Bz Treatment Research Articles

Benzonidazole treatment has a beneficial effect on cells infected with the Colombian strain of Trypanosoma cruzi.

Benznidazole (Bz) is the recommended drug for the treatment of Chagas disease; however, its efficacy may vary according to the sensitivity of Trypanosoma cruzi strains to the drug and host immune background. The study evaluated the immune response of peripheral blood mononuclear cells (PBMC) that were infected in vitro with the Colombian strain (Col) and treated with Bz. The co-cultures were incubated for 24 h, 5 and 10 days, where cytokine dosage was performed in the supernatant and evaluation of the cells for CD28+ and CTLA-4+ molecules in CD4+ and CD8+ lymphocytes, and CD80+ , CD86+ and HLA-DR+ in CD14+ cells. The results showed that Col induced a strong inflammatory response, with an increase in IFN-γ and TNF early in the infection (24 h), however, from 5 days of infection on, TNF production declined, and IL-10 production increased, which may be associated with a control mechanism of the exacerbated inflammatory response. The Bz treatment did not significantly alter the frequencies of the phenotypes evaluated both T cell subsets and CD14+ cells. Therefore, this study reinforces the need for typing the patient's strain to guide therapy and promote individualized treatment protocols due to the heterogeneous genetic background among T. cruzi strains.

P860: THE DE NOVO DNA METHYLTRANSFERASE DNMT3B PLAYS AN IMPORTANT ROLE IN MM CELL GROWTH, CLONOGENICITY AND DRUG RESPONSE

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy due to the development of drug resistance (DR). In about half of the MM patients, modifications are observed in epigenetic modifiers (epiplayers) at the time of diagnosis and this frequency is further increased at relapse, indicating an important role for epiplayers in MM cell DR. However, so far, only for two epiplayers, MMSET and EZH2, a clear role in MM cell DR development has been established. With the aim to identify new, clinically relevant epiplayers involved in MM progression and relapse, we recently compared the RNASeq data from matched newly diagnosed and relapsed patients from the MMRF CoMMpass study. We found that the epiplayer DNMT3B is significantly increased in relapsed patients, suggesting a possible role for DNMT3B in MM relapse. Aims: The aim of this study is to explore the role of DNMT3B in MM cell biology and drug response. Methods: Publicly available gene expression profiling data of three independent cohorts of newly diagnosed MM patients (GSE4581, E-MTAB-372, MMRF-COMMPASS), one cohort of relapsed patients (GSE9782), BM plasma cells (E-MTAB-372), primary MM cells (E- MTAB-372), and human MM cell lines (HMCLs; E-TABM-1088 and E-TABM-937) was used. DNMT3B specific targeting was achieved by using the DNMT3B specific inhibitor Nanaomycin A and genetic inhibition using doxycycline inducible shRNA against DNMT3B. Viability and apoptosis were assessed using a CellTiter-Glo assay and an AnnexinV/7AAD staining respectively. In addition, BrdU incorporation and cell cycle analysis was evaluated to assess cell proliferation. Clonogenic capacity was evaluated by a colony formation assay. Results: We found that DNMT3B mRNA levels increase during disease progression and high DNMT3B mRNA expression correlates with a worse disease outcome in both newly diagnosed and relapsed patients, indicating a role for DNMT3B in MM progression and DR. In line, Nanaomycin A treatment (up to 72h) led to a significant and dose-dependent decrease in cell viability and proliferation and increase in apoptosis in the XG-2, XG-7 and AMO-1 HMCLs. We also validated the anti-MM activity of Nanaomycin A on human primary MM cells. Since DNMT3B is thought to play a significant role in cancer cell stemness, we next evaluated the effect on clonogenicity. A significant and dose-dependent decrease in the number of colonies was observed when low doses of Nanaomycin A were added to AMO-1 (100 and 200 nM) and XG-2 (30 and 50 nM) cells on the day of plating (day 0), but not when added 7 days after plating (day 7). In contrast, treatment with a high Nanaomycin A concentration (800 nM) significantly reduced the number of colonies at both timepoints, indicating that high Nanaomycin A doses are cytotoxic whereas low doses impair the proliferation and clonogenicity of the MM cells. The anti-clonogenic effect of DNMT3B targeting was confirmed by DNMT3B knockdown in AMO-1 cells. Finally, combining Nanaomycin A (100 nM) with bortezomib (Bz, 4 nM) resulted in a significant decrease in colony formation compared to either Nanaomycin A or Bz treatment alone. Summary/Conclusion: Taken together, our findings indicate that DNMT3B is a novel promising target to overcome or delay relapse in MM. DNMT3B targeting impairs MM cell proliferation and clonogenicity and sensitizes the cells to the proteasome inhibitor Bz. In the near future, the anti-myeloma activity of DNMT3B targeting will be validated in vivo using the 5TMM murine MM models and the underlying mechanisms will be further determined.

