TdR Incorporation Research Articles

Regulation of muscle cell proliferation by extracts from crushed muscle

Cell culture studies were conducted to determine whether myotrophic factors were released from mature murine or bovine muscle following a crush injury. Murine crushed muscle extract (mCME) was added to C2 muscle (satellite) cell cultures at concentrations of 0, 50, 100, 200, and 400 micrograms of total protein/mL. Bovine crushed muscle extract (bCME) was added at concentrations of 0, 100, 200, 400, and 500 micrograms/mL. Murine CME and bCME at each concentration caused an increase (P < .01) in [3H]TdR incorporation into muscle cells compared to control cultures. The saturating concentrations (P < .01) of CME in the presence of 2% FBS were approximately 200 and 400 micrograms/mL for murine and bovine extracts, respectively. Murine CME or bCME acted in an additive fashion with independent, saturating concentrations of the insulin-like growth factors (IGF-I and IGF-II), basic fibroblast growth factor (bFGF), and transforming growth factor-beta (TGF-beta) to increase (P < .01) C2 muscle cell proliferation. Subsequently, in separate experiments, mCME or bCME acted additively with a combination of all growth factors to increase (P < .01) cell proliferation. Combining mCME and bCME at saturating levels in one treatment was not (P > .05) additive to that elicited by either CME alone. These results suggest that myotrophic factors are released following injury in mature skeletal muscle, and they are not species-specific.

Effects of β1‐integrin antisense phosphorothioate‐modified oligonucleotide on myoblast behaviour in vitro

Myoblasts gene-engineered in vitro and then injected in vivo are safe, efficient options for gene therapy. While isolation of satellite cells is routinely achieved, their proliferation potential in vitro remains a limiting factor for cell transplantation under clinical conditions. We have studied the role of reversible inhibition of gene expression by antisense oligonucleotides on the proliferation of the myogenic cells. Addition of antisense oligonucleotides to myoblast cultures has been used to inhibit specifically the expression of the beta 1-integrin subunit gene. Here we show that the effects of multiple pulses of a phosphorothioate oligodeoxinucleotide antisense on the attachment to substrata and on the proliferation of myoblasts are dose-dependent. The addition of antisense to rat myoblasts caused rounding up of the cells and most of the cells became detached after several days in culture. A single pulse did not show any consistent effect, while in the presence of continuously administered antisense, the relative numbers of myoblasts in the treated muscle culture increased. We have no evidence of inhibition of myoblast fusion under these conditions. On the other hand, [3H]-TdR incorporation, total DNA and total number of cells decreased in antisense-treated cultures thus demonstrating an inhibitory effect of the phosphorothioate oligonucleotides on DNA synthesis. These side-effects could be overcome by substituting the phosphorothioate by unmodified oligonucleotides, so decreasing the half-life of the antisense, but also its toxicity. The overall results suggest a potential role of integrin antisense strategy in modulating the potential of myoblasts to proliferate.

Immunosuppressive activity of a porcine high-molecular weight uterine macromolecule is associated with transforming growth factor-β

A ≥230 kDa lymphocyte suppressor component recovered from uterine luminal protein (ULP) secretions of Landrace and Yorkshire gilts on day 15 of pregnancy was further purified by anion-exchange (DEAE Sepharose CL-6B) and gel filtration (Sepharose CL-6B) chromatography. Macromolecules within each peak fraction were tested for suppression of thymidine (TdR) incorporation into phytohemagglutinin (PHA)-treated peripheral blood lymphocytes (PBL). A neutralizing antibody to transforming growth factor-β (TGF-β) was added to additional cultures in order to determine whether suppressor activity of high-molecular weight macromolecules was associated with TGF-β. The ≥230 kDa component suppressed ( P < 0.0005) TdR incorporation into PHA-treated PBL and anti-TGF-β reversed ( P < 0.034) the suppressor response. Following anion-exchange chromatography of this component, suppressor activity was greatest ( P < 0.05) for an acidic component (fraction III), which comprised 45.9% of eluted macromolecules. Sepharose CL-6B chromatography of fraction III resulted in a major (58.7% of eluted macromolecules) component (≥4 × 10 6 Da (≥4 MDa), eluted at the void volume) which suppressed ( P < 0.05) TdR incorporation into PHA-treated PBL. Anti-TGF-β reduced the suppressor activity of this macromolecule by 34.2%. Suppressor responses were not associated with viabilities of PBL. These data demonstrate that ULP secretions from gilts on day 15 of pregnancy contain a ≥ 4 MDa macromolecule which suppresses proliferative responses of PBL and likely serves as a carrier for TGF-β.

