HMGA2, but not HMGA1, is overexpressed in human larynx carcinomas.

AIMS: Malignant tumours from the upper aerodigestive tract are grouped collectively in the class of head and neck squamous cell carcinoma (HNSCC). The head and neck tumours were responsible for more than 500 000 cancer cases in 2012, accounting for the sixth highest incidence rate and mortality worldwide among all tumour types. Laryngeal squamous cell carcinoma (LSCC) possesses the second highest incidence rate among all HNSCC. Despite significant advances in surgery and radiotherapy during the last few decades, no treatment has been shown to achieve a satisfactory therapeutic outcome and the mortality rate of LSCC is still high, with a 5-year survival rate of 64%. Therefore, further investigations are required to identify the pathogenesis of LSCC. METHODS AND RESULTS: In order to search for new LSCC biomarkers, we have analysed the expression of the HMGA family members, HMGA1 and HMGA2, by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry. HMGA proteins are usually absent in the healthy adult tissues. In contrast, their constitutive expression is a feature of several neoplasias, being associated with a highly malignant phenotype and reduced survival. Here, we report HMGA2 overexpression in larynx carcinomas. Conversely, HMGA1 does not show any differences in its expression between normal and carcinoma tissues. Interestingly, HMGA2 overexpression appears associated with that of two HMGA1-pseudogenes, HMGA1P6 and HMGA1P7, acting as a sponge for HMGA1- and HMGA2-targeting microRNAs and involved in several human cancers. CONCLUSIONS: Therefore, HMGA2 overexpression appears to be a strong feature of larynx carcinoma, supporting its detection as a valid tool for the diagnosis of these malignancies.

Gastrospheres of human gastric mucosa cells: an in vitro model of stromal and epithelial stem cell niche reconstruction.

The molecular characterization of mechanisms involved in the gastrointestinal tract disorders needs an in vitro 3D culture model able to mimic the in vivo gastric microenvironment. Herein, we propose a 3D coculture system where gastric epithelial and stromal cells are grown together building spherical and solid structures using the NASA bioreactor - cell culture system (RCCS), a bioreactor. Epithelial and stromal cells from human antral gastric mucosa were isolated from endoscopic gastric biopsies. Thereafter, these cells were mechanically and enzymatically dispersed by treatment with dispase and collagenase, respectively. Using specific culture procedures, these cells formed 3D structures by using a RCCS, named "gastrospheres". Briefly, gastrospheres were obtained by initial seeding of 2.5x104 cells/well in 96 well culture plates. At 24 h after their formation, they were transferred into RCCS, and maintained for 7, 14, 21, and 28 days. The gastrospheres were morphologically characterized by immunocytochemisty to evaluate extracellular matrix (ECM), and by electron microscopy. These analysis of gastrospheres revealed that the epithelial cells were cytokeratin (CK) and lectin reactive and were arranged in the outer layer; stromal cells presented long cytoplasmic processes and were localized inside the gastrosphere. They were vimentin (VIM) and α-smooth muscle actin (α-SMA) positive and expressed ECM components such as laminin (LN), fibronectin (FN), and type IV collagen (CIV). Electron microscopy revealed groups of cohesive gastric cells surrounded by complex stromal structures, with multiple microvilli, and tight cellular junctions interspersed with extracellular matrix fibrils and fibers. The presence of some nestin-positive cells was observed in the inner region of the gastrospheres, suggesting an intermediary localization between epithelial and stromal cells. Altogether, our data suggest that in vitro gastrospheres recapitulate the in vivo gastric niche microenvironment.

Experimental diabetes induces structural, inflammatory and vascular changes of Achilles tendons.

This study aims to demonstrate how the state of chronic hyperglycemia from experimental Diabetes Mellitus can influence the homeostatic imbalance of tendons and, consequently, lead to the characteristics of tendinopathy. Twenty animals were randomly divided into two experimental groups: control group, consisting of healthy rats and diabetic group constituted by rats induced to Diabetes Mellitus I. After twenty-four days of the induction of Diabetes type I, the Achilles tendon were removed for morphological evaluation, cellularity, number and cross-sectional area of blood vessel, immunohistochemistry for Collagen type I, VEGF and NF-κB nuclear localization sequence (NLS) and nitrate and nitrite level. The Achilles tendon thickness (µm/100g) of diabetic animals was significantly increased and, similarly, an increase was observed in the density of fibrocytes and mast cells in the tendons of the diabetic group. The average number of blood vessels per field, in peritendinous tissue, was statistically higher in the diabetic group 3.39 (2.98) vessels/field when compared to the control group 0.89 (1.68) vessels/field p = 0.001 and in the intratendinous region, it was observed that blood vessels were extremely rare in the control group 0.035 (0.18) vessels/field and were often present in the tendons of the diabetic group 0.89 (0.99) vessels/field. The immunohistochemistry analysis identified higher density of type 1 collagen and increased expression of VEGF as well as increased immunostaining for NFκB p50 NLS in the nucleus in Achilles tendon of the diabetic group when compared to the control group. Higher levels of nitrite/nitrate were observed in the experimental group induced to diabetes. We conclude that experimental DM induces notable structural, inflammatory and vascular changes in the Achilles tendon which are compatible with the process of chronic tendinopathy.

