Effect of a single high dose of vitamin D3 on cytokines, chemokines, and growth factor in patients with moderate to severe COVID-19.

BACKGROUND: The modulating effect of vitamin D on cytokine concentrations in severe coronavirus disease 2019 (COVID-19) remains unknown. OBJECTIVES: We aimed to investigate the effect of a single high dose of vitamin D3 on cytokines, chemokines, and growth factor in hospitalized patients with moderate to severe COVID-19. METHODS: This is a post hoc, ancillary, and exploratory analysis from a multicenter, double-blind, placebo-controlled, randomized clinical trial. Patients with moderate to severe COVID-19 were recruited from 2 hospitals in São Paulo, Brazil. Of 240 randomly assigned patients, 200 were assessed in this study and randomly assigned to receive a single oral dose of 200,000 IU vitamin D3 (n = 101) or placebo (n = 99). The primary outcome was hospital length of stay, which has been published in our previous study. The prespecified secondary outcomes were serum concentrations of IL-1ß, IL-6, IL-10, TNF-α, and 25-hydroxyvitamin D. The post hoc exploratory secondary outcomes were IL-4, IL-12p70, IL-17A, IFN-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, IFN-inducible protein-10 (IP-10), macrophage inflammatory protein-1ß (MIP-1ß), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and leukocyte count. Generalized estimating equations for repeated measures, with Bonferroni's adjustment, were used for testing all outcomes. RESULTS: The study included 200 patients with a mean ± SD age of 55.5 ± 14.3 y and BMI of 32.2 ± 7.1 kg/m2, of which 109 (54.5%) were male. GM-CSF concentrations showed a significant group-by-time interaction effect (P = 0.04), although the between-group difference at postintervention after Bonferroni's adjustment was not significant. No significant effects were observed for the other outcomes. CONCLUSIONS: The findings do not support the use of a single dose of 200,000 IU vitamin D3, compared with placebo, for the improvement of cytokines, chemokines, and growth factor in hospitalized patients with moderate to severe COVID-19.This trial was registered at clinicaltrials.gov as NCT04449718.

Methotrexate limits inflammation through an A20-dependent cross-tolerance mechanism.

OBJECTIVES: Methotrexate (MTX) is the anchor drug for treatment of rheumatoid arthritis (RA), but the mechanism of its anti-inflammatory action is not fully understood. In RA, macrophages display a proinflammatory polarisation profile that resembles granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated macrophages and the response to MTX is only observed in thymidylate synthase+ GM-CSF-dependent macrophages. To determine the molecular basis for the MTX anti-inflammatory action, we explored toll-like receptor (TLR), RA synovial fluid (RASF) and tumour necrosis factor receptor (TNFR)-initiated signalling in MTX-exposed GM-CSF-primed macrophages. METHODS: Intracellular responses to TLR ligands, TNFα or RASF stimulation in long-term low-dose MTX-exposed human macrophages were determined through quantitative real-time PCR, western blot, ELISA and siRNA-mediated knockdown approaches. The role of MTX in vivo was assessed in patients with arthritis under MTX monotherapy and in a murine sepsis model. RESULTS: MTX conditioned macrophages towards a tolerant state, diminishing interleukin (IL)-6 and IL-1ß production in LPS, LTA, TNFα or RASF-challenged macrophages. MTX attenuated LPS-induced MAPK and NF-κB activation, and toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF1)-dependent signalling. Conversely, MTX increased the expression of the NF-κB suppressor A20 (TNFAIP3), itself a RA-susceptibility gene. Mechanistically, MTX-induced macrophage tolerance was dependent on A20, as siRNA-mediated knockdown of A20 reversed the MTX-induced reduction of IL-6 expression. In vivo, TNFAIP3 expression was significantly higher in peripheral blood cells of MTX-responsive individuals from a cohort of patients with arthritis under MTX monotherapy, whereas MTX-treated mice exhibited reduced inflammatory responses to LPS. CONCLUSIONS: MTX impairs macrophage proinflammatory responses through upregulation of A20 expression. The A20-mediated MTX-induced innate tolerance might limit inflammation in the RA synovial context, and positions A20 as a potential MTX-response biomarker.

Mast cells decrease efficacy of anti-angiogenic therapy by secreting matrix-degrading granzyme B.

