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

Decomposing oceanic temperature and salinity change using ocean carbon change

As the planet warms due to the accumulation of anthropogenic CO2 in the atmosphere, the interaction of surface ocean carbonate chemistry and the radiative forcing of atmospheric CO2 leads to the global ocean sequestering heat and carbon in a ratio that is nearly constant in time. This ratio has been approximated as globally uniform, enabling the intimately linked patterns of ocean heat and carbon uptake to be derived. Patterns of ocean salinity also change as the Earth system warms due to hydrological cycle intensification and perturbations to air–sea freshwater fluxes. Local temperature and salinity change in the ocean may result from perturbed air–sea fluxes of heat and fresh water (excess temperature, salinity) or from reorganisation of the preindustrial temperature and salinity fields (redistributed temperature, salinity), which are largely due to circulation changes. Here, we present a novel method in which the redistribution of preindustrial carbon is diagnosed and the redistribution of temperature and salinity is estimated using only local spatial information. We demonstrate this technique in the NEMO ocean general circulation model (OGCM) coupled to the MEDUSA-2 biogeochemistry model under an RCP8.5 scenario over 1860–2099. The excess changes (difference between total and redistributed property changes) are thus calculated. We demonstrate that a global ratio between excess heat and temperature is largely appropriate regionally with key regional differences consistent with reduced efficiency in the transport of carbon through the mixed layer base at high latitudes. On centennial timescales, excess heat increases everywhere, with the North Atlantic being a key site of excess heat uptake over the 21st century, accounting for 25 % of the total. Excess salinity meanwhile increases in the Atlantic but is generally negative in other basins, consistent with increasing atmospheric transport of fresh water out of the Atlantic. In the North Atlantic, changes in the inventory of excess salinity are detectable in the late 19th century, whereas increases in the inventory of excess heat do not become significant until the early 21st century. This is consistent with previous studies which find salinification of the subtropical North Atlantic to be an early fingerprint of anthropogenic climate change. Over the full simulation, we also find the imprint of Atlantic meridional overturning circulation (AMOC) slowdown through significant redistribution of heat away from the North Atlantic and of salinity to the South Atlantic. Globally, temperature change at 2000 m is accounted for by both redistributed and excess heat, but for salinity the excess component accounts for the majority of changes at the surface and at depth. This indicates that the circulation variability contributes significantly less to changes in ocean salinity than to heat content. By the end of the simulation excess heat is the largest contribution to density change and steric sea level rise, while excess salinity greatly reduces spatial variability in steric sea level rise through density compensation of excess temperature patterns, particularly in the Atlantic. In the Atlantic, redistribution of the preindustrial heat and salinity fields also produces generally compensating changes in sea level, though this compensation is less clear elsewhere. The regional strength of excess heat and salinity signals grows through the model run in response to the evolving forcing. In addition, the regional strength of the redistributed temperature and salinity signals also grows, indicating increasing circulation variability or systematic circulation change on timescales of at least the model run

The effect of high intakes of casein and casein phosphopeptide on calcium absorption in the rat

peer-reviewedThe effect of the level or source of dietary protein or protein-derived peptides on Ca absorption is not well understood. We determined, therefore, the influence of habitual dietary casein level, meal casein and meal casein phosphopeptide (CPP) on Ca absorption in the rat. True fractional Ca absorption was investigated in male 7-week-old rats, Wistar strain, in three separate studies using a faecal 47Sc : 47Ca ratio method. In studies A and C, rats (n 8 per group) were fed on a purified diet containing 200 g casein/kg for 2 weeks. Rats were then given a 47Ca-labelled meal (10 g) containing (per kg) either 0, 100, 200, or 300 g casein (study A) or 0, 100, 200, 350 or 500 g CPP (study C). In study B, rats (n 24 per group) were fed on a purified diet containing (per kg) either 200, 350 or 500 g casein for 2 weeks. Each group was then further randomized into three groups (n 8 per group) and given a 47Ca-labelled meal (10 g of the same diet) containing (per kg) either 200, 350 or 500 g casein. Ca absorption from a meal was unaffected by increasing meal casein concentration from 0 to 300 g/kg (study A), but was increased with a meal casein content of 500 g/kg (study B). Fractional Ca absorption decreased with increasing usual dietary casein intake in the range 200±500 g/kg (study B), suggesting intestinal adaptation. Ca absorption was unaffected by inclusion of 100 g CPP/kg in a single meal but was significantly (P,0×001) reduced by 200, 350 and 500 g CPP/kg meal, with no evident dose-relationship. Thus, while Ca absorption was enhanced by high-casein meals, the mechanism remains unclear

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

Teagasc submission made in response to the Discussion document for the preparation of a National Policy Statement on the Bioeconomy

Teagasc SubmissionThis document is Teagasc’s response to the “Discussion Document for the Preparation of a National Policy Statement on the Bioeconomy” issued by the Department of the Taoiseach’s Economic Division in July 2017. It recognises the potential significance of the bioeconomy to Ireland, offers some policy and strategic insights from other countries, and identifies Teagasc’s role in supporting the development of the bioeconomy in Ireland