IL-17F co-expression improves cell growth characteristics and enhances recombinant protein production during CHO cell line engineering.

The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL-17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL-17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL-17F expression improves the efficiency of cell line subcloning processes. IL-17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame.

Regions of mouse mammary tumor virus superantigen involved in interaction with the major histocompatibility complex class II I-A molecule.

To study the major histocompatibility complex class II I-E dependence of mouse mammary tumor virus (MMTV) superantigens, we constructed hybrids between the I-E-dependent MMTV(GR) and the I-E-independent mtv-7 superantigens and tested them in vivo. Our results suggest that, although the C-terminal third mediates I-A interaction, additional binding sites are located elsewhere in the superantigen.

Histone deacetylase inhibitors impair antibacterial defenses of macrophages.

Histone deacetylases (HDACs) control gene expression by deacetylating histones and nonhistone proteins. HDAC inhibitors (HDACi) are powerful anticancer drugs that exert anti-inflammatory and immunomodulatory activities. We recently reported a proof-of-concept study demonstrating that HDACi increase susceptibility to bacterial infections in vivo. Yet, still little is known about the effects of HDACi on antimicrobial innate immune defenses. Here we show that HDACi belonging to different chemical classes inhibit at multiple levels the response of macrophages to bacterial infection. HDACi reduce the phagocytosis and the killing of Escherichia coli and Staphylococcus aureus by macrophages. In line with these findings, HDACi decrease the expression of phagocytic receptors and inhibit bacteria-induced production of reactive oxygen and nitrogen species by macrophages. Consistently, HDACi impair the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and inducible nitric oxide synthase. These data indicate that HDACi have a strong impact on critical antimicrobial defense mechanisms in macrophages.

Termination of major ductile strike-slip shear and differential cooling along the Insubric line (Central Alps): U-Pb, Rb-Sr and 40Ar/39Ar ages of cross-cutting pegmatites

To constrain the age of strike-slip shear, related granitic magmatism, and cooling along the Insubric line, 29 size fractions of monazite and xenotime were dated by the U-Pb method, and a series of 25 Rb-Sr and Ar-40/Ar-39 ages were measured on different size fractions of muscovite and biotite. The three pegmatitic intrusions analyzed truncate high-grade metamorphic mylonite gneisses of the Simplon shear zone, a major Alpine structure produced in association with dextral strike-slip movements along the southern edge of the European plate, after collision with its Adriatic indenter. Pegmatites and aplites were produced between 29 and 25 Ma in direct relation to right-lateral shear along the Insubric line, by melting of continental crust having Sr-87/Sr-86 between 0.7199 and 0.7244 at the time of melting. High-temperature dextral strike-slip shear was active at 29.2 +/- 0.2 (2 sigma) Ma, and it terminated before 26.4 +/- 0.1 Ma. During dike injection, temperatures in the country rocks of the Isorno-Orselina and Monte Rosa structural units did not exceed approximate to 500 degrees C, leading to fast initial cooling, followed by slower cooling to approximate to 350 degrees C within several million years. In one case, initial cooling to approximate to 500 degrees C was significantly delayed by about 4 m.y., with final cooling to approximate to 300 degrees C at 20-19 Ma in all units. For the period between 29 and 19 Ma, cooling of the three sample localities was non-uniform in space and time, with significant variations on the kilometre scale. These differences are most likely due to strongly varying heat flow, and/or heterogeneous distribution of unroofing rates within the continuously deforming Insubric line. If entirely ascribed to differences in unroofing, corresponding rates would vary between 0.5 and 2.5 mm/y, for a thermal gradient of 30 degrees/km.

Blocking hypoxia-induced autophagy in tumors restores cytotoxic T-cell activity and promotes regression.

