RESUMEN

Plazomicin is an aminoglycoside with in vitro activity against multidrug-resistant Enterobacteriaceae. A phase 1, randomized, double-blind, crossover study assessed the potential effects of plazomicin on cardiac repolarization (NCT01514929). Fifty-six healthy adults (24 men, 32 women) received a single therapeutic dose of plazomicin (15 mg/kg administered by 30-minute intravenous infusion), a single supratherapeutic dose of plazomicin (20 mg/kg administered by 30-minute intravenous infusion), placebo, or oral moxifloxacin (400 mg). The primary end point was the baseline-adjusted, placebo-corrected QTc interval using the Fridericia formula (ΔΔQTcF). Assay sensitivity was concluded if the lower limit of a 1-sided 95%CI (adjusted for multiplicity using the Hochberg procedure) for moxifloxacin ΔΔQTcF was >5 milliseconds at ≥1 prespecified time points. No QT prolongation effect for plazomicin was concluded if the largest mean effect was <5 milliseconds, and the upper limit of a 2-sided 90%CI for plazomicin ΔΔQTcF was <10 milliseconds at all time points. Assay sensitivity was demonstrated based on moxifloxacin ΔΔQTcF. No QT prolongation effect for plazomicin was concluded because the largest mean ΔΔQTcF for plazomicin was 3.5 milliseconds, and the highest upper limit was 5.6 milliseconds. No clinically relevant changes were observed in electrocardiograms. For the 15- and 20-mg/kg dose levels of plazomicin, mean peak plasma concentration values were 76.0 and 96.6 mg/L, and mean values of the area under the concentration-time curve over 24 hours were 263 and 327 mg·h/L, respectively. Model-derived pharmacokinetic parameters and safety findings were generally consistent with previously reported plazomicin studies. In conclusion, therapeutic and supratherapeutic doses of plazomicin had no clinically significant effect on cardiac repolarization and were generally well tolerated.

Asunto(s)

Antibacterianos , Síndrome de QT Prolongado/inducido químicamente , Sisomicina/análogos & derivados , Adulto , Antibacterianos/administración & dosificación , Antibacterianos/efectos adversos , Antibacterianos/sangre , Área Bajo la Curva , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Electrocardiografía/efectos de los fármacos , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Infusiones Intravenosas , Síndrome de QT Prolongado/sangre , Síndrome de QT Prolongado/epidemiología , Masculino , Sisomicina/administración & dosificación , Sisomicina/efectos adversos , Sisomicina/sangre

RESUMEN

The objective of this study was to determine the comparative pharmacodynamics of four different carbapenems in combination with polymyxin B (PMB) against carbapenem-resistant Acinetobacter baumannii isolates using time-kill experiments at two different inocula. Two A. baumannii strains (03-149-1 and N16870) with carbapenem minimum inhibitory concentrations (MICs) ranging from 8 to 64 mg/L were investigated in 48-h time-kill experiments using starting inocula of 106 CFU/mL and 108 CFU/mL. Concentration arrays of ertapenem, doripenem, meropenem and imipenem at 0.25×, 0.5×, 1×, 1.5× and 2× published maximum serum concentration (Cmax) values (Cmax concentrations of 12, 21, 48 and 60 mg/L, respectively) were investigated in the presence of 1.5 mg/L PMB. Use of carbapenems without PMB resulted in drastic re-growth. All carbapenem combinations were able to achieve a ≥3 log10 CFU/mL reduction by 4 h against both strains at 106 CFU/mL, whereas maximum reductions against strain 03-149-1 at 108 CFU/mL were 1.0, 3.2, 2.2 and 3.3 log10 CFU/mL for ertapenem, doripenem, meropenem and imipenem, respectively. None of the combinations were capable of reducing 108 CFU/mL of N16870 by ≥2 log10 CFU/mL. Ertapenem combinations consistently displayed the least activity, whereas doripenem, meropenem and imipenem combinations had similar activities that were poorly predicted by carbapenem MICs. As doripenem, meropenem, or imipenem displayed similar pharmacodyanmics in combination, the decision of which carbapenem to use in combination with PMB may be based on toxicodynamic profiles if drastic discordance in MICs is not present.

