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1.
Immunol Res ; 70(6): 817-828, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36222965

RESUMO

Coronavirus disease 2019 (COVID-19) is associated with immune dysregulation, severe respiratory failure, and multiple organ dysfunction caused by a cytokine storm involving high blood levels of ferritin and IL-18. Furthermore, there is a resemblance between COVID-19 and macrophage activation syndrome (MAS) characterized by high concentrations of soluble CD163 (sCD163) receptor and IL-18. High levels of ferritin, IL-18, and sCD163 receptor are associated with "hyperferritinemic syndrome", a family of diseases that appears to include COVID-19. In this retrospective cohort study, we tested the association and intercorrelations in the serum levels of ferritin, sCD163, and IL-18 and their impact on the prognosis of COVID-19. We analyzed data of 70 hospitalized patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The levels of sCD163, ferritin, and IL-18 were measured and the correlation of these parameters with the respiratory deterioration and overall 30-day survival was assessed. Among the 70 patients, 60 survived 30 days from hospitalization. There were substantial differences between the subjects who were alive following 30 days compared to those who expired. The differences were referring to lymphocyte and leukocyte count, CRP, D-dimer, ferritin, sCD163, and IL-18. Results showed high levels of IL-18 (median, 444 pg/mL in the survival group compared with 916 pg/mL in the mortality group, p-value 8.54 × 10-2), a statistically significant rise in levels of ferritin (median, 484 ng/mL in the survival group compared with 1004 ng/mL in the mortality group p-value, 7.94 × 10-3), and an elevated value of in sCD163 (mean, 559 ng/mL in the survival group compared with 840 ng/mL in the mortality group, p-value 1.68 × 10-2). There was no significant correlation between the rise of ferritin and the levels sCD163 or IL-18. Taken together, sCD163, ferritin, and IL-18 were found to correlate with the severity of COVID-19 infection. Although these markers are associated with COVID-19 and might contribute to the cytokine storm, no intercorrelation was found among them. It cannot be excluded though that the results depend on the timing of sampling, assuming that they play distinct roles in different stages of the disease course. The data represented herein may provide clinical benefit in improving our understanding of the pathological course of the disease. Furthermore, measuring these biomarkers during the disease progression may help target them at the right time and refine the decision-making regarding the requirement for hospitalization.


Assuntos
COVID-19 , Humanos , Biomarcadores , Síndrome da Liberação de Citocina , Ferritinas , Interleucina-18 , Prognóstico , Estudos Retrospectivos , SARS-CoV-2
2.
J Exp Clin Cancer Res ; 41(1): 243, 2022 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-35953814

