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1.
PLoS One ; 16(11): e0258743, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34758029

RESUMO

BCG vaccination is known to induce innate immune memory, which confers protection against heterologous infections. However, the effect of BCG vaccination on the conventional adaptive immune cells subsets is not well characterized. We investigated the impact of BCG vaccination on the frequencies of T cell subsets and common gamma c (γc) cytokines in a group of healthy elderly individuals (age 60-80 years) at one month post vaccination as part of our clinical study to examine the effect of BCG on COVID-19. Our results demonstrate that BCG vaccination induced enhanced frequencies of central (p<0.0001) and effector memory (p<0.0001) CD4+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001), stem cell memory (p = 0.0001) CD4+ T cells and regulatory T cells. In addition, BCG vaccination induced enhanced frequencies of central (p = 0.0008), effector (p<0.0001) and terminal effector memory (p<0.0001) CD8+ T cells and diminished frequencies of naïve (p<0.0001), transitional memory (p<0.0001) and stem cell memory (p = 0.0034) CD8+T cells. BCG vaccination also induced enhanced plasma levels of IL-7 (p<0.0001) and IL-15 (p = 0.0020) but diminished levels of IL-2 (p = 0.0033) and IL-21 (p = 0.0020). Thus, BCG vaccination was associated with enhanced memory T cell subsets as well as memory enhancing γc cytokines in elderly individuals, suggesting its ability to induce non-specific adaptive immune responses.


Assuntos
Vacina BCG/imunologia , Citocinas/imunologia , Memória Imunológica/imunologia , Subunidade gama Comum de Receptores de Interleucina/imunologia , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Feminino , Humanos , Interleucinas/imunologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Vacinação/métodos
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(5): 1429-1435, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34627421

RESUMO

OBJECTIVE: To establish the in vivo traceable acute myeloid leukemia mice model with Luciferase-Expressing KG1a Cells. METHODS: KG1a cells with stable luciferase gene expression (called as KG1a-Luc cells) were constructed by lentivirus transfection, then sifted out by puromycin. Eighteen male NOD-SCID-IL2rg-/-mice aged 8 to 12 weeks were randomly and equally divided into two groups: the control group and the KG1a-Luc group. The mice in KG1a-Luc group were injected with 200 µl PBS containing 5×106 KG1a-Luc cells through tail veins, and the mice in control group were injected with 200 µl PBS only. The bioluminescence imaging technology was used to monitor the tumor burden in vivo. The peripheral blood of the mice in both groups was analyzed by flow cytometry. After the mice were sacrificed, there were pathologic evaluations: bone marrow and spleens made into smears, and livers sliced to get paraffin sections. The survival time of the mice in the two groups was recorded and compared. RESULTS: KG1a cells expressing luciferase stably were successfully obtained. The tumor luminescence wildly spread at day 17 captured by in vivo imaging. The KG1a-Luc tumor cells could be detected in the peripheral blood of the mice, with the average percentage of (16.27±6.66)%. The morphology and pathology result showed that KG1a-Luc cells infiltrate was detected in bone marrow, spleens and livers. The survival time of the KG1a-Luc mice was notably shorter as compared with those in the control group, the median survival time was 30.5 days (95%CI: 0.008-0.260). CONCLUSION: The acute myeloid leukemia NOD-SCID-IL2rg-/-mouse model was successfully established by tail vein injection of 5×106 KG1a-Luc cells.


Assuntos
Leucemia Mieloide Aguda , Animais , Modelos Animais de Doenças , Subunidade gama Comum de Receptores de Interleucina , Luciferases/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
3.
Oncoimmunology ; 10(1): 1981049, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34616589

RESUMO

Combining natural killer (NK) cell adoptive transfer with tumor-sensitizing chemotherapy is an attractive approach against recurrent ovarian cancer (OC), as OC is sensitive to NK cell-mediated immunity. Previously, we showed that CD34+ hematopoietic progenitor cell (HPC)-derived NK cells can kill OC cells in vitro and inhibit OC tumor growth in mice. Here, we investigated the potential of HPC-NK cell therapy combined with chemotherapeutic gemcitabine (used in recurrent OC patients) against OC. We examined the phenotypical, functional, and cytotoxic effects of gemcitabine on HPC-NK cells and/or OC cells in vitro and in OC-bearing mice. To this end, we treated OC cells and/or HPC-NK cells with or without gemcitabine and analyzed the phenotype, cytokine production, and anti-tumor reactivity. We found that gemcitabine did not affect the phenotype and functionality of HPC-NK cells, while on OC cells expression of NK cell activating ligands and death receptors was upregulated. Although gemcitabine pre-treatment of OC cells did not improve the functionality of HPC-NK cells, importantly, HPC-NK cells and gemcitabine additively killed OC cells in vitro. Similarly, combined HPC-NK cell and gemcitabine treatment additively decreased tumor growth in OC-bearing mice. Collectively, our results indicate that combination therapy of HPC-NK cells and gemcitabine results in augmented OC killing in vitro and in vivo. This provides a rationale for exploring this therapeutic strategy in patients with recurrent OC.


