Changes In Secretion Research Articles

Airway Secretory microRNAome Changes during Rhinovirus Infection in Early Childhood.

BackgroundInnate immune responses are fine-tuned by small noncoding RNA molecules termed microRNAs (miRs) that modify gene expression in response to the environment. During acute infections, miRs can be secreted in extracellular vesicles (EV) to facilitate cell-to-cell genetic communication. The purpose of this study was to characterize the baseline population of miRs secreted in EVs in the airways of young children (airway secretory microRNAome) and examine the changes during rhinovirus (RV) infection, the most common cause of asthma exacerbations and the most important early risk factor for the development of asthma beyond childhood.MethodsNasal airway secretions were obtained from children (≤3 yrs. old) during PCR-confirmed RV infections (n = 10) and age-matched controls (n = 10). Nasal EVs were isolated with polymer-based precipitation and global miR profiles generated using NanoString microarrays. We validated our in vivo airway secretory miR data in an in vitro airway epithelium model using apical secretions from primary human bronchial epithelial cells (HBEC) differentiated at air-liquid interface (ALI). Bioinformatics tools were used to determine the unified (nasal and bronchial) signature airway secretory miRNAome and changes during RV infection in children.ResultsMultiscale analysis identified four signature miRs comprising the baseline airway secretory miRNAome: hsa-miR-630, hsa-miR-302d-3p, hsa- miR-320e, hsa-miR-612. We identified hsa-miR-155 as the main change in the baseline miRNAome during RV infection in young children. We investigated the potential biological relevance of the airway secretion of hsa-mir-155 using in silico models derived from gene datasets of experimental in vivo human RV infection. These analyses confirmed that hsa-miR-155 targetome is an overrepresented pathway in the upper airways of individuals infected with RV.ConclusionsComparative analysis of the airway secretory microRNAome in children indicates that RV infection is associated with airway secretion of EVs containing miR-155, which is predicted in silico to regulate antiviral immunity. Further characterization of the airway secretory microRNAome during health and disease may lead to completely new strategies to treat and monitor respiratory conditions in all ages.

A Word About Bioidenticals

A Word About Bioidenticals

Perspectives in Polycystic Ovary Syndrome: From Hair to Eternity.

Polycystic ovary syndrome (PCOS) is a common complex genetic disease. It is characterized by hyperandrogenism, gonadotropin secretory changes, polycystic ovarian morphology, and insulin resistance. The etiology of PCOS remains unknown, but modern genetic approaches, such as genome-wide association studies (GWAS), Mendelian randomization, and next-generation sequencing, promise to identify the pathways that are primarily disrupted. The literature on PCOS, including the author's research, is discussed. Recent genetic analyses are reviewed. Considerable progress has been made mapping PCOS susceptibility genes. GWAS have implicated gonadotropin secretion and action as important primary defects in disease pathogenesis in European and Han Chinese PCOS cohorts, respectively. European women with the National Institutes of Health and Rotterdam phenotypes as well as those with self-reported PCOS have some gene regions in common, such as chromosome 11p14.1 region containing the FSH B polypeptide (FSHB) gene, suggesting shared genetic susceptibility. Several chromosomal signals are significant in both Han Chinese and European PCOS cohorts, suggesting that the susceptibility genes in these regions are evolutionarily conserved. In addition, GWAS have suggested that DENND1A, epidermal growth factor signaling, and DNA repair pathways play a role in PCOS pathogenesis. Only a small amount of the heritability of PCOS is accounted for by the common susceptibility variants mapped so far. Future studies should clarify the contribution of rare genetic variants and epigenetic factors to the PCOS phenotype. Furthermore, Mendelian randomization can be used to clarify causal relationships, and phenome-wide association studies can provide insight into health risks associated with PCOS susceptibility variants.

