Antitumor effects of redox-responsive nanoparticles containing platinum（Ⅳ）in ovarian cancer / 中华肿瘤杂志

Objectives: To explore the antitumor effects of redox-responsive nanoparticles containing platinum(Ⅳ)-NP@Pt(Ⅳ) in ovarian cancer. Methods: Redox-responsive polymer carriers were synthesized. Polymer carriers and platinum(Ⅳ)-Pt(Ⅳ) can self-assemble into NP@Pt(Ⅳ). Inductively coupled plasma mass spectrometry was performed to detect the platinum release from NP@Pt(Ⅳ) in reducing environment and the platinum content in ovarian cancer cells ES2 treated with cisplatin, Pt(Ⅳ) and NP@Pt(Ⅳ). The proliferation ability of the ovarian cancer cells were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was assessed by flow cytometry. Collection of primary ovarian cancer tissues from patients with primary high-grade serous ovarian cancer who were surgically treated at the Cancer Hospital of the Chinese Academy of Medical Sciences from October to December 2022. The high-grade serous ovarian cancer patient-derived xenograft (PDX) mice were intravenously injected with Cy7.5 labeled NP@Pt(Ⅳ) followed by in vivo imaging system. Mice were treated with PBS, cisplatin and NP@Pt(Ⅳ). Tumor volume and weight were measured in each group. Necrosis, apoptosis and cell proliferation of tumor tissues were detected by hematoxylin-eosin (HE) staining, TUNEL fluorescence staining and Ki-67 immunohistochemistry staining. Body weight and HE staining of heart, liver, spleen, lung and kidney of mice in each group were measured. Results: The platinum release of NP@Pt(Ⅳ) after 48 hours in reducing environment was 76.29%, which was significantly higher than that of 26.82% in non-reducing environment (P<0.001). The platinum content in ES2 cells after 4 hours and 7 hours of treatment with NP@Pt(Ⅳ) (308.59, 553.15 ng/million cells) were significantly higher than those of Pt(Ⅳ) (100.21, 180.31 ng/million cells) and cisplatin (43.36, 50.36 ng/million cells, P＜0.05). The half inhibitory concentrations of NP@Pt(Ⅳ) in ovarian cancer cells ES2, A2780, A2780DDP were 1.39, 1.42 and 4.62 μmol/L, respectively, which were lower than those of Pt(IV) (2.89, 7.27, and 16.74 μmol/L) and cisplatin (5.21, 11.85, and 71.98 μmol/L). The apoptosis rate of ES2 cells treated with NP@Pt(Ⅳ) was (33.91±3.80)%, which was significantly higher than that of Pt(Ⅳ) [(16.28±2.41)%] and cisplatin [(15.01±1.17)%, P＜0.05]. In high-grade serous ovarian cancer PDX model, targeted accumulation of Cy7.5 labeled NP@Pt(Ⅳ) at tumor tissue could be observed. After the treatment, the tumor volume of mice in NP@Pt(IV) group was (130±98) mm3, which was significantly lower than those in control group [(1 349±161) mm3, P<0.001] and cisplatin group [(715±293) mm3, P=0.026]. The tumor weight of mice in NP@Pt(IV) group was (0.17±0.09)g, which was significantly lower than those in control group [(1.55±0.11)g, P<0.001] and cisplatin group [(0.82±0.38)g, P=0.029]. The areas of tumor necrosis and apoptosis in mice treated with NP@Pt(Ⅳ) were higher than those in mice treated with cisplatin. Immunohistochemical staining revealed that there were low expressions of Ki-67 at tumor tissues of mice treated with NP@Pt(Ⅳ) compared with cisplatin. The change in body weight of mice in NP@Pt(Ⅳ) group was not significantly different from that of the control group [(18.56±2.04)g vs.(20.87±0.79)g, P=0.063]. Moreover, the major organs of the heart, liver, spleen, lung, and kidney were also normal by HE staining. Conclusion: Redox-responsive NP@Pt(Ⅳ), produced in this study can enhance the accumulation of cisplatin in ovarian cancer cells and improve the efficacy of ovarian cancer chemotherapy.

Antitumor effects of redox-responsive nanoparticles containing platinum（Ⅳ）in ovarian cancer / 中华肿瘤杂志

