Magnus Gidlund

Biologically modified LDL increases the adhesive properties of endothelial cells.

Adhesion of monocytes to the arterial endothelium is an important early event in atherosclerosis. Several lines of evidence have suggested that oxidation of low density lipoprotein (LDL) in the arterial wall may initiate the inflammatory-like process that generally is present in atherosclerotic lesions. In vitro, oxidation of LDL can be obtained both by exposure to divalent ions, such as Cu2+, or by incubation with different cell types, including monocytes and endothelial cells. The present study was designed to investigate the possible influence of oxidized LDL on the adhesive properties of endothelial cells. We report here that Cu(2+)-oxidized LDL is as effective as interleukin 1 beta in stimulating the ability of cultured human endothelial cells to bind U937 monocytic cells. The stimulation was inhibited by cycloheximide, indicating that de novo protein synthesis is required. Biologically modified LDL, obtained by incubation with human peripheral blood monocytes, also enhanced the adhesiveness of endothelial cells. This effect was not due to an increased secretion of interleukin 1 beta from the monocytes exposed to LDL. Treatment of endothelial cells for 24 h with native LDL was also found to increase the adhesion of U937 cells. Exposure of endothelial cells to LDL for 24 h resulted in an oxidative modification of LDL. Furthermore, the antioxidant butylated hydroxytoluene inhibited both the endothelial-dependent oxidation of LDL as well as the increased adhesion of U937 cells, suggesting a coupling between these two processes. The results indicate that LDL, modified by exposure to monocytes or endothelial cells in the arterial wall, may increase the adhesive properties of the endothelium.

Susceptibility to infection by the human immunodeficiency virus (HIV) correlates with T4 expression in a parental monocytoid cell line and its subclones

The monocytoid tumor cell line U-937 and five derived subclones were infected with the HTLV-IIIB isolate of the human immunodeficiency virus (HIV). Susceptibility to infection and sensitivity to the cytopathic effects correlated with the expression of the T4 antigen on the cell surface. On the basis of these characteristics the lines could be divided into three groups. Less than 10% T4 positive cells were present in the parental line and clone 4; hence productive infection could only be established after a long latency or with a high virus inoculum. These lines showed no or only marginal cytopathic effect. Clone 16 contained more than 95% T4 positive cells and was the most sensitive line to infection with HTLV-IIIB and its cytopathic effect. Cell death was so extensive following infection that no continuous virus producer line could ever be established from clone 16 cells. Cultures with intermediate T4 expression (50-70% T4+ cells) also had intermediate susceptibility to virus infection. Cytopathic changes, even if pronounced, could be overcome in the infected cultures by the addition of uninfected cells and, in each case, a producer line could be established. HTLV-IIIB infection of clone 16 cells could be blocked by preincubation with monoclonal anti-T4 antibodies indicating a close similarity between the HTLV-IIIB receptor on T4 positive T cells and monocytoid cells. The results thus show that T4 positive monocytoid cells can function as target cells for the HIV.

Insulin like growth factor-1 and -2 and their role in the re-epithelialisation of wounds; interactions with insulin like growth factor binding protein type 1.

Insulin like growth factor (IGF) 1 and 2 which are present and actively synthesised in the wound fluid stimulate several cell types involved in the process of wound healing. To investigate the role of IGF-1 and 2 and in addition, the association between IGF and their carrier proteins, IGF binding proteins (IGFBP), we have used a newly established model for human wound healing in fresh biopsy material. Histological examination shows that IGF-1 stimulates efficient reepithelialisation of the wounds both alone and in the presence of recombinant IGFBP-1. In contrast, IGF-2 stimulates healing only when used in combination with IGFBP-1. These findings suggest that the two IGFs and their carrier proteins may function during different phases of wound healing and that both IGF-1 and 2 act as potent inducers of wound healing; this may have direct clinical implications.

