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1.
Am J Dermatopathol ; 45(2): 107-112, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36669074

RESUMO

BACKGROUND: Staged excision has emerged as a superior treatment option for lentigo maligna (LM) of the head and neck when compared with conventional wide local excision. Assessing surgical excision margins for remaining LM poses a diagnostic challenge. OBJECTIVES: To determine whether immunohistochemical (IHC) staining with SOX10 and preferentially expressed antigen in melanoma (PRAME) aids in diagnosing LM on excision margins compared with conventional hematoxylin and eosin and Melan A IHC staining. METHODS: This study included cases of LM of the head and neck treated with staged excision. Histological findings were reviewed according to standard criteria for the diagnosis of LM and compared with the results after IHC staining for Melan A, SOX10, and PRAME. RESULTS: The cohort consisted of 35 sections. Based on hematoxylin and eosin and Melan A IHC staining, 23 sections were diagnosed as LM by the initial pathologist. Further staining with SOX10 IHC showed only 8 to be consistent with a diagnosis of LM and 9 revealing features of actinic melanocyte hyperplasia. PRAME was positive in 5 of the 8 cases of LM and negative in all 9 cases of actinic melanocyte hyperplasia (P = 0.009). The presence of melanocyte nests (P = 0.29) and pagetoid spread (P = 0.003) was the most reliable histological findings distinguishing LM from its mimics. CONCLUSION: SOX10 is a more specific and sensitive marker for melanocytes when assessing for LM on excision margins compared with Melan A. The addition of PRAME can be useful to confirm or exclude the diagnosis in challenging cases.


Assuntos
Sarda Melanótica de Hutchinson , Neoplasias Cutâneas , Humanos , Sarda Melanótica de Hutchinson/patologia , Neoplasias Cutâneas/patologia , Margens de Excisão , Imuno-Histoquímica , Antígeno MART-1 , Hiperplasia , Amarelo de Eosina-(YS) , Hematoxilina , Antígenos de Neoplasias , Fatores de Transcrição SOXE
2.
Asian Pac J Cancer Prev ; 24(1): 257-266, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36708575

RESUMO

BACKGROUND: Due to many uses of cell culture in cell biology, biotechnology, and medical research, this technique has evolved into a widely used and accepted methodology. The isolation of primary cells from primary cancer tissue is a crucial step in cell culture technology since it offers a trustworthy source for studying the biology, morphology, and molecular evaluation of cancer cells, just like in the oral cavity tissue of patients. Therefore, the technique used for the isolation, culture, and evaluation of these cells is crucial. AIM: The aim of the present study is to isolate and culture the cells from human primary Oral Squamous Cell Carcinoma [OSCC] tissue and evaluate them for morphological variations using an explant method. MATERIALS AND METHODS: The patients with OSCC who were undergoing surgery provided the tissue samples. An explant technique was used to achieve the isolation of cells from tissue samples. Following that, the cells were maintained, subcultured, and stored in accordance with the standard American Type Culture Collection [ATCC] protocol. Routine Hematoxylin & Eosin and crystal violet stains were used. These cells were morphologically studied, and the results were assessed for further studies. RESULTS: We were able to successfully isolate and culture cells from 4 different tissue samples using the explant method. Morphological analysis revealed that one tissue had a significantly distinct presentation of epithelial and stromal cells, whereas the other three tissues had only minor morphological differences predominantly stromal cells. Two tissues were discarded after showing contamination. CONCLUSION: Tissue culture should be done very meticulously specially when oral cavity tissue is used as it is house for millions of microorganisms. The technique must also be thoroughly followed and adjusted accordingly. Using common, inexpensive stains like Hematoxylin and Eosin and crystal violet, which are of great help for examining the morphology of cells routinely.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas/cirurgia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço , Neoplasias Bucais/patologia , Hematoxilina , Amarelo de Eosina-(YS) , Violeta Genciana , Corantes , Linhagem Celular
4.
Arch Oral Biol ; 146: 105601, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36521282

RESUMO

OBJECTIVE: Patients with diabetes are known to have high salivary glucose levels. But the mechanisms are still unclear. We hypothesized that the topological changes of glucose transporters affect the salivary glucose level. METHODS: We used adult Goto-Kakizaki (GK) rats, an animal model of advanced diabetes, and Wistar rats as a control, with or without glucose load. The sections of salivary glands from the animals were processed for standard histological, immunohistochemical, and immunofluorescent staining. RESULTS: Parotid acinar cells of GK rats appeared like mucous filled with low-eosin-stained granules and possessing a flat nucleus located basally, whereas those of Wistar rats appeared as a typical serous gland with eosin-rich cytoplasm and a spherical nucleus. Cytoplasmic granules of GK rat parotid acinar cells showed no reaction of polysaccharide staining. In acinar cell cytoplasm of GK rats, intense GLUT1 immunoreactivity was observed compared to Wistar rats. By double immunostaining for GLUT1 and Golgi apparatus-specific markers, it was determined that GLUT1 was localized to the Golgi apparatus. By glucose loading in starved GK rats, the distribution of GLUT1-immunoreactive signals was spread out clearly from the apical side of the nucleus to the basolateral side. CONCLUSIONS: In rat model of diabetes, highly localized GLUT1 at Golgi apparatus in acinar cells seems to increase taking up cytoplasmic glucose to form exocytotic vesicles. This phenomenon may transform parotid glands from serous to mucous-like and result in saccharide-rich saliva.


