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1.
Sci Immunol ; 6(62)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376481

RESUMO

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+ T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+ T lymphocytes, while CD4+ T cells were less expanded and skewed toward TCM and TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+ GZMB+ effector cells were clonally expanded both during the infection and post-infection, while CD8+ GZMK+ lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+ GZMB+ and GZMK+ subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+ T cell population with memory precursor-like features.


Assuntos
COVID-19/genética , COVID-19/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunofenotipagem , SARS-CoV-2/imunologia , Transcriptoma , Adulto , Idoso , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , COVID-19/virologia , Plasticidade Celular/genética , Plasticidade Celular/imunologia , Evolução Clonal/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Isotipos de Imunoglobulinas/imunologia , Memória Imunológica , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
2.
Biomed Res Int ; 2021: 5520053, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34337017

RESUMO

Background: Treponema pallidum subspecies pallidum (T. pallidum) infection induces significant immune responses, resulting in tissue damage. Gene expression plays an essential role in regulating the progression of syphilis infection. However, little is known about the regulatory role of microRNAs (miRNAs) in the immune response to T. pallidum infection. Here, we analyze the differential expression of miRNAs in peripheral blood mononuclear cells (PBMCs) between untreated secondary syphilis patients and healthy controls and study the correlation between miRNA expression and clinical features with bioinformatics. Methods: The expression profile of miRNAs was measured by microarray analysis in PBMCs of untreated secondary syphilis patients and healthy controls. Weighted Gene Coexpression Network Analysis (WGCNA) was used to construct the expression of miRNAs and the clinical data of secondary syphilis patients. Gene ontology (GO) and KEGG enrichment analyses were performed on target genes of miR-142-3p. Results: 244 miRNAs exhibited at least 1.0-fold differential expression between secondary syphilis patients and healthy controls. The miRNAs were divided into three modules by WGCNA. The blue module was positively correlated with TPHA, TRUST, duration of disease, and erythema. And in the blue module, the expression of miR-142-3p was significantly higher in secondary syphilis patients than in healthy controls (p = 0.02), which is also close to the clinical features of secondary syphilis. GO and KEGG pathway analyses showed that these target genes of miR-142-3p are correlated with endocytosis and positive regulation of the apoptotic process. Conclusion: The elevated miR-142-3p expression in PBMCs may play an important role in the immune response to T. pallidum infection and may be a potential biomarker for secondary syphilis.


Assuntos
Regulação da Expressão Gênica , Genoma Humano , Leucócitos Mononucleares/metabolismo , MicroRNAs/genética , Sífilis/sangue , Sífilis/genética , Adolescente , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Imunidade/genética , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Fenótipo , Mapas de Interação de Proteínas/genética , Sífilis/imunologia , Adulto Jovem
3.
Molecules ; 26(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34443554

RESUMO

Rheumatoid arthritis (RA) is an autoimmune inflammatory joint disease with complex pathogenesis associated with cytokine dysregulation. Macrophage migration inhibitory factor (MIF) plays a role in systemic inflammation and joint destruction in RA and could be associated with the secretion of other immune-modulatory cytokines such as IL-25, IL-31, and IL-33. For the above, our main aim was to evaluate the IL-25, IL-31, and IL-33 secretion from recombinant human MIF (rhMIF)-stimulated peripheral blood mononuclear cells (PBMC) of RA patients. The rhMIF and lipopolysaccharide (LPS) plus rhMIF stimuli promote the secretion of IL-25, IL-31, and IL-33 (p < 0.05) from PBMC of RA patients. The study groups, the different stimuli, and the interaction between both showed a statistically significant effect on the secretion of IL-25 (p < 0.05) and IL-31 (p < 0.01). The study of the effect of the RA patient treatments and their interaction with the effect of stimuli did not show an interaction between them. In conclusion, our study generates new evidence for the role of MIF in the secretion of IL-25, IL-31, and IL-33 and its immunomodulatory effect on RA.


Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Interleucina-17/metabolismo , Interleucina-33/metabolismo , Interleucinas/metabolismo , Oxirredutases Intramoleculares/metabolismo , Leucócitos Mononucleares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Adulto , Feminino , Humanos , Imunomodulação/efeitos dos fármacos , Oxirredutases Intramoleculares/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Fatores Inibidores da Migração de Macrófagos/farmacologia , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/farmacologia
4.
Nutrients ; 13(7)2021 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-34371884

RESUMO

The dietary supplement, trans-resveratrol and hesperetin combination (tRES-HESP), induces expression of glyoxalase 1, countering the accumulation of reactive dicarbonyl glycating agent, methylglyoxal (MG), in overweight and obese subjects. tRES-HESP produced reversal of insulin resistance, improving dysglycemia and low-grade inflammation in a randomized, double-blind, placebo-controlled crossover study. Herein, we report further analysis of study variables. MG metabolism-related variables correlated with BMI, dysglycemia, vascular inflammation, blood pressure, and dyslipidemia. With tRES-HESP treatment, plasma MG correlated negatively with endothelial independent arterial dilatation (r = -0.48, p < 0.05) and negatively with peripheral blood mononuclear cell (PBMC) quinone reductase activity (r = -0.68, p < 0.05)-a marker of the activation status of transcription factor Nrf2. For change from baseline of PBMC gene expression with tRES-HESP treatment, Glo1 expression correlated negatively with change in the oral glucose tolerance test area-under-the-curve plasma glucose (ΔAUGg) (r = -0.56, p < 0.05) and thioredoxin interacting protein (TXNIP) correlated positively with ΔAUGg (r = 0.59, p < 0.05). Tumor necrosis factor-α (TNFα) correlated positively with change in fasting plasma glucose (r = 0.70, p < 0.001) and negatively with change in insulin sensitivity (r = -0.68, p < 0.01). These correlations were not present with placebo. tRES-HESP decreased low-grade inflammation, characterized by decreased expression of CCL2, COX-2, IL-8, and RAGE. Changes in CCL2, IL-8, and RAGE were intercorrelated and all correlated positively with changes in MLXIP, MAFF, MAFG, NCF1, and FTH1, and negatively with changes in HMOX1 and TKT; changes in IL-8 also correlated positively with change in COX-2. Total urinary excretion of tRES and HESP metabolites were strongly correlated. These findings suggest tRES-HESP counters MG accumulation and protein glycation, decreasing activation of the unfolded protein response and expression of TXNIP and TNFα, producing reversal of insulin resistance. tRES-HESP is suitable for further evaluation for treatment of insulin resistance and related disorders.


Assuntos
Hesperidina/administração & dosagem , Resistência à Insulina , Obesidade/terapia , Sobrepeso/terapia , Resveratrol/administração & dosagem , Adulto , Pressão Sanguínea/efeitos dos fármacos , Índice de Massa Corporal , Proteínas de Transporte/sangue , Correlação de Dados , Estudos Cross-Over , Suplementos Nutricionais , Método Duplo-Cego , Quimioterapia Combinada , Dislipidemias/sangue , Dislipidemias/terapia , Feminino , Transtornos do Metabolismo de Glucose/sangue , Transtornos do Metabolismo de Glucose/terapia , Glicosilação/efeitos dos fármacos , Humanos , Inflamação , Mediadores da Inflamação/sangue , Leucócitos Mononucleares/metabolismo , Masculino , Obesidade/sangue , Sobrepeso/sangue , Aldeído Pirúvico/sangue , Fator de Necrose Tumoral alfa/sangue
5.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360753

