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2.
Blood Adv ; 4(22): 5755-5761, 2020 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-33216889

RESUMO

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell (HSC) disorder characterized by defective synthesis of the glycosylphosphatidylinositol (GPI) anchors as a result of somatic mutations in the X-linked PIGA gene. The disease is acquired. No constitutional PNH has been described. Here, we report familial PNH associated with unusual inflammatory symptoms. Genetic analysis revealed a germline heterozygous PIGB mutation on chromosome 15 without mutations in PIGA or any of the other genes involved in GPI biosynthesis. In vitro data confirmed that transfection of the mutant PIGB could not restore the surface expression of GPI-anchored proteins (APs) in PIGB-deficient Chinese hamster ovary cells. Homozygosity was caused by copy number-neutral loss of heterozygosity (CN-LOH) of the germline PIGB mutation, leading to deficient expression of GPI-APs in the affected blood cells of the index patient and her mother. The somatic event leading to homozygosity of the germline mutant PIGB gene involved a 70-kbp microdeletion of chromosome 15q containing the TM2D3 and TARSL2 genes, which was implicated in chromosome 15q mosaicism. Interestingly, we detected the deletion in both the patient and her mother. A sister of the mother, who carried the same germline PIGB mutation but without this microdeletion involving TM2D3 and TARSL2, did not have a PNH clone or CN-LOH. In conclusion, we describe PNH caused by CN-LOH of a germline heterozygous PIGB mutation in a patient and her mother and hypothesize that the 70-kbp microdeletion may have contributed to the PNH clone in both.

3.
Gan To Kagaku Ryoho ; 47(1): 6-10, 2020 Jan.
Artigo em Japonês | MEDLINE | ID: mdl-32381853

RESUMO

Reprogramming of glucose metabolism in tumor cells is referred to as the Warburg effect. The Warburg effect is caused by tumor cells not only to adapt their metabolism to the demand for and limited supply of oxygen but also to obtain large amounts of nucleotides, amino acids and lipids for excessive proliferation of tumor cells. The Warburg effect results in increased production of lactic acid, as the final product of glycolysis, in the tumor microenvironment. Lactic acid secreted by tumor cells functions as an immunosuppressive mediator and converts macrophages into M2 macrophages. M2 macrophages reduce inflammatory responses and adaptive Th1 immunity, and promote angiogenesis and tissue remodeling. Tumor-associated macrophages(TAMs)polarize into the M2 phenotype and suppress the host anti-cancer immune response, leading to tumor progression. We have demonstrated that tumor-secreted lactic acid is linked to the induction of M2-macrophage polarization in human head and neck squamous cell carcinoma(HNSCC). FDG, which is a glucose analog, uptake measured by positron emission tomography/computed tomography(PET/CT)indicates the Warburg effect in tumor tissue. M2-macrophage polarization is promoted in HNSCC with increased glucose uptake, maximum standardized uptake value(SUVmax), mean SUV(SUVmean). Tumor cells mediate an immunosuppressive microenvironment via inducing M2-macrophage polarization by reprogramming of glucose metabolism, called Warburg effect.


Assuntos
Neoplasias de Cabeça e Pescoço , Humanos , Macrófagos , Neovascularização Patológica , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Microambiente Tumoral
4.
Auris Nasus Larynx ; 47(4): 658-667, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32089351

