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1.
Med Mycol ; 59(5): 505-509, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33336238

RESUMO

Activation of mucosal-associated invariant T cells (MAIT cells) by certain bacteria, viruses, and yeast is well studied, but the activation potential of filamentous moulds from the order Mucorales is not known. Here, we show a rapid response of human MAIT cells against the Mucorales species Mucor circinelloides, Rhizopus arrhizus, and Rhizopus microsporus. This activation included upregulation of CD69 and degranulation marked by increased CD107a expression, while intracellular perforin and granzyme A expression were reduced. Furthermore, blocking of the antigen-presenting molecule major histocompatibility complex class I-related abrogated MAIT cell activation demonstrating a T cell receptor-dependent stimulation by Mucorales.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Mucorales/imunologia , Mucormicose/imunologia , Mucormicose/metabolismo , Células T Invariantes Associadas à Mucosa/imunologia , Células T Invariantes Associadas à Mucosa/metabolismo , Riboflavina/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Granzimas/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Mucor/imunologia , Mucormicose/microbiologia , Perforina/metabolismo , Rhizopus/imunologia , Rhizopus oryzae/imunologia , Regulação para Cima
2.
Eur J Immunol ; 48(10): 1698-1706, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30059139

RESUMO

Mucosal associated invariant T cells (MAIT cells) are innate-like T cells (TC) which are known to be activated by several bacteria and viruses. However, activation of MAIT cells by moulds, such as the opportunistic human pathogen Aspergillus, is not well described. Stimulation of human PBMC with A. fumigatus, A. flavus, or A. terreus conidia revealed that in contrast to conventional CD4+ and CD8+ TC, MAIT cells responded already after 4 h of coincubation with upregulation of CD69. Furthermore, concurrent increase of CD107a expression and reduced intracellular expression of cytolytic proteins like perforin and granzyme indicated degranulation of intracellular vesicles. MAIT cell activation only occurred in the presence of APC and was dependent on cell-cell contact as separation of TC and APC abrogated MAIT cell activation. Furthermore, we observed that MAIT cell activation by moulds requires presentation of riboflavin metabolites and depends on TCR engagement as antibody blocking of MR1, the antigen presenting molecule for MAIT cells, prevented upregulation of CD69 and CD107a. In summary, we could demonstrate that MAIT cells are activated by Aspergillus conidia in a TCR-dependent manner by APC. These findings reveal MAIT cells as an interesting new target in antifungal defense.


Assuntos
Apresentação de Antígeno , Aspergillus/imunologia , Ativação Linfocitária , Células T Invariantes Associadas à Mucosa/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Células Cultivadas , Granzimas/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Lectinas Tipo C/genética , Proteína 1 de Membrana Associada ao Lisossomo/genética , Perforina/genética , Esporos Fúngicos/imunologia
3.
Cytometry A ; 95(3): 332-338, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30450827

RESUMO

Systemic infections with the opportunistic mold Aspergillus fumigatus are a great threat to immunocompromised patients such as transplant recipients. Immunological research on A. fumigatus involves the measurement of phagocytosis of fungal conidia (spores) by human phagocytes. Here, we present a fast and flexible way to analyze phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC) labeling of conidia prior to co-incubation with human leukocytes and an anti-FITC counterstaining step postincubation to allow the discrimination of internalized and adherent conidia. In contrast to many other protocols, this method can be combined with further surface marker analyses. We sought to determine phagocytosis rates of A. fumigatus conidia in different stages and after several incubation times using this method. Moreover, we provide an example of application by comparing phagocytosis of A. fumigatus mutants to the wild type. © 2018 International Society for Advancement of Cytometry.


