Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 195
Filtrar
1.
PLoS One ; 16(8): e0245361, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34428207

RESUMO

OBJECTIVE: The molecular mechanisms of the degeneration of the aortic wall in abdominal aortic aneurysm (AAA) are poorly understood. The monomeric form of C-reactive protein (mCRP) is deposited in damaged cardiovascular organs and aggravates the prognosis; however, it is unknown whether mCRP is deposited in the degenerated aorta of abdominal aortic aneurysm (AAA). We investigated whether mCRP is deposited in AAA and examined the associated pathogenic signaling pathways. METHODS: Twenty-four cases of AAA were analyzed and their histological features were compared according to the level of serum CRP and the degree of mCRP deposition. Proteomic analysis was performed in AAA cases with strong and diffuse CRP immunopositivity (n = 7) and those with weak, focal, and junctional CRP immunopositivity (n = 3). RESULTS: mCRP was deposited in the aortic specimens of AAA in a characteristic pattern that coincided with the lesion of the diminished elastic layer of the aortic wall. High serum CRP level was associated with stronger mCRP immunopositivity and a larger maximal diameter of aortic aneurysm. Proteomic analysis in AAA showed that multiple proteins were differentially expressed according to mCRP immunopositivity. Also, ingenuity pathway analysis showed that pathways associated with atherosclerosis, acute phase response, complement system, immune system, and coagulation were enriched in AAA cases with high mCRP immunopositivity. CONCLUSIONS: AAA showed a characteristic deposition of mCRP, and multiple potentially pathologic signaling pathways were upregulated in AAA cases with strong CRP immunopositivity. mCRP and the aforementioned pathological pathways may serve as targets for managing the progression of AAA.

2.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204265

RESUMO

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.


Assuntos
ADP Ribose Transferases , Antineoplásicos Imunológicos/farmacologia , Toxinas Bacterianas , Exotoxinas , Imunotoxinas/farmacologia , Proteínas Ligantes de Maltose , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única , Fatores de Virulência , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Exotoxinas/genética , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Humanos , Imunotoxinas/genética , Imunotoxinas/isolamento & purificação , Proteínas Ligantes de Maltose/genética , Espectrometria de Massas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Fatores de Virulência/genética
3.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198626

RESUMO

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a') tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.


Assuntos
Fator de Células-Tronco/biossíntese , Sequência de Aminoácidos , Ciclo Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Fator de Células-Tronco/química
4.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067755

RESUMO

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.


Assuntos
Cromatografia em Gel/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Isomerases de Dissulfetos de Proteínas/metabolismo , Diferenciação Celular , Eletroforese em Gel de Poliacrilamida/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Proteínas Ligantes de Maltose/metabolismo , Células Procarióticas/metabolismo , Isomerases de Dissulfetos de Proteínas/fisiologia , Transporte Proteico , Solubilidade
5.
Genes (Basel) ; 12(2)2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671400

RESUMO

Defects in the mitochondrial genome (mitochondrial DNA (mtDNA)) are associated with both congenital and acquired disorders in humans. Nuclear-encoded DNA polymerase subunit gamma (POLG) plays an important role in mtDNA replication, and proofreading and mutations in POLG have been linked with increased mtDNA deletions. SSBP1 is also a crucial gene for mtDNA replication. Here, we describe a patient diagnosed with Pearson syndrome with large mtDNA deletions that were not detected in the somatic cells of the mother. Exome sequencing was used to evaluate the nuclear factors associated with the patient and his family, which revealed a paternal POLG mutation (c.868C > T) and a maternal SSBP1 mutation (c.320G > A). The patient showed lower POLG and SSBP1 expression than his healthy brothers and the general population of a similar age. Notably, c.868C in the wild-type allele was highly methylated in the patient compared to the same site in both his healthy brothers. These results suggest that the co- deficient expression of POLG and SSBP1 genes could contribute to the development of mtDNA deletion.


