[Recombinant human alpha-fetoprotein as a regulator of adipose tissue stromal cell activity].

Recombinant human alpha-fetoprotein (rhAFP) expressed in yeast system as a glycoprotein, was isolated and purified to 98% by multistep method. The testing of the rhAFP in the culture of adipose tissue stromal cells (hASC) has revealed its ability to enhance hASC proliferation and migration as well as vascular endothelial growth factor production, with no significant influence on cell invasion and matrix metalloproteinase-2 and -9 secretion. It has been also estimated that rhAFP is internalized in hASC via clathrin-dependent mechanism. A study in the murine experimental model of hindlimb ischemia has shown the capability of rhAFP to enhance blood flow recovery. These data suggest that rhAFP is a promising agent for enhancement of the hASC regenerative ability.

alpha-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes.

BACKGROUND AND PURPOSE: The immunomodulatory effects of alpha-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. EXPERIMENTAL APPROACH: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFkappaB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFkappaB DNA binding activity was measured by specific assays. Nitric oxide and H(2)O(2) production and redox status were assessed by fluorescent probe and biochemical methods. KEY RESULTS: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H(2)O(2) and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFkappaB was activated by AFP alone or by its combination with UVA. CONCLUSIONS AND IMPLICATIONS: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of pro-inflammatory mediators and modulation of their pro-inflammatory response to cytokines or UVA. AFP may modulate inflammatory events in human skin.

Alpha-fetoprotein as a TNF resistance factor for the human hepatocarcinoma cell line HepG2.

Human hepatocarcinoma HepG2 cells are known to be insensitive to tumor necrosis factor (TNF) cytotoxicity. In this report, preliminary washing of HepG2 cells with serum-free medium to remove endogenous and exogenous alpha-fetoprotein (AFP) from the cultivation medium transfers cells from the TNF-resistant to the TNF-sensitive state without addition of any transcriptional inhibitors. HepG2 cells sensitized to by washing again became TNF-resistant after their treatment with exogenous AFP. Protective AFP activity against TNF-induced cytotoxicity directly depends on the AFP/TNF concentration ratio, demonstrating biphasic AFP activity. Our data show that 0.2 mg/ml of AFP acts synergistically to enhance cytotoxicity of suboptimal TNF doses. In contrast, the same AFP dose significantly attenuates the cytotoxicity of high TNF doses. It is concluded that AFP can function as a protective factor against TNF cytotoxicity in human hepatoma cells. These observations suggest that AFP secretion by certain tumor cells allows a highly flexible regulation of TNF cytotoxicity, dependent on the amount of endogenous AFP.

Induction of apoptosis in human hepatoma cells by alpha-fetoprotein.

We have investigated the effects of purified human alpha-fetoprotein (AFP) on the growth of the human hepatocarcinoma-cells HepG2 in culture. Cancer-derived AFP (cAFP), isolated from the culture medium of AFP-secreting HepG2 cells and embryonal AFP (eAFP), isolated from human cord serum, were used for these studies. Both AFP pre parations studied were shown to induce strong dose-dependent inhibition of HepG2 cell proliferation and complete growth arrest at high protein concentrations (more than 0.1 mg/ml). To test whether AFP may trigger an endogenous suicide program in hepatoma cells, we examined whether DNA fragmentation preceded cell death. After exposure of the cells of the high AFP dose (1.0 mg/ml), DNA fragmentation was detected as early as 2 h after treatment, and 70% of cells were apoptotic by 24 h. DNA fragmentation was shown to precede other signs of cell death for several hours. Typical morphological changes of apoptosis were observed after 4 h of exposure of cells to high AFP doses. Low concentrations of cAFP and eAFP (less than 0.1 mg/ml) failed to induce growth inhibition of HepG2 cells, rather showing a weak stimulative effect, demonstrating a biphasic AFP activity. Cell pretreatment with the transcriptional inhibitor actinomycin D had no measurable influence on AFP cytotoxicity. These findings demonstrate that protein synthesis is not required for this mechanism of cell death. The charcoal-treated ligand-free eAFP (eAFPp) had a dose-dependent growth-inhibitory activity, similar to intact protein, but slightly less intensive. The similar growth-inhibitory activities of cAFP, eAFP and eAFPp, which have a significant difference in bound-ligand content, provide evidence that the main role in cell growth regulation may be attributed to the protein moiety of the entire AFP molecule, but not to its ligands. These biologically active AFP ligands could, however, modulate AFP-growth-regulating activity. Growth factor deprivation distinctly enhanced the cytostatic activity of high AFP concentrations and also increased the mitogenic activity of low AFP levels, showing the interdependence of the growth-regulative activity of AFP and growth factors. The findings of this study demonstrated that AFP is directly introduced into the intracellular pathways of cell growth regulation and programmed cell death.