Assuntos
Aspergillus fumigatus/metabolismo , Citometria de Fluxo/métodos , Fluoresceína-5-Isotiocianato/análise , Leucócitos/imunologia , Fagocitose/imunologia , Esporos Fúngicos/metabolismo , Aspergilose/imunologia , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Fluoresceína-5-Isotiocianato/química , Fluorescência , Humanos , Leucócitos/microbiologia , Fagocitose/genética
4.
Biochim Biophys Acta ; 1861(11): 1605-1613, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27477678

RESUMO

Cysteinyl leukotrienes (cys-LTs) cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A4 (LTA4) that is subsequently conjugated with glutathione (GSH) by LTC4 synthase (LTC4S). Cys-LT receptor antagonists and LTC4S inhibitors have been developed, but only the former have reached the market. High structural homology to related enzymes and lack of convenient test systems due to instability of added LTA4 have hampered the development of LTC4S inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA4 that allow studying LTC4S activity and investigating LTC4S inhibitors. Co-incubations of microsomes from HEK293 cells expressing LTC4S with isolated 5-LOX efficiently converted exogenous AA to LTC4 (~1.3µg/200µg protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC4S with Ca2+-ionophore A23187 and 20µM AA resulted in strong LTC4 formation (~250ng/106 cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTC4S, consistently inhibited LTC4 formation in all assay types (IC50=3.1-3.5µM) and we successfully confirmed TK04a as potent LTC4S inhibitor in these assay systems (IC50=17 and 300nM, respectively). We demonstrated transcellular LTC4 biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA4 from AA and HEK293 cells expressing LTC4S that transform LTA4 to LTC4. In conclusion, our assay approaches are advantageous as the substrate LTA4 is generated in situ and are suitable for studying enzymatic functionality of LTC4S including site-directed mutations and evaluation of LTC4S inhibitors.


Assuntos
Bioensaio/métodos , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Sistema Livre de Células , Cromatografia Líquida de Alta Pressão , Células HEK293 , Humanos , Leucotrieno C4/metabolismo , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Espectrometria de Massas em Tandem
5.
FASEB J ; 30(1): 276-85, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26396238

RESUMO

5-Lipoxygenase (5-LO) catalyzes the initial steps in the biosynthesis of proinflammatory leukotrienes. Upon cell activation, 5-LO translocates to the nuclear membrane where arachidonic acid is transferred by 5-LO-activating protein (FLAP) to 5-LO for metabolism. Although previous data indicate association of 5-LO with FLAP, the in situ assembly of native 5-LO/FLAP complexes remains elusive. Here, we show time-resolved 5-LO/FLAP colocalization by immunofluorescence microscopy and in situ 5-LO/FLAP interaction by proximity ligation assay at the nuclear membrane of Ca(2+)-ionophore A23187-activated human monocytes and neutrophils in relation to 5-LO activity. Although 5-LO translocation and product formation is completed within 1.5-3 min, 5-LO/FLAP interaction is delayed and proceeds up to 30 min. Though monocytes and neutrophils contain comparable amounts of 5-LO protein, neutrophils produce 3-5 times higher levels of 5-LO products due to prolonged activity, accompanied by delayed 5-LO nuclear membrane translocation. Arachidonic acid seemingly acts as adaptor for 5-LO/FLAP assembly, whereas FLAP inhibitors (MK886, 100 nM; BAY X 1005, 3 µM) disrupt the complex. We conclude that FLAP may regulate 5-LO activity in 2 ways: first by inducing an initial flexible association for efficient 5-LO product synthesis, followed by the formation of a tight 5-LO/FLAP complex that terminates 5-LO activity.


Assuntos
Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Araquidonato 5-Lipoxigenase/metabolismo , Leucócitos/metabolismo , Adulto , Ácido Araquidônico/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Feminino , Células HEK293 , Humanos , Ligação Proteica , Transporte Proteico
6.
Transfus Med Hemother ; 44(1): 46-51, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28275333

RESUMO

BACKGROUND: Living donor liver transplantation (LDLT) is an option to expand the donor organ pool for patients with life-threatening diseases who cannot be supplied with a cadaver organ. Next to the donor risks, complications after ABO-incompatible LDLT (ABOi LDLT) in the recipient are subject to controversial discussion. Improvement in ABOi graft survival rates have been achieved with plasma treatment procedures (PTP) and immunosuppression but antibody-mediated rejection (AMR) and graft loss still occur. METHODS: Since 2008, we have prepared 10 patients for ABOi LDLT. Seven of the 10 patients for transplantation had hepatocellular carcinoma (HCC). RESULTS: All patients underwent PTP before and after ABOi LDLT as well as immunosuppression according to the treatment schedule. We did not use anti-CD20 monoclonal antibodies in the transplant setting. We transplanted 6 of 10 preconditioned patients. After 3 years, 5 of the 6 transplanted patients were still alive. CONCLUSION: Even if B-cell depletion with anti-CD 20 treatment in the setting of ABOi LDLT is commonly accepted, our center successfully administered only quadruple drug immunosuppression combined with PTP. Especially patients with HCC had a high titer increment also pre-transplantation and were at high risk for arterial thrombosis and graft loss.