RESUMO

OBJECTIVE: Cancer cells secrete large amounts of lactic acid via aerobic glycolysis. We have shown that lactic acid plays an important role as a proinflammatory and immunosuppressive mediator and promotes tumor progression. Fluorine-18 fluorodeoxyglucose (FDG) uptake detected by positron emission tomography/computed tomography (PET/CT) is considered as a good indicator of aerobic glycolysis in cancer. In this study, we examined the relationships between systemic inflammatory parameters and FDG-PET/CT parameters in advanced head and neck squamous cell carcinoma (HNSCC). Furthermore, we investigated the relationships between FDG-PET/CT parameters and M2-macrophage polarization in HNSCC by assessing the ratio of CD163, a M2-macrophage marker, to CD68, a pan-macrophage marker. METHODS: This study included 73 advanced HNSCC patients. We assessed the C-reactive protein (CRP) level, white blood cell (WBC) count, neutrophil count, lymphocyte count, and monocyte count as systemic inflammatory markers. Additionally, we assessed the maximum standardized uptake value (SUVmax), mean SUV (SUVmean), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) as FDG-PET/CT parameters. RESULTS: The CRP level, WBC count, and neutrophil count were correlated with whole-body FDG-PET/CT parameters. The CD163/CD68 ratio was correlated with SUVmax and SUVmean. Our results suggest that systemic inflammation, which is associated with neutrophils, develops in patients with HNSCC having tumors with a larger volume and increased glucose uptake and that M2-macrophage polarization is promoted in HNSCC with increased glucose uptake, SUVmax, and SUVmean. FDG-PET/CT has the potential to reflect cancer-related chronic inflammation and immunosuppressive conditions in cancer patients. CONCLUSIONS: FDG-PET/CT parameters appear to be useful in assessing the immune status in HNSCC.

5.
Intern Med ; 59(1): 93-99, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31902910

RESUMO

Atypical hemolytic uremic syndrome (aHUS) is an extremely rare condition caused by an excessive activation of the complement pathway based on genetic or acquired dysfunctions in complement regulation, leading to thrombotic microangiopathy (TMA). A complement-amplifying condition (CAC) can trigger aHUS occurrence along with complement abnormality. We herein report a case of severe TMA after laparoscopic myomectomy in a healthy woman. This case was eventually diagnosed as complement-mediated TMA secondary to surgical invasive stress as a CAC, with no definitive diagnosis of aHUS despite a genetic test. The patient fully recovered after several eculizumab administrations.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Laparoscopia/efeitos adversos , Hemorragia Pós-Operatória/complicações , Microangiopatias Trombóticas/tratamento farmacológico , Miomectomia Uterina/efeitos adversos , Adulto , Inativadores do Complemento/uso terapêutico , Feminino , Humanos , Doenças Raras , Microangiopatias Trombóticas/diagnóstico , Microangiopatias Trombóticas/etiologia
6.
ACS Chem Biol ; 15(2): 360-368, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31841301

RESUMO

Molecular-targeting peptides and mini-proteins are promising alternatives to antibodies in a wide range of applications in bioscience and medicine. We have developed a helix-loop-helix (HLH) peptide as an alternative to antibodies to inhibit specific protein interactions. Cytotoxic T lymphocyte antigen-4 (CTLA-4) downregulates immune responses of cytotoxic T-cells by interaction with B7-1, a co-stimulatory molecule expressed on antigen presenting cells (APCs). To induce immune stimulatory activity, we used directed evolution methods to generate a HLH peptide that binds to CTLA-4, inhibiting the CTLA-4-B7-1 interaction and inducing immune stimulatory activity. Yeast-displayed libraries of HLH peptides were constructed and screened against CTLA-4 and identified the binding peptide Y-2, which exhibits a moderate affinity. The affinity of Y-2 was improved by in vitro affinity maturation to afford a stronger binder, ERY2-4. Peptide ERY2-4 specifically bound to CTLA-4 with a KD of 196.8 ± 2.3 nM, comparable to the affinity of the CTLA-4-B7-1 interaction. Furthermore, ERY2-4 inhibited the CTLA-4-B7-1 interaction with an IC50 of 1.1 ± 0.03 µM and blocked the interaction between CTLA-4 and dendritic cells (DCs) presenting B7 on their surface. Importantly, ERY2-4 showed no cross-reactivity against CD28, suggesting it does not suppress T-cell activation. Finally, in a mixed lymphocyte reaction assay with DCs and T cells, ERY2-4 enhanced an allogeneic lymphocyte response. Since CTLA-4 is a critical immune checkpoint for restricting the cancer immune response, this inhibitory HLH peptide represents a new class of drug candidates for immunotherapy.

