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1.
Virol J ; 20(1): 203, 2023 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-37661270

RESUMO

BACKGROUND: Inflammation affecting the heart and surrounding tissues is a clinical condition recently reported following COVID-19 mRNA vaccination. Assessing trends of these events related to immunization will improve vaccine safety surveillance and best practices for forthcoming vaccine campaigns. However, the causality is unknown, and the mechanisms associated with cardiac myocarditis are not understood. CASE PRESENTATION: After the first dose, we reported an mRNA vaccine-induced perimyocarditis in a young patient with a history of recurrent myocardial inflammation episodes and progressive loss of cardiac performance. We tested this possible inflammatory cytokine-mediated cardiotoxicity after vaccination in the acute phase (ten days), and we found a significant elevation of MCP-1, IL-18, and IL-8 inflammatory mediators. Still, these cytokines decreased considerably at the recovery phase (42 days later). We used the cardiomyoblasts cell line to test the effect of serum on cell viability, observing that serum from the acute phase reduced the cell viability to 75%. We did not detect this toxicity in cells when we tested serum from the patient in the recovery phase. We also tested serum-induced hypertrophy, a phenomenon in myocarditis and heart failure. We found that acute phase-serum has hypertrophy effects, increasing 25% of the treated cardiac cells' surface and significantly increasing B-type natriuretic peptide. However, we did not observe the hypertrophic effect in the recovery phase or sera from healthy controls. CONCLUSION: Our results opened the possibility of the inflammatory cytokines or serum soluble mediators as key factors for vaccine-associated myocarditis. In this regard, identifying anti-inflammatory molecules that reduce inflammatory cytokines could help avoid vaccine-induced myocardial inflammation.


Assuntos
COVID-19 , Miocardite , Humanos , Miocardite/etiologia , Vacinas contra COVID-19/efeitos adversos , COVID-19/prevenção & controle , Hipertrofia , Inflamação , Citocinas , Vacinas de mRNA
2.
BMC Med ; 20(1): 388, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36316769

RESUMO

BACKGROUND: Breastmilk is a dynamic fluid whose initial function is to provide the most adapted nutrition to the neonate. Additional attributes have been recently ascribed to breastmilk, with the evidence of a specific microbiota and the presence of various components of the immune system, such as cytokines and leukocytes. The composition of breastmilk varies through time, according to the health status of mother and child, and altogether contributes to the future health of the infant. Obesity is a rising condition worldwide that creates a state of systemic, chronic inflammation including leukocytosis. Here, we asked whether colostrum, the milk produced within the first 48 h post-partum, would contain a distinct leukocyte composition depending on the body mass index (BMI) of the mother. METHODS: We collected peripheral blood and colostrum paired samples from obese (BMI > 30) and lean (BMI < 25) mothers within 48 h post-partum and applied a panel of 6 antibodies plus a viability marker to characterize 10 major leukocyte subpopulations using flow cytometry. RESULTS: The size, internal complexity, and surface expression of CD45 and CD16 of multiple leukocyte subpopulations were selectively regulated between blood and colostrum irrespective of the study groups, suggesting a generalized cell-specific phenotype alteration. In obesity, the colostrum B lymphocyte compartment was significantly reduced, and CD16+ blood monocytes had an increased CD16 expression compared to the lean group. CONCLUSIONS: This is the first characterization of major leukocyte subsets in colostrum of mothers suffering from obesity and the first report of colostrum leukocyte subpopulations in Latin America. We evidence various significant alterations of most leukocyte populations between blood and colostrum and demonstrate a decreased colostrum B lymphocyte fraction in obesity. This pioneering study is a stepping stone to further investigate active immunity in human breastmilk.


Assuntos
Colostro , Leucócitos , Leite Humano , Obesidade , Feminino , Humanos , Lactente , Recém-Nascido , Gravidez , Colostro/citologia , Estudos Transversais , Leite Humano/citologia , Mães
3.
Cytometry A ; 85(12): 1057-64, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24995861

RESUMO

Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens.


Assuntos
Citometria de Fluxo/métodos , Contagem de Leucócitos/métodos , Neutrófilos , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C
4.
BMJ Open Diabetes Res Care ; 12(4)2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39122366

