Survival or death: a dual role of autophagy in stress-induced pericyte loss in diabetic retinopathy

Aims/hypothesis
Intra-retinal extravasation and modification of LDL have been implicated in diabetic retinopathy: autophagy may mediate these effects.
Methods
Immunohistochemistry was used to detect autophagy marker LC3B in human and murine diabetic and non-diabetic retinas. Cultured human retinal capillary pericytes (HRCPs) were treated with in vitro-modified heavily-oxidised glycated LDL (HOG-LDL) vs native LDL (N-LDL) with or without autophagy modulators: green fluorescent protein–LC3 transfection; small interfering RNAs against Beclin-1, c-Jun NH(2)-terminal kinase (JNK) and C/EBP-homologous protein (CHOP); autophagy inhibitor 3-MA (5 mmol/l) and/or caspase inhibitor Z-VAD-fmk (100 μmol/l). Autophagy, cell viability, oxidative stress, endoplasmic reticulum stress, JNK activation, apoptosis and CHOP expression were assessed by western blots, CCK-8 assay and TUNEL assay. Finally, HOG-LDL vs N-LDL were injected intravitreally to STZ-induced diabetic vs control rats (yielding 50 and 200 mg protein/l intravitreal concentration) and, after 7 days, retinas were analysed for ER stress, autophagy and apoptosis.
Results
Intra-retinal autophagy (LC3B staining) was increased in diabetic vs non-diabetic humans and mice. In HRCPs, 50 mg/l HOG-LDL elicited autophagy without altering cell viability, and inhibition of autophagy decreased survival. At 100–200 mg/l, HOG-LDL caused significant cell death, and inhibition of either autophagy or apoptosis improved survival. Further, 25–200 mg/l HOG-LDL dose-dependently induced oxidative and ER stress. JNK activation was implicated in autophagy but not in apoptosis. In diabetic rat retina, 50 mg/l intravitreal HOG-LDL elicited autophagy and ER stress but not apoptosis; 200 mg/l elicited greater ER stress and apoptosis.
Conclusions
Autophagy has a dual role in diabetic retinopathy: under mild stress (50 mg/l HOG-LDL) it is protective; under more severe stress (200 mg/l HOG-LDL) it promotes cell death.

A bioavailable cathepsin S nitrile inhibitor abrogates tumor development

BACKGROUND: Cathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models.

METHODS: Validation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true K i values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors.

RESULTS: We confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis.

CONCLUSIONS: In summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential.

Refining the diagnosis and EGFR status of non-small cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis.

INTRODUCTION: The dichotomization of non-small cell carcinoma (NSCLC) subtype into squamous (SQCC) and adenocarcinoma (ADC) has become important in recent years and is increasingly required with regard to management. The aim of this study was to determine the utility of a panel of commercially available antibodies in refining the diagnosis on small biopsies and also to determine whether cytologic material is suitable for somatic EGFR genotyping in a prospectively analyzed series of patients undergoing investigation for suspected lung cancer. METHODS: Thirty-two consecutive cases of NSCLC were first tested using a panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, 34betaE12, and a D-PAS stain for mucin, to determine their value in refining diagnosis of NSCLC. After this test phase, two further pathologists independently reviewed the cases using a refined panel that excluded 34betaE12 because of its low specificity for SQCC, and refinement of diagnosis and concordance were assessed. Ten cases of ADC, including eight derived from cytologic samples, were sent for EGFR mutation analysis. RESULTS: There was refinement of diagnosis in 65% of cases of NSCLC to either SQCC or ADC in the test phase. This included 10 of 13 cases where cell pellets had been prepared from transbronchial needle aspirates. Validation by two further pathologists with varying expertise in lung pathology confirmed increased refinement and concordance of diagnosis. All samples were adequate for analysis, and they all showed a wild-type EGFR genotype. CONCLUSION: A panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, and a D-PAS stain for mucin increases diagnostic accuracy and agreement between pathologists when faced with refining a diagnosis of NSCLC to SQCC or ADC. These small samples, even cell pellets derived from transbronchial needle aspirates, seem to be adequate for EGFR mutation analysis.

