Ki-67 Immunostaining Research Articles

Independent Validation of the International Grading System for Medullary Thyroid Carcinoma: A Single Institution Experience

Medullary thyroid carcinoma (MTC), an uncommon C cell thyroid malignancy, accounts for a disproportionate number of thyroid cancer deaths. To predict MTC clinical behavior, the recent international MTC grading system (IMTCGS) was published combining features from the Memorial Sloan Kettering Cancer Center and Royal North Shore Hospital grading systems that incorporates mitotic count, necrosis, and Ki67 proliferative index (Ki67PI). The IMTCGS appears promising, but independent validation data are limited. Here, we applied the IMTCGS to our institutional MTC cohort and assessed its ability to predict clinical outcomes. Our cohort comprised 87 MTCs (30 germline and 57 sporadic). Slides for each case were reviewed by 2 pathologists and histologic features recorded. Ki67 immunostaining was performed on all cases. Each MTC was graded with the IMTCGS based on tumor necrosis, Ki67PI, and mitotic count. Cox regression analysis was performed to assess the impact of various clinical and pathological data on disease outcomes, including overall survival (OS), disease-free survival, disease-specific survival (DSS), and distant metastasis-free survival. In our MTC cohort, 18.4% (n = 16/87) were IMTCGS high grade. IMTCGS grade was strongly prognostic for OS, disease-free survival, DSS, and distant metastasis-free survival on univariate analysis and multivariable analysis in both the entire MTC cohort and in the sporadic subset. Among the individual IMTCGS parameters, while all 3 were associated with poorer survival outcomes on univariate analysis, necrosis had the strongest association with all survival parameters on multivariable analysis, whereas Ki67PI or mitotic count was associated only with OS and DSS. This retrospective study independently demonstrates that the IMTCGS is valid for grading MTCs. Our findings support incorporating IMTCGS into routine pathology practice. IMTCGS grading may help clinicians to better predict the prognosis of MTC. Future studies may shed light on how MTC grading should impact treatment protocols.

Medicinal and edible plant Allium macrostemon Bunge for the treatment of testosterone-induced androgenetic alopecia in mice

Ethnopharmacological relevanceAllium macrostemon Bunge (AMB), a widely distributed wild garlic plant, possesses a variety of health-promoting properties. Androgenetic alopecia (AGA) is a common disorder that affects quality of life. Aim of the studyWe sought to investigate whether AMB stimulates hair regrowth in AGA mouse model, and clarify the underlying molecular mechanisms. Materials and methodsThe chemical constituents of AMB water extract were identified by ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q/TOF-MS) analysis. Cell viability assay and Ki-67 immunostaining were undertaken to evaluate the impacts of AMB on human hair dermal papilla cell (HDPC) proliferation. Wound-healing assay was undertaken to assess cell migration. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay were performed to examine cell apoptosis. Western blotting, real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and immunostaining assays were undertaken to determine the impacts of AMB on Wnt/β-catenin signaling and growth factors expression in HDPC cells. AGA mouse model was induced by testosterone treatment. The effects of AMB on hair regeneration in AGA mice were demonstrated by hair growth measuring and histological scoring. The levels of β-catenin, p-GSK-3β, and Cyclin D1 in dorsal skin were measured. ResultsAMB promoted proliferation and migration, as well as the expression of growth factors in cultured HDPC cells. Meanwhile, AMB restrained apoptosis of HDPC cells by increasing the ratio of anti-apoptotic Bcl-2/pro-apoptotic Bax. Besides, AMB activated Wnt/β-catenin signaling and thereby enhancing growth factors expression as well as proliferation of HDPC cells, which was abolished by Wnt signaling inhibitor ICG-001. In addition, an increase of hair shaft elongation was observed in mice suffering from testosterone-induced AGA upon the treatment of AMB extract (1% and 3%). Consistent with the in vitro assays, AMB upregulated the Wnt/β-catenin signaling molecules in dorsal skin of AGA mice. ConclusionThis study demonstrated that AMB promoted HDPC cell proliferation and stimulated hair regrowth in AGA mice. Wnt/β-catenin signaling activation, which induced production of growth factors in hair follicles and, eventually, contributed to the influence of AMB on the hair regrowth. Our findings may contribute to effective utilization of AMB in alopecia treatment.

