Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral supernatant

Abstract We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.http://deepblue.lib.umich.edu/bitstream/2027.42/109454/1/13075_2000_Article_409.pd

Phase I study of MLN8237—investigational Aurora A kinase inhibitor—in relapsed/refractory multiple myeloma, Non-Hodgkin lymphoma and chronic lymphocytic leukemia

Purpose Amplification or over-expression of the mitotic Aurora A kinase (AAK) has been reported in several heme-lymphatic malignancies. MLN8237 (alisertib) is a novel inhibitor of AAK that is being developed for the treatment of advanced malignancies. The objectives of this phase I study were to establish the safety, tolerability, and pharmacokinetic profiles of escalating doses of MLN8237 in patients with relapsed or refractory heme-lymphatic malignancies. Methods Sequential cohorts of patients received MLN8237 orally as either a powder-in-capsule (PIC) or enteric-coated tablet (ECT) formulation. Patients received MLN8237 PIC 25–90 mg for 14 or 21 consecutive days plus 14 or 7 days’ rest, respectively, or MLN8237 ECT, at a starting dose of 40 mg/day once-daily (QD) for 14 days plus 14 days’ rest, all in 28-day cycles. Subsequent cohorts received MLN8237 ECT 30–50 mg twice-daily (BID) for 7 days plus 14 days’ rest in 21-day cycles. Results Fifty-eight patients were enrolled (PIC n = 28, ECT n = 30). The most frequent grade ≥3 drug-related toxicities were neutropenia (45 %), thrombocytopenia (28 %), anemia (19 %), and leukopenia (19 %). The maximum tolerated dose on the ECT 7-day schedule was 50 mg BID. The terminal half-life of MLN8237 was approximately 19 h. Six (13 %) patients achieved partial responses and 13 (28 %) stable disease. Conclusion The recommended phase II dose of MLN8237 ECT is 50 mg BID for 7 days in 21-day cycles, which is currently being evaluated as a single agent in phase II/III trials in patients with peripheral T-cell lymphoma. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10637-013-0050-9) contains supplementary material, which is available to authorized users

Antigen-specific tumor vaccine efficacy in vivo against prostate cancer with low class I MHC requires competent class II MHC

BACKGROUND Cancers can escape immune recognition by means of evading class I major histocompatibility complex (MHC) -mediated recognition by cytotoxic T lymphocytes. However, immunization strategies targeting defined tumor-associated antigens have not been extensively characterized in murine prostate cancer models. Therefore, we evaluated antigen-specific, antitumor immunity after antigen-encoding vaccinia immunization against mouse prostate cancer cells expressing a model tumor-associated antigen (Β-galactosidase) and exhibiting partially deficient class I MHC. METHODS AND RESULTS Low class I MHC expression in Β-galactosidase–expressing D7RM-1 prostate cancer cells was shown by fluorescence activated cell sorting, and deficient class I MHC-mediated antigen presentation was shown in resistance of D7RM-1 to cytolysis by Β-galactosidase–specific cytotoxic T lymphocytes (CTL). Despite partially deficient class I MHC presenting function, immunization with vaccinia encoding Β-galactosidase conferred antigen-specific protection against D7RM-1 cancer. Antigen-specific immunity was recapitulated in Β 2 m knockout mice (with deficient class I MHC and CTL function), confirming that class I MHC antigen presentation was not required for immunity against tumor partially deficient in class I MHC. Conversely, antigen-specific antitumor immunity was abrogated in A b Β knockout mice (with deficient class II MHC and helper T cell function), demonstrating a requirement for functional class II MHC. Resistant tumors from the otherwise effectively immunized Β 2 m knockout mice (among which tumor progression had been reduced or delayed) showed reduced target antigen expression, corroborating antigen-specificity (and showing an alternative immune escape mechanism), whereas antigen expression (like tumor growth) was unaffected among A b Β knockout mice. CONCLUSION Our results demonstrate that class I MHC-restricted antigen presentation and CTL activity is neither necessary nor sufficient for antigen-encoding vaccinia immunization to induce protective immunity against class I MHC-low tumors, whereas host class II MHC-mediated antigen presentation facilitates antigen-specific immunity against prostate cancer in vivo. Reduced expression of the target antigen developed rapidly in vivo as an immune escape mechanism for such cancers. Prostate 53: 183–191, 2002. © 2002 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34762/1/10136_ftp.pd

HacA-Independent Functions of the ER Stress Sensor IreA Synergize with the Canonical UPR to Influence Virulence Traits in Aspergillus fumigatus

Endoplasmic reticulum (ER) stress is a condition in which the protein folding capacity of the ER becomes overwhelmed by an increased demand for secretion or by exposure to compounds that disrupt ER homeostasis. In yeast and other fungi, the accumulation of unfolded proteins is detected by the ER-transmembrane sensor IreA/Ire1, which responds by cleaving an intron from the downstream cytoplasmic mRNA HacA/Hac1, allowing for the translation of a transcription factor that coordinates a series of adaptive responses that are collectively known as the unfolded protein response (UPR). Here, we examined the contribution of IreA to growth and virulence in the human fungal pathogen Aspergillus fumigatus. Gene expression profiling revealed that A. fumigatus IreA signals predominantly through the canonical IreA-HacA pathway under conditions of severe ER stress. However, in the absence of ER stress IreA controls dual signaling circuits that are both HacA-dependent and HacA-independent. We found that a ΔireA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasts the partial virulence of a ΔhacA mutant, suggesting that IreA contributes to pathogenesis independently of HacA. In support of this conclusion, we found that the ΔireA mutant had more severe defects in the expression of multiple virulence-related traits relative to ΔhacA, including reduced thermotolerance, decreased nutritional versatility, impaired growth under hypoxia, altered cell wall and membrane composition, and increased susceptibility to azole antifungals. In addition, full or partial virulence could be restored to the ΔireA mutant by complementation with either the induced form of the hacA mRNA, hacAi, or an ireA deletion mutant that was incapable of processing the hacA mRNA, ireAΔ10. Together, these findings demonstrate that IreA has both HacA-dependent and HacA-independent functions that contribute to the expression of traits that are essential for virulence in A. fumigatus

A proteomic approach to investigating gene cluster expression and secondary metabolite functionality in Aspergillus fumigatus.

A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism

The influence of solid state information and descriptor selection on statistical models of temperature dependent aqueous solubility.

Predicting the equilibrium solubility of organic, crystalline materials at all relevant temperatures is crucial to the digital design of manufacturing unit operations in the chemical industries. The work reported in our current publication builds upon the limited number of recently published quantitative structure-property relationship studies which modelled the temperature dependence of aqueous solubility. One set of models was built to directly predict temperature dependent solubility, including for materials with no solubility data at any temperature. We propose that a modified cross-validation protocol is required to evaluate these models. Another set of models was built to predict the related enthalpy of solution term, which can be used to estimate solubility at one temperature based upon solubility data for the same material at another temperature. We investigated whether various kinds of solid state descriptors improved the models obtained with a variety of molecular descriptor combinations: lattice energies or 3D descriptors calculated from crystal structures or melting point data. We found that none of these greatly improved the best direct predictions of temperature dependent solubility or the related enthalpy of solution endpoint. This finding is surprising because the importance of the solid state contribution to both endpoints is clear. We suggest our findings may, in part, reflect limitations in the descriptors calculated from crystal structures and, more generally, the limited availability of polymorph specific data. We present curated temperature dependent solubility and enthalpy of solution datasets, integrated with molecular and crystal structures, for future investigations