RESUMO

Activation of innate immunity and low-grade inflammation contributes to hyperglycemia and an onset of Type 2 Diabetes Mellitus (T2DM). Interleukin-2 (IL-2), leptin, High Mobility Group Box-1 (HMGB-1), and increased glucose concentrations are mediators of these processes also by modulating peripheral blood mononuclear cells (PBMCs) response. The aim of this study was to investigate if HMGB-1 and IL-2 turn on PBMCs and their leptin secretion. In isolated human PBMCs and their subpopulations from healthy individuals and naïve T2DM patients, leptin release, pro-inflammatory response and Toll-like Receptors (TLRs) activation was measured. After treatment with IL-2 and HMGB1, NK (Natural Killer) have the highest amount of leptin secretion, whilst NK-T have the maximal release in basal conditions. TLR4 (TAK242) and/or TLR2 (TLR2-IgA) inhibitors decreased leptin secretion after IL-2 and HMGB1 treatment. A further non-significant increase in leptin secretion was reported in PBMCs of naive T2DM patients in response to IL-2 and HMGB-1 stimulation. Finally, hyperglycemia or hyperinsulinemia might stimulate leptin secretion from PBMCs. The amount of leptin released from PBMCs after the different treatments was enough to stimulate the secretion of IL-1ß from monocytes. Targeting leptin sera levels and secretion from PBMCs could represent a new therapeutic strategy to counteract metabolic diseases such as T2DM.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proteína HMGB1/farmacologia , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Interleucina-2/farmacologia , Leptina/metabolismo , Leucócitos Mononucleares/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Hiperglicemia/patologia , Hiperinsulinismo/patologia , Leucócitos Mononucleares/patologia
6.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360706

RESUMO

For the treatment of severe COVID-19, supplementation with human plasma-purified α-1 antitrypsin (AAT) to patients is currently considered. AAT inhibits host proteases that facilitate viral entry and possesses broad anti-inflammatory and immunomodulatory activities. Researchers have demonstrated that an interaction between SARS-CoV-2 spike protein (S) and lipopolysaccharides (LPS) enhances pro-inflammatory responses in vitro and in vivo. Hence, we wanted to understand the potential anti-inflammatory activities of plasma-derived and recombinant AAT (recAAT) in a model of human total peripheral blood mononuclear cells (PBMCs) exposed to a combination of CHO expressed trimeric spike protein and LPS, ex vivo. We confirmed that cytokine production was enhanced in PBMCs within six hours when low levels of LPS were combined with purified spike proteins ("spike"). In the presence of 0.5 mg/mL recAAT, however, LPS/spike-induced TNF-α and IL-1ß mRNA expression and protein release were significantly inhibited (by about 46-50%) relative to LPS/spike alone. Although without statistical significance, recAAT also reduced production of IL-6 and IL-8. Notably, under the same experimental conditions, the plasma-derived AAT preparation Respreeza (used in native and oxidized forms) did not show significant effects. Our findings imply that an early pro-inflammatory activation of human PBMCs is better controlled by the recombinant version of AAT than the human plasma-derived AAT used here. Considering the increasing clinical interest in AAT therapy as useful to ameliorate the hyper-inflammation seen during COVID-19 infection, different AAT preparations require careful evaluation.


Assuntos
Anti-Inflamatórios/farmacologia , Leucócitos Mononucleares/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , alfa 1-Antitripsina/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/imunologia , Células CHO , COVID-19/terapia , Células Cultivadas , Cricetulus , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/toxicidade , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , alfa 1-Antitripsina/química , alfa 1-Antitripsina/imunologia
7.
Nutrients ; 13(7)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34371879

RESUMO

Sepsis is an extremely complex clinical syndrome, usually involving an excessive inflammatory response including an overshooting cytokine release that damages tissue and organs of the patient. Due to the severity of this condition, it is estimated that over 11 million people die from sepsis each year. Despite intensive research in the field, there is still no specific therapy for sepsis. Many sepsis patients show a marked deficiency of vitamin C. 9 out of 10 sepsis patients have a hypovitaminosis C, and every third patient even shows a clinical deficiency in the scurvy range. In addition, low vitamin C levels of intensive care sepsis patients correlate with a higher need for vasopressors, higher Sequential Organ Failure Assessment (SOFA) scores, and increased mortality. Based on this observation and the conducted clinical trials using vitamin C as sepsis therapy in intensive care patients, the aim of the present ex vivo study was to evaluate the effects of high-dose vitamin C alone and in a triple combination supplemented with vitamin B1 (thiamine) and hydrocortisone on the lipopolysaccharide (LPS)-induced cytokine response in peripheral blood mononuclear cells (PBMCs) from healthy human donors. We found that all corticosteroid combinations strongly reduced the cytokine response on RNA- and protein levels, while high-dose vitamin C alone significantly diminished the PBMC mediated secretion of the cytokines interleukin (IL)-10, IL-23, and monocyte chemo-attractant protein (MCP-1), which mediate the inflammatory response. However, vitamin C showed no enhancing effect on the secretion of further cytokines studied. This data provides important insights into the possible immunomodulatory function of vitamin C in an ex vivo setting of human PBMCs and the modulation of their cytokine profile in the context of sepsis. Since vitamin C is a vital micronutrient, the restoration of physiologically adequate concentrations should be integrated into routine sepsis therapy, and the therapeutic effects of supraphysiological concentrations of vitamin C in sepsis patients should be further investigated in clinical trials.


