RESUMO
BACKGROUND: Differential leukocyte counts are usually measured based on cellular morphology or surface marker expression. It has recently been shown that leukocyte counts can also be determined by cell-type-specific DNA methylation (DNAm). Such epigenetic leukocyte counting is applicable to small blood volumes and even frozen material, but for clinical translation, the method needs to be further refined and validated. METHODS: We further optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, we tested 36 samples from ring trials and venous blood from 266 patients diagnosed with different hematological diseases. Deconvolution of cell types was determined with various models using DNAm values obtained by pyrosequencing or digital droplet PCR (ddPCR). RESULTS: Relative leukocyte quantification correlated with conventional blood counts for granulocytes, lymphocytes, B cells, T cells (CD4 or CD8), natural killer cells, and monocytes with pyrosequencing (r = 0.84; r = 0.82; r = 0.58; r = 0.50; r = 0.70; r = 0.61; and r = 0.59, respectively) and ddPCR measurements (r = 0.65; r = 0.79; r = 0.56; r = 0.57; r = 0.75; r = 0.49; and r = 0.46, respectively). In some patients, particularly with hematopoietic malignancies, we observed outliers in epigenetic leukocyte counts, which could be discerned if relative proportions of leukocyte subsets did not sum up to 100%. Furthermore, absolute quantification was obtained by spiking blood samples with a reference plasmid of known copy number. CONCLUSIONS: Targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization.
Assuntos
Metilação de DNA , Epigenômica , Granulócitos , Humanos , Contagem de Leucócitos , LeucócitosRESUMO
PURPOSE: The trace element zinc is essential for immune function and its regulation. Since zinc deficiency and allergic hyperresponsive reactions are often accompanied, the influence of zinc on allergen-induced cell growth, CD4+ regulatory T (Treg) cell numbers and cytokine expression during allergic immune reactions was investigated. METHODS: Peripheral blood mononuclear cells (PBMCs) from non-atopic and atopic subjects were treated with timothy grass allergen pre-incubated with or without zinc. Proliferation was determined by analyzing the incorporation of 3H-thymidine. Intracellular zinc and Foxp3 levels and cell surface antigens were measured by FACS, cytokine expression by ELISA and real-time PCR. RESULTS: Incubation with 50 µM zinc sulfate (Zn50) enhances cytosolic zinc concentrations in CD3+ T cells. The data also reveal that the combination of Zn50 plus allergen significantly reduces PBMC proliferation of atopic subjects. Additionally, Zn50 plus allergen enhances Th1 cytokine responses shown by increased interferon (IFN)-γ/interleukin (IL)-10 ratios as well as enhanced tumor necrosis factor-α release. In response to allergen, zinc increases Treg cells and upregulates the mRNA expression of cytotoxic T-lymphocyte antigen-4 in atopic subjects. Interestingly, Zn50 alone leads to an increase of CD4+CD25high(hi)+ cells in atopic and non-atopic subjects. CONCLUSIONS: Zinc may regulate unwanted hyperresponsive immune reactions by suppressing proliferation through a significant shift from IL-10 to the Th1 cytokine IFN-γ, and enhanced regulatory T cell numbers. Therefore, zinc supplementation may be a promising tool for the therapy of allergies, without negatively affecting the immune system.
