Purification and partial characterization of serum monocytotropic factor, a platelet-derived cyclooxygenase-inducing polypeptide.

It has previously been shown that a heat- and acid-stable component of human and animal sera was capable of stimulating prostanoid biosynthesis in human blood monocytes, very probably by a mechanism involving cyclooxygenase induction. Many physico-chemical characteristics of this factor are similar to those of identified platelet factors. Here we show that human platelets are a rich source of this factor (serum monocytotropic factor) and that results from experiments using arachidonic acid or thrombin as releasers are consistent with its presence in platelet membranes. Serum monocytotropic factor has been purified 1500-fold by three chromatographic steps. Purification was more difficult when starting from platelet releasates or lysates. The purified serum monocytotropic factor had an apparent molecular mass of 70,000 as judged by Sephadex G-75 chromatography and by polyacrylamide gel electrophoresis; however, when subjected to HPLC on a gel permeation column in the presence of 6 M urea, one major peak corresponding to a relative molecular mass (Mr) of 30,000-35,000 was observed, which suggests a homodimeric structure. It is therefore very likely that human platelets store, in addition to the two well-identified polypeptide growth factors, platelet-derived growth factor and transforming growth factor-beta, a third polypeptide capable of regulating prostanoid production in monocytes.

Regulation of thromboxane A2 biosynthesis in platelet-free human monocytes and the possible role of polypeptide growth factor(s) in the induction of cyclooxygenase system.

It has previously been shown that platelet-free human monocytes, when properly incubated in the presence of animal and human sera, became capable of producing large amounts of thromboxane A2 and prostaglandin E2. The characteristics of these processes are reported here. Prostaglandin biosynthesis was time and cell concentration dependent; 24 h of incubation at 37 degrees C and 0.5 X 10(6) cells per ml medium were found to give the most reproducible results. Human monocytes produced thromboxane A2 and prostaglandin E2 in a typical ratio which ranged from 2.0 to 5.0 (28 experiments). Animal and human sera were similarly effective, while serum obtained from platelet-free blood was much less active. The activity of all sera tested was stable to heating (100 degrees C for 2-10 min) and extreme pH values (pH 2 and 11). It was unstable when the serum was heated at pH 11 and after 2-mercaptoethanol treatment. These observations prompted us to check the effect of polypeptide growth factors having properties similar to those reported above, such as platelet-derived growth factor, fibroblast growth factor, epidermal growth factor as well as insulin and transferrin. None of these, alone or in various combinations, was capable of eliciting a stimulation comparable with that of serum. Stimulation due to sera was, as expected, dose dependently inhibited by acetylsalicylic acid and more efficiently by indomethacin; unexpectedly it was also inhibited by protein synthesis inhibitors such as actinomycin D and cycloheximide in conditions under which no toxic effect of the drugs was evident. On the basis of these results we conclude that: (a) polypeptide growth factor(s) with a molecular weight at least 30 000 (as judged by Amicon ultrafiltration) is involved in the regulation of prostaglandin biosynthesis); (b) such a factor(s) acts by inducing rather than by activating the cyclooxygenase system.

Cytotoxicity of, and DNA damage by, active oxygen species produced by xanthine oxidase.

Toxicity to Raji cells of the xanthine oxidase/hypoxanthine system is related to the formation of single-strand DNA breaks. DNA damage was proportional to the concentration of xanthine oxidase and to the time of exposure. It was prevented by the absence of hypoxanthine, or by the presence of allopurinol, or both superoxide dismutase and catalase. The release of 51Cr from damaged cells was detectable 12 h after the inhibition of cloning efficiency and the production of DNA breakage. These data suggest that DNA damage induced by the oxygen products precedes the severe lesion to the cellular membrane.

Antibody response in partridge (Perdix perdix L.) 1. Effect of sex and age on the immune response to sheep red blood cells (SRBC), Newcastle disease virus (NDV) and Brucella abortus (Buck 19).