RESUMO

BACKGROUND: Solid tumors subjected to intermittent hypoxia are characterized by resistance to chemotherapy and immune-killing by effector T-lymphocytes, particularly tumor-infiltrating Vγ9Vδ2 T-lymphocytes. The molecular circuitries determining this double resistance are not known. METHODS: We analyzed a panel of 28 human non-small cell lung cancer (NSCLC) lines, using an in vitro system simulating continuous and intermittent hypoxia. Chemosensitivity to cisplatin and docetaxel was evaluated by chemiluminescence, ex vivo Vγ9Vδ2 T-lymphocyte expansion and immune-killing by flow cytometry. Targeted transcriptomics identified efflux transporters and nuclear factors involved in this chemo-immuno-resistance. The molecular mechanism linking Hypoxia-inducible factor-1α (HIF-1α), CCAAT/Enhancer Binding Protein-ß (C/EBP-ß) isoforms LAP and LIP, ABCB1, ABCC1 and ABCA1 transporters were evaluated by immunoblotting, RT-PCR, RNA-IP, ChIP. Oxidative phosphorylation, mitochondrial ATP, ROS, depolarization, O2 consumption were monitored by spectrophotometer and electronic sensors. The role of ROS/HIF-1α/LAP axis was validated in knocked-out or overexpressing cells, and in humanized (Hu-CD34+NSG) mice bearing LAP-overexpressing tumors. The clinical meaning of LAP was assessed in 60 NSCLC patients prospectively enrolled, treated with chemotherapy. RESULTS: By up-regulating ABCB1 and ABCC1, and down-regulating ABCA1, intermittent hypoxia induced a stronger chemo-immuno-resistance than continuous hypoxia in NSCLC cells. Intermittent hypoxia impaired the electron transport chain and reduced O2 consumption, increasing mitochondrial ROS that favor the stabilization of C/EBP-ß mRNA mediated by HIF-1α. HIF-1α/C/EBP-ß mRNA binding increases the splicing of C/EBP-ß toward the production of LAP isoform that transcriptionally induces ABCB1 and ABCC1, promoting the efflux of cisplatin and docetaxel. LAP also decreases ABCA1, limiting the efflux of isopentenyl pyrophosphate, i.e. the endogenous activator of Vγ9Vδ2 T-cells, and reducing the immune-killing. In NSCLC patients subjected to cisplatin-based chemotherapy, C/EBP-ß LAP was abundant in hypoxic tumors and was associated with lower response to treatment and survival. LAP-overexpressing tumors in Hu-CD34+NSG mice recapitulated the patients' chemo-immuno-resistant phenotype. Interestingly, the ROS scavenger mitoquinol chemo-immuno-sensitized immuno-xenografts, by disrupting the ROS/HIF-1α/LAP cascade. CONCLUSIONS: The impairment of mitochondrial metabolism induced by intermittent hypoxia increases the ROS-dependent stabilization of HIF-1α/LAP complex in NSCLC, producing chemo-immuno-resistance. Clinically used mitochondrial ROS scavengers may counteract such double resistance. Moreover, we suggest C/EBP-ß LAP as a new predictive and prognostic factor in NSCLC patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Docetaxel , Humanos , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo
3.
J Exp Clin Cancer Res ; 37(1): 286, 2018 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-30482226

RESUMO

BACKGROUND: Triple negative breast cancer (TNBC) easily develops resistance to the first-line drug doxorubicin, because of the high levels of the drug efflux transporter P-glycoprotein (Pgp) and the activation of pro-survival pathways dependent on endoplasmic reticulum (ER). Interfering with these mechanisms may overcome the resistance to doxorubicin, a still unmet need in TNBC. METHODS: We analyzed a panel of human and murine breast cancer cells for their resistance to doxorubicin, Pgp expression, lysosome and proteasome activity, nitrite production, ER-dependent cell death and immunogenic cell death parameters. We evaluated the efficacy of genetic (C/EBP-ß LIP induction) and pharmacological strategies (lysosome and proteasome inhibitors), in restoring the ER-dependent and immunogenic-dependent cell death induced by doxorubicin, in vitro and in syngeneic mice bearing chemoresistant TNBC. The results were analyzed by one-way analysis of variance test. RESULTS: We found that TNBC cells characterized by high levels of Pgp and resistance to doxorubicin, had low induction of the ER-dependent pro-apoptotic factor C/EBP-ß LIP upon doxorubicin treatment and high activities of lysosome and proteasome that constitutively destroyed LIP. The combination of chloroquine and bortezomib restored doxorubicin sensitivity by activating multiple and interconnected mechanisms. First, chloroquine and bortezomib prevented C/EBP-ß LIP degradation and activated LIP-dependent CHOP/TRB3/caspase 3 axis in response to doxorubicin. Second, C/EBP-ß LIP down-regulated Pgp and up-regulated calreticulin that triggered the dendritic cell (DC)-mediated phagocytosis of tumor cell, followed by the activation of anti-tumor CD8+T-lymphocytes upon doxorubicin treatment. Third, chloroquine and bortezomib increased the endogenous production of nitric oxide that further induced C/EBP-ß LIP and inhibited Pgp activity, enhancing doxorubicin's cytotoxicity. In orthotopic models of resistant TNBC, intratumor C/EBP-ß LIP induction - achieved by a specific expression vector or by chloroquine and bortezomib - effectively reduced tumor growth and Pgp expression, increased intra-tumor apoptosis and anti-tumor immune-infiltrate, rescuing the efficacy of doxorubicin. CONCLUSIONS: We suggest that preventing C/EBP-ß LIP degradation by lysosome and proteasome inhibitors triggers multiple virtuous circuitries that restore ER-dependent apoptosis, down-regulate Pgp and re-activate the DC/CD8+T-lymphocytes response against TNBC. Lysosome and proteasome inhibitors associated with doxorubicin may overcome the resistance to the drug in TNBC.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Doxorrubicina/farmacologia , Retículo Endoplasmático/metabolismo , Óxido Nítrico/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias de Mama Triplo Negativas/patologia
4.
Lung Cancer ; 120: 34-45, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29748013