Assuntos
Alérgenos/imunologia , Hipersensibilidade Imediata/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Zinco/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Interferon gama/análise , Interferon gama/genética , Interleucina-10/análise , Interleucina-10/genética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Contagem de Linfócitos , Phleum/imunologia , RNA Mensageiro/análise , Linfócitos T Reguladores/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Zinco/metabolismoRESUMO
The essential trace element zinc is indispensable for proper immune function as zinc deficiency accompanies immune defects and dysregulations like allergies, autoimmunity and an increased presence of transplant rejection. This point to the importance of the physiological and dietary control of zinc levels for a functioning immune system. This study investigates the capacity of zinc to induce immune tolerance. The beneficial impact of physiological zinc supplementation of 6 µg/day (0.3mg/kg body weight) or 30 µg/day (1.5mg/kg body weight) on murine experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis with a Th1/Th17 (Th, T helper) cell-dominated immunopathogenesis, was analyzed. Zinc administration diminished EAE scores in C57BL/6 mice in vivo (P<.05), reduced Th17 RORγT(+) cells (P<.05) and significantly increased inducible iTreg cells (P<.05). While Th17 cells decreased systemically, iTreg cells accumulated in the central nervous system. Cumulatively, zinc supplementation seems to be capable to induce tolerance in unwanted immune reactions by increasing iTreg cells. This makes zinc a promising future tool for treating autoimmune diseases without suppressing the immune system.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Zinco/administração & dosagem , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
SCOPE: Zinc is an essential trace element, regulating immune function. Its deficiency results in immune dysfunction and transplant rejection. In here, a benefit of zinc supplementation for the induction of tolerance was investigated, focusing on the TH 1-dominated allogeneic immune reaction. METHODS AND RESULTS: Allogeneic immune reaction was modeled by mixed lymphocyte culture (MLC). The effect of zinc supplementation was monitored via expression of cytokines and surface lineage markers using ELISA and flow cytometry. Epigenetic analyses were performed to investigate mechanisms underlying zinc-induced changes in regulatory T cell (Treg) activation. Results reveal that Tregs are induced when MLCs are treated with 50 µM zinc causing a decrease in IFNγ production. IL-2 and IL-10 expression were not affected. The teleology of this effect includes the inhibition of histone deacetylase Sirt-1-mediated Foxp3 deacetylation, resulting in its decreased degradation. CONCLUSION: In conclusion, zinc should be considered to prevent graft-versus-host disease (GVHD) as it is capable of stabilizing iTregs, resulting in increased numbers of this cell type while not suppressing the immune system.
Assuntos
Sirtuína 1/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Zinco/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Doença Enxerto-Hospedeiro/prevenção & controle , Inibidores de Histona Desacetilases/farmacologia , Humanos , Sirtuína 1/metabolismo , Linfócitos T Reguladores/imunologia , Células Th1/efeitos dos fármacosRESUMO
AIM: The purpose of this in vivo study was quantification of inflammatory reaction to ceramic restorations made from lithium disilicate and zirconia by measurement of the concentration of indicators of inflammation in the gingival crevicular fluid (GCF). MATERIAL AND METHODS: Patients out of three prospective cohort-studies investigating three different all-ceramic restoration materials for crowns and fixed dental prostheses were included. Patients needed an associated, unrestored tooth to serve as control. GCF samples were taken from the sulcus of the restored teeth and the related controls (n = 59 pairs) and the concentrations of IL1-beta, IL-1ra and aMMP-8, as indicators of inflammation, were determined by use of ELISA tests. Periodontal status was also assessed clinically by measurement of pocket depth (PD), plaque index (PI) and bleeding on probing (BOP). RESULTS: The concentrations of the inflammation indicators were not significantly different between restored teeth and controls or between lithium disilicate and zirconia restorations (p > 0.05). Furthermore, no significant difference between PD of restored teeth and controls or between groups could be shown. CONCLUSION: Within the limitation of the study, treatment with all-ceramic restorations did not induce inflammatory reactions in a group of periodontal healthy patients. No differences between the gingiva reactions of lithium disilicate and zirconia restorations could be shown.
