Messenger RNA Production Research Articles

Utilization of bone morphogenetic protein receptors during chondrocyte maturation.

Cartilage from the upper, cephalic portion of embryonic chick sternums undergoes hypertrophy, while the lower, caudal portion of the sternum remains as cartilage. Bone morphogenetic proteins (BMPs) induce type X collagen (colX) in cultured upper but not lower sternal chondrocytes (LSCs). We have examined the utilization of BMP receptors (BMPRs) by upper sternal chondrocytes (USCs) and LSCs both by analyzing receptor expression and by overexpressing mutant BMPRs. Reverse-transcription polymerase chain reaction (RT-PCR) analyses indicate that both upper and lower chondrocytes produce messenger RNA (mRNA) for all three receptors: BMPR type IA (BMPR-IA), BMPR type IB (BMPR-IB), and BMPR type II (BMPR-II). Infection of USC with retroviral vectors expressing constitutively active (CA) BMPRs showed that CA-BMPR-IB, like exogenous BMP-4, induced both colX mRNA and elevated alkaline phosphatase (AP), while CA-BMPR-IA was markedly less potent. However, expression of activated receptors in LSC cultures resulted in only minimal induction of hypertrophic markers. Consistent with the results seen for CA receptors, dominant negative (DN) BMPR-IB blocked BMP-induced hypertrophy in USCs more effectively than DN-BMPR-IA. These results imply that the major BMPR required for BMP induction of chondrocyte hypertrophy is BMPR-IB, and that difference between permanent and prehypertrophic chondrocytes is not caused by absence of receptors required for BMP signaling.

Correlation of Transforming Growth Factor-β Messenger RNA (TGF-β mRNA) Expression with Cellular Immunoassays in Triamcinolone-Treated Captive Hybrid Striped Bass.

Assessing fish immune status with molecular markers has been hampered by a lack of specific reagents. A quantitative polymerase chain reaction (PCR) method (reverse transcription quantitative-competitive PCR, RT-qcPCR) for measuring transforming growth factor-β (TGF-β) transcription from a broad range of teleost fish has recently been developed. The quantitative PCR now permits monitoring production of this important immunosuppressive cytokine in response to immunomodulating agents and conditions. We examined anterior kidney and spleen mononuclear cells from hybrid striped bass (female striped bass Morone saxatilis × male white bass M. chrysops) for production of TGF-β messenger RNA (mRNA) in response to administration of the synthetic glucocorticoid triamcinolone. We also compared TGF-β transcription with anterior kidney macrophage bactericidal activity and splenic lymphocyte blastogenesis. Anterior kidney mononuclear cell TGF-β mRNA levels decreased, whereas bactericidal activity increased. Spleen TGF-β mRNA levels did not change significantly, and splenic lymphocyte pokeweed mitogen stimulation index increased in triamcinolone-treated fish. Since triamcinolone is used therapeutically as a suppressive immunomodulator, the enhanced immune functions indicated by the cellular immunoassays were unexpected; however, the inverse response of TGF-β production and macrophage bactericidal activity was consistent with the known relationship between TGF-β and macrophage activation in mammals. Induced immunomodulation in hybrid striped bass was detectable by both traditional cellular immunoassays and the new RT-qcPCR for TGF-β.

