RESUMEN
Red blood cell (RBC) metabolic reprogramming upon exposure to high altitude contributes to physiological human adaptations to hypoxia, a multifaceted process critical to health and disease. To delve into the molecular underpinnings of this phenomenon, first, we performed a multi-omics analysis of RBCs from six lowlanders after exposure to high-altitude hypoxia, with longitudinal sampling at baseline, upon ascent to 5,100 m and descent to sea level. Results highlighted an association between erythrocyte levels of 2,3-bisphosphoglycerate (BPG), an allosteric regulator of hemoglobin that favors oxygen off-loading in the face of hypoxia, and expression levels of the Rhesus blood group RHCE protein. We then expanded on these findings by measuring BPG in RBCs from 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. These data informed a genome-wide association study using BPG levels as a quantitative trait, which identified genetic polymorphisms in the region coding for the Rhesus blood group RHCE as critical determinants of BPG levels in erythrocytes from healthy human volunteers. Mechanistically, we suggest that the Rh group complex, which participates in the exchange of ammonium with the extracellular compartment, may contribute to intracellular alkalinization, thus favoring BPG mutase activity.
Asunto(s)
Altitud , Antígenos de Grupos Sanguíneos , Hipoxia , Sistema del Grupo Sanguíneo Rh-Hr , Humanos , 2,3-Difosfoglicerato/metabolismo , Eritrocitos/metabolismo , Estudio de Asociación del Genoma Completo , Hipoxia/genética , Hipoxia/metabolismo , Polimorfismo Genético , Sistema del Grupo Sanguíneo Rh-Hr/genética , Sistema del Grupo Sanguíneo Rh-Hr/metabolismoRESUMEN
ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.
Asunto(s)
Donantes de Sangre , Hemólisis , Humanos , Quinurenina/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Eritrocitos/metabolismo , Metabolómica , Conservación de la Sangre/métodosRESUMEN
ABSTRACT: Recent large-scale multiomics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on 2 separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured l-carnitine and acyl-carnitines in 13 091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation study. Genome-wide association studies against 879 000 polymorphisms identified critical genetic factors contributing to interdonor heterogeneity in end-of-storage carnitine levels, including common nonsynonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, and SLC16A9); carnitine synthesis (FLVCR1 and MTDH) and metabolism (CPT1A, CPT2, CRAT, and ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying 2 alleles of the rs12210538 SLC22A16 single-nucleotide polymorphism exhibited the lowest l-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice, and Percoll density gradients of human RBCs, showed age-dependent depletions of l-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process after chemically induced membrane lipid damage. Supplementation of stored murine RBCs with l-carnitine boosted posttransfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.
Asunto(s)
Carnitina , Eritrocitos , Hemólisis , Carnitina/metabolismo , Humanos , Animales , Ratones , Eritrocitos/metabolismo , Polimorfismo de Nucleótido Simple , Envejecimiento Eritrocítico , Estudio de Asociación del Genoma Completo , Masculino , Femenino , Miembro 5 de la Familia 22 de Transportadores de Solutos/genética , Miembro 5 de la Familia 22 de Transportadores de Solutos/metabolismo , Conservación de la Sangre/métodosRESUMEN
Serial blood and mucosal samples were characterized for 102 participants enrolled a median of 7.0 days after coronavirus disease 2019 diagnosis. Mucosal RNA was detectable for a median of 31.5 (95% confidence interval [CI], 20.5-63.5) days, with persistence ≥1 month associated with obesity (body mass index [BMI] ≥30 kg/m2; odds ratio [OR], 3.9 [95% CI, 1.2-13.8]) but not age, sex, or chronic conditions. Fifteen participants had likely reinfection; lower serum anti-spike IgG levels were associated with reinfection risk. Nearly half of participants (47%) reported symptoms lasting ≥2-3 months; persistence ≥3 months was associated with BMI ≥30 kg/m2 (OR, 4.2 [95% CI, 1.1-12.8]) and peak anti-spike and anti-nucleocapsid antibody levels.