Influence of postnatal overfeeding on postnatal heart development in juvenile mice

Shortly after birth, cardiomyocytes progressively lose their ability to proliferate to switch towards a hypertrophic growth and this ability is known to be influenced by nutritional factors. Postnatal overfeeding (PNOF) induced by litter size reduction in rodents reproduces the impact of childhood overnutrition and was shown to alter levels of circulating metabolites (lipids and glucose). Moreover, PNOF is known to have a major impact on metabolic status and cardiac function in adulthood. The aim of this work was to study the impact of PNOF on postnatal cardiac proliferation and apoptosis in juvenile mice. C57BL/6 male pups were raised in litter adjusted either to 9 or 3 pups for control (NF) and PNOF group respectively. At 7, 10 or 24 days after birth, hearts were harvested for immunostaining of proliferation marker Ki67 or apoptosis marker caspase 3. Cardiomyocyte number was also counted in hearts of 24 days-old mice and RT-qPCR were performed to study different cyclin mRNA expressions. At 7, 10 and 24 days, body weight and heart mass of PNOF mice was significantly higher than control mice. The proliferation rate of cardiomyocytes in PNOF hearts was decreased at 7 and 10 days with no signs of apoptosis at 7 days. At 24 days-old, even though the proliferation rate was not different between the two groups, the number of cardiomyocytes per mg of tissue was smaller in PNOF hearts and associated to a decreased expression of cyclins D1 and A2. In this study we observed that PNOF does impact the postnatal proliferative window of cardiomyocytes by reducing their proliferation rate 7 and 10 days after birth. This leads to a diminution of cardiomyocytes number in PNOF hearts at weaning and is associated to a decreased expression of specific cyclins essential for the cell cycle progression. Such a decrease could later be involved in the long-term cardiac alterations observed in PNOF mice.

The IBD-candidate gene, PTPN2, Regulates Goblet Cell Number and Expression of Intestinal Epithelial Cell Markers in a Regional-specific Manner in Mice

Loss-of-function variants in the Protein Tyrosine Phosphatase non-receptor Type 2 ( PTPN2) gene are associated with increased risk of Inflammatory Bowel Disease (IBD). PTPN2 encodes T Cell Protein Tyrosine Phosphatase (TCPTP), a negative regulator of intracellular signaling pathways including JAK-STAT. PTPN2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage crosstalk, and modulating the gut microbiota in mice and IBD patients. Aim: To determine if Ptpn2 loss compromises IEC subtypes that restrict gut dysbiosis. Methods: Ileal and colonic IEC subtypes and their function were assessed by PAS staining of goblet cells and immunostaining. RNA isolated from colonic IECs of whole-body Ptpn2-wild-type (WT), heterozygous (Het) and KO mice was assayed by two separate Nanostring ® panels: PanCancer and AutoImmune totaling >1500 targets. Flow cytometry was performed to evaluate abundance of mucosal-associated immune cells. Results: Goblet cells were reduced in the ileal and cecal crypts compared with WT mice (p<0.05; n=5) while numbers in the proximal and distal colon were variable. Ki-67 immunostaining revealed increased proliferating cells in the proximal and distal colon (p<0.01; n=3). Nanostring analysis had 1399 targets with detectable expression. Notably, expression of growth factors Egf and Fgfr3, differentiation factors Atoh1 and Mycn, tuft cell marker Dclk1, and intestinal stem cell marker Lgr5, were all downregulated in colonic IECs from Ptpn2-KO mice compared with WT (p<0.05; FDR<0.1; n=4). Moreover, abundance of mucosal neutrophils, macrophages, CD4 + expressing IFN-γ, CD8 + expressing TNF/IFN-γ, and CD4 + expressing IL-22 T-cells were higher in colon and cecum of Ptpn2-KO mice compared with WT and HET mice (p<0.01; n=5), whereas abundance of IL-4 expressing CD4 + cells, and Tregs expressing IL-10 were decreased in all intestinal segments of Ptpn2-KO mice (p<0.01; n=5). Conclusion: Ptpn2 loss negatively impacts abundance of IEC subtypes in a region-specific manner, affecting epithelial proliferation and expression of critical differentiation/cell markers. The elevated abundance of immune cells expressing pro-inflammatory signals accompanied by decreased levels of cells expressing IL-4 and IL-10, could contribute to a skewed abundance of IEC subtypes and their function. The observed effects may contribute to the increased susceptibility to infection and the dysbiotic microbiota in Ptpn2-deficient mice, and partly explain association of PTPN2 variants with dysbiosis in clinical IBD. Supported by NIH 2R01DK091281, 1R01AI153314-01, R21AI152017 and R01AI165490 (DFM). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Homogenous subpopulation of human mesenchymal stem cells and their extracellular vesicles restore function of endometrium in an experimental rat model of Asherman syndrome

