RESUMO
The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several in vitro/ex vivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel in vitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.
Assuntos
Fator H do Complemento , Receptores de Complemento 3b , Proteínas Recombinantes de Fusão , Humanos , Fator H do Complemento/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/química , Fator H do Complemento/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Ativação do Complemento/efeitos dos fármacos , Antígenos CD55/genética , Antígenos CD55/metabolismo , Hemólise/efeitos dos fármacos , Via Alternativa do Complemento/efeitos dos fármacos , Inativadores do Complemento/farmacologia , Eritrócitos/metabolismoRESUMO
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.
Assuntos
Antígeno B7-H1/metabolismo , Ativação do Complemento/imunologia , Complemento C3/antagonistas & inibidores , Complemento C5/antagonistas & inibidores , Hemoglobinúria Paroxística/patologia , Antígeno B7-H1/sangue , Antígenos CD55/genética , Antígenos CD59/genética , Complemento C3/imunologia , Complemento C5/imunologia , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/citologia , Hemoglobinúria Paroxística/imunologia , Humanos , Evasão da Resposta Imune/imunologia , Proteínas de Membrana/genética , Monócitos/metabolismoRESUMO
OBJECTIVES: Immune checkpoints play an important role in maintaining the balance of the immune system and in the development of autoimmune diseases. A central checkpoint molecule is the programmed cell death protein 1 (PD-1, CD279) which is typically located on the surface of T cells. Its primary ligand PD-L1 is expressed on antigen presenting cells and on cancer cells. Several variants of PD-L1 exist, among these soluble molecules (sPD-L1) present in serum at low concentrations. sPD-L1 was found elevated in cancer and several other diseases. sPD-L1 in infectious diseases has received relatively little attention so far and is therefore subject of this study. METHODS: sPD-L1 serum levels were determined in 170 patients with viral infections (influenza, varicella, measles, Dengue fever, SARS-CoV2) or bacterial sepsis by ELISA and compared to the levels obtained in 11 healthy controls. RESULTS: Patients with viral infections and bacterial sepsis generally show significantly higher sPD-L1 serum levels compared to healthy donors, except for varicella samples where results do not reach significance. sPD-L1 is increased in patients with impaired renal function compared to those with normal renal function, and sPD-L1 correlates significantly with serum creatinine. Among sepsis patients with normal renal function, sPD-L1 serum levels are significantly higher in Gram-negative sepsis compared to Gram-positive sepsis. In addition, in sepsis patients with impaired renal function, sPD-L1 correlates positively with ferritin and negatively with transferrin. CONCLUSIONS: sPD-L1 serum levels are significantly elevated in patients with sepsis, influenza, mesasles, Dengue fever or SARS-CoV2. Highest levels are detectable in patients with measles and Dengue fever. Also impaired renal function causes an increase in levels of sPD-L1. As a consequence, renal function has to be taken into account in the interpretation of sPD-L1 levels in patients.
Assuntos
Varicela , Dengue , Influenza Humana , Sarampo , Sepse , Humanos , Antígeno B7-H1/metabolismo , Doadores de Sangue , RNA Viral , Rim/fisiologia , PrognósticoRESUMO
BACKGROUND: In recent years, there has been increasing evidence that asthma is associated with atherosclerosis and cardiovascular disease. However, data in children and adolescents are scarce and conflicting. We aimed to assess the impact of asthma with and without an allergic component on the carotid intima-media thickness in a large pediatric population. METHODS: The community-based early vascular ageing-Tyrol cohort study was performed between May 2015 and July 2018 in North, East (Austria) and South Tyrol (Italy) and recruited youngster aged 14 years and above. Medical examinations included anthropometric measurements, fasting blood analysis, measurement of the carotid intima-media thickness by high-resolution ultrasound, and a physician guided interview. RESULTS: The mean age of the 1506 participants was 17.8 years (standard deviation 0.90). 851 (56.5%) participants were female. 22 subjects had a physician diagnosis of non-allergic asthma, 268 had inhalative allergies confirmed by a positive radio-allergo-sorbent-test and/or prick test, and 58 had allergic asthma. Compared to healthy controls, participants with non-allergic asthma (411.7 vs. 411.7 µm; p = 0.932) or inhalative allergy (420.0 vs. 411.7 µm; p = 0.118) did not have significantly higher carotid intima-media thickness (cIMT). However, participants with allergic asthma had significantly higher cIMT (430.8 vs. 411.7; p = 0.004) compared to those without and this association remained significant after multivariable adjustment for established cardiovascular risk factors. CONCLUSION: Allergic asthma in the youth is associated with an increased carotid intima-media thickness. Physicians should therefore be aware of allergic asthma as a potential cardiovascular risk factor in children and adolescents. Trial Registration Number The EVA-Tyrol Study has been retrospectively registered at clinicaltrials.gov under NCT03929692 since April 29, 2019.