A Validation Study for the Use of ROS1 Immunohistochemical Staining in Screening for ROS1 Translocations in Lung Cancer

INTRODUCTION: The presence of ROS proto-oncogene 1, receptor tyrosine kinase gene (ROS1) rearrangements in lung cancers confers sensitivity to ROS kinase inhibitors, including crizotinib. However, they are rare abnormalities (in ∼1% of non-small cell lung carcinomas) that are typically identified by fluorescence in situ hybridization (FISH), and so screening using immunohistochemical (IHC) staining would be both cost- and time-efficient.

METHODS: A cohort of lung tumors negative for other common mutations related to targeted therapies were screened to assess the sensitivity and specificity of IHC staining in detecting ROS1 gene rearrangements, enriched by four other cases first identified by FISH. A review of published data was also undertaken.

RESULTS: IHC staining was 100% sensitive (95% confidence interval: 48-100) and 83% specific (95% confidence interval: 86-100) overall when an h-score higher than 100 was used. Patients with ROS1 gene rearrangements were younger and typically never-smokers, with the tumors all being adenocarcinomas with higher-grade architectural features and focal signet ring morphologic features (two of five). Four patients treated with crizotinib showed a partial response, with three also showing a partial response to pemetrexed. Three of four patients remain alive at 13, 27, and 31 months, respectively.

CONCLUSION: IHC staining can be used to screen for ROS1 gene rearrangements, with patients herein showing a response to crizotinib. Patients with tumors that test positive according to IHC staining but negative according to FISH were also identified, which may have implications for treatment selection.

Trasporte eléctrico y magnetismo en sistemas electrodo - aislante - electrodo: hacia el desarrollo de dispositivos del tipo juntura tunel.

La presente tesis es un estudio dedicado a la optimización y desarrollo de sistemas del tipo juntura túnel. La metodología utilizada para la realización de la tesis consistió, en primer lugar, en la optimización de las componentes independientes de la juntura túnel: electrodo y barrera aislante. Posteriormente se optimizaron los procesos de fabricación para el desarrollo y caracterización de dispositivos del tipo juntura túnel en su forma final. En la primera parte de la tesis se analizan detalladamente los resultados obtenidos de la caracterización eléctrica y topografica de barreras aislantes en sistemas electrodo - barrera. Los sistemas bicapas estudiados, GdBa_2Cu_3_7/SrTiO_3, Nb/Ba_0,05Sr_0,95TiO_3 y YBa_2Cu_3O_7/SrTiO_3, fueron caracterizados utilizando un microscopio de fuerza atómica en modo conductor. Se propuso un modelo fenomenológico basado en los resultados experimentales, que permitió la obtención de parámetros críticos para el desarrollo de dispositivos del tipo juntura túnel con nuevas funcionalidades. La información obtenida de la caracterización de los sistemas bicapas (homogeneidad de crecimiento, baja densidad de defectos y de pinholes) indican un muy buen control de los parámetros de crecimiento de las barreras. Por otro lado, se obtuvo un buen comportamiento aislante para espesores mayores a 2 nm sin la presencia de pinholes en la barrera. La similitud en la estequiometría de las barreras (SrTiO_3) permitió comparar los distintos sistemas estudiados en términos de conductividad eléctrica. Se verificó que el modelo fenomenológico permite comparar la conductividad eléctrica de los sistemas mediante uno de los parámetros