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Modulating SYK has been demonstrated to have impacts on pathogenic neutrophil responses in COVID-19. During sepsis, neutrophils are vital in early bacterial clearance but also contribute to the dysregulated immune response and organ injury when hyperactivated. Here, we evaluated the impact of R406, the active metabolite of fostamatinib, on neutrophils stimulated by LPS. We demonstrate that R406 was able to effectively inhibit NETosis, degranulation, ROS generation, neutrophil adhesion, and the formation of CD16low neutrophils that have been linked to detrimental outcomes in severe sepsis. Further, the neutrophils remain metabolically active, capable of releasing cytokines, perform phagocytosis, and migrate in response to IL-8. Taken together, this data provides evidence of the potential efficacy of utilizing fostamatinib in bacterial sepsis.
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The COVID-19 pandemic has brought biosafety to the forefront of many life sciences. The outbreak has compelled research institutions to re-evaluate biosafety practices and potential at-risk areas within research laboratories and more specifically within Shared Resource Laboratories (SRLs). In flow cytometry facilities, biological safety assessment encompasses known hazards based on the biological sample and associated risk group, as well as potential or unknown hazards, such as aerosol generation and instrument "failure modes." Cell sorting procedures undergo clearly defined biological safety assessments and adhere to well-established biosafety guidelines that help to protect SRL staff and users against aerosol exposure. Conversely, benchtop analyzers are considered low risk due to their low sample pressure and enclosed fluidic systems, although there is little empirical evidence to support this assumption of low risk. To investigate this, we evaluated several regions on analyzers using the Cyclex-d microsphere assay, a recently established method for cell sorter aerosol containment testing. We found that aerosol and/or droplet hazards were detected on all benchtop analyzers predominantly during operation in "failure modes." These results indicate that benchtop analytical cytometers present a more complicated set of risks than are commonly appreciated.
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COVID-19/prevención & control , Separación Celular/instrumentación , Contención de Riesgos Biológicos , Contaminación de Equipos/prevención & control , Citometría de Flujo/instrumentación , Personal de Laboratorio , Exposición Profesional/efectos adversos , Salud Laboral , Aerosoles , COVID-19/transmisión , Humanos , Medición de Riesgo , Factores de RiesgoRESUMEN
Biosafety has always been an important aspect of daily work in any research institution, particularly for cytometry Shared Resources Laboratories (SRLs). SRLs are common-use spaces that facilitate the sharing of knowledge, expertise, and ideas. This sharing inescapably involves contact and interaction of all those within this working environment on a daily basis. The current pandemic caused by SARS-CoV-2 has prompted the re-evaluation of many policies governing the operations of SRLs. Here we identify and review the unique challenges SRLs face in maintaining biosafety standards, highlighting the potential risks associated with not only cytometry instrumentation and samples, but also the people working with them. We propose possible solutions to safety issues raised by the COVID-19 pandemic and provide tools for facilities to adapt to evolving guidelines and future challenges.
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COVID-19/epidemiología , Contención de Riesgos Biológicos/tendencias , Laboratorios/tendencias , COVID-19/prevención & control , COVID-19/transmisión , Contención de Riesgos Biológicos/normas , Citometría de Flujo , Humanos , Laboratorios/normas , Medición de Riesgo/normas , Medición de Riesgo/tendenciasRESUMEN
Mutations in the RNA splicing gene SF3B1 are found in >80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS). We investigated the origin of SF3B1 mutations within the bone marrow hematopoietic stem and progenitor cell compartments in patients with MDS-RS. Screening for recurrently mutated genes in the mononuclear cell fraction revealed mutations in SF3B1 in 39 of 40 cases (97.5%), combined with TET2 and DNMT3A in 11 (28%) and 6 (15%) patients, respectively. All recurrent mutations identified in mononuclear cells could be tracked back to the phenotypically defined hematopoietic stem cell (HSC) compartment in all investigated patients and were also present in downstream myeloid and erythroid progenitor cells. While in agreement with previous studies, little or no evidence for clonal (SF3B1 mutation) involvement could be found in mature B cells, consistent involvement at the pro-B-cell progenitor stage was established, providing definitive evidence for SF3B1 mutations targeting lymphomyeloid HSCs and compatible with mutated SF3B1 negatively affecting lymphoid development. Assessment of stem cell function in vitro as well as in vivo established that only HSCs and not investigated progenitor populations could propagate the SF3B1 mutated clone. Upon transplantation into immune-deficient mice, SF3B1 mutated MDS-RS HSCs differentiated into characteristic ring sideroblasts, the hallmark of MDS-RS. Our findings provide evidence of a multipotent lymphomyeloid HSC origin of SF3B1 mutations in MDS-RS patients and provide a novel in vivo platform for mechanistically and therapeutically exploring SF3B1 mutated MDS-RS.
