RESUMO
To date only four recombinant growth factors, including Filgrastim (rhG-CSF), have been approved by FDA as radiomitigators to ameliorate hematopoietic acute radiation syndrome (H-ARS). These approved agents are not stable under room-temperature, needing to be stored at 2-8 °C, and would not be feasible in a mass casualty scenario where rapid and cost-effective intervention is crucial. Delta-tocotrienol (δ-T3H), the most potent G-CSF-inducing agent among vitamin E isoforms, exhibited efficiency and selectivity on G-CSF production in comparison with TLR and STING agonists in mice. Five-dose δ-T3H was utilized as the optimal therapeutic regimen due to long-term G-CSF production and the best peripheral blood (PB) recovery of irradiated mice. Comparable with rhG-CSF, sequential administration of δ-T3H post-irradiation improved hematologic recovery and accelerated the regeneration of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in the bone marrow (BM) and spleen of 6.5Gy irradiated mice; and consistently enhanced repopulation of BM-HSCs. In 4.0Gy irradiated nonhuman primates, δ-T3H exhibited comparable efficacy as rhG-CSF to promote PB recovery and colony-formation of BM-HPCs. Altogether, we demonstrated that sequential administration of delta-tocotrienol ameliorates radiation-induced myelosuppression in mice and non-human primates through inducing G-CSF production, indicated δ-T3H as a promising radiomitigator for the management of H-ARS, particularly in a mass casualty scenario.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Vitamina E , Animais , Camundongos , Medula Óssea/patologia , Medula Óssea/efeitos da radiação , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Primatas , Proteínas Recombinantes/farmacologia , Vitamina E/análogos & derivados , Vitamina E/uso terapêuticoRESUMO
Hematopoietic stem cells (HSCs) must ensure adequate blood cell production following distinct external stressors. A comprehensive understanding of in vivo heterogeneity and specificity of HSC responses to external stimuli is currently lacking. We performed single-cell RNA sequencing (scRNA-Seq) on functionally validated mouse HSCs and LSK (Lin-, c-Kit+, Sca1+) progenitors after in vivo pharmacological perturbation of niche signals interferon, granulocyte colony-stimulating factor (G-CSF), and prostaglandin. We identified six HSC states that are characterized by enrichment but not exclusive expression of marker genes. External signals induced rapid transitions between HSC states but transcriptional response varied both between external stimulants and within the HSC population for a given perturbation. In contrast to LSK progenitors, HSCs were characterized by a greater link between molecular signatures at baseline and in response to external stressors. Chromatin analysis of unperturbed HSCs and LSKs by scATAC-Seq suggested some HSC-specific, cell intrinsic predispositions to niche signals. We compiled a comprehensive resource of HSC- and LSK progenitor-specific chromatin and transcriptional features that represent determinants of signal receptiveness and regenerative potential during stress hematopoiesis.
Most organs in the human body are maintained by a type of immature cells known as adult stem cells, which ensure a constant supply of new, mature cells. Adult stem cells monitor their environment through external signalling molecules and replace damaged cells as needed. Stem cell therapy takes advantage of the regenerative ability of immature stem cells and can be helpful for conditions such as blood diseases, autoimmune diseases, neurodegeneration and cancer. For example, hematopoietic stem-cell transplantation is a treatment for some types of cancer and blood disorders, in which stem cells are harvested from the blood or bone marrow and reintroduced into the body, where they can develop into all types of blood cells, including white blood cells, red blood cells and platelets. Hematopoietic stem-cell transplants have been in use for over 30 years, but they remain a highly risky procedure. One of the challenges is that outcomes can vary between patients and many of the factors that can influence the 'regenerative' potential of hematopoietic stem cells, such as external signalling molecules, are not well understood. To fill this gap, Fast et al. analysed which genes are turned on and off in hematopoietic stem cells in response to several external signalling molecules. To do so, three signalling pathways in mice were altered by injecting them with different chemicals. After two hours, the hematopoietic stem cells were purified and the gene expression for each cell was analysed. This revealed that the types of genes and the strength at which they were affected by each chemical was unique. Moreover, hematopoietic stem cells responded rapidly to external signals, with substantial differences in gene expression between individual groups of cells. Contrary to more specialised cells, the external signalling genes in some hematopoietic stem cells were already activated without being injected with external signalling molecules. This suggest that low levels of external signalling molecules released from their microenvironment may prepare stem cells to better respond to future stress or injuries. These results help to better understand stem cells and to evaluate how the signalling state of hematopoietic stem cells affects regeneration, and ultimately improve hematopoietic stem cell transplantation for patients.