definidos en el modelo fenomenológico (obtenido de los ajustes lineales de las curvas I(V)). De los 3 sistemas estudiados, las bicapas GdBa_2Cu_3O_7/SrTiO_3 presentaron un mayor valor de longitud de atenuación de los portadores de carga a través de la barrera y una muy baja densidad de defectos superficiales. Las bicapas YBa_2Cu_3O_7/SrTiO_3 y Nb/Ba_0,05Sr_0,95TiO_3 permitieron validar el modelo fenomenológico propuesto para el análisis de la respuesta corriente - voltaje obtenida con el microscopio de fuerza atómica en modo conductor. La segunda parte de la tesis abarca conceptos de magnetismo y microfabricación para el desarrollo de junturas túnel magnéticas. Durante la caracterización de las películas ferromagnéticas individuales de Co_90Fe_10 (CoFe) se logró aumentar valor del campo coercitivo de films de 10 nm de espesor al incrementar la temperatura de depósito. Esto se debe a un aumento del tamaño de grano de los films. El aumento de la temperatura del sustrato durante el crecimiento influye en la morfología y las propiedades magnéticas de los films de CoFe favoreciendo la formación de granos y la pérdida del eje preferencial de magnetización. Estos resultados permitieron la fabricación de sistemas Co_90Fe_10/M_gO/Co_90Fe_10 con distintas orientaciones relativas accesibles con campo magnético para el estudio del acople magnético entre los films de CoFe. La caracterización eléctrica de estos sistemas, particularmente la respuesta corriente - voltaje obtenida con el microscopio de fuerza atómica en modo conductor, indicó que las propiedades de transporte eléctrico de las junturas presentan un alto grado de reproducibilidad. Se analizó además la inuencia del sustrato utilizado en la corriente túnel que atraviesa la barrera aislante. Por otro lado, se discuten los fenómenos relacionados a la optimización de las propiedades magnéticas de electrodos ferromagnéticos para la fabricación de junturas túnel Co_90Fe_10/MgO/Co_90Fe_10 y Co_90Fe_10/MgO /Fe_20Ni_80. En particular, se estudió el acople magnético entre capas ferromagnéticas y la inuencia del sustrato utilizado para el crecimiento de las tricapas. La optimización de los electrodos magnéticos involucró el análisis de la inuencia de la presencia de un aislante entre dos capas magnéticas en el acople de los electrodos. Se logró el desacople de films de 10 nm de Co_90Fe_10 y Fe_20Ni_80 separados por un espaciador de MgO de 2 nm. Finalmente se detallan los pasos para la fabricación de una red de junturas túnel magnéticas y su caracterización eléctrica a bajas temperaturas. El sistema estudiado fue la tricapa Co_90Fe_10 (10 nm)/M_gO (8 nm)/ Fe_20Ni_80 (10 nm) crecido sobre un sustrato de M_gO. La caracterización eléctrica confirmó la buena calidad de la junturas fabricadas. Las junturas obtenidas presentaron un comportamiento altamente resistivo (~ MΩ). Las mediciones de la corriente túnel en función de la temperatura permitieron descartar la presencia de pinholes en la barrera. El transporte de los portadores de carga es por efecto túnel a través de la barrera aislante. Las curvas de conductancia diferencial permitieron calcular el valor medio de la altura de la barrera de potencial (φ = 3.1 eV) a partir del modelo de Brinkman. Los resultados obtenidos en cada uno de los capítulos se complementan y son relevantes para la optimización de junturas túnel, debido a que brindan información crítica para su correcto funcionamiento. En la presente tesis se lograron obtener los primeros avances para la fabricación de arreglos de junturas túnel que permitan el desarrollo de dispositivos.