Assuntos
Anti-Inflamatórios/farmacologia , Ácido Ascórbico/farmacologia , Hidrocortisona/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Sepse/tratamento farmacológico , Tiamina/farmacologia , Adulto , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Quimioterapia Combinada , Feminino , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Sepse/metabolismo , Adulto Jovem
8.
Stem Cell Res ; 54: 102432, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34214899

RESUMO

Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a basic helix-loop-helix (bHLH/PAS) transcription factor involved in the development of paraventricular nucleus of the hypothalamus (PVH) through the heterodimerization with Single-minded 1 (SIM1) (Michaud et al., 2000). Using a Sendai virus-based approach, the four reprogramming factors OCT3/4, SOX2, KLF4 and C-MYC were delivered into Peripheral Blood Mononuclear Cell (PBMCs) from a 14-year-old girl with early onset obesity carrying a de novo variant (p.P130A) in ARNT2. The resulting iPSC line CUIMCi003-A had a normal karyotype, showed pluripotency and three germ layer differentiation capacity in vitro and was heterozygous for the de novo ARNT2 variant.


Assuntos
Células-Tronco Pluripotentes Induzidas , Adolescente , Translocador Nuclear Receptor Aril Hidrocarboneto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/metabolismo , Obesidade/genética
9.
Free Radic Biol Med ; 172: 441-450, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34197940

RESUMO

The deficiency of dead cell clearance is a prominent pathogenic factor in systemic lupus erythematosus (SLE). In this study, the overexpression of miR-210-5p resulted in the accumulation of secondary necrotic cells (SNECs) in macrophages through the reduction of protein degradation. The upreguation of miR-210-5p inhibited NADPH oxidase (NOX) activation, reactive oxygen species (ROS) generation, and SNEC clearance. miR-210-5p overexpression suppressed Sp1 and HSCARG expression, and the knockdown of SP1 and HSCARG inhibited NOX expression and superoxide production in macrophages. Furthermore, patients with active SLE expressed a higher level of miR-210-5p and lower expression of SP1 and HSCARG in peripheral blood mononuclear cells. In summary, our findings indicate that the upregulation of miR-210-5p increases the accumulation of SNECs through a decrease in the Sp1-and HSCARG-mediated NOX activity and ROS generation in macrophages. Our results also suggest that targeting miR-210-5p may have therapeutic potential for SLE.


Assuntos
Macrófagos , MicroRNAs , NADPH Oxidases , Fator de Transcrição Sp1 , Fatores de Transcrição/genética , Humanos , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico , Macrófagos/citologia , MicroRNAs/genética , NADPH Oxidases/genética , Oxirredutases , Fator de Transcrição Sp1/genética , Regulação para Cima
10.
Nucleic Acids Res ; 49(15): 8505-8519, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34320202

RESUMO

The transcriptomic diversity of cell types in the human body can be analysed in unprecedented detail using single cell (SC) technologies. Unsupervised clustering of SC transcriptomes, which is the default technique for defining cell types, is prone to group cells by technical, rather than biological, variation. Compared to de-novo (unsupervised) clustering, we demonstrate using multiple benchmarks that supervised clustering, which uses reference transcriptomes as a guide, is robust to batch effects and data quality artifacts. Here, we present RCA2, the first algorithm to combine reference projection (batch effect robustness) with graph-based clustering (scalability). In addition, RCA2 provides a user-friendly framework incorporating multiple commonly used downstream analysis modules. RCA2 also provides new reference panels for human and mouse and supports generation of custom panels. Furthermore, RCA2 facilitates cell type-specific QC, which is essential for accurate clustering of data from heterogeneous tissues. We demonstrate the advantages of RCA2 on SC data from human bone marrow, healthy PBMCs and PBMCs from COVID-19 patients. Scalable supervised clustering methods such as RCA2 will facilitate unified analysis of cohort-scale SC datasets.