Autologous stem cells therapy, The first baby of idiopathic premature ovarian failure

Introduction: Stem cells (sc) are the foundation cells for every organ, tissue and cell in the body. They are self sustaining and can replicate themselves for long periods of time. Stem cells can differentiate into different types of cells. Women below the age of forty and showing ovarian function loss are defined to have premature ovarian failure (POF). It is associated with sex steroid deficiency, amenorrhea, infertility, and elevated serum gonadotropins. Aim: To evaluate the therapeutic potential of autologous Mesenchymal sc (MSC) transplantation in women suffering from POF. Out of 112 high risk patients for POF(cases with amenorrhoea befor the age of forty), diagnosis was confirmed in10 cases. The ten POF patients were scheduled for MSC transplantation at Al- Azhar University Hospitals. MSC preparation from the bone marrow of the iliac crest was laparoscopically injected into the ovaries. Endometrial fractional biopsy was histopathologicaly (HP) and Immunohistochemically (IH) stained and evaluated according to Edessy stem cells score (ESS). Ovarian reserve was evaluated according to Edessy ovarian reserve score (EORS). Results: Showed that after transplantation two cases (20%) (ESS = 5 and 6) resumed menstruation after 3 months, one of them (10%) (Case no 5) (ESS = 6) got pregnancy after 11 months and delivered a healthy full term baby (Zeinab).Ten months after transplantation EORS of patient who developed pregnancy (case no 6) was found to be 7 after being 0 before therapy. EORS of the other menstruating case (case no 10) was 5 after being 0. The 2 menstruating cases showed focal secretory changes after being atrophic endometrium in case 5 and distorted proliferative endometrium in case 10. Conclusion: Stem cell transplantation is a good procedure and regarded as a real and hope to get healthy pregnancy and baby for cases of POF. It showed good clinical, HP and IH outcome.

In silico, in vitro and in vivo analysis identifies a potential role for steroid hormone regulation of FOXD3 in endometriosis-associated genes.

Can bioinformatics analysis of publically available microarray datasets be utilized in identifying potentially important transcription factors (TF) in the hormonal regulation of the endometrium? Systems integration and analysis of existing complex (published) datasets, predicted a role for the novel transcription factor, Forkhead Box D3 (FOXD3) in healthy endometrium and in endometriosis, which was followed by the demonstration of decreased levels of the protein upon decidualisation of normal human endometrial stromal cells in vitro and differential endometrial expression in the stroma in endometriosis. The reported endometriosis-associated endometrial aberrations are most pronounced in the progesterone-dominant secretory phase and progesterone resistance is a proposed causative factor. The study was initially an 'in silico' study, with confirmatory 'wet lab' data from western blotting (WB), qPCR and Immunohistochemistry (IHC) on endometrial biopsies obtained from 142 women undergoing gynaecological surgery. The study was conducted at a University Research Institute. Bioinformatic analysis of selected published microarray datasets identified differentially regulated genes for the early and mid-secretory phases relative to the proliferative phase. Diseases and Functions categories were identified with Ingenuity (IPA) 'core analysis' software. The key transcription factors controlling secretory phase gene changes were revealed with oPOSSUM software. FOXD3 expression levels were examined in human endometrial samples from women aged 18-55 years by WB, IHC, and qPCR. The progesterone regulation of endometrial FOXD3 levels was examined in vivo and in cultured primary human endometrial stromal cells in vitro. Initial data mining and subsequent bioinformatics analysis of human endometrial microarray datasets identified FOXD3 to be a key regulator of gene expression specific to secretory phase/endometriosis. FOXD3 was dynamically expressed in healthy endometrium and differentially expressed in endometriosis. In vitro decidualisation of primary endometrial stromal cells significantly decreased FOXD3 protein (P = 0.0005) and progestagen (Levonorgestrel) treatment also reduced the high endometrial FOXD3 protein (P = 0.0001) and mRNA levels (P = 0.04) seen in untreated women with endometriosis, with a shift of FOXD3 from the nucleus to the cytoplasm. The quality of Bioinformatics analysis and results depends on the published micro-array data. An in depth analysis of FOXD3 function and its relationship with estrogen and progesterone might provide insights into its potential deregulation in proliferative disorders of the endometrium including endometrial cancer where its expression is also deregulated. Further, FOX transcription factors are increasingly seen as novel therapeutic targets in disease. We acknowledge the support by Wellbeing of Women project grant RG1073 (D.K.H., A.J.V.). We also acknowledge the support of Liverpool Women's Hospital Foundation Trust (J.A.D.), Institute of Translational Medicine (D.M., A.J.V., D.K.H.) and the Institute of Integrative Biology (O.V.), University of Liverpool. All authors declare no conflict of interest. N/A.

