RESUMO
BACKGROUND: Pivotal randomized trials demonstrating efficacy, safety and good tolerance, of two new potassium binders (patiromer and sodium zirconium cyclosilicate) led to their recent approval. A major hurdle to the implementation of these potassium-binders is understanding how to integrate them safely and effectively into the long-term management of cardiovascular and kidney disease patients using renin angiotensin aldosterone system inhibitors (RAASi), the latter being prone to induce hyperkalaemia. METHODS: A multidisciplinary academic panel including nephrologists and cardiologists was convened to develop consensus therapeutic algorithm(s) aimed at optimizing the use of the two novel potassium binders (patiromer and sodium zirconium cyclosilicate) in stable adults who require treatment with RAASi and experience(d) hyperkalaemia in a non-emergent setting. RESULTS: Two dedicated pragmatic algorithms are proposed. The lowest intervention threshold (i.e. 5.1â¯mmol/L or greater) was the one used in the patiromer and sodium zirconium cyclosilicate) pivotal trials, both drugs being indicated to treat hyperkalaemia in a non -emergent setting. Acknowledging the heterogeneity across specialty guidelines in hyperkalaemia definition and thresholds to intervene when facing hyperkalaemia, we have been mindful to use soft language i.e. "it is to consider", not necessarily "to do". CONCLUSIONS: Providing the clinical community with pragmatic algorithms may help optimize the management of high-risk patients by avoiding the risks of both hyper and hypokalaemia and of suboptimal RAASi therapy.
Assuntos
Cardiopatias , Insuficiência Cardíaca , Hiperpotassemia , Insuficiência Renal Crônica , Adulto , Algoritmos , Humanos , Hiperpotassemia/diagnóstico , Hiperpotassemia/tratamento farmacológico , Hipertensão Renal , Nefrite , Potássio , Insuficiência Renal Crônica/tratamento farmacológico , Sistema Renina-AngiotensinaRESUMO
Objective To study the influence of Maghrebian ethnicity on lupus nephritis. Methods We retrospectively reviewed the files of a cohort of 194 patients with proliferative lupus nephritis followed in seven lupus centres belonging to three groups: Europeans living in Belgium/France (E; n = 111); Maghrebians living in Europe, in casu Belgium/France (ME; n = 43); and Maghrebians living in Morocco (MM; n = 40). Baseline presentation was compared between these three groups but complete long-term outcome data were available only for E and ME patients. Results At presentation, the clinical and pathological characteristics of lupus nephritis did not differ between E, ME and MM patients. Renal relapses were more common in ME patients (54%) than in E patients (29%) ( P < 0.01). Time to renal flare and to end-stage renal disease was shorter in ME patients compared to E patients ( P < 0.0001 and P < 0.05, respectively). While proteinuria measured at month 12 accurately predicted a serum creatinine value of less than 1 mg/dl at 7 years in E patients, this was not the case in the ME group, in whom serum creatinine at month 12 performed better. Conclusion Despite a similar disease profile at onset, the prognosis of lupus nephritis is more severe in Maghrebians living in Europe compared to native Europeans, with a higher relapse rate.
