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1.
Molecules ; 25(12)2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32604797

RESUMO

Viruses can be spread from one person to another; therefore, they may cause disorders in many people, sometimes leading to epidemics and even pandemics. New, previously unstudied viruses and some specific mutant or recombinant variants of known viruses constantly appear. An example is a variant of coronaviruses (CoV) causing severe acute respiratory syndrome (SARS), named SARS-CoV-2. Some antiviral drugs, such as remdesivir as well as antiretroviral drugs including darunavir, lopinavir, and ritonavir are suggested to be effective in treating disorders caused by SARS-CoV-2. There are data on the utilization of antiretroviral drugs against SARS-CoV-2. Since there are many studies aimed at the identification of the molecular mechanisms of human immunodeficiency virus type 1 (HIV-1) infection and the development of novel therapeutic approaches against HIV-1, we used HIV-1 for our case study to identify possible molecular pathways shared by SARS-CoV-2 and HIV-1. We applied a text and data mining workflow and identified a list of 46 targets, which can be essential for the development of infections caused by SARS-CoV-2 and HIV-1. We show that SARS-CoV-2 and HIV-1 share some molecular pathways involved in inflammation, immune response, cell cycle regulation.


Assuntos
Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/metabolismo , Mineração de Dados/métodos , Infecções por HIV/epidemiologia , Infecções por HIV/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/metabolismo , Anti-Inflamatórios/uso terapêutico , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Proteínas do Sistema Complemento/genética , Proteínas do Sistema Complemento/imunologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Bases de Dados Genéticas , Regulação da Expressão Gênica , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/uso terapêutico , Inflamação , Interferons/genética , Interferons/imunologia , Interleucinas/genética , Interleucinas/imunologia , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/imunologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia
2.
Nat Commun ; 11(1): 581, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996683

RESUMO

Cancer cells are poorly immunogenic and have a wide range of mutations, which makes them unsuitable for use in vaccination treatment. Here, we show that elimination of CD47, a ligand for the myeloid cell inhibitory receptor SIRPα, from tumor cells by genetic deletion or antibody blocking, significantly improves the effectiveness of the immune response to tumour cells. In both solid and hematopoietic mouse tumor models, vaccination with tumor cells or tumor antigen-expressing cells, that lack CD47 or were pre-coated with anti-CD47 antibodies, achieved an antitumor immune response. The efficacy of this approach was synergistically enhanced when used in combination with anti-PD-1 antibodies. The induction of antitumor responses depends on SIRPα+CD11c+ DCs, which exhibit rapid expansion following introduction of CD47-deficient tumor cells. Our results indicate that CD47-deficient whole tumor cells can induce antitumor responses.


Assuntos
Anticorpos Antineoplásicos/efeitos dos fármacos , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Antígeno CD47/genética , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Vacinação , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Antineoplásicos/imunologia , Antígenos de Diferenciação/imunologia , Antígenos de Neoplasias , Antígeno CD11c , Antígeno CD47/imunologia , Feminino , Transplante de Células-Tronco Hematopoéticas , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Células Mieloides/imunologia
3.
Iran J Immunol ; 16(4): 291-298, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31885006

RESUMO

BACKGROUND: NK (natural killer) and NKT (natural killer T) cells, as components of innate immune system, play a crucial role in tumor progression and dissemination. OBJECTIVE: To investigate the percentages of NK cells, NKT cells, iNKT (invariant natural killer T) cells, total T lymphocytes as well as activated T lymphocytes, in tumor draining lymph nodes (TDLNs) of patients with breast cancer (BC) and their association with different clinic-pathological features of the patients. METHODS: Axillary lymph nodes were obtained from 30 Iranian women with breast cancer. After routine pathological evaluations, mononuclear cells were separated from their lymph nodes and incubated with appropriate fluorochrome conjugated monoclonal antibodies specific for CD3, HLA-DR, CD16/56, and Vα24Jα18-TCR. Data were collected on a four-color flow cytometer and analyzed by CellQuest software. RESULTS: The mean percentages of NK (CD3-CD16/56+), NKT (CD3+CD16/56+) and iNKT (Vα24Jα18-TCR+) cells in TDLNs mononuclear cells of BC patients were 2.04%, 2.44% and 0.1%, respectively. A significant decrease in the percentages of NK and iNKT subsets in patients with grade I was observed compared to grade III (p=0.03 and p=0.01, respectively). Moreover, NK cells were increased in patients with grade III of BC compared to grade II (p= 0.003). CONCLUSION: The increase in the percentage of NK and iNKT cells in TDLNs of patients with higher grade of BC might suggest a suppressive phenotype for these cells in breast cancer, which merit more functional investigation.