Experimental Combination Therapy with Amiodarone and Low-Dose Benznidazole in a Mouse Model of Trypanosoma cruzi Acute Infection.

ABSTRACTChagas disease (CD), caused by Trypanosoma cruzi, affects approximately 6 to 7 million people in Latin America, with cardiomyopathy being the clinical manifestation most commonly associated with patient death during the acute phase. The etiological treatment of CD is restricted to benznidazole (Bz) and nifurtimox (Nif), which involve long periods of administration, frequent side effects, and low efficacy in the chronic phase. Thus, combined therapies emerge as an important tool in the treatment of CD, allowing the reduction of Bz dose and treatment duration. In this sense, amiodarone (AMD), the most efficient antiarrhythmic drug currently available and prescribed to CD patients, is a potential candidate for combined treatment due to its known trypanocidal activity. However, the efficacy of AMD during the acute phase of CD and its interaction with Bz or Nif are still unknown. In the present study, using a well-established murine model of the acute phase of CD, we observed that the Bz/AMD combination was more effective in reducing the peak parasitemia than both monotherapy treatments. Additionally, the Bz/AMD combination reduced (i) interleukin-6 (IL-6) levels in cardiac tissue, (ii) P-wave duration, and (iii) frequency of arrhythmia in infected animals and (iv) restored gap junction integrity in cardiac tissue. Therefore, our study validates AMD as a promising candidate for combined therapy with Bz, reinforcing the strategy of combined therapy for CD.IMPORTANCE Chagas disease affects approximately 6 to 7 million people worldwide, with cardiomyopathy being the clinical manifestation that most commonly leads to patient death. The etiological treatment of Chagas disease is limited to drugs (benznidazole and nifurtimox) with relatively high toxicity and therapeutic failures. In this sense, amiodarone, the most effective currently available antiarrhythmic drug prescribed to patients with Chagas disease, is a potential candidate for combined treatment due to its known trypanocidal effect. In the present study, we show that combined treatment with benznidazole and amiodarone improves the trypanocidal effect and reduces cardiac damage in acutely T. cruzi-infected mice.

Effect of Buzhong Yiqi decoction on anti-acetylcholine receptor antibody and clinical status in juvenile ocular myasthenia gravis.

Ocular myasthenia gravis (MG) is the mainly widespread type of MG among juveniles. Buzhong Yiqi decoction (BZ) is a well-known traditional Chinese medicine prescription for treating MG. It has rarely been discussed whether the concentration of anti-acetylcholine receptor (AChR) antibodies is related to the clinical status of juvenile ocular myasthenia gravis (JOMG) treated with BZ.The patients with JOMG who had more than once AChR-antibody (ab) test and treated with BZ were retrospectively identified from June 2013 to January 2020 in the first hospital in Shijiazhuang. The presence or absence of ocular symptoms was used to grade the effectiveness of treatment. Generalized estimating equations logistic regression analysis was used to evaluate the effect of AChR ab concentration on the clinical status of MG.A total of 549 AChR-ab tests were performed in 135 patients, and the corresponding clinical status was recorded. One hundred two patients received treatment with BZ only and 33 combined with immunosuppressive drugs. In the group receiving only BZ treatment, the anti-acetylcholine receptor ab concentration was positively correlated with the clinical score after treatment.The results suggest that BZ could affect the AChR-ab. Repeated AChR-ab assays can provide information about the clinical status. For JOMG patients who only receive Buzhong Yiqi Decoction treatment, this can support treatment decisions.

CD8low T cells expanded following acute Trypanosoma cruzi infection and benznidazole treatment are a relevant subset of IFN-γ producers.

