RESUMEN
Clozapine is the most effective antipsychotic for patients with treatment-resistant schizophrenia. However, response is highly variable and possible genetic underpinnings of this variability remain unknown. Here, we performed polygenic risk score (PRS) analyses to estimate the amount of variance in symptom severity among clozapine-treated patients explained by PRSs (R2) and examined the association between symptom severity and genotype-predicted CYP1A2, CYP2D6, and CYP2C19 enzyme activity. Genome-wide association (GWA) analyses were performed to explore loci associated with symptom severity. A multicenter cohort of 804 patients (after quality control N = 684) with schizophrenia spectrum disorder treated with clozapine were cross-sectionally assessed using the Positive and Negative Syndrome Scale and/or the Clinical Global Impression-Severity (CGI-S) scale. GWA and PRS regression analyses were conducted. Genotype-predicted CYP1A2, CYP2D6, and CYP2C19 enzyme activities were calculated. Schizophrenia-PRS was most significantly and positively associated with low symptom severity (p = 1.03 × 10-3; R2 = 1.85). Cross-disorder-PRS was also positively associated with lower CGI-S score (p = 0.01; R2 = 0.81). Compared to the lowest tertile, patients in the highest schizophrenia-PRS tertile had 1.94 times (p = 6.84×10-4) increased probability of low symptom severity. Higher genotype-predicted CYP2C19 enzyme activity was independently associated with lower symptom severity (p = 8.44×10-3). While no locus surpassed the genome-wide significance threshold, rs1923778 within NFIB showed a suggestive association (p = 3.78×10-7) with symptom severity. We show that high schizophrenia-PRS and genotype-predicted CYP2C19 enzyme activity are independently associated with lower symptom severity among individuals treated with clozapine. Our findings open avenues for future pharmacogenomic projects investigating the potential of PRS and genotype-predicted CYP-activity in schizophrenia.
Asunto(s)
Antipsicóticos , Clozapina , Citocromo P-450 CYP2C19 , Esquizofrenia , Antipsicóticos/uso terapéutico , Clozapina/uso terapéutico , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2D6/genética , Estudio de Asociación del Genoma Completo , Humanos , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/genéticaRESUMEN
As a result of the CD28 superagonist biotherapeutic monoclonal antibody (TGN 1412) "cytokine storm" incident, cytokine release assays (CRA) have become hazard identification and prospective risk assessment tools for screening novel biotherapeutics directed against targets having a potential risk for eliciting adverse pro-inflammatory clinical infusion reactions. Different laboratories may have different strategies, assay formats, and approaches to the reporting, interpretation, and use of data for either decision making or risk assessment. Additionally, many independent contract research organizations (CROs), academic and government laboratories are involved in some aspect of CRA work. As a result, while some pharmaceutical companies are providing CRA data as part of the regulatory submissions when necessary, technical and regulatory practices are still evolving to provide data predictive of cytokine release in humans and that are relevant to safety. This manuscript provides an overview of different approaches employed by the pharmaceutical industry and CROs, for the use and application of CRA based upon a survey and post survey follow up conducted by ILSI-Health and Environmental Sciences Institute (HESI) Immunotoxicology Committee CRA Working Group. Also discussed is ongoing research in the academic sector, the regulatory environment, current limitations of the assays, and future directions and recommendations for cytokine release assays.
Asunto(s)
Bioensayo/métodos , Citocinas/sangre , Anticuerpos Monoclonales Humanizados , Antígenos CD28/inmunología , Citocinas/inmunología , Evaluación Preclínica de Medicamentos , Humanos , Inflamación/sangre , Inflamación/inmunología , Insuficiencia Multiorgánica/inmunologíaRESUMEN
Cirrhosis represents a late stage of hepatic fibrosis and leads to high morbidity and mortality, and the most frequent complication is ascites. Only a few patients with advanced cirrhosis have 'refractory ascites' and do not respond to conventional treatment. Repeated paracentesis for evacuation is considered the treatment of choice in these cases. A large proportion of these patients have associated chronic kidney disease (CKD), which may require renal replacement therapy (RRT). Due to the complications associated with liver disease with coagulation disorders and tendencies towards spontaneous hypotension, there are significant problems associated to RRT, especially haemodialysis (HD). On the contrary, peritoneal dialysis (PD) offers several advantages over HD in cirrhotic patients (with or without ascites) thanks to better haemodynamic tolerance, as it is a continuous and slow technique. Furthermore, PD has a low rate of infection and bleeding.