Assuntos
Antígenos de Diferenciação/imunologia , Neoplasias da Mama/imunologia , Citometria de Fluxo , Células Matadoras Naturais/imunologia , Linfonodos/imunologia , Células T Matadoras Naturais/imunologia , Adulto , Neoplasias da Mama/patologia , Feminino , Humanos , Irã (Geográfico) , Células Matadoras Naturais/patologia , Linfonodos/patologia , Pessoa de Meia-Idade , Células T Matadoras Naturais/patologia
4.
Immunology ; 158(3): 161-170, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31517385

RESUMO

Regulatory T (Treg) cells are a subset of CD4+ T cells that are critical for the maintenance of self-tolerance. The forkhead box transcription factor Foxp3 is a master regulator for the Treg phenotype and function and its expression is essential in Treg cells, as the loss of Foxp3 results in lethal autoimmunity. Two major subsets of Treg cells have been described in vivo; thymus-derived Treg (tTreg) cells that develop in the thymus and peripherally induced Treg (pTreg) cells that are derived from conventional CD4+  Foxp3- T cells and are converted in peripheral tissues to cells that express Foxp3 and acquire suppressive ability. The transcription factor Helios, a member of the Ikaros transcription factor family, is expressed in 60-70% of Treg cells in both mouse and man, and is believed to be a marker of tTreg cells. In this review, we discuss the role and function of Helios in Treg cells, the controversy surrounding the use of Helios as a marker of tTreg cells, and how Helios controls specific aspects of the Treg cell program.


Assuntos
Antígenos de Diferenciação/imunologia , Proteínas de Ligação a DNA/imunologia , Fatores de Transcrição Forkhead/imunologia , Fator de Transcrição Ikaros/imunologia , Linfócitos T Reguladores/imunologia , Fatores de Transcrição/imunologia , Animais , Humanos , Camundongos
5.
Blood ; 134(17): 1430-1440, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31383641

RESUMO

Antibodies that bind CD47 on tumor cells and prevent interaction with SIRPα on phagocytes are active against multiple cancer types including T-cell lymphoma (TCL). Here we demonstrate that surface CD47 is heterogeneously expressed across primary TCLs, whereas major histocompatibility complex (MHC) class I, which can also suppress phagocytosis, is ubiquitous. Multiple monoclonal antibodies (mAbs) that block CD47-SIRPα interaction promoted phagocytosis of TCL cells, which was enhanced by cotreatment with antibodies targeting MHC class I. Expression levels of surface CD47 and genes that modulate CD47 pyroglutamation did not correlate with the extent of phagocytosis induced by CD47 blockade in TCL lines. In vivo treatment of multiple human TCL patient-derived xenografts or an immunocompetent murine TCL model with a short course of anti-CD47 mAb markedly reduced lymphoma burden and extended survival. Depletion of macrophages reduced efficacy in vivo, whereas depletion of neutrophils had no effect. F(ab')2-only fragments of anti-CD47 antibodies failed to induce phagocytosis by human macrophages, indicating a requirement for Fc-Fcγ receptor interactions. In contrast, F(ab')2-only fragments increased phagocytosis by murine macrophages independent of SLAMF7-Mac-1 interaction. Full-length anti-CD47 mAbs also induced phagocytosis by Fcγ receptor-deficient murine macrophages. An immunoglobulin G1 anti-CD47 mAb induced phagocytosis and natural killer cell-mediated cytotoxicity of TCL cells that was augmented by cotreatment with mogamulizumab, an anti-CCR4 mAb, or a mAb blocking MHC class I. These studies help explain the disparate activity of monotherapy with agents that block CD47 in murine models compared with patients. They also have direct translational implications for the deployment of anti-CD47 mAbs alone or in combination.