Objectives: To explore the antitumor effects of redox-responsive nanoparticles containing platinum(Ⅳ)-NP@Pt(Ⅳ) in ovarian cancer. Methods: Redox-responsive polymer carriers were synthesized. Polymer carriers and platinum(Ⅳ)-Pt(Ⅳ) can self-assemble into NP@Pt(Ⅳ). Inductively coupled plasma mass spectrometry was performed to detect the platinum release from NP@Pt(Ⅳ) in reducing environment and the platinum content in ovarian cancer cells ES2 treated with cisplatin, Pt(Ⅳ) and NP@Pt(Ⅳ). The proliferation ability of the ovarian cancer cells were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was assessed by flow cytometry. Collection of primary ovarian cancer tissues from patients with primary high-grade serous ovarian cancer who were surgically treated at the Cancer Hospital of the Chinese Academy of Medical Sciences from October to December 2022. The high-grade serous ovarian cancer patient-derived xenograft (PDX) mice were intravenously injected with Cy7.5 labeled NP@Pt(Ⅳ) followed by in vivo imaging system. Mice were treated with PBS, cisplatin and NP@Pt(Ⅳ). Tumor volume and weight were measured in each group. Necrosis, apoptosis and cell proliferation of tumor tissues were detected by hematoxylin-eosin (HE) staining, TUNEL fluorescence staining and Ki-67 immunohistochemistry staining. Body weight and HE staining of heart, liver, spleen, lung and kidney of mice in each group were measured. Results: The platinum release of NP@Pt(Ⅳ) after 48 hours in reducing environment was 76.29%, which was significantly higher than that of 26.82% in non-reducing environment (P<0.001). The platinum content in ES2 cells after 4 hours and 7 hours of treatment with NP@Pt(Ⅳ) (308.59, 553.15 ng/million cells) were significantly higher than those of Pt(Ⅳ) (100.21, 180.31 ng/million cells) and cisplatin (43.36, 50.36 ng/million cells, P＜0.05). The half inhibitory concentrations of NP@Pt(Ⅳ) in ovarian cancer cells ES2, A2780, A2780DDP were 1.39, 1.42 and 4.62 μmol/L, respectively, which were lower than those of Pt(IV) (2.89, 7.27, and 16.74 μmol/L) and cisplatin (5.21, 11.85, and 71.98 μmol/L). The apoptosis rate of ES2 cells treated with NP@Pt(Ⅳ) was (33.91±3.80)%, which was significantly higher than that of Pt(Ⅳ) [(16.28±2.41)%] and cisplatin [(15.01±1.17)%, P＜0.05]. In high-grade serous ovarian cancer PDX model, targeted accumulation of Cy7.5 labeled NP@Pt(Ⅳ) at tumor tissue could be observed. After the treatment, the tumor volume of mice in NP@Pt(IV) group was (130±98) mm3, which was significantly lower than those in control group [(1 349±161) mm3, P<0.001] and cisplatin group [(715±293) mm3, P=0.026]. The tumor weight of mice in NP@Pt(IV) group was (0.17±0.09)g, which was significantly lower than those in control group [(1.55±0.11)g, P<0.001] and cisplatin group [(0.82±0.38)g, P=0.029]. The areas of tumor necrosis and apoptosis in mice treated with NP@Pt(Ⅳ) were higher than those in mice treated with cisplatin. Immunohistochemical staining revealed that there were low expressions of Ki-67 at tumor tissues of mice treated with NP@Pt(Ⅳ) compared with cisplatin. The change in body weight of mice in NP@Pt(Ⅳ) group was not significantly different from that of the control group [(18.56±2.04)g vs.(20.87±0.79)g, P=0.063]. Moreover, the major organs of the heart, liver, spleen, lung, and kidney were also normal by HE staining. Conclusion: Redox-responsive NP@Pt(Ⅳ), produced in this study can enhance the accumulation of cisplatin in ovarian cancer cells and improve the efficacy of ovarian cancer chemotherapy.

Application of Rapid HE Staining in Cytological Rapid On-site Evaluation of Peripheral Lung Cancer Needle Biopsy / 中国肺癌杂志

BACKGROUND@#Rapid on-site evaluation (ROSE) is a technique used for simultaneous evaluation of biopsy specimens through rapid cytology staining. Diff-Quik (DQ) staining is the most commonly employed method for cytological rapid on-site evaluation (C-ROSE). However, the utilization of DQ staining for on-site cytological interpretation remains uncommon among pathologists in China, posing challenges to the implementation of C-ROSE. This study aims to assess the application of rapid hematoxylin-eosin (HE) staining and DQ staining for C-ROSE during percutaneous needle biopsy of peripheral lung cancer and evaluate the value of rapid HE staining in C-ROSE.@*METHODS@#Computed tomography (CT)-guided lung biopsies were conducted on 300 patients diagnosed with peripheral lung cancer. The patients were randomly assigned to two groups for C-ROSE using either rapid HE staining or DQ staining, and subsequently the two methods were compared and evaluated.@*RESULTS@#The concordance rate between C-ROSE and histopathological diagnosis was 96.7%. The median staining time for rapid HE staining was 160 s, while that for DQ staining was 120 s, representing a significant difference between the two groups (P<0.001). However, there were no significant differences observed in terms of total biopsy time, concordance rate with histopathology, cytology specimen peeling rate, and incidence of serious adverse reactions between the two groups (P>0.05).@*CONCLUSIONS@#Both staining methods comply with C-ROSE criteria in the biopsy setting of peripheral lung cancer. Rapid HE staining is more aligned with domestic clinical requirements and holds potential for further promotion and adoption in C-ROSE.

A prospective study on application of human umbilical cord mesenchymal stem cells combined with autologous Meek microskin transplantation in patients with extensive burns / 中华烧伤杂志

