RESUMEN

DDT, a persistent organic pollutant, remains affecting human health worldwide. DDT and its most persistent metabolite (p,p'-DDE) negatively affect the immune response regulation and mechanisms involved in protecting against pathogens Such metabolite decreases the capability to limit intracellular growth of Mycobacterium microti and yeast. However, the effect on unstimulated (M0) and anti-inflammatory macrophages (M2) has been evaluated scanty. Herein, we evaluated the impact of p,p'-DDE at environmentally relevant concentrations (0.125, 1.25, 2.5, and 5 µg/mL) on bone marrow-derived macrophages stimulated with IFNγ+LPS to M1 or with IL-4 +IL-13 to M2. Thus we study whether the p,p'-DDE induces M0 to a specific phenotype or modulates activation of the macrophage phenotypes and explains, at least partly, the reported effects of p,p'-DDE on the M1 function. The p,p'-DDE did not affect the cell viability of M0 or the macrophage phenotypes. In M1, the p,p'-DDE decreased NO•- production and IL-1ß secretion, but increasing cellular ROS and mitochondrial O2•-, but did not alter iNOS, TNF-α, MHCII, and CD86 protein expression nor affect M2 markers arginase activity, TGF-ß1, and CD206; p,p'-DDE, did not affect marker expression in M0 or M2, supporting that its effects on M1 parameters are not dependent on M0 nor M2 modulation. The decreasing of NO•- production by the p,p'-DDE without altering iNOS levels, Arginase activity, or TNF-α, but increasing cellular ROS and mitochondrial O2 suggests that p,p'-DDE interferes with the iNOS function but not with its transcription. The p,p'-DDE decreasing of IL-1ß secretion, without any effect on TNF-α, suggest that an alteration of specific targets involved in IL-1ß secretion may be affected and related to ROS induction. The p,p'-DDE effect on iNOS function and the IL-1ß secretion process, as the NLRP3 activation, deserves further study.

Asunto(s)

Diclorodifenil Dicloroetileno , Macrófagos , Animales , Humanos , Ratones , Arginasa/genética , Arginasa/metabolismo , Arginasa/farmacología , DDT/metabolismo , DDT/farmacología , Diclorodifenil Dicloroetileno/toxicidad , Diclorodifenil Dicloroetileno/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones Endogámicos BALB C , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/genética

RESUMEN

Leishmania (Viannia) braziliensis is the main species responsible for American tegumentary leishmaniasis in Brazil. Nevertheless, the use of this parasite species to study Leishmania infection in the murine model has been less conducted when compared with other Leishmania species. The control of murine infection with Leishmania has been associated with nitric oxide (NO) produced by inducible NO synthase (iNOS) from M1 macrophages, while arginase expressed by M2 macrophages is related to Leishmania proliferation. Here we use three different strains of L. (V.) braziliensis and one strain of L. (L.) major to study a 9-day infection of macrophages in vitro. Wild-type bone marrow-derived macrophages (BMDM) supported the proliferation of L. (L) major amastigotes from the 3rd day after infection, while all strains of L. (V.) braziliensis did not proliferate even inside IL-4-treated or iNOS knockout (KO) macrophages. The arginase activity was higher in iNOS KO than IL-4-treated macrophage showing that the absence of proliferation is independent of arginase. Importantly, L. (V.) braziliensis was able to cause uncontrolled disease in iNOS KO mice in vivo demonstrating that murine macrophages present at the site of infection have additional changes beyond inhibition of NO production or stimulation of arginase activity to support parasite proliferation.

Asunto(s)

Leishmania braziliensis , Leishmania , Leishmaniasis Cutánea , Animales , Arginasa/genética , Proliferación Celular , Interleucina-4 , Leishmaniasis Cutánea/parasitología , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Óxido Nítrico

RESUMEN

Aim: This work examined whether ARG1 (rs2781659, rs2781667, rs2246012 and rs17599586) and ARG2 (rs3742879 and rs10483801) single-nucleotide polymorphisms (SNPs) are associated with antihypertensive therapy responsiveness in preeclampsia (PE) and their effects on arginase isoforms and nitrite concentrations in responsive and nonresponsive patients. Methods: SNP genotypes were determined by TaqMan assays. Plasma arginase levels were measured by ELISA and nitrite concentrations were measured using an ozone-based chemiluminescence assay. Results: The G allele for ARG2 rs3742879 (A>G) was less frequent in nonresponsive compared with responsive patients (15.5% vs 24.7%, respectively) and the G carriers of the nonresponsive subgroup had lower arginase 2 (9.2 ± 7.5 ng/ml vs 19.1 ± 17.3 ng/ml) and higher nitrite concentrations (110.2 ± 52.8 nM vs 78.5 ± 37.9 nM) than carriers of the AA genotype (all p < 0.05). Conclusion: ARG2 SNP rs3742879 is associated with diminished arginase 2 levels and increased nitric oxide formation in nonresponsive PE patients.