To ascertain the immune response capacity of Partridges to classical experimental and natural antigens, dose and time response curves to sheep red blood cells (SRBC), Newcastle disease virus (NDV) and Brucella abortus (Buck 19 strain), were determined in two strains reared in two different farms. 1 ml of 20% SRBC, 10(9) NDV particles and 10(7) Buck 19 microorganisms, gave optimal antibody responses seven (SRBC and Buck 19) or fourteen days after immunization. As far as SRBC is concerned, the antibody response was sex independent, but declined markedly in partridges older than 1 year. The immunization schedule used by us allow the identification of high and low responder animals. In fact, highly significant correlation in individual partridges between antibody titers obtained after two successive immunizations to an optimal SRBC dose given three weeks apart, was found. This fact, together with the observed great variability in the antibody response to the three antigens considered, should allow a selection for high and low responder lines, in order to improve the fitness toward environmental pathogens.

Prostaglandin and thromboxane biosynthesis in isolated platelet-free human monocytes. III. The induction of cycloxygenase by colony stimulating factor-1.

Previous observations showing the presence in the serum of a component capable of regulating prostanoid biosynthesis in human cultured monocytes, have led us to suspect its presence in human platelets. We have purified this serum monocytotropic factor (SMF) and have shown its identity with a component of platelet membranes. Surprisingly its structure appeared to be very similar to that of a polypeptide growth factor never before identified in platelets: the colony stimulating factor-1 (CSF-1 or M-CSF). Here we show that SMF and CSF-1 have very similar biological properties. Thus, CSF-1 when released from human platelets is capable of triggering the differentiation pathway leading from blood monocytes to resident macrophages. It is likely that cycloxygenase induction plays a pivotal role in these events.

Prostaglandins and cyclic nucleotides as effectors in non-lymphocyte mediated surveillance processes ( short review).

This paper reviews the evidence pointing to a role of prostaglandins and cyclic AMP in the regulation of surveillance processes against transformed cells carried out by activated monocytes macrophages and natural killer (NK) cells. Specific topics of discussion include: (a) the regulation of monocyte/macrophage system and NK cells by prostaglandins and cyclic AMP; and (b) the possible immunomodulatory role of thromboxanes generated by activated monocytes and macrophages. Also the role of cyclic AMP dependent and independent protein kinases as well as their link with oncogenes is briefly reviewed.

Enhanced DNA repair in lymphocytes of Down syndrome patients: the influence of zinc nutritional supplementation.

Oral zinc supplementation is able to correct zinc deficiency and some immune defects present in Down's syndrome (DS), while other beneficial effects can be predicted because of the broad spectrum of biochemical pathways and the great variety of enzymes which depend on zinc bio-availability. To test if the maintenance of DNA integrity is also affected by zinc supplementation, DNA damage and repair after gamma-radiation was studied by alkaline elution assay in phytohemagglutinin-stimulated lymphocytes from Down's syndrome children before and after an oral zinc supplementation given for 4 months to correct their immune defects. In comparison with lymphocytes from normal children the DNA damage induction after ionizing radiation in DS lymphocytes both before and after zinc supplementation was normal. On the other hand, the rate of DNA repair in DS was highly and significantly accelerated before zinc treatment. After supplementation with zinc sulfate, the DNA repair rate was consistently slowed down becoming similar to that of control subjects. This is the first demonstration that a nutritional intervention in humans is apparently able to modify the biochemical steps which control the rate of DNA repair.

Possible involvement of prostaglandin H synthase-1 induction in the differentiation of U-937 cells.

The promyelocytic human cell line U937, cultured in the presence of TPA and/or vit. D3, differentiates to monocytes and to macrophage-like cells. A potent stimulus for differentiation is represented also by colony stimulating factor-1 (CSF-1). Since this factor is a strong inducer of PGH synthase in human monocytes, we have investigated whether this event may be connected to the differentiation of U937. We have found that TPA, in the presence of serum, increased the production of thromboxane B2 (TXB2) 4-5 fold, while DMSO, which induced differentiation to neutrophils, was not active. Here we report studies indicating that the effect of protein and RNA synthesis inhibitors on prostanoid production, in cells incubated in the presence of CSF-1 (or FCS), can be correlated with an inductive event carried out by the growth factor, as demonstrated by the use of Western and Northern blotting procedures. However, while in human monocytes PGH-s and its mRNA are absent in controls and are expressed at high levels in CSF-1 stimulated cells, in U937 cells exposed to TPA, PGH-s mRNA was clearly detected by Northern blots, but its translation product was expressed at low level, and cells generated low amounts of TXA2 (13% of maximal production). After incubation with CSF-1 (or FCS) mRNA levels were only slightly modified, but large amounts of TXA2 accumulated in the medium. We have interpreted these findings by suggesting that CSF-1 is capable not only of regulating the expression of the gene encoding PGHs, but also of acing translationally to regulate the expression of its mature mRNA.