Objective: To investigate the effects of human umbilical cord mesenchymal stem cells (hUCMSCs) combined with autologous Meek microskin transplantation on patients with extensive burns. Methods: The prospective self-controlled study was conducted. From May 2019 to June 2022, 16 patients with extensive burns admitted to the 990th Hospital of PLA Joint Logistics Support Force met the inclusion criteria, while 3 patients were excluded according to the exclusion criteria, and 13 patients were finally selected, including 10 males and 3 females, aged 24-61 (42±13) years. A total of 20 trial areas (40 wounds, with area of 10 cm×10 cm in each wound) were selected. Two adjacent wounds in each trial area were divided into hUCMSC+gel group applied with hyaluronic acid gel containing hUCMSCs and gel only group applied with hyaluronic acid gel only according to the random number table, with 20 wounds in each group. Afterwards the wounds in two groups were transplanted with autologous Meek microskin grafts with an extension ratio of 1∶6. In 2, 3, and 4 weeks post operation, the wound healing was observed, the wound healing rate was calculated, and the wound healing time was recorded. The specimen of wound secretion was collected for microorganism culture if there was purulent secretion on the wound post operation. In 3, 6, and 12 months post operation, the scar hyperplasia in wound was assessed using the Vancouver scar scale (VSS). In 3 months post operation, the wound tissue was collected for hematoxylin-eosin (HE) staining to observe the morphological changes and for immunohistochemical staining to observe the positive expressions of Ki67 and vimentin and to count the number of positive cells. Data were statistically analyzed with paired samples t test and Bonferronni correction. Results: In 2, 3, and 4 weeks post operation, the wound healing rates in hUCMSC+gel group were (80±11)%, (84±12)%, and (92±9)%, respectively, which were significantly higher than (67±18)%, (74±21)%, and (84±16)% in gel only group (with t values of 4.01, 3.52, and 3.66, respectively, P<0.05). The wound healing time in hUCMSC+gel group was (31±11) d, which was significantly shorter than (36±13) d in gel only group (t=-3.68, P<0.05). The microbiological culture of the postoperative wound secretion specimens from the adjacent wounds in 2 groups was identical, with negative results in 4 trial areas and positive results in 16 trial areas. In 3, 6, and 12 months post operation, the VSS scores of wounds in gel only group were 7.8±1.9, 6.7±2.1, and 5.4±1.6, which were significantly higher than 6.8±1.8, 5.6±1.6, and 4.0±1.4 in hUCMSC+gel group, respectively (with t values of -4.79, -4.37, and -5.47, respectively, P<0.05). In 3 months post operation, HE staining showed an increase in epidermal layer thickness and epidermal crest in wound in hUCMSC+gel group compared with those in gel only group, and immunohistochemical staining showed a significant increase in the number of Ki67 positive cells in wound in hUCMSC+gel group compared with those in gel only group (t=4.39, P<0.05), with no statistically significant difference in the number of vimentin positive cells in wound between the 2 groups (P>0.05). Conclusions: The application of hyaluronic acid gel containing hUCMSCs to the wound is simple to perform and is therefore a preferable route. Topical application of hUCMSCs can promote healing of the autologous Meek microskin grafted area in patients with extensive burns, shorten wound healing time, and alleviate scar hyperplasia. The above effects may be related to the increased epidermal thickness and epidermal crest, and active cell proliferation.

Myocardial biopsy of Liwen procedure: representability and etiological diagnostic value of cardiac samples obtained by a novel technique in patients with hypertrophic cardiomyopathy / 中华心血管病杂志

Objective: To investigate the representability and etiological diagnostic value of myocardium samples obtained from patients with hypertrophic cardiomyopathy (HCM) by transthoracic echocardiography-guided percutaneous intramyocardial septal biopsy (myocardial biopsy of Liwen procedure). Methods: This study was a retrospective case-series analysis. Patients with HCM, who underwent myocardial biopsy of Liwen procedure and radiofrequency ablation in Xijing Hospital, Air Force Military Medical University from July to December 2019, were included. Demographic data (age, sex), echocardiographic data and complications were collected through electronic medical record system. The histological and echocardiographic features, pathological characteristics of the biopsied myocardium of the patients were analyzed. Results: A total of 21 patients (aged (51.2±14.5) years and 13 males (61.9%)) were enrolled. The thickness of ventricular septum was (23.3±4.5)mm and the left ventricular outflow tract gradient was (78.8±42.6)mmHg (1 mmHg=0.133 kPa). Eight patients (38.1%) were complicated with hypertension, 1 patient (4.8%) had diabetes, and 2 patients (9.5%) had atrial fibrillation. Hematoxylin-eosin staining of myocardial samples of HCM patients before radiofrequency ablation evidenced myocytes hypertrophy, myocytes disarray, nuclear hyperchromatism, hypertrophy, atypia, coronary microvessel abnormalities, adipocyte infiltration, inflammatory cell infiltration, cytoplasmic vacuoles, lipofuscin deposition. Interstitial fibrosis and replacement fibrosis were detected in Masson stained biopsy samples. Hematoxylin-eosin staining of myocardial samples of HCM patients after radiofrequency ablation showed significantly reduced myocytes, cracked nuclear in myocytes, coagulative necrosis, border disappearance and nuclear fragmentation. Quantitative analysis of myocardial specimens of HCM patients before radiofrequency ablation showed that there were 9 cases (42.9%) with mild myocardial hypertrophy and 12 cases (57.1%) with severe myocardial hypertrophy. Mild, moderate and severe fibrosis were 5 (23.8%), 9 (42.9%) and 7 (33.3%), respectively. Six cases (28.6%) had myocytes disarray. There were 11 cases (52.4%) of coronary microvessel abnormalities, 4 cases (19.0%) of adipocyte infiltration, 2 cases (9.5%) of inflammatory cell infiltration,6 cases (28.5%) of cytoplasmic vacuole, 16 cases (76.2%) of lipofuscin deposition. The diameter of cardiac myocytes was (25.2±2.8)μm, and the percentage of collagen fiber area was 5.2%(3.0%, 14.6%). One patient had severe replacement fibrosis in the myocardium, with a fibrotic area of 67.0%. The rest of the patients had interstitial fibrosis. The myocardial specimens of 13 patients were examined by transmission electron microscopy. All showed increased myofibrils, and 9 cases had disorder of myofibrils. All patients had irregular shape of myocardial nucleus, partial depression, mild mitochondrial swelling, fracture and reduction of mitochondrial crest, and local aggregation of myofibrillary interfascicles. One patient had hypertrophy of cardiomyocytes, but the arrangement of muscle fibers was roughly normal. There were vacuoles in the cytoplasm, and Periodic acid-Schiff staining was positive. Transmission electron microscopy showed large range of glycogen deposition in the cytoplasm, with occasional double membrane surround, which was highly indicative of glycogen storage disease. No deposition of glycolipid substance in lysozyme was observed under transmission electron microscope in all myocardial specimens, which could basically eliminate Fabry disease. No apple green substance was found under polarized light after Congo red staining, which could basically exclude cardiac amyloidosis. Conclusion: Myocardium biopsied samples obtained by Liwen procedure of HCM patients are representative and helpful for the etiological diagnosis of HCM.

Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice / 中华烧伤杂志

Objective: To prepare graphene oxide (GO)-containing gelatin methacrylate anhydride (GelMA) hydrogel and to investigate the effects of in situ photopolymerized GO-GelMA composite hydrogel in wound vascularization of full-thickness skin defect in mice. Methods: The experimental study method was used. The 50 μL of 0.2 mg/mL GO solution was evenly applied onto the conductive gel, and the structure and size of GO were observed under field emission scanning electron microscope after drying. Human skin fibroblasts (HSFs) were divided into 0 μg/mL GO (without GO solution, the same as below) group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, 5.0 μg/mL GO group, and 10.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the absorbance value was detected using a microplate analyzer after 48 h of culture to reflect the proliferation activity of cells (n=6). HSFs and human umbilical vein vascular endothelial cells (HUVECs) were divided into 0 μg/mL GO group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the migration rates of HSFs at 24 and 36 h after scratching (n=5) and HUVECs at 12 h after scratching (n=3) were detected by scratch test, and the level of vascular endothelial growth factor (VEGF) secreted by HSFs after 4, 6, and 8 h of culture was detected by enzyme-linked immunosorbent assay method (n=3). The prepared GO-GelMA composite hydrogels containing GO of the corresponding final mass concentration were set as 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group to observe their properties before and after cross-linking, and to detect the release of GO after soaking with phosphate buffer solution for 3 and 7 d (n=3). The full-thickness skin defect wounds were made on the back of 16 6-week-old female C57BL/6 mice. The mice treated with in situ cross-linked GO-GelMA composite hydrogel containing GO of the corresponding final mass concentration were divided into 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group according to the random number table, with 4 mice in each group. The general condition of wound was observed and the wound healing rate was calculated on 3, 7, and 14 d of treatment, the wound blood perfusion was detected by laser Doppler flowmetry on 3, 7, and 14 d of treatment and the mean perfusion unit (MPU) ratio was calculated, and the wound vascularization on 7 d of treatment was observed after hematoxylin-eosin staining and the vascular density was calculated (n=3). The wound tissue of mice in 0 μg/mL GO composite hydrogel group and 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was collected to observe the relationship between the distribution of GO and neovascularization by hematoxylin-eosin staining (n=3) and the expression of VEGF by immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey's method. Results: GO had a multilayered lamellar structure with the width of about 20 μm and the length of about 50 μm. The absorbance value of HSFs in 10.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group after 48 h of culture (q=7.64, P<0.01). At 24 h after scratching, the migration rates of HSFs were similar in the four groups (P>0.05); at 36 h after scratching, the migration rate of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.48, 10.81, and 10.20, respectively, P<0.01). At 12 h after scratching, the migration rate of HUVECs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.11, 8.99, and 14.92, respectively, P<0.01), and the migration rate of HUVECs in 5.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group and 1.0 μg/mL GO group (with q values of 7.81 and 5.33, respectively, P<0.05 or P<0.01 ). At 4 and 6 h of culture, the VEGF expressions of HSFs in the four groups were similar (P>0.05); at 8 h of culture, the VEGF expression of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group and 5.0 μg/mL GO group (with q values of 4.75 and 4.48, respectively, P<0.05). The GO-GelMA composite hydrogels in the four groups were all red liquid before cross-linking, which turned to light yellow gel after cross-linking, with no significant difference in fluidity. The GO in the GO-GelMA composite hydrogel of 0 μg/mL GO composite hydrogel group had no release of GO at all time points; the GO in the GO-GelMA composite hydrogels of the other 3 groups was partially released on 3 d of soaking, and all the GO was released on 7 d of soaking. From 3 to 14 d of treatment, the wounds of mice in the 4 groups were covered with hydrogel dressings, kept moist, and gradually healed. On 3, 7, and 14 d of treatment, the wound healing rates of mice in the four groups were similar (P>0.05). On 3 d of treatment, the MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 10.70, 11.83, and 10.65, respectively, P<0.05 or P<0.01). On 7 and 14 d of treatment, the MPU ratios of wound of mice in the four groups were similar (P>0.05). The MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was significantly lower than that on 3 d of treatment (q=14.38, P<0.05), and that on 14 d of treatment was significantly lower than that on 7 d of treatment (q=27.78, P<0.01). On 7 d of treatment, the neovascular density of wound of mice on 7 d of treatment was 120.7±4.1 per 200 times of visual field, which was significantly higher than 61.7±1.3, 77.7±10.2, and 99.0±7.9 per 200 times of visual field in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 12.88, 7.79, and 6.70, respectively, P<0.01), and the neovascular density of wound of mice in 1.0 μg/mL GO composite hydrogel group and 5.0 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group (with q values of 5.10 and 6.19, respectively, P<0.05). On 7 d of treatment, cluster of new blood vessels in wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly more than that in 0 μg/mL GO composite hydrogel group, and the new blood vessels were clustered near the GO; a large amount of VEGF was expressed in wound of mice in 0.1 μg/mL GO composite hydrogel group in the distribution area of GO and new blood vessels. Conclusions: GO with mass concentration lower than 10.0 μg/mL had no adverse effect on proliferation activity of HSFs, and GO of 0.1 μg/mL can promote the migration of HSFs and HUVECs, and can promote the secretion of VEGF in HSFs. In situ photopolymerized of GO-GelMA composite hydrogel dressing can promote the wound neovascularization of full-thickness skin defect in mice and increase wound blood perfusion in the early stage, with GO showing an enrichment effect on angiogenesis, and the mechanism may be related to the role of GO in promoting the secretion of VEGF by wound cells.