Assuntos
Imunossupressores/uso terapêutico , Falência Renal Crônica/mortalidade , Rim/patologia , Nefrite Lúpica/tratamento farmacológico , Proteinúria/etnologia , Adulto , África do Norte/etnologia , Creatinina/sangue , Europa (Continente) , Feminino , Taxa de Filtração Glomerular , Humanos , Estimativa de Kaplan-Meier , Falência Renal Crônica/etnologia , Nefrite Lúpica/complicações , Nefrite Lúpica/etnologia , Masculino , Pessoa de Meia-Idade , Curva ROC , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
Kidney transplant recipients (KTR) are subjected to immunosuppressive therapy that can enhance hepatitis B and C virus replication, leading to cirrhosis and hepatocellular carcinoma (HCC). The aim of this study was to assess the prevalence and outcome of HCC in KTR. Case-control study. Patients with chronic HBV and/or HCV infection who underwent kidney transplantation between 1976 and 2011 and subsequently developed HCC were compared to a control group of patients with chronic HBV and/or HCV infection, matched for gender and age at HCC diagnosis, who did not receive kidney transplantation. Among 2944 KTR, 330 had hepatitis B and/or C. Fourteen developed HCC, a period prevalence of 4.2%. Age at HCC diagnosis was 52.6 ± 6.5 years (53.5 ± 5.7 in controls, P=.76). Time between transplantation and HCC diagnosis was 16.7 ± 2.7 years. Six HCCs were related to HBV, six to HCV and two to co-infection with HBV and HCV. Immunosuppressive therapy was comparable in HBV, HCV and HBV+HCV patients. At diagnosis, 71% of patients met Milan criteria (65% in the control group, P=.4). Alpha-fetoprotein levels, tumour characteristics and treatment modalities were comparable between both groups. Patient survival 2 years after HCC diagnosis was 28% in KTR, compared to 68% in controls (P=.024). Survival after HCC diagnosis is significantly worse in KTR compared to nontransplanted patients with HBV and/or HCV. Prevention is crucial and should be based on viral eradication/suppression before or after transplantation.
Assuntos
Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/mortalidade , Hepatite B Crônica/complicações , Hepatite C Crônica/complicações , Transplante de Rim , Transplantados , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Resultado do TratamentoRESUMO
OBJECTIVE: The objective of this paper is to evaluate whether the different International Society of Nephrology/Renal Pathology Society (ISN/RPS) classes of proliferative lupus nephritis (LN) have a distinct baseline presentation, short-term response to immunosuppression (IS) and long-term prognosis. METHODS: Ninety-eight patients with new onset (first renal biopsy) ISN/RPS proliferative LN (Class III: n=24; IV-S: n=23; IV-G: n=51) were diagnosed at our institution between 1995 and 2012 (Louvain Lupus Nephritis inception Cohort). Their baseline renal parameters, primary response to IS at one year, survival and long-term renal outcome (mean follow-up: 77 months) were compared. RESULTS: At baseline, serum creatinine and 24-hour proteinuria were higher in Class IV-G, as was activity index on renal biopsy in Class IV-S and IV-G compared to III. Upon treatment, renal parameters improved with the same kinetics and to the same extent in the three pathological classes. On repeat renal biopsies (n=43), activity indices dropped similarly. Poor outcomes (death, end-stage renal disease, renal impairment defined by an eGFR <60 ml/min/1.73 m(2)) did not statistically differ between groups, although there was a trend toward more renal impairment at follow-up in Class IV-G compared to IV-S and III. Finally, the presence of even mild chronic lesions on baseline biopsy was clearly predictive of late renal outcome. CONCLUSION: Subsetting proliferative LN into Class III, IV-S and IV-G provides less clinically discriminant prognostic information than baseline chronicity index.
Assuntos
Nefrite Lúpica/classificação , Nefrite Lúpica/patologia , Adulto , Bélgica , Estudos de Coortes , Feminino , Seguimentos , Humanos , Imunossupressores/uso terapêutico , Rim/patologia , Nefrite Lúpica/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Proteinúria/tratamento farmacológico , Resultado do Tratamento , Adulto JovemRESUMO
We report the case of a 53-year-old woman treated for 8 years with Betaferon® (interferon-ß-1b), who developed mild renal failure with hypertension, proteinuria and glomerular hematuria. Kidney biopsy was consistent with thrombotic microangiopathy (TMA). Considering the strong evidence of interferon-α causing TMA and the numerous immunomodulatory activities shared by INF-α and -ß, we incriminated Betaferon as the etiological agent of TMA in our patient. To our knowledge, it is the first time such an association has been published.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Adjuvantes Imunológicos/efeitos adversos , Interferon beta/efeitos adversos , Esclerose Múltipla/tratamento farmacológico , Microangiopatias Trombóticas/induzido quimicamente , Feminino , Humanos , Interferon beta-1b , Pessoa de Meia-IdadeRESUMO
The combination of Pleth Variability Index (PVI) and passive leg raising (PLR)-induced pulse pressure variation may help to diagnose hypovolemia in spontaneously breathing patients. In 44 subjects, PVI and Pulse Pressure (PP) variation after PLR were measured before and after induced hypovolemia (blood gift or hemodialysis session). PVI values were significantly greater after hemodialysis session or blood gift (22% vs 18%, P = 0.03); in contrast PP variation did not change significantly (7% vs 4%, P = 0.49). The accuracy of these parameters or of their combination to identify the "after hypovolemia induction" period was weak. In spontaneous ventilation, PVI value is greater after induced hypovolemia, whereas PP variation does not change significantly. The combination of PVI and PLR does not improve the accuracy of the detection of induced hypovolemia.