Assuntos
Antígenos de Diferenciação/imunologia , Antineoplásicos Imunológicos/farmacologia , Antígeno CD47/imunologia , Linfoma de Células T/tratamento farmacológico , Receptores de IgG/imunologia , Receptores Imunológicos/imunologia , Animais , Antineoplásicos Imunológicos/uso terapêutico , Antígeno CD47/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Camundongos , Receptores Fc/imunologia
6.
Science ; 365(6449): 176-180, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31296770

RESUMO

Elevated levels of type I interferon (IFN) during pregnancy are associated with intrauterine growth retardation, preterm birth, and fetal demise through mechanisms that are not well understood. A critical step of placental development is the fusion of trophoblast cells into a multinucleated syncytiotrophoblast (ST) layer. Fusion is mediated by syncytins, proteins deriving from ancestral endogenous retroviral envelopes. Using cultures of human trophoblasts or mouse cells, we show that IFN-induced transmembrane proteins (IFITMs), a family of restriction factors blocking the entry step of many viruses, impair ST formation and inhibit syncytin-mediated fusion. Moreover, the IFN inducer polyinosinic:polycytidylic acid promotes fetal resorption and placental abnormalities in wild-type but not in Ifitm-deleted mice. Thus, excessive levels of IFITMs may mediate the pregnancy complications observed during congenital infections and other IFN-induced pathologies.


Assuntos
Antígenos de Diferenciação/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Fusão Celular , Morte Fetal/etiologia , Interferon Tipo I/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Ligação a RNA/imunologia , Trofoblastos/imunologia , Animais , Feminino , Reabsorção do Feto/imunologia , Produtos do Gene env/imunologia , Humanos , Camundongos , Poli I-C/farmacologia , Gravidez , Proteínas da Gravidez/imunologia , Trofoblastos/efeitos dos fármacos
7.
MAbs ; 11(6): 1036-1052, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257988

RESUMO

Targeting the CD47-signal-regulatory protein α (SIRPα) pathway represents a novel therapeutic approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses. Unlike CD47, which is expressed ubiquitously, SIRPα expression is mainly restricted to myeloid cells and neurons. Therefore, compared to CD47-targeted therapies, targeting SIRPα may result in differential safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics and pharmacodynamics. The development of effective SIRPα antagonists is restricted by polymorphisms within the CD47-binding domain of SIRPα, necessitating pan-allele reactive anti-SIRPα antibodies for therapeutic intervention in diverse patient populations. We immunized wild-type and human antibody transgenic chickens with a multi-allele and multi-species SIRPα regimen in order to discover pan-allelic and pan-mammalian reactive anti-SIRPα antibodies suitable for clinical translation. A total of 200 antibodies were isolated and screened for SIRPα reactivity from which approximately 70 antibodies with diverse SIRPα binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRPα antibodies bound to both human SIRPα v1 and v2 alleles with high affinity ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRPα interaction, and potentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal structures of five anti-SIRPα antigen-binding fragments, each with unique epitopes, in complex with SIRPα (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the anti-SIRPα antibodies cross-react with cynomolgus SIRPα and various mouse SIRPα alleles (BALB/c, NOD, BL/6), which can facilitate preclinical to clinical development. These properties provide an attractive rationale to advance the development of these anti-SIRPα antibodies as a novel therapy for advanced malignancies. Abbreviations: ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; CFSE: carboxyfluorescein succinimidyl ester; Fab: fragment antigen binding; Fc: fragment crystallizable; FcγR: Fcγ receptor; Ig: immunoglobulin; IND: investigational new drug; MDM⊘: monocyte-derived macrophage; NOD: non-obese diabetic; scFv: single chain fragment variable; SCID: severe combined immunodeficiency; SIRP: signal-regulatory protein.


Assuntos
Anticorpos Monoclonais , Especificidade de Anticorpos , Antígenos de Diferenciação , Receptores Imunológicos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação/química , Antígenos de Diferenciação/imunologia , Antígeno CD47/imunologia , Galinhas , Cristalografia por Raios X , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Imunoterapia , Masculino , Neoplasias/imunologia , Neoplasias/terapia , Domínios Proteicos , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/química , Receptores Imunológicos/imunologia
8.
PLoS One ; 14(6): e0217548, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31185026