7.
J Nat Prod ; 79(3): 590-7, 2016 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-26918635

RESUMO

Humulus lupulus (hop plant) has long been used in traditional medicine as a sedative and antimicrobial agent. More recently, attention has been devoted to the phytoestrogenic activity of the plant extracts as well as to the anti-inflammatory and chemopreventive properties of the prenylated chalcones present. In this study, an Italian sample of H. lupulus cv. "Cascade" has been investigated and three new compounds [4-hydroxycolupulone (6), humudifucol (7) and cascadone (8)] have been purified and identified by means of NMR spectroscopy along with four known metabolites. Notably, humudifucol (7) is the first prenylated dimeric phlorotannin discovered in nature. Because structurally related phloroglucinols from natural sources were found previously to inhibit microsomal prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), the isolated compounds were evaluated for their bioactivity against these pro-inflammatory target proteins. The prenylated chalcone xanthohumol inhibited both enzymes at low µM concentrations.


Assuntos
Chalconas/isolamento & purificação , Chalconas/farmacologia , Humulus/química , Fitoestrógenos/isolamento & purificação , Fitoestrógenos/farmacologia , Plantas Medicinais/química , Polifenóis/isolamento & purificação , Polifenóis/farmacologia , Araquidonato 5-Lipoxigenase , Chalconas/química , Flavonoides , Oxirredutases Intramoleculares/antagonistas & inibidores , Itália , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Fitoestrógenos/química , Extratos Vegetais/química , Polifenóis/química , Prenilação , Propiofenonas , Prostaglandina-E Sintases
8.
J Cancer Res Clin Oncol ; 150(5): 224, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38693452

RESUMO

BACKGROUND: Incorporating chimeric antigen receptor (CAR)-T cell therapy into relapsed or refractory large B-cell lymphoma (rr LBCL) treatment algorithms has yielded remarkable response rates and durable remissions, yet a substantial portion of patients experience progression or relapse. Variations in outcomes across treatment centers may be attributed to different bridging strategies and remission statuses preceding CAR-T cell therapy. PATIENTS: Twenty-nine consecutive adult patients receiving tisagenlecleucel (tisa-cel) for rr LBCL from December 2019 to February 2023 at Jena University Hospital were analyzed. RESULTS: The median age was 63, with a median of 3 prior treatments. Twenty patients (69%) were refractory to any systemic therapy before CAR-T cell treatment. Following leukapheresis, 25 patients (86%) received bridging therapy with the majority undergoing chemotherapy (52%) or combined modality therapy (32%). Radiotherapy (RT) was part of the bridging strategy in 44%, with moderately hypofractionated involved site RT (30.0 Gy/2.5 Gy) being applied most frequently (64%). Post-CAR-T infusion, the objective response rate at 30 days was 83%, with 55% achieving complete response. Twelve-month progression-free (PFS) and overall survival (OS) were 60% and 74%, respectively, with a median follow up of 11.1 months for PFS and 17.9 months for OS. Factors significantly associated with PFS were chemotherapy sensitivity pre-leukapheresis and response to bridging. CONCLUSION: The study underscores the importance of minimal tumor burden at CAR-T initiation, emphasizing the need for suitable bridging regimens. The findings advocate for clinical trials and further real-world analyses to optimize CAR-T cell therapy outcomes by identifying the most effective bridging strategies.