Asunto(s)
Ascitis/terapia , Diálisis Peritoneal , Ascitis/etiología , Ascitis/fisiopatología , Trastornos de la Coagulación Sanguínea/etiología , Enfermedad Crónica , Hemodinámica , Hepatitis Viral Humana/epidemiología , Hepatitis Viral Humana/etiología , Hepatitis Viral Humana/transmisión , Humanos , Hipoproteinemia/etiología , Hipotensión/etiología , Enfermedades Renales/complicaciones , Enfermedades Renales/terapia , Cirrosis Hepática/complicaciones , Desnutrición/etiología , Diálisis Peritoneal/efectos adversos , Diálisis Peritoneal/métodos , Peritonitis/etiología , Pronóstico , Diálisis Renal/efectos adversos , Riesgo , Análisis de SupervivenciaRESUMEN
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumour cells through activation of TRAIL-R1 and TRAIL-R2 death signalling receptors. Here, we describe the characterisation and activity of HGS-ETR1, the first fully human, agonistic TRAIL-R1 mAb that is being developed as an antitumour therapeutic agent. HGS-ETR1 showed specific binding to TRAIL-R1 receptor. HGS-ETR1 reduced the viability of multiple types of tumour cells in vitro, and induced activation of caspase 8, Bid, caspase 9, caspase 3, and cleavage of PARP, indicating activation of TRAIL-R1 alone was sufficient to induce both extrinsic and intrinsic apoptotic pathways. Treatment of cell lines in vitro with HGS-ETR1 enhanced the cytotoxicity of chemotherapeutic agents (camptothecin, cisplatin, carboplatin, or 5-fluorouracil) even in tumour cell lines that were not sensitive to HGS-ETR1 alone. In vivo administration of HGS-ETR1 resulted in rapid tumour regression or repression of tumour growth in pre-established colon, non-small-cell lung, and renal tumours in xenograft models. Combination of HGS-ETR1 with chemotherapeutic agents (topotecan, 5-fluorouracil, and irinotecan) in three independent colon cancer xenograft models resulted in an enhanced antitumour efficacy compared to either agent alone. Pharmacokinetic studies in the mouse following intravenous injection showed that HGS-ETR1 serum concentrations were biphasic with a terminal half-life of 6.9-8.7 days and a steady-state volume of distribution of approximately 60 ml kg(-1). Clearance was 3.6-5.7 ml(-1) day(-1) kg(-1). These data suggest that HGS-ETR1 is a specific and potent antitumour agent with favourable pharmacokinetic characteristics and the potential to provide therapeutic benefit for a broad range of human malignancies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Antineoplásicos/inmunología , Apoptosis/fisiología , Western Blotting , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Semivida , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Receptores del Ligando Inductor de Apoptosis Relacionado con TNFRESUMEN
TR6, a member of the tumor necrosis factor (TNF) receptor superfamily, has recently been shown to bind to Fas ligand (FasL) and inhibit FasL-mediated cell killing in vitro. In the current study, we demonstrate that TR6 can block the lethal activity of FasL in multiple in vitro systems, and extend this finding to an in vivo model of hepatitis. The binding of human TR6 to human FasL was verified with BIAcore chip technology. Human primary hepatocytes, HT-29 cells and Jurkat cells were assayed for viability to demonstrate TR6 inhibition of FasL-mediated cytotoxicity in vitro. Human TR6 was also shown to cross-react with membrane-bound mouse FasL, since the in vitro cytotoxic activity of L929 cells transfected with murine FasL was inhibited in the presence of human TR6. In vivo, FasL-induced acute, lethal, fulminant hepatic apoptosis resulting in death within 2 h of intravenous injection into Fas+ mice, but not Fas- MRL/lpr mice. Pretreatment of mice with TR6 blocked FasL-induced mortality, presumably by attenuating FasL-induced hepatic apoptosis. Thus, in both in vitro and in vivo systems, TR6 acts as a functional FasL decoy receptor and may be clinically useful in the treatment of hepatitis and other diseases associated with FasL-mediated tissue injury.
