Transport of human and mouse major histocompatibility complex class I glycoproteins has been examined in a transport deficient B-lymphoblastoid cell line X T-lymphoblastoid cell line (B-LCL X T-LCL) hybrid, 174 X CEM.T2 (T2). This cell line expresses no detectable endogenous HLA-B5 and reduced levels of HLA-A2 on its surface although these molecules are synthesized. In order to study this defect further, either HLA-Bw58 or HLA-B7 genomic clones were transfected into T2. Metabolic labeling and immune precipitation demonstrated biosynthesis of the Bw58 or B7 glycoprotein. However, like the endogenous HLA-B5 molecule, neither HLA-Bw58 nor HLA-B7 was expressed at the cell surface. The cloned genes were properly expressed on the surface of C1R, a control B-LCL. To determine if mouse class I alleles had the same transport requirements as the human class I glycoproteins, either mouse H-2Dp or H-2Kb class I genes were introduced into T2. Surprisingly, the H-2 class I glycoproteins were transported to the cell surface normally. These data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport.
Particular subsets of T cells expressing the NK1.1 antigen have been proposed to play an immune regulatory role by their fast and strong production of cytokines, in particular IL-4. We sought to determine factors driving the functional differentiation of NK1.1+ T cells. Since NK1.1+ T cells are exquisitely sensitive to IL-7 stimulation, we analyzed the development, selection and IL-4 production of NK1.1+ T cells in IL-7-deficient mice (IL-7-/-m mice). Besides a sharp reduction of all T cell subsets, NK1.1+ T cells develop at normal relative frequencies in IL-7-/- mice. They also undergo a normal selection process, as revealed by the biased V beta TCR repertoire identical to the one in IL-7+/+ mice. However, NK1.1+ T cells from IL-7-/- mice were found to be impaired in IL-4 and IFN-gamma production in in vitro and in vivo models. In addition, IL-7 was able to restore IL-4 production by NK1.1+ thymocytes from IL-7-/- mice. Finally, IL-7 but not IL-2 or IL-4 was able to maintain and increase IL-4 production by NK1.1+ thymocytes from normal mice. These data suggest that the functional maturation of NK1.1+ T cells requires a cytokine-driven differentiation process, in which IL-7 plays a major role.
The interactive association between T lymphocytes and their target cells is an important system of cell-cell interactions. Major histocompatibility complex class I molecules are the cell surface structures recognized by cytolytic T lymphocytes. To define the molecular structures recognized by cytotoxic T lymphocytes, we have saturated the 270-basepair al exon of the H-2DP gene with point mutations, rapidly producing a "library" of 2.5 x 103 independent mutants. The library contains enough recombinant clones (each clone encoding approximately one amino acid replacement mutation) to predict a mutation at each nucleotide position of the al exon. The functional analysis of the first five transfected gene products tested has shown that mutation of a conserved tyrosine at position 27 to asparagine destroys recognition of the H-2DP gene product by polyclonal alloreactive cytotoxic T lymphocytes. Recognition of the same mutant molecule by three monoclonal antibodies and H-2-restricted lymphocytic choriomenengitis virus-specific cytotoxic T lymphocytes is unaffected.Mutagenesis has been a useful tool for understanding both gene function and regulation. Some biological systems have allowed the production and selection of a large number of point mutations that approaches saturation of a gene. Classic examples are the definition of the bacteriophage T4 rII region (1) and the estimation of the white locus gene size of Drosophila (2). The generation of a similar spectrum of mutants in mammalian genes, however, is severely limited by diploidy, genome complexity, and lack of suitable selection schemes. Automated DNA synthesis has allowed the rapid in vitro generation of point mutant libraries that contain all the possible single-base substitutions of a small defined "target" sequence (3, 4). We have applied this strategy to the much larger coding sequence target of a major histocompatibility complex (MHC) class I gene for a detailed structure-function analysis. The application of other conventional mutagenesis approaches requires stringent selection to find mutations that occur at a rate of 1O-4 to 10-6. Our synthetic approach ensures that mutations are recovered at a rate of at least 25% with a controllable frequency per clone. This changes the major obstacle in mutagenesis experiments from the production of mutants to the detailed characterization of systems with mutant phenotypes. We believe the spectrum of mutants created by saturation techniques allows for sampling mutations that would not have been easily recovered by other conventional mutagenesis procedures.Cytotoxic Assembly of Mutant Oligonucleotides into a1 Restriction Fragments. Oligonucleotides that had a 5' internal end, in relation to the final double-stranded structure, were phosphorylated. All 12 oligonucleotides were mixed in equimolar amounts in an Eppendorf tube and placed in 1 liter of boiling water. The water was allowed to cool to room temperature, during which time the oligonucleotides annealed to form Abbreviations: MHC, major histocompatibility...
Background: Breast cancer is the most common malignancy to affect women of reproductive age. As more women are delaying fertility into the 4th and 5th decade of life, more women face the possibility of a breast cancer diagnosis prior to accomplishing their reproductive goals. Methods: Literature review PubMed, EMBASE. Conclusions: From expectant management, hormonal suppression, in vitro fertilization with embryo freezing, to egg and ovarian tissue cryopreservation, numerous fertility sparing options are available to women with breast cancer. Therapy will be individualized based on a patient's age, financial resources, availability of services, cancer prognosis, ER/PR receptor, HER2 and BRCA status.
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