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Células Madre Hematopoyéticas/metabolismo , Linfocitos/metabolismo , Mutación/genética , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Células Mieloides/metabolismo , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , Anciano , Anciano de 80 o más Años , Animales , Diferenciación Celular , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Empalmosomas/metabolismoRESUMEN
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system involving dysregulated encephalitogenic T cells. Myeloid-derived suppressor cells (MDSCs) have been recognized for their important function in regulating T-cell responses. Recent studies have indicated a role for MDSCs in autoimmune diseases, but their significance in MS is not clear. Here, we assessed the frequencies of CD14+ HLA-DRlow monocytic MDSCs (Mo-MDSCs) and CD33+ CD15+ CD11b+ HLA-DRlow granulocytic MDSCs (Gr-MDSCs) and investigated phenotypic and functional differences of Mo-MDSCs at different clinical stages of MS and in healthy subjects (HC). Increased frequencies of Mo-MDSCs (P < 0.05) and Gr-MDSCs (P < 0.05) were observed in relapsing-remitting MS patients during relapse (RRMS-relapse) compared to stable RRMS (RRMS-rem). Secondary progressive MS (SPMS) patients displayed a decreased frequency of Mo-MDSCs and Gr-MDSCs compared to HC (P < 0.05). Mo-MDSCs within RRMS patients expressed significantly higher cell surface protein levels of CD86 and CD163 compared to SPMS patients. Mo-MDSCs within SPMS exhibited decreased mRNA expression of interleukin-10 and heme oxygenase 1 compared to RRMS and HC. Analysis of T-cell regulatory function of Mo-MDSCs demonstrated T-cell suppressive capacity in RRMS and HCs, while Mo-MDSCs of SPMS promoted autologous T-cell proliferation, which aligned with a differential cytokine profile compared to RRMS and HCs. This study is the first to show phenotypic and functional shifts of MDSCs between clinical stages of MS, suggesting a role for MDSCs as a therapeutic target to prevent MS disease progression.
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Esclerosis Múltiple/inmunología , Células Supresoras de Origen Mieloide/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Antígenos CD/metabolismo , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Inmunomodulación , Inmunofenotipificación , Inmunoterapia/tendencias , Masculino , Persona de Mediana Edad , Células Supresoras de Origen Mieloide/trasplanteRESUMEN
A high proportion of patients with lower-risk del(5q) myelodysplastic syndromes will respond to treatment with lenalidomide. The median duration of transfusion-independence is 2 years with some long-lasting responses, but almost 40% of patients progress to acute leukemia by 5 years after starting treatment. The mechanisms underlying disease progression other than the well-established finding of small TP53-mutated subclones at diagnosis remain unclear. We studied a longitudinal cohort of 35 low- and intermediate-1-risk del(5q) patients treated with lenalidomide (n=22) or not (n=13) by flow cytometric surveillance of hematopoietic stem and progenitor cell subsets, targeted sequencing of mutational patterns, and changes in the bone marrow microenvironment. All 13 patients with disease progression were identified by a limited number of mutations in TP53, RUNX1, and TET2, respectively, with PTPN11 and SF3B1 occurring in one patient each. TP53 mutations were found in seven of nine patients who developed acute leukemia, and were documented to be present in the earliest sample (n=1) and acquired during lenalidomide treatment (n=6). By contrast, analysis of the microenvironment, and of hematopoietic stem and progenitor cells by flow cytometry was of limited prognostic value. Based on our data, we advocate conducting a prospective study aimed at investigating, in a larger number of cases of del(5q) myelodysplastic syndromes, whether the detection of such mutations before and after lenalidomide treatment can guide clinical decision-making.