Asunto(s)
Apoptosis/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Glicoproteínas de Membrana/antagonistas & inhibidores , Receptores de Superficie Celular/fisiología , Animales , Apoptosis/fisiología , Relación Dosis-Respuesta a Droga , Proteína Ligando Fas , Femenino , Células HT29/efectos de los fármacos , Células HT29/fisiología , Hepatocitos/patología , Humanos , Inmunoglobulina G/farmacología , Células Jurkat/efectos de los fármacos , Células Jurkat/fisiología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Oligopéptidos , Péptidos/farmacología , Receptores del Factor de Necrosis Tumoral , Miembro 6b de Receptores del Factor de Necrosis TumoralRESUMEN
TR6 (DcR3) is a new member of the TNF receptor (TNFR) family that lacks a transmembrane domain in its sequence, indicating that it is a secreted molecule. TR6 can bind to FasL and prevent FasL-induced apoptosis; it can also associate with LIGHT, another TNF family member. The role of TR6 in immune responses was investigated in this study. According to flow cytometry, recombinant human TR6-Fc binds to human LIGHT expressed on 293 cells or on activated human T cells and competes with the LIGHT receptor TR2 for the binding to LIGHT on these cells. Human TR6 could cross-react with mouse LIGHT in immunoprecipitation. TR6-Fc also downregulates cytotoxic T lymphocyte activity in vitro and graft-versus-host responses in mice. Moreover, TR6-Fc modulates lymphokine production by alloantigen-stimulated mouse T cells. TR6-Fc ameliorated rejection response to mouse heart allograft. These results indicate that TR6 can dampen T-cell responses to alloantigens. Such regulatory effects of TR6 probably occur via interference with interaction between pairs of related TNF and TNFR family members, LIGHT/TR2 being one of the possible candidate pairs.
Asunto(s)
Isoantígenos/inmunología , Glicoproteínas de Membrana , Receptores de Superficie Celular/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Linfocitos T/inmunología , Animales , Línea Celular , Femenino , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Trasplante de Corazón/fisiología , Humanos , Immunoblotting , Interferón-alfa/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Isoantígenos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Pruebas de Precipitina , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Necrosis Tumoral/inmunología , Miembro 6b de Receptores del Factor de Necrosis Tumoral , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Bazo/citología , Bazo/metabolismo , Linfocitos T/fisiología , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Myeloid progenitor inhibitory factor (MPIF)-2 is a beta-chemokine with select and potent activities on eosinophils and myeloid progenitors. In the beta-chemokine family, biological activity is modulated by differential processing of the amino-terminus. Here, for MPIF-2, we describe the biological activities of NH(2)-terminal deletion mutants and compare regions necessary for eosinophil and myeloid progenitor activities. Five MPIF-2 proteins with deletions at the amino-terminus were produced in Escherichia coli and assayed for calcium mobilization, chemotaxis and receptor binding activities on eosinophils, and for their ability to inhibit colony formation of human myeloid bone marrow progenitors. For eosinophils, deletion of the first two amino acids did not markedly alter activity, while subsequent truncations result in a complete loss of activity. One of the MPIF-2 mutants, MPIF-2 (P30-R99) was converted from an agonist to an antagonist of eotaxin, MPIF-2 and MCP-4 functional responses in eosinophil calcium flux and chemotaxis assays. Surprisingly, while displaying a complete loss of agonist activity toward eosinophils, MPIF-2 (P30-R99) retains ability to inhibit human bone marrow myeloid progenitor cell colony formation. In addition, processing at the amino terminus of MPIF-2 in vivo, may result in a chemokine with altered biological activities.