Stingless bee propolis, metformin, and their combination alleviate diabetic cardiomyopathy

Abstract Background and aim: Stingless bee propolis, a resinous compound processed by mandibular secretion of stingless bees, is used for maintenance of hygiene and stability of beehives. Research on stingless bee propolis shows therapeutic properties attributed to polyphenols exhibiting antioxidative, antihyperglycemic and antiischemic effect. However, the cardioprotective effect of stingless bee propolis on diabetic cardiomyopathy is unknown. Methods: Adult male Sprague Dawley rats were randomised to five groups: normal group, diabetic group, diabetic given metformin (DM+M), diabetic given propolis (DM+P) and diabetic given combination therapy (DM+M+P) and treated for four weeks. Body weight, fasting blood glucose, food and water intake were taken weekly. At the end of experiment, biomarkers of oxidative damage were measured in serum and heart tissue. Antioxidants in heart tissue were quantified. Part of left ventricle of heart was processed for histological staining including Haematoxylin and Eosin (H&E) stain for myocyte size and Masson's Trichrome (MT) stain for heart fibrosis and perivascular fibrosis. Results: Propolis alleviated features of diabetic cardiomyopathy such as myocyte hypertrophy, heart fibrosis and perivascular fibrosis associated with improvement in antioxidative status. Conclusion: This study reports beneficial effect of propolis and combination with metformin in alleviating histopathological feature of diabetic cardiomyopathy by modulating antioxidants, making propolis an emerging complementary therapy.

Fibroblast growth factor receptor 3 gene (FGFR3) mutations in high-grade muscle-invasive urothelial bladder cancer in a Brazilian population: evaluation and prevalence

ABSTRACT Objective To understand the feasibility of FGFR3 tests in the Brazilian public health context, and to sample the mutational burden of this receptor in high-grade muscle invasive bladder cancer. Methods A total of 31 patients with high-grade muscle-invasive bladder cancer were included in the present study. Either transurethral resection of bladder tumor or radical cystectomy specimens were analyzed. Formalin-fixed paraffin-embedded tissue blocks were sectioned, hematoxylin and eosin stained, and histologic sections were reviewed. Total RNA was extracted using the RNeasy DSP formalin-fixed paraffin-embedded kit. Qualitative results were displayed in Rotor-Gene AssayManager software. Results Six patients were excluded. From the samples analyzed, four (16.7%) were considered inadequate and could not have their RNA extracted. Two patients presented FGFR3 mutations, accounting for 9.5% of material available for adequate analysis. The two mutations detected included a Y373C mutation in a male patient and a S249C mutation in a female patient. Conclusion FGFR3 mutations could be analyzed in 84% of our cohort and occurred in 9.5% of patients with high-grade muscle invasive bladder cancer in this Brazilian population. FGFR3 gene mutations are targets for therapeutic drugs in muscle-invasive bladder cancer. For this reason, know the frequency of these mutations can have a significant impact on public health policies and costs provisioning.

Efectos de la adición de probiótico Saccharomyces cerevisiae sobre histomorfología intestinal en pollos de engorde / Effects of addition of probiotic Saccharomyces cerevisiae on intestinal histomorphology in broilers

RESUMEN Este trabajo describe los efectos del probiótico Saccharomyces cerevisiae sobre el área, número de criptas de Lieberkühn en duodeno y yeyuno, y producción de moco en ambas secciones intestinales de pollos de engorde. Fueron empleados los tejidos de un total de 27 individuos clasificados en un grupo control GC (n=12) y un grupo suplementado con probióticos GP (n=15). Los resultados revelaron que los grupos suplementados con el S. cerevisiae presentaron una mayor amplitud del área de las criptas en duodeno (p= 0,0119) y yeyuno (p= 0,0355), menor número de criptas por milímetro en duodeno (p= 0,0420) y mayor producción de moco en duodeno respecto al grupo control (p= 0,0185), mientras que en yeyuno no se observaron diferencias significativas. Se concluyó que el uso de Saccharomyces cerevisiae aumentó el tamaño del área de las criptas en ambas secciones intestinales y aumentó la producción de moco en duodeno; lo cual, al aumentar la superficie de absorción intestinal, seguramente podría resultar en mejoras de los parámetros productivos.

ABSTRACT This work describes the effects of the probiotic Saccharomyces cerevisiae on the area, number of Lieberkühn crypts in the duodenum and jejunum, and mucus production in both intestinal sections. Tissues from a total of 27 individuals were used, classified in control group - GC (n = 12) and group supplemented with probiotics - GP (n = 15). The results revealed that the group supplemented with S. cerevisiae presented a greater area of the crypts in the duodenum (p = 0.0119) and jejunum (p = 0.0355), less number of crypts per millimeter in the duodenum (p = 0.0420) and higher mucus production in the duodenum compared to the control group (p = 0.0185), while in the jejunum no significant differences were observed. It was concluded that the use of Saccharomyces cerevisiae increased the size of the crypt area in both intestinal sections and increased mucus production in the duodenum; which by increasing the intestinal absorption surface could surely result in improvements in the productive parameters.

Evaluation of Histological Properties of Human Meniscal Grafts Stored in a Tissue Bank / Avaliação das propriedades histológicas de enxertos meniscais humanos armazenados em banco de tecido

Abstract Objectives The present paper aims to evaluate and compare the histological features of fresh and frozen menisci stored in a tissue bank for 1 month and for 5 years. Methods The meniscal grafts were subjected to a histological study. A total of 10 menisci were evaluated; 2 were frozen for 5 years, 4 were frozen for 1 month, and 4 were fresh, recently harvested specimens. Histological properties were evaluated in sections stained with hematoxylin and eosin and Masson trichrome methods. Results The menisci frozen for 1 month showed partially preserved collagen fiber structure and no significant hydropic tissue degeneration. The menisci frozen for 5 years presented an evident dissociation of collagen fibers and multiple foci of hydropic degeneration. Discussion Degeneration was much more significant in menisci stored for 5 years, indicating that a long freezing period results in substantial progression of tissue deterioration. This may suggest that the 5-year period, considered the maximum time for graft storage before transplant, is too long. Conclusion Grafts stored for 1 month showed a slight degenerative change in collagen fibers, whereas menisci frozen for 5 years presented significant tissue degeneration.