CD8 T cells are regarded as pivotal players in both immunoprotection and immunopathology following Trypanosoma cruzi infection. Previously, we demonstrated the expansion of CD8+ T lymphocytes in the spleen of T. cruzi-infected mice under treatment with benznidazole (N-benzyl-2-nitroimidazole acetamide; Bz), a drug available for clinical therapy. This finding underlies the concept that the beneficial effects of Bz on controlling acute T. cruzi infection are related to a synergistic process between intrinsic trypanocidal effect and indirect triggering of the active immune response. In the present study, we particularly investigated the effect of Bz treatment on the CD8+ T cell subset following T. cruzi infection. Herein we demonstrated that, during acute T. cruzi infection, Bz treatment reduces and abbreviates the parasitemia, but maintains elevated expansion of CD8+ T cells. Within this subset, a remarkable group of CD8low cells was found in both Bz-treated and non-treated infected mice. In Bz-treated mice, early pathogen control paralleled the lower frequency of recently activated CD8low cells, as ascertained by CD69 expression. However, the CD8low subset sustains significant levels of CD44highCD62Llow and CD62LlowT-bethigh effector memory T cells, in both Bz-treated and non-treated infected mice. These CD8low cells also comprise the main group of spontaneous interferon (IFN)-γ-producing CD8+ T cells. Interestingly, following in vitro anti-CD3/CD28 stimulation, CD8+ T cells from Bz-treated T. cruzi-infected mice exhibited higher frequency of IFN-γ+ cells, which bear mostly a CD8low phenotype. Altogether, our results point to the marked presence of CD8low T cells that arise during acute T. cruzi infection, with Bz treatment promoting their significant expansion along with a potential effector program for high IFN-γ production.

Benznidazole: Hero or villain of cellular immune response in chronic Chagas disease patients?

Although the treatment of chronic Chagas disease (CCD) patients with Benznidazole (Bz) is still controversial, its use may prevent or delay the progression of the disease to the most severe forms. One of the main factors that can influence the effectiveness of the treatment is the possible cooperation between drug effect and the host immune response. Herein, we evaluated the immune response of peripheral blood mononuclear cells (PBMCs) infected with Trypanosoma cruzi and submitted to Bz treatment. Blood samples of CCD patients (n = 7) and non-infected individuals (n = 6) were drawn to obtain PBMCs. After cell culture, the supernatants were harvested and stored, and the cell analyzed by flow cytometer. The results showed that Bz positively regulated the molecular process of cell activation (CD80) and antigen presentation (HLA-DR), increased phagocytosis receptor and macrophage activation (CD64), and did not induce an exacerbated immune response. In conclusion, these results highlight the relevance of using Bz that, despite not being a true hero, it is also not a villain, as it presents a wide range of pharmacological/immunological response interactions, important for the immune balance in the clinical progression of CCD.

PRODUCTIVE AND REPRODUCTIVE PERFORMANCE OF MAGHREBI SHE-CAMELS FED WITH ORGANIC ZINC DIET UNDER SEMI-ARID EGYPTIAN CONDITIONS

Organic zinc-binding with probiotic (Biogen-Zn®; BZ) is a Zn-methionine combined with Bacillus subtilis capable of improving zinc bioavailability, animal health and ameliorating reproductive stress. The present experiment was performed to evaluate the effect of addition BZ to she-camels on their physiological status, reproductive and productive traits under semi-arid Egyptian conditions. Twenty pregnant Maghrebi camels (4–6 parities) with average body weight of 519.96±19.98 kg were randomly divided into four groups (n=5 in each) according to live body weight and parity. The 1st group, camels were fed basal ration and served as control (without supplementation). The 2nd, 3rd and 4th groups of camels were fed the basal ration supplied with BZ at doses of 5, 7, and 10 g BZ/head/day, respectively. BZ was added daily during a period from the first day of the 10th month of pregnancy period and continued up to seven months of lactation period. Blood samples were collected biweekly during different she-camels’ reproductive stages at 10th monthly of pregnancy, at 1st day after calving, and during seven months of lactation period. The results indicated that BZ significantly increased glucose concentrations in serum, total protein, globulin in dose dependent manner. While, serum concentrations of cholesterol, triglycerides, low density lipoprotein and very low-density lipoprotein were decreased in treated groups compared to the control one. Liver and kidney functions were improved at different sampling stages with 10 g of BZ treatment. Moreover, camels treated with 10 g of BZ revealed a significant effect on placental characteristics fluids including, the shortest duration drop of placenta and the heaviest weights of placenta treated group. As well as, live body weight at 1st service,number of services/conception and service period length during the post-partum period were improved by BZ supplementation. In conclusion, organic zinc-binding with probiotic (BZ) has a potential use for improving physiological status, reproductive and productive performance of she-camels, to particularly with a dose of 10g/h/d of BZ.