Development and characterization of a new oral dapsone nanoemulsion system: permeability and in silico bioavailability studies.

BACKGROUND: Dapsone is described as being active against Mycobacterium leprae, hence its role in the treatment of leprosy and related pathologies. Despite its therapeutic potential, the low solubility of dapsone in water results in low bioavailability and high microbial resistance. Nanoemulsions are pharmaceutical delivery systems derived from micellar solutions with a good capacity for improving absorption. The aim of this work was to develop and compare the permeability of a series of dapsone nanoemulsions in Caco-2 cell culture against that of effective permeability in the human body simulated using Gastroplus™ software. METHODS AND RESULTS: The release profiles of the dapsone nanoemulsions using different combinations of surfactants and cosolvent showed a higher dissolution rate in simulated gastric and enteric fluid than did the dispersed dapsone powder. The drug release kinetics were consistent with a Higuchi model. CONCLUSION: This comparison of dapsone permeability in Caco-2 cells with effective permeability in the human body simulated by Gastroplus showed a good correlation and indicates potential improvement in the biodisponibility of dapsone using this new system.

Inhibition of tissue factor by ixolaris reduces primary tumor growth and experimental metastasis in a murine model of melanoma.

Melanoma is a highly metastatic cancer and there is strong evidence that the clotting initiator protein, tissue factor (TF), contributes to its aggressive pattern. TF inhibitors may attenuate primary tumor growth and metastasis. In this study, we evaluated the effect of ixolaris, a TF inhibitor, on a murine model of melanoma B16F10 cells. Enzymatic assays performed with B16F10 and human U87-MG tumor cells as the TF source showed that ixolaris inhibits the generation of FX in either murine, human or hybrid FVIIa/TF complexes. The effect of ixolaris on the metastatic potential was further estimated by intravenous injection of B16F10 cells in C57BL/6 mice. Ixolaris (250 µg/kg) dramatically decreased the number of pulmonary tumor nodules (4 ± 1 compared to 47 ± 10 in the control group). Furthermore, a significant decrease in tumor weights was observed in primary tumor growth assays in animals treated with ixolaris (250 µg/kg) from days 3 to 18 after a subcutaneous inoculation of melanoma cells. Remarkably, immunohistochemical analyses showed that inhibition of melanoma growth by ixolaris is accompanied by a significant downregulation of both vascular endothelial growth factor (VEGF) expression and microvascular density in the tumor mass. Our data demonstrate that ixolaris targets B16F10 cell-derived TF, resulting in the reduction of both the primary tumor growth and the metastatic potential of melanoma, as well as the inhibition of tumor angiogenesis. Therefore TF may be a potential target for the treatment of this aggressive malignancy.

Extracellular matrix secreted by reactive stroma is a main inducer of pro-tumorigenic features on LNCaP prostate cancer cells.

Tumor microenvironment modifications are related to the generation of reactive stroma and to critical events in cancer progression, such as proliferation, migration and apoptosis. In order to clarify these cellular interactions mediated by reactive stroma, we investigated the effects of cell-cell contacts, and the influence of soluble factors and extracellular matrix (ECM) secreted by Benign Prostate Hyperplasia (BPH) reactive stroma over LNCaP prostate tumor cells. Using in vitro functional assays, we demonstrated that ECM strongly stimulated LNCaP cell proliferation and migration, while inhibiting apoptosis, and inducing a deregulated expression pattern of several genes related to prostate cancer (PCa) progression. Conversely, reactive stromal cells per se and their secreted conditioned medium partially modulated these pro-tumorigenic events. These data indicate that secreted ECM in reactive stroma microenvironment contains key molecules that positively modulate important cancer hallmarks.

Metalloproteinases 2 and 9 and their tissue inhibitors 1 and 2 are increased in vulvar lichen sclerosus.

