Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines

Pancreatic beta-cell apoptosis is known to participate in the beta-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB).

Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

BACKGROUND. Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. METHODS. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13) was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radiation-induced apoptosis (RIA) at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. RESULTS. Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively). We observed that those "expected resistant patients" (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively) were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively) in multivariate analysis. CONCLUSIONS. A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low risk of suffer severe subcutaneous late toxicity after clinical treatment at high radiation doses in our series. However, due to the small sample size, other prospective studies with higher number of patients are needed to validate these results.

The benefits of using selenium in the treatment of Chagas disease: prevention of right ventricle chamber dilatation and reversion of Trypanosoma cruzi-induced acute and chronic cardiomyopathy in mice

Cardiac damage is a frequent manifestation of Chagas disease, which is caused by the parasite Trypanosoma cruzi. Selenium (Se) is an essential micronutrient, the deficiency of which has been implicated in the development of cardiomyopathy. Our group has previously demonstrated that Se supplementation prevents myocardial damage during acute T. cruzi infection in mice. In this study, we analyzed the effect of Se treatment in cases of T. cruzi infection using prevention and reversion schemes. In the Se prevention scheme, mice were given Se supplements (2 ppm) starting two weeks prior to inoculation with T. cruzi(Brazil strain) and continuing until 120 days post-infection (dpi). In the Se reversion scheme, mice were treated with Se (4 ppm) for 100 days, starting at 160 dpi. Dilatation of the right ventricle was observed in the infected control group at both phases of T. cruzi infection, but it was not observed in the infected group that received Se treatment. Surviving infected mice that were submitted to the Se reversion scheme presented normal P wave values and reduced inflammation of the pericardium. These data indicate that Se treatment prevents right ventricular chamber increase and thus can be proposed as an adjuvant therapy for cardiac alterations already established by T. cruziinfection.

GLUT3 is induced during epithelial-mesenchymal transition and promotes tumor cell proliferation in non-small cell lung cancer.

BACKGROUND: Alterations in glucose metabolism and epithelial-mesenchymal transition (EMT) constitute two important characteristics of carcinoma progression toward invasive cancer. Despite an extensive characterization of each of them separately, the links between EMT and glucose metabolism of tumor cells remain elusive. Here we show that the neuronal glucose transporter GLUT3 contributes to glucose uptake and proliferation of lung tumor cells that have undergone an EMT. RESULTS: Using a panel of human non-small cell lung cancer (NSCLC) cell lines, we demonstrate that GLUT3 is strongly expressed in mesenchymal, but not epithelial cells, a finding corroborated in hepatoma cells. Furthermore, we identify that ZEB1 binds to the GLUT3 gene to activate transcription. Importantly, inhibiting GLUT3 expression reduces glucose import and the proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. Using a large microarray data collection of human NSCLCs, we determine that GLUT3 expression correlates with EMT markers and is prognostic of poor overall survival. CONCLUSIONS: Altogether, our results reveal that GLUT3 is a transcriptional target of ZEB1 and that this glucose transporter plays an important role in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Moreover, these findings emphasize the development of GLUT3 inhibitory drugs as a targeted therapy for the treatment of patients with poorly differentiated tumors.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches.

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Laser-ultraviolet-A induced ultra weak photon emission in human skin cells: A biophotonic comparison between keratinocytes and fibroblasts.

Photons participate in many atomic and molecular interactions and processes. Recent biophysical research has discovered an ultraweak radiation in biological tissues. It is now recognized that plants, animal and human cells emit this very weak biophotonic emission which can be readily measured with a sensitive photomultiplier system. UVA laser induced biophotonic emission of cultured cells was used in this report with the intention to detect biophysical changes between young and adult fibroblasts as well as between fibroblasts and keratinocytes. With suspension densities ranging from 1-8 x 106 cells/ml, it was evident that an increase of the UVA-laser-light induced photon emission intensity could be observed in young as well as adult fibroblastic cells. By the use of this method to determine ultraweak light emission, photons in cell suspensions in low volumes (100 microl) could be detected, in contrast to previous procedures using quantities up to 10 ml. Moreover, the analysis has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 micros instead of more than 100 milliseconds. These significant changes lead to an improvement factor up to 106 in comparison to classical detection procedures. In addition, different skin cells as fibroblasts and keratinocytes stemming from the same donor were measured using this new highly sensitive method in order to find new biophysical insight of light pathways. This is important in view to develop new strategies in biophotonics especially for use in alternative therapies.

