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OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.
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In cancer, elevated activin levels promote cachectic wasting of muscle, irrespective of tumor progression. In excess, activins A and B use the myostatin signaling pathway in muscle, triggering a decrease in protein synthesis and an increase in protein degradation, which ultimately leads to atrophy. Recently, we demonstrated that local delivery of engineered activin and myostatin propeptides (natural inhibitors of these growth factors) could induce profound muscle hypertrophy in healthy mice. Additionally, the expression of these propeptides effectively attenuated localized muscle wasting in models of dystrophy and cancer cachexia. In this study, we examined whether a systemically administered recombinant propeptide could reverse activin A-induced cachectic wasting in mice. Chinese hamster ovary cells stably expressing activin A were transplanted into the quadriceps of nude mice and caused an 85-fold increase in circulating activin A levels within 12 days. Elevated activin A induced a rapid reduction in body mass (-16%) and lean mass (-10%). In agreement with previous findings, we demonstrated that adeno-associated virus-mediated delivery of activin propeptide to the tibialis anterior muscle blocked activin-induced wasting. In addition, despite massively elevated levels of activin A in this model, systemic delivery of the propeptide significantly reduced activin-induced changes in lean and body mass. Specifically, recombinant propeptide reversed activin-induced wasting of skeletal muscle, heart, liver, and kidneys. This is the first study to demonstrate that systemic administration of recombinant propeptide therapy effectively attenuates tumor-derived activin A insult in multiple tissues.
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Activinas/toxicidad , Caquexia/inducido químicamente , Péptidos/farmacología , Animales , Células CHO , Caquexia/prevención & control , Cricetinae , Cricetulus , Riñón/efectos de los fármacos , Riñón/patología , Hígado/patología , Masculino , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Miocardio , Tamaño de los Órganos/efectos de los fármacos , Péptidos/químicaRESUMEN
Systemic endothelial dysfunction is a key characteristic of preeclampsia (PE), which is a serious disorder of human pregnancy. We have previously reported that high-temperature requirement factor (Htr)A4 is a placenta-specific protease that is secreted into the maternal circulation and significantly up-regulated in PE, especially early-onset PE. We have also demonstrated that high levels of HtrA4 detected in the early onset PE circulation induce endothelial dysfunction in HUVECs. In the current study, we investigated whether HtrA4 could cleave the main receptor of VEGFA, the kinase domain receptor (KDR), thereby inhibiting VEGFA signaling. We first demonstrated that HtrA4 cleaved recombinant KDR in vitro. We then confirmed that HtrA4 reduced the level of KDR in HUVECs and inhibited the VEGFA-induced phosphorylation of Akt kinase, which is essential for downstream signaling. Further functional studies demonstrated that HtrA4 prevented the VEGFA-induced tube formation in HUVECs and dose-dependently inhibited the VEGFA-induced angiogenesis in explants of mouse aortic rings. These data strongly suggest that high levels of HtrA4 in the maternal circulation could cleave the main receptor of VEGFA in endothelial cells to induce a wide-spread impairment of angiogenesis. Our studies therefore suggest that HtrA4 is a potential causal factor of early onset PE.-Wang, Y., La, M., Pham, T., Lovrecz, G. O., Nie, G. High levels of HtrA4 detected in preeclamptic circulation may disrupt endothelial cell function by cleaving the main VEGFA receptor KDR.
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Células Endoteliales de la Vena Umbilical Humana/metabolismo , Preeclampsia/metabolismo , Serina Proteasas/metabolismo , Animales , Western Blotting , Femenino , Células HEK293 , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Fosforilación , Embarazo , Serina Proteasas/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Factor A de Crecimiento Endotelial Vascular , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Herein we describe production of purified equine IgG obtained from horses immunized with plasmid DNA followed by boosting with Kunjin replicon virus-like particles both encoding a modified Ebola glycoprotein. Administration of the equine IgG over 5 days to cynomolgus macaques infected 24 hours previously with a lethal dose of Ebola virus suppressed viral loads by more than 5 logs and protected animals from mortality. Animals generated their own Ebola glycoprotein-specific IgG responses 9-15 days after infection, with circulating virus undetectable by day 15-17. Such equine IgG may find utility as a post-exposure prophylactic for Ebola infection and provides a low cost, scalable alternative to monoclonal antibodies, with extensive human safety data and WHO-standardized international manufacturing capability available in both high and low income countries.
