During central nervous system (CNS) development, glial precursors proliferate in subventricular zones and then migrate throughout the CNS to adopt their final destinations and differentiate into various types of mature glial cells. Although several growth factors promoting the proliferation and/or differentiation of glial precursors have been identified, very little is known about the nature of signals that guide glial cell migration in the CNS. Therefore, we have investigated whether polypeptide growth factors and/or extracellular matrix molecules may mediate the migration of two major glial cell types, type 1 astrocytes and oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells. We show that, in a microchemotaxis chamber assay, type 1 astrocytes move toward laminin and complement-derived C5a. Astrocyte migration toward laminin is inhibited by a laminin-specific pentapeptide, YIGSR-NH2. In contrast, O-2A progenitors migrate toward platelet-derived growth factor (PDGF), which also functions as a mitogen for these cells. Using a new method to simultaneously assay migration and DNA synthesis, we also demonstrate that O-2A progenitors can migrate toward PDGF even when DNA replication is inhibited with an antimitotic agent. Thus, migration of different types of glial cells can be induced in vitro by specific signaling molecules, which are present in the developing brain and may stimulate migration of glial cells prior to CNS myelination.

A neutrophil chemotaxis assay was developed which permits rapid, quantitative assessment of migration across a membrane filter. The critical factor in the assay was the use of a 10 pm thick polycarbonate membrane without the usual polyvinylpyrrolidone coating. Migrated neutrophils remain adherent to the uncoated membrane, whereas 20--50% fall off polyvinylpyrrolidone-coated membranes. A major advantage of the method is that neutrophil chemotaxis can be readily quantified, since the migrated cells adhere to the membrane surface and are in one optical plane for counting. A 25 mm X 80 mm membrane sheet was used in a 48-well micro chemotaxis assembly, which requires only 20,000 neutrophils and 25 pl of attractant per assay well. Neutrophil chemotaxis was complete within 10--20 min at 37°C, with 20--30% of the cells migrating to N-formylmethionyl-leucyl-phenylalanine and 40--50% migrating to complement derived C5a.

Microglia demonstrate many characteristics similar to those seen in monocytes and tissue-specific macrophages, including phagocytosis, production of oxygen radicals, and growth factors and expression of MHC antigens. We have examined the ability of microglia, cultured from the cerebral cortices of neonatal rats, to demonstrate another important functional characteristic of monocytic-derived cells, that is, chemotaxis. Our results show that cultured rat microglia demonstrate chemotaxis to complement dependent chemoattractants such as recombinant C5a, zymosan activated serum, and to rat serum as well as to transforming growth factor-beta, a chemoattractant produced by platelets. Microglia fail to migrate to bacterial dependent chemoattractants such as the N-formyl peptides. The failure to respond is not dependent on maturational state of the microglia. Treatment with DMSO or casein, agents known to induce morphological and functional changes in cultured microglia reminescent of a "resting" and an "activated" macrophage, respectively, do not alter the response to fMet-Leu-Phe. In addition, the chemotactic response to serum in DMSO or casein-treated cells is the same as the response seen in untreated day 10 cultured microglia or untreated age-matched controls. The ability of microglia to migrate in response to inflammatory mediators suggests that these cells can move to sites of injury, thereby enabling them to participate in an inflammatory response.

T-cel migration into tissue depends on a cascade of rapid and selective adhesive Intactios with endothellum. "Triggering" is a step in that cascade required to activate T-cell integrins. Hepatocyte The recruitment of T lymphocytes into tissue is regulated by a cascade of molecular events resulting in adhesion to endothelium followed by migration into tissue (1, 2). Initially, selectin-mediated interactions cause T cells to roll along the vessel wall, where they contact factors that trigger strong binding to endothelium by activating T-cell integrins. Thereafter migration into tissue is directed by locally active promigratory factors (1). Although the selectin and integrin components are relatively well defined, the factors responsible for triggering T-cell adhesion and directing migration into tissue are not. A recent model has proposed that a family of cytokines, the chemokines, trigger adhesion and migration of leukocyte subsets when they are immobilized on the endothelial surface by binding to proteoglycan (3,4).Two aspects of the adhesion cascade model are poorly understood. First, there must be diversity in the triggering step to account for the specificity of leukocyte subset recruitment (1) and it is unclear whether chemokines alone can provide this. Second, if they are to be relevant physiologically, trigger factors must activate integrins within seconds of interacting with the leukocyte (5,6). Such rapid effects of pro-adhesive cytokines on T cells have not been demonstrated. Here we report that a structurally distinct cytokine, hepatocyte growth factor (HGF), can induce adhesion and migration of T-cell subsets and that HGF and the chemokine macrophage inflammatory protein 113 (MEP-118) can induce cytoskeletal changes within seconds of T-cell exposure to them.HGF (also known as scatter factor) is a heparin-binding growth factor with structural homology to plasminogen that causes epithelial cells to proliferate, differentiate, and scatter by activating the tyrosine kinase receptor c-Met (7-10). Although HGF has not previously been shown to affect T lymphocytes, several findings suggest a potential role for HGF in leukocyte recruitment: (i) it is released by inflammatory cells (11,12), (ii) it can enhance both neutrophil (12) and B-lymphocyte (13) functions, (iii) it can induce motility in the J-111 monocyte cell line (14), and (iv) it is detected immunohistochemically on endothelium in tissue (15, 16). These properties of HGF led us to investigate its effect on T-cell migration and adhesion. METHODSIsolation of Resting Human T Cells. T cells and the following T-cell subsets, CD4+, CD8+, CD45RA-, and CD45RO-, were prepared by negative selection from peripheral blood of normal donors by using a mixture of monoclonal antibodies (mAbs) (17). Purity of the T cells was >95%.Migration Assays. Three assays were used. (i) Migration through protein-coated 5-pm-pore polycarbonate membranes was assessed with 48-well microchemotaxis chambers (4, 18). Membranes were coated by floating overnight at 40C on a s...