Assuntos
Diabetes Mellitus Experimental , Glândula Parótida , Ratos , Animais , Ratos Wistar , Glândula Parótida/metabolismo , Células Acinares , Transportador de Glucose Tipo 1/metabolismo , Diabetes Mellitus Experimental/metabolismo , Amarelo de Eosina-(YS)/metabolismo , Glucose/metabolismo , Complexo de Golgi
5.
Theriogenology ; 198: 12-18, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36529107

RESUMO

To achieve optimal vitrification, tissue structure and fragment size represent a challenge for obtaining sufficient cooling velocity. Theoretically, thin ovarian tissue fragments lead to higher surface contact, hence higher solute penetration. Another critical factor is the concentration of cryoprotectants (CPA): CPA toxicity may occur with high concentrations, and as such, this may induce local apoptosis. Therefore two experiments were conducted: In experiment I, we compared the effect of sucrose supplementation in vitrification solution along with ovarian fragments of different sizes on post-warming tissue viability and follicle architecture. Fragments of two different sizes, with a thickness and radius of 1.5 × 0.75 mm and 3 × 1.5 mm respectively were vitrified in vitrification solution without sucrose and with 0.5 M sucrose supplementation. Post-warming, fragments of ovarian tissue (fresh and vitrified) were evaluated for viability (Calcein AM/Propidium Iodide) and for morphology (hematoxylin-eosin). In experiment II, we aimed to reduce cryoprotectant toxicity by using lower CPA concentrations in combination with an optimized carrier medium (HypThermosol®; HTS). Ovarian tissue fragments were randomly allocated to five groups (A: fresh controls; B: vitrified in GLOBAL® TOTAL® LP w/HEPES with 15% ethylene glycol (EG) and 15% DMSO; C: vitrified in HTS with 5% EG and 5% DMSO; D: vitrified in HTS with 10% EG and 10% DMSO; E: vitrified in HTS with 15% EG and 15% DMSO). Fragments (fresh and vitrified) were evaluated for morphology (hematoxylin-eosin) and for apoptosis through the activity of caspase-3. Results showed that follicular morphology was affected by the size of the fragment; smaller sized fragments contained a greater proportion of intact follicles (53.8 ± 2.0%) compared to the larger fragments (40.3 ± 2.0%). Our results demonstrated that 1.5 × 0.75 mm sized pieces vitrified in a vitrification solution supplemented with 0.5 M sucrose had more intact follicles (54.8 ± 1.3%; P = 0.0002) after vitrification. In addition, HTS presented no additional protective effect as a base medium, neither for follicular morphology nor apoptotic rate.


Assuntos
Criopreservação , Vitrificação , Feminino , Gatos , Animais , Criopreservação/veterinária , Dimetil Sulfóxido/farmacologia , Amarelo de Eosina-(YS) , Hematoxilina , Crioprotetores/farmacologia , Etilenoglicol/farmacologia , Sacarose/farmacologia
6.
Lancet Digit Health ; 5(2): e71-e82, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36496303

RESUMO

BACKGROUND: Endometrial cancer can be molecularly classified into POLEmut, mismatch repair deficient (MMRd), p53 abnormal (p53abn), and no specific molecular profile (NSMP) subgroups. We aimed to develop an interpretable deep learning pipeline for whole-slide-image-based prediction of the four molecular classes in endometrial cancer (im4MEC), to identify morpho-molecular correlates, and to refine prognostication. METHODS: This combined analysis included diagnostic haematoxylin and eosin-stained slides and molecular and clinicopathological data from 2028 patients with intermediate-to-high-risk endometrial cancer from the PORTEC-1 (n=466), PORTEC-2 (n=375), and PORTEC-3 (n=393) randomised trials and the TransPORTEC pilot study (n=110), the Medisch Spectrum Twente cohort (n=242), a case series of patients with POLEmut endometrial cancer in the Leiden Endometrial Cancer Repository (n=47), and The Cancer Genome Atlas-Uterine Corpus Endometrial Carcinoma cohort (n=395). PORTEC-3 was held out as an independent test set and a four-fold cross validation was performed. Performance was measured with the macro and class-wise area under the receiver operating characteristic curve (AUROC). Whole-slide images were segmented into tiles of 360 µm resized to 224 × 224 pixels. im4MEC was trained to learn tile-level morphological features with self-supervised learning and to molecularly classify whole-slide images with an attention mechanism. The top 20 tiles with the highest attention scores were reviewed to identify morpho-molecular correlates. Predictions of a nuclear classification deep learning model serve to derive interpretable morphological features. We analysed 5-year recurrence-free survival and explored prognostic refinement by molecular class using the Kaplan-Meier method. FINDINGS: im4MEC attained macro-average AUROCs of 0·874 (95% CI 0·856-0·893) on four-fold cross-validation and 0·876 on the independent test set. The class-wise AUROCs were 0·849 for POLEmut (n=51), 0·844 for MMRd (n=134), 0·883 for NSMP (n=120), and 0·928 for p53abn (n=88). POLEmut and MMRd tiles had a high density of lymphocytes, p53abn tiles had strong nuclear atypia, and the morphology of POLEmut and MMRd endometrial cancer overlapped. im4MEC highlighted a low tumour-to-stroma ratio as a potentially novel characteristic feature of the NSMP class. 5-year recurrence-free survival was significantly different between im4MEC predicted molecular classes in PORTEC-3 (log-rank p<0·0001). The ten patients with aggressive p53abn endometrial cancer that was predicted as MMRd showed inflammatory morphology and appeared to have a better prognosis than patients with correctly predicted p53abn endometrial cancer (p=0·30). The four patients with NSMP endometrial cancer that was predicted as p53abn showed higher nuclear atypia and appeared to have a worse prognosis than patients with correctly predicted NSMP (p=0·13). Patients with MMRd endometrial cancer predicted as POLEmut had an excellent prognosis, as do those with true POLEmut endometrial cancer. INTERPRETATION: We present the first interpretable deep learning model, im4MEC, for haematoxylin and eosin-based prediction of molecular endometrial cancer classification. im4MEC robustly identified morpho-molecular correlates and could enable further prognostic refinement of patients with endometrial cancer. FUNDING: The Hanarth Foundation, the Promedica Foundation, and the Swiss Federal Institutes of Technology.