Asunto(s)

Antihipertensivos , Arginasa , Preeclampsia , Femenino , Humanos , Embarazo , Antihipertensivos/uso terapéutico , Arginasa/sangre , Arginasa/genética , Óxido Nítrico/metabolismo , Nitritos , Polimorfismo de Nucleótido Simple , Preeclampsia/tratamiento farmacológico , Preeclampsia/genética

RESUMEN

Leprosy household contacts are generally more prone to develop the disease compared to the general population. Previous studies have demonstrated that genes related to the alternative activation (M2) profile in macrophages are associated with the increased bacillary load in multibacillary leprosy patients (MB), and that contacts of MB patients have a higher risk of contracting the disease. In addition, positive serological responses to PGL-1 or LID-1 are associated with a higher risk of disease. We performed a 5-year follow-up of contacts of leprosy patients and evaluated the pattern of gene and protein expression in cells from contacts that developed leprosy during this period. Leprosy household contacts had decreased soluble CD163 and heme oxygenase 1 (HO-1) serum levels when compared with healthy donors and leprosy patients. In contrast, arginase 1 activities were higher in contacts when compared with both healthy donors and leprosy patients. Of the contacts, 33 developed leprosy during the follow-up. Gene expression analysis revealed reduced ARG1 expression in these contacts when compared with contacts that did not develop disease. Arginase activity was a good predictive marker of protection in contacts (sensitivity: 90.0%, specificity: 96.77%) and the association with serology for anti-PGL-1 and anti-LID-1 increased the sensitivity to 100%. Altogether, the data presented here demonstrate a positive role of arginase against leprosy and suggest that the evaluation of arginase activity should be incorporated into leprosy control programs in order to aid in the decision of which contacts should receive chemoprophylaxis.

Asunto(s)

Lepra , Mycobacterium leprae , Anticuerpos Antibacterianos , Antígenos Bacterianos , Arginasa/genética , Biomarcadores , Ensayo de Inmunoadsorción Enzimática , Glucolípidos , Humanos

RESUMEN

The metalloenzyme arginase hydrolyzes l-arginine to produce l-ornithine and urea. In bacteria, arginase has important functions in basic nitrogen metabolism and redistribution, production of the key metabolic precursor l-ornithine, stress resistance and pathogenesis. We describe the regulation and specific functions of the arginase pathway as well as summarize key characteristics of related arginine catabolic pathways. The use of arginase-derived ornithine as a precursor molecule is reviewed. We discuss the biochemical and transcriptional regulation of arginine metabolism, including arginase, with the latter topic focusing on the RocR and AhrC transcriptional regulators in the model organism Bacillus subtilis. Finally, we consider similarities and contrasts in the structure and catalytic mechanism of the arginases from Bacillus caldovelox and Helicobacter pylori. The overall aim of this review is to provide a panorama of the diversity of physiological functions, regulation and biochemical features of arginases in a variety of bacterial species.

Asunto(s)

Arginasa , Helicobacter pylori , Arginasa/genética , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Helicobacter pylori/genética , Ornitina

RESUMEN

Previously, we reported that the presence of multiple day 7 (D7) bovine embryos in the uterus induces systemic immune responses in circulating polymorphonuclear neutrophils (PMNs), but with unknown mechanism. Thus, this study aimed to investigate the direct impact of D7 bovine embryo on PMNs' immune responses in vitro and whether these PMNs can amplify and transfer embryo signals further to another PMN population. PMNs were directly stimulated by embryo culture media (ECM) or interferon tau (IFNT) (10 ng/ml) followed by evaluating mRNA expression by real-time PCR and phenotypic analysis by flow cytometry. To test whether PMNs can transfer embryo signals to a new PMN population, PMNs triggered by ECM or IFNT, were thoroughly washed and diluted to remove any media components, and again were incubated in fresh culture media for 3 h, from which culture supernatants were collected and used as PMN conditioned media (CM) to stimulate a new PMN population. Similar to ECM, IFNT directly stimulated expressions of IFNs (IFNA, IFNG), interferon-stimulated genes (ISGs; OAS1, ISG15, MX1), STAT1, TGFB and IL8, and downregulated TNFA in PMNs. Flow cytometrical analyses demonstrated that IFNT stimulated expressions of pregnancy-related phenotypic markers, CD16 and arginase-1 (ARG1), in PMNs. Most importantly, PMN CM induced ISGs and STAT1 mRNA in fresh PMNs. Since IFNT directly upregulated IFNA expression in PMNs, the impact of IFNA on PMNs' immune responses was further tested. Stimulation of PMNs with IFNA, especially at a low level (1 pg/ml), induced IFNT-like immune responses comparable to those induced by PMN CM. Together, these findings indicated that D7 bovine embryos induce direct anti-inflammatory responses with upregulation of ISGs expressions in PMNs mainly via IFNT. Additionally, PMNs can amplify and transfer embryo signals to a new PMN population in a cell-to-cell communication mechanism possibly mediated in part by IFNA. Such a novel immunological crosstalk might contribute to embryo tolerance and pregnancy establishment in cattle.