Assuntos
Envelhecimento/fisiologia , Asma/complicações , Doenças Cardiovasculares/etiologia , Artérias Carótidas/diagnóstico por imagem , Espessura Intima-Media Carotídea , Adolescente , Asma/diagnóstico , Asma/epidemiologia , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/epidemiologia , Criança , Feminino , Humanos , Incidência , Masculino , Estudos Prospectivos , Fatores de Risco , Taxa de Sobrevida/tendênciasRESUMO
OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections cause coronavirus disease 2019 (COVID-19) and induce a specific antibody response. Serological assays detecting IgG against the receptor binding domain (RBD) of the spike (S) protein are useful to monitor the immune response after infection or vaccination. The objective of our study was to evaluate the clinical performance of the Siemens SARS-CoV-2 IgG (sCOVG) assay. METHODS: Sensitivity and specificity of the Siemens sCOVG test were evaluated on 178 patients with SARS-CoV-2-infection and 160 pre-pandemic samples in comparison with its predecessor test COV2G. Furthermore, correlation with virus neutralization titers was investigated on 134 samples of convalescent COVID-19 patients. RESULTS: Specificity of the sCOVG test was 99.4% and sensitivity was 90.5% (COV2G assay 78.7%; p<0.0001). S1-RBD antibody levels showed a good correlation with virus neutralization titers (r=0.843; p<0.0001) and an overall qualitative agreement of 98.5%. Finally, median S1-RBD IgG levels increase with age and were significantly higher in hospitalized COVID-19 patients (median levels general ward: 25.7 U/mL; intensive care: 59.5 U/mL) than in outpatients (3.8 U/mL; p<0.0001). CONCLUSIONS: Performance characteristics of the sCOVG assay have been improved compared to the predecessor test COV2G. Quantitative SARS-CoV-2 S1-RBD IgG levels could be used as a surrogate for virus neutralization capacity. Further harmonization of antibody quantification might assist to monitor the humoral immune response after COVID-19 disease or vaccination.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/diagnóstico , Imunoglobulina G/imunologia , Testes de Neutralização , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/patologia , COVID-19/virologia , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Subunidades Proteicas/imunologia , Kit de Reagentes para Diagnóstico , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Adulto JovemRESUMO
OBJECTIVES: Serological tests detect antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in the ongoing coronavirus disease-19 (COVID-19) pandemic. Independent external clinical validation of performance characteristics is of paramount importance. METHODS: Four fully automated assays, Roche Elecsys Anti-SARS-CoV-2, Abbott SARS-CoV-2 IgG, Siemens SARS-CoV-2 total (COV2T) and SARS-CoV-2 IgG (COV2G) were evaluated using 350 pre-pandemic samples and 700 samples from 245 COVID-19 patients (158 hospitalized, 87 outpatients). RESULTS: All tests showed very high diagnostic specificity. Sensitivities in samples collected at least 14 days after disease onset were slightly lower than manufacturers' claims for Roche (93.0%), Abbott (90.8%), and Siemens COV2T (90.3%), and distinctly lower for Siemens COV2G (78.8%). Concordantly negative results were enriched for immunocompromised patients. ROC curve analyses suggest a lowering of the cut-off index for the Siemens COV2G assay. Finally, the combination of two anti-SARS-CoV-2 antibody assays is feasible when considering borderline reactive results. CONCLUSIONS: Thorough on-site evaluation of commercially available serologic tests for detection of antibodies against SARS-CoV-2 remains imperative for laboratories. The potentially impaired sensitivity of the Siemens COV2G necessitates a switch to the company's newly filed SARS-CoV-2 IgG assay for follow-up studies. A combination of tests could be considered in clinical practice.