Assuntos
Síndrome Congênita de Insuficiência da Medula Óssea/genética , DNA Polimerase gama/genética , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Erros Inatos do Metabolismo Lipídico/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Doenças Musculares/genética , Adolescente , Adulto , Criança , Pré-Escolar , Síndrome Congênita de Insuficiência da Medula Óssea/patologia , Replicação do DNA/genética , Feminino , Humanos , Erros Inatos do Metabolismo Lipídico/patologia , Masculino , Doenças Mitocondriais/patologia , Doenças Musculares/patologia , Linhagem , Deleção de Sequência/genética , Sequenciamento Completo do Exoma
6.
Scand J Gastroenterol ; 56(5): 598-603, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33764846

RESUMO

OBJECTIVES: Circumferential endoscopic submucosal dissection (ESD) for large lesions induces severe stricture, requiring subsequent treatment. We aimed to evaluate the efficacy of allogeneic epithelial cell sheet transplantation in preventing esophageal stricture after circumferential ESD in a porcine model. MATERIALS AND METHODS: A total of 15 conventional pigs underwent a 4 cm long circumferential ESD in the mid-esophagus. Out of these animals, 11 were immediately subjected to allogeneic oral mucosal cell sheet transplantation at the resection site, whereas four pigs underwent circumferential ESD only. We performed upper endoscopy 1 and 2 weeks after ESD and assessed the degree of esophageal stricture and histologic characteristics. RESULTS: Dysphagia scores and weight change ratios recorded 1 and 2 weeks after ESD did not differ between the two groups. The stricture rate 2 weeks after ESD was 100% in the control group and 90.9% in the cell sheet group (p = 1.000). The median mucosal constriction rates of the control and cell sheet groups were 73.5% (range 63.0-80.0%) and 53.8% (37.5-73.3%, p = .018), respectively. With regard to microscopic measurements, the length of re-epithelialization was greater in the cell sheet group than in the control group (2,495 µm vs. 369 µm, p = .008). Median fibrosis thickness and degree of muscle damage were not significantly different between groups. CONCLUSIONS: Although allogeneic epithelial cell sheet transplantation showed greater re-epithelialization and less mucosal constriction of post-ESD ulcers, it was not sufficiently effective in preventing post-ESD stricture.


Assuntos
Ressecção Endoscópica de Mucosa , Neoplasias Esofágicas , Estenose Esofágica , Transplante de Células-Tronco Hematopoéticas , Animais , Ressecção Endoscópica de Mucosa/efeitos adversos , Células Epiteliais , Estenose Esofágica/etiologia , Estenose Esofágica/prevenção & controle , Complicações Pós-Operatórias/prevenção & controle , Suínos
7.
J Perinat Med ; 49(3): 275-298, 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33544519

RESUMO

OBJECTIVES: Clinical chorioamnionitis at term is considered the most common infection-related diagnosis in labor and delivery units worldwide. The syndrome affects 5-12% of all term pregnancies and is a leading cause of maternal morbidity and mortality as well as neonatal death and sepsis. The objectives of this study were to determine the (1) amniotic fluid microbiology using cultivation and molecular microbiologic techniques; (2) diagnostic accuracy of the clinical criteria used to identify patients with intra-amniotic infection; (3) relationship between acute inflammatory lesions of the placenta (maternal and fetal inflammatory responses) and amniotic fluid microbiology and inflammatory markers; and (4) frequency of neonatal bacteremia. METHODS: This retrospective cross-sectional study included 43 women with the diagnosis of clinical chorioamnionitis at term. The presence of microorganisms in the amniotic cavity was determined through the analysis of amniotic fluid samples by cultivation for aerobes, anaerobes, and genital mycoplasmas. A broad-range polymerase chain reaction coupled with electrospray ionization mass spectrometry was also used to detect bacteria, select viruses, and fungi. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin-6 (IL-6) concentration ≥2.6 ng/mL. RESULTS: (1) Intra-amniotic infection (defined as the combination of microorganisms detected in amniotic fluid and an elevated IL-6 concentration) was present in 63% (27/43) of cases; (2) the most common microorganisms found in the amniotic fluid samples were Ureaplasma species, followed by Gardnerella vaginalis; (3) sterile intra-amniotic inflammation (elevated IL-6 in amniotic fluid but without detectable microorganisms) was present in 5% (2/43) of cases; (4) 26% of patients with the diagnosis of clinical chorioamnionitis had no evidence of intra-amniotic infection or intra-amniotic inflammation; (5) intra-amniotic infection was more common when the membranes were ruptured than when they were intact (78% [21/27] vs. 38% [6/16]; p=0.01); (6) the traditional criteria for the diagnosis of clinical chorioamnionitis had poor diagnostic performance in identifying proven intra-amniotic infection (overall accuracy, 40-58%); (7) neonatal bacteremia was diagnosed in 4.9% (2/41) of cases; and (8) a fetal inflammatory response defined as the presence of severe acute funisitis was observed in 33% (9/27) of cases. CONCLUSIONS: Clinical chorioamnionitis at term, a syndrome that can result from intra-amniotic infection, was diagnosed in approximately 63% of cases and sterile intra-amniotic inflammation in 5% of cases. However, a substantial number of patients had no evidence of intra-amniotic infection or intra-amniotic inflammation. Evidence of the fetal inflammatory response syndrome was frequently present, but microorganisms were detected in only 4.9% of cases based on cultures of aerobic and anaerobic bacteria in neonatal blood.