Asunto(s)

Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Carbapenémicos/farmacología , Interacciones Farmacológicas , Polimixina B/farmacología , Resistencia betalactámica , Pruebas de Sensibilidad Microbiana

RESUMEN

Proximal tubule (PT) cells are critical targets of acute ischemic injury. Elimination of the mitochondrial fusion protein mitofusin 2 (Mfn2) sensitizes PT cells to apoptosis in vitro. However, the role of PT Mfn2 in ischemic AKI in vivo is unknown. To test its role, we evaluated the effects of conditional KO of PT Mfn2 (cKO-PT-Mfn2) on animal survival after transient bilateral renal ischemia associated with severe AKI. Forty-eight hours after ischemia, 28% of control mice survived compared with 86% of cKO-PT-Mfn2 animals (P<0.001 versus control). Although no significant differences in histologic injury score, apoptosis, or necrosis were detected between genotypes, cKO-PT-Mfn2 kidneys exhibited a 3.5-fold increase in cell proliferation restricted to the intrarenal region with Mfn2 deletion. To identify the signals responsible for increased proliferation, primary PT cells with Mfn2 deficiency were subjected to stress by ATP depletion in vitro. Compared with normal Mfn2 expression, Mfn2 deficiency significantly increased PT cell proliferation and persistently activated extracellular signal-regulated kinase 1/2 (ERK1/2) during recovery from stress. Furthermore, stress and Mfn2 deficiency decreased the interaction between Mfn2 and Ras detected by immunoprecipitation, and purified Mfn2 dose-dependently decreased Ras activity in a cell-free assay. Ischemia in vivo also reduced the Mfn2-RAS interaction and increased both RAS and p-ERK1/2 activity in the renal cortical homogenates of cKO-PT-Mfn2 mice. Our results suggest that, in contrast to its proapoptotic effects in vitro, selective PT Mfn2 deficiency accelerates recovery of renal function and enhances animal survival after ischemic AKI in vivo, partly by increasing Ras-ERK-mediated cell proliferation.

Asunto(s)

Lesión Renal Aguda/metabolismo , GTP Fosfohidrolasas/metabolismo , Túbulos Renales Proximales/fisiología , Regeneración , Lesión Renal Aguda/etiología , Lesión Renal Aguda/mortalidad , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Isquemia/complicaciones , Isquemia/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones Noqueados , Recuperación de la Función

RESUMEN

We hypothesized that nucleophosmin (NPM), a nucleolar phosphoprotein, is critical for Bax-mediated cell death. To test this hypothesis, Bax activation was induced by metabolic stress. During stress, nucleolar NPM translocated into the cytosol, NPM-Bax complexes formed, and both NPM and Bax accumulated in mitochondria. Expression of a cytosol-restricted NPM mutant (NPM-ΔNLS), but not a nucleus-restricted NPM mutant, increased NPM-Bax complex formation, mitochondrial NPM and Bax accumulation, mitochondrial membrane injury, caspase 3 activation, and ischemia-induced cell death. Coexpression of NPM-ΔNLS with constitutively active Bax mutants caused nearly universal cell death in the absence of metabolic stress, whereas expression of active Bax or NPM-ΔNLS alone did not. A Bax peptide that disrupts NPM-Bax interaction significantly reduced cell death caused by exposure to metabolic inhibitors in vitro and preserved kidney function after ischemia in vivo. Thus, NPM-Bax interaction enhances mitochondrial Bax accumulation, organelle injury, and cell death. NPM-Bax complex formation is a novel target for preventing ischemic tissue injury.

Asunto(s)

Apoptosis , Isquemia/patología , Riñón/irrigación sanguínea , Proteínas Nucleares/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Técnicas de Silenciamiento del Gen , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/fisiología , Nucleofosmina , Cultivo Primario de Células , Unión Proteica , Transporte de Proteínas , ARN Interferente Pequeño/genética , Daño por Reperfusión/metabolismo , Estrés Fisiológico , Proteína X Asociada a bcl-2/genética