Assuntos
Cerâmica , Adulto , Coroas , Feminino , Líquido do Sulco Gengival , Humanos , Inflamação , Proteína Antagonista do Receptor de Interleucina 1 , Masculino , Pessoa de Meia-Idade , Índice Periodontal , Estudos ProspectivosRESUMO
BACKGROUND: Regulatory T cells (Tregs, CD4(+)CD25(hi)) are specialized in steering the immune response and cytokine release to maintain tolerance to self-antigens. As cytokines such as interleukin (IL)-1ß, IL-6 and interferon (IFN)-α have been shown to be involved in the pathophysiology of depression and cytokine levels have been shown to change during successful antidepressant treatment, we tested the involvement CD4(+)CD25(hi) Tregs in these immunological processes during antidepressant therapy. METHODS: 16 patients suffering from a depressive episode were included into the study and treated with antidepressants according to their doctor's choice. Blood samples were collected during the first week after admission and after 6 weeks of treatment. Therein, we determined plasma levels of IL-1ß, and measured IL-1ß, IL-6 and IFN-α levels in the stimulated blood by performing a whole blood assay. We distinguished lymphocytes and identified CD4(+)CD25(hi) Tregs by multiparameter flow cytometry. The psychopathological status was assessed using the Hamilton Depression Rating Scale (HAMD-21). RESULTS: HAMD-21 score, IL-1ß serum levels as well as LPS-stimulated IL-1ß and IL-6 production had decreased significantly at the end of treatment. In contrast, the amount of CD4(+)CD25(hi) cells increased significantly from 2.74% ± 0.88 (mean value ± standard deviation) to 3.54% ± 1.21; p = 0.007. No significant changes in virus-induced IFN-α production was observed. CONCLUSIONS: The increase in CD4(+)CD25(hi) Tregs during antidepressant therapy may be the reason for the decrease in cytokine production and the recovery from depression.
Assuntos
Antidepressivos/farmacologia , Transtorno Depressivo/metabolismo , Transtorno Depressivo/patologia , Interleucina-1beta/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Adulto , Antidepressivos/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Citocinas/metabolismo , Transtorno Depressivo/tratamento farmacológico , Transtorno Depressivo/imunologia , Feminino , Citometria de Fluxo/métodos , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Escalas de Graduação PsiquiátricaRESUMO
OBJECTIVE: A growing body of data indicates that an activation of proinflammatory cytokines such as interferon-gamma (IFN-gamma) is involved in the pathophysiology of depression and that the suppression of pro-inflammatory cytokine production by antidepressants may lead to an improvement of depressive symptoms. However, the influence of the serotonin and noradrenalin reuptake inhibitor (SNRI) venlafaxine and its metabolite O-desmethylvenlafaxine on the stimulated blood cell secretion of IFN-gamma has not been studied so far. METHOD: We measured IFN-gamma levels in the stimulated blood of healthy female subjects in a whole blood assay using the toxic shock syndrome toxin TSST-1 as stimulant. Blood was either supplemented with antidepressants or not. RESULTS: Mean IFN-gamma concentrations differed between blood with and without antidepressant supplements (p = 0.026). Planned contrasts revealed that compared to non-supplemented blood, four of the blood samples supplemented with the tricyclic antidepressants (TCAs) reduced IFN-gamma levels: amitriptyline (adjusted p-value (p = 0.004), nortriptyline (p = 0.037), imipramine (p = 0.021), and desipramine (p = 0.048). There was no significant difference between the control condition and the venlafaxine or O-desmethylvenlafaxine condition. CONCLUSIONS: TCAs might, among other mechanisms, act as antidepressants by suppressing the production of pro-inflammatory cytokines, whereas no significant effect of venlafaxine and O-desmethylvenlafaxine on IFN-gamma secretion could be demonstrated.
Assuntos
Amitriptilina/farmacologia , Antidepressivos de Segunda Geração/farmacologia , Antidepressivos Tricíclicos/farmacologia , Cicloexanóis/farmacologia , Transtorno Depressivo/imunologia , Imipramina/farmacologia , Interferon gama/sangue , Citocinas/sangue , Desipramina/farmacologia , Succinato de Desvenlafaxina , Feminino , Humanos , Técnicas In Vitro , Masculino , Nortriptilina/farmacologia , Cloridrato de VenlafaxinaRESUMO
Aging is associated with low-grade inflammation on the one hand and mild zinc deficiency on the other. These conditions contribute to decreased immune functions, resulting in increased incidences of infections and autoimmune diseases. The aim of this study was to give more insight into the question, to what extent is low-grade inflammation caused by zinc deficient status. Here we report the effect of improved intracellular zinc status on low-grade inflammatory activity in 19 healthy elderly subjects. Our experiments show that adjustment of labile zinc by moderate zinc supplementation reduces spontaneous cytokine release and defects in termination of inflammatory activity. This results in reduced amounts of unspecific preactivated T cells and leads to improved T cell response upon mitogenic stimulation. Therefore, in contrast to other anti-inflammatory drugs, zinc does not suppress, but improves immune reaction upon pathogen invasion. These results suggest that mildly zinc-deficient, healthy elderly subjects might benefit from moderate zinc supplementation due to a more balanced immune response with reduced incidences of infections and autoimmune diseases.