RESUMO

INTRODUCTION: Chronic hyperglycemia affects neutrophil functions, leading to reduced pathogen killing and increased morbidity. This impairment has been directly linked to increased glycemia, however, how this specifically affects neutrophils metabolism and their differentiation in the bone marrow is unclear and difficult to study. RESEARCH DESIGN AND METHODS: We used high-resolution respirometry to investigate the metabolism of resting and activated donor neutrophils, and flow cytometry to measure surface CD15 and CD11b expression. We then used HL-60 cells differentiated towards neutrophil-like cells in standard media and investigated the effect of doubling glucose concentration on differentiation metabolism. We measured the oxygen consumption rate (OCR), and the enzymatic activity of carnitine palmitoyl transferase 1 (CPT1) and citrate synthase during neutrophil-like differentiation. We compared the surface phenotype, functions, and OCR of neutrophil-like cells differentiated under both glucose concentrations. RESULTS: Donor neutrophils showed significant instability of CD11b and OCR after phorbol 12-myristate 13-acetate stimulation at 3 hours post-enrichment. During HL-60 neutrophil-like cell differentiation, there was a significant increase in surface CD15 and CD11b expression together with the loss of mitochondrial mass. Differentiated neutrophil-like cells also exhibited higher CD11b expression and were significantly more phagocytic. In higher glucose media, we measured a decrease in citrate synthase and CPT1 activities during neutrophil-like differentiation. CONCLUSIONS: HL-60 neutrophil-like differentiation recapitulated known molecular and metabolic features of human neutrophil differentiation. Increased glucose concentrations correlated with features described in hyperglycemic donor neutrophils including increased CD11b and phagocytosis. We used this model to describe metabolic features of neutrophil-like cell differentiation in hyperglycemia and show for the first time the downregulation of CPT1 and citrate synthase activity, independently of mitochondrial mass.


Assuntos
Diferenciação Celular , Hiperglicemia , Neutrófilos , Humanos , Neutrófilos/metabolismo , Células HL-60 , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Antígeno CD11b/metabolismo , Glucose/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Consumo de Oxigênio , Antígenos CD15/metabolismo , Citrato (si)-Sintase/metabolismo
5.
Sci Rep ; 12(1): 17966, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36289285

RESUMO

The gold-standard method to evaluate a functional antiviral immune response is to titer neutralizing antibodies (NAbs) against a viral pathogen. This is historically performed using an in vitro assay of virus-mediated infection, which requires BSL-3 facilities. As these are insufficient in Latin American countries, including Mexico, scant information is obtained locally about viral pathogens NAb, using a functional assay. An alternative solution to using a BSL-3 assay with live virus is to use a BSL-2-safe assay with a non-replicative pseudovirus. Pseudoviral particles can be engineered to display a selected pathogen's entry protein on their surface, and to deliver a reporter gene into target cells upon transduction. Here we comprehensively describe the first development of a BSL-2 safe NAbs-measuring functional assay in Mexico, based on the production of pseudotyped lentiviral particles. As proof-of-concept, the assay is based on Nanoluc luciferase-mediated luminescence measurements from target cells transduced with SARS-CoV-2 Spike-pseudotyped lentiviral particles. We applied the optimized assay in a BSL-2 facility to measure NAbs in 65 serum samples, which evidenced the assay with 100% sensitivity, 86.6% specificity and 96% accuracy. Overall, this is the first report of a BSL-2 safe pseudovirus-based functional assay developed in Mexico to measure NAbs, and a cornerstone methodology necessary to measure NAbs with a functional assay in limited resources settings.


Assuntos
Anticorpos Neutralizantes , COVID-19 , Humanos , SARS-CoV-2 , Testes de Neutralização/métodos , Glicoproteína da Espícula de Coronavírus/metabolismo , Anticorpos Antivirais , México , Luciferases/genética , Antivirais
6.
Lab Chip ; 10(20): 2655-8, 2010 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-20820632

RESUMO

Minimally invasive biosensors are of great interest for rapid detection of disease biomarkers for diagnostic screening at the point-of-care. Here we introduce a device which extracts disease-specific biomarkers directly from the upper dermis, without the needle and syringe or resource-intensive blood processing. Using antigen-specific antibodies raised in mice as a model system, we confirm the analytical specificity and sensitivity of the antibody capture and extraction in comparison to the conventional methods based on needle/syringe blood draw followed by processing and antigen-specific ELISAs.


Assuntos
Biomarcadores/análise , Técnicas Biossensoriais/instrumentação , Coleta de Amostras Sanguíneas/instrumentação , Imunoensaio/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Análise Serial de Proteínas/instrumentação , Pele/metabolismo , Animais , Ação Capilar , Desenho de Equipamento , Análise de Falha de Equipamento , Camundongos , Camundongos Endogâmicos C57BL
7.
Artigo em Inglês | MEDLINE | ID: mdl-32903834

RESUMO

[This corrects the article DOI: 10.3389/fbioe.2020.00620.].

8.
Artigo em Inglês | MEDLINE | ID: mdl-32637403

RESUMO

Cell therapy products exhibit great therapeutic potential but come with a deterring price tag partly caused by their costly manufacturing processes. The development of strategies that lead to cost-effective cell production is key to expand the reach of cell therapies. Growth factors are critical culture media components required for the maintenance and differentiation of cells in culture and are widely employed in cell therapy manufacturing. However, they are expensive, and their common use in soluble form is often associated with decreased stability and bioactivity. Immobilization has emerged as a possible strategy to optimize growth factor use in cell culture. To date, several immobilization techniques have been reported for attaching growth factors onto a variety of biomaterials, but these have been focused on tissue engineering. This review briefly summarizes the current landscape of cell therapy manufacturing, before describing the types of chemistry that can be used to immobilize growth factors for cell culture. Emphasis is placed to identify strategies that could reduce growth factor usage and enhance bioactivity. Finally, we describe a case study for stem cell factor.