Assuntos
Anticorpos Antivirais/sangue , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Imunoglobulina G/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/epidemiologia , Feminino , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Pandemias , Curva ROC , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Adulto JovemRESUMO
Eculizumab inhibits the terminal, lytic pathway of complement by blocking the activation of the complement protein C5 and shows remarkable clinical benefits in certain complement-mediated diseases. However, several reports suggest that activation of C5 is not always completely suppressed in patients even under excess of eculizumab over C5, indicating that residual C5 activity may derogate the drug's therapeutic benefit under certain conditions. By using eculizumab and the tick-derived C5 inhibitor coversin, we determined conditions ex vivo in which C5 inhibition is incomplete. The degree of such residual lytic activity depended on the strength of the complement activator and the resulting surface density of the complement activation product C3b, which autoamplifies via the alternative pathway (AP) amplification loop. We show that at high C3b densities required for binding and activation of C5, both inhibitors reduce but do not abolish this interaction. The decrease of C5 binding to C3b clusters in the presence of C5 inhibitors correlated with the levels of residual hemolysis. However, by employing different C5 inhibitors simultaneously, residual hemolytic activity could be abolished. The importance of AP-produced C3b clusters for C5 activation in the presence of eculizumab was corroborated by the finding that residual hemolysis after forceful activation of the classical pathway could be reduced by blocking the AP. By providing insights into C5 activation and inhibition, our study delivers the rationale for the clinically observed phenomenon of residual terminal pathway activity under eculizumab treatment with important implications for anti-C5 therapy in general.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Ativação do Complemento/efeitos dos fármacos , Complemento C5/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/uso terapêutico , Complemento C3b/imunologia , Complemento C5/imunologia , Via Alternativa do Complemento/efeitos dos fármacos , Hemoglobinúria Paroxística/tratamento farmacológico , Hemoglobinúria Paroxística/imunologia , Hemólise/efeitos dos fármacos , HumanosRESUMO
CONCLUSIONS: This update of the CD59 blood group system (Weinstock C, Anliker M, von Zabern I. CD59: a long-known complement inhibitor has advanced to a blood group system. Immunohematology 2015;31:145-51) increases the number of reported patients with CD59 deficiency from 10 to 14. All of these 14 patients suffered from severe illness. Recently, a new variant allele was found in heterozygosity. Flow cytometry data suggest that this variant was expressed on the red blood cells of the propositus. Although additional alleles have been found, the CD59 system (International Society of Blood Transfusion system 35) continues to have one antigen.
Assuntos
Antígenos de Grupos Sanguíneos/análise , Anemia Hemolítica , Antígenos CD59 , Eritrócitos , Citometria de Fluxo , Hemoglobinúria , HumanosRESUMO
BACKGROUND: Therapeutic intervention strategies in complement-mediated hemolytic diseases are still inappropriate, and lethal events cannot be reliably prevented. As an in vitro model of intravascular hemolysis, a sensitive flow cytometric assay was designed using red blood cells (RBCs) of patients with paroxysmal nocturnal hemoglobinuria (PNH) as target cells. Complement activation by human allo- and autoantibodies directed against RBC antigens and the effect of different complement inhibitors were studied. STUDY DESIGN AND METHODS: RBCs of patients with a PNH III RBC clone of more than 20% were coated with different human allo- or autoantibodies. Hemolysis was initiated with pooled normal human AB serum with or without the addition of complement inhibitors. Loss of PNH III RBCs was estimated by flow cytometry. RESULTS: RBC antibodies of 174 different patients representing 37 different specificities were tested for their potency to activate complement. In correlation with blood group specificities roughly three different patterns were observed: 1) strong and regular, 2) sporadic, and 3) weak or absent complement activation. Remarkably strong complement activators were among antibodies directed against high-prevalence blood group antigens. The C5 inhibitor eculizumab abrogated mild but not strong complement activation, even in presence of excess inhibitor. However, this residual complement activity could be further depressed by combining eculizumab with other inhibitors. CONCLUSION: The PNH hemolysis assay offers a sensitive tool for in vitro analyses of classical pathway-mediated complement activation. The recognition of additive effects of complement inhibitors may guide novel intervention strategies against unwanted complement damage.