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Anticuerpos Antivirales/administración & dosificación , Antígenos Virales/inmunología , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Inmunoglobulina G/administración & dosificación , Profilaxis Posexposición , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Especificidad de Anticuerpos/inmunología , Glicoproteínas/inmunología , Caballos , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Macaca fascicularisRESUMEN
UNLABELLED: The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine and are platforms for the delivery of foreign antigenic sequences. To investigate structure-immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified virus-like particles (VLPs) exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wild-type (WT) HBsAgS N146 glycosylation site was converted to N146Q; for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced T116N and G130N sites were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs, with minor variations regarding the degree of fucosylation, bisecting N-acetylglucosamines, and sialylation. Antigenic fingerprints for the WT and hypo- and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their ability to bind to the different VLP glyco-analogues, suggesting that the additional N-glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N-glycan abundance and antibody titers. The T116N VLPs induced earlier and longer-lasting antibody responses than did the hypoglycosylated and WT VLPs. The ability of nonnative VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with cells of the innate immune system illustrates pathways for the design of immunogens for superior preventive applications. IMPORTANCE: The use of biochemically modified, nonnative immunogens represents an attractive strategy for the generation of modulated or enhanced immune responses possibly due to differences in their interaction with immune cells. We have generated virus-like particles (VLPs) composed of hepatitis B virus envelope proteins (HBsAgS) with additional N-glycosylation sites. Hyperglycosylated VLPs were synthesized and characterized, and the results demonstrated that they carry the same types of glycans as wild-type VLPs. Comparative immunization studies demonstrated that the VLPs with the highest N-glycan density induce earlier and longer-lasting antibody immune responses than do wild-type or hypoglycosylated VLPs, possibly allowing reduced numbers of vaccine injections. The ability to modulate the immunogenicity of an immunogen will provide opportunities to develop optimized vaccines and VLP delivery platforms for foreign antigenic sequences, possibly in synergy with the use of suitable adjuvanting compounds.
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Antígenos de Superficie de la Hepatitis B/inmunología , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Polisacáridos/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Asparagina/química , Línea Celular , Femenino , Glicosilación , Células HEK293 , Hepatitis B/inmunología , Hepatitis B/prevención & control , Humanos , Ratones , Ratones Endogámicos BALB C , Vacunas Sintéticas/inmunologíaRESUMEN
Soluble activin type II receptors (ActRIIA/ActRIIB), via binding to diverse TGF-ß proteins, can increase muscle and bone mass, correct anemia or protect against diet-induced obesity. While exciting, these multiple actions of soluble ActRIIA/IIB limit their therapeutic potential and highlight the need for new reagents that target specific ActRIIA/IIB ligands. Here, we modified the activin A and activin B prodomains, regions required for mature growth factor synthesis, to generate specific activin antagonists. Initially, the prodomains were fused to the Fc region of mouse IgG2A antibody and, subsequently, "fastener" residues (Lys(45), Tyr(96), His(97), and Ala(98); activin A numbering) that confer latency to other TGF-ß proteins were incorporated. For the activin A prodomain, these modifications generated a reagent that potently (IC(50) 5 nmol/l) and specifically inhibited activin A signaling in vitro, and activin A-induced muscle wasting in vivo. Interestingly, the modified activin B prodomain inhibited both activin A and B signaling in vitro (IC(50) ~2 nmol/l) and in vivo, suggesting it could serve as a general activin antagonist. Importantly, unlike soluble ActRIIA/IIB, the modified prodomains did not inhibit myostatin or GDF-11 activity. To underscore the therapeutic utility of specifically antagonising activin signaling, we demonstrate that the modified activin prodomains promote significant increases in muscle mass.
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Activinas/metabolismo , Ingeniería Genética/métodos , Músculo Esquelético/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Activinas/antagonistas & inhibidores , Activinas/genética , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Dependovirus/genética , Regulación de la Expresión Génica , Vectores Genéticos/genética , Factores de Diferenciación de Crecimiento/genética , Factores de Diferenciación de Crecimiento/metabolismo , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/patología , Miostatina/genética , Miostatina/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Transfección , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Cardiovascular diseases are the leading cause of death and disability worldwide, yet we do not fully understand their underlying causes to reliably identify and treat, let alone prevent, these diseases. The majority of therapeutic approaches are symptom orientated, and current practice often follows a 'one-fits-all' approach. New strategies are needed, which harness the potential of individualized medicine with its three major pillars: in vitro diagnostics for early identification of individuals at risk and monitoring of drug efficacy; molecular imaging for disease localization and monitoring; and innovative, mechanism-based drugs. One so far untargeted mechanism of cardiovascular disease is oxidative stress, that is, the increased occurrence of reactive oxygen species in the vascular wall that leads to endothelial dysfunction. We outline why previous antioxidant supplements do not work and suggest an alternative approach targeting the enzymatic sources of oxidative stress and using emerging biomarkers of oxidative stress. These and similar approaches may be applied to fewer patients but in a much more individualized, effective and cost-saving manner.