BackgroundAsherman syndrome (AS), or intrauterine adhesions, is a main cause of infertility in reproductive age women after endometrial injury. Mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) are promising candidates for therapies that repair damaged endometria. However, concerns about their efficacy are attributed to heterogeneity of the cell populations and EVs. A homogenous population of MSCs and effective EV subpopulation are needed to develop potentially promising therapeutic options in regenerative medicine.MethodsAS model was induced by mechanical injury in adult rat uteri. Then, the animals were treated immediately with homogeneous population of human bone marrow-derived clonal MSCs (cMSCs), heterogenous parental MSCs (hMSCs), or cMSCs-derived EV subpopulations (EV20K and EV110K). The animals were sacrificed two weeks post-treatment and uterine horns were collected. The sections were taken, and hematoxylin–eosin was used to examine the repair of endometrial structure. Fibrosis was measured by Masson’s trichrome staining and α-SMA and cell proliferation by Ki67 immunostaining. The function of the uteri was explored by the result of mating trial test. Expression changes of TNFα, IL-10, VEGF, and LIF were assayed by ELISA.ResultsHistological analysis indicated fewer glands, thinner endometria, increased fibrotic areas, and decreased proliferation of epithelial and stroma of the uteri in the treated compared with intact and sham-operated animals. However, these parameters improved after transplantation of both types of cMSCs and hMSCs and/or both cryopreserved EVs subpopulations. The cMSCs demonstrated more successful implantation of the embryos in comparison with hMSCs. The tracing of the transplanted cMSCs and EVs showed that they migrated and localized in the uteri. Protein expression analysis results demonstrated downregulation of proinflammatory factor TNFα and upregulation of anti-inflammatory cytokine IL-10, and endometrial receptivity cytokines VEGF and LIF in cMSC- and EV20K-treated animals.ConclusionTransplantation of MSCs and EVs contributed to endometrial repair and restoration of reproductive function, likely by inhibition of excessive fibrosis and inflammation, enhancement of endometrial cell proliferation, and regulation of molecular markers related to endometrial receptivity. Compared to classical hMSCs, cMSCs were more efficient than hMSCs in restoration of reproductive function. Moreover, EV20K is more cost-effective and feasible for prevention of AS in comparison with conventional EVs (EV110K).

Automated Ki-67 labeling index assessment in prostate cancer using artificial intelligence and multiplex fluorescence immunohistochemistry.

The Ki-67 labeling index (Ki-67 LI) is a strong prognostic marker in prostate cancer, although its analysis requires cumbersome manual quantification of Ki-67 immunostaining in 200-500 tumor cells. To enable automated Ki-67 LI assessment in routine clinical practice, a framework for automated Ki-67 LI quantification, which comprises three different artificial intelligence analysis steps and an algorithm for cell-distance analysis of multiplex fluorescence immunohistochemistry (mfIHC) staining, was developed and validated in a cohort of 12,475 prostate cancers. The prognostic impact of the Ki-67 LI was tested on a tissue microarray (TMA) containing one 0.6mmsample per patient. A 'heterogeneity TMA' containing three to six samples from different tumor areas in each patient was used to model Ki-67 analysis of multiple different biopsies, and 30 prostate biopsies were analyzed to compare a 'classical' bright field-based Ki-67 analysis with the mfIHC-based framework. The Ki-67 LI provided strong and independent prognostic information in 11,845 analyzed prostate cancers (p< 0.001 each), and excellent agreement was found between the framework for automated Ki-67 LI assessment and the manual quantification in prostate biopsies from routine clinical practice (intraclass correlation coefficient: 0.94 [95% confidence interval: 0.87-0.97]). The analysis of the heterogeneity TMA revealed that the Ki-67 LI of the sample with the highest Gleason score (area under the curve [AUC]: 0.68) was as prognostic as the mean Ki-67 LI of all six foci (AUC: 0.71 [p= 0.24]). The combined analysis of the Ki-67 LI and Gleason score obtained on identical tissue spots showed that the Ki-67 LI added significant additional prognostic information in case of classical International Society of Urological Pathology grades (AUC: 0.82 [p= 0.002]) and quantitative Gleason score (AUC: 0.83 [p= 0.018]). The Ki-67 LI is a powerful prognostic parameter in prostate cancer that is now applicable in routine clinical practice. In the case of multiple cancer-positive biopsies, the sole automated analysis of the worst biopsy was sufficient. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Parathyroid Cancer—A Rare Finding during Parathyroidectomy in High Volume Surgery Centre