Assuntos
Recidiva Local de Neoplasia , Neoplasias Ovarianas , Animais , Desoxicitidina/análogos & derivados , Feminino , Homicídio , Humanos , Subunidade gama Comum de Receptores de Interleucina , Células Matadoras Naturais , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Ovarianas/tratamento farmacológico
4.
Cancer Sci ; 112(11): 4478-4489, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34545658

RESUMO

IL-2 is a pleiotropic cytokine that regulates immune cell homeostasis. Its immunomodulatory function has been used clinically as an active immunotherapy agent for metastatic cancers. However, severe adverse effects, including the vascular leak syndrome and the preferential stimulation of anti-immunogenic Treg rather than effector T cells, have been obstacles. We newly designed a mutein IL-2, Mutakine-6 (MK-6), with reduced IL-2Rα-binding capability. MK-6 induced comparable cell growth potential toward IL-2Rßγ-positive T cells but was far less efficient in in vitro Treg proliferation and STAT5 activation. Unlike IL-2, in vivo administration of MK-6 produced minimal adverse effects. Using CT26 and B16F10-syngeneic tumor models, we found MK-6 was highly efficacious on tumor regression. Serum albumin conjugation to MK-6 prolonged in vivo half-life and accumulated in CT26 tumors, showing enhanced antitumor effect. Tumor-infiltrating leukocytes analysis revealed that albumin-fused MK-6 increased the ratio of effector CD8+ T cells to CD4+ Treg cells. These results demonstrated that MK-6 is an efficient immunomodulator potentially used for improved immunotherapy with decreased adverse effects and attenuated Treg stimulation.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/citologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Meia-Vida , Imunidade Celular , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-2/efeitos adversos , Interleucina-2/metabolismo , Interleucina-2/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade beta de Receptor de Interleucina-2/metabolismo , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação , Fator de Transcrição STAT5/metabolismo , Albumina Sérica/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/fisiologia , Proteínas Supressoras de Tumor/metabolismo
5.
Sci Rep ; 11(1): 16951, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417497

RESUMO

T-cell activation and cellular expansion by common gamma chain cytokines such as Interleukin-2 is necessary for adaptive immunity. However, when unregulated these same pathways promote pathologies ranging from autoimmune disorders to cancer. While the functional role of Interleukin-2 and downstream effector molecules is relatively clear, the repertoire of phosphoregulatory proteins downstream of this pathway is incomplete. To identify phosphoproteins downstream of common gamma chain receptor, YT cells were radiolabeled with [32P]-orthophosphate and stimulated with Interleukin-2. Subsequently, tyrosine phosphorylated proteins were immunopurified and subjected to tandem mass spectrometry-leading to the identification of CrkL. Phosphoamino acid analysis revealed concurrent serine phosphorylation of CrkL and was later identified as S114 by mass spectrometry analysis. S114 was inducible through stimulation with Interleukin-2 or T-cell receptor stimulation. Polyclonal antibodies were generated against CrkL phospho-S114, and used to show its inducibility by multiple stimuli. These findings confirm CrkL as an Interleukin-2 responsive protein that becomes phosphorylated at S114 by a kinase/s downstream of PI3K and MEK/ERK signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/metabolismo , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-2/metabolismo , Fosfosserina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Anticorpos/metabolismo , Linhagem Celular , Humanos , Sistema de Sinalização das MAP Quinases , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Processamento de Proteína Pós-Traducional , Serina-Treonina Quinases TOR/metabolismo
6.
Front Immunol ; 12: 677970, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34248959