Assuntos
Regulação da Expressão Gênica/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Transcriptoma , Animais , Linhagem da Célula , Feminino , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Interferons/efeitos dos fármacos , Masculino , Camundongos , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/metabolismo , Prostaglandinas/metabolismo , Análise de Sequência de RNA , Transdução de SinaisRESUMO
BACKGROUND: Kawasaki disease (KD) is the most common pediatric systemic vasculitides of unknown etiology. Recent clinical studies led to reappraisal of the usefulness of initial combination therapy of intravenous immunoglobulin (IVIG) plus a corticosteroid for patients with severe KD. However, the molecular mechanisms underlying the clinical benefits of that combination therapy remain unclear. Here, we used cultured human coronary artery endothelial cells (HCAECs), as a mimic of KD, to study the possible mechanisms responsible for the clinical benefits of adding a corticosteroid to standard IVIG therapy for patients with severe KD. METHODS: HCAECs were stimulated with TNF-α, IL-1α or IL-1ß in the presence and absence of high-dose IgG and/or dexamethasone (DEX). The mRNA and protein concentrations for high-mobility group box-1 (HMGB1), IL-1α, IL-6 and granulocyte-colony stimulating factor (G-CSF) in the culture supernatants were measured by quantitative PCR (qPCR) and ELISA, respectively. Apoptosis was evaluated by the caspase 3/7 activities. RESULTS: DEX, but not IgG, significantly inhibited apoptosis caused by inflammatory stimuli, resulting in effective reduction of HMGB1 and IL-1α protein release by HCAECs. As previously reported, DEX or IgG alone significantly suppressed TNF-α-induced production of IL-6 and G-CSF and mRNA expression, but induction of those cytokines by IL-1 s (IL-1α and IL-1ß) was resistant to high-dose IgG. CONCLUSIONS: A corticosteroid can effectively inhibit the release of HMGB1 and IL-1α, which may be involved in IVIG resistance in KD. Since high-dose IgG does not have such beneficial anti-cytotoxic effects, adding a corticosteroid to standard IVIG therapy may help prevent the progression of IVIG resistance in KD.
Assuntos
Vasos Coronários/citologia , Dexametasona/farmacologia , Células Endoteliais/efeitos dos fármacos , Glucocorticoides/farmacologia , Imunoglobulina G/farmacologia , Fatores Imunológicos/farmacologia , Síndrome de Linfonodos Mucocutâneos/tratamento farmacológico , Apoptose/efeitos dos fármacos , Células Cultivadas , Dexametasona/uso terapêutico , Quimioterapia Combinada , Células Endoteliais/metabolismo , Glucocorticoides/uso terapêutico , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteína HMGB1/efeitos dos fármacos , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Técnicas In Vitro , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1alfa/farmacologia , Interleucina-1beta/farmacologia , Interleucina-6/genética , Interleucina-6/metabolismo , Modelos Cardiovasculares , Síndrome de Linfonodos Mucocutâneos/genética , Síndrome de Linfonodos Mucocutâneos/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
A 67-year-old man with relapsed anaplastic large cell lymphoma received salvage chemotherapy, and pegfilgrastim was used to prevent febrile neutropenia. On day 18 of chemotherapy, he developed a pseudogout attack. Although the first symptoms improved, another pseudogout attack occurred when he received the second course of chemotherapy and pegfilgrastim. Filgrastim was then used for the third course of chemotherapy, and a pseudogout attack did not occur. The serum granulocyte-stimulating factor (G-CSF) level was extremely elevated only when pegfilgrastim was used, suggesting a relationship between pseudogout and G-CSF. Pseudogout should be recognized as an adverse effect of pegfilgrastim.
