Cancer-associated mesothelial cells are regulated by the anti-Müllerian hormone axis.

Cancer-associated mesothelial cells (CAMCs) in the tumor microenvironment are thought to promote growth and immune evasion. We find that, in mouse and human ovarian tumors, cancer cells express anti-Müllerian hormone (AMH) while CAMCs express its receptor AMHR2, suggesting a paracrine axis. Factors secreted by cancer cells induce AMHR2 expression during their reprogramming into CAMCs in mouse and human in vitro models. Overexpression of AMHR2 in the Met5a mesothelial cell line is sufficient to induce expression of immunosuppressive cytokines and growth factors that stimulate ovarian cancer cell growth in an AMH-dependent way. Finally, syngeneic cancer cells implanted in transgenic mice with Amhr2-/- CAMCs grow significantly slower than in wild-type hosts. The cytokine profile of Amhr2-/- tumor-bearing mice is altered and their tumors express less immune checkpoint markers programmed-cell-death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Taken together, these data suggest that the AMH/AMHR2 axis plays a critical role in regulating the pro-tumoral function of CAMCs in ovarian cancer.

Physico-Chemical Characterization and Assessment of Cytotoxic and Genotoxic Effects of Poly-Ethylene-Glycol Coated and Uncoated Gold Nanoparticles on Human Kidney (HK-2) Cells.

Although gold nanoparticles (Au-NPs) have been widely used in medicine for the diagnosis and treatment of patients due to their unique physicochemical properties, chemical stability and biocompatibility, recent reports have also highlighted their potential to induce toxicity to humans. In the present study, we investigated the toxic effects of uncoated and polyethylene glycol (PEG)-coated AuNPs on human kidney (HK-2) cells. Both forms of AuNP were synthesized and characterized using standard protocols. Dynamic Light Scattering (DLS), Zeta Sizer Nano ZS analyzer, Transmission Electron Microscopy (TEM), and Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) were used to measure their distribution, zeta potential/surface charge, morphological size, and Au concentrations, respectively. Cytotoxicity was measured by Cyto-Tox assay and trypan blue exclusion test. Oxidative stress (OS) was assessed by quantifying the levels of Glutathione (GSH), and Mitochondria Membrane Potential (MMP). Genotoxicity was assessed by single cell gel electrophoresis (Comet assay) and Chromosomal Aberration (CA) assay. Uncoated AuNPs significantly reduced cell viability, increased ROS, decreased GSH, depolarized the MMP, and induced significant DNA damage and chromosomal alterations including chromosome gaps, centric rings, breaks, deletions, and intra and inter-chromosome exchanges, in a concentration-dependent manner. PEG-coated AuNPs displayed lower cytotoxic and genotoxic effects, and did not produce any significant increase in ROS or significant decrease in GSH along with negligible polarization of the MMP. Hence, PEG-coated AuNPs are relatively less toxic than uncoated AuNPs and therefore, may have potential applications in nanomedicine.

Differential expression of interferon-induced genes and other tissue-based biomarkers in acute graft-versus-host disease vs. lupus erythematosus in skin.

BACKGROUND: In both acute graft-versus-host disease (GVHD) and lupus erythematosus (LE), the patient's own tissues are subjected to immunological assault via complex mechanisms influenced by interferon (IFN) and other cytokines. Although not typically confused clinically, these entities have overlapping histopathological findings in the skin. AIM: To assess whether GVHD can be differentiated from LE using molecular methods on skin specimens. METHODS: We developed a quantitative reverse transcription PCR assay based on previously identified tissue-based biomarkers of cutaneous GVHD, and compared gene expression in GVHD with that in LE. RESULTS: Both entities showed robust expression of IFN-induced genes and of genes encoding proteins involved in antigen presentation, cell signalling and tissue repair. Levels of gene expression differed significantly in GVHD compared with LE, particularly those of IFN-induced genes such as MX1, OAS3, TAP1 and STAT3 (P < 0.01). Three logistic regression models could differentiate the two entities with a high degree of certainty (receiver operating characteristic area under the curve of 1.0). CONCLUSION: The study demonstrates the feasibility of distinguishing between microscopically similar inflammatory dermatoses using tissue-based molecular techniques.