Characterization of TcSTI-1, a homologue of stress-induced protein-1, in Trypanosoma cruzi

The life cycle of the protozoan Trypanosoma cruzi exposes it to several environmental stresses in its invertebrate and vertebrate hosts. Stress conditions are involved in parasite differentiation, but little is known about the stress response proteins involved. We report here the first characterization of stress-induced protein-1 (STI-1) in T. cruzi (TcSTI-1). This co-chaperone is produced in response to stress and mediates the formation of a complex between the stress proteins HSP70 and HSP90 in other organisms. Despite the similarity of TcSTI-1 to STI-1 proteins in other organisms, its expression profile in response to various stress conditions, such as heat shock, acidic pH or nutrient starvation, is quite different. Neither polysomal mRNA nor protein levels changed in exponentially growing epimastigotes cultured under any of the stress conditions studied. Increased levels of TcSTI-1 were observed in epimastigotes subjected to nutritional stress in the late growth phase. Co-immunoprecipitation assays revealed an association between TcSTI-1 and TcHSP70 in T. cruzi epimastigotes. Immunolocalization demonstrated that TcSTI-1 was distributed throughout the cytoplasm and there was some colocalization of TcSTI-1 and TcHSP70 around the nucleus. Thus, TcSTI-1 associates with TcHSP70 and TcSTI-1 expression is induced when the parasites are subjected to stress conditions during specific growth phase.

VEGF gene expression in adult human thymus fat: a correlative study with hypoxic induced factor and cyclooxygenase-2.

It is well known that the adult human thymus degenerates into fat tissue; however, it has never been considered as a potential source of angiogenic factors. Recently, we have described that this fat (TAT) produces angiogenic factors and induces human endothelial cell proliferation and migration, indicating its potential angiogenic properties. DESIGN Adult thymus fat and subcutaneous adipose tissue specimens were obtained from 28 patients undergoing cardiac surgery, making this tissue readily available as a prime source of adipose tissue. We focused our investigation on determining VEGF gene expression and characterizing the different genes, mediators of inflammation and adipogenesis, and which are known to play a relevant role in angiogenesis regulation. RESULTS We found that VEGF-A was the isoform most expressed in TAT. This expression was accompanied by an upregulation of HIF-1alpha, COX-2 and HO-1 proteins, and by increased HIF-1 DNA binding activity, compared to SAT. Furthermore, we observed that TAT contains a high percentage of mature adipocytes, 0.25% of macrophage cells, 15% of endothelial cells and a very low percentage of thymocyte cells, suggesting the cellular variability of TAT, which could explain the differences in gene expression observed in TAT. Subsequently, we showed that the expression of genes known as adipogenic mediators, including PPARgamma1/gamma2, FABP-4 and adiponectin was similar in both TAT and SAT. Moreover the expression of these latter genes presented a significantly positive correlation with VEGF, suggesting the potential association between VEGF and the generation of adipose tissue in adult thymus. CONCLUSION Here we suggest that this fat has a potential angiogenic function related to ongoing adipogenesis, which substitutes immune functions within the adult thymus. The expression of VEGF seems to be associated with COX-2, HO-1 and adipogenesis related genes, suggesting the importance that this new fat has acquired in research in relation to adipogenesis and angiogenesis.

Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans.

Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody-induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that maybe responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody-induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family,which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody-induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms,some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody-induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody-induced nephritis and lupus.

Assessment of drug-induced hepatotoxicity in clinical practice: a challenge for gastroenterologists.

Currently, pharmaceutical preparations are serious contributors to liver disease; hepatotoxicity ranking as the most frequent cause for acute liver failure and post-commercialization regulatory decisions. The diagnosis of hepatotoxicity remains a difficult task because of the lack of reliable markers for use in general clinical practice. To incriminate any given drug in an episode of liver dysfunction is a step-by-step process that requires a high degree of suspicion, compatible chronology, awareness of the drug's hepatotoxic potential, the exclusion of alternative causes of liver damage and the ability to detect the presence of subtle data that favors a toxic etiology. This process is time-consuming and the final result is frequently inaccurate. Diagnostic algorithms may add consistency to the diagnostic process by translating the suspicion into a quantitative score. Such scales are useful since they provide a framework that emphasizes the features that merit attention in cases of suspected hepatic adverse reaction as well. Current efforts in collecting bona fide cases of drug-induced hepatotoxicity will make refinements of existing scales feasible. It is now relatively easy to accommodate relevant data within the scoring system and to delete low-impact items. Efforts should also be directed toward the development of an abridged instrument for use in evaluating suspected drug-induced hepatotoxicity at the very beginning of the diagnosis and treatment process when clinical decisions need to be made. The instrument chosen would enable a confident diagnosis to be made on admission of the patient and treatment to be fine-tuned as further information is collected.