Assuntos
Aprendizado Profundo , Neoplasias do Endométrio , Feminino , Humanos , Amarelo de Eosina-(YS) , Hematoxilina , Projetos Piloto , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia
7.
Immunol Invest ; 51(6): 1529-1547, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34544313

RESUMO

Circular RNAs (circRNAs) titrate the function of microRNAs (miRNAs), regulate transcription, and interfere with splicing. This study attempted to confirm the role of a novel circRNA circ_0128846 during osteoarthritis (OA) progression. Tissues and chondrocytes were isolated from OA patients. Overexpression and knockdown of target genes were generated using cell transfection and siRNA interference. Expression levels of genes were measured by qRT-PCR, Western blot, and immunohistochemistry, respectively. The interactions among circ_0128846, miR-140-3p, and JAK2 were verified by bioinformatics prediction, a dual-luciferase reporter assay, and RNA immunoprecipitation assay. The role of circ_0128846 in vivo was confirmed by the construction of experimental OA rats. Pathological changes were evaluated by hematoxylin and eosin and Safranin O staining. In OA patients, the level of circ_0128846 and JAK2 were up-regulated with down-regulated level of miR-140-3p. Circ_0128846 was principally located in the cytoplasm. Circ_0128846 silence enhanced cells viability, but reduced apoptosis rate and inflammatory response, which was obviously reversed by miR-140-3p knockdown. The overexpression of JAK2 reversed the effects of miR-140-3p on cell phenotypes. Circ_0128846 silence suppressed the level of MMP-13 and promoted the expression of collagen II by up-regulating miR-140-3p and down-regulating JAK2 in OA cells. Results of animal experiments demonstrated that circ_0128846 silence promoted collagen II expression and attenuated the OA progression by regulating the miR-140-3p/JAK2 axis. Circ_0128846 contributes to OA development through acting as a sponge RNA for miR-140-3p and thereby increasing JAK2 expression. Results indicated that targeting circ_0128846 may have the potential to alleviate OA progression.Abbreviations:circRNAs: Circular RNAs; miRNAs: microRNAs; OA: osteoarthritis; RIP: RNA immunoprecipitation; H&E: hematoxylin and eosin; ncRNAs: noncoding RNAs; ceRNA: competitive endogenous RNA; DMEM: Dulbecco's modified Eagle's medium; PBS: phosphate buffered saline; OE-circ_0128846: overexpression vector for circ_0128846; pcDNA3.1-JAK2: pcDNA3.1 overexpression vector for Janus kinase 2; NC: negative control; CCK-8: Cell Counting Kit-8; PI: propidium iodide; WT: Wild-type; mutants (MUT); SD rats: Sprague Dawley rats; DMM: destabilization of medial meniscus; IHC: immunohistochemistry; DAB: diaminobenzene; pre-Mrna: precursor mRNA.


Assuntos
MicroRNAs , Osteoartrite , Animais , Apoptose/genética , Proliferação de Células/genética , Amarelo de Eosina-(YS) , Hematoxilina , Janus Quinase 2/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoartrite/genética , RNA Circular/genética , Ratos , Ratos Sprague-Dawley
8.
Front Immunol ; 13: 1033498, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36466901

RESUMO

Objective: To investigate the efficacy of indirubin combined with human umbilical cord mesenchymal stem cells (hUC-MSCs) in the treatment of psoriatic lesions in BALB/c mice and to explore the related mechanism of indirubin in the treatment of psoriasis. Methods: A BALB/c mouse psoriasis model induced by imiquimod was established and randomly divided into the control group, model group, indirubin group, hUC-MSCs group, and indirubin combined with hUC-MSCs group. Psoriasis area and severity index (PASI) score was used to observe skin lesion changes in the psoriasis-like mouse model. The epidermal scale, the degree of keratinization, and the infiltration of inflammatory cells were observed by hematoxylin eosin (HE) staining. The concentrations of TNF-α, IFN-γ, IL-17A, and IL-23 in serum of mice were measured using enzyme-linked immunosorbent assay (ELISA). Results: The PASI integral trend chart indicates that hUC-MSCs and indirubin and the combination of drugs could relieve the appearance of skin lesions and accelerate the recovery of skin lesions. The indirubin group had the best effect in improving the scale of skin lesions. HE staining showed that the number of parakeratosis cells in the three treatment groups was significantly reduced, the degree of erythrocyte extravasation dermis hyperplasia and inflammatory cell infiltration was significantly lower than that in the model group, and the skin thickness and spleen index of the combined treatment group exhibited the most noticeable improvement. ELISA showed that the concentrations of TNF-α, IFN-γ, IL-17A, and IL-23 in serum of mice in the hUC-MSCs treatment group, indirubin group, and combined administration group were all decreased compared with those in the model group, and the concentrations of IFN-γ, IL-17A, and IL-23 could be decreased significantly in the indirubin group. Conclusions: Both hUC-MSCs and indirubin can effectively reduce psoriasis-like lesions in BALB/c mice, and the combined administration of these drugs has the best effect.