Assuntos
Autoanticorpos/imunologia , Ativação do Complemento/imunologia , Inativadores do Complemento/farmacologia , Eritrócitos/imunologia , Citometria de Fluxo , Hemoglobinúria Paroxística/imunologia , Hemólise/imunologia , Isoanticorpos/imunologia , Ativação do Complemento/efeitos dos fármacos , Eritrócitos/patologia , Feminino , Hemoglobinúria Paroxística/patologia , Hemólise/efeitos dos fármacos , Humanos , MasculinoAssuntos
COVID-19 , Vacinas , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Monócitos/metabolismo , RNA Viral/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , COVID-19/prevenção & controle , SARS-CoV-2 , Vacinas/metabolismo , Granulócitos/metabolismo , VacinaçãoRESUMO
The serum proteins factor H (FH), consisting of 20 complement control protein modules (CCPs), and its splice product FH-like protein 1 (FHL-1; consisting of CCPs 1-7) are major regulators of the alternative pathway (AP) of complement activation. The engineered version of FH, miniFH, contains only the N- and C-terminal portions of FH linked by an optimized peptide and shows â¼ 10-fold higher ex vivo potency. We explored the hypothesis that regulatory potency is enhanced by unmasking of a ligand-binding site in the C-terminal CCPs 19-20 that is cryptic in full-length native FH. Therefore, we produced an FH variant lacking the central domains 10-15 (FHΔ10-15). To explore how avidity affects regulatory strength, we generated a duplicated version of miniFH, termed midiFH. We compared activities of FHΔ10-15 and midiFH to miniFH, FH, and FHL-1. Relative to FH, FHΔ10-15 exhibited an altered binding profile toward C3 activation products and a 5-fold-enhanced complement regulation on a paroxysmal nocturnal hemoglobinuria patient's erythrocytes. Contrary to dogma, FHL-1 and FH exhibited equal regulatory activity, suggesting that the role of FHL-1 in AP regulation has been underestimated. Unexpectedly, a substantially increased avidity for complement opsonins, as seen in midiFH, did not potentiate the inhibitory potential on host cells. In conclusion, comparisons of engineered and native FH-based regulators have identified features that determine high AP regulatory activity on host cells. Unrestricted availability of FH CCPs 19-20 and an optimal spatial orientation between the N- and C-terminal FH regions are key.
Assuntos
Proteínas Inativadoras do Complemento C3b/imunologia , Fator H do Complemento/imunologia , Inativadores do Complemento/farmacologia , Via Alternativa do Complemento/imunologia , Proteínas Recombinantes/farmacologia , Sequência de Aminoácidos , Fator H do Complemento/química , Inativadores do Complemento/síntese química , Inativadores do Complemento/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/imunologiaRESUMO
The blood group system number 35 is based on CD59, a 20-kDa membrane glycoprotein present on a large number of different cells, including erythrocytes. The major function of CD59 is to protect cells from complement attack. CD59 binds to complement components CS and C9 and prevents the polymerization of C9, which is required for the formation of the membrane attack complex (MAC). Other functions of CD59 in cellular immunity are less well defined. CD59 is inserted into the membrane by a glycosylphosphatidylinositol (GPI) anchor. A defect of this anchor causes lack of this protein from the cell membrane, which leads to an enhanced sensitivity towards complement attack. Patients with paroxysmal nocturnal hemoglobinuria (PNH) harbor a varying percentage of red blood cell clones with a defect in GPI-anchored proteins, including CD59. The most characteristic symptoms of this disease are episodes of hemolysis and thromboses. Although CD59 has been classified as a membrane protein for more than 25 years, an alloantibody directed against CD59 was found only recently. So far, the first and sole alloantibody described was detected in a CD59-deficient child. In 2014, CD59 received the status of a blood group system by the International Society for Blood Transfusion Red Cell Immunogenetics and Blood Group Terminology Working Party. Among a variety of almost 20 synonyms, the designation CD59 was chosen for the blood group system and CD59.l for the wild-type protein. The only three alleles published to date are null alleles. All CD59-deficient individuals recognized so far were severely ill, two of whom have died. Most of the reported cases present with a typical clinical picture within the first year of life that includes neuropathy, strokes, and mild Coombs-negative hemolysis. In one published case, the application of the complement inhibitor eculizumab caused a pronounced improvement of the clinical situation.
