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Background: Serum concentrations of some hormones are risk factors for certain cancers, but little is known about their familial associations especially for females., Methods: We measured serum concentrations of estradiol (E(2)), testosterone (T), SHBG, prolactin, and IGF-I for 645 Australian female postmenopausal twins and their sisters [182 monozygotic (MZ) and 107 dizygotic (DZ) pairs and 67 nontwin sisters] using well-established immunoassays. After suitable transformation and adjusting for age, body mass index (BMI), and time since menopause, familial correlations and proportions of variance attributed to genetic (h(2)) and nongenetic factors common to sisterships (c(2)) were estimated under the classic twin multivariate normal model using FISHER., Results: For all serum concentrations except prolactin, MZ, DZ, and sister pairs were correlated (P < 0.001). MZ correlations were in the range 0.5-0.7, and for all serum concentrations, there were no differences between DZ and sister correlations. MZ correlations were greater than DZ and sister correlations for log SHBG (P = 0.0001), IGF-I (P = 0.0002), and square-root T (P = 0.007) but not log E(2) (P = 0.3), and the respective h(2) estimates were 0.56 (SE = 0.14), 0.53 (0.17), 0.39 (0.14), and 0.14 (0.16). For log E(2) and square-root T, c(2) estimates were 0.39 (0.14) and 0.22 (0.12)., Conclusion: There are strong familial correlations in postmenopausal SHBG, IGF-I, and to a lesser extent T, which are consistent with a genetic etiology. For E(2), and to a lesser extent T, correlations are consistent with substantial nongenetic familial factors. The latter might include maternal effects.

Objectives: The present study investigates serum hepatocyte growth factor (HGF) levels in patients with acute and chronic hepatitis B and the relation between these levels and intrahepatic inflammatory markers of the liver and fibrosis, as well as the cerebrospinal fluid (CSF) HGF levels in patients with meningitis and the relation between these levels and CSF findings. To our knowledge this is the first study regarding CSF HGF levels in tuberculous meningitis., Patients and Methods: The study consisted of 35 patients with chronic hepatitis B (HbeAg and HBV-DNA positive), 20 with acute hepatitis B, 20 with acute bacterial meningitis and 15 having tuberculous meningitis. HGF levels in the serum and CSF samples were measured by using the ELISA method., Results: The mean serum HGF levels in acute hepatitis B group were found statistically significantly higher than those in the control group and chronic hepatitis B group (p<0.0001). It was established that serum HGF levels in patients with chronic hepatitis B were significantly correlated with serum alanine aminotransferase (ALT) and HBV-DNA levels (r: 0.816, 0.951; p<0.05, respectively). Similarly, serum HGF levels of patients with chronic hepatitis B were correlated with fibrosis score and hepatic activity index of the liver histopathology (r: 0.750, 0.459; p<0.05, respectively). The mean CSF HGF levels of patients with acute bacterial meningitis and tuberculous meningitis were higher than those in the control group (p<0.05). In addition, it was observed that mean CSF HGF levels in patients suffered from tuberculous meningitis were statistically significantly higher than those in acute bacterial meningitis (p<0.05)., Conclusions: We suggest that serum HGF level in patients with chronic hepatitis B might reflect viral load, necro-inflammatory activity in the liver and the degree of structural progression. Our findings have demonstrated that tuberculous meningitis cause increased HGF concentrations in CSF. It is, therefore, suggested that examination of HGF levels in CSF may provide additional information in the differential diagnosis.

Hepatocyte growth factor-scatter factor (HGF-SF) is a potent hepatic mitogen yet inhibits hepatocellular carcinoma (HCC) cell growth in vitro. Insulin-like growth factor I (IGF-I) is a pleiotropic growth factor shown to be important in cell growth and differentiation in other tumors. We hypothesized that IGF-I may play a role in regulating HGF-SF activity and HCC progression. Using an in vivo model of HCC, we showed elevated IGF-I messenger RNA (mRNA) expression in normal liver from tumor-burdened animals in the absence of changes in circulating IGF-I levels. Analysis of IGF-I receptor (IGF-IR) and HGF-SF (c-met) receptor expression showed significantly higher expression of both receptors in normal liver compared with an HCC specimen. Using cultured HCC cells from this model, we next showed that treatment with IGF-I led to significant increases in mitogen-activated protein kinase (MAPK) activity. Furthermore, we observed significant time-dependent increases in the expression of the c-fos and c-jun proto-oncogenes after addition of IGF-I (n = 5 per group, P <.05). Despite activation of a MAPK pathway and increased proto-oncogene expression, IGF-I failed to significantly affect cell mitogenesis. In contrast, HGF significantly inhibited cell mitogenesis in HCC lines (68.4% +/- 9.4% vs. control, n = 4, P <.05). Pretreatment of HCC cells with IGF-I (60 minutes) led to significant HGF-SF stimulation of total cell mitogenesis dependent on both IGF-I and HGF-SF dose (194% +/- 8% increase vs. control, n = 4, P <.05). In conclusion, tumor burden is important in altering intrahepatic growth factor synthesis. Signal cooperation between multiple cytokine pathways is an important factor in the progression of HCC.

