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1.
Science ; 365(6455): 786-793, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31395745

RESUMO

How cellular and organismal complexity emerges from combinatorial expression of genes is a central question in biology. High-content phenotyping approaches such as Perturb-seq (single-cell RNA-sequencing pooled CRISPR screens) present an opportunity for exploring such genetic interactions (GIs) at scale. Here, we present an analytical framework for interpreting high-dimensional landscapes of cell states (manifolds) constructed from transcriptional phenotypes. We applied this approach to Perturb-seq profiling of strong GIs mined from a growth-based, gain-of-function GI map. Exploration of this manifold enabled ordering of regulatory pathways, principled classification of GIs (e.g., identifying suppressors), and mechanistic elucidation of synergistic interactions, including an unexpected synergy between CBL and CNN1 driving erythroid differentiation. Finally, we applied recommender system machine learning to predict interactions, facilitating exploration of vastly larger GI manifolds.


Assuntos
Epistasia Genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Apoptose/genética , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Células Eritroides/citologia , Eritropoese/genética , Feminino , Perfilação da Expressão Gênica , Granulócitos/citologia , Humanos , Proteínas dos Microfilamentos/genética , Proteínas Proto-Oncogênicas c-cbl/genética
2.
Ann Lab Med ; 39(5): 438-446, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31037862

RESUMO

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired pluripotent hematopoietic stem cell disorder associated with an increase in the number of glycosyl-phosphatidyl inositol (GPI)-deficient blood cells. We investigated PNH clonal proliferation in the three cell lineages-granulocytes, T lymphocytes, and red blood cells (RBCs)-by analyzing PIGA gene mutations and T-cell receptor (TCR) clonality. METHODS: Flow cytometry was used on peripheral blood samples from 24 PNH patients to measure the GPI-anchored protein (GPI-AP) deficient fraction in each blood cell lineage. PIGA gene mutations were analyzed in granulocytes and T lymphocytes by Sanger sequencing. A TCR clonality assay was performed in isolated GPI-AP deficient T lymphocytes. RESULTS: The GPI-AP deficient fraction among the three lineages was the highest in granulocytes, followed by RBCs and T lymphocytes. PIGA mutations were detected in both granulocytes and T lymphocytes of 19 patients (79.2%), with a higher mutation burden in granulocytes. The GPI-AP deficient fractions of granulocytes and T lymphocytes correlated moderately (rs=0.519, P=0.049) and strongly (rs=0.696, P=0.006) with PIGA mutation burden, respectively. PIGA mutations were more frequently observed in patients with clonal rearrangements in TCR genes (P=0.015). The PIGA mutation burden of T lymphocytes was higher in patients with clonal TCRB rearrangement. CONCLUSIONS: PIGA mutations were present in approximately 80% of PNH patients. PNH clone size varies according to blood cell lineage, and clonal cells may obtain proliferation potential or gain a survival advantage over normal cells.


Assuntos
Proliferação de Células , Granulócitos/citologia , Hemoglobinúria Paroxística/diagnóstico , Proteínas de Membrana/genética , Linfócitos T/citologia , Adolescente , Adulto , Idoso , Linhagem da Célula , Criança , Feminino , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Rearranjo Gênico , Granulócitos/metabolismo , Hemoglobinúria Paroxística/genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Adulto Jovem
3.
Vet Immunol Immunopathol ; 210: 46-54, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30947979

RESUMO

The blood cell counting methods used for diagnostic and research purposes in turkeys are, up to date, inferior to the techniques established for mammals and chickens. While microscopic counts are time consuming, previous flow cytometric approaches did not cover all blood cell types of interest due to the lack of turkey-specific markers for the different cell populations. Moreover, it is unknown to what extent the different leukocyte populations are affected by host and environmental factors including age, breed of the bird, and housing environment, respectively. In this study we established a whole blood based flow cytometric analysis method for turkeys. This method was used to determine baseline values depending on the age of the birds as well as under consideration of variations between trials and animal room effects. During three trials whole blood samples of B.U.T. 6 female turkeys were collected to analyze different leukocyte concentrations (cells/µl whole blood). In the first trial one group and in the second and third trial two groups with 22 birds/group were housed. Blood samples were collected at days one, 23, 43, 60, and 88 post hatch and concentrations of monocytes, MHC class II-positive, CD4+, and CD8+ lymphocytes, as well as thrombocytes and granulocytes were determined by flow cytometric analysis. Concentrations of all identified populations were not only influenced by the bird's age (p < 0.05), they varied also among trials (p < 0.05) and even for some of the populations between animal rooms within the same trial despite comparable housing and management conditions. Therefore, for the establishment of baseline values for leucocyte concentrations in whole blood effects of age and housing have to be considered. In addition, our data emphasize the importance of the establishment of baseline values for different age groups, as age had the strongest effect on the blood cell numbers in this study.