OBJECTIVES: To evaluate the expression of different matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in vulvar lichen sclerosus (LS), a chronic dermatosis in women, histologically characterized by a zone of collagen remodeling in the superior dermis. STUDY DESIGN: Analysis of the expression of different MMPs (MMP-1, -2, -9 and -13) and TIMPs (TIMP-1 and -2) by reverse transcriptase-polymerase chain reaction (RT-PCR) in vulvar biopsies from patients with LS (n=11), classified according to Hewitt histological criteria and compared with clinically normal vulvar tissue (n=5), and the immunohistochemistry of MMP-2 and -9 and TIMP-1 and -2 distribution in the remodeling zone of LS (n=31) and in clinically normal vulvar tissue (n=28). RESULTS: Although no statistically significant difference between LS and normal skin groups at the mRNA level of MMP and TIMP transcripts was shown, an increase in the immunodistribution of MMP-2 and -9 and TIMP-1 and -2 in LS compared to normal vulvar skin was observed. CONCLUSIONS: These results suggest that these molecules could be related to the process of cutaneous collagen remodeling in LS pathology.

Effects of a nanocomposite containing Orbignya speciosa lipophilic extract on Benign Prostatic Hyperplasia.

ETHNOPHARMACOLOGICAL RELEVANCE: Lower urinary tract symptoms (LUTS) are a common complaint among aging men and are usually caused by Benign Prostatic Hyperplasia (BPH). A number of medical treatments for LUTS/BPH exist, such as α-blockers, 5α-reductase inhibitors, phytotherapeutical drugs and combination therapies. Babassu is the common name of a Brazilian native palm tree called Orbignya speciosa, whose kernels are commonly used (eaten entirely or as a grounded powder), in parts of Brazil for the treatment of urinary disorders. This study investigates the effects of Orbignya speciosa nanoparticle extract, a newly developed phytotherapic formulation derived from the kernels of babassu, in the treatment of BPH. MATERIALS AND METHODS: Orbignya speciosa extract was obtained from the kernels, a nanoparticulate system was developed and acute toxicity test was performed. BPH primary stromal cell and tissue cultures were established and treated with 300µg/mL Orbignya speciosa nanoparticle (NanoOse) extract in order to evaluate its effects on apoptosis induction, cytotoxicity, cell morphology and proliferation. RESULTS: Our results indicated that NanoOSE shows no toxicity in animals and acts incisively by promoting morphological cell changes, reducing cell proliferation as well as inducing necrosis/apoptosis on BPH cells and tissues. CONCLUSIONS: This study provided the first report of the successful use of NanoOSE on BPH treatment which corroborates with the popular use of the kernels of this plant. The results also suggest the potential of NanoOSE as a candidate new phytotherapeutic agent on the management of BPH.

Localization and characterization of sulfated glycosaminoglycans in the body of the earthworm Eisenia andrei (Oligochaeta, Annelida).

The aim of this study was to characterize the compartmental distribution of sulfated glycosaminoglycans (S-GAGs) in adults and their occurrence during the development of the earthworm Eisenia andrei. S-GAGs were extracted from the body of earthworms to identify their composition and the time of their appearance and disappearance in embryonic, newborn, juvenile, and adult earthworms. S-GAGs were also analyzed in earthworm tissue using histochemical metachromatic staining. Purified S-GAGs obtained from the whole body of adult earthworms were composed of chondroitin sulfate (CS) and heparan sulfate (HS). In addition, an unknown, highly sulfated polysaccharide (HSP) was detected. In order to characterize specifically the S-GAG composition in the integument, earthworms were dissected and as much as possible of their viscera was removed. HS and CS were the predominant sulfated polysaccharides in the dissected integument, whereas in viscera, CS, HS and the HSP were found in proportions similar to those identified in the body. The qualitative S-GAG composition in juveniles was similar to that obtained from adult earthworms. CS was the predominant S-GAG in newborn earthworms, accompanied by lesser amounts of HS and by tiny amounts of the HSP. This study provides a detailed descriptive account of the pattern of S-GAG synthesis during development, and also the characterization of the tissue distribution of these compounds in the body of earthworms.

Higher expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) and metalloproteinase-9 (MMP-9) in a rat model of peritoneal endometriosis is similar to cancer diseases.