Objective: The association between major depression (MD) and altered immunity appears to be age-related, with differing immune changes found in prepubertal children, young adults, and older adults. There is limited information concerning immunity in adolescents with MD., Method: Thirty-six otherwise healthy medication-free adolescents (aged 14-20; 23 female) from a community sample, meeting Diagnostic Interview Schedule for Children DSM-III-R criteria for unipolar MD, were compared with 36 nondepressed adolescents matched by gender, age, and racial background. A battery of quantitative and functional immune measures was obtained., Results: MD adolescents had increased (p < .05) circulating lymphocytes and lymphocyte subsets; however, altered distribution of lymphocyte subsets was found only for activated T (HLA-DR+) cells (p < .004) and, possibly, natural killer (NK) (CD56+) cells (p < .06), each showing lower percentages in the MD adolescents. Concanavalin A (but not phytohemagglutinin or pokeweed mitogen) mitogen response was lower in the MD adolescents (p < .02). NK cell activity was elevated at higher effector-target ratios (p < .001), an effect not associated with the number of circulating CD56+ (NK) cells., Conclusions: Depressed adolescents showed changes in immune measures that have been found to be altered in other MD groups, although the pattern of effects differs.

Human thioredoxin (Trx) catalyzes intracellular disulfide reductions but has also co-cytokine activity with interleukins after leaderless secretion. A recombinant truncated form of thioredoxin with the 80 N-terminal residues (Trx80) was purified to homogeneity. We discovered that Trx80 by itself is a potent mitogenic cytokine stimulating growth of resting human peripheral blood mononuclear cells. No effect was seen by Trx, but Trx80 at 50-100 nm induced cell proliferation of human peripheral blood mononuclear cells in serum-free synthetic medium, measured as [(3)H]thymidine incorporation after 72 h, with a maximum effect being comparable with that of 5 units/ml of interleukin-2. Trx80 lacked redox activity, but CD spectra suggested a secondary structure similar to Trx. Reduced Trx80 had an M(r) of 25,000, indicating that it is a dimer in solution. We also developed two different sandwich enzyme-linked immunosorbent assays that distinguish between full-length Trx and Trx80 and determined plasma levels of Trx and Trx80 in blood donors. The levels of Trx80 varied from 2 to 175 ng/ml; in comparison levels of Trx varied from 16 to 55 ng/ml without correlation to Trx80. In conclusion, the naturally occurring Trx80 is a novel mitogenic cytokine for normal resting human blood mononuclear cells.

In this study the routine use of different parameters for evaluation of the overall therapeutic outcome in atopic dermatitis was investigated. The disease activity of 117 randomly selected hospitalized patients suffering from atopic dermatitis was routinely assessed using the Severity Scoring of Atopic Dermatitis (SCORAD) index on admission and at discharge. Serum levels of eosinophil cationic protein and mast cell tryptase were determined in parallel both on admission and at discharge. After a mean treatment period of 24+/-12 days a decrease in the SCORAD index from 47.6+/-19.5 to 7.7+/-8.2 was achieved (p<0.001). Serum levels of eosinophil cationic protein decreased from 22.8+/-19.7 microg/l to 15.4+/-17.5 microg/l, whereas serum tryptase levels did not change. However, there was no significant correlation between the changes in SCORAD, eosinophil cationic protein and tryptase in our cohort. Thus, routine determination of serum eosinophil cationic protein or tryptase levels, in addition to evaluation of disease activity using the SCORAD index, is not recommended in unselected patients with atopic dermatitis.

Objective: Superantigens (SAg) are potent immunomodulatory microbial proteins that can activate T cells, B cells, natural killer cells, and monocytes and are known to trigger experimental autoimmune disease. We investigated whether sera from patients with rheumatic diseases contained elevated antibodies to Mycoplasma arthritidis mitogen (MAM) or staphylococcal enterotoxins A and B (SEA and SEB)., Methods: Standard ELISA were used to measure IgG responses to SAg and IgM and IgG rheumatoid factors and total IgM and IgG levels. Modifications of standard lymphocyte proliferation assays were used to determine functional consequences of the observed antibodies., Results: Antibodies to MAM were elevated in sera from patients with rheumatoid arthritis (RA) compared to sera from patients with systemic lupus erythematosus, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, or healthy controls. Responses to other SAg were also elevated in rheumatic disease sera, but the levels were not specific for a given rheumatic disease. Anti-superantigen antibody levels did not correlate with the presence of rheumatoid factor., Conclusion: The selected elevation of antibodies to MAM in RA sera suggests that MAM or a MAM-like molecule might be associated with RA, whereas elevation of antibodies to SEA and SEB in sera from patients with rheumatic diseases was less specific.

Objective: To determine the serum levels and clinical correlation of connective tissue growth factors (CTGF) in patients with systemic sclerosis (SSc)., Methods: Serum samples from patients with limited cutaneous SSc (lSSc, n = 32), diffuse cutaneous SSc (dSSc, n = 28), systemic lupus erythematosus (SLE, n = 30), polymyositis/dermatomyositis (PM/DM, n = 20), and healthy control subjects (n = 30) were examined by ELISA for detection of CTGF., Results: Serum CTGF levels in patients with SSc were significantly higher than those in patients with SLE or PM/DM, and in controls. CTGF levels in patients with dSSc were significantly higher than those in patients with lSSc. As for clinical correlation of CTGF, SSc patients with elevated CTGF had pulmonary fibrosis, decreased DLCO, and decreased vital capacity more frequently than those with normal CTGF levels. Further, DLCO and vital capacity were inversely and directly correlated with serum CTGF levels in patients with SSc. The dSSc patients with disease duration of 1-3 years had significantly elevated levels of CTGF compared with dSSc patients with duration < 1 year or more than 3 years., Conclusion: Serum CTGF levels were increased in patients with SSc, and correlated with the extent of skin sclerosis and the severity of pulmonary fibrosis. In addition, it appears that production of CTGF is involved in the development or maintenance of fibrosis rather than in initiation of fibrosis in SSc. These data suggest that CTGF plays a critical role in the development of fibrosis in SSc.