RESUMO

OBJECTIVES: Cisplatin-based chemotherapy is moderately active in malignant pleural mesothelioma (MPM) due to intrinsic drug resistance and to low immunogenicity of MPM cells. CAAT/enhancer binding protein (C/EBP)-ß LIP is a pro-apoptotic and chemosensitizing transcription factor activated in response to endoplasmic reticulum (ER) stress. MATERIALS AND METHODS: We investigated if LIP levels can predict the clinical response to cisplatin and survival of MPM patients receiving cisplatin-based chemotherapy. We studied the LIP-dependent mechanisms determining cisplatin-resistance and we identified pharmacological approaches targeting LIP, able to restore cisplatin sensitiveness, in patient-derived MPM cells and animal models. Results were analyzed by a one-way analysis of variance test. RESULTS: We found that LIP was degraded by constitutive ubiquitination in primary MPM cells derived from patients poorly responsive to cisplatin. LIP ubiquitination was directly correlated with cisplatin chemosensitivity and was associated with patients' survival after chemotherapy. Overexpression of LIP restored cisplatin's pro-apoptotic effect by activating CHOP/TRB3/caspase 3 axis and up-regulating calreticulin, that triggered MPM cell phagocytosis by dendritic cells and expanded autologous anti-tumor CD8+CD107+T-cytotoxic lymphocytes. Proteasome inhibitor carfilzomib and lysosome inhibitor chloroquine prevented LIP degradation. The triple combination of carfilzomib, chloroquine and cisplatin increased ER stress-triggered apoptosis and immunogenic cell death in patients' samples, and reduced tumor growth in cisplatin-resistant MPM preclinical models. CONCLUSION: The loss of LIP mediates cisplatin resistance, rendering LIP a possible predictor of cisplatin response in MPM patients. The association of proteasome and lysosome inhibitors reverses cisplatin resistance by restoring LIP levels and may represent a new adjuvant strategy in MPM treatment.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos/uso terapêutico , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linfócitos T CD8-Positivos/imunologia , Cisplatino/uso terapêutico , Células Dendríticas/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Neoplasias Pleurais/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose , Proteína beta Intensificadora de Ligação a CCAAT/genética , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Ativação Linfocitária , Mesotelioma/genética , Mesotelioma/mortalidade , Mesotelioma Maligno , Oligopeptídeos/farmacologia , Neoplasias Pleurais/mortalidade , Prognóstico , Proteólise , Análise de Sobrevida , Células Tumorais Cultivadas , Ubiquitinação
6.
Curr Opin Nephrol Hypertens ; 27(1): 42-48, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29059080

RESUMO

PURPOSE OF REVIEW: This review will critically highlight the role of leukotrienes as mediators of renal diseases and drug nephrotoxicity. It will also discuss the recently identified mechanism of cysteinyl leukotrienes induction and action, and will propose clinical implementation of these findings. RECENT FINDINGS: Since last reviewed in 1994, leukotrienes were shown to mediate drug-associated nephrotoxicity, transplant rejection and morbidity in several models of renal diseases. Although leukotrienes may be released by various infiltrating leukocytes, a recent study demonstrated that cytotoxic agents trigger production of leukotriene C4 (LTC4) in mouse kidney cells by activating a biosynthetic pathway based on microsomal glutathione-S-transferase 2 (MGST2). LTC4 then elicits nuclear accumulation of hydrogen peroxide-generating NADPH oxidase 4, leading to oxidative DNA damage and cell death. LTC4 inhibitors, commonly used as systemic asthma drugs, alleviated drug-associated damage to proximal tubular cells and attenuated mouse morbidity. SUMMARY: Cysteinyl leukotrienes released by mast cells trigger the symptoms of asthma, including bronchoconstriction and vasoconstriction. Therefore, effective leukotriene inhibitors were approved as orally administered asthma drugs. The findings that leukotrienes mediate the cytotoxicity of nephrotoxic drugs, and are involved in numerous renal diseases, suggest that such asthma drugs may ameliorate drug-induced nephrotoxicity, as well as some renal diseases.