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Deleción Cromosómica , Cromosomas Humanos Par 5 , Mutación , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/genética , Anciano , Anciano de 80 o más Años , Biomarcadores , Biología Computacional/métodos , Progresión de la Enfermedad , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Lenalidomida , Masculino , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Síndromes Mielodisplásicos/terapia , Pronóstico , Nicho de Células Madre , Talidomida/análogos & derivados , Talidomida/uso terapéutico , Resultado del TratamientoRESUMEN
AIMS/HYPOTHESIS: We aimed to elucidate the impact of fat cell size and inflammatory status of adipose tissue on the development of type 2 diabetes in non-obese individuals. METHODS: We characterised subcutaneous abdominal adipose tissue by examining stromal cell populations by 13 colour flow cytometry, measuring expression of adipogenesis genes in the progenitor cell fraction and determining lipolysis and adipose secretion of inflammatory proteins in 14 non-obese men with type 2 diabetes and 13 healthy controls matched for age, sex, body weight and total fat mass. RESULTS: Individuals with diabetes had larger fat cells than the healthy controls but stromal cell population frequencies, adipose lipolysis and secretion of inflammatory proteins did not differ between the two groups. However, in the entire cohort fat cell size correlated positively with the ratio of M1/M2 macrophages, TNF-α secretion, lipolysis and insulin resistance. Expression of genes encoding regulators of adipogenesis and adipose morphology (BMP4, CEBPα [also known as CEBPA], PPARγ [also known as PPARG] and EBF1) correlated negatively with fat cell size. CONCLUSIONS/INTERPRETATION: We show that a major phenotype of white adipose tissue in non-obese individuals with type 2 diabetes is adipocyte hypertrophy, which may be mediated by an impaired adipogenic capacity in progenitor cells. Consequently, this could have an impact on adipose tissue inflammation, release of fatty acids, ectopic fat deposition and insulin sensitivity.
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Adipocitos/metabolismo , Adipocitos/patología , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Grasa Subcutánea/metabolismo , Grasa Subcutánea/patología , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Lipólisis/fisiología , Macrófagos/metabolismo , Macrófagos/fisiología , Masculino , Persona de Mediana Edad , Obesidad/metabolismo , Obesidad/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In development, epigenetic mechanisms such as DNA methylation have been suggested to provide a cellular memory to maintain multipotency but also stabilize cell fate decisions and direct lineage restriction. In this study, we set out to characterize changes in DNA methylation and gene expression during granulopoiesis using 4 distinct cell populations ranging from the oligopotent common myeloid progenitor stage to terminally differentiated neutrophils. We observed that differentially methylated sites (DMSs) generally show decreased methylation during granulopoiesis. Methylation appears to change at specific differentiation stages and overlap with changes in transcription and activity of key hematopoietic transcription factors. DMSs were preferentially located in areas distal to CpG islands and shores. Also, DMSs were overrepresented in enhancer elements and enriched in enhancers that become active during differentiation. Overall, this study depicts in detail the epigenetic and transcriptional changes that occur during granulopoiesis and supports the role of DNA methylation as a regulatory mechanism in blood cell differentiation.
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Metilación de ADN , Elementos de Facilitación Genéticos , Granulocitos/citología , Transcriptoma , Diferenciación Celular , Separación Celular , Islas de CpG , Citosina/metabolismo , Epigénesis Genética , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genómica , Humanos , Neutrófilos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismoRESUMEN
Phosphorylcholine (PC) is a classic T-independent Ag that is exposed on apoptotic cells, oxidized phospholipids, and bacterial polysaccharides. Experimental as well as epidemiological studies have over the past decade implicated Abs against PC (anti-PC) as anti-inflammatory and a strong protective factor in cardiovascular disease. Although clinically important, little is known about the development of anti-PC in humans. This study was conceived to dissect the human anti-PC repertoire and generate human mAbs. We designed a PC-specific probe to identify, isolate, and characterize PC-reactive B cells from 10 healthy individuals. The donors had all mounted somatically mutated Abs toward PC using a broad variety of Ig genes. PC-reactive B cells were primarily found in the IgM(+) memory subset, although significant numbers also were detected among naive, IgG(+), and CD27(+)CD43(+) B cells. Abs from these subsets were clonally related, suggesting a common origin. mAbs derived from the same donors exhibited equivalent or higher affinity for PC than the well-characterized murine T-15 clone. These results provide novel insights into the cellular and molecular ontogeny of atheroprotective PC Abs, thereby offering new opportunities for Ab-based therapeutic interventions.
