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OBJECTIVES: Sepsis and sterile both release "danger signals' that induce the systemic inflammatory response syndrome (SIRS). So differentiating infection from SIRS can be challenging. Precision diagnostic assays could limit unnecessary antibiotic use, improving outcomes. METHODS: After surveying human leukocyte cytokine production responses to sterile damage-associated molecular patterns (DAMPs), bacterial pathogen-associated molecular patterns, and bacteria we created a multiplex assay for 31 cytokines. We then studied plasma from patients with bacteremia, septic shock, "severe sepsis," or trauma (ISS ≥15 with circulating DAMPs) as well as controls. Infections were adjudicated based on post-hospitalization review. Plasma was studied in infection and injury using univariate and multivariate means to determine how such multiplex assays could best distinguish infective from noninfective SIRS. RESULTS: Infected patients had high plasma interleukin (IL)-6, IL-1α, and triggering receptor expressed on myeloid cells-1 (TREM-1) compared to controls [false discovery rates (FDR) <0.01, <0.01, <0.0001]. Conversely, injury suppressed many mediators including MDC (FDR <0.0001), TREM-1 (FDR <0.001), IP-10 (FDR <0.01), MCP-3 (FDR <0.05), FLT3L (FDR <0.05), Tweak, (FDR <0.05), GRO-α (FDR <0.05), and ENA-78 (FDR <0.05). In univariate studies, analyte overlap between clinical groups prevented clinical relevance. Multivariate models discriminated injury and infection much better, with the 2-group random-forest model classifying 11/11 injury and 28/29 infection patients correctly in out-of-bag validation. CONCLUSIONS: Circulating cytokines in traumatic SIRS differ markedly from those in health or sepsis. Variability limits the accuracy of single-mediator assays but machine learning based on multiplexed plasma assays revealed distinct patterns in sepsis- and injury-related SIRS. Defining biomarker release patterns that distinguish specific SIRS populations might allow decreased antibiotic use in those clinical situations. Large prospective studies are needed to validate and operationalize this approach.
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Citocinas/sangue , Sepse/sangue , Sepse/diagnóstico , Síndrome de Resposta Inflamatória Sistêmica/sangue , Síndrome de Resposta Inflamatória Sistêmica/diagnóstico , Relatórios Anuais como Assunto , Diagnóstico Diferencial , Cirurgia Geral , Testes Hematológicos/métodos , Humanos , Estudos Prospectivos , Sepse/imunologia , Sociedades Médicas , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Estados UnidosRESUMO
Members of the Burkholderia cepacia complex (Bcc) cause chronic opportunistic lung infections in people with cystic fibrosis (CF), resulting in a gradual lung function decline and, ultimately, patient death. The Bcc is a complex of 20 species and is rarely eradicated once a patient is colonized; therefore, vaccination may represent a better therapeutic option. We developed a new proteomics approach to identify bacterial proteins that are involved in the attachment of Bcc bacteria to lung epithelial cells. Fourteen proteins were reproducibly identified by two-dimensional gel electrophoresis from four Bcc strains representative of two Bcc species: Burkholderia cenocepacia, the most virulent, and B. multivorans, the most frequently acquired. Seven proteins were identified in both species, but only two were common to all four strains, linocin and OmpW. Both proteins were selected based on previously reported data on these proteins in other species. Escherichia coli strains expressing recombinant linocin and OmpW showed enhanced attachment (4.2- and 3.9-fold) to lung cells compared to the control, confirming that both proteins are involved in host cell attachment. Immunoproteomic analysis using serum from Bcc-colonized CF patients confirmed that both proteins elicit potent humoral responses in vivo Mice immunized with either recombinant linocin or OmpW were protected from B. cenocepacia and B. multivorans challenge. Both antigens induced potent antigen-specific antibody responses and stimulated strong cytokine responses. In conclusion, our approach identified adhesins that induced excellent protection against two Bcc species and are promising vaccine candidates for a multisubunit vaccine. Furthermore, this study highlights the potential of our proteomics approach to identify potent antigens against other difficult pathogens.
