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The application of nitrification inhibitors (nitrapyrin) and urease inhibitors (N-(N-butyl) thiophosphoric triamide) under conventional water resources has been considered as an effective means to improve nitrogen utilization efficiency and mitigate soil greenhouse gas emissions. However, it is not known whether the inhibitors still have an inhibitory effect under unconventional water resources (reclaimed water and livestock wastewater) irrigation and whether their use in combination with biochar improves the mitigation effect. Therefore, unconventional water resources were used for irrigation, with groundwater (GW) control. Nitrapyrin and N-(N-butyl) thiophosphoric triamide were used alone or in combination with biochar in a pot experiment, and CO2, N2O, and CH4 emissions were measured. The results showed that irrigation of unconventional water resources exacerbated global warming potential (GWP). All exogenous substance treatments increased CO2 and CH4 emissions and suppressed N2O emissions, independent of the type of water, compared to no substances (NS). The inhibitors were ineffective in reducing the GWP whether or not in combination with biochar, and the combined application of inhibitors with biochar further increased the GWP. This study suggests that using inhibitors and biochar in combination to regulate the greenhouse effect under unconventional water resources irrigation should be done with caution.
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Agricultura , Carbón Orgánico , Ganado , Compuestos Organofosforados , Animales , Agricultura/métodos , Aguas Residuales , Calentamiento Global , Dióxido de Carbono/análisis , Óxido Nitroso/análisis , Suelo , Fertilizantes , MetanoRESUMEN
Over-exploitation of groundwater due to intensive irrigation and anticipated climate change pose severe threats to the water and food security worldwide, particularly in the North China Plain (NCP). Limited irrigation has been recognized as an effective way to improve crop water productivity and slow the rapid decline of groundwater levels. Whether optimized limited irrigation strategies could achieve a balance between groundwater pumping and grain production in the NCP under future climate change deserves further study. In this study, an improved Soil and Water Assessment Tool (SWAT) model was used to simulate climate change impacts on shallow groundwater levels and crop production under limited irrigation strategies to suggest optimal irrigation management practices under future climate conditions in the NCP. The simulations of eleven limited irrigation strategies for winter wheat with targeted irrigations at different growth stages and with irrigated or rainfed summer maize were compared with future business-as-usual management. Climate change impacts showed that mean wheat (maize) yield under adequate irrigation was expected to increase by 13.2% (4.9%) during the middle time period (2041-2070) and by 11.2% (4.6%) during the late time period (2071-2100) under three SSPs compared to the historical period (1971-2000). Mean decline rate of shallow groundwater level slowed by approximately 1 m a-1 during the entire future period (2041-2100) under three SSPs with a greater reduction for SSP5-8.5. The average contribution rate of future climate toward the balance of shallow groundwater pumping and replenishment was 62.9%. Based on the simulated crop yields and decline rate of shallow groundwater level under the future climate, the most appropriate limited irrigation was achieved by applying irrigation during the jointing stage of wheat with rainfed maize, which could achieve the groundwater recovery and sustainable food production.
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Cambio Climático , Agua Subterránea , Producción de Cultivos , Agua , China , Triticum , Riego AgrícolaRESUMEN
Soil contamination by Cd has drawn global attention, while how irrigation waters modulate Cd sorption and mobility in soil remains obscure. We address this by investigating how cropped sandy soil irrigated with different waters altered Cd sorption and mobility using a rhizobox experiment followed by a batch experiment. Maize were planted in the rhizoboxes and irrigated by reclaimed water (RW), livestock wastewater (LW) and deionized water (CK), respectively. The bulk soil sampled from each treatment after 60 days of growth was employed to measure the Cd sorption and mobility using the isothermal adsorption and desorption experiments. The results showed that, in a small rhizobox experiment, the adsorption rate of Cd by the bulk soil in the adsorption phase was much faster than the desorption rate in desorption phase. Irrigation with RW and LW both reduced the Cd adsorption capacity of soil, and the reducing degree brought by LW was more obvious. Cd desorption rate was very low but keep increasing in the desorption stage, and pre-RW irrigation had the potential to increase Cd desorption from soil. Although the results were obtained based on the bulk soil sampled from a rhizobox experiment, our study strongly suggests that the altered Cd adsorption and desorption behavior in the soil caused by the RW and LW irrigation may risk the farmland ecosystem and deserve more concern.