Asunto(s)
Quimiocinas CC/genética , Eosinófilos/metabolismo , Células Progenitoras Mieloides/metabolismo , Sitios de Unión , Calcio/metabolismo , Señalización del Calcio , Quimiocina CCL24 , Quimiocinas CC/biosíntesis , Quimiocinas CC/fisiología , Vectores Genéticos/metabolismo , Humanos , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/fisiología , Receptores CCR3 , Receptores de Quimiocina/metabolismo , Receptores del VIH/metabolismo , Eliminación de SecuenciaRESUMEN
B lymphocyte stimulator (BLyS) is a novel member of the TNF family of proteins expressed by myeloid cells as membrane-bound and soluble forms. BLyS was shown to act specifically on B cells, inducing proliferation and immunoglobulin production both in vitro and in vivo. The present study was undertaken to characterize binding of radiolabeled BLyS to its cognate receptor on human B lymphocytes and examine intracellular events initiated by BLyS binding. Similar to other TNF family members, BLyS is present in solution as a homotrimer as determined by gel filtration chromatography and light scattering analysis. BLyS binding to B cells is specific as other TNF family members tested did not compete for(125)I-BLyS binding. Analysis of equilibrium binding of(125)I-labeled BLyS to purified human tonsillar B cells demonstrated saturable binding. Scatchard analysis of the binding data revealed a single class of high-affinity binding on human B cells with approximately 2600 binding sites per cell and an apparent dissociation constant (K(D)) of about 0.1 nM. In addition we report that BLyS binding to B cells results in the activation of NF-kappaB and the Ets family transcription factor, ELF-1, and in the induction of mRNA for Polo-like kinase (PLK).
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor Activador de Células B , Sitios de Unión , Reactivos de Enlaces Cruzados , Humanos , Técnicas In Vitro , Radioisótopos de Yodo , Cinética , Proteínas de la Membrana/química , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/químicaRESUMEN
The human beta chemokine known as LEC (also called NCC-4, HCC-4, or LMC) displays chemotactic activity for monocytes and dendritic cells. The possibility that its local presence increases tumor immunogenicity is addressed in this paper. TSA parental cells (TSA-pc) are poorly immunogenic adenocarcinoma cells that grow progressively, kill both nu/nu and syngeneic BALB/c mice, and give rise to lung metastases. TSA cells engineered to release LEC (TSA-LEC) are still able to grow in nu/nu mice, but are promptly rejected and display a marginal metastatic phenotype in BALB/c mice. Rejection is associated with a marked T lymphocyte and granulocyte infiltration, along with extensive macrophage and dendritic cell recruitment. NK cells and CD4+ T lymphocytes are uninfluential in TSA-LEC cell rejection, whereas both CD8+ lymphocytes and polymorphonuclear leukocytes play a major role. An antitumor immune memory is established very quickly after rejection, since 6 days later 75% of BALB/c mice were already resistant to a TSA-pc challenge. Spleen cells from rejecting mice display specific cytotoxic activity against TSA-pc and secrete IFN-gamma and IL-2 when restimulated by TSA-pc. The ability of LEC to markedly improve recognition of poorly immunogenic cells by promoting APC-T cell cross-talk suggests that it could be an effective component of antitumor vaccines.