Interleukins and interferons: yin-yang modulators of PGH synthase in human macrophages.

Prostaglandin H synthase (PGHs) not only is an unstable enzyme, mainly when it is challenged with substrate, but its mRNA is one of the shortest lived species so far identified in mammalian cells. Therefore, signals regulating its level are critical for the role it plays in many cells. The expression of genes coding for PGHs appears to be under the control of polypeptide growth factors. This is in accordance with our previous data showing that a colony stimulating factor-1 (CSF-1)-like factor induces the de novo synthesis of PGHs in human monocytes, as demonstrated by immunoblotting. Here we extend this concept by showing that interleukin (IL)-1 alpha behaves as a potent inducer of PGHs in human macrophages, as indicated by the block in its action due to the addition of RNA and protein synthesis inhibitors. Interferons (IFNs) alpha and beta, however, inhibit prostanoid production in a dose-dependent fashion mainly when macrophages activated by serum are tested. Thus, the PGHs system appears to be under a fine control, CSF-1 being the main regulator during the differentiation from pro-monocyte to monocyte and from monocyte to macrophage, and IL-1 (and perhaps IL-2) as well as IFN alpha and beta, the regulators during differentiation and/or proliferation of human macrophages.

Functional assessment of cellular non-specific and specific immunity in selected healthy elderly.

Thirteen healthy elderly were selected according to a simplified SENIEUR admission protocol including clinical, hematological and biochemical parameters. The goal of this protocol was to limit the influence of diseases and/or medications on the assessment of immune functions in the elderly. Plasma zinc levels of healthy elderly were comparable to those of young subjects. Cellular nonspecific immunity was determined by measuring chemiluminescence (CL) of peripheral blood granulocytes activated by opsonized zymosan particles. CL of granulocytes from healthy elderly was delayed in comparison to that of young controls when autologous serum was used. Lymphocyte proliferation induced by phytohemagglutinin-P (PHA-P) or zinc chloride (ZnCl(2)) in a serum free medium was lower in the elderly than in young controls. Preincubation of lymphocytes with ZnCl(2) before PHA-P stimulation did not restore the impaired proliferative activity of cells from old donors.

Sialyltransferases in cancer.

It has long been known that cancer cells often express more heavily sialylated glycans on their surface and that this feature sometimes correlates with invasion. It is now well established that specific sialylated structures, such as the Thomsen-Friedenreich-related antigens, the sialyl Lewis antigens, the sialyl alpha2-6 lactosaminyl structure, the polysialic acid or some gangliosides, can mediate cellular interactions and are altered in cancer cells. This review summarizes the current knowledge on the cancer-associated alterations in sialyltransferase expression which are often at the basis of the deranged expression of sialylated structures.

Simple sugars inhibit proliferation of human T lymphocytes in autologous and allogeneic mixed lymphocyte reactions.

Several oligo- and monosaccharides were studied for their capacity to modulate lymphocyte proliferation in human allogeneic and autologous mixed lymphocyte reactions (MLR). A defined subset of sugars showed a marked inhibitory effect on lymphocyte proliferative response in the majority of the allogeneic MLR combinations studied. The inhibitory effect disappeared when sugars were added to allogeneic MLR 96 hr after the beginning of culture. These sugars also showed a significant inhibitory power on autologous MLR, performed by using T- and non-T-enriched lymphocytes from the same donor. The reported data suggest that carbohydrate determinants are involved in the proliferative response of human lymphocytes in both autologous and allogeneic MLR.

Biosynthesis of the cancer-related sialyl-alpha 2,6-lactosaminyl epitope in colon cancer cell lines expressing beta-galactoside alpha 2,6-sialyltransferase under a constitutive promoter.