Rapid Anti-Inflammatory Effects of Gonadotropin-Releasing Hormone Antagonism in Rheumatoid Arthritis Patients with High Gonadotropin Levels in the AGRA Trial.

ObjectivesGonadotropin-releasing hormone (GnRH) and pituitary gonadotropins, which appear to be proinflammatory, undergo profound secretory changes during events associated with rheumatoid arthritis (RA) onset, flares, or improvement e.g. menopausal transition, postpartum, or pregnancy. Potential anti-inflammatory effects of GnRH-antagonists may be most pronounced in patients with high GnRH and gonadotropin levels. Therefore, we investigated the efficacy and safety of a GnRH-antagonist, cetrorelix, in RA patients with high gonadotropin levels.MethodsWe report intention-to-treat post hoc analyses among patients with high gonadotropin levels (N = 53), i.e. gonadotropin levels>median, from our proof-of-concept, double-blind AGRA-study (N = 99). Patients with active longstanding RA, randomized to subcutaneous cetrorelix (5mg days1–2; 3mg days 3–5) or placebo, were followed through day 15. Only predefined primary and secondary endpoints were analyzed.ResultsThe primary endpoint, Disease Activity Score of 28-joint counts with C-reactive protein (DAS28-CRP), improved with cetrorelix compared with placebo by day 5 (-1.0 vs. -0.4, P = 0∙010). By day 5, more patients on cetrorelix achieved at least a 20% improvement in the American College of Rheumatology scale (44% vs. 19%, P = 0.049), DAS28-CRP≤3.2 (24% vs. 0%, P = 0.012), and European League against Rheumatism ‘Good-responses’ (19% vs. 0%, P = 0.026). Tumor necrosis factor-α, interleukin-1β, interleukin-10, and CRP decreased with cetrorelix (P = 0.045, P = 0.034, P = 0.020 and P = 0.042 respectively) compared with placebo by day 15. Adverse event rates were similar between groups.ConclusionsGnRH-antagonism produced rapid anti-inflammatory effects in RA patients with high gonadotropin levels. GnRH should be investigated further in RA.Trial RegistrationClinicalTrials.gov NCT00667758

Cambios endometriales anormales producidos en la rata por estrógenos y hormona coriónica

La administración de hormona coriónica humana y estrógeno, a ratas normales, produce un cuadro histológico endometrial atípico, caracterizado por presencia simultánea de cambios proliferativos y secretorios. El núcleo de las células epiteliales muestra en general un aspecto más vesicular que lo normal y ocasionalmente alcanza gran tamaño. El cuadro anormal no puede ser inducido tratando a las ratas normales con estrógenos u hormonas coriónicas aisladamente o administrando ambas a animales castrados. Una alteración similar se ve, sin embargo, cuando ratas ooforectomizadas son tratadas con grandes dosis de estrógenos y progesterona. Se discute brevemente la patogenia y la posible relación de esta alteración con ciertos cambios endometriales observados en seres humanos.

Endometrial evaluation by ultrasonography, hysteroscopy and histopathology in cases of breast carcinoma on Tamoxifen therapy.