RESUMEN

HBO1 acetylates lysine residues of histones and is involved in DNA replication and gene transcription. Two isoforms of JADE1, JADE1S and JADE1L, bind HBO1 and promote acetylation of histones in chromatin context. We characterized the role of JADE1-HBO1 complexes in vitro and in vivo during epithelial cell replication. Down-regulation of JADE1 by siRNA diminished the rate of DNA synthesis in cultured cells, decreased endogenous HBO1 protein expression, and prevented chromatin recruitment of replication factor Mcm7, demonstrating that JADE1 is required for cell proliferation. We used a murine model of acute kidney injury to examine expression of HBO1-JADE1S/L in injured and regenerating epithelial tissue. In control kidneys, JADE1S, JADE1L, and HBO1 were expressed in nuclei of proximal and distal tubular epithelial cells. Ischemia and reperfusion injury resulted in an initial decrease in JADE1S, JADE1L, and HBO1 protein levels, which returned to baseline during renal recovery. HBO1 and JADE1S recovered as cell proliferation reached its maximum, whereas JADE1L recovered after bulk proliferation had ceased. The temporal expression of JADE1S correlated with the acetylation of histone H4 on lysines 5 and 12, but not with acetylation of histone H3 on lysine 14, demonstrating that the JADE1S-HBO1 complex specifically marks H4 during epithelial cell proliferation. These data implicate JADE1-HBO1 complex in acute kidney injury and suggest distinct roles for JADE1 isoforms during epithelial cell recovery.

Asunto(s)

Células Epiteliales/fisiología , Histona Acetiltransferasas/metabolismo , Proteínas de Homeodominio/biosíntesis , Proteínas Supresoras de Tumor/biosíntesis , Lesión Renal Aguda/etiología , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Animales , Línea Celular , Núcleo Celular/metabolismo , Proliferación Celular , Replicación del ADN , Regulación hacia Abajo , Células Epiteliales/metabolismo , Silenciador del Gen , Histona Acetiltransferasas/biosíntesis , Histona Acetiltransferasas/genética , Proteínas de Homeodominio/genética , Humanos , Túbulos Renales/metabolismo , Ratones , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , ARN Interferente Pequeño/genética , Regeneración/genética , Regeneración/fisiología , Daño por Reperfusión/complicaciones , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Proteínas Supresoras de Tumor/genética

RESUMEN

The role of mitofusin 2 (MFN2), a key regulator of mitochondrial morphology and function in the renal stress response is unknown. To assess its role, the MFN2 floxed gene was conditionally deleted in the kidney of mice (MFN2 cKO) by Pax2 promoter driven Cre expression (Pax2Cre). MFN2 cKO caused severe mitochondrial fragmentation in renal epithelial cells that are critical for normal kidney tubular function. However, despite a small (20%) decrease in nephron number, newborn cKO pups had organ or tubular function that did not differ from littermate Cre-negative pups. MFN2 deficiency in proximal tubule epithelial cells in primary culture induced mitochondrial fragmentation but did not significantly alter ATP turnover, maximal mitochondrial oxidative reserve capacity, or the low level of oxygen consumption during cyanide exposure. MFN2 deficiency also did not increase apoptosis of tubule epithelial cells under non-stress conditions. In contrast, metabolic stress caused by ATP depletion exacerbated mitochondrial outer membrane injury and increased apoptosis by 80% in MFN2 deficient vs. control cells. Despite similar stress-induced Bax 6A7 epitope exposure in MFN2 deficient and control cells, MFN2 deficiency significantly increased mitochondrial Bax accumulation and was associated with greater release of both apoptosis inducing factor and cytochrome c. In conclusion, MFN2 deficiency in the kidney causes mitochondrial fragmentation but does not affect kidney or tubular function during development or under non-stress conditions. However, MFN2 deficiency exacerbates renal epithelial cell injury by promoting Bax-mediated mitochondrial outer membrane injury and apoptosis.