Exacerbation of Multiple Sclerosis Following Wasp Stings

To the Editor: We saw a 41-year-old man first diagnosed as having multiple sclerosis (MS) in 1993 after complaints of diplopia, hand tremors, and lower extremity ataxia. The diagnosis was confirmed by cerebrospinal fluid analysis and magnetic resonance imaging, He was treated effectively with interferon beta until 1994 at which time it was discontinued for financial reasons. In spring 1996, he had a 3-week episode of dysarthria, but since that time had remained symptom-free. In August 1998, he presented to our clinic with a complaint of paresthesia in the right leg and gluteal region. Two days previous, he had been working in his yard and was stung in the face multiple times by wasps (Vespula), and his symptoms began the next morning. He denied any history of wasp or bee sting allergy. On examination, he had decreased strength throughout in his right lower extremity (4/5), 3+ patellar reflexes bilaterally, and T10 sensory level on the right to all modalities. He was admitted for observation and treatment with intravenous methylprednisolone, had slightly improved symptoms within 24 hours, and was discharged to home. Three weeks later, his lower extremity strength had improved, but he had developed new symptoms in his right arm and was treated again with corticosteroids. In 1960, Goldstein et al1Goldstein NP Rucker CW Woltman HW Neuritis occurring after insect stings.JAMA. 1960; 173: 181-184Google Scholar reported a case of optic neuritis and another with a sensorimotor syndrome consistent with MS, both following bee sting. Means et al,2Means ED Barron KD Van Dyne BJ Nervous system lesions after sting hy yellow jacket: a case report.Neurology. 1973; 23: 881-890Crossref PubMed Google Scholar in 1973, reported a 52-year-old woman with no history of neurologic disease who presented with multiple relapsing-remitting neurologic complaints following a yellow jacket sting. The patient died of a cardiac arrest within 2 months of the sting, and autopsy showed multiple areas of necrosis and demyelination in the brain and spinal cord. Relapses of MS have been reported with other immunologic insults, notably tetanus toxoid, rabies, and other immunizations.3Annunziata P Traversi C Fanctti G Conli T Multiple sclerosis onset after anti-tetanus toxoid immunoglobulin prophylaxis [letter].Ital J Neural Sci. 1993; 14: 270PubMed Google Scholar Bee venom has been studied for its role in inflammatory processes, and bee venom therapy, also known as apitherapy, is being heralded by some as a complementary treatment in MS. There are no reports of MS-like exacerbations resulting from this technique, and while bee sting is common, we found no report of MS exacerbation following single or multiple bee stings. It is interesting to postulate on possible mechanisms that could have occurred, albeit cause and effect have not been proven. The role of interferon gamma has been well established in the pathogenesis of MS.4Panitch HS Interferons in multiple sclerosis: a review of the evidence.Drugs. 1992; 44: 946-962Crossref PubMed Scopus (104) Google Scholar Interestingly, a 1997 study of reaction to wasp stings by Bonay et al5Bonay M Echchakir H Lecossier D et al.Characterization of prolif-erative responses and cytokine mRNA profiles induced by Vespula venom in patients with severe reactions to wasp slings.Clin Exp Immunol. 1997; 109: 342-350Crossref PubMed Scopus (11) Google Scholar showed that even the T cells from nonallergic control patients had a marked increase in production of messenger RNA coding for interferon gamma after exposure to Vespula venom. Is it possible that a wasp sting in a nonatlergic patient could raise serum levels of interferon gamma that might, in turn, lead to a flare of MS symptoms? A search for similar cases, with analyses of symptoms and laboratory data, may help to elucidate possible mechanisms underlying this case and the immunologic pathogenesis of MS.

Targeted disruption of Stat6 DNA binding activity by an oligonucleotide decoy blocks IL-4–driven TH2 cell response

The transcription factor, signal transducer and activator of transcription (Stat) 6, regulates T(H)2-lymphocyte activity by controlling the expression and responsiveness to interleukin (IL)-4, which plays a key role in numerous allergic maladies. Therefore, we sought to use a phosphorothiolate cis-element decoy to target disruption of Stat6 transcriptional activity. Here we showed that the Stat6 decoy potently ablated the messenger RNA expression and production of IL-4, but not of several other cytokines. The Stat6 decoy functionally disrupted IL-4-inducible cell proliferation of murine T(H)2 cells and primary human CD4(+) T lymphocytes. Specificity of the decoy was demonstrated by its ability to directly block Stat6 binding to a cis-element probe and transactivation, but not affect Stat6 tyrosine phosphorylation or expression of the IL-4 receptor chains. Moreover, the decoy failed to inhibit non-Stat6-dependent signaling pathways since IL-2 was competent to induce cell proliferation and activation of Stats 1, 3, and 5a/b. With the use of laser scanning confocal microscopy, fluorescently tagged Stat6 decoy was detectable in the cytoplasm and nucleus; however, greater levels of oligonucleotide were present in the latter following IL-4 treatment. Taken together, these data suggest that IL-4-driven T(H)2 cell activity can be preferentially restricted via targeted disruption of Stat6 by a novel and specific decoy strategy that may possess gene therapeutic potential. (Blood. 2000;95:1249-1257)

