Citations

ABSTRACT The abnormal accumulation of the amyloid β protein (Aβ) has been implicated as an early and critical event in the etiology and pathogenesis of Alzheimer's disease (AD). Compounds that reduce Aβ accumulation may therefore be useful therapeutically. In cell‐based screens we detected a significant reduction in Aβ concentration after treatment with the phosphatidylinositol kinase inhibitors wortmannin and LY294002. To determine the effect of this class of compounds on in vivo Aβ accumulation, we administered wortmannin to the Tg2576 mouse model of AD. Oral administration of wortmannin over four months resulted in a significant, non‐overlapping 40%–50% reduction in the number of senile plaques, one of the pathological hallmarks of AD. Sandwich ELISA analysis of formic acid extractable Aβ in the brain of treated animals indicates that both Aβ40 and the longer, more amyloidogenic form of the peptide, Aβ42, were significantly reduced. These data provide the first direct evidence that compounds identified by their ability to reduce Aβ concentration in vitro can reduce Aβ accumulation and deposition in the brain, thus establishing a basic paradigm for the identification and evaluation of additional compounds that lower Aβ accumulation.

The Rac1 guanine nucleotide exchange factor, Vav, is activated in hematopoietic cells in response to a large variety of stimuli. The downstream signaling events derived from Vav have been primarily characterized as leading to transcription or transformation. However, we report here that Vav and Rac1 in natural killer (NK) cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing. In addition, overexpression of Vav, but not of a mutant lacking exchange factor activity, enhances both forms of killing by NK cells. Furthermore, dominant-negative Rac1 inhibits the development of NK cell–mediated cytotoxicity by two mechanisms: (a) conjugate formation between NK cells and target cells is decreased; and (b) those NK cells that do form conjugates have decreased ability to polarize their granules toward the target cell. Therefore, our results suggest that in addition to participating in the regulation of transcription, Vav and Rac1 are pivotal regulators of adhesion, granule exocytosis, and cellular cytotoxicity.

Natural killer (NK) cells are named based on their natural cytotoxic activity against a variety of target cells. However, the mechanisms by which sensitive targets activate killing have been difficult to study due to the lack of a prototypic NK cell triggering receptor. Pharmacologic evidence has implicated protein tyrosine kinases (PTKs) in natural killing; however, Lck-deficient, Fyn-deficient, and ZAP-70–deficient mice do not exhibit defects in natural killing despite demonstrable defects in T cell function. This discrepancy implies the involvement of other tyrosine kinases. Here, using combined biochemical, pharmacologic, and genetic approaches, we demonstrate a central role for the PTK Syk in natural cytotoxicity. Biochemical analyses indicate that Syk is tyrosine phosphorylated after stimulation with a panel of NK-sensitive target cells. Pharmacologic exposure to piceatannol, a known Syk family kinase inhibitor, inhibits natural cytotoxicity. In addition, gene transfer of dominant-negative forms of Syk to NK cells inhibits natural cytotoxicity. Furthermore, sensitive targets that are rendered NK-resistant by major histocompatibility complex (MHC) class I transfection no longer activate Syk. These data suggest that Syk activation is an early and requisite signaling event in the development of natural cytotoxicity directed against a variety of cellular targets.

The major histocompatibility complex class II genes play an important role in the genetic predisposition to many autoimmune diseases. In the case of rheumatoid arthritis (RA), the human leukocyte antigen (HLA)-DRB1 locus has been implicated in the disease predisposition. The "shared epitope" hypothesis predicts that similar motifs within the third hypervariable (HV3) regions of some HLA-DRB1 alleles are responsible for the class II-associated predisposition to RA. Using a line of transgenic mice expressing the DQB1*0302/DQA1*0301 (DQ8) genes in the absence of endogenous mouse class II molecules, we have analyzed the antigenicity of peptides covering the HV3 regions of RA-associated and nonassociated DRB1 molecules. Our results show that a correlation exists between proliferative response to peptides derived from the HV3 regions of DRB1 chains and nonassociation of the corresponding alleles with RA predisposition. While HV3 peptides derived from nonassociated DRB1 molecules are highly immunogenic in DQ8 transgenic mice, all the HV3 peptides derived from RA-associated DRB1 alleles fail to induce a DQ8-restricted T-cell response. These data suggest that the role of the "shared epitope" in RA predisposition may be through the shaping of the T-cell repertoire.

Cellular immunity is mediated by the interaction of an αβ T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have αβ TCRs that can recognize both self- and foreign peptide–MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive αβ T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired αβ heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2K b -dEV8, H-2K bm3 -dEV8, H-2K b -SIYR, and H-2L d -p2Ca spans a range of affinities from K d = 10 −4 to 10 −6 M for the syngeneic (H-2K b ) and allogeneic (H-2K bm3 , H-2L d ) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the α and β chains. X-ray quality crystals of complexes of 2C with H-2K b -dEV8, H-2K bm3 -dEV8 and H-2K b -SIYR have been grown.