Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 µg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.

Treg-therapy allows mixed chimerism and transplantation tolerance without cytoreductive conditioning.

Establishment of mixed chimerism through transplantation of allogeneic donor bone marrow (BM) into sufficiently conditioned recipients is an effective experimental approach for the induction of transplantation tolerance. Clinical translation, however, is impeded by the lack of feasible protocols devoid of cytoreductive conditioning (i.e. irradiation and cytotoxic drugs/mAbs). The therapeutic application of regulatory T cells (Tregs) prolongs allograft survival in experimental models, but appears insufficient to induce robust tolerance on its own. We thus investigated whether mixed chimerism and tolerance could be realized without the need for cytoreductive treatment by combining Treg therapy with BM transplantation (BMT). Polyclonal recipient Tregs were cotransplanted with a moderate dose of fully mismatched allogeneic donor BM into recipients conditioned solely with short-course costimulation blockade and rapamycin. This combination treatment led to long-term multilineage chimerism and donor-specific skin graft tolerance. Chimeras also developed humoral and in vitro tolerance. Both deletional and nondeletional mechanisms contributed to maintenance of tolerance. All tested populations of polyclonal Tregs (FoxP3-transduced Tregs, natural Tregs and TGF-beta induced Tregs) were effective in this setting. Thus, Treg therapy achieves mixed chimerism and tolerance without cytoreductive recipient treatment, thereby eliminating a major toxic element impeding clinical translation of this approach.

Electronic monitoring of compliance to lipid-lowering therapy in clinical practice

Nonadherence to treatment is a common problem in the clinical management of hypercholesterolemic patients. This study was carried out with the aim of monitoring the daily compliance to a 6-month course of lipid-lowering therapy, using a microelectronic device, the Medication Event Monitoring System (MEMS), versus pill count. Forty men with primary hypercholesterolemia were prescribed fluvastatin 1 x 40 mg daily, provided in a MEMS package to record the date and time of each opening of the pillbox. Thirty-nine of 40 patients (98%) completed the study. Total cholesterol and LDL cholesterol levels decreased significantly (18% and 25%, p < 0.001) during the 6-month therapy period. A high mean rate of compliance was achieved by MEMS using the following three indexes--compliance to total prescribed dose (88.8% +/- 13.5%), compliance to prescribed days (82.4% +/- 19.5%), and compliance to prescribed time of day (81.86% +/- 19.5%)--and by pill count (93.4% +/- 9.5%). In addition, the MEMS provided some patterns of nonadherence to medication, undetectable by pill count alone, such as a drug holiday in 38% of cases, a drug omission for more than 7 consecutive days in 9% of cases, and, conversely, use of more than the one prescribed daily dose in 47% of cases. A significant correlation between the rate of compliance and the decrease in LDL cholesterol was observed only when the compliance was assessed by MEMS. The results indicate that MEMS is a useful tool for monitoring compliance in clinical practice and may possibly increase adherence to long-term lipid-lowering therapy.

Treatment of chronic hepatitis c in 3 patients with hcv-induced severe thrombocytopenia

BACKGROUND: Thrombocytopenia has been described in HCV infection even in the absence of cirrhosis and splenomegaly. Different mechanisms have been proposed, including immunemediated platelet (plt) destruction. Here, we report on the treatment of 3 patients with HCV-HIV coinfection and HCVinduced severe thrombocytopenia. PATIENTS AND TREATMENT: All patients had an infection with HCV genotype 3, an intermediate fibrosis stage (Metavir F2 or F3), HIV infection controlled by antiretroviral combination therapy, and severe, steroid-refractory thrombocytopenia. Pegylated interferon-α2a (PEG-IFN-α2a) was started at 45 or 90 μg per week and doses were rapidly increased in the following, while ribavirin (RBV) was prescribed at standard doses. Treatment was pursued for 48 weeks. Two patients received intravenous immunoglobulins (IVIG) during the first weeks of PEG-IFN-α2a and RBV combination therapy. RESULTS: A significant increase in plt counts (from 17, 39 and 37 G/l, respectively, to > 100 G/l) was observed in the 3 patients while they experienced a virological response. Thrombocytopenia relapsed in one patient together with a relapse of chronic hepatitis C. The other 2 patients achieved a sustained virological response (SVR), with normal plt counts at follow-up in one and persistent mild thrombocytopenia in the other. CONCLUSIONS: Carefully titrated PEG-IFN-α2a and RBV combination therapy may be performed safely in this difficult-totreat patient population, with close monitoring and eventually concomitant IVIG during the first weeks. SVR can lead to normalization or significant improvement of plt counts, suggesting a causative role of HCV in this condition.