Eicosanoid-induced growth and signaling events in rat glomerular mesangial cells

Renal glomerular injury frequently results in proliferation of a specialized supporting cell of the glomerular capillary known as the mesangial cell. In various forms of renal injury there is enhanced glomerular synthesis of specific eicosanoids of the arachidonic cyclooxygenase and lipoxygenase pathways including prostaglandin (PG) F 2α, thromboxane (Tx) A 2, the hydroxyeicosatetraenoic acids 12-HETE and 5-HETE, and the leukotrienes LTB 4 and LTD 4 and attempts have been made to link these eicosanoids with injury-induced mesangial cell growth. In this study, the growth promoting effect of these eicosanoids on glomerular mesangial cells was correlated with activation of two growth regulatory enzymes: phospholipase C (PLC) and protein kinase C (PKC). PGF 2α, and TxA 2 endoperoxide analog U-46619, and LTD 4 significantly enhanced DNA synthesis ((as assessed by [ 3H]thymidine (TdR) incorporation)] in relatively quiescent (0.5% serum) mesangial cells, activated PLC [as assessed by increased 1,4,5-inositol tris-phosphate (IP 3) generation and diacylglycerol (DAG) synthesis], and activated PKC (as assessed by translocation of the enzyme activity from the cytosol to the membrane). The effect of PGF 2α on IP 3 generation was not blocked by the TxA 2 receptor antagonist, SQ-29,548. PGF 2α was the most effective eicosanoid in inducing all three events, and concentrations that enhanced TdR incorporation (1 μM) also activated PLC and PKC. In contrast, concentrations of U-46619 and LTD 4 which enhanced TdR incorporation (1 μM), also activated PLC, but were insufficient to also activate PKC. Our observations indicate that the growth-promoting effect of PGF 2α, U-46619, and LTD 4 can best be correlated with LPC activation. In addition, PGF 2α does not mediate PLC activation through binding to the TxA 2 receptor.

Ligand binding to Fc gamma R induces c-myc-dependent apoptosis in IL-2-stimulated NK cells.

The role of signals transduced via Fc gamma RIIIA in the modulation of the proliferative potential of NK cells has been investigated. Fc gamma R stimulation does not induce NK cell proliferation, and inhibits that induced by IL-2, but not by IL-12, as measured by [3H]TdR incorporation, without affecting entrance or progression through cell cycle. The inhibitory effect depends, at least in part, on induced apoptosis of the cells, detected by both light and electron microscopy examination. Fc gamma R stimulation induces apoptosis only in NK cells that have been previously activated by IL-2: this occurs within 3 h from receptor stimulation and is independent from de novo receptor-induced RNA or protein synthesis, but requires receptor-induced activation of protein tyrosine kinases and extracellular Ca2+ influx. IL-2 induces accumulation of c-myc mRNA in NK cells, and treatment of the cells with c-myc antisense oligodeoxyribonucleotides during the IL-2 stimulation phase inhibits the susceptibility to Fc gamma RIIIA-induced cell death, indicating that the induction of sustained levels of this proto-oncogene is necessary for the phenomenon. Thus, a two-step model is suggested for the Fc gamma R-induced apoptosis in IL-2 activated NK cells: the first step involves induced expression of c-myc, and possibly other permissive factors, upon IL-2 prestimulation; the second depends directly on the stimulation of the receptor, independently of additional gene induction. The evidence presented here suggests a mechanism of control of NK cell expansion at the latest stages of Ab-dependent immune responses.