Resistance towards VEGF-centered anti-angiogenic therapy still represents a substantial clinical challenge. We report here that mast cells alter the proliferative and organizational state of endothelial cells which reduces the efficacy of anti-angiogenic therapy. Consequently, absence of mast cells sensitizes tumor vessels for anti-angiogenic therapy in different tumor models. Mechanistically, anti-angiogenic therapy only initially reduces tumor vessel proliferation, however, this treatment effect was abrogated over time as a result of mast cell-mediated restimulation of angiogenesis. We show that mast cells secrete increased amounts of granzyme b upon therapy, which mobilizes pro-angiogenic laminin- and vitronectin-bound FGF-1 and GM-CSF from the tumor matrix. In addition, mast cells also diminish efficacy of anti-angiogenic therapy by secretion of FGF-2. These pro-angiogenic factors act beside the targeted VEGFA-VEGFR2-axis and reinduce endothelial cell proliferation and angiogenesis despite the presence of anti-angiogenic therapy. Importantly, inhibition of mast cell degranulation with cromolyn is able to improve efficacy of anti-angiogenic therapy. Thus, concomitant mast cell-targeting might lead to improved efficacy of anti-angiogenic therapy.Resistance towards VEGF-centered anti-angiogenic therapy is an important clinical challenge. Here, the authors show that mast cells mediate resistance to anti-angiogenetic inhibitors by altering the proliferative and organizational state of endothelial cells through mobilization of FGF-1 and GM-CSF from the tumor matrix and secretion of FGF-2.

[PDGF-BB Promotes CFU-GM and CFU-MK Formation and Its Possible Mechnism].

OBJECTIVE: To investigate the effect of platelet-derived growth factor (PDGF-BB) on the formation of granulocyte-monocyte colony forming unit (CFU-GM) and megakaryocyte colony forming unit (CFU-MK) and its anti-apoptotic effect on CHRF cells. METHODS: The CFU-GM and CFU-MK of murine and human bone marrow cells were cultured in vitro by using plasma clot culture system. The anti-apoptotic effect of PDGF-BB on CHRF cells and its mechanism were clarified by flow cytometry. RESULTS: PDGF-BB 0-100 ng/ml stimulated the proliferation of murine and human CFU-GM and CFU-MK in a dose-dependent manner. The maximal stimulation effect was obtained at 50 ng/ml of PDGF-BB (P<0.01). Furthermore, PDGF-BB had an anti-apoptotic effect on CHRF cells as shown by the flow cytometry with AnnexinV/PI double staining, Caspase-3 expression and JC-1 detection (P<0.05). CONCLUSION: PDGF-BB significantly stimulates the proliferation of CFU-GM and CFU-MK in vitro, and has an anti-apoptotic effect on CHRF cells.

Effects of Ambient Fine Particles PM2.5 on Human HaCaT Cells.

The current study was conducted to observe the effects of fine particulate matter (PM2.5) on human keratinocyte cell line (HaCaT) cells. The potential mechanism linking PM2.5 and skin was explored. HaCaT cells were cultured and then accessed in plate with PM2.5. Cell viability was tested by Cell Counting Kit-8. The mRNA and protein expression of Filaggrin, Loricrin, Involucrin, and Repetin were analyzed. The levels of Granulocyte-macrophage Colony Stimulating Factor, Thymic Stromal Lymphopoietin, Tumor Necrosis Factor-α, Interleukin-1α, and Interleukin-8 were detected in the supernatant of the HaCaT cell with enzyme-linked immunosorbent assay kits. Cell viability decreased with the increase in PM2.5. Compared with the control group, the protein expression of Filaggrin, Repetin, Involucrin, and Loricrin showed different expression patterns in PM2.5 treatment groups. The level of Tumor Necrosis Factor-α, Thymic Stromal Lymphopoietin, Interleukin-1α, and Interleukin-8 significantly increased in the cells treated with PM2.5. Ambient PM2.5 may increase the risk of eczema and other skin diseases. The relative mechanism may be associated with the impairment of the skin barrier and the elevation of inflammatory responses.

IL-17A synergistically stimulates TNF-α-induced IL-8 production in human airway epithelial cells: A potential role in amplifying airway inflammation.