Resumo Objetivos Avaliar e comparar as características histológicas de meniscos frescos e meniscos congelados armazenados em banco de tecidos por 1 mês e por 5 anos. Métodos Foi feito um estudo histológico com enxertos meniscais. Avaliamos 10 meniscos, sendo 2 que ficaram armazenados sob congelamento por 5 anos, 4 armazenados congelados por 1 mês, e 4 frescos, recém captados. Foram feitos cortes histológicos corados com hematoxilina e eosina e Tricrômico de Masson, para avaliação das propriedades histológicas. Resultados Os meniscos congelados por 1 mês apresentaram preservação parcial da estrutura das fibras colágenas, sem degeneração hidrópica significativa do tecido. Nos meniscos congelados por 5 anos, observamos dissociação evidente das fibras colágenas, com presença de múltiplos focos de degeneração hidrópica. Discussão Encontramos degeneração bem mais significativa nos meniscos armazenados por 5 anos, o que indica que o longo período de congelamento leva à progressão significativa da degeneração do tecido. Isto pode sugerir que o período de 5 anos, considerado período máximo que o enxerto pode permanecer armazenado antes de ser transplantado, é um período muito longo. Conclusão Nos enxertos armazenados por 1 mês, existiu apenas discreta alteração degenerativa das fibras colágenas, enquanto que nos meniscos com 5 anos de congelamento foi observada degeneração significativa do tecido. Tibiais

Estudio histomorfométrico de folículos tiroideos humanos con microscopía holográfica digital / Histomorphometric study of human thyroid follicles by digital holographic microscopy

Introducción: La microscopía holográfica digital ha permitido a la microscopía óptica hacer uso de herramientas numéricas y computacionales; y esto, a su vez, ha favorecido múltiples avances en el estudio de las células y los tejidos en diferentes campos de la medicina y otras ciencias afines. Objetivo: Describir las características histológicas y morfométricas de los folículos tiroideos humanos con la microscopía holográfica digital. Métodos: Se realizó, desde el punto de vista histomorfométrico, un estudio descriptivo y transversal de folículos tiroideos humanos utilizando una instalación de microscopía holográfica digital. Se empleó la técnica de inclusión en parafina y tinción de hematoxilina-eosina para el procesamiento de las muestras. Se realizaron de 10 a 12 capturas de hologramas por muestra y el método de doble propagación para la reconstrucción de los hologramas. Se calculó el área, el perímetro, el diámetro mayor y menor de los folículos y cavidades foliculares y se realizaron reconstrucciones de imágenes holográficas en tres dimensiones. Se determinó como medida de tendencia central la media aritmética y como medida de dispersión la desviación típica o estándar. Resultados: Parámetros foliculares: área (5140,31 ± 1126,71 µm2); perímetro (2961,54 ± 71,2 µm); diámetro mayor:(921,17 ± 24,34 µm); diámetro menor: (746,67 ± 18,08 µm); altura del epitelio (7,92 ± 0,96). Cavidades foliculares: área (3686,18 ±1023,52 µm2); diámetro mayor: (698,86 ± 19,55 µm) y diámetro menor: (581,15 ± 13,82 µm). Conclusiones: Existen parámetros foliculares, determinados mediante la microscopía holográfica digital, no reportados por la literatura consultada, que resultan de interés en el estudio histológico de los folículos tiroideos humanos(AU)

Introduction: Digital holographic microscopy has made it possible to incorporate the use of numerical and computer tools into optical microscopy. This in turn has led to great progress in the study of cells and tissues in several fields of medicine and related sciences. Objective: Describe the histological and morphometric characteristics of human thyroid follicles using digital holographic microscopy. Methods: A descriptive cross-sectional histomorphometric study was conducted of human thyroid follicles using a digital holographic microscopy facility. Sample processing was based on inclusion technique by paraffin and hematoxylin-eosin staining. Ten to twelve holographic captures were made per sample, and the double propagation method was used for holographic reconstruction. Estimation was carried out of the area, perimeter, and greatest and smallest diameter of follicles and follicular cavities, and tri-dimensional reconstructions were made of holographic images. Arithmetic mean was determined as the measure of central tendency, and typical or standard deviation as the measure of dispersion. Results: Follicular parameters: area (5 140.31 ± 1 126.71 µm2); perimeter (2 961.54 ± 71.2 µm); greatest diameter (921.17 ± 24.34 µm); smallest diameter (746.67 ± 18.08 µm); epithelial height (7.92 ± 0.96). Follicular cavities: area (3 686.18 ± 1 023.52 µm2); greatest diameter (698.86 ± 19.55 µm); smallest diameter (581.15 ± 13.82 µm). Conclusions: A number of follicular parameters determined by digital holographic microscopy have not been reported by the literature consulted, and they are of interest to the histological study of human thyroid follicles(AU)

Histological analysis of elastic cartilages treated with alkaline solution / Análise histológica de cartilagens elásticas tratadas com solução alcalina

