A new method to detect directly in culture cell surface membrane immunoglobulins

An ELISA assay is described for the measurement of the smIgG. The method is based on the detection of cell-smIgG directly on the same microplate used for the culture. The cells, preincubated at 37 degrees C for one hour, were cultured in the presence of S-ConA and serum-free medium for two days. Using this strategy, the background noise due to non specific adsorption of IgG to plastic wells and cytophilic antibodies was eliminated. The cells in the presence of S-ConA and serum-free medium adhered to the plastic wells, and the cell-smIgG were detected using an anti-human IgG covalently linked to alkaline phosphatase or its F(ab')2 fragment. The possibility of measuring the modulation of the expression of the cell-smIgG without any additional manipulation is stressed.     

A Serum-Free System for Primary Cultures of Human Pituitary Adenomas

We report the successful use of a serum-free culture system for primary cultures of human pituitary adenomas. The system utilizes histiotypic suspension culture with low protein-binding membrane inserts that enable cells to retain their three-dimensional tissue configuration, closely mimicking the growth pattern in vivo. A serum-free defined medium was developed with CMRL-1969 (Connaught, Willowdale, Ontario, Canada) supplemented with 0.375% albumin bovine Fraction V, 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL sodium selenite, 30 μg/mL putrescine, 6.85 × 10⁻¹¹ M hydrocortisone, and 3.7 × 10⁻¹¹ M tri-iodothyronine (T₃). We analyzed eight surgically resected human pituitary adenomas. Basal pituitary hormone secretion measured by radioimmunoassay of pituitary hormones was compared with hormone hypersecretion in vivo and with control cells of the same tumors cultured in CMRL-1969 with 10% fetal calf serum. The light microscopic, immunocytochemical, and ultrastructural morphology of cells cultured in this serum-free histiotypic system was compared with cells cultured in serum-supplemented media and with cells cultured on collagen-coated plastic; all cultured cells were compared with the morphology of surgically resected tissues of the same specimens. Basal pituitary hormone secretion during 24-hour incubations correlated with the clinical patterns of hormone excess; the data were similar in serum-enriched and serum-free cultures, however, hormone secretion decreased less rapidly in the serum-free cultures. Cells maintained in the histiotypic culture system closely resembled the corresponding surgically resected tumor using the morphologic parameters and were better preserved than those plated in collagen-coated plastic wells. This comparative study indicates that this serum-free histiotypic culture system provides an ideal method of examining pituitary adenomas in vitro without altering the profile of hormone secretion and cell morphology documented in vivo. This system can be used to examine the production and effects of a wide range of hormones and growth factors that have been implicated as causative agents in pituitary tumorigenesis.     

Survival, proliferation and morphological specialization of mouse Schwann cells in a serum-free, fully defined medium

Summary Neonatal mouse dorsal root ganglionic (DRG) cell dissociates were cultured in a synthetic medium with horse serum or the serum-free supplement N₁ (insulin, transferrin, progesterone, putrescine, selenium). Serum-supplemented cultures with added nerve growth factor (NGF) yielded neurons, small flat and spindle cells (Schwann) and large flat cells (fibroblastic elements). However, in serum-free, N₁-supplemented medium plus exogenous NGF, neurons and Schwann cells predominated, with very few large flat cells. In the N₁ medium most Schwann cells assumed a typical spindle shape and were associated with neuritic processes when neurons were present. Upon addition of serum, virtually all of the Schwann cells appeared to abandon physical contact with the neurites and develop a more flattened morphology. In N₁ medium without NGF (no neurites), most Schwann cells still assumed a spindle shape and formed characteristic chain-like associations. Autoradiographic techniques, as well as numerical analyses, demonstrated that in N₁ medium Schwann cells were able to proliferate when associated with neurites but only slightly so in their absence. These Schwann cells showed a marked increase in proliferation when serum was added regardless of the presence or absence of neurites. The above observations may provide a basis for the preparation of purified Schwann cells, alone or in combination with their neurons.     