Patients with uraemia have abnormal platelet function that may be partially corrected by haemodialysis, cryoprecipitate or 1-desamino-8-D-arginine vasopressin (DDAVP). We studied the platelet von Willebrand factor receptor, glycoprotein Ib (GPIb), and plasma von Willebrand factor (vWF) in uraemic patients undergoing chronic haemodialysis. Using the slope of agglutination of formalin-fixed platelets as an index of response to ristocetin (with a constant amount of normal plasma as a source of vWF), we found the response of platelets from uraemic patients, both before (2.7 +/- 1.5, n = 40) and after dialysis (1.2 +/- 1.2, n = 40) to be significantly less than that for normal controls (14.1 +/- 10.2, n = 20; P less than 0.001). In addition, the agglutination response of platelets obtained after dialysis was less than that of platelets obtained before dialysis (P less than 0.001). Immunoblotting demonstrated decreased or absent staining of glycocalicin, a subunit of GPIb, in platelet lysates from 25 patients. All platelet samples with reduced glycocalicin also had decreased responses to ristocetin. Tritium-labelled platelets from seven patients showed decreased labelling of a protein with an electrophoretic mobility equivalent to that of GPIb (140,000 daltons). In addition, platelets with the lowest levels of surface GPIb, as demonstrated by flow cytometry, also had decreased ristocetin agglutination and decreased staining on immunoblot. Levels of von Willebrand factor antigen and ristocetin cofactor in plasma from 10 patients were generally within the normal range, although postdialysis levels tended to be higher than pre-dialysis levels. The pre- and post-dialysis plasma vWF multimeric patterns were normal.

In the present study inactivated human immunodeficiency virus type 1 (HIV-1) was conjugated to Brucella abortus and tested for immunogenicity in normal and anti-L3T4-treated BALB/c mice. HIV-BA was more immunogenic than uncoupled HIV in normal mice, since 6-fold less virus in HIV-BA preparations elicited higher titer responses than HIV-1 alone. Furthermore, the HIV-BA antibody response reached higher levels before the HIV-1 response. Immunoblot analysis showed that most of the HIV-1 antigens were recognized by antibodies induced by either HIV-1 or HIV-BA. Isotype analysis revealed that HIV-1 induced similar levels of IgG1 and IgG2a antibodies, whereas the IgG2a responses to HIV-BA were more pronounced than the IgG1 response. These different IgG subclass patterns suggest that conjugation of HIV-1 to BA changed the immunogenic nature of HIV-1. The requirement for helper T cells was examined by immunizing mice that were depleted of CD4+ T cells by in vivo anti-L3T4 treatment. Under these conditions the IgG responses to HIV-1 were completely eliminated. Although HIV-BA antibody responses were markedly reduced in anti-L3T4-treated mice, anti-HIV-1 antibodies, mainly of the IgG2a isotype, were produced. The antibodies generated by HIV-1 and HIV-BA immunization were also tested for their ability to inhibit syncytia formed by infecting CD4 + CEM cells with gp160 vaccinia. Sera from normal mice, immunized with either HIV-1 or HIV-BA were capable of inhibiting syncytia. In contrast, following anti-L3T4 treatment, only mice immunized with HIV-BA, but not HIV-1, produced antibodies capable of inhibiting syncytia.

IntroductionC5a is an 1 1,000-D fragment of the fifth component of complement (C5)

Of 693,000 volunteer blood donors in Washington, D.C., who were screened for infection with human immunodeficiency virus type 1 (HIV-1) from July 1985 through December 1988, 284 tested positive on both enzyme immunoassay and Western blot assay. To determine the clinical importance of confirmed positive test results in asymptomatic blood donors, we followed 156 donors with positive Western blot assays and 80 donors with positive enzyme immunoassays but negative or indeterminate Western blots at 6-month intervals for a mean of 28 months. As compared with Western blot-negative persons, those with positive Western blots were significantly more likely to be black, male, and first-time donors and to have a history of venereal disease, generalized lymphadenopathy on examination, CD4-cell counts lower than 0.4 x 10(9) per liter, IgG levels higher than 18 g per liter, and antibody to hepatitis B core antigen on initial evaluation. In 17 (11 percent) of the Western blot-positive donors, the disease progressed to Class IV (symptomatic disease), according to the Centers for Disease Control system. CD4 counts below 0.2 x 10(9) per liter, IgA levels above 4 g per liter, abnormal proliferative responses to tetanus toxoid, and positive viral cultures were the strongest predictors of disease progression. Among the 80 donors with repeatedly reactive assay results but either negative or indeterminate Western blot assays, there was no evidence of HIV exposure in their histories, physical examinations, or laboratory evaluations, and manifestations of HIV infection developed in none of them. We conclude that a small number of persons with HIV infection continue to donate blood, despite attempts to exclude them, but that donors who test positive on enzyme immunoassay but persistently negative or indeterminate on Western blot assay probably do not represent a risk for the transmission of HIV.