Coordinated expression of oestrogen and androgen receptors in HER2-positive breast carcinomas: impact on proliferative activity

Aims Human epidermal growth factor receptor 2 (HER2)-positive breast cancers are aggressive neoplasms associated with a variable response to systemic therapies. Therefore, the identification of biomarkers to better characterise this heterogeneity would improve treatment efficacy. The aim of this study was to evaluate the influence of androgen receptor (AR) and oestrogen receptor (ER) on clinicopathological features in a series of HER2-positive breast carcinomas. Methods A total of 104 carcinomas were selected and reviewed. Immunohistochemical studies for ER, progesterone receptor and Ki-67 were analysed on tumour whole histological sections. AR expression was analysed on samples represented on tissue microarrays. According to steroid receptor expression, cases were classified into three groups: AR positive/ER positive (48 cases), AR positive/ER negative (41 cases) and AR negative/ER negative (13 cases). Results AR-positive tumours corresponded to 89 (85.6%) of 104 carcinomas. AR-positive carcinomas were associated with a higher frequency of ER and progesterone receptor co-expression and lower proliferative activity determined by the expression of Ki-67. AR-negative carcinomas were more often high grade. The group of AR-positive/ER-negative carcinomas was associated with the highest frequency of apocrine morphological features. The group of AR-negative/ER-negative carcinomas was associated with the highest proliferative activity and the highest frequency of high histological and nuclear grade. The lowest frequency of high-grade tumours and the lowest proliferative activity were seen among tumours with expression of both receptors. Conclusions These results suggest that co-expression of AR and ER can provide a protective effect based on phenotypical presentation of HER2-positive carcinomas. Furthermore, lack of both steroid hormone receptors characterises the most aggressive phenotype.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

Biotic versus abiotic pollination in Oeceoclades maculata (Lindl.) Lindl. (Orchidaceae)

Geographical variation in the reproductive biology of widespread species often occurs at their distributional boundaries. We sought to determine whether such variation has occurred in an invasive orchid, Oeceoclades maculata, across its naturalized range. We compared its reproductive biology in a Brazilian population with that published for a population on the Caribbean island of Puerto Rico. In the state of Sao Paulo, O. maculata flowers between December and February, at the height of the rainy season. Similar fruit sets were observed in manual self (76%) and cross (70.4%) pollination treatments. The fruit set of plants protected from both pollinators and rainfall was 6.1%, whereas plants exposed only to rainfall had a fruit set of 41.4%, slightly less than the controls (48.3%). Like the Puerto Rico population, reproduction is primarily through rain-assisted autogamy, but unlike observations made on the island, outcrossing can eventually occur. We observed two butterfly species (Heliconius ethilla narcaea and Heliconius erato phyllis) pollinating O. maculata. Secretory epidermal cells and trichomes of the spur lumen produced 0.7 mu L of 25% (sucrose equivalents) nectar per flower each morning, which was stored in a dilated basal portion of the spur and reabsorbed by the afternoon. Thus, geographical variation in reproductive biology exists across the broad invasive range of O. maculata.

Early IL-10 production is essential for syngeneic graft acceptance

We performed a comparative study and evaluated cellular infiltrates and anti-inflammatory cytokine production at different time-points after syngeneic or allogeneic skin transplantation. We observed an early IL-10 production in syngeneic grafts compared with allografts. This observation prompted us to investigate the role of IL-10 in isograft acceptance. For this, we used IL-10 KO and WT mice to perform syngeneic transplantation, where IL-10 was absent in the graft or in the recipient. The majority of syngeneic grafts derived from IL-10 KO donors did not engraft or was only partially accepted, whereas IL-10 KO mice transplanted with skin from WT donors accepted the graft. We evaluated IL-10 producers in the transplanted skin and observed that epithelial cells were the major source. Taken together, our data show that production of IL-10 by donor cells, but not by the recipient, is determinant for graft acceptance and strongly suggest that production of this cytokine by keratinocytes immediately upon transplantation is necessary for isograft survival. J. Leukoc. Biol. 92: 259-264; 2012.

