Amino acid metabolism in the inflammatory niche

Stroma and parenchyma represent the supportive and functional components in every organ of the body, respectively. Beyond their ability to produce structural support and to differentiate into tissues of mesodermal origin, mesenchymal stromal cells (MSC) exhibit potent immunomodulatory properties. Such a function requires an activation step (‘licensing signal’) provided by the inflammatory microenvironment to which MSC are exposed. My results have attributed immunosuppressive effects of MSC to essential amino acid (EAA) deprivation. Amongst the EAA consuming enzymes examined, blocking nitric oxide synthase 2 (NOS2) and histidine decarboxylase (HDC) both resulted in impaired anti-proliferative activity of MSC, while NOS2 appeared to be a more prominent effector. My results have also demonstrated that TNF-α and IFN-γ differently account for NOS2 and HDC up-regulation, respectively. Furthermore, MyD88 and NF-ĸB were identified as upstream mediators for initiating NOS2 production. The role of TNF-α and NOS2 in MSC-mediated immunosuppression was assessed in vivo using a murine model of peritonitis. MSC treatment remarkably reduced the local inflammatory response during acute peritoneal inflammation. Nevertheless, both Nos2-/- and Tnfr1/r2-/- MSC delivered similar effects compared to WT MSC, indicating the presence of other complementary mechanisms in MSC-mediated immunosuppression in vivo. In addition to their immunomodulatory properties, MSC are fundamental in regulating self-renewal and differentiation of haematopoietic stem cells (HSC). MSC protect HSC from potential damage by maintaining their quiescence. My results have revealed that the ability of MSC to enhance the quiescence of HSC was associated with cell-cycle arrest induced by NOS2. As striking parallels exist between the normal and malignant stem cell niche, I investigated the ability of MSC to protect haematopoietic malignant cells from chemotherapy-induced apoptosis. MSC were observed to confer protection from etoposide-induced necrosis of EL4 cells, possibly due to their ability to suppress EL4 proliferation. Collectively, my results have demonstrated the role of MSC across the fields of immunomodulation, niche-supporting and anti-apoptotic effects.Open Acces

Quality of life measurement in women with cervical cancer: implications for Chinese cervical cancer survivors

<p>Abstract</p> <p>Background</p> <p>Women with cervical cancer now have relatively good 5-year survival rates. Better survival rates have driven the paradigm in cancer care from a medical illness model to a wellness model, which is concerned with the quality of women's lives as well as the length of survival. Thus, the assessment of quality of life among cervical cancer survivors is increasingly paramount for healthcare professionals. The purposes of this review were to describe existing validated quality of life instruments used in cervical cancer survivors, and to reveal the implications of quality of life measurement for Chinese cervical cancer survivors.</p> <p>Methods</p> <p>A literature search of five electronic databases was conducted using the terms <it>cervical/cervix cancer</it>, <it>quality of life</it>, <it>survivors</it>, <it>survivorship</it>, <it>measurement</it>, and <it>instruments</it>. Articles published in either English or Chinese from January 2000 to June 2009 were searched. Only those adopting an established quality of life instrument for use in cervical cancer survivors were included.</p> <p>Results</p> <p>A total of 11 validated multidimensional quality of life instruments were identified from 41 articles. These instruments could be classified into four categories: generic, cancer-specific, cancer site-specific and cancer survivor-specific instruments. With internal consistency varying from 0.68-0.99, the test-retest reliability ranged from 0.60-0.95 based on the test of the Pearson coefficient. One or more types of validity supported the construct validity. Although all these instruments met the minimum requirements of reliability and validity, the original versions of these instruments were mainly in English.</p> <p>Conclusion</p> <p>Selection of an instrument should consider the purpose of investigation, take its psychometric properties into account, and consider the instrument's origin and comprehensiveness. As quality of life can be affected by culture, studies assessing the quality of life of cervical cancer survivors in China or other non-English speaking countries should choose or develop instruments relevant to their own cultural context. There is a need to develop a comprehensive quality of life instrument for Chinese cervical cancer survivors across the whole survivorship, including immediately after diagnosis and for short- (less than 5 years) and long-term (more than 5 years) survivorship.</p

FAM129B, an antioxidative protein, reduces chemosensitivity by competing with Nrf2 for Keap1 binding.