Background and Objectives: Parathyroid cancer is a very rare endocrine tumor, especially in patients with secondary hyperparathyroidism due to end stage renal disease failure. This pathology is difficult to diagnose preoperatively because it has nonspecific clinical manifestations and paraclinical aspects. Our study of the literature identified 34 reported cases of parathyroid carcinoma over the last 40 years in patients undergoing dialysis. We present our experience as illustrative of the features of clinical presentation and histopathological findings of parathyroid carcinoma and assess its management considering the recent relevant literature. Materials and Methods: From January 2012 to November 2022, 650 patients with secondary hyperparathyroidism undergoing dialysis were treated at our academic Department of General Surgery and only two cases of parathyroid carcinoma were diagnosed on histopathological examination. Results: All patients presented with symptomatic hypercalcemia, with no clinical or imaging suspicion of malignant disease and were surgically treated by total parathyroidectomy. Histopathological examination revealed morphologic aspects of parathyroid carcinoma in two cases and immunostaining of Ki-67 was performed for diagnostic confirmation. Postoperative follow-up showed no signs of recurrence and no oncological adjuvant treatment or surgical reinterventions were needed. Conclusions: Parathyroid neoplasia is a particularly rare disease, that remains a challenge when it comes to diagnosis and proper management. Surgical approach is the only valid treatment to remove the malignant tissue and thus improve the patient's prognosis. Medical and oncologic treatment may be beneficial to control hypercalcemia in case of tumor recurrence.

Cu,Zn-Superoxide Dismutase has Minimal Effects Against Cuprizone-Induced Demyelination, Microglial Activation, and Neurogenesis Defects in the C57BL/6 Mouse Hippocampus.

Cuprizone causes consistent demyelination and oligodendrocyte damage in the mouse brain. Cu,Zn-superoxide dismutase 1 (SOD1) has neuroprotective potential against various neurological disorders, such as transient cerebral ischemia and traumatic brain injury. In this study, we investigated whether SOD1 has neuroprotective effects against cuprizone-induced demyelination and adult hippocampal neurogenesis in C57BL/6 mice, using the PEP-1-SOD1 fusion protein to facilitate the delivery of SOD1 protein into hippocampal neurons. Eight weeks feeding of cuprizone-supplemented (0.2%) diets caused a significant decrease in myelin basic protein (MBP) expression in the stratum lacunosum-moleculare of the CA1 region, the polymorphic layer of the dentate gyrus, and the corpus callosum, while ionized calcium-binding adapter molecule 1 (Iba-1)-immunoreactive microglia showed activated and phagocytic phenotypes. In addition, cuprizone treatment reduced proliferating cells and neuroblasts as shown using Ki67 and doublecortin immunostaining. Treatment with PEP-1-SOD1 to normal mice did not show any significant changes in MBP expression and Iba-1-immunoreactive microglia. However, Ki67-positive proliferating cells and doublecortin-immunoreactive neuroblasts were significantly decreased. Simultaneous treatment with PEP-1-SOD1 and cuprizone-supplemented diets did not ameliorate the MBP reduction in these regions, but mitigated the increase of Iba-1 immunoreactivity in the corpus callosum and alleviated the reduction of MBP in corpus callosum and proliferating cells, not neuroblasts, in the dentate gyrus. In conclusion, PEP-1-SOD1 treatment only has partial effects to reduce cuprizone-induced demyelination and microglial activation in the hippocampus and corpus callosum and has minimal effects on proliferating cells in the dentate gyrus.

Chlorogenic acid improves intestinal morphology by enhancing intestinal stem-cell activity.