Asunto(s)

Embrión de Mamíferos/inmunología , Embrión de Mamíferos/metabolismo , Regulación de la Expresión Génica , Interferón Tipo I/inmunología , Neutrófilos/inmunología , Proteínas Gestacionales/inmunología , Embarazo/genética , Embarazo/inmunología , Animales , Arginasa/genética , Bovinos , Medios de Cultivo Condicionados/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Inmunidad Innata , Técnicas In Vitro , Interferón Tipo I/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fenotipo , Proteínas Gestacionales/farmacología , Receptores de IgG/genética

RESUMEN

BACKGROUND AND AIMS: Preeclampsia is associated with reduced nitric oxide (NO) bioavailability. Arginase is related to NO synthesis, but relatively unexplored in preeclampsia. However, no previous study has examined whether variations in ARG1 and ARG2 genes affect NO bioavailability and the risk of preeclampsia. Here, we compared the alleles and genotypes of single nucleotide polymorphisms (SNPs) in ARG1 (rs2781659; rs2781667; rs2246012; rs17599586) and ARG2 (rs3742879; rs10483801) in healthy pregnant women and preeclampsia, and examined whether these SNPs affect plasma nitrite concentrations (a marker of NO formation) in these groups. METHODS: Genotypes for the ARG1 and ARG2 SNPs were determined by Taqman probe and plasma nitrite by an ozone-based chemiluminescence assay. RESULTS: Regarding ARG1 SNPs, the GG genotype and G allele frequencies for rs2781659, and the C allele frequencies for rs2246012 were higher in preeclampsia compared to healthy pregnant women. Moreover, the GG genotype for rs2781659 and the TT genotype for rs2781667 were associated with higher plasma nitrite in healthy pregnant. We found no association of ARG2 polymorphisms with preeclampsia or nitrite levels in the study groups. CONCLUSIONS: Our results suggest that SNPs of ARG1 increase the risk of preeclampsia and modulate plasma nitrite levels in healthy pregnant women.

Asunto(s)

Arginasa/genética , Óxido Nítrico/metabolismo , Preeclampsia/genética , Embarazo/genética , Adulto , Femenino , Frecuencia de los Genes , Humanos , Óxido Nítrico/sangre , Nitritos/sangre , Nitritos/metabolismo , Polimorfismo de Nucleótido Simple , Preeclampsia/sangre , Preeclampsia/metabolismo , Adulto Joven

RESUMEN

PURPOSE: Propofol anesthesia is usually accompanied by hypotensive responses, which are at least in part mediated by nitric oxide (NO). Arginase I (ARG1) and arginase II (ARG2) compete with NO synthases for their common substrate L-arginine, therefore influencing the NO formation. We examined here whether ARG1 and ARG2 genotypes and haplotypes affect the changes in blood pressure and NO bioavailability in response to propofol. METHODS: Venous blood samples were collected from 167 patients at baseline and after 10 min of anesthesia with propofol. Genotypes were determined by polymerase chain reaction. Nitrite concentrations were measured by using an ozone-based chemiluminescence assay, while NOx (nitrites + nitrates) levels were determined by using the Griess reaction. RESULTS: We found that patients carrying the AG + GG genotypes for the rs3742879 polymorphism in ARG2 gene and the ARG2 GC haplotype show lower increases in nitrite levels and lower decreases in blood pressure after propofol anesthesia. On the other hand, subjects carrying the variant genotypes for the rs10483801 polymorphism in ARG2 gene show more intense decreases in blood pressure (CA genotype) and/or higher increases in nitrite levels (CA and AA genotypes) in response to propofol. CONCLUSION: Our results suggest that ARG2 variants affect the hypotensive responses to propofol, possibly by modifying NO bioavailability. TRIAL REGISTRATION: NCT02442232.