Assuntos
Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Leucotrieno C4/metabolismo , Animais , Antiasmáticos/uso terapêutico , Morte Celular , Cisteína/metabolismo , Dano ao DNA , Glutationa Transferase/metabolismo , Humanos , Antagonistas de Leucotrienos/uso terapêutico , Leucotrieno C4/antagonistas & inibidores , Leucotrieno C4/biossíntese , Leucotrienos/metabolismo , NADPH Oxidase 4/metabolismo , Estresse Oxidativo
7.
Mol Cancer ; 16(1): 91, 2017 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-28499449

RESUMO

BACKGROUND: Nutrient deprivation, hypoxia, radiotherapy and chemotherapy induce endoplasmic reticulum (ER) stress, which activates the so-called unfolded protein response (UPR). Extensive and acute ER stress directs the UPR towards activation of death-triggering pathways. Cancer cells are selected to resist mild and prolonged ER stress by activating pro-survival UPR. We recently found that drug-resistant tumor cells are simultaneously resistant to ER stress-triggered cell death. It is not known if cancer cells adapted to ER stressing conditions acquire a chemoresistant phenotype. METHODS: To investigate this issue, we generated human cancer cells clones with acquired resistance to ER stress from ER stress-sensitive and chemosensitive cells. RESULTS: ER stress-resistant cells were cross-resistant to multiple chemotherapeutic drugs: such multidrug resistance (MDR) was due to the overexpression of the plasma-membrane transporter MDR related protein 1 (MRP1). Gene profiling analysis unveiled that cells with acquired resistance to ER stress and chemotherapy share higher expression of the UPR sensor protein kinase RNA-like endoplasmic reticulum kinase (PERK), which mediated the erythroid-derived 2-like 2 (Nrf2)-driven transcription of MRP1. Disrupting PERK/Nrf2 axis reversed at the same time resistance to ER stress and chemotherapy. The inducible silencing of PERK reduced tumor growth and restored chemosensitivity in resistant tumor xenografts. CONCLUSIONS: Our work demonstrates for the first time that the adaptation to ER stress in cancer cells produces a MDR phenotype. The PERK/Nrf2/MRP1 axis is responsible for the resistance to ER stress and chemotherapy, and may represent a good therapeutic target in aggressive and resistant tumors.


Assuntos
Neoplasias do Colo/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fator 2 Relacionado a NF-E2/genética , eIF-2 Quinase/genética , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Doxorrubicina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HT29 , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genética , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/antagonistas & inibidores
8.
Cell Death Dis ; 8(4): e2733, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28383550

RESUMO

Many types of tumor cell are devoid of the extracellular matrix proteoglycan osteoglycin (Ogn), but its role in tumor biology is poorly studied. Here we show that RNAi of Ogn attenuates stress-triggered cell death, whereas its overexpression increases cell death. We found that the transcription factor C/EBPß regulates the expression of Ogn. C/EBPß is expressed as a full-length, active form (LAP) and as a truncated, dominant-negative form (LIP), and the LIP/LAP ratio is positively correlated with the extent of cell death under stress. For example, we reported that drug-resistant tumor cells lack LIP altogether, and its supplementation abolished their resistance to chemotherapy and to endoplasmic reticulum (ER) stress. Here we further show that elevated LIP/LAP ratio robustly increased Ogn expression and cell death under stress by modulating the mitogen-activated protein kinase/activator protein 1 pathway (MAPK/AP-1). Our findings suggest that LIP deficiency renders tumor cell resistant to ER stress by preventing the induction of Ogn.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Estresse do Retículo Endoplasmático , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Proteínas de Neoplasias/genética , Neoplasias/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
10.
Nat Commun ; 6: 10112, 2015 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-26656251