Chlorogenic acid (CGA), as one of the most abundant naturally occurring phenolic acids, has been documented to be beneficial for intestinal health. However, the underlying mechanism is still not fully understood. The adult intestinal stem cell is the critical driver of epithelial homeostasis and regeneration. This study hypothesized that CGA exerted intestinal health effects by modulating intestinal stem-cell functions. Lgr5-EGFP mice were treated for 14 days, and intestinal organoids derived from these mice were treated for 3 days, using CGA solution. In comparison with the control group, CGA treatment increased intestinal villous height and crypt depth in mice and augmented the area expansion and the number of budding intestinal organoids. Quantitative polymerase chain reaction (qPCR) analysis revealed that CGA treatment significantly increased the expression of genes coding intestinal stem-cell markers in intestinal tissue and organoids, and upregulated the expression of genes coding secretory cell lineages and enterocytes, although not statistically significantly. Fluorescence-activated cell-sorting analysis further confirmed that CGA augmented the number of stem cells. 5-Ethynyl-2'-deoxyuridine (EdU) incorporation and Ki67 immunostaining results also demonstrated that CGA treatment enhanced intestinal stem-cell proliferation. Altogether, our findings indicate that CGA could activate intestinal stem-cell and epithelial regeneration, which could contribute to the improvement of intestinal morphology or organoid growth of mice. This highlights a promising mechanism for CGA as an excellent candidate for the formulation of dietary supplements and functional foods for intestinal protection. © 2023 Society of Chemical Industry.

The Role of P16, P53, KI-67 and PD-L1 Immunostaining in Primary Vaginal Cancer.

To analyze clinical, pathological and immunohistochemical correlates of survival in vaginal cancer patients. Retrospective analysis of primary vaginal cancer patients, treated at the Department of Gynecology and Gynecological Oncology of the University Hospital Bonn between 2007 and 2021. The study cohort comprised 22 patients. The median age was 63 years (range: 32-87 years). Squamous cell histology was present in 20 patients. Five-year OS in Stage I, II, III and IV was 100%, 56.25%, 0% and 41.67%, respectively (p = 0.147). Five-year DFS was 100%, 50%, 0% and 20.83%, respectively (p = 0.223). The 5-year OS was significantly reduced in the presence of nodal metastasis (p = 0.004), lymphangiosis (p = 0.009), hemangiosis (p = 0.002) and an age above 64 years (p = 0.029). Positive p 16 staining was associated with significantly improved OS (p = 0.010). Tumoral and immune cell PD-L1 staining was positive in 19 and in 16 patients, respectively, without significant impact on OS; 2 patients with metastastic disease are long-term survivors treated with either bevacizumab or pembrolizumab. P16 expression, absence of lymph- or hemangiosis, nodal negative disease and an age below 64 years show improved survival rates in PVC. Tumoral PD-L1 expression as well as PD-L1 expression on immune cells is frequent in PVC, without impacting survival. Within our study cohort, long-term survivors with recurrent PVC are treated with anti-VEGF and immunotherapy.

Loss of RanGAP1 drives chromosome instability and rapid tumorigenesis of osteosarcoma.

Chromothripsis is a catastrophic event of chromosomal instability that involves intensive fragmentation and rearrangements within localized chromosomal regions. However, its cause remains unclear. Here, we show that reduction and inactivation of Ran GTPase-activating protein 1 (RanGAP1) commonly occur in human osteosarcoma, which is associated with a high rate of chromothripsis. In rapidly expanding mouse osteoprogenitors, RanGAP1 deficiency causes chromothripsis in chr1q, instant inactivation of Rb1 and degradation of p53, consequent failure in DNA damage repair, and ultrafast osteosarcoma tumorigenesis. During mitosis, RanGAP1 anchors to the kinetochore, where it recruits PP1-γ to counteract the activity of the spindle-assembly checkpoint (SAC) and prevents TOP2A degradation, thus safeguarding chromatid decatenation. Loss of RanGAP1 causes SAC hyperactivation and chromatid decatenation failure. These findings demonstrate that RanGAP1 maintains mitotic chromosome integrity and that RanGAP1 loss drives tumorigenesis through its direct effects on SAC and decatenation and secondary effects on DNA damage surveillance.

316Engineered extracellular vesicle-mediated delivery of miR-199a-3p increases the viability of 3D-printed cardiac patches.