Serum tryptase was measured with the B12 and G5 antibody-based immunoassays in 25 adult patients with mastocytosis and in 18 controls. Twelve patients had uncomplicated cutaneous mastocytosis (urticaria pigmentosa) and 13 had urticaria pigmentosa with systemic symptoms. Tryptase levels were compared with histamine turnover estimated as urinary excretion of the main histamine metabolite tele-methylimidazoleacetic acid. Elevated B12 tryptase levels (> 20 microg/L) were found in most mastocytosis patients, including five of eight patients with only cutaneous manifestations who had a low urinary histamine metabolite excretion. This indicated a higher sensitivity for diagnosing mild mastocytosis on the basis of levels of serum tryptase as opposed to urinary methylimidazoleacetic acid. However, the serum B12 tryptase assay could not differentiate between urticaria pigmentosa patients with and without systemic disease: the measurement of histamine metabolite excretion probably reflects the mast cell burden more accurately. Serum G5 tryptase levels were generally low in both controls and mastocytosis patients.

Human chorionic gonadotropin (hCG) is a differentiating agent and chemopreventive in rat DMBA-induced breast carcinogenesis. Insulin-like growth factors (IGFs) are potent mitogens for breast cancer cells. We report that the administration of hCG to female rats resulted in a significant increase in serum IGFBP-3 levels at doses greater than 10 UI and a significant decrease in serum IGF-I levels. Northern blot analysis revealed that hCG inhibited hepatic and mammary IGF-I gene expression at doses greater than 10 UI. Mammary IGFBP-5 and IGFBP-2 gene expression significantly increased at high doses of hCG while IGFBP-4 slightly decreased. Treatments greater than 10 UI of hCG resulted in a significant suppression of mammary IGFBP-3 and IGF-I receptor expression. Since IGFs are potent mitogens and antiapoptotic agents for breast cancer, this newly described activity of hCG may contribute to its antiproliferative properties, particularly with regard to the differentiation and inhibition of mammary tumours seen in animal models.

Glial mitogenic effect was investigated in sera from the following groups of subjects: group (1) 31 patients clinically and genetically affected by Neurofibromatosis type 1 (NF1) belonging to different families; group (2) 42 patients without family history of NF1 affected by sporadic neoplasms of the same histogenetic origin as the proliferative lesions that are present in NF1; group (3) 51 healthy volunteers without family history of NF1 nor of neoplastic disease; group (4) 54 clinically healthy relatives of the NF1 patients included in the first group. All NF1 patients and 3/54 healthy relatives had alterations of exons 31 or 32 of NF1 gene. Glial proliferation, measured by [3H]thymidine incorporation, was significantly increased by sera from all NF1 patients and from 23/54 of clinically healthy relatives, as compared to sera from healthy volunteers. This serum mitogenic activity strongly suggests the existence of soluble glial proliferating molecules in NF1 families. The molecular weight (3-30 kDa), the heat- and freeze-stability and the specificity for glial cells, suggest that the molecules responsible for this mitogenic effect are different from the growth factors previously described in NF1-associated tumor extracts and from lymphokines. Within each NF1 family, the maximal serum dilution stimulating glial proliferation was similar both in affected members and in their clinically healthy relatives. Since none of the clinically healthy relatives showing serum mitogenic activity was positive for the NF1 mutation analysis and, conversely, those having altered exons 31 or 32 of NF1 gene did not show any mitogenic activity; these results suggest that the phenotype expression of NF1 might depend not only on the NF1 mutations per se, but also on other genetic or epigenetic factors, such as serum glial proliferating molecules., (Copyright 1998 Elsevier Science B.V.)

3H-thymidine incorporation into isolated ring dove lymphocytes in vitro was used as a measure of lymphocyte proliferation. Lymphocytes taken from doves with increased plasma concentrations of prolactin demonstrated significantly increased 3H-thymidine incorporation. In vitro incubation with mitogens significantly increased incorporation of 3H-thymidine into lymphocytes from non-breeding doves. However, similar treatment of lymphocytes taken from doves which had elevated levels of plasma prolactin failed to induce any further increase in the stimulation index. Antigen caused a significant increase in 3H-thymidine incorporation in non-breeding doves. Antigen administration also led to the production of specific antibodies. The titre of specific anti-human red blood cell (HRBC) agglutinins was greatest in those birds which also had elevated levels of plasma prolactin, reaching significance in the group of incubating doves with naturally occurring increased concentrations of plasma prolactin. The results presented here may be relevant to our understanding of the role of hormones such as prolactin on lymphocyte activation.

Thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+ pump, has been shown to deplete inositol-1,4,5-trisphosphate-sensitive Ca2+ stores. Here we report that when thapsigargin was introduced to serum-stimulated human fibroblasts at a time point just before the G1/S boundary, it completely inhibited expression of cyclin A, activation of p33CDK2 cyclin-dependent kinase and initiation of DNA synthesis. In contrast, the Ca2+ mobilizing ionophore ionomycin was without effect. These findings indicate that Ca2+ inside the inositol-1,4,5-trisphosphate-sensitive Ca2+ stores plays a pivotal role for traverse across the G1/S transition point.

