Improvement of the HIV/AIDS vaccine candidate NYVAC-C by deletion of the viral type I IFN inhibitor and/or acquisition of replication competence

Background: Recombinant viruses based on the attenuated vaccinia virus strain NYVAC are promising HIV vaccine candidates as phase I/II clinical trials have shown good safety and immunogenicity profiles. However, this NYVAC strain is non-replicating in most human cell lines and encodes viral inhibitors of the immune system. Methods: With the aim to increase the immune potency of the current NYVAC-C vector (expressing the codon optimized clade C HIV-1 genes encoding gp120 and Gag-Pol-Nef polyprotein), we have generated and characterized three NYVAC-C-based vectors by, 1) deletion of the viral type I IFN inhibitor gene (NYVAC-CdeltaB19R), 2) restoration of virus replication competence in human cells by re-inserting K1L and C7L host range genes (NYVAC-C-KC) and, 3) combination of both strategies (NYVACC- KC-deltaB19R). Results: Insertion of the KC fragment restored the replication competence of the viruses in human cells (HeLa cells and primary dermal fibroblasts and keratinocytes), increased the expression of HIV antigens by more than 3-fold compared to the non-replicating homologs, inhibited apoptosis induced by the parental NYVAC-C and retained attenuation in a newborn mouse model. In adult mice, replication-competent viruses showed a limited capacity to replicate in tissues surrounding the inoculation site (ovaries and lymph nodes). After infection of keratinocytes, PBMCs and dendritic cells these viruses induced differential modulation in specific host cell signal transduction pathways, triggering genes important in immune modulation. Conclusion: We have developed improved NYVAC-C-based vectors with enhanced HIV-1 antigen expression, with the ability to replicate in cultured human cells and partially in some tissues, with an induced expression of cellular genes relevant to immune system activation, and which trigger IFN-dependent and independent signalling pathways, while maintaining a safety phenotype. These new vectors are promising new HIV vaccine candidates. These studies were performed within the Poxvirus Tcell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Role of the transcriptional factor C/EBPbeta in free fatty acid-elicited beta-cell failure.

Fatty acids can favour the development of Type 2 diabetes by reducing insulin secretion and inducing apoptosis of pancreatic beta-cells. Here, we show that sustained exposure of the beta-cell line MIN6 or of isolated pancreatic islets to the most abundant circulating fatty acid palmitate increases the level of C/EBPbeta, an insulin transcriptional repressor. In contrast, two unsaturated fatty acids, oleate and linoleate were without effect. The induction of C/EBPbeta elicited by palmitate was prevented by inhibiting the ERK1/2 MAP kinase pathway or by reducing mitochondrial fatty acid oxidation with an inhibitor of Carnitine Palmitoyl Transferase-1. Overexpression of C/EBPbeta mimicked the detrimental effects of palmitate and resulted in a drastic reduction in insulin promoter activity, impairment in the capacity to respond to secretory stimuli and an increase in apoptosis. Our data suggest a potential involvement of C/EBPbeta as mediator of the deleterious effects of unsaturated free fatty acids on beta-cell function.

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor.

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 "clients". We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538.

Pathway analysis of glioblastoma tissue after preoperative treatment with the EGFR tyrosine kinase inhibitor gefitinib--a phase II trial.

Amplification of the epidermal growth factor receptor (EGFR) gene is one of the most common oncogenic alterations in glioblastoma (45%) making it a prime target for therapy. However, small molecule inhibitors of the EGFR tyrosine kinase showed disappointing efficacy in clinical trials for glioblastoma. Here we aimed at investigating the molecular effects of the tyrosine kinase inhibitor gefitinib on the EGFR signaling pathway in human glioblastoma. Twenty-two patients selected for reoperation of recurrent glioblastoma were treated within a phase II trial for 5 days with 500 mg gefitinib before surgery followed by postoperative gefitinib until recurrence. Resected glioblastoma tissues exhibited high concentrations of gefitinib (median, 4.1 μg/g), 20 times higher than respective plasma. EGFR-pathway activity was evaluated with phosphorylation-specific assays. The EGFR was efficiently dephosphorylated in treated patients as compared to a control cohort of 12 patients. However, no significant effect on 12 pathway constituents was detected. In contrast, in vitro treatment of a glioblastoma cell line, BS-153, with endogenous EGFRwt amplification and EGFRvIII expression resulted not only in dephosphorylation of the EGFR, but also of key regulators in the pathway such as AKT. Treating established xenografts of the same cell line as an in vivo model showed dephosphorylation of the EGFR without affecting downstream signal transductors, similar to the human glioblastoma. Taken together, gefitinib reaches high concentrations in the tumor tissue and efficiently dephosphorylates its target. However, regulation of downstream signal transducers in the EGFR pathway seems to be dominated by regulatory circuits independent of EGFR phosphorylation.