Assuntos
Idoso , Suplementos Nutricionais , Mediadores da Inflamação/metabolismo , Linfócitos T/efeitos dos fármacos , Zinco/farmacologia , Adulto , Idoso de 80 Anos ou mais , Proteínas de Transporte de Cátions/genética , Células Cultivadas , Citocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Absorção Intestinal/genética , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Linfócitos T/fisiologia , Zinco/metabolismoRESUMO
Elderly subjects suffer from increased levels of activated T cells and a TH1/TH2 imbalance. Zinc deficiency of the aged is correlated with decreased cell-mediated immune responses. The association of age and zinc adjustment with the amounts of TH1 (CCR5+) and TH2 (CCR4+) cell populations in healthy aged old donors enrolled in the European ZINCAGE project was examined. Old and nonagenarian individuals revealed increased TH1, TH2 cell numbers and a decreased TH2/TH1 ratio in comparison to young individuals. The differences between TH2/TH1 ratios of young and old/nonagenarians arose from young females. Adjusted zinc status led to enhanced TH2 and TH1 amounts in fresh whole blood and thawed cells of aged donors whereas increased HLA-DR+ expression and a generally lower CCR5 expression was observed on thawed PBMC. In conclusion, aging is associated with an increase in T helper cell polarization, and changes in TH2/TH1 subsets are more obvious in women than in men. Advanced healthy aging is accompanied by TH cell polarization, too. Moderate zinc supplementation in vivo alters TH proportions. Longer zinc treatment will give more insight into the beneficial effect of zinc on T helper cell modulation.
Assuntos
Envelhecimento/imunologia , Células Th1/metabolismo , Células Th2/metabolismo , Oligoelementos/farmacologia , Zinco/farmacologia , Idoso , Idoso de 80 Anos ou mais , Suplementos Nutricionais , Feminino , Humanos , Ativação Linfocitária/fisiologia , Masculino , Receptores CCR4/metabolismo , Receptores CCR6/metabolismo , Subpopulações de Linfócitos T , Oligoelementos/administração & dosagem , Zinco/administração & dosagemRESUMO
Excessive and permanent cytokine production in response to bacterial LPS causes cell and tissue damage, and hence organ failure during sepsis. We have previously demonstrated that zinc treatment prevents LPS-induced TNF-alpha expression and production in human monocytes by inhibiting cyclic nucleotide phosphodiesterase (PDE) activity and expression, and subsequent elevation of the cyclic nucleotide cGMP. In the present study, we investigated the molecular mechanism by which cGMP signaling affects the LPS-induced signaling cascade to suppress TNF-alpha transcription and release from monocytes. Zinc-mediated cGMP elevation led to cross activation of protein kinase A. This zinc-induced protein kinase A activation inhibited Raf-1 activity by phosphorylation at serine 259, preventing activation of Raf-1 by phosphorylation of serine 338. By this mechanism, zinc suppressed LPS-induced activation of IkappaB kinase beta (IKKbeta) and NF-kappaB, and subsequent TNF-alpha production. Our study shows that PDE inhibition by zinc modulates the monocytic immune response by selectively intervening in the Raf-1/IKKbeta/NF-kappaB pathway, which may constitute a common mechanism for the anti-inflammatory action of PDE inhibitors.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Regulação para Baixo/imunologia , Quinase I-kappa B/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Zinco/fisiologia , Animais , Cátions Bivalentes , Linhagem Celular , Células Cultivadas , GMP Cíclico/fisiologia , Ativação Enzimática/imunologia , Humanos , Quinase I-kappa B/fisiologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , NF-kappa B/fisiologia , Fosforilação , Inibidores de Proteínas Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/fisiologiaRESUMO
Superantigens (SAgs) are very potent microbial toxins that are involved in severe diseases such as necrotizing fasciitis and toxic shock syndrome. There are currently 11 different SAgs that have been identified from Streptococcus pyogenes. In the present study, two sets of multiplex PCRs were developed for detection of these 11 SAg genes. The first group comprises spea1-3+5, spec, speg, spej, spek, and spel. The second group consists of spea1-4, speh, spei, spem, ssa, and smez. The presence of Streptococcus pyogenes SAg genes can be immediately identified using a real-time method with SYBR-Green, thus providing an excellent tool in clinical diagnostics. After testing more than 300 clinical isolates, we identified one strain without any SAg gene. This finding contrasts with previous reports describing SAg genes located on every Streptococcus pyogenes genome. This SAg gene-negative strain also did not show any mitogenic activity. It is hypothesized that clinical isolates from patients may overrepresent bacterial strains with pathogenic factors, such as SAgs.