Soluble Fas in serum from patients with renal cell carcinoma

Objectives. Fas/APO-1 is an apoptosis-signaling cell-surface receptor belonging to the tumor necrosis factor receptor family. The Fas-Fas ligand system plays an important role in cytotoxic T-lymphocyte-mediated or natural killer cell-mediated cytotoxicity against tumor cells. Soluble Fas (sFas), generated by alternative splicing, has been reported to antagonize the interaction of cell-surface Fas with Fas ligand. This study examined the level of sFas in the serum of patients with renal cell carcinoma (RCC) and investigated the correlation between the sFas level and clinicopathologic parameters of RCC. Methods. Using reverse transcriptase-polymerase chain reaction, we examined the production of sFas messenger RNA (mRNA) from the cultured human RCC cell lines ACHN and OUR-10 and from surgical specimens. We also measured sFas levels in the serum of 31 patients with RCC before and after nephrectomy using an sFas-specific enzyme-linked immunosorbent assay. Results. mRNA of sFas was identified both in cultured ACHN cells and human RCC tissues, although mRNA of wild-type Fas was exclusively predominant. The level of sFas in the serum of patients with RCC was significantly higher than that of normal controls, but sFas was not detectable in the supernatant of cultured renal cancer cells. Preoperative and postoperative serum sFas levels did not clearly correlate with the patients’ age or sex or with histologic stage, grade, or cell type of RCC. The serum sFas level in patients with RCC correlated with tumor size. In 24 of the 31 cases, radical nephrectomy reduced the serum sFas level within 3 months. Conclusions. Our results suggest that the elevated serum sFas level in patients with RCC might not be derived from the tumor itself but might reflect an immune response to the tumor burden. Serum sFas may be a useful indicator of tumor burden in patients with RCC.

Molecular Biology of the Androgen Receptor

The androgen receptor is a member of a superfamily of nuclear transcription factors that mediate the action of steroid hormones. When activated by ligand binding, these transcription factors bind to specific DNA sequences on target genes and regulate the transcriptional activity of those genes. Various genes have been identified that are regulated by androgens, including prostate-specific antigen (PSA). PSA contains an upstream glucocorticoid response element/androgen response element, so it is believed that androgens directly regulate its transcription rate. Despite this, posttranscriptional regulation through stability of the messenger RNA product is believed to be a major mechanism of androgens' effects.

Type-specific induction of interleukin-8 by adenovirus.

Infection with adenovirus (Ad) causes acute pneumonia in a type-specific fashion because type 7 but not type 5 Ad has been isolated as a causative agent. We postulated that the type specificity of induction of pneumonia may be related to type-specific cytokine induction in lung cells. To test this hypothesis, we infected human fetal lung fibroblasts and the lung epithelial cell line A549 with live type 5 and type 7 Ad. Virus inactivated by irradiation was used as a control. Type 7 but not type 5 Ad induced interleukin (IL)-8 protein production in both cell types in a dose- and time-dependent manner. Inactivated virus had no effect on the production of IL-8 protein. Type 7 but not type 5 virus also stimulated IL-8-specific messenger RNA (mRNA) production in these cells. Because half-life of IL-8 mRNA was prolonged in both type 5- and type 7-infected A549 cells, induction likely involves enhancement of message stability as well as other effects. Virus early gene expression did not consistently correlate with IL-8 message induction and followed induction in fibroblasts. These results suggest that there is type-specific induction of IL-8 production during infection of lung cells with Ad. Induction involves message stabilization and may not require viral gene expression. Because IL-8 is one of the important mediators of lung inflammation, type-specific induction of this and other cytokines may account for the different consequences of lung infection with different types of Ad.