The costimulatory and differentiating activity of soluble class I MHC antigens for an autologous thymocyte population.

Combined cultivation of macrophages with syngeneic thymocytes resulted in accumulation of soluble H-2Kk antigens in culture medium. Incubation of intact autologous thymocytes with these soluble class I MHC molecules was shown to induce functional maturation of thymocytes assayed in local graft-vs-host reaction. Similar thymocyte costimulating activity was detected for the papain-solubilized purified H-2Kk antigens. Soluble class I antigens were shown to costimulate IL2 production by thymocytes in response to submitogenic doses of exogenous IL2 and to increase PHA-induced thymocyte proliferation. Soluble class I molecules were shown to increase the level of expression of function-associated membrane antigens, H-2Kk, CD8 and CD4, and to trigger thymocyte differentiation. The expression of I-Ak antigens remained invariable. It was also shown that soluble autologous class I molecules may function as direct amplifiers of thymocyte proliferation in autologous, but not allogeneic, mixed leukocyte reactions. It is concluded that soluble MHC class I molecules are capable of triggering functional and phenotype differentiation of syngeneic thymocytes and acting as restricted coaccessory molecules when thymocyte activation is induced by a submitogenic dose of different stimuli.

[Immunobiologic characteristics of hybrid proteins consisting of tumor necrosis factor alpha and thymosin alpha 1]. / Izuchenie immunobiologicheskikh svoistv gibridnykh belkov sostoiashchikh iz faktora nekroza opukholei alpha i timozina alpha 1.

The preparations of alpha 1-thymosin (T), alpha-tumor necrotic factor (TNF) and their based hybrid proteins: T-TNF, TNF-T, and T-TNF-T were studied by using a wide spectrum of immunobiological tests. In the L-929 cells, T-TNF preserved cytotoxicity unique to TNF; TNF-T preserved it 10 times less, and T-NTF-T was completely inactive. TNF-T inhibited the growth of Molt-4, Jm-9, Raji cells by 63 = 84%, and TNF suppressed only Raji cells by 50%. Hybrid proteins caused cell proliferation of the thymus, spleen, and lymph nodes; H-2K, CD4, and CD8 differently increased the expression of thymocytic antigens. The authors found the different effects of the drugs on phagocytosis, the production of antibody-forming cells, delayed reaction, and activation of natural killer cells. The protein T-TNF-T produced the most pronounced action.

[Study of the structural properties of recombinant leukocyte interferon A by means of fluorescence polarization, circular dichroism, and differential microcalorimetry]. / Issledovanie strukturnykh svoistv rekombinantnogo leikotsitarnogo interferon A metodami poliarizatsii fluorestsentsii, krugovogo dikhroizma i differentsial'noi mikrokalorimetryii.