An elevation of beta-galactoside alpha 2,6-sialyltransferase (ST6Gal.I) enzyme activity and an increased alpha 2,6-sialylation of cell membranes are among the most prominent glycosylation changes associated with colon cancer; both modifications correlate with a worse prognosis. In our previous studies, we have frequently observed a discrepancy between the ST6Gal.I level within a colon cancer sample or cell line and the respective level of reactivity with the alpha 2,6-sialyl-specific lectin from Sambucus nigra (SNA). In this study, we have investigated quantitatively the biosynthesis of the sialyl-alpha 2,6-lactosaminyl epitope in two colon cancer cell types expressing the ST6Gal.I cDNA under the control of a constitutive promoter. By measuring the amount of ST6Gal.I mRNA using competitive RT-PCR, the expression of alpha 2,6-sialylated lactosaminic structures with SNA and anti-CDw75 Ig, and the presence of unsubstituted lactosaminic termini by Erythrina cristagalli lectin, we reached the following conclusions: (a) a high proportion of the cell surface lactosaminic termini remains unsubstituted, even in the presence of a very high ST6Gal.I activity. This proportion is strongly dependent on the cell type; (b) ST6Gal.I-transfected colon cancer cells do not express the CDw75 epitope; (c) the level of ST6Gal.I enzyme activity only partially correlates with the mRNA level; (d) despite the control by a constitutive promoter, the ST6Gal.I mRNA is not constantly expressed over time; and (e) a very large portion of the enzyme molecules is secreted in the extracellular milieu. These results indicate that post-transcriptional and post-translational mechanisms play a pivotal role in the control of alpha 2,6-sialylation in colon cancer cells.

Differential expression of the hepatic transcript of beta-galactoside alpha2,6-sialyltransferase in human colon cancer cell lines.

The activity of beta-galactoside alpha2,6-sialyltransferase (ST6Gal.1), the enzyme responsible for the addition of sialic acid in alpha2,6-linkage to N-acetyllactosaminic (Gal beta1,4GlcNAc) units of glycoconjugates, is increased in the vast majority of colon cancer specimens, and a positive correlation with an invasive phenotype has been suggested by several studies. In many tissues, ST6Gal.1 is regulated mainly at the transcriptional level through the use of different cell-specific promoters which generate transcripts differing in their 5'-untranslated regions. With the aim of understanding the molecular bases of the increased ST6Gal.1 expression in colon cancer, we investigated the expression of mRNA species in colon cancer cell lines and the relationship with enzyme activity and extent of alpha2,6-sialylation of cell glycoproteins. All cell lines examined express the form containing the 5'-untranslated exons Y and Z, typical of the "basal" expression of the gene, while others express also the liver transcript. This indicates that colon cancer cell lines can be grouped according to expression of the liver transcript of ST6Gal.1. The cell lines expressing only the Y+Z form display, in general, a lower activity:mRNA ratio, which might indicate reduced translational efficiency. The level of alpha2,6-sialylation of cell glycoproteins, as determined by reactivity with the Sambucus nigra lectin, is closely associated with the level of enzyme activity.

Oral zinc supplementation in Down's syndrome: restoration of thymic endocrine activity and of some immune defects.

Eighteen non-institutionalized Down's syndrome (DS) children (mean age: 7.0 +/- 10/12 years) with a history of respiratory tract, auditory and skin infections, low plasma levels of a nonapeptide thymic hormone, i.e. Serum Thymic Factor (STF), high plasma levels of inactive zinc-unbound STF molecules, and reduced absolute number of circulating T-lymphocytes, were given an oral non-pharmacological supplementation of zinc sulphate (1 mg Zn++/kg body weight/day for 2 months; two cycles, 10 months apart) and monitored immunologically before and after each cycle. A dramatic increase of plasma STF level and concomitantly an almost complete disappearance of inactive STF molecules was observed after each cycle. The absolute number of circulating T-lymphocytes was significantly increased by zinc treatment. The marginal zinc deficiency was also corrected without any appreciable influence on copper plasma levels. A reduction of recurrent infections and an improvement in school attendance after zinc supplementation were recorded. These beneficial effects of zinc supplementation were also noted in those DS children who did not show an apparent zinc deficiency, as assessed by measuring zinc plasma level. The reduced number of circulating B lymphocytes and the impaired lymphocyte responsiveness to phytohaemagglutinin and concanavalin A were not restored. On the whole, these findings suggest that there exists a defect in the bio-availability and/or in the utilization of zinc in DS. This alteration, of unknown origin, can be underestimated on the simple basis of the zinc plasma level and can be corrected with moderate nutritional zinc supplementation.