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Given that Cd pollution in dry land has aroused wide public concern, numerous remediation technologies has been utilized, yet there are limited cost-effective techniques that do not affect the original planting patterns. Fortunately, irrigation management can meet these requirements, while the effects of irrigation practices on Cd uptake by crops in slightly Cd-polluted upland soil remain elusive. Here, we aimed to investigate how the irrigation methods altered the Cd availability in soil, Cd accumulation in plants, microorganism population in soil, root morphology, and enzyme activities in soil and plants. We examined three irrigation treatments - surface drip irrigation (DI), subsurface drip irrigation (SDI), alternate-rows irrigation (ARI), and the control conventional furrow irrigation (CFI). The results showed that SDI remarkably reduced Cd content in roots, shoots and fruits, increased yield, and improved root growth and activity in soil of 20-40 cm compared to other treatments, though the Cd concentration in rhizosphere was not decreased significantly. The microbial population and enzyme activities in rhizosphere and enzyme activities in leaves and roots in SDI and ARI were basically higher than DI and CFI. Therefore, SDI has the prominent potential to reduce Cd uptake by crops in upland soil polluted with low Cd.
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Capsicum , Contaminantes del Suelo , Riego Agrícola , Cadmio/análisis , Rizosfera , Suelo , Contaminantes del Suelo/análisis , VerdurasRESUMEN
OBJECTIVE: To explore the clinical outcomes of open reduction and internal fixation by posterolateral and posteromedial approaches for treating posterior Pilon fractures in elderly patients. METHODS: Between August 2009 and August 2014, 20 elderly patients with posterior Pilon fractures were treated with open reduction and internal fixation by posterolateral and posteromedial approaches. There were 14 males and 6 females, aged from 66 to 83 years (mean, 72.7 years). The causes were falling injury in 11 cases and traffic accident injury in 9 cases. All the patients had lateral malleolus and medial malleolus fractures. The time from injury to operation was 7-14 days (mean, 8.6 days). The posterolateral incision was made to expose the posterolateral bone fragments of posterior malleolus and lateral malleolus fracture, and the posteromedial incision was made to expose the posteromedial fracture fragments of posterior malleolus and medial malleolus fracture. After reduction, fracture was fixed with locking plate or cannulated screw. All the patients began to functional exercise at 1 day after operation. RESULTS: The operation time was 60-110 minutes (mean, 92 minutes). The incisions healed primarily in all patients. There were no complications of incision dehiscence, infection, implant exposure, and nerve damage. No irritation sign of tendon was observed. All 20 cases were followed up for 12-18 months (mean, 13 months). The X-ray films showed that fracture healed at 3-9 months, with an average of 5.2 months. During follow-up period, no loosening or breakage of the implant was observed. The other patients could walk normally except 2 patients (over 80 years old) who could walk with crutch. According to American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot score, the results were excellent in 12 cases, good in 4 cases, and fair in 4 cases; the excellent and good rate was 80%. CONCLUSIONS: A combination of posterolateral approach and posteromedial approach for open reduction and fixation of posterior Pilon fractures can achieve satisfactory effect in elderly patients. It has the advantages of protecting ankle blood supply and avoiding the soft tissue necrosis and implants exposure.