Asunto(s)
Anticuerpos Antivirales , COVID-19 , ARN Viral , SARS-CoV-2 , Humanos , COVID-19/inmunología , COVID-19/diagnóstico , Masculino , Femenino , SARS-CoV-2/inmunología , Persona de Mediana Edad , ARN Viral/sangre , Adulto , Anticuerpos Antivirales/sangre , Anciano , Inmunoglobulina G/sangre , Reinfección/inmunología , Reinfección/virologíaRESUMEN
BACKGROUND: The safety of transfusion of SARS-CoV-2 antibodies in high plasma volume blood components to recipients without COVID-19 is not established. We assessed whether transfusion of plasma or platelet products during periods of increasing prevalence of blood donor SARS-CoV-2 infection and vaccination was associated with changes in outcomes in hospitalized patients without COVID-19. METHODS: We conducted a retrospective cohort study of hospitalized adults who received plasma or platelet transfusions at 21 hospitals during pre-COVID-19 (3/1/2018-2/29/2020), COVID-19 pre-vaccine (3/1/2020-2/28/2021), and COVID-19 post-vaccine (3/1/2021-8/31/2022) study periods. We used multivariable logistic regression with generalized estimating equations to adjust for demographics and comorbidities to calculate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: Among 21,750 hospitalizations of 18,584 transfusion recipients without COVID-19, there were 697 post-transfusion thrombotic events, and oxygen requirements were increased in 1751 hospitalizations. Intensive care unit length of stay (n = 11,683) was 3 days (interquartile range 1-5), hospital mortality occurred in 3223 (14.8%), and 30-day rehospitalization in 4144 (23.7%). Comparing the pre-COVID, pre-vaccine and post-vaccine study periods, there were no trends in thromboses (OR 0.9 [95% CI 0.8, 1.1]; p = .22) or oxygen requirements (OR 1.0 [95% CI 0.9, 1.1]; p = .41). In parallel, there were no trends across study periods for ICU length of stay (p = .83), adjusted hospital mortality (OR 1.0 [95% CI 0.9-1.0]; p = .36), or 30-day rehospitalization (p = .29). DISCUSSION: Transfusion of plasma and platelet blood components collected during the pre-vaccine and post-vaccine periods of the COVID-19 pandemic was not associated with increased adverse outcomes in transfusion recipients without COVID-19.
Asunto(s)
Transfusión de Componentes Sanguíneos , Donantes de Sangre , COVID-19 , Transfusión de Plaquetas , Adulto , Humanos , COVID-19/epidemiología , Oxígeno , Transfusión de Plaquetas/efectos adversos , Estudios Retrospectivos , Vacunación , Vacunas contra la COVID-19 , Transfusión de Componentes Sanguíneos/efectos adversos , Plasma , HospitalizaciónRESUMEN
Extracellular vesicles (EVs) are nanoparticles with a role in intercellular communication. Cell-free mitochondrial DNA (cf-mtDNA) has been associated with cognitive dysfunction in people with HIV (PWH). We conducted a nested case-control study to test the hypothesis that plasma EVs are associated with cf-mtDNA and cognitive dysfunction in older PWH. A machine learning-based model identified total EVs, including select EV subpopulations, as well as urine cf-mtDNA and 4-meter walk time carry power to predict the neurocognitive impairment. These features resulted in an AUC-ROC of 0.845 + / - 0.109 (0.615, 1.00).
Asunto(s)
Ácidos Nucleicos Libres de Células , Disfunción Cognitiva , Vesículas Extracelulares , Infecciones por VIH , Humanos , Anciano , Ácidos Nucleicos Libres de Células/genética , Estudios de Casos y Controles , Disfunción Cognitiva/genética , Disfunción Cognitiva/complicaciones , ADN Mitocondrial/genética , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológicoRESUMEN
BACKGROUND AIMS: Culture-derived mesenchymal stromal cells (MSCs) exhibit variable characteristics when manufactured using different methods, source material and culture media. The purpose of this multicenter study was to assess the impact on MSC expansion, gene expression and other characteristics when different laboratories expanded MSCs from cultures initiated with bone marrow-MSC aliquots derived from the same donor source material yet with different growth media. METHODS: Eight centers expanded MSCs using four human platelet lysate (HPL) and one fetal bovine serum (FBS) products as media supplements. The expanded cells were taken through two passages then assessed for cell count, viability, doubling time, immunophenotype, cell function, immunosuppression and gene expression. Results were analyzed by growth media and by center. RESULTS: Center methodologies varied by their local seeding density, feeding regimen, inoculation density, base media and other growth media features (antibiotics, glutamine, serum). Doubling times were more dependent on center than on media supplements. Two centers had appropriate immunophenotyping showing all MSC cultures were positive for CD105, CD73, CD90 and negative for CD34, CD45, CD14, HLA-DR. MSCs cultured in media supplemented with FBS compared with HPL featured greater T-cell inhibition potential. Gene expression analysis showed greater impact of the type of media supplement (HPL versus FBS) than the manufacturing center. Specifically, nine genes were decreased in expression and six increased when combining the four HPL-grown MSCs versus FBS (false discovery rate [FDR] <0.01), however, without significant difference between different sources of HPL (FDR <0.01). CONCLUSIONS: Local manufacturing process plays a critical role in MSC expansion while growth media may influence function and gene expression. All HPL and FBS products supported cell growth.