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Cardiovascular diseases remain the leading cause of death in industrialised nations. Since the pathomechanisms of most cardiovascular diseases are not understood, the majority of therapeutic approaches are symptom-orientated. Knowing the molecular mechanism of disease would enable more targeted therapies. One postulated underlying mechanism of cardiovascular diseases is oxidative stress, i.e. the increased occurrence of reactive oxygen species such as superoxide. Oxidative stress leads to a dysfunction of vascular endothelium-dependent protective mechanisms. There is growing evidence that this scenario also involves impaired nitric oxide (NO)-cyclic GMP signalling. Out of a number of enzyme families that can produce reactive oxygen species, NADPH oxidases stand out, as they are the only enzymes whose sole purpose is to produce reactive oxygen species. This review focuses on the clinically validated targets of oxidative stress, NO synthase (NOS) and the NO receptor, soluble guanylate cyclase as well as the source of ROS, e.g. NADPH oxidases. We place recent knowledge in the function and regulation of these enzyme families into clinical perspective. For a comprehensive overview of the biology and pharmacology of oxidative stress and possible other sources and targets, we refer to other literature overviews.
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NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Estrés Oxidativo/efectos de los fármacos , Animales , Guanilato Ciclasa/metabolismo , Humanos , Especies Reactivas de Oxígeno/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Guanilil Ciclasa SolubleRESUMEN
Annexin 1 (ANXA1), galectin-1 (Gal-1) and galectin-3 (Gal-3) proteins have been identified as important mediators that promote or inhibit leukocyte migration. The expression of these proteins was studied in human neutrophils and endothelial cells (ECs) during a transmigration process induced by IL-8. Upon neutrophil adhesion to EC, a significant increase in the cleaved ANXA1 (LCS3, raised against all ANXA1 isoforms) expression was detected in the plasma membrane of adhered neutrophils and ECs compared to intact ANXA1 isoform (LCPS1, against N-terminus of protein). Adherent neutrophils had elevated Gal-3 levels in the nucleus and cytoplasm, and ECs in their plasma membranes. In contrast, a decrease in the total amounts of Gal-1 was detected in migrated compared to non-migrated neutrophils. Therefore, ANXA1 and Gal-3 changed in their content and localization when neutrophils adhere to endothelia, suggesting a process of sensitive-balance between two endogenous anti- and pro-inflammatory mediators.
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Anexina A1/metabolismo , Comunicación Celular , Células Endoteliales/citología , Galectina 1/metabolismo , Galectina 3/metabolismo , Regulación de la Expresión Génica , Neutrófilos/citología , Adhesión Celular , Movimiento Celular , Células Cultivadas , Células Endoteliales/ultraestructura , Humanos , Inmunohistoquímica , Neutrófilos/ultraestructuraRESUMEN
OBJECTIVE: The authors investigated whether the anti-inflammatory protein tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6) and its Link module (Link_TSG6) could affect the complex multistep process of leukocyte/endothelial cell (EC) interaction. METHODS: Mouse mesenteries were inflamed with interleukin (IL)-1beta and the extent of leukocyte rolling, adhesion, and emigration was determined after 2 h. Link_TSG6 and a single-point mutant (termed K13E) were given intraperitoneally together with the cytokine. Human neutrophil chemotaxis and transmigration were determined in vitro in response to IL-8 and/or TNF-alpha. TSG-6, Link_TSG6, and K13E were added to the leukocytes or the EC monolayers. RESULTS: Co-injection of Link_TSG6 with IL-1beta selectively inhibited cell flux, adhesion, and emigration as analyzed in mesenteric postcapillary venules. The fewer cells that rolled in the animals treated with Link_TSG6 displayed a velocity similar to that measured in vehicle-treated mice. In vitro, Link_TSG6 did not affect neutrophil chemotaxis or EC activation but did inhibit neutrophil transmigration across EC monolayers. The latter effect was shared by full-length TSG-6 and observed equally in response to IL-8 or TNF-alpha. CONCLUSIONS: These data restrict the site of action for at least some of the anti-inflammatory effects ascribed to TSG-6/Link_TSG6 to the microenvironment of the extravasating leukocyte.
