Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production.

Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.     

Growth, morphology, function, and morphogenetic properties of rat renal glomerular epithelial cells in vitro: effects of retinyl acetate

We report here that retinyl acetate (RA) regulates growth, morphology, function and cell organization in rat renal glomerular epithelial cells (SGE1). SGE1 cells are able to grow in a serum-free medium (DHFs medium) which is supplemented with insulin, transferrin, selenium, bovine serum albumin (BSA), linoleic acid and epidermal growth factor (EGF). When 0.1 and 1 micrograms/ml RA were added to the medium, the growth rates in the sparse culture were noticeably increased, compared to those in DHFs alone, whereas more than 10 micrograms/ml RA was cytotoxic to the cells. In the confluent culture, addition of 0.1, 1.0 and 10 micrograms/ml RA prolonged the cell survival. Since 10 micrograms/ml RA is not cytotoxic to the confluent culture, the cytotoxic action of RA seems to be dependent on cell density as well as RA dose. Ultrastructural observation revealed that RA treatment caused an increase of microvilli and alteration of cell shape, from flattened to columnar. Biochemical and immunological studies revealed that RA treatment increased the activity of r-glutamyl transpeptidase (GGT) and an amount of the membrane component with molecular mass (Mr) of 108,000 which is identical to one of nephritogenic antigens, Fx1A. By using fluorescence phalloidin stain, it was found that RA treatment increased content and organization of F-actin fibers. Furthermore, in collagen-embedding culture, RA induced 3-dimensional (3D) growth of SGE1 cells leading to the formation of organoids, cystic spheres with central lumen, in a serum-free condition; the addition of DHFs to collagen gel alone was ineffective for the 3D growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sera from lipopolysaccharide (LPS)-injected mice exhibit complement-dependent cytotoxicity against syngeneic and autologous spleen cells.

To determine if lymphocytes are able to discriminate between self and nonself, the polyclonal B-cell activator lipopolysaccharide (LPS) was injected into mice, and sera from those mice were tested at different times for their cytotoxic effect against autologous and syngeneic isotope-labeled spleen cells in the presence of complement. It was regularly found that LPS caused the appearance of cytotoxic activity in sera detectable against autologous and syngeneic spleen cells. This cytotoxicity was found to be complement dependent, and it was abolished by absorbing the sera with the target cells. LPS did not induce cytotoxic serum activity in the LPS nonresponder strain C3H/HeJ. When the serum was passed through an anti-mouse Ig column, the eluted sample completely lost its cytotoxicity. It is likely, therefore, that these cytotoxic factors are immunoglobulins with specificity for self, suggesting that tolerance to thymus-dependent autoantigens does not exist at the B-cell level. The implications of this possibility for the understanding of the triggering mechanism of B lymphocytes and for self-nonself discrimination are discussed.     

Immune complexes from rheumatoid arthritis synovial fluid induce FcgammaRIIa dependent and rheumatoid factor correlated production of tumour necrosis factor-alpha by peripheral blood mononuclear cells

Immune complexes (ICs) can induce production of cytokines by peripheral blood mononuclear cells via Fc receptors. Rheumatoid factor (RF) develop in response to ICs in many clinical and experimental settings. We investigated whether and how polyethylene glycol (PEG) precipitated ICs from rheumatoid arthritis (RA) sera and synovial fluid (SF) can influence cytokine production by peripheral blood mononuclear cells. We also examined the relationship between RF and IC induced cytokine production. Parallel sera and SF from 47 RA patients and sera from 15 healthy control individuals were PEG precipitated. The precipitates were added to serum-free peripheral blood mononuclear cell cultures and tumour necrosis factor (TNF)-alpha levels were measured after 20 hours. In separate cell culture experiments FcgammaRIIa and FcgammaRIII were blocked and monocytes were depleted or enriched. RF in serum was determined by nephelometry, and IgG levels in precipitates and anti-cyclic citrullinated peptide antibodies in serum were measured using ELISA. Clinical data were collected from the patients' charts. In two separate investigations, we demonstrated a correlation between RF, PEG-precipitated IgG levels and induction of the proinflammatory cytokine TNF-alpha by PEG-precipitated SF ICs. No such correlation was found for serum ICs. TNF-alpha levels induced by SF precipitates, but not serum precipitates, correlated with the number of swollen and tender joints. Monocytes/macrophages were shown to be the main responder cells, and blockade of FcgammaRIIa, but not blockade of FcgammaRIII, inhibited TNF-alpha production in cultures stimulated with precipitated ICs. Anti-cyclic citrullinated peptide correlated with RF but exhibited no association with IgG content in PEG precipitates or with precipitate-induced TNF-alpha levels. These findings support the hypothesis that SF ICs and correlated RF production are directly linked to cytokine-dependent inflammation in RA. Suppression of monocytes/macrophages in RA joints or blockade of the primate-specific activating FcgammaRIIa receptor might be ways to reduce IC-induced TNF-alpha production in the joints of seropositive RA patients.     