Anti-TNF Therapy in the Treatment of Psoriasis in a Patient with Acute-on-Chronic Pancreatitis.

Background: Psoriasis is accepted as a multisystemic disease with several important systemic manifestations. Thus, underlying comorbidities have to be taken into account in the choice of treatment. Objective: To explore the role of anti-TNF therapy in the treatment of psoriasis in a patient with acute-on-chronic pancreatitis. Methods: Here, we present the case of a 75-year-old patient with severe psoriasis also suffering from chronic alcohol-induced pancreatitis with recurrent acute flares. A recent life-threatening episode of acute pancreatitis and ischemic liver precluded the reintroduction of methotrexate. Cyclosporine was also excluded as it has been reported to induce acute pancreatitis. Thus, an anti-TNF treatment was initiated in close collaboration with a gastroenterologist. Results: A year after starting anti-TNF therapy the patient continues to show complete clinical remission of his psoriasis. No side effects, particularly no bacterial infections, were reported. No relapses of the patient's underlying chronic pancreatitis were observed throughout the entire treatment with regular clinical and laboratory monitoring, suggesting that chronic pancreatitis is not per se a contraindication for anti-TNF therapy. Conclusion: This case study opens the way for further questioning on the role of TNF in the pathogenesis of chronic and acute pancreatitis and the use of anti-TNF therapy in its treatment. © 2013 S. Karger AG, Basel.

Abnormal humoral immune response to influenza vaccination in pediatric type-1 human immunodeficiency virus infected patients receiving highly active antiretroviral therapy

Given that highly active antiretroviral therapy (HAART) has been demonstrated useful to restore immune competence in type-1 human immunodeficiency virus (HIV-1)-infected subjects, we evaluated the specific antibody response to influenza vaccine in a cohort of HIV-1-infected children on HAART so as to analyze the quality of this immune response in patients under antiretroviral therapy. Sixteen HIV-1-infected children and 10 HIV-1 seronegative controls were immunized with a commercially available trivalent inactivated influenza vaccine containing the strains A/H1N1, A/H3N2, and B. Serum hemagglutinin inhibition (HI) antibody titers were determined for the three viral strains at the time of vaccination and 1 month later. Immunization induced a significantly increased humoral response against the three influenza virus strains in controls, and only against A/H3N2 in HIV-1-infected children. The comparison of post-vaccination HI titers between HIV-1+ patients and HIV-1 negative controls showed significantly higher HI titers against the three strains in controls. In addition, post vaccination protective HI titers (defined as equal to or higher than 1:40) against the strains A/H3N2 and B were observed in a lower proportion of HIV-1+ children than in controls, while a similar proportion of individuals from each group achieved protective HI titers against the A/H1N1 strain. The CD4+ T cell count, CD4/CD8 T cells ratio, and serum viral load were not affected by influenza virus vaccination when pre- vs post-vaccination values were compared. These findings suggest that despite the fact that HAART is efficient in controlling HIV-1 replication and in increasing CD4+ T cell count in HIV-1-infected children, restoration of immune competence and response to cognate antigens remain incomplete, indicating that additional therapeutic strategies are required to achieve a full reconstitution of immune functions.

The advantage of letrozole over tamoxifen in the BIG 1-98 trial is consistent in younger postmenopausal women and in those with chemotherapy-induced menopause.