In recent years, extrusion-based three-dimensional (3D) bioprinting is employed for engineering cardiac patches (CP) due to its ability to assemble complex structures from hydrogel-based bioinks. However, the cell viability in such CPs is low due to shear forces applied on the cells in the bioink, inducing cellular apoptosis. Herein, we investigated whether the incorporation of extracellular vesicles (EVs) in the bioink, engineered to continually deliver the cell survival factor miR-199a-3p would increase the viability within the CP. EVs from THP-1-derived activated macrophages (MΦ) were isolated and characterized by nanoparticle tracking analysis (NTA), cryogenic electron microscopy (cryo-TEM), and Western blot analysis. MiR-199a-3p mimic was loaded into EVs by electroporation after optimization of applied voltage and pulses. Functionality of the engineered EVs was assessed in neonatal rat cardiomyocyte (NRCM) monolayers using immunostaining for the proliferation markers ki67 and Aurora B kinase. To examine the effect of engineered EVs on 3D-bioprinted CP viability, the EVs were added to the bioink, consisting of alginate-RGD, gelatin, and NRCM. Metabolic activity and expression levels of activated-caspase 3 for apoptosis of the 3D-bioprinted CP were evaluated after 5 days. Electroporation (850 V with 5 pulses) was found to be optimal for miR loading; miR-199a-3p levels in EVs increased fivefold compared to simple incubation, with a loading efficiency of 21.0%. EV size and integrity were maintained under these conditions. Cellular uptake of engineered EVs by NRCM was validated, as 58% of cTnT+ cells internalized EVs after 24 h. The engineered EVs induced CM proliferation, increasing the ratio of cell-cycle re-entry of cTnT+ cells by 30% (Ki67) and midbodies+ cell ratio by twofold (Aurora B) compared with the controls. The inclusion of engineered EVs in bioink yielded CP with threefold greater cell viability compared to bioink with no EVs. The prolonged effect of EVs was evident as the CP exhibited elevated metabolic activities after 5 days, with less apoptotic cells compared to CP with no EVs. The addition of miR-199a-3p-loaded EVs to the bioink improved the viability of 3D-printed CP and is expected to contribute to their integration in vivo.

Efficacy of Ki-67 immunostaining in the diagnosis of primary myoepithelial carcinoma of the parotid gland after surgery for metastatic lung tumor: A case report

Efficacy of Ki-67 immunostaining in the diagnosis of primary myoepithelial carcinoma of the parotid gland after surgery for metastatic lung tumor: A case report

Evaluation of variable p16 immunostaining patterns, Ki-67 indices and HPV status in cervical SILs and squamous cell carcinomas: An institutional experience.

The present study evaluates the immunoexpression of p16 and Ki-67 in cervical squamous intraepithelial lesion (SIL) and carcinomas and correlates their expression with clinicopathological features and HPV-DNA status. A total 36 included cases of SIL and squamous cell carcinoma (SCC) were subjected to p16 and Ki-67 immunostaining. p16 staining was evaluated depending on grading, distribution, localization pattern, intensity and IHC score. Ki-67 expression was graded based on percentage of positive cells. Incidence of HSIL and SCC cases was found to be significantly increased with parity > 5. p16 grade III diffuse nucleocytoplasmic immunostaining was observed in 62.5% LSIL, 80% HSIL and 87% SCC cases. Significant association of p16 staining intensity, IHC score and Ki-67 indices was noted with increasing grades of SILs and carcinomas. Our experience indicates that a combination of p16 and Ki-67 immunostaining may be useful to determine the severity of dysplastic change.

Pioglitazone Induces Dysplastic Urothelial Changes in Urinary Bladder of Experimental Diabetes.

Pioglitazone (PIO) is a widely prescribed oral antidiabetic drug that has concerns regarding a potential risk of developing carcinoma of the urinary bladder. The objective of the current study was to assess this potential risk. The potential risk of PIO-induced urinary bladder carcinoma was assessed in the current study by examining urinary bladder of rats for urothelial cytokeratin (CK) expression and proliferative activity by Ki67 immunostaining. Histological examination revealed dysplastic urothelial changes in PIO per se and diabetes mellitus + PIO (diabetic rats receiving PIO). In addition, a significantly (P < 0.05) decreased CK7 and CK8 expression together with a significantly increased CK20 as well as Ki67 expression was detected in the urothelial cells of groups administrated PIO, contrary to those which did not. The manifestations of urothelial dysplasia evidenced by histological examination as well as by the aberrant expression in CK and Ki67 after PIO administration add supporting evidence at cellular and experimental level to the previous clinical suspicions.

Evaluation of Nuclear Morphometry and Ki-67 Proliferative Marker in Astrocytomas: A Ambispective Study