RESUMO

Objective: The contribution of sustained autologous autoantibody production by B cells to the pathogenesis of systemic sclerosis (SSc) and granulomatosis with polyangiitis (GPA) is not fully understood. To investigate this, a humanized mouse model was generated by transferring patient-derived peripheral blood mononuclear cells (PBMC) into immunocompromised mice. Methods: PBMC derived from patients with SSc and GPA as well as healthy controls (HD) were isolated, characterized by flow cytometry, and infused into Rag2-/-/IL2rg-/- mice. In addition, PBMC from SSc patients treated with rituximab were transferred into mice. Twelve weeks later, human autoantibodies were determined in blood of the recipient mice and affected tissues were analyzed for pathological changes by histology and immunohistochemistry. Results: Mice engrafted with PBMC derived from SSc patients developed autoantibodies such as antinuclear antibodies (ANA) mimicking the pattern of the respective donors. Moreover, cellular infiltrates dominated by B cells were observed in lung, kidney and muscles of the recipient mice. By contrast, PBMC derived from HD or GPA patients survived in recipient mice after transfer, but neither human autoantibodies nor inflammatory infiltrates in tissues were detected. Furthermore, these pathological changes were absent in mice transferred with PBMC from rituximab-treated SSc patients. Conclusion: This humanized mouse model is indicative for cross-reactivity of human lymphocytes to murine autoantigens and argues for a pivotal role of B cells as well as of sustained autoimmunity in the pathogenesis of SSc. It provides a powerful tool to study interstitial lung disease and so far, under-recognized disease manifestations such as myositis and interstitial nephritis.


Assuntos
Anticorpos Antinucleares/imunologia , Proteínas de Ligação a DNA/metabolismo , Granulomatose com Poliangiite/sangue , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/transplante , Escleroderma Sistêmico/sangue , Adulto , Idoso , Animais , Anticorpos Antinucleares/sangue , Linfócitos B/imunologia , Reações Cruzadas , Proteínas de Ligação a DNA/genética , Feminino , Granulomatose com Poliangiite/imunologia , Humanos , Hospedeiro Imunocomprometido , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Fatores Imunológicos/uso terapêutico , Inflamação/imunologia , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Modelos Animais , Rituximab/uso terapêutico , Escleroderma Sistêmico/tratamento farmacológico , Escleroderma Sistêmico/imunologia , Resultado do Tratamento , Adulto Jovem
7.
Science ; 372(6543)2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33986151

RESUMO

Reactive oxygen species (ROS) increase in activated T cells because of metabolic activity induced to support T cell proliferation and differentiation. We show that these ROS trigger an oxidative stress response that leads to translation repression. This response is countered by Schlafen 2 (SLFN2), which directly binds transfer RNAs (tRNAs) to protect them from cleavage by the ribonuclease angiogenin. T cell-specific SLFN2 deficiency results in the accumulation of tRNA fragments, which inhibit translation and promote stress-granule formation. Interleukin-2 receptor ß (IL-2Rß) and IL-2Rγ fail to be translationally up-regulated after T cell receptor stimulation, rendering SLFN2-deficient T cells insensitive to interleukin-2's mitogenic effects. SLFN2 confers resistance against the ROS-mediated translation-inhibitory effects of oxidative stress normally induced by T cell activation, permitting the robust protein synthesis necessary for T cell expansion and immunity.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Imunidade Celular , Estresse Oxidativo , RNA de Transferência/metabolismo , Linfócitos T/imunologia , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células , Feminino , Deleção de Genes , Infecções por Herpesviridae/imunologia , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-2/metabolismo , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Muromegalovirus , Ligação Proteica , Biossíntese de Proteínas , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/metabolismo , Transdução de Sinais
8.
Transplant Cell Ther ; 27(8): 658.e1-658.e10, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33964513