Human leukocyte interferon-A1 (IFN-alpha A) structure in solution was investigated by fluorescence polarization, circular dichroism and scanning microcalorimetry techniques. Using gel-filtration it was established that at neutral pH values and at concentration not exceeding 0.3 mg/ml IFN-alpha A has a dimeric configuration in solution. At pH below 5, IFN-alpha A exists as a monomer. Using circular dichroism technique the IFN-alpha A molecule was shown to preserve a native structure upon decreasing pH to 3.5. The rotational correlation time of IFN-alpha A molecule in dimeric and monomeric form was measured using fluorescence of DNS, conjugated with the protein, and fluorescence of tryptophan residues. Our data indicate that the shape of IFN-alpha A molecule may be approximated by the rigid ellipsoid of revolution with the axis ratio = 4:1. The intramolecular melting of IFN-alpha A was studied by scanning microcalorimetry and circular dichroism in the acidic pH range. Thermodynamic analysis reveals two independent cooperative transitions. These transitions can be explained by assuming that the IFN-alpha A molecule consists of two structural domains.

[Macrophages and the functional differentiation of thymocytes (a review of the authors' own data)]. / Makrofagi i funktsional'naia differentsirovka timotsitov (obzor sobstvenennykh dannykh).

Cellular, humoral, and genetic mechanisms of induction of T-effectors of the transplant vs. host reaction (THR) have been studied in two-cell culture of phagocyte mononuclears and thymocytes. A direct physical contact and similarity of H-2K locus of major histocompatibility complex between the cooperating cells in culture is required for successful induction of T-effectors of THR. Contact interaction of macrophages with thymocytes leads to accumulation of a 67 KDa humoral factor in the culture medium. Incubation of intact thymocytes with this factor leads to functional transformation of immature thymocytes into corresponding effector cells. Similarity of H-2K locus of the factor producers and intact lymphocytes is also required for successful humoral induction of the T-effectors. The surface H-2K antigen is able to induce formation of THR t-effectors from non-reactive thymocytes. The H2-K-specific mediator, affinity-isolated from the supernatant of the macrophage-thymocyte culture can also cause this induction.

Polarization fluorescence, spin label and ultracentrifugal studies of specific interaction of low molecular weight proteins with the Fc fragment of human immunoglobulin G.

Low mol. wt (about 2000) proteins which were found in normal human serum formed complexes with the Fc fragment of IgG. The interaction constant was not less than 10(6) l/mole. These complexes dissociated on dilution of the protein solution to below 2 microM or decreasing the pH below 6. The binding site of these proteins was located in the CH2 domains in close proximity to the carbohydrate moieties. The dissociation of IgG complexes with these proteins was accompanied by a conformational change in the Fc fragment.

Release of low molecular weight proteins from the Fc fragment of rabbit immunoglobulin G induced by formation of insoluble complexes with antigen.

Serum rabbit IgG was found to be in complexes, with low mol. wt proteins (mol. wt about 1500) which are noncovalently associated with the Fc fragment. The formation of precipitating antigen-antibody complexes resulted in the dissociation of IgG complexes with these proteins and their release in solution. On formation of non-precipitating antigen-antibody complexes, the dissociation of low mol. wt proteins from IgG was not observed.

[Study of binding of spin probes of the carboline and benzocarboline series to bovine serum albumin]. / Issledovanie sviazyvaniia spinovykh zondov karbolinovogo i benzokarbolinovogo riada na bych'em syvorotochnom al'bumine.

The dependence of the external and internal wide hyperfine extreme shifts of the ESR spectra on temperature and viscosity for spin-probes in solution of BSA was studied. Seven homologous spin-probes of carboline and benzocarboline derivatives were used. The obtained dependences are a consequence of the involvement of the spin-probe in two types of rotation: an anisotropic fast reorientation with tau > 10(-9) s with respect to a macromolecule and the isotropic one with tau > 10(-8) s due to rotation of the macromolecule itself. It was shown, that extrapolation values of a separation between hyperfine extreme do not reflect the degree of the immediate spin-probe environment polarity, but are determined by the hyperfine tenzor partial averaging as a result of the rapid anisotropic reorientation of the spin-probe. All spin-probes used were shown to be bound by the BSA molecule in the near vicinity of the tryptophan residue. The rotation correlation time of the BSA molecule was determined to be equal to 40 ns.