RESUMO

Endoplasmic reticulum (ER) stress and major chemotherapeutic agents damage DNA by generating reactive oxygen species (ROS). Here we show that ER stress and chemotherapy induce leukotriene C4 (LTC4) biosynthesis by transcriptionally upregulating and activating the enzyme microsomal glutathione-S-transferase 2 (MGST2) in cells of non-haematopoietic lineage. ER stress and chemotherapy also trigger nuclear translocation of the two LTC4 receptors. Acting in an intracrine manner, LTC4 then elicits nuclear translocation of NADPH oxidase 4 (NOX4), ROS accumulation and oxidative DNA damage. Mgst2 deficiency, RNAi and LTC4 receptor antagonists abolish ER stress- and chemotherapy-induced ROS and oxidative DNA damage in vitro and in mouse kidneys. Cell death and mouse morbidity are also significantly attenuated. Hence, MGST2-generated LTC4 is a major mediator of ER stress- and chemotherapy-triggered oxidative stress and oxidative DNA damage. LTC4 inhibitors, commonly used for asthma, could find broad clinical use in major human pathologies associated with ER stress-activated NOX4.


Assuntos
Dano ao DNA , Leucotrieno C4/metabolismo , Estresse Oxidativo , Animais , Brefeldina A/toxicidade , Sobrevivência Celular , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/fisiologia , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Nefropatias/induzido quimicamente , Leucotrieno C4/genética , Camundongos , Camundongos Knockout , Interferência de RNA , Tunicamicina/toxicidade
11.
J Natl Cancer Inst ; 107(5)2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25766403

RESUMO

BACKGROUND: Chemotherapy triggers endoplasmic reticulum (ER) stress, which in turn regulates levels of the active (LAP) and the natural dominant-negative (LIP) forms of the transcription factor C/EBP-ß. LAP upregulates and LIP downregulates the multidrug resistance (MDR) protein P-glycoprotein (Pgp), but it is not known how critical is their role in establishing MDR. METHODS: Cell viability was quantitated by crystal violet staining and measuring absorbance at 540nm. Expression of various proteins was determined by immunoblotting. mRNA levels were determined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). LIP and LAP were overexpressed using expression plasmids followed by selection with blasticidin. Tumor cells expressing doxycycline-inducible LIP were orthotopically implanted in mice (n = 15 mice per group), and tumor size was measured daily by caliper. Tumor sections were stained with hematoxylin and eosin and immunostained for Pgp, proliferation, and ER stress markers. RESULTS: MDR cells do not express basal, chemotherapy-triggered, or ER stress-triggered LIP and fail to activate the CHOP-caspase-3 death-triggering axis upon ER stress or chemotherapy challenge. Overexpression of LIP reversed the MDR phenotype in vitro and in tumors implanted in mice. LIP was undetectable in MDR cells, probably due to its ubiquitination, which was 3.56-fold higher, resulting in lysosomal and proteasomal degradation of LIP. CONCLUSIONS: Spontaneous and drug-selected MDR cells lack LIP, which is eliminated by ubiquitin-mediated degradation. Loss of LIP drives MDR not only by increasing Pgp expression but also by a two-fold attenuation of ER stress-triggered cell death.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Estresse do Retículo Endoplasmático , Proteínas de Neoplasias/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Apoptose , Células CACO-2 , Proliferação de Células , Regulação para Baixo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
12.
Diabetes ; 63(3): 900-11, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24319114