RESUMO

Graft-versus-host disease (GVHD) is one of the major complications following hematopoietic stem cell transplantation, which remains the sole curative therapy for many malignant diseases of the hematopoietic system. The immunomodulatory potential of mesenchymal stromal cells (MSCs) to treat GVHD is currently being tested in various preclinical and clinical trials. Because the results of the preclinical and clinical trials on the use of MSCs to treat GVHD have not been consistent, we analyzed the potential beneficial effects of syngeneic versus allogenic treatment, culture expansion of MSCs, and various MSC cell doses and time points of MSC transplantation in a murine GVHD model. We established the murine GVHD model based on the transplantation of umbilical cord blood-derived hematopoietic stem cells (UC-HSCs) and used this model to assess the therapeutic potential of umbilical cord blood-derived MSCs (UC-MSCs). The use of HSC and MSC populations derived from the same donor allowed us to exclude third-party cells and test the UC-HSCs and UC-MSCs in a matched setting. Moreover, we were able to compare various doses, transplantation time points, and the influence of culture expansion of MSCs on the impact of treatment. This resulted in 16 different treatment groups. The most efficient setting for treatment of UC-HSC-induced GVHD reactions was based on the simultaneous administration of 1 × 106 culture-expanded, syngeneically matched UC-MSCs. This therapy effectively reduced the number of CD8+ T cells in the blood, protected the mice from weight loss, and prolonged their survival until the end of observation period. Taken together, our data show beneficial effects of (1) syngeneic over allogeneic UC-HSCs and UC-MSCs, (2) culture-expanded cells over freshly isolated primary cells, (3) simultaneous over sequential administration, and (4) high doses of UC-MSCs. The animal model of GVHD established here is now available for more detailed studies, including a comparative analysis of the efficacy of MSCs derived from alternative sources, such as adipose tissue and bone marrow.


Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Linfócitos T CD8-Positivos , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA , Humanos , Subunidade gama Comum de Receptores de Interleucina , Camundongos , Camundongos Endogâmicos NOD , Cordão Umbilical
9.
Front Immunol ; 12: 644687, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33959125

RESUMO

Mutations of the interleukin 2 receptor γ chain (IL2RG) result in the most common form of severe combined immunodeficiency (SCID), which is characterized by severe and persistent infections starting in early life with an absence of T cells and natural killer cells, normal or elevated B cell counts and hypogammaglobulinemia. SCID is commonly fatal within the first year of life, unless the immune system is reconstituted by hematopoietic stem cell transplantation (HSCT) or gene therapy. We herein describe a male infant with X-linked severe combined immunodeficiency (X-SCID) diagnosed at 5 months of age. Genetic testing revealed a novel C to G missense mutation in exon 1 resulting in a 3' splice site disruption with premature stop codon and aberrant IL2 receptor signaling. Following the diagnosis of X-SCID, the patient subsequently underwent a TCRαß/CD19-depleted haploidentical HSCT. Post transplantation the patient presented with early CD8+ T cell recovery with the majority of T cells (>99%) being non-donor T cells. Genetic analysis of CD4+ and CD8+ T cells revealed a spontaneous 14 nucleotide insertion at the mutation site resulting in a novel splice site and restoring the reading frame although defective IL2RG function was still demonstrated. In conclusion, our findings describe a spontaneous second-site mutation in IL2RG as a novel cause of somatic mosaicism and early T cell recovery following haploidentical HSCT.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Transplante de Células-Tronco Hematopoéticas , Mutação , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Aloenxertos , Humanos , Lactente , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/imunologia , Masculino , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia
10.
Sci Rep ; 11(1): 10592, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011961

RESUMO

The use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαßγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rßγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule's in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Subunidade gama Comum de Receptores de Interleucina/agonistas , Subunidade beta de Receptor de Interleucina-2/agonistas , Linfócitos/efeitos dos fármacos , Animais , Células CHO , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Subunidade gama Comum de Receptores de Interleucina/imunologia , Subunidade beta de Receptor de Interleucina-2/imunologia , Macaca fascicularis , Masculino , Camundongos Endogâmicos BALB C
11.
PLoS One ; 16(4): e0249340, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33793647

RESUMO

Many human viruses, including Epstein-Barr virus (EBV), do not infect mice, which is challenging for biomedical research. We have previously reported that EBV infection induces erosive arthritis, which histologically resembles rheumatoid arthritis, in humanized NOD/Shi-scid/IL-2Rγnull (hu-NOG) mice; however, the underlying mechanisms are not known. Osteoclast-like multinucleated cells were observed during bone erosion in this mouse model, and therefore, we aimed to determine whether the human or mouse immune system activated bone erosion and analyzed the characteristics and origin of the multinucleated cells in hu-NOG mice. Sections of the mice knee joint tissues were immunostained with anti-human antibodies against certain osteoclast markers, including cathepsin K and matrix metalloproteinase-9 (MMP-9). Multinucleated cells observed during bone erosion stained positively for human cathepsin K and MMP-9. These results indicate that human osteoclasts primarily induce erosive arthritis during EBV infections. Human osteoclast development from hematopoietic stem cells transplanted in hu-NOG mice remains unclear. To confirm their differentiation potential into human osteoclasts, we cultured bone marrow cells of EBV-infected hu-NOG mice and analyzed their characteristics. Multinucleated cells cultured from the bone marrow cells stained positive for human cathepsin K and human MMP-9, indicating that bone marrow cells of hu-NOG mice could differentiate from human osteoclast progenitor cells into human osteoclasts. These results indicate that the human immune response to EBV infection may induce human osteoclast activation and cause erosive arthritis in this mouse model. Moreover, this study is the first, to our knowledge, to demonstrate human osteoclastogenesis in humanized mice. We consider that this model is useful for studying associations of EBV infections with rheumatoid arthritis and human bone metabolism.