Assuntos
Condrocalcinose/induzido quimicamente , Filgrastim/efeitos adversos , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Polietilenoglicóis/efeitos adversos , Idoso , Filgrastim/uso terapêutico , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Humanos , Masculino , Neutropenia/tratamento farmacológico , Polietilenoglicóis/uso terapêutico , Proteínas Recombinantes/uso terapêuticoRESUMO
Cocaine addiction is characterized by dysfunction in reward-related brain circuits, leading to maladaptive motivation to seek and take the drug. There are currently no clinically available pharmacotherapies to treat cocaine addiction. Through a broad screen of innate immune mediators, we identify granulocyte-colony stimulating factor (G-CSF) as a potent mediator of cocaine-induced adaptations. Here we report that G-CSF potentiates cocaine-induced increases in neural activity in the nucleus accumbens (NAc) and prefrontal cortex. In addition, G-CSF injections potentiate cocaine place preference and enhance motivation to self-administer cocaine, while not affecting responses to natural rewards. Infusion of G-CSF neutralizing antibody into NAc blocks the ability of G-CSF to modulate cocaine's behavioral effects, providing a direct link between central G-CSF action in NAc and cocaine reward. These results demonstrate that manipulating G-CSF is sufficient to alter the motivation for cocaine, but not natural rewards, providing a pharmacotherapeutic avenue to manipulate addictive behaviors without abuse potential.
Assuntos
Comportamento Animal/efeitos dos fármacos , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Cocaína/farmacologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Plasticidade Neuronal/efeitos dos fármacos , Animais , Comportamento Aditivo/tratamento farmacológico , Comportamento Aditivo/fisiopatologia , Cocaína/administração & dosagem , Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Condicionamento Operante , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
OBJECTIVE: To determine the effect of unfractionated heparin (UFH) on lipopolysaccharide (LPS)-induced expression of granulocyte colony-stimulating factor (G-CSF), and the role of Toll-like receptor 4 (TLR4) signaling pathway in this process. METHODS: Human pulmonary microvascular endothelial cells (HPMECs) were cultured in vitro, and the cells between passages 3 and 5 were used in the experiments. Experiment I: the cells were divided into four groups as follows: control group, LPS stimulation group (LPS 10 µg/mL), LPS + 0.1 U/mL UFH group, and LPS + 1 U/mL UFH group. HPMECs in UFH groups were treated with 0.1 U/mL or 1 U/mL UFH 15 minutes before LPS stimulation, and HPMECs in control group were treated with an equal volume of phosphate-buffered saline (PBS) instead. The concentrations of interleukin-6 (IL-6) and G-CSF in cell culture supernatants were determined by enzyme linked immunosorbent assay (ELISA) 24 hours after LPS challenge to detect the effect of UFH on HPMECs. Experiment II: HPMECs were treated with 5 µg/mL of rhodobacter sphaeroides LPS (LPS-RS, antagonist for TLR4) 4 hours before the addition of PBS or LPS. The concentrations of IL-6 and G-CSF in cell culture supernatants were determined 24 hours after LPS stimulation to detect the effect of TLR4 on LPS-induced HPMEC injury. Experiment III: HPMECs were divided into four groups as before: control group, LPS stimulation group, LPS + 0.1 U/mL UFH group, LPS + 1 U/mL UFH group. Treatments to cells were the same as experiment I. The protein expression of TLR4 in HPMECs was determined by Western Blot 1 hour after LPS stimulation to detect the effect of UFH on TLR4. RESULTS: (1) Compared with control group, the levels of IL-6 and G-CSF in LPS stimulation group were increased [IL-6 (ng/L): 655.9±58.3 vs. 75.5±18.2, G-CSF (ng/L): 388.7±36.2 vs. 35.3±12.6, both P < 0.05]. Compared with those of LPS stimulation group, in