Prostanoids as second messengers of polypeptide growth factors.

Prostaglandin H synthase (PGHs), also known as cyclooxygenase, is an unstable enzyme whose mRNA has an half life of 10 minutes. Some polypeptide factors have been reported to induce the enzyme in target cells. We have purified and characterized a component of animal sera which behaves as a potent inducer of human monocyte PGHs. This factor, called serum monocytotropic factor, has been identified in human platelets and it appears to be structurally and biochemically different from identified platelet factors, such as platelet derived growth factor (PDGF) and transforming growth factor beta (TGF-beta), while showing strong similarities to colony stimulating factor 1 (CSF-1), so far undetected in platelets. Moreover, we have shown, by immunoblot analysis, that CSF-1 behaves as a potent and specific inducer of monocyte PGHs. The hypothesis that prostanoids may be considered as second messengers of platelet CSF-1 like factor, as well as of other growth factors and that PGHs induction plays a pivotal role in this process, will be illustrated.

Sister chromatid exchanges and DNA repair capability in sanitary workers exposed to ethylene oxide: evaluation of the dose-effect relationship.

Determination of ethylene oxide (EtO) in the working environment and induction of sister chromatid exchanges (SCE) and unscheduled DNA synthesis (UDS) in peripheral lymphocytes of 10 exposed sanitary workers and 10 control subjects matched for sex, age, and smoking habits are reported. The relationship between the external dose of EtO and the frequency of SCE was determined in the above group and in a group of 41 sanitary workers previously studied. The 10 newly examined workers were exposed to EtO concentrations (1.84 ppm as time-weighted average) intermediate between the high (10.7 ppm) and low (0.35 ppm) levels of exposure of the two previously examined groups (19 and 22 workers, respectively). A statistically significant (p less than 0.002) increase of SCE frequency was observed between the present control and exposed groups. The inducibility of unscheduled DNA synthesis by gamma rays was lower in the lymphocytes of the exposed workers than in controls, but the difference was not statistically significant. A significant relationship between the frequency of SCE and the level of EtO exposure for the three exposed groups was demonstrated by two different statistical methods. It is suggested that the present Italian threshold limit value for EtO (3 ppm) may not protect the exposed workers against possible genotoxic effects and that even a chronic exposure to 1 ppm may not be devoid of genotoxic risk.

Immunosuppressive activity of Tamm-Horsfall glycoprotein oligosaccharides: effect of removal of outer sugars and conjugation with a protein carrier.

Tamm-Horsfall (TH) glycoprotein, the major protein of human urine, is, in vitro, a powerful immunosuppressive agent and the activity resides in its oligosaccharide chains. In this study we investigated structural features required for the inhibitory activity of TH glycoprotein oligosaccharides in the one-way mixed lymphocyte reaction (MLR). We found that both high-mannose and complex-type TH glycopeptides, fractionated from Pronase-digested TH glycoprotein, behaved as inhibitors. Sequential exoglycosidase digestion of complex-type TH glycopeptide results in a slight increase of the inhibitory activity, with a maximum after desialylation and beta-galactosidase treatment. These results suggest that the immunosuppressive activity resides in the central portion of TH glycoprotein N-linked oligosaccharides. The conjugation of complex-type TH glycopeptides to a protein carrier, such as bovine serum albumin, greatly enhanced the inhibitory activity. This effect occurred if the TH-glycopeptide conjugate was added to MLR within the first 24 hr. These results indicate that (i) the immunosuppressive activity is strongly dependent on a multivalent interaction between TH oligosaccharides and ligand(s) at the lymphocyte surface; (ii) an early step of cell-cell recognition is the target of the immunosuppressive conjugate; (iii) TH oligosaccharides compete with a carbohydrate recognition system between effector and stimulator cells which contributes to the MLR-induced blastogenesis.