Requirement for a serum component in cytotoxin ("lymphotoxin")-mediated target cell lysis

Serum was found to play an essential role in cytotoxin-mediated target cell lysis. The degree of cytotoxic activity on target L cells was directly related to the concentration of sera in the media incubating the L cells. Furthermore, when ether-ethanol-extracted sera were used in the incubation media, almost no cytotoxic activity was found, indicating that a lipid component of sera, removed by the extraction procedure, might be involved in the action of cytotoxin. The effects of increased sera concentrations and extracted sera occur primarily in the initial 24-hr incubation of L cells (prior to the addition of cytotoxin) rather than when cytotoxin is added, indicating that serum acts by influencing target cell susceptibility to cytotoxin rather than on cytotoxin itself.     

Neutralization by human serum samples of a transmissible agent isolated from the cerebrospinal fluid of neurological patients

A transmissible cytotoxic agent thought to be associated with one or more misfolded protein(s) was found in several cerebrospinal fluid (CSF) samples from neurological patients. Since some experiments carried out to identify this unusual infectious factor showed the block of its propagation by rabbit gammaglobulins (IgGs), the search for such an activity by human IgGs was programmed. Neutralizing assays carried out using human sera as IgGs source showed a blocking property displayed by: twenty serum samples from as many patients with a diagnosis of acute infection, two of ten sera from healthy subjects and four serum samples from patients with lupus erythematous (SLE). When neutralizing sera were tested on cell cultures in immunofluorescence assays for the serum ability to label specific protein( s), similar fluorescent pictures resulted in treated and control cells. On the other hand, the SLE serum samples disclosed a granulosity of the nuclear material of cytotoxic cells in accordance with the DNA apoptotic laddering reported in previous papers. Oxidative disorders, as suggested by the immunoblotting analysis of the antioxidant enzymes Mn-superoxide dismutase (SOD2) and heme-oxygenase 1 (HO-1), point to an alteration of the oxidative pathway among the causes of the DNA damage induced by the cytotoxic transmissible agent under study.     

Inhibition of chondrogenesis by normal mouse serum in cultured chick limb cells

Chick limb-bud mesoderm cells from embryonic stages 22–25 were cultured at high cell densities in media known to support chondrogenesis. Under these conditions the continuous presence of normal mouse serum, at a concentration of 10%, inhibits the ability of the cells to produce toluidine blue-stainable cartilage matrix materials. In contrast, mesodermal cells treated with comparable concentrations of other heterologous sera continue to differentiate much like the control cultures while growing in the presence of the test sera. The inhibitory effect of the serum was shown not to be the result of a general cytotoxic effect on protein synthesis or the inability of the cells to incorporate [³H]d-glucosamine. There was a significant difference however, in the distribution of the incorporated glucosamine. Less label was associated with the cell layer of the treated series, while a greater amount of the incorporated material was found to be secreted into the medium when compared with the control cultures. Studies have shown also that the serum inhibitory response is dose dependent, while the factor(s) itself is non-dialysable, stable to heat and repeated freezing and is not a conventional serum lipoprotein. Following the addition of whole or delipidated mouse serum, a significant increase in lipid droplets appears in the cytoplasm of the cells. Biochemical analyses of mouse serum-treated cells indicate that there is a marked increase in their triglyceride content as compared to the control cells. While the nature of the serum inhibitory factor remains to be determined, the accumulation of triglyceride following mouse serum treatment suggests that this may play a role in modulating the expression of the chondrogenic phenotype.     