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Anticuerpos Antifosfolípidos/inmunología , Subgrupos de Linfocitos B/inmunología , Inmunoglobulina M/inmunología , Memoria Inmunológica/fisiología , Fosforilcolina/inmunología , Adulto , Animales , Subgrupos de Linfocitos B/citología , Femenino , Humanos , Inmunoglobulina G/inmunología , Masculino , RatonesRESUMEN
BACKGROUND: Chronic granulomatous disease (CGD) is a primary immune deficiency characterized by a defect in reactive oxygen species production. Although the effect of CGD mainly reflects on the phagocytic compartment, B-cell responses are also impaired in patients with CGD. OBJECTIVE: We sought to investigate how defective gp91(phox) expression in patients with CGD and CGD carriers might affect the B-cell compartment and maintenance of long-term memory. METHODS: We studied the B-cell compartment of patients with CGD in terms of phenotype and ability to produce reactive oxygen species and proliferate on stimuli differently directed to the B-cell receptor and Toll-like receptor 9. We further studied their capacity to maintain long-term memory by measuring cellular and serologic responses to measles. RESULTS: We show that the memory B-cell compartment is impaired among patients with CGD, as indicated by reduced total (CD19(+)CD27(+)) and resting (CD19(+)CD27(+)CD21(+)) memory B cells in parallel to increased naive (CD19(+)CD27(-)IgD(+)) B-cell frequencies. Data on CGD carriers reveal that such alterations are related to gp91(phox) expression. Moreover, proliferative capabilities of B cells on selective in vitro stimulation of B-cell receptor or Toll-like receptor 9 pathways were reduced in patients with CGD compared with those seen in age-matched healthy control subjects. Significantly lower measles-specific antibody levels and antibody-secreting cell numbers were also observed, indicating a poor ability to maintain long-term memory in these patients. CONCLUSION: Altogether, our data suggest that patients with CGD present a defective B-cell compartment in terms of frequencies of memory B cells, response to in vitro stimulation, and maintenance of long-term antigen-specific memory.
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Linfocitos B/inmunología , Enfermedad Granulomatosa Crónica/inmunología , Memoria Inmunológica/efectos de los fármacos , Sarampión/prevención & control , Glicoproteínas de Membrana/inmunología , NADPH Oxidasas/inmunología , Adolescente , Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Estudios de Casos y Controles , Proliferación Celular/efectos de los fármacos , Preescolar , Femenino , Expresión Génica , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/patología , Humanos , Inmunofenotipificación , Lactante , Masculino , Sarampión/inmunología , Glicoproteínas de Membrana/genética , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Fenotipo , Mitógenos de Phytolacca americana/farmacología , Cultivo Primario de Células , Especies Reactivas de Oxígeno/inmunología , Especies Reactivas de Oxígeno/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/inmunología , Vacunación , Vacunas Virales/administración & dosificación , Adulto JovenRESUMEN
BACKGROUND: In connection with platelet (PLT) production, random but transient aggregates sometimes form in the newly produced units. The underlying mechanisms as well as the impact on cellular level of this phenomenon are unknown. Hypothetically, random occurrence of aggregates may induce biochemical changes leading to PLT activation and release of immunomodulatory factors from the PLTs. STUDY DESIGN AND METHODS: PLTs were aliquoted and prepared with an automated system for PLT pooling (OrbiSac, Terumo BCT) for a three-arm nonpaired study design (n=8). Initially aggregated PLT units in SSP+ were selected by visual inspection and compared to unaffected PLT units stored in SSP+ or 100% plasma. Cellular, metabolic, and functional variables were analyzed, including the concentrations of RANTES, sCD40L, and sTWEAK in the bags during a 9-day storage period. RESULTS: Isolated aggregated PLTs show signs of spontaneous activation and respond less efficiently to TRAP stimulation. RANTES, sCD40L, and sTWEAK accumulated in the various PLT units during storage but sCD40L and RANTES accumulated in the initially aggregated PLT units to higher concentrations than the reference units and PLTs stored in 100% plasma (p<0.001). Over time, the levels of sTWEAK increased more in the plasma storage environment compared with PLT units stored in SSP+ (p<0.001). CONCLUSION: Our data indicate that random occurrence of aggregates may lead to higher activation level and increased release of immunomodulatory factors from the PLTs.