Genome-wide methylation analysis identifies a core set of hypermethylated genes in CIMP-H colorectal cancer.

BACKGROUND: Aberrant DNA methylation profiles are a characteristic of all known cancer types, epitomized by the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). Hypermethylation has been observed at CpG islands throughout the genome, but it is unclear which factors determine whether an individual island becomes methylated in cancer. METHODS: DNA methylation in CRC was analysed using the Illumina HumanMethylation450K array. Differentially methylated loci were identified using Significance Analysis of Microarrays (SAM) and the Wilcoxon Signed Rank (WSR) test. Unsupervised hierarchical clustering was used to identify methylation subtypes in CRC. RESULTS: In this study we characterized the DNA methylation profiles of 94 CRC tissues and their matched normal counterparts. Consistent with previous studies, unsupervized hierarchical clustering of genome-wide methylation data identified three subtypes within the tumour samples, designated CIMP-H, CIMP-L and CIMP-N, that showed high, low and very low methylation levels, respectively. Differential methylation between normal and tumour samples was analysed at the individual CpG level, and at the gene level. The distribution of hypermethylation in CIMP-N tumours showed high inter-tumour variability and appeared to be highly stochastic in nature, whereas CIMP-H tumours exhibited consistent hypermethylation at a subset of genes, in addition to a highly variable background of hypermethylated genes. EYA4, TFPI2 and TLX1 were hypermethylated in more than 90% of all tumours examined. One-hundred thirty-two genes were hypermethylated in 100% of CIMP-H tumours studied and these were highly enriched for functions relating to skeletal system development (Bonferroni adjusted p value =2.88E-15), segment specification (adjusted p value =9.62E-11), embryonic development (adjusted p value =1.52E-04), mesoderm development (adjusted p value =1.14E-20), and ectoderm development (adjusted p value =7.94E-16). CONCLUSIONS: Our genome-wide characterization of DNA methylation in colorectal cancer has identified 132 genes hypermethylated in 100% of CIMP-H samples. Three genes, EYA4, TLX1 and TFPI2 are hypermethylated in >90% of all tumour samples, regardless of CIMP subtype.

Differential expression of estrogen receptor alpha in the embryonic adrenal-kidney-gonadal complex of the oviparous lizard, Calotes versicolor (Daud.).

Estrogen signalling is critical for ovarian differentiation in reptiles with temperature-dependent sex determination (TSD). To elucidate the involvement of estrogen in this process, adrenal-kidney-gonadal (AKG) expression of estrogen receptor (ERα) was studied at female-producing temperature (FPT) in the developing embryos of the lizard, Calotes versicolor which exhibits a distinct pattern of TSD. The eggs of this lizard were incubated at 31.5±0.5°C (100% FPT). The torso of embryos containing adrenal-kidney-gonadal complex (AKG) was collected during different stages of development and subjected to Western blotting and immunohistochemistry analysis. The ERα antibody recognized two protein bands with apparent molecular weight ∼55 and ∼45kDa in the total protein extracts of embryonic AKG complex of C. versicolor. The observed results suggest the occurrence of isoforms of ERα. The differential expression of two different protein isoforms may reveal their distinct role in cell proliferation during gonadal differentiation. This is the first report to reveal two isoforms of the ERα in a reptile during development. Immunohistochemical studies reveal a weak, but specific, cytoplasmic ERα immunostaining exclusively in the AKG during late thermo-sensitive period suggesting the responsiveness of AKG to estrogens before gonadal differentiation at FPT. Further, cytoplasmic as well as nuclear expression of ERα in the medulla and in oogonia of the cortex (faint activity) at gonadal differentiation stage suggests that the onset of gonadal estrogen activity coincides with sexual differentiation of gonad. Intensity and pattern of the immunoreactions of ERα in the medullary region at FPT suggest endogenous production of estrogen which may act in a paracrine fashion to induce neighboring cells into ovarian differentiation pathway.

Reproductive outcomes after resection of intrauterine septum.