Asunto(s)

Anestésicos Intravenosos/efectos adversos , Arginasa/genética , Hipotensión/inducido químicamente , Óxido Nítrico/metabolismo , Propofol/efectos adversos , Adulto , Anciano , Anestésicos Intravenosos/farmacocinética , Femenino , Genotipo , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Nitratos/sangre , Nitritos/sangre , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Propofol/farmacocinética

RESUMEN

The outcome of Leishmania infection is strongly influenced by the host's genetic background. BALB/c mice are susceptible to Leishmania infection, while C57BL/6 mice show discrete resistance. Central to the fate of the infection is the availability of l-arginine and the related metabolic processes in the host and parasite. Depending on l-arginine availability, nitric oxide synthase 2 (NOS2) of the host cell produces nitric oxide (NO) controlling the parasite growth. On the other hand, Leishmania can also use host l-arginine for the production of polyamines through its own arginase activity, thus favouring parasite replication. Considering RNA-seq data, we analysed the dual modulation of host and parasite gene expression of BALB/c or C57BL/6 mouse bone marrow-derived macrophages (BMDMs) after 4 h of infection with Leishmania amazonensis wild-type (La-WT) or L. amazonensis arginase knockout (La-arg-). We identified 12 641 host transcripts and 8282 parasite transcripts by alignment analysis with the respective Mus musculus and L. mexicana genomes. The comparison of BALB/c_La-arg-versus BALB/c_La-WT revealed 233 modulated transcripts, with most related to the immune response and some related to the amino acid transporters and l-arginine metabolism. In contrast, the comparison of C57BL/6_La-arg-vs. C57BL/6_La-WT revealed only 30 modulated transcripts, including some related to the immune response but none related to amino acid transport or l-arginine metabolism. The transcriptome profiles of the intracellular amastigote revealed 94 modulated transcripts in the comparison of La-arg-_BALB/c vs. La-WT_BALB/c and 45 modulated transcripts in the comparison of La-arg-_C57BL/6 vs. La-WT_C57BL/6. Taken together, our data present new insights into the impact of parasite arginase activity on the orchestration of the host gene expression modulation, including in the immune response and amino acid transport and metabolism, mainly in susceptible BALB/c-infected macrophages. Moreover, we show how parasite arginase activity affects parasite gene expression modulation, including amino acid uptake and amastin expression.

Asunto(s)

Arginasa/genética , Perfilación de la Expresión Génica/métodos , Leishmania/genética , Óxido Nítrico Sintasa de Tipo II/genética , Animales , Femenino , Regulación de la Expresión Génica , Antecedentes Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Protozoarias/genética , Análisis de Secuencia de ARN

RESUMEN

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and requires a bivalent cation, especially Mn2+ for its catalytic activity. It is a component of the urea cycle and regulates the intracellular levels of l-arginine, which makes the arginase a target for treatment of vascular diseases and asthma. Mammalian arginases contain an unusual S-shaped motif located at the intermonomeric interface. Until now, the studies were limited to structural role of the motif. Then, our interest was focused on functional aspects and our hypothesis has been that the motif is essential for maintain the oligomeric state, having Arg308 as a central axis. Previously, we have shown that the R308A mutant is monomeric and re-associates to the trimeric-cooperative state in the presence of low concentrations of guanidine chloride. We have now mutated Asp204 that interacts with Arg308 in the neighbor subunit, and also we mutated Glu256, proposed as important for oligomerization. Concretely, the human arginase I mutants D204A, D204E, E256A, E256Q and E256D were generated and examined. No differences were observed in the kinetic parameters at pH 9.5 or in tryptophan fluorescence. However, the D204A and E256Q variants were monomeric. On the other hand, D204E and E256D proved to be trimeric and kinetically cooperative at pH 7.5, whereas hyperbolic kinetics was exhibited by E256A, also trimeric. The results obtained strongly support the importance of the interaction between Arg255 and Glu256 in the cooperative properties of arginase, and Asp204 would be relevant to maintain the oligomeric state through salt bridges with Arg255 and Arg308.

Asunto(s)

Arginasa/ultraestructura , Arginina/genética , Ácido Aspártico/genética , Conformación Proteica , Arginasa/química , Arginasa/genética , Arginina/química , Ácido Aspártico/química , Ácido Glutámico/química , Ácido Glutámico/genética , Humanos , Cinética , Sustancias Macromoleculares , Modelos Moleculares , Mutación/genética , Multimerización de Proteína/genética

RESUMEN

In bacteria, l-arginine is a precursor of various metabolites and can serve as a source of carbon and/or nitrogen. Arginine catabolism by arginase, which hydrolyzes arginine to l-ornithine and urea, is common in nature but has not been studied in symbiotic nitrogen-fixing rhizobia. The genome of the alfalfa microsymbiont Sinorhizobium meliloti 1021 has two genes annotated as arginases, argI1 (smc03091) and argI2 (sma1711). Biochemical assays with purified ArgI1 and ArgI2 (as 6His-Sumo-tagged proteins) showed that only ArgI1 had detectable arginase activity. A 1021 argI1 null mutant lacked arginase activity and grew at a drastically reduced rate with arginine as sole nitrogen source. Wild-type growth and arginase activity were restored in the argI1 mutant genetically complemented with a genomically integrated argI1 gene. In the wild-type, arginase activity and argI1 transcription were induced several fold by exogenous arginine. ArgI1 purified as a 6His-Sumo-tagged protein had its highest in vitro enzymatic activity at pH 7.5 with Ni2+ as cofactor. The enzyme was also active with Mn2+ and Co2+, both of which gave the enzyme the highest activities at a more alkaline pH. The 6His-Sumo-ArgI1 comprised three identical subunits based on the migration of the urea-dissociated protein in a native polyacrylamide gel. A Lrp-like regulator (smc03092) divergently transcribed from argI1 was required for arginase induction by arginine or ornithine. This regulator was designated ArgIR. Electrophoretic mobility shift assays showed that purified ArgIR bound to the argI1 promoter in a region preceding the predicted argI1 transcriptional start. Our results indicate that ArgI1 is the sole arginase in S. meliloti, that it contributes substantially to arginine catabolism in vivo and that argI1 induction by arginine is dependent on ArgIR.

