Defects in dendritic spines are common to several forms of cognitive deficits, including mental retardation and Alzheimer disease. Because mutation of p21-activated kinase (PAK) can lead to mental retardation and because PAK-cofilin signaling is critical in dendritic spine morphogenesis and actin dynamics, we hypothesized that the PAK pathway is involved in synaptic and cognitive deficits in Alzheimer disease. Here, we show that PAK and its activity are markedly reduced in Alzheimer disease and that this is accompanied by reduced and redistributed phosphoPAK, prominent cofilin pathology and downstream loss of the spine actin-regulatory protein drebrin, which cofilin removes from actin. We found that beta-amyloid (Abeta) was directly involved in PAK signaling deficits and drebrin loss in Abeta oligomer-treated hippocampal neurons and in the Appswe transgenic mouse model bearing a double mutation leading to higher Abeta production. In addition, pharmacological PAK inhibition in adult mice was sufficient to cause similar cofilin pathology, drebrin loss and memory impairment, consistent with a potential causal role of PAK defects in cognitive deficits in Alzheimer disease.

Neutrophil migration inhibition factor from T lymphocytes (NIF-T) is a lymphokine that acts to localize granulocytes. Medium conditioned by the Mo human T-lymphoblast cell line was used to purify NIF-T, a glycoprotein with a molecular weight of 22,000. The NIF-T was found to potently stimulate the growth of granulocyte and macrophage colonies from human bone marrow and colony formation by the KG-1 myeloid leukemia cell line. Thus a human lymphokine (NIF-T) that modulates the activities of mature neutrophilic granulocytes is also a colony-stimulating factor acting on precursors to induce growth and differentiation of new effector cells.

The polymorphonuclear leukocyte (PMN), or neutrophil, is the major host defence cell protecting the body against invasion by bacteria and fungi. Products of oxidative metabolism mediate PMN microbicidal and tumoricidal activity but the mechanisms by which these pathways become activated are not well understood. We have previously described a human granulocyte-macrophage colony-stimulating factor (GM-CSF) of relative molecular mass (Mr) 22,000 that also inhibits neutrophil motility (NIF-T activity). Because of its direct action on granulocytes, this lymphokine is a candidate for a neutrophil-activating factor. We have studied the effect of GM-CSF/NIF-T on superoxide anion generation in response to the bacterial chemo-attractant N-formylmethionyl-leucylphenylalanine (f-MLP), and report here that PMNs preincubated with either purified natural GM-CSF or biosynthetic (recombinant) GM-CSF showed increased (as much as fourfold) superoxide anion production in response to f-MLP. These results indicate that human GM-CSF is a neutrophil-activating factor.

Systemic lupus erythematosus (SLE) is distinct among autoimmune diseases due to its association with circulating autoantibodies reactive against host DNA. The precise role that anti-DNA antibodies play in SLE pathophysiology remains to be elucidated, and potential applications of lupus autoantibodies in cancer therapy have not previously been explored. Here we report the unexpected finding that a cell-penetrating lupus autoantibody, 3E10, has potential as a targeted therapy for DNA-repair deficient malignancies. We find that 3E10 preferentially binds DNA single-strand tails, inhibits key steps in DNA single-strand and double-strand break repair, and sensitizes cultured tumor cells and human tumor xenografts to DNA-damaging therapy, including doxorubicin and radiation. Moreover, we demonstrate that 3E10 alone is synthetically lethal to BRCA2-deficient human cancer cells and selectively sensitizes such cells to low dose doxorubicin. Our results establish an approach to cancer therapy that we expect will be particularly applicable to BRCA2-related malignancies such as breast, ovarian, and prostate cancers. In addition, our findings raise the possibility that lupus autoantibodies may be partly responsible for the intrinsic deficiencies in DNA repair and the unexpectedly low rates of breast, ovarian, and prostate cancers observed in SLE patients. In summary, this study provides the basis for the potential use of a lupus anti-DNA antibody in cancer therapy and identifies lupus autoantibodies as a potentially rich source of therapeutic agents.