A decapeptide (Gln-Asp-Leu-Thr-Met-Lys-Tyr-Gln-Ile-Phe) from human melanoma is recognized by CTL in melanoma patients.

The decapeptide 810 (QDLTMKYQIF) contains the antigenic epitope (KYQI) recognized by human mAb L92. This sequence is found in a 43-kDa protein associated with human melanoma M14. We examined the expression of 810 in human cells and its involvement in the cellular immune responses of melanoma patients. Nineteen stage III melanoma patients and 19 normal donors were studied for their responses to 810. All patients were immunized in vivo with an allogeneic melanoma cell vaccine. PBMC cytotoxicity was tested on autologous EBV-transformed B lymphoblastoid cell lines (BCL) pulsed with 810 and autologous melanomas. Proliferative responses of PBMC to 810 were evaluated by using [3H]Tdr incorporation assays. Western blotting revealed that the 43-kDa protein was not specific to melanoma but was common to various cells. However, the percentage of cytotoxicity of PBMC against autologous 810-pulsed BCL was significantly greater in melanoma patients than in normal controls (p < 0.005). Cytotoxicity was increased after melanoma cell vaccine immunization in 15 patients (78%). Proliferative responses to 810 were observed only in melanoma patients and were enhanced in 12 patients (63%) after vaccination. Restimulation of PBMC from vaccinated patients with 810 increased cytotoxicity against both autologous 810-pulsed BCL and melanomas. These targets were lysed by CD8+ T lymphocytes in an HLA class I-restricted manner. HLA-A2 and -A11 seemed to serve as the 810-presenting molecule. Our findings indicate that 810 may function as an epitope for CTL on human melanoma and can be used as a vaccine.

High performance liquid chromatography of Carbon-11 labeled thymidine and its major catabolites for clinical PET studies

The identity and pharmacology of the rapidly formed radiolabeled metabolites formed following i.v. injection of C-11 thymidine (TdR) labeled in the 5-methyl-position are being studied in animal models and patients with cancer. A high performance liquid chromatographic (HPLC) procedure has been developed for the separation of these metabolites including thymine, dihydrothymine, β-ureidoisobutyric acid, and β-aminoisobutyric acid from plasma and tissue extracts. Information obtained regarding the pharmacokinetics of the metabolites are being used to generate mathematical models for C-11 TdR incorporation rates into DNA.

Modulatory effects of eicosanoids on mesangial cell growth in response to immune injury

In a rat model of glomerular mesangial cell immune injury by a monoclonal antibody (ER4) against the mesangial cell membrane antigen Thy 1.1 and in which mesangial cell proliferation is a prominent feature, we examined the role of arachidonate 5- and 12-lipoxygenation (LO) eicosanoids and of thromboxane (Tx) in modulating the proliferative response. Significant increments in glomerular cell proliferation, assessed by counting glomerular cells positive for the Proliferating Cell Nuclear Antigen (PCNA) and by the incorporation of [3H]thymidine ([3H]TdR) in mesangial cell outgrowths from explanted glomeruli, occurred during the mesangioproliferative phase of injury. This event was abrogated in animals depleted of leukocytes or platelets prior to administration of ER4 and in animals pretreated with the arachidonate 5-LO inhibitor MK886. Pretreatment with the Tx synthase inhibitor, Furegrelate, or the arachidonate 12-LO inhibitor, Baicalein, had no effect, indicating that eicosanoids of arachidonate 5-LO but not those of 12-LO or Tx modulate mesangial cell proliferation following immune injury. We further identified those 5-lipoxygenation eicosanoids with growth modulatory effects on cultured mesangial cells. Leukotriene (LT)C4 and D4 but not LTB4 or 5-hydroxyeicosatetraenoic (HETE) acid enhanced [3H]TdR incorporation in growth-arrested mesangial cells. This effect of LTC4 and LTD4 was abrogated by the specific protein kinase C (PKC) inhibitor calphostin C, indicating a PKC-dependent mechanism. LTC4 and LTD4 but not 5-HETE or LTB4 also increased mesangial cell mass levels of the endogenous PKC activator diacylglycerol. The observations indicate that leukocyte-derived arachidonate 5-LO eicosanoids modulate mesangial cell proliferation following immune injury. Of these LTC4 and LTD4 are the likely candidates as they promote mesangial cell growth via a PKC-dependent mechanisms.