Assuntos
Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/imunologia , Antígenos CD59/química , Antígenos CD59/imunologia , Inativadores do Complemento/química , Inativadores do Complemento/imunologia , HumanosRESUMO
The resident assessment instrument (RAI) as a standardized assessment has become established as the gold standard in homes for the elderly and resident nursing homes in Switzerland. The RAI is an important tool with respect to the clarification of needs in long-term outpatient and inpatient nursing care in compliance with the requirements of the Health Insurance Act. The information collated in the initial assessment, the minimum data set (MDS), can be reliably utilized for the nursing process and measurement of quality. The key to success in long-term inpatient nursing can be attributed to the generalized financial solution of resource utilization groups (RUG), which is generated directly from the assessment. Due to standardized training and documentation and in the inpatient area, a uniform electronic data processing solution, a vast amount of data from the last 15 years of experience are available also for scientific use.
Assuntos
Avaliação Geriátrica/métodos , Geriatria/normas , Cuidados de Enfermagem/normas , Casas de Saúde/normas , Guias de Prática Clínica como Assunto , Inquéritos e Questionários/normas , Idoso , Idoso de 80 Anos ou mais , Feminino , Serviços de Assistência Domiciliar , Humanos , Masculino , Planejamento de Assistência ao Paciente/normas , Garantia da Qualidade dos Cuidados de Saúde/normas , Suíça , Estados UnidosRESUMO
BACKGROUND: CD59 is a cell surface glycoprotein of approximately 20 kDa limiting the lytic activity of the terminal complement complex C5b-9. Although CD59 is known as a red blood cell (RBC) antigen defined by monoclonal antibodies, it so far has not been identified as a blood group antigen, since the description of a human alloantibody was missing. In this study we show the presence of an anti-CD59 in a patient affected by a homozygous CD59 deficiency. STUDY DESIGN AND METHODS: RBC CD59 and CD55 were determined by flow cytometry or by the column agglutination technique using monoclonal antisera. Commercially available His-tagged recombinant soluble CD59 protein was used to inhibit anti-CD59. RESULTS: Seven cases of an isolated CD59 deficiency due to three distinct null alleles of the CD59 gene have been published so far. Recently we described the CD59-null allele c.146delA in a young child of heterozygous parents. Her plasma contained an alloantibody directed against the high-prevalence RBC antigen CD59. The antibody specificity was identified using soluble recombinant human CD59 protein, which blocked the reactivity of the patient's antibody and of monoclonal anti-CD59 but not of monoclonal anti-CD55. In addition, RBC alloantibodies such as anti-K, anti-C, anti-c, or anti-Fy(a) remained unaffected. Therefore, inhibition by recombinant CD59 is a useful diagnostic tool to detect alloantibodies in the presence of anti-CD59. CONCLUSION: This is the first demonstration of a human anti-CD59 alloantibody, which defines CD59 as an RBC blood group antigen. CD59 represents a candidate for a new blood group system.
Assuntos
Anemia Hemolítica/imunologia , Antígenos de Grupos Sanguíneos/imunologia , Antígenos CD59/imunologia , Hemoglobinúria/imunologia , Isoanticorpos/sangue , Anemia Hemolítica/genética , Antígenos de Grupos Sanguíneos/genética , Antígenos CD59/genética , Pré-Escolar , Feminino , Mutação da Fase de Leitura , Hemoglobinúria/genética , HumanosRESUMO
Post-hepatectomy liver failure (PHLF) remains the major contributor to death after liver resection. Oxidative stress is associated with postoperative complications, but its impact on liver function is unclear. This first in-human, prospective, single-center, observational pilot study evaluated perioperative oxidative stress and PHLF according to the ISGLS (International Study Group for Liver Surgery). Serum 8-isoprostane, 4-hydroxynonenal (4-HNE), total antioxidative capacity, vitamins A and E, and intraoperative, sequential hepatic tissue 4-HNE and UCP2 (uncoupling protein 2) immunohistochemistry (IHC) were assessed. The interaction with known risk factors for PHLF and the predictive potential of oxidative stress markers were analyzed. Overall, 52 patients were included (69.2% major liver resection). Thirteen patients (25%) experienced PHLF, a major factor for 90-day mortality (23% vs. 0%; p = 0.013). Post-resection, pro-oxidative 8-isoprostane significantly increased (p = 0.038), while 4-HNE declined immediately (p < 0.001). Antioxidative markers showed patterns of consumption starting post-resection (p < 0.001). Liver tissue oxidative stress increased stepwise from biopsies taken after laparotomy to post-resection in situ liver and resection specimens (all p < 0.001). Cholangiocarcinoma patients demonstrated significantly higher serum and tissue oxidative stress levels at various timepoints, with consistently higher preoperative values in advanced tumor stages. Combining intraoperative, post-resection 4-HNE serum levels and in situ IHC early predicted PHLF with an AUC of 0.855 (63.6% vs. 0%; p < 0.001). This was also associated with grade B/C PHLF (36.4% vs. 0%; p = 0.021) and 90-day mortality (18.2% vs. 0%; p = 0.036). In conclusion, distinct patterns of perioperative oxidative stress levels occur in patients with liver dysfunction. Combining intraoperative serum and liver tissue markers predicts subsequent PHLF. Cholangiocarcinoma patients demonstrated pronounced systemic and hepatic oxidative stress, with increasing levels in advanced tumor stages, thus representing a worthwhile target for future exploratory and therapeutic studies.