RESUMEN
BACKGROUND: Human leukocyte antigen (HLA) alloimmunization can occur after platelet transfusion. These antibodies can complicate future platelet transfusions or organ transplantation. Animal data suggest that Mirasol pathogen reduction treatment (PRT) can prevent alloimmunization after transfusion. STUDY DESIGN AND METHODS: The MIPLATE trial enrolled 330 of a planned 660 participants with hematological malignancies at risk for grade 2 or greater bleeding. The study was halted early for futility after a planned interim analysis. Participants were randomized to receive PRT versus standard control platelets. Serum samples were collected from participants at baseline (pretransfusion), weekly for the first 4 weeks, then at days 42 and 56. HLA antibody levels were determined using a commercial multianalyte bead-based assay. HLA antibody levels were analyzed using low, medium, and high cutoffs based on prior studies. RESULTS: The rate of alloimmunization was low in both arms of the study, particularly at the high HLA antibody cutoff (total of 6 of 277 subjects at risk, or 2.2%). The risk of alloimmunization did not differ between study arms, nor did the risk of immune refractoriness to platelet transfusion. CONCLUSIONS: The data do not support the conclusion that Mirasol exerted a protective effect against alloimmunization after platelet transfusion in the MIPLATE trial.
Asunto(s)
Plaquetas , Isoanticuerpos , Animales , Humanos , Transfusión de Plaquetas/efectos adversos , Antígenos HLA , Antígenos de Histocompatibilidad Clase IRESUMEN
Human immunodeficiency virus (HIV) infection is associated with increased systemic microbial translocation, neuroinflammation, and occasionally, neuronal injury. Whether systemic lipopolysaccharide (LPS) penetrates into the brain and contributes to neuroinflammation remain unknown in HIV. Here, we measured plasma and cerebrospinal fluid (CSF) LPS levels along with biomarkers of neuroinflammation (white blood cell counts and 40 soluble markers) and neurofilament light chain (NfL). Notably, CSF LPS was undetectable in all samples, including 3 HIV-infected individuals with dementia. Increased plasma LPS, neuroinflammation, and blood-brain barrier (BBB) dysfunction were found in untreated HIV-infected individuals, but not in healthy or treated HIV-infected individuals. Plasma LPS levels were directly correlated with various markers of inflammation in both plasma and CSF, as well as with degree of BBB permeability but not with CSF NfL in HIV-infected subjects. These results suggest that the magnitude of microbial translocation associates with neuroinflammation and BBB permeability in HIV without direct penetration into the central nervous system.