Asunto(s)

GTP Fosfohidrolasas/fisiología , Riñón/metabolismo , Estrés Fisiológico/genética , Animales , Animales Recién Nacidos , Apoptosis/genética , Nitrógeno de la Urea Sanguínea , Células Cultivadas , Femenino , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Hematócrito , Riñón/lesiones , Riñón/patología , Masculino , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Mitocondrias/patología , Consumo de Oxígeno/genética , Consumo de Oxígeno/fisiología , Transporte de Proteínas , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

Hexokinase (HK), the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. Since HK isoforms I and II have mitochondrial targeting sequences, we attempted to separate the protective effects of HK on cell metabolism from those on apoptosis. We exposed renal epithelial cells to metabolic stress causing ATP depletion in the absence of glucose and found that this activated glycogen synthase kinase 3ß (GSK3ß) and Bax caused mitochondrial membrane injury and apoptosis. ATP depletion led to a progressive HK II dissociation from mitochondria, released mitochondrial apoptosis inducing factor and cytochrome c into the cytosol, activated caspase-3, and reduced cell survival. Compared with control, adenoviral-mediated HK I or II overexpression improved cell survival following stress, but did not prevent GSK3ß or Bax activation, improve ATP content, or reduce mitochondrial fragmentation. HK I or HK II overexpression increased mitochondria-associated isoform-specific HK content, and decreased mitochondrial membrane injury and apoptosis after stress. In vivo, HK II localized exclusively to the proximal tubule. Ischemia reduced total renal HK II content and dissociated HK II from proximal tubule mitochondria. In cells overexpressing HK II, Bax and HK II did not interact before or after stress. While the mechanism by which HK antagonizes Bax-mediated apoptosis is unresolved by these studies, one possible scenario is that the two proteins compete for a common binding site on the outer mitochondrial membrane.

Asunto(s)

Células Epiteliales/enzimología , Hexoquinasa/metabolismo , Enfermedades Renales/enzimología , Túbulos Renales Proximales/enzimología , Membranas Mitocondriales/enzimología , Daño por Reperfusión/enzimología , Estrés Fisiológico , Proteína X Asociada a bcl-2/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Caspasa 3/metabolismo , Supervivencia Celular , Células Cultivadas , Citocromos c/metabolismo , Modelos Animales de Enfermedad , Células Epiteliales/patología , Glucosa/deficiencia , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hexoquinasa/genética , Enfermedades Renales/patología , Túbulos Renales Proximales/irrigación sanguínea , Túbulos Renales Proximales/patología , Ratones , Membranas Mitocondriales/patología , Zarigüeyas , Transporte de Proteínas , Daño por Reperfusión/patología , Transducción de Señal , Factores de Tiempo , Transfección

RESUMEN

Heat shock protein 70 (Hsp70) is a potent antiapoptotic agent. Here, we tested whether it directly regulates renal cell survival and organ function in a model of transient renal ischemia using Hsp70 knockout, heterozygous, and wild-type mice. The kidney cortical Hsp70 content inversely correlated with tubular injury, apoptosis, and organ dysfunction after injury. In knockout mice, ischemia caused changes in the activity of Akt and glycogen synthase kinase 3-ß (kinases that regulate the proapoptotic protein Bax), increased active Bax, and activated the proapoptotic protease caspase 3. As these changes were significantly reduced in the wild-type mice, we tested whether Hsp70 influences ischemia-induced apoptosis. An Hsp70 inducer, geranylgeranylacetone, increased Hsp70 expression in heterozygous and wild-type mice, and reduced both ischemic tubular injury and organ dysfunction. When administered after ischemia, this inducer also decreased tubular injury and organ failure in wild-type mice but did not protect the knockout mice. ATP depletion in vitro caused greater mitochondrial Bax accumulation and death in primary proximal tubule cells harvested from knockout compared with wild-type mice and altered serine phosphorylation of a Bax peptide at the Akt-specific target site. In contrast, lentiviral-mediated Hsp70 repletion decreased mitochondrial Bax accumulation and rescued Hsp70 knockout cells from death. Thus, increasing Hsp70 either before or after ischemic injury preserves renal function by attenuating acute kidney injury.