RESUMO

PURPOSE: Retinal detachment (RD) is one of the most frequently diagnosed ophthalmologic conditions requiring prompt surgical intervention. Combination of proper surgical technique and new diagnostic markers, both clinical and molecular, can help improve the diagnosis and prognosis of RD treatment. METHODS: 12 patients with rhegmatogenous RD (rRD) were included into the study after obtaining patient consent and Regional Ethical Approval (average age: 58.1 ± 17.4 years). OCT was performed before and after 23G vitrectomy for RD. Pure subretinal fluid (SRF) was collected during surgery and analyzed by protein array profiling on a panel of 105 inflammatory cytokines (Human XL Cytokine Array), while the effect of SRF upon human macrophages-driven phagocytosis of apoptotic retinal pigment epithelial (RPE) cells ex vivo was quantified by flow cytometry. Immunohistochemistry (IHC) of retinectomized tissue due to PVR caused by RD was performed to determine presence of markers for microglial cells (CD34), macrophages and activated microglia (CD68), regulator of the immune response to infection (NFkB), progenitor and stem cell marker (Sox2), pluripotency marker (Oct4) and intermediate filament markers (GFAP and Nestin). RESULTS: OCT of fresh RD patients contained pre-operatively hyper reflective points (HRPs) at the detached neuroretina border and proximal to the RPE layer-their size and number decreased following successful reattachment surgery. IHC of the retinectomized tissue from detached retina due to severe PVR showed presence of cell conglomerates at the detached neuroretina border which were positive for CD68, NFkB, Sox2 and GFAP, less positive for CD47 and Nestin and negative for Oct4 and CD34. The SRF contained at least 37 cytokines with higher, and 4 cytokine with lower concentration compared to that in vitreous from non-RD pathology; when used as conditional medium to human macrophages ex vivo, the SRF doubled their capacity for engulfing dying RPEs. CONCLUSIONS: Fresh RD can be hallmarked by presence of HRPs at the detached neuroretina border on OCT; the HRPs decrease in size and number after successful reattachment surgery, and likely resemble the macrophage conglomerates seen by IHC. The neuroretina in RD contains progenitor/stem-like cells and signs of inflammatory reaction, while the SRF contains inflammatory cytokines and other factors which increase the ability of professional phagocytes to engulf dying RPE, or for that matter, other dying cells in the retina.


Assuntos
Antígenos de Diferenciação/imunologia , Proteínas do Olho/imunologia , Descolamento Retiniano/imunologia , Epitélio Pigmentado da Retina/imunologia , Células-Tronco/imunologia , Adulto , Idoso , Apoptose/imunologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/cirurgia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Microglia/imunologia , Microglia/patologia , Pessoa de Meia-Idade , Fagocitose , Descolamento Retiniano/patologia , Descolamento Retiniano/cirurgia , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/cirurgia , Células-Tronco/patologia
9.
Immunopharmacol Immunotoxicol ; 41(3): 455-462, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31142168

RESUMO

Objective: Dendritic cells (DCs) are professional antigen presenting cells majorly modulated by various environmental factors. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine from interleukin-6 family. Previous studies demonstrate that LIF is associated with several tolerogenic events; yet the exact effect of this cytokine on the generation and function of DCs was not explicitly identified. Materials and methods: To clarify the role of LIF in DCs development, immature DCs were differentiated from mouse bone marrow (BM) in a GM-CSF and IL-4 containing medium with or without LIF. Afterwards, in maturation process, the differentiated DCs were exposed to TNF-α in the presence or absence of LIF. Results: Immature DCs differentiated in the presence of LIF, proved a significant enhancement in the expression of MHCII, CD40, or CD86 molecules and in the antigen uptake function. LIF treatment of normal DCs while stimulating for maturation, caused a significant decrement in the expression of phenotypic markers as well as an increment in the antigen uptake function in comparison with TNF-α-only stimulated cells; however, the reduced ability for induction of allogenic T-cell proliferation proved no statistical significance. Conclusions: Our results can reflect a role for LIF in the generation and particularly maturation of DCs. It can be assumed that LIF rather modulates the maturation level, leading to the development of semi-mature and tolerogenic DCs. According to the high levels of LIF in immune-privileged sites like brain and uterine, it seems that the cytokine may account for the formation of local DCs that help the establishment of immunosuppressive environments.


Assuntos
Células da Medula Óssea/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Fator Inibidor de Leucemia/imunologia , Animais , Antígenos de Diferenciação/imunologia , Células da Medula Óssea/citologia , Células Dendríticas/citologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Fator Inibidor de Leucemia/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/imunologia
10.
Vet Immunol Immunopathol ; 211: 1-5, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084887

RESUMO

Previous studies on the immune system of water buffalo (Bubalus bubalis) using cross-reactive monoclonal antibodies (mAbs) revealed significant similarities and differences to the bovine immune system. Herein, we extend these studies and document the pattern of expression of CD14, CD16, CD163 and CD172a on buffalo leukocytes using a set of cross-reactive mAbs that are known to recognize conserved epitopes within orthologous molecules in cattle, sheep and goats. Buffalo leukocytes were isolated and subjected to mAb labelling for flow cytometry. Single color flow cytometry confirmed mAbs recognition of buffalo orthologues of CD14, CD16, CD163 and CD172a, and revealed consistent patterns of expression similar to that reported in other ruminants. Multicolor flow cytometry revealed that buffalo CD14+ monocytes uniquely co-express CD16, CD163 and CD172a, whereas buffalo granulocytes co-express CD16 and CD172a. This study expands mAbs available to define and study the buffalo monocytes, and also extends information available on the unique features of the buffalo immune system.


Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Búfalos/imunologia , Leucócitos/imunologia , Receptores de Lipopolissacarídeos/imunologia , Receptores de Superfície Celular/imunologia , Receptores de IgG/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação/imunologia , Citometria de Fluxo/veterinária , Granulócitos/imunologia , Monócitos/imunologia
11.
Cell Physiol Biochem ; 52(5): 1178-1192, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30990587

RESUMO

BACKGROUND/AIMS: Rheumatoid arthritis (RA) is a progressive, chronic, even disabling systemic autoimmune disease. Imbalance between pathogenic immune cells and immunosuppressive cells is associated with the pathogenesis and development of RA and other autoimmune diseases. As Foxp3 is also expressed on activated CD4+ cells in the presence of inflammation, the identification of Treg cells in patients with RA remains a challenge. METHODS: Comprehensive analyses were carried out by Flow cytometry. Expression of Helios, CD226, T cell immunoreceptor with Ig and ITIM domains clinical samples and healthy controls. RESULTS: We have systemically examined three potential markers, Helios, CD226 and TIGIT, that are possibly related to Treg identification, and found that Helios expression on CD4+Foxp3+cells was decreased and negatively correlated with the disease activity of RA patients, while CD226 and TIGIT both showed elevated expression levels in CD4+Foxp3+cells in RA patients and they were not associated with disease activity of RA patients. CONCLUSION: Taken together, our findings indicate that CD4+CD25hiCD127low/-Foxp3+Helios+ may represent the real Treg cell population in patients with RA.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação/imunologia , Artrite Reumatoide/imunologia , Fatores de Transcrição Forkhead/imunologia , Fator de Transcrição Ikaros/imunologia , Receptores Imunológicos/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Artrite Reumatoide/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/patologia
12.
J Immunol ; 202(9): 2700-2709, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30867240

RESUMO

GM-CSF is required for alveolar macrophage (AM) development shortly after birth and for maintenance of AM functions throughout life, whereas M-CSF is broadly important for macrophage differentiation and self-renewal. However, the comparative actions of GM-CSF and M-CSF on AMs are incompletely understood. Interstitial macrophages (IMs) constitute a second major pulmonary macrophage population. However, unlike AMs, IM responses to CSFs are largely unknown. Proliferation, phenotypic identity, and M1/M2 polarization are important attributes of all macrophage populations, and in this study, we compared their modulation by GM-CSF and M-CSF in murine primary AMs and IMs. CSFs increased the proliferation capacity and upregulated antiapoptotic gene expression in AMs but not IMs. GM-CSF, but not M-CSF, reinforced the cellular identity, as identified by surface markers, of both cell types. GM-CSF, but not M-CSF, increased the expression of both M1 and M2 markers exclusively in AMs. Finally, CSFs enhanced the IFN-γ- and IL-4-induced polarization ability of AMs but not IMs. These first (to our knowledge) data comparing effects on the two pulmonary macrophage populations demonstrate that the activating actions of GM-CSF and M-CSF on primary AMs are not conserved in primary IMs.


Assuntos
Proliferação de Células/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interferon gama/imunologia , Interleucina-4/imunologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos Alveolares/imunologia , Animais , Antígenos de Diferenciação/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Macrófagos/imunologia , Macrófagos Alveolares/citologia , Masculino , Camundongos
13.
J Immunol ; 202(8): 2493-2501, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30842275

RESUMO

The limited number of hematopoietic stem cells (HSC) within a single unit of human cord blood currently limits its use as an alternate graft source. However, we have developed a strategy using 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), which expands transplantable HSC 7- to 10-fold. In our current studies, we have assessed the allostimulatory capacity of the 5azaD/TSA-expanded grafts. The coexpression of immunophenotypic dendritic cell (DC) markers, such as HLA-DR/CD86 and HLA-DR/CD11c as determined by flow cytometry, and the allostimulatory capacity of 5azaD/TSA-expanded cells as determined by MLC were both significantly lower than control. It has been previously demonstrated that STAT3 is indispensable for the differentiation of DC from HSC. Real-time quantitative PCR analysis revealed that 5azaD/TSA-expanded cells expressed more STAT3 transcript than control while also expressing increased transcripts for STAT3 inhibitors including SHP1, p21, and GATA1. Western blot analysis indicates that chromatin-modifying agent-expanded grafts displayed a reduced ratio of p-STAT3 to total STAT3 than control cultures, which is likely indicative of STAT3 inactivity in 5azD/TSA-expanded grafts. Culturing 5azaD/TSA-expanded cord blood cells in extended cultures reveals that they are still capable of generating DC. Notably, STAT3 inactivity was transient because the transcript levels of STAT3 and its inhibitors, including SHP1, were comparable between 5azaD/TSA and control cultures following extended culture. Taken together, our studies indicate that the reduced allostimulatory capacity of 5azaD/TSA-expanded cells is likely because of reversible inhibition of STAT3-dependent DC differentiation. These results suggest that a graft composed of 5azaD/TSA-expanded cells possesses relatively less allostimulatory response but is still capable of generating DC in permissive conditions.