Combination therapy with benznidazole and doxycycline shows no additive effect to monotherapy with benznidazole in mice infected with the VL-10 strain of the Trypanosoma cruzi

BackgroundChagas heart disease is the most important clinical manifestation of Trypanosoma cruzi infection. Pharmacological therapies have been proposed aiming to reduce inflammatory response and cardiac damage in infected hosts. In this study, we investigated the use of doxycycline (Dox), in a sub-antimicrobial dose, in monotherapy and in combination with benznidazole (Bz) during the acute phase of infection with the VL-10 strain of T. cruzi, evaluating the therapeutic effect during the acute and chronic phases of the infection. Methods and resultsC57BL/6 mice were treated for 20 days with Dox (30 mg/kg), Bz (100 mg/kg), or both drugs in combination starting 9 days after infection. Parasitemia was measured during the acute phase and the animals were monitored for 12 months, after which echocardiography analysis was performed. Blood samples were obtained from euthanized mice for CCL2, CCL5, IL-10 analysis, and cardiac fragments were collected for histopathological evaluation. Dox treatment did not ameliorate parasitological/inflammatory parameters but reduced the cardiac collagen neoformation (CN) in 35%. In contrast, Bz administration reduced parasitemia, plasma levels of CCL2 and CCL5, and cardiac infiltration during acute infection, and reduced the level of IL-10 and CN (95%) at 12 months. Dox was unable to improve ejection fraction, while Bz treatment ameliorated the ejection fraction. No additive effect was observed in combination therapy. ConclusionDox monotherapy is not effective in the acute or chronic phases of experimental cardiomyopathy induced by the VL-10 strain of T. cruzi. Furthermore, combination therapy with Dox does not potentiate the effects of Bz monotherapy.

Elevated IL-17 levels and echocardiographic signs of preserved myocardial function in benznidazole-treated individuals with chronic Chagas’ disease

BackgroundThe immunological and clinical impact of trypanocidal treatment in chronic Chagas’ disease (CCD) is unclear. Methodology and findingsSeveral cytokines were measured in plasma of 66 patients with CCD. Thirty-three patients had been previously treated with benznidazole and 33 had never been treated. The treated group exhibited higher levels of IL-17 (median 142.45×1.22pg/ml, P=0.025), which was the only one significantly associated with Bz treatment, especially after adjusting for time of disease and NYHA class (P=0.024; OR 1.006, 95% CI 1.001-1.010). Compared to untreated patients, the treated group exhibited higher median values of mitral annular E’ lateral (13.0×10.0cm/s, P=0.038), S’ infero-lateral (8.9×7.6cm/s, P=0.013), S’ septal (8.5×7.4cm/s, P=0.034), mean S’ (9.0×7.9cm/s, P=0.013) and tricuspid annular S’ (13.3×11.1cm/s, P=0.001) and lower values of E/E’ septal (7.2×9.5cm/s, P=0.049). After adjustment for time of disease and NYHA class, S’ infero-lateral (P=0.031), mean S’ (P=0.049) and S’ tricuspid (P=0.024) persisted as significantly associated with treatment. ConclusionThe present findings suggest that the group of CCD patients treated with Bz displayed increased plasma levels of IL-17 and preserved myocardial function, reinforcing the idea that Bz treatment may be beneficial.

Effects of gut microbiota disturbance induced in early life on the expression of extrasynaptic GABA-A receptor α5 and δ subunits in the hippocampus of adult rats.

Previous studies have demonstrated that gut microbiota disturbance significantly increases the risk of emotional disorders via the gut-brain axis, but the mechanism is unclear. Furthermore, gamma-aminobutyric acid (GABA) deficits were reported to be implicated in the development of depression and amnesia, but the alterations in the GABA-A receptor subunits that are involved in the pathogenetic process have not been fully elucidated. This study used juvenile rats that were fed ampicillin-Na to establish degree III dysbiosis of the intestinal flora and examined emotional change via the tail suspension test, forced swim test and Morris water maze. Additionally, our study investigated the expression of GABA-A receptor α5 and δ subunits in the hippocampus in adulthood via q-PCR and immunohistochemistry. We focused on the role of GABA-A receptor α5 and δ subunit changes induced by juvenile gut microbiota disturbance in the pathogenesis of emotional disorders in adulthood. In addition, we investigated the protective effects of probiotics and benzodiazepine (clonazepam). Findings showed that juvenile gut microbiota disturbances induced chronic depression and memory loss and reduced the expression of GABA-A receptor α5 and δ subunits in the hippocampus of the adult rat. Furthermore, moderate probiotic administration led to a significant improvement compared to short-term BZ treatment. However, we are aware that these results have been established with a single animal experiment and will require further confirmation with a larger group of individuals. Future directions for the exploration of the effects of gut microbiota disturbance on GABA-A receptor α5 and δ subunits are discussed.