RESUMO
Febrile neutropenia secondary to chemotherapy is one of the most critical complications in cancer treatment. The aim of this study was to determine if an increase in the percentage of immature platelet fraction (IPF%) might predict early neutrophil recovery following cytostatic-dependent aplasia. A retrospective cohort study compared serial complete blood counts and the level of C-reactive protein (CRP) following induction chemotherapy for Ewing sarcoma and Non-Ewing sarcoma patients. The measurements were taken on a Sysmex XE-2100 instrument. A total of 287 paired samples from 28 children after the first cycle of chemotherapy were analyzed to test if an increase in the IPF% anticipated the CRP peak and recovery of neutrophil count. The chemotherapy associated nadir of neutrophils, reticulocytes and platelets was reached at 9.7 ± 1.5, 11.0 ± 1.7 and 11.9 ± 0.9 days (mean ± SD) respectively, in Ewing sarcoma patients. Still in severe neutropenia, IPF% was the first parameter that significantly increased and anticipated the CRP peak (11.9 ± 1.6 days, mean ± SD). The IPF% continuously increased (maximum = 6.56% ± 2.8%, mean ± SD) and peaked at 12.2 ± 1.4 days (mean ± SD) after commencement of chemotherapy. Compared to neutrophil recovery (14.6 ± 1.4 days, mean ± SD), the IPF% peak was anticipated by 2.4 days (p = 0.0085). Although variably treated, in non-Ewing sarcoma patients the effect was similar and the IPF% peak anticipated neutrophil recovery by 6.8 ± 4.7 days (p < 0.01). IPF% increased significantly at > 48 h before neutrophil recovery in patients treated with chemotherapy. IPF% is an inexpensive parameter and may be valuable in the management of febrile neutropenia.
Assuntos
Neutropenia Febril , Tumores Neuroectodérmicos Primitivos Periféricos , Sarcoma de Ewing , Criança , Humanos , Medula Óssea , Estudos Retrospectivos , Plaquetas , Sarcoma de Ewing/tratamento farmacológico , Proteína C-Reativa , VerdurasRESUMO
INTRODUCTION: The objective of the present study was to evaluate and compare the validity and utility of two fully automated ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity assays for clinical diagnostic decision-making and to compare their performance. METHODS: Two automated ADAMTS13 activity assays (Werfen HemosIL® AcuStar ADAMTS13 Activity, Technoclone Technofluor ADAMTS13 Activity) were compared with a manual FRET assay (BioMedica ACTIFLUOR ADAMTS13 Activity). The following samples were used: 13 acute phase TTP (thrombotic thrombocytopenic purpura) samples from 11 different patients, one sample from a patient with congenital ADAMTS13 deficiency, 16 samples from control patients, three follow-up samples from TTP patients in long-term remission and one sample from a patient with stem cell transplantation related thrombotic microangiopathy (TMA). The WHO 1st International Standard for ADAMTS13 and several dilutions of normal plasma with ADAMTS13-depleted normal plasma were also tested. Statistical analysis included descriptive statistics, sensitivity and specificity, Passing & Bablok regression and Bland-Altman plot. RESULTS: The quantitative comparison between the HemosIL® (x) and Technofluor (y) methods showed a strong correlation (Pearson r = 0.98, n = 49). When considering an ADAMTS13 activity of <10% as a hallmark for the diagnosis of TTP, two fully automated assays were both able to identify all TTP- and non-TTP-samples correctly, resulting in sensitivities and specificities of 100%. CONCLUSION: Both fully automated ADAMTS13 activity assays showed a good diagnostic performance and quantitative correlation among themselves, discriminating reliably between TTP- and non-TTP-patients.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Púrpura Trombocitopênica Trombótica , Microangiopatias Trombóticas , Humanos , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Transferência Ressonante de Energia de Fluorescência , Púrpura Trombocitopênica Trombótica/diagnósticoRESUMO