Assuntos
Pressão Sanguínea , Hipovolemia/diagnóstico , Respiração , Idoso , Algoritmos , Feminino , Humanos , Perna (Membro) , Masculino , Pessoa de Meia-Idade , Diálise RenalRESUMO
Paecilomyces lilacinus is a saprophytic mould which rarely causes infection in humans. We report a case of Paecilomyces lilacinus catheter-related fungemia in a chronic hemodialyzed patient. Blood cultures remained positive for 8 weeks. The infection was cured after eventual acceptance by the patient of oral voriconazole treatment for 6 weeks and removal of the tunneled catheter. The literature on Paecilomyces fungemia in humans is reviewed.
Assuntos
Antifúngicos/uso terapêutico , Infecções Relacionadas a Cateter/tratamento farmacológico , Infecções Relacionadas a Cateter/microbiologia , Cateteres de Demora/efeitos adversos , Fungemia/tratamento farmacológico , Fungemia/microbiologia , Paecilomyces/isolamento & purificação , Pirimidinas/uso terapêutico , Diálise Renal , Triazóis/uso terapêutico , Idoso , Feminino , Humanos , VoriconazolRESUMO
We describe an HIV1-positive patient under long-term tenofovir treatment who developed a severe, biopsy-proven, acute tubular necrosis with proximal tubule (PT) dysfunction, precipitated by the very recent start of diclofenac, a nonsteroidal antiinflammatory drug (NSAID). Recent studies show that NSAIDs not only alter glomerular filtration but also multidrug resistance protein (MRP) 4-mediated PT secretion of several substrates. Since the patient tolerated tenofovir well for several years prior to diclofenac use, our observation suggests that diclofenac interfered with tenofovir clearance, thereby favoring its nephrotoxicity. NSAIDs should be avoided in patients under tenofovir.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Adenina/análogos & derivados , Diclofenaco/efeitos adversos , Síndrome de Fanconi/induzido quimicamente , Anticorpos Anti-HIV/imunologia , Soropositividade para HIV/tratamento farmacológico , HIV-1/imunologia , Organofosfonatos/efeitos adversos , Injúria Renal Aguda/patologia , Adenina/efeitos adversos , Adenina/uso terapêutico , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/uso terapêutico , Anti-Inflamatórios não Esteroides/efeitos adversos , Anti-Inflamatórios não Esteroides/uso terapêutico , Biópsia , Diagnóstico Diferencial , Diclofenaco/uso terapêutico , Sinergismo Farmacológico , Quimioterapia Combinada , Síndrome de Fanconi/patologia , Feminino , Soropositividade para HIV/virologia , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Pessoa de Meia-Idade , Organofosfonatos/uso terapêutico , TenofovirRESUMO
We report moderate renal failure in a 50-year-old man with a history of recent colonoscopy after oral sodium phosphate purgative use. We initially missed the correct diagnosis, but renal biopsy revealed signs of acute phosphate nephropathy. The patient had residual renal impairment at 8-month follow-up. Greater awareness of this complication is needed amongst health care professionals. The preventive strategies are discussed.