Assuntos
Antígenos de Diferenciação/imunologia , Cromatina/imunologia , Decitabina/farmacologia , Sangue Fetal/imunologia , Células-Tronco Hematopoéticas/imunologia , Ácidos Hidroxâmicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/citologia , Humanos
14.
PLoS Biol ; 17(2): e3000137, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30726215

RESUMO

Tripartite motif (TRIM) proteins belong to a large family with many roles in host biology, including restricting virus infection. Here, we found that TRIM2, which has been implicated in cases of Charcot-Marie-Tooth disease (CMTD) in humans, acts by blocking hemorrhagic fever New World arenavirus (NWA) entry into cells. We show that Trim2-knockout mice, as well as primary fibroblasts from a CMTD patient with mutations in TRIM2, are more highly infected by the NWAs Junín and Tacaribe virus than wild-type mice or cells are. Using mice with different Trim2 gene deletions and TRIM2 mutant constructs, we demonstrate that its antiviral activity is uniquely independent of the RING domain encoding ubiquitin ligase activity. Finally, we show that one member of the TRIM2 interactome, signal regulatory protein α (SIRPA), a known inhibitor of phagocytosis, also restricts NWA infection and conversely that TRIM2 limits phagocytosis of apoptotic cells. In addition to demonstrating a novel antiviral mechanism for TRIM proteins, these studies suggest that the NWA entry and phagocytosis pathways overlap.


Assuntos
Antígenos de Diferenciação/genética , Arenavirus do Novo Mundo/genética , Doença de Charcot-Marie-Tooth/genética , Interações Hospedeiro-Patógeno/genética , Proteínas Nucleares/genética , Receptores Imunológicos/genética , Animais , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Apoptose , Arenavirus do Novo Mundo/crescimento & desenvolvimento , Arenavirus do Novo Mundo/patogenicidade , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/virologia , Linhagem Celular Tumoral , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Chlorocebus aethiops , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/imunologia , Proteínas de Neurofilamentos/metabolismo , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Osteoblastos/imunologia , Osteoblastos/metabolismo , Osteoblastos/virologia , Cultura Primária de Células , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais , Células Vero , Internalização do Vírus
15.
Viruses ; 11(2)2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30704088

RESUMO

The proteins IFITM1, IFITM2, and IFITM3 are host effectors against a broad range of RNA viruses whose roles in classical swine fever virus (CSFV) infection had not yet been reported. We investigated the effect of these proteins on CSFV replication in mammalian cells. The proteins were overexpressed and silenced using lentiviruses. Confocal microscopy was used to determine the distribution of these proteins in the cells, and immunofluorescence colocalization analysis was used to evaluate the relationship between IFITMs and the CSFV endosomal pathway, including early endosomes, late endosomes, and lysosomes. IFITM1, IFITM2, or IFITM3 overexpression significantly inhibited CSFV replication, whereas protein knockdown enhanced CSFV replication. In porcine alveolar macrophages (PAMs), IFITM1 was mainly located at the cell surface, whereas IFITM2 and IFITM3 were mainly located in the cytoplasm. Following CSFV infection, the distribution of IFITM1 changed. IFITM1, IFITM2, and IFITM3 colocalization with Lamp1, IFITM2 with Rab5 and Rab7, and IFITM3 with Rab7 were observed in CSFV-infected cells. Collectively, these results provide insights into the possible mechanisms associated with the anti-CSFV action of the IFITM family.