Effect of physiological concentrations of estradiol on PGI2 and NO in endothelial cells

Objectives: To elucidate the mechanisms by which estrogens protect against occlusive vascular disorders, we studied the effect of 17β-estradiol on the production of prostacyclin (PGI₂) and nitric oxide (NO) in primary cultures of human umbilical vein endothelial cells (HUVECs). Methods: To study the effect of 17β-estradiol on PGI₂ production, HUVECs were incubated in the absence and presence of 17β-estradiol (0.01–10 nmol/l) encapsulated within β-cyclodextrin for 12 h in serum-free medium. To study the effect of 17β-estradiol (100 nmol/l) on maximal calcium-dependent NO production, we used different approaches. First, HUVECs were incubated with 2 μmol/l calcium ionophore A23187 with or without 17β-estradiol (100 nmol/l) for 24 h in serum-free medium. Second, HUVECs were preincubated with or without 17β-estradiol (100 nmol/l) for 12 h in medium supplemented with 2% fetal calf serum, and thereafter incubated in serum-free medium with 2 μmol/l of A23187 and with 100 nmol/l of 17β-estradiol (cells which contained 17β-estradiol during the preincubation period as well as cells which did not) or without it (only cells which did not contain 17β-estradiol during the preincubation period) for 6 h or 24 h. Results: 17β-Estradiol (0.1 nmol/l) increased the concentration of 6-keto-prostaglandin F_1α, a stable metabolite of PGI₂ in the incubation medium, by 16%, and no further increase occurred with higher 17β-estradiol concentrations. The stimulation was prevented by tamoxifen. 17β-Estradiol did not affect NO production in any of our experiments measured as accumulation of nitrate and nitrite in the experimental medium. Conclusions: The stimulatory effect on PGI₂ production of physiological concentrations of 17β-estradiol, shown now for the first time, may provide one explanation for the ability of 17β-estradiol to protect against occlusive vascular disorders.     

An Autocrine Activity Capable of Substituting for Serum in Cell Cultures

Abstract

Provided that high cell densities (above 10⁶/ml) are maintained, a factor-dependent murine hemopoietic progenitor cell line (FDC-PI) will proliferate in serum-free medium. The conditioned medium (CM) from high-density FDC-PI cells permits the serum-free survival of FDC-PI cells even at low density, indicating the existence of a diffusible autocrine factor. The requirement of FDC-PI for a colony-stimulating factor (either IL-3 or GM-CSF) is not abrogated by culturing the cells at high cell density or in the conditioned medium. Furthermore, the CM from FDC-PI enhances the mitogenic stimulation of normal human skin fibroblasts (HSF) by epidermal growth factor (EGF): i.e., the lag period before entry into the cell cycle is shortened by up to 6 hr. The fibroblasts themselves secrete an activity into serum-free medium that appears to be required during mitogenic stimulation by EGF. The HSF-CM also allows FDC-PI cells to survive and proliferate serum-free at low cell densities. Low concentrations of fetal calf serum or human plasma (0.2-2%) have the same effect as FDC-PI-CM and HSF-CM. We have tested many of the known growth factors, and none of them mimicked the autocrine serum replacing activity (ASRA). The activity in human plasma elutes from a gel-filtration column with an apparent molecular weight of 60,000. It appears as if cultured normal cells and cell lines produce molecules capable of complementing the growth factors required for the survival and proliferation of a range of cells in serum-free cultures.     

Induction of immunoglobulin-producing human peripheral blood lymphocytes in serum-free medium

Induction of immunoglobulin-secreting cells from human peripheral blood lymphocytes in a serum-free culture medium was studied. Albumin, transferrin, insulin and fibronectin can replace serum entirely for support of pokeweed mitogen (PWM)-stimulated B lymphocytes, measured by a reverse hemolytic plaque assay using protein A-coated red cells. In this serum-free system, growth and maturation to IgM and IgG secretion occur at the same or higher efficiency as in conventional serum-containing medium, with maximum numbers of plaque-forming cells on day 6 at optimal dose of PWM, 0.5 ～ 5 μg/ml. This system can be used to avoid the interference from undefined serum components.     

Dense IgG4 plasma cell infiltrates associated with chronic infectious aortitis: implications for the diagnosis of IgG4-related disease