Assuntos
Algoritmos , Análise por Conglomerados , RNA Citoplasmático Pequeno/genética , RNA-Seq/métodos , Análise de Célula Única/métodos , Animais , Artrite Reumatoide/genética , Células da Medula Óssea/metabolismo , COVID-19/sangue , COVID-19/patologia , Estudos de Coortes , Conjuntos de Dados como Assunto , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Camundongos , Especificidade de Órgãos , Controle de Qualidade , RNA-Seq/normas , Análise de Célula Única/normas , Transcriptoma
11.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298935

RESUMO

In vitro evaluation of bone graft materials is generally performed by analyzing the interaction with osteoblasts or osteoblast precursors. In vitro bone models comprising different cell species can give specific first information on the performance of those materials. In the present study, a 3D co-culture model was established comprising primary human osteoblasts, osteoclasts and osteocytes. Osteocytes were differentiated from osteoblasts embedded in collagen gels and were cultivated with osteoblast and osteoclasts seeded in patterns on a porous membrane. This experimental setup allowed paracrine signaling as well as separation of the different cell types for final analysis. After 7 days of co-culture, the three cell species showed their typical morphology and gene expression of typical markers like ALPL, BSPII, BLGAP, E11, PHEX, MEPE, RANKL, ACP5, CAII and CTSK. Furthermore, relevant enzyme activities for osteoblasts (ALP) and osteoclasts (TRAP, CTSK, CAII) were detected. Osteoclasts in triple culture showed downregulated TRAP (ACP5) and CAII expression and decreased TRAP activity. ALP and BSPII expression of osteoblasts in triple culture were upregulated. The expression of the osteocyte marker E11 (PDPN) was unchanged; however, osteocalcin (BGLAP) expression was considerably downregulated both in osteoblasts and osteocytes in triple cultures compared to the respective single cultures.


Assuntos
Osso e Ossos/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteócitos/metabolismo , Idoso , Diferenciação Celular/genética , Células Cultivadas , Técnicas de Cocultura , Colágeno/metabolismo , Regulação para Baixo/genética , Feminino , Expressão Gênica/genética , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Regulação para Cima/genética
12.
Molecules ; 26(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202367

RESUMO

Carvacrol (CV) is an essential oil with numerous therapeutic properties, including immunomodulatory activity. However, this effect has not been studied in nanoemulsion systems. The objective of this study was to develop an innovative carvacrol-loaded nanoemulsion (CVNE) for immunomodulatory action. The developed CVNE comprised of 5% w/w oily phase (medium chain triglycerides + CV), 2% w/w surfactants (Tween 80®/Span 80®), and 93% w/w water, and was produced by ultrasonication. Dynamic light scattering over 90 days was used to characterize CVNE. Cytotoxic activity and quantification of cytokines were evaluated in peripheral blood mononuclear cell (PBMC) culture supernatants. CVNE achieved a drug loading of 4.29 mg/mL, droplet size of 165.70 ± 0.46 nm, polydispersity index of 0.14 ± 0.03, zeta potential of -10.25 ± 0.52 mV, and good stability for 90 days. CVNE showed no cytotoxicity at concentrations up to 200 µM in PBMCs. CV diminished the production of IL-2 in the PBMC supernatant. However, CVNE reduced the levels of the pro-inflammatory cytokines IL-2, IL-17, and IFN-γ at 50 µM. In conclusion, a stable CVNE was produced, which improved the CV immunomodulatory activity in PBMCs.