7.
J Clin Invest ; 129(12): 5123-5136, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31430258

RESUMO

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a clonal population of blood cells deficient in glycosylphosphatidylinositol-anchored (GPI-anchored) proteins, resulting from a mutation in the X-linked gene PIGA. Here we report on a set of patients in whom PNH results instead from biallelic mutation of PIGT on chromosome 20. These PIGT-PNH patients have clinically typical PNH, but they have in addition prominent autoinflammatory features, including recurrent attacks of aseptic meningitis. In all these patients we find a germ-line point mutation in one PIGT allele, whereas the other PIGT allele is removed by somatic deletion of a 20q region comprising maternally imprinted genes implicated in myeloproliferative syndromes. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. From studies of patients' leukocytes and of PIGT-KO THP-1 cells we show that, through increased IL-1ß secretion, activation of the lectin pathway of complement and generation of C5b-9 complexes, free GPI is the agent of autoinflammation. Eculizumab treatment abrogates not only intravascular hemolysis, but also autoinflammation. Thus, PIGT-PNH differs from PIGA-PNH both in the mechanism of clonal expansion and in clinical manifestations.


Assuntos
Proteínas do Sistema Complemento/imunologia , Hemoglobinúria Paroxística/imunologia , Inflamassomos/imunologia , Inflamação/imunologia , Proteínas de Membrana/genética , Idoso , Alelos , Anticorpos Monoclonais Humanizados/uso terapêutico , Feminino , Deleção de Genes , Genes Ligados ao Cromossomo X , Alemanha , Glicosilfosfatidilinositóis/metabolismo , Hemólise/efeitos dos fármacos , Humanos , Japão , Leucócitos/imunologia , Masculino , Pessoa de Meia-Idade , Mutação , Mutação Puntual , Células THP-1
8.
PLoS One ; 14(6): e0219065, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31247017

RESUMO

BACKGROUND: Few randomized controlled trials have assessed the effects of laughter therapy on health-related quality of life (QOL) in cancer patients. This study aimed to evaluate these effects as an exploratory endpoint in cancer patients as part of a randomized controlled trial conducted at a single institution in Japan. METHODS: The Initiative On Smile And CAncer (iOSACA) study was an open-label randomized controlled trial conducted in 2017 in which participants aged 40-64 years with cancer were randomly assigned to either an intervention group (laughter therapy) or control group (no laughter therapy). Each participant in the intervention group underwent a laughter therapy session once every two weeks for seven weeks (total of four sessions). Each session involved a laughter yoga routine followed by Rakugo or Manzai traditional Japanese verbal comedy performances. We assessed QOL as a secondary endpoint in this intention-to-treat population using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ-C30). The questionnaire was completed at baseline (Week 0) and at Weeks 3 and 7. Mixed-effects models for repeated measures were developed to compare time-dependent changes in each QOL domain from baseline between the intervention and control groups. RESULTS: Four participants retracted consent and one participant was retrospectively excluded from analysis due to unmet inclusion criteria. The analysis was conducted using 56 participants, with 26 in the intervention group and 30 in the control group. Questionnaire completion rates were high (>90%), with similar QOL scores reported at baseline in both groups. The mixed-effects models showed that the intervention group had significantly better cognitive function and less pain than the control group for a short period. CONCLUSION: Laughter therapy may represent a beneficial, noninvasive complementary intervention in the clinical setting. Further studies are needed to verify the hypotheses generated from this exploratory study.


Assuntos
Terapia do Riso , Neoplasias/terapia , Adulto , Cognição , Estudos Cross-Over , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/fisiopatologia , Neoplasias/psicologia , Medição da Dor , Estudos Prospectivos , Qualidade de Vida , Inquéritos e Questionários
9.
Brain Nerve ; 71(6): 555-564, 2019 Jun.
Artigo em Japonês | MEDLINE | ID: mdl-31171752

RESUMO

The complement was named after "a complement", a protein molecule that supports an antibody. It was considered previously that the complement mainly participates in protecting against microbial infections. But later, as research on biological functions in complement activation advanced drastically, it was elucidated that the complement could be involved in the onset of various diseases. In 2007, eculizumab (ECZ), an anti-C5 (complement factor 5) monoclonal antibody, was approved as a drug for paroxysmal nocturnal hemoglobinuria (PNH) in the United States. In Japan, ECZ was approved for PNH and atypical hemolytic uremic syndrome (aHUS) in 2010 and 2013, respectively. The success of ECZ created an opportunity for drug companies to develop new therapeutics targeting the complement system; development of complement therapeutics is now a major venture of pharmaceutical companies worldwide. Here, I will provide an outline of the approved complement therapeutics and those that are in development and clinical trial phase currently.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Inativadores do Complemento/uso terapêutico , Proteínas do Sistema Complemento , Hemoglobinúria Paroxística/tratamento farmacológico , Ensaios Clínicos como Assunto , Complemento C5/antagonistas & inibidores , Humanos , Japão
10.
Clin Exp Nephrol ; 23(1): 65-75, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29959568