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Moléculas de Adhesión Celular/farmacología , Endotelio Vascular/efectos de los fármacos , Leucocitos/efectos de los fármacos , Animales , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/fisiología , Movimiento Celular/efectos de los fármacos , Endotelio Vascular/patología , Endotelio Vascular/fisiología , Humanos , Técnicas In Vitro , Inflamación/patología , Inflamación/prevención & control , Interleucina-1/administración & dosificación , Leucocitos/patología , Leucocitos/fisiología , Masculino , Ratones , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Neutrófilos/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Peritonitis/etiología , Peritonitis/patología , Peritonitis/prevención & control , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Neutrophil elastase (NE) remains a controversial player in the process of leukocyte transmigration and much of this controversy stems from conflicting reports on the effects of NE inhibitors. The availability of NE-deficient mice (NE(-/-)) provides a clean and elegant tool for the study of leukocyte migration in vivo. In this study, NE(-/-) mice were used to investigate the role of NE in leukocyte migration through cremasteric venules, as observed by intravital microscopy, induced by locally administered cytokines IL-1beta and TNF-alpha and the particulate stimulus, zymosan. Although no defects in leukocyte responses induced by the cytokines were observed, zymosan-induced leukocyte firm adhesion and transmigration was suppressed in NE(-/-) mice. These responses were also inhibited in wild-type mice when zymosan was coinjected with a specific NE inhibitor. Quantification of inflammatory mediator levels in homogenates of zymosan-stimulated tissues indicated reductions in levels of IL-1beta, KC, and macrophage inflammatory protein-1alpha in NE(-/-) mice. Furthermore, phagocytosis of fluorescent zymosan particles, as observed by intravital microscopy, was diminished in NE-deficient animals. Collectively, the findings of this study indicate a nonredundant role for NE in zymosan-induced leukocyte firm adhesion and transmigration, and that this defect is associated with impaired generation of proinflammatory mediators as well as phagocytosis of zymosan particles in vivo.
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Mediadores de Inflamación/metabolismo , Elastasa de Leucocito/deficiencia , Elastasa de Leucocito/fisiología , Infiltración Neutrófila/inmunología , Neutrófilos/enzimología , Neutrófilos/inmunología , Fagocitosis/inmunología , Zimosan/administración & dosificación , Animales , Moléculas de Adhesión Celular/biosíntesis , Mediadores de Inflamación/antagonistas & inhibidores , Interleucina-1/farmacología , Elastasa de Leucocito/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía por Video , Músculo Esquelético/irrigación sanguínea , Infiltración Neutrófila/genética , Cavidad Peritoneal/citología , Fagocitosis/genética , Escroto , Factor de Necrosis Tumoral alfa/farmacología , Vénulas/citología , Vénulas/inmunologíaRESUMEN
Galectin-1 (Gal-1), the prototype of a family of beta-galactoside-binding proteins, has been shown to attenuate experimental acute and chronic inflammation. In view of the fact that endothelial cells (ECs), but not human polymorphonuclear leukocytes (PMNs), expressed Gal-1 we tested here the hypothesis that the protein could modulate leukocyte-EC interaction in inflammatory settings. In vitro, human recombinant (hr) Gal-1 inhibited PMN chemotaxis and trans-endothelial migration. These actions were specific as they were absent if Gal-1 was boiled or blocked by neutralizing antiserum. In vivo, hrGal-1 (optimum effect at 0.3 micro g equivalent to 20 pmol) inhibited interleukin-1beta-induced PMN recruitment into the mouse peritoneal cavity. Intravital microscopy analysis showed that leukocyte flux, but not their rolling velocity, was decreased by an anti-inflammatory dose of hrGal-1. Binding of biotinylated Gal-1 to resting and postadherent human PMNs occurred at concentrations inhibitory in the chemotaxis and transmigration assays. In addition, the pattern of Gal-1 binding was differentially modulated by PMN or EC activation. In conclusion, these data suggest the existence of a previously unrecognized function of Gal-1, that is inhibition of leukocyte rolling and extravasation in experimental inflammation. It is possible that endogenous Gal-1 may be part of a novel anti-inflammatory loop in which the endothelium is the source of the protein and the migrating PMNs the target for its anti-inflammatory action.