Bortezomib initiates endoplasmic reticulum stress, elicits autophagy and death in Echinococcus granulosus larval stage.

Cystic echinococcosis (CE) is a worldwide distributed helminthic zoonosis caused by Echinococcus granulosus. Benzimidazole derivatives are currently the only drugs for chemotherapeutic treatment of CE. However, their low efficacy and the adverse effects encourage the search for new therapeutic targets. We evaluated the in vitro efficacy of Bortezomib (Bz), a proteasome inhibitor, in the larval stage of the parasite. After 96 h, Bz showed potent deleterious effects at a concentration of 5 μM and 0.5 μM in protoscoleces and metacestodes, respectively (P < 0.05). After 48 h of exposure to this drug, it was triggered a mRNA overexpression of chaperones (Eg-grp78 and Eg-calnexin) and of Eg-ire2/Eg-xbp1 (the conserved UPR pathway branch) in protoscoleces. No changes were detected in the transcriptional expression of chaperones in Bz-treated metacestodes, thus allowing ER stress to be evident and viability to highly decrease in comparison with protoscoleces. We also found that Bz treatment activated the autophagic process in both larval forms. These facts were evidenced by the increase in the amount of transcripts of the autophagy related genes (Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16) together with the increase in Eg-Atg8-II detected by western blot and by in toto immunofluorescence labeling. It was further confirmed by direct observation of autophagic structures by electronic microscopy. Finally, in order to determine the impact of autophagy induction on Echinococcus cell viability, we evaluated the efficacy of Bz in combination with rapamycin and a synergistic cytotoxic effect on protoscolex viability was observed when both drugs were used together. In conclusion, our findings demonstrated that Bz induced endoplasmic reticulum stress, autophagy and subsequent death allowing to identify unstudied parasite-host pathways that could provide a new insight for control of parasitic diseases.

Bortezomib treatment induces a higher mortality rate in lupus model mice with a higher disease activity

BackgroundBortezomib (Bz) is a proteasome inhibitor that directly targets antibody-producing plasma cells. We recently reported the first randomized control trial that evaluated the effects of Bz in patients with systemic lupus erythematosus (SLE). In that study, we demonstrated that Bz treatment is associated with many adverse reactions in patients with refractory disease. In the present study, we examine the therapeutic and toxic effects of Bz on MRL/MpJ-lpr/lpr (MRL/lpr) mice with severe disease activity.MethodsFemale MRL/lpr mice at 10 and 14 weeks of age were treated with phosphate buffered saline (PBS) (n = 19), Bz (750 μg/kg twice weekly) (n = 27), or cyclophosphamide (Cyc) (1 mg/body, once in 2 weeks) (n = 20). Cellular subsets, serum immunoglobulin, anti-double-stranded DNA (anti-dsDNA) antibody titer, and a pathological index of glomerulonephritis were then analyzed at 22 weeks of age. Survival curves of the 10-week-old and 14-week-old Bz-treated groups were compared. Blood counts, creatinine, liver enzymes, and serum cytokine levels were measured 1 week after Bz treatment. Gene expression profiling of spleens from Bz and Cyc treatment mice were compared with those from control mice.ResultsThe anti-dsDNA antibody levels were significantly higher in 14-week-old than in 10-week-old mice, indicating a higher disease activity at 14 weeks. A significant decrease in the number of splenic cells and glomerulonephritis index was observed in Bz-treated and Cyc-treated mice. Bz, but not Cyc, significantly decreased serum immunoglobulin and anti-dsDNA antibody titer levels. Survival curve analysis revealed a significantly higher mortality rate in 14-week-old than in 10-week-old Bz-treated and control groups. Following two injections of Bz, serum IL-6 and TNF-α levels were significantly more elevated in 14-week-old than in 10-week-old mice. Potentially immunogenic molecules, such as heat shock proteins, were characteristically upregulated in spleens of Bz-treated but not Cyc-treated mice.ConclusionsIn spite of its therapeutic effect, Bz treatment had more toxic effects associated with increased proinflammatory cytokine levels in mice with a higher disease activity. Understanding the mechanism of the toxicity and developing preventive strategies against it is important for the safe clinical application of Bz in human SLE.