Introduction: Astrocytomas are the commonest primary Central Nervous System (CNS) tumours. Its diagnosis is based on histopathological criteria defined by the World Health Organization (WHO) 2016 that grades astrocytoma into four grades. The subjective nature of WHO grading has prompted for more objective methods to evaluate nuclear features. Furthermore, Ki-67, a marker of cellular proliferation is a useful diagnostic tool that also helps in prognostic evaluation and to plan adjuvant therapy in astrocytomas. Aim: To evaluate the nuclear morphometry and Ki-67 proliferative marker in astrocytomas and to assess the relationship of WHO grade with proliferative activity using Ki-67 immunostaining. Materials and Methods: This ambispective study was conducted in the Department of Pathology, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India, with a total duration of four years (October 2017 to September 2021). From 35 astrocytoma cases, Haematoxylin and Eosin (H&amp;E) stained slides were retrieved and reviewed appropriately by two pathologists and graded using the WHO criteria. Nuclear morphometric analysis was performed on the H&amp;E slides using Olympus BX-41 research microscope. The parameters evaluated were mean nuclear length, mean nuclear diameter, mean nuclear perimeter, mean nuclear area, Mean Nuclear Roundness Factor (MNRF) and mean nuclear ellipse form. The cases were then stained with Ki-67 antibody. The relationships between WHO grade of astrocytoma and nuclear morphometry and WHO grade with proliferative index was analysed. Results: According to WHO grading, four cases were Grade 1 (11.4%), 12 grade 2 (34.3%), six grade 3 (17.1%) and 13 grade 4 (37.1%). Significant correlation was seen between WHO grading and mean nuclear length, diameter, perimeter, area and Ki-67 with p-value of less than 0.001. Conclusion: Astrocytoma is an extremely heterogeneous disease with unpredictable outcome. The widely used WHO grading is subjective, while nuclear morphometry, using computer assisted image analysis, can ensure more objective assessment. The Ki-67 index could provide valuable information and may compliment the other parameters.

Gastroprotective and anti-Helicobacter pylori potentials of essential oils from the oleoresins of Araucaria bidwillii and Araucaria heterophylla

Plant resins or oleoresins comprise a chemically complex mixture of different classes of compounds. Oleoresin of the genus Araucaria combines essential oil (EO) and resin. It possesses gastroprotective, cytotoxic, and timicrobial, antipyretic, and anti-inflammatory activities. The study aimed to investigate the EOs from the oleoresins of two Araucaria species, A. bidwillii and A. heterophylla, chemically and biologically for their gastroprotective, anti-inflammatory, antioxidant, and anti-Helicobacter pylori potentials. The chemical composition of both species cultivated in Egypt was analyzed with GC-MS and compared with those cultivated abroad using principal component analysis (PCA). There were 37 and 17 secondary metabolites identified in A. heterophylla and A. bidwillii, respectively. The EOs of both species showed a pronounced inhibitory effect on Helicobacter pylori activity in vitro. The gastroprotective effect was assessed in vivo using ethanol-induced gastric ulcer model in rats. Inflammatory cytokines, oxidative stress, and the nuclear factor-kappa B (NF-κB) biomarkers were assessed in the stomach tissues. The ulcer index and percentage of ulcer protection were determined. Stomach sections were examined histopathologically by staining with (H/E) and periodic acid Schiff (PAS). Moreover, the proliferative index was determined using the Ki-67 immunostaining. The treatment of rats with EOs (50, 100, and 200 mg/kg, orally) 1 hour prior to ethanol administration showed promising gastroprotective, anti-inflammatory, and antioxidant potentials. These findings declared the gastroprotective role played by both EOs with the superiority of A. bidwillii over A. heterophylla via modulation of oxidative stress/NF-κB/inflammatory cytokines. Their use can be recommended to protect against the recurrence of peptic ulcers.

T3 and glucose increase expression of phosphoenolpyruvate carboxykinase (PCK1) leading to increased β-cell proliferation

ObjectivesThyroid hormone (T3) and high glucose concentrations are critical components of β-cell maturation and function. In the present study, we asked whether T3 and glucose signaling pathways coordinately regulate transcription of genes important for β-cell function and proliferation. MethodsRNA-seq analysis was performed on cadaveric human islets from five different donors in response to low and high glucose concentrations and in the presence or absence of T3. Gene expression was also studies in sorted human β-cells, mouse islets and Ins-1 cells by RT-qPCR. Silencing of the thyroid hormone receptors (THR) was conducted using lentiviruses. Proliferation was assessed by ki67 immunostaining in primary human/mouse islets. Chromatin immunoprecipitation and proximity ligation assay were preformed to validate interactions of ChREBP and THR. ResultsWe found glucose-mediated expression of carbohydrate response element binding protein alpha and beta (ChREBPα and ChREBPβ) mRNAs and their target genes are highly dependent on T3 concentrations in rodent and human β-cells. In β-cells, T3 and glucose coordinately regulate the expression of ChREBPβ and PCK1 (phosphoenolpyruvate carboxykinase-1) among other important genes for β-cell maturation. Additionally, we show the thyroid hormone receptor (THR) and ChREBP interact, and their relative response elements are located near to each other on mutually responsive genes. In FACS-sorted adult human β-cells, we found that high concentrations of glucose and T3 induced the expression of PCK1. Next, we show that overexpression of Pck1 together with dimethyl malate (DMM), a substrate precursor, significantly increased β-cell proliferation in human islets. Finally, using a Cre-Lox approach, we demonstrated that ChREBPβ contributes to Pck1-dependent β-cell proliferation in mouse β-cells. ConclusionsWe conclude that T3 and glucose act together to regulate ChREBPβ, leading to increased expression and activity of Pck1, and ultimately increased β-cell proliferation.