Effect of recombinant IGF binding protein-1 on primary cultures of human keratinocytes and fibroblasts : selective enhancement of IGF-1 but not IGF-2-induced cell proliferation

The present report describes the mitogenic effect of recombinant IGF-2 on cultured human keratinocytes and fibroblasts compared to that of IGF-1. Furthermore, the modulating effect of a recently expressed recombinant form of placental-derived IGF-binding protein 1 (IGFBP-1) on IGF-induced proliferation was examined. A dose-dependent increase, up to 100%, in cell proliferation was seen in cultured human keratinocytes with IGF-2 and -1 and the proliferative response was comparable to the effect of epidermal growth factor (EGF). In human fibroblasts, IGF-1 stimulated DNA synthesis up to 300% for IGF-1 and up to 200% for IGF-2. The mitogenic effect of IGF-1 was enhanced by IGFBP-1 in both cell types. In contrast, the IGF-2-induced mitogenic effect was unperturbed. These findings indicate that the interaction between IGFs and their binding proteins may induce different responses depending upon the ligand and the target cell.

T and B cell responses to chimeric proteins containing heterologous T helper epitopes inserted at different positions.

The ability of a T helper (Th) epitope to induce help for B cells recognizing different determinants within a multideterminant antigen was investigated. Chimeric fusion proteins, containing inserts of single or multiple copies of the Th epitope ovalbumin 323-339 (ova) at two different positions, were compared with respect to their ability to induce specific antibody production and ova-specific T cell activation. The antibody responses against B cell determinants at the amino and carboxy terminus, respectively was differently influenced by the molecular positioning of the inserted Th determinant. All ova-containing fusion proteins induced antibody production against the B cell determinant at the amino terminal end irrespective of the positioning of ova. In addition, multiple copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against the determinant at the carboxy terminus was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. Furthermore a fusion protein, containing four copies of ova effectively elicited T cell help for high levels of antibody production against both examined B cell determinants, showing that activated Th cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein. Immunodominant T cell recognition of ova in all chimeric peptides, independently of its position, was demonstrated by lymph node cell (LNC) proliferation of primed BALB/c mice. The level of ova-specific T cell proliferation was similar, irrespective of which chimeric peptide that had been used for priming, and thus did not reveal any differences in T cell priming efficiencies related to the number of ova copies in the fusion proteins. However, when the peptides were presented to a ova-specific T cell line by A20 B lymphoma cells, a close correlation between IL-2 production by the clonal T cells and the number of ova epitopes in the chimeric peptides was observed. Thus, increased cytokine production by ova-specific T cells may be important for the increased level of in vivo antibody production observed in response to multiple copies of ova in the chimeric antigens.

A specific assay measuring binding of 125I-Gp 120 from HIV to T4+/CD4+ cells

The HIV (HTLV-III) envelope glycoprotein, Gp120, was isolated from virus-infected tissue culture cells using affinity chromatography. A radioimmunoassay was developed to determine the degree of iodinated Gp120 to target CD4+ (T4+) cells. 125I-Gp120 could be shown to selectively bind to CD4+ cells only. The Gp120 remained bound to these cells after repeated washes. Monoclonal anti-CD4 antibodies block the binding of Gp120 to CD4+ cells. Monoclonal antibodies to other cell surface components do not interfere with 125I-Gp120 binding. All IgG antibodies from HIV seropositive donors tested block 125I-Gp120 binding, though with variable titers. We believe that this assay provides further proof for the use of CD4 (T4) as a component of the receptor for HIV. It represents a safe, objective and sensitive method for the analysis of Gp120-CD4 interactions, as well as the potential of antibodies to interfere with this binding.