Assuntos
Artrite/patologia , Diferenciação Celular , Herpesvirus Humano 4/fisiologia , Osteogênese , Animais , Artrite/metabolismo , Artrite/virologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/virologia , Catepsina K/imunologia , Catepsina K/metabolismo , Modelos Animais de Doenças , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/patologia , Metaloproteinase 9 da Matriz/imunologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Osteoclastos/citologia , Osteoclastos/metabolismo , Microtomografia por Raio-X
12.
Sci Rep ; 11(1): 7584, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33828203

RESUMO

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients' anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.


Assuntos
Adenocarcinoma/terapia , Eletroporação/métodos , Neoplasias Pancreáticas/terapia , Adenocarcinoma/patologia , Adenocarcinoma/fisiopatologia , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Condutividade Elétrica , Feminino , Técnicas de Inativação de Genes , Humanos , Hospedeiro Imunocomprometido , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/imunologia , Masculino , Camundongos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Estudo de Prova de Conceito , Suínos , Pesquisa Médica Translacional , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Chem Inf Model ; 61(4): 1913-1920, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33765385

RESUMO

Interleukin (IL) 2 and IL15 are two members of the common gamma chain cytokine family, involved in the regulation of the T cell differentiation process. Both molecules use a specific alpha subunit, IL2Rα and IL15Rα, and share the same beta and gamma chains signaling receptors. The presence of the specific alpha subunit modulates the T cell ability to compete for both soluble cytokines while the beta and gamma subunits are responsible for the signal transduction. Recent experimental results point out that the specific alpha subunits modulate the capacity of IL2 and IL15 to induce the differentiation of stimulated T cells. In other membrane receptors, the outcome of the signal transduction has been associated with the strength of the interaction of the signaling subunits. Here, we investigate how IL2Rα and IL15Rα modulate the stability of their signaling complexes by combining molecular dynamics simulations and free energy calculations. Our simulations predict that IL2Rα binding destabilizes the ß-γc interaction mediated by IL2, while IL15Rα has the opposite effect. These results explain the ability of IL2Rα and IL15Rα to modulate the signaling outcome and suggest new strategies for the development of better CD8+ T cell differentiation protocols for adoptive cell transfer (ACT).


Assuntos
Subunidade alfa de Receptor de Interleucina-15 , Interleucina-2 , Subunidade gama Comum de Receptores de Interleucina/genética , Interleucina-15 , Subunidade alfa de Receptor de Interleucina-2 , Transdução de Sinais
14.
Int J Radiat Oncol Biol Phys ; 110(5): 1341-1349, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33647370

RESUMO

Outcomes for triple negative breast cancer (TNBC) are poor and may be improved by increasing CD8+ tumor infiltrating lymphocytes (TIL) to augment antitumor immunity. Radiation (RT) can promote immunogenic cell death with increased antitumor T cell activity but also stimulates suppressive regulatory T cells (Tregs). Because metabolic alterations affect immune homeostasis and prior studies show caloric restriction (CR) combined with RT improves preclinical TNBC outcomes, we hypothesized that CR augments RT, in part, by altering intratumoral immunity. Using an in vivo model of TNBC, we treated mice with ad libitum (AL) diet, radiation, a CR diet, or CR + RT, and demonstrated an immune suppressive environment with a significant increase in CD4+ CD25+Foxp3+ Tregs after RT but not in CR-fed mice. CD8:Treg ratio in CR + RT TIL increased 4-fold compared with AL + RT mice. In vivo CD8 depletion was performed to assess the role of effector T cells in mitigating the effects of CR, and it was found that in mice undergoing CR, depletion of CD8 T cells resulted in increased tumor progression and decreased median survival compared with isotype control-treated mice. In addition, PD-1 expression on CD3+CD8+ T cells within the tumor microenvironment was significantly increased in CR + RT versus AL + RT treated mice as per immunofluorescence. Serum from breast cancer patients undergoing RT alone or CR and RT was collected pre- and postintervention, and a cytokine array demonstrated that patients treated with CR + RT had notable decreases in immunosuppressive cytokines such as IL-2Rγ, IL-10Rß, and TGF-ß2 and 3 compared with patients receiving RT alone. In conclusion, combining CR with RT decreases intratumoral Tregs, increases CD8:Treg, and increases PD-1 expression via a process dependent on CD8 T cells in a TNBC model. Breast cancer patients undergoing CR concurrently with RT also had significant reduction in immunosuppressive cytokine levels compared with those receiving RT alone.


