is a highly coordinated host cell response dependent on host Nlrc4 and caspase-1 and on bacterial flagellin and type IV secretion system.

In Vitro Phototoxicity of Liposomes and Nanocapsules Containing Chloroaluminum Phthalocyanine on Human Melanoma Cell Line

In this study, the photodynamic action of liposomes (LP) and nanocapsules (NC) containing Chloroaluminum phthalocyanine (CIAIPc), on the human melanoma cell (WM 1552C), was assessed. The light source was setup at 672 nm, which corresponds to the maximum absorption wavelength of the CIAIPc. Both colloidal carriers presented size in nanometric scale as well as negative zeta potential. The cellular damage was light dose dependent ranging from 30% of cell death at 70 mJ.cm(-2) to 90% of death at 700 mJ.cm(-2). However, the photocytotoxic effect of LP at 70 mJ.cm(-2) was slightly more efficient to induce cellular death than NC formulation. At 140 mJ.cm(-2), and 700 mJ.cm(-2) both nanocarriers were equally efficient to induce cellular damage. Therefore, in the present work, the maximum phototoxic effect was obtained with 700 mJ.cm(-2) of light dose, in combination with 0.29 mu g.mL(-1) of CIAIPc encapsulated into LP and NC. The cells were also positive to annexin V, after the PDT treatment with LP and NC, showing that one of the mechanisms of cellular death involved is apoptosis. In summary, the potential of LP and NC as a drug delivery system, in Photodynamic Therapy (PDT) against melanoma, has been confirmed using a lower concentration of the photosensitizer and lower light doses than that applied in current protocols. This is an innovative proposal to treat melanoma cell lines that until now have not received the benefit of the PDT protocol for treatment.

Tentorial meningiomas: follow-up review

Tentorial meningiomas comprise 3-5% of the intracranial meningiomas. Different locations and sinus invasion require special surgical skills. This study aimed to analyze factors influencing the outcome of 29 patients (30 tumors) with tentorial meningiomas surgically treated. The study included 22 female and seven male patients, with age of 18-76 years old, and a follow-up of 6-179 months. Eight tumors were located on the inner tentorial ring, 15 on the outer ring, four were falcotentorial, and three attached/invading the torcula. Outcome was analyzed using survival and recurrence-free survival (RFS) curves. Twenty-seven tumors were WHO grade I and three were grades II-III. Total and subtotal resections were reached in 87.5% and 12.5% of tumors. Survival was better for patients with grade I tumors and similar according to sex, location, size, and extent of resection. Recurrence/regrowing rate was 12.5%. RFS curves were better for patients with grade I or with radical resection and similar according to sex, location, and size. There was no operative mortality. Permanent postoperative cranial nerve deficits occurred in 9.7% (all inner ring tumors). Despite being many times large-sized, surgical treatment of tentorial meningiomas gives good results. Prognostic factors for recurrence were histopathologic grade III and subtotal resection. Radical resection allowed better results. Nevertheless, subtotal resection may be acceptable for cases with cranial nerves or sinuses invasions.

Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA(1,2). Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in E mu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc- overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap- dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site ( IRES)- dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic- specific expression of the endogenous IRES- dependent form of Cdk11 ( also known as Cdc21 and PITSLRE)(3-5), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in E mu-Myc/+ mice. When accurate translational control is re- established in E mu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post- genomic level.

Predictors of Mortality in Patients with Chagas` Cardiomyopathy and Ventricular Tachycardia Not Treated with Implantable Cardioverter-Defibrillators

Methods: We assessed the outcome of 56 patients with Chagas` cardiomyopathy ([31 men]; mean age of 55 years; mean left ventricular ejection fraction [LVEF] 42%) presenting with either sustained ventricular tachycardia (VT) or nonsustained VT (NSVT), before therapy with implantable cardioverter-defibrillator was available at our center. Results: Over a mean follow-up of 38 +/- 16 months (range, 1-61 months), 16 patients (29%) died, 11 due to sudden cardiac death (SCD), and five from progressive heart failure. Survivors and nonsurvivors had comparable baseline characteristics, except for a lower LVEF (46 +/- 7% vs 31 +/- 9%, P < 0.001) and a higher New York Heart Association class (P = 0.003) in those who died during follow-up. Receiver-operator characteristic curve analysis showed that an LVEF cutoff value of 38% had the best accuracy for predicting all-cause mortality and an LVEF cutoff value of 40% had the best accuracy for prediction of SCD. Using the multivariate Cox regression analysis, LVEF < 40% was the only predictor of all-cause mortality (hazard ratio [HR] 12.22, 95% confidence interval [CI] 3.46-43.17, P = 0.0001) and SCD (HR 6.58, 95% CI 1.74-24.88, P = 0.005). Conclusions: Patients with Chagas` cardiomyopathy presenting with either sustained VT or NSVT run a major risk for mortality when had concomitant severe or even moderate LV systolic dysfunction. (PACE 2011; 54-62).

Food energy content influences food portion size estimation by nutrition students

Background: Food portion size estimation involves a complex mental process that may influence food consumption evaluation. Knowing the variables that influence this process can improve the accuracy of dietary assessment. The present study aimed to evaluate the ability of nutrition students to estimate food portions in usual meals and relate food energy content with errors in food portion size estimation. Methods: Seventy-eight nutrition students, who had already studied food energy content, participated in this cross-sectional study on the estimation of food portions, organised into four meals. The participants estimated the quantity of each food, in grams or millilitres, with the food in view. Estimation errors were quantified, and their magnitude were evaluated. Estimated quantities (EQ) lower than 90% and higher than 110% of the weighed quantity (WQ) were considered to represent underestimation and overestimation, respectively. Correlation between food energy content and error on estimation was analysed by the Spearman correlation, and comparison between the mean EQ and WQ was accomplished by means of the Wilcoxon signed rank test (P < 0.05). Results: A low percentage of estimates (18.5%) were considered accurate (+/- 10% of the actual weight). The most frequently underestimated food items were cauliflower, lettuce, apple and papaya; the most often overestimated items were milk, margarine and sugar. A significant positive correlation between food energy density and estimation was found (r = 0.8166; P = 0.0002). Conclusions: The results obtained in the present study revealed a low percentage of acceptable estimations of food portion size by nutrition students, with trends toward overestimation of high-energy food items and underestimation of low-energy items.