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Fracturas de Tobillo/cirugía , Fijación Interna de Fracturas , Huesos Tarsianos/cirugía , Fracturas de la Tibia/cirugía , Anciano , Articulación del Tobillo/cirugía , Placas Óseas , Tornillos Óseos , Femenino , Peroné , Humanos , Masculino , Huesos Tarsianos/lesiones , Tibia , Resultado del TratamientoRESUMEN
OBJECTIVE: To observe the effects of electroacupuncture (EA) of different intensities on lactate dehydrogernase (LDH), succinate dehydrogenase (SDH) and ATPase in brain tissue of rats with cerebral ischemia-reperfusion injury (CI/R). METHODS: Forty male SD rats were uniformly randomized into sham operation group (group A), CI/R group (group B), CI/R+5 mA EA (group C), CI/R+3 mA EA (group D) and CI/R+1 mA EA (group E) groups with eight rats in each group. Transient general brain ischemia was induced by four-vessel occlusion and reperfusion. The rats in group C, group D and group E were punctured and stimulated at Baihui (GV20), Mingmen (GV4) and Zusanli (ST36) with the same intermittent and rarefaction-dense wave (30 to 50 Hz) and different electric current intensities: 5 mA, 3 mA and 1 mA for 20 min after CI/R. Then the activities of Na(+)-K(+)-ATPase, SDH and LDH in mitochondria of brain tissue were measured by spectrophotometry. The ischemic cerebral cortex tissue was taken for observing the ultrastructure changes of impaired nerve cells. RESULTS: Compared with group A, the activities of LDH, SDH and Na(+)-K(+)-ATPase were lowerer in the group B (P<0.05 or P<0.01). However, the activities of LDH, SDH and Na(+)-K(+)-ATPase were higher in the group D than those in the group B (P<0.05 orP<0.01). In group A, the anatomical structure of the cerebral cortex cells was basically normal; in group B, the neuronal cellular structures were severely damaged, the neuronal mitochondria got swelling, the mitochondrial cristae were broken, the medullated nerve fifibers were not integrated. In group C, group D and group E, the ultrastructure of impaired neuron were improved. Group D was the best among three groups above. CONCLUSION: EA of 3 mA intensity could strengthen aerobic metabolism by elevating the activities of SDH and LDH, meanwhile maintaining the ionic equilibrium in the exterior and interior brain cell and relieving the cellular edema by reinforcing the activities of Na(+)-K(+)-ATPase.
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Encéfalo/metabolismo , Electroacupuntura , Metabolismo Energético , Mitocondrias/metabolismo , Daño por Reperfusión/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: We investigated the hypothesis that Foxp3+ cells are an integral component of antiallograft immunity but are dominated by pathogenic effectors. METHODS: Wild-type H-2b C57BL/6 (B6) mice or B6 mice with a targeted disruption of c-Rel gene (c-Rel-/-) were used as recipients of islet grafts from allogeneic DBA/2 (H-2d) mice or syngeneic B6 mice. We developed kinetic quantitative polymerase chain reaction assays and measured intragraft expression of mRNA for Foxp3, IDO, cytolytic molecules, proinflammatory cytokines, and chemokines/receptors. RESULTS: Intraislet levels of mRNA for Foxp3, IDO, CD3, CD25, tumor necrosis factor-alpha, RANTES, IP-10, and CXCR3 were highest in DBA/2 islet allografts from WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or syngeneic B6 islet grafts from WT B6 mice. The ratio of granzyme B or IFN-gamma to Foxp3 was higher with the DBA/2 islet allografts from the WT B6 recipients compared to DBA/2 islet allografts from c-Rel-/- B6 recipients or B6 islet grafts from WT B6 recipients. CONCLUSIONS: Foxp3+ cells are an integral component of acute rejection of allografts but may be dominated by pathogenic effectors.
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Factores de Transcripción Forkhead/genética , Rechazo de Injerto/patología , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Trasplante de Islotes Pancreáticos/fisiología , Animales , Granzimas/genética , Trasplante de Islotes Pancreáticos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Proteínas Proto-Oncogénicas c-rel/deficiencia , Proteínas Proto-Oncogénicas c-rel/genética , ARN Mensajero/genéticaRESUMEN
BACKGROUND: The outcome of renal transplantation after an episode of acute rejection is difficult to predict, even with an allograft biopsy. METHODS: We studied urine specimens from 36 subjects with acute rejection, 18 subjects with chronic allograft nephropathy, and 29 subjects with normal biopsy results. Levels of messenger RNA (mRNA) for FOXP3, a specification and functional factor for regulatory T lymphocytes, and mRNA for CD25, CD3epsilon, perforin, and 18S ribosomal RNA (rRNA) were measured with a kinetic, quantitative polymerase-chain-reaction assay. We examined associations of mRNA levels with acute rejection, rejection reversal, and graft failure. RESULTS: The log-transformed mean (+/-SE) ratio of FOXP3 mRNA copies to 18S ribosomal RNA copies was higher in urine from the group with acute rejection (3.8+/-0.5) than in the group with chronic allograft nephropathy (1.3+/-0.7) or the group with normal biopsy results (1.6+/-0.4) (P<0.001 by the Kruskal-Wallis test). FOXP3 mRNA levels were inversely correlated with serum creatinine levels measured at the time of biopsy in the acute-rejection group (Spearman's correlation coefficient = -0.38, P=0.02) but not in the group with chronic allograft nephropathy or the group with normal biopsy results. Analyses of receiver-operating-characteristic curves demonstrated that reversal of acute rejection can be predicted with 90 percent sensitivity and 73 percent specificity with use of the optimal identified cutoff for FOXP3 mRNA of 3.46 (P=0.001). FOXP3 mRNA levels identified subjects at risk for graft failure within six months after the incident episode of acute rejection (relative risk for the lowest third of FOXP3 mRNA levels, 6; P=0.02). None of the other mRNA levels were predictive of reversal of acute rejection or graft failure. CONCLUSIONS: Measurement of FOXP3 mRNA in urine may offer a noninvasive means of improving the prediction of outcome of acute rejection of renal transplants.