Pre-and post-septal resection reproductive outcomes were compared in women with infertility and prior pregnancy losses operated on for uterine septum from 2003 to 2010. The major indications for surgery were primary infertility (n = 41, 33.1%), secondary infertility (n = 21, 16.9%) and prior pregnancy losses (n = 52, 41.9%). A total of 74 women (59.7%) had an incomplete septum and 50 (40.3%) had a complete septum. Re-hysteroscopy was done in two women (1.6%); 40 (32.3%) women were lost to follow-up after resection; 63 of the remaining 84 (75.0%) conceived and there were a total of 98 pregnancies. A cerclage was done in 28 (32.9%) of these pregnancies. Miscarriage and live birth rates were 92.9% and 4.1% pre- and 32.1% and 61.2% post-resection, respectively. Hysteroscopic septal resection significantly improves reproductive performance in women with infertility and prior pregnancy losses.

Collagen type XI α1 facilitates head and neck squamous cell cancer growth and invasion.

BACKGROUND: Although it is well established that the extracellular matrix affects tumour progression, not much is known about the various components and their effect on head and neck squamous cell carcinoma (HNSCC) progression. Levels of collagen type XI α1 (colXIα1), a minor fibrillar collagen, have been shown to be increased in tumour compared with normal tissue in several cancers, including colorectal, breast, and non-small cell lung cancer. Currently, the functional significance of colXIα1 is not understood. METHODS: We examined the expression levels of colXIα1 mRNA and elucidated the functional role of colXIα1 in HNSCC. Cell proliferation, invasion, and migration were examined with and without colXIα1 knockdown with siRNA in HNSCC cells. RESULTS: Our data demonstrate that colXIα1 expression is increased in tumour samples compared with levels in normal adjacent tissue in 16/23 HNSCC patients. In addition, colα11 is increased in HNSCC cell lines compared with normal immortalised epithelial cells and is increased in tumour-derived fibroblasts compared with normal fibroblasts. Using an siRNA approach, we demonstrate that colXIα1 contributes to proliferation, migration, and invasion of HNSCC. CONCLUSION: Our cumulative findings suggest that colXIα1 contributes to HNSCC tumorigenesis and may serve as a potential therapeutic target.

Andrographolide inhibits osteopontin expression and breast tumor growth through down regulation of PI3 kinase/Akt signaling pathway.

Breast cancer is one of the most common cancers among women in India and around the world. Despite recent advancement in the treatment of breast cancer, the results of chemotherapy to date remain unsatisfactory, prompting a need to identify natural agents that could target cancer efficiently with least side effects. Andrographolide (Andro) is one such molecule which has been shown to possess inhibitory effect on cancer cell growth. In this study, Andro, a natural diterpenoid lactone isolated from Andrographis paniculata has been shown to inhibit breast cancer cell proliferation, migration and arrest cell cycle at G2/M phase and induces apoptosis through caspase independent pathway. Our experimental evidences suggest that Andro attenuates endothelial cell motility and tumor-endothelial cell interaction. Moreover, Andro suppresses breast tumor growth in orthotopic NOD/SCID mice model. The anti-tumor activity of Andro in both in vitro and in vivo model was correlated with down regulation of PI3 kinase/Akt activation and inhibition of pro-angiogenic molecules such as OPN and VEGF expressions. Collectively, these results demonstrate that Andro may act as an effective anti-tumor and anti-angiogenic agent for the treatment of breast cancer.

Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate.

PURPOSE: The retinal pigment epithelium (RPE) and underlying Bruch's membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell-substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch's membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this "aged" substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. METHODS: Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. RESULTS: Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. CONCLUSIONS: This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch"s membrane could play a significant role in age-related dysfunction of the RPE.

Sphenoid masses in children: radiologic differential diagnosis with pathologic correlation.

Childhood central skull base masses are rare, often difficult to diagnose, and have overlapping imaging findings. In this review, we provide an overview of the epidemiology, clinical findings, and management of pediatric sphenoid bone and sphenoid sinus masses with an emphasis on imaging findings that may help to differentiate lesions. Radiologic-pathologic correlation is provided. Finally, an imaging-based algorithm is presented as a guide to help radiologists narrow their differential diagnoses. Some of the entities discussed are virtually unique to the pediatric population; others occur rarely in this age group but should be considered in the appropriate clinical setting. Entities included in the discussion are grouped into 2 categories: those that cause nonaggressive osseous remodeling and those that are more commonly associated with aggressive bone changes. Mucocele, aneurysmal bone cyst, giant cell lesions, meningioma, and fibrous dysplasia tend to remodel bone, while entities such as chordoma, craniopharyngioma, rhabdomyosarcoma, sinonasal carcinoma, and neuroblastoma may cause more aggressive local bone changes.