8.
J Perinat Med ; 49(4): 412-430, 2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-33554577

RESUMO

OBJECTIVES: Spontaneous preterm labor is an obstetrical syndrome accounting for approximately 65-70% of preterm births, the latter being the most frequent cause of neonatal death and the second most frequent cause of death in children less than five years of age worldwide. The purpose of this study was to determine and compare to uncomplicated pregnancies (1) the frequency of placental disorders of villous maturation in spontaneous preterm labor; (2) the frequency of other placental morphologic characteristics associated with the preterm labor syndrome; and (3) the distribution of these lesions according to gestational age at delivery and their severity. METHODS: A case-control study of singleton pregnant women was conducted that included (1) uncomplicated pregnancies (controls, n=944) and (2) pregnancies with spontaneous preterm labor (cases, n=438). All placentas underwent histopathologic examination. Patients with chronic maternal diseases (e.g., chronic hypertension, diabetes mellitus, renal disease, thyroid disease, asthma, autoimmune disease, and coagulopathies), fetal malformations, chromosomal abnormalities, multifetal gestation, preeclampsia, eclampsia, preterm prelabor rupture of the fetal membranes, gestational hypertension, gestational diabetes mellitus, and HELLP (hemolysis, elevated liver enzymes and low platelet count) syndrome were excluded from the study. RESULTS: Compared to the controls, the most prevalent placental lesions among the cases were the disorders of villous maturation (31.8% [106/333] including delayed villous maturation 18.6% [62/333] vs. 1.4% [6/442], q<0.0001, prevalence ratio 13.7; and accelerated villous maturation 13.2% [44/333] vs. 0% [0/442], q<0.001). Other lesions in decreasing order of prevalence included hypercapillarized villi (15.6% [68/435] vs. 3.5% [33/938], q<0.001, prevalence ratio 4.4); nucleated red blood cells (1.1% [5/437] vs. 0% [0/938], q<0.01); chronic inflammatory lesions (47.9% [210/438] vs. 29.9% [282/944], q<0.0001, prevalence ratio 1.6); fetal inflammatory response (30.1% [132/438] vs. 23.2% [219/944], q<0.05, prevalence ratio 1.3); maternal inflammatory response (45.5% [195/438] vs. 36.1% [341/944], q<0.01, prevalence ratio 1.2); and maternal vascular malperfusion (44.5% [195/438] vs. 35.7% [337/944], q<0.01, prevalence ratio 1.2). Accelerated villous maturation did not show gestational age-dependent association with any other placental lesion while delayed villous maturation showed a gestational age-dependent association with acute placental inflammation (q-value=0.005). CONCLUSIONS: Disorders of villous maturation are present in nearly one-third of the cases of spontaneous preterm labor.