BackgroundIgG4-related aortitis is a newly recognized form of noninfectious aortitis that occurs as part of the spectrum of a systemic disease referred to as IgG4-related disease. IgG4-related aortitis is distinguished from giant cell aortitis and Takayasu aortitis in part by the presence of increased numbers of IgG4-expressing plasma cells. Chronic infectious aortitis can also display lymphoplasmacytic infiltrates, but the degree of IgG4 expression in these cases has not been specifically evaluated.MethodsTwo cases of chronic active infectious abdominal aortitis were prospectively identified. Both were due to gram-positive bacteria, and at least one of the cases was due to chronic active Staphylococcus aureus infection. The degree of IgG4 plasma cell infiltration was assessed by immunohistochemistry.ResultsBoth cases of chronic infectious aortitis focally displayed high levels of IgG4-expressing plasma cells, greater than 50% of the IgG-expressing plasma cells, and greater than 50 IgG4-expressing plasma cells per high-power field.ConclusionsFocal dense IgG4 plasma cell infiltrates can be seen in association with chronic infectious aortitis due to gram-positive bacteria, including Staphylococcus aureus. This observation supports the proposal that chronic Staphylococcus aureus infection may stimulate a Th2-mediated elevation in IgG4. The pathologic diagnosis of IgG4-related aortitis should not be based solely on the presence of increased IgG4 plasma cell counts from immunohistochemistry, but requires consideration of the overall pathology, including careful exclusion of infectious aortitis.     

Isolation and cultivation of microvascular endothelial cells from rat lungs: effects of gelatin substratum and serum.

Microvascular endothelial cells from rat lungs were cultured in serum-free medium supplemented with an endothelial growth substance, insulin, hydrocortisone and so on. Five to seven days after plating, cultured cells formed a monolayer. They were identified as endothelial cells by morphology and by positive immunohistochemistry for factor VIII-related antigen, a marker for endothelia cells. Differences between gelatin coated culture plates and plastic culture plates in endothelial cell proliferation were evaluated. Cells plated on uncoated plastic plates had a spindle-shaped morphology and did not express factor VIII-related antigen. Two types of medium, serum-free medium containing endothelial growth substance and basal medium supplemented with 20% fetal calf serum, were also compared in primary culture. In contrast with the serum-free medium, cells cultured in the serum-containing medium showed fibroblast-like morphology and did not express factor VIII-related antigen. These results suggest that a gelatin substratum and serum-free medium containing endothelial growth supplement are necessary for in vitro proliferation of microvascular endothelial cells isolated from rat lungs. The culture method and conditions outlined here allow the proliferation of pure microvascular endothelial cells from rat lungs. It may be useful in studying hematogenous metastasis to the lung and the role of microvascular endothelium in other pulmonary disease.     

B cells capable of spontaneous IgG secretion in cerebrospinal fluid from patients with multiple sclerosis: dependency on local IL-6 production.

Cerebrospinal fluid (CSF) from multiple sclerosis (MS) patients contains B cells capable of spontaneous IgG secretion in vitro. This study analyses the function and regulation of these cells. CSF cells obtained from nine MS patients actively produced IgG during 2-3 days in culture, and the activity decreased when CSF cells were cultured in serum-free medium. CSF cells from four controls did not secrete detectable IgG in vitro. Further experiments revealed that IL-6 played a role on MS CSF IgG-secreting cells, as can be deduced from the following findings: (i) the addition of exogenous IL-6, but not of other cytokines, to serum-free cultures restored missing CSF cell IgG secretion (ii) the inclusion of anti-IL-6, but not of control, blocking MoAb reduced IgG secretion by CSF cells in fetal calf serum (FCS)-containing cultures; and (iii) CSF cells were capable of active IL-6 production in the presence of FCS. These results suggest that endogenous IL-6 production by MS CSF cells seems to be responsible for inducing CSF IgG-secreting B cells to reach terminal differentiation.     

The failure to show a necessary role for C3 in the in vitro antibody response

The in vitro antibody response of mouse spleen cells to TNP coupled to both T-dependent and T-independent carriers as well as to sheep erythrocytes has been studied to investigate the possible role of complement activation in the induction of antibody formation. The following has been found. (1) In vitro responses of both IgM and IgG can be obtained to both T-dependent and T-independent antigens in serum-free media, although they are smaller than those found in serum-containing media. This shows that no exogenous source of complement is necessary for in vitro antibody formation by spleen cells. (2) Similarly, normal antibody responses are obtained if the cultures are grown in human serum depleted of C3b-inactivator, which contains high concentrations of C3b. (3) In the presence of antibody to mouse C3 the response to the T-independent antigen is reduced, the IgM responses being more affected than the IgG. However, purified F(ab′)₂ anti-C3 has no inhibitory effect and it therefore seems likely that it is the formation of intact immune complexes containing Fc rather than the interference with C3 function that is responsible for the inhibition seen. (4) The conventionally purified anti-complementary factor from cobra venom has no effect on the antibody response in serum-free culture or when human or fetal calf sera are used. In no experiment was any potentiation of T-dependent responses observed. However, the presence of quite small concentrations (2 %) of cobra venom factor (CVF)-treated normal mouse serum produced a profound inhibition of the antibody responses, affecting IgG responses more than IgM, and being T-dependent more than T-independent, but no part of the response being unaffected. However, CVF heated to 70 °C which destroys the anticomplementary activity but not the contaminating phospholipase A was equally effective. More highly purified CVF, on the other hand, was not. These findings strongly suggest that the inhibition observed is due to residual phospholipase A reacting with lecithin in the mouse serum rather than to any anticomplementary effect. (5) Spleen cells taken from mice treated with CVF in vivo were also unable to give a full IgG response to the T-dependent antigens if challenged 24 h after CVF treatment. However, after 48 h no inhibition was observed. It therefore seems to us that it is unlikely that C3 plays any real part in the in vitro antibody response. Our data – like the occurrence of genetically C3-deficient humans with apparently normal antibody responses – would seem to preclude any necessary role for C3 in antibody production.     

Development of a novel explant culture method for the isolation of mesenchymal stem cells from human breast tumor

Background: Mesenchymal stem cells (MSCs) were isolated from various sources, including various types of tumors. However choosing an appropriate isolation method is an important step in obtaining cells with optimal quality and yield in companion with economical considerations. The purpose of this study was to isolate more pure MSCs from human breast tumor tissue by a modified explant culture method.

Methods and Materials: The tumor tissues (n = 8) were cut into 1 to 3-mm cube-like pieces (explant). Each explant was placed in a well of 24-well format plates, cultured in Dulbecco’s Modified Eagle’s medium (DMEM), and maintained at 37°C with 5% humidified incubator. Morphological phenotypes of the cells were surveyed by an inverted microscope and wells with rather homogenous fibroblast-like morphology cell were considered as positive and selected for more expansion and characterization.

Results: A total of 185 wells, 63.7% of wells were positive that were chosen for expansion. Flowcytometry analysis demonstrated that isolated cells were positive for CD73, CD44, CD29, CD105, and CD90 but negative for CD11b, CD45, CD34, and HLA?DR. In addition, cells possessed the capability of multipotential differentiation into osteoblasts and adipocytes.     

Improved serum-free culture conditions for spleen-derived murine fibrocytes

Both wound repair and fibrosing diseases involve circulating monocytes entering a tissue and differentiating into fibroblast-like cells called fibrocytes. Fibrocyte biology has been extensively studied in both humans and mice. However, current in vitro techniques to culture murine fibrocytes can take up to two weeks and can require multiple mice to obtain enough circulating monocytes for a single experiment. An alternative source of fibrocytes is the splenic reservoir of monocytes, where one can obtain significantly more cells compared to the peripheral blood. We found that in serum-free medium, fibrocytes differentiate from murine spleen cells within 5 days. To maximize fibrocyte yield, we found the optimal purification technique was to digest the spleen with a collagenase/DNase cocktail, pass the cells through a cell strainer, and lyse the red blood cells. We found that IL-13 and M-CSF significantly enhanced fibrocyte differentiation and that the optimal cell density to promote differentiation was 1.75 × 10⁶ cells/ml. Serum amyloid P (SAP) and cross-linked IgG are two factors known to inhibit the differentiation of human monocytes into fibrocytes. We found that SAP and cross-linked IgG also inhibited the differentiation of murine spleen cells into fibrocytes. These results suggest that culturing murine spleen cells in serum-free medium is a rapid and efficient system to study factors that can affect fibrocyte differentiation.     

In Vitro Studies of Human Prostatic Epithelial Cells: Attempts to Identify Distinguishing Features of Malignant Cells

Abstract

Recent advances in culture techniques have enabled routine establishment and propagation of epithelial cells derived from normal and malignant tissues of the human prostate. Comparative studies of the responses of normal and cancer-derived cell populations to various growth and differentiation factors in vitro were undertaken to examine the possibility that cancer cells might respond differentially. Clonal growth assays in serum-free medium demonstrated that optimal proliferation of normal as well as cancer cell strains was generally dependent on the presence of cholera toxin, epidermal growth factor, pituitary extract, hydrocortisone, insulin, and high levels of calcium in the culture medium, and on the use of collagen-coated dishes. Only one cancer strain responded aberrantly to epidermal growth factor and hydrocortisone. Putative differentiation factors (transforming growth factor-β and vitamin A) inhibited the growth of all normal and cancer strains. The origin of a cancer-derived cell strain that responded similarly to normal strains was verified by positive labeling with a prostate cancer-specific antibody, validating the conclusion from these studies that normal and cancer prostatic epithelial cells are not distinguishable on the basis of responses to the tested factors.     