Assuntos
Catárticos/efeitos adversos , Nefrocalcinose/induzido quimicamente , Biópsia , Colonoscopia , Creatinina/sangue , Humanos , Hiperfosfatemia/induzido quimicamente , Doença Iatrogênica , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The serum level of C-reactive protein, an acute-phase marker of systemic inflammation, has been shown to predict cardiovascular events in the general population and cardiovascular and total mortality in hemodialysis patients. High-sensitivity CRP assays (hs-CRP) have been used in numerous studies. We hypothesized that the level of CRP as measured by the conventional assay (c-CRP) would predict mortality in hemodialysis patients with an accuracy similar to that of high-sensitivity assays. METHODS: In April 2001 CRP serum level was measured with both a conventional and a high-sensitivity assay in 102 prevalent hemodialysis patients. Mortality was prospectively monitored over 6 years. RESULTS: 49 patients (48%) died during follow-up. With both assays, almost 2/3 of patients had high CRP levels (> 1 mg/dl). Survival at 6 years was significantly lower in patients with high CRP levels, no matter which assay was used (31.5% for patients with high hs-CRP and 27.3% for patients with high c-CRP vs 48.4% for patients with low hs-CRP and 47.1% for patients with low c-CRP). Cardiovascular mortality was also higher in patients with high CRP levels, whatever the type of assay (conventional or high sensitivity) used. The correlation between the two tests was excellent. CONCLUSION: CRP level, measured by a conventional inexpensive assay, is predictive of mortality in hemodialysis patients.
Assuntos
Proteína C-Reativa/metabolismo , Doenças Cardiovasculares/sangue , Falência Renal Crônica/sangue , Diálise Renal , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bélgica/epidemiologia , Biomarcadores/sangue , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/etiologia , Feminino , Seguimentos , Humanos , Incidência , Falência Renal Crônica/complicações , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Nefelometria e Turbidimetria , Prognóstico , Estudos Prospectivos , Fatores de Risco , Taxa de Sobrevida/tendências , Fatores de Tempo , Adulto JovemRESUMO
We report the case of a 42-year-old man who developed biopsy-confirmed acute interstitial nephritis (AIN) after cocaine sniffing. He required a few hemodialysis sessions but fully recovered within 3 weeks after cocaine withdrawal and a short course of corticosteroids. AIN should be recognized as a potential cause of acute renal failure in cocaine users, and a history of cocaine use should be carefully elicited in patients with unexplained AIN.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/complicações , Nefrite Intersticial/etiologia , Doença Aguda , Adulto , Biópsia , Diagnóstico Diferencial , Humanos , Masculino , Nefrite Intersticial/patologia , Nefrite Intersticial/terapiaRESUMO
BACKGROUND: Tacrolimus is metabolized by members of the cytochrome p450 3A subfamily, and its bioavailability depends also on P-glycoprotein. We have observed that some patients admitted for infection presented with increased tacrolimus trough levels (TLs). The aim of the study was to assess the impact of infection on tacrolimus TLs and to determine the factors involved in TL fluctuations. METHODS: This retrospective cohort study included patients transplanted with a kidney between 2009 and 2011 who were hospitalized for an acute infection. Tacrolimus TLs and dosages were recorded before hospitalization, at admission, and 1 month after discharge. Increased levels of tacolimus were defined as TL 25% higher on admission than those recorded at the last visit before hospitalization. RESULTS: Seventy-seven patients were hospitalized 138 times for infection. More than two thirds of first hospitalizations occurred during the first post-transplant year. Causes of hospitalization were urinary (33%), cytomegalovirus (27%), digestive (15%), and pulmonary (12%) infections. Thirty-five percent of kidney transplant recipients had increased tacrolimus TLs (27/77 patients) in 24% of the hospitalizations (34/138). In 34 hospitalizations occurring in 27 patients, TL at admission was ≥25% compared with the last visit before admission. Comparing these 34 hospitalizations with the other 104, no significant differences were noted, except for a greater fraction of digestive infections in the group with elevated tacrolimus TLs, independent of diarrhea occurrence. CONCLUSIONS: Up to 35% of kidney transplant recipients admitted for acute infection present with high tacrolimus TLs, requiring a dose reduction. How acute infection precisely affects metabolism and bioavailability of tacrolimus remains to be investigated.