Asunto(s)
Células Presentadoras de Antígenos/patología , Movimiento Celular/inmunología , Quimiocinas CC/fisiología , Rechazo de Injerto/inmunología , Granulocitos/patología , Memoria Inmunológica , Linfocitos/patología , Neoplasias Mamarias Experimentales/inmunología , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Células Presentadoras de Antígenos/inmunología , División Celular/inmunología , Quimiocinas CC/metabolismo , Femenino , Rechazo de Injerto/patología , Reacción Injerto-Huésped/inmunología , Granulocitos/inmunología , Humanos , Linfocitos/inmunología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales CultivadasRESUMEN
Allergic reactions are characterized by the infiltration of tissues by activated eosinophils, Th2 lymphocytes, and basophils. The beta-chemokine receptor CCR3, which recognizes the ligands eotaxin, eotaxin-2, monocyte chemotactic protein (MCP) 3, MCP4, and RANTES, plays a central role in this process, and antagonists to this receptor could have potential therapeutic use in the treatment of allergy. We describe here a potent and specific CCR3 antagonist, called Met-chemokine beta 7 (Ckbeta7), that prevents signaling through this receptor and, at concentrations as low as 1 nM, can block eosinophil chemotaxis induced by the most potent CCR3 ligands. Met-Ckbeta7 is a more potent CCR3 antagonist than Met- and aminooxypentane (AOP)-RANTES and, unlike these proteins, exhibits no partial agonist activity and is highly specific for CCR3. Thus, this antagonist may be of use in ameliorating leukocyte infiltration associated with allergic inflammation. Met-Ckbeta7 is a modified form of the beta-chemokine macrophage inflammatory protein (MIP) 4 (alternatively called pulmonary and activation-regulated chemokine (PARC), alternative macrophage activation-associated C-C chemokine (AMAC) 1, or dendritic cell-derived C-C chemokine (DCCK) 1). Surprisingly, the unmodified MIP4 protein, which is known to act as a T cell chemoattractant, also exhibits this CCR3 antagonistic activity, although to a lesser extent than Met-Ckbeta7, but to a level that may be of physiological relevance. MIP4 may therefore use chemokine receptor agonism and antagonism to control leukocyte movement in vivo. The enhanced activity of Met-Ckbeta7 is due to the alteration of the extreme N-terminal residue from an alanine to a methionine.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Alanina/metabolismo , Quimiocinas CC/fisiología , Metionina/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Receptores de Quimiocina/antagonistas & inhibidores , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Adulto , Alanina/genética , Calcio/antagonistas & inhibidores , Calcio/metabolismo , Señalización del Calcio/inmunología , Inhibición de Migración Celular , Quimiocina CCL11 , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Quimiotaxis de Leucocito/inmunología , Clonación Molecular , Citocinas/metabolismo , Eosinófilos/metabolismo , Humanos , Radioisótopos de Yodo/metabolismo , Ligandos , Metionina/genética , Fragmentos de Péptidos/genética , Receptores CCR3 , Receptores de Quimiocina/metabolismo , Proteínas Recombinantes/biosíntesis , Células Tumorales CultivadasRESUMEN
BALB/c mammary adenocarcinoma cells engineered to express TNF-related apoptosis-inducing ligand (TRAIL)/APO-2 ligand (APO-2L) on their membrane (TSA-TRAIL) grow with kinetics similar to that of parental cells (TSA-pc) in vitro and in nu/nu mice. In contrast, TSA-TRAIL cells grow faster than TSA-pc in normal BALB/c mice. In DBA/2 mice, which differ from BALB/c mice at minor histocompatibility Ags, they also grow faster and display a higher percentage of tumor takes than TSA-pc. In fully histoincompatible C57BL/6 (B6) mice, TSA-TRAIL cells form evident tumors that are slowly rejected by most mice, but outgrow in a few. In contrast, TSA-pc cells are rejected at once by B6 mice. Since TRAIL/APO-2L induces apoptosis by interacting with a variety of specific receptors, this rapid growth in both syngeneic and allogeneic mice may be the result of an immunosuppressive mechanism. The following evidence supports this hypothesis: 1) TSA-TRAIL cells overcome the strong immunity against TSA-pc cells elicited in BALB/c mice by preimmunization with TSA cells engineered to release IL-4; 2) their rejection by B6 mice does not prime a CTL-mediated memory; 3) thymidine uptake by T lymphocytes unstimulated or stimulated by allogeneic cells is inhibited when TSA-TRAIL cells are added as third party cells; 4) CTL kill TSA-pc but not TSA-TRAIL cells in 48-h assays; and 5) activated lymphocytes interacting with TSA-TRAIL cells in vivo and in vitro undergo apoptosis.