Mitogenic effects of fetal calf serum (FCS), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta), and forskolin to adult mouse Schwann cells were examined by bromodeoxyuridine (BrdU) incorporation and double immunofluorescence for S100 and BrdU. PDGF-BB, basic FGF, and TGF-beta 1 and beta 2 were all mitogenic for Schwann cells in media containing FCS. Forskolin suppressed the mitogenic activity of these factors. In serum-free media, PDGF-BB and bFGF were also mitogenic, but TGF-beta 1 and beta 2 were not. Heparin-binding fractions of FCS obtained by heparin-Sepharose chromatography synergized with TGF-beta 1 and beta 2 to produce a mitogenic response. Since PDGF-BB, acidic FGF, and basic FGF were not detected in these fractions by immunoabsorption and immunoblot assays, the presence of unidentified heparin-binding molecules in FCS bioactive for adult mouse Schwann cells is suggested.

A bioassay system was developed to measure porcine serum mitogenic activity, which is an indicator of growth factor activity. A MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide++ +] cell proliferation assay was used to monitor cell growth. In comparison with electronic cell counting, this bioassay system is sensitive, economical, and semi-automatic. A non-fusing myoblast cell line, BC3H1, was used, making it possible to increase seeding density and thus improve precision. The assay was carried out in 96-well microplates with automatic reading and data processing by a computer-controlled ELISA reader. Results were highly correlated with Kotts' method. This assay system is of potential value in studying growth regulation in pigs.

Sera from rats of either sex and different ages were examined for their ability to stimulate DNA synthesis in BALB/c 3T3 cells. The activity levels of sera from male and female rats were almost the same, with age-related changes in activity also being quite similar. Activity was considerably higher in infant rats (1-month-old), but then, at a young age (6-7 months), decreased drastically for male rats, but not significantly for female rats. It increased again in middle-aged rats (12-13 months old) and was maintained at the same level toward old age (24-26 months old) for both sexes. In order to determine what kinds of growth factors were responsible for these changes, we carried out heparin affinity chromatography on the sera of male rats. Four peaks were obtained for all sera, with individual peaks exhibiting specific age-related changes in activity. Among them a peak which was eluted at 1.1 M NaCl had very high activity. It showed a similar age-related change to that of the whole sera, except for a significant increase at old age, and the factor(s) included in the peak was found to be derived from platelets. These results suggested that the factor(s) in the peak was responsible for maintaining serum mitogenic activity at an old age. The experiments undertaken to characterize this factor suggested that it is a novel one.

An increase in functional sarcolemmal Na(+)-K(+)-ATPase (Na+ pump) precedes proliferation in vascular smooth muscle cells (VSMCs) seeded in 10% fetal bovine serum (FBS), but its role in mitogenesis is unresolved. Enzymatically dispersed canine coronary artery VSMCs were seeded in FBS and studied through confluence. Before a shift in cell cycle (G1-->S, G2 + M) and appearance of the nonmuscle isoform of myosin (MHCnm), intracellular Na+ content (Na+i) and cell volume (CV) increased (day 0 through day 3). Na+ pump number ([3H]-ouabain binding) increased at day 4 followed by a decrease in Na+i and CV. When Na+ pumps were inhibited by the addition of ouabain to FBS, VSMCs were arrested in G1, and MHCnm was not upregulated. Na+i increased similarly to that in FBS but failed to correct to day 0 levels. Withdrawal of ouabain at day 4 in culture led to an increase in Na+ pump number, a decrease in Na+i, entry of cells into S and G2 + M, and upregulation of MHCnm. These data suggest that Na+i, phenotypic modulation, and entry of cells into the cell cycle are temporally related, with Na+ pump-mediated correction of increased Na+i as a key event in the VSMC mitogenic process.

In studies performed on 30 MS cases, as compared to 16 healthy individuals, significantly higher expression of HLA class II antigens, interleukin 2 receptor and antigens specific for lymphocytes B was observed. In MS patients after stimulation by non specific mitogens increased expression of HLA class II antigens and increased proliferation induced by PMA was found. The obtained results suggest absence of any greater modulation of surface antigens on the lymphocytes stimulated by non-specific mitogens. Our observations indicate that the number of activated T cells, as assessed by expression of Il-2 receptor, is in MS patients relatively low.

Serum and skeletal muscle-derived extracts (MDE) were bioassayed for their ability to promote [3H]thymidine incorporation in L6 myoblasts to determine if exercise-training increases mitogenic growth factor activity. Rats were trained by climbing a 60 cm vertical grid with progressively greater weight for 7 and 28 days. Serum from 7 day trained rats and vastus MDE from 7 and 28 day trained rats, but not rectus femoris MDE, had increased mitogenic activity compared to serum and MDE from untrained rats. These data suggest that exercise may increase mitogenic growth factors in some skeletal muscles.