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Comunicación Celular , Endotelio Vascular/fisiopatología , Galectina 1/metabolismo , Neutrófilos , Peritonitis/fisiopatología , Animales , Sitios de Unión , Comunicación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Endotelio Vascular/patología , Citometría de Flujo , Galectina 1/administración & dosificación , Galectina 1/farmacología , Humanos , Técnicas In Vitro , Inyecciones , Interleucina-1/administración & dosificación , Interleucina-8/farmacología , Rodamiento de Leucocito/efectos de los fármacos , Masculino , Ratones , Neutrófilos/patología , Peritonitis/inducido químicamente , Peritonitis/patología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Aspirin (ASA) and dexamethasone (DEX) are widely used anti-inflammatory agents yet their mechanism(s) for blocking polymorphonuclear neutrophil (PMN) accumulation at sites of inflammation remains unclear. Here, we report that inhibition of PMN infiltration by ASA and DEX is a property shared by aspirin-triggered lipoxins (ATL) and the glucocorticoid-induced annexin 1 (ANXA1)-derived peptides that are both generated in vivo and act at the lipoxin A(4) receptor (ALXR/FPRL1) to halt PMN diapedesis. These structurally diverse ligands specifically interact directly with recombinant human ALXR demonstrated by specific radioligand binding and function as well as immunoprecipitation of PMN receptors. In addition, the combination of both ATL and ANXA1-derived peptides limited PMN infiltration and reduced production of inflammatory mediators (that is, prostaglandins and chemokines) in vivo. Together, these results indicate functional redundancies in endogenous lipid and peptide anti-inflammatory circuits that are spatially and temporally separate, where both ATL and specific ANXA1-derived peptides act in concert at ALXR to downregulate PMN recruitment to inflammatory loci.
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Antiinflamatorios/farmacología , Aspirina/farmacología , Dexametasona/farmacología , Metabolismo de los Lípidos , Péptidos/metabolismo , Receptores de Superficie Celular/agonistas , Receptores de Formil Péptido , Receptores de Lipoxina , Animales , Línea Celular , Células Cultivadas , Humanos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Activación NeutrófilaRESUMEN
BACKGROUND: The "L-arginine paradox" refers to situations where L-arginine (L-Arg) supplementation stimulates nitric oxide (NO) synthesis, despite saturating intracellular concentrations. This paradox is frequently observed in acute renal failure (ARF). First, the effects of L-Arg on renal function of rats with ARF were studied. Based on the promising results from these initial studies, the second part of our study searched for a form of ARF in humans that could be studied easily under conditions with little variance and yet was linked with endothelial dysfunction. Thus, we investigated the effects of L-Arg supplementation immediately after kidney transplantation in 54 patients. METHODS: In uranyl nitrate-induced ARF in rats the effects of L-Arg and L-NNA (inhibitor of nitric oxide synthase; NOS) on glomerular filtration rate (GFR), renal plasma flow (RPF), blood pressure (BP) and NOx (NO2- +NO3-) excretion were examined. Tissue L-Arg levels, NOS activities, immunodetection of NOS and superoxide dismutase (SOD), activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase, and nitrotyrosine immunoreactive protein (NT-IR) were determined and compared to sham operated animals. Secondly, in a randomized, double-blind study, the effects of L-Arg on GFR and RPF were investigated in 54 kidney transplant recipients, receiving IV L-Arg for three days. GFR and RPF were measured on days 1, 3, 5 and 10 by scintigraphy. RESULTS: In experimental ARF, decreased RPF and GFR were associated with reduced tissue L-Arg levels, endothelial NOS-III expression, NO formation and NOx excretion. Reduction in GFR, RPF and NOx excretion were reversed upon administration of exogenous L-Arg. There also was a loss of Cu,Zn-SOD, a key enzyme against oxidative stress, and an elevation of NT-IR, an indicator of nitrosative stress and suggested marker for pathological actions of NO. However, NT-IR was not dependent on de novo NO synthesis and not related to the functional effects of l-Arg administration. In kidney transplant recipients receiving organs with a short cold ischemia time (CIT) and from young donors, that is, those with a higher likelihood of a functional endothelium, early administration of L-Arg improved renal function. CONCLUSION: Both experimental and clinical data show that \L-Arg deficiency and endothelial dysfunction are pathomechanistically relevant in ARF. The data suggest a therapeutic potential for the administration of L-Arg in ARF and kidney transplantation, at least in patients receiving kidneys with shorter CIT and from younger donors.