BACKGROUND: Endometriosis is a common disease characterized by the presence of a functional endometrium outside the uterine cavity, causing pelvic pain, dysmenorrheal, and infertility. This disease has been associated to development of different types of malignancies; therefore new blood vessels are essential for the survival of the endometrial implant. Our previous observations on humans showed that angiogenesis is predominantly found in rectosigmoid endometriosis, a deeply infiltrating disease. In this study, we have established the experimental model of rat peritoneal endometriosis to evaluate the process of angiogenesis and to compare with eutopic endometrium. METHODS: We have investigated the morphological characteristics of these lesions and the vascular density, VEGF and its receptor Flk-1 and MMP-9 expression, and activated macrophage distribution, using immunohistochemistry and RT-PCR. RESULTS: As expected, the auto-transplantation of endometrium pieces into the peritoneal cavity is a well-established method for endometriosis induction in rats. The lesions were cystic and vascularized, and demonstrated histological hallmarks of human pathology, such as endometrial glands and stroma. The vascular density and the presence of VEGF and Flk-1 and MMP-9 were significantly higher in endometriotic lesions than in eutopic endometrium, and confirmed the angiogenic potential of these lesions. We also observed an increase in the number of activated macrophages (ED-1 positive cells) in the endometriotic lesions, showing a positive correlation with VEGF. CONCLUSION: The present endometriosis model would be useful for investigation of the mechanisms of angiogenesis process involved in the peritoneal attachment of endometrial cells, as well as of the effects of therapeutic drugs, particularly with antiangiogenic activity.

Composition of sulfated glycosaminoglycans and immunodistribution of chondroitin sulfate in deeply infiltrating endometriosis affecting the rectosigmoid.

The composition of sulfated glycosaminoglycans (GAGs) and the tissue distribution of chondroitin sulfate (CS) were analyzed in deeply infiltrating endometriosis (DIE) of rectosigmoid, using metachromatic staining, and biochemical analysis employing electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against CS. The sulfated GAGs were characterized as dermatan sulfate (DS), heparan sulfate (HS) and CS; and DS strongly predominated compared to HS and CS. Immunostaining procedures showed that CS was concentrated in the endometriosis foci, distributed throughout the stroma around the glands. This is the first report describing the composition of sulfated GAGs and the tissue location of CS in DIE by means of histochemical, biochemical and immunohistochemical analyses. These results confirmed that in DIE of rectosigmoid, as in eutopic endometrium [Nasciutti, L.E., Ferrari, R., Berardo, P.T., Souza, M.L.S., Takiya, C.M., Borojevic, R., Abrao, M.S., Silva, L.C.F., 2006. Distribution of chondroitin sulfate in human endometrium. Micron 37, 544-550], CS was the dominant sulfated GAG in stroma of the lesion foci.

Identification and distribution of chondroitin sulfate in the three electric organs of the electric eel, Electrophorus electricus (L.).

The electrogenic tissue of the electric eel Electrophorus electricus (L.) is distributed in three well-defined electric organs, the Main electric organ, Sach's organ and Hunter's organ. Sulfated glycosaminoglycan (GAG) composition was characterized in the three electric organs of the electric eel. Sulfated GAGs were analyzed in the electric organs using metachromatic staining, biochemical analysis including electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against chondroitin sulfate (CS). Our results showed in the three electric organs that CS was the main sulfated GAG species detected, accompanied by small and diminutive amounts of CS/dermatan sulfate hybrid chains and heparan sulfate (HS), respectively. However, HS was not detected in the Sach's organ. CS was predominantly detected in the innervated membrane face of the electroplaques in the three electric organs. Our findings extend previous observations on the GAG composition in the electric organs of E. electricus and provide new information regarding the tissue distribution and location of CS.

Distribution of chondroitin sulfate in human endometrium.

Sulfated glycosaminoglycan (GAG) composition was characterized in the human endometrium during proliferative and secretory phases of the menstrual cycle. Sulfated GAGs were analyzed in endometrium tissue using metachromatic staining, biochemical analysis including electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against chondroitin sulfate (CS). Our results showed that CS was the main sulfated GAG species detected, accompanied by small amounts of heparan sulfate and dermatan sulfate. CS was distributed overall the connective stroma, around arteriole vessels and glands, and there was no important difference in the immunostaining between the proliferative and secretory endometrium phases. Our findings extend previous observations on the GAG composition in the human endometrium providing new information regarding the tissue distribution and location of endometrial CS.

Acharan sulfate, the new glycosaminoglycan from Achatina fulica Bowdich 1822. Structural heterogeneity, metabolic labeling and localization in the body, mucus and the organic shell matrix.