It has been postulated that platelet-derived growth factor and platelet factor 4 (PF4) are involved in the imbalance of the mechanism of medullar stroma maintenance which triggers off the bone marrow myelofibrotic process. In this work we compare the PF4 and the beta-thromboglobulin (beta-TG) and mitogenic activity in platelet-poor plasma (PPP) and platelet extracts (PE) from patients with myeloproliferative disorders (MPD) with those of secondary thrombocytosis (ST) and normal volunteers. Statistically significant differences were found between MPD and ST patients or controls, but none between ST and controls in all the parameters studied. Maximal differences in platelet-derived factors (PDFs) between MPD and control groups were found in polycythemia vera patients. However, the relationship between the presence of myelofibrosis and abnormal levels of beta-TG, PF4 and mitogenic activity in PPP and PE was only observed in patients with agnogenic myeloid metaplasia (AMM). These results show that PDFs are specifically decreased in MPD platelets. Furthermore, no statistical correlation was found between PDFs and the number of platelets. However, other unknown factors or conditions would be necessary to develop myelofibrosis in MPD, which is present in AMM.

Ionizing irradiation induces chromosomal aberrations in directly exposed cells and is known to have mutagenic and carcinogenic potential for the exposed host. Under controlled conditions, we examined whether such clastogenic effects of irradiation might be due in part to radiation-induced plasma factors. Irradiated cells and sera from CF-Nelson rats were used at 15 min, and 1, 7, 14, and 56-70 days after total body irradiation (250 R, n = 67 or 400 R, n = 39). Control rats (n = 44) served as donors of nonirradiated sera and cells. In addition, sera from six rats were irradiated (250 R or 400 R) in vitro. On the average, 298 metaphases from six rats were studied at each time-point. Cytogenetic abnormalities observed included chromatid- and chromosome-type lesions and hyperdiploidy. The frequency of abnormalities was comparable at both radiation doses. Nonirradiated cells exposed in vitro to irradiated serum (15 min postirradiation) exhibited a 36- to 48-fold increment in hyperdiploidy (p = 0.0001) and a 2.- to 2.2-fold rise in chromatid gaps and breaks (p less than 0.01), but none of the chromosome-type aberrations seen in cells exposed to radiation. The clastogenic activity of irradiated plasma persisted in circulation for the 10-wk duration of the study and was not abrogated by dilution with nonirradiated serum. Serum irradiated in vitro was not clastogenic. This study shows that irradiation of rats results in the prompt appearance of clastogenic activity in their plasma. This activity is not due to radiation-induced depletion of protective factors nor to chemical-physical changes of normal plasma components, but results from circulating factors released by irradiated cells.

1. The authors have searched for the existence of mitogenic factor for macrophages in the serums of rats having undergone diverse immune and non-immune inflammatory reactions. 2. The evolution of the mitogenic activity of the inflammatory serums have been studied over a 48 hour period. This study underlined that the appearance of the mitogenic factor is very rapid since it can be found as early as 30 minutes after the injection of the irritant. 3. In most cases the evolution of the mitogenic activity of serums reveals a biphasic aspect: a strong activity is observed 1 to 2 hours after the injection of the irritant followed by a decrease of this activity which becomes weak or even null towards the sixth hour. It then increases to attain, after about 24 hours, values almost equal to those seen for the first peak.

Mitogenic activities on confluent fibroblasts by sera from patients with scleroderma and normal controls were studied. In experiments using eight different fibroblast strains (one human fetal lung fibroblast, one foreskin fibroblast, three adult skin fibroblast, and three scleroderma fibroblast), pooled scleroderma serum showed lower mitogenic activity by 3H-thymidine incorporation assay than pooled normal serum. Individual serum investigations revealed that 11 of 33 patients (33%) showed low mitogenic activities; all 11 patients were receiving dipyridamole. Of 15 patients, 11 (73%) receiving dipyridamole showed low mutagenic activities; none of 18 patients not receiving dipyridamole showed low mitogenic activities. Approximately 70% of this serum activity was inhibited by the addition of anti-platelet-derived growth factor (PDGF) antibody indicating that most of this serum activity seemed to be derived from PDGF. Western blot analysis of extracts of normal sera before and after administration of dipyridamole with antihuman PDGF antibody showed a large decrease in the amount of immunoreactive PDGF present. These data indicate that dipyridamole is an effective drug to lower release of PDGF during blood clotting.

Using human fibroblast cultures in vitro, a study was carried out on the effect of sera from patients with psoriasis and from healthy volunteers on DNA and protein synthesis. Thymidine incorporation was enhanced under the influence of psoriatic sera whereas there was no difference in amino acid incorporation in the control sera. Based on these results, we conclude that systemic psoriasis may be under humoral control.

The abilities of plasma and serum to support the growth of vascular smooth muscle cells maintained on uncoated tissue culture dishes or dishes coated with an extracellular matrix (ECM) have been compared. Vascular smooth muscle cells maintained on plastic dishes and exposed to plasma proliferate poorly; when exposed to serum they proliferate actively. Addition of fibroblast growth factor (FGF) incrases the growth rate of the cultures in both cases. In contrast, when vascular smooth muscle cells are maintained on an ECM, they proliferate equally well exposed to either plasma or serum. Because the cultures had an average doubling time (15 hr) that was already at a minimum, FGF no longer had an effect on vascular smooth muscle cell proliferation. These results raise the possibility that the lack of response of vascular smooth muscle cells, as well as that of other cell types in vitro, to plasma factors is not an intrinsic property of the cells but is rather due to the substrate upon which the cells rest. Because cells maintained on an ECM respond to plasma factors, it is likely that the close contact of the cells with the ECM restores their sensitivity to physiological factors present in plasma.