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Factores de Transcripción Forkhead/genética , Rechazo de Injerto/orina , Trasplante de Riñón , ARN Mensajero/orina , Enfermedad Aguda , Biomarcadores/sangre , Biomarcadores/orina , Complejo CD3/orina , Creatinina/sangre , Factores de Transcripción Forkhead/orina , Expresión Génica , Rechazo de Injerto/sangre , Rechazo de Injerto/diagnóstico , Supervivencia de Injerto/fisiología , Humanos , Glicoproteínas de Membrana/orina , Perforina , Proteínas Citotóxicas Formadoras de Poros , ARN Ribosómico 18S/orina , Curva ROC , Receptores de Interleucina-2/análisis , Riesgo , Linfocitos T/inmunología , Trasplante HomólogoRESUMEN
BACKGROUND: We explored the hypothesis that measurements of mRNA encoding interferon-inducible protein-10 (IP-10) or the chemokine receptor CXCR3 in urinary cells offer a noninvasive means of elucidating cellular traffic causing acute rejection of human renal allografts. METHODS: We obtained 63 urine specimens from 58 renal allograft recipients who underwent 63 allograft biopsies to resolve the basis for graft dysfunction, and 27 additional urine samples from 24 other patients with stable allograft function. Twenty-seven of the 63 biopsies were classified as acute rejection, 20 as other, and 16 as chronic allograft nephropathy. We measured the levels of transcripts for IP-10 and CXCR3, and a constitutively expressed gene 18S rRNA in the urine specimens and correlated transcript levels with renal allograft diagnosis. RESULTS: mRNA levels of IP-10 (P < 0.0001) or CXCR3 (P < 0.0001) but not the levels of 18S rRNA (P= 0.56) predicted intragraft cellular traffic causing acute rejection. Receiver-operating characteristic curve analysis demonstrated that acute rejection can be predicted with a sensitivity of 100% and a specificity of 78% using the (log-transformed) cutoff value of 9.11 copies of IP-10, and with a sensitivity of 63% and a specificity of 83% using the cutoff value of 11.59 copies of CXCR3. Immunohistologic analysis of allograft biopsies showed exuberant expression of IP-10 and CXCR3 during acute rejection whereas both were absent in grafts with stable function. CONCLUSION: Our investigation demonstrates that intragraft cellular events associated with acute rejection of human renal allografts can be noninvasively identified by measurements of mRNA for IP-10 and CXCR3 in urinary cells.
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Quimiocinas CXC/genética , Rechazo de Injerto/diagnóstico , Inflamación/diagnóstico , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/inmunología , ARN Mensajero/orina , Receptores de Quimiocina/genética , Adulto , Secuencia de Bases , Quimiocina CXCL10 , ADN/genética , Femenino , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Trasplante de Riñón/patología , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , Curva ROC , Receptores CXCR3RESUMEN
BACKGROUND: Immunosuppressive therapy is a risk factor for the increased incidence and metastatic progression of malignancies in organ graft recipients. Transforming growth factor (TGF)-beta(1) has been associated with tumor invasion and metastasis, and we have implicated cyclosporine-associated TGF-beta(1) hyperexpression in tumor progression in mice. METHODS: BALB/c mice or severe combined immunodeficient-beige mice were treated with 2 or 4 mg/kg of tacrolimus, and the effect of treatment on mouse renal cancer cell pulmonary metastasis was investigated. We also determined whether tacrolimus induces TGF-beta(1) expression. Spleens from tacrolimus-treated mice were analyzed for level of expression of TGF-beta(1) mRNA with the use of competitive-quantitative polymerase chain reaction assay, and circulating levels of TGF-beta(1) protein were measured with the use of an enzyme-linked immunosorbent assay. RESULTS: Treatment with tacrolimus resulted in a dose-dependent increase in the number of pulmonary metastases in the BALB/c mice (197+/-16 in untreated mice, 281+/-26 in mice treated with 2 mg/kg of tacrolimus, and 339+/-25 in mice treated with 4 mg/kg of tacrolimus; no treatment vs. 4 mg/kg tacrolimus, Bonferroni's P<0.001) and in the severe combined immunodeficient-beige mice (117+/-18 in untreated mice, 137+/-19 in mice treated with 2 mg/kg of tacrolimus, and 216+/-29 in mice treated with 4 mg/kg of tacrolimus; no treatment vs. 4 mg/kg tacrolimus, P<0.05). Treatment with 4 mg/kg but not 2 mg/kg of tacrolimus resulted in a significant increase in the levels of expression of TGF-beta(1) mRNA and circulating levels of TGF-beta(1) protein. CONCLUSIONS: Tacrolimus has a dose-dependent effect on tumor progression and TGF-beta(1) expression, and tacrolimus-induced TGF-beta(1) overexpression may be a pathogenetic mechanism in tumor progression.