9.
Semin Fetal Neonatal Med ; 25(4): 101146, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-33164775

RESUMO

The fetus can deploy a local or systemic inflammatory response when exposed to microorganisms or, alternatively, to non-infection-related stimuli (e.g., danger signals or alarmins). The term "Fetal Inflammatory Response Syndrome" (FIRS) was coined to describe a condition characterized by evidence of a systemic inflammatory response, frequently a result of the activation of the innate limb of the immune response. FIRS can be diagnosed by an increased concentration of umbilical cord plasma or serum acute phase reactants such as C-reactive protein or cytokines (e.g., interleukin-6). Pathologic evidence of a systemic fetal inflammatory response indicates the presence of funisitis or chorionic vasculitis. FIRS was first described in patients at risk for intraamniotic infection who presented preterm labor with intact membranes or preterm prelabor rupture of the membranes. However, FIRS can also be observed in patients with sterile intra-amniotic inflammation, alloimmunization (e.g., Rh disease), and active autoimmune disorders. Neonates born with FIRS have a higher rate of complications, such as early-onset neonatal sepsis, intraventricular hemorrhage, periventricular leukomalacia, and death, than those born without FIRS. Survivors are at risk for long-term sequelae that may include bronchopulmonary dysplasia, neurodevelopmental disorders, such as cerebral palsy, retinopathy of prematurity, and sensorineuronal hearing loss. Experimental FIRS can be induced by intra-amniotic administration of bacteria, microbial products (such as endotoxin), or inflammatory cytokines (such as interleukin-1), and animal models have provided important insights about the mechanisms responsible for multiple organ involvement and dysfunction. A systemic fetal inflammatory response is thought to be adaptive, but, on occasion, may become dysregulated whereby a fetal cytokine storm ensues and can lead to multiple organ dysfunction and even fetal death if delivery does not occur ("rescued by birth"). Thus, the onset of preterm labor in this context can be considered to have survival value. The evidence so far suggests that FIRS may compound the effects of immaturity and neonatal inflammation, thus increasing the risk of neonatal complications and long-term morbidity. Modulation of a dysregulated fetal inflammatory response by the administration of antimicrobial agents, anti-inflammatory agents, or cell-based therapy holds promise to reduce infant morbidity and mortality.


Assuntos
Corioamnionite/imunologia , Corioamnionite/fisiopatologia , Trabalho de Parto Prematuro/fisiopatologia , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Síndrome de Resposta Inflamatória Sistêmica/fisiopatologia , Adulto , Corioamnionite/diagnóstico , Corioamnionite/terapia , Citocinas/sangue , Feminino , Feto , Humanos , Recém-Nascido , Doenças do Prematuro/imunologia , Doenças do Prematuro/fisiopatologia , Interleucina-6/sangue , Gravidez , Síndrome de Resposta Inflamatória Sistêmica/diagnóstico
10.
PLoS One ; 15(5): e0233057, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32396545

RESUMO

Poor survival of human pluripotent stem cells (hPSCs) following freezing, thawing, or passaging hinders the maintenance and differentiation of stem cells. Rho-associated kinases (ROCKs) play a crucial role in hPSC survival. To date, a typical ROCK inhibitor, Y-27632, has been the primary agent used in hPSC research. Here, we report that another ROCK inhibitor, fasudil, can be used as an alternative and is cheaper than Y-27632. It increased hPSC growth following thawing and passaging, like Y-27632, and did not affect pluripotency, differentiation ability, and chromosome integrity. Furthermore, fasudil promoted retinal pigment epithelium (RPE) differentiation and the survival of neural crest cells (NCCs) during differentiation. It was also useful for single-cell passaging of hPSCs and during aggregation. These findings suggest that fasudil can replace Y-27632 for use in stem research.


Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Pesquisa com Células-Tronco
11.
J Perinat Med ; 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238609