BACKGROUND: Recent reports have suggested an involvement of neutrophilic inflammation driven by interleukin (IL)-17 from Th17 cells, especially in severe, refractory asthma. It remains unknown about the possible interactions of this cytokine and other proinflammatory cytokines to direct neutrophilic airway inflammation. MATERIALS AND METHODS: We evaluated the effects of IL-17A, IL-17E, and IL-17F in combination with other stimuli such as tumor necrosis factor (TNF) -α on the production and expression of IL-8 in human bronchial epithelial cells. We also studied their effects on other cytokine production. The possible role of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling pathways was evaluated by specific inhibitors. We examined the effects of anti-asthma drugs, such as steroids or salmeterol. RESULTS: IL-17A alone induced only a minimal effect on IL-8 expression. IL-17A, but not IL-17E or IL-17F, in combination with TNF-α showed a synergistic effect on IL-8 expression. Similar findings were found when combination with IL-1ß and IL-17A were used, but such was not the case with lipopolysaccharide (LPS). In addition, we further found such synergy on GM-CSF production. The synergy with TNF-α and IL-17A was significantly inhibited by MAPKs inhibitors. Corticosteroids such as fluticasone propionate and dexamethasone, but not salmeterol, partially suppressed the IL-17A and TNF-α-induced IL-8 production. CONCLUSIONS: IL-17A in the combination with TNF-α or IL-1ß showed a synergistic augmenting effect on IL-8 and GM-CSF production in human airway epithelial cells.

Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma.

PURPOSE: Talimogene laherparepvec (T-VEC) is a herpes simplex virus type 1-derived oncolytic immunotherapy designed to selectively replicate within tumors and produce granulocyte macrophage colony-stimulating factor (GM-CSF) to enhance systemic antitumor immune responses. T-VEC was compared with GM-CSF in patients with unresected stage IIIB to IV melanoma in a randomized open-label phase III trial. PATIENTS AND METHODS: Patients with injectable melanoma that was not surgically resectable were randomly assigned at a two-to-one ratio to intralesional T-VEC or subcutaneous GM-CSF. The primary end point was durable response rate (DRR; objective response lasting continuously ≥ 6 months) per independent assessment. Key secondary end points included overall survival (OS) and overall response rate. RESULTS: Among 436 patients randomly assigned, DRR was significantly higher with T-VEC (16.3%; 95% CI, 12.1% to 20.5%) than GM-CSF (2.1%; 95% CI, 0% to 4.5%]; odds ratio, 8.9; P < .001). Overall response rate was also higher in the T-VEC arm (26.4%; 95% CI, 21.4% to 31.5% v 5.7%; 95% CI, 1.9% to 9.5%). Median OS was 23.3 months (95% CI, 19.5 to 29.6 months) with T-VEC and 18.9 months (95% CI, 16.0 to 23.7 months) with GM-CSF (hazard ratio, 0.79; 95% CI, 0.62 to 1.00; P = .051). T-VEC efficacy was most pronounced in patients with stage IIIB, IIIC, or IVM1a disease and in patients with treatment-naive disease. The most common adverse events (AEs) with T-VEC were fatigue, chills, and pyrexia. The only grade 3 or 4 AE occurring in ≥ 2% of T-VEC-treated patients was cellulitis (2.1%). No fatal treatment-related AEs occurred. CONCLUSION: T-VEC is the first oncolytic immunotherapy to demonstrate therapeutic benefit against melanoma in a phase III clinical trial. T-VEC was well tolerated and resulted in a higher DRR (P < .001) and longer median OS (P = .051), particularly in untreated patients or those with stage IIIB, IIIC, or IVM1a disease. T-VEC represents a novel potential therapy for patients with metastatic melanoma.

Simvastatin inhibits LPS-induced alveolar bone loss during metabolic syndrome.