Combined cetuximab and trastuzumab are superior to gemcitabine in the treatment of human pancreatic carcinoma xenografts.

BACKGROUND: Pancreatic carcinoma remains a treatment-refractory cancer with a poor prognosis. Here, we compared anti-epidermal growth factor receptor (EGFR) and anti-HER2 monoclonal antibodies (2mAbs) injections with standard gemcitabine treatment on human pancreatic carcinoma xenografts. MATERIALS AND METHODS: Nude mice, bearing human pancreatic carcinoma xenografts, were treated with either combined anti-EGFR (cetuximab) and anti-HER2 (trastuzumab) or gemcitabine, and tumor growth was observed. RESULTS AND CONCLUSION: In first-line therapy, mice survival was significantly longer in the 2mAbs group compared with gemcitabine (P < 0.0001 for BxPC-3, P = 0.0679 for MiaPaCa-2 and P = 0.0019 for Capan-1) and with controls (P < 0.0001). In second-line therapy, tumor regressions were observed after replacing gemcitabine by 2mAbs treatment, resulting in significantly longer animal survival compared with mice receiving continuous gemcitabine injections (P = 0.008 for BxPC-3, P = 0.05 for MiaPaCa-2 and P < 0.001 for Capan-1). Therapeutic benefit of 2mAbs was observed despite K-Ras mutation. Interestingly, concerning the mechanism of action, coinjection of F(ab')(2) fragments from 2mAbs induced significant tumor growth inhibition, compared with controls (P = 0.001), indicating that the 2mAbs had an Fc fragment-independent direct action on tumor cells. This preclinical study demonstrated a significant improvement of survival and tumor regression in mice treated with anti-EGFR/anti-HER2 2mAbs in first- and second-line treatments, compared with gemcitabine, independently of the K-Ras status.

Genomic organization, fine-mapping, and expression of the human islet-brain 1 (IB1)/c-Jun-amino-terminal kinase interacting protein-1 (JIP-1) gene.

Islet-brain 1 (IB1), a regulator of the pancreatic beta-cell function in the rat, is homologous to JIP-1, a murine inhibitor of c-Jun amino-terminal kinase (JNK). Whether IB1 and JIP-1 are present in humans was not known. We report the sequence of the 2133-bp human IB1 cDNA, the expression, structure, and fine-mapping of the human IB1 gene, and the characterization of an IB1 pseudogene. Human IB1 is 94% identical to rat IB1. The tissue-specific expression of IB1 in human is similar to that observed in rodent. The IB1 gene contains 12 exons and maps to chromosome 11 (11p11.2-p12), a region that is deleted in DEFECT-11 syndrome. Apart from an IB1 pseudogene on chromosome 17 (17q21), no additional IB1-related gene was found in the human genome. Our data indicate that the sequence and expression pattern of IB1 are highly conserved between rodent and human and provide the necessary tools to investigate whether IB1 is involved in human diseases.