Introduction:Tamoxifen, a nonsteroidal antiestrogenic agent, is used widely as adjunctive therapy for women with breast cancer. Most studies have found that the increased relative risk of developing endometrial cancer for women taking Tamoxifen is two to three times higher than that of an age-matched population. So we designed this study to assess the endometrial status in patients taking Tamoxifen for breast carcinoma.Material and Methods:The study was conducted at Govt. Medical College and Rajindra Hospital, Patiala, India. A total of 50 patients of Ca Breast taking Tamoxifen were selected as per study criterion and TVS performed. If endometrial thickness was more than 5 mm hysteroscopy and endometrial HPE was done and data analysed.Results:On ultrasonography 35 patients (70%) had an endometrial thickness up to 5 mm. 15 patients (30%) had an endometrial thickness more than 5mm. Out of these, 11 patients, i.e. 22% of total, had an endometrial thickness of 5.1 to 10 mm and 2 patients, i.e. 4% of total had an endometrial thickness of more than 20 mm. Hysteroscopy was done on 11 patients. Out of these 8 patients had a normal hysteroscopic appearance whereas 3 patients had an abnormal hysteroscopic picture. Endometrial HPE of these 11 patients revealed 2 patients had secretory changes, 1 had polyp change, 1 had atrophic endometrium, 3 had simple endometrial hyperplasia, 1 had endometrial adenocarcinoma and 4 patients were reported to have scanty curetting.Conclusion:The duration of Tamoxifen therapy turned out to have a relationship with the incidence of endometrial carcinoma (P < 0.0001). Also, a relationship was observed between the duration of Tamoxifen therapy and symptom status of the patients (P < 0.0001). This correlation did not extend to duration of Tamoxifen therapy and endometrial thickness. (P = 0.190). This correlation did not extend to duration of Tamoxifen therapy and endometrial thickness. (P = 0.190).

Insulin replacement restores the vesicular secretory apparatus in the diabetic rat lacrimal gland.

In the lacrimal gland (LG) acinar cells, signaling regulates the release of secretory vesicles through specific Rab and SNARE exocytotic proteins. In diabetes mellitus (DM), the LGs are dysfunctional. The aim of this work was to determine if secretory apparatus changes were associated with any effects on the secretory vesicles (SV) in diabetic rats as well as the expression levels of constituent Rab and members of the SNARE family, and if insulin supplementation reversed those changes. DM was induced in male Wistar rats with an intravenous dose of streptozotocin (60 mg/kg). One of the two diabetic groups was then treated every other day with insulin (1 IU). A third control group was injected with vehicle. After 10 weeks, Western blotting and RT-PCR were used to compared the Rab and SNARE secretory factor levels in the LGs. Transmission electron microscopy evaluated acinar cell SV density and integrity. In the diabetes mellitus group, there were fewer and enlarged SV. The Rab 27b, Rab 3d, and syntaxin-1 protein expression declined in the rats with diabetes mellitus. Insulin treatment restored the SV density and the Rab 27b and syntaxin expression to their control protein levels, whereas the Vamp 2 mRNA expression increased above the control levels. Diabetes mellitus LG changes were associated with the declines in protein expression levels that were involved in supporting exocytosis and vesicular formation. They were partially reversed by insulin replacement therapy. These findings may help to improve therapeutic management of dry eye in diabetes mellitus.

Cambios endometriales anormales producidos en la rata por estrógenos y hormona criónica

La administración de hormona coríónica humana y estrógeno a ratas normales, produce un cuadro histológico endometrial atípico caracterizado por presencia simultánea de cambios proliferativos y secretorios. El núcleo de las células epiteliales muestra en general un aspecto más vesicular que lo normal y ocasionalmente alcanza gran tamaño. El cuadro anormal no puede ser inducido tratando a las ratas normales con estróqenos u hormones coriónicos aisladamente o administrando ambas a animales castrados. Una alteración similar se ve, sin embargo; cuando ratas ooforectomizadas son tratadas con grandes dosis de estrógenos y progesterona. Se discute brevemente la patogenia y la posible relacíón de esta alteración con ciertos cambios endometriales observados en seres humanos.

Growth and development of the barnacle Amphibalanus amphitrite: time and spatially resolved structure and chemistry of the base plate

The radial growth and advancement of the adhesive interface to the substratum of many species of acorn barnacles occurs underwater and beneath an opaque, calcified shell. Here, the time-dependent growth processes involving various autofluorescent materials within the interface of live barnacles are imaged for the first time using 3D time-lapse confocal microscopy. Key features of the interface development in the striped barnacle, Amphibalanus (= Balanus) amphitrite were resolved in situ and include advancement of the barnacle/substratum interface, epicuticle membrane development, protein secretion, and calcification. Microscopic and spectroscopic techniques provide ex situ material identification of regions imaged by confocal microscopy. In situ and ex situ analysis of the interface support the hypothesis that barnacle interface development is a complex process coupling sequential, timed secretory events and morphological changes. This results in a multi-layered interface that concomitantly fulfills the roles of strongly adhering to a substratum while permitting continuous molting and radial growth at the periphery.