Assuntos
Imunoterapia Adotiva , Linfoma Difuso de Grandes Células B , Humanos , Masculino , Pessoa de Meia-Idade , Feminino , Idoso , Imunoterapia Adotiva/métodos , Linfoma Difuso de Grandes Células B/terapia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Adulto , Indução de Remissão , Recidiva Local de Neoplasia/terapia , Recidiva Local de Neoplasia/patologia , Alemanha , Receptores de Antígenos de Linfócitos T/uso terapêutico , Estudos Retrospectivos , Terapia Combinada
9.
Front Nutr ; 10: 1095245, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36819683

RESUMO

Background: Regular consumption of the soluble dietary fiber ß-glucan is associated with decreased total cholesterol (TC), low-density lipoprotein (LDL) cholesterol and blood glucose. Barley and oat flakes as natural sources of ß-glucan were roasted to improve sensory quality. The aim of this study was to investigate whether roasting of barley and oat flakes changes the physiological impact of the ß-glucan-rich flakes on glucose and lipid metabolism. Method: A five-armed randomized crossover trial design was used. The intervention study was conducted from May 2018 to May 2019 and included 32 healthy subjects with moderately increased LDL cholesterol (≥2.5 mmol/L). During the 3-week intervention periods, 80 g of roasted or traditional barley or oat flakes, or four slices of white toast bread per day were consumed for breakfast. At the start and the end of each intervention, fasting and postprandial blood was taken. The intervention periods were separated by 3-week wash-out periods. Results: During the interventions with the cereal flakes, TC and LDL cholesterol concentrations were significantly reduced compared to baseline values by mean differences of 0.27-0.33 mmol/L and 0.21-0.30 mmol/L, respectively (p < 0.05), while high-density lipoprotein (HDL) cholesterol was only reduced after the intervention with barley flakes (p < 0.05). After the intervention period with toast, TC and HDL cholesterol increased (p < 0.05). The fasting levels of triglycerides, fasting blood glucose and insulin did not change in any group. The effects of traditional and roasted varieties on blood lipids did not differ between the groups. Conclusion: The regular consumption of traditional or roasted barley and oat flakes contributes to the management of cardiovascular diseases by improving TC and LDL cholesterol. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03648112, identifier NCT03648112.

10.
Ther Apher Dial ; 27(4): 790-801, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36719401

RESUMO

INTRODUCTION: Following SARS-CoV-2-infection up to 21% of patients will develop post-COVID-syndrome. Autoantibodies (AAbs) targeting neuronal-ß-adrenergic and muscarinic receptors may provide crucial contributions to the pathophysiology of this condition. Immunoadsorption (IA) has been identified as an effective means of removing AAbs and has resulted in clinical improvements of other autoantibody-associated diseases. METHODS: We determined AAb-levels (anti-ß1/ß2 and anti-M3/M4 receptor) in 178 patients diagnosed with post-COVID-syndrome and described the clinical courses of two patients with elevated AAb-levels that underwent IA-treatment. RESULTS: AAbs were detected in 57% (101/178) of patients diagnosed with post-COVID-syndrome. Substantial reductions in AAb-levels and clinical remission were achieved in one of two patients who was treated with IA. However, this patient relapsed within 6 weeks with a concomitant increase in AAb-levels. CONCLUSION: Collectively, AAbs may play a pathophysiologic role in post-COVID and their removal provide transient benefits in some patients. However, these findings should be further investigated in randomized-controlled-trials.


Assuntos
COVID-19 , Humanos , Autoanticorpos , COVID-19/terapia , SARS-CoV-2 , Síndrome
11.
Transfus Med Hemother ; 39(4): 234-240, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22969692

RESUMO

Primary organ failure after transplantation (TX) remains a serious complication and leads to a high percentage of lethality. It is known, however, that the speed of rejection and tissue destruction depends on 3 main factors: antibody titer, the ability of the tissue to repair itself, and immunosuppressive measures. Especially with evidence for antibodies against human leukocyte antigen (HLA-ab), the immunological risk of persistent and acute episodes of rejection increases. The role of non-HLA-ab in rejection episodes is often underestimated and should be studied further. Antibody-mediated rejection (AMR) is still an unsolved problem in thoracic organ TX. An essential pillar of antihumoral therapy are the extracorporeal procedures like plasmapheresis (PP), therapeutic plasma exchange (TPE), and immunoadsorption (IA), because only they have the ability to remove preformed or de novo developed antibodies quickly and effectively. The quick removal of antibodies and other plasma factors through TPE or IA remains an effective and supportive method for treating AMR and allows the TX despite preformed antibodies. The pertinent literature does not disclose, however, how often and for how long treatment should be administered. It is known, that repeated treatment cycles with adequately processed plasma volume must be used to overcome redistribution of pathological antibodies. Based on our experience in heart transplant recipients with compromised graft function due to non-HLA-ab and HLA-ab, IA seems to be more effective.