Effects of Traumatic Stress Induced in the Juvenile Period on the Expression of Gamma-Aminobutyric Acid Receptor Type A Subunits in Adult Rat Brain.

Studies have found that early traumatic experience significantly increases the risk of posttraumatic stress disorder (PTSD). Gamma-aminobutyric acid (GABA) deficits were proposed to be implicated in development of PTSD, but the alterations of GABA receptor A (GABAAR) subunits induced by early traumatic stress have not been fully elucidated. Furthermore, previous studies suggested that exercise could be more effective than medications in reducing severity of anxiety and depression but the mechanism is unclear. This study used inescapable foot-shock to induce PTSD in juvenile rats and examined their emotional changes using open-field test and elevated plus maze, memory changes using Morris water maze, and the expression of GABAAR subunits (γ2, α2, and α5) in subregions of the brain in the adulthood using western blotting and immunohistochemistry. We aimed to observe the role of GABAAR subunits changes induced by juvenile trauma in the pathogenesis of subsequent PTSD in adulthood. In addition, we investigated the protective effects of exercise for 6 weeks and benzodiazepine (clonazepam) for 2 weeks. This study found that juvenile traumatic stress induced chronic anxiety and spatial memory loss and reduced expression of GABAAR subunits in the adult rat brains. Furthermore, exercise led to significant improvement as compared to short-term BZ treatment.

Monoclonal Gammopathy of Renal Significance (MGRS). Rapid Hematological and Renal complete response to Bortezomib

The term monoclonal gammopathy of renal significance (MGRS) was introduced to distinguish monoclonal gammopathies that result in the development of kidney disease from those that are benign. The term is controlversial for some authors and the optimal treatment is not well established. Therapy should be based in the control of clonal plasma cell disorder. There is few clinical reports regarding evolution of MGRS with novel drugs for GM. We present a clinical case of severe MGRS with a rapid hematological and renal response to Bortezomib (Bz) treatment with complet recovery of kydney function.

Identification of markers that functionally define a quiescent multiple myeloma cell sub-population surviving bortezomib treatment.

BackgroundThe mechanisms allowing residual multiple myeloma (MM) cells to persist after bortezomib (Bz) treatment remain unclear. We hypothesized that studying the biology of bortezomib-surviving cells may reveal markers to identify these cells and survival signals to target and kill residual MM cells.MethodsWe used H2B-GFP label retention, biochemical tools and in vitro and in vivo experiments to characterize growth arrest and the unfolded protein responses in quiescent Bz-surviving cells. We also tested the effect of a demethylating agent, 5-Azacytidine, on Bz-induced quiescence and whether inhibiting the chaperone GRP78/BiP (henceforth GRP78) with a specific toxin induced apoptosis in Bz-surviving cells. Finally, we used MM patient samples to test whether GRP78 levels might associate with disease progression. Statistical analysis employed t-test and Mann-Whitney tests at a 95% confidence.ResultsWe report that Bz-surviving MM cells in vitro and in vivo enter quiescence characterized by p21CIP1 upregulation. Bz-surviving MM cells also downregulated CDK6, Ki67 and P-Rb. H2B-GFP label retention showed that Bz-surviving MM cells are either slow-cycling or deeply quiescent. The Bz-induced quiescence was stabilized by low dose (500nM) of 5-azacytidine (Aza) pre-treatment, which also potentiated the initial Bz-induced apoptosis. We also found that expression of GRP78, an unfolded protein response (UPR) survival factor, persisted in MM quiescent cells. Importantly, GRP78 downregulation using a specific SubAB bacterial toxin killed Bz-surviving MM cells. Finally, quantification of Grp78high/CD138+ MM cells from patients suggested that high levels correlated with progressive disease.ConclusionsWe conclude that Bz-surviving MM cells display a GRP78HIGH/p21HIGH/CDK6LOW/P-RbLOW profile, and these markers may identify quiescent MM cells capable of fueling recurrences. We further conclude that Aza + Bz treatment of MM may represent a novel strategy to delay recurrences by enhancing Bz-induced apoptosis and quiescence stability.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1460-1) contains supplementary material, which is available to authorized users.