Role and regulation of islet-Brain 1 in pancreatic β-cells

Résumé : L'insuline est produite et sécrétée par la cellule ß-pancréatique. Son rôle est de régler le taux de sucre dans le sang. Si ces cellules meurent ou échouent à produire suffisamment de l'insuline, les sujets développent le diabète de type 2 (DT2), une des maladies les plus communes dans les pays développés. L'excès chronique des lipoprotéines LDL oxydés (oxLDL) et/ou des cytokines pro-inflammatoires comme l'interleukine-1ß (IL-1ß) participent au dérèglement et à la mort des cellules ß. Nous avons montré qu'une chute des niveaux d'expression de la protéine nommée «mitogen activated protein kinase 8 interacting protein 1» ou «islet brain 1 (IB 1)» est en partie responsable des effets provoqués par les oxLDL ou IL-1ß. IB1 régule l'expression de l'insuline et la survie cellulaire en inhibant la voie de signalisation « c-jun N-terminal Kinase (JNK)». La réduction des niveaux d'expression d'IB1 provoque l'activation de la voie JNK en réponse aux facteurs environnementaux, et ainsi initie la réduction de l'expression de l'insuline et l'induction du programme de mort cellulaire. Les mimétiques de l'hormone "Glucagon-like peptide 1", tel que l'exendin-4 (ex-4), sont une nouvelle classe d'agents hypoglycémiants utilisés dans le traitement du DT2. Les effets bénéfiques de l'ex-4 sont en partie accomplis en préservant l'expression de l'insuline et la survie des cellules ß contre les stress associés au DT2. La restauration des niveaux d'expression d'IB1 est un des mécanismes par lequel l'ex-4 prodigue son effet sur la cellule. En effet, cette molécule stimule l'activité du promoteur du gène et ainsi compense la réduction du contenu en IB1 causée par le stress. Outre ce rôle anti-apoptotique, dans ce travail de thèse nous avons mis en évidence une autre fonction d'IB1 dans la cellule ß. La réduction de l'activité ou des niveaux d'expression d'IB1 induisent une réduction importante de la sécrétion de l'insuline en réponse au glucose. Le mécanisme par lequel IB1 régule la sécrétion de l'insuline implique à la fois le métabolisme du glucose et éventuellement le transport vésiculaire en contrôlant l'expression de la protéine annexin A2. En résumé, IB 1 est une molécule clé à travers laquelle l'environnement du diabétique pourrait exercer un effet délétère sur la cellule ß. L'amélioration de l'activité d'IB1 et/ou de son expression devrait être considérée dans les approches thérapeutiques futures visant à limiter la perte des cellules ß dans le diabète. Abstract : ß-cells of the pancreatic islets of Langerhans produce and secrete insulin when blood glucose rises. In turn, insulin ensures that plasma glucose concentrations return within a relatively narrow physiological range. If ß-cells die or fail to produce enough insulin, individuals develop one of the most common diseases in Western countries, namely type 2 diabetes (T2D). Chronic excess of oxidized low density lipoproteins (oxLDL) and/or pro-inflammatory cytokines such as interleukin 1-ß (IL-1ß) contribute to decline of ß-cells and thereby are thought to accelerate progression of the disease overtime. We showed that profound reduction in the levels of the mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) causes ß-cell failure accomplished by oxLDL or IL-1 ß. IB1 regulates insulin expression and cell survivals by inhibiting the c-Jun N-terminal Kinase pathway. Diminution in IB 1 levels leads to an increase in activation of the JNK pathway induced by environmental stressors, and thus initiates loss of insulin expression and programmed cell death. The mimetic agents of the glucoincretin glucagon-like peptide 1 such as exendin-4 (ex-4) are new class of hypoglycaemic medicines for treatment of T2D. The beneficial property is in part achieved by preserving insulin expression and ß-cell survival against stressors related to diabetes. Restored levels in IB 1 account for the cytoprotective effect of the ex-4. In fact, the latter molecule .stimulates the promoter activity of the gene and thus compensates loss of IB1 content triggered by stress. Beside of the anti-apoptotic role, an additional leading function for IB 1 in ß-cells was highlighted in this thesis. Impairment in IB1 activity or silencing of the gene in ß-cells revealed a major reduction in insulin secretion elicited by glucose. The mechanisms whereby IB 1 couples glucose to insulin release involve glucose metabolism and potentially, vesicles trafficking by maintaining the levels of annexin A2. IB 1 is therefore a key molecule through which environmental factors related to diabetes may exert harmful effects on ß-cells. Improvement in IB 1 activity and/or expression should be considered as a target for therapeutic purpose.

Absence of mTOR Inhibitor Effect on Hepatic Cyst Growth: A Case Report of a Kidney Transplant Recipient with Autosomal Dominant Polycystic Kidney Disease.

Some experimental studies have suggested a beneficial effect of the mammalian target of rapamycin (mTOR) inhibitor use on hepatic and renal cyst growth in patients with autosomal dominant polycystic kidney disease (ADPKD). However, the results of clinical studies are conflicting and the role of mTOR inhibitors is still uncertain. We report the case of a patient with ADPKD who underwent deceased kidney transplantation because of an end-stage renal disease. The evolution was uneventful with an excellent graft function under cyclosporine (CsA) monotherapy. Some years later, the patient developed a symptomatic hepatomegaly due to growth of cysts. CsA was replaced by sirolimus, an mTOR inhibitor, in order to reduce or control the increase in the cyst and liver volume. Despite the switch, the hepatic volume increased by 25% in two years. Finally sirolimus was stopped because of the lack of effect on hepatic cyst growth and the presence of sirolimus side effects. The interest of our case resides in the followup by MRI imaging during the mTOR inhibitor treatment and 15 months after the restart of the initial immunosuppressive therapy. This observation indicates that mTOR inhibitors did not have significant effect on cyst-associated hepatic growth in our patient, which is consistent with some results of recent large clinical studies.