Asunto(s)
Barrera Hematoencefálica , Infecciones por VIH , Inflamación , Lipopolisacáridos , Enfermedades Neuroinflamatorias , Biomarcadores , Infecciones por VIH/complicaciones , VIH-1 , Humanos , Inflamación/complicaciones , Inflamación/virología , Lipopolisacáridos/sangre , Lipopolisacáridos/líquido cefalorraquídeo , Enfermedades Neuroinflamatorias/complicaciones , Enfermedades Neuroinflamatorias/virología , PermeabilidadRESUMEN
The continuing spread of HIV/AIDS is predominantly fueled by sexual exposure to HIV-contaminated semen. Seminal plasma (SP), the liquid portion of semen, harbors a variety of factors that may favor HIV transmission by facilitating viral entry into host cells, eliciting the production of proinflammatory cytokines, and enhancing the translocation of HIV across the genital epithelium. One important and abundant class of factors in SP is extracellular vesicles (EVs), which, in general, are important intercellular signal transducers. Although numerous studies have characterized blood plasma-derived EVs from both uninfected and HIV-infected individuals, little is known about the properties of EVs from the semen of HIV-infected individuals. We report here that fractionated SP enriched for EVs from HIV-infected men induces potent transcriptional responses in epithelial and stromal cells that interface with the luminal contents of the female reproductive tract. Semen EV fractions from acutely infected individuals induced a more proinflammatory signature than those from uninfected individuals. This was not associated with any observable differences in the surface phenotypes of the vesicles. However, microRNA (miRNA) expression profiling analysis revealed that EV fractions from infected individuals exhibit a broader and more diverse profile than those from uninfected individuals. Taken together, our data suggest that SP EVs from HIV-infected individuals exhibit unique miRNA signatures and exert potent proinflammatory transcriptional changes in cells of the female reproductive tract, which may facilitate HIV transmission.IMPORTANCE Seminal plasma (SP), the major vehicle for HIV, can modulate HIV transmission risk through a variety of mechanisms. Extracellular vesicles (EVs) are extremely abundant in semen, and because they play a key role in intercellular communication pathways and immune regulation, they may impact the likelihood of HIV transmission. However, little is known about the properties and signaling effects of SP-derived EVs in the context of HIV transmission. Here, we conduct a phenotypic, transcriptomic, and functional characterization of SP and SP-derived EVs from uninfected and HIV-infected men. We find that both SP and its associated EVs elicit potent proinflammatory transcriptional responses in cells that line the genital tract. EVs from HIV-infected men exhibit a more diverse repertoire of miRNAs than EVs from uninfected men. Our findings suggest that EVs from the semen of HIV-infected men may significantly impact the likelihood of HIV transmission through multiple mechanisms.
Asunto(s)
Vesículas Extracelulares/genética , MicroARNs/genética , Semen/metabolismo , Adulto , Estudios de Cohortes , Citocinas/metabolismo , Vesículas Extracelulares/metabolismo , Femenino , Genitales Femeninos , Infecciones por VIH/inmunología , VIH-1/fisiología , Humanos , Masculino , Conducta Sexual , Transcriptoma/genéticaRESUMEN
BACKGROUND AIMS: The cryopreservation of hematopoietic stem cells (HSCs) in dimethyl sulfoxide (DMSO) is used widely, but DMSO toxicity in transplant patients and the effects of DMSO on the normal function of cryopreserved cells are concerns. To address these issues, in vitro and clinical studies have explored using reduced concentrations of DMSO for cryopreservation. However, the effect of reducing DMSO concentration on the efficient cryopreservation of HSCs has not been directly measured. METHODS: Cryopreservation of human bone marrow using 10%, 7.5% and 5% DMSO concentrations was examined. Cell counting, flow cytometry and colony assays were used to analyze different cell populations. The recovery of stem cells was enumerated using extreme limiting dilution analysis of long-term multi-lineage engraftment in immunodeficient mice. Four different methods of analyzing human engraftment were compared to ascertain stem cell engraftment: (i) engraftment of CD33+ myeloid, CD19+ B-lymphoid, CD235a+ erythroid and CD34+ progenitors; (ii) engraftment of the same four populations plus CD41+CD42b+ platelets; (iii) engraftment of CD34++CD133+ cells; and (iv) engraftment of CD34++CD38- cells. RESULTS: Hematopoietic colony-forming, CD34++/+, CD34++CD133+ and CD34++CD38- cells were as well preserved with 5% DMSO as they were with the higher concentrations tested. The estimates of stem cell frequencies made in the xenogeneic transplant model did not show any significant detrimental effect of using lower concentrations of DMSO. Comparison of the different methods of gauging stem cell engraftment in mice led to different estimates of stem cell numbers, but overall, all measures found that reduced concentrations of DMSO supported the cryopreservation of HSCs. CONCLUSION: Cryopreservation of HSCs in DMSO concentrations as low as 5% is effective.
