Normal and tumor-bearing host macrophage responses: variability in accessory function, surface markers, and cell-cycle kinetics.

Normal and tumor-bearing host (TBH) peritoneal macrophage (M phi) responses to in vitro lipopolysaccharide (LPS) treatment were measured by assessing functional and phenotypic changes. Both normal and TBH untreated M phi suppressed mixed lymphocyte reaction (MLR) reactivity at all concentrations. Normal host M phi treated with LPS for 3 h were suppressive at all concentrations. TBH M phi treated with LPS for 3 h were not suppressive in the MLR until more than 5% were added. Surprisingly, 24 h treatment of normal and TBH M phi with LPS induced cells that significantly enhanced MLR reactivity when added at 2% or 5%. These cells were not suppressive until a 20% M phi concentration was reached. LPS treatment of normal and TBH M phi changed the percentage of cells expressing the surface markers Mac-1, -2, -3, and Ia as determined by flow cytometry. Normal host peritoneal M phi treated with LPS for 3 h had decreased Mac-1 and -3 expression, but there was no change in Mac-2 or Ia. Plating for 24 h did not change the percentage of M phi expressing Mac-1, -3, or Ia but did cause an increase in Mac-2+ M phi. Treatment of normal host M phi with LPS for 24 h led to a decrease in Mac-1+ and Ia+ M phi, no change in Mac-3+ M phi, but an increase in Mac-2+ M phi. LPS treatment of TBH M phi for 3 h decreased the number of Mac-1+ M phi, but Mac-2+, -3+, or Ia+ M phi numbers did not change. Plating TBH M phi for 24 h caused a decrease in the number of Mac-1+ M phi, no change in Mac-3+ or Ia+ M phi, but an increase in Mac-2+ M phi. Treatment with LPS for 24 h led to no change in the number of Mac-1+, -3+, or Ia+ TBH M phi, but Mac-2+ M phi increased. The phenotypic and functional changes after LPS treatment led us to ask if these changes were detectable at the level of DNA and RNA. Flow cytometric analysis of acridine orange-stained M phi was used to measure DNA and RNA levels. This analysis determines M phi cell-cycle kinetics and estimates their RNA synthesis. In normal host M phi, a 3-h LPS treatment caused a decrease of cells in G0/G1 but an insignificant change in RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD4+ helper T cell depression in autism

CD4+ (helper) T cells are a heterogenous population of lymphocytes including at least two distinct subpopulations. To investigate the possibility that immune abnormalities in some subjects with autism may involve abnormal distributions of CD4+ and/or CD8+ cells, (suppressor) T cells, peripheral blood lymphocytes of 25 autistic subjects were characterized with monoclonal antibodies and flow cytometry. The autistic subjects had a significantly lower percentage and number of CD4+ cells, a lower number of T cells (CD2+ cells) and B cells (CD20+ cells), and a lower percentage and number of total lymphocytes than siblings and normal subjects. The level of blood values for female subjects appeared lower than those for males as compared to normal subjects of the same sex. These results suggest that a decrease in CD4+ cells is associated with autism.     

Molecular interactions of Leishmania promastigote surface protease with human alpha 2-macroglobulin

The interaction of Leishmania promastigote surface protease (PSP) with the plasmatic protease inhibitor alpha 2-macroglobulin (alpha 2M) was investigated. In plasma, solubilized PSP forms covalent complexes only with alpha 2M, at the exclusion of other protease inhibitors. The formation of complexes is accompanied by the proteolytic cleavage of the alpha 2M subunit and by the transition from the 'slow' to the 'fast' form of alpha 2M. The proteolytic activity of solubilized PSP on azocasein is inhibited by alpha 2M. In contrast, we found no evidence for a specific interaction of alpha 2M with the surface of promastigotes and PSP proteolytic activity on intact cells was not inhibited by alpha 2M.     

PMN chemotactic factor produced by glass-adherent cells in the acute inflammation caused by Paracoccidioides brasiliensis

Intraperitoneal inoculation of BIO.A mice with P. brasiliensis induces an acute inflammatory infiltrate in which 40-50% of the cells are PMN leucocytes. Previous depletion of serotonin, prostaglandin, histamine and complement does not alter the course of inflammation. Complement-derived factors appear to have no active participation in the process since C5-deficient mice depleted or not by Cobra venom factor (CoF) show the same kind of cellular influx. On the other hand, peritoneal cells incubated (6 h) with the fungus release a soluble factor that induces in vivo an active chemotaxis of PMN cells when inoculated i.p. The factor has the following characteristics: a) it is produced by adherent cells; b) it is protein in nature; c) its production is inhibited by incubation of peritoneal cells with 10 micrograms/ml puromycin and d) it has a molecular weight less than 15 000 daltons, as determined by gel filtration through a Sephadex G-75 column.     

Susceptibility and resistance of inbred mice to Paracoccidioides brasiliensis.