Assuntos
Antígenos de Diferenciação/imunologia , Vírus da Febre Suína Clássica/imunologia , Interações entre Hospedeiro e Microrganismos , Macrófagos Alveolares/virologia , Proteínas de Membrana/imunologia , Proteínas de Ligação a RNA/imunologia , Animais , Linhagem Celular , Vírus da Febre Suína Clássica/fisiologia , Endossomos/imunologia , Endossomos/virologia , Interferons/farmacologia , Macrófagos Alveolares/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Internalização do Vírus , Replicação Viral
16.
J Pediatr Hematol Oncol ; 41(8): 648-652, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-29912035

RESUMO

Adoptive cell therapy (ACT) of chimeric antigen receptor T cells has demonstrated remarkable success for the treatment of pediatric B-cell leukemia. For patients who are not candidates for chimeric antigen receptor T-cell therapy, ACT using tumor antigen-experienced polyclonal T cells may be a treatment option. Since leukemic blasts reside in the bone marrow and bone marrow is a preferred site for homeostatic proliferation of cytotoxic memory CD8 T cells, we hypothesized that bone marrow would be a source of activated T cells. The aim of this study was to determine the feasibility of using bone marrow-derived T cells following postinduction chemotherapy for use in adoptive cell transfer. Matched patient samples of bone marrow and peripheral blood-derived T cells expanded ex vivo and displayed similar apoptotic profiles. Before activation and expansion, there was a significant increase in the percentage of bone marrow-derived CD8 T cells expressing activation markers PD1, CD45RO, and CD69 as compared with peripheral blood CD8 T cells. Considering, melanoma-reactive CD8 T cells reside in the subset of PD1CD8 T cells, the bone marrow may be an enriched source leukemic-specific T cells that can be used for ACT.


Assuntos
Apoptose/imunologia , Células da Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Regulação Leucêmica da Expressão Gênica/imunologia , Memória Imunológica , Quimioterapia de Indução , Leucemia , Proteínas de Neoplasias/imunologia , Receptor de Morte Celular Programada 1/imunologia , Antígenos de Diferenciação/imunologia , Células da Medula Óssea/patologia , Linfócitos T CD8-Positivos/patologia , Criança , Feminino , Humanos , Leucemia/tratamento farmacológico , Leucemia/imunologia , Leucemia/patologia , Masculino
17.
J Allergy Clin Immunol ; 143(1): 213-228.e10, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29596938

RESUMO

BACKGROUND: A delicate balance between cell death and keratinocyte proliferation is crucial for normal skin development. Previous studies have reported that cellular FLICE (FADD-like ICE)-inhibitory protein plays a crucial role in prevention of keratinocytes from TNF-α-dependent apoptosis and blocking of dermatitis. However, a role for cellular FLICE-inhibitory protein in TNF-α-independent cell death remains unclear. OBJECTIVE: We investigated contribution of TNF-α-dependent and TNF-α-independent signals to the development of dermatitis in epidermis-specific Cflar-deficient (CflarE-KO) mice. METHODS: We examined the histology and expression of epidermal differentiation markers and inflammatory cytokines in the skin of CflarE-KO;Tnfrsf1a+/- and CflarE-KO;Tnfrsf1a-/- mice. Mice were treated with neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand to block TNF-α-independent cell death of CflarE-KO;Tnfrsf1a-/- mice. RESULTS: CflarE-KO;Tnfrsf1a-/- mice were born but experienced severe dermatitis and succumbed soon after birth. CflarE-KO;Tnfrsf1a+/- mice exhibited embryonic lethality caused by massive keratinocyte apoptosis. Although keratinocytes from CflarE-KO;Tnfrsf1a-/- mice still died of apoptosis, neutralizing antibodies against Fas ligand and TNF-related apoptosis-inducing ligand substantially prolonged survival of CflarE-KO;Tnfrsf1a-/- mice. Expression of inflammatory cytokines, such as Il6 and Il17a was increased; conversely, expression of epidermal differentiation markers was severely downregulated in the skin of CflarE-KO;Tnfrsf1a-/- mice. Treatment of primary keratinocytes with IL-6 and, to a lesser extent, IL-17A suppressed expression of epidermal differentiation markers. CONCLUSION: TNF receptor superfamily 1 (TNFR1)-dependent or TNFR1-independent apoptosis of keratinocytes promotes inflammatory cytokine production, which subsequently blocks epidermal differentiation. Thus blockade of both TNFR1-dependent and TNFR1-independent cell death might be an alternative strategy to treat skin diseases when treatment with anti-TNF-α antibody alone is not sufficient.