Resistance of Malignant Trophoblast Cells to both the Anti-proliferative and Anti-invasive Effects of Transforming Growth Factor-β

Human placental trophoblast invasion of the uterus is a highly controlled event. We had shown that transforming growth factor-β (TGF-β) produced in the pregnant uterus controls invasiveness and reduces proliferation of first trimester placental trophoblasts in vitro. The anti-invasive effect of TGF-β was due, at least in part, to induction of tissue inhibitor of metalloproteinases (TIMP)-1. In the present study we compared the effects of TGF-β on proliferation ([3H]-TdR incorporation) and invasiveness (3-day Matrigel invasion assay) of JAR and JEG-3 choriocarcinoma cells vs normal first trimester human trophoblast cells. Transcripts of type IV collagenases (72- and 92-kDa enzymes, i.e., gelatinases A and B) and their inhibitors (TIMP-1 and TIMP-2) in these cells were measured by Northern analysis, and secretion of gelatinases and plasminogen activators (PAs) was evaluated by gel zymography. The results revealed that: (a) TGF-β inhibited invasiveness and proliferation of normal trophoblast but not JAR and JEG-3 choriocarcinoma cells; (b) gelatinase A mRNA, expressed by the normal trophoblast and JAR cells, was upregulated in the presence of TGF-β; (c) gelatinase B mRNA was not detected in the total RNA preparations of treated or untreated normal trophoblast or choriocarcinoma cells; (d) TGF-β significantly upregulated the levels of TIMP-1 mRNA in the normal trophoblasts, but this transcript was very low in treated as well as untreated choriocarcinoma cells; TGF-β also upregulated the 3.5-kb TIMP-2 message in the normal trophoblast; (e) gelatin zymography revealed a distinct band of approximately 68-kDa (gelatinase A) in the conditioned media of normal trophoblast and JAR cells; however, TGF-β did not change the level of secretion of this gelatinase; and (f) the normal trophoblast also exhibited significant PA secretion (casein zymography) which was reduced in the presence of TGF-β. PA secretion by the malignant trophoblast cells was low and unaffected by TGF-β. These findings suggest that choriocarcinoma cells may become refractory to the mechanisms which control normal trophoblast proliferation and invasiveness. Concurrent resistance to antiproliferative and anti-invasive molecules such as TGF-β may be highly relevant to tumor progression.

Immunosuppressive Effects of Azelastine Hydrochloride on Contact Hypersensitivity and T-Cell Proliferative Response: A Comparative Study with FK-506

Azelastine hydrochloride (AZE) is an anti-allergic drug that inhibits the release of various chemical mediators from mast cells. We compared the immunosuppressive effects of AZE and FK-506 in vivo and in vitro. Topical application of AZE strongly inhibited the efferent phase of contact hypersensitivity, as did application of FK-506. In in vitro experiments, we found that 1) the suppression by AZE on interleukin (IL)-2 production from splenic T cells was partial and considerably large amounts of IL-2 were still produced, even in the presence of 10(-5) M of AZE, which was in sharp contrast to the observed marked inhibition of [3H]-TdR incorporation; 2) AZE significantly inhibited the phorbol myristate acetate-induced IL-2 responsiveness; 3) AZE did not inhibit the IL-2 receptor alpha expression of activated T cells; and 4) the significant inhibitory action was still observed even when AZE was added at 48 h after the initiation of culture. In regard to FK-506, we found that 1) FK-506 completely blocked the production of IL-2; 2) exogeneous IL-2 consistently restored the FK-506-induced inhibition; 3) FK-506 affected the phorbol myristate acetate-induced IL-2 responsiveness very little, if any; and 4) the significant suppression was observed only when FK-506 was added within 24 h after the initiation of culture. Thus, AZE exerts its in vitro immunosuppressive activity preferentially by interfering with the IL-2 responsiveness, with partial inhibition of IL-2 production. Conversely, FK-506 acts as a strong inhibitor of IL-2 production without a prominent effect on IL-2 responsiveness. The immunosuppressive activity of AZE shown in vitro may also be operative in vivo and may be applicable for topical use.