Assuntos
Restrição Calórica , Linfócitos do Interstício Tumoral/efeitos da radiação , Linfócitos T Reguladores/efeitos da radiação , Neoplasias de Mama Triplo Negativas/radioterapia , Microambiente Tumoral/efeitos da radiação , Adulto , Idoso , Animais , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/efeitos da radiação , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos da radiação , Terapia Combinada/métodos , Progressão da Doença , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead , Humanos , Subunidade gama Comum de Receptores de Interleucina/sangue , Subunidade beta de Receptor de Interleucina-10/sangue , Subunidade alfa de Receptor de Interleucina-2 , Depleção Linfocítica/métodos , Linfócitos do Interstício Tumoral/citologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Distribuição Aleatória , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta2/sangue , Fator de Crescimento Transformador beta3/sangue , Neoplasias de Mama Triplo Negativas/sangue , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/mortalidade , Microambiente Tumoral/imunologia
15.
PLoS One ; 16(3): e0248352, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33750947

RESUMO

Hepatocellular carcinoma (HCC) is the fifth most common primary tumor and the third leading cause of cancer-related deaths worldwide. Rodent models of HCC have contributed to the advancement of studies investigating liver carcinogenesis, tumor-host interactions, and drug screening. However, their small size renders them unsuitable for surgical or clinical imaging studies, necessitating the development of larger-size HCC models. Here, we developed a xenograft model of human HCC in X-linked interleukin-2 receptor gamma chain gene (Il2rg)-targeted severe combined immunodeficient (SCID) pigs. HepG2 cell suspension in serum-free medium containing 50% membrane matrix was directly injected into the liver parenchyma of eight X-linked Il2rg-targeted SCID pigs (6.6-15.6 kg) via ultrasonography-guided percutaneous puncture. Tumor engraftment was evaluated weekly using ultrasonography, and cone-beam computed tomography was performed during arterial portography (CTAP) and hepatic arteriography (CTHA) to evaluate the hemodynamics of engrafted tumors. The engrafted tumors were histologically analyzed following necropsy and assessed for pathological similarities to human HCCs. Macroscopic tumor formation was observed in seven of the eight pigs (simple nodular tumors in three and multinodular tumors in four). Engrafted tumors were identified as low-echoic upon ultrasonography and as perfusion-defect nodules on the CTAP images. Meanwhile, CTHA showed that the tumors were hyperattenuating. Further, histopathological findings of the engrafted tumors were consistent with those of human HCC. In conclusion, the porcine model of human HCC, successfully generated herein, might help develop more effective therapeutic strategies for HCC.


Assuntos
Carcinoma Hepatocelular/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Neoplasias Hepáticas/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Animais , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/patologia , Meios de Contraste/farmacologia , Modelos Animais de Doenças , Células Hep G2 , Xenoenxertos , Humanos , Fígado/diagnóstico por imagem , Fígado/patologia , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/patologia , Suínos/genética , Tomografia Computadorizada por Raios X , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/diagnóstico por imagem , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/patologia
16.
Cancer Res ; 81(9): 2510-2521, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33637565

RESUMO

Prostate cancer is one of the most common malignancies worldwide, yet limited tools exist for prognostic risk stratification of the disease. Identification of new biomarkers representing intrinsic features of malignant transformation and development of prognostic imaging technologies are critical for improving treatment decisions and patient survival. In this study, we analyzed radical prostatectomy specimens from 422 patients with localized disease to define the expression pattern of methionine aminopeptidase II (MetAP2), a cytosolic metalloprotease that has been identified as a druggable target in cancer. MetAP2 was highly expressed in 54% of low-grade and 59% of high-grade cancers. Elevated levels of MetAP2 at diagnosis were associated with shorter time to recurrence. Controlled self-assembly of a synthetic small molecule enabled design of the first MetAP2-activated PET imaging tracer for monitoring MetAP2 activity in vivo. The nanoparticles assembled upon MetAP2 activation were imaged in single prostate cancer cells with post-click fluorescence labeling. The fluorine-18-labeled tracers successfully differentiated MetAP2 activity in both MetAP2-knockdown and inhibitor-treated human prostate cancer xenografts by micro-PET/CT scanning. This highly sensitive imaging technology may provide a new tool for noninvasive early-risk stratification of prostate cancer and monitoring the therapeutic effect of MetAP2 inhibitors as anticancer drugs. SIGNIFICANCE: This study defines MetAP2 as an early-risk stratifier for molecular imaging of aggressive prostate cancer and describes a MetAP2-activated self-assembly small-molecule PET tracer for imaging MetAP2 activity in vivo.