BackgroundThe transcription factor Nrf2 is a master regulator of antioxidant response. While Nrf2 activation may counter increasing oxidative stress in aging, its activation in cancer can promote cancer progression and metastasis, and confer resistance to chemotherapy and radiotherapy. Thus, Nrf2 has been considered as a key pharmacological target. Unfortunately, there are no specific Nrf2 inhibitors for therapeutic application. Moreover, high Nrf2 activity in many tumors without Keap1 or Nrf2 mutations suggests that alternative mechanisms of Nrf2 regulation exist.MethodsInteraction of FAM129B with Keap1 is demonstrated by immunofluorescence, colocalization, co-immunoprecipitation and mammalian two-hybrid assay. Antioxidative function of FAM129B is analyzed by measuring ROS levels with DCF/flow cytometry, Nrf2 activation using luciferase reporter assay and determination of downstream gene expression by qPCR and wester blotting. Impact of FAM129B on in vivo chemosensitivity is examined in mice bearing breast and colon cancer xenografts. The clinical relevance of FAM129B is assessed by qPCR in breast cancer samples and data mining of publicly available databases.FindingsWe have demonstrated that FAM129B in cancer promotes Nrf2 activity by reducing its ubiquitination through competition with Nrf2 for Keap1 binding via its DLG and ETGE motifs. In addition, FAM129B reduces chemosensitivity by augmenting Nrf2 antioxidative signaling and confers poor prognosis in breast and lung cancer.InterpretationThese findings demonstrate the important role of FAM129B in Nrf2 activation and antioxidative response, and identify FMA129B as a potential therapeutic target. FUND: The Chang Gung Medical Foundation (Taiwan) and the Ministry of Science and Technology (Taiwan)

Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells.

IntroductionAlthough breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors.MethodsParaffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation.ResultsImmunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH+ cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH- cells. GD2+ cells showed a 3.9-fold greater capacity than GD2- cells. ALDH+/GD2+cells displayed 12.8-fold greater mammosphere forming ability than ALDH-/GD2- cells. In vivo, the tumor-initiating frequency of ALDH+/GD2+ cells were up to 33-fold higher than that of ALDH+ cells, with as few as 50 ALDH+/GD2+ cells being sufficient for engraftment. Moreover, we provided the first evidence for the induction of ALDH+/GD2+ cells to differentiate into neural cells of various lineages, along with the observation of neural differentiation in clinical specimens and xenografts of malignant phyllodes tumors. ALDH+ or ALDH+/GD2+ cells could also be induced to differentiate into adipocytes, osteocytes or chondrocytes.ConclusionsOur findings revealed that malignant phyllodes tumors possessed many characteristics of MSC, and their CSCs were enriched in ALDH+ and ALDH+/GD2+ subpopulations

BRANCHLESS TRICHOMES links cell shape and cell cycle control in Arabidopsis trichomes

Endoreplication, also called endoreduplication, is a modified cell cycle in which DNA is repeatedly replicated without subsequent cell division. Endoreplication is often associated with increased cell size and specialized cell shapes, but the mechanism coordinating DNA content with shape and size remains obscure. Here we identify the product of the BRANCHLESS TRICHOMES (BLT) gene, a protein of hitherto unknown function that has been conserved throughout angiosperm evolution, as a link in coordinating cell shape and nuclear DNA content in endoreplicated Arabidopsis trichomes. Loss-of-function mutations in BLT were found to enhance the multicellular trichome phenotype of mutants in the SIAMESE (SIM) gene, which encodes a repressor of endoreplication. Epistasis and overexpression experiments revealed that BLT encodes a key regulator of trichome branching. Additional experiments showed that BLT interacts both genetically and physically with STICHEL, another key regulator of trichome branching. Although blt mutants have normal trichome DNA content, overexpression of BLT results in an additional round of endoreplication, and blt mutants uncouple DNA content from morphogenesis in mutants with increased trichome branching, further emphasizing its role in linking cell shape and endoreplication. © 2011. Published by The Company of Biologists Ltd

SENP3 Promotes an Mff-Primed Bcl-x L -Drp1 Interaction Involved in Cell Death Following Ischemia

Dysregulation of the mitochondrial fission machinery has been linked to cell death following ischemia. Fission is largely dependent on recruitment of Dynamin-related protein 1 (Drp1) to the receptor Mitochondrial fission factor (Mff) located on the mitochondrial outer membrane (MOM). Drp1 is a target for SUMOylation and its deSUMOylation, mediated by the SUMO protease SENP3, enhances the Drp1-Mff interaction to promote cell death in an oxygen/glucose deprivation (OGD) model of ischemia. Another interacting partner for Drp1 is the Bcl-2 family member Bcl-x(L), an important protein in cell death and survival pathways. Here we demonstrate that preventing Drp1 SUMOylation by mutating its SUMO target lysines enhances the Drp1-Bcl-x(L) interaction in vivo and in vitro. Moreover, SENP3-mediated deSUMOylation of Drp1 promotes the Drp1-Bcl-x(L) interaction. Our data suggest that Mff primes Drp1 binding to Bcl-x(L) at the mitochondria and that Mff and Bcl-x(L) can interact directly, independent of Drp1, through their transmembrane domains. Importantly, SENP3 loss in cells subjected to OGD correlates with reduced Drp1-Bcl-x(L) interaction, whilst recovery of SENP3 levels in cells subjected to reoxygenation following OGD correlates with increased Drp1-Bcl-x(L) interaction. Expressing a Bcl-x(L) mutant with defective Drp1 binding reduces OGD plus reoxygenation-evoked cell death. Taken together, our results indicate that SENP3-mediated deSUMOlyation promotes an Mff-primed Drp1-Bcl-x(L) interaction that contributes to cell death following ischemia