Assuntos
Células-Tronco Mesenquimais , Psoríase , Dermatopatias , Humanos , Camundongos , Animais , Interleucina-17 , Camundongos Endogâmicos BALB C , Fator de Necrose Tumoral alfa , Cordão Umbilical , Psoríase/terapia , Dermatopatias/terapia , Amarelo de Eosina-(YS) , Interleucina-23
9.
Arkh Patol ; 84(6): 67-73, 2022.
Artigo em Russo | MEDLINE | ID: mdl-36469721

RESUMO

OBJECTIVE: Development of original methodological approaches to annotation and labeling of histological images in relation to the problem of automatic segmentation of the layers of the stomach wall. MATERIAL AND METHODS: Three image collections were used in the study: NCT-CRC-HE-100K, CRC-VAL-HE-7K, and part of the PATH-DT-MSU collection. The used part of the original PATH-DT-MSU collection contains 20 histological images obtained using a high performance digital scanning microscope.Each image is a fragment of the stomach wall, cut from the surgical material of gastric cancer and stained with hematoxylin and eosin. Images were obtained using a scanning microscope Leica Aperio AT2 (Leica Microsystems Inc., Germany), annotations were made using Aperio ImageScope 12.3.3 (Leica Microsystems Inc., Germany). RESULTS: A labeling system is proposed that includes 5 classes (tissue types): areas of gastric adenocarcinoma (TUM), unchanged areas of the lamina propria (LP), unchanged areas of the muscular lamina of the mucosa (MM), a class of underlying tissues (AT), including areas of the submucosa, own muscular layer of the stomach and subserous sections, image background (BG). The advantage of this marking technique is to ensure high efficiency of recognition of the muscularis lamina (MM) - a natural «line¼ separating the lamina propria of the mucous membrane and all other underlying layers of the stomach wall. The disadvantages of the technique include a small number of classes, which leads to insufficient detailing of automatic segmentation. CONCLUSION: In the course of the study, an original technique for labeling and annotating images was developed, including 5 classes (types of tissues). This technique is effective at the initial stages of teaching mathematical algorithms for the classification and segmentation of histological images. Further stages in the development of a real diagnostic algorithm to automatically determine the depth of invasion of gastric cancer require the correction and development of the presented method of marking and annotation.


Assuntos
Neoplasias Gástricas , Humanos , Neoplasias Gástricas/patologia , Estômago/patologia , Amarelo de Eosina-(YS)
10.
Zhonghua Fu Chan Ke Za Zhi ; 57(11): 821-829, 2022 Nov 25.
Artigo em Chinês | MEDLINE | ID: mdl-36456478

RESUMO

Objective: To investigate the application of sentinel lymph node biopsy (SLNB) in early-staged cervical cancer by laparoscopy. Methods: It was a prospective, single-arm, single-center clinical study. Seventy-eight cases of cervical cancer patients were collected from July 2015 to December 2018 at the Fourth Hospital of Hebei Medical University. All the patients were injected with tracer into the disease-free block of cervical tissue after anesthesia by the same surgeon who learned sentinel lymph node (SLN) mapping technique in Memorial Sloan-Kettering Cancer Center, and underwent SLN mapping followed by complete pelvic lymphadenectomy. Moreover, all the dissected lymph nodes were stained with hematoxylin eosin staining (HE) pathological examination. Besides, the negative SLN on hematoxylin-eosin staining were detected by immunohistochemistry cytokeratin staining micro-metastasis. To analyze the distribution, detection rate, false negative rate the sensitivity and negative predictive value of the SLN in early-staged cervical cancer by laparoscopy, and explore the value of SLN mapping in predicting the lymph nodes metastasis in early-staged cervical cancer. Results: The overall detection rate of SLN in cervical cancer was 99% (77/78), bilateral detection rate was 87% (68/78). The average of 12.4 lymph node (LN) and 3.6 SLN were dissected for each patients each side. SLN of cervical cancer were mainly distributed in the obturator space (61.5%, 343/558), followed by external iliac (23.5%, 131/558), common iliac (7.3%, 41/558), para-uterine (3.8%, 21/558), internal iliac (2.2%, 12/558), para abdominal aorta (1.1%, 6/558), and anterior sacral lymphatic drainage area (0.7%, 4/558). Fourteen cases of LN metastasis were found among all 78 cases. There were a total of 38 positive LN, including 26 SLN metastasis and 12 none sentinel LN metastasis. Through immunohistochemical staining and pathological ultra-staging, 1 SLN was found to be isolated tumor cells (ITC), and 5 SLNs were found to be micro-metastases (MIC), accounting for 23% (6/26) of positive SLN. SLN mapping with pathological ultra-staging improved the prediction of LN metastasis in cervical cancer (2/14). Metastatic SLN mainly distributed in the obturator space (65%, 17/26), peri-uterine region (12%, 3/26), common iliac region (15%, 4/26), and external iliac region (8%, 2/26). The consistency of the diagnosis of lymph node metastasis by SLN biopsy and postoperative retroperitoneal lymph node metastasis showed that the Kappa value was 1.000 (P<0.001), indicated that the metastasis status of SLN and retroperitoneal lymph node were completely consistent. The sensitivity, specificity, accuracy, false-negative rate, and negative predictive value of SLN biopsy in the diagnosis of lymph node metastasis were 100%, 100%, 100%, 0, and 100%, respectively. Conclusions: SLN in early-staged cervical cancer patients were mainly distributed in the obturator and external iliac space, pathalogical ultra-staging of SLN could improve the prediction of LN metastasis. Intraoperative SLN mapping is safe, feasible and could predict the state of retroperitoneal LN metastasis in early-staged cervical cancer. SLNB may replace systemic pelvic lymphadenectomy.