RESUMO

Objective The aims of this study were to ascertain the frequency of disorders of villous maturation in fetal death and to also delineate other placental histopathologic lesions in fetal death. Methods This was a retrospective observational cohort study of fetal deaths occurring among women between January 2004 and January 2016 at Hutzel Women's Hospital, Detroit, MI, USA. Cases comprised fetuses with death beyond 20 weeks' gestation. Fetal deaths with congenital anomalies and multiple gestations were excluded. Controls included pregnant women without medical/obstetrical complications and delivered singleton, term (37-42 weeks) neonate with 5-min Apgar score ≥7 and birthweight between the 10th and 90th percentiles. Results Ninety-two percent (132/143) of placentas with fetal death showed placental histologic lesions. Fetal deaths were associated with (1) higher frequency of disorders of villous maturation [44.0% (64/143) vs. 1.0% (4/405), P < 0.0001, prevalence ratio, 44.6; delayed villous maturation, 22% (31/143); accelerated villous maturation, 20% (28/143); and maturation arrest, 4% (5/143)]; (2) higher frequency of maternal vascular malperfusion lesions [75.5% (108/143) vs. 35.7% (337/944), P < 0.0001, prevalence ratio, 2.1] and fetal vascular malperfusion lesions [88.1% (126/143) vs. 19.7% (186/944), P < 0.0001, prevalence ratio, 4.5]; (3) higher frequency of placental histologic patterns suggestive of hypoxia [59.0% (85/143) vs. 9.3% (82/942), P < 0.0001, prevalence ratio, 6.8]; and (4) higher frequency of chronic inflammatory lesions [53.1% (76/143) vs. 29.9% (282/944), P < 0.001, prevalence ratio 1.8]. Conclusion This study demonstrates that placentas of women with fetal death were 44 times more likely to present disorders of villous maturation compared to placentas of those with normal pregnancy. This suggests that the burden of placental disorders of villous maturation lesions is substantial.

12.
Stem Cell Res Ther ; 11(1): 1, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900237

RESUMO

BACKGROUND: Human placenta-derived mesenchymal stem cells (PD-MSCs) are powerful sources for cell therapy in regenerative medicine. However, a limited lifespan by senescence through mechanisms that are well unknown is the greatest obstacle. In the present study, we first demonstrated the characterization of replicative senescent PD-MSCs and their possible mitochondrial functional alterations. METHODS: Human PD-MSCs were cultured to senescent cells for a long period of time. The cells of before passage number 8 were early cells and after passage number 14 were late cells. Also, immortalized cells of PD-MSCs (overexpressed hTERT gene into PD-MSCs) after passage number 14 were positive control of non-senescent cells. The characterization and mitochondria analysis of PD-MSCs were explored with long-term cultivation. RESULTS: Long-term cultivation of PD-MSCs exhibited increases of senescent markers such as SA-ß-gal and p21 including apoptotic factor, and decreases of proliferation, differentiation potential, and survival factor. Mitochondrial dysfunction was also observed in membrane potential and metabolic flexibility with enlarged mitochondrial mass. Interestingly, we founded that fatty acid oxidation (FAO) is an important metabolism in PD-MSCs, and carnitine palmitoyltransferase1A (CPT1A) overexpressed in senescent PD-MSCs. The inhibition of CPT1A induced a change of energy metabolism and reversed senescence of PD-MSCs. CONCLUSIONS: These findings suggest that alteration of FAO by increased CPT1A plays an important role in mitochondrial dysfunction and senescence of PD-MSCs during long-term cultivation.


Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Ácidos Graxos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Placenta/metabolismo , Carnitina O-Palmitoiltransferase/genética , Proliferação de Células/fisiologia , Senescência Celular/fisiologia , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Oxirredução , Gravidez
13.
Protein Expr Purif ; 167: 105530, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31698036

RESUMO

Human serum albumin (HSA), the most abundant serum protein in healthy humans, plays important roles in many physiological processes and has wide clinical and research applications. Despite several efforts to obtain recombinant HSA (rHSA) from bacterial and eukaryotic expression systems, a low-cost and high-yield method for rHSA production is not available. The large molecular weight and high disulphide content hamper the expression and production of rHSA using bacterial hosts. Hence, a strategy that uses a fusion technique and engineered Escherichia coli strains was employed to improve the expression of soluble rHSA in the bacterial cytoplasm. The solubilities of the b'a' domain of human protein disulphide isomerase (PDIb'a')- and maltose-binding protein (MBP)-tagged rHSA expressed in Origami 2 at 18 °C were notably increased by up to 90.1% and 96%, respectively. A simple and efficient protocol for rHSA purification was established and approximately 9.46 mg rHSA was successfully obtained from a 500-mL culture at 97% purity. However, rHSA was mostly obtained in soluble oligomeric form. By introducing a simple refolding and size-exclusion chromatography step, monomeric rHSA was obtained at 34% yield. Native polyacrylamide gel electrophoresis confirmed the similarity in the molecular weights between E. coli-derived monomeric rHSA and commercial monomeric HSA.