Fetal calf serum (SVF), insolubilized on a sepharose matrix, retains mitogenic factors (FM) from horse serum (SC) which can be eluted by molar acetic acid. The FM can: 1 degree initiate the synthesis of DNA in serum starved, contact inhibited 3T3 BALB/c fibroblasts and 2 degrees allow at least three generations to take place. The FM have no antitryptic activity and do not allow cell attachment. Weight to weight, this fraction is 10 to 50 times more active than horse serum. Polyacrylamide gel electrophoresis shows that various proteins are present, particularly immunoglobulins. The intervention of epidermal growth factor (EGF), fibroblast growth factor (FGF) or the platelet-derived growth factor (PDGF) have been excluded.

We have previously shown that serum from young women receiving the same combined mestranol-norethindrone containing oral contraceptive (OC) preparation accelerated the proliferation of arterial smooth muscle cells (SMC) in tissue culture, and this in vitro effect was not a direct action of either of its estrogenic or progestogenic constituents. To identify the substance(s) which might contribute to this potentially atherogenic action, blood was obtained from 20 OC users and control women for the measurement of growth hormone, insulin, somatomedins (insulin-like growth factor IGF-I and IGF-II), and the platelet alpha-granule constituents platelet-derived growth factor (PDGF), beta-thromboglobulin, and platelet factor 4 (PF4). No difference was demonstrable between OC users and controls in the levels of any of these growth-promoting hormones, nor in plasma concentrations of any of the platelet alpha-granule proteins. These studies indicate that the enhanced mitogenicity found in OC sera is probably not attributable directly to these hormones or PDGF, and may instead result from an in vivo OC-induced alteration in other as yet unidentified mediators of cellular growth.

Serine proteinase activity has been identified in purified human lymphocyte membranes, and the corresponding enzymes have been isolated from human leukocyte extracts by affinity chromatography. The localization of these enzymes on lymphocyte and granulocyte membranes has been immunochemically demonstrated. Mitogenic activity towards human lymphocytes has been demonstrated for these enzymes, and anti proteinase antibodies inhibit the growth of transformed lymphocytes. The proteinases are similar in many properties to enzymes previously isolated from human leukocytes, and reported to be involved in a wide variety of leukocyte functions.

Multiplication-stimulating activity (MSA), purified from medium conditioned by the BRL-3A rat liver cell line, previously has been shown to be closely related to the human somatomedins or insulin-like growth factors. A radioimmunoassay was utilized to measure MSA levels in sera from fetal, maternal, and young rats. A serum somatomedin-binding protein was found to interfere in the radioimmunoassay by competing with antibody for binding 125I-labeled MSA. Therefore, prior to radioimmunoassay, sera were filtered on Sephadex G-75 in 1 M acetic acid to dissociate and separate somatomedin activity from the binding protein. Concentrations of MSA by radioimmunoassay were 20- to 100-fold higher in feta rat sera (1.8-4.4 micrograms/ml) than in maternal sera. MSA levels gradually decreased after birth, reaching maternal levels by day 25 of extrauterine life. MSA concentrations in feta rat sera also were found to be correspondingly high by a rat liver membrane radioreceptor assay and a competitive binding protein assay using rat serum somatomedin-binding protein. The findings of higher levels of MSA in fetal than in maternal rat sera and the gradual decline in MSA serum concentrations after birth are in direct contrast to total somatomedin activities measured by biosassay. Thus, MSA may function as a growth factor in the fetal rat whereas other somatomedins may play a role in stimulating growth during extrauterine life.

There is increasing evidence that endothelial cell injury and altered endothelial cell function play an important role in the pathogenesis of preeclampsia. Endothelial cell injury can lead to the secretion of potent mitogens by activating platelets and directly through the increased production of peptide growth factors by endothelial cells themselves. This study was undertaken to test the hypotheses that increased secretion of mitogenic factors is a feature of preeclampsia and that this activity could be detected in the serum of preeclamptic women. Paired serum samples were collected in early labor and again at 24 to 48 hours post partum from term patients with preeclampsia (n = 15) and normal pregnant controls (n = 14). A bioassay was used to quantify mitogenic activity in these paired samples by assessing their ability to stimulate the incorporation of tritiated thymidine into deoxyribonucleic acid of confluent, quiescent (GO stage) human fibroblasts in monolayer culture. Mitogenic activity was significantly increased in prepartum, preeclamptic sera compared with normal controls and diminished rapidly postpartum to levels equivalent to normal pre- and postpartum serum. These findings are consistent with endothelial cell injury, a process that we believe plays a central role in the pathophysiology of the preeclamptic syndrome.

Platelet basic protein (PBP) (isoelectric point, 10.0-10.5; apparent Mr, 11,000-15,000) has been purified to homogeneity from material secreted by fresh human platelets after stimulation by thrombin. The purification, using preparative isoelectric focusing and chromatography on heparin-Sepharose, yielded two additional peptides with antiheparin activity that were immunologically identical with PBP: low-affinity platelet factor 4 and beta-thromboglubulin. The purity of the peptides was confirmed by immuoelectrophoresis and by NH2-terminal amino acid analysis. Dansyl chloride-treated PBP yielded a single dansylated amino acid residue (glycine). By using a specific radioimmunoassay it was shown that 10(9) human platelets contain 2-3 microgram of PBP which can be released in response to specific stimulation. PBP is associated with mitogenic activity as assayed in Swiss 3T3 mouse cells cultured in low-serum (0.4-1.5%) medium at levels of about 1 ng/ml and saturating at 10-40 ng/ml. The biological activity of different PBP preparations was variable, presumably due to inhibition by the varying amounts of ampholytes that interfered with the mitogenic activity of the peptide. Mitogenic activity was eluted from NaDodSO4/polyacrylamide gels and shown to comigrate with immunoreactive material and with conventional marker proteins of 14,000-17,000 daltons or with histones of 11,000-15,000 daltons. Evidence is presented that PBP is different from cationic platelet-derived growth factor which has an apparent Mr of 30,000.