Assuntos
Metionil Aminopeptidases/metabolismo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/enzimologia , Transdução de Sinais/genética , Animais , Antibióticos Antineoplásicos/administração & dosagem , Seguimentos , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Metionil Aminopeptidases/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , O-(Cloroacetilcarbamoil)fumagilol/administração & dosagem , Células PC-3 , Neoplasias da Próstata/patologia , Medição de Risco/métodos , Distribuição Tecidual , Transfecção , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Am Assoc Lab Anim Sci ; 60(1): 4-17, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33046180

RESUMO

Highly immunodeficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) are commonly used as a models in preclinical studies for patient-derived engraftment. However, despite the frequency of their use, reference values for their clinical pathology markers have not been determined. In accordance with the American Society of Veterinary Clinical Pathology (ASVCP) recommendations, we established de novo reference values for hematologic and biochemical variables and evaluated bone marrow cytology and histology in forty 9-wk-old male and female NSG mice. Hematologic analyses were performed using 2 separate analyzers (IDEXX ProCyte Dx, Sysmex XT-2000iV) and biochemical values were measured using a Scil VetScan2. The primary hematologic characteristic seen in NSG mice was a very low white blood cell (WBC) count (below 1.6 109/L). Lymphocyte and monocyte counts were respectively over- and under-estimated by the analyzers, as compared with manual counts, likely due to misidentification of the very low concentrations of these cell types by the analyzers. This analytical bias highlights the need for confirmatory microscopic observation of blood smears from these mice for WBC differential identification. Results for all other hematology and biochemistry variables were similar to those previously reported in inbred mice, except for MPV and an unexpectedly high glucose concentration (11.5 to 19.0 mmol/L), potentially due to the nonfasting status of the animals. The differential bone marrow cell count and Myeloid:Erythroid ratio (median 1.76) were also established. Megakaryocyte and adipocyte count differed significantly between the femoral diaphysis and metaphysis and between genders. These results provide a reliable resource of baseline data for hematologic variables for researchers monitoring graft rejection studies in NSG mice.


Assuntos
Medula Óssea , Hematologia , Animais , Proteína Quinase Ativada por DNA , Proteínas de Ligação a DNA , Feminino , Humanos , Subunidade gama Comum de Receptores de Interleucina , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Valores de Referência
18.
Clin Exp Immunol ; 203(3): 448-457, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33040328

RESUMO

Severe combined immunodeficiency (SCID) is fatal if not treated with immune reconstitution. In Egypt, T- B+ SCID accounts for 38·5% of SCID diagnoses. An accurate genetic diagnosis is essential for choosing appropriate treatment modalities and for offering genetic counseling to the patient's family. The objectives of this study were to describe the clinical, immunological and molecular characteristics of a cohort of twenty Egyptian patients with T- B+ SCID. The initial diagnosis (based on clinical features and flow cytometry) was followed by molecular investigation (whole-exome sequencing). All patients had the classic clinical picture for SCID, including failure to thrive (n = 20), oral candidiasis (n = 17), persistent diarrhea (n = 14), pneumonia (n = 13), napkin dermatitis (n = 10), skin rash (n = 7), otitis media (n = 3) and meningitis (n = 2). The onset of manifestations was at the age of 2·4 ± 1·6 months and diagnosis at the age of 6·7 ± ·5 months, giving a diagnostic delay of 4·3 months. JAK3 gene variants were most frequent (n = 12) with three novel variants identified, followed by IL2Rγ variants (n = 6) with two novel variants. IL7Rα and CD3ε variants were found once, with a novel variant each. T- B+ NK- SCID accounted for approximately 90% of the Egyptian patients with T- B+ SCID. Of these T- B+ NK- SCID cases, 60% were autosomal recessive syndromes caused by JAK3 mutations and 30% were X-linked syndromes. It might be useful to sequence the JAK3 gene (i.e. targeted Sanger sequencing) in all T- B+ SCID patients, especially after X-linked SCID has been ruled out. Hence, no more than 10% of T- B+ SCID patients might require next-generation for a molecular diagnosis.


