Effect of heparin on bovine epithelial lens cell proliferation induced by heparin affin regulatory peptide.

HARP (heparin affin regulatory peptide) is an 18 kDa heparin binding protein, also known as HB-GAM or pleiotrophin (PTN) which has been primarily isolated from brain and uterus, and displays neurite outgrowth, angiogenic and mitogenic activities. Previously, we have expressed the human cDNA encoding human HARP in NIH 3T3 cells. Purified recombinant HARP displayed mitogenic activity for endothelial cells. Its NH2-terminal sequence indicates that the HARP molecule possesses a three amino acid extension from the signal peptide more than the NH2-terminal described. For HB-GAM or PTN, these three amino acids may be essential for the stability and the mitogenic activity of this growth factor. In an attempt to further study the mode of action of this growth factor, we have investigated the mitogenic effect of HARP on various cell types. In contrast to FGF-2, HARP failed to induce stimulation of DNA synthesis on a CCL39 cell line. However, we found that in quiescent bovine epithelial lens (BEL) cells, the stimulation of DNA synthesis induced by HARP is dose-dependent (EC50: 2.5 ng/ml) and maximal stimulation is as potent as that induced by FGF-2 (EC50: 25 pg/ml). Interestingly, when BEL cells were allowed to quiesce in the presence of serum, the stimulation induced by HARP is considerably less potent. In this highly responsive cell system, heparin could potentiate the mitogenic activity of HARP at very low doses (0.1-1 microgram/ml) and inhibit this activity at concentrations of 10 micrograms/ml. In contrast to its protective effect on FGF-1 and -2, heparin was unable to preserve HARP from tryptic and chymotryptic degradations.

Production and purification of active FGF2 via recombinant fusion protein.

Basic fibroblast growth factor (FGF2) is involved in both cell proliferation and differentiation processes. Heparin may interfere in the stability and biological activities of FGFs. However, it is difficult to obtain FGF preparation without traces of heparin since heparin affinity chromatographies are routinely used to prepare this growth factor. We have therefore devised a means of production of active recombinant FGF2 devoid of heparin traces. The bovine FGF2 gene was inserted into the pMAL-c prokaryotic expression vector and the recombinant protein was synthesised as a fusion product between the maltose binding protein (MBP) and FGF2. Purification of the FGF2 fusion protein was performed by an amylose affinity chromatography. Yields were similar to those obtained by using a traditional heparin affinity column purification procedure. The fusion protein (MBP-FGF2) and the cleaved-off FGF2 were tested for some of their biological properties and compared to recombinant FGF2 purified by heparin affinity chromatography. Mitogenic activity on Chinese hamster lung fibroblasts (CCL39) and neurite outgrowth on pheochromocytoma culture cells (PC12) were used as biological assays. The cleaved-off FGF2 was as active as commercially available recombinant FGF2 (ED50 at 0.16 and 0.04 nM respectively). However MBP-FGF2 was less active (ED50 at 0.9 nM) in both tests.

Biochemical and mitogenic properties of the heparin-binding growth factor HARP.

Heparin affin regulatory peptide (HARP), also called Pleiotrophin (PTN), is a polypeptide that displays a high affinity for heparin and that shares approximately 50% sequence homology with Midkine (MK). According to this structural homology, these two molecules constitute a new family of heparin-binding proteins. The biological properties of HARP and MK remain largely a subject of debate. Both proteins have been described as neurite outgrowth promoting agents whereas until recently the mitogenic activity has been controversial. The aim of this review is to summarize the information on HARP with special focus on the recent data relating to its mitogenic properties.

Mitogenic and in vitro angiogenic activity of human recombinant heparin affin regulatory peptide.

We have previously described the purification of a heparin binding growth factor from adult bovine brain named heparin affin regulatory peptide (HARP), which was identical to an uterus derived growth factor named pleiotrophin and to a developmentally regulated neurite promoting factor named heparin-binding growth associated molecule. However, for yet unclear reasons, the mitogenic activity of this purified polypeptide following isolation from animal tissue extracts is a subject of controversy, due to conflicting and irreproducible data when produced by recombinant DNA technologies in E. coli or insect cells. The purified protein was inactive in mitogenic assays but the natural molecule was active in assay of neurite outgrowth. In order to clarify these conflicting results and to obtain a recombinant protein free from other contaminating heparin-binding growth factors, we have cloned human cDNA encoding human HARP, engineered its expression in NIH 3T3 cells and characterised the resulting recombinant polypeptide. Purified recombinant HARP displayed mitogenic activity for capillary endothelial cells with half-maximal stimulation at approximately 1 ng/ml (55 pM) and induced angiogenesis in an in vitro model. Interestingly, while the NH2 terminal sequence of tissue purified HARP was NH2-GKKEKPEKK, the NH2 terminal sequence of the biologically active recombinant protein was NH2-AEAGKKEKPEKK, corresponding to a three amino acid extended form.