LPS + 0.1 U/mL UFH group and LPS + 1 U/mL UFH group, the levels of IL-6 and G-CSF were significantly decreased [IL-6 (ng/L): 518.2±64.6, 489.1±75.6 vs. 655.9±58.3, G-CSF (ng/L): 298.8±41.0, 273.4±33.2 vs. 388.7±36.2, all P < 0.05]. The results indicated that 1 U/mL UFH had better results, though there was no statistical significance between the results of two UFH groups. (2) LPS-induced up-regulation of IL-6 and G-CSF levels was prevented by LPS-RS [IL-6 (ng/L): 139.1±37.6 vs. 655.9±58.3, G-CSF (ng/L): 73.7±19.7 vs. 388.7±36.2, both P < 0.05]. LPS-RS alone had no effect on cytokines [IL-6 (ng/L): 118.2±42.1 vs. 75.5±18.2, G-CSF (ng/L): 48.4±26.8 vs. 35.3±12.6, both P > 0.05]. (3) Compared with control group, the protein expression of TLR4 (grey value) in LPS stimulation group was significantly upregulated after 1 hour (0.87±0.23 vs. 0.36±0.12, P < 0.05). UFH with 0.1 U/mL and 1 U/mL lowered TLR-4 protein expression induced by LPS (0.68±0.18, 0.62±0.26 vs. 0.87±0.23, both P < 0.05). CONCLUSIONS: The expressions of IL-6 and G-CSF were increased obviously in LPS treated HPMECs. UFH might take its therapeutic effect through TLR4-dependent pathway.
Assuntos
Células Endoteliais/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Heparina/farmacologia , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Células Cultivadas , Citocinas , Humanos , Interleucina-6 , Lipopolissacarídeos , PulmãoRESUMO
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.
Assuntos
Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/terapia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos da radiação , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Fluoruracila/farmacologia , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fator Estimulador de Colônias de Granulócitos/efeitos da radiação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos da radiação , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/radioterapia , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/efeitos da radiação , Interleucina-6/metabolismo , Interleucina-6/efeitos da radiação , Interleucina-8/metabolismo , Interleucina-8/efeitos da radiação , Equilíbrio Th1-Th2/efeitos dos fármacos , Equilíbrio Th1-Th2/efeitos da radiação , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/efeitos da radiaçãoRESUMO
Dietary n-3 polyunsaturated fatty acids (PUFA) influence the inductive phase of inflammation but less is known about their effects on the resolution phase. This study examined the effects of dietary fish oil on induction and resolution of antigen-induced inflammation in mice. Mice were fed a control diet with or without 2.8% fish oil, immunized twice with methylated BSA (mBSA) and inflammation induced by intraperitoneal injection of mBSA. Prior to and at different time points after mBSA administration, peritoneal cells were analyzed and expression of surface molecules determined by flow cytometry. Concentration of chemokines, cytokines and soluble cytokine receptors was determined by ELISA. Mice fed the fish oil diet had fewer peritoneal neutrophils, shorter resolution interval and lower levels of pro-inflammatory cytokines and chemokines than mice fed the control diet. In mice fed the fish oil diet there was an early peak in peritoneal levels of the immunosuppressive molecules sIL-6R and TGF-ß, that was not seen in mice fed the control diet. In the resolution phase, peritoneal macrophages from mice fed the fish oil diet expressed more of the atypical chemokine receptor D6 and peritoneal TGF-ß levels were higher than that in mice fed the control diet. Furthermore, in the late-resolution phase there were more peritoneal eosinophils and macrophages in mice fed the fish oil diet than in mice fed the control diet. These results demonstrate a suppressive effect of n-3 PUFA on the inductive phase of inflammation and indicate an enhancing effect of n-3 PUFA on resolution of inflammation.