HOXA10 Regulates Expression of Cytokeratin 15 in Endometrial Epithelial Cytoskeletal Remodeling

The mammalian cytoskeleton is composed in part from keratin filaments which form a complex, highly dynamic intracellular network. We investigate the expression of cytokeratin 15 (CK15) in human endometrium and its regulation by HOXA10 in the human endometrial cell lines. Endometrial biopsies from throughout the menstrual cycle (N = 32) were evaluated for CK15 protein expression by immunohistochemistry using a mouse monoclonal antibody. The human endometrial epithelial cell line (Ishikawa) was transfected with pcDNA/HOXA10. Total RNA was isolated and quantitative real-time polymerase chain reaction was performed to determine expression levels of CK15. In the peri-implantation window (days 16 through 23) CK15 protein expression in glandular epithelium of human endometrium decreased to 50% of proliferative phase expression levels. Expression of CK15 messenger RNA decreased by 99% (P < .05) after pcDNA/HOXA10 transfection of Ishikawa cells. The CK15 expression corresponded to the time of maximal secretory epithelial remodeling. Gene expression of CK15 is decreased in a HOXA10-dependent fashion in human endometrial epithelial cells. Expression decreases in the peri-implantation period concurrent with maximal HOXA10 expression. Dramatic changes in cellular architecture are necessary to achieve the secretory changes in the endometrial epithelium that bring about the implantation window. Alterations in CK15 likely facilitate these cytoskeletal changes, ultimately promoting endometrial receptivity.

Progesterone Threshold Determines Nucleolar Channel System Formation in Human Endometrium

Nucleolar channel systems (NCSs), micron-sized organelles specific to nuclei of human endometrial epithelial cells (EECs), are robust markers of the midluteal phase under the apparent control of progesterone. To gain further insight into the role of progesterone in NCS formation, we quantitatively assessed their sensitivity to oral contraceptive pills (OCPs) using immunofluorescence-based detection of NCSs. Comparison of endometrial biopsies and serum progesterone levels on cycle day (CD) 10 and 20 (LH +6/7) of 6 naturally cycling women and 6 OCP users demonstrated that OCPs interfered with NCS formation on CD20, their natural peak presence. Although this confirmed prior observation based on electron microscopic sampling, OCPs unexpectedly induced limited but distinct amounts of NCSs already on CD10, when they are never present in natural cycles. Thus, OCPs can cause secretory changes in the endometrium during the proliferative phase. In a novel finding, robust NCS formation on CD20 was dependent on a 4 ng/mL progesterone threshold but did not correlate linearly with serum progesterone levels. Given the threshold being close to that serving as evidence for ovulation, NCSs can serve as ovulation markers.

Loss of Slc26a9 anion transporter alters intestinal electrolyte and HCO3(-) transport and reduces survival in CFTR-deficient mice.

Slc26a9 is an anion transporter that is strongly expressed in the stomach and lung. Slc26a9 variants were recently found associated with a higher incidence of meconium ileus in cystic fibrosis (CF) infants, raising the question whether Slc26a9 is expressed in the intestine and what its functional role is. Slc26a9 messenger RNA (mRNA) was found highly expressed in the mucosae of the murine and human upper gastrointestinal tract, with an abrupt decrease in expression levels beyond the duodenum. Absence of SLC26a9 expression strongly increased the intestinally related mortality in cystic fibrosis transmembrane conductance regulator (CFTR)-deficient mice. Proximal duodenal JHCO3 − and fluid secretion were reduced in the absence of Slc26a9 expression. In the proximal duodenum of young Slc26a9 KO mice, the glands and villi/crypts were elongated and proliferation was enhanced. This difference was lost with ageing, as were the alterations in fluid movement, whereas the reduction in JHCO3 - remained. Laser dissection followed by qPCR suggested Slc26a9 expression to be crypt-predominant in the duodenum. In summary, deletion of Slc26a9 caused bicarbonate secretory and fluid absorptive changes in the proximal duodenal mucosa and increased the postweaning death rates in CFTR-deficient mice. Functional alterations in the duodenum were most prominent at young ages. We assume that the association of meconium ileus and Slc26a9 variants may be related to maldigestion and impaired downstream signaling caused by loss of upper GI tract digestive functions, aggravating the situation of lack of secretion and sticky mucus at the site of obstruction in CF intestine.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-014-1543-x) contains supplementary material, which is available to authorized users.