Kirsten sarcoma virus-immortalized mast cell lines. Reversible inhibition of growth by dexamethasone and evidence for the presence of an autocrine growth factor.

Expansion of mast cell numbers occurs in vivo during certain inflammatory reactions, including active fibrosis, parasite infestations, and immediate hypersensitivity reactions. T cell-produced cytokines, including IL-3 and IL-4, are thought to control this mast cell proliferation in part, and glucocorticoid regulation of T cell-produced cytokines is thought to account for diminished mast cell proliferation during administration of glucocorticoids in vivo. Here we show that glucocorticoids have a direct inhibitory effect on proliferation of Kirsten sarcoma virus-immortalized mast cells (KiSV-MC) in vitro, with an ID50 of 1.0 +/- 0.2 nM dexamethasone (mean +/- SD, n = 4). At 10 nM dexamethasone, KiSV-MC proliferation was inhibited by 83 +/- 5% (mean +/- SD, n = 4). As determined by trypan blue staining and [3H]TdR incorporation, the glucocorticoid-mediated growth inhibition was due to diminished mast cell proliferation rather than cell death and was completely reversible after 6 days of glucocorticoid treatment. By cell cycle analysis, glucocorticoids diminished the percentage of mast cells in S phase and increased the percentage in G0-G1 phase. Although we show that the KiSV-MC proliferate via an autocrine mechanism, glucocorticoid treatment of the KiSV-MC did not inhibit their production of the autocrine growth factor. During 6 days of treatment with 1 to 1000 nM dexamethasone, mast cell carboxypeptidase activity increased by a maximum of 3.5-fold. In contrast, total chymotryptic and tryptic esterase activities diminished by as much as 40% with dexamethasone treatment. We conclude that glucocorticoids directly affect mast cell growth and differentiation at levels equal to the reported Kd for glucocorticoid receptors on other immune cells.

Thiol-mediated redox regulation of lymphocyte proliferation. Possible involvement of adult T cell leukemia-derived factor and glutathione in transferrin receptor expression.

The proliferative response of PBMC to PHA, Con A, OKT3 mAb and IL-2-dependent proliferation of PHA-blasts was examined in a thiol-free environment (cultured in a L-cystine- and GSH-free medium). [3H]TdR incorporation assay and cell cycle analysis revealed that stimulated PBMC could not enter the S phase when deprived of these thiol compounds. In thiol-free cultures, an increase in intracellular free Ca2+ concentration and IL-2R alpha-chain/p 55 (Tac) induction was still observed, whereas transferrin receptor induction was markedly reduced, suggesting that the proliferative response of mitogenically stimulated PBMC was arrested in the late G1 phase in which transferrin receptor is induced. In GSH-depleted cultures, a similar reduction of the proliferative response of PBMC and PHA-blasts was observed when the concentration of L-cystine was lowered, in a dose-dependent manner. Each reduction or loss of proliferative response was partially restored by supplementation of 2-ME or adult T cell leukemia-derived factor (ADF)/human thioredoxin which is considered to be an endogenous dithiol-reducing factor. L-Cystine transport analysis showed that mitogenically stimulated PBMC and PHA blasts incorporated L-cystine, whereas resting PBMC did not. Furthermore, ADF as well as 2-ME exhibited an enhancing activity on the L-cystine transport in PHA blasts. Together with the fact that L-cystine transport is a limiting step in glutathione synthesis, these findings suggest that GSH and ADF might cooperate in the thiol-mediated redox regulation process and might also play key roles in cell cycle (late G1 to S) progression of activated lymphocytes.