Asunto(s)

Arginasa/fisiología , Arginina/metabolismo , Proteínas Bacterianas/fisiología , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/fisiología , Arginasa/genética , Proteínas Bacterianas/genética , Regulación de la Expresión Génica , Prueba de Complementación Genética , Genoma Bacteriano , Concentración de Iones de Hidrógeno , Mutación , Nitrógeno/metabolismo , Ornitina/metabolismo , Proteínas Recombinantes , Sinorhizobium meliloti/enzimología , Urea/metabolismo

Asunto(s)

Arginasa/metabolismo , Criptococosis/inmunología , Cryptococcus gattii , Pulmón/inmunología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Animales , Arginasa/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Citocinas/metabolismo , Inflamación/metabolismo , Pulmón/enzimología , Pulmón/metabolismo , Pulmón/microbiología , Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Óxido Nítrico Sintasa de Tipo II/genética

RESUMEN

Viral persistence alters cellular antiviral activities. Nitric oxide (NO), a highly reactive free radical and a potent antiviral molecule, can inhibit replication of RNA and DNA viruses, but its production and effect during viral persistence are largely unknown. NO synthesis is stimulated in epithelial cells during acute infection with respiratory syncytial virus (RSV) and interferes with viral replication. In this study, we compared the levels of production of NO and expression of its regulatory enzymes, inducible nitric oxide synthase (NOS II) and arginase 1 (Arg-1), during acute and persistent RSV infection in a macrophage cell line to investigate their role in the control and maintenance of viral infection. We observed that NO and NOS II mRNA were induced at higher levels in acutely infected macrophages than in persistently infected macrophages, while the kinetics of NOS II protein expression were similar in both types of infected cultures, except that its disappearance was delayed during acute infection. Thus, NOS II was inducible and expressed at high levels during persistent infection, but production of NO was low relative to acute infection. This was not associated with a lack of enzymatic activity but instead was due to constitutive expression of the Arg-1 enzyme at the mRNA and protein levels, suggesting that arginase restricts availability of L-arginine as a substrate for NOS II to synthesize NO. This hypothesis was supported by showing that arginase enzymatic activity was inhibited in persistently RSV-infected cells by Nω-hydroxy-nor-L-arginine, increasing L-arginine availability in conditioned medium and producing increased levels of nitrites, concurrently with a significant reduction in virus genome replication, implying that Arg-1 overexpression contributes to the maintenance of the RSV genome in the host in persistent infection.

Asunto(s)

Arginasa/metabolismo , Óxido Nítrico/metabolismo , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/fisiología , Arginasa/genética , Arginina/metabolismo , Regulación hacia Abajo , Humanos , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Infecciones por Virus Sincitial Respiratorio/enzimología , Infecciones por Virus Sincitial Respiratorio/genética , Virus Sincitial Respiratorio Humano/genética , Replicación Viral

RESUMEN

Elderly organisms are more susceptible to infectious diseases. However, the impact of aging on antiparasitic mechanisms, especially the nitric oxide pathway, is poorly understood. Using an integrated in vivo and in vitro model, we compared the severity of Trypanosoma cruzi infection in young and elderly (8 or 72 weeks old) mice. Forty C57BL/6 mice were randomized into four groups: Y-inf, young infected; Yn-inf, young uninfected; A-inf, aged infected; An-inf, aged uninfected. Parasitemia was measured daily, and animals were euthanized after 15 days of infection. Trypanosoma cruzi-induced inflammatory processes were analyzed in blood and heart samples, as well as in bone marrow-derived macrophages (BMDMs) co-cultured with splenocytes isolated from young or elderly mice. Our results indicated upregulated IgG2b and IL-17 production in elderly animals, which was not sufficient to reduce parasitemia, parasitic load and myocarditis to levels observed in young animals. The higher susceptibility of elderly mice to T. cruzi infection was accompanied by reduced cardiac inducible nitric oxide synthase (iNOS) gene expression, nitric oxide (NO) and IFN-γ levels, as well as an antagonistic upregulation of arginase-1 expression and arginase activity. The same responses were observed when BMDMs co-cultured with splenocytes from elderly mice were stimulated with T. cruzi antigens. Our findings indicate that elderly mice were more susceptible to T. cruzi infection, which was potentially related to an attenuated response to antigenic stimulation, inhibition of iNOS gene expression and NO production, and antagonistic upregulation of arginase gene expression and activity, which created favorable conditions for heart parasitism and myocarditis development.