Effect of the chitin synthesis inhibitor triflumuron on the development, viability and reproduction of Aedes aegypti

The control of Aedes aegypti is impaired due to the development of resistance to chemical insecticides. Insect Growth Regulators (IGR) exhibit distinct mechanisms of action and are considered potential vector control alternatives. Studies regarding the effects of sublethal IGR doses on the viability of resulting adults will contribute to eval-uating their impact in the field. We analyzed several aspects of Ae. aegypti adults surviving exposure to a partially lethal dose of triflumuron, a chitin synthesis inhibitor. A highly significant difference in the proportion of males and females was noted in the triflumuron-exposed group (65.0% males) compared to the controls (50.2% males). Triflumuron affected adult longevity, particularly for females; after 16 days, only 29.2% of males and 13.8% of females were alive, in contrast with 94% survival of the control mosquitoes. The locomotor activity was reduced and the blood-feeding ability of the treated females was also affected (90.4% and 48.4% of the control and triflumuron-exposed females, respectively, successfully ingested blood). Triflumuron-surviving females ingested roughly 30% less blood and laid 25% fewer eggs than the control females. The treated males and females exhibited a diminished ability to copulate, resulting in less viable eggs.

Analysis of TNFAIP3, a feedback inhibitor of nuclear factor-κB and the neighbor intergenic 6q23 region in rheumatoid arthritis susceptibility

INTRODUCTION Genome-wide association studies of rheumatoid arthritis (RA) have identified an association of the disease with a 6q23 region devoid of genes. TNFAIP3, an RA candidate gene, flanks this region, and polymorphisms in both the TNFAIP3 gene and the intergenic region are associated with systemic lupus erythematosus. We hypothesized that there is a similar association with RA, including polymorphisms in TNFAIP3 and the intergenic region. METHODS To test this hypothesis, we selected tag-single nucleotide polymorphisms (SNPs) in both loci. They were analyzed in 1,651 patients with RA and 1,619 control individuals of Spanish ancestry. RESULTS Weak evidence of association was found both in the 6q23 intergenic region and in the TNFAIP3 locus. The rs582757 SNP and a common haplotype in the TNFAIP3 locus exhibited association with RA. In the intergenic region, two SNPs were associated, namely rs609438 and rs13207033. The latter was only associated in patients with anti-citrullinated peptide antibodies. Overall, statistical association was best explained by the interdependent contribution of SNPs from the two loci TNFAIP3 and the 6q23 intergenic region. CONCLUSIONS Our data are consistent with the hypothesis that several RA genetic factors exist in the 6q23 region, including polymorphisms in the TNFAIP3 gene, like that previously described for systemic lupus erythematosus.

Incretin receptor null mice reveal key role of GLP-1 but not GIP in pancreatic beta cell adaptation to pregnancy.

Islet adaptations to pregnancy were explored in C57BL6/J mice lacking functional receptors for glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). Pregnant wild type mice and GIPRKO mice exhibited marked increases in islet and beta cell area, numbers of medium/large sized islets, with positive effects on Ki67/Tunel ratio favouring beta cell growth and enhanced pancreatic insulin content. Alpha cell area and glucagon content were unchanged but prohormone convertases PC2 and PC1/3 together with significant amounts of GLP-1 and GIP were detected in alpha cells. Knockout of GLP-1R abolished these islet adaptations and paradoxically decreased pancreatic insulin, GLP-1 and GIP. This was associated with abolition of normal pregnancy-induced increases in plasma GIP, L-cell numbers, and intestinal GIP and GLP-1 stores. These data indicate that GLP-1 but not GIP is a key mediator of beta cell mass expansion and related adaptations in pregnancy, triggered in part by generation of intra-islet GLP-1.

Hyperglycemia downregulates Connexin36 in pancreatic islets via the upregulation of ICER-1/ICER-1γ.

Channels formed by the gap junction protein Connexin36 (CX36) contribute to the proper control of insulin secretion. We previously demonstrated that chronic exposure to glucose decreases Cx36 levels in insulin-secreting cells in vitro. Here, we investigated whether hyperglycemia also regulates Cx36 in vivo. Using a model of continuous glucose infusion in adult rats, we showed that prolonged (24-48 h) hyperglycemia reduced the Cx36 gene Gjd2 mRNA levels in pancreatic islets. Accordingly, prolonged exposure to high glucose concentrations also reduced the expression and function of Cx36 in the rat insulin-producing INS-1E cell line. The glucose effect was blocked after inhibition of the cAMP/PKA pathway and was associated with an overexpression of the inducible cAMP early repressor ICER-1/ICER-1γ, which binds to a functional cAMP-response element in the promoter of the Cx36 gene Gjd2. The involvement of this repressor was further demonstrated using an antisense strategy of ICER-1 inhibition, which prevented glucose-induced downregulation of Cx36. The data indicate that chronic exposure to glucose alters the in vivo expression of Cx36 by the insulin-producing β-cells through ICER-1/ICER-1γ overexpression. This mechanism may contribute to the reduced glucose sensitivity and altered insulin secretion, which contribute to the pathophysiology of diabetes.