Single‐cell RNA content of natural marine planktonic bacteria measured by hybridization with multiple 16S rRNA‐targeted fluorescent probes

Despite the potential of cellular RNA as a tool for the study of bacterial activities in natural environments, cellular RNA has rarely been measured because of methodological limitations. In a previous study, we quantitatively analyzed single‐cell RNA content with multiple 16S rRNA‐targeted fluorescent probes. In the present study, we explore the potential of this approach with 12 samples of natural planktonic bacteria collected from coastal water over a 6‐month period (late winter to summer). We measured fluorescence from single cells hybridized with multiple probes, cellular DNA and RNA by ethidium bromide (EtBr) fluorometry, and [3H]thymidine (TdR) incorporation rates. Fluorescence of the probe‐hybridized cells was highly correlated to cellular RNA determined by EtBr fluorometry. Winter and summer samples showed different patterns of fluorescence‐frequency distribution. Temperature had a large positive effect on TdR incorporation rates, but there was a negative correlation with cellular RNA content; only the samples taken at elevated summer temperatures showed a positive relationship between TdR incorporation and cellular RNA content. We estimated that the RNA content of the cells not visibly labeled with five probes was &lt;0.3 fg.

Phenotypic and functional cellular differences between human CD3- decidual and peripheral blood leukocytes.

CD3- leukocyte clones derived from human decidualized endometrial tissue of first trimester pregnancy have been compared with CD3- PBL clones. Most CD3- decidual granulated leukocyte (DGL) clones were CD16- CD56+, whereas most CD3- PBL clones were CD16+ CD56+. CD3- DGL and PBL clones, whether CD16+ or not, showed MHC-nonrestricted NK cell activity. However, CD3- CD16- DGL clones had low cytotoxic activity against the NK-resistant cell line BSM, whereas CD3- CD16+ DGL and CD3- PBL clones were strongly cytotoxic. Cytolytic activity has also been investigated in respect of target cell HLA-G expression, because this nonpolymorphic class I MHC molecule is expressed selectively by invasive fetal cytotrophoblast. Class I HLA Ag loss cell mutants were killed efficiently by CD3- DGL clones. Expression of transfected HLA-B8 increased their sensitivity to lysis by most CD3- DGL clones, whereas expression of transfected HLA-G commonly led to decreased target cell killing. In addition, the effects of uncloned CD3- DGL on the one-way MLR have been examined. These cells were very poor responders and, unless cultured to induce expression of class II MHC molecules, were also very poor stimulators. When fresh CD3- DGLs were added as third-party cells, either autologous or allogeneic to responder cells, [3H]TdR incorporation was decreased in the MLR. Thus, CD3- DGL clones express MHC-nonrestricted cytolytic activity, notably against HLA-negative cells, but expression of HLA-G offers protection to target cells. In addition, CD3- DGL may function to suppress allogeneic responses.

3Hthymidine incorporation of rhizosphere bacteria influenced by plant N-status

The effect of plant-root N-status on bacterial growth in the rhizosphere was studied with 5-week-old wheat plants grown in soil with low N content obtained by mixing 9:1 gravel:sandy loam. As a consequence of N limitation, significant increase in3Hthymidine (Tdr) incorporation rate occured 3 days after addition of 30 mM ammonium compared to controls without ammonium. Plants were grown with split-roots to separate the effect of soil N from effect of plant root derived organic matter-N on bacterial activity. The increase in nitrate concentration from 10 mM to 30 mM at one part of the root system led to significant increased3HT dr incorporation in the rhizosphere at the other part of root system after 4 days showing that the composition of root exudates became more favourable for bacterial growth when plants were fertilized with the higher level of nitrate.

Costimulation of cutaneous T-cell lymphoma cells by interleukin-7 and interleukin-2: potential autocrine or paracrine effectors in the Sézary syndrome.