Letrozole, an aromatase inhibitor, is ineffective in the presence of ovarian estrogen production. Two subpopulations of apparently postmenopausal women might derive reduced benefit from letrozole due to residual or returning ovarian activity: younger women (who have the potential for residual subclinical ovarian estrogen production), and those with chemotherapy-induced menopause who may experience return of ovarian function. In these situations tamoxifen may be preferable to an aromatase inhibitor. Among 4,922 patients allocated to the monotherapy arms (5 years of letrozole or tamoxifen) in the BIG 1-98 trial we identified two relevant subpopulations: patients with potential residual ovarian function, defined as having natural menopause, treated without adjuvant or neoadjuvant chemotherapy and age ≤ 55 years (n = 641); and those with chemotherapy-induced menopause (n = 105). Neither of the subpopulations examined showed treatment effects differing from the trial population as a whole (interaction P values are 0.23 and 0.62, respectively). Indeed, both among the 641 patients aged ≤ 55 years with natural menopause and no chemotherapy (HR 0.77 [0.51, 1.16]) and among the 105 patients with chemotherapy-induced menopause (HR 0.51 [0.19, 1.39]), the disease-free survival (DFS) point estimate favoring letrozole was marginally more beneficial than in the trial as a whole (HR 0.84 [0.74, 0.95]). Contrary to our initial concern, DFS results for young postmenopausal patients who did not receive chemotherapy and patients with chemotherapy-induced menopause parallel the letrozole benefit seen in the BIG 1-98 population as a whole. These data support the use of letrozole even in such patients.

The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell linesvia arrest of cell cycle and induction of apoptosis

BACKGROUND Protein-bound polysaccharide (PSK) is derived from the CM-101 strain of the fungus Coriolus versicolor and has shown anticancer activity in vitro and in in vivo experimental models and human cancers. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the adjuvant treatment of gastric, esophageal, colorectal, breast and lung cancers. These studies have suggested the efficacy of PSK as an immunomodulator of biological responses. The precise molecular mechanisms responsible for its biological activity have yet to be fully elucidated. METHODS The in vitro cytotoxic anti-tumour activity of PSK has been evaluated in various tumour cell lines derived from leukaemias, melanomas, fibrosarcomas and cervix, lung, pancreas and gastric cancers. Tumour cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of PSK on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in PSK-treated cells. RESULTS PSK showed in vitro inhibition of tumour cell proliferation as measured by BrdU incorporation and viable cell count. The inhibition ranged from 22 to 84%. Inhibition mechanisms were identified as cell cycle arrest, with cell accumulation in G0/G1 phase and increase in apoptosis and caspase-3 expression. These results indicate that PSK has a direct cytotoxic activity in vitro, inhibiting tumour cell proliferation. In contrast, PSK shows a synergistic effect with IL-2 that increases PBL proliferation. CONCLUSION These results indicate that PSK has cytotoxic activity in vitro on tumour cell lines. This new cytotoxic activity of PSK on tumour cells is independent of its previously described immunomodulatory activity on NK cells.

Early and late skin reactions to radiotherapy for breast cancer and their correlation with radiation-induced DNA damage in lymphocytes

INTRODUCTION Radiotherapy outcomes might be further improved by a greater understanding of the individual variations in normal tissue reactions that determine tolerance. Most published studies on radiation toxicity have been performed retrospectively. Our prospective study was launched in 1996 to measure the in vitro radiosensitivity of peripheral blood lymphocytes before treatment with radical radiotherapy in patients with breast cancer, and to assess the early and the late radiation skin side effects in the same group of patients. We prospectively recruited consecutive breast cancer patients receiving radiation therapy after breast surgery. To evaluate whether early and late side effects of radiotherapy can be predicted by the assay, a study was conducted of the association between the results of in vitro radiosensitivity tests and acute and late adverse radiation effects. METHODS Intrinsic molecular radiosensitivity was measured by using an initial radiation-induced DNA damage assay on lymphocytes obtained from breast cancer patients before radiotherapy. Acute reactions were assessed in 108 of these patients on the last treatment day. Late morbidity was assessed after 7 years of follow-up in some of these patients. The Radiation Therapy Oncology Group (RTOG) morbidity score system was used for both assessments. RESULTS Radiosensitivity values obtained using the in vitro test showed no relation with the acute or late adverse skin reactions observed. There was no evidence of a relation between acute and late normal tissue reactions assessed in the same patients. A positive relation was found between the treatment volume and both early and late side effects. CONCLUSION After radiation treatment, a number of cells containing major changes can have a long survival and disappear very slowly, becoming a chronic focus of immunological system stimulation. This stimulation can produce, in a stochastic manner, late radiation-related adverse effects of varying severity. Further research is warranted to identify the major determinants of normal tissue radiation response to make it possible to individualize treatments and improve the outcome of radiotherapy in cancer patients.