The elastic cartilage is composed by chondroblasts and chondrocytes, extracellular matrix and surrounded by perichondrium. It has a low regeneration capacity and is a challenge in surgical repair. One of obstacles in engineering a structurally sound and long-lasting tissue is selecting the most appropriate scaffold material. One of the techniques for obtaining biomaterials from animal tissues is the decellularization that decreases antigenicity. In this work, alkaline solution was used in bovine ear elastic cartilages to evaluate the decellularization and the architecture of the extracellular matrix. The cartilages were treated in alkaline solution (pH13) for 72 hours and lyophilized to be compared with untreated cartilages by histological analysis (hematoxylin-eosin, Masson's trichrome and Verhoeff slides). Areas of interest for cell counting and elastic fiber quantification were delineated, and the distribution of collagen and elastic fibers and the presence of non-fibrous proteins were observed. The results demonstrated that the alkaline solution caused 90% decellularization in the middle and 13% in the peripheral region, and maintenance of the histological characteristics of the collagen and elastic fibers and non-fibrous protein removal. It was concluded that the alkaline solution was efficient in the decellularization and removal of non-fibrous proteins from the elastic cartilages of the bovine ear.(AU)

A cartilagem elástica é composta por condroblastos e condrócitos, matriz extracelular e envolta por pericôndrio. Possui uma baixa capacidade de regeneração e é um desafio em reparos cirúrgicos. Um dos obstáculos na engenharia de tecido estruturalmente sólido e de longa duração é a seleção do material de arcabouço mais adequado. Uma das técnicas para obtenção de biomateriais oriundos de tecidos animais é a descelularização, que diminui a antigenicidade. Neste trabalho, foi utilizada solução alcalina em cartilagem elástica auricular bovina para avaliar a descelularização e a arquitetura da matriz extracelular. As cartilagens foram tratadas em solução alcalina (pH13) durante 72 horas e liofilizadas, e comparadas com cartilagens não tratadas por análise histológica (hematoxilina-eosina, tricrômio de Masson e Verhoeff). Foram determinadas as áreas de interesse para contagem celular e quantificação de fibras elásticas, observada a distribuição de colágeno e fibras elásticas e a presença de proteínas não fibrosas. Os resultados demonstraram que a solução alcalina causou 90% de descelularização na região central e 13% na região periférica, manutenção das características histológicas do colágeno e fibras elásticas e remoção das proteínas não fibrosas. Concluiu-se que a solução alcalina foi eficiente na descelularização e retirada de proteínas não fibrosas de cartilagens elásticas da orelha de bovinos.(AU)

Improvement program on pretreatment of acid decalcified tissue in hematoxylin-eosin staining / 华西口腔医学杂志

OBJECTIVE@#To explore the treatment conditions of acid decalcified specimens and improve the poor quality of sections and unclear structure of hematoxylin-eosin (HE) staining caused by the change in pH in tooth and hard tissue after acid decalcification.@*METHODS@#A total of 20 cases of oral pathological specimens that contain hard tissues were decalcified and treated with routine treatment, concentrated ammonia water immersion treatment, and saturated lithium carbonate solution immersion treatment. The quality and HE staining effects of hard tissue sections treated with different methods were compared.@*RESULTS@#Compared with routine treatment, lithium carbonate saturated solution treatment showed complete sections. Hematoxylin is strongly stained, the nucleus is clear, and the cytoplasm is bright.@*CONCLUSIONS@#Soaking acid decalcified specimens in lithium carbonate saturated solution before embedding in dehydration can neutralize the acidic environment of the tissue. The quality of sections and HE staining effect are improved and are suitable for the pretreatment of acid decalcified tissue samples of oral pathology.

Salivary Duct Cancer Metastasis Mimicking Primary Breast Cancer: A Case Report and Review / 한국유방암학회지

Microelectric Treatment by Transcutaneous Electrical Nerve Stimulation in a Rat Model of Acute Spinal Cord Injury

STUDY DESIGN: Animal study. OBJECTIVES: To investigate the effects of microelectric treatment by transcutaneous electrical nerve stimulation (TENS) on functional recovery and histological changes in a rat model of spinal cord injury (SCI). SUMMARY OF LITERATURE REVIEW: The effects of TENS on spasticity and its underlying mechanisms remain unclear. MATERIALS AND METHODS: SCI was induced by a 1.5-mm impactor with 200,000–260,000 dyne after laminectomy. Rats were divided into the following groups: group I (normal control), group II (microelectric treatment of 0 A), group III (microelectric treatment of 100 µA for 1 hr/day), group IV (microelectric treatment of 400 µA for 1 hr/day), and group V (microelectric treatment of 400 µA for 24 hr/day). After inducing SCI, rats were assessed by a sensory test with von Frey filaments and the locomotor recovery test (BBB rating scale) at 1, 4, 7, 14, 21, and 28 days. To evaluate spinal cord damage, histopathological studies were performed with hematoxylin and eosin. Brain-derived neurotrophic factor (BDNF) and TrkB immunohistochemistry studies were performed at 28 days. RESULTS: In groups IV and V, the BBB score had significantly improved on days 21 and 28 after SCI, and the TENS-treated groups showed significant neuronal recovery. After SCI, groups IV and V showed a significant recovery of locomotor function and the motor sensory response of the withdrawal threshold to 3.5 g. In addition, necrotic tissue and cystic spaces in the spinal cord were significantly reduced and BDNF/TrkB-positive cells were highly expressed in groups III, IV, and V. CONCLUSIONS: Microelectric treatment can play a role in facilitating the recovery of locomotion following SCI.