9.
Front Med (Lausanne) ; 6: 21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30881955

RESUMO

Neutropenia is a common side-effect of acute myeloid leukemia (AML) chemotherapy characterized by a critical drop in neutrophil blood concentration. Neutropenic patients are prone to infections, experience poorer clinical outcomes, and require expensive medical care. Although transfusions of donor neutrophils are a logical solution to neutropenia, this approach has not gained clinical traction, primarily due to challenges associated with obtaining sufficiently large numbers of neutrophils from donors whilst logistically managing their extremely short shelf-life. A protocol has been developed that produces clinical-scale quantities of neutrophils from hematopoietic stem and progenitor cells (HSPC) in 10 L single-use bioreactors (1). This strategy could be used to mass produce neutrophils and generate sufficient cell numbers to allow decisive clinical trials of neutrophil transfusion. We present a bioprocess model for neutrophil production at relevant clinical-scale. We evaluated two production scenarios, and the impact on cost of goods (COG) of multiple model parameters including cell yield, materials costs, and process duration. The most significant contributors to cost were consumables and raw materials, including the cost of procuring HSPC-containing umbilical cord blood. The model indicates that the most cost-efficient culture volume (batch size) is ~100 L in a single bioreactor. This study serves as a framework for decision-making and optimization strategies when contemplating the production of clinical quantities of cells for allogeneic therapy.

10.
Virol J ; 5: 77, 2008 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-18613963

RESUMO

BACKGROUND: The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes. RESULTS: Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y. CONCLUSION: Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.


Assuntos
Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Neuraminidase/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Charadriiformes/virologia , Galinhas/virologia , Criança , Pré-Escolar , Primers do DNA , Patos/virologia , Feminino , Cavalos/virologia , Humanos , Lactente , Vírus da Influenza A Subtipo H9N2 , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Infecções por Orthomyxoviridae/veterinária , Infecções por Orthomyxoviridae/virologia , RNA Viral/análise , RNA Viral/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA
11.
Sci Transl Med ; 7(290): 290ra87, 2015 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-26041704

RESUMO

In animals, immunomodulatory dendritic cells (DCs) exposed to autoantigen can suppress experimental arthritis in an antigen-specific manner. In rheumatoid arthritis (RA), disease-specific anti-citrullinated peptide autoantibodies (ACPA or anti-CCP) are found in the serum of about 70% of RA patients and are strongly associated with HLA-DRB1 risk alleles. This study aimed to explore the safety and biological and clinical effects of autologous DCs modified with a nuclear factor κB (NF-κB) inhibitor exposed to four citrullinated peptide antigens, designated "Rheumavax," in a single-center, open-labeled, first-in-human phase 1 trial. Rheumavax was administered once intradermally at two progressive dose levels to 18 human leukocyte antigen (HLA) risk genotype-positive RA patients with citrullinated peptide-specific autoimmunity. Sixteen RA patients served as controls. Rheumavax was well tolerated: adverse events were grade 1 (of 4) severity. At 1 month after treatment, we observed a reduction in effector T cells and an increased ratio of regulatory to effector T cells; a reduction in serum interleukin-15 (IL-15), IL-29, CX3CL1, and CXCL11; and reduced T cell IL-6 responses to vimentin(447-455)-Cit450 relative to controls. Rheumavax did not induce disease flares in patients recruited with minimal disease activity, and DAS28 decreased within 1 month in Rheumavax-treated patients with active disease. This exploratory study demonstrates safety and biological activity of a single intradermal injection of autologous modified DCs exposed to citrullinated peptides, and provides rationale for further studies to assess clinical efficacy and antigen-specific effects of autoantigen immunomodulatory therapy in RA.


Assuntos
Artrite Reumatoide/terapia , Citrulina/química , Células Dendríticas/imunologia , Antígenos HLA/genética , Imunoterapia , Peptídeos/química , Idoso , Artrite Reumatoide/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
12.
Stem Cells Transl Med ; 3(4): 541-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24598780

RESUMO

High-dose chemotherapy is accompanied by an obligate period of neutropenia. Resulting bacterial and fungal infections are the leading cause of morbidity and mortality in neutropenic patients despite prophylactic antimicrobials and hematopoietic growth factor supplements. Replacing neutrophils in the patient through transfusion of donor cells is a logical solution to prevent fulminant infections. In the past, this strategy has been hampered by poor yield, inability to store collected cells, and possible donor morbidity caused by granulocyte colony-stimulating factor injections and apheresis. Today, neutrophil-like cells can be manufactured in the laboratory at the clinical scale from hematopoietic stem and progenitor cells enriched from umbilical cord blood. This article reviews the rationale for focusing research efforts toward ex vivo neutrophil production and explores clinical settings for future trials.


Assuntos
Transplante de Células , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Controle de Infecções , Infecções , Aloenxertos , Anti-Infecciosos/uso terapêutico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Humanos , Neutropenia/etiologia , Neutropenia/mortalidade
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