Defects in dendritic spines and synapses contribute to cognitive deficits in mental retardation syndromes and, potentially, Alzheimer disease. p21-activated kinases (PAKs) regulate actin filaments and morphogenesis of dendritic spines regulated by the Rho family GTPases Rac and Cdc42. We previously reported that active PAK was markedly reduced in Alzheimer disease cytosol, accompanied by downstream loss of the spine actinregulatory protein Drebrin. ␤-Amyloid (A␤) oligomer was implicated in PAK defects. Here we demonstrate that PAK is aberrantly activated and translocated from cytosol to membrane in Alzheimer disease brain and in 22-month-old Tg2576 transgenic mice with Alzheimer disease. This active PAK coimmunoprecipitated with the small GTPase Rac and both translocated to granules. A␤ 42 oligomer treatment of cultured hippocampal neurons induced similar effects, accompanied by reduction of dendrites that were protected by kinase-active but not kinase-dead PAK. A␤ 42 oligomer treatment also significantly reduced N-methyl-D-aspartic acid receptor subunit NR2B phosphotyrosine labeling. The Src family tyrosine kinase inhibitor PP2 significantly blocked the PAK/Rac translocation but not the loss of p-NR2B in A␤ 42 oligomer-treated neurons. Src family kinases are known to phosphorylate the Rac activator Tiam1, which has recently been shown to be A␤-responsive. In addition, anti-oligomer curcumin comparatively suppressed PAK translocation in aged Tg2576 transgenic mice with Alzheimer amyloid pathology and in A␤ 42 oligomer-treated cultured hippocampal neurons. Our results implicate aberrant PAK in A␤ oligomer-induced signaling and synaptic deficits in Alzheimer disease. Cognitive deficits in Alzheimer disease (AD)2 correlate with progressive synaptic dysfunction and loss that may be initiated by soluble ␤-amyloid peptide 1-42 (A␤ 42 ) and driven further by the accumulating neuropathological hallmarks, including intraneuronal neurofibrillary tangles, extracellular amyloid plaques, and neuron loss. Soluble A␤ or A␤ oligomers correlate highly with synapse loss (1, 2) and the degree of dementia (3). They also potently inhibit long term potentiation (LTP) in vivo (4). A␤-induced synaptic dysfunction likely contributes to cognitive deficits in several different AD transgenic mouse models (5-7).Both dystrophic neurites and dendritic spine loss are observed in AD and many mental retardation syndromes (8 -10). Dendritic spines, major sites of synaptic contacts, are structurally reliant on the actin cytoskeleton. p21-activated kinases (PAK) (11) are a family of serine/threonine protein kinases involved in regulating the actin-severing protein cofilin, the actin cytoskeleton, and dendritic function as downstream effectors of Rac1/Cdc42 (12). Thus, the small GTPases (Rho, Rac, and Cdc42) play critical roles in regulating dendrite initiation, growth, branching, spinogenesis, and spine maintenance (13-15). Mutations in PAK3 are associated with X-linked nonsyndromic forms of mental retardation, in which the only distinctive clinica...

Immunoglobulin A is the primary immunoglobulin isotype in tears, saliva, breast milk and other mucosal secretions, constituting between 6% and 15% of the total serum immunoglobulins. Human peripheral blood neutrophils have IgA receptors, but these cells do not normally participate in IgA-mediated phagocytosis. The haematopoietic factors granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) prime neutrophils to be more responsive to a variety of stimuli. We therefore studied their effect on IgA-mediated phagocytosis. GM-CSF and G-CSF both induce a change from low to high-affinity neutrophil IgA Fc crystallizable fragment receptors within 30 min; a change which is associated with the development of IgA-mediated phagocytosis. Human IL-3, which does not affect neutrophil function, is inactive in this system. These results define a new mechanism for CSF-augmented host defence whereby neutrophil function can be modulated by CSF-mediated IgA Fc receptor activation.

Purified natural and biosynthetic (recombinant) human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate colony formation by myeloid progenitor cells and enhance the function of mature neutrophils. Both of these actions occur at concentrations between 1 and 100 pM, with half-maximal stimulation at 10-20 pM. We have examined

Regulation of gene expression by intranuclear transduction of macromolecules such as transcription factors is an alternative to gene therapy for the treatment of numerous diseases. The identification of an effective intranuclear delivery vehicle and pathway for the transport of therapeutic macromolecules across plasma and nuclear membranes, however, has posed a significant challenge. The anti-DNA antibody fragment 3E10 Fv has received attention as a novel molecular delivery vehicle due to its penetration into living cells with specific nuclear localization, absence of toxicity, and successful delivery of therapeutic cargo proteins in vitro and in vivo. Elucidation of the pathway that allows 3E10 Fv to cross cell membranes is critical to the development of new molecular therapies. Here we show that 3E10 Fv penetrates cells through a nucleoside salvage transporter. 3E10 Fv is unable to penetrate into cells deficient in the equilibrative nucleoside transporter ENT2, and reconstitution of ENT2 into ENT2-deficient cells restores 3E10 Fv transport into cell nuclei. Our results represent the first demonstration of protein transport through a nucleoside salvage pathway. We expect that our finding will facilitate a variety of methods of gene regulation in the treatment of human diseases, open up new avenues of research in nucleoside salvage pathways, and enhance our understanding of the pathophysiology of autoimmune diseases.The ability to regulate gene expression through intranuclear delivery of macromolecules would significantly impact the treatment of a multitude of human diseases. Effective macromolecular therapy is dependent upon molecular delivery vehicles to circumvent the plasma membrane barrier and facilitate intracellular transport of cargo molecules. The single chain Fv fragment of the 3E10 anti-DNA autoantibody (3E10 Fv) has recently been harnessed as a novel molecular delivery vehicle due to its specific nuclear localization and apparent lack of toxicity (1). 3E10 Fv and Fv fusion proteins readily transduce across cell membranes and penetrate into cell nuclei, and 3E10 Fv has successfully delivered biologically active proteins such as Hsp70 (2) and p53 (3) into living cells in vitro. Moreover, 3E10 Fv mediated full-length p53 protein therapy in vivo (4). The pathway that carries 3E10 Fv across cell membranes and into cell nuclei, however, has not been identified.Previous studies implicated DNA binding as important in 3E10 Fv transduction into cell nuclei. Specifically, mutations that abrogate DNA binding by the antibody render it incapable of cellular penetration (5). The association between cellular penetration and DNA binding distinguished 3E10 Fv from other protein transduction domains and implied that nucleoside salvage pathways might be involved in 3E10 Fv transport. Both concentrative (CNT) 3 and equilibrative (ENT) nucleoside salvage transporters mediate the uptake of nucleobases and nucleosides by mammalian cells (6). Since any major role of CNTs in 3E10 Fv transport was excluded by previous studie...