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Inmunosupresores/efectos adversos , Neoplasias/patología , Tacrolimus/efectos adversos , Factor de Crecimiento Transformador beta/análisis , Animales , Carcinoma de Células Renales/patología , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Inmunosupresores/administración & dosificación , Inmunosupresores/farmacología , Neoplasias Renales/patología , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Metástasis de la Neoplasia/patología , Bazo/química , Tacrolimus/administración & dosificación , Tacrolimus/farmacología , Factor de Crecimiento Transformador beta1RESUMEN
Acute rejection (AR) and urinary tract infection (UTI) continue to plague renal transplantation. We tested the hypotheses that UTI does not increase granzyme B mRNA levels in urinary cells, and that the levels distinguish AR from UTI. We measured the levels of granzyme B mRNA in 15 urine specimens from renal allograft recipients with UTI, 29 specimens from patients with AR but without UTI, and 14 specimens from patients without AR and without UTI. We also measured transcript levels in urine specimens from 41 nontransplant individuals, 11 with UTI and 30 without UTI. UTI did not increase granzyme B mRNA levels. Granzyme B mRNA levels were lower in renal allograft recipients with UTI compared with those with AR (P<0.0001). We conclude that bacterial UTI is unlikely to confound AR diagnosis made by measurement of granzyme B mRNA levels in urinary cells.
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Rechazo de Injerto/diagnóstico , Trasplante de Riñón , Serina Endopeptidasas/orina , Infecciones Urinarias/diagnóstico , Enfermedad Aguda , Adulto , Diagnóstico Diferencial , Femenino , Rechazo de Injerto/orina , Granzimas , Humanos , Masculino , Persona de Mediana Edad , Infecciones Urinarias/orina , Orina/citologíaRESUMEN
BACKGROUND: Serine proteinase inhibitor (PI)-9 with a reactive center P1 (Glu)-P1' is a natural antagonist of granzyme B and is expressed in high levels in cytotoxic T lymphocytes (CTL). In view of the role of CTL in acute rejection, we explored the hypothesis that PI-9 would be hyperexpressed during acute rejection. Because PI-9 can protect CTL from its own fatal arsenal and potentially enhance the vitality of CTL, we examined whether PI-9 levels correlate with the severity of rejection as well as predict subsequent graft function. METHODS: We obtained 95 urine specimens from 87 renal allograft recipients. RNA was isolated from the urinary cells and mRNA encoding PI-9, granzyme B, or perforin and a constitutively expressed 18S rRNA was measured with the use of real-time quantitative polymerase chain reaction assay, and the level of expression was correlated with allograft status. RESULTS: The levels of PI-9 (P=0.001), granzyme B (P<0.0001), and perforin mRNAs (P<0.0001), but not the levels of 18S rRNA (P=0.54), were higher in the urinary cells from the 29 patients with a biopsy-confirmed acute rejection than in the 58 recipients without acute rejection. PI-9 levels were significantly higher in patients with type II or higher acute rejection changes compared with those with less than type II changes (P=0.01). Furthermore, PI-9 levels predicted subsequent graft function (r=0.43, P=0.01). CONCLUSIONS: PI-9 mRNA levels in urinary cells are diagnostic of acute rejection, predict renal allograft histology grade, and predict functional outcome following an acute rejection episode.