RESUMO

BACKGROUND: Eculizumab has been available for the treatment of atypical hemolytic-uremic syndrome (aHUS) in Japan since 2013. To assess safety and effectiveness of eculizumab in adult aHUS patients in the real-life setting, we performed interim analysis of a post-marketing surveillance mandated by Japanese regulations. METHODS: This study enrolled any patient who was diagnosed with TMA excluding Shiga toxin-producing Escherichia coli-HUS or thrombotic thrombocytopenic purpura based on Japanese clinical guide published in 2013 as inclusion criteria and treated with eculizumab. Although the term aHUS was redefined to denote only complement-mediated HUS in the guide revised in 2016, the patients with TMA caused by other causes (secondary TMA) were included. Patient outcomes and safety were evaluated at 6 months, 12 months, and annually thereafter. RESULTS: Thirty-three patients with aHUS and 27 patients with secondary TMA were enrolled. Median treatment duration of aHUS was 24weeks. Complement genes variants were detected in 11 of 18 patients with aHUS (61.1%). Among the 29 aHUS patients with available baseline data, platelet count (PLT), lactic dehydrogenase and serum creatinine (SCr) improved within 1-month after eculizumab initiation. TMA event-free status, complete TMA response, PLT normalization, and SCr decrease were achieved in 67.9% (19/28), 27.8% (5/18), 56.5% (13/23), and 57.1% (16/28) of patients, respectively. Thirty-three and 11 adverse reactions were observed in patients with aHUS (13/33 patients) and secondary TMA (6/27 patients), respectively. CONCLUSIONS: This interim analysis confirmed the acceptable safety profile and effectiveness of eculizumab for Japanese adult aHUS patients in real-world settings.


Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/uso terapêutico , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Inativadores do Complemento/efeitos adversos , Inativadores do Complemento/uso terapêutico , Adulto , Idoso , Síndrome Hemolítico-Urêmica Atípica/genética , Complemento C5/antagonistas & inibidores , Proteínas do Sistema Complemento/genética , Feminino , Variação Genética/genética , Humanos , Japão , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , Segurança do Paciente , Vigilância de Produtos Comercializados , Resultado do Tratamento , Adulto Jovem
11.
Clin Exp Nephrol ; 23(1): 112-121, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30039480

RESUMO

BACKGROUND: In 2013, eculizumab was approved for treatment of the atypical hemolytic-uremic syndrome (aHUS) in Japan, which was defined as a thrombotic microangiopathy (TMA) excluding Shiga toxin-producing Escherichia coli-HUS and thrombotic thrombocytopenic purpura. Simultaneously, post-marketing surveillance was started to assess its safety and effectiveness. In 2016, Japanese clinical guide redefined terms to limit the use of "aHUS" to complement-mediated HUS only. Accordingly, TMA with other causes was defined as secondary TMA. Here we report the interim analysis of post-marketing surveillance of pediatric patients with aHUS and secondary TMA. METHODS: Pediatric patients treated with eculizumab from approval to 15 March 2017 were included in this observational real-world study. Clinical endpoints of effectiveness were TMA event-free status, complete TMA response, platelet count normalization, and improvement of estimated glomerular filtration rate (eGFR). Adverse reactions to eculizumab were also analyzed. RESULTS: In 27 pediatric patients with aHUS, median age at diagnosis was 4 years. Complement genes' variants were detected in 14 of 21 patients (66.7%). Median time from diagnosis to eculizumab initiation was 2.0 days. TMA event-free status, complete TMA response, platelet normalization, and improvement in eGFR were achieved in 85.2, 36.4, 78.3, and 75.0% of patients, respectively. Three patients with aHUS died. Twenty-four and 10 adverse reactions were reported in 31 aHUS patients and 17 secondary TMA patients, respectively; however, no eculizumab-related death or meningococcal infection was reported. CONCLUSIONS: This interim analysis confirmed that eculizumab is well-tolerated and effective for Japanese pediatric patients with aHUS in a real-world setting.


Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/uso terapêutico , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Inativadores do Complemento/efeitos adversos , Inativadores do Complemento/uso terapêutico , Adolescente , Síndrome Hemolítico-Urêmica Atípica/genética , Criança , Pré-Escolar , Complemento C5/antagonistas & inibidores , Proteínas do Sistema Complemento/genética , Feminino , Variação Genética/genética , Taxa de Filtração Glomerular , Humanos , Lactente , Recém-Nascido , Japão , Testes de Função Renal , Masculino , Segurança do Paciente , Contagem de Plaquetas , Vigilância de Produtos Comercializados , Resultado do Tratamento
12.
Cancer Sci ; 109(5): 1319-1329, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29575556

RESUMO

For a successful tumor vaccine, it is necessary to develop effective immuno-adjuvants and identify specific tumor antigens. Tumor cells obtained from surgical or biopsy tissues are a good source of tumor antigens but, unlike bacteria, they do not induce strong immune responses. Here, we designed 2 novel lipopeptides that coat tumor cell surfaces and mimic bacterial components. Tumor cells coated with these lipopeptides (called bacteria-mimicking tumor cells [BMTC]) were prepared and their efficacy as a tumor vaccine examined. Natural bacterial lipopeptides act as ligands for toll-like receptor 2 (TLR2) and activate dendritic cells (DC). To increase the affinity of the developed lipopeptides for the negatively charged plasma membrane, a cationic polypeptide was connected to Pam2Cys (P2C), which is the basic structure of the TLR2 ligand. This increased the non-specific binding affinity of the peptides for the cell surface. Two such lipopeptides, P2CSK11 (containing 1 serine and 11 lysine residues) and P2CSR11 (containing 1 serine and 11 arginine residues) bound to irradiated tumor cells via the long cationic polypeptides more efficiently than the natural lipopeptide MALP2 (P2C-GNNDESNISFKEK) or a synthetic lipopeptide P2CSK4 (a short cationic polypeptide containing 1 serine and 4 lysines). BMTC coated with P2CSR11 or P2CSK11 were efficiently phagocytosed by DC and induced antigen cross-presentation in vitro. They also induced effective tumor-specific cytotoxic T cell responses and inhibited tumor growth in in vivo mouse models. P2CSR11 activated DC but induced less inflammation-inducing cytokines/interferons than other lipopeptides. Thus, P2CSR11 is a strong candidate antigen-specific immuno-adjuvant, with few adverse effects.


Assuntos
Vacinas Anticâncer/administração & dosagem , Lipopeptídeos/administração & dosagem , Neoplasias/tratamento farmacológico , Receptor 2 Toll-Like/imunologia , Animais , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Lipopeptídeos/imunologia , Lipopeptídeos/farmacologia , Camundongos , Neoplasias/imunologia , Fagocitose , Ensaios Antitumorais Modelo de Xenoenxerto
13.
PLoS One ; 12(11): e0188738, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29190690

RESUMO

Although dendritic cell (DC)-based immunotherapy shows little toxicity, improvements should be necessary to obtain satisfactory clinical outcome. Using interferon-gamma injection along with DCs, we previously obtained significant clinical responses against small or early stage malignant tumors in dogs. However, improvement was necessary to be effective to largely developed or metastatic tumors. To obtain effective methods applicable to those tumors, we herein used a DC-targeting Toll-like receptor ligand, h11c, and examined the therapeutic effects in murine subcutaneous and visceral tumor models and also in the clinical treatment of canine cancers. In murine experiments, most and significant inhibition of tumor growth and extended survival was observed in the group treated with the combination of h11c-activated DCs in combination with interferon-gamma and a cyclooxygenase2 inhibitor. Both monocytic and granulocytic myeloid-derived suppressor cells were significantly reduced by the combined treatment. Following the successful results in mice, the combined treatment was examined against canine cancers, which spontaneously generated like as those in human. The combined treatment elicited significant clinical responses against a nonepithelial malignant tumor and a malignant fibrous histiocytoma. The treatment was also successful against a bone-metastasis of squamous cell carcinoma. In the successful cases, the marked increase of tumor-responding T cells and decrease of myeloid-derived suppressor cells and regulatory T cells was observed in their peripheral blood. Although the combined treatment permitted the growth of lung cancer of renal carcinoma-metastasis, the marked elevated and long-term maintaining of the tumor-responding T cells was observed in the patient dog. Overall, the combined treatment gave rise to emphatic amelioration in DC-based cancer therapy.