Assuntos
Laparoscopia , Linfonodo Sentinela , Neoplasias do Colo do Útero , Humanos , Feminino , Linfonodo Sentinela/cirurgia , Metástase Linfática , Neoplasias do Colo do Útero/cirurgia , Amarelo de Eosina-(YS) , Hematoxilina , Estudos Prospectivos
11.
Cells ; 11(24)2022 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-36552789

RESUMO

Background: As a fibrotic disease with a high incidence, the pathogenesis of hypertrophic scarring is still not fully understood, and the treatment of this disease is also challenging. In recent years, human adipose-derived mesenchymal stem cells (AD-MSCs) have been considered an effective treatment for hypertrophic scars. This study mainly explored whether the therapeutic effect of AD-MSCs on hypertrophic scars is associated with oxidative-stress-related proteins. Methods: AD-MSCs were isolated from adipose tissues and characterized through flow cytometry and a differentiation test. Afterwards, coculture, cell proliferation, apoptosis, and migration were detected. Western blotting and a quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect oxidative stress-related genes and protein expression in hypertrophic scar fibroblasts (HSFs). Flow cytometry was used to detect reactive oxygen species (ROS). A nude mouse animal model was established; the effect of AD-MSCs on hypertrophic scars was observed; and hematoxylin and eosin staining, Masson's staining, and immunofluorescence staining were performed. Furthermore, the content of oxidative-stress-related proteins, including nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), B-cell lymphoma 2(Bcl2), Bcl2-associated X(BAX) and caspase 3, was detected. Results: Our results showed that AD-MSCs inhibited HSFs' proliferation and migration and promoted apoptosis. Moreover, after coculture, the expression of antioxidant enzymes, including HO-1, in HSFs decreased; the content of reactive oxygen species increased; and the expression of Nrf2 decreased significantly. In animal experiments, we found that, at 14 days after injection of AD-MSCs into human hypertrophic scar tissue blocks that were transplanted onto the dorsum of nude mice, the weight of the tissue blocks decreased significantly. Hematoxylin and eosin staining and Masson's staining demonstrated a rearrangement of collagen fibers. We also found that Nrf2 and antioxidant enzymes decreased significantly, while apoptotic cells increased after AD-MSC treatment. Conclusions: Our results demonstrated that AD-MSCs efficiently cured hypertrophic scars by promoting the apoptosis of HSFs and by inhibiting their proliferation and migration, which may be related to the inhibition of Nrf2 expression in HSFs, suggesting that AD-MSCs may provide an alternative therapeutic approach for the treatment of hypertrophic scars.


Assuntos
Cicatriz Hipertrófica , Fibroblastos , Células-Tronco Mesenquimais , Animais , Humanos , Camundongos , Antioxidantes/metabolismo , Apoptose , Cicatriz Hipertrófica/genética , Cicatriz Hipertrófica/metabolismo , Cicatriz Hipertrófica/terapia , Amarelo de Eosina-(YS) , Fibroblastos/metabolismo , Hematoxilina , Células-Tronco Mesenquimais/metabolismo , Camundongos Nus , Fator 2 Relacionado a NF-E2/metabolismo , Espécies Reativas de Oxigênio/metabolismo
12.
Inorg Chem ; 61(51): 20719-20724, 2022 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-36516228

RESUMO

Nanoscale zerovalent iron (NZVI) features potential application to biomedicine, (electro-/photo)catalysis, and environmental remediation. However, multiple-synthetic steps and limited ZVI content prompt the development of a novel strategy for efficient preparation of NZVI composites. Herein, a dinitrosyl iron complex [(N3MDA)Fe(NO)2] (1-N3MDA) was explored as a molecular precursor for one-pot photosynthesis of a cubic Fe@Fe3O4 core-shell nanoparticle (ZVI% = 60%) well-dispersed in an N-doping carbonaceous polymer (NZVI@NC). Upon photolysis of 1-N3MDA, photosensitizer Eosin Y, and sacrificial reductant TEA, the α-diimine N3MDA and noninnocent NO ligands (1) enable the slow reduction of 1-N3MDA into an unstable [(N3MDA)Fe(NO)2]- species, (2) serve as a capping reagent for controlled nucleation of zerovalent Fe atom into Fe nanoparticle, and (3) promote the polymerization of degraded Eosin Y with N3MDA yielding an N-doping carbonaceous matrix in NZVI@NC. This discovery of a one-pot photosynthetic process for NZVI@NC inspires continued efforts on its application to photolytic water splitting and ferroptotic chemotherapy in the near future.


Assuntos
Nanopartículas , Poluentes Químicos da Água , Amarelo de Eosina-(YS) , Ferro , Água , Substâncias Redutoras
13.
J Craniofac Surg ; 33(8): 2704-2710, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36409848

RESUMO

OBJECTIVE: To investigate the correlation between the features of basement membranes (BMs) of residual myofibers in the sternocleidomastoid muscle (SCM) and fibrosis of congenital muscular torticollis (CMT) in children, and to evaluate the relationship between BM changes and appearance of CMT. MATERIALS AND METHODS: We reviewed the CMT patients from 2017 to 2018 and performed pathological studies. Forty resected specimens were stained by hematoxylin and eosin and Masson collagen staining. Immunohistochemical staining of collagen IV and laminin was also performed. Five adductor muscle specimens from patients with developmental dysplasia of the hip were used as the control group. RESULTS: Hematoxylin and eosin staining revealed apparent interstitial fibrosis around residual myofibers in lesion specimens. However, the severity of fibrosis differed within the same samples. The average percent area of fibrous tissue in affected SCMs and controls were different significantly. Immunohistochemical staining of collagen IV and laminin showed these proteins were mostly expressed in the BM and vascular wall of affected SCM. However, BMs and myofibers from three different areas within the same SCM tissue exhibited significant differences in proteins expression. CONCLUSIONS: Therefore, the defective BMs are associated with myofiber and mesenchyme fibrosis in patients with CMT, which is crucial for understanding the histopathology of SCM.