Asunto(s)
Dimetilsulfóxido , Trasplante de Células Madre Hematopoyéticas , Animales , Antígenos CD34 , Recuento de Células , Criopreservación , Dimetilsulfóxido/farmacología , Células Madre Hematopoyéticas , Humanos , RatonesRESUMEN
BACKGROUND: Removal of cytokines, chemokines, and microvesicles from the supernatant of allogeneic erythrocytes may help mitigate adverse transfusion reactions. Blood bank-based washing procedures present logistical difficulties; therefore, we tested the hypothesis that on-demand bedside washing of allogeneic erythrocyte units is capable of removing soluble factors and is feasible in a clinical setting. METHODS: There were in vitro and prospective, observation cohort components to this a priori planned substudy evaluating bedside allogeneic erythrocyte washing, with a cell saver, during cardiac surgery. Laboratory data were collected from the first 75 washed units given to a subset of patients nested in the intervention arm of a parent clinical trial. Paired pre- and postwash samples from the blood unit bags were centrifuged. The supernatant was aspirated and frozen at -70°C, then batch-tested for cell-derived microvesicles, soluble CD40 ligand, chemokine ligand 5, and neutral lipids (all previously associated with transfusion reactions) and cell-free hemoglobin (possibly increased by washing). From the entire cohort randomized to the intervention arm of the trial, bedside washing was defined as feasible if at least 75% of prescribed units were washed per protocol. RESULTS: Paired data were available for 74 units. Washing reduced soluble CD40 ligand (median [interquartile range]; from 143 [1 to 338] ng/ml to zero), chemokine ligand 5 (from 1,314 [715 to 2,551] to 305 [179 to 488] ng/ml), and microvesicle numbers (from 6.90 [4.10 to 20.0] to 0.83 [0.33 to 2.80] × 106), while cell-free hemoglobin concentration increased from 72.6 (53.6 to 171.6) mg/dl to 210.5 (126.6 to 479.6) mg/dl (P < 0.0001 for each). There was no effect on neutral lipids. Bedside washing was determined as feasible for 80 of 81 patients (99%); overall, 293 of 314 (93%) units were washed per protocol. CONCLUSIONS: Bedside erythrocyte washing was clinically feasible and greatly reduced concentrations of soluble factors thought to be associated with transfusion-related adverse reactions, increasing concentrations of cell-free hemoglobin while maintaining acceptable (less than 0.8%) hemolysis.
Asunto(s)
Eliminación de Componentes Sanguíneos/métodos , Quimiocinas , Citocinas , Transfusión de Eritrocitos/métodos , Eritrocitos/química , Reacción a la Transfusión/prevención & control , Conservación de la Sangre , Estudios de Cohortes , Eritrocitos/citología , Humanos , Sistemas de Atención de Punto , Estudios ProspectivosRESUMEN
Pathogen inactivation of platelet concentrates reduces the risk for blood-borne infections. However, its effect on platelet function and hemostatic efficacy of transfusion is unclear. We conducted a randomized noninferiority trial comparing the efficacy of pathogen-inactivated platelets using riboflavin and UV B illumination technology (intervention) compared with standard plasma-stored platelets (control) for the prevention of bleeding in patients with hematologic malignancies and thrombocytopenia. The primary outcome parameter was the proportion of transfusion-treatment periods in which the patient had grade 2 or higher bleeding, as defined by World Health Organization criteria. Between November 2010 and April 2016, 469 unique patients were randomized to 567 transfusion-treatment periods (283 in the control arm, 284 in the intervention arm). There was a 3% absolute difference in grade 2 or higher bleeding in the intention-to-treat analysis: 51% of the transfusion-treatment periods in the control arm and 54% in the intervention arm (95% confidence interval [CI], -6 to 11; P = .012 for noninferiority). However, in the per-protocol analysis, the difference in grade 2 or higher bleeding was 8%: 44% in the control arm and 52% in the intervention arm (95% CI -2 to 18; P = .19 for noninferiority). Transfusion increment parameters were â¼50% lower in the intervention arm. There was no difference in the proportion of patients developing HLA class I alloantibodies. In conclusion, the noninferiority criterion for pathogen-inactivated platelets was met in the intention-to-treat analysis. This finding was not demonstrated in the per-protocol analysis. This trial was registered at The Netherlands National Trial Registry as #NTR2106 and at www.clinicaltrials.gov as #NCT02783313.