The relationship between hypoxic stress, autophagy, and specific cell-mediated cytotoxicity remains unknown. This study shows that hypoxia-induced resistance of lung tumor to cytolytic T lymphocyte (CTL)-mediated lysis is associated with autophagy induction in target cells. In turn, this correlates with STAT3 phosphorylation on tyrosine 705 residue (pSTAT3) and HIF-1α accumulation. Inhibition of autophagy by siRNA targeting of either beclin1 or Atg5 resulted in impairment of pSTAT3 and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Furthermore, inhibition of pSTAT3 in hypoxic Atg5 or beclin1-targeted tumor cells was found to be associated with the inhibition Src kinase (pSrc). Autophagy-induced pSTAT3 and pSrc regulation seemed to involve the ubiquitin proteasome system and p62/SQSTM1. In vivo experiments using B16-F10 melanoma tumor cells indicated that depletion of beclin1 resulted in an inhibition of B16-F10 tumor growth and increased tumor apoptosis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine in B16-F10 tumor-bearing mice and mice vaccinated with tyrosinase-related protein-2 peptide dramatically increased tumor growth inhibition. Collectively, this study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen-specific T-cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.

Dicionários disponíveis on-line para aprendizes de inglês: estruturação e recursos

O artigo descreve a estrutura e as formas de acesso características dos dicionários eletrônicos do tipo on-line disponíveis ao aprendiz de língua inglesa, comparando o conteúdo e os recursos de dois dicionários monolíngues e dois dicionários bilíngües - o Cambridge Advanced Learner's Dictionary (CALD, 2009), o Merriam-Webster Learner's Dictionary (MWLD, 2009), o Reverso Dictionary English/Portuguese (RDEP, 2008) e o Wiktionary Inglês/Português (WIP, 2009). Discute-se a funcionalidade desses instrumentos em relação às demandas do aprendiz.

Distribution of free and liposomal doxorubicin after isolated lung perfusion in a sarcoma model.

BACKGROUND: Isolated lung perfusion (ILP) with free and a novel liposomal-encapsulated doxorubicin (Liporubicin, CT Sciences SA, Lausanne, Switzerland) was compared with respect to drug uptake and distribution in rat lungs bearing a sarcomatous tumor. METHODS: A single sarcomatous tumor was generated in the left lung of 39 Fischer rats, followed 10 days later by left-sided ILP (n = 36) with free and equimolar-dosed liposomal doxorubicin at doses of 100 microg (n = 9) and 400 microg (n = 9) for each doxorubicin formulation. In each perfused lung, the drug concentration and distribution were assessed in the tumor and in three areas of normal lung parenchyma by high-performance liquid chromatography (n = 6) and fluorescence microscopy (n = 3). Histologic assessment and immunostaining with von Willebrand factor was performed in 3 animals with untreated tumors. RESULTS: The sarcomatous tumors in controls were well vascularized with fine branching capillaries present throughout the tumors. Isolated lung perfusion resulted in a heterogeneous drug distribution within the perfused lung and a consistently lower drug uptake in tumors than in lung parenchyma for both doxorubicin formulations and both drug doses applied. Isolated lung perfusion with free doxorubicin resulted in a significantly higher drug uptake than Liporubicin in both the tumor and lung tissue for both drug doses applied (p < 0.01). However, the tumor/normal tissue drug ratio was lower for free than for liposomal doxorubicin at a drug dose of 100 microg (0.27 +/- 0.1 vs 0.53 +/- 0.5; p = 0.225) and similar for both doxorubicin formulations at a drug dose of 400 microg (0.67 +/- 0.2 vs 0.54 +/- 0.2; p = 0.335). Both doxorubicin formulations resulted in fluorescence signaling emerging from all tissue compartments of normal lung parenchyma but only in weak and sporadic signaling from the tumors confined to the tumor periphery and vessels situated within the tumor for both drug doses assessed. CONCLUSIONS: Isolated lung perfusion with free and liposomal doxorubicin resulted in a heterogeneous drug distribution within the perfused lung and in a lower drug uptake in tumors than in lung tissue for both doxorubicin formulations and drug doses applied.