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Adesinas Bacterianas/metabolismo , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriocinas/metabolismo , Infecções por Burkholderia/prevenção & controle , Complexo Burkholderia cepacia/fisiologia , Células Epiteliais/microbiologia , Adesinas Bacterianas/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Bacteriocinas/imunologia , Infecções por Burkholderia/imunologia , Fibrose Cística/imunologia , Fibrose Cística/microbiologia , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/fisiologia , Feminino , Expressão Gênica , Humanos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resultado do TratamentoRESUMO
INTRODUCTION: Multiple large clinical trauma trials have documented an increased susceptibility to infection after injury. Although neutrophils (polymorphonuclear leukocytes [PMNs]) were historically considered a homogeneous cell type, we hypothesized that injury could alter neutrophil heterogeneity and predispose to dysfunction. To explore whether trauma modifies PMN heterogeneity, we performed an observational mass-spectrometry-based cytometry study on total leukocytes and low-density PMNs found in the peripheral blood mononuclear cell fraction of leukocytes from healthy controls and trauma patients. METHODS: A total of 74 samples from 12 trauma patients, each sampled at 1 or more time points, and matched controls were fractionated and profiled by mass-spectrometry-based cytometry using a panel of 44 distinct markers. After deconvolution and conservative gating on neutrophils, data were analyzed using Seurat, followed by clustering of principal components. RESULTS: Eleven distinct neutrophil populations were resolved in control and trauma neutrophils based on differential protein surface marker expression. Trauma markedly altered the basal heterogeneity of neutrophil subgroups seen in the control samples, with loss of a dominant population of resting neutrophils marked by high expression of C3AR and low levels of CD63, CD64, and CD177 (cluster 1), and expansion of two alternative neutrophil populations, one of which is marked by high expression of CD177 with suppression of CD10, CD16, C3AR, CD63, and CD64 (cluster 6). Remarkably, following trauma, a substantially larger percentage of neutrophils sediment in the monocyte fraction. These low-density neutrophils bear markers of functional exhaustion and form a unique trauma-induced population (cluster 9) with markedly upregulated expression of active surface adhesion molecules (activated CD11b/CD18), with suppression of nearly all other surface markers, including receptors for formyl peptides, leukotrienes, chemokines, and complement. CONCLUSION: Circulating neutrophils demonstrate considerable evidence of functional heterogeneity that is markedly altered by trauma. Trauma induces evolution of a novel, exhausted, low-density neutrophil population with immunosuppressive features.
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Antígenos CD18 , Neutrófilos , Humanos , Neutrófilos/metabolismo , Antígenos CD18/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos/metabolismo , QuimiocinasRESUMO
CD4+ regulatory T cells (Tregs) activate and expand in response to different types of injuries, suggesting that they play a critical role in controlling the immune response to tissue and cell damage. This project used multi-dimensional profiling techniques to comprehensively characterize injury responsive Tregs in mice. We show that CD44high Tregs expand in response to injury and were highly suppressive when compared to CD44low Tregs. T cell receptor (TCR) repertoire analysis revealed that the CD44high Treg population undergo TCRαß clonal expansion as well as increased TCR CDR3 diversity. Bulk RNA sequencing and single-cell RNA sequencing with paired TCR clonotype analysis identified unique differences between CD44high and CD44low Tregs and specific upregulation of genes in Tregs with expanded TCR clonotypes. Gene ontology analysis for molecular function of RNA sequencing data identified chemokine receptors and cell division as the most enriched functional terms in CD44high Tregs versus CD44low Tregs. Mass cytometry (CyTOF) analysis of Tregs from injured and uninjured mice verified protein expression of these genes on CD44high Tregs, with injury-induced increases in Helios, Galectin-3 and PYCARD expression. Taken together, these data indicate that injury triggers the expansion of a highly suppressive CD44high Treg population that is transcriptionally and phenotypically distinct from CD44low Tregs suggesting that they actively participate in controlling immune responses to injury and tissue damage.
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Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T Reguladores , Animais , Proliferação de Células , CamundongosRESUMO
CD4+ regulatory T cells (Tregs) are acutely activated by traumatic injury, which suggests that they may react to injury with similar kinetics as memory T cells. Here, we used a mouse burn trauma model to screen for memory-like T cell responses to injury by transferring T cells from sham or burn CD45.1 mice into CD45.2 mice and performing secondary injuries in recipient mice. Among all T cell subsets that were measured, only Tregs expanded in response to secondary injury. The expanded Tregs were a CD44high /CD62Llow subpopulation, markers indicative of memory T cells. CyTOF (cytometry by time-of-flight) mass cytometry was used to demonstrate that injury-expanded Tregs expressed higher levels of CD44, CTLA-4, ICOS, GITR, and Helios than Tregs from noninjured mice. Next, we tested whether a similar population of Tregs might react acutely to burn trauma. We observed that Tregs with a phenotype that matched the injury-expanded Tregs were activated by 6 h after injury. To test if Treg activation by trauma requires functional MHC class II, we measured trauma-induced Treg activation in MHC class II gene deficient (MHCII-/- ) mice or in mice that were given Fab fragment of anti-MHC class II antibody to block TCR activation. Injury-induced Treg activation occurred in normal mice but only partial activation was detected in MHCII-/- mice or in mice that were given Fab anti-MHCII antibody. These findings demonstrate that trauma activates a memory-like Treg subpopulation and that Treg activation by injury is partially dependent on TCR signaling by an MHC class II dependent mechanism.