RESUMO

Fatty acid binding protein 4 (FABP4, also known as aP2) is a cytoplasmic fatty acid chaperone expressed primarily in adipocytes and myeloid cells and implicated in the development of insulin resistance and atherosclerosis. Here we demonstrate that FABP4 triggers the ubiquitination and subsequent proteasomal degradation of peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and insulin responsiveness. Importantly, FABP4-null mouse preadipocytes as well as macrophages exhibited increased expression of PPARγ, and complementation of FABP4 in the macrophages reversed the increase in FABP4 expression. The FABP4-null preadipocytes exhibited a remarkably enhanced adipogenesis compared with wild-type cells, indicating that FABP4 regulates adipogenesis by downregulating PPARγ. We found that the FABP4 level was higher and PPARγ level was lower in human visceral fat and mouse epididymal fat compared with their subcutaneous fat. Furthermore, FABP4 was higher in the adipose tissues of obese diabetic individuals compared with healthy ones. Suppression of PPARγ by FABP4 in visceral fat may explain the reported role of FABP4 in the development of obesity-related morbidities, including insulin resistance, diabetes, and atherosclerosis.


Assuntos
Adipogenia , Tecido Adiposo/metabolismo , Proteínas de Ligação a Ácido Graxo/fisiologia , PPAR gama/fisiologia , Animais , Epididimo/metabolismo , Proteínas de Ligação a Ácido Graxo/análise , Humanos , Gordura Intra-Abdominal/metabolismo , Masculino , Camundongos , PPAR gama/análise , Complexo de Endopeptidases do Proteassoma/fisiologia , Gordura Subcutânea/metabolismo , Ubiquitinação
13.
Proc Natl Acad Sci U S A ; 110(18): 7306-11, 2013 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-23589850

RESUMO

Vesicular stomatitis virus (VSV) exhibits a remarkably robust and pantropic infectivity, mediated by its coat protein, VSV-G. Using this property, recombinant forms of VSV and VSV-G-pseudotyped viral vectors are being developed for gene therapy, vaccination, and viral oncolysis and are extensively used for gene transduction in vivo and in vitro. The broad tropism of VSV suggests that it enters cells through a highly ubiquitous receptor, whose identity has so far remained elusive. Here we show that the LDL receptor (LDLR) serves as the major entry port of VSV and of VSV-G-pseudotyped lentiviral vectors in human and mouse cells, whereas other LDLR family members serve as alternative receptors. The widespread expression of LDLR family members accounts for the pantropism of VSV and for the broad applicability of VSV-G-pseudotyped viral vectors for gene transduction.


Assuntos
Receptores de LDL/metabolismo , Receptores Virais/metabolismo , Vírus da Estomatite Vesicular Indiana/metabolismo , Animais , Bovinos , Endocitose , Fibroblastos/citologia , Fibroblastos/virologia , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Camundongos , Ligação Proteica , Solubilidade , Transdução Genética , Vírus da Estomatite Vesicular Indiana/patogenicidade , Internalização do Vírus
14.
Bioconjug Chem ; 23(8): 1577-86, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22759320

RESUMO

We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO(3)S-(CH(2))(15)-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K(a) value of 0.83 ± 0.08 × 10(6) M(-1), a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH(2))(15)-SO(3)(-) into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn(2+)-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH(2))(15)-SO(3)(-) to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH(2))(15)-SO(3)(-)](3) to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.


Assuntos
Desenho de Fármacos , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacocinética , Peptídeos/metabolismo , Peptídeos/farmacocinética , Albumina Sérica/metabolismo , Sequência de Aminoácidos , Animais , Disponibilidade Biológica , Glicemia/metabolismo , Ácidos Graxos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Masculino , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , Ligação Proteica , Especificidade por Substrato
15.
Methods Mol Biol ; 820: 195-214, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22131033

RESUMO

Ligand affinity chromatography separation is based on unique interaction between the target analyte and a ligand, which is coupled covalently to a resin. It is a simple, rapid, selective, and efficient purification procedure of proteins providing tens of thousands fold purification in one step. The biological activity of the isolated proteins is retained in most cases thus function is revealed concomitantly with the isolation. Prior to the completion of the genome project this method facilitated rapid and reliable cloning of the corresponding gene. Upon completion of this project, a partial protein sequence is enough for retrieving its complete mRNA and hence its complete protein sequence. This method is indispensable for the isolation of both expected (e.g. receptors) but mainly unexpected, unpredicted and very much surprising binding proteins. No other approach would yield the latter. This chapter provides examples for both the expected target proteins, isolated from rich sources of human proteins, as well as the unexpected binding proteins, found by serendipity.