Assuntos
Albumina Sérica Humana/biossíntese , Cromatografia em Gel , Escherichia coli/genética , Escherichia coli/metabolismo , Etiquetas de Sequências Expressas/metabolismo , Humanos , Proteínas Ligantes de Maltose/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/isolamento & purificação , Albumina Sérica Humana/metabolismo , Solubilidade
14.
Growth Factors ; 37(3-4): 131-145, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31542979

RESUMO

Despite strong evidence for the involvement of PDGF signaling in breast cancer, little is known about the PDGF ligand responsible for PDGFR activation during breast cancer progression. Here, we found PDGF-C to be highly expressed in breast carcinoma cell lines. Immunohistochemical analysis of invasive breast cancer revealed an association between increased PDGF-C expression and lymph node metastases, Ki-67 proliferation index, and poor disease-free survival. We also identified a PDGF-C splice variant encoding truncated PDGF-C (t-PDGF-C) isoform lacking the signal peptide and the N-terminal CUB domain. While t-PDGF C homodimer is retained intracellularly, it can be secreted as a heterodimer with full-length PDGF-C (FL-PDGF-C). PDGF-C downregulation reduced anchorage-independent growth and matrigel invasion of MDA-MB-231 cells. Conversely, ectopic expression of t-PDGF-C enhanced phenotypic transformation and invasion in BT-549 cells expressing endogenous FL-PDGF-C. The present study provides new insights into the functional significance of PDGF-C and its splice variant in human breast cancer.


Assuntos
Neoplasias da Mama/patologia , Metástase Linfática/genética , Linfocinas/genética , Linfocinas/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Metástase Linfática/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais
15.
Sci Rep ; 9(1): 13706, 2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31548569

RESUMO

Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, liver regeneration, cardiovascular and metabolic diseases. Cytoplasmic expression of OSM in Escherichia coli results in inclusion bodies, and complex solubilisation, refolding and purification is required to prepare bioactive protein. Herein, eight N-terminal fusion variants of OSM with hexahistidine (His6) tag and seven solubility-enhancing tags, including thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (Nusa), human protein disulphide isomerase (PDI) and the b'a' domain of PDI (PDIb'a'), were tested for soluble OSM expression in E. coli. The His6-OSM plasmid was also introduced into genetically engineered Origami 2 and SHuffle strains to test expression of the protein. At 18 °C, MBP-tagged OSM was highly expressed and solubility was dramatically enhanced. In addition, His6-OSM was more highly expressed and soluble in Origami 2 and SHuffle strains than in BL21(DE3). MBP-OSM and His6-OSM were purified more than 95% with yields of 11.02 mg and 3.27 mg from a 500 mL culture. Protein identity was confirmed by mass spectroscopy, and bioactivity was demonstrated by in vitro inhibition of Th17 cell differentiation.


Assuntos
Oncostatina M/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Escherichia coli , Expressão Gênica , Engenharia Genética , Histidina , Humanos , Proteínas Ligantes de Maltose/metabolismo , Oligopeptídeos , Oncostatina M/genética , Proteínas Recombinantes de Fusão/genética , Solubilidade
16.
J Hum Genet ; 64(11): 1117-1125, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31451716