Assuntos
Cimenos , Citocinas/metabolismo , Fatores Imunológicos , Leucócitos Mononucleares/metabolismo , Nanopartículas/química , Cimenos/química , Cimenos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Emulsões , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Polissorbatos/química , Polissorbatos/farmacologia
13.
Front Immunol ; 12: 693269, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34220854

RESUMO

Chronic immune activation has been considered as the driving force for CD4+ T cell depletion in people infected with HIV-1. Interestingly, the normal immune profile of adult HIV-negative individuals living in Africa also exhibit chronic immune activation, reminiscent of that observed in HIV-1 infected individuals. It is characterized by increased levels of soluble immune activation markers, such as the cytokines interleukin (IL)-4, IL-10, TNF-α, and cellular activation markers including HLA-DR, CD-38, CCR5, coupled with reduced naïve and increased memory cells in CD4+ and CD8+ subsets. In addition, it is accompanied by low CD4+ T cell counts when compared to Europeans. There is also evidence that mononuclear cells from African infants secrete less innate cytokines than South and North Americans and Europeans in vitro. Chronic immune activation in Africans is linked to environmental factors such as parasitic infections and could be responsible for previously observed immune hypo-responsiveness to infections and vaccines. It is unclear whether the immunogenicity and effectiveness of anti-SARS-CoV-2 vaccines will also be reduced by similar mechanisms. A review of studies investigating this phenomenon is urgently required as they should inform the design and delivery for vaccines to be used in African populations.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas contra COVID-19/imunologia , Imunogenicidade da Vacina/imunologia , Ativação Linfocitária/imunologia , SARS-CoV-2/imunologia , ADP-Ribosil Ciclase 1/sangue , África , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/imunologia , COVID-19/prevenção & controle , Antígenos HLA-DR/sangue , Humanos , Interleucina-10/sangue , Interleucina-4/sangue , Leucócitos Mononucleares/metabolismo , Glicoproteínas de Membrana/sangue , Receptores CCR5/sangue , Fator de Necrose Tumoral alfa/sangue
14.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299155

RESUMO

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Citocinas/metabolismo , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/farmacologia , Modelos Biológicos , Glicoproteína da Espícula de Coronavírus/farmacologia , Voluntários Saudáveis , Humanos , Técnicas In Vitro , Leucócitos Mononucleares/efeitos dos fármacos
15.
Sci Data ; 8(1): 177, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267220

RESUMO

Bovine mammary function at molecular level is often studied using mammary tissue or primary bovine mammary epithelial cells (pbMECs). However, bulk tissue and primary cells are heterogeneous with respect to cell populations, adding further transcriptional variation in addition to genetic background. Thus, understanding of the variation in gene expression profiles of cell populations and their effect on function are limited. To investigate the mononuclear cell composition in bovine milk, we analyzed a single-cell suspension from a milk sample. Additionally, we harvested cultured pbMECs to characterize gene expression in a homogeneous cell population. Using the Drop-seq technology, we generated single-cell RNA datasets of somatic milk cells and pbMECs. The final datasets after quality control filtering contained 7,119 and 10,549 cells, respectively. The pbMECs formed 14 indefinite clusters displaying intrapopulation heterogeneity, whereas the milk cells formed 14 more distinct clusters. Our datasets constitute a molecular cell atlas that provides a basis for future studies of milk cell composition and gene expression, and could serve as reference datasets for milk cell analysis.


Assuntos
Leucócitos Mononucleares/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Leite/citologia , Leite/metabolismo , Análise de Sequência de RNA , Análise de Célula Única , Animais , Bovinos , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Cultura Primária de Células
16.
Int J Mol Sci ; 22(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299256

RESUMO

To investigate the effect of leptin in childhood ITP, we measured plasma leptin in 39 children with acute ITP, after treatment and in remission, and in 33 healthy age/BMI-matched controls. We also cultured ITP and control peripheral blood mononuclear cells (PBMCs) with recombinant leptin to assess its direct effect on pro/anti-inflammatory cytokine gene expression. A significant increase in leptin was observed in children with active disease compared to controls. A significant inverse correlation of leptin with platelet count was also observed in children with acute ITP. Leptin remained high after treatment with IVIg, whereas steroid treatment lowered leptin below control levels. In remission, leptin was in the control range. Cytokine gene expression was significantly increased in children with acute ITP compared with controls, with highest expression for IFN-γ and IL-10. IVIg/steroid treatment significantly decreased IFN-γ and IL-10 expression. In remission, IFN-γ and IL-10 expression remained low. Addition of leptin to PBMCs isolated from patients in remission resulted in a significant increase in IL-10 gene expression compared to controls. Further experiments with purified T-cells and monocytes identified monocytes as the source of leptin-induced IL-10. We suggest that leptin acts as an active anti-inflammatory agent in childhood ITP by promoting IL-10 secretion by monocytes.