Involvement of microRNAs in the cytotoxic effects exerted by proinflammatory cytokines on pancreatic beta-cells.

OBJECTIVE: Pancreatic beta-cells exposed to proinflammatory cytokines display alterations in gene expression resulting in defective insulin secretion and apoptosis. MicroRNAs are small noncoding RNAs emerging as key regulators of gene expression. Here, we evaluated the contribution of microRNAs to cytokine-mediated beta-cell cytotoxicity. RESEARCH DESIGN AND METHODS: We used global microarray profiling and real-time PCR analysis to detect changes in microRNA expression in beta-cells exposed to cytokines and in islets of pre-diabetic NOD mice. We assessed the involvement of the microRNAs affected in cytokine-mediated beta-cell failure by modifying their expression in insulin-secreting MIN6 cells. RESULTS: We found that IL-1beta and TNF-alpha induce the expression of miR-21, miR-34a, and miR-146a both in MIN6 cells and human pancreatic islets. We further show an increase of these microRNAs in islets of NOD mice during development of pre-diabetic insulitis. Blocking miR-21, miR-34a, or miR-146a function using antisense molecules did not restore insulin-promoter activity but prevented the reduction in glucose-induced insulin secretion observed upon IL-1beta exposure. Moreover, anti-miR-34a and anti-miR-146a treatment protected MIN6 cells from cytokine-triggered cell death. CONCLUSIONS: Our data identify miR-21, miR-34a, and miR-146a as novel players in beta-cell failure elicited in vitro and in vivo by proinflammatory cytokines, notably during the development of peri-insulitis that precedes overt diabetes in NOD mice.

Coexpression of the T-cell receptor constant alpha domain triggers tumor reactivity of single-chain TCR-transduced human T cells.

Transfer of tumor antigen-specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRalpha/beta chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRalpha constant alpha (Calpha) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Calpha domain preceded by a TCRalpha signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Calpha were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector zeta-chain-associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Calpha transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Combination of bevacizumab and 2-weekly pegylated liposomal doxorubicin as first-line therapy for locally recurrent or metastatic breast cancer. A multicenter, single-arm phase II trial (SAKK 24/06).

BACKGROUND: Pegylated liposomal doxorubicin (PLD) and bevacizumab are active agents in the treatment of metastatic breast cancer (MBC). We carried out a multicenter, single-arm phase II trial to evaluate the toxicity and efficacy of PLD and bevacizumab as first-line treatment in MBC patients. METHODS: Bevacizumab (10 mg/kg) and PLD (20 mg/m(2)) were infused on days 1 and 15 of a 4-week cycle for a maximum of six cycles. Thereafter, bevacizumab monotherapy was continued at the same dose until progression or toxicity. The primary objective was safety and tolerability, and the secondary objective was to evaluate efficacy of the combination. RESULTS: Thirty-nine of 43 patients were assessable for the primary end point. Eighteen of 39 patients (46%, 95% confidence interval 30% to 63%) had a grade 3 toxicity. Sixteen (41%) had grade 3 palmar-plantar erythrodysesthesia, one had grade 3 mucositis, and one severe cardiotoxicity. Secondary end point of overall response rate among 43 assessable patients was 21%. CONCLUSIONS: In this nonrandomized single-arm trial, the combination of bimonthly PLD and bevacizumab in locally recurrent and MBC patients demonstrated higher than anticipated toxicity while exhibiting only modest activity. Based on these results, we would not consider this combination for further investigation in this setting.