Optimization and scale-up of cell culture and purification processes for production of an adenovirus-vectored tuberculosis vaccine candidate

Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. The only available TB vaccine is the Bacille Calmette-Guerin (BCG). However, parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A, an adenoviral vector expressing the mycobacterial protein Ag85A, is a new tuberculosis vaccine candidate, and has shown promising results in pre-clinical studies and phase I trial. This adenovirus vectored vaccine is produced using HEK 293 cell culture. Here we report on the optimization of cell culture conditions, scale-up of production and purification of the AdAg85A at different scales. Four commercial serum-free media were evaluated under various conditions for supporting the growth of HEK293 cell and production of AdAg85A. A culturing strategy was employed to take advantages of two culture media with respective strengths in supporting the cell growth and virus production, which enabled to maintain virus productivity at higher cell densities and resulted in more than two folds of increases in culture titer. The production of AdAg85A was successfully scaled up and validated at 60L bioreactor under the optimal conditions. The AdAg85A generated from the 3L and 60L bioreactor runs was purified through several purification steps. More than 98% of total cellular proteins was removed, over 60% of viral particles was recovered after the purification process, and purity of AdAg85A was similar to that of the ATCC VR-1516 Ad5 standard. Vaccination of mice with the purified AdAg85A demonstrated a very good level of Ag85A-specific antibody responses. The optimized production and purification conditions were transferred to a GMP facility for manufacturing of AdAg85A for generation of clinical grade material to support clinical trials

Physical activity as a modifiable risk factor for periodontal disease

Non-communicable diseases (NCDs), which contribute significantly to global morbidity, are largely preventable through behavioral changes. As with other NCDs, periodontitis is associated with modifiable risk factors such as smoking and stress and is linked to multiple adverse health outcomes through a shared pathway of chronic systemic inflammation. While the health benefits of physical activity have been widely promoted in public health and extensively studied for other systemic conditions, its impact on periodontal health has only recently started to gain attention. This article critically evaluates the current literature on the relationship between physical activity and periodontitis. While cross-sectional studies have shown an inverse association between physical activity levels and periodontitis risk in the general population, clinical oral health surveys of elite athletes with high levels of physical activity have nonetheless revealed poor periodontal conditions. Although causality has not been determined, physical activity could positively impact periodontitis directly, by reducing inflammatory biomarkers, and indirectly, through its modulatory effects on insulin sensitivity, obesity, bone density, stress, and other health promoting behaviors. Given the importance of risk factor control during initial periodontal therapy, understanding the role of physical activity as a potential behavioral risk modifier is paramount. The findings of this review provide an evidence-based overview of how physical activity could influence periodontitis. There is a need for longitudinal cohort studies to verify the temporality of the reported associations and exclude confounders, while interventions are needed to assess the efficacy of physical activity on periodontal treatment outcomes

Development and validation of a risk score for hospitalization for heart failure in patients with Type 2 Diabetes Mellitus

<p>Abstract</p> <p>Background</p> <p>There are no risk scores available for predicting heart failure in Type 2 diabetes mellitus (T2DM). Based on the Hong Kong Diabetes Registry, this study aimed to develop and validate a risk score for predicting heart failure that needs hospitalisation in T2DM.</p> <p>Methods</p> <p>7067 Hong Kong Chinese diabetes patients without history of heart failure, and without history and clinical evidence of coronary heart disease at baseline were analyzed. The subjects have been followed up for a median period of 5.5 years. Data were randomly and evenly assigned to a training dataset and a test dataset. Sex-stratified Cox proportional hazard regression was used to obtain predictors of HF-related hospitalization in the training dataset. Calibration was assessed using Hosmer-Lemeshow test and discrimination was examined using the area under receiver's operating characteristic curve (aROC) in the test dataset.</p> <p>Results</p> <p>During the follow-up, 274 patients developed heart failure event/s that needed hospitalisation. Age, body mass index (BMI), spot urinary albumin to creatinine ratio (ACR), HbA_1c, blood haemoglobin (Hb) at baseline and coronary heart disease during follow-up were predictors of HF-related hospitalization in the training dataset. HF-related hospitalization risk score = 0.0709 × age (year) + 0.0627 × BMI (kg/m²) + 0.1363 × HbA_1c(%) + 0.9915 × Log₁₀(1+ACR) (mg/mmol) - 0.3606 × Blood Hb(g/dL) + 0.8161 × CHD during follow-up (1 if yes). The 5-year probability of heart failure = 1-S₀(5)^{EXP{0.9744 × (Risk Score - 2.3961)}}. Where S₀(5) = 0.9888 if male and 0.9809 if female. The predicted and observed 5-year probabilities of HF-related hospitalization were similar (p > 0.20) and the adjusted aROC was 0.920 for 5 years of follow-up.</p> <p>Conclusion</p> <p>The risk score had adequate performance. Further validations in other cohorts of patients with T2DM are needed before clinical use.</p