Assuntos
Gorduras Insaturadas na Dieta/uso terapêutico , Ácidos Graxos Ômega-3/uso terapêutico , Óleos de Peixe/uso terapêutico , Peritonite/prevenção & controle , Animais , Quimiocina CCL11/efeitos dos fármacos , Quimiocina CXCL1/efeitos dos fármacos , Feminino , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Inflamação/prevenção & controle , Interleucina-6/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/dietoterapia , Peritonite/imunologia , Receptores de Interleucina-6/efeitos dos fármacos , Soroalbumina Bovina/imunologia , Fator de Crescimento Transformador beta/efeitos dos fármacosRESUMO
In the experiments on F1 (CBA x C57BL) and BALB mice irradiated by 137Cs gamma-rays, preparations of unglycosilated G-SCF such as Neupogen and their domestic analogs Leucostim and Neupomax were investigated. The tests such as 9-day bone marrow cellularity (BMC) and endogenous CFUs, the neutrophile number restoration, the 30-day survival index have shown that all three preparations have an approximately equal effectiveness relating to acute radiation disease treatment and granulopoiesis stimulation after a 5-10 day consecutive administration following irradiation of mice at lethal and sublethal doses. We have come to the conclusion that Leucostim and Neupomax can be regarded as adequate substitutes for Neupogen.
Assuntos
Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Neutrófilos , Protetores contra Radiação/administração & dosagem , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/efeitos da radiação , Feminino , Filgrastim , Raios gama , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Leucopoese/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos da radiação , Doses de Radiação , Proteínas Recombinantes/administração & dosagemRESUMO
OBJECTIVE: Fetal alcohol syndrome (FAS) is associated with intellectual disability and neurodevelopmental abnormalities. Neuroprotective peptides NAPVSIPQ (NAP) and SALLRSIPA (SAL) can prevent some of the alcohol-induced teratogenesis including fetal death, growth abnormalities, and learning impairment in part by preventing alcohol-induced alterations in N-methyl-D-aspartate receptor gene expression in a mouse model for FAS. We evaluated a panel of cytokines and chemokines to determine whether NAP plus SAL work through a cytokine/chemokine-mediated pathway in preventing these alterations. STUDY DESIGN: Using a well-characterized FAS model, timed, pregnant C57BL6/J mice were treated on gestational day (E) 8 with alcohol (0.03 mL/g), placebo, or alcohol plus peptides. Embryos were evaluated at 2 time points: after 6 hours and 10 days later at E18. A panel of cytokines/chemokines was measured using a microsphere-based multiplex immunoassay (Luminex xMAP; Millipore, Billerica, MA). Statistical analysis included Kruskal-Wallis, with P < .05 considered significant. RESULTS: Six hours after treatment, interleukin (IL)-6 and keratinocyte chemoattractant cytokine (KC) were not detectable in the control embryos. Alcohol treatment resulted in detectable levels and significant increases in IL-6 (median, 15.7; range, 10.1-45.9 pg/mL) and KC (median, 45.9; range, 32.5-99.1 pg/mL). Embryos exposed to alcohol plus NAP plus SAL had undetectable IL-6 and KC (both P < .003), similar to the controls. Alcohol exposure resulted in a significant increase of granulocyte colony-stimulating factor (G-CSF) (P < .003) as compared with controls, and treatment with NAP plus SAL prevented the alcohol-induced increase. IL-13 and IL-1ß were decreased 6 hours after alcohol exposure, and exposure to alcohol plus NAP plus SAL did not completely ameliorate the decrease. At E18, 10 days after exposure, these alterations were no longer present. Several analytes (regulated upon activation, normal T cell expressed, and secreted, tumor necrosis factor-α, interferon-γ, and IL-4) were not detectable at either time point in any of the groups. CONCLUSION: Prenatal alcohol exposure acutely results in a significant elevation of IL-6, G-CSF and the KC, which are known to affect N-methyl-D-aspartate receptors. NAP plus SAL treatment prevented alcohol-induced increases. This provides additional insight into the mechanism of alcohol damage in FAS and NAP plus SAL prevention of neurodevelopmental anomalies.
Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Transtornos do Espectro Alcoólico Fetal/tratamento farmacológico , Oligopeptídeos/farmacologia , Animais , Quimiocinas/efeitos dos fármacos , Modelos Animais de Doenças , Etanol/toxicidade , Feminino , Transtornos do Espectro Alcoólico Fetal/metabolismo , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Fatores Imunológicos/metabolismo , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , GravidezRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 8/efeitos dos fármacos , Caspase 9/efeitos dos fármacos , Halitose/metabolismo , Sulfeto de Hidrogênio/efeitos adversos , Osteoblastos/efeitos dos fármacos , Células 3T3 , Animais , Caspase 3/efeitos dos fármacos , Citocromos c/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Proteína Ligante Fas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Interferon gama/efeitos dos fármacos , Interleucina-10/análise , Interleucina-1alfa/análise , Interleucina-1beta/efeitos dos fármacos , Interleucina-2/análise , Interleucina-4/análise , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Compostos Orgânicos Voláteis/efeitos adversos , Proteína X Associada a bcl-2/efeitos dos fármacos , Receptor fas/efeitos dos fármacosRESUMO
This paper explores the effect of granulocyte colony-stimulating factor (G-CSF) on mouse myocardial microvascular endothelial cell (CMECs) proliferation. CMECs were harvested from C57/BL6 mice. CMECs were cultured in medium containing G-CSF (0 ng/mL, 20 ng/mL, 40 ng/mL, 60 ng/mL) for five days. Proliferative activity of CMECs was examined by CCK-8 method. Hypoxia inducible factor-1 (HIF-1) and p53 expression levels was determined from the mRNA obtained by reverse transcription polymerase chain reaction (RT-PCR). Results showed that the purity quotient of the CMECs, which were cultured by the method of modified myocardial tissue explant culture, was higher than 95%. Compared with control untreated cells, the proliferative activity of CMECs and the expression level of HIF-1 mRNA in these cells were enhanced by G-CSF treatment, whereas the expression level of p53 mRNA was markedly reduced. It may be concluded that G-CSF could promote the proliferative activity of CMECs, which might be mediated by upregulation of HIF-1 and downregulation of p53.
Assuntos
Proliferação de Células , Células Endoteliais/metabolismo , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Animais , Células Cultivadas , Vasos Coronários/citologia , Regulação para Baixo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
We present the first case report of the use of sorafenib and S-1 for the treatment of renal cell carcinoma (RCC) producing granulocyte colony-stimulating factor (G-CSF). This entity is clinically rare and has a poor outcome. A 78-year-old Japanese man presented with macrohematuria, left flank pain, and a palpable mass. Laboratory data showed marked leukocytosis with increased serum and urinary G-CSF. The histopathological diagnosis was unclassified RCC. New combination therapy with sorafenib and S-1 exerted a therapeutic effect and apparently decreased serum and urinary G-CSF levels, although the patient died of gastrointestinal perforation. The use of combined sorafenib and S-1 may be worthy of consideration in the treatment of RCC producing G-CSF.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Fator Estimulador de Colônias de Granulócitos/biossíntese , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Idoso , Benzenossulfonatos/administração & dosagem , Carcinoma de Células Renais/patologia , Combinação de Medicamentos , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/urina , Humanos , Neoplasias Renais/patologia , Leucocitose , Masculino , Futilidade Médica , Niacinamida/análogos & derivados , Ácido Oxônico/administração & dosagem , Compostos de Fenilureia , Piridinas/administração & dosagem , Sorafenibe , Tegafur/administração & dosagemRESUMO
Clinical application of retinoic acids (RAs) and demethylation agents has proven to be effective in treating certain myeloid leukemia patients. However, the target genes that mediate these antileukemia activities are still poorly understood. In this study, we identified olfactomedin 4 (OLFM4), a myeloid-lineage-specific gene from the olfactomedin family, as a novel target gene for RAs and the demethylation agent, 5-aza-2'-deoxycytidine. We demonstrated that the retinoic acid receptor alpha/retinoic X receptor alpha heterodimer binds to a retinoic acid response-element (DR5) site in the OLFM4 promoter and mediates all-trans-retinoic acid (ATRA)-induced transactivation of the OLFM4 gene. OLFM4 overexpression in HL-60 cells led to growth inhibition, differentiation, and apoptosis, and potentiated ATRA induction of these effects. Conversely, down-regulation of endogenous OLFM4 in acute myeloid leukemia-193 cells compromised ATRA-induced growth inhibition, differentiation, and apoptosis. Overexpression of OLFM4 in HL-60 cells inhibited constitutive and ATRA-induced phosphorylation of the eukaryote initiation factor 4E-binding protein 1 (4E-BP1), whereas down-regulation of OLFM4 protein in acute myeloid leukemia-193 cells increased 4E-BP1 phosphorylation, suggesting that OLFM4 is a potent upstream inhibitor of 4E-BP1 phosphorylation/deactivation. Thus, our study demonstrates that OLFM4 plays an important role in myeloid leukemia cellular functions and induction of OLFM4-mediated effects may contribute to the therapeutic value of ATRA.