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Rechazo de Injerto/metabolismo , Trasplante de Riñón/inmunología , Glicoproteínas de Membrana/antagonistas & inhibidores , Serina Endopeptidasas , Serpinas/biosíntesis , Enfermedad Aguda , Adulto , Anciano , Femenino , Granzimas , Humanos , Masculino , Glicoproteínas de Membrana/genética , Persona de Mediana Edad , Perforina , Proteínas Citotóxicas Formadoras de Poros , ARN Mensajero/análisis , Serina Endopeptidasas/genética , Serpinas/genética , Linfocitos T Citotóxicos/inmunología , Trasplante HomólogoRESUMEN
BACKGROUND: CD103 is displayed on the cell surface of alloreactive CD8 cytotoxic T lymphocytes (CTLs) and is a critical component for the intraepithelial homing of T cells. Because intratubular localization of mononuclear cells is a feature of acute cellular rejection of renal allografts, we explored the hypothesis that CD103 messenger (m)RNA levels in urinary cells predict acute rejection. METHODS: We collected 89 urine specimens from 79 recipients of renal allografts. RNA was isolated from the urinary cells, and we measured CD103 mRNA levels and a constitutively expressed 18S ribosomal (r)RNA with the use of real-time quantitative polymerase chain reaction assay. RESULTS: CD103 mRNA levels, but not 18S rRNA levels, were higher in urinary cells from 30 patients with an episode of acute rejection (32 biopsies and 32 urine samples) compared with the levels in 12 patients with other findings on allograft biopsy (12 biopsies and 12 urine samples), 12 patients with biopsy evidence of chronic allograft nephropathy (12 biopsies and 12 urine samples), and 25 patients with stable graft function after renal transplantation (0 biopsies and 33 urine samples) (P = 0.001; one-way analysis of variance). Acute rejection was predicted with a sensitivity of 59% and a specificity of 75% using natural log-transformed value 8.16 CD103 copies per microgram as the cutoff value (P = 0.001). CONCLUSION: CD103 mRNA levels in urinary cells are diagnostic of acute rejection of renal allografts. Because CD103 is a cell surface marker of intratubular CD8 CTLs, a noninvasive assessment of cellular traffic into the allograft may be feasible by the measurement of CD103 mRNA levels in urinary cells.
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Antígenos CD/orina , Rechazo de Injerto , Trasplante de Riñón , Enfermedad Aguda , Adulto , Antígenos CD/genética , Biopsia , Femenino , Predicción , Humanos , Cadenas alfa de Integrinas/genética , Riñón/patología , Masculino , Persona de Mediana Edad , ARN Mensajero/orina , Curva ROC , Trasplante Homólogo , Orina/citologíaRESUMEN
BACKGROUND: Apoptosis is a well-documented pathway for islet cell death. One potential mechanism is overexpression of death-promoting Bax compared with antiapoptotic Bcl-2 in islets. METHODS: We isolated islets from 10 human pancreata and measured the expression of Bax mRNA and Bcl-2 mRNA by real-time quantitative polymerase chain reaction; islet and pancreas expression of Bax, Bcl-2, activated caspase-3, and cleaved poly (ADP-ribose) polymerase were also assessed by immunohistochemistry. Islet cell apoptosis was evaluated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometry. RESULTS: The mean (+/-SE) level of Bax mRNA was 336+/-79 copies per nanogram of total RNA, and the level of Bcl-2 mRNA was 36+/-10 (P=0.001). A positive correlation existed between islet expression of Bax mRNA and Bcl-2 mRNA (P=0.001). The islet Bax to Bcl-2 ratio was 10.8+/-1.3 and 1.71+/-0.3 for the spleens (P=0.0001). Bax mRNA (P=0.04), but not Bcl-2 mRNA, was expressed at a higher level in islets compared with spleens. Human islets contained large numbers of cells expressing Bax protein, whereas only infrequent islet cells expressed Bcl-2 protein, activated caspase-3, and poly (ADP-ribose) polymerase. The apoptotic index was 5% by TUNEL assay, and the percentage of apoptotic islet cells was 9.7+/-2.5% by flow cytometry. Sections of human pancreas before islet isolation showed islet staining for Bax but not Bcl-2. CONCLUSIONS: Our finding that isolated human islets express Bax at a higher level compared with Bcl-2 suggests a molecular mechanism for islet cell death by apoptosis. We hypothesize that reducing islet expression of Bax, or regulating its activation, will help preserve islet cell mass after islet transplantation.