Immune complexes from rheumatoid arthritis synovial fluid induce FcγRIIa dependent and rheumatoid factor correlated production of tumour necrosis factor-α by peripheral blood mononuclear cells

Immune complexes (ICs) can induce production of cytokines by peripheral blood mononuclear cells via Fc receptors. Rheumatoid factor (RF) develop in response to ICs in many clinical and experimental settings. We investigated whether and how polyethylene glycol (PEG) precipitated ICs from rheumatoid arthritis (RA) sera and synovial fluid (SF) can influence cytokine production by peripheral blood mononuclear cells. We also examined the relationship between RF and IC induced cytokine production. Parallel sera and SF from 47 RA patients and sera from 15 healthy control individuals were PEG precipitated. The precipitates were added to serum-free peripheral blood mononuclear cell cultures and tumour necrosis factor (TNF)-α levels were measured after 20 hours. In separate cell culture experiments FcγRIIa and FcγRIII were blocked and monocytes were depleted or enriched. RF in serum was determined by nephelometry, and IgG levels in precipitates and anti-cyclic citrullinated peptide antibodies in serum were measured using ELISA. Clinical data were collected from the patients' charts. In two separate investigations, we demonstrated a correlation between RF, PEG-precipitated IgG levels and induction of the proinflammatory cytokine TNF-α by PEG-precipitated SF ICs. No such correlation was found for serum ICs. TNF-α levels induced by SF precipitates, but not serum precipitates, correlated with the number of swollen and tender joints. Monocytes/macrophages were shown to be the main responder cells, and blockade of FcγRIIa, but not blockade of FcγRIII, inhibited TNF-α production in cultures stimulated with precipitated ICs. Anti-cyclic citrullinated peptide correlated with RF but exhibited no association with IgG content in PEG precipitates or with precipitate-induced TNF-α levels. These findings support the hypothesis that SF ICs and correlated RF production are directly linked to cytokine-dependent inflammation in RA. Suppression of monocytes/macrophages in RA joints or blockade of the primate-specific activating FcγRIIa receptor might be ways to reduce IC-induced TNF-α production in the joints of seropositive RA patients.     

Characterization of an antiserum specific for cell surface antigens of rat mast cells.

Rabbits were immunized with rat peritoneal mast cells (RMC) in complete Freund's adjuvant. The antisera (anti-RMC) were checked for their reactivity with RMC by intradermal skin tests in rats. The best serum was selected and absorbed with rat liver cells and rat immunoglobulins, including IgE. The absorbed serum (anti-RMCabs), as well as the anti-RMC serum, were then tested for their reactivity with RMC. Both sera were cytotoxic to RMC but only anti-RMC was cytotoxic for rat lymph node cells. Both sera gave positive reactions in rat skin, as seen by the permeability to Evan's blue dye. The binding of rat IgE to RMC was also inhibited by both sera. A control rabbit anti-rat sarcoma serum absorbed with liver cells did not show any interaction with RMC. When 125I-labeled RMC surface antigens were precipitated with anti-RMCabs and analyzed by SDS polyacrylamide gel electrophoresis, several components were observed. Among these was one with a mobility identical to that of a mast cell surface component that had previously been identified as the receptor for IgE or at least a component thereof.     

Natural killer activity in man under different experimental conditions]

The cytotoxic activity of natural killer (NK) cells against labelled with 3H-uridine target cells of standard human erythromyeloblast line K562 in 63 healthy donors was studied in 14-hr cytotoxic test. The cytotoxic reaction was realized in complete medium supplemented with different types of serum such as foetal calf serum (FCS), autologous or homologous sera in different schemes of incubation. It has been shown that NK cell activity was augmented by 50 per cent (p < 0.05) in the presence of homologous serum added to mononuclear cell suspension for 1 hr at 37 degrees C in comparison with the effect of FCS presence. Thus, the serum of healthy donors contains some factors which not only reflect an individual genotypic information of a donor, but also can be recognized by NK cells and significantly change the cytotoxic NK cell activity. The data obtained exclude possibility of using a homologous serum when positive controls for natural cytotoxic reactions are planned.     