Studies in recent years have shown a positive relationship between metabolic syndrome (MS) and periodontal disease (PD). Given that patients with MS take statins to reduce cholesterol, and statins also have anti-inflammatory effects, it is important to determine if statin intake hinders the progression of MS-associated PD. In this study, PD was induced in Zucker fat rats (ZFRs), an animal model for MS, and in control lean rats by periodontal injection of Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS), while simvastatin was given to some of the rats via gavage. After 4 wk of treatment, alveolar bone loss was determined by micro-computed tomography. To explore the underlying mechanisms, we determined the effect of simvastatin on tissue inflammation and the expression of molecules involved in osteoclastogenesis. Results showed that while bone loss was increased by LPS in both ZFRs and the control lean rats, it was significantly more in the former than the latter. Simvastatin effectively alleviated bone loss in both ZFRs and the control rats. Results also showed that LPS stimulated leukocyte tissue infiltration and expression of molecules for osteoclastogenesis, but simvastatin significantly modulated the stimulation. This study demonstrated that simvastatin inhibited LPS-induced alveolar bone loss and periodontal tissue inflammation in rats with MS.

Cytokine changes in response to radio-/chemotherapeutic treatment in head and neck cancer.

BACKGROUND: Radiation and systemic chemotherapy are standard treatment strategies for advanced or metastatic head and neck cancer. However, little is known about the implications and changes in the tumor microenvironment, including the T-helper (TH)1/TH2 balance in response to these treatment regimens. The aim of the current study was to unravel the effects of chemotherapeutic drugs and radiation on cytokine changes. MATERIALS AND METHODS: In this study, the effect of radiation and chemotherapeutic treatment (5-fluorouracil and cisplatin) on eight cell lines was determined. Before and after exposure, cytokine levels in culture supernatants of cell lines were evaluated using the Bio-Plex Assay (Bio-Rad) and the Human TH1/TH2 Cytometric Bead Array (Becton Dickinson). Results were correlated with parallel measurements for cellular proliferation assessed by cytotoxicity assay. RESULTS: Seven out of eight cell lines of primary tumors or metastases demonstrated an enhanced level of the cytokines interleukin (IL)-1ß, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α), after sub-lethal radiation doses. Under treatment with low concentrations of 5-fluorouracil and cisplatin, all examined cell lines showed an increasing secretion of the cytokines IL-6 and G-CSF. In contrast, sub-lethal doses of both cytostatic drugs revealed a dose-dependent decrease in secretion IL-1ß. Regarding GM-CSF and TNF-α, we demonstrated an increase in secretion by the primary tumors under low doses of 5-fluorouracil and cisplatin, whereas the metastases showed a sharp drop of GM-CSF and TNF-α secretion. Chemotherapeutic treatment led to no changes of the IL-8 cytokine profile. CONCLUSION: The results suggest complex cytokine changes of the tumor microenvironment and more aberrant expression profiles under treatment with radiation and the chemotherapeutic drugs 5-fluorouracil and cisplatin.

Effects of tobacco on cytokine expression from human endothelial cells.

OBJECTIVE: To investigate the effects of nicotine and cigarette smoke condensate (CSC) exposure on cytokine expression from human endothelial cells in order to identify one possible mechanism that smoking plays in the pathogenesis of both periodontal disease (PDD) and cardiovascular disease (CVD). METHODS: Human endothelial cells (HUVECs) were exposed to different concentrations of nicotine and CSC to examine the effects that they have on cell proliferation and cytotoxicity. Non-toxic levels were then used to examine cytokine expression using cytokine protein arrays. RESULTS: Exposure to nicotine caused significant down-regulation in the expression of IL-10 (P = 0.046), growth-regulated oncogene (GRO)α (P = 0.036), MCP-1 (P = 0.046), and GMCSF (P = 0.004) compared with the control untreated HUVECs. Exposure to CSC caused significant down-regulation in the expression of GRO (P = 0.04), GROα (P = 0.01), IL-6 (P = 0.03), and MCP-1 (P = 0.04) compared with the control untreated HUVECs. CONCLUSIONS: Exposure of HUVECs to nicotine or CSC affects the levels of cytokine expression including reduction in anti-inflammatory and chemoattractant cytokines. This may subsequently affect the host defensive mechanisms of the tissues. The action of toxic chemicals in tobacco smoke on endothelial cells is a potential pathogenic mechanism that may in part explain the association between tobacco, PDD, and CVD.

Oral malodorous compound causes caspase-8 and -9 mediated programmed cell death in osteoblasts.