Asunto(s)

Proteínas HSP70 de Choque Térmico/biosíntesis , Isquemia/prevención & control , Riñón/irrigación sanguínea , Lesión Renal Aguda/tratamiento farmacológico , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Lesión Renal Aguda/prevención & control , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Diterpenos/administración & dosificación , Expresión Génica , Técnicas de Inactivación de Genes , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Proteínas HSP70 de Choque Térmico/deficiencia , Proteínas HSP70 de Choque Térmico/genética , Isquemia/tratamiento farmacológico , Isquemia/fisiopatología , Riñón/efectos de los fármacos , Riñón/lesiones , Riñón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Recombinantes/metabolismo , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

The mechanism by which the serine-threonine kinase glycogen synthase kinase-3beta (GSK3beta) affects survival of renal epithelial cells after acute stress is unknown. Using in vitro and in vivo models, we tested the hypothesis that GSK3beta promotes Bax-mediated apoptosis, contributing to tubular injury and organ dysfunction after acute renal ischemia. Exposure of renal epithelial cells to metabolic stress activated GSK3beta, Bax, and caspase 3 and induced apoptosis. Expression of a constitutively active GSK3beta mutant activated Bax and decreased cell survival after metabolic stress. In contrast, pharmacologic inhibition (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione [TDZD-8]) or RNA interference-mediated knockdown of GSK3beta promoted cell survival. Furthermore, RNA interference-mediated knockdown of Bax abrogated the cell death induced by constitutively active GSK3beta. In a cell-free assay, TDZD-8 inhibited the phosphorylation of a peptide containing the Bax serine(163) site targeted by stress-activated GSK3beta. In rats, TDZD-8 inhibited ischemia-induced activation of GSK3beta, Bax, and caspase 3; ameliorated tubular and epithelial cell damage; and significantly protected renal function. Taken together, GSK3beta-mediated Bax activation induces apoptosis and tubular damage that contribute to acute ischemic kidney injury.

Asunto(s)

Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Apoptosis , Glucógeno Sintasa Quinasa 3/metabolismo , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Animales , Caspasa 3/metabolismo , Línea Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3 beta , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Masculino , Ratones , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Tiadiazoles/farmacología , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/metabolismo

RESUMEN

Ischemia activates Bax, a proapoptotic BCL2 protein, as well as the prosurvival beta-catenin/Wnt signaling pathway. To test the hypothesis that beta-catenin/Wnt signaling regulates Bax-mediated apoptosis after induction of metabolic stress, which occurs during renal ischemia, we infected immortalized and primary proximal tubular epithelial cells with adenovirus to express either constitutively active or dominant negative beta-catenin constructs. Constitutively active beta-catenin significantly decreased apoptosis and improved cell survival after metabolic stress. Furthermore, active beta-catenin decreased Bax activation, oligomerization, and translocation to mitochondria, and reduced both organelle membrane injury and apoptosis. Dominant negative beta-catenin had the opposite effects. Because Akt regulates Bax, we examined the effects of the beta-catenin mutants on Akt expression and activation. Constitutively active beta-catenin increased Akt-1 expression and activation before and after stress, and treatment with a phosphatidylinositol-3 kinase inhibitor antagonized the protective effects of beta-catenin on Akt activation, Bax inhibition, and cell survival. In addition, beta-catenin significantly increased the rate of phosphorylation at Bax serine(184), an Akt-specific target. Taken together, these results suggest that beta-catenin/Wnt signaling promotes survival of renal epithelial cells after metabolic stress, in part by inhibiting Bax in a phosphatidylinositol-3 kinase/Akt-dependent manner.

Asunto(s)

Células Epiteliales/citología , Células Epiteliales/metabolismo , Túbulos Renales Proximales/citología , Proteína X Asociada a bcl-2/metabolismo , beta Catenina/metabolismo , Adenoviridae/genética , Animales , Apoptosis/fisiología , Línea Celular Transformada , Supervivencia Celular/fisiología , Genes Reporteros , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Estrés Fisiológico/fisiología , Proteínas Wnt/metabolismo , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/genética , beta Catenina/genética