Assuntos
Reação em Cadeia da Polimerase/métodos , Streptococcus pyogenes/genética , Superantígenos/genética , Proteínas de Bactérias/genética , Benzotiazóis , Diaminas , Humanos , Compostos Orgânicos/metabolismo , Quinolinas , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/classificação , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/isolamento & purificaçãoRESUMO
Bacterial superantigens are potent stimulators of the immune system. In this study, we expressed recombinant superantigens, which were then affinity purified and used for growth curves and DNase activity assays. Overexpression of Mycoplasma arthritidis-derived superantigen in Escherichia coli reduced bacterial growth. This is unique, as staphylococcal enterotoxin A and toxic shock syndrome toxin-1, expressed in the same vector system, showed no growth impairment. The observed growth inhibition was caused by the DNase activity of recombinant M. arthritidis-derived superantigen, thus describing the first superantigen showing enzymatic activity, which may be a result of the separate evolution of this toxin.
Assuntos
Antígenos de Bactérias/metabolismo , DNA Bacteriano/metabolismo , DNA/metabolismo , Desoxirribonucleases/metabolismo , Mycoplasma arthritidis/enzimologia , Mycoplasma arthritidis/imunologia , Superantígenos/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/genética , Desoxirribonucleases/genética , Desoxirribonucleases/isolamento & purificação , Desoxirribonucleases/toxicidade , Enterotoxinas/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Staphylococcaceae , Superantígenos/genética , Superantígenos/isolamento & purificação , Superantígenos/toxicidadeRESUMO
OBJECTIVE: We examined the influence of zinc on T-helper type 1 (Th1)/T-helper type 2 (Th2) balance in human lymphocytes. METHODS: Human peripheral blood mononuclear cells or diluted whole blood were cultured for 8 d in the presence of zinc (30 or 60 microM) or 1 microM of N, N, N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (a zinc-specific chelator). Phytohemagglutinin-induced cytokine release was measured by enzyme-linked immunosorbent assay, and expression of CD56/CD69, CCR4/CD3, and CCR5/CD3 and intracellular labile zinc were detected by flow cytometry. RESULTS: We found that our in vitro supplementation resulted in an increase of intracellular labile zinc comparable to that of a 7-wk administration of 10 mg of zinc per day in vivo. Zinc triggered interferon-gamma release and impaired interleukin-10 release. Phenotypically, a Th2/Th1 shift could not be confirmed after detecting the Th1-specific chemokine receptor CCR5 or CCR4 for Th2 cells. Surprisingly, we detected a larger amount of CD56+ cells after zinc stimulation, leading us to the conclusion that the amount of interferon-gamma release after zinc supplementation might be attributed to the upregulation of natural killer cells after in vitro zinc supplementation rather than to a Th2/Th1 shift. CONCLUSION: We suggest that a nutritional intake of 10 mg of zinc increases the quantity of interferon-gamma-producing natural killer cells and strengthens the immune system against neoplasms and viral infections.