Asunto(s)
Plaquetas/metabolismo , Hemostasis , Transfusión de Plaquetas , Coagulación Sanguínea , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Estudios Multicéntricos como Asunto , Evaluación del Resultado de la Atención al Paciente , Pruebas de Función Plaquetaria , Transfusión de Plaquetas/efectos adversos , Transfusión de Plaquetas/métodos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
STUDY QUESTION: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)? SUMMARY ANSWER: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization. WHAT IS KNOWN ALREADY: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization. PARTICIPANTS/MATERIALS, SETTING, METHODS: eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs. MAIN RESULTS AND THE ROLE OF CHANCE: SP promoted decidualization both in the absence and presence of steroid hormones (P < 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization. LARGE SCALE DATA: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640. LIMITATIONS, REASONS FOR CAUTION: This study is limited to in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS: Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest.
Asunto(s)
Decidua , Fibroblastos/citología , Interleucina-11/fisiología , Semen , Estudios Transversales , Decidua/fisiología , Endometriosis , Endometrio/citología , Femenino , Humanos , Interleucina-11/genética , Síndrome del Ovario PoliquísticoRESUMEN
BACKGROUND AND OBJECTIVES: Red-blood-cell (RBC) transfusion is associated with lung injury, which is further exacerbated by mechanical ventilation. Manufacturing methods of blood products differ globally and may play a role in the induction of pulmonary cell activation through alteration of the immunomodulatory property of the products. Here, the effect of different manufacturing methods on pulmonary cell activation was investigated in an in vitro model of mechanical ventilation. MATERIALS AND METHODS: Pulmonary type II cells were incubated with supernatant from fresh and old RBC products obtained via whole blood filtration (WBF), red cell filtration (RCF), apheresis-derived (AD) or whole blood-derived (WBD) methods. Lung cells were subjected to 25% stretch for 24 h. Controls were non-stretched or non-incubated cells. RESULTS: Fresh but not old AD products and WBF products induce lung cell production of pro-inflammatory cytokines and chemokines, which was not observed with WBD or RCF products. Effects were associated with an increased amount of platelet-derived vesicles and an increased thrombin-generating capacity. Mechanical stretching of lung cells induced more severe cell injury compared to un-stretched controls, including alterations in the cytoskeleton, which was further augmented by incubation with AD products. In all read-out parameters, RCF products seemed to induce less injury compared to the other products. CONCLUSIONS: Our findings show that manufacturing methods of RBC products impact pulmonary cell activation, which may be mediated by the generation of vesicles in the product. We suggest RBC manufacturing method may be an important factor in understanding the association between RBC transfusion and lung injury.
Asunto(s)
Conservación de la Sangre , Transfusión de Eritrocitos/efectos adversos , Inflamación , Pulmón/patología , Citocinas , Eritrocitos , Humanos , Respiración Artificial , TrombinaRESUMEN
BACKGROUND: Cell based therapies, such as bone marrow derived mesenchymal stem cells (BM-MSCs; also known as mesenchymal stromal cells), are currently under investigation for a number of disease applications. The current challenge facing the field is maintaining the consistency and quality of cells especially for cell dose production for pre-clinical testing and clinical trials. Here we determine how BM-donor variability and thus the derived MSCs factor into selection of the optimal primary cell lineage for cell production and testing in a pre-clinical swine model of trauma induced acute respiratory distress syndrome. METHODS: We harvested bone marrow and generated three different primary BM-MSCs from Yorkshire swine. Cells from these three donors were characterized based on (a) phenotype (morphology, differentiation capacity and flow cytometry), (b) in vitro growth kinetics and metabolic activity, and (c) functional analysis based on inhibition of lung endothelial cell permeability. RESULTS: Cells from each swine donor exhibited varied morphology, growth rate, and doubling times. All expressed the same magnitude of standard MSC cell surface markers by flow cytometry and had similar differentiation potential. Metabolic activity and growth potential at each of the passages varied between the three primary cell cultures. More importantly, the functional potency of the MSCs on inhibition of endothelial permeability was also cell donor dependent. CONCLUSION: This study suggests that for production of MSCs for cell-based therapy, it is imperative to examine donor variability and characterize derived MSCs for marker expression, growth and differentiation characteristics and testing potency in application dependent assays prior to selection of the optimal cell lineage for large scale expansion and dose production.