Assuntos
Citometria de Fluxo , Contagem de Leucócitos/veterinária , Leucócitos/citologia , Fatores Etários , Animais , Feminino , Granulócitos/citologia , Abrigo para Animais , Contagem de Leucócitos/métodos , Monócitos/citologia , Perus
4.
Contrast Media Mol Imaging ; 2019: 4368342, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019414

RESUMO

The scintigraphy with radiolabelled autologous leukocytes (WBCs) is considered the gold-standard technique for imaging infections. Leukokit® is a commercially available, disposable, sterile kit for labelling WBCs ex vivo. In this kit, WBCs isolation from red blood cells (RBCs) was performed using poly(O-2-hydroxyethyl)starch (HES) as the RBCs sedimentation agent. Due to its poor availability, HES has been recently replaced by Gelofusine as the RBC sedimentation agent. The aim of this study was to compare the labelling efficiency and the diagnostic accuracy of WBCs labelled with Leukokit® with HES vs Leukokit® with Gelofusine. WBCs were isolated using HES or Gelofusine for 45 minutes and then purified from platelets (PLTs) and labelled with 1.1 ± 0.3 GBq of freshly prepared 99mTc-HMPAO. The following parameters were evaluated: the number and type of recovered WBCs, RBCs contamination, PLTs contamination, vitality of neutrophils, and chemotactic properties of neutrophils. Clinical comparison was performed between 80 patients (33 males; age 67.5 ± 14.2) injected with 99mTc-HMPAO-WBCs, using HES as the sedimentation agent, and 92 patients (38 males; age 68.2 ± 12.8) injected with 99mTc-HMPAO-WBCs using Gelofusine as the sedimentation agent. Patients were affected by prosthetic joint infections, peripheral bone osteomyelitis, or vascular graft infection. We compared radiolabelling efficiency (LE), final recovery yield (RY), and diagnostic outcome based on microbiology or 2-year follow-up. Results showed that HES provides the lowest RBCs and PLTs contamination, but Gelofusine provides the highest WBC recovery. Both agents did not influence the chemotactic properties of WBCs, and no differences were found in terms of LE and RY. Sensitivity, specificity, and accuracy were also not significantly different for WBCs labelled with both agents (diagnostic accuracy 90.9%, CI = 74.9-96.1 vs 98.3%, CI = 90.8-100, for HES and Gelofusine, respectively). In conclusion, Gelofusine can be considered a suitable alternative of HES for WBCs separation and labelling.


Assuntos
Poligelina/química , Tecnécio Tc 99m Exametazima/química , Idoso , Idoso de 80 Anos ou mais , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Feminino , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Humanos , Marcação por Isótopo/métodos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade
5.
Ther Apher Dial ; 23(3): 210-216, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31025504

RESUMO

Adacolumn is a therapeutic mode for ulcerative colitis that achieves therapeutic efficacy through the adhesion of leukocytes to cellulose acetate beads. We used scanning electron microscopy and observed leukocyte adsorption on Adacolumn beads after granulocytapheresis/granulocyte and monocyte adsorption apheresis. We then compared results between two patients with a low and high C-reactive protein (CRP) levels to determine whether adhesion is affected by a difference in leukocyte activity depending on the level of inflammation. We found that the surface layers of the beads from both patients were covered by a clay-like layer, and spherical granulocytes were adsorbed here and there on top of it. In cross-section the adsorbed granulocytes were visible in the clay-like layer and the surface layer alike. The clay-like layer had a maximum thickness of approximately 12 µm in the low CRP patient and approximately 50 µm in the high CRP patient, so in the high CRP patient the clay-like adsorption layer was thicker. Taken together, adsorption onto beads is considered to involve an immunological mechanism. Our findings suggest that granulocytes contact and adhere to each other at the surface layer after adsorption, and that granulocyte-granulocyte adhesion is enhanced by a higher inflammatory response.


Assuntos
Proteína C-Reativa/metabolismo , Colite Ulcerativa/terapia , Granulócitos/citologia , Leucaférese/métodos , Adsorção/fisiologia , Adulto , Estudos Transversais , Eletroforese em Acetato de Celulose , Humanos , Masculino , Amostragem , Sensibilidade e Especificidade , Adulto Jovem
6.
Ther Apher Dial ; 23(3): 217-223, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31025815

RESUMO

In patients with active ulcerative colitis (UC), adsorptive granulocyte/monocyte apheresis (GMA) is expected to promote remission. We conducted a retrospective cohort study to evaluate the efficacy and safety of GMA in patients with active UC. Twenty-one UC patients including five pregnant or lactating mothers and four elderly patients (aged >60 years) received up to 10 GMA sessions. UC severity was evaluated at baseline and after GMA therapy according to Lichtiger's Clinical Activity Index (CAI). We defined clinical remission as CAI ≤4. Overall, the median CAI score after GMA therapy had decreased from 9 to 4 (P < 0.001). The clinical remission rate was 62%, but in the elderly and pregnant or lactating mothers, the remission rates were 100% and 60%, respectively. No severe adverse effects were seen in this study. Our results may support GMA as an effective and safe treatment for active UC patients, including elderly patients and pregnant cases.