Nine different inbred strains of mice inoculated intraperitoneally with yeast cells of Paracoccidioides brasiliensis showed significantly varying patterns of susceptibility. The A/SN strain was found to be the most resistant, while BIOD2/nSn, BIO.A and BIOD2/oSn the most susceptible strains. These susceptibility differences were not dependent on the size of challenge inocula and sex of animals. All strains studied showed a mean survival time proportional to the size of inocula used. Although almost all infected male mice presented a shorter survival time when compared with females, significant mortality differences between sexes were found only in two of the strains studied, namely BALB/c and BIOD2/nSn. The H-2 region did not influence the susceptibility pattern since the A/SN and BIO.A strains share the same H-2 haplotype and were respectively highly resistant and susceptible to P. brasiliensis. Furthermore, the presence of C5 and unresponsiveness to lipopolysaccharide had no influence on the mortality data observed. Specific antibodies were detected only in a small number of animals and titres were consistently low, appearing later in the resistant (A/SN) than in a susceptible strain (BIO.A). Omentum, spleen and liver were the most affected organs in both strains, but the susceptible mice had more granulomatous lesions and earlier dissemination of the fungus.     

Autoradiographic study of corneal neovascularization induced by chemical cautery

The corneas of adult rats were cauterized chemically, and the responses of the pericorneal blood vessels and the cellular constituents of the cornea were followed by light microscopic autoradiography after labeling with 3H-thymidine. As in previous experiments, this injury elicited a neovascularization as capillaries sprouted and extended centripetally from the corneoscleral limbus to the cautery site. Chemical cautery induced a response in the epithelium, endothelium, and fibroblasts of the cornea as well as in the vascular cells. Elevated labeling indices for the corneal epithelium and endothelium began at 18 and 21 hours after injury, respectively. In all of these corneal cell types, the labeling index returned to control values by 75 hours. The onset and decline of DNA synthesis in corneal fibroblasts paralleled that of the corneal epithelium and endothelium. Labeling indices of vascular cells (endothelial cells and pericytes) increased 21 hours after injury, reached a maximal level at 45 hours, and returned to control values by 1 month after cautery. The first mitoses in vascular endothelial cells and pericytes were noted 36 hours after injury, and the initial capillary sprouts appeared at 39 hours. This study demonstrates that the thymidine incorporation by cells in the pericorneal blood vessels occurs early within the postcauterization period, at least 15 hours before the first mitotic figures are detected in these same vascular cells. The significance of the temporally related elevations in labeling indices of the vascular cells and the cellular constituents of the corneal cells is uncertain, but there are many potential interrelationships between the controls of cell division and migration for cells of the vessels, epithelium, endothelium, and corneal stroma.     

Insoluble polyanions as activators of both pathways of complement

Soluble polyanions, e.g. dextran sulphate, are known to interact with components of the classical pathway of complement and also to activate the alternative pathway (APC).† Insoluble polyanions offer the opportunity to isolate and to characterize intermediates of the reaction sequence. Sephadex, an insoluble, crosslinked dextran, was substituted with sulphate groups using chlorosulphonic acid. The sulphated Sephadex (SS) activated the APC in normal and in C4-deficient-guinea-pig serum as shown by haemolytic and immunochemical methods. After incubation of SS with normal guinea-pig serum, a C3-cleaving enzyme bound to the SS particles was present. This enzyme was inhibited by antisera against the components C2 and C4. Anti-serum against factor B or anti-C3-Fab had no inhibitory effect. Incubation at 37° inactivated the enzyme; activity was restored by incubation with C2, but not with factors B and D of the APC. These results suggest the presence of the C[unk]42-enzyme bound to the SS particles after incubation with normal serum. However, preincubation of SS with C4 deficient serum did not yield an enzyme which could act on purified C3, but enzymatic activity cleaving C4 and C2 was present, indicating that binding and activation of C1 had occurred. Utilizing purified C[unk]1, it was shown that SS binds purified C[unk]1 in a functionally active state. These data indicate that the polyanion SS has a dual function: SS activates both the APC and the classical sequence. Thus, the chemically simple, ester-linked, anionic sulphate groups, distributed along crosslinked polysaccharide chains, are sufficient to be recognized as initiating signal for both pathways of complement.     

The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Cell adhesion-related proteins as specific markers of sponge cell types involved in allogeneic recognition

Sponge immunocyte identification is of interest to comparative immunologists since characterizing these cells will allow investigations into the mechanisms of non-self recognition in the oldest animal phylum. Here, we report that polyclonal antibodies raised against the core protein of a proteoglycan involved in cell adhesion in the marine sponge Microciona prolifera are specific markers for archaeocytes, the totipotent sponge cells. Archaeocytes are mobilized upon allogeneic contact and they accumulate in the contact zone. A second type of cell, the gray cells, are specifically recognized by monoclonal antibodies raised against CD44, a hyaluronan receptor. Gray cells do also accumulate in the contact area. Specific staining of a third sponge cell type, the rhabdiferous cells, shows that these do not accumulate upon allografting. These specific cell markers allow tracking of archaeocytes and gray cells, and show that they play an active role in sponge allogeneic reactions.