Partial Characterization of Animal Growth Inhibitors from Soybeans

The pH 4. 4 supernatant from raw (unheated) soybean meal was separated into 5 fractions on a Sephadex G-25 column in efforts to isolate the animal growth inhibitors present soybeans. Each fraction was added to the diet of growing mice and growth inhibition (GI) activity was determined by comparing their_ growth rates to g1 \u27owth rates achieved on an autoclaved soybean meal diet. Fractions I and II each contained about 50% of the GI activity, with a trace in fraction III and none in fractions IV and V. Only fraction I contained trypsin inhibitor and caused pancreatic enlargement. Fraction I was further separated into 2 fractions (Ia and Ib) on a Sephadex G-50 column. Fraction Ib contained trypsin inhibitor and caused pancreatic enlargement. Both fractions Ia and Ib contained GI activity, with most of the GI activity present in Ib. Fraction II was further separated into 2 fractions (IIa and IIb) on a phosphocellulose column eluted with a KCl gradient. Most of the GI activity was present in fraction IIb. Amino acid analysis of G-25 - II indicated all amino acids present, with the sulfur-containing amino acids, methionine and half cystine, present in only very small amount

How two business models respond to current chalenges of agrowood production: the case of Brandenburg, Germany

PosterThe agrowood acreage in Brandenburg has increased fifteen-fold since 2008 reaching in 2013 1819ha (Ministerium für Infrastruktur und Landwirtschaft 2013) and therefore the leading position of all federal states in Germany. Despite of the constant rise and the chances associated with the production of agrowood, potential producers have to face a wide range of challenges: uncertainties about yields, high initial investments, a fixation of land for the 20 years` lifetime of an agrowood plantation and an irregular cash flow all 3 or 5 years. A non-transparent market in addition to lacking long term experiences and machinery available, affects the decision-making process of potential producers negatively. In Brandenburg this innovative crop, growing even under harsh conditions, matches the unfavorable agricultural conditions (Murach et al. 2008). Our subjects of investigation are the two prevailing business models in the agrowood sector in Brandenburg: comprehensive cooperation agreements and independent farming. They coexist and mutually interact with synergies as well as obstructions. We combine a qualitative method with guided interviews focusing on planting decision making processes with a modeling approach using different risk levels and yield expectations to analyze transaction and opportunity costs of those two models. Thereby the characteristics and effects of the business models are compared and analyzed. Our results show that cooperation agreements have effects on the actor`s decision by motivating farmers to decide in favor of agrowood. They also contribute to an expansion of agrowood acreage accounting for almost 40% of the total area in Brandenburg in 2012 (Ehm 2013). Furthermore cooperation agreements are affirmed by interviewees to overcome economic, trade and machinery related constraints of agrowood, secure long-term incomes and increase creditworthiness of producers. In contrast, independent producers are acknowledged to have the burden of higher risks, but may benefit from governmental support programs, which not apply for contract farmers

Targeting human melanoma neoantigens by T cell receptor gene therapy

In successful cancer immunotherapy, T cell responses appear to be directed toward neoantigens created by somatic mutations; however, direct evidence that neoantigen-specific T cells cause regression of established cancer is lacking. Here, we generated T cells expressing a mutation-specific transgenic T cell receptor (TCR) to target different immunogenic mutations in cyclin-dependent kinase 4 (CDK4) that naturally occur in human melanoma. Two mutant CDK4 isoforms (R24C, R24L) similarly stimulated T cell responses in vitro and were analyzed as therapeutic targets for TCR gene therapy. In a syngeneic HLA-A2-transgenic mouse model of large established tumors, we found that both mutations differed dramatically as targets for TCR-modified T cells in vivo. While T cells expanded efficiently and produced IFN-γ in response to R24L, R24C failed to induce an effective antitumor response. Such differences in neoantigen quality might explain why cancer immunotherapy induces tumor regression in some individuals, while others do not respond, despite similar mutational load. We confirmed the validity of the in vivo model by showing that the melan-A-specific (MART-1-specific) TCR DMF5 induces rejection of tumors expressing analog, but not native, MART-1 epitopes. The described model allows identification of those neoantigens in human cancer that serve as suitable T cell targets and may help to predict clinical efficacy

Do CARs need a driver's license? Adoptive cell therapy with chimeric antigen receptor-redirected T cells caused serious adverse events

Adoptive transfer of genetically retargeted T cells provides promise in the therapy of malignant diseases; two severe adverse events, however, due to "on-targeted" activation of modified T cells occurred in recent trials. We here discuss the challenge to balance targeted anti-tumor responses whilst avoiding destruction of healthy tissues and the need of continued and carefully designed pre-clinical and clinical evaluations