Regulation of urokinase production by androgens in human prostate cancer cells: effect on tumor growth and metastases in vivo.

During the complex multistep process of tumor progression, prostate cancer is initiated as an androgen-sensitive, nonmetastatic cancer, followed by a gradual transition into a highly metastatic and androgen-insensitive variety that lacks the expression of functional androgen receptors (AR). Urokinase (uPA), a member of the serine protease family, has been implicated in the progression of various human malignancies, including prostate cancer. Although uPA production is regulated by various growth factors and cytokines, the role of sex steroids (androgens) in regulating uPA gene expression in prostate cancer is poorly understood. In the current study, we have examined the role of androgens in regulating uPA production and the invasive capacity of the androgen insensitive PC-3 cells transfected with the full-length human AR complementary DNA (PC-3T). Restoration of androgen responsiveness in PC-3T cells caused a marked decrease in cell doubling time. Treatment of PC-3T cells with dihydroxytestosterone (DHT) caused a dose-dependent decrease in uPA messenger RNA and protein production, resulting in their decreased ability to invade through the Matrigel. Nuclear runoff assays revealed that these effects were attributable to the ability of DHT to inhibit uPA gene transcription. AR antagonist flutamide (Flu) reversed the effect of DHT on proliferation and invasion of PC-3T cells. Both control (PC-3) and experimental (PC-3T) cells were injected into the right flank of male BALB/c nu/nu mice. Control animals developed palpable tumors and microscopic tumor metastases at lymph nodes, lungs, and liver at 6-week posttumor cell inoculation. In contrast to this, because of androgen sensitivity of PC-3T cells, palpable tumors were observed only at week 12, with occasional tumor metastases in lungs. Furthermore, inoculation of PC-3T cells into surgically castrated host animals resulted in the development of tumors at a much earlier time (week 10) and a high incidence of metastases, compared with regular animals receiving PC-3T cells. Collectively, these results demonstrate the ability of androgen to regulate uPA production, which may directly effect prostate cancer growth, invasion, and metastasis in vitro and in vivo.

Inhibition of cytokine-induced nitric oxide synthase expression by gene transfer of adenoviral IκBα

Background: Nitric oxide is overexpressed in nearly every organ during sepsis and it has profound biologic effects. Previously, we showed that maximal inducible nitric oxide synthase (iNOS) expression is up-regulated by a combination of cytokines and that this effect is mediated by the transcription factor NF-κB. Therefore the purpose of this study was to establish whether gene transfer of the inhibitory molecule IκB would result in the abrogation of cytokine-induced iNOS expression. Methods: Cultured hepatocytes were infected with an adenoviral vector containing the IκBα gene (Ad5IκB) and after an 18-hour recovery period were stimulated with the cytokine mixture of tumor necrosis factor-α (500 U/mL) plus interleukin 1β (200 U/mL) plus interferon gamma (100 U/mL). Results: As expected, cytokine mixture induced significant hepatocyte nitrite (NO 2 –) and iNOS messenger RNA production. Cells infected with the IκBα gene showed a dose-dependent decrease in NO 2 – and iNOS messenger RNA levels. Western blot analysis showed a marked decrease in iNOS protein levels in the presence of Ad5IκBα. Gel shift assays of nuclear extracts demonstrated that Ad5IκBα decreased the cytokine-induced DNA binding activity for NFκB. Conclusions: NFκB is an important regulator of cytokine-induced NO expression. These results identify a novel therapeutic approach where gene transfer of the inhibitory molecule IκBα can be used to down-regulate cytokine-induced iNOS expression as well as other NFκB-dependent genes that are up-regulated during the inflammatory response. (Surgery 1999;126:142-7.)