12.
Ther Apher Dial ; 26(1): 229-241, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33914397

RESUMO

Immunoadsorption is well known to selectively remove immunoglobulins and immune complexes from plasma and is applied in a variety of autoimmune diseases and for desensitization before, or at acute rejection after organ transplantation. Performance, safety, and clinical effectiveness of immunoadsorption were the aim of this study. This prospective, noninterventional, multicentre cohort study included patients treated with immunoadsorption (Immunosorba or GLOBAFFIN adsorbers) for any indication. Clinical effectiveness was assessed after termination of the patient's individual treatment schedule. Eighty-one patients were included, 69 were treated with Immunosorba, 11 with GLOBAFFIN, one patient with both adsorbers. A majority of patients was treated for neurological indications, dilated cardiomyopathy, and before or after kidney or heart transplantation. Mean IgG reduction from pre- to post-treatment was 69.9% ± 11.5% for Immunosorba and 74.1% ± 5.0% for GLOBAFFIN, respectively. The overall IgG reduction over a complete treatment block was 68%-93% with Immunosorba and 62%-90% with GLOBAFFIN depending on the duration of the overall treatment. After termination of the immunoadsorption therapy, an improvement of clinical status was observed in 63.0%, stabilization of symptoms in 29.6%, and a deterioration in 4.9% of patients. Changes in fibrinogen, thrombocytes, and albumin were mostly classified as noncritical. Overall, the treatments were well tolerated. Immunoadsorption in routine clinical practice with both GLOBAFFIN and Immunosorba has been safely performed, was well tolerated by patients, and effective in lowering immunoglobulins with an improvement or maintenance of clinical status, thus represents an additional therapeutic option for therapy refractory immune disorders.


Assuntos
Doenças Autoimunes/terapia , Cardiomiopatia Dilatada/terapia , Técnicas de Imunoadsorção , Doenças do Sistema Nervoso/terapia , Cuidados Pós-Operatórios/métodos , Cuidados Pré-Operatórios/métodos , Doenças Autoimunes/imunologia , Cardiomiopatia Dilatada/imunologia , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso/imunologia , Estudos Prospectivos , Transplantados/estatística & dados numéricos , Resultado do Tratamento
13.
Acta Pharm Sin B ; 11(6): 1629-1647, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34221873

RESUMO

Chronic inflammation results from excessive pro-inflammatory signaling and the failure to resolve the inflammatory reaction. Lipid mediators orchestrate both the initiation and resolution of inflammation. Switching from pro-inflammatory to pro-resolving lipid mediator biosynthesis is considered as efficient strategy to relieve chronic inflammation, though drug candidates exhibiting such features are unknown. Starting from a library of Vietnamese medical plant extracts, we identified isomers of the biflavanoid 8-methylsocotrin-4'-ol from Dracaena cambodiana, which limit inflammation by targeting 5-lipoxygenase and switching the lipid mediator profile from leukotrienes to specialized pro-resolving mediators (SPM). Elucidation of the absolute configurations of 8-methylsocotrin-4'-ol revealed the 2S,γS-isomer being most active, and molecular docking studies suggest that the compound binds to an allosteric site between the 5-lipoxygenase subdomains. We identified additional subordinate targets within lipid mediator biosynthesis, including microsomal prostaglandin E2 synthase-1. Leukotriene production is efficiently suppressed in activated human neutrophils, macrophages, and blood, while the induction of SPM biosynthesis is restricted to M2 macrophages. The shift from leukotrienes to SPM was also evident in mouse peritonitis in vivo and accompanied by a substantial decrease in immune cell infiltration. In summary, we disclose a promising drug candidate that combines potent 5-lipoxygenase inhibition with the favorable reprogramming of lipid mediator profiles.

14.
J Vis Exp ; (154)2019 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-31868176

RESUMO

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.