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Plaquetas/metabolismo , Ligando de CD40/metabolismo , Quimiocina CCL5/metabolismo , Activación Plaquetaria/fisiología , Humanos , Inmunomodulación/fisiologíaRESUMEN
The emergence of new pathogens continues to fuel the need for advanced high-containment laboratories across the globe. Here we explore challenges and opportunities for integration of cytometry, a central technology for cell analysis, within high-containment laboratories. We review current applications in infectious disease, vaccine research, and biosafety. Considerations specific to cytometry within high-containment laboratories, such as biosafety requirements, and sample containment strategies are also addressed. We further tour the landscape of emerging technologies, including combination of cytometry with other omics, the application of automation, and artificial intelligence. Finally, we propose a framework to fast track the immersion of advanced technologies into the high-containment research setting to improve global preparedness for new emerging diseases.
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Investigación Biomédica , Laboratorios , Inteligencia Artificial , Contención de Riesgos Biológicos , TecnologíaRESUMEN
Self-reactive and polyreactive B cells generated during B cell development are silenced by either apoptosis, clonal deletion, receptor editing or anergy to avoid autoimmunity. The specific contribution of apoptosis to normal B cell development and self-tolerance is incompletely understood. Here, we quantify self-reactivity, polyreactivity and apoptosis during physiologic B lymphocyte development. Self-reactivity and polyreactivity are most abundant in early immature B cells and diminish significantly during maturation within the bone marrow. Minimal apoptosis still occurs at this site, however B cell receptors cloned from apoptotic B cells show comparable self-reactivity to that of viable cells. Apoptosis increases dramatically only following immature B cells leaving the bone marrow sinusoids, but above 90% of cloned apoptotic transitional B cells are not self-reactive/polyreactive. Our data suggests that an apoptosis-independent mechanism, such as receptor editing, removes most self-reactive B cells in the bone marrow. Mechanistically, lack of survival signaling rather than clonal deletion appears to be the underpinning cause of apoptosis in most transitional B cells in the periphery.
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Apoptosis , Linfocitos B , Supresión Clonal , Ratones Endogámicos C57BL , Animales , Apoptosis/inmunología , Supresión Clonal/inmunología , Linfocitos B/inmunología , Ratones , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/genética , Diferenciación Celular/inmunología , Médula Ósea/inmunología , Femenino , Células Precursoras de Linfocitos B/inmunologíaRESUMEN
Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is the most widely used application of flow cytometry. Here, we present protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, we provide a procedure for preparing a PE-Texas Red tandem conjugate dye that can then be used for antibody conjugation. These protocols enable investigators to label antibodies of their choice with multiple fluorochromes and permit more combinations of antibodies for multicolor flow applications. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Labeling an antibody with fluorescein isothiocyanate (FITC) Basic Protocol 2: Labeling an antibody with long-armed biotin Basic Protocol 3: Labeling an antibody with Texas Red-X Basic Protocol 4: Labeling an antibody with a synthetic organic fluor kit Basic Protocol 5: Labeling an antibody with phycobiliproteins Basic Protocol 6: Conjugation of Texas Red to R-phycoerythrin to produce an energy transfer fluorochrome.