Effects of diesel organic extracts on chemokine production by peripheral blood mononuclear cells

Background: Polyaromatic hydrocarbons (PAHs) associated with diesel exhaust particles (DEPs) are found in the atmospheric urban pollution. Such compounds have been shown to favor IgE production, bronchial hyperresponsiveness, and airway inflammation. Chemokines are a group of chemotactic cytokines involved in the recruitment of inflammatory cells. Objective: We investigated the effect of DEP-PAHs on the release and mRNA expression of IL-8, MCP-1, and RANTES by PBMCs obtained from healthy subjects. Methods: Protein production in supernatants was assessed by ELISA, and mRNA expression was evaluated by semiquantitative RT-PCR. Results: Secretion of IL-8 and RANTES increased in a dose-dependent manner with increasing concentrations of DEP-PAHs (range, 0.5 ng to 50 ng/mL). On the contrary, the release of MCP-1 was significantly inhibited, also in a dose-dependent manner. Messenger RNA production coding for IL-8, RANTES, and MCP-1 showed parallel variations to the production of the correspondent proteins. Effects of DEP-PAHs became significant at 7 hours and up to 48 hours time culture for MCP-1, and up to 24 hours time culture for IL-8 and RANTES. Moreover, supernatants from DEP-PAH–activated cells, compared with those of controls, exhibited a significantly enhanced chemotactic activity for neutrophils and eosinophils, which was significantly inhibited by pretreatment with anti-IL-8 and anti-RANTES neutralizing antibodies, respectively. Conclusion: These findings suggest that the chemokine pathways are modulated by DEP-PAHs at the transcriptional level, reinforcing the idea that the development of inflammatory reactions might be affected by diesel exhaust emission. (J Allergy Clin Immunol 1999;103:1115-24.)

Cytokine profiles in Wegener's granulomatosis: predominance of type 1 (Th1) in the granulomatous inflammation.

To determine whether a specific cytokine pattern (type 1 [Th1] or type 2 [Th2]) predominates in Wegener's granulomatosis (WG), by evaluating interferon-gamma (IFNgamma) and interleukin-4 (IL-4) expression in different compartments of the body (i.e., biopsied nasal mucosal tissue [NBS], bronchoalveolar lavage [BAL] fluid, and peripheral blood [PB]) and comparing the findings with those in disease and healthy control subjects. Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to assess IFNgamma and IL-4 expression in T cell clones (TCC), T cell lines (TCL), and polyclonal CD4+ and CD8+ cells derived from NBS, BAL, and PB. Patients with WG and chronic rhinitis were found to share in situ production of messenger RNA (mRNA) specific for IFNgamma (Th1). Only 2 patients with WG expressed IL-4, whereas IL-4 mRNA PCR products were found in inflamed tissues of the disease control patients. The granuloma-derived T cells of WG patients produced only IFNgamma, while TCC, TCL, and CD4+ and CD8+ T cells from BAL and PB produced mainly IFNgamma. Our data indicate that a Thl cytokine pattern predominates in the granulomatous inflammation in patients with WG.

Cytokine profiles in Wegener's granulomatosis: Predominance of type 1 (Th1) in the granulomatous inflammation

Objective To determine whether a specific cytokine pattern (type 1 [Th1] or type 2 [Th2]) predominates in Wegener's granulomatosis (WG), by evaluating interferon-γ (IFNγ) and interleukin-4 (IL-4) expression in different compartments of the body (i.e., biopsied nasal mucosal tissue [NBS], bronchoalveolar lavage [BAL] fluid, and peripheral blood [PB]) and comparing the findings with those in disease and healthy control subjects. Methods Competitive reverse transcriptase–polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to assess IFNγ and IL-4 expression in T cell clones (TCC), T cell lines (TCL), and polyclonal CD4+ and CD8+ cells derived from NBS, BAL, and PB. Results Patients with WG and chronic rhinitis were found to share in situ production of messenger RNA (mRNA) specific for IFNγ (Th1). Only 2 patients with WG expressed IL-4, whereas IL-4 mRNA PCR products were found in inflamed tissues of the disease control patients. The granuloma-derived T cells of WG patients produced only IFNγ, while TCC, TCL, and CD4+ and CD8+ T cells from BAL and PB produced mainly IFNγ. Conclusion Our data indicate that a Th1 cytokine pattern predominates in the granulomatous inflammation in patients with WG.