Polymerase chain reaction amplified HTLV-I, HIV-1 and HIV-2 DNA fragments in subjects with mixed retroviral infections.

Serum and peripheral blood mononuclear cells from eight patients from the Ivory Coast with positive screening test results for retroviral infections were studied by serology (ELISA, Western blot (WB), synthetic peptide test), cell co-culture, and polymerase chain reaction (PCR). Two HIV-2 infections with indeterminate interpretation on HIV-1 WB were detected, two were clear dual HIV-1/HIV-2 infections, three were ambiguous mixed HIV-1/HIV-2 infections, and one was a triple retroviral infection by HTLV-I, HIV-1 and HIV-2. Four slow/low HIV-1 strains were isolated at the expense of HTLV-I and HIV-2 strains. The ELISA tests were found to be very sensitive. Indeterminate WB interpretations were frequent (HTLV-I, four; HIV-1, three; HIV-2, two). PCR provided clear evidence of multiple retroviral infections in three cases and enabled interpretation of indeterminate WB samples in three cases. One sample presented a puzzling pattern with positive PCR results for HIV-1 and HIV-2 associated with negative or indeterminate serological results. Thus, our data emphasise the need to analyse serological as well as virological markers to gain better insight on mixed retroviral infections, especially in endemic areas such as West Africa.

Immunogenicity of combined anti-HIV and anti-suppressive vaccine preparations.

HIV-1 antigens generate in man both a humoral and cellular immune reaction. However, in ARC/AIDS patients, the cellular response is inhibited by HIV-1 which induces an antiproliferative (suppressive) effect on activated T cells. To overcome this inhibition and up-regulate the cellular response, we designed a new vaccine strategy directed both against HIV-1 and immunosuppression and we used an immunizing preparation composed of HIV-1 antigens combined with immunoregulatory peptides prepared in a biologically inactivated but immunogenic form. In mice, this preparation induced anti-HIV-1 antibodies and a cell-mediated cytotoxicity directed against H2 restricted cells carrying HIV-1 antigens.

Cell-mediated immunity against HGP-30, a group-specific peptide of HIV p17 in individuals infected with the AIDS virus.

HGP-30, the synthetic peptide analogue and active component in an HIV-1 (human immunodeficiency virus, type 1) p 17 core-based experimental vaccine, has previously been shown to induce cytotoxic and helper T-lymphocyte responses. In order to further define the T-helper cell responses which are known to play a role in enhancing the immunological response to foreign antigens, we studied the response of individuals infected with HIV to HGP-30 at various stages of disease progression. We have investigated the proliferative cellular response of peripheral blood mononuclear cells (PBMCs) derived from individuals infected with HIV-1 to HGP-30. We have found a PBMC proliferative response to HGP-30 in 40% of the healthy seroconverted patients, in 35% of the CDC stage III patients and in 18% of the CDC stage IV patients. There was no correlation between the proliferative response to HGP-30 and other antigens such as HIV-like proteins or tetanus toxoid not to CD4 cell count. HLA-DR typing revealed the possible presentation of HGP-30 by several different class II molecules. Since these class II molecules occur frequently in the general population, HGP-30 appears to contain broadly reactive epitopes and thus is not restricted as are many peptide vaccines. Due to its broad reactivity and extreme conservation in many HIV-1 strains. HGP-30 is one of the promising candidates for inclusion as a subunit vaccine against HIV-1.

Variable stringency hybridization of polymerase chain reaction amplified HIV-1 DNA fragments.

DNA isolated from the peripheral blood mononuclear cells of HIV-1 seropositive individuals was used for polymerase chain reaction (PCR) amplification of gag and envelope regions. Eight aliquots of the amplified DNA fragments have been subjected to Southern/dot blot analysis, hybridizing with 32P-labelled-BH10 (HIV-1 strain IIIB) at low stringency. After the filters had been autoradiographed, they were cut so that each hybridized band/dot could be subject to variable stringency washing using various ionic concentrations at a fixed temperature. The filter was reconstructed so that the effect of the variable stringency wash might be visualized following a second exposure to Kodak film. The level of activity for each band/dot was measured by counting the 32P or by densitometry analysis of the photographic record. The results allow a plot to be made of the decrease in bound radioactivity against ionic strength. By comparison with a standard curve obtained for HIV-1 strain IIIB amplified fragments subject to similar hybridization and analysis, an estimation of the degree of nucleotide mismatch relative to the BH10 DNA probe can be obtained. The technique provides a rapid means of characterizing PCR amplified fragments.