Assuntos
Células Dendríticas/imunologia , Imunoterapia , Neoplasias/terapia , Receptores Toll-Like/metabolismo , Animais , Doenças do Cão/terapia , Cães , Ligantes , Camundongos , Camundongos Endogâmicos , Neoplasias/veterinária
14.
Cancer Sci ; 108(6): 1128-1134, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28370718

RESUMO

Reprogramming of glucose metabolism in tumor cells is referred to as the Warburg effect and results in increased lactic acid secretion into the tumor microenvironment. We have previously shown that lactic acid has important roles as a pro-inflammatory and immunosuppressive mediator and promotes tumor progression. In this study, we examined the relationship between the lactic acid concentration and expression of LDHA and GLUT1, which are related to the Warburg effect, in human head and neck squamous cell carcinoma (HNSCC). Tumors expressing lower levels of LDHA and GLUT1 had a higher concentration of lactic acid than those with higher LDHA and GLUT1 expression. Lactic acid also suppressed the expression of LDHA and GLUT1 in vitro. We previously reported that lactic acid enhances expression of an M2 macrophage marker, ARG1, in murine macrophages. Therefore, we investigated the relationship between the lactic acid concentration and polarization of M2 macrophages in HNSCC by measuring the expression of M2 macrophage markers, CSF1R and CD163, normalized using a pan-macrophage marker, CD68. Tumors with lower levels of CD68 showed a higher concentration of lactic acid, whereas those with higher levels of CSF1R showed a significantly higher concentration of lactic acid. A similar tendency was observed for CD163. These results suggest that tumor-secreted lactic acid is linked to the reduction of macrophages in tumors and promotes induction of M2-like macrophage polarization in human HNSCC.


Assuntos
Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Ácido Láctico/metabolismo , Macrófagos/patologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Humanos , Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço
15.
Biochim Biophys Acta Gen Subj ; 1861(2): 1-14, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27864148

RESUMO

BACKGROUND: Pentraxins (PTXs) are a superfamily of multifunctional conserved proteins involved in acute-phase responses. Recently, we have shown that collectin placenta 1 (CL-P1) and C-reactive protein (CRP) mediated complement activation and failed to form terminal complement complex (TCC) in normal serum conditions because of complement factor H inhibition. METHODS: We used CL-P1 expressing CHO/ldlA7 cells to study the interaction with PTXs. Soluble type CL-P1 was used in an ELISA assay for the binding, C3 and TCC deposition experiments. Furthermore, we used our previously established CL-P1 expressing HEK293 cells for the C3 fragment and TCC deposition assay. RESULTS: We demonstrated that CL-P1 also bound serum amyloid p component (SAP) and pentraxin 3 (PTX3) to activate the classical pathway and the alternative pathway using factor B. CRP and PTX3 further amplified complement deposition by properdin. We found that CRP and PTX3 recruit CFH, whereas SAP recruits C4 binding protein on CL-P1 expressing cell surfaces to prevent the formation of TCC in normal serum conditions. In addition, depletion of CFH, C4BP and complement factor I (CFI) failed to prevent TCC formation both in ELISA and cell experiments. Furthermore, soluble complement receptor 1, an inhibitor of all complement pathways prevents PTX induced TCC formation. CONCLUSION: Our current study hypothesizes that the interaction of pentraxins with CL-P1 is involved in complement activation. GENERAL SIGNIFICANCE: CL-P1 might generally inhibit PTX induced complement activation and host damage to protect self-tissues.