Interleukin-4 (IL-4) and IL-13 Enhance the Effect of IL-1β on Production of IL-1 Receptor Antagonist by Human Primary Hepatocytes and Hepatoma HepG2 Cells: Differential Effect on C-Reactive Protein Production

Interleukin-1 receptor antagonist (IL-1Ra) is produced by hepatocytes with characteristics of an acute-phase protein. To examine the role of IL-4 and IL-13 in production of IL-1Ra, human primary hepatocytes and HepG2 human hepatoma cells were cultured in the presence of IL-4 or IL-13 in combination with IL-1beta and/or IL-6. The results indicated that both IL-4 and IL-13 amplified the stimulatory effect of IL-1beta on production of IL-1Ra protein and messenger RNA (mRNA) by both human primary hepatocytes and HepG2 cells. IL-1Ra refers to three different peptides, one secreted (sIL-1Ra) and two intracellular (icIL-1RaI and icIL-1RaII), derived from the same gene. sIL-1Ra and icIL-1RaI are the products of two different mRNA, whereas icIL-1RaII is synthesized by alternative translation initiation mainly from sIL-1Ra mRNA. Our results show that both sIL-1Ra and icIL-1RaII, but not icIL-1RaI, are produced by HepG2 cells and human hepatocytes. Transient transfection experiments as well as mRNA stability studies indicated that IL-4 stimulated sIL-1Ra production primarly at the level of transcription. Gel retardation assays showed that IL-4 induced the formation of a STAT6-DNA complex with a STAT6 binding element within the sIL-1Ra promoter, but had no effect on IL-1-induced NF-kappaB binding activity. In contrast to IL-1Ra, production of C-reactive protein by human primary hepatocytes was stimulated by IL-6 and decreased by the addition of IL-4.

Interleukin-4 (IL-4) and IL-13 Enhance the Effect of IL-1β on Production of IL-1 Receptor Antagonist by Human Primary Hepatocytes and Hepatoma HepG2 Cells: Differential Effect on C-Reactive Protein Production

AbstractInterleukin-1 receptor antagonist (IL-1Ra) is produced by hepatocytes with characteristics of an acute-phase protein. To examine the role of IL-4 and IL-13 in production of IL-1Ra, human primary hepatocytes and HepG2 human hepatoma cells were cultured in the presence of IL-4 or IL-13 in combination with IL-1β and/or IL-6. The results indicated that both IL-4 and IL-13 amplified the stimulatory effect of IL-1β on production of IL-1Ra protein and messenger RNA (mRNA) by both human primary hepatocytes and HepG2 cells. IL-1Ra refers to three different peptides, one secreted (sIL-1Ra) and two intracellular (icIL-1RaI and icIL-1RaII), derived from the same gene. sIL-1Ra and icIL-1RaI are the products of two different mRNA, whereas icIL-1RaII is synthesized by alternative translation initiation mainly from sIL-1Ra mRNA. Our results show that both sIL-1Ra and icIL-1RaII, but not icIL-1RaI, are produced by HepG2 cells and human hepatocytes. Transient transfection experiments as well as mRNA stability studies indicated that IL-4 stimulated sIL-1Ra production primarly at the level of transcription. Gel retardation assays showed that IL-4 induced the formation of a STAT6-DNA complex with a STAT6 binding element within the sIL-1Ra promoter, but had no effect on IL-1–induced NF-κB binding activity. In contrast to IL-1Ra, production of C-reactive protein by human primary hepatocytes was stimulated by IL-6 and decreased by the addition of IL-4.

Effect of pentoxifylline on survival and intestinal cytokine messenger RNA transcription in a rat model of ongoing peritoneal sepsis.