GATA3 ameliorates melanocyte injuries in vitiligo through SIRT3-mediated HMGB1 deacetylation.

Vitiligo is a skin depigmentation disorder. GATA3 expression is downregulated in vitiligo patients, and its role and regulatory mechanism in vitiligo are unclear. GATA3 and HMGB1 levels were detected by qRT-PCR in peripheral blood cells of vitiligo patients and healthy controls, as well as H2 O2 -treated PIG1 cells. Their expression correlation was assessed by Pearson analysis. qRT-PCR, MTT assay, Ki67 immunostaining, flow cytometry, ELISA and Western blot were applied to determine GATA3 expression, cell survival, cell proliferation, cell apoptosis, melanin contents, and melanin-related protein expressions. The cellular distributions of HMGB1 and its deacetylation levels were detected by Western blot. The binding of GATA3 to SIRT3 promoter and effects on SIRT3 expression and HMGB1 deacetylation was determined by dual-luciferase assay, ChIP assay, and Western blot. GATA3 was decreased, and HMGB1 was increased in vitiligo. Pearson correlation assay showed that they were negatively correlated. H2 O2 significantly inhibited cell survival, proliferation, melanin secretion, and melanin-related protein expressions but remarkably increased cell apoptosis. GATA3 overexpression could distinctly reverse the effects of H2 O2 through decreasing HMGB1 expression and retained HMGB1 in nuclear due to the decreased HMGB1 acetylation. GATA3 bound to the SIRT3 and subsequently decreased H2 O2 -induced HMGB1 acetylation. Overexpressing HMGB1 or knockdown of SIRT3 could reverse the effects of GATA3 overexpression. GATA3 inhibited H2 O2 -induced injury in PIG1 cells and enhanced melanin secretion by SIRT3-regulated HMGB1 deacetylation, which might provide new evidence to treat vitiligo.

Smoking habits do not affect biological responses induced by leucocyte and platelet-rich fibrin in periodontal ligament cells.

Leucocyte- and platelet-rich fibrin has been developed to stimulate wound healing response. However, it is currently unknown whether smoking affects the biological responses elicited by leucocyte- and platelet-rich fibrin on periodontal ligament-derived mesenchymal stromal cells. This study analyzes the kinetics of biomolecule release from leucocyte- and platelet-rich fibrin derived from smokers and nonsmokers and their effect on periodontal ligament cell proliferation and migration as essential biological activities during wound healing. Biomolecules present in leucocyte- and platelet-rich fibrin exudates and conditioned media collected from smokers and nonsmokers were analyzed by Luminex arrays. Periodontal ligament-derived mesenchymal stromal cell obtained from one nonsmoker were treated with leucocyte- and platelet-rich fibrin exudates or leucocyte- and platelet-rich fibrin conditioned media derived from both smokers and nonsmokers. The parameters evaluated included cell proliferation, determined by Ki67 immunostaining and migration assessed using transwell assays. Also, cells were treated with nicotine in the presence of fetal bovine serum 10% or leucocyte- and platelet-rich fibrin conditioned media. A similar biomolecular profile was detected in leucocyte- and platelet-rich fibrin exudates and leucocyte- and platelet-rich fibrin conditioned media from smokers and nonsmokers, stimulating (periodontal ligament-derived mesenchymal stromal cell) proliferation, and migration to a comparable degree. Nicotine reduced cell proliferation and migration of periodontal cells; however, this effect was recovered in the presence of leucocyte- and platelet-rich fibrin conditioned media. Leucocyte- and platelet-rich fibrin derived from smokers could be an autologous source of biomolecules to stimulate cell biological activities involved in wound healing in smokers who have difficulties in ceasing this habit. Clinical trials are required to evaluate the impact of leucocyte- and platelet-rich fibrin on healing responses in smokers.