Synchronization of Rous sarcoma virus production in chick embryo cells

The growth of chick embryo fibroblasts transformed by and producing the Schmidt-Ruppin strain of Rous sarcoma virus was synchronized by exposure to serum-free medium. It was found that virus-specific RNA and protein synthesis and virus release are synchronized in these cells and occur in the early S and G₁ phases of the cell cycle, respectively. These findings support the hypothesis that RSV synthesis is dependent upon specific host cell processes. The duration of the phases of the cell cycle in transformed cells could not be distinguished from the pattern in uninfected cells. Evidence is presented which indicates that the synchrony of transformed cells is achieved by the low pH of the serum-free medium rather than by the absence of serum.     

Staphylococcus aureus cowan I and Branhamella Catarrhalis as B lymphocyte mitogens. Culture conditions for optimal DNA synthesis and selective stimulation of human B lymphocytes

Staphylococcus aureus Cowan I and Branhamella catarrhalis that selectively stimulate human B lymphocytes to increased DNA synthesis in serum-free culture media were shown to do so also in 3 day cultures in medium containing human serum or albumin. After this time the transformed cells expressed little or no surface-bound immunoglobulin and did not form rosettes with sheep erythrocytes. When lymphocytes were cultured with bacterial mitogens in the presence of serum or albumin for more than 3 days transformed T cells appeared. The bacteria induced antibody production mainly of IgM class in lymphocytes cultured in albumin supplemented medium.     

Essential role of tumor necrosis factor-α in the differentiation of human tonsil in vivo induced B cells capable of spontaneous and high-rate immunoglobulin secretion

Human tonsils contain B cells capable of spontaneous and high-rate immunoglobulin (Ig) secretion in vitro. These cells are in vivo induced mature B cells, and, as such, they provide an adequate model for studying tonsil B cell differentiation. The present report analyzes the effect of a variety of factors on purified tonsil B cells capable of spontaneous IgG secretion in fetal calf serum (FCS)-containing and serum-free supplemented cultures. Tumor necrosis factor-(TNF)α was found to be important for these B cells to reach the high-rate IgG-secreting stage, as is indicated by the following findings: (a) none of the factors used modified tonsil B cell IgG secretion in FCS-containing cultures; (b) TNF-α (5-20 ng/ml), but not other cytokines or factors including interleukin (IL)-6, was capable of restoring missing IgG production in serum-free supplemented cultures of tonsil B cells; and (c) IgG secretion in FCS-containing cultures was inhibited by the addition of blocking anti-TNF-α antibodies, but not anti-IL-6 antibodies, and this inhibition could be specifically reversed by exogenous TNF-α. TNF-α was actively produced by tonsil B cells (range 120-750 pg/ml) in the presence, but not in the absence, of FCS. The TNF-α inductive effect occurred during the first 12 h of culture and did not require DNA synthesis. These results indicate that the early and endogenous generation of TNF-α seems to be essential for tonsil in vivo induced B cells to differentiate into the high-rate Ig-secreting stage.     

Studying the Proliferation of Human Peripheral Blood T Lymphocytes in Serum-Free Medium

We compared the cultivation of human peripheral blood lymphocytes in serum-free medium Hybris-2 and RPMI 1640 medium with 10% fetal bovine serum in the presence of phytohemagglutinin and interleukin-2. The optimal concentration of phytohemagglutinin significantly differed in serum-free and serum-containing media (0.5 and 5 mg/ml, respectively). Both mitogens were more potent in stimulating the proliferation of lymphocytes in serum-free medium than in serum-containing medium. Strong proliferation of CD3⁺ and CD4⁺ T lymphocytes was observed in both media. The dynamics of other markers was similar in serum-free and serum-containing media. However, significant differences were revealed between individual donors. Our results indicate that the developed serum-free medium may be used in lymphocyte cultivation for scientific, diagnostic, and therapeutic purposes. Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 10-15, 2009 An erratum to this article can be found at     