Assuntos
Aspergillus fumigatus/fisiologia , Citometria de Fluxo , Leucócitos/imunologia , Leucócitos/microbiologia , Fagocitose , Esporos Fúngicos/fisiologia , Humanos , Leucócitos/citologia
15.
Transfus Apher Sci ; 38(2): 149-57, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18348910

RESUMO

The aim of this investigation was to compare 11 different production procedures for human blood plasma, using preparative apheresis and whole blood. We intended to determine the plasma quality in regard to the number of residual cells or cell fragments. Cells were analysed using the Cell-Dyn 4000 blood cell counter and cell fragments were detected by an immunoassay. Antigenic structure could be detected in clearly different quantities corresponding to the separation technique used. Methods which separated blood components only with centrifugation produced plasma with more detectable cellular material than plasma produced with additional membrane filtration. No remaining antigenic fragments were detected when an additional plasma filter was used.


Assuntos
Antígenos/metabolismo , Remoção de Componentes Sanguíneos/métodos , Separação Celular/métodos , Plasma/citologia , Anticorpos Monoclonais/química , Antígenos/química , Remoção de Componentes Sanguíneos/instrumentação , Doadores de Sangue , Preservação de Sangue/métodos , Transfusão de Sangue , Centrifugação , Humanos , Imunoensaio/métodos , Leucócitos/citologia , Plasma/imunologia , Plasma/metabolismo , Risco
16.
Nat Commun ; 9(1): 3834, 2018 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-30237488

RESUMO

Systemic vitamin E metabolites have been proposed as signaling molecules, but their physiological role is unknown. Here we show, by library screening of potential human vitamin E metabolites, that long-chain ω-carboxylates are potent allosteric inhibitors of 5-lipoxygenase, a key enzyme in the biosynthesis of chemoattractant and vasoactive leukotrienes. 13-((2R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2,6,10-trimethyltridecanoic acid (α-T-13'-COOH) can be synthesized from α-tocopherol in a human liver-on-chip, and is detected in human and mouse plasma at concentrations (8-49 nM) that inhibit 5-lipoxygenase in human leukocytes. α-T-13'-COOH accumulates in immune cells and inflamed murine exudates, selectively inhibits the biosynthesis of 5-lipoxygenase-derived lipid mediators in vitro and in vivo, and efficiently suppresses inflammation and bronchial hyper-reactivity in mouse models of peritonitis and asthma. Together, our data suggest that the immune regulatory and anti-inflammatory functions of α-tocopherol depend on its endogenous metabolite α-T-13'-COOH, potentially through inhibiting 5-lipoxygenase in immune cells.


Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Inflamação/patologia , Vitamina E/metabolismo , Adolescente , Adulto , Idoso , Animais , Araquidonato 5-Lipoxigenase/química , Hiper-Reatividade Brônquica/patologia , Sobrevivência Celular/efeitos dos fármacos , Sistema Livre de Células , Humanos , Concentração Inibidora 50 , Leucócitos/metabolismo , Inibidores de Lipoxigenase/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metaboloma , Camundongos , Pessoa de Meia-Idade , Peritonite/patologia , Proteínas Recombinantes/metabolismo , Vitamina E/química , Adulto Jovem
17.
Sci Rep ; 7(1): 3759, 2017 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-28630405

RESUMO

The severity and course of inflammatory processes differ between women and men, but the biochemical mechanisms underlying these sex differences are elusive. Prostaglandins (PG) and leukotrienes (LT) are lipid mediators linked to inflammation. We demonstrated superior LT biosynthesis in human neutrophils and monocytes, and in mouse macrophages from females, and we confirmed these sex differences in vivo where female mice produced more LTs during zymosan-induced peritonitis versus males. Here, we report sex differences in PG production in neutrophils during acute inflammation. In the late phase (4-8 hrs) of mouse zymosan-induced peritonitis and rat carrageenan-induced pleurisy, PG levels in males were higher versus females, seemingly due to higher PG production in infiltrated neutrophils. Accordingly, human neutrophils from males produced more PGE2 than cells from females. Increased PG biosynthesis in males was accompanied by elevated cyclooxygenase (COX)-2 expression connected to increased nuclear factor-kappa B activation, and was abolished when LT synthesis was pharmacologically blocked, suggesting that elevated PG production in males might be caused by increased COX-2 expression and by shunting phenomena due to suppressed LT formation. Conclusively, our data reveal that the biosynthesis of pro-inflammatory PGs and LTs is conversely regulated by sex with consequences for the inflammatory response.