Assuntos
Fibroma , Laminina , Criança , Humanos , Hematoxilina , Amarelo de Eosina-(YS) , Músculos do Pescoço , Fibrose , Fibroma/patologia , Membrana Basal/patologia
14.
Sci Rep ; 12(1): 19913, 2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36402802

RESUMO

Cell segmentation is a key step for a wide variety of biological investigations, especially in the context of muscle science. Currently, automated methods still struggle to perform skeletal muscle fiber quantification on Hematoxylin-Eosin (HE) stained histopathological whole slide images due to low contrast. On the other hand, the Deep Learning algorithm Cellpose offers new perspectives considering its increasing adoption for segmentation of a wide range of cells. Combining two open-source tools, Cellpose and QuPath, we developed MyoSOTHES, an automated Myofibers Segmentation wOrkflow Tuned for HE Staining. MyoSOTHES enables solving segmentation inconsistencies encountered by default Cellpose model in presence of large range size cells and provides information related to muscle Feret's diameter distribution and Centrally Nucleated Fibers, thus depicting muscle health and treatment effects. MyoSOTHES achieves high quality segmentation compared to baseline workflow with a detection F1-score increasing from 0.801 to 0.919 and a Root Mean Square Error (RMSE) on diameter improved by 31%. MyoSOTHES was validated on an animal study featuring gene transfer in [Formula: see text]-Sarcoglycanopathy, for which dose-response effect is visible and conclusions drawn are consistent with those previously published. MyoSOTHES thus paves the way for wide quantification of HE stained muscle sections and retrospective analysis of HE labeled slices used in laboratories for decades.


Assuntos
Inteligência Artificial , Fibras Musculares Esqueléticas , Animais , Hematoxilina , Amarelo de Eosina-(YS) , Fluxo de Trabalho , Estudos Retrospectivos , Fenótipo
15.
Crit Rev Biomed Eng ; 50(2): 1-19, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36374820

RESUMO

Many researchers have developed computer-assisted diagnostic (CAD) methods to diagnose breast cancer using histopathology microscopic images. These techniques help to improve the accuracy of biopsy diagnosis with hematoxylin and eosin-stained images. On the other hand, most CAD systems usually rely on inefficient and time-consuming manual feature extraction methods. Using a deep learning (DL) model with convolutional layers, we present a method to extract the most useful pictorial information for breast cancer classification. Breast biopsy images stained with hematoxylin and eosin can be categorized into four groups namely benign lesions, normal tissue, carcinoma in situ, and invasive carcinoma. To correctly classify different types of breast cancer, it is important to classify histopathological images accurately. The MobileNet architecture model is used to obtain high accuracy with less resource utilization. The proposed model is fast, inexpensive, and safe due to which it is suitable for the detection of breast cancer at an early stage. This lightweight deep neural network can be accelerated using field-programmable gate arrays for the detection of breast cancer. DL has been implemented to successfully classify breast cancer. The model uses categorical cross-entropy to learn to give the correct class a high probability and other classes a low probability. It is used in the classification stage of the convolutional neural network (CNN) after the clustering stage, thereby improving the performance of the proposed system. To measure training and validation accuracy, the model was trained on Google Colab for 280 epochs with a powerful GPU with 2496 CUDA cores, 12 GB GDDR5 VRAM, and 12.6 GB RAM. Our results demonstrate that deep CNN with a chi-square test has improved the accuracy of histopathological image classification of breast cancer by greater than 11% compared with other state-of-the-art methods.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/diagnóstico por imagem , Hematoxilina , Aprendizado de Máquina , Amarelo de Eosina-(YS) , Redes Neurais de Computação
16.
Dis Markers ; 2022: 1927688, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36284987

RESUMO

Purpose: Gegen Qinlian Decoction (GGQL) has been employed to treat type 2 diabetes mellitus (T2DM) in the clinical practice of traditional Chinese medicine. However, the underlying mechanism of GGQL in the treatment of T2DM remains unknown. This study was aimed at exploring the pharmacological mechanisms of GGQL against T2DM via network pharmacology analysis combined with experimental validation. Methods: The effective components of GGQL were screened, and the target was predicted by using traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP). The candidate targets of GGQL were predicted by network pharmacological analysis, and crucial targets were chosen by the protein-protein interaction (PPI) network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed to predict the core targets and pathways of GGQL against T2DM. Then, T2DM mice were induced by a high-fat diet combined with streptozotocin. The model and GGQL groups were given normal saline and GGQL aqueous solution (10 and 20 g/kg/d) intragastric administration, respectively, for 8 weeks. The mice in the GGQLT groups were administered with GGQLT at 10 and 20 g/kg/d, respectively. The pathological changes in liver tissues were observed by hematoxylin-eosin staining. The protein expression of TNF-α and NF-κB was verified by western blotting. Results: A total of 204 common targets of GGQL for the treatment of T2DM were obtained from 140 active ingredients and 212 potential targets of T2DM. GO and KEGG enrichment analysis involved 119 signaling pathways, mainly in inflammatory TNF signaling pathways. Animal experiments showed that GGQL significantly reduced the serum levels of body mass, fasting blood glucose, fasting insulin, HOMA-IR, TNF-α, and IL-17. The liver pathological section showed that GGQL could improve the vacuolar degeneration and lipid deposition in the liver of T2DM mice. Mechanistically, GGQL downregulated the mRNA expression of TNF-α and NF-κB. Conclusions: This study demonstrated that GGQL may exert antidiabetic effects against T2DM by suppressing TNF-α signaling pathway activation, thus providing a basis for its potential use in clinical practice and further study in treating T2DM.