Assuntos
Colite Ulcerativa/diagnóstico , Colite Ulcerativa/terapia , Leucaférese/métodos , Segurança do Paciente , Adsorção/fisiologia , Adulto , Fatores Etários , Idoso , Estudos de Coortes , Feminino , Granulócitos/citologia , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Gravidez , Estudos Retrospectivos , Medição de Risco , Índice de Gravidade de Doença , Fatores Sexuais , Resultado do Tratamento
7.
Ther Apher Dial ; 23(3): 224-232, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31025824

RESUMO

Twice-weekly intensive granulocyte/monocyte adsorptive apheresis is effective and safe for ulcerative colitis, but maintaining two blood access routes is problematic. We previously reported that intensive granulocyte/monocyte adsorptive apheresis using a single needle in ulcerative colitis is effective and safe. We hypothesized that the efficacy and safety of single-needle intensive granulocyte/monocyte adsorptive apheresis for ulcerative colitis would especially benefit the elderly. We enrolled 17 elderly ulcerative colitis patients to receive single-needle intensive granulocyte/monocyte adsorptive apheresis, 27 elderly ulcerative colitis patients to receive double-needle intensive granulocyte/monocyte adsorptive apheresis, and 52 nonelderly ulcerative colitis patients to receive single-needle intensive granulocyte/monocyte adsorptive apheresis. Remission and mucosal healing rates after treatment did not differ significantly between elderly ulcerative colitis patients receiving single-needle apheresis and the other two groups. In addition, no serious adverse effects, including blood clots, were observed in single-needle intensive granulocyte/monocyte adsorptive apheresis patients. Single-needle intensive granulocyte/monocyte adsorptive apheresis might be a novel alternative therapeutic option for elderly ulcerative colitis patients before considering corticosteroids.


Assuntos
Colite Ulcerativa/diagnóstico , Colite Ulcerativa/terapia , Leucaférese/métodos , Segurança do Paciente , Corticosteroides , Adsorção , Idoso , Idoso de 80 Anos ou mais , Produtos Biológicos , Estudos de Coortes , Feminino , Avaliação Geriátrica , Granulócitos/citologia , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Agulhas , Índice de Gravidade de Doença , Resultado do Tratamento
8.
Ulus Travma Acil Cerrahi Derg ; 25(2): 177-182, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30892679

RESUMO

BACKGROUND: Acute necrotizing pancreatitis (ANP) is the most severe form of acute pancreatitis (AP), and it has high mortality rates. Therefore, early diagnosis and treatment are of critical importance for the prognosis. The aim of this study was to investigate the effectiveness of immature granulocyte percentage (IG%) in the early prediction of ANP. METHODS: This retrospective study included 96 adult patients hospitalized with a diagnosis of AP. Demographic data of the patients were recorded. The white blood cell (WBC) count, neutrophil-to-lymphocyte ratio (NLR), IG%, C-reactive protein (CRP), and amylase levels were determined. Furthermore, computed abdominal tomography was applied to the patients, and the length of hospital stay was recorded. Patients were divided into two groups as those with acute edematous pancreatitis and ANP, according to the tomography results. The differences between the groups were analyzed statistically. RESULTS: The WBC count, NLR, CRP, and IG% were significant markers in the prediction of ANP. However, IG% had higher values with regard to the sensitivity, specificity, AUROC, and negative and positive predictive values (100%, 95%, 0.982, 78.9%, and 100%, respectively). CONCLUSION: An increased IG% is a simple, fast, and effective marker in the early prediction of ANP. METHODS: This retrospective study was carried out on 96 adult patients who were hospitalized with a diagnosis of acute pancreatitis. Demographic data of the patients were recorded. White blood cell count, neutrophil/lymphocyte ratio, IG%, C-reactive protein and amylase levels were determined. Furthermore computed abdominal tomography was applied to the patients and the length of hospital stay was recorded. The patients were divided into two groups as acute edematous pancreatitis and acute necrotizing pancreatitis according to the tomography results. The differences between the groups were analyzed statistically. RESULTS: White blood cell count, neutrophil/lymphocyte ratio, C-reactive protein and IG% were significant markers in the prediction of acute necrotizing pancreatitis. However, IG% had higher values of sensitivity, specificity, AUROC, negative and positive predictive values ( 100%, 95%, 0.982, 78.9%, 100%,respectively). CONCLUSION: Increased IG% is a simple, fast, and effective marker in the early prediction of acute necrotizing pancreatitis.