Designer T cells - New possibilities for immunotherapy of cancer [Designer-T-Zellen - neue Möglichkeiten für die Immuntherapie von Krebs]

The genetic engineering of T cells with T cell or chimeric antigen receptors generates tumor-specific designer T cells for immunotherapy of cancer. The clinical use of these cells requires (1) careful selection of the target antigen that should be tumor-specific, (2) an optimized configuration of therapeutic genes, to generate T cells of high functional activity and (3) efficient vector systems that allow the generation of sufficient numbers of engineered T cells within a short period of time

NY-ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability

The cancer-testis antigen NY-ESO-1 has been used as a target for different immunotherapies like vaccinations and adoptive transfer of antigen-specific cytotoxic T cells, as it is expressed in various tumor types and has limited expression in normal cells. The in vitro generation of T cells with defined antigen specificity by T cell receptor (TCR) gene transfer is an established method to create cells for immunotherapy. However, an extensive characterization of TCR which are candidates for treatment of patients is crucial for successful therapies. The TCR has to be efficiently expressed, their affinity to the desired antigen should be high enough to recognize low amounts of endogenously processed peptides on tumor cells, and the TCR should not be cross-reactive to other antigens. We characterized three NY-ESO-1 antigen-reactive cytotoxic T lymphocyte (CTL) clones which were generated by different approaches of T cell priming (autologous, allogeneic), and transferred their TCR into donor T cells for more extensive evaluations. Although one TCR most efficiently bound MHC-multimers loaded with NY-ESO-1 peptide, T cells expressing this transgenic TCR were not able to recognize endogenously processed antigen. A second TCR recognized HLA-A2 independent of the bound peptide beside of its much stronger recognition of NY-ESO-1 bound to HLA-A2. A third TCR displayed an intermediate but peptide-specific performance in all functional assays and, therefore, is the most promising candidate TCR for further clinical development. Our data indicate that multiple parameters of TCR gene-modified T cells have to be evaluated to identify an optimal TCR candidate for adoptive therapy

TCR-engineered T cells: a model of inducible TCR expression to dissect the interrelationship between two TCRs

TCR gene-modified T cells for adoptive therapy simultaneously express the transgenic (tg) TCR and the endogenous TCR which might lead to mispaired TCRs with harmful unknown specificity and to a reduced function of TCR-tg T cells. We generated dual TCR T cells in two settings in which either TCR was constitutively expressed by a retroviral promoter while the second TCR expression was regulable by a tet-on system. Constitutively expressed TCR molecules were reduced on the cell surface depending on the induced TCR expression leading to strongly hampered function. Besides that, using fluorescence resonance energy transfer (FRET) we detected mispaired TCR dimers and different pairing behaviors of individual TCR chains with a mutual influence on TCR chain expression. The loss of function and mispairing could not be avoided by changing the TCR expression level or by introduction of an additional cysteine bridge. However, in polyclonal T cells, optimized TCR formats (cysteineization, codon optimization) enhanced correct pairing and function. We conclude from our data that (i) the level of mispairing depends on the individual TCRs and is not reduced by increasing the level of one TCR, and (ii) modifications (cysteineization, codon optimization) improve correct pairing but do not completely exclude mispairing (cysteineization)

TCR-engineered T cells: a model of inducible TCR expression to dissect the interrelationship between two TCRs

TCR gene-modified T cells for adoptive therapy simultaneously express the transgenic (tg) TCR and the endogenous TCR which might lead to mispaired TCRs with harmful unknown specificity and to a reduced function of TCR-tg T cells. We generated dual TCR T cells in two settings in which either TCR was constitutively expressed by a retroviral promoter while the second TCR expression was regulable by a tet-on system. Constitutively expressed TCR molecules were reduced on the cell surface depending on the induced TCR expression leading to strongly hampered function. Besides that, using fluorescence resonance energy transfer (FRET) we detected mispaired TCR dimers and different pairing behaviors of individual TCR chains with a mutual influence on TCR chain expression. The loss of function and mispairing could not be avoided by changing the TCR expression level or by introduction of an additional cysteine bridge. However, in polyclonal T cells, optimized TCR formats (cysteineization, codon optimization) enhanced correct pairing and function. We conclude from our data that (i) the level of mispairing depends on the individual TCRs and is not reduced by increasing the level of one TCR, and (ii) modifications (cysteineization, codon optimization) improve correct pairing but do not completely exclude mispairing (cysteineization)

an In Vitro Study

The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP) was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL) could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP); RegenLab (PRP-BCT)), platelet concentrate (apheresis, PC), and PL (freezing-thawing destruction of PC). Additionally, ten commercial allogenic PLs (AlloPL) from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP- BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1β, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration