In vitro modulation of differentiation by calcium in organ cultures of human and murine epithelial tissue

Summary The differentiation of epithelial tissue in organ cultures of murine buccal mucosa, various human oral mucosa, and human newborn foreskin was found to be dependent on the calcium concentration of the culture media. In low calcium medium (≤0.07 mM) epithelial differentiation was inhibited. The original stratifying layers separate and can be removed, producing a destratified explant. Histologically such an explant consits of a dorsal epithelial layer of basal keratinocytes resting on an intact basal lamina with subjacent stroma. At 0.01 mM calcium, the epithelial layer was one to two cells thick whereas at 0.07 mM it could be three or more layers in thickness with the most superficial cells being spread over the underlying cells. In addition to differentiation, keratinocyte migration over the sides of the explant (epiboly) and epithelial proliferation as determined by [³H]thymidine autoradiography were reduced by culture in low calcium medium. Redifferentiation occurs upon return to normal calcium levels (1.8 mM); addition of hydrocortisone to low calcium media was found to facilitate this redifferentiation. This investigation was supported by NIH Grant CA29255 from the National Cancer Institute, PHS/DHHS, and by NIH Grant RR01219 supporting the New York State High-Voltage Electron Microscope as a National Biotechnology Resource, awarded by the Division of Research Resources, PHS/DHHS.     

In vitro modulation of proliferation and melanization of melanoma cells by citrate

B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.     

In vitro modulation of normal and diseased human neutrophil function by pentoxifylline

The influence of pentoxifylline on normal and diseased neutrophil function has been studied in vitro. In high concentrations pentoxifylline stimulated human neutrophil chemotaxis toward both bacterial oligopeptides and complement components. Pentoxifylline was also shown in vitro to restore the normal chemotactic capacity of neutrophils from patients with known functional defects, i.e. myelodysplastic syndromes, lazy leucocyte syndrome, juvenile parodontitis, hyper-IgE-syndrome and liver cirrhosis. Pentoxifylline was also shown to strongly inhibit the release of primary and secondary granule release of granulocytes. Moreover, pentoxifylline inhibits both basal and stimulated neutrophil adhesion to both aortic and pulmonary artery calf endothelium. The mechanism whereby pentoxifylline exerts this action is not adequately understood. While our results partially imply interference of pentoxifylline with neutrophil cyclic AMP and/or prostaglandin metabolism, down-regulation of neutrophil functional antigen (e.g. CD11, CD18) expression seems to play a key role in the observed drug effects. Finally, these results indicate that pentoxifylline may be useful in the treatment of granulocyte mediated diseases and symptoms.     

In vitro modulation of cytokine production by lymphocytes in human coccidioidomycosis

The modulation of the cytokine response to coccidioidal antigen by lymphocytes from donors with coccidioidomycosis was examined. In initial experiments, samples from 13 healthy immune donors and seven donors with active coccidioidomycosis anergic to the coccidioidal antigen T27K were assessed for CD3 lymphocyte expression of intracellular IFN-gamma using whole blood analysis. Addition of 10 ng/ml of recombinant IL-12 significantly increased response to T27K among immune and anergic subjects (p<0.05), but the percent of cells expressing IFN-gamma was still significantly greater for immune subjects. Among immune donors, the percentage of CD3 lymphocytes expressing IFN-gamma was significantly reduced with the addition of 10 ng/ml of recombinant IL-4, IL-10, TGF-beta, or their combination (for all, p<0.05). Among anergic donors, addition of 10 ng/ml of anti-IL-10 significantly increased IFN-gamma production (p<0.05), but addition of anti-IL-4 or anti-TGF-beta did not. Among immune donors, the percent of both CD3 lymphocytes and NK cells expressing IFN-gamma after 24h of T27K was increased above control (p<0.05), while the percent of NK cells producing TNF-alpha in response to T27K was not greater than control. Depletion of NK cells from peripheral blood mononuclear cells resulted in significant increases in TNF-alpha and IL-10 (for both, p<0.05) but resulted in no significant decrease in IFN-gamma or IL-2. These data demonstrate a differential response to stimulation with the coccidioidal antigen T27K among donors with coccidioidomycosis that can be manipulated by cell type and cytokine environment.     

In vitro growth properties of Xenopus retinal neurons undergo developmental modulation

To determine whether Xenopus retinal neurons undergo intrinsic developmental changes in growth properties, retinal explants from embryos and tadpoles of different stages were grown on laminin, fibronectin, and collagen I in serum-free media. Growth was assayed in terms of a neurite growth index (NGI) and the appearance of clockwise bundles, or a clockwise growth index (CGI). The first neurites from stage 25 optic vesicles are pioneers and display a unique growth phenotype; they emerge rapidly, survive for a short time, show little substrate preferences for growth (they grow almost as well on BSA as they do on laminin and fibronectin), and form no clockwise bundles under any conditions. Neurites from progressively older retinas (stages 32-37) share with stage 25 neurites the rapid outgrowth pattern, but begin to show substrate preferences and clockwise growth. From stage 40 to 50, the mature growth pattern is expressed; a lag in initial outgrowth, long-term survival, distinct substrate preferences (they grow 10 times better on laminin and fibronectin than on BSA) and display robust clockwise growth patterns on laminin and fibronectin. The acquisition of clockwise growth is independent of optic fiber contact with the tectum or exposure to diffusible factors from mature brain tissues. The results suggest that retinal neurons undergo developmental modulation of surface adhesive properties and/or cytoskeletal organization.     

In vitro differentiation and antigenic changes in human melanoma cell lines

Summary Malignant transformation of melanocytes may be associated with changes in the expression of HLA antigens and melanoma-associated antigens (MAA). To determine whether these changes reflect the differential expression of HLA antigens and MAA by melanocytes at different stages of differentiation, we have studied the effect of the reversible induction of differentiation by fibroblast interferon (interferon ) and/or 12-O-tetradecanoyl-phorbol 13-acetate (TPA) on the expression of HLA antigens and MAA by the melanoma cell lines DU-2, FO-1 and HO-1. The three melanoma cell lines differed in their sensitivity to the differentiating and antiproliferative activity of these two compounds and displayed an increased growth suppression and induction of differentiation, when incubated with the combination of TPA and interferon . Incubation of the three melanoma cell lines with interferon , TPA or their combination resulted in a differential modulation of the expression of membrane-bound high-molecular-mass melanoma-associated antigen, 115-kDa MAA, 100-kDa MAA, intercellular adhesion molecule 1, HLA class I antigens and gene products of the HLA-D region. Each melanoma cell line displayed a unique pattern of antigenic modulation when exposed to the two differentiating agents alone or in combination. No direct relationship was found between the effects of interferon and/or TPA on the growth and differentiation of the three melanoma cell lines and the expression of HLA antigens or the MAA evaluated in the present study. These findings argue against a direct role of any of the antigens tested in the reversible induction of human melanoma cell differentiation in the in vitro system.     

Growth factor modulation of melanoma growth stimulatory activity mRNA expression in human malignant melanoma cells correlates with cell growth

This report demonstrates that the expression of melanoma growth stimulatory activity (MGSA) mRNA can be modulated in a positive fashion in the Hs294T human melanoma cell line by PDGF and MGSA. There is close correlation between MGSA expression and the pattern of cell growth in Hs294T cells.     

Glutamate modulation of human lymphocyte growth: in vitro studies

Peripheral blood mononuclear cell (PBMC) proliferation induced by phytohemagglutinin, or by anti-CD3 alone or plus anti-CD28 monoclonal antibodies (mAb) was inhibited by glutamate (Glu) in a concentration-dependent manner. This inhibition was not reproduced by selective ionotropic Glu receptor agonists, whereas it was potentiated by l-buthionine-(S,R)-sulfoximine, which depletes glutathione (GSH) stores, and counteracted by 2-mercaptoethanol, a preserver of cell thiols. The inhibitory effects of Glu were related to depletion of intracellular GSH stores, since it decreased GSH levels in a concentration-dependent manner. Furthermore, Glu modulated cytokine secretion by anti-CD3 mAb activated PBMC: it increased IFN-gamma (+44.3+/-8.2%) and IL-10 (+31.6+/-9.7%) secretion, whereas that of IL-2, IL-4, IL-5, and TNF-alpha was not affected. These data suggest that high levels of Glu, which can be reached in damaged tissues, modulate lymphocyte responses to activating stimuli by favouring polarization of the T helper effector response.     

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

In vitro modulation of fish phagocytic cells by beta-endorphin

The activation of rainbow trout, Oncorhynchus mykiss, and carp, Cyprinus carpio, phagocytic cells by synthetic chum salmon, O. keta, beta-endorphin was analysed in vitro. Rainbow trout head kidney leukocytes were cultured in RPMI 1640 medium containing 1, 10, 50 or 100 ng ml-1 of chum salmon beta-endorphin and the production of superoxide anion was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with 10 ng ml-1 up to 100 ng ml-1 of beta-endorphin showed an increase in their production of superoxide anion in comparison with control macrophages which were cultured without hormone. beta-endorphin also increased the production of superoxide anion in phagocytic cells prepared from kidney of carp. This stimulation was inhibited by naloxone. Phagocytic cells treated with beta-endorphin also displayed increased phagocytic activity and phagocytic index. These results showed that beta-endorphin in lower vertebrates activates the function of phagocytic cells in vitro.     

In vitro modulation of cisplatin resistance by cytokines.

We have previously demonstrated that treatment of wild-type (wt) T98G malignant glioma cells with Cisplatin (CDDP) led to a resistant phenotype. It has been demonstrated that interleukin 1 (IL-1) potentiates the cytotoxic effect of CDDP and that IL-6 decreases cytotoxicity by inhibition of apoptosis in cancer cells. Here we examined the influence of IL-1 and IL-6 on the sensitivity of resistant and wt T98G cells. Using semi-quantitative PCR reactions in three independent experiments, resistant glioma cells revealed a decreased IL-1alpha (50.3+/-7.2), IL-1beta (56.0+/-4.0) and IL-6 (44. 3+/-18.2) mRNA content compared to wt cells (100%;P<0.05). Resistant and wt cells were positive for the receptors IL-1RI and IL-6R (PCR). To investigate whether IL-1alpha, IL-1beta or IL-6 changes the sensitivity of the resistant and wt cells towards CDDP, cells were incubated up to 7 days with 10(-5) M CDDP and with different concentrations (0, 0.01, 0.1, 1 ng/ml) of cytokine. Sensitivity was tested in a colorimetric assay (MTT). IL-6 did not influence the sensitivity towards CDDP of either wt or resistant cells, while IL-1alpha and IL-1beta enhanced sensitivity of resistant cells to CDDP. These data suggest that autocrine IL-1 production is involved in the mechanisms of resistance in T98G cells.     

In vitro growth inhibition of human cancer cells by novel honokiol analogs

Honokiol possesses many pharmacological activities including anti-cancer properties. Here in, we designed and synthesized honokiol analogs that block major honokiol metabolic pathway which may enhance their effectiveness. We studied their cytotoxicity in human cancer cells and evaluated possible mechanism of cell cycle arrest. Two analogs, namely 2 and 4, showed much higher growth inhibitory activity in A549 human lung cancer cells and significant increase of cell population in the G0-G1 phase. Further elucidation of the inhibition mechanism on cell cycle showed that analogs 2 and 4 inhibit both CDK1 and cyclin B1 protien levels in A549 cells.     

In vitro reconstruction of malignant melanoma

It is report here that three dimensional melanocytic tumors may be reconstituted when adding collagen to the cells harvested during the under covering lattice culture of malignant melanoma explants (cutaneous metastasis). The cells issued from these rebuilt malignant melanoma may be used following the same methods. Thus, the serial making of melanocytic tumors becomes possible. The problem of the relation between cellular differentiation and invasive potential of the cells may be studied using as an original model the rebuilt tumors and the cellular population which are issued from them.     

In vitro modulation of human keratinocyte pro- and anti-inflammatory cytokine production by the capsule of Malassezia species

Malassezia spp. are commensal, cutaneous fungi that are implicated in seborrhoeic dermatitis. We hypothesize that the lipid-rich capsule of Malassezia spp. masks the organism from host detection, and depletion of this layer elicits an inflammatory response. To test this, preparations of capsulated or acapsular [10% (v/v) Triton X-100 treated], viable and nonviable, exponential or stationary phase Malassezia furfur, Malassezia globosa, Malassezia obtusa, Malassezia restricta, Malassezia slooffiae and Malassezia sympodialis, were incubated with normal human keratinocytes. Proinflammatory (IL-6, IL-8, IL-1alpha and tumour necrosis factor-alpha) and anti-inflammatory cytokine (IL-10) release and intracellular IL-10 concentrations were quantified using enzyme-linked immunosorbent assays. Capsulated Malassezia yeasts stimulated limited or no production of inflammatory cytokines, and increased intracellular IL-10 (P < 0.05). Removal of the capsule of many Malassezia preparations caused a significantly increased production of IL-6, IL-8 and IL-1alpha, and a decrease in intracellular IL-10. Notably, acapsular viable, stationary phase M. globosa caused a 66-fold increase in IL-8 production (P < 0.001) and acapsular nonviable, stationary phase M. furfur caused a 38-fold increase in IL-6 production (P < 0.001) and a 12-fold decrease in intracellular IL-10 (P < 0.001). These results support the hypothesis that the lipid layer of Malassezia spp. modulates cytokine production by keratinocytes. This has implications in the pathogenesis of seborrhoeic dermatitis.     