RESUMEN

Disruption of cell contact sites in renal epithelial cells contributes to organ dysfunction after ischemia. We hypothesized that heat shock protein 27 (Hsp27), a known cytoprotectant protein, preserves cell architecture and cell contact site function during ischemic stress. To test this hypothesis, renal epithelial cells were subjected to transient ATP depletion, an in vitro model of ischemia-reperfusion injury. Compared with control, selective Hsp27 overexpression significantly preserved cell-cell junction function during metabolic stress as evidenced by reduced stress-mediated redistribution of the adherens junction protein E-cadherin, higher transepithelial electrical resistance, and lower unidirectional flux of lucifer yellow. Hsp27 overexpression also preserved paxillin staining within focal adhesion complexes and significantly decreased cell detachment during stress. Surprisingly, Hsp27, an F-actin-capping protein, only minimally reduced stress induced actin cytoskeleton collapse. In contrast to Hsp27 overexpression, siRNA-mediated knockdown had the opposite effect on these parameters. Since ischemia activates c-Src, a tyrosine kinase that disrupts both cell-cell and cell-substrate interactions, the relationship between Hsp27 and c-Src was examined. Although Hsp27 and c-Src did not coimmunoprecipitate and Hsp27 overexpression failed to inhibit whole cell c-Src activation during injury, manipulation of Hsp27 altered active c-Src accumulation at cell contact sites. Specifically, Hsp27 overexpression reduced, whereas Hsp27 knockdown increased active p-(416)Src detected at contact sites in intact cells as well as in a purified cell membrane fraction. Together, this evidence shows that Hsp27 overexpression prevents sublethal REC injury at cell contact sites possibly by a c-Src-dependent mechanism. Further exploration of the biochemical link between Hsp27 and c-Src could yield therapeutic interventions for ameliorating ischemic renal cell injury and organ dysfunction.

Asunto(s)

Células Epiteliales/enzimología , Proteínas de Choque Térmico HSP27/metabolismo , Isquemia/prevención & control , Riñón/enzimología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Actinas/metabolismo , Adenosina Trifosfato/deficiencia , Uniones Adherentes/enzimología , Animales , Adhesión Celular , Línea Celular , Membrana Celular/enzimología , Citoprotección , Activación Enzimática , Células Epiteliales/patología , Proteínas de Choque Térmico HSP27/genética , Humanos , Isquemia/enzimología , Isquemia/patología , Riñón/irrigación sanguínea , Riñón/patología , Ratones , Permeabilidad , Fosforilación , Interferencia de ARN , Fibras de Estrés/metabolismo , Estrés Fisiológico , Transducción Genética

RESUMEN

Freshwater turtles as a group are more resistant to anoxia than other vertebrates, but some species, such as painted turtles, for reasons not fully understood, can remain anoxic at winter temperatures far longer than others. Because buffering of lactic acid by the shell of the painted turtle is crucial to its long-term anoxic survival, we have tested the hypothesis that previously described differences in anoxia tolerance of five species of North American freshwater turtles may be explained at least in part by differences in their shell composition and buffering capacity. All species tested have large mineralized shells. Shell comparisons included 1) total shell CO2 concentration, 2) volume of titrated acid required to hold incubating shell powder at pH 7.0 for 3 h (an indication of buffer release from shell), and 3) lactate concentration of shell samples incubated to equilibrium in a standard lactate solution. For each measurement, the more anoxia-tolerant species (painted turtle, Chrysemys picta; snapping turtle, Chelydra serpentina) had higher values than the less anoxia-tolerant species (musk turtle, Sternotherus odoratus; map turtle, Graptemys geographica; red-eared slider, Trachemys scripta). We suggest that greater concentrations of accessible CO2 (as carbonate or bicarbonate) in the more tolerant species enable these species, when acidotic, to release more buffer into the extracellular fluid and to take up more lactic acid into their shells. We conclude that the interspecific differences in shell composition and buffering can contribute to, but cannot explain fully, the variations observed in anoxia tolerance among freshwater turtles.

Asunto(s)

Hipoxia/fisiopatología , Tortugas/fisiología , Animales , Tampones (Química) , Dióxido de Carbono/sangre , Dióxido de Carbono/metabolismo , Agua Dulce , Concentración de Iones de Hidrógeno , Ácido Láctico/metabolismo , Minerales/metabolismo , Especificidad de la Especie