Asunto(s)
Células de la Médula Ósea/citología , Selección de Donante , Células Madre Mesenquimatosas/citología , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Forma de la Célula , Medios de Cultivo Condicionados/farmacología , Impedancia Eléctrica , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Humanos , Inmunofenotipificación , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , PorcinosRESUMEN
BACKGROUND: Following transfusion, donor white blood cells (WBCs) can persist long-term in the recipient, a phenomenon termed transfusion-associated microchimerism (TA-MC). Prior studies suggest TA-MC is limited to transfusion following traumatic injury, and is not prevented by leukoreduction. STUDY DESIGN AND METHODS: We conducted a prospective cohort study at a major trauma center to evaluate TA-MC following injury. Index samples were collected upon arrival, prior to transfusion. Follow-up samples were collected at intervals up to one year, and beyond for those testing positive for TA-MC. TA-MC was detected by real-time quantitative allele-specific polymerase chain reaction assays at the HLA-DR locus and several polymorphic insertion deletion sites screening for non-recipient alleles. RESULTS: A total of 378 trauma patients were enrolled (324 transfused cases and 54 non-transfused controls). Mean age was 42 ± 18 years, 74% were male, and 80% were injured by blunt mechanism. Mean Injury Severity Score was 20 ± 12. Among transfused patients, the median (interquartile range) number of red cell units transfused was 6 (3,12), and median time to first transfusion was 9 (0.8,45) hours. Only one case of long-term TA-MC was confirmed in our cohort. We detected short-term TA-MC in 6.5% of transfused subjects and 5.6% on non-transfused controls. CONCLUSIONS: In contrast to earlier studies, persistent TA-MC was not observed in our cohort of trauma subjects. Short-term TA-MC was detected, but at a lower frequency than previously observed, and rates were not significantly different than what was observed in non-transfused controls. The reduction in TA-MC occurrence may be attributable to changes in leukoreduction or other blood processing methods.
Asunto(s)
Quimerismo , Reacción a la Transfusión/genética , Heridas y Lesiones/terapia , Adulto , Femenino , Humanos , Puntaje de Gravedad del Traumatismo , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
BACKGROUND: Platelet transfusions can induce alloimmunization against HLA antigens. The use of pathogen-reduced platelet concentrates (PCs) was suggested to reduce HLA alloimmunization and concomitant transfusion refractoriness. METHODS: This study investigated HLA alloimmunization in available samples from 448 hemato-oncological patients who were randomized for the Pathogen Reduction Evaluation and Predictive Analytical Rating Score (PREPAReS) trial to receive either untreated or pathogen-reduced PCs (Mirasol, Terumo BCT Inc.). Anti-HLA Class I and II antibodies were determined before the first platelet transfusion and weekly thereafter using multiplex assay with standard cutoffs to detect low- as well as high-level antibodies. RESULTS: When using the lower cutoff, in patients who were antibody negative at enrollment, 5.4% (n = 12) developed anti-HLA Class I antibodies after receiving untreated PCs, while this was significantly higher in patients receiving pathogen-reduced PCs, 12.8% (n = 29; p = 0.009, intention-to-treat [ITT] analysis). A similar but nonsignificant trend was observed in the per-protocol (PP) analysis (5.4% vs. 10.1%; p = 0.15). HLA class II antibody formation was similar between both types of PCs in the ITT analysis, while the PP analysis showed a trend toward lower immunization after receiving pathogen-reduced PCs. Multivariate analysis identified receiving pathogen-reduced platelets as an independent risk factor for HLA Class I alloimmunization (ITT: odds ratio [95% confidence interval] = 3.02 [1.42-6.51], PP: odds ratio [95% confidence interval] = 2.77 [1.00-5.40]), without affecting HLA Class II alloimmunization. When using the high cutoff value, the difference in HLA Class I alloimmunization between study arms remained significant in the ITT analysis and again was not significant in the PP analysis. CONCLUSION: Our data clearly indicate that Mirasol pathogen inactivation does not prevent HLA Class I or II alloimmunization after platelet transfusions.