Irinotecan-induced central nervous system toxicity: a case report.

Here we describe the first case, to our knowledge, of CNS [Central nervous system] toxicity in a patient treated with irinotecan.

Genetic polymorphisms of NAT2, CYP2E1 and GST enzymes and the occurrence of antituberculosis drug-induced hepatitis in Brazilian TB patients

Isoniazid (INH), one of the most important drugs used in antituberculosis (anti-TB) treatment, is also the major drug involved in hepatotoxicity. Differences in INH-induced toxicity have been attributed to genetic variability at several loci, such as NAT2, CYP2E1, GSTM1 and GSTT1, that code for drug-metabolising enzymes. Our goal was to examine the polymorphisms in these enzymes as susceptibility factors to anti-TB drug-induced hepatitis in Brazilian individuals. In a case-control design, 167 unrelated active tuberculosis patients from the University Hospital of the Federal University of Rio de Janeiro, Brazil, were enrolled in this study. Patients with a history of anti-TB drug-induced acute hepatitis (cases with an increase to 3 times the upper limit of normal serum transaminases and symptoms of hepatitis) and patients with no evidence of anti-TB hepatic side effects (controls) were genotyped for NAT2, CYP2E1, GSTM1 and GSTT1 polymorphisms. Slow acetylators had a higher incidence of hepatitis than intermediate/rapid acetylators [22% (18/82) vs. 9.8% (6/61), odds ratio (OR), 2.86, 95% confidence interval (CI), 1.06-7.68, p = 0.04). Logistic regression showed that slow acetylation status was the only independent risk factor (OR 3.59, 95% CI, 2.53-4.64, p = 0.02) for the occurrence of anti-TB drug-induced hepatitis during anti-TB treatment with INH-containing schemes in Brazilian individuals.

Trial of recombinant follicle-stimulating hormone pretreatment for gnrh-induced fertility in patients with congenital hypogonadotropic hypogonadism.

CONTEXT AND OBJECTIVE: The optimal strategy for inducing fertility in men with congenital hypogonadotropic hypogonadism (CHH) is equivocal. Albeit a biologically plausible approach, pretreatment with recombinant FSH (rFSH) before GnRH/human chorionic gonadotropin administration has not been sufficiently assessed. The objective of the study was to test this method. DESIGN AND SETTING: This was a randomized, open-label treatment protocol at an academic medical center. PATIENTS AND INTERVENTIONS: GnRH-deficient men (CHH) with prepubertal testes (<4 mL), no cryptorchidism, and no prior gonadotropin therapy were randomly assigned to either 24 months of pulsatile GnRH therapy alone (inducing endogenous LH and FSH release) or 4 months of rFSH pretreatment followed by 24 months of GnRH therapy. Patients underwent serial testicular biopsies, ultrasound assessments of testicular volume, serum hormone measurements, and seminal fluid analyses. RESULTS: rFSH treatment increased inhibin B levels into the normal range (from 29 ± 9 to 107 ± 41 pg/mL, P < .05) and doubled testicular volume (from 1.1 ± 0.2 to 2.2 ± 0.3 mL, P < .005). Histological analysis showed proliferation of both Sertoli cells (SCs) and spermatogonia, a decreased SC to germ cell ratio (from 0.74 to 0.35), and SC cytoskeletal rearrangements. With pulsatile GnRH, the groups had similar hormonal responses and exhibited significant testicular growth. All men receiving rFSH pretreatment developed sperm in their ejaculate (7 of 7 vs 4 of 6 in the GnRH-only group) and showed trends toward higher maximal sperm counts. CONCLUSIONS: rFSH pretreatment followed by GnRH is successful in inducing testicular growth and fertility in men with CHH with prepubertal testes. rFSH not only appears to maximize the SC population but also induces morphologic changes, suggesting broader developmental roles.