Assuntos
Diabetes Mellitus Tipo 2 , Medicamentos de Ervas Chinesas , Camundongos , Animais , Interleucina-17 , Glicemia , Estreptozocina/uso terapêutico , NF-kappa B/genética , Fator de Necrose Tumoral alfa/genética , Solução Salina/uso terapêutico , Amarelo de Eosina-(YS)/uso terapêutico , Hematoxilina/uso terapêutico , Farmacologia em Rede , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Insulina , RNA Mensageiro , Lipídeos
17.
Ren Fail ; 44(1): 1780-1790, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36285371

RESUMO

PURPOSE: Glomerular mesangial cell (GMC) dysfunction plays a vital role in the pathogenesis of diabetic kidney disease (DKD). Transient receptor potential canonical 6 (TRPC6) has been demonstrated to be involved in the development of DKD. However, the underlying mechanism remains unclear. The present study investigated the role of TRPC6 in GMC dysfunction and the related mechanism. METHODS: Diabetic rats and cultured GMCs were used in the experiment. The diabetic rat model was created by intraperitoneal injection of streptozotocin. Protein and mRNA levels were assessed by Western blotting and quantitative RT-PCR, respectively. Histological changes in the kidneys were observed by immunochemistry and hematoxylin and eosin. TRPC6 knockdown was achieved by adenovirus-mediated TRPC6 shRNA delivery in vivo and TRPC6 siRNA transfection in vitro. RESULTS: TRPC6 expression was increased in diabetic rat kidneys. Knockdown of TRPC6 attenuated diabetes-induced kidney functional deterioration. In addition, the increases in extracellular matrix components, including collagen IV, collagen I, and fibronectin production, as well as NFAT2 expression were also suppressed. In cultured GMCs, high glucose (25 mM, HG) treatment increased the expression of TRPC6. Knockdown of TRPC6 alleviated HG-induced increases in collagen IV, fibronectin, and NFAT2 expression. Knockdown of NFAT2 also inhibited the upregulation of proteins, including collagen IV and fibronectin, in HG-treated GMCs. CONCLUSION: These results demonstrate that inhibition of TRPC6/NFAT2 signaling attenuates GMC dysfunction and the development of DKD and suggest that pharmacological targeting of TRPC6/NFAT2 in GMCs may provide beneficial effects for DKD.


Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Ratos , Animais , Células Mesangiais , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Fibronectinas/metabolismo , Canal de Cátion TRPC6/genética , Canal de Cátion TRPC6/metabolismo , RNA Interferente Pequeno/metabolismo , Diabetes Mellitus Experimental/metabolismo , Estreptozocina , Hematoxilina/metabolismo , Hematoxilina/farmacologia , Amarelo de Eosina-(YS)/metabolismo , Amarelo de Eosina-(YS)/farmacologia , Linfócitos T , Glucose/metabolismo , RNA Mensageiro/metabolismo , Colágeno/metabolismo , Células Cultivadas
18.
Biomed Pharmacother ; 155: 113778, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36271559

RESUMO

BACKGROUND: The intestinal tract is considered the body's "engine" and the most impacted organ during sepsis. In this study, we explored toll-like receptor 4 (TLR4) functions in sepsis-induced intestinal barrier dysfunction. METHODS: Wild-type and TLR4-knockout (KO) mice were used to establish a sepsis-induced dysfunctional intestinal barrier model via the intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). Hematoxylin and eosin staining, Transmission electron microscope, enzyme linked immunosorbent assay, western blot, quantitative real-time polymerase chain reaction, TdT-mediated dUTP nick end labeling staining, 16 S rRNA gene sequencing were used to explore differences in inflammatory cytokines, apoptosis, tight junction (TJ) protein expression, and intestinal flora diversity between groups. RESULTS: TLR4-deficiency reduced procalcitonin and C-reactive protein to prevent sepsis, and also inhibited inflammatory response by decreasing interleukin (IL)- 1ß, IL-6 and tumor necrosis factor-α levels. Also, BAX/Bcl2 and cleaved-caspase 3 expressions were decreased in TLR4-KO mice to suppress the intestinal mucosal cell apoptosis. TJ proteins, including zonula occludens protein, Occludin and Claudin-5 were significantly increased and intestinal fatty acid binding protein, myosin light chain and myosin light chain kinase were reduced in TLR4-KO mice. Additionally, 16 S rRNA gene sequencing indicated that TLR4-deficiency improved flora diversity and altered normal and abnormal bacterial proportions. CONCLUSIONS: TLR4 deficiency alleviated LPS-induced intestinal barrier dysfunction by reducing inflammatory responses and apoptosis, impairing intestinal damage, and regulating intestinal flora disturbance.