Assuntos
Granulócitos/citologia , Contagem de Leucócitos/estatística & dados numéricos , Pancreatite Necrosante Aguda , Adulto , Humanos , Pancreatite Necrosante Aguda/sangue , Pancreatite Necrosante Aguda/diagnóstico , Pancreatite Necrosante Aguda/epidemiologia , Valor Preditivo dos Testes , Estudos Retrospectivos
9.
Cell Tissue Res ; 377(2): 245-257, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30919048

RESUMO

Despite extensive investigation focused on both the molecular characteristics and the expression level of Toll-like receptors (TLRs) during the inflammatory response in vertebrates, few data are available in the literature on the role of these proteins in invertebrate's immune response. Here, we propose the medicinal leech as a valuable model to better elucidate the role of TLR4 and its related products, such as tumor necrosis factor (TNF-α), after activation of the leech peripheral immune system with the endogenous medicinal leech recombinant allograft inflammatory factor-1 (rHmAIF-1) or with an exogenous stimulus, such as lipopolysaccharide (LPS). Our results indicate that activated macrophages (HmAIF-1+) and granulocytes (CD11b+) express both TLR4 and its coreceptor CD14. Moreover, functional studies performed by injecting a cyanobacterium selective TLR4 antagonist CyP demonstrated that only the TLR4 pathway was blocked, while the immune response caused by lipoteichoic acid (LTA) treatment is not affected. These results are consistent with literature on vertebrates, indicating that TLR4 functions as a LPS receptor while the recognition of LTA may involve other pathways.


Assuntos
Modelos Animais de Doenças , Granulócitos/imunologia , Inflamação/imunologia , Sanguessugas , Macrófagos/imunologia , Receptor 4 Toll-Like , Animais , Proteínas de Ligação ao Cálcio/imunologia , Granulócitos/citologia , Aplicação de Sanguessugas , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Proteínas dos Microfilamentos/imunologia , Ácidos Teicoicos/farmacologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/imunologia
10.
Environ Toxicol ; 34(5): 666-673, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30794351

RESUMO

Fluorotelomer alcohols (FTOHs) are fluorinated intermediates used in manufacturing specialty polymer and surfactants, with 8:2 FTOH the homologue of largest production. FTOHs were found to pose acute toxicity, hepatotoxicity, nephrotoxicity, developmental toxicity and endocrine-disrupting risks, whereas research regarding immunotoxicity and its underlying mechanism, especially on specific immune cells is limited. Here, we investigated the immunotoxicity of 8:2 FTOH on immature immune cells in an in vitro system. We observed that exposure of HL-60 cells, a human promyelocytic leukemic cell line, to 8:2 FTOH reduced cell viability in a dose- and time-dependent manner. In addition, 8:2 FTOH exposure caused G1 cell cycle arrest in HL-60 cells, while it showed no effect on apoptosis. Exposure to 8:2 FTOH inhibited the mRNA expression of cell cycle-related genes, including CCNA1, CCNA2, CCND1, and CCNE2. Moreover, exposure to 8:2 FTOH inhibited the mRNA expression of granulocytic differentiation-related genes of CD11b, CSF3R, PU.1, and C/EPBε in HL-60 cells . Furthermore, 8:2 FTOH exhibited no effect on intracellular ROS level, while hydralazine hydrochloride (Hyd), one reactive carbonyl species (RCS) scavenger, partially blocked 8:2 FTOH-caused cytotoxicity in HL-60 cells. Overall, the results obtained in the study show that 8:2 FTOH poses immunotoxicity in immature immune cells and RCS may partially underline its mechanism.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fluorcarbonetos/toxicidade , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Granulócitos/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/imunologia , Genes cdc/efeitos dos fármacos , Genes cdc/imunologia , Granulócitos/citologia , Granulócitos/imunologia , Células HL-60 , Humanos , Fatores de Tempo
11.
Fish Shellfish Immunol ; 87: 96-104, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30633961

RESUMO

Granulocytes are known as the main immunocompetent hemocytes that play important roles in the immune defense of oyster Crassostrea gigas. In the present study, an alcohol acyltransferase (designed as CgAATase) with specific expression pattern was identified from oyster C. gigas, and it could be employed as a potential marker for the isolation of oyster granulocytes. The open reading frame (ORF) of CgAATase was of 1431 bp, encoding a peptide of 476 amino acids with a typically conserved AATase domain. The mRNA transcripts of CgAATase were highest expressed in hemocytes, lower expressed in hepatopancreas, mantle, gonad, gill, ganglion, adductor muscle, and labial palp. The mRNA expression level of CgAATase in hemocytes was significantly up-regulated at 3-12 h and reached the highest level (27.40-fold compared to control group, p < 0.05) at 6 h after Vibrio splendidus stimulation. The total hemocytes were sorted as granulocytes, semi-granulocytes and agranulocytes by Percoll® density gradient centrifugation. CgAATase transcripts were dominantly observed in granulocytes, which was 8.26-fold (p < 0.05) and 2.80-fold (p < 0.05) of that in agranulocytes and semi-granulocytes, respectively. The monoclonal antibody against CgAATase was produced and employed for the isolation of granulocytes with the immunomagnetic bead. CgAATase protein was mainly detected on the cytomembrane of granulocytes. About 85.7 ±â€¯4.60% of the granulocytes were positive for CgAATase and they could be successfully separated by flow cytometry with immunomagnetic bead coated with anti-CgAATase monoclonal antibody, and 97.7 ±â€¯1.01% of the rest hemocytes (agranulocytes and semi-granulocytes) were negative for CgAATase. The isolated primary granulocytes could maintain cell activity for more than one week in vitro culture that exhibited numerous filopodia. These results collectively suggested that CgAATase was a potential marker of oyster granulocytes, and the granulocytes could be effectively isolated from total circulating hemocytes by immunomagnetic bead coated with the anti-CgAATase monoclonal antibody.