Asunto(s)

Envejecimiento/genética , Arginasa/genética , Cardiomiopatía Chagásica/genética , Enfermedad de Chagas/genética , Óxido Nítrico Sintasa de Tipo II/genética , Parasitemia/genética , Trypanosoma cruzi/patogenicidad , Envejecimiento/inmunología , Animales , Antígenos de Protozoos/farmacología , Arginasa/sangre , Cardiomiopatía Chagásica/inmunología , Cardiomiopatía Chagásica/parasitología , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/parasitología , Técnicas de Cocultivo , Regulación de la Expresión Génica , Corazón/parasitología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/genética , Interferón gamma/sangre , Interferón gamma/genética , Interleucina-10/sangre , Interleucina-10/genética , Interleucina-17/sangre , Interleucina-17/genética , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/sangre , Parasitemia/inmunología , Índice de Severidad de la Enfermedad , Transducción de Señal , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/parasitología , Trypanosoma cruzi/inmunología

RESUMEN

Keloid scars are currently considered a chronic inflammatory process and no longer a benign skin tumor. Keloids are defined as highly inflamed, hyperproliferative pathological scars. Growth factors and cytokines have important functions in the keloid inflammatory etiopathogenesis. The aim of this study was to analyze the in situ expression of cytokines and growth factors in keloid scars in comparison with that in normal scars. Among them, we specifically assessed TGF-ß, FGF, IL-33, IL-22, ARG-1, ARG-2, iNOS, VIP, VIP-R1, TAC, and TAC-R1. A total of 98 biopsies were evaluated, including of 53 keloid and 45 normal scars. The age of patients with keloids ranged from 11 to 73 years, with a mean age of 28 years and predominance of the female gender (58.5% of the total patients). Around 64.15% of the patients belonged to the black ethnic group. Evaluated keloids were most commonly located in the earlobe because of ear piercing, representing 73.6% of the cases. We found significantly greater expression of TGF-ß, IL-22, and ARG-1 in keloids when compared with that in normal scars. As for IL-33, ARG-2, and VIP-R1, despite the higher number of mRNA copies found in keloids, this difference was not significant. Furthermore, FGF, iNOS, VIP, TAC, and TAC-R1 mRNA levels were not detectable, and therefore these results were inconclusive in this study. Considering these results, understanding the cellular and molecular mechanisms that control the inflammatory response during cutaneous healing may promote the development of strategies to improve the treatment of patients with keloids.

Asunto(s)

Arginasa/genética , Expresión Génica , Interleucinas/genética , Queloide/genética , ARN Mensajero/genética , Factor de Crecimiento Transformador beta/genética , Adulto , Biomarcadores , Biopsia , Estudios de Casos y Controles , Femenino , Humanos , Hibridación in Situ , Interleucinas/metabolismo , Queloide/metabolismo , Queloide/patología , Masculino , Factor de Crecimiento Transformador beta/metabolismo , Interleucina-22

RESUMEN

BACKGROUND: Treatment of spinal cord injury is dependent on neuronal survival, appropriate synaptic circuit preservation, and inflammatory environment management. In this sense, mesenchymal stem cell (MSC) therapy is a promising tool that can reduce glial reaction and provide trophic factors to lesioned neurons. METHODS: Lewis adult female rats were submitted to a unilateral ventral funiculus cut at the spinal levels L4, L5, and L6. The animals were divided into the following groups: IA (intramedullary axotomy), IA + DMEM (Dulbecco's modified Eagle's medium), IA + FS (fibrin sealant), IA + MSC (106 cells), and IA + FS + MSC (106 cells). Seven days after injury, qPCR (n = 5) was performed to assess gene expression of VEGF, BDNF, iNOS2, arginase-1, TNF-α, IL-1ß, IL-6, IL-10, IL-4, IL-13, and TGF-ß. The cellular infiltrate at the lesion site was analyzed by hematoxylin-eosin (HE) staining and immunohistochemistry (IH) for Iba1 (microglia and macrophage marker) and arginase-1. Fourteen days after injury, spinal alpha motor neurons (MNs), evidenced by Nissl staining (n = 5), were counted. For the analysis of astrogliosis in spinal lamina IX and synaptic detachment around lesioned motor neurons (GAP-43-positive cells), anti-GFAP and anti-synaptophysin immunohistochemistry (n = 5) was performed, respectively. Twenty-eight days after IA, the gait of the animals was evaluated by the walking track test (CatWalk; n = 7). RESULTS: The site of injury displayed strong monocyte infiltration, containing arginase-1-expressing macrophages. The FS-treated group showed upregulation of iNOS2, arginase-1, proinflammatory cytokine (TNF-α and IL-1ß), and antiinflammatory cytokine (IL-10, IL-4, and IL-13) expression. Thus, FS enhanced early macrophage recruitment and proinflammatory cytokine expression, which accelerated inflammation. Rats treated with MSCs displayed high BDNF-positive immunolabeling, suggesting local delivery of this neurotrophin to lesioned motoneurons. This BDNF expression may have contributed to the increased neuronal survival and synapse preservation and decreased astrogliosis observed 14 days after injury. At 28 days after lesion, gait recovery was significantly improved in MSC-treated animals compared to that in the other groups. CONCLUSIONS: Overall, the present data demonstrate that MSC therapy is neuroprotective and, when associated with a FS, shifts the immune response to a proinflammatory profile.