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Memória Imunológica , Ativação Linfocitária/imunologia , Linfócitos T Reguladores/imunologia , Ferimentos e Lesões/imunologia , Animais , Biomarcadores/metabolismo , Queimaduras/imunologia , Queimaduras/patologia , Proliferação de Células , Antígenos de Histocompatibilidade Classe II/metabolismo , Linfonodos/patologia , Camundongos Endogâmicos C57BL , Baço/patologia , Ferimentos e Lesões/patologiaRESUMO
Patients with immune deficiencies from cancers and associated treatments represent a growing population within the intensive care unit with increased risk of morbidity and mortality from sepsis. Mesenchymal stromal cells (MSCs) are an integral part of the hematopoietic niche and express toll-like receptors, making them candidate cells to sense and translate pathogenic signals into an innate immune response. In this study, we demonstrate that MSCs administered therapeutically in a murine model of radiation-associated neutropenia have dual actions to confer a survival benefit in Pseudomonas aeruginosa pneumo-sepsis that is not from improved bacterial clearance. First, MSCs augment the neutrophil response to infection, an effect that is enhanced when MSCs are preconditioned with CpG oligodeoxynucleotide, a toll-like receptor 9 agonist. Using cytometry by time of flight, we identified proliferating neutrophils (Ly6GlowKi-67+) as the main expanded cell population within the bone marrow. Further analysis revealed that CpG-MSCs expand a lineage restricted progenitor population (Lin-Sca1+C-kit+CD150-CD48+) in the bone marrow, which corresponded to a doubling in the myeloid proliferation and differentiation potential in response to infection compared with control. Despite increased neutrophils, no reduction in organ bacterial count was observed between experimental groups. However, the second effect exerted by CpG-MSCs is to attenuate organ damage, particularly in the lungs. Neutrophils obtained from irradiated mice and cocultured with CpG-MSCs had decreased neutrophil extracellular trap formation, which was associated with decreased citrullinated H3 staining in the lungs of mice given CpG-MSCs in vivo. Thus, this preclinical study provides evidence for the therapeutic potential of MSCs in neutropenic sepsis.
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Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Neutropenia , Sepse , Animais , Hematopoese , Humanos , Camundongos , Neutropenia/terapia , Sepse/terapiaRESUMO
BACKGROUND: Trauma causes tissue injury that results in the release of damage associated molecular patterns (DAMPs) and other mediators at the site of injury and systemically. Such mediators disrupt immune system homeostasis and may activate multicellular immune responses with downstream complications such as the development of infections and sepsis. To characterize these alterations, we used time-of-flight mass cytometry to determine how trauma plasma affects normal peripheral blood mononuclear cell (PBMC) activation to gain insights into the kinetics and nature of trauma-induced circulating factors on human immune cell populations. A better understanding of the components that activate cells in trauma may aid in the discovery of therapeutic targets. METHODS: PBMCs from healthy volunteers were cultured with 5% plasma (healthy, trauma-1day, or trauma-3day) or known DAMPs for 24 h. Samples were stained with a broad immunophenotyping CyTOF antibody panel. Multiplex (Luminex) cytokine assays were used to measure differences in multiple cytokine levels in healthy and trauma plasma samples. RESULTS: Plasma from day 1, but not day 3 trauma patients induced the acute expansion of CD11c+ NK cells and CD73+/CCR7+ CD8 T cell subpopulations. Additionally, trauma plasma did not induce CD4+ T cell expansion but did cause a phenotypic shift towards CD38+/CCR7+ expressing CD4+ T cells. Multiplex analysis of cytokines by Luminex showed increased levels of IL-1RA, IL-6 and IL-15 in trauma-1day plasma. Similar to trauma day 1 plasma, PBMC stimulation with known DAMPs showed activation and expansion of CD11c+ NK cells. CONCLUSIONS: We hypothesized that circulating factors in trauma plasma would induce phenotypic activation of normal human immune cell subsets. Using an unbiased approach, we identified specific changes in immune cell subsets that respond to trauma plasma. Additionally, CD11c+ NK cells expanded in response to DAMPs and LPS, suggesting they may also be responding to similar components in trauma plasma. Collectively, our data demonstrate that the normal PBMC response to trauma plasma involves marked changes in specific subsets of NK and CD8+ T cell populations. Future studies will target the function of these trauma plasma reactive immune cell subsets. These findings have important implications for the field of acute traumatic injuries.