Peripheral blood mononuclear cells from a patient with Sézary syndrome which lacked E-rosette-forming ability and surface immunoglobulins, and which displayed a markedly depressed response to a variety of mitogens, were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) on 3 occasions. The first peripheral blood sample (smooth) differed significantly from two later samples (moderate numbers of microvilli) when surface characteristics were examined by SEM; these differences were confirmed by TEM. The Sézary syndrome cells in this patient may be related to a T lymphocyte which has lost certain surface markers and mitogen response characteristics through a process of de-differentiation.

Peritoneal macrophages in culture could be stimulated to divide after treatment with serum or exudate from animals presenting an acute inflammatory reaction. This stimulation is still detected up to 1/4096 dilution for exudate and up to 1/8 192 for serum. This factor can be evidenced in athymic mice presenting an acute inflammatory reaction.

Smooth muscle cells from the aortic media of adult pigs and monkeys have been grown in primary culture by plating cells enzymatically dissociated from the intact aorta. During the first 6 d these cells are in the "contractile" phenotype. That is, they contract slowly in response to angiotensin II and their cytoplasm is filled with both thick and thin myofilaments. In this state they do not incorporate [3H]thymidine into DNA or proliferate in response to normolipemic or hyperlipemic whole blood serum (WBS). After 7 d in culture the cells undergo a spontaneous modulation of phenotype to a "synthetic" state where they cannot be stimulated to contract and their cytoplasm is filled with organelles usually associated with synthesis of secretory protein. Thick myosin-containing filaments can no longer be demonstrated. When challenged with normolipemic or hyperlipemic WBS the cells incorporate [3H]thymidine into DNA and undergo logarithmic growth. It is suggested that when smooth muscle is the contractile phenotype (as normally exists for most cells in the aortic media of adult animals) it does not divide when challenged with serum mitogens but can undergo a change of phenotype to a synthetic state in which division can be stimulated.

In culture medium containing heparinized, heat-inactivated, chicken plasma, normal chicken heart mesenchymal cells do not proliferate but their Rous sarcoma virus-infected counterparts proliferate maximally. In medium containing serum derived from chicken whole blood or plasma, on the other hand, normal chicken heart mesenchymal cells proliferate actively, at similar overall rates and to similar extents. The rate and extent of normal cell proliferation are decreased by a factor of approximately 1/2 with whole blood-derived serum that is heparinized and inactivated; proliferation ceases in plasma-derived serum that is heparinized and inactivated. Heparinization and inactivation of serum does not affect the proliferation of Rous sarcoma virus-infected cells, indicating that this combined treatment eliminates a mitogenic (regulatory) rather than a supportive (nutrient) factor(s) for cell replication. We hypothesize that mitogen(s) is released from plasma protein precursors when plasma clots in the presence of formed elements of the blood or when plasma-derived serum is exposed to cultured cells; heparinization and inactivation, within the framework of this hypothesis, would render nonfunctional the plasma protein precursor(s) from which the mitogen(s) is generated. Alternatively, our data are consistent with the release of two mitogens during blood clotting, one from plasma protein precursors and the other from formed elements of the blood. We also have studied the proliferative behavior of Swiss and BALB/c 3T3 cells in whole blood-derived and plasma-derived human serum. Our studies suggest that the platelet-derived growth factor has an artifactual supportive (nutrient) role, rather than an authentic mitogenic role, in cell replication.

B lymphocytes from patients suffering from systemic lupus erythematosus show increasing rates of spontaneous transformation in prolonged cultivation (144 hr). An additional B cell stimulation could be detected following the addition of sera from lupus erythematosus patients (isolated during the acute phase of disease) to the culture medium. In addition enriched mitogenic serum components from lupus sera increase the 3H-thymidine uptake of Brucella abortus primed B cells of healthy controls. The incorporation of 3H-thymidine with respect to the Brucella antigen concentration is described.

Chick embryo fibroblasts serially propagated in media containing division rate-limiting amounts of fetal bovine serum underwent premature culture senescence as illustrated by accelerated declines in the number of cells incorporating 3H-thymidine, increased population doubling times, reduced cell densities at subcultivation, and reduced replicative life-spans compared to cells grown in medium containing non-rate-limiting amounts of serum. Low serum serially propagated "senescent" cultures returned to 10% serum containing medium had proliferative rates, incorporated 3H-thymidine, and attained saturation densities at confluency similar to younger cells. "Senescent" cells serially propagated in low serum and returned to 10% serum, achieved life-spans similar to cells continuously grown in the presence of 10% serum. The results of these and other studies show that cells serially propagated in the presence of division rate-limiting amounts of fetal bovine serum, or at high inoculation densities, accumulate a substantial number of cells in the population during exponential growth conditions that are not senescent but are prevented from entering DNA synthesis because of mitogen limitations. Our results indicate that the amount of serum mitogen in the growth medium affects only the rate at which cells express their genetically predetermined replicative potential and not the replicative life-span per se. These results are discussed in relation to the techniques that should be employed for studying cellular aging and the mechanism of senescent cell formation.