Assuntos
Imunossupressores/sangue , Infecções/metabolismo , Transplante de Rim , Tacrolimo/sangue , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Doença Aguda , Adulto , Idoso , Citocromo P-450 CYP3A/metabolismo , Feminino , Hospitalização , Humanos , Imunossupressores/metabolismo , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Tacrolimo/metabolismo , Tacrolimo/uso terapêuticoRESUMO
AIM: This report describes a case of kidney failure secondary to orlistat, a lipase inhibitor commonly used in the treatment of obesity. CASE REPORT: An 80-year-old man with type 2 diabetes who was being treated with orlistat developed rapidly progressive kidney failure. Low-grade albuminuria argued against diabetic nephropathy. Renal biopsy showed tubulointerstitial nephritis associated with numerous calcium oxalate crystals. Enteric hyperoxaluria was attributed to the orlistat treatment. The latter was stopped and the patient received calcium supplements. Six months after orlistat withdrawal, oxaluria was normalized and kidney function stabilized. CONCLUSION: Oxalate nephropathy may result from hyperoxaluria secondary to orlistat treatment. This suggests that kidney function and oxaluria be closely monitored in patients taking orlistat.
Assuntos
Fármacos Antiobesidade/efeitos adversos , Diabetes Mellitus Tipo 2/tratamento farmacológico , Nefropatias/induzido quimicamente , Lactonas/efeitos adversos , Idoso de 80 Anos ou mais , Fármacos Antiobesidade/uso terapêutico , Humanos , Hiperoxalúria , Lactonas/uso terapêutico , Masculino , OrlistateRESUMO
AIMS: The use of ultrapure dialysate decreases hemodialysis patients' morbidity. Bacterial and endotoxin content of reverse osmosis (RO) water is usually lowered or eliminated by a combination of bacterial filtration and regular disinfection of the distribution. Whether bacterial filtration may be replaced by UV irradiation is unknown. MATERIALS AND METHODS: One, and subsequently two UV lamps were inserted in a complex RO water distribution circuit, devoid of bacterial filters. RO water bacterial content was checked weekly between RO water distribution disinfections. RESULTS: With one UV lamp on the departure of the RO water circuit, bacterial results remained negative (< 1 cfu/ml) till the second week after disinfection. Disinfection of the circuit was required every four weeks to comply with the AAMI Guidelines (< 200 cfu/ml). Failure of the lamp was followed by bacterial growth (up to 500 cfu/ml), promptly aborted after replacement of the failed lamp. Subsequent addition of a second UV lamp on the return line of the water circuit kept bacteria < 1 cfu/ml for up to five weeks. Endotoxin levels remained < 0.125 EU. CONCLUSIONS: UV irradiation preserves a low RO water bacterial/endotoxin content in the distribution line and is not associated with a measurable endotoxin increase.
Assuntos
Desinfecção/métodos , Diálise Renal/instrumentação , Raios Ultravioleta , Água , Endotoxinas/análise , Endotoxinas/biossíntese , Osmose , Microbiologia da ÁguaRESUMO
AIMS: Depression and anxiety are so common in hemodialysis (HD) patients that we found it useful to study the respective contributions of the subjective somatic sensations and of the objective medical comorbidity to psychological distress. We also hypothesized that denial has a protective effect against anxiety and depression, and that alexithymia is, on the contrary, a risk factor. MATERIAL AND METHODS: In a cross-sectional design, we investigated relationships between psychological distress and somatic complaints, Charlson comorbidity index, denial and alexithymia, in a group of 54 patients on incenter HD. They filled psychometric self-rated questionnaires in (State Anxiety Inventory, Hospital Anxiety and Depression Scale, 13-item Short Beck Depression Inventory, Kidney Disease Quality of Life Short Form, 20-item Toronto Alexithymia Scale). A principal component analysis allowed us to focus on HADS-total score, which was confirmed to be representative of anxio-depression. Then, correlational analyses and a stepwise regression analysis were performed. RESULTS: HADS-total score is inversely associated with the use of denial as a psychological defence mechanism (p < 0.001), and positively correlated with difficulties in identifying emotions (p < 0.001), with difficulties in expressing feelings (p < 0.05), and with the intensity of subjective somatic complaints (p < 0.001). On the contrary, it is not related to the somatic comorbidity. In the stepwise regression, the somatic complaints, the denial and the difficulties in recognizing emotions emerge as the three main variables related to the HADS-total score (p < 0.001). CONCLUSIONS: Subjective physical complaints are here associated with psychological distress in chronic HD patients, while objective organic comorbidity does not seem to influence their mood and anxiety status. Denial is an efficient coping style against negative emotions, but it can diminish compliance. So, the subjective perception of the disease seems to have an important impact on the anxiety and mood levels, which can also be influenced by the emotional regulation abilities.