RESUMO

Whole exome sequencing (WES) is an effective tool for the genetic diagnosis of mitochondrial disorders due to various nuclear genetic defects. In this study, three patients affected by extremely rare mitochondrial disorders caused by nuclear genetic defects are described. The medical records of each patient were reviewed to obtain clinical symptoms, results of biochemical and imaging studies, and muscle biopsies. WES and massive parallel sequencing of whole mtDNA were performed for each patient. The oxygen consumption rate (OCR) and complex activity I and IV was measured. Patients 1 and 2 had exhibited global developmental delay and seizure since early infancy. Blood lactate, the lactate-to-pyruvate ratio, and urinary excretion of Krebs cycle intermediates were markedly elevated. Patient 1 also was noted for ophthalmoplegia. Patient 2 had left ventricular hypertrophy and ataxia. Patient 3 developed dysarthria, gait disturbance, and right-side weakness at age 29. Brain magnetic resonance imaging demonstrated abnormal signal intensity involving the bilateral thalami, midbrain, or pons. Based on WES, patient 1 had p.Glu415Gly and p.Arg484Trp variants in MTO1. In patient 2, p.Gln111ThrfsTer5 and RNA mis-splicing were identified in TSFM. Patient 3 carried p.Met151Thr and p.Met246Lys variants in AARS2. Skin fibroblasts of three patients exhibited decreased OCRs and complex 1 activity, and mitochondrial DNA was normal. These results demonstrate the utility of WES for identifying the genetic cause of extremely rare mitochondrial disorders, which has implications for genetic counseling.


Assuntos
Alanina-tRNA Ligase/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Fatores de Alongamento de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Doenças Raras/genética , Adulto , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Criança , DNA Mitocondrial/genética , Disartria/genética , Disartria/fisiopatologia , Exoma/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/fisiopatologia , Imageamento por Ressonância Magnética , Masculino , Mitocôndrias/genética , Doenças Mitocondriais/diagnóstico por imagem , Doenças Mitocondriais/fisiopatologia , Mutação , Oftalmoplegia/genética , Oftalmoplegia/fisiopatologia , Linhagem , Doenças Raras/diagnóstico por imagem , Doenças Raras/fisiopatologia , Sequenciamento Completo do Exoma
17.
Placenta ; 86: 45-51, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31326089

RESUMO

INTRODUCTION: Massive perivillous fibrin deposition (MPFD) is frequently associated with detrimental pregnancy outcomes, and extensive perivillous fibrin deposition results in severe placental dysfunction and loss of maternofetal interface. Unfortunately, the fundamental pathogenesis of MPFD remains unknown, and systematic analyses of MPFD in miscarriage is lacking. We analyzed the frequency and clinicopathological characteristics of MPFD in first trimester miscarriages. METHODS: We analyzed a consecutive series of miscarriages (n = 582) gathered between March 2012 and June 2016. MPFD was classified as fibrin-type (f-MPFD) and matrix-type (m-MPFD) by immunostaining for fibrin and collagen type IV. The control group consisted of miscarriage cases (MC, n = 18) that were matched to f-MPFD with normal chromosome (f-MPFD-nc) for number of previous miscarriages and placental chromosomal status. RESULTS: MPFD was identified in 2.7% of miscarriages. f-MPFD was associated with recurrent abortions. Compared with miscarriages without fibrin deposition, MPFD cases had higher proportion of those with normal placental chromosome (69.2% vs. 27.4%, P < 0.005) and higher frequency of villous syncytiotrophoblast C4d deposition (73.3% vs. 33.9%, P < 0.005). All C4d(+) f-MPFD patients had more than three recurrent miscarriages, whereas C4d(-) f-MPFD patients had no history of recurrent miscarriage (P < 0.05). Patients with f-MPFD-nc had significantly higher HLA PRA immunopositivity rate than did MC patients (P = 0.005). DISCUSSION: MPFD was more common in miscarriages than in preterm and term pregnancies. Placental massive fibrin-type fibrinoid deposition and villous C4d immunoreactivity were associated with recurrent miscarriage.


Assuntos
Aborto Habitual/patologia , Fibrina/metabolismo , Placenta/patologia , Aborto Habitual/imunologia , Aborto Habitual/metabolismo , Adulto , Estudos de Coortes , Feminino , Humanos , Placenta/imunologia , Placenta/metabolismo , Gravidez , Primeiro Trimestre da Gravidez
18.
PLoS One ; 14(5): e0216610, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31063484