Structure-activity relationship of cinnamic acylsulfonamide analogues on the human EP3 prostanoid receptor

Potent and selective antagonists of the human EP3 receptor have been identified. The structure-activity relationship of the chemical series was conducted and we found several analogues displaying sub-nanomolar K(i) values at the EP3 receptor and micromolar activities at the EP1, EP2 and EP4 receptors. The effect of added human serum albumin (HSA) on the binding affinity at the EP3 receptor was also investigated.     

In vitro modulation of rat liver glucocorticoid receptor by urea

We have examined the influence of urea on the properties of the rat liver glucocorticoid receptor (GR). A 1-h incubation of hepatic cytosol with 1-3 M urea at 0 or at 23 degrees C caused a progressive decrease in the steroid binding efficiency of GR. Urea treatment of cytosol incubated with 20 nM [3H]triamcinolone acetonide caused transformation of glucocorticoid-receptor complexes (GRc) and resulted in an increase in the binding of GRc to DNA-cellulose and ATP-Sepharose. The transforming effect was maximal with 2.5 M urea at 0 degrees C for 1 h, and it caused a shift in the rate of sedimentation of the 9 S untransformed GRc to a 4 S form, similar to that observed upon incubation of the cytosol GRc at 23 degrees C. This 9 to 4 S transformation could also be observed in the presence of Na2MoO4. The Stokes radii of the GRc eluted from a Bio-Gel-A-0.5m agarose column were determined to be 5.9 and 4.9 nm in the absence and presence of 2.5 M urea. The aqueous two-phase partitioning analysis revealed a significant change in surface properties of GR following urea treatment; the observed partition coefficient values (cpm upper phase/bottom phase) were 0.022, 0.208, and 0.60 for GRc, GRc + 23 degrees C, and GRc + 2.5 M urea, respectively. Furthermore, the urea treatment rendered the GRc less negatively charged, forcing their appearance in the flow-through fractions of a DEAE-Sephacel column. These results suggest that urea is a potent in vitro modulator of the physicochemical behavior of GR, influencing both the steroid binding and the process of receptor transformation.     

In vitro thymidine labelling in human and porcine growth plates

An in vitro technique has been used to label dividing cells in the growth plates of human bones with tritiated thymidine. The patterns of labelling in autoradiographs of human plates are described and values given for the labelling index, the number of cells in the proliferation zones and the heights of hypertrophied cells. In two of the four subjects no labelled cells were found in the growth plates and possible causes for these failures are discussed. In vitro labelling data on five porcine growth plates are also presented for comparison with the human data. In both structure and cell kinetics the epiphyseal cartilage plate in the pig is intermediate between the human and rodent plates.     

In vitro growth of B lymphocytes infiltrating human melanoma tissue by transformation with EBV: evidence for secretion of anti-melanoma antibodies by some transformed cells

Epstein Barr virus was used to transform the B lymphocytes infiltrating metastatic tumor tissue from seven patients with melanoma. In this way it was possible to establish continuously growing B lymphoblastoid cell lines (LCL) derived from the tumor-infiltrating B lymphocytes from each of the seven patients. Antibody production of up to 50 micrograms/ml could be achieved by such cultures, and the lymphoblastoid cells could be cloned readily by limit dilution on a feeder layer of irradiated fetal fibroblasts. Preliminary analysis of the antibodies produced by lymphoblastoid cell lines established from tumors from two of the patients indicated that most were of IgM type and bound to a panel of melanoma cell target cells, as well as to some nonmelanoma tumor cell lines. Cloned LCL were produced from the tumor-infiltrating B cells from one of the patients, and of 100 such clones tested, 9% secreted antibody that bound to autologous tumor cells, and one of these clones produced antibody that appeared to be melanoma specific.     

In vitro modulation of the P450 activities of hamster and human lung slices

The respiratory tract is a portal of entry for many environmental chemicals. The respiratory tract plays an important role in the detoxification or metabolic activation of these chemicals, e.g., via cytochrome P450 enzymes. Alterations in the capabilities of these enzymes to metabolize inhaled compounds can, therefore, affect the toxicity of the chemicals. The pulmonary cytochrome P450 activity has been studied in many species, but relatively little is known about this activity in the human lung tissue. In this limited study, we have investigated the possibility of modulating in vitro the P450 activity in lung slices from hamsters and humans. The alkoxyresorufin-O-dealkylase activity was measured in the S9 fraction of lung slices incubated for 24 h with 106 mol/L 20-methylcholanthrene (3MC) or -naphthoflavone (N). The ethoxyresorufin-O-deethylase (EROD) activity was increased by 3MC and N in lung slices of both species. The benzyloxyresorufin-O-deethylase (BROD) activity was decreased after incubation with 3MC but increased with N. These data show that in vitro modulation in lung slices is feasible, although technical improvement is still needed, particularly in relation to the viability of the slices.