人畸胎瘤细胞株PA-1的体外诱导分化

PA-1细胞是一株从人卵巢性畸胎瘤衍生的细胞株,经10~(-5)mol/L视黄酸诱导后,部分细胞的形态和排列方式发生一定的改变。通过细胞免疫荧光染色,发现诱导后细胞的结蛋白和胞外基质(纤粘连蛋白、层粘连蛋白和腱粘连蛋白)表达与分布模式有较大的变化,并且这些变化与细胞形态改变相关。但大部分PA-1细胞诱导后的生长状况并没有较大的改变,仍分泌与表皮生长因子、类胰岛素生长因子-Ⅰ相关的活性物质。以上结果提示PA-1细胞经视黄酸诱导后一部分细胞可能向肌细胞方向分化。     

Correlations between the presence of islet cell surface antibodies in human serum and the cytotoxic effects on rat islet beta-cells

Isolated pancreatic islets from 7-10 days old Wistar rats were incubated in freshly prepared human serum. The cytotoxic insulin leakage from the islets was correlated with the presence in the sera of islet cell surface antibodies (ICSA) determined by indirect immunofluorescence. Such cytotoxic ICSA were found to be common in Type-I diabetics as well as in nondiabetic subjects who had contact with laboratory rats.     

Culturing of HepG2 cells with human serum improve their functionality and suitability in studies of lipid metabolism

Primary human hepatocytes are considered to be the “gold standard” in studies of lipid metabolism despite a number of disadvantages like large inter-donor differences and inability to proliferate. Human hepatoma HepG2 cells retain many hepatocyte-specific functions but do also exhibit disadvantages like secretion of lipoproteins and bile acids that do not emulate human hepatocytes in vivo. The aim of this study was to investigate whether supplementation of the culturing media with human serum could improve the functionality of HepG2 cells and thereby make them more apposite in studies of lipid metabolism. The cells were cultured with human sera (2%) from three healthy individuals or with fetal bovine serum (10%). Lipoprotein, apolipoprotein, bile acid, albumin, and proprotein subtilisin/kexin type 9 (Pcsk9) concentrations in the cell media, as well as gene and protein expressions were then measured. We found apoB-containing LDL-sized but also apoA1-containing HDL-sized particles, increased bile acid and Pcsk9 concentrations in the cell media, as well as increased expression of genes involved in lipid metabolism and differentiation in HepG2 cells cultured with human sera. Thus, supplementation of the culturing media with human serum improves the functionality of HepG2 cells and makes them more apposite in studies of lipid metabolism.     

Humoral immune response to melanoma-associated membrane antigen and fetal brain antigen demonstrated by indirect membrane immunofluorescence. I

Summary A melanoma-associated membrane antigen and a fetal brain antigen were identified on the surface of a human melanoma cell line by indirect membrane immunofluorescence techniques. The target melanoma cells were grown in gamma globulin-depleted human serum. Sera from melanoma patients were used as the source of antimelanoma antibodies. To remove alloantibodies, the allogeneic sera were preabsorbed with cultured lymphoblastoid cells derived from the peripheral lymphocytes of the donor of the target cell line. To further define the antigen responsible for antibody activity, sequential absorption tests were performed with fetal brain cells, cultured sarcomas, and breast carcinomas. Some antibody activity was removed by fetal brain tissues. Further absorption with fetal brain or the cultured sarcoma or breast carcinoma did not remove additional activity. However, antibody activity was completely removed by either cultured or biopsy-derived melanoma cells. A serum autochthonous to the target cell line was also tested. The antibody titer of the serum was completely removed by absorption with either autochthonous biopsied tumor or an allogeneic melanoma cell line, but not with the normal tissues. Thus it appeared that sera from melanoma patients contained antibody to both a melanoma-associated membrane antigen and a fetal brain antigen.     

Does hydatid disease have protective effects against lung cancer?

We hypothesized that solid tumors rarely occur in patients with hydatid disease. We obtained the serum of 14 patients diagnosed with hydatid disease, the serum of 10 patients who did not have a history of hydatid disease, and the hydatid cyst fluid from six patients. These sera and fluid samples were added at different concentrations to NCI-H209/An1 human lung small cell carcinoma cells and L929 mouse fibroblasts as a control group. Sera of patients with hydatid diseases had cytotoxic effects on NCI-H209/An1 cells, but they did not have cytotoxic effects on fibroblast cells. Sera from healthy subjects did not have a cytotoxic effect on the tumor cell line or control fibroblasts. Cyst fluid, also, did not have toxic effects on the NCI-H209/An1 cell line, but was toxic to fibroblasts up to a 1:32 dilution. Sera from patients with hydatid disease had cytotoxic effects on human small cell lung cancer cells in vitro.     

Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen I (OFA-I)

Summary Oncofetal antigen I (OFA-I) has been identified by immunofluorescence and immune adherence (IA) as a membrane antigen on human tumor cells, which cross-reacts with fetal brain tissue. This antigen induces humoral antibody in patients with cancer. The present work was designed to evaluate the complement-dependent cytotoxic potential of anti-OFA-I antibody produced in melanoma patients against an OFA-I-positive melanoma cell line, UCLA SO M14 (M14). Patients' sera were chosen on the basis of anti-OFA-I activity by IA. Alloantibodies to M14 were removed by absorption of the sera with lymphoblastoid cells autologous to M14. Fourteen sera were tested, and all demonstrated cytotoxic activity in the presence of rabbit complement. Human complement was also shown to mediate cytotoxicity, although less effectively than rabbit complement. The specificity of the reaction was confirmed when the cytotoxic capability could be absorbed by fetal brain, but not by autologous fetal liver tissues. These results indicate that a patient's serum antibody to OFA-I can lyse tumor cells expressing this antigen.     

Circulating immune complexes as possible cause for anticomplementary activity in humans with malignant melanoma

Summary Sera from 98 melanoma patients, 20 noncancer patients with immune complex-associated diseases, and 90 normal donors were analyzed for anticomplementary (AC) activity by the complement consumption method. Some of these sera were also tested for immune complex-like materials by the Raji cell radioimmune assay. In addition, serum samples from ten melanoma patients were analyzed serially to correlate the AC activity with clinical course. Significant levels of Ac activity were found in 45% of melanoma sera, 75% of nonmalignant immune complex-associated disease sera, and 10% of normal donors' sera. In some patients, AC activity decreased and became undetectable as their disease progressed. AC-negative serum samples taken from melanoma patients late in the course of disease when the tumor burden was large became anticomplementary when mixed with autologous or allogeneic serum samples taken earlier at the time of little or no tumor burden. The early serum samples contained antibodies against autologous tumor extracts, as shown by a complement fixation test. Absorption of early serum samples with cultured allogeneic melanoma cells reduced their ability to consume complement when mixed with autologous late serum samples, suggesting the presence of free antigen in the latter. The mixed samples of early and late sera and the sera positive in the complement consumption test contained heavy nonmonomeric IgG. Therefore, the AC activity of melanoma sera could be due to tumor-associated antigen-antibody complexes.     

The contact-mediated response of peripheral-blood monocytes to preactivated T cells is suppressed by serum factors in rheumatoid arthritis

Stimulation of monocytes/macrophages after cell contact with preactivated T cells has been suggested to contribute to the excessive TNF-α production in rheumatoid arthritis (RA). In this study, T cell-contact-dependent TNF-α production by peripheral-blood monocytes in vitro was investigated and found to be significantly lower in treated and untreated patients with RA than in healthy controls. This suppression was not due to a general deficiency of monocytes to respond, because responses to lipopolysaccharide were comparable in patients and controls. In agreement with the pivotal role of TNF-α in RA, T cell-dependent induction of TNF-α in synovial macrophages was fivefold to tenfold higher than in peripheral-blood monocytes from either patients or controls. The decreased response of peripheral-blood monocytes from patients with RA was found to be mediated by inhibitory serum factors, because the addition of patient sera to monocytes from healthy controls suppressed TNF-α response in the co-culture assay. Preincubation of monocytes from healthy controls with RA serum was sufficient to suppress the subsequent TNF-α response in T cell co-cultures, indicating that inhibitory factors do indeed bind to monocyte surfaces, which might represent a regulatory counter-action of the immune system to the long-standing and consuming autoimmune process in RA. There are some indications that apolipoprotein A-1 might be part of this regulatory system.     

Effects of Bacillus sphaericus 1593 and 2362 spore/crystal toxin on cultured mosquito cells

An in vitro assay system for the toxin of Bacillus sphaericus strains 1593 and 2362 has been developed utilizing cultured Culex quinquefasciatus cells. The cytotoxic activity of extracts of B. sphaericus strain 1593 did not necessarily correlate with insecticidal activity. Cytotoxicity and larvicidal activity were neutralized by immune rabbit serum prepared against crude toxin extracts as well as by serum prepared against purified toxin from strain 2362. This purified toxin was also found to be cytotoxic. Activation with mosquito larval gut homogenates enhanced cytotoxicity of both 1593 extracts and purified toxin from 2362. The activity of cytotoxic preparations against three mosquito cell lines paralleled the activity of B. sphaericus spores against larvae of these mosquito species. The results suggest the presence of a protoxin and one or more cytotoxic proteins derived from it.