Assuntos
Janus Quinase 3/genética , Mutação , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/imunologia , Sequenciamento Completo do Exoma/métodos , Consanguinidade , Egito , Saúde da Família , Feminino , Humanos , Lactente , Recém-Nascido , Subunidade gama Comum de Receptores de Interleucina/genética , Janus Quinase 3/deficiência , Contagem de Linfócitos , Masculino , Linhagem , Imunodeficiência Combinada Severa/patologia , Linfócitos T/metabolismo
19.
Dent Mater J ; 40(1): 209-219, 2021 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-33162457

RESUMO

The effect of various pretreatments on the bonding of a resin cement to resin-composite CAD/CAM blocks (RCBs) was examined. The surface of dispersed-filler RCBs (DF-RCBs) and a polymer infiltrated ceramic network RCB (PICN-RCB) was roughened using hydrofluoric acid etching (HF) or sandblasting, and followed by silanization and/or universal adhesive (UA) application. Microtensile bond strength (µTBS), surface roughness parameters (arithmetical mean height (Sa); developed interfacial area ratio (Sdr)), and critical surface energy (γc) were determined. For most DF-RCBs, the highest µTBS was obtained using HF+UA. UA application to DF-RCBs resulted in similar or higher µTBS compared to silanization, which indicates that silane treatment is not crucial for DF-RCBs, especially after HF. In contrast, the highest µTBS to PICN-RCB was obtained with silanization. Both roughening pretreatments significantly increased the surface roughness parameters and the γc of all RCBs. The γc was positively correlated with Sa (r=0.756, p<0.001) and Sdr (r=0.837, p<0.001).


Assuntos
Colagem Dentária , Cerâmica , Protocolos Clínicos , Resinas Compostas , Ácido Fluorídrico , Subunidade gama Comum de Receptores de Interleucina , Teste de Materiais , Cimentos de Resina , Silanos , Propriedades de Superfície , Resistência à Tração
20.
Res Vet Sci ; 134: 137-146, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33383491

RESUMO

"Humanized" immunodeficient mice generated via the transplantation of CD34+ human hematopoietic stem cells (hHSC) are an important preclinical model system. The triple transgenic NOD.Cg-PrkdcscidIl2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSGS) mouse line is increasingly used as recipient for CD34+ hHSC engraftment. NSGS mice combine the features of the highly immunodeficient NSG mice with transgenic expression of the human myeloid stimulatory cytokines GM-CSF, IL-3, and Kit ligand. While generating humanized NSGS (huNSGS) mice from two independent cohorts, we encountered a fatal macrophage activation syndrome (MAS)-like phenotype resulting from the transplantation of CD34+ hHSC. huNSGS mice exhibiting this phenotype declined clinically starting at approximately 10 weeks following CD34+ hHSC engraftment, with all mice requiring euthanasia by 16 weeks. Gross changes comprised small, irregular liver, splenomegaly, cardiomegaly, and generalized pallor. Hematological abnormalities included severe thrombocytopenia and anemia. Pathologically, huNSGS spontaneously developed a disseminated histiocytosis with infiltrates of activated macrophages and hemophagocytosis predominantly affecting the liver, spleen, bone marrow, and pancreas. The infiltrates were chimeric with a mixture of human and mouse macrophages. Immunohistochemistry suggested activation of the inflammasome in both human and murine macrophages. Active Epstein-Barr virus infection was not a feature. Although the affected mice exhibited robust chimerism of the spleen and bone marrow, the phenotype often developed in the face of low chimerism of the peripheral blood. Given the high penetrance and early lethality associated with the MAS-like phenotype here described, we urge caution when considering the use of huNSGS mice for the development of long-term studies.


Assuntos
Síndrome de Ativação Macrofágica/patologia , Macrófagos/microbiologia , Animais , Antígenos CD34 , Proteína Quinase Ativada por DNA/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Herpesvirus Humano 4 , Histiocitose/imunologia , Humanos , Subunidade gama Comum de Receptores de Interleucina/imunologia , Síndrome de Ativação Macrofágica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Recombinantes/imunologia , Fator de Células-Tronco/imunologia
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