Trasporte eléctrico y magnetismo en sistemas electrodo - aislante - electrodo: hacia el desarrollo de dispositivos del tipo juntura tunel.

La presente tesis es un estudio dedicado a la optimización y desarrollo de sistemas del tipo juntura túnel. La metodología utilizada para la realización de la tesis consistió, en primer lugar, en la optimización de las componentes independientes de la juntura túnel: electrodo y barrera aislante. Posteriormente se optimizaron los procesos de fabricación para el desarrollo y caracterización de dispositivos del tipo juntura túnel en su forma final. En la primera parte de la tesis se analizan detalladamente los resultados obtenidos de la caracterización eléctrica y topografica de barreras aislantes en sistemas electrodo - barrera. Los sistemas bicapas estudiados, GdBa_2Cu_3_7/SrTiO_3, Nb/Ba_0,05Sr_0,95TiO_3 y YBa_2Cu_3O_7/SrTiO_3, fueron caracterizados utilizando un microscopio de fuerza atómica en modo conductor. Se propuso un modelo fenomenológico basado en los resultados experimentales, que permitió la obtención de parámetros críticos para el desarrollo de dispositivos del tipo juntura túnel con nuevas funcionalidades. La información obtenida de la caracterización de los sistemas bicapas (homogeneidad de crecimiento, baja densidad de defectos y de pinholes) indican un muy buen control de los parámetros de crecimiento de las barreras. Por otro lado, se obtuvo un buen comportamiento aislante para espesores mayores a 2 nm sin la presencia de pinholes en la barrera. La similitud en la estequiometría de las barreras (SrTiO_3) permitió comparar los distintos sistemas estudiados en términos de conductividad eléctrica. Se verificó que el modelo fenomenológico permite comparar la conductividad eléctrica de los sistemas mediante uno de los parámetros definidos en el modelo fenomenológico (obtenido de los ajustes lineales de las curvas I(V)). De los 3 sistemas estudiados, las bicapas GdBa_2Cu_3O_7/SrTiO_3 presentaron un mayor valor de longitud de atenuación de los portadores de carga a través de la barrera y una muy baja densidad de defectos superficiales. Las bicapas YBa_2Cu_3O_7/SrTiO_3 y Nb/Ba_0,05Sr_0,95TiO_3 permitieron validar el modelo fenomenológico propuesto para el análisis de la respuesta corriente - voltaje obtenida con el microscopio de fuerza atómica en modo conductor. La segunda parte de la tesis abarca conceptos de magnetismo y microfabricación para el desarrollo de junturas túnel magnéticas. Durante la caracterización de las películas ferromagnéticas individuales de Co_90Fe_10 (CoFe) se logró aumentar valor del campo coercitivo de films de 10 nm de espesor al incrementar la temperatura de depósito. Esto se debe a un aumento del tamaño de grano de los films. El aumento de la temperatura del sustrato durante el crecimiento influye en la morfología y las propiedades magnéticas de los films de CoFe favoreciendo la formación de granos y la pérdida del eje preferencial de magnetización. Estos resultados permitieron la fabricación de sistemas Co_90Fe_10/M_gO/Co_90Fe_10 con distintas orientaciones relativas accesibles con campo magnético para el estudio del acople magnético entre los films de CoFe. La caracterización eléctrica de estos sistemas, particularmente la respuesta corriente - voltaje obtenida con el microscopio de fuerza atómica en modo conductor, indicó que las propiedades de transporte eléctrico de las junturas presentan un alto grado de reproducibilidad. Se analizó además la inuencia del sustrato utilizado en la corriente túnel que atraviesa la barrera aislante. Por otro lado, se discuten los fenómenos relacionados a la optimización de las propiedades magnéticas de electrodos ferromagnéticos para la fabricación de junturas túnel Co_90Fe_10/MgO/Co_90Fe_10 y Co_90Fe_10/MgO /Fe_20Ni_80. En particular, se estudió el acople magnético entre capas ferromagnéticas y la inuencia del sustrato utilizado para el crecimiento de las tricapas. La optimización de los electrodos magnéticos involucró el análisis de la inuencia de la presencia de un aislante entre dos capas magnéticas en el acople de los electrodos. Se logró el desacople de films de 10 nm de Co_90Fe_10 y Fe_20Ni_80 separados por un espaciador de MgO de 2 nm. Finalmente se detallan los pasos para la fabricación de una red de junturas túnel magnéticas y su caracterización eléctrica a bajas temperaturas. El sistema estudiado fue la tricapa Co_90Fe_10 (10 nm)/M_gO (8 nm)/ Fe_20Ni_80 (10 nm) crecido sobre un sustrato de M_gO. La caracterización eléctrica confirmó la buena calidad de la junturas fabricadas. Las junturas obtenidas presentaron un comportamiento altamente resistivo (~ MΩ). Las mediciones de la corriente túnel en función de la temperatura permitieron descartar la presencia de pinholes en la barrera. El transporte de los portadores de carga es por efecto túnel a través de la barrera aislante. Las curvas de conductancia diferencial permitieron calcular el valor medio de la altura de la barrera de potencial (φ = 3.1 eV) a partir del modelo de Brinkman. Los resultados obtenidos en cada uno de los capítulos se complementan y son relevantes para la optimización de junturas túnel, debido a que brindan información crítica para su correcto funcionamiento. En la presente tesis se lograron obtener los primeros avances para la fabricación de arreglos de junturas túnel que permitan el desarrollo de dispositivos.

Comparative assessment of seller’s staining test (SST) and direct fluorescent antibody test for rapid and accurate laboratory diagnosis of rabies.