To examine interleukin-7 (IL7)- and interleukin-2 (IL2)-induced proliferation of Sézary lymphoma cells and to consider if an autocrine or paracrine growth-stimulatory circuit involving IL7 exists in the Sézary syndrome (SS). Fresh Sézary lymphoma cells were maintained in short-term culture in the presence of cytokines, and growth was measured by incorporation of (3H)-thymidine (TdR). Expression of IL7 and IL7 and IL2 receptors (IL7-R and IL2-R, respectively) was assessed by polymerase chain amplification of first-strand complementary DNA (RT-PCR), by affinity cross-linking of radioactive iodine-125-IL7, and by dual-color fluorescence-activated cell analysis. IL-7 production was measured by immunoassay. Sézary lymphoma cells from seven patients showed synergistic (five of seven) or additive (two of seven) proliferation when cultured in the presence of IL2 and IL7, as compared with culture with either cytokine alone. Two patients with evidence of synergistic stimulation of [3H]-TdR incorporation showed IL7-R gene expression by RT-PCR and IL7 affinity cross-linking. Incubation of all seven patients' cells with IL7 induced coexpression to varying degrees of IL7-R and IL2-R. Sézary lymphoma cells from at least three of five patients studied expressed IL7 mRNA, and skin from three of five patients studied, as well as normal skin, expressed IL7 mRNA by RT-PCR. Sézary lymphoma cells respond by proliferation to IL7 plus IL2, and in some instances produce IL7. Therapeutic maneuvers should be pursued to take advantage of this potential autocrine or paracrine growth-stimulatory mechanism.

Induction of IL-6 gene expression in Kaposi's sarcoma cells.

IL-6 is a multifunctional cytokine that functions as an autocrine growth factor for AIDS-derived Kaposi's sarcoma (KS) cells. We report that IL-6 is highly inducible at both the mRNA and protein levels in cultured KS cells by multiple agents, yet the effect of the IL-6 on the proliferation of KS cells is dependent on the agent responsible for its induction. Both IL-1 beta and the synthetic dsRNA, poly (I:C), induced high levels of IL-6 mRNA and protein expression, whereas LPS and TNF-alpha led to only modest increases in IL-6 protein and mRNA. When KS cells were incubated with poly (I:C) in combination with either IL-1 beta or TNF-alpha, there was a synergistic increase in the level of IL-6 production, whereas LPS and TNF-alpha in combination led to only an additive increase in the level of IL-6 production. Exogenous IL-6 was shown to induce proliferation in KS cells, yet there was a dramatic inhibition of proliferation in response to poly (I:C), despite the high levels of IL-6 produced. This inhibition of proliferation by poly (I:C) was unlikely as a result of expression of class I IFN in response to the poly (I:C) because high concentrations of exogenous IFN-alpha had no demonstrable effect on [3H]TdR incorporation under conditions in which poly (I:C) caused a 90% decrease in [3H]TdR incorporation. Pretreatment of KS cells with poly (I:C) for 24 h followed by removal of the poly (I:C) led to high levels of IL-6 secreted into medium that induced proliferation in KS cells. These data suggest that in vivo, multiple agents that occur in response to infection and systemic disease could induce IL-6 production by KS cells, yet the ability of the IL-6 to influence proliferation of KS cells is dependent on the context in which the IL-6 is induced.

Lymph node cell proliferation assay in guinea pigs for the assessment of sensitizing potentials of chemical compounds

The efficacy of a lymph node cell proliferation assay in the guinea pig as a first stage screening method of predicting sensitizing potentials of chemicals was studied by using several haptens. Animals were sensitized by a single 24-hr occlusive patch (24 cp), intradermal injection (id) and a combination of id and 24 cp, at a concentration used for guinea pig conventional contact hypersensitivity assay methods. Control animals were treated with vehicle(s) only. Suspensions of the lymph node cells (LNC) were individually prepared and cultured with [ 3H]methyl thymidine ([ 1H]TdR). [ 1H]TdR incorporation was measured and a stimulation index (SI) was calculated as a ratio of the mean [ 3H]TdR incorporation in sensitized animals and the mean [ 3H]TdR incorporation in control animals. LNC sensitized by 24 cp with 2,4-dinitrochlorobenzene proliferated maximally and significantly at day 5, whereas this occurred at day 7 after id sensitization. Significant LNC proliferation and high SI values were obtained successively by a combination of 24 cp and id. Moreover, strongly sensitizing chemicals increased significant LNC proliferation (SI > 2.0); weakly to moderately sensitizing chemicals also induced significant LNC proliferation (SI = 1.3 1.7). On the other hand, a primary irritant, sodium dodecyl sulfate, failed to encourage LNC proliferation (SI ≈ 1.0).