Assuntos
Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/genética , Leucemia Mieloide/genética , Tretinoína/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Azacitidina/farmacologia , Western Blotting , Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Decitabina , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Células HL-60 , Humanos , Imuno-Histoquímica , Leucemia Mieloide/metabolismo , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Elementos de Resposta/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We investigated the ability of granulocyte-colony stimulating factor (G-CSF) on inhibiting glutamate release by microdialysis in a rat stroke model. Male Wistar rats (n=15) were treated with either intravenous saline or G-CSF (60 microg/kg) 30 min after temporary middle cerebral artery occlusion (MCAO). G-CSF significantly attenuated the release of glutamate in the infarcted striatum from 30 minutes to 180 minutes after tMCAO compared with control (p<0.05). Infarct volume in G-CSF treated group (135+/-13 mm(3)) reduced significantly compared to control (181+/-10mm(3)) at 24 hours after tMCAO. The result of present study show that G-CSF possess an ability to inhibit excitotoxicity after ischemic stroke.
Assuntos
Ácido Glutâmico/metabolismo , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Infarto da Artéria Cerebral Média/metabolismo , Animais , Infarto Encefálico/etiologia , Infarto Encefálico/metabolismo , Infarto Encefálico/patologia , Infarto Encefálico/prevenção & controle , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Masculino , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Hematopoiesis-modulating action of meloxicam, a cyclooxyge-nase-2 inhibitor, has been evaluated in mice. Increased serum level of granulocyte colony-stimulating factor (G-CSF) after meloxicam administration has been found in sublethally gamma-irradiated animals. In further experiments hematopoiesis-stimulating effects of meloxicam and G-CSF given alone or in combination have been investigated. Granulocyte/macrophage progenitor cells counts were used to monitor these effects. Meloxicam and exogenous G-CSF did not act synergistically when given in combination, but could be mutually substituted during their repeated administration. The results suggest a promising possibility of using meloxicam as an auxiliary drug reducing the high costs of G-CSF therapy of myelosuppression.
Assuntos
Medula Óssea/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Fator Estimulador de Colônias de Granulócitos/sangue , Hematopoese/efeitos dos fármacos , Tiazinas/farmacologia , Tiazóis/farmacologia , Animais , Medula Óssea/efeitos da radiação , Diferenciação Celular , Interações Medicamentosas , Quimioterapia Combinada , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Granulócitos/efeitos da radiação , Leucopenia/prevenção & controle , Masculino , Meloxicam , Camundongos , Camundongos Endogâmicos CBA , Irradiação Corporal TotalRESUMO
Arginine is one of the most favorable additives in protein refolding. However, arginine does not work for certain disulfide-bond-containing proteins, which is not yet well explained. In this work, refolding of three proteins in the presence of 0-2 M arginine was investigated and compared. Bovine carbonic anhydrase B (CAB), containing no cysteine, was successfully refolded with the help of arginine. The refolding yield could reach almost 100% in the presence of 0.75 M arginine. However, recombinant human colony stimulating factor (rhG-CSF), containing five cysteines, could only achieve 65% refolding yield. The formation of aggregates was found. Blocking of free SH groups of the denatured rhG-CSF by iodoacetamide and subsequently refolding of the protein could reduce the aggregate formation substantially. Further investigation on recombinant green fluorescence protein (GFP), containing two cysteines, also revealed the accumulation of oligomers. The content of oligomers increased with the concentration of arginine, reaching about 30% at 2 M arginine. Comparison of reduced and nonreduced SDS-PAGE revealed that the oligomers were formed through intermolecular disulfide binding. Analysis of the refolding kinetics indicated that intermolecular disulfide bonds were probably formed in the intermediate stage where arginine slowed down the refolding rate and stabilized the intermediates. The accumulated intermediates with unpaired cysteine possessed more chances to react with each other to form oligomers, whereas arginine failed to inhibit disulfide bond formation.