Asunto(s)
Antígenos HLA , Neoplasias Hematológicas , Inmunización , Isoanticuerpos , Transfusión de Plaquetas/efectos adversos , Reacción a la Transfusión , Anciano , Femenino , Antígenos HLA/sangre , Antígenos HLA/inmunología , Neoplasias Hematológicas/sangre , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/terapia , Humanos , Isoanticuerpos/sangre , Isoanticuerpos/inmunología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Reacción a la Transfusión/sangre , Reacción a la Transfusión/inmunologíaRESUMEN
BACKGROUND: Several retrospective studies have suggested that transfusion with red blood cells (RBCs) stored for longer periods is associated with increased mortality. The Age of Blood Evaluation (ABLE) study randomized subjects to receive fresh vs. standard issue RBC units and showed no difference in the primary or secondary endpoints of mortality or change in multi-organ dysfunction syndrome (MODS) score. METHODS: In this study a subset of 100 ABLE subjects were enrolled to measure coagulation and immune parameters. Samples were collected pre-transfusion and on days 2, 6, 28, and 180 post-transfusion. Levels of 16 coagulation parameters, regulatory and functional T cells, 25 cytokines, and 16 markers of extracellular vesicles (EVs) were determined. RESULTS: Changes from baseline in levels of protein C, factor V, and EVs expressing phosphatidyl serine and CTLA-4 (CD152) differed between recipients of fresh and standard storage age RBC units, with the vast majority of coagulation and EV markers and all cytokines tested showing no difference between study arms. Although most analytes showed no difference between subjects in the fresh and standard arms of the study, 6 coagulation parameters, 15 cytokines, and 7 EV parameters changed significantly in the period post-transfusion. DISCUSSION: Transfusion of fresh vs. standard issue RBC units does not result in substantial changes in coagulation or immune parameters, up to day 35 of RBC storage. Furthermore, significant changes in multiple coagulation and immune parameters are detectable post-transfusion, though causality cannot be determined based on the current study.
Asunto(s)
Coagulación Sanguínea/inmunología , Conservación de la Sangre , Citocinas , Transfusión de Eritrocitos , Eritrocitos , Vesículas Extracelulares , Biomarcadores/sangre , Enfermedad Crítica , Citocinas/sangre , Citocinas/inmunología , Eritrocitos/inmunología , Eritrocitos/metabolismo , Vesículas Extracelulares/inmunología , Vesículas Extracelulares/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de TiempoRESUMEN
BACKGROUND: Retrospective studies suggested that storage age of RBCs is associated with inflammation and thromboembolism. The Red Cell Storage Duration Study (RECESS) trial randomized subjects undergoing complex cardiac surgery to receive RBCs stored for shorter versus longer periods, and no difference was seen in the primary outcome of change in multiple organ dysfunction score. STUDY DESIGN AND METHODS: In the current study, 90 subjects from the RECESS trial were studied intensively using a range of hemostasis, immunologic, and nitric oxide parameters. Samples were collected before transfusion and on Days 2, 6, 28, and 180 after transfusion. RESULTS: Of 71 parameters tested, only 4 showed a significant difference after transfusion between study arms: CD8+ T-cell interferon-γ secretion and the concentration of extracellular vesicles bearing the B-cell marker CD19 were higher, and plasma endothelial growth factor levels were lower in recipients of fresh versus aged RBCs. Plasma interleukin-6 was higher at Day 2 and lower at Days 6 and 28 in recipients of fresh versus aged RBCs. Multiple parameters showed significant modulation after surgery and transfusion. Most analytes that changed after surgery did not differ based on transfusion status. Several extracellular vesicle markers, including two associated with platelets (CD41a and CD62P), decreased in transfused patients more than in those who underwent surgery without transfusion. CONCLUSIONS: Transfusion of fresh versus aged RBCs does not result in substantial changes in hemostasis, immune, or nitric oxide parameters. It is possible that transfusion modulates the level of platelet-derived extracellular vesicles, which will require study of patients randomly assigned to receipt of transfusion to define.