Improvement of pharmacokinetic profile and efficacy of sesquiterpene lactone in nanocarriers in the experimental Chagas disease in mice

Chagas disease (CD), present in Latin America, is neglected and autochthone in other Continents [1]. Objetive: (i) evaluate the pharmacokinetic of LYC, a sesquiterpene lactone extracted from Lychnophora trichocarpha and LYC-NC formulated in polymeric nanocapsules (NC) and (ii) its efficacy in Swiss mice infected with Trypanosoma cruzi strains of different patterns of drugs resistance. NC was produced [2] at different concentrations of LYC and LYC-NCs were fully characterized, rending high percentages of encapsulation [3,4] and showing high stability. A new fast and precise bioanalytical method was developed (HPLC-UV detection), validated following FDA and National Agency of Sanitary Vigilance/Brazil guidelines [5] and further applied to LYC pharmacokinetic evaluation. It was observed an increasing in AUC (14 fold) and t1/2 (2,4 fold) and reduction of the Clearance (124 fold) of LYC when associated to PLA-PEG-NC x PLC-NC. The efficacy of free LYC, LYC-NC, BZ and controls was studied in mice inoculated with 1.0 × 104 trypomastigotes (acute phase) or 500 (chronic phase) of the CL, Y, Colombiana, VL10 T. cruzi strains. The cure rate in mice infected with all strains, acute and chronic phases, treated by IV/oral routes decreases in the following order: LYC-PLA-PEG-NC>LYC-PCL-NC>LYC free>BZ and was 0% for all controls. Cure rate of 88% was found with LYC-PLAPEG-NC treatment by oral route for resistant T. cruzi strain in chronic model (VL10 strain). This study is pioneer in the demonstration of better efficacy of LYC in vivo, when associated with NC, compared with BZ treatment of experimental CD, in both phases of infection with T. cruzi strains totally resistant to BZ/Nifurtimox. Furthermore, PLA-PEG-NC improved the pharmacokinetic parameters of LYC, increasing its plasma AUC and mean residence time, which contributes to the improvement of the efficacy in mice and showing the great potential of LYC for further clinical studies of CD treatment.

Proteasome inhibition in skeletal muscle cells unmasks metabolic derangements in type 2 diabetes.

Two-dimensional difference gel electrophoresis (2-D DIGE)-based proteome analysis has revealed intrinsic insulin resistance in myotubes derived from type 2 diabetic patients. Using 2-D DIGE-based proteome analysis, we identified a subset of insulin-resistant proteins involved in protein turnover in skeletal muscle of type 2 diabetic patients, suggesting aberrant regulation of the protein homeostasis maintenance system underlying metabolic disease. We then validated the role of the ubiquitin-proteasome system (UPS) in myotubes to investigate whether impaired proteasome function may lead to metabolic arrest or insulin resistance. Myotubes derived from muscle biopsies obtained from people with normal glucose tolerance (NGT) or type 2 diabetes were exposed to the proteasome inhibitor bortezomib (BZ; Velcade) without or with insulin. BZ exposure increased protein carbonylation and lactate production yet impaired protein synthesis and UPS function in myotubes from type 2 diabetic patients, marking the existence of an insulin-resistant signature that was retained in cultured myotubes. In conclusion, BZ treatment further exacerbates insulin resistance and unmasks intrinsic features of metabolic disease in myotubes derived from type 2 diabetic patients. Our results highlight the existence of a confounding inherent abnormality in cellular protein dynamics in metabolic disease, which is uncovered through concurrent inhibition of the proteasome system.

Reduced CXCR4 expression is associated with extramedullary disease in a mouse model of myeloma and predicts poor survival in multiple myeloma patients treated with bortezomib

Reduced CXCR4 expression is associated with extramedullary disease in a mouse model of myeloma and predicts poor survival in multiple myeloma patients treated with bortezomib

In Silico Prediction of Novel Drug Combinations to Combat Bortezomib-Resistant Multiple Myeloma