Immune complex assays and stored normal human sera

The aim of this study was to determine if rabbit lymphocytes were activated with a specific ligand, anti-immunoglobulin (anti-Ig) in a well-defined serum-free medium. A modified medium, composed of Neuman-Tytell (NT) basal medium supplemented with BSA, transferrin (Tf), and fatty acids (FA), was found to support a vigorous anti-Ig-induced proliferative response of rabbit lymphocytes. The optimal concentrations of BSA, Tf and FA supplements were determined for the mitogen-induced response. The mitogenesis was enhanced by addition of 2-mercaptoethanol. The proliferation could be maintained for 10 days provided cells received fresh medium and mitogen on day 5. The presence of anti-Ig throughout the culture period is required for this extended proliferation. The response did not require the Fc portion of the anti-Ig and was blocked by soluble rabbit F(ab′)₂ fragments. The anti-Ig-activated blasts lacked a T cell surface marker and about half of them re-expressed sIg when anti-Ig was washed out (assayed 24 h later). The viability of the mitogen-induced cells on day 5 was above 55%, whereas the viability of the cells without mitogen was 36%. The supplemented NT medium could also support Con A-induced mitogenesis of rabbit lymphocytes. Additionally, Con A- and LPS-stimulated murine lymphocytes showed considerable proliferation for up to 5 days in the supplemented NT medium.     

Continuous in vitro cultivation of Babesia caballi in serum-free medium

Experiments were undertaken to develop a serum-free medium for the in vitro cultivation of Babesia caballi, a tick-borne hemoprotozoan parasite, one of the causative agents of equine piroplasmosis. A modified HL-1 medium supplemented with horse serum, L-glutamine, antibiotics, and hypoxanthine was used. B. caballi organisms were continuously cultivated at 37 °C in microaerophilous stationary-phase culture in a humidified atmosphere containing 5% CO₂ in air before exposure to serum-free culture conditions. For serum-free propagation, lipid-rich bovine serum albumin (LR-BSA), alone or with chemically defined lipids (CDL), were added instead of serum. Media containing LR-BSA alone or LR-BSA and CDL in various amounts supported the in vitro propagation of B. caballi. Growth was maintained for more than 6 months. The growth rates obtained in serum-free media were similar to those previously obtained in traditional serum-containing medium. Received: 30 June 1998 / Accepted: 29 September 1998     

Terminal Differentiation of Mouse Preadipocyte Cells: The Mitogenic-Adipogenic Role of Growth Hormone is Mediated by the Protein Kinase C Signalling Pathway

Abstract

The role of growth hormone (GH) in the differentiation process of Obi 771 mouse preadipocyte cells has been studied under culture conditions that were serum-free and hormone-supplemented and which were previously shown to lead to terminal differentiation. In the absence of GH, a dramatic decrease in the adipogenic activity of the culture medium could be observed, as indicated 12 days after confluence by the low levels of glycerol-3-phosphate dehydrogenase activity and the sharp reduction of the number of triacylglycerol-containing cells. This decrease in adipogenic activity was accompanied by a parallel loss of the mitogenic potency of the culture medium. Determination of the half-maximal and maximal concentrations of GH required for the restoration of growth and differentiation were identical, 0.5 and 2 nM, respectively. Despite the presence of insulin-like growth factor-I (IGF-I) to substitute for supraphysiological concentrations of insulin and to saturate IGF-I receptor, GH was still required to induce terminal differentiation of a maximal number of cells. However, protein kinase C activators such as prostaglandin F₂a, phorbol esters and diacylglycerol were able to mimic GH in promoting a maximal mitogenic-adipogenic response, indicating that the ability of GH to induce diacylglycerol production (Doglio et al., 1989; Catalioto et al., 1990) plays a prominent role in this process. Furthermore, in agreement with the fact that the mitoses which precede terminal differentiation of Obi 771 preadipocytes are strictly controlled by cAMP and only modulated by protein kinase C., terminal differentiation of Ob 1771 preadipocytes occured in the absence of GH upon supplementation with high concentrations of carbaprostacyclin, added as a cAMP-elevating agent or with 8-Br-cAMP, added as a cAMP analogue. It is concluded that the control exerted by GH on terminal differentiation of mouse preadipocytes corresponds to a modulating mitogenic effect mediated through protein kinase C activation and leading to a potentiation of the cAMP and IGF-I mitogenic signalling pathways.