Assuntos
Crassostrea/imunologia , Granulócitos/imunologia , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Crassostrea/citologia , Crassostrea/enzimologia , Citometria de Fluxo/métodos , Granulócitos/citologia , Granulócitos/enzimologia , Hemócitos/citologia , Separação Imunomagnética/métodos , Proteínas/genética , Vibrio/imunologia
12.
Transfusion ; 59(1): 259-266, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30444537

RESUMO

BACKGROUND: Short- and long-term effects of mobilization regimens in hematopoietic stem cell and granulocyte donors have been well characterized. In this study, we examined the longitudinal hematopoietic changes related to repeat stimulated granulocyte donation. STUDY DESIGN AND METHODS: Complete blood counts for consecutive granulocyte donors between October 1994 and May 2017 were compared to unstimulated granulocyte donors. Plateletpheresis donors served as controls. The longitudinal change in precollection white blood cell (WBC) counts for these donor groups were modeled using a linear mixed-effects model. The investigated variables were granulocyte, lymphocyte, and monocyte counts and the granulocyte collection yield. Contrasts were performed to explore the effect of donation number on precollection counts. RESULTS: For the granulocyte-colony-stimulating factor plus dexamethasone (G-CSF/Dex)-stimulated group, both the granulocyte and the lymphocyte counts decreased 6.51 × 109 /L (-23.1%, p < 0.001) and 0.21 × 109 /L (-20.4%, p < 0.001), respectively, between Donation 1 and Donation 20. This effect was still present at the 3- to 4-year interval (b = -0.0008313, SE = 0.00029, p = 0.004). For the unstimulated donor group between Donation 1 and Donation 20, the lymphocyte count decreased by 0.62 × 109 /L (-51.5%, p < 0.001). This effect was only significant up to Year 2 (b = -0.0026, SE = 0.0010, p = 0.013). CONCLUSIONS: Past granulocyte donations were found to have a statistically strong negative effect on precollection granulocyte counts and lymphocyte counts and decreased granulocyte yield both in the G-CSF/Dex-stimulated donors and the unstimulated donors. In this statistical model, for both these groups, the effect of past donations on granulocyte and WBC counts were still detectable 2 years later.


Assuntos
Doadores de Sangue/estatística & dados numéricos , Granulócitos/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Hematopoese/fisiologia , Mobilização de Células-Tronco Hematopoéticas , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
13.
J Clin Apher ; 34(4): 359-366, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30549092

RESUMO

BACKGROUND: Extracorporeal photopheresis (ECP) is an efficient and established therapy to treat acute and chronic graft vs host disease (GVHD). Using an "off-line" method, the first step (mononuclear cell [MNC] collection) is decisive, as long as a high MNC yield and purity in the collected product is desirable. Two "off-line" devices were compared: the COBE Spectra and the Spectra Optia (Terumo BCT), using both continuous and intermittent protocols. PATIENTS AND METHODS: Twelve patients with GvHD (7 acute/5 chronic) were enrolled between June 2014 and May 2015 and were alternatively assigned for each procedure to either the COBE Spectra or the Spectra Optia cell separator. Patients characteristics and procedure/product parameters were analyzed. RESULTS: Two hundred procedures (100 per device) were included. The Spectra Optia system showed higher total nucleated cells and MNC collection efficiencies (18.6(10.2-29.7) vs 7.9(4.1-14.8)% and 43.6(20.3-59.5) vs 23.3(11.4-37.1)%, P < .001) and monocyte and lymphocyte collection efficiencies (55.2(17.7-83.2) vs 22.8(9-38.9)% and 38.3(26.7-53.4) vs 22.2(9-38.9)%, respectively, P < .001). Absolute platelet loss (PL) and PL per liter of blood processed were significantly lower in the Spectra Optia group (22.9(18.3-28.1) vs 33.6(26.5-41.1)%, P < .001 and 3.7(3.1-4.5) vs 4.3(3.5-4.2)%, P = .01, respectively). However, granulocyte contamination was higher (4.5(1.3-36) vs 1.2(0.4-5.7)%, P < .001) and a higher product haematocrit was obtained with the Spectra Optia (1(0.5-1.6) vs 0.3(0.2-0.5)%, P < .001), without an impact on irradiation time. CONCLUSIONS: In our study, Spectra Optia proved to be safe and effective in collecting MNC with high yield and purity for ECP in GvHD.