Background: Rabies causes 55, 000 annual human deaths globally and about 10,000 people are exposed annually in Nigeria. Diagnosis of animal rabies in most African countries has been by direct microscopic examination. In Nigeria, the Seller’s stain test (SST) was employed until 2009. Before then, both SST and dFAT were used concurrently until the dFAT became the only standard method. Objective: This study was designed to assess the sensitivity and specificity of the SST in relation to the ‘gold standard’ dFAT in diagnosis of rabies in Nigeria. Methods: A total of 88 animal specimens submitted to the Rabies National Reference Laboratory, Nigeria were routinely tested for rabies by SST and dFAT. Results: Overall, 65.9% of the specimens were positive for rabies by SST, while 81.8% were positive by dFAT. The sensitivity of SST in relation to the gold standard dFAT was 81.0% (95% CIs; 69.7% - 88.6%), while the specificity was 100% (95% CIs; 76% - 100%). Conclusion: The relatively low sensitivity of the SST observed in this study calls for its replacement with the dFAT for accurate diagnosis of rabies and timely decisions on administration of PEP to prevent untimely deaths of exposed humans.

The Role of Epstein-Barr Virus LMP-1 Immunohistochemical Staining in Childhood Hodgkin Lymphoma

Background: There are a few published studies about prognostic markers of Epstein-B virus (EBV) related to outcomes in pediatric Hodgkin Lymphoma (HL). Objectives: We aimed to investigate the prognostic value and effect of EBV on survival by using biopsy materials in children and adolescents diagnosed with HL. Patients and Methods: EBV LMP-1 expression was examined using immunohistochemical methods in 58 tumor samples. Clinical features, overall survival (OS) and failure free survival time (FFS) were compared between EBV LMP-1 positive and negative patients. Results: In 20 (35%) patients tumors were LMP-1 positive. When compared with patients above 10 years old, EBV LMP-1 was often positive in patients under 10 years old (30% vs. 70%, P = 0.02). In our most cases having B symptoms and advanced stage, EBV positiveness in Hodgkin Reed-Stenberg cells (H-RS) was not a significant determinant for survival (P = 0.78). Half of the past clinical trials in childhood HL reported longer survival rates in EBV LMP-1 positive patients. In some trials similar to our results there was no significant relationship between EBV and prognosis. Conclusions: The reason of diminished EBV positiviness may be related to technical methods such as not using immunohistochemical and in situ hybridization for EBER antigen but in laboratory conditions painting of control tissues with EBV impair this probability. In addition, cases enrolled to our study were living in Istanbul where social and economical factors are improved rather than generally.

Desquamation Is A Novel Phenomenon For Collective Prostate Epithelial Cell Deletion After Castration.

The mechanism underlying castration-induced prostate regression, which is a classical physiological concept translated into the therapeutic treatment of advanced prostate cancer, involves epithelial cell apoptosis. In searching for events and mechanisms contributing to prostate regression in response to androgen modulation, we have frequently observed the collective deletion of epithelial cells. This work was undertaken to characterize this phenomenon hereafter named desquamation and to verify its presence after 17β-estradiol (E2) administration. Electron microscopy revealed that the desquamating cells had preserved cell-cell junctions and collapsed nuclear contents. The TUNEL reaction was negative for these cells, which were also negative for cleaved caspases-8, -9, -3 and nuclear apoptosis-inducing factor. Detailed analyses revealed that the condensed chromatin was first affected detaching from the nuclear lamina, which was observable after lamin A immunohistochemistry, suggesting the lack of lamin A degradation. A search in animals treated with supraphysiological E2 employed as an alternative anti-androgen treatment revealed no desquamation. The combined treatment (Cas + E2 group) caused changes particular to each treatment, including desquamation. In conclusion, desquamation appeared as a novel phenomenon contributing to collective prostate epithelial cell deletion, distinct from the classical castration-induced apoptosis and particular to the androgen deprivation resulting from surgical castration, and should be considered as part of the mechanisms promoting organ regression.

Hepatic Involvement In Paediatric Patients With Paracoccidioidomycosis: A Histological Study.