Effects of MDL 100,240, a dual inhibitor of angiotensin-converting enzyme and neutral endopeptidase on the vasopressor response to exogenous angiotensin I and angiotensin II challenges in healthy volunteers.

MDL 100,240, a dual inhibitor of angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP), was administered intravenously to two panels of four healthy males in a four-period, dose-increasing (0, 1.56, 6.25, and 25 mg, and 0, 3.13, 12.5, and 50 mg, respectively) double-blind, placebo-controlled study. Plasma ACE activity and blood-pressure response to exogenous angiotensin I and angiotensin II i.v. challenges and safety and tolerance were assessed over a 24-h period. MDL 100,240 induced a rapid, dose-related, and sustained inhibition of ACE (>70% over 24 h at doses > or =12.5 mg). The time integral of ACE inhibition was related to the dose but with near-maximal values already attained at doses > or =12.5 mg. Systolic and diastolic blood-pressure responses to exogenous angiotensin I challenges were inhibited in a dose-dependent fashion, whereas the effects of angiotensin II remained unaffected. Mean supine blood pressure decreased transiently (3 h) at doses > or =3.125 mg and < or =24 h with the 25- and 50-mg doses, but not significantly. MDL 100,240 was well tolerated. In healthy subjects, MDL 100,240 exerts a dose-dependent and long-lasting ACE-blocking activity, also expressed by the inhibition of the pressor responses to exogenous angiotensin I challenges. The baroreceptor reflex, assessed by the response to exogenous angiotensin II challenge, remains unaltered.

Targeting renal cell carcinoma with NVP-BEZ235, a dual PI3K/mTOR inhibitor, in combination with sorafenib.

Background: Targeted therapies for metastatic renal cell carcinoma (RCC), including mammalian target of rapamycin (mTOR) inhibitors and small-molecule multikinase inhibitors, have produced clinical effects. However, most patients acquire resistance over time. Thus, new therapeutic strategies need to be developed. Here, we evaluated the effect of the dual PI3K/mTOR inhibitor NVP-BEZ235, in combination with the multikinase inhibitor sorafenib on renal cancer cell proliferation and survival in vitro as well as on tumor growth in vivo.Methods: The renal carcinoma cell lines 786-0 and Caki-1 were treated with NVP-BEZ235 or sorafenib, either alone or in combination. Tumor cell proliferation and apoptosis were investigated in vitro. The anticancer efficacy of NVP-BEZ235 alone, or in combination with sorafenib, was also evaluated on RCC xenografts in nude mice.Results: Treatment of 786-0 and Caki-1 cells with NVP-BEZ235 or sorafenib resulted in reduced tumor cell proliferation and increased tumor cell apoptosis in vitro. The combination of NVP-BEZ235 and sorafenib was more effective than each compound alone. Similarly, in vivo, NVP-BEZ235 or sorafenib reduced the growth of xenografts generated from 786-0 or Caki-1 cells. The antitumor efficacy of NVP-BEZ235 in combination with sorafenib was superior to NVP-BEZ235 or sorafenib alone.Conclusions: Our findings indicate that the simultaneous use of NVP-BEZ235 and sorafenib has greater antitumor benefit compared to either drug alone and thus provides a treatment strategy in RCC.

The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil

The mosquito Aedes aegypti is the main focus of dengue control campaigns. Because of widespread resistance against conventional chemical insecticides, chitin synthesis inhibitors (CSIs) are considered control alternatives. We evaluated the resistance status of four Brazilian Ae. aegypti populations to both the organophosphate temephos and the pyrethroid deltamethrin, which are used in Brazil to control larvae and adults, respectively. All vector populations exhibited high levels of temephos resistance and varying rates of alterations in their susceptibility to pyrethroids. The effect of the CSI novaluron on these populations was also investigated. Novaluron was effective against all populations under laboratory conditions. Field-simulated assays with partial water replacement were conducted to evaluate novaluron persistence. Bioassays were continued until an adult emergence inhibition of at least 70% was attained. We found a residual effect of eight weeks under indoor conditions and novaluron persisted for five-six weeks in assays conducted in an external area. Our data show that novaluron is effective against the Ae. aegypti populations tested, regardless of their resistance to conventional chemical insecticides.