Assuntos
Arginina/farmacologia , Anidrase Carbônica I/metabolismo , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Proteínas de Fluorescência Verde/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Animais , Arginina/química , Sítios de Ligação , Anidrase Carbônica I/química , Bovinos , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos/isolamento & purificação , Proteínas de Fluorescência Verde/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
We previously reported that alginate oligomers, prepared by specific enzymatic digestion of alginate polymer, induced cytokine secretion from mouse macrophage cell line RAW264.7. In the present study, we examined the cytokine levels in the mouse serum after intraperitoneal (ip) administration of a mixture of alginate oligomers. After ip injection of 700 mg/kg of oligomers, the serum level of G-CSF increased promptly and reached the maximum level after 2 h and this high level was sustained until 6 h, and then gradually decreased, whereas injection of 700 mg/kg of alginate polymer had no effect. The effect of alginate oligomer mixture was dose-dependent, and 70 mg/kg was sufficient to attain the maximum serum level of G-CSF. A Bio-Plex bead assay that can detect 23 cytokines at the same time revealed that ip administration of alginate oligomer mixture induced an increase in 20 cytokines in the serum at different levels and with different kinetics depending on the cytokine. Among the cytokines detected the level of G-CSF was the highest. The levels of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, keratinocyte-derived chemokine (KC), IL-12 (p40), and regulated upon activation normal T cell expressed and secreted (RANTES) were also relatively high and exceeded 5000 pg/mL serum at the peak point.
Assuntos
Alginatos/farmacologia , Citocinas/biossíntese , Fator Estimulador de Colônias de Granulócitos/sangue , Oligossacarídeos/farmacologia , Alginatos/administração & dosagem , Animais , Ensaio de Imunoadsorção Enzimática , Fator Estimulador de Colônias de Granulócitos/efeitos dos fármacos , Injeções Intraperitoneais , Cinética , Masculino , Camundongos , Camundongos Endogâmicos , Oligossacarídeos/administração & dosagemRESUMO
OBJECTIVES: The main adverse effect of deferiprone is the development of neutropenia, which occurs via an unknown mechanism. We aimed to gain insight into the pathogenesis of deferiprone-induced neutropenia by assessing the peripheral blood haematopoietic progenitor cells. METHODS: Sixteen patients with beta thalassaemia were studied; nine (Group A) were receiving desferrioxamine and seven (Group B) deferiprone. Ten healthy individuals comprised the control group (Group C). RESULTS: Granulocyte-erythrocyte-monocyte-megakaryocyte colony forming units were significantly more in Groups A and B compared with Group C. Granulocyte-macrophage colony forming units (CFU-GM) were significantly more in Group B compared with Group C. Macrophage colony forming units were significantly less in Group B compared with Group C. Granulocyte colony forming units (CFU-G) were significantly more in Group A compared with Group C. We found a trend in the difference in the number of CFU-G between patients' groups (P = 0.123). Adding serum from patients receiving deferiprone to cultures of controls resulted in a maturation arrest of the granulocytic lineage. CONCLUSION: Our findings point to a maturation arrest at the level of CFU-GM as a potential mechanism of deferiprone-induced neutropenia.