BACKGROUND AND OBJECTIVE: Hydrogen sulfide (H(2) S) is one of two volatile sulfur compounds that are known to be the main cause of oral malodor; the other is methyl mercaptan. Other known volatiles existing in mouth air do not contribute significantly to oral malodor originating in the oral cavity. Hydrogen sulfide is also known to be an etiological factor in periodontal disease. However, the effects of H(2) S on alveolar bone remain unclear. The objectives of this study were to determine the apoptotic effects of H(2) S on osteoblasts and to verify the apoptotic molecular pathways. MATERIAL AND METHODS: A clonal murine calvaria cell line was incubated with 50 ng/mL of H(2) S. To detect apoptosis, the cells were analysed by flow cytometry and ELISA. Mitochondrial membrane depolarization was assessed using flow cytometry as well. ELISA was used to evaluate the release of cytochrome c into the cytosol and to assess Fas ligand, p53, tumor necrosis factor α, interleukin IL1-α IL-ß, IL-2, IL-4, IL-10, interferon-γ, granulocyte-colony stimulating factor and granulocyte-macrophage colony stimulating factor. Caspase-3, -8 and -9 activities were estimated. Expression of BAX and Bcl-2 was assessed by real-time quantitative RT-PCR. DNA fragmentation was detected by single-cell gel electrophoresis. Fas receptors were evaluated by western blotting. RESULTS: After H(2) S incubation, apoptotic levels increased significantly in a time-dependent manner. Mitochondrial membrane depolarization, the release of cytochrome c, p53 and caspase-3, -8 and -9 and DNA fragmentation were all significantly greater. BAX gene activity was upregulated, whereas Bcl-2 remained low. Fas ligand/Fas receptor, tumor necrosis factor α and other cytokines were not increased to a significant degree. CONCLUSION: At less-than-pathological concentrations in gingival crevicular fluid, H(2) S induces apoptosis in osteoblasts. The molecular mechanisms underlying the apoptotic process include p53, a mitochondrial pathway and caspase-8 activation.

Zinc supplementation restores PU.1 and Nrf2 nuclear binding in alveolar macrophages and improves redox balance and bacterial clearance in the lungs of alcohol-fed rats.

BACKGROUND: Chronic alcohol abuse causes oxidative stress, impairs alveolar macrophage immune function, and increases the risk of pneumonia and acute lung injury. Recently we determined that chronic alcohol ingestion in rats decreases zinc levels and macrophage function in the alveolar space; provocative findings in that zinc is essential for normal immune and antioxidant defenses. Alveolar macrophage immune function depends on stimulation by granulocyte/monocyte colony-stimulating factor, which signals via the transcription factor PU.1. In parallel, the antioxidant response element signals via the transcription factor Nrf2. However, the role of zinc bioavailability on these signaling pathways within the alveolar space is unknown. METHODS: To determine the efficacy of dietary zinc supplementation on lung bacterial clearance and oxidative stress, we tested 3 different groups of rats: control-fed, alcohol-fed, and alcohol-fed with zinc supplementation. Rats were then inoculated with intratracheal Klebsiella pneumoniae, and lung bacterial clearance was determined 24 hours later. Isolated alveolar macrophages were isolated from uninfected animals and evaluated for oxidative stress and signaling through PU.1 and Nrf2. RESULTS: Alcohol-fed rats had a 5-fold decrease in lung bacterial clearance compared to control-fed rats. Dietary zinc supplementation of alcohol-fed rats normalized bacterial clearance and mitigated oxidative stress in the alveolar space, as reflected by the relative balance of the thiol redox pair cysteine and cystine, and increased nuclear binding of both PU.1 and Nrf2 in alveolar macrophages from alcohol-fed rats. CONCLUSIONS: Dietary zinc supplementation prevents alcohol-induced alveolar macrophage immune dysfunction and oxidative stress in a relevant experimental model, suggesting that such a strategy could decrease the risk of pneumonia and lung injury in individuals with alcohol use disorders.

APOE genotype affects outcome in a murine model of sepsis: implications for a new treatment strategy.