Paracoccidioidomycosis is a systemic mycosis that is endemic to certain countries in Latin America. This study aimed to describe the histological features of liver involvement in patients with paracoccidioidomycosis aged <16 years of age who were treated between 1980 and 2010, with a diagnosis that was confirmed by detection of the fungus by pathological examination. Liver tissue was obtained from one necropsy and 12 biopsies. Throughout 2007, biopsies were taken from patients with persistent jaundice or portal hypertension, after which biopsies became indicated due to elevated aminotransferase and low albumin levels. Using haematoxylin and eosin (H&E), Masson's trichrome and immunohistochemical (CK7 and CK19) staining, we noted degenerative alterations in bile duct cells and inflammatory injury to the bile ducts in 10 biopsies. Using immunohistochemistry for CK7 and CK19, we observed ductal proliferation in all 12 samples. Bile duct injuries by inflammatory cells might explain the predominant increase in canalicular enzymes; immunohistochemistry is more sensitive in demonstrating ductular reactions and might show changes that are not apparent on H&E staining.

Defective Apoptosis In Intestinal And Mesenteric Adipose Tissue Of Crohn's Disease Patients.

Crohn's disease (CD) is associated with complex pathogenic pathways involving defects in apoptosis mechanisms. Recently, mesenteric adipose tissue (MAT) has been associated with CD ethiopathology, since adipose thickening is detected close to the affected intestinal area. However, the potential role of altered apoptosis in MAT of CD has not been addressed. To evaluate apoptosis in the intestinal mucosa and MAT of patients with CD. Samples of intestinal mucosa and MAT from patients with ileocecal CD and from non-inflammatory bowel diseases patients (controls) were studied. Apoptosis was assessed by TUNEL assay and correlated with the adipocytes histological morphometric analysis. The transcriptional and protein analysis of selected genes and proteins related to apoptosis were determined. TUNEL assay showed fewer apoptotic cells in CD, when compared to the control groups, both in the intestinal mucosa and in MAT. In addition, the number of apoptotic cells (TUNEL) correlated significantly with the area and perimeter of the adipose cells in MAT. Transcriptomic and proteomic analysis reveal a significantly lower transcript and protein levels of Bax in the intestinal mucosa of CD, compared to the controls; low protein levels of Bax were found localized in the lamina propria and not in the epithelium of this tissue. Furthermore, higher level of Bcl-2 and low level of Caspase 3 were seen in the MAT of CD patients. The defective apoptosis in MAT may explain the singular morphological characteristics of this tissue in CD, which may be implicated in the pathophysiology of the disease.

The Influence Of Altered Occlusion On Pro-inflammatory Cytokine Levels In The Tmj Synovial Tissues Of Rats.

The aim of this study was to evaluate whether altered occlusion affects both the condylar cartilage thickness and the cytokine levels of the TMJs of rats. Thirty adult-male rats (n=30) were randomly assigned to three experimental conditions: a control group that underwent sham operations with unaltered occlusion; an FPDM group that underwent functional posterior displacement of the mandible that was induced by an incisor guiding appliance; and an iOVD group in which the increased occlusal vertical dimension was induced in the molars. The rats were subjected to the FPDM or iOVD model for 14 days and then killed. Both the right and left TMJs were removed and randomly assigned to examination with staining or immunoassay techniques. Toluidine blue staining was used to measure the thicknesses of the four layers of the articular cartilage (i.e., the fibrous, proliferating, mature, and hypertrophic layers). ELISA assays were used to assess the concentrations of the pro-inflammatory cytokines IL-1α, IL-1β, IL-6, and tumour necrosis factor (TNF-α). The measurements of the articular cartilage layers and cytokine concentrations were analyzed with ANOVA and Tukey's tests and Kruskal-Wallis and Dunn tests, respectively (α=5%). The thickness of articular cartilage in the FPDM group (0.3±0.03mm) was significantly greater than those of the control (0.2±0.01mm) and iOVD (0.25±0.03mm) groups. No significant difference was observed between the control and iOVD groups. The four articular cartilage layers were thicker in the FPDM group than in the control and iOVD groups, and the latter two groups did not differ one from each other. Both the FPDM and iOVD groups exhibited higher cytokine levels than did the control (p<0.05) group. Compared to the FPDM group, the iOVD group exhibited significantly higher levels of IL-1β and TNF-α. Both models induced inflammation in the TMJ and caused significant structural changes in the TMJ and surrounding tissues.

Analysis Of The Contribution Of Immunologically-detectable Her2, Steroid Receptors And Of The Triple-negative" Tumor Status To Disease-free And Overall Survival Of Women With Epithelial Ovarian Cancer."