Assuntos
Doença Enxerto-Hospedeiro/terapia , Leucaférese/instrumentação , Fotoferese/instrumentação , Adulto , Plaquetas/citologia , Contagem de Células , Feminino , Granulócitos/citologia , Humanos , Leucaférese/métodos , Leucaférese/normas , Leucócitos Mononucleares/citologia , Linfócitos/citologia , Masculino , Pessoa de Meia-Idade , Fotoferese/métodos , Resultado do Tratamento
14.
Mater Sci Eng C Mater Biol Appl ; 94: 318-332, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30423714

RESUMO

Our study reports the fabrication and characterization (surface morphology, hydrophobicity/hydrophilicity, photocatalytic efficiency) of cotton fibers treated by various methods with graphene oxide decorated with Fe, N-doped TiO2 nanoparticles. Designed as prospective industrial self-cleaning, antimicrobial and biocompatible textiles, microbiological and cytotoxicity tests were performed on these particles-treated fibers to validate their qualities. The photocatalytic effect was dependent on chemicals used to disperse the nanoparticles, the parameters of the treatment, the fiber structure and composition of the material. The double and triple treatment of the textiles with the same particle dispersion resulted in a relatively uniform coverage of cotton fibers with relatively large amounts of particles. A larger amount of doped TiO2 particles demonstrated a better photocatalytic effect under visible light. The material's hydrophobicity increased with the number of treatments due to the deposition of successive layers of reduced graphene, ensuring self-cleaning properties. The photocatalyst-treated cotton fabrics exhibited an increased resistance to Enterococcus faecalis and Escherichia coli colonization, and also high biocompatibility, as they did not affect the cell viability, membrane integrity and morphology, nor induce inflammation. All these data confirm the improved properties of cotton fibers treated with graphene oxide decorated with Fe, N-doped TiO2 particles in order to be used as industrial self-cleaning and biocompatible textiles.


Assuntos
Materiais Biocompatíveis/química , Fibra de Algodão , Grafite/química , Ferro/química , Luz , Nanopartículas/química , Nitrogênio/química , Titânio/química , Citoesqueleto de Actina/metabolismo , Catálise , Fibroblastos/citologia , Granulócitos/citologia , Humanos , Lisossomos/metabolismo , Masculino , Nanopartículas/ultraestrutura , Tamanho da Partícula , Fagocitose , Espectrometria por Raios X , Análise Espectral Raman , Molhabilidade
15.
J Clin Apher ; 34(1): 39-43, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30426567

RESUMO

BACKGROUND: Mobilization regimen choice is a significant contributing factor for successful hematopoietic progenitor cell (HPC) collection by leukocytapheresis and reaching the target CD34+ cell dose. How mobilization regimen affects collection efficiency and the quality of products collected using the Spectra Optia apheresis instrument is not fully known. METHODS: We evaluated the impact of granulocyte-colony stimulating factor (GCSF) and GCSF/plerixafor mobilization regimens on CE and product composition. We studied 373 leukocytapheresis HPC collections for 147 autologous transplants from January 1, 2010 to December 31, 2014. Patients were categorized in two groups; good mobilizers, mobilized with GCSF only (GM) and poor mobilizers, mobilized with GCSF and Plerixafor (PM). RESULTS: Overall, compared with PM group, total nucleated cell (TNC) yield was significantly lower in GM group (P = <.001). In contrast, median percent mononuclear cell (MNC) collected from GM (86.5%) was significantly higher than products collected from PM group (79.5%; P < .001). Compared with GM group, CD34+ cell CE was about 10% lower in PM group (P < .008). In addition, daily CD34+ cell/Kg yield was significantly higher in GM (2.08 × 10/Kg) compared with PM group (1.64 x 10/Kg, P = .019). Overall, the median number of collections per patient was two for GM and three for PM (P = .004). CONCLUSION: Products collected from PM group contained higher TNC content relative to GM group but had lower MNC enrichment, CD34+ cell CE and daily CD34+ cell yield per Kg.