In this study, we assessed whether apolipoprotein E (APOE) polymorphism affects inflammatory responses and mortality in the caecal ligation and puncture model of peritonitis. In addition, we determined the effects of APOE mimetic peptide administration in this sepsis model. Differences in survival between targeted replacement mice expressing the human APOE3 allele (APOE3TR) and the APOE4 allele (APOE4TR) mice were assessed. In a separate series of experiments, COG1410, an apoE-mimetic peptide, was administered intravenously at 12-hour intervals for 72 hours and compared to vehicle-treated control animals. End-points included mortality and serum levels of interleukin-1beta, interleukin-6, interleukin-12 and tumour necrosis factor-alpha. Mice expressing the human APOE4 allele (n = 16) demonstrated an increase in mortality following caecal ligation and puncture compared with APOE3TR mice (n = 22; P = 0.039). Administration of the apolipoprotein E mimetic COG1410 was well tolerated and APOE3TR mice treated with peptide (n = 20) demonstrated a significant reduction in mortality compared with vehicle treated animals (n = 20; P = 0.007). A similar effect was also observed in APOE4TR animals, in which treatment with COG1410 was associated with reduced mortality compared with vehicle treatment (n =16 animals/group; P = 0.027). COG1410 was also associated with a reduction in TNFalpha, interleukin-1beta, interleukin-6 and interleukin-12 levels in both APOE3TR and APOE4TR (n = 5 animals/group) assessed at 24 hours. Thus, administration of an apolipoprotein E-mimetic peptide is well tolerated, suppresses inflammatory responses, and improves mortality in a caecal ligation and puncture model of sepsis.

[Immuno-modulating effects of eukaryotic expressing vectors of IL-12 and GM-CSF associated to DNA-based vaccination against experimental cutaneous leishmaniasis in BALB/c mouse]. / Effets immuno-modulateurs des vecteurs d'expressions eucaryotes de l'il-12 et du GM-CSF en association avec la vaccination à base d'ADN contre la leishmaniose cutanée expérimentale chez la souris BALB/c.

Different works of DNA based vaccination against leishmaniasis highlight the complexity of the induced immune responses to fight against the disease. In this work, we exploited the capacity of IL-12 and GMC-SF to activate immune cell mediators and effectors to induce a Th1 response, more capable of clearing the parasite. To generate these immunomodulating activities, we associated eukaryotic expressing vectors of murine IL-12 and GMC-SF to several DNA based vaccine candidates encoding to several L. (L.) major antigens, in the BALB/c mouse. When mice were challenged with a high parasitic load in the hind footpad, no additional protective effect could be generated. However, when the challenge was carried out in the inner face of the ear with a small parasitic load, the association of plasmids encoding to IL-12 and GMC-SF to DNA based vaccination, the protective effects were increased.

Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-kappaB translocation.

Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 microM after 48 h incubation. Pretreatment with 100 microM PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstrated that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-alpha, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and IkappaBalpha, as well as the nuclear translocation of NF-kappaB primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-kappaB nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers.

Gene therapy of beta(c)-deficient pulmonary alveolar proteinosis (beta(c)-PAP): studies in a murine in vivo model.

Pulmonary alveolar proteinosis (PAP) due to deficiency of the common beta-chain (beta(c)) of the interleukin-3 (IL-3)/IL-5/granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors is a rare monogeneic disease characterized by functional insufficiency of pulmonary macrophages. Hematopoietic stem cell gene therapy for restoring expression of beta(c)-protein in the hematopoietic system may offer a curative approach. Toward this end, we generated a retroviral construct expressing the murine beta(c) (mbeta(c)) gene and conducted investigations in a murine model of beta(c)-deficient PAP. Functional correction of mbeta(c) activity in mbeta(c)(-/-) bone marrow (BM) cells was demonstrated by restoration of in vitro colony formation in response to GM-CSF. In addition, in a murine in vivo model of mbeta(c)-deficient PAP mbeta(c) gene transfer to hematopoietic stem cells not only restored the GM-CSF-sensitivity of hematopoietic progenitor cells but also, within a period of 12 weeks, almost completely reversed the morphologic features of surfactant accumulation. These results were obtained despite modest transduction levels (10-20%) and, in comparison to wild-type mice, clearly reduced beta(c) expression levels were detected in hematopoietic cells. Therefore, our data demonstrating genetic and functional correction of mbeta(c)(-/-) deficiency in vitro as well as in a murine in vivo model of PAP strongly suggest gene therapy as a potential new treatment modality in beta(c)-deficient PAP.

Inhibitory effects of thunberginols A and B isolated from Hydrangeae Dulcis Folium on mRNA expression of cytokines and on activation of activator protein-1 in RBL-2H3 cells.