Assuntos
Proteína C-Reativa/metabolismo , Colectinas/metabolismo , Ativação do Complemento/fisiologia , Componente Amiloide P Sérico/metabolismo , Reação de Fase Aguda/metabolismo , Animais , Células CHO , Linhagem Celular , Fator H do Complemento/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/fisiologia , Cricetulus , Células HEK293 , Humanos , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia
16.
Cell Rep ; 16(11): 2819-2828, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27626653

RESUMO

During the development of mammalian embryonic germ cells, global demethylation and de novo DNA methylation take place. In mouse embryonic germ cells, two PIWI family proteins, MILI and MIWI2, are essential for the de novo DNA methylation of retrotransposons, presumably through PIWI-interacting RNAs (piRNAs). Although piRNA-associated MIWI2 has been reported to play critical roles in the process, its molecular mechanisms have remained unclear. To identify the mechanism, transgenic mice were produced; they contained a fusion protein of MIWI2 and a zinc finger (ZF) that recognized the promoter region of a type A LINE-1 gene. The ZF-MIWI2 fusion protein brought about DNA methylation, suppression of the type A LINE-1 gene, and a partial rescue of the impaired spermatogenesis of MILI-null mice. In addition, ZF-MIWI2 was associated with the proteins involved in DNA methylation. These data indicate that MIWI2 functions as an effector of de novo DNA methylation of the retrotransposon.


Assuntos
Proteínas Argonauta/metabolismo , Metilação de DNA/genética , Embrião de Mamíferos/citologia , Inativação Gênica , Espermatozoides/citologia , Espermatozoides/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Argonauta/química , Proteínas Argonauta/genética , Sequência de Bases , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Transgênicos , Ligação Proteica/genética , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/metabolismo , Espermatogênese , Testículo/metabolismo , Dedos de Zinco
17.
Cancer Med ; 5(9): 2513-21, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27465739

RESUMO

Tumor-infiltrating lymphocytes (TILs) are direct effectors of tumor immunity, and their characterization is important for further development of immunotherapy. Recent advances in high-throughput sequencing technologies have enabled a comprehensive analysis of T-cell receptor (TCR) complementarity-determining region 3 (CDR3) sequences, which may provide information of therapeutic importance. We developed a high-fidelity target sequencing method with the ability for absolute quantitation, and performed large-scale sequencing of TCR beta chain (TCRB) CDR3 regions in TILs and peripheral blood lymphocytes (PBLs). The estimated TCRB repertoire sizes of PBLs from four healthy individuals and TILs from four colorectal cancer tissue samples were 608,664-1,003,098 and 90,228-223,757, respectively. The usage of J- and V-regions was similar in PBLs and TILs. Proportions of CDR3 amino acid (aa) sequences occupying more than 0.01% of the total molecular population were 0.33-0.43% in PBLs and 1.3-3.6% in TILs. Additional low coverage sequencing of 15 samples identified five CDR3 aa sequences that were shared by nine patients, one sequence shared by 10 patients, and one sequence shared by 12 patients. The estimated size of the TCRB repertoire in TILs was significantly smaller than that in PBLs. The proportion of abundant species (>0.01%) in TILs was larger than that in PBLs. Shared CDR3 aa sequences represent a response to common antigens, and the identification of such CDR3 sequences may be beneficial in developing clinical biomarkers.


Assuntos
Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Estudos de Casos e Controles , Regiões Determinantes de Complementaridade/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfócitos do Interstício Tumoral/imunologia , Masculino , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Éxons VDJ
18.
Biochim Biophys Acta ; 1860(6): 1118-28, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26922829

RESUMO

BACKGROUND: C-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown. METHODS: We prepared CHO/ldlA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1. RESULTS: Here, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions. GENERAL SIGNIFICANCE: The interaction of collectin CL-P1 with CFH might be key for preventing attack on "self" as a result of complement activation induced by the CL-P1 and CRP interaction.