Assuntos
Leptina/análise , Púrpura Trombocitopênica Idiopática/metabolismo , Adolescente , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Criança , Pré-Escolar , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Feminino , Expressão Gênica , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Lactente , Interferon gama/metabolismo , Interleucina-10/metabolismo , Leptina/sangue , Leptina/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Monócitos/metabolismo , Plasma/química , Contagem de Plaquetas , Púrpura Trombocitopênica Idiopática/genética , Púrpura Trombocitopênica Idiopática/fisiopatologia , Células Th2/imunologia
17.
Nat Commun ; 12(1): 4175, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234126

RESUMO

Although we can now measure single-cell signaling responses with multivariate, high-throughput techniques our ability to interpret such measurements is still limited. Even interpretation of dose-response based on single-cell data is not straightforward: signaling responses can differ significantly between cells, encompass multiple signaling effectors, and have dynamic character. Here, we use probabilistic modeling and information-theory to introduce fractional response analysis (FRA), which quantifies changes in fractions of cells with given response levels. FRA can be universally performed for heterogeneous, multivariate, and dynamic measurements and, as we demonstrate, quantifies otherwise hidden patterns in single-cell data. In particular, we show that fractional responses to type I interferon in human peripheral blood mononuclear cells are very similar across different cell types, despite significant differences in mean or median responses and degrees of cell-to-cell heterogeneity. Further, we demonstrate that fractional responses to cytokines scale linearly with the log of the cytokine dose, which uncovers that heterogeneous cellular populations are sensitive to fold-changes in the dose, as opposed to additive changes.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Interferon Tipo I/metabolismo , Leucócitos Mononucleares/metabolismo , Modelos Imunológicos , Células 3T3 , Animais , Voluntários Saudáveis , Humanos , Interferon Tipo I/imunologia , Leucócitos Mononucleares/imunologia , Camundongos , Modelos Estatísticos , Cultura Primária de Células , Transdução de Sinais/imunologia , Análise de Célula Única , Software
18.
Aging (Albany NY) ; 13(11): 14687-14708, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088884

RESUMO

Bone marrow mononuclear cell therapy improves cardiac repair after myocardial infarction (MI), in-part through signaling to resident cardiac cells, such as fibroblasts, which regulate scar formation. The efficacy of cell therapy declines with age, as aging of both donor and recipient cells decreases repair responses. Autophagy regulates the microenvironment by both extracellular vesicle (EV)-dependent and independent secretion pathways. We hypothesized that age-related autophagy changes in bone marrow cells (BMCs) alter paracrine signaling, contributing to lower cell therapy efficacy. Here, we demonstrate that young Sca-1+ BMCs exhibited a higher LC3II/LC3I ratio compared to old Sca-1+ BMCs, which was accentuated when BMCs were cultured under hypoxia. To examine the effect on paracrine signaling, old cardiac fibroblasts were cultured with conditioned medium (CM) from young and old Sca-1+ BMCs. Young, but not old CM, enhanced fibroblast proliferation, migration, and differentiation, plus reduced senescence. These beneficial effects were lost when autophagy or EV secretion in BMCs was blocked pharmacologically, or by siRNA knockdown of Atg7. Therefore, both EV-dependent and -independent paracrine signaling from young BMCs is responsible for paracrine stimulation of old cardiac fibroblasts. In vivo, bone marrow chimerism of old mice with young BMCs increased the number of LC3b+ cells in the heart compared to old mice reconstituted with old BMCs. These data suggest that the deterioration of autophagy with aging negatively impacts the paracrine effects of BMCs, and provide mechanistic insight into the age-related decline in cell therapy efficacy that could be targeted to improve the function of old donor cells.