AbstractAbstract 1344Bortezomib/VELCADE® (Bz) is a proteasome inhibitor that has been used successfully in the treatment of multiple myeloma (MM) patients. However, acquired resistance to Bz is an emerging problem. Thus, there is a need for novel therapeutic combinations that enhance Bz sensitivity or re-sensitize Bz resistant MM cells to Bz. The Connectivity Map (CMAP; Broad Institute) database contains treatment-induced transcriptional signatures from 1,309 bioactive compounds in 4 human cancer cell lines. An input signature can be used to query the database for correlated drug signatures, a technique that has been used previously to identify drugs that combat chemoresistance in cancer (Wei, et al. Cancer Cell (2006) 10:331). In this study we used in silico bioinformatic screening of gene expression profiles from isogenic pairs of Bz sensitive and resistant mouse cell lines derived from the iMycCα/Bcl-xL mouse model of plasma cell malignancy to identify compounds that combat Bz resistance.We established Bz-induced kinetic gene expression profiles (GEPs) in 3 pairs of Bz sensitive and resistant mouse cell lines over the course of 24 hours. GEPs were collected in the absence of large-scale cell death. The 16 and 24 hour time points were averaged and compared between each Bz sensitive and resistant pair. Genes in the sensitive cell line with a fold change greater than 2, relative to the resistant line, were given the binary distinction of “up” or “down” depending on the direction of change. Genes that met these criteria were assembled into signatures, and then used as inputs for CMAP queries to identify compounds that induce similar transcriptional responses. In all pairs, treatment of the Bz sensitive line correlated with GEPs of drugs that target the proteasome, NF-κB, HSP90 and microtubules, as indicated by positive connectivity scores. However eight compounds, all classified as Topoisomerase (Topo) I and/or II inhibitors, were negatively correlated to our input signature. A negative connectivity score could have two interpretations: (1) this could indicate simply that Topos are upregulated by Bz treatment in Bz sensitive lines, which has been previously reported (Congdan, et al. Biochem. Pharmacol. (2008) 74: 883); or (2) this score could be interpreted as Topos are inhibited in Bz resistant cells upon Bz treatment. This led us to ask whether Topo inhibitors could target Bz resistant MM cells and re-sensitize them to Bz. Indeed, we found that multiple Topo inhibitors were significantly more active against Bz resistant cells as single agents and restored sensitivity to Bz when combined with Bz as a cocktail regimen. This work demonstrates the potential of this in silico bioinformatic approach for identifying novel therapeutic combinations that overcome Bz resistance in MM. Furthermore, it identifies Topo inhibitors – drugs that are already approved for clinical use – as agents that may have utility in combating Bz resistance in refractory MM patients. Disclosures:Stessman:Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

Myocardial scars correlate with eletrocardiographic changes in chronic Trypanosoma cruzi infection for dogs treated with Benznidazole

The cardiac form of Chagas disease is evidenced by a progressive cardiac inflammation that leads to myocarditis, fibrosis and electrocardiographic (ECG) conduction abnormalities. Considering these characteristics, the aim of this study was to prospectively evaluate the early ECG changes in dogs that were experimentally inoculated with Benznidazole (Bz)-susceptibly (Berenice-78) and Bz-resistant (VL-10, and AAS) Trypanosoma cruzi strains and, later, evaluate the efficacy of Bz treatment for preventing these ECG alterations. Electrocardiographic changes of treated and untreated animals were prospectively evaluated for up to 270 days after infection, at which point collagen (right atrium) quantification was performed. All infected dogs had a high intensity of heart fibrosis (4616.00 ± 1715.82 collagen/74931 μm(2) in dogs infected with Berenice-78 strain, 5839.2 ± 1423.49 collagen/74931 μm(2) in infected by AAS and 6294.40 ± 896.04 collagen/74931 μm(2) in animals infected with VL-10 strain), while 78.57% of all infected dogs showed ECG alterations. Bz Therapy reduced or prevented fibrosis in Bz-susceptible Berenice-78 (2813.00 ± 607.13 collagen/74931 μm(2) ) and Bz-resistant AAS strains (4024 ± 1272.44 collagen/74931 μm(2) ), coincident with only 10% de ECG alterations at 270 days. However, in those animals infected with a Bz-resistant VL-10 strain, specific treatment did not alter collagen deposition (6749.5 ± 1596.35 collagen/74931 μm(2) ) and there was first atrioventricular block and chamber overload at 120 and 270 days after infection, with 75% abnormal ECG exams. These findings indicate that an effective antiparasitic treatment in the early stage of Chagas disease can lead to a significant reduction in the frequency and severity of the parasite-induced cardiac disease, even if parasites are not completely eliminated.