Assuntos
Transtornos de Ansiedade/etiologia , Transtorno Depressivo/etiologia , Falência Renal Crônica/psicologia , Transtornos Psicofisiológicos/etiologia , Diálise Renal/psicologia , Estresse Psicológico/etiologia , Adaptação Psicológica , Sintomas Afetivos/psicologia , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Negação em Psicologia , Feminino , Humanos , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Testes PsicológicosRESUMO
CONTEXT: Vascular calcification (VC) is prevalent and progressive in renal transplant recipients (RTRs). Recent cross-sectional data suggest that activated Wnt signaling contributes to VC. OBJECTIVE: The objective was to investigate whether circulating levels of the Wnt antagonist sclerostin associate with progression of VC. DESIGN: This was a post hoc analysis of the longitudinal observational Brussels Renal Transplant Cohort study. SETTING: The setting was a tertiary care academic hospital. PATIENTS: Coronary artery calcification and aorta calcification were measured by multislice spiral computerized tomography in 268 prevalent RTRs (age, 53 ± 13 y; 61% male) at baseline and remeasured in 189 patients after a median follow-up of 4.4 years. Baseline serum sclerostin levels were assessed on stored blood samples. Regression analysis was performed to identify determinants of baseline VC and progression. MAIN OUTCOME MEASURE: The main outcome measure was progression of VC. RESULTS: VC was present in up to 84% of participants at baseline. Almost half of the patients showed progression of VC, according to Hokanson criteria. The cross-sectional analysis at baseline demonstrated a direct association between sclerostin levels and VC score in univariate analysis, which became inverse after adjustment for age, gender and PTH level. Remarkably, a lower sclerostin level was identified as an independent determinant of a higher baseline aorta calcification score in the final regression model. Moreover, baseline sclerostin levels showed an inverse association with VC progression, at least after adjustment for traditional risk factors. CONCLUSIONS: Serum sclerostin levels inversely associated with VC burden and progression in prevalent RTRs after adjustment for traditional risk factors. Our data corroborate previous findings in nontransplanted chronic kidney disease patients and support the notion that sclerostin may be up-regulated in the vascular wall during the VC process as part of a local counterregulatory mechanism directed to suppress VC. Additional clinical and experimental data are required for confirmation.
Assuntos
Proteínas Morfogenéticas Ósseas/sangue , Transplante de Rim , Transplantados , Calcificação Vascular/sangue , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Aorta/patologia , Estudos de Coortes , Progressão da Doença , Feminino , Marcadores Genéticos , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Tomografia Computadorizada por Raios X , Calcificação Vascular/patologiaRESUMO
A new one-step culture-immunoassay procedure is described for testing cytokine production by immunocompetent cells in whole blood (WB) without the need for an isolation step. Briefly, WB samples or distilled water were added to RPMI medium containing specific anti-cytokine peroxidase-labelled monoclonal antibodies and incubated in micro-well plates coated with specific capture monoclonal antibodies, directed against distinct epitopes of the cytokine, and containing dried polyclonal activators (5.625 micrograms LPS + 1.125 micrograms PHA) or dried standards respectively. The optimalisation of the assay is described for an extended measurement range. The best compromise between sensitivity and linearity was obtained with the addition of 50 ng/well for TNF-alpha and IL-6 or 100 ng/well for IFN-gamma of unconjugate antibodies to the corresponding conjugate. The kinetics of individual production of each cytokine in WB of normal healthy donors showed values entering the standard range following incubation times of between 2 and 8 h for TNF-alpha, 2 and 4 h for IL-6, and 4 and 24 h for IFN-gamma. The sensitivity, the precision (intra-assay CVs) and the reproducibility (interassay CVs) of the assays were as follows: 70 pg/ml, < or = 14% and < or = 11% for TNF-alpha; 25 pg/ml, < or = 11% and < or = 16% for IL-6; 25 pg/ml, < or = 19% and < or = 20% for IFN-gamma. The accuracy (% of recovery) of the assays was in the order of 100% and between 40 and 60% in the absence or presence of polyclonal activators, reflecting the occurrence of an active production/consumption mechanism during the activation.