Assuntos
Proteína C-Reativa/química , Colectinas/química , Ativação do Complemento , Receptores Depuradores/química , Animais , Proteína C-Reativa/fisiologia , Células CHO , Colectinas/fisiologia , Fator H do Complemento/química , Cricetulus , Células HEK293 , Humanos , Receptores Depuradores/fisiologia
19.
Int J Cancer ; 135(12): 2847-56, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24789268

RESUMO

Materials used for the past 30 years as immunoadjuvants induce suboptimal antitumor immune responses and often cause undesirable local inflammation. Some bacterial lipopeptides that act as Toll-like receptor (TLR) 2 ligands activate immune cells as immunoadjuvants and induce antitumor effects. Here, we developed a new dendritic cell (DC)-targeting lipopeptide, h11c (P2C-ATPEDNGRSFS), which uses the CD11c-binding sequence of intracellular adhesion molecule-1 to selectively and efficiently activate DCs but not other immune cells. Although the h11c lipopeptide activated DCs similarly to an artificial lipopeptide, P2C-SKKKK (P2CSK4), via TLR2 in vitro, h11c induced more effective tumor inhibition than P2CSK4 at low doses in vivo with tumor antigens. Even without tumor antigens, h11c lipopeptide significantly inhibited tumor growth and induced tumor-specific cytotoxic T cells. P2CSK4 was retained subcutaneously at the vaccination site and induced severe local inflammation in in vivo experiments. In contrast, h11c was not retained at the vaccination site and was transported into the tumor within 24 hr. The recruitment of DCs into the tumor was induced by h11c more effectively, while P2CSK4 induced the accumulation of neutrophils leading to severe inflammation at the vaccination site. Because CD11b+ cells, but not CD11c+ cells, produced neutrophil chemotactic factors such as macrophage inflammatory protein (MIP)-2 in response to stimulation with TLR2 ligands, the DC-targeting lipopeptide h11c induced less MIP-2 production by splenocytes than P2CSK4. In this study, we succeeded in developing a novel immunoadjuvant, h11c, which effectively induces antitumor activity without adverse effects such as local inflammation via the selective activation of DCs.


Assuntos
Adjuvantes Imunológicos/química , Células Dendríticas/citologia , Lipopeptídeos/química , Neoplasias/imunologia , Animais , Antígenos de Neoplasias/metabolismo , Antígeno CD11c/metabolismo , Antígenos CD18/metabolismo , Citocinas/metabolismo , Humanos , Imunoterapia Adotiva/métodos , Inflamação , Cinética , Ligantes , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Baço/citologia , Linfócitos T Citotóxicos/citologia
20.
J Med Genet ; 51(3): 203-7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24367057

RESUMO

BACKGROUND: Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors 150 or more kinds of proteins to the human cell surface. There are at least 26 genes involved in the biosynthesis and remodelling of GPI anchored proteins (GPI-APs). Recently, inherited GPI deficiencies (IGDs) were reported which cause intellectual disability often accompanied by epilepsy, coarse facial features and multiple anomalies that vary in severity depending upon the degree of defect and/or step in the pathway of affected gene. METHODS AND RESULTS: A patient born to non-consanguineous parents developed intractable seizures with typical hypsarrhythmic pattern in electroencephalography, and was diagnosed as having West syndrome. Because the patient showed severe developmental delay with dysmorphic facial features and hyperphosphatasia, characteristics often seen in IGDs, the patient was tested for GPI deficiency. The patient had decreased surface expression of GPI-APs on blood granulocytes and was identified to be compound heterozygous for NM_178517:c.211A>C and c.499A>G mutations in PIGW by targeted sequencing. CONCLUSION: Here we describe the first patient with deficiency of PIGW, which is involved in the addition of the acyl-chain to inositol in an early step of GPI biosynthesis. Therefore, IGD should be considered in West syndrome and flow cytometric analysis of blood cells is effective in screening IGD.


Assuntos
Anormalidades Múltiplas/genética , Glicosilfosfatidilinositóis/deficiência , Hemoglobinúria Paroxística/genética , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Distúrbios do Metabolismo do Fósforo/genética , Espasmos Infantis/genética , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Glicosilfosfatidilinositóis/genética , Humanos , Lactente , Dados de Sequência Molecular , Convulsões , Alinhamento de Sequência
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