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Apoptosis/fisiología , Islotes Pancreáticos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Proteínas Proto-Oncogénicas/fisiología , Adulto , Criopreservación , Femenino , Humanos , Islotes Pancreáticos/fisiología , Masculino , Persona de Mediana Edad , Páncreas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Proteína X Asociada a bcl-2RESUMEN
BACKGROUND: Polyoma virus type BK (BKV) nephritis has emerged as an important cause of renal allograft dysfunction and graft failure. Its diagnosis is contingent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for BKV nephritis could improve clinical outcome. METHODS: We obtained 25 urine specimens from 8 renal allograft recipients with biopsy-confirmed BKV nephritis, 31 samples from 28 recipients in whom BKV nephritis was excluded by allograft biopsy, and 74 specimens from 34 patients with stable allograft function. RNA was isolated from the urinary cells and reverse transcribed to complementary DNA. We designed gene-specific oligonucleotide primers and probes for the measurement of messenger RNA (mRNA) encoding BKV VP1 protein and a constitutively expressed 18S ribosomal RNA (rRNA) by real-time quantitative polymerase chain reaction. We explored the hypothesis that BKV VP1 mRNA levels predict BKV nephritis. RESULTS: The levels of BKV VP1 mRNA but not the levels of 18S rRNA predicted BKV nephritis. Analysis involving the receiver operating characteristic curve demonstrated that BKV nephritis can be predicted with a sensitivity of 93.8% and a specificity of 93.9% with the use of a cutoff value of 6.5 x 10 BKV VP1 mRNA copy number per nanogram of total RNA ( <0.00001). In the receiver operating characteristic curve analysis, the calculated area under the curve was 0.949 (95% confidence interval, 0.912 to 0.987, <0.00001) for BKV VP1 mRNA levels and 0.562 (95% confidence interval, 0.417 to 0.708, >0.2) for 18S rRNA. CONCLUSIONS: Measurement of BKV VP1 mRNA in urinary cells offers a noninvasive and accurate means of diagnosing BKV nephritis.
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Virus BK/genética , Proteínas de la Cápside , Cápside/fisiología , ADN Viral/orina , Nefritis/virología , Infecciones por Polyomavirus/diagnóstico , Infecciones Tumorales por Virus/diagnóstico , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nefritis/orina , Infecciones por Polyomavirus/orina , Curva ROC , Infecciones Tumorales por Virus/orina , Orina/citologíaRESUMEN
BACKGROUND: The NF-kappaB/Rel family of transcription factors regulates biologic processes ranging from apoptosis to inflammation and innate immunity. Whether c-Rel, a lymphoid-predominant member of the NF-kappaB/Rel family, is essential for transplantation immunity is not known. METHODS: We explored the role of c-Rel in the anti-allograft repertory using mice with targeted disruption of the c-Rel gene (c-Rel-/-) as recipients of H-2 mismatched islet allografts. Allogeneic DBA/2 (H-2d) islets were transplanted into the renal subcapsular space of diabetic c-Rel-/- C57BL/6 (H-2b) mice or the c-Rel +/+ C57BL/6 wild-type mice. Islet graft survival, cellular traffic into the islet grafts and their phenotype, and intragraft expression of cytokines and cytotoxic attack molecules were determined at the protein (by immunohistochemistry) and mRNA (by real-time quantitative polymerase chain reaction) levels. RESULTS: We found superior islet graft survival in the c-Rel-/- recipients compared to c-Rel+/+ C57BL/6 recipients. Splenocytes from c-Rel-/- mice proliferated poorly compared to splenocytes from the c-Rel+/+ mice on stimulation with anti-CD3 mAbs or Con A. Peri-islet infiltration composed of T lymphocytes and macrophages was found in both c-Rel+/+ recipients and c-Rel-/- recipients, but intra-islet infiltration was observed only in c-Rel+/+ recipients. Immunohistologic and molecular studies showed impaired T helper-type 1 immunity and decreased intragraft expression of cytotoxic attack molecules perforin and granzyme B in c-Rel-/- recipients as compared to wild-type recipients. CONCLUSIONS: Our results demonstrate that c-Rel is essential for robust rejection of islet allografts and support the idea that strategies that impair c-Rel function may be of value for constraining alloimmunity and facilitating survival of allogafts.