Asunto(s)

Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Regulación de la Expresión Génica/fisiología , Inmunomodulación/fisiología , Células Madre Mesenquimatosas/fisiología , Neuronas Motoras/metabolismo , Neuroprotección/fisiología , Traumatismos de la Médula Espinal , Animales , Arginasa/genética , Arginasa/metabolismo , Axotomía/métodos , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Adhesivo de Tejido de Fibrina/uso terapéutico , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/terapia , Adhesivos Tisulares/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

The urea cycle disorder argininemia is caused by a defective arginase 1 (ARG1) enzyme resulting from mutations in the ARG1 gene. Patients generally develop hyperargininemia, spastic paraparesis, progressive neurological and intellectual impairment, and persistent growth retardation. Interestingly, in contrast to other urea cycle disorders, hyperammonemia is rare. We report here 66 mutations (12 of which are novel), including 30 missense mutations, seven nonsense, 10 splicing, 15 deletions, two duplications, one small insertion, and one translation initiation codon mutation. For the most common mutations (p.Thr134Ile, p.Gly235Arg and p.Arg21*), which cluster geographically in Brazil, China, or Turkey, a structural rationalization of their effect has been included. In order to gain more knowledge on the disease, we have collected clinical and biochemical information of 112 patients, including the patients' genetic background and ethnic origin. We have listed as well the missense variants with unknown relevance. For all missense variants (of both known and unknown relevance), the conservation, severity prediction, and ExAc scores have been included. Lastly, we review ARG1 regulation, animal models, diagnostic strategies, newborn screening, prenatal testing, and treatment options.

Asunto(s)

Arginasa/genética , Mutación/genética , Brasil , China , Codón sin Sentido/genética , Humanos , Mutación Missense/genética , Turquía

RESUMEN

Arginase 1 (ARG1) and arginase 2 (ARG2) compete with nitric oxide synthases for the substrate l-arginine. Here we aim to assess whether arginase 1 and 2 plasma levels, plasma arginase activity or genetic factors are associated with altered responsiveness to sildenafil. We studied 71 post-prostatectomy erectile dysfunction (ED) patients (PED group) and 72 clinical ED patients (CED). Patients responded to the International Index of Erectile Function questionnaire before and after the treatment. We found positive and negative correlations between plasma levels of arginase 1 and sildenafil responsiveness in the PED and CED groups, respectively. PED group also presented negative correlation between plasma arginase activity and sildenafil responsiveness. Sildenafil poor responders have shown higher plasma arginase activity in PED and higher arginase 1 levels on CED groups. In addition, variant genotypes for the rs2781659, rs2781667 and rs17599586 polymorphisms were associated with reduced arginase activity, as well as the GTTT ARG1 haplotype in CED group.

Asunto(s)

Arginasa/sangre , Arginasa/genética , Disfunción Eréctil/sangre , Disfunción Eréctil/genética , Citrato de Sildenafil/sangre , Vasodilatadores/sangre , Adulto , Anciano , Arginasa/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Disfunción Eréctil/tratamiento farmacológico , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético/genética , Citrato de Sildenafil/farmacología , Citrato de Sildenafil/uso terapéutico , Resultado del Tratamiento , Vasodilatadores/farmacología , Vasodilatadores/uso terapéutico