Septic animals receiving high-protein liquid diets have increased mortality and increased production of cytokines by the gut compared with animals receiving low-protein diets. The purpose of this study was to evaluate the ability of pentoxifylline to alter gut cytokine production in a rat model of prolonged acute peritonitis, to determine its effect on survival in such animals, and to determine whether alteration of gut cytokine production was associated with survival. Prospective, randomized animal study. Research laboratory. Male Lewis rats weighing between 250 and 300 g. Anesthetized rats had placement of a gastrostomy, followed 1 wk later by implantation of a bacteria-filled osmotic minipump into the peritoneal cavity. Rats were fed a high-protein (20% total energy) enteral diet. Saline or pentoxifylline (5 or 20 mg/kg im) was administered daily beginning at the time of pump implantation. Septic rats fed the high-protein liquid diet and given pentoxifylline in a dose of 5 mg/kg/day demonstrated improved survival compared with saline-treated animals or animals given the high dose (20 mg/kg/day) of pentoxifylline (p< .05). Administration of pentoxifylline at 5 mg/kg/day also down regulated the production of IL-6 messenger RNA (mRNA) in liver and lipopolysaccharide binding protein mRNA in the liver and intestine of septic animals given the high-protein liquid diet. Low-dose (but not high-dose) pentoxifylline administration reduced production of some, but not all, cytokines studied in the gut and liver in a rat model of acute peritonitis and this reduced production was associated with an improved survival in such animals.

Human semen induces interleukin 10 and 70 kDa heat shock protein gene transcription and inhibits interferon-gamma messenger RNA production in peripheral blood mononuclear cells.

The influence of semen on immunity in sexually active women has been scarcely studied. The effect of human semen on production of messenger RNA (mRNA) for the anti-inflammatory TH2-related cytokine, interleukin-10 (IL-10), the 70 kDa heat shock protein (HSP70) and the pro-inflammatory TH1-related cytokine, interferon-gamma (IFN-gamma) was examined. Co-incubation of peripheral blood mononuclear cells (PBMC) from 10 women with a non-cytotoxic 1:50 dilution of semen lead to induction of IL-10 mRNA. Semen from each of seven different men tested induced IL-10 mRNA in PBMC. IL-10 protein was also released into the culture supernatant after PBMC-semen co-culture. Similarly, semen induced transcription of the HSP70 gene in PBMC obtained from 10 women. In contrast, semen did not induce IFN-gamma mRNA in any of the female PBMC donors. Furthermore, semen markedly inhibited IFN-gamma mRNA production without affecting cell viability in PBMC that were cocultured with phytohaemagglutinin, a potent IFN-gamma-inducing T-cell mitogen. Thus, human semen is both an inducer of an anti-inflammatory (TH2) immune response and an inhibitor of pro-inflammatory (TH1) cell-mediated immunity.

Effects of colony stimulating factor-1 on human extravillous trophoblast growth and invasion.

Colony stimulating factor (CSF)-1 has been localized in a variety of tissues and shown to influence proliferation and differentiation of numerous cell types. Messenger RNA and protein products of CSF-1 and its receptor (c-fms) have been identified in the human placenta and decidua. We examined whether CSF-1 and c-fms mRNA and protein are expressed by normal human first trimester invasive extravillous trophoblast (EVT) cells propagated in culture and whether CSF-1 influences proliferation and/or invasion of these cells. CSF-1 mRNA and protein expression was determined by RT-PCR and immunofluorescence microscopy. Proliferation was assessed by the cellular uptake of tritiated thymidine and invasion was evaluated by Matrigel invasion assay as well as Northern blot analysis of mRNA expression for invasion-associated enzymes and their inhibitors. Results revealed that normal invasive EVT cells in culture express both CSF-1 and c-fms mRNA and protein. Under serum-free conditions, exogenous CSF-1 greatly stimulated the proliferation of these cells. CSF-1 neutralizing and c-fms receptor blocking antibody (Ab) each abolished the growth stimulatory effects of CSF-1, indicating that CSF-1 and c-fms interaction was responsible for these effects. In fact, c-fms Ab alone reduced proliferation to below background levels. While exogenous CSF-1 failed to influence EVT cell invasiveness, Northern blot analysis of mRNA indicated a slight upregulation of the invasion-associated enzyme 72 kDa type IV collagenase as well as its natural inhibitor tissue inhibitor of metalloprotease (TIMP)-1, so that the balance between the two remained unaltered. These findings suggest that CSF-1 may represent an autocrine (and possibly paracrine) growth stimulatory factor for the invasive trophoblast cells in situ with no net effect on their invasiveness.