B cell mitogenic activity of sea squirt antigen.

The activity of sea squirt antigen, one of the allergy-inducing substances for humans, on murine and human lymphocytes was studied in vitro. Sea squirt antigen stimulated normal mouse spleen cells to proliferate, as detected by [3H]-TdR incorporation, in a dose-dependent manner. The responder cells are B cells because the response was reduced by the treatment of spleen cells with anti-immunoglobulin antibody and complement and passing through a nylon wool column, but not with anti-Thy-1 antibody and complement. Spleen cells of C3H/HeJ mice, which are lipopolysaccharide low responders, were also stimulated as well as spleen cells of C3H/HeN mice, suggesting that this response is not due to lipopolysaccharide in the antigen fraction. Sea squirt antigen stimulated not only proliferative response of B cells, but also polyclonal immunoglobulin production. Furthermore, sea squirt antigen also stimulated human lymphocytes to proliferate and to produce immunoglobulin. All these results suggest that sea squirt antigen has mitogenic activity on B cells, and this ability is concerned with the induction of allergic reaction.

IL-2-induced expression of TTK, a serine, threonine, tyrosine kinase, correlates with cell cycle progression.

We have recently isolated the cDNA for a unique human 97-kDa kinase, TTK, by expression screening of a cDNA expression library using anti-phosphotyrosine antibodies. When expressed in Escherichia coli, TTK can phosphorylate serine, threonine, and tyrosine residues. Thus TTK appears to belong to a newly described family of kinases able to phosphorylate all three hydroxy amino acids. This family of multispecific kinases includes several other kinases involved in cell cycle progression. In support of a possible role in regulating cell cycle progression, TTK message is readily detected in rapidly proliferating tissues in vivo including testes, thymus, bone marrow, and many malignant tumors, but not in benign tissues with a low proliferative rate in vivo. To determine the effect of cell activation and cell cycle progression on TTK expression, we measured TTK mRNA and protein levels as well as kinase activity in freshly isolated T cells or IL-2-expanded T cell blasts activated to proliferate by the addition of a variety of mitogens. TTK mRNA levels, protein levels, and kinase activity were greatly enhanced when either freshly isolated PBL or T cell blasts were activated by cross-linking the TCR complex by mitogenic lectins or by bypassing the TCR with phorbol esters and cation ionophores. Incubation with IL-2 increased TTK expression in PBL blasts, which proliferate in response to IL-2, but not in fresh PBL, which do not proliferate in response to IL-2. TTK expression was blocked by either cyclosporin A or FK520, which inhibit IL-2 production and could be recovered by the addition of exogenous IL-2. Furthermore, TTK expression was prevented by incubation of the cells with rapamycin, which blocks IL-2 signaling. Thus, TTK expression in T cells appears to be a consequence of IL2-induced cell proliferation. Agonist-induced TTK expression was a delayed event occurring 12 to 24 h after activation of PBL blasts and 48 to 72 h after activation of fresh PBL. TTK protein and mRNA expression increased in both fresh PBL and T cell blasts concurrently with passage of cells through S phase as indicated by [3H]TdR incorporation and cell cycle analysis of propidium iodide-stained cells. TTK mRNA and protein levels reached a maximum as cells entered the G2 phase of the cell cycle. These results were confirmed by cell cycle blockade studies with aphidicolin and nocodazole wherein TTK protein levels are not detected in cells in G1 and are readily detectable in cells in the S and G2 phases of the cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)