Assuntos
Lipopolissacarídeos , Sepse , Camundongos , Animais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Caspase 3/metabolismo , Ocludina/metabolismo , Cadeias Leves de Miosina/metabolismo , Interleucina-6/metabolismo , Pró-Calcitonina/metabolismo , Proteína C-Reativa/metabolismo , Claudina-5/metabolismo , Hematoxilina , Amarelo de Eosina-(YS) , Proteína X Associada a bcl-2/metabolismo , Proteínas de Junções Íntimas/metabolismo , Citocinas/metabolismo , Sepse/induzido quimicamente , Proteínas de Ligação a Ácido Graxo , Proteínas da Zônula de Oclusão/metabolismo
19.
BMC Pulm Med ; 22(1): 388, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36289489

RESUMO

BACKGROUND: Neutrophil infiltration accelerates the inflammatory response and is highly correlated to the development of acute lung injury (ALI). Budesonide (BUD) and N-acetylcysteine (NAC) both inhibit the inflammatory response to alleviate ALI, so we further investigated whether their combination is better for ALI. METHODS: In this study, we investigated the effect and mechanism of Combined BUD and NAC therapy on LPS-induced ALI. Rat ALI model and neutrophil abnormal activation model were established by lipopolysaccharide (LPS). BUD and NAC were treated alone or in combination, or cells were transfected with miR-196b-5p mimic or si-Socs3 to evaluate the efficacy and mechanism of BUD and NAC alone or in combination. Histopathological observation of lungs was performed by Hematoxylin Eosin (HE) staining. The quantity of neutrophils and inflammatory factors level in bronchoalveolar lavage fluid (BALF) were determined by Richter-Gimza complex stain and Enzyme-Linked Immunosorbnent Assay (ELISA), respectively. ReverseTranscription-PolymeraseChainReaction (RT-qPCR) was utilized to assess miR-196b-5p and inflammatory factor mRNA levels. The expression level of Socs3 was detected by immunohistochemistry or Western Blot. RESULTS: BUD and NAC combined treatment had a better effect on neutrophil recruitment and inflammatory response in LPS-induced ALI than did BUD and NAC alone. Transfection of the miR-196b-5p mimic reversed the effect of combined BUD and NAC. In conclusion, the combination of BUD and NAC is a better treatment for ALI. CONCLUSIONS: Combination therapy with BUD and NAC ameliorates LPS-induced ALI by attenuating neutrophil recruitment through the miR-196b-5p/Socs3 molecular axis.


Assuntos
Lesão Pulmonar Aguda , MicroRNAs , Ratos , Animais , Lipopolissacarídeos , Acetilcisteína , Infiltração de Neutrófilos , Budesonida/farmacologia , Amarelo de Eosina-(YS)/efeitos adversos , Hematoxilina , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo
20.
J Neuroinflammation ; 19(1): 262, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36289519

RESUMO

BACKGROUND: Glaucoma, the major cause of irreversible blindness worldwide, is characterized by progressive degeneration of retinal ganglion cells (RGCs). Current treatments for glaucoma only slow or partially prevent the disease progression, failing to prevent RGCs death and visual field defects completely. Glutamate excitotoxicity via N-methyl-D-aspartic acid (NMDA) receptors plays a vital role in RGCs death in glaucoma, which is often accompanied by oxidative stress and NLRP3 inflammasome activation. However, the exact mechanisms remain unclear. METHODS: The glutamate-induced R28 cell excitotoxicity model and NMDA-induced mouse glaucoma model were established in this study. Cell counting kit-8, Hoechst 33342/PI dual staining and lactate dehydrogenase release assay were performed to evaluate cell viability. Annexin V-FITC/PI double staining was used to detect apoptosis and necrosis rate. Reactive oxygen species (ROS) and glutathione (GSH) were used to detect oxidative stress in R28 cells. Levels of proinflammatory cytokines were measured by qRT-PCR. Transmission electron microscopy (TEM) was used to detect necroptotic morphological changes in RGCs. Retinal RGCs numbers were detected by immunofluorescence. Hematoxylin and eosin staining was used to detect retinal morphological changes. The expression levels of RIP1, RIP3, MLKL and NLRP3 inflammasome-related proteins were measured by immunofluorescence and western blotting. RESULTS: We found that glutamate excitotoxicity induced necroptosis in RGCs through activation of the RIP1/RIP3/MLKL pathway in vivo and in vitro. Administration of the RIP3 inhibitor GSK872 and RIP1 inhibitor necrostatin-1 (Nec-1) prevented glutamate-induced RGCs loss, retinal damage, neuroinflammation, overproduction of ROS and a decrease in GSH. Furthermore, after suppression of the RIP1/RIP3/MLKL pathway by GSK872 and Nec-1, glutamate-induced upregulation of key proteins involved in NLRP3 inflammasome activation, including NLRP3, pro-caspase-1, cleaved-caspase-1, and interleukin-1ß (IL-1ß), was markedly inhibited. CONCLUSIONS: Our findings suggest that the RIP1/RIP3/MLKL pathway mediates necroptosis of RGCs and regulates NLRP3 inflammasome activation induced by glutamate excitotoxicity. Moreover, GSK872 and Nec-1 can protect RGCs from necroptosis and suppress NLRP3 inflammasome activation through inhibition of RIP1/RIP3/MLKL pathway, conferring a novel neuroprotective treatment for glaucoma.


Assuntos
Glaucoma , Necroptose , Camundongos , Animais , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Interleucina-1beta/metabolismo , N-Metilaspartato , Inflamassomos/metabolismo , Caspase 1/metabolismo , Células Ganglionares da Retina/metabolismo , Ácido Glutâmico/toxicidade , Hematoxilina , Amarelo de Eosina-(YS) , Apoptose , Glaucoma/induzido quimicamente , Glaucoma/tratamento farmacológico , Glutationa/metabolismo , Lactato Desidrogenases/metabolismo
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