Assuntos
Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Dermatite/imunologia , Epiderme/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Apoptose/genética , Apoptose/imunologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Dermatite/genética , Dermatite/patologia , Epiderme/patologia , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia
18.
J Innate Immun ; 11(1): 99-108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30408777

RESUMO

Early exposure to inflammatory signals may have a lasting impact on immune function. Present throughout embryogenesis, macrophages are key cells providing innate immune protection to the developing fetus and newborn. Here, we have used an established model of macrophage development to test how early inflammatory signals can impact cellular differentiation and function. Bone marrow-derived macrophages were treated with Escherichia coli lipopolysaccharide (LPS) 2 days after initial isolation and culture. LPS treatment during this early stage of differentiation decreased the expression of CSF1R and increased that of the mature macrophage marker F4/80. These early changes in macrophage differentiation were also measured in cells from mice lacking IKKß, but the change in CSF1R expression after LPS treatment was blocked with MAPK inhibition. LPS-induced changes in macrophage marker expression persisted following LPS removal, suggesting that early inflammatory activation could induce a lasting developmental impact. Early LPS exposure inhibited macrophage phagocytosis of labeled E. coli while LPS had no effect on fully differentiated macrophages. Our data demonstrate that early inflammatory exposure to a microbial stimulus induce lasting phenotypic changes in macrophages.


Assuntos
Diferenciação Celular , Ativação de Macrófagos , Macrófagos , Receptores Toll-Like/metabolismo , Animais , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Escherichia coli , Quinase I-kappa B/metabolismo , Imunidade Inata , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fagocitose/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Transdução de Sinais
19.
Methods Mol Biol ; 1884: 335-347, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30465214

RESUMO

Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting, with antibodies such as rituximab, to blocking of immune checkpoints to reactivate antitumor immunity. In addition, bispecific antibodies/antibody fragments are also of great interest in cancer therapy, as these constructs have the ability to redirect immune effector cells to cancer targets and, thereby, enhance therapeutic efficacy. A number of bispecific antibody formats have been reported, with the first FDA-approved bispecific antibody being blinatumomab, a so-called bispecific T cell engager (BiTE), which redirects and potently activates T cell immune responses. Recently, we described an additional novel bispecific antibody derivative, termed RTX-CD47, which was designed to inhibit the innate immune checkpoint CD47-SIRPα only on -positive cancer cells. RTX-CD47 contains two antibody fragments in tandem and has monovalent binding specificity for CD47 and . Only upon dual binding to and CD47 RTX-CD47 blocks CD47 "Don't eat me" signaling. Here, we provide a detailed protocol for the construction and functional evaluation of such a bispecific antibody derivative.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Bioensaio/métodos , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Biespecíficos/uso terapêutico , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Antineoplásicos/uso terapêutico , Bioensaio/instrumentação , Antígeno CD47/genética , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Células CHO , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Separação Celular/instrumentação , Separação Celular/métodos , Cromatografia de Afinidade/instrumentação , Cromatografia de Afinidade/métodos , Cricetulus , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/patologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
20.
Front Immunol ; 9: 2728, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30534127

RESUMO

Checkpoint inhibitors target the inhibitory receptors expressed by tumor-infiltrating T cells in order to reinvigorate an anti-tumor immune response. Therefore, understanding T cell composition and phenotype in human tumors is crucial. We analyzed by flow cytometry tumor-infiltrating lymphocytes (TILs) from two independent cohorts of patients with different cancer types, including RCC, lung, and colon cancer. In healthy donors, peripheral T cells are usually either CD4+ or CD8+ with a small percentage of CD4+ CD8+ DP cells (<5%). Compared to several other cancer types, including lung, and colorectal cancers, TILs from about a third of RCC patients showed an increased proportion of DP CD4+CD8+ T cells (>5%, reaching 30-50% of T cells in some patients). These DP T cells have an effector memory phenotype and express CD38, 4-1BB, and HLA-DR, suggesting antigen-driven expansion. In fact, TCR sequencing analysis revealed a high degree of clonality in DP T cells. Additionally, there were high levels of PD-1 and TIM-3 expression on DP T cells, which correlated with higher expression of PD-1 and TIM-3 in conventional single positive CD8 T cells from the same patients. These results suggest that DP T cells could be dysfunctional tumor-specific T cells with the potential to be reactivated by checkpoint inhibitors.


Assuntos
Antígenos de Diferenciação/imunologia , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Carcinoma de Células Renais/imunologia , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Proteínas de Neoplasias/imunologia , Linfócitos T/imunologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Neoplasias Renais/patologia , Linfócitos do Interstício Tumoral/patologia , Masculino , Pessoa de Meia-Idade , Linfócitos T/patologia
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