Assuntos
Antígenos CD34/sangue , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Controle de Qualidade , Adulto , Autoenxertos , Contagem de Células , Feminino , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Granulócitos/citologia , Compostos Heterocíclicos/uso terapêutico , Humanos , Leucaférese , Leucócitos Mononucleares/citologia , Masculino , Pessoa de Meia-Idade
16.
Epigenomics ; 11(1): 95-109, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30427216

RESUMO

AIM: To determine whether epigenetic modifications of DNA regulate gene expression and alternative splicing during terminal granulopoiesis. MATERIALS & METHODS: Using whole genome bisulfite sequencing, reduced representation hydroxymethylation profiling and mRNA sequencing, we compare changes in DNA methylation, DNA hydroxymethylation, gene expression and alternative splicing in mouse promyelocytes and granulocytes. RESULTS & CONCLUSION: We show reduced DNA methylation at the promoters and enhancers of key granulopoiesis genes, indicating a regulatory role in the activation of lineage-specific genes during differentiation. Notably, increased DNA hydroxymethylation in exons is associated with preferential inclusion of specific exons in granulocytes. Overall, DNA methylation and hydroxymethylation changes at particular genomic loci may play specific roles in gene regulation or alternative splicing during terminal granulopoiesis. Data deposition: Whole genome bisulfite sequencing of mouse promyelocytes and granulocytes: Gene Expression Omnibus (GSE85517); mRNA sequencing of mouse promyelocytes and granulocytes: Gene Expression Omnibus (GSE48307); reduced representation 5-hydroxymethylation profiling of mouse promyelocytes and granulocytes: Bioproject (PRJNA495696).


Assuntos
Processamento Alternativo , Metilação de DNA , Granulócitos/citologia , Granulócitos/metabolismo , Mielopoese/genética , Animais , Diferenciação Celular/genética , Biologia Computacional/métodos , Ilhas de CpG , Elementos Facilitadores Genéticos , Epigênese Genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Íntrons , Masculino , Camundongos
17.
Transfusion ; 59(1): 160-168, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30383912

RESUMO

BACKGROUND: Patients with prolonged neutropenia caused by chemotherapy or underlying marrow disorders are at risk of invasive bacterial and fungal infections. New treatment options alongside targeted antimicrobial therapy that might improve outcomes include granulocyte transfusions (GTX). To inform the research agenda, a prospective observational cohort study was performed in the Netherlands and United Kingdom. The aim was to describe the incidence, characteristics, and outcomes of patients developing invasive infections and assess patients fulfilling criteria for GTX. STUDY DESIGN AND METHODS: All patients receiving myeloablative chemotherapy and anticipated to develop 7 or more days of neutropenia (<0.5 × 109 /L) were eligible and followed for the development of invasive infections according to a defined algorithm and mortality up to 100 days. Secondary outcomes were types of infection and eligibility for GTX. RESULTS: A total of 471 patients enrolled at six hematology-oncology departments were followed for 569 neutropenic episodes. Overall, 32.5% of patients developed invasive infections during their first episode. Significant baseline risk factors for developing infections were high comorbidity scores (WHO performance status ≥ 2, hazard ratio [HR], 2.6 [1.7-3.9]; and hematopoietic cell transplantation-comorbidity index score ≥ 2 HR 1.3 [0.9-1.8]). Infections were bacterial (59.4%) and fungal (22.3%). Despite 34 patients (6.3% of all episodes) appearing to meet criteria to receive GTX, only nine patients received granulocytes. The HR for death was 5.8 (2.5-13.0) for patients with invasive infections. CONCLUSION: This study documents that invasive infections are associated with significant mortality. There is a need for new strategies to prevent and treat infections, which may include better understanding of use GTX.


Assuntos
Granulócitos/citologia , Transfusão de Leucócitos/métodos , Neutropenia/epidemiologia , Neutropenia/terapia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Criança , Pré-Escolar , Feminino , Humanos , Incidência , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Estudos Prospectivos , Fatores de Risco , Adulto Jovem
18.
J Clin Apher ; 34(1): 51-60, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30407662

RESUMO

Ulcerative colitis (UC) and Crohn's disease (CD) are major phenotypes of the chronic inflammatory bowel disease (IBD), which afflicts millions of individuals throughout the world with debilitating symptoms. The chronic nature of IBD means that patients require life-long medications, and this may lead to drug dependency, loss of response together with adverse side effects as additional morbidity factors. The efficacy of antitumour necrosis factor (TNF)-α biologics has validated the role of inflammatory cytokines notably TNF-α in the exacerbation and perpetuation of IBD. However, cytokines are released by myeloid lineage leucocytes like the CD14+ CD16+ monocyte phenotype. Additionally in IBD, myeloid leucocytes are elevated with activation behavior, while lymphocytes are compromised. Therefore, patients' leucocytes appear logical targets of therapy. Adsorptive granulomonocytapheresis (GMA) with an Adacolumn uses carriers, which interact with the Fcγ receptor expressing leucocytes and deplete the elevated myeloid leucocytes, while the neutrophils, which re-enter the circulation via the Adacolumn outflow (≥40%) are phagocytosed by CD19 B-cells to become interleukin (IL)-10 producing Bregs or CD19high CD1Dhigh B-cells. IL-10 is an anti-inflammatory cytokine. GMA has been applied to treat patients with IBD. The efficacy outcomes have been impressive as well as disappointing, the clinical response to GMA defines the patients' disease course and severity at entry. Efficacy outcomes in patients with deep ulcers together with extensive loss of the mucosal tissue are not encouraging, while patients without these features respond well and attain a favorable long-term disease course. Accordingly, for responder patients, GMA fulfills a desire to be treated without drugs.