Growth factor activity, as determined by the stimulation of [3H]thymidine incorporation into the DNA of quiescent 3T3 cells in culture, was found in lysates of guinea pig platelets and megakaryocytes. Quantitative dilution studies demonstrated that, of the cells present in the guinea pig bone marrow, only the megakaryocyte possessed quantitatively significant growth factor activity. The amount of activity present in one megakaryocyte was equivalent to that present in 1,000-5,000 platelets, a value approximately comparable to the number of platelets shed from a single megakaryocyte. It is suggested that guinea pig platelet-derived growth factor has its origin in the megakaryocyte.

Experiments on Wistar rats were carried out to examine the changes in protein, glycoprotein, enzymatic and antigenic blood content after sialotomy of the salivary gland with an object of detecting mitogens. It was demonstrated that injury is accompanied by a moderately pronounced dysproteinemia, an increase in the seromucoid fraction, 48 hours after injury, and by variation in the ratio of LDH isozyme fractions, characteristic of the anaerobic metabolic pattern. No antigens of the injured gland were seen in the blood. The evidence obtained does not confirm the participation of high-molecular proteins of the injured gland in the activation of distant proliferation in the corneal epithelium.

The mitogenic effect of serum pulses, observed previously in quiescent BP-A31 cells, is an artifact due to adsorption of (unknown) serum mitogens to the culture dish; the continuous presence of growth factors is necessary for these cells to traverse the G1 phase. When pretreated with cycloheximide (CH) during the last 8-24 h of quiescence, the BP-A31 cells are particularly sensitive to the adsorbed serum mitogens, as well as to low concentrations of fetal calf serum (less than 0.25%) and to basic fibroblast growth factor. In contrast, the mitogenic activity of insulin was not influenced by CH pretreatment. The expression of the "competence genes" c-myc and JE was only transiently elevated in quiescent BP-A31 cells during CH exposure and did not correlate with the enhancement of the cells' sensitivity to mitogens.

Sera from normal and psoriatic subjects had similar mitogenic effects when added to monolayer cultures of human skin fibroblasts. Fibroblasts derived from a psoriasis patient proliferated faster in both types of sera than fibroblasts from a normal subject, despite similar rates of proliferation in foetal calf serum. The psoriatic fibroblasts appeared to be inherently capable of a greater response to human serum mitogens than the normal fibroblasts.

The mitotic activity of the adrenal cortex shows diurnal variations. Irrespective of these fluctuations, single doses of 2-acetylaminofluorene selectively stimulate cell division in the zona fasciculata. The serum from 2-acetylaminofluorene-treated rats also acts mitogenically after transfer to intact recipients. After simultaneous administration of 3-methylcholanthrene and 2-acetylaminofluorene the mitotic action of the serum is lost. The proliferative response of the adrenal cortex is supposed to be systemically mediated by growth factors which are induced by the carcinogen.

Mitogenic activities for adult skin fibroblasts in 17 sera from untreated patients with scleroderma at the early stage were studied. Mitogenic activities in 10% test sera were measured by the [3H]-thymidine incorporation assay using confluent fibroblasts; there was no significant difference between scleroderma-affected and normal controls. Growth-stimulatory activities for subconfluent fibroblasts in 10% test sera were measured by increased cell numbers; again, there was no significant difference between scleroderma-affected and normal controls. There have been contradictory reports on growth-stimulatory activities in scleroderma sera; our data do not suggest the presence of a specific mitogen in scleroderma sera.

Fibroblast mitogenic activity (MA) has been identified in scleroderma (SD) sera. Control and scleroderma skin fibroblasts in early passage were observed for replication (cell counts) after 72 h of serum exposure. SD sera at 15% concentration induced a significant increase in control fibroblast numbers when compared with control sera; this effect was not seen with SD cells while at higher serum concentrations (30%); SD cells were slightly responsive to the MA. MA was completely abrogated by the proteinase inhibitors STI and TLCK which did not affect mitogens in healthy sera. Circulating mitogenic proteinases selective for fibroblasts could play a role in the fibrosis of SD by modulating fibroblast replication.

We previously proposed that the decreased rates of synthesis of collagen and proteoglycans in vitamin C-deficient guinea pigs were unrelated to the role of ascorbate in proline hydroxylation but might result from modulation of hormones known to change during fasting. In the present studies, we found that sera from guinea pigs on an ascorbate-free diet for 24-28 days or from those fasted for 4 days, with vitamin C supplementation, showed similar changes in the concentrations of several hormones. EGF and IGF-II concentrations were unchanged, but cortisol was increased 3-5 times and growth hormone was increased to approximately twice normal levels. Thyroxine and IGF-I concentrations were decreased to 40% and 25-33% of normal levels, respectively. The decrease in serum IGF-I must occur by a growth hormone-independent pathway. The extent of changes in hormone concentrations in sera from ascorbate-deficient guinea pigs was correlated with the extent of weight loss. Sera from scorbutic and fasted guinea pigs failed to stimulate DNA synthesis in quiescent BALB 3T3 cells in the presence of saturating concentrations of EGF and PDGF. Addition of experimental sera to normal serum showed that lack of mitogenic activity was due to the presence of an inhibitor. Inhibition was not related to IGF-I concentrations in the sera, although it was reversed by the addition of IGF-I to sera from scorbutic or fasted animals. These results support our proposed model and suggest that IGF-I, as well as an inhibitor of its activity, plays a role in the regulation of growth by vitamin C and other nutrients.