Asunto(s)
Adenocarcinoma/inmunología , Regulación Neoplásica de la Expresión Génica/inmunología , Neoplasias Mamarias Experimentales/inmunología , Glicoproteínas de Membrana/genética , Linfocitos T/inmunología , Escape del Tumor/inmunología , Factor de Necrosis Tumoral alfa/genética , Adenocarcinoma/genética , Animales , Proteínas Reguladoras de la Apoptosis , División Celular/genética , División Celular/inmunología , Citotoxicidad Inmunológica/genética , Citotoxicidad Inmunológica/inmunología , Femenino , Neoplasias Mamarias Experimentales/genética , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Desnudos , Trasplante de Neoplasias , Ingeniería de Proteínas , Especificidad de la Especie , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas , Escape del Tumor/genética , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Neutrophils release elastase, which is known secondarily to cause tissue damage. However, it is rapidly inactivated by the endogenous alpha1-proteinase inhibitor (alpha1Pi). Nevertheless, under pathological conditions, alpha1i is inactivated by oxidants released from neutrophils, resulting in an excess of elastase at the site of inflammation. This elastase/alpha1Pi imbalance has been implicated as a pathogenic factor in cystic fibrosis, acute respiratory distress syndrome, and emphysema. Elastase inhibitors, which do not interfere with the microbicidal activity of neutrophils and are resistant to neutrophil-released oxidants, would undoubtedly represent an important advance in the management of neutrophil-mediated tissue injury. We report that a new family of elastase inhibitors ICI200355 and ZD0892 was found to be resistant toward superoxide, hypochlorous acid, hydrogen peroxide, hydroxyl radical, and peroxynitrite mediated degradation as well as having no effect on the formation of these oxidants by activated neutrophils. More importantly, we found that these inhibitors did not interfere with the ability of human neutrophils to phagocytose and to kill Staphylococcus aureus. In conclusion, a new potent class of elastase inhibitors, while blocking the effects of neutrophil elastase, was found not to impede various physiological functions of human neutrophils, in particular the ability of these phagocytic cells to phagocytose and kill bacteria.
Asunto(s)
Elastasa de Leucocito/antagonistas & inhibidores , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Oligopéptidos/farmacología , Pirroles/farmacología , Inhibidores de Serina Proteinasa/farmacología , Sulfonamidas/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Cinética , Elastasa de Leucocito/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacología , Activación Neutrófila/efectos de los fármacos , Activación Neutrófila/fisiología , Neutrófilos/enzimología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
This paper describes the development a series of peptidyl trifluoromethyl ketone inhibitors of human leukocyte elastase which are found to have excellent pharmacological profiles. Methods have been developed that allow for the synthesis of these inhibitors in stereochemically pure form. Two of these compounds, 1k and 1l, have high levels of oral bioavailability in several species. Compound 1l has entered development as ZD8321 and is presently undergoing clinical evaluation. These compounds demonstrate that peptidyl trifluoromethyl ketone inhibitors can achieve high levels of oral activity and bioavailability, and therefore they may prove useful as therapeutic agents in the treatment of diseases in which elastase is implicated.
Asunto(s)
Elastasa de Leucocito/antagonistas & inhibidores , Oligopéptidos/farmacología , Inhibidores de Serina Proteinasa/síntesis química , Administración Oral , Animales , Disponibilidad Biológica , Cricetinae , Perros , Humanos , Isomerismo , Oligopéptidos/administración & dosificación , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/farmacocinética , Ratas , Inhibidores de Serina Proteinasa/administración & dosificación , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
Two novel human beta-chemokines, Ck beta-8 or myeloid progenitor inhibitory factor 1 (MPIF-1), and Ck beta-6 or MPIF-2, were discovered as part of a large scale cDNA sequencing effort. The MPIF-1 and MPIF-2 cDNAs were isolated from aortic endothelium and activated monocyte libraries, respectively. Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells. The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1alpha, showing 51% identity. It displays chemotactic activity on resting T lymphocytes and monocytes, a minimal but significant activity on neutrophils, and is negative on activated T lymphocytes. MPIF-1 is also a potent suppressor of bone marrow low proliferative potential colony-forming cells, a committed progenitor that gives rise to granulocyte and monocyte lineages. The mature recombinant MPIF-2 has 93 amino acid residues and shows 39 and 42% identity with monocyte chemoattractant protein (MCP)-3 and MIP-1alpha, respectively. It displays chemotactic activity on resting T lymphocytes, a minimal activity on neutrophils, and is negative on monocytes and activated T lymphocytes. On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4. In hematopoietic assays, MPIF-2 strongly suppressed the colony formation by the high proliferative potential colony-forming cell (HPP-CFC), which represents a multipotential hematopoietic progenitor.