Assuntos
Neutrófilos/metabolismo , Peritonite/metabolismo , Prostaglandinas/biossíntese , Caracteres Sexuais , Doença Aguda , Animais , Ciclo-Oxigenase 2/biossíntese , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Neutrófilos/patologia , Peritonite/induzido quimicamente , Peritonite/patologia , Zimosan/toxicidade
18.
Biochem Pharmacol ; 130: 71-82, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28189727

RESUMO

Pharmacological interference with vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, is considered a potential strategy for cancer therapy. Macrophages are critically involved in tumor progression and may occur as pro-tumoral M2 phenotype, whereas classically-activated M1 can inhibit tumor development for example by releasing tumor-suppressing molecules, including tumor necrosis factor (TNF)α. Here, we show that targeting V-ATPase by selective inhibitors such as archazolid upregulates the expression and secretion of TNFα in lipopolysaccharide (LPS)- or LPS/interferon (INF)γ-activated M1-like macrophages derived from human blood monocytes. In contrast, archazolid failed to elevate TNFα production from uncommitted (M0) or interleukin (IL)-4-treated M2-like macrophages. Secretion of other relevant cytokines (i.e., IL-1ß, IL-6, IL-10) or chemokines (i.e. IL-8 and monocyte chemotactic protein-1) from M1 was not affected by archazolid. Though V-ATPase inhibitors elevated the lysosomal pH in M1 comparable to chloroquine or ammonium chloride, the latter agents suppressed TNFα secretion. Archazolid selectively increased TNFα mRNA levels, which was abolished by dexamethasone. Interestingly, archazolid enhanced the phosphorylation and nuclear translocation of the p65 subunit of NFκB and stimulated phosphorylation of SAPK/JNK. In a microfluidically-supported human tumor biochip model, archazolid-treated M1 significantly reduced tumor cell viability. Together, our data show that V-ATPase inhibition selectively upregulates TNFα production in classically-activated macrophages along with NFκB and SAPK/JNK activation. Such increased TNFα release caused by V-ATPase inhibitors may contribute to tumor suppression in addition to direct targeting cancer cells.


Assuntos
Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Células MCF-7 , Macrolídeos/farmacologia , Macrófagos/enzimologia , Tiazóis/farmacologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores
19.
Sci Rep ; 7: 40599, 2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-28094291

RESUMO

Vitamin A and vitamin D are essential nutrients with a wide range of pleiotropic effects in humans. Beyond their well-documented roles in cellular differentiation, embryogenesis, tissue maintenance and bone/calcium homeostasis, both vitamins have attracted considerable attention due to their association with-immunological traits. Nevertheless, our knowledge of their immunomodulatory potential during infection is restricted to single gene-centric studies, which do not reflect the complexity of immune processes. In the present study, we performed a comprehensive RNA-seq-based approach to define the whole immunomodulatory role of vitamins A and D during infection. Using human monocytes as host cells, we characterized the differential role of both vitamins upon infection with three different pathogens: Aspergillus fumigatus, Candida albicans and Escherichia coli. Both vitamins showed an unexpected ability to counteract the pathogen-induced transcriptional responses. Upon infection, we identified 346 and 176 immune-relevant genes that were regulated by atRA and vitD, respectively. This immunomodulatory activity was dependent on the inflammatory stimulus, allowing us to distinguish regulatory patterns which were specific for each stimulatory setting. Moreover, we explored possible direct and indirect mechanisms of vitamin-mediated regulation of the immune response. Our findings highlight the importance of vitamin-monitoring in critically ill patients. Moreover, our results underpin the potential of atRA and vitD as therapeutic options for anti-inflammatory treatment.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Infecções/genética , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Transcrição Gênica , Vitamina A/farmacologia , Vitamina D/farmacologia , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Fatores Imunológicos , Imunomodulação/efeitos dos fármacos , Infecções/imunologia , Infecções/microbiologia , Monócitos/imunologia , Transcriptoma
20.
Sci Rep ; 7: 41434, 2017 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-28134280

RESUMO

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells.


Assuntos
Actinas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Microambiente Tumoral , Caspases/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citotoxicidade Imunológica , Depsipeptídeos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Imunomodulação , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Neoplasias/patologia , Transporte Proteico , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/metabolismo
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