Cystein 402 of HIV gp120 is essential for CD4-binding and resistance of gp120 to intracellular degradation

SummaryA DNA fragment encoding the CD4-binding region of human immunodeficiency virus type 1 (HIV) gp120 was excised from an SV40-based expression vector containing gp160, and subcloned into phage M13 for site-directed mutagenesis. Mutant vectors were constructed and CV-1 cells were transfected with constructs, where Cys402 was substituted for a serine, and metabolically labelled with [3H]-N-acetylglucosamine (GlcN). Radioimmunoprecipitation with an hyperimmunserum, specific for gp120/gp160, and subsequent SDS-polyacrylamide gel electrophoresis demonstrated presence of gp160, whereas gp120 was replaced by [3H]-GlcN-labelled material, migrating as a diffuse band corresponding to 80–105k, suggesting increased sensitivity of mutantenv gene products to proteolysis after cleavage to gp120. Wild type gp120 and gp160 bound to CD4, whereas neither gp160 nor gp120 from mutant-transfected cell lysates did bind to CD4. Altogether the results indicated that Cys402, probably by participating in a disulfide bridge, is essential for (i) the CD4-binding ability ofenv gene products and for (ii) the physical stability of gp120.

Enzyme immunoassay (ELISA) for the evaluation of antibodies directed to the CD4 receptor-binding site of the HIV gp120 molecule

The interaction between the HIV envelope glycoprotein gp120 and the CD4 molecule is probably the most important primary event determining HIV infection. Reactivity with the native viral envelope has been difficult to measure due to the lack of gp120 ligand purified directly from primary virus cultures. We have developed an ELISA, utilizing Galanthus nivalis agglutinin (GNA) which selectively binds native HIV envelope gp120 in culture medium. The GNA-based ELISA eliminates the need for isotope-labelled reagents, live cells and recombinant non-natively glycosylated envelope proteins and offers an easy way of using gp120 directly from crude HIV culture medium. The reactivities of sera from several categories of HIV infected individuals were assayed for inhibition of the HIV-1 gp120-CD4 binding. 19/32 (59.3%) sera from asymptomatic individuals and 7/10 (70%) sera from ARC/AIDS patients blocked the CD4-gp120 binding. 20 serum samples from uninfected individuals showed a gp120-CD4 interaction blocking capacity of 0-15%. Two monoclonal antibodies, T4.2 directed to CD4 and 1171 directed to the CD4 binding site of gp120 were used as positive controls. Both Mabs inhibited CD4-gp120 binding by 66-90%.

Human α-fetoprotein (AFP) causes a selective down regulation of monocyte MHC class II molecules without altering other induced or noninduced monocyte markers or functions in monocytoid cell lines

Human alpha-fetoprotein (AFP) purified from human amniotic fluid was investigated for its effect on human monocytoid cell lines, including U 937 cells with established subclones. The impact of AFP on the expression of surface markers (MHC class I and II, CD4, CD18, CD45, Fc receptors for IgG) was analyzed using known inducers of monocyte-macrophage differentiation such as phorbol esters and IFN-gamma. Furthermore we investigated the effect of AFP on the induction of macrophage antibody-dependent cell-mediated cytolytic activity (ADCC). AFP did selectively induce a rapid down regulation of surface MHC class II expression. No evidence of alterations was found in the endogenous or differentiation-induced expression of other markers on the surface on monocytes, nor did AFP affect the functional maturation of surface Fc receptors or the ability to express ADCC.

Production and characterization of a fragment containing the HIV-gp120 binding region of CD4 using a bovine papilloma virus (BPV) vector

SummaryWe have used a bovine papilloma virus (BPV) based mammalian cell expression vector consisting of the complete BPV genome and a human cytomegalovirus transcription unit for the production of soluble CD4. Mouse C-127 cells were transfected with vector DNA together with a selectable G418 resistance plasmid. Surviving clones were selected for high production using a solid phase ELISA based on the immobilization of supernatant-derived CD4 onto nitrocellulose paper and subsequent detection with anti-CD4 antibodies. The expressed protein was shown to bind HIV-gp120 and efficiently block HIV-1 infection in vitro. The possibility to use the above system for rapid production of defined glycoprotein fragments harboring defined functional regions, for the further elucidation of the functional role of CD4 in antigen presentation and cell to cell contact, and for possible intervention during HIV infection is discussed.