Asunto(s)
Quimiocinas CC , Quimiocinas/aislamiento & purificación , Células Madre Hematopoyéticas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Quimiocina CCL24 , Quimiocinas/genética , Quimiocinas/farmacología , Quimiotaxis de Leucocito , Clonación Molecular , Citosol/metabolismo , ADN/genética , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Perforin and granzymes are proteins thought to play a relevant role in cell-mediated cytotoxicity. These molecules are constitutively expressed in NK cells and their level of expression in cytotoxic T lymphocytes is regulated by several cytokines. We analyzed the mechanisms by which cytokines and cellular ligands known to modulate NK cell-mediated cytotoxicity affect the expression of the mRNA encoding granzyme A and B and perforin in NK cells. Our data indicate that IL-2 and IL-12 induce increased accumulation of both perforin and, to a higher degree, granzyme B mRNA. In contrast, binding of target cells or immune complexes up-regulates expression of granzyme B mRNA without altering that of perforin. Results of in situ hybridization experiments confirm that mRNA for both molecules are expressed at low levels in most NK cells, and that both are induced to accumulate by the two cytokines in the majority of the cells. The mechanisms by which IL-2 and IL-12 regulate expression of the two molecules are, in part, distinct: both cytokines increase the transcriptional rate of the encoding genes, whereas only IL-2 acts also at a post-transcriptional level to increase the stability of their mRNA.
Asunto(s)
Regulación de la Expresión Génica , Células Asesinas Naturales/metabolismo , Glicoproteínas de Membrana/genética , ARN Mensajero/análisis , Serina Endopeptidasas/genética , Calcio/fisiología , Línea Celular , Granzimas , Humanos , Interleucina-12 , Interleucina-2/farmacología , Interleucinas/farmacología , Perforina , Proteínas Citotóxicas Formadoras de Poros , Biosíntesis de Proteínas , Receptores de IgG/fisiología , Células Tumorales CultivadasRESUMEN
The transmembrane receptor for immunoglobulin G immune complexes on natural killer (NK) cells and macrophages, Fc gamma RIIIA (CD16), mediates cellular activation through a tyrosine kinase-dependent pathway. We show that Fc gamma RIII crosslinking results in activation of the src-related kinase p56lck in NK cells and demonstrate a physical association of p56lck with Fc gamma RIIIA in immunoprecipitates from NK cells obtained using anti-Fc gamma RIII antibodies or immune complexes. Our studies show that the zeta chain, the signal transducing subunit of Fc gamma RIIIA and of T cell receptor, associates with p56lck and, in NK cells, is a substrate for this kinase. Such direct association of p56lck with the zeta subunit as confirmed by demonstrating the interaction in heterologous cells transfected with cDNA expressing p56lck and zeta. Our findings demonstrate both functional and physical association of p56lck with Fc gamma RIIIA, through direct interaction of the kinase with the zeta and/or the gamma signal transducer subunits of the receptor. These data suggest a possible mechanism by which activation via Fc gamma RIIIA occurs.