RESUMO

OBJECTIVE: Prognosis of myocardial infarction tends to be worse when serum C-reactive protein (CRP) level is high. miRNAs are also known to be involved in different pathogeneses of heart diseases such as myocardial infarction. However, how CRP is involved in myocardial infarction has not been fully elucidated. We hypothesized that serum CRP changes the miRNA profile during ischemia-reperfusion injury (IRI) of the myocardium. To confirm this hypothesis, we performed global miRNA expression profiling of myocardium using IRI and CRP infusion rat model. METHODS: After ligation of the coronary artery of rat hearts, human serum CRP was intravenously injected, and reperfusion was performed (I/R+CRP group, n = 6). Control group consisted of the sham group (n = 3), IV CRP infusion group (CRP only, n = 3), and the I/R-only group (I/R only, n = 5). We evaluated 423 miRNA expression in non-ischemic areas and areas at risk (AAR) of each group using NanoString nCounter miRNA expression assay. RESULTS: MiR-124 was downregulated in non-ischemic myocardium in CRP-only group. In AAR, 7 miRNAs were commonly upregulated in both I/R-only and I/R+CRP groups. And additional 6 miRNAs were upregulated in the I/R+CRP group (miR-33, miR-409-3p, miR-384-3p, miR-3562, miR-101a, and miR-340-5p). Similarly, in the non-ischemic areas, 6 miRNAs were commonly upregulated in both I/R-only and I/R+CRP groups, and additional 5 miRNAs changed in the I/R+CRP group (upregulation of miR-3559-5p, miR-499, and miR-21 and downregulation of miR-500 and miR-532-3p). CONCLUSION: We showed that when serum CRP level is high, IRI results in multiple miRNA profile changes not only in ischemic areas but also in non-ischemic myocardium. Our results may provide a strong basis for studying the role of CRP and miRNAs in ischemic heart disease.


Assuntos
Proteína C-Reativa/análise , Proteína C-Reativa/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , MicroRNAs/genética , Traumatismo por Reperfusão Miocárdica/patologia , Animais , Feminino , Humanos , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Ratos , Ratos Sprague-Dawley
19.
Sci Rep ; 9(1): 6623, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036853

RESUMO

Mesenchymal stem cells derived from Wharton's jelly of the umbilical cord (UC-MSCs) have immunomodulatory properties. The aim of this study was to explore whether extracts of MSCs (MSC-Ex) could augment the low therapeutic efficacy of the whole cells in an Aspergillus fumigatus (Af)-induced atopic dermatitis (AD) model. LPS- or TNF-α/IFN-γ-stimulated keratinocytes (HaCaT cells) were treated with MSC-Ex, and the Af-induced AD model was established in BALB/c mice. In HaCaT cells, MSC-Ex treatment significantly reduced the inflammatory cytokine (IL-6, IL-1ß, IL-4, IL-5 and TNF-α), iNOS and NF-κB levels, and upregulated the anti-inflammatory cytokines (IL-10 and TGF-ß1). In the AD mice, the MSC-Ex group showed greatly reduced dermatitis, and lower clinical symptom scores and IgE levels. The histological dermatitis scores were also markedly lower in the MSC-Ex-treated animals compared with the MSC-treated group. Decreased levels of IFN-γ (Th1) and IL-17 (Th17), IL-4 and IL-13 (Th2) were detected in T cells and the skin tissue from the MSC-Ex treated AD mice. The therapeutic capacity of MSC-Ex was preserved after lyophilization and reconstitution. MSC-Ex treatment reproducibly suppresses dermatitis and inhibits the induction of inflammatory cytokines in the skin of AD mice. MSC-Ex is therefore a potential new treatment agent for AD.


Assuntos
Aspergillus fumigatus/imunologia , Aspergillus fumigatus/patogenicidade , Dermatite Atópica/microbiologia , Dermatite Atópica/terapia , Animais , Linhagem Celular , Interleucina-13/metabolismo , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Interleucina-4/metabolismo , Interleucina-6/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
BMB Rep ; 52(8): 496-501, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30670149

RESUMO

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins. [BMB Reports 2019; 52(8): 496-501].


Assuntos
ADP Ribose Transferases/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Exotoxinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Fatores de Virulência/farmacologia , ADP Ribose Transferases/química , ADP Ribose Transferases/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antineoplásicos/química , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Exotoxinas/química , Exotoxinas/metabolismo , Feminino , Humanos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...