Generation of Organotypic Multicellular Spheres by Magnetic Levitation: Model for the Study of Human Hematopoietic Stem Cells Microenvironment

BACKGROUND AND OBJECTIVE: The characteristics of human hematopoietic stem cells are conditioned by the microenvironment of the bone marrow, where they interact with other cell populations, such as mesenchymal stem cells and endothelial cells; however, the study of this microenvironment is complex. The objective of this work was to develop a 3D culture system by magnetic levitation that imitates the microenvironment of human HSC. METHODS AND RESULTS: Human bone marrow-mesenchymal stem cells, umbilical cord blood-hematopoietic stem cells and a non-tumoral endothelial cell line (CC2811, Lonza®) were used to develop organotypic multicellular spheres by the magnetic levitation method. We obtained viable structures with an average sphericity index greater than 0.6, an average volume of 0.5 mm3 and a percentage of aggregation greater than 70%. Histological studies of the organotypic multicellular spheres used hematoxylin and eosin stains, and an evaluation of vimentin expression by means of immunohistochemistry demonstrated an organized internal structure without picnotic cells and a high expression of vimentin. The functional capacity of human hematopoietic stem cells after organotypic multicellular spheres culture was evaluated by multipotency tests, and it was demonstrated that 3D structures without exogenous Flt3L are autonomous in the maintenance of multipotency of human hematopoietic stem cells. CONCLUSIONS: We developed organotypic multicellular spheres from normal human cells that mimic the microenvironment of the human hematopoietic stem cells. These structures are the prototype for the development of complex organoids that allow the further study of the biology of normal human stem cells and their potential in regenerative medicine.

Degranulated Eosinophils Contain More Fine Nerve Fibers in the Duodenal Mucosa of Patients With Functional Dyspepsia

BACKGROUND/AIMS: Functional dyspepsia (FD) is characterized as chronic recurrent upper gastrointestinal symptoms in the absence of any organic disorder. We hypothesized that duodenal low-grade inflammation activates superficial afferent nerve sprouting, thereby contributing to hypersensitivity in patients with FD. METHODS: A prospective case-control study was conducted in a tertiary referral center. FD was defined using the Rome III criteria. Standardized endoscopic biopsies were performed in the stomach and duodenum. Hematoxylin and eosin staining and immunohistochemical staining for major basic proteins were performed to detect granulated eosinophil-derived granules, and S-100 staining was performed to detect fine nerve fibers. RESULTS: A total of 51 patients with FD (82% female; mean age 35.8 ± 13.4 years) and 35 controls were enrolled. Activated eosinophil counts in the duodenum were significantly higher in patients with FD than in controls (41.4% vs 17.1%, P = 0.005). Microscopic duodenitis was more frequently detected in patients with FD than in controls. Fine nerve fibers were more abundant in patients with FD than in controls (45.1% vs 11.4%, P = 0.029). The abundance of fine nerve fibers highly correlated with the degree of activated eosinophils. CONCLUSION: Duodenal low-grade inflammation, such as mucosal eosinophilic accumulation with degranulation, promoted mucosal enteric nerve fiber density and sprouting in patients with FD.

Matrix Production in Chondrocytes Transfected with Sex Determining Region Y-Box 9 and Telomerase Reverse Transcriptase Genes: An In Vitro Evaluation from Monolayer Culture to Three-Dimensional Culture

BACKGROUND: This study aimed to observe the cartilaginous matrix production in SRY (sex determining region Y)-box 9 (SOX9)- and/or telomerase reverse transcriptase (TERT)-transfected chondrocytes from monolayer to three-dimensional (3D) culture.

Stimulatory Effects of KPR-A148 on Osteoblast Differentiation and Bone Regeneration

BACKGROUND: Xanthine derivatives have been used to treat a variety of medical conditions including respiratory disease and neural degeneration. However, few studies have reported their effects on bone regeneration. Therefore, we investigated the effects of KPR-A148, a synthetic xanthine derivative on osteoblast differentiation in vitro and bone regeneration in vivo. METHODS: The cytotoxicity of KPR-A148 was evaluated using MC3T3-E1 cells by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltertrazolium bromide assay. The effects of KPR-A148 on osteoblast differentiation were examined by alkaline phosphatase staining, Alizarin red S staining, and real-time PCR of osteoblast differentiation marker genes. To investigate the effects of KPR-A148 on in vivo bone regeneration, a KPR-A148-containing collagen sponge was implanted into a mouse calvarial defect and KPR-A148 was injected twice, weekly. Bone regeneration was evaluated quantitatively by micro-CT and qualitatively by hematoxylin and eosin, as well as Masson's Trichrome staining. RESULTS: KPR-A148 did not show toxicity in the MC3T3-E1 cells and promoted osteoblast differentiation in a concentration-dependent manner. 10 µM of KPR-A148 showed the most significant effect on alkaline phospatase staining and matrix mineralization. KPR-A148 increased the expression of osteoblast marker genes in both the early and late stages of differentiation. In addition, KPR-A148 significantly induced new bone formation in the calvarial defect model. CONCLUSION: These results demonstrate that KPR-A148 strongly induces osteoblast differentiation and new bone formation. Therefore, it could be used as a potential therapeutic agent for regenerating bone following its destruction by disease or trauma.

Chondroid metaplasia of paraspinal connective tissue in the degenerative spine / 대한해부학회지

A 51-year-old male was routinely biopsied during a paraspinal muscle study. The biopsy sample was taken from the right erector spinae muscle at the fourth lumbar vertebra. The patient had no history of (diagnosed) major back trauma. The obtained sample was histologically analyzed (hematoxylin and eosin, safranin O), and complementary magnetic resonance imaging was performed. The biopsied sample contained chondroid tissue. Based on its location, the biopsy sample was appointed as chondroid metaplasia. Although chondroid metaplasia is not uncommon in humans, this is the first report of chondroid metaplasia within the paraspinal connective tissue. We propose a novel mechanism to explain the paraspinal chrondrogenic changes, related to spinal degeneration.