Assuntos
Citocinas/sangue , Imunoensaio/métodos , Linfócitos/imunologia , Adulto , Anticorpos Monoclonais , Células Cultivadas , Feminino , Humanos , Hibridomas , Interferon gama/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fator de Necrose Tumoral alfa/análiseRESUMO
Biological and biochemical characteristics of monoclonal antibodies (MABs) raised against human interleukin-10 (IL-10) are described as well as their use in the design of a specific ELISA for the measurement of the cytokine. 21 murine anti-human interleukin-10 (IL-10) MABs were obtained by fusion of splenocytes from mice immunized against human recombinant IL-10 with SP2/0 myelomatous cells. These antibodies define three major antigenic areas on the IL-10 molecule, one of which comprises epitopes involved in receptor binding and induction of biological activity. They recognize recombinant human IL-10 with affinities ranging from 1.3 x 10(-7) to 3 x 10(-11), as well as natural IL-10. Most of them also recognize viral IL-10 (vIL-10) encoded by the Epstein-Barr virus (EBV). A specific human-IL-10 ELISA has been developed using two MABs (18 and 19) as capture antibody and one MAB (17) as detector. The sensitivity (3 pg/ml), precision (intra-assays < 4%), reproducibility (interassay < 3%), and accuracy (recoveries, ranging between 84 and 107%, in several fluids) of the assay, plus its excellent performance in dilution tests, and the lack of interference when in the presence of possible cross-reactive substances, permits accurate cytokine measurement in biological fluids such as serum, plasma, bronchoalveolar lavage, urine and culture supernatants. Using the assay, IL-10 was measurable in the plasma of patients with septic shock (range 11-2740 pg/ml) whereas IL-10 plasma levels were < 7.8 pg/ml in healthy volunteers.
Assuntos
Anticorpos Monoclonais/imunologia , Interleucina-10/imunologia , Sequência de Bases , Bioensaio , Primers do DNA/química , Ensaio de Imunoadsorção Enzimática/métodos , Mapeamento de Epitopos , Humanos , Interleucina-10/análise , Dados de Sequência Molecular , Choque Séptico/sangueRESUMO
A new monoclonal antibody-based ELISA for leukaemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) measurements is described. The sensitivity (56 pg/ml after 4 h incubation, 14 pg/ml after 24 h incubation), precision (intra-assays < 5%), reproducibility (interassay < 10%), and accuracy (recoveries, ranging between 98 and 119%, in several fluids) of the assay, plus its excellent performance in dilution tests, and the lack of interference when in the presence of possible cross-reactive substances guarantee accurate cytokine measurement in biological fluids such as serum, plasma, synovial fluid, follicular fluid, urine and culture supernatants. Using the assay, LIF/HILDA was measurable in supernatants after in vitro whole blood stimulation with phytohemagglutinin (PHA), OKT3, and phorbol myristate acetate (PMA) but not with lipopolysaccharide (LPS) or Ca ionophore. LIF/HILDA production was not measurable until after 24 h of culture, when cytokine levels were seen to increase linearly in the supernatant to reach values of up to 40 ng/ml after 96 h of culture. Finally, a good correlation was found (r = 0.96; p < 0.0001; y = 23.1x + 233) between the LIF/HILDA values obtained using the ELISA and DA-1a bioassay.