5-Fluorouracil-loaded poly(ε-caprolactone) nanoparticles combined with phage E gene therapy as a new strategy against colon cancer

This work aimed to develop a new therapeutic approach to increase the efficacy of 5-fluorouracil (5-FU) in the treatment of advanced or recurrent colon cancer. 5-FU-loaded biodegradable poly(ε-caprolactone) nanoparticles (PCL NPs) were combined with the cytotoxic suicide gene E (combined therapy). The SW480 human cancer cell line was used to assay the combined therapeutic strategy. This cell line was established from a primary adenocarcinoma of the colon and is characterized by an intrinsically high resistance to apoptosis that correlates with its resistance to 5-FU. 5-FU was absorbed into the matrix of the PCL NPs during synthesis using the interfacial polymer disposition method. The antitumor activity of gene E from the phage ϕX174 was tested by generating a stable clone (SW480/12/E). In addition, the localization of E protein and its activity in mitochondria were analyzed. We found that the incorporation of 5-FU into PCL NPs (which show no cytotoxicity alone), significantly improved the drug's anticancer activity, reducing the proliferation rate of colon cancer cells by up to 40-fold when compared with the nonincorporated drug alone. Furthermore, E gene expression sensitized colon cancer cells to the cytotoxic action of the 5-FU-based nanomedicine. Our findings demonstrate that despite the inherent resistance of SW480 to apoptosis, E gene activity is mediated by an apoptotic phenomenon that includes modulation of caspase-9 and caspase-3 expression and intense mitochondrial damage. Finally, a strongly synergistic antiproliferative effect was observed in colon cancer cells when E gene expression was combined with the activity of the 5-FU-loaded PCL NPs, thereby indicating the potential therapeutic value of the combined therapy.

Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases.

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1alpha, IL-1beta, CCL20/MIP-3alpha, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation.

Decrease in beta-Cell Proliferation Precedes Apoptosis during Diabetes Development in Bio-Breeding/Worcester Rat: Beneficial Role of Exendin-4

In autoimmune type 1 diabetes mellitus, proinflammatory cytokine-mediated apoptosis of beta-cells has been considered to be the first event directly responsible for beta-cell mass reduction. In the Bio-Breeding (BB) rat, an in vivo model used in the study of autoimmune diabetes, beta-cell apoptosis is observed from 9 wk of age and takes place after an insulitis period that begins at an earlier age. Previous studies by our group have shown an antiproliferative effect of proinflammatory cytokines on cultured beta-cells in Wistar rats, an effect that was partially reversed by Exendin-4, an analogue of glucagon-like peptide-1. In the current study, the changes in beta-cell apoptosis and proliferation during insulitis stage were also determined in pancreatic tissue sections in normal and thymectomized BB rats, as well as in Wistar rats of 5, 7, 9, and 11 wk of age. Although stable beta-cell proliferation in Wistar and thymectomized BB rats was observed along the course of the study, a decrease in beta-cell proliferation and beta-cell mass from the age of 5 wk, and prior to the commencement of apoptosis, was noted in BB rats. Exendin-4, in combination with anti-interferon-gamma antibody, induced a near-total recovery of beta-cell proliferation during the initial stages of insulitis. This highlights the importance of early intervention and, as well, the possibilities of new therapeutic approaches in preventing autoimmune diabetes by acting, initially, in the insulitis stage and, subsequently, on beta-cell regeneration and on beta-cell apoptosis.

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.