PURPOSE: Standard therapy for lupus nephritis is based on non-specific immunosuppression. We aimed to identify specific alterations in T cell and cytokine homeostasis and possible associations with disease activity in children with lupus nephritis (LN). METHODS: The phenotype of circulating T cells from children with LN and healthy controls (HC) was analyzed by flow cytometry. Intracellular expression of IL-17 and INF-γ was assessed after stimulation with anti-CD3 and anti-CD28. Serum concentrations of IP10, CCL2, TGF-ß, IL-17, and IL-23 were measured by ELISA. Disease activity was determined using the Systemic Lupus Erythematosus Disease Activity Index 2000 update (SLEDAI-2K). RESULTS: Children with active LN displayed increased frequencies of effector memory CD4(+)CD45RO(+)CCR7(-) and terminal differentiated CD4(+)CD45RA(+)CCR7(-) T cells and reduced naive CD4(+)CD45RA(+)CCR7(+) T cells compared to those with inactive LN or HC. Circulating CD4(+)CXCR3(+) and CD4(+)CCR2(+) T cells correlated inversely with the renal SLEDAI-2K, whereas IP10 and CCL2 showed a positive correlation. Reduced CD4(+)Foxp3(+) T cells and serum TFG-ß levels in active LN were associated with high serum IL-17 and IL-23 levels and correlated inversely with the renal SLEDAI-2K (r = -0.5855, p = 0.0013 and r = -0.6246, p = 0.0005, respectively), whereas IL-17 and IL-23 correlated positively (r = 0.5516, p = 0.0029 and r = 0.6116, p = 0.0007, respectively). Expansion of Th17 and Th1/Th17 cells in children with LN was significantly greater than in HC (p = 0.0304 and p = 0.0067, respectively). CONCLUSION: Children with active LN display high levels of pro-inflammatory cytokines associated with an increase in effector T cells and reduction of regulatory T cells. Therapeutic regulation of the aberrant cytokine profile might specifically interrupt pathogenic mechanisms.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Nefrite Lúpica/imunologia , Adolescente , Quimiocina CCL2/imunologia , Criança , Citocinas/sangue , Feminino , Homeostase , Humanos , Imunofenotipagem , Leucócitos Mononucleares/imunologia , Nefrite Lúpica/sangue , Masculino , Receptores CCR2/imunologia , Receptores CXCR3/imunologia , Receptores de Citocinas/imunologiaRESUMO
PURPOSE: We performed a single-day cross-sectional study to assess the prevalence of vitamin D deficiency as well as folate status in healthy young female volunteers well educated with respect to health information. METHODS: We assessed dietary intake of vitamin D and calcium, serum concentrations of 25-OH-vitamin D(3), folate, red blood cell folate and other dietary, laboratory, and lifestyle parameters in 215 young healthy women (age 18-30 years) on a single day at the end of the winter months. Primary aim was to investigate the prevalence of hypovitaminosis D. Folic acid status was a secondary study aim. RESULTS: Mean daily ingestion of vitamin D was 2.25 µg/day with a daily calcium intake of 749 mg/day. 6.9% had hypovitaminosis D (25-OH-vitamin D(3) <30 nmol/L) and 89.3% were vitamin D insufficient (<75 nmol/L). Preplanned subpopulation comparison (lower vs. upper quartile) revealed a significant negative correlation (P = 0.048) between plasma PTH and 25-OH-vitamin D(3) levels. Fifteen individuals (6.9%) were folic acid deficient (<140 ng/mL RBC folate). Only 9.3% reached RBC folate concentrations regarded as optimal for the prevention of fetal neural tube defects (>400 ng/mL). CONCLUSIONS: The prevalence of hypovitaminosis D in healthy young women trained in health care professions is low but 89.3% can be classified as vitamin D insufficient in spring. Folate status can also be considered not sufficient. Considering the emerging role of higher vitamin D plasma levels for many health conditions, a timely correction of vitamin D status in the general Austrian population appears appropriate.