Assuntos
Envelhecimento/patologia , Autofagia , Células da Medula Óssea/metabolismo , Leucócitos Mononucleares/metabolismo , Comunicação Parácrina , Animais , Antígenos Ly/metabolismo , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia/metabolismo , Meios de Cultivo Condicionados/farmacologia , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Comunicação Parácrina/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia
19.
J Cancer Res Clin Oncol ; 147(10): 2983-2991, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34165589

RESUMO

BACKGROUND: While molecular testing is a promising strategy for preoperative assessment of cytologically indeterminate thyroid nodules, thyroid fine needle aspiration biopsy (FNA) presents unique challenges for molecular assays, including contaminating peripheral blood mononuclear cells (PBMC) and variable numbers of evaluable epithelial thyroid cells. Moreover, the newly recognized entity, noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), has added an additional challenge to the currently available molecular diagnostic platforms. New diagnostic tools are still needed to correctly distinguish benign and malignant thyroid nodules preoperatively. METHODS: Twenty-two transcript splice variants from 12 genes we previously identified as discriminating benign from malignant thyroid nodules were characterized in 80 frozen thyroid tumors from 8 histological subtypes. Isoforms detectable in PBMC were excluded, and the 5 most discriminating isoforms were further validated by real-time quantitative PCR (qPCR) on intraoperative FNA samples from 59 malignant tumors, 55 benign nodules, and 23 NIFTP samples. The qPCR threshold cycle values for each transcript were normalized to the thyrocyte-specific thyroid peroxidase isoform 1 (TPO1) and z-transformed. Receiver operating characteristic (ROC) analyses of the composite transcript scores were used to evaluate classification of thyroid FNAs by the 5-gene isoform expression panel. RESULTS: A molecular signature was developed by combining expression levels of specific isoforms of CDH3, FNDC4, HMGA2, KLK7, and PLAG1. FNAs containing at least 12-36 thyrocytes were sufficient for this assay. The 5-gene composite score achieved an area under the ROC curve (AUC) of 0.86 for distinguishing malignant from benign nodules, with a specificity of 91%, sensitivity of 75%, negative predictive value of 91%, and positive predictive value of 74%. CONCLUSION: Our newly developed 5-gene isoform expression panel distinguishes benign from malignant thyroid tumors and, may help distinguish benign from malignant thyroid nodules in the context of the new NIFTP subtype.


Assuntos
Biomarcadores Tumorais/metabolismo , Leucócitos Mononucleares/patologia , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Nódulo da Glândula Tireoide/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biópsia por Agulha Fina , Feminino , Seguimentos , Humanos , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular , Prognóstico , Estudos Retrospectivos , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/cirurgia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/cirurgia , Nódulo da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/cirurgia , Adulto Jovem
20.
Immunity ; 54(7): 1561-1577.e7, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34102100

RESUMO

A common metabolic alteration in the tumor microenvironment (TME) is lipid accumulation, a feature associated with immune dysfunction. Here, we examined how CD8+ tumor infiltrating lymphocytes (TILs) respond to lipids within the TME. We found elevated concentrations of several classes of lipids in the TME and accumulation of these in CD8+ TILs. Lipid accumulation was associated with increased expression of CD36, a scavenger receptor for oxidized lipids, on CD8+ TILs, which also correlated with progressive T cell dysfunction. Cd36-/- T cells retained effector functions in the TME, as compared to WT counterparts. Mechanistically, CD36 promoted uptake of oxidized low-density lipoproteins (OxLDL) into T cells, and this induced lipid peroxidation and downstream activation of p38 kinase. Inhibition of p38 restored effector T cell functions in vitro, and resolution of lipid peroxidation by overexpression of glutathione peroxidase 4 restored functionalities in CD8+ TILs in vivo. Thus, an oxidized lipid-CD36 axis promotes intratumoral CD8+ T cell dysfunction and serves as a therapeutic avenue for immunotherapies.


Assuntos
Antígenos CD36/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Peroxidação de Lipídeos/fisiologia , Lipoproteínas LDL/metabolismo , Neoplasias/metabolismo , Receptores Depuradores/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microambiente Tumoral/fisiologia
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