Previously, thunberginols A and B from the processed leaves of Hydrangeae macrophylla var. thunbergii (Hydrangea dulcis folium) substantially inhibited the degranulation caused by antigen and calcium ionophore A23187, and the release of tumor necrosis factor (TNF)-alpha and interleukin (IL)-4 by antigen in RBL-2H3 cells. In the present study, we examined the effect of thunberginol B on the expression of mRNA of several cytokines [ILs-2, 3, 4 and 13, TNF-alpha and granulocyte/macrophage-colony stimulating factor (GM-CSF)] and effects of thunberginols A and B on activator protein (AP)-1 composed of c-jun and c-fos, which is essential for the expression of the cytokine mRNA, in RBL-2H3 cells. Thunberginol B inhibited up-regulated genes of all cytokines, and thunberginols A and B (30 microM) inhibited the phosphorylation of c-jun and expression of c-fos mRNA and phosphorylation of extracellular signal-regulated kinases (ERK1/2). In addition, the profile of gene expression by thunberginol B was similar to that by luteolin, a natural flavone with a potent anti-allergic effect.

Calcium dependent and independent cytokine synthesis by air pollution particle-exposed human bronchial epithelial cells.

UNLABELLED: Exposure to ambient air pollution particles with a diameter of <10 microm (PM(10)) has been associated with increased cardiopulmonary morbidity and mortality. We have shown that human bronchial epithelial cells (HBECs) exposed to PM(10) produce pro-inflammatory mediators that contribute to a local and systemic inflammatory response. Changes in intracellular calcium concentrations ([Ca(2+)](i)) have been demonstrated to regulate several functions of the airway epithelium including the production of pro-inflammatory mediators. The aim of the present study was to determine the nature and mechanism of calcium responses induced by PM(10) in HBECs and its relationship to cytokine synthesis. METHODS: Primary HBECs were exposed to urban air pollution particles (EHC-93) and [Ca(2+)](i) responses were measured using the fluoroprobe (Fura-2). Cytokine levels were measured at mRNA and protein levels using real-time PCR and ELISA. RESULTS: PM(10) increased [Ca(2+)](i) in a dose-dependent manner. This calcium response was reduced by blocking the influx of calcium into cells (i.e. calcium-free medium, NiCl(2), LaCl(3)). PM(10) also decreased the activity of calcium pumps. PM(10) increased the production of IL-1beta, IL-8, GM-CSF and LIF. Preincubation with intracellular calcium chelator (BAPTA-AM) attenuated IL-1beta and IL-8 production, but not GM-CSF and LIF production. CONCLUSION: We conclude that exposure to PM(10) induces an increase in cytosolic calcium and cytokine production in bronchial epithelial cells. Our results also suggest that PM(10) induces the production of pro-inflammatory mediators via either intracellular calcium-dependent (IL-1beta, IL-8) or -independent (GM-CSF, LIF) pathways.

Theoretical basis for herbal medicines, Tokishakuyaku-San and Sairei-To, in the treatment of recurrent abortion: enhancing the production of granulocyte-macrophage colony-stimulating factor in decidual stromal cells.

PROBLEM: To get insight into the basis for the empirical usage of herbal medicines, such as Tokishakuyaku-san (Toki) and Sairei-to (Sai) in the treatment of recurrent abortion and intrauterine growth restriction, we examined whether these medicines modulate the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine working as an important mediator for intercellular communication in the embryonic development, in decidual stromal cells (DSCs). METHOD OF STUDY: Human DSCs were cultured with either Toki or Sai at several different concentrations. The effect on cell proliferation was assessed by WST-8 assay. GM-CSF released into culture medium was analyzed using enzyme-linked immunosorbent assay, and semi-quantitative polymerase chain reaction was carried out to see GM-CSF mRNA expression in DSCs. RESULTS: Sai inhibited the proliferation of cultured DSCs, while no interference was observed in the presence of Toki. Both Toki and Sai enhanced the release of GM-CSF into culture medium. The amount of GM-CSF mRNA in cultured DSCs was as well increased by either Toki or Sai. CONCLUSION: Considering the significance of GM-CSF in embryonic development, clinical benefit of these herbal medicines in the treatment of recurrent abortion might be based on the shown pharmacological reaction related to GM-CSF.