RESUMEN

BACKGROUND: Leishmania uses the amino acid L-arginine as a substrate for arginase, enzyme that produces urea and ornithine, last precursor of polyamine pathway. This pathway is used by the parasite to replicate and it is essential to establish the infection in the mammalian host. L-arginine is not synthesized by the parasite, so its uptake occurs through the amino acid permease 3 (AAP3). AAP3 is codified by two copies genes (5.1 and 4.7 copies), organized in tandem in the parasite genome. One copy presents the expression regulated by L-arginine availability. METHODOLOGY/PRINCIPAL FINDINGS: RNA-seq data revealed 14 amino acid transporters differentially expressed in the comparison of La-WT vs. La-arg- promastigotes and axenic amastigotes. The 5.1 and 4.7 aap3 transcripts were down-regulated in La-WT promastigotes vs. axenic amastigotes, and in La-WT vs. La-arg- promastigotes. In contrast, transcripts of other transporters were up-regulated in the same comparisons. The amount of 5.1 and 4.7 aap3 mRNA of intracellular amastigotes was also determined in sample preparations from macrophages, obtained from BALB/c and C57BL/6 mice and the human THP-1 lineage infected with La-WT or La-arg-, revealing that the genetic host background is also important. We also determined the aap3 mRNA and AAP3 protein amounts of promastigotes and axenic amastigotes in different environmental growth conditions, varying pH, temperature and L-arginine availability. Interestingly, the increase of temperature increased the AAP3 level in plasma membrane and consequently the L-arginine uptake, independently of pH and L-arginine availability. In addition, we demonstrated that besides the plasma membrane localization, AAP3 was also localized in the glycosome of L. amazonensis promastigotes and axenic amastigotes. CONCLUSIONS/SIGNIFICANCE: In this report, we described the differential transcriptional profiling of amino acids transporters from La-WT and La-arg- promastigotes and axenic amastigotes. We also showed the increased AAP3 levels under amino acid starvation or its decrease in L-arginine supplementation. The differential AAP3 expression was determined in the differentiation of promastigotes to amastigotes conditions, as well as the detection of AAP3 in the plasma membrane reflecting in the L-arginine uptake. Our data suggest that depending on the amino acid pool and arginase activity, Leishmania senses and could use an alternative route for the amino acid transport in response to stress signaling.

Asunto(s)

Sistemas de Transporte de Aminoácidos/clasificación , Sistemas de Transporte de Aminoácidos/metabolismo , Arginasa/metabolismo , Arginina/metabolismo , Leishmania/enzimología , Macrófagos/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Animales , Arginasa/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células THP-1 , Transcriptoma

RESUMEN

BACKGROUND: Leishmania is a protozoan parasite that alternates its life cycle between the sand-fly vector and the mammalian host. This alternation involves environmental changes and leads the parasite to dynamic modifications in morphology, metabolism, cellular signaling and regulation of gene expression to allow for a rapid adaptation to new conditions. The L-arginine pathway in L. amazonensis is important during the parasite life cycle and interferes in the establishment and maintenance of the infection in mammalian macrophages. Host arginase is an immune-regulatory enzyme that can reduce the production of nitric oxide by activated macrophages, directing the availability of L-arginine to the polyamine pathway, resulting in parasite replication. In this work, we performed transcriptional profiling to identify differentially expressed genes in L. amazonensis wild-type (La-WT) versus L. amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes. METHODOLOGY/PRINCIPAL FINDINGS: A total of 8253 transcripts were identified in La-WT and La-arg- promastigotes and axenic amastigotes, about 60% of them codifying hypothetical proteins and 443 novel transcripts, which did not match any previously annotated genes. Our RNA-seq data revealed that 85% of genes were constitutively expressed. The comparison of transcriptome and metabolome data showed lower levels of arginase and higher levels of glutamate-5-kinase in La-WT axenic amastigotes compared to promastigotes. The absence of arginase activity in promastigotes increased the levels of pyrroline 5-carboxylate reductase, but decreased the levels of arginosuccinate synthase, pyrroline 5-carboxylate dehydrogenase, acetylornithine deacetylase and spermidine synthase transcripts levels. These observations can explain previous metabolomic data pointing to the increase of L-arginine, citrulline and L-glutamate and reduction of aspartate, proline, ornithine and putrescine. Altogether, these results indicate that arginase activity is important in Leishmania gene expression modulation during differentiation and adaptation to environmental changes. Here, we confirmed this hypothesis with the identification of differential gene expression of the enzymes involved in biosynthesis of amino acids, arginine and proline metabolism and arginine biosynthesis. CONCLUSIONS/SIGNIFICANCE: All data provided information about the transcriptomic profiling and the expression levels of La-WT and La-arg- promastigotes and axenic amastigotes. These findings revealed the importance of arginase in parasite survival and differentiation, and indicated the existence of a coordinated response in the absence of arginase activity related to arginine and polyamine pathways.

Asunto(s)

Arginasa/metabolismo , Regulación de la Expresión Génica , Leishmania mexicana/genética , Análisis de Secuencia de ARN , Arginasa/genética , Arginina/biosíntesis , Arginina/metabolismo , Expresión Génica , Perfilación de la Expresión Génica/métodos , Técnicas de Inactivación de Genes , Leishmania mexicana/enzimología , Leishmania mexicana/crecimiento & desarrollo , Leishmania mexicana/metabolismo , Macrófagos/parasitología , Óxido Nítrico/metabolismo , Poliaminas/metabolismo