ABSTRACT
Genetic engineering of T cells for adoptive immunotherapy in cancer patients has shown significant promise. To ensure optimal antitumor activity and safety, the simultaneous expression of multiple genes is frequently required, and short viral-derived 2A sequences are increasingly preferred for this purpose. Concerns exist, however, that these virus-derived sequences may induce unwanted immune responses, and thus diminish persistence of the gene-modified cells after adoptive transfer. Whereas such responses were absent in immunocompromised recipients, potential immunogenicity in immunocompetent individuals remains a concern. We now address whether ex vivo T cell responses can be elicited against the most widely used 2A sequences (2A-Thosea asigna virus (TAV) or 2A-equine rhinitis virus (ERAV), specifically) in immunocompetent individuals. We used a potent ex vivo culture system previously validated to induce T cell responses even against weakly immunogenic antigens. Of the sixteen donors tested, only five released very low levels of interferon-γ in response to 2A-TAV peptide mixtures (single peptide specificity in three donors, adjacent self-antigen peptide specificity in one donor and nonspecific reactivity in one donor). None of them produced cytotoxic activity or responded to 2A-ERAV. These results suggest that exposure to viral-derived 2A sequences is unlikely to produce unwanted T cell responses in immunocompetent individuals and further supports their continued use for studies of human gene therapy.
Subject(s)
Aphthovirus/immunology , Peptides/immunology , RNA Viruses/immunology , T-Lymphocytes/immunology , Amino Acid Sequence , Antigens/immunology , Aphthovirus/genetics , Cell Line , Genetic Vectors , Healthy Volunteers , Humans , Immunocompetence , Immunotherapy, Adoptive , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Lymphocyte Activation , Molecular Sequence Data , Peptides/chemistry , RNA Viruses/genetics , T-Lymphocytes/metabolismSubject(s)
Amylases/metabolism , Semen/enzymology , Amylases/blood , Humans , Male , Oligospermia/enzymology , VasectomyABSTRACT
Urinary excretion of fructose during three hours following the ingestion of 100 g of hydrolysed sucrose (in 200 ml) was studied in 85 normal men. This solution was better tolerated than a solution of 50 g of pure fructose and gave higher urinary excretion of fructose than with 100 g of unhydrolysed sucrose. The mean fructose excretion was 54.0 mg (S.D. 40.6 MG). The majority of subjects (63) excreted between 1 and 75 mg and the highest value was 187mg. This suggests a relatively inexpensive method for assessing the existence of porta-systemic shunts beyond normal.