RESUMEN

Hematopoietic stem cell (HSC) therapy for myocardial repair is limited by the number of stem cells that migrate to, engraft in, and proliferate at sites of injured myocardium. To alleviate this limitation, we studied whether a strategy using a bispecific antibody (BiAb) could target human stem cells specifically to injured myocardium and preserve myocardial function. Using a xenogeneic rat model whereby ischemic injury was induced by transient ligation of the left anterior descending artery (LAD), we determined the ability of a bispecific antibody to target human CD34+ cells to specific antigens expressed in ischemic injured myocardium. A bispecific antibody comprising an anti-CD45 antibody recognizing the common leukocyte antigen found on HSCs and an antibody recognizing myosin light chain, an organ-specific injury antigen expressed by infarcted myocardium, was prepared by chemical conjugation. CD34+ cells armed and unarmed with this BiAb were injected intravenously in rats 2 days postmyocardial injury. Immunohistochemistry studies showed that the armed CD34+ cells specifically localized to the infarcted region of the heart, colocalized with troponin T-stained cells, and colocalization with vascular structures. Compared to unarmed CD34+ cells, the bispecific antibody improved delivery of the stem cells to injured myocardium, and such targeted delivery was correlated with improved myocardial function 5 weeks after infarction (p < .01). Bispecific antibody targeting offers a unique means to improve the delivery of stem cells to facilitate organ repair and a tool to study stem cell biology.

Asunto(s)

Anticuerpos Antineoplásicos/uso terapéutico , Trasplante de Células Madre Hematopoyéticas/métodos , Infarto del Miocardio/terapia , Trasplante de Células Madre/métodos , Animales , Anticuerpos Biespecíficos/inmunología , Separación Celular , Ecocardiografía , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Antígenos Comunes de Leucocito/inmunología , Ratas , Células Madre/citología , Células Madre/inmunología , Trasplante Heterólogo

RESUMEN

Cytokine-induced killer (CIK) cells are ex vivo activated and expanded CD8+ natural killer T cells that have been shown to have antitumor activity. This is the first study exploring cell killing of primary ovarian carcinoma cells with and without bispecific antibodies. Primary cancer cells and autologous CIK cells were collected from women with epithelial ovarian cancer. Bispecific antibodies against cancer antigen-125 (BSAbxCA125) and Her2 (BSAbxHer2) were developed using chemical heteroconjugation. On fluorescence-activated cell sorting analysis, the expansion of CIK cells resulted in a significant increase of CD3+CD8+ and CD3+CD56+ T cells. With enhancement by bispecific antibodies, the mean percent lysis in a 51Cr release assay of fresh ovarian cancer cells exposed to autologous CIK cells increased from 21.7 +/- 0.3% to 89.4 +/- 2.1% at an E:T ratio of 100:1 (P < 0.001). Anti-NKG2D antibodies attenuated the CIK activity by 56.8% on primary cells (P < 0.001). In a xenograft severe combined immunodeficient mouse model, real-time tumor regression and progression was visualized using a noninvasive in vivo bioluminescence imaging system. Four hours after CIK cell injection, we were able to visualize CD8+NKG2D+ CIK cells infiltrating Her2-expressing cancer cells on fluorescence microscopy. Mice that underwent adoptive transfer of CIK cells redirected with BSAbxCA125 and BSAbxHer2 had significant reduction in tumor burden (P < 0.001 and P < 0.001) and improvement in survival (P = 0.05 and P = 0.006) versus those treated with CIK cells alone. Bispecific antibodies significantly enhanced the cytotoxicity of CIK cells in primary ovarian cancer cells and in our in vivo mouse model. The mechanism of cytolysis seems to be mediated in part by the NKG2D receptor.

Asunto(s)

Anticuerpos Biespecíficos/farmacología , Linfocitos T CD8-positivos/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Ováricas/inmunología , Animales , Anticuerpos Biespecíficos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Linfocitos T CD8-positivos/citología , Línea Celular Tumoral , Citocinas/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Citotoxicidad Inmunológica/inmunología , Femenino , Humanos , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/citología , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones SCID , Microscopía Fluorescente , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto/métodos