Assuntos
Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade/métodos , Receptores de Citocinas/isolamento & purificação , Receptores de Citocinas/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Humanos , Ligantes , Camundongos , Células NIH 3T3 , Análise de Sequência de DNA
16.
PLoS One ; 5(3): e9516, 2010 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-20209087

RESUMO

Endoplasmic reticulum (ER) stress elicits the unfolded protein response (UPR), initially aimed at coping with the stress, but triggering cell death upon further stress. ER stress induces the C/EBP-beta variant Liver-enriched Activating Protein (LAP), followed by the dominant-negative variant, Liver Inhibitory Protein (LIP). However, the distinct role of LAP and LIP in ER stress is unknown. We found that the kinetics of the ER stress-induced expression of LIP overlapped with that of the cell death in mouse B16 melanoma cells. Furthermore, inducible over-expression of LIP augmented ER stress-triggered cell death whereas over-expression of LAP attenuated cell death. Similar results were obtained in human 293T cells. Limited vasculature in tumors triggers hypoxia, nutrient shortage and accumulation of toxic metabolites, all of which eliciting continuous ER stress. We found that LAP promoted and LIP inhibited B16 melanoma tumor progression without affecting angiogenesis or accelerating the cell cycle. Rather, LAP attenuated, whereas LIP augmented tumor ER stress. We therefore suggest that C/EBP-beta regulates the transition from the protective to the death-promoting phase of the UPR. We further suggest that the over-expression of LAP observed in many solid tumors promotes tumor progression by attenuating ER stress-triggered tumor cell death [corrected].


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Retículo Endoplasmático/metabolismo , Animais , Morte Celular , Progressão da Doença , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Hipóxia , Antígeno Ki-67/biossíntese , Cinética , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Neoplasias/patologia , Desnaturação Proteica , Dobramento de Proteína
17.
J Autoimmun ; 34(2): 121-6, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19699611

RESUMO

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies particularly to nuclear antigens and by an abnormal production of proinflammatory cytokines. In the present study, we measured the levels of the proinflammatory cytokine IL-18 and its natural inhibitor, the IL-18 binding protein (IL-18BP), in sera of SLE patients at various stages of the disease. This is the first study to present IL-18BP levels in sera of SLE patients as well as the calculated, biologically active, free IL-18 concentrations that are most probably more relevant to the pathology of SLE. Sera from 48 unselective SLE patients (total of 195 samples) were obtained longitudinally with a mean follow-up period of 11.1 +/- 8.9 years and were compared to sera from 100 healthy volunteers. Circulating levels of IL-18, IL-18BP and free IL-18 in the SLE patients were significantly higher than the levels of healthy controls (5 fold, 6 fold and 3 fold for IL-18, IL-18BP and free IL-18, respectively) and correlated with disease activity as scored by SLEDAI-2K. Furthermore, these levels during active disease (SLEDAI-2K > or = 6) were higher compared to the levels measured in the sera of the same patients during remission or during mild disease (SLEDAI-2K 0-5). The high levels of IL-18 and IL-18BP in sera of active SLE patients suggest their possible role in the pathogenesis and course of the disease. However, despite the elevated levels of IL-18BP during active disease, free IL-18 remained more than 2 fold higher than the levels in healthy controls suggesting a potential benefit of administration of exogenous IL-18BP as a novel therapeutic approach for active SLE.


Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/sangue , Interleucina-18/sangue , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/fisiopatologia , Adolescente , Adulto , Idoso , Criança , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Seguimentos , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/terapia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença
18.
Arterioscler Thromb Vasc Biol ; 27(12): 2743-9, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17951325

RESUMO

OBJECTIVE: To investigate free interleukin-18 (fIL-18) levels, and variation within the IL-18 system genes, in heart surgery patients, and healthy men. METHODS AND RESULTS: fIL-18 was calculated from IL-18 and IL-18 binding protein (BP) levels, in 421 healthy men and 196 post-coronary artery bypass graft (CABG) patients. After surgery, fIL-18 peaked at 6 hours (from 117 to 331 pg/mL) but fell to below presurgery levels at 24 hours (99 pg/mL), because of changes in total IL-18 and IL-18BP. fIL-18 24 hours postsurgery was significantly higher in those who suffered a major complication after surgery (125 versus 80 pg/mL, P<0.01). Baseline total IL-18 was also higher in healthy men who went on to suffer an MI over 17 years of prospective study (276 versus 240 pg/mL, P=0.01). Tagging SNPs for IL18 (n=5) and IL18BP (n=3) were determined, in both studies the IL18 HapIII haplotype (frequency 30%) was associated with 36% lower baseline fIL-18 levels before surgery (P<0.01), and 7% lower in healthy men (P=0.04). The frequency of HapIII was lower in CABG patients than in healthy men (20.7 versus 29.8%, P<0.01). CONCLUSIONS: IL-18 levels, which are determined in part by variation in IL18, play a role in CHD development and postsurgery outcome.


Assuntos
Ponte de Artéria Coronária , Doença das Coronárias/sangue , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Interleucina-18/sangue , Infarto do Miocárdio/sangue , Polimorfismo de Nucleotídeo Único , Complicações Pós-Operatórias/sangue , Idoso , Estudos de Casos e Controles , Doença das Coronárias/genética , Doença das Coronárias/cirurgia , Europa (Continente) , Seguimentos , Frequência do Gene , Predisposição Genética para Doença , Haplótipos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interleucina-18/genética , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/genética , Fenótipo , Estudos Prospectivos , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento
19.
Nat Immunol ; 8(10): 1123-31, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17828268

RESUMO

Catecholamines are important regulators of homeostasis, yet their functions in hematopoiesis are poorly understood. Here we report that immature human CD34+ cells dynamically expressed dopamine and beta2-adrenergic receptors, with higher expression in the primitive CD34+CD38(lo) population. The myeloid cytokines G-CSF and GM-CSF upregulated neuronal receptor expression on immature CD34+ cells. Treatment with neurotransmitters increased the motility, proliferation and colony formation of human progenitor cells, correlating with increased polarity, expression of the metalloproteinase MT1-MMP and activity of the metalloproteinase MMP-2. Treatment with catecholamines enhanced human CD34+ cell engraftment of NOD-SCID mice through Wnt signaling activation and increased cell mobilization and bone marrow Sca-1+c-Kit+Lin- cell numbers. Our results identify new functions for neurotransmitters and myeloid cytokines in the direct regulation of human and mouse progenitor cell migration and development.


Assuntos
Antígenos CD34/análise , Catecolaminas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Neurotransmissores/farmacologia , Transdução de Sinais/fisiologia , Proteínas Wnt/fisiologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dopamina/farmacologia , Agonistas de Dopamina/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Norepinefrina/farmacologia , Receptores Dopaminérgicos/análise
20.
Cytokine Growth Factor Rev ; 18(5-6): 519-24, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17683975

RESUMO

In 1976-1977, I adapted reversed-phase HPLC (RP-HPLC) to peptide and protein purification, starting with pituitary proteins and continuing with the first successful purification to homogeneity of human leukocyte interferon (IFN-alpha). Using this technology, I isolated and characterized 6-8 different leukocyte interferon subtypes, which were later identified as products of the IFN-alpha gene family. Since then, RP-HPLC became a standard procedure for isolation and analysis of proteins. The successful purification of IFN-alpha led to the development of Roferon-A, a drug used for the treatment of hairy cell leukemia, hepatitis C and a variety of other diseases. Later studies with my colleagues in Israel and abroad led to isolation and discovery of several cytokine receptors and binding proteins, including those of Type I IFNs, TNF and IL-18. The use of HPLC was indispensable in most of these studies.


Assuntos
Interferon-alfa/isolamento & purificação , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Receptores de Citocinas/isolamento & purificação
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