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Anticuerpos Monoclonales , Colorantes Fluorescentes , Humanos , Biotina , Fluoresceína , FicoeritrinaRESUMEN
Autoreactive B cells generated during B cell development are inactivated by clonal deletion, receptor editing or anergy. Up to 97% of immature B cells appear to die before completing maturation, but the anatomic sites and reasons underlying this massive cell loss are not fully understood. Here, we directly quantitated apoptosis and clonal deletion during physiologic B lymphocyte development using Rosa26INDIA apoptosis indicator mice. Immature B cells displayed low levels of apoptosis in the bone marrow but started dying at high levels in the periphery upon release from bone marrow sinusoids into the blood circulation. Clonal deletion of self-reactive B cells was neither a major contributor to apoptosis in the bone marrow nor the periphery. Instead, most peripheral transitional 1 B cells did not encounter the signals required for positive selection into the mature B cell compartments. This study sheds new light on B cell development and suggests that receptor editing and/or anergy efficiently control most primary autoreactivity in mice.
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Humanity has faced three recent outbreaks of novel betacoronaviruses, emphasizing the need to develop approaches that broadly target coronaviruses. Here, we identify 55 monoclonal antibodies from COVID-19 convalescent donors that bind diverse betacoronavirus spike proteins. Most antibodies targeted an S2 epitope that included the K814 residue and were non-neutralizing. However, 11 antibodies targeting the stem helix neutralized betacoronaviruses from different lineages. Eight antibodies in this group, including the six broadest and most potent neutralizers, were encoded by IGHV1-46 and IGKV3-20. Crystal structures of three antibodies of this class at 1.5-1.75-Å resolution revealed a conserved mode of binding. COV89-22 neutralized SARS-CoV-2 variants of concern including Omicron BA.4/5 and limited disease in Syrian hamsters. Collectively, these findings identify a class of IGHV1-46/IGKV3-20 antibodies that broadly neutralize betacoronaviruses by targeting the stem helix but indicate these antibodies constitute a small fraction of the broadly reactive antibody response to betacoronaviruses after SARS-CoV-2 infection.
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COVID-19 , SARS-CoV-2 , Animales , Cricetinae , Anticuerpos Monoclonales , Brotes de Enfermedades , Mesocricetus , Anticuerpos Antivirales , Anticuerpos Neutralizantes , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Crimean-Congo hemorrhagic fever virus (CCHFV) causes viral hemorrhagic fever with high case-fatality rates and is geographically widely distributed. Due to the requirement for a biosafety level 4 (BSL-4) laboratory and the lack of an animal model, knowledge of the viral pathogenesis is limited. Crimean-Congo hemorrhagic fever (CCHF) is characterized by hemorrhage and vascular permeability, indicating the involvement of endothelial cells (ECs). The interplay between ECs and CCHFV is therefore important for understanding the pathogenesis of CCHF. In a previous study, we found that CCHFV-infected monocyte-derived dendritic cells (moDCs) activated ECs; however, the direct effect of CCHFV on ECs was not investigated. Here, we report that ECs are activated upon infection, as demonstrated by upregulation of mRNA levels for E-selectin, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule 1 (ICAM1). Protein levels and cell surface expression of ICAM1 responded in a dose-dependent manner to increasing CCHFV titers with concomitant increase in leukocyte adhesion. Furthermore, we examined vascular endothelial (VE) cadherin in CCHFV-infected ECs by different approaches. Infected ECs released higher levels of interleukin 6 (IL-6) and IL-8; however, stimulation of resting ECs with supernatants derived from infected ECs did not result in increased ICAM1 expression. Interestingly, the moDC-mediated activation of ECs was abrogated by addition of neutralizing tumor necrosis factor alpha (TNF-α) antibody to moDC supernatants, thereby identifying this soluble mediator as the key cytokine causing EC activation. We conclude that CCHFV can exert both direct and indirect effects on ECs.
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Endotelio Vascular/virología , Virus de la Fiebre Hemorrágica de Crimea-Congo/fisiología , Animales , Antígenos CD/metabolismo , Western Blotting , Cadherinas/metabolismo , Células Cultivadas , Chlorocebus aethiops , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Reacción en Cadena de la Polimerasa , Células VeroRESUMEN
Today, HIV-infected children who have access to treatment face a chronic rather than a progressive and fatal disease. As a result, new challenges are emerging in the field. Recent lines of evidence outline several factors that can differently affect the ability of the immune system to fully reconstitute and to mount specific immune responses in children receiving HAART. In this paper, we review the underlying mechanisms of immune reconstitution after HAART initiation among vertically HIV-infected children analyzing the possible causes of suboptimal responses.