Asunto(s)
Células Asesinas Naturales/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de IgG/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Humanos , Células Asesinas Naturales/enzimología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Datos de Secuencia Molecular , Fosforilación , Transducción de SeñalRESUMEN
Binding of ligand to the alpha subunit of Fc gamma RIIIA(CD16), expressed at the natural killer (NK) cell membrane in association with homo or heterodimers of proteins of the zeta family, results in phosphorylation of several proteins on tyrosine residues. We have analyzed the role of protein tyrosine phosphorylation in the regulation of molecular events induced upon stimulation of Fc gamma RIIIA in NK cells and in T cells expressing the Fc gamma RIII alpha chain in association with endogenous zeta 2 homodimers and devoid of other (CD3, CD2) transducing molecules. Our data indicate that treatment of these cells with protein tyrosine kinase inhibitors prevents not only Fc gamma RIIIA-induced protein tyrosine phosphorylation but also phosphatidylinositol 4,5 diphosphate hydrolysis and increased intracellular Ca2+ concentration, indicating a primary role of tyrosine kinase(s) in the induction of these early activation events. Occupancy of Fc gamma RIIIA by ligand results in phospholipase C (PLC)-gamma 1 tyrosine phosphorylation in NK cells and in Fc gamma RIIIA-transfected CD3-/CD2- T cells, and induces functional activation of p56lck in Fc gamma RIIIA alpha/zeta 2-transfected T cells, suggesting the possibility that the receptor-induced PLC-gamma 1 activation occurs upon phosphorylation of its tyrosine residues mediated by this kinase and is, at least in part, responsible for the signal transduction mediated via CD16 upon ligand binding.
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Isoenzimas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de IgG/metabolismo , Fosfolipasas de Tipo C/metabolismo , Anticuerpos Monoclonales , Membrana Celular/inmunología , Humanos , Células Asesinas Naturales/inmunología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Sustancias Macromoleculares , Fosforilación , Fosfotirosina , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores de IgG/genética , Transfección , Células Tumorales Cultivadas , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
A simple technique has been used to observe the effects of rTNF alpha on nuclear morphology. Using the intercalating dye p-phenylenediamine to stain nuclei, we detected TNF alpha-induced nuclear alterations which characteristically occur during apoptosis in the TNF alpha sensitive U937 cell line. Nuclear alterations were visible prior to the loss of plasma membrane integrity and subsequent cell death. A subclone of U937 cells was isolated in which TNF alpha failed to alter either cell viability or nuclear morphology. TNF alpha resistant U937 cells, however, retained the ability to bind, internalize and degrade TNF alpha. These results suggest that nuclear damage induced by TNF alpha in sensitive U937 cells occurs early and precedes cell death as measured by dye exclusion assays. Staining cells with p-phenylenediamine and visualization with a 520 nm fluorescence filter provides a rapid and simple method to monitor apoptotic cell death.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Fenilendiaminas , Coloración y Etiquetado/métodos , Factor de Necrosis Tumoral alfa/farmacología , División Celular/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Humanos , Microscopía Fluorescente , Membrana Nuclear/efectos de los fármacos , Proteínas Recombinantes/farmacología , Timidina/metabolismoRESUMEN
Transferrin receptor expression in the monocyte-like cell line U937 was investigated during in vitro cultivation. U937 cells expressed a single class of high affinity surface transferrin receptors (KD approximately 4 nM), with apparent subunit Mr of 90-95,000 Da as determined by SDS-reducing PAGE. [125I]-transferrin binding studies on detergent-solubilized cells revealed that half to two-thirds of the total functional binding sites were located intracellularly. Radioligand binding, immunofluorescence and flow cytometry studies were performed on intact, detergent-solubilized, or saponin-permeabilized cells, using either transferrin or the anti-transferrin receptor monoclonal antibody OKT9 IgG. These studies demonstrated that functional and antigenic transferrin receptor levels were maximal on cells 24 h after subculture at low density and declined during the culture period. Scatchard analysis of radioligand binding data suggested that the decline in functional transferrin binding sites resulted from a decline in the number of available receptors. These results demonstrate that in U937 cells there is a density-dependent regulation of transferrin receptor expression, resulting in a loss of functional and antigenic receptors from both plasma membrane and intracellular locations.