A rare case of Moraxella lacunata producing combined native mitral and aortic valve endocarditis.

We describe a case of infective endocarditis due to Moraxella lacunata involving the native mitral and aortic valves, complicated by cerebral emboli and resultant hemiparesis. The patient was treated with ceftriaxone and gentamicin and improved. This appears to be the first case reported in the medical literature of native multivalvular endocarditis produced by this rare organism.

Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?

We have employed recently developed blind modification search techniques to generate the most comprehensive map of post-translational modifications (PTMs) in human lens constructed to date. Three aged lenses, two of which had moderate cataract, and one young control lens were analyzed using multidimensional liquid chromatography mass spectrometry. In total, 491 modification sites in lens proteins were identified. There were 155 in vivo PTM sites in crystallins: 77 previously reported sites and 78 newly detected PTM sites. Several of these sites had modifications previously undetected by mass spectrometry in lens including carboxymethyl lysine (+58 Da), carboxyethyl lysine (+72 Da), and an arginine modification of +55 Da with yet unknown chemical structure. These new modifications were observed in all three aged lenses but were not found in the young lens. Several new sites of cysteine methylation were identified indicating this modification is more extensive in lens than previously thought. The results were used to estimate the extent of modification at specific sites by spectral counting. We tested the long-standing hypothesis that PTMs contribute to age-related loss of crystallin solubility by comparing spectral counts between the water-soluble and water-insoluble fractions of the aged lenses and found that the extent of deamidation was significantly increased in the water-insoluble fractions. On the basis of spectral counting, the most abundant PTMs in aged lenses were deamidations and methylated cysteines with other PTMs present at lower levels.

Actions of MDMA at glutamatergic neuromuscular junctions.

3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") compels mammalian serotonergic neurons to release serotonin (5-HT). In this study, MDMA altered synaptic transmission presynaptically by enhancing quantal release in two model glutamatergic synapses-the neuromuscular junction (NMJ) of the crayfish opener muscle, which is enhanced by exogenous 5-HT application, and the NMJ of a larval body wall muscle in Drosophila melanogaster, which is insensitive to exogenous 5-HT application. At the crayfish NMJ, MDMA mimicked the actions of 5-HT but only at a substantially higher concentration. At the Drosophila NMJ, MDMA altered synaptic transmission but not through a 5-HT receptor. Using simple invertebrate preparations, we have demonstrated an additional non-serotonergic mechanism of MDMA activity that has not yet been addressed in vertebrate systems and that may play an important role in understanding the mechanism of action for a commonly abused drug.

Antimicrobial effect of ciprofloxacin, povidone-iodine, and gentamicin in the decontamination of human donor globes.

PURPOSE: Clinical research addressing the issue of donor globe decontamination is yet to establish convincing data for the optimal choice of an antimicrobial agent. METHODS: In a donor-globe decontamination study, the antimicrobial effectiveness of a fluoroquinolone antibiotic (ciprofloxacin, 0.3%) was evaluated for the first time and compared with povidone-iodine (P-I, 5%) and gentamicin (0.3%). RESULTS: Ciprofloxacin and gentamicin were found to be less effective than P-I (p < 0.05) in converting culture-positive donor globes to culture negative. In eliminating coagulase-negative staphylococci that predominated the bacterial spectrum, again P-I scored better than ciprofloxacin (p = 0.003) and gentamicin (p = 0.006). Overall, P-I performed better than the other two in the 3-min decontamination procedure. Decontamination was carried out with the same agent for 15 min to assess the effect of duration of decontamination on the antimicrobial activity of P-I. With time, there was no significant increase in the antimicrobial efficacy of the agent except for Corynebacterium species. CONCLUSION: P-I continues to be the preferred agent for decontaminating donor globes. Whereas a contact of 3-min duration between P-I and donor globe remains satisfactory in decontamination procedures, corneal tolerance of this procedure needs investigation.