Pre- and postsynaptic effects of SKF64139, a phenylethanolamine N-methyltransferase inhibitor, in conscious normotensive rats

We investigated in conscious normotensive rats the effect of SKF64139 (2 mg i.v.), a potent phenylethanolamine N-methyltransferase (PNMT) inhibitor, on blood pressure responses to norepinephrine (40, 80, and 160 ng i.v.); methoxamine (2.5, 5 and 10 micrograms i.v.), a directly active sympathomimetic agent that is not taken up by adrenergic nerves; and tyramine (20, 40, and 80 micrograms i.v.), an indirectly acting sympathomimetic amine. The pressor effect of norepinephrine was not changed by 2 mg of SKF64139, while those of methoxamine and tyramine were significantly reduced. The dose-response curve to exogenous norepinephrine was also evaluated following blockade of norepinephrine uptake in the nerve endings using 0.25 mg desipramine i.v. This dose of desipramine had no effect on blood pressure increase induced by methoxamine. In rats pretreated with the neuronal uptake inhibitor desipramine in a dose that did not affect alpha-adrenoceptors, SKF64139 significantly decreased the pressor responses to norepinephrine. Increasing the dose of SKF64139 to 8 mg i.v. resulted in a significant fall in base-line blood pressure and in a blunted blood pressure response to norepinephrine. These data demonstrate that in vivo the PNMT inhibitor SKF64139 blocks alpha-adrenoceptors and inhibits neuronal uptake. The alpha-adrenoceptor blocking properties of SKF65139 are masked by simultaneous blockade of norepinephrine uptake when agonists with affinity for the uptake system are used. These findings need to be taken into account when interpreting cardiovascular effects of the PNMT inhibitor SKF64139.

Different antigen-presenting cells differ in their capacity to induce lymphokine production and proliferation of an apo-cytochrome c-specific T cell clone.

The activation of an apo-cytochrome c-specific T cell clone was found to differ, depending on the antigen-presenting cell population. Whereas total syngeneic spleen cells and bone marrow macrophages could be shown to trigger proliferation, IL 2, and MAF production by the T cell clone, a B cell lymphoma only induced MAF secretion. Further studies demonstrated that this effect was not due to a different antigen processing by the B lymphoma or to limiting amounts of Ia and antigen molecules on the B lymphoma cell surface. The dissociation of induction of MAF production from IL-2 production/proliferation found with the different antigen-presenting cells indicates strongly that molecules other than Ia and antigen may be required for the complete functional activation of antigen-specific T cell clones.

An EORTC Phase II study of caspofungin as first-line therapy of invasive aspergillosis in haematological patients.

OBJECTIVES: Caspofungin was evaluated as first-line monotherapy of invasive aspergillosis (IA) in patients with haematological malignancies and undergoing autologous transplants. METHODS: Adults with proven or probable IA, defined strictly according to EORTC-MSG criteria, were eligible. Those with possible IA were enrolled, but were not evaluable for efficacy unless upgraded to proven/probable disease within 7 days of registration based on investigations performed within 48 h after enrolment. Caspofungin dosage was 70 mg (day 1) followed by 50 mg/day. The primary endpoint was the proportion of patients with complete or partial response at the end of caspofungin therapy in the modified intention to treat (MITT) group; secondary endpoints were response and survival at day 84 and safety. RESULTS: In the MITT group (n = 61), 75% of patients had cancer not in remission (relapsing or refractory), 85% were neutropenic at enrolment and 49% had a Karnofsky score of < or =50. At end of treatment, 1 and 19 patients had complete and partial response, respectively [success rate 33% (20/61)], 9 (15%) achieved stabilization and 31 (51%) had disease progression. One patient was not evaluable. The 6 and 12 week survival rates were 66% (40/61) and 53% (32/60), respectively. Baseline characteristics associated with survival at day 84 were an underlying disease in remission (not relapsing or refractory) and Karnofsky score. Recovery from neutropenia at the end of treatment was also significantly associated with survival. No serious drug-related adverse events or discontinuations due to drug-related adverse events were observed. CONCLUSIONS: Caspofungin provided an observed response rate compatible with the null hypothesis of a true response rate of < or =35%. Underlying disease-related factors had a major impact on results.