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Linfócitos T CD8-Positivos/imunologia , Citocinas/biossíntese , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/citologia , Ferimentos e Lesões/imunologia , Adulto , Antígenos CD11/biossíntese , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Plasma , Fatores de Tempo , Adulto JovemRESUMO
This study aims to explore correlates between body appreciation and mental health among a racially and ethnically diverse sample of adult women using validated measures. The sample included racially and ethnically diverse women (N = 497) from various socioeconomic levels. Linear regression results indicated that body appreciation was significantly and inversely associated with depression (b = -3.68; p < 0.001). In this sample, as body appreciation increased, depression scores decreased. Similarly, body appreciation was significantly and inversely associated with anxiety among this sample (b = -1.78; p < 0.001). This article concludes with a discussion of findings and implications.
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Ansiedade/etnologia , Imagem Corporal , Depressão/etnologia , Adulto , Feminino , HumanosRESUMO
INTRODUCTION: Significant health disparities exist in asthma and obesity for African American youths. Successful interventions present an opportunity to address these disparities but require detailed study in order to ensure generalizability. This study investigated the intersection of obesity, neighbourhood disadvantage, and asthma. METHODS: Data were extracted from 129 African American females ages 13 to 19 years (mean = 15.6 years [SD = 1.9]). Obesity was measured via body mass index (BMI). Asthma status was based on clinical diagnosis and/or results of the International Study of Asthma and Allergies during Childhood (ISAAC) questionnaire. The concentrated disadvantage index (CDI) assessed neighbourhood disadvantage. RESULTS: Findings showed that 21.5% (n = 28) of participants were clinically defined as having asthma, 76.2% (n = 99) had obesity, and 24.9% (n = 31) were classified without obesity. The mean BMI was 35.1 (SD = 9.1) and the mean CDI was 1.0 (SD = 0.9). CDI and obesity were significantly associated in participants without asthma, but not in those with asthma. Multivariable linear regression results showed a significant interaction between CDI and asthma (t value = 2.2, P = .03). CONCLUSION: In sum, results from this study found that asthma moderated the relationship between neighbourhood disadvantage and obesity.
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High-risk injection related behavior including use of non-sterile syringes is associated with negative health outcomes among people who inject drugs (PWID). Drug treatment programs have been reported to curb hepatitis C (HCV) transmission. This study aims to assess the role of drug treatment programs and knowledge of HCV status, and how they influence current injection-related risk. Data were collected in 2012 by the New Orleans arm of the CDC funded National HIV Behavioral Surveillance. Respondent driven sampling was used to recruit a sample of PWID. The analytic sample consisted of 473 participants. Univariate, bivariate, and linear regression analyses were performed. Findings indicated that history of drug treatment is associated with sterile syringe use among PWID. Further, knowledge of HCV status modifies the relationship between history of drug treatment and sterile syringe use in this sample. These findings highlight the importance of scaling up prevention efforts by expanding testing, counselling, and treatment for HCV among PWID who enter drug treatment facilities.
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Conhecimentos, Atitudes e Prática em Saúde , Hepatite C/tratamento farmacológico , Abuso de Substâncias por Via Intravenosa/epidemiologia , Adulto , Feminino , Hepatite C/psicologia , Humanos , Masculino , Programas de Troca de Agulhas , Assunção de Riscos , Abuso de Substâncias por Via Intravenosa/prevenção & controle , Inquéritos e Questionários , SeringasRESUMO
Wild male roach (Rutilus rutilus) living in U.K. rivers contaminated with estrogenic effluents from wastewater treatment works show feminized responses and have a reduced reproductive capability, but the chemical causation of sexual disruption in the roach has not been established. Feminized responses were induced in male roach exposed to environmentally relevant concentrations of the pharmaceutical estrogen 17alpha-ethinylestradiol, EE2 (up to 4 ng/ L), during early life (from fertilization to 84 days posthatch, dph), and these effects were signaled by altered patterns of expression of two cloned roach estrogen receptor (ER) subtypes, ERalpha. and ERbeta, in the brain and gonad/ liver. Transactivation assays were developed for both roach ER subtypes and the estrogenic potencies of steroidal estrogens differed markedly at the different ER subtypes. EE2 was by far the most potent chemical, and estrone (E1, the most prevalent environmental steroid in wastewater discharges) was equipotent with estradiol (E2) in activating the ERs. Comparison of the EC50 values for the compounds tested showed that ERbeta was 3-21-fold more sensitive to natural steroidal estrogens and 54-fold more sensitive to EE2 as compared to ERalpha. These findings add substantial support to the hypothesis that steroidal estrogens play a significant role in the induction of intersex in roach populations in U.K. rivers and that the molecular approach described could be usefully applied to understand interspecies sensitivity to xenoestrogens.