Fine needle aspiration of breast masses in pregnant and lactating women: experience with 28 cases emphasizing Thinprep findings.

Fine needle aspiration (FNA) of breast masses in pregnant or lactating women is an uncommon procedure, and cytological interpretation is considered problematic due to atypia inherent to secretory change in glandular epithelia. Previous descriptions of "lactating adenoma" (LAd), the most common tumor in this population, have been on direct smears (DS), while ThinPrep (TP, Hologic, Boxborough, MA) findings therein remain largely uncharacterized. FNA cases from breast masses in pregnant or lactating women (2005-2012), processed as TP and/or DS were retrospectively reviewed. 28 cases from as many patients (mean age 36 years), at 23 weeks of pregnancy to 10 months postpartum, were reviewed. Size of mass ranged from 1.0 to 4.5 cm. Corresponding histopathology was available in 21/28 cases. TP was available in 24/28 cases. Relative to DS, in TP, LAd showed "lacy" fragments, tissue paper-like texture, and globular clumps of "milky" background material, with embedded singly dispersed "bare" epithelial cell nuclei containing cherry-red macronucleoli. Architecture appeared disrupted in TP with isolated cells, smaller cell clusters, and lobules in LAd. Cellular morphology was better preserved in TP. Cytological features of carcinoma on TP were similar to DS. There were no false-positive cases. In this series, LAd was the most common diagnosis for breast masses in pregnant and lactating women (78.5%) and demonstrated background, architectural, and cellular alterations on TP. In this setting, malignancy is an important consideration (encountered in 3/28, 11% of cases, including one false-negative angiosarcoma case).

Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes

Purpose: Recently, it has been suggested that chondrocyte senescence plays an important role in the pathogenesis and development of Osteoarthritis (OA). SIRT6 is a member of sirtuin family of NAD+-dependent protein deacetylase. Recently, SIRT6 has been implicated in DNA repair, telomere maintenance, attenuation of inflammation and glucose homeostasis. Notably studies in mice have shown that SIRT6 deficiency produces premature aging phenotypes, whereas SIRT6 overexpression causes a moderate increase in the lifespan of male mice, strongly suggesting an important role of SIRT6 in aging and aging-associated diseases. However, its role in chondrocytes has so far not been explored. The purpose of this study is to examine the role of SIRT6 in human chondrocytes by inhibiting SIRT6 with a RNA interference (RNAi) technique. The hypothesis of the present study is that SIRT6 has a protective function toward human chondrocytes Methods: OA cartilage tissues were obtained from varus knee OA during total knee arthroplasty. Lateral and medial femoral condyles were used as cartilage with mild and severe OA, respectively. Articular cartilage tissues without OA were obtained from the patients underwent surgery for femoral neck fracture. These were used for histological experiments. Next, SIRT6 was depleted by RNAi and examined the influence of SIRT6 depletion on gene expression changes, proliferation, and senescence in normal human chondrocytes. Normal Human Articular Chondrocytes-knee (NHAC-kn) was used as a normal human chondrocytes. RNA or proteins were extracted 48 hours after the lipofection, and real-time PCR and western blotting analysis were respectively performed. The proliferation activity was examined using a Cell Counting Kit-8. Optical density was measured at 0, 24, 48, 72, 96 hours after the lipofection. Senescence associated β-Galactosidase (SA-β-Gal) staining was examined using a SA-β-Gal staining kit. Furthermore, to detect DNA damage and telomere dysfunction, the γH2AX foci and telomere dysfunction-induced foci (TIFs) where γH2AX foci co-localized with telomere repeat binding factor-1 (TRF-1) were examined using an immunofluorescence confocal microscopy. Results: Immunohistochemical analysis showed that SIRT6-positive cells were observed in normal cartilage, and mild and severe OA cartilage. In real-time PCR analysis, the depletion of SIRT6 significantly increased expression of matrix metalloproteinase (MMP) -1 and 13 mRNA. The proliferation assay showed that the absorbance was significantly decreased by the depletion of SIRT6 at 72 and 96 hours after lipofection, indicating reduced proliferation by the depletion of SIRT6. SA-β-Gal assay showed that the percentage of SA-β-Gal positive cells was significantly increased by the depletion of SIRT6, indicating that the depletion of SIRT6 induced premature senescence. Immunofluorescence microscopic analysis showed that γH2AX foci and TIFs was significantly increased in the SIRT6-depleted chondrocytes compared with control. Furthermore, to examine the downstream signaling pathways that mediate the induction of senescence by the depletion of SIRT6, the protein level of mediators for DNA damage induced-senescence, p16 and p21, was examined by western blotting. p16 protein level was increased while p21 protein level was reduced in the in SIRT6-depleted chondrocytes compared with control. Conclusions: The present study demonstrated that the depletion of SIRT6 in human chondrocytes caused increased expression of MMP-1 and 13, reduced proliferation and increased senescent cells. In addition, the depletion of SIRT6 caused accumulation of γH2AX and TIFs. These observations suggested that the depletion of SIRT6 induced premature senescence associated with a secretory phenotypic change and that the induction of the premature senescence may be caused by increased DNA damages and telomere dysfunction. Moreover, we observed p16 was increased by SIRT6 depletion. The p16-pRB pathway has been reported to be a major pathways that mediate premature senescence and proliferation arrest. Therefore taken together, these observations suggest the depletion of SIRT6 in human chondrocytes caused DNA damage and telomere dysfunction, followed by a premature senescence possibly via the p16-pRB pathway. SIRT6 may play an important role to protect chondrocytes from premature senescence, DNA damage, and telomere dysfunction.

Ki-67 Index as an Ancillary Tool in the Differential Diagnosis of Proliferative Endometrial Lesions With Secretory Change

"Secretory change" can accompany a variety of proliferative endometrial lesions, ranging from hyperplasia to carcinoma. It is characterized by subnuclear or supranuclear vacuolization, mimicking early secretory endometrium (SEM). As an additional diagnostic challenge, mitotic activity and cytologic atypia are often low in hyperplastic lesions with secretory change. As mitotic activity in lesions with secretory change is decreased, the mitotic index may not be useful to distinguish SEM with glandular crowding from hyperplasia with secretory change. We therefore hypothesized that Ki-67 immunohistochemistry, an alternative marker of proliferative activity, might be useful in this setting. Forty-four endometrial lesions with secretory change and 30 controls were stained for Ki-67. Three "hot spot" areas per case were photographed and 200 to 300 cells were manually counted to obtain the ratio of Ki-67-positive cells versus total cells. A second pathologist performed an independent review of the same preselected fields and estimates without preselection. There was an incremental increase in the Ki-67 index from 2.6% in SEM to 17% in nonatypical hyperplasia, 36% in atypical hyperplasia, and 60% in endometrial carcinoma. The Ki-67 index for SEM was significantly (P<0.01) lower than hyperplastic lesions and carcinoma with secretory change. Similar, statistically significant results were obtained by independent estimates of Ki-67 immunopositivity. In the setting of secretory morphology, the Ki-67 index was highly sensitive and specific (>90%) for the differential diagnosis of SEM with crowding versus nonatypical hyperplasia, atypical hyperplasia, and endometrial carcinoma. In summary, the Ki-67-labeling index is a useful technique to distinguish SEM with crowding, an exaggerated physiological condition, from cancer precursors.