We assessed associations between steroid receptors including: estrogen-alpha, estrogen-beta, androgen receptor, progesterone receptor, the HER2 status and triple-negative epithelial ovarian cancer (ERα-/PR-/HER2-; TNEOC) status and survival in women with epithelial ovarian cancer. The study included 152 women with primary epithelial ovarian cancer. The status of steroid receptor and HER2 was determined by immunohistochemistry. Disease-free and overall survival were calculated and compared with steroid receptor and HER2 status as well as clinicopathological features using the Cox Proportional Hazards model. A mean follow-up period of 43.6 months (interquartile range=41.4 months) was achieved where 44% of patients had serous tumor, followed by mucinous (23%), endometrioid (9%), mixed (9%), undifferentiated (8.5%) and clear cell tumors (5.3%). ER-alpha staining was associated with grade II-III tumors. Progesterone receptor staining was positively associated with a Body Mass Index≥25. Androgen receptor positivity was higher in serous tumors. In stand-alone analysis of receptor contribution to survival, estrogen-alpha positivity was associated with greater disease-free survival. However, there was no significant association between steroid receptor expression, HER2 status, or TNEOC status, and overall survival. Although estrogen-alpha, androgen receptor, progesterone receptor and the HER2 status were associated with key clinical features of the women and pathological characteristics of the tumors, these associations were not implicated in survival. Interestingly, women with TNEOC seem to fare the same way as their counterparts with non-TNEOC.

Design And Synthesis Of N-acylated Aza-goniothalamin Derivatives And Evaluation Of Their In Vitro And In Vivo Antitumor Activity.

Herein we describe the synthesis of a focused library of compounds based on the structure of goniothalamin (1) and the evaluation of the potential antitumor activity of the compounds. N-Acylation of aza-goniothalamin (2) restored the in vitro antiproliferative activity of this family of compounds. 1-(E)-But-2-enoyl-6-styryl-5,6-dihydropyridin-2(1H)-one (18) displayed enhanced antiproliferative activity. Both goniothalamin (1) and derivative 18 led to reactive oxygen species generation in PC-3 cells, which was probably a signal for caspase-dependent apoptosis. Treatment with derivative 18 promoted Annexin V/7-aminoactinomycin D double staining, which indicated apoptosis, and also led to G2 /M cell-cycle arrest. In vivo studies in Ehrlich ascitic and solid tumor models confirmed the antitumor activity of goniothalamin (1), without signs of toxicity. However, derivative 18 exhibited an unexpectedly lower in vivo antitumor activity, despite the treatments being administered at the same site of inoculation. Contrary to its in vitro profile, aza-goniothalamin (2) inhibited Ehrlich tumor growth, both on the ascitic and solid forms. Our findings highlight the importance of in vivo studies in the search for new candidates for cancer treatment.

Granulocyte Colony-stimulating Factor (g-csf) Positive Effects On Muscle Fiber Degeneration And Gait Recovery After Nerve Lesion In Mdx Mice.

G-CSF has been shown to decrease inflammatory processes and to act positively on the process of peripheral nerve regeneration during the course of muscular dystrophy. The aims of this study were to investigate the effects of treatment of G-CSF during sciatic nerve regeneration and histological analysis in the soleus muscle in MDX mice. Six-week-old male MDX mice underwent left sciatic nerve crush and were G-CSF treated at 7 days prior to and 21 days after crush. Ten and twenty-one days after surgery, the mice were euthanized, and the sciatic nerves were processed for immunohistochemistry (anti-p75(NTR) and anti-neurofilament) and transmission electron microscopy. The soleus muscles were dissected out and processed for H&E staining and subsequent morphologic analysis. Motor function analyses were performed at 7 days prior to and 21 days after sciatic crush using the CatWalk system and the sciatic nerve index. Both groups treated with G-CSF showed increased p75(NTR) and neurofilament expression after sciatic crush. G-CSF treatment decreased the number of degenerated and regenerated muscle fibers, thereby increasing the number of normal muscle fibers. The reduction in p75(NTR) and neurofilament indicates a decreased regenerative capacity in MDX mice following a lesion to a peripheral nerve. The reduction in motor function in the crushed group compared with the control groups may reflect the cycles of muscle degeneration/regeneration that occur postnatally. Thus, G-CSF treatment increases motor function in MDX mice. Nevertheless, the decrease in baseline motor function in these mice is not reversed completely by G-CSF.