Assuntos
Granulócitos/citologia , Doenças Inflamatórias Intestinais/terapia , Leucaférese/métodos , Monócitos/citologia , Colite Ulcerativa/terapia , Doença de Crohn/terapia , Humanos , Resultado do Tratamento
19.
Cell Biol Int ; 43(2): 192-206, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30571852

RESUMO

Myeloproliferative neoplasms (MPNs) are developing resistance to therapy by JAK1/2 inhibitor ruxolitinib. To explore the mechanism of ruxolitinib's limited effect, we examined the JAK1/2 mediated induction of proliferation related ERK1/2 and AKT signaling by proinflammatory interleukin-6 (IL-6) in MPN granulocytes and JAK2V617F mutated human erythroleukemia (HEL) cells. We found that JAK1/2 or JAK2 inhibition prevented the IL-6 activation of STAT3 and AKT pathways in polycythemia vera and HEL cells. Further, we showed that these inhibitors also blocked the IL-6 activation of the AKT pathway in primary myelofibrosis (PMF). Only JAK1/2 inhibitor ruxolitinib largely activated ERK1/2 signaling in essential thrombocythemia and PMF (up to 4.6 fold), with a more prominent activation in JAK2V617F positive granulocytes. Regarding a cell cycle, we found that IL-6 reduction of HEL cells percentage in G2M phase was reversed by ruxolitinib (2.6 fold). Moreover, ruxolitinib potentiated apoptosis of PMF granulocytes (1.6 fold). Regarding DNA replication, we found that ruxolitinib prevented the IL-6 augmentation of MPN granulocytes frequency in the S phase of the cell cycle (up to 2.9 fold). The inflammatory stimulation induces a cross-talk between the proliferation linked pathways, where JAK1/2 inhibition is compensated by the activation of the ERK1/2 pathway during IL-6 stimulation of DNA replication.


Assuntos
Replicação do DNA/efeitos dos fármacos , Interleucina-6/farmacologia , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Transtornos Mieloproliferativos/patologia , Adulto , Idoso , Antígenos CD34/metabolismo , Linhagem Celular Tumoral , Feminino , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Humanos , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/metabolismo , Fosforilação/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único , Pirazóis/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Fatores de Transcrição STAT/metabolismo
20.
PLoS One ; 13(12): e0209832, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30589898

RESUMO

Bacterial and fungal infections still remain an important cause of mortality in patients with hematological malignancies and in recipients of hematopoietic stem cell transplants (HSCT) especially in developing countries like India. Granulocyte transfusions (GTX) from healthy donors may lead to early clearance of index infection and thus prevent mortality. The aim of the present study was to evaluate the efficacy of GTX in combating life-threatening infections and preventing mortality in patients of hematological disorders/recipients of HSCT with severe neutropenia. This study was a prospective, observational analysis of patients with different hematological disorders/recipients of HSCT, who received GTX from January 2014 to December 2017. All patients had an Absolute neutrophil Count (ANC) < 0.5 x 109/L and a life threatening sepsis defined by presence of hemodynamic instability/ impending septic shock/ continuous high fever despite the use of the highest line of antimicrobials. A total of 143 granulocyte collections were done for 66 infectious episodes (IEs) in 60 patients. Multidrug resistant organisms (MDROs) were observed in 47/66 IEs (71.2%) and fungal infections were seen in 9/66 IEs (13.6%). Resolution of index infection after GTX was seen in 45/66 IEs (68.2%), and the 30 day overall survival (OS) was 67.7%. OS was significantly higher in patients who received GTX within 7 days of neutropenic sepsis (p = 0.01). Patients with MDROs who received early GTX therapy had a better OS as compared to those who received late GTX (p = 0.02). GTX were well tolerated and only 6 patients' developed mild features of transfusion related acute lung injury (TRALI) which was managed conservatively, and 1 patient demonstrated hypocalcemic tetany. GTX may be of particular relevance in countries like India, where the incidence of infections is very high in neutropenic patients and there is an increasing emergence of MDROs.


Assuntos
Transfusão de Leucócitos/métodos , Adolescente , Adulto , Feminino , Granulócitos/citologia , Granulócitos/fisiologia , Neoplasias Hematológicas/terapia , Humanos , Índia , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Neutropenia/terapia , Estudos Prospectivos , Centros de Atenção Terciária/estatística & dados numéricos , Adulto Jovem
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