Endometrial Hyperplasia and Carcinoma With Superimposed Secretory Changes

Endometrial Hyperplasia and Carcinoma With Superimposed Secretory Changes

Hyperplasia and Carcinoma in Secretory Endometrium

The diagnosis of endometrial hyperplasia or carcinoma in a background of secretory endometrium can be difficult. We attempt to establish the diagnostic criteria to be used in such cases. We examined 80 cases of endometrial hyperplasia, carcinoma, and other conditions with glandular crowding arising in secretory endometrium, analyzed their morphologic features, assessed the volume percentage stroma in each case and performed Ki67 immunostaining on 27 cases. Thirteen cases each of secretory and gestational endometrium served as controls. The mean age of the patients was 45 yr. The non-neoplastic diseases included simple hyperplasia without atypia (56%), endometrial polyps (12.5%), and chronic endometritis with glandular crowding (3%). The proportion of cases with complex hyperplasia without atypia was 10%. Neoplastic diseases included atypical complex hyperplasia (12.5%) and endometrioid carcinoma (6%). The secretory changes were usually less advanced in the hyperplastic glands than in the background endometrium. The morphologic features that best distinguished hyperplasia or carcinoma from secretory endometrium included glandular crowding that stood out from the background; architectural disorder (the long axes of the glands pointing in different directions or parallel to the endometrial surface); dilated, irregularly shaped glands, including budding or branching glands and staghorn-shaped glands; stroma of a polyp; cribriform or confluent glands in cases of carcinoma; nuclear atypia in cases of atypical hyperplasia and carcinoma; and crowded nonsecretory glands. The volume percentage stroma of neoplastic lesions was less than that of non-neoplastic ones (34% vs. 61%, P=0.000001) and that of secretory endometrium (34% vs. 68%, P=0.000038). Non-neoplastic lesions did not have significantly more crowded glands than secretory endometrium (61% vs. 68%, P=0.11). Gestational endometrium had more crowded glands than non-neoplastic lesions (39% vs. 61%, P=0.000004), an approximately equal volume percentage stroma with complex hyperplasia without atypia (39% vs. 43%, P=0.51), and less crowded glands than neoplastic lesions (39% vs. 34%, P=0.03). The Ki67 index of the neoplastic lesions was higher than that of the controls, including secretory and gestational endometria (positive nuclei per 100 epithelial cells, 44.8 vs. 4.6, P=0.0004), of the non-neoplastic lesions (44.8 vs. 5.4, P=0.002) and of complex hyperplasia without atypia (44.8 vs. 9.3, P=0.007). Hyperplasia and carcinoma in secretory endometrium can be diagnosed on the basis of increased glandular crowding, architectural irregularity, nuclear atypia, and increased Ki67 index.

35. Clear cell carcinoma arising within an endometrioma

There is an increasing awareness that malignant transformation of endometriosis is an uncommon but distinct entity. We review one such case of a clear cell carcinoma arising within an endometrioma of a rectus sheath scar. These malignancies have a favourable prognosis, with extra-gonadal disease commonly associated with unopposed estrogen replacement therapy. Presentation A 56-year-old female presented to TQEH for management of post menopausal bleeding. She proceeded to undergo a hysterectomy and bilateral salpingo-oopgerectomy. She underwent a simultaneous excision of an endometrioma arising in a rectus sheath scar in the setting of previous endometritis. Her previous medical history is otherwise unremarkable with no other recorded malignancies or family history of endometrial adenocarcinoma. Findings 5-10% of the endometrium showed complex endometrial hyperplasia with secretory change. There was also a background of early mid-secretory change with corpus luteum formation in the right ovary. There were five intramural and subserosal leiomyomas (5-80mm in diameter) with no evidence of ovarian endometriosis. A single focus of cervical endometriosis was identified. Sectioning through the endometrioma from the rectus sheath showed a clear cell carcinoma arising in the setting of endometriosis. The tumour was ER negative, CK 7 positive, CK 20 negative and CDX2 negative. It measured 12 mm maximally with the closest soft tissue margin 3-4 mm. No origin from the ovaries or endometrium was identified. Conclusion Carcinoma arising in extra-ovarian foci of endometriosis is a rare but recognised phenomenon. Most of these tumours are either endometrioid or clear cell, as in this case. Radiation and/or chemotherapy is offered with favourable prognosis.