RESUMEN

PURPOSE: Cancer immunotherapy has been limited by anergy of patient T cells, inadequate numbers of precursor tumor-specific CTL, and difficulty in producing therapeutic doses of CTL. To overcome these limitations, bispecific antibodies have been used to create artificial antibody receptors that direct polyclonal activated T cells (ATC) to target tumor antigens. Studies reported herein were designed to characterize bispecific antibody-armed ATC functions during multiple rounds of targeted cell stimulation. EXPERIMENTAL DESIGN: ATCs were generated from human peripheral blood mononuclear cells (PBMC) by culture with anti-CD3 and interleukin 2 for 14 days and armed with anti-CD3 x anti-Her2 bispecific antibody (Her2Bi). In vitro, Her2Bi-armed ATC were examined for a range of functions after repeated stimulation with the Her2/neu-expressing breast cancer cell line SK-BR-3. PBMC isolated from cancer patients treated with Her2Bi-armed ATC were tested ex vivo for cytotoxicity against SK-BR-3. RESULTS: In vitro, armed ATC divided, maintained surface Her2Bi, and expressed a range of activities for extended periods of time. Perforin-mediated cytotoxic activity by armed ATC continued for at least 336 hours, and cytokines and chemokines (i.e., IFN-gamma and regulated on activation, normal T-cell expressed and secreted protein [RANTES]) were secreted during successive rounds of stimulation. Furthermore, PBMC isolated from patients over their courses of immunotherapy exhibited significant cytolytic activity against SK-BR-3 as a function of Her2Bi-armed ATC infusions. CONCLUSIONS: These studies show that armed ATC are specific, durable, and highly functional T-cell populations in vitro. These previously unappreciated broad and long-term functions of armed ATC are encouraging for their therapeutic use in treating cancer.

Asunto(s)

Anticuerpos Biespecíficos/farmacología , Neoplasias de la Mama/terapia , Complejo CD3/inmunología , Citotoxicidad Inmunológica , Receptor ErbB-2/inmunología , Linfocitos T/inmunología , Neoplasias de la Mama/inmunología , Línea Celular Tumoral , Quimiocina CCL5/metabolismo , Femenino , Humanos , Inmunoterapia , Interferón gamma/metabolismo , Interleucina-2/inmunología , Activación de Linfocitos , Glicoproteínas de Membrana/farmacología , Perforina , Proteínas Citotóxicas Formadoras de Poros

RESUMEN

OBJECTIVE: Resistance to rituximab, a chimeric monoclonal antibody that binds to CD20, is a major limitation for the successful treatment of patients with non-Hodgkin lymphoma and other CD20+ B-cell malignancies. To circumvent rituximab resistance in these patient populations, we have constructed a bispecific antibody (BiAb), anti-CD3 x anti-CD20 (CD20Bi), that combines rituximab targeting with non-major histocompatibility complex (non-MHC)-restricted cytotoxicity mediated by activated T cells (ATC). MATERIALS AND METHODS: Activated T cells were obtained from anti-CD3 activated peripheral blood mononuclear cells (PBMC) of normal donors or the leukapheresis products of patients by culturing in the presence of interleukin-2 for 6-14 days. After ATC expansion, the cells were armed with CD20Bi. Killing activity was evaluated by 51Cr-release assay. RESULTS: Arming ATC with as little as 5 ng CD20Bi/10(6) cells significantly increased cytotoxicity above unarmed ATC. CD20Bi-armed ATC (50 ng/10(6) cells) efficiently lysed CD20+ cell lines at E:T of 6.25-50, but not the nonhematologic, CD20- SK-BR-3 cell line. High levels of cytotoxicity mediated by CD20Bi-armed ATC (p < 0.05) could not be blocked by an 8000-fold excess of soluble rituximab. CD20Bi-armed ATC in the presence of complement killed ARH-77 cells, a rituximab-complement pathway-resistant multiple myeloma, significantly (p < 0.05) better than rituximab or unarmed ATC, suggesting that CD20Bi-armed ATC may be clinically effective for treatment of rituximab-resistant CD20+ hematologic malignancies. CONCLUSIONS: Our findings demonstrate that CD20Bi-armed ATC enhance cytotoxicity against CD20+ B-cell lines and circumvent complement-mediated rituximab resistance, providing a strong rationale for this immune-based strategy for the treatment of rituximab-refractory CD20+ B-cell malignancies.

Asunto(s)

Anticuerpos Biespecíficos/farmacología , Antígenos CD20/inmunología , Linfocitos B/patología , Complejo CD3/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales , Anticuerpos Monoclonales de Origen Murino , Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Transformada , Línea Celular Tumoral , Proteínas del Sistema Complemento , Citocinas/metabolismo , Pruebas Inmunológicas de Citotoxicidad , Resistencia a Antineoplásicos , Femenino , Humanos , Técnicas In Vitro , Rituximab , Linfocitos T/metabolismo