Acharan sulfate, a recently discovered glycosaminoglycan isolated from Achatina fulica, has a major disaccharide repeating unit of -->4)-2-acetyl,2-deoxy-alpha-d-glucopyranose(1-->4)-2-sulfo-alpha-l-idopyranosyluronic acid (1-->, making it structurally related to both heparin and heparan sulfate. It has been suggested that this glycosaminoglycan is polydisperse, with an average molecular mass of 29 kDa and known minor disaccharide sequence variants containing unsulfated iduronic acid. Acharan sulfate was found to be located in the body of this species using alcian blue staining and it was suggested to be the main constituent of the mucus. In the present work, we provide further information on the structure and compartmental distribution of acharan sulfate in the snail body. Different populations of acharan sulfate presenting charge and/or molecular mass heterogeneities were isolated from the whole body, as well as from mucus and from the organic shell matrix. A minor glycosaminoglycan fraction susceptible to degradation by nitrous acid was also purified from the snail body, suggesting the presence of N-sulfated glycosaminoglycan molecules. In addition, we demonstrate the in vivo metabolic labeling of acharan sulfate in the snail body after a meal supplemented with [35S]free sulfate. This simple approach might be applied to the study of acharan sulfate biosynthesis. Finally, we developed histochemical assays to localize acharan sulfate in the snail body by metachromatic staining and by histoautoradiography following metabolic radiolabeling with [35S]sulfate. Our results show that acharan sulfate is widely distributed among several organs.

Identification and tissue-specific distribution of sulfated glycosaminoglycans in the blood-sucking bug Rhodnius prolixus (Linnaeus).

We have previously characterized heparan sulfate (HS) as the major ovarian sulfated glycosaminoglycan (GAG) in females of Rhodnius prolixus, while chondroitin sulfate (CS) was the minor component. Using histochemical procedures we found that GAGs were concentrated in the ovarian tissue but not found inside the oocytes. Here, we extend our initial observations of GAG expression in R. prolixus by characterizing these molecules in other organs: the fat body, intestinal tract, and the reproductive tracts. Only HS and CS were found in the three organs analyzed, however CS was the major GAG species in these tissues. We also determined the compartmental distribution of GAGs in these organs by histochemical analysis using 1,9-dimethylmethylene blue, and evaluated the specific distribution of CS within both male and female reproductive tracts by immunohistochemistry using an anti-CS antibody. We also determined the GAG composition in eggs at days 0 and 6 of embryonic development. Only HS and CS were found in eggs at day 6, while no sulfated GAGs were detected at day 0. Our results demonstrate that HS and CS are the only sulfated GAG species expressed in the fat body and in the intestinal and reproductive tracts of Rhodnius male and female adults. Both sulfated GAGs were also identified in Rhodnius embryos. Altogether, these results show no qualitative differences in the sulfated GAG composition regarding tissue-specific or development-specific distribution.

A continuous lineage of rat adenohypophysis stromal cells: characterisation and effects on GH(3)B(6) prolactin-secreting cell behaviour.

Although some studies have shown a possible modulation of the stroma on the hormonal secretion, it is not clear as to what are the requirements for these cellular interactions. In the present work, a homogeneous and continuous lineage of rat adenohypophysis stromal cells (APS9 cells) obtained from rat adenohypophysis primary culture was established. Using immunocytochemical methods and electron microscopy, we have characterised APS9 cells as elongated fibroblastoid-like cells with intercellular contacts, expressing alpha-smooth muscle actin, type IV collagen and laminin. By biochemical procedures, higher amounts of chondroitin sulphate and heparan sulphate were found in the pericellular and extracellular compartments of APS9 cell culture. In order to evaluate the possible effects of APS9 cell on GH(3)B(6) prolactin-secreting cell survival and/or proliferation, we established co-culture and proliferation assays. When GH(3)B(6) cells were cultivated on APS9 cell substrate, they displayed an organisation of many cellular cords strongly attached and covering all the stromal cell area, establishing punctual interactions or extensive surface associations between adjacent cells. Prolactin immunoreactivity appeared to be more scattered throughout the cytoplasm and accumulated in its periphery. When plated on glass coverslips, on newborn rat skin fibroblasts, on murine haematopoietic bone marrow stroma cell line or on murine foetal liver stroma cell line, GH(3)B(6) cells changed their organisation and presented a decrease in cell number and adherence to the substrate. Our results showed that the APS9 cell/GH(3)B(6) cell interactions favour cell growth and probably PRL secretion, and raises questions about the specificity of different organs and/or animal species stromas on the hormone secretion.