RNA polymerase II is an essential mRNA polyadenylation factor.

Production of messenger RNA in eukaryotic cells is a complex, multistep process. mRNA polyadenylation, or 3' processing, requires several protein factors, including cleavage/polyadenylation-specificity factor (CPSF), cleavage-stimulation factor, two cleavage factors and poly(A) polymerase. These proteins seem to be unnecessary for other steps in mRNA synthesis such as transcription and splicing, and factors required for these processes were not considered to be essential for polyadenylation. Nonetheless, these reactions may be linked so that they are effectively coordinated in vivo. For example, the CTD carboxy-terminal domain of the largest subunit of RNA polymerase II (RNAP II) is required for efficient splicing and polyadenylation in vivo, and CPSF is brought to a promoter by the transcription factor TFIID and transferred to RNAP II at the time of transcription initiation. These findings suggest that polyadenylation factors can be recruited to an RNA 3'-processing signal by RNAP II, where they dissociate from the polymerase and initiate polyadenylation. Here we present results that extend this model by showing that RNAP II is actually required, in the absence of transcription, for 3' processing in vitro.

Multisystemic production of interleukin 10 limits the severity of acute pancreatitis in mice

Background—Interleukin 10 (IL-10) decreases the severity of experimental acute pancreatitis. The role of endogenous IL-10 in modulating the course of pancreatitis is currently unknown.Aims—To examine the systemic release of IL-10...

Inhibition of GABAergic inhibitory postsynaptic currents by cannabinoids in rat corpus striatum

Electrophysiological consequences of activation of cannabinoid receptors have been mostly investigated on neuronal cell lines and on cells transfected with cannabinoid receptors. The aim of the present experiments was to study cannabinoid effects on identified neurons in situ. Electrically-evoked postsynaptic currents and voltage-dependent calcium currents were investigated in the principal neurons of the corpus striatum, the medium spiny neurons, with the patch-clamp method for brain slices. These neurons were chosen because they produce messenger RNA for cannabinoid receptors and because the density of cannabinoid binding sites in the striatum is high. Activation of muscarinic receptors by carbachol (10 −5 M) reduced inhibitory postsynaptic current amplitude by 67%. The synthetic cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphtalenyl)methanone (WIN55212-2; 10 −8 to 10 −5 M) dose-dependently reduced striatal inhibitory postsynaptic currents; the maximum effect, inhibition by 52%, was observed at 10 −6 M. Another cannabinoid agonist, (−)- cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]- trans-4-(3-hydroxypropyl)cyclohexanol (CP55940; 10 −6 M), also reduced inhibitory postsynaptic currents, by 50%. The CB1 cannnabinoid receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide (SR141716A; 10 −6 M) had no effect when given alone but abolished the effect of WIN55212-2 (10 −6 M). WIN55212-2 (10 −6 M) did not change the current evoked by the GABA A-receptor agonist muscimol (10 −6 M). Activation of muscarinic receptors by carbachol (10 −5 M) inhibited voltage-dependent calcium currents by 21%, but the cannabinoid receptor agonist WIN55212-2 (10 −6 M) was without effect. The results show that activation of CB1 cannabinoid receptors reduces GABAergic inhibitory postsynaptic currents in medium spiny neurons of the corpus striatum: the likely mechanism is presynaptic inhibition of GABA release from terminals of recurrent axons of the medium spiny neurons themselves.