Characterization of a Novel Oral Glucocorticoid System and Its Possible Role in Disease

Synthetic corticosteroids are used widely for the treatment of a variety of diseases of the mouth. However, little is known as to whether the oral mucosa is able to modulate the local concentration of active corticosteroids or to produce steroids de novo. This has important clinical implications, because tissue-specific regulation of glucocorticoids is a key determinant of the clinical efficacy of these drugs. In the present study, we show that oral fibroblasts and keratinocytes expressed ACTH receptor (MC2R), glucocorticoid receptor (GR), and 11 beta-hydroxysteroid dehydrogenases (11 beta-HSDs). Unlike keratinocytes, fibroblasts lacked 11 beta-HSD2 and could not effectively deactivate exogenously administered cortisol. However, both cell types were able not only to activate cortisone into the active form cortisol, but also to synthesize cortisol de novo following stimulation with ACTH. 11 beta-HSD2, the enzyme controlling cortisol deactivation, exhibited different patterns of expression in normal (squamous epithelium and salivary glands) and diseased oral mucosa (squamous cell carcinoma and mucoepidermoid carcinoma). Blocking of endogenous cortisol catabolism in keratinocytes with the 11 beta-HSD2 inhibitor 18 beta-glycyrrhetinic acid mimicked the effect of exogenous administration of hydrocortisone and partially prevented the detrimental effects induced by pemphigus vulgaris sera. Analysis of the data demonstrates that a novel, non-adrenal glucocorticoid system is present in the oral mucosa that may play an important role in disease.

Histopathology of black spot symptoms in sweet oranges

In Brazil, citrus black spot (CBS) caused by Guignardia citricarpa is a major disease that has different symptoms on fruit. In this study, fruit of Citrus sinensis infected by G. citricarpa and showing the symptoms false melanosis, freckle spot and hard spot were cross-sectioned and analysed anatomically and histochemically by light microscopy. Immuno-histological assays were performed. All symptoms were accompanied by a thickening of the cuticle. False melanosis lesions did not contain pycnidia and remained restricted to the epicarp or to the first layers of the mesocarp. The stomata in this type of lesion showed phenolic compounds in the guard cells and in the sub-stomatal chamber. In some samples, the guard cells and their surrounding cells lysed, and a wound meristem began to form underneath them. Freckle spot and hard spot lesions had very similar histological alterations to the epicarp and mesocarp, but in our samples only hard spot lesions contained pycnidia. Both of these symptoms were accompanied by protein inclusions. Epidermal and sub-epidermal cells located in the oil-gland region were obliterated, causing alterations in these structures. All symptoms had regions that stained strongly for lipids and phenols.

An in vitro study showing the three-dimensional microenvironment influence over the behavior of head and neck squamous cell carcinoma

Objectives: The Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth worldwide. The mechanisms of growth, invasion and metastasis of this pathology are extensively studied and generally related to specific variations in signaling pathways like the PI3K-Akt; however most of these competent studies have been performed bidimensionally, which may hide important questions. This study sought to analyze the influence of the microenvironment upon the behavior of HNSCC. Study Design: The status of pAkt, NF-kappa B and Cyclin D1 proteins was accessed through immunofluorescence and western blot methods in HNSCC cell lines originating from tongue, pharynx and metastatic lymph node when submitted to a three-dimensional culture model utilizing a matrix system. A bidimensional culture model (monolayer) was used as control. Results: The HNSCC cell lines cultured three-dimensionally exhibited a growth pattern characterized by small isolated islands, different from the control group. When the three-dimensional model was applied, two of the studied cell lines showed the same expression pattern as the bidimensional model regarding nuclear or cytoplasmatic localization, as well as reduction of all protein levels; however, the cell line originated from tongue, which specially has the epidermal growth factor receptor constitutively activated, demonstrated nuclear translocation of pAkt and also an increase in the levels of Cyclin D1. Conclusions: The results suggest the influence of the microenvironment upon the behavior of HNSCC cells due to the changed expression of proteins related to tumor growth and cellular invasion. Furthermore, intrinsically genetic conditions also played important roles over the cells, despite the culture model employed.

Transforming growth factor alpha immunoreactivity. A study in hepatocellular carcinoma and in non-neoplastic liver tissue

Background. Transforming growth factor alpha (TGF alpha) is an important mitogen that binds to epidermal growth factor receptor and is associated with the development of several tumors. Aims. Assessment of the immunoexpression of TGF alpha in hepatocellular carcinoma (HCC) and in non-neoplastic liver tissue and its relationship to morphological patterns of HCC. Material and methods. The immunohistochemical expression of TGF alpha was studied in 47 cases of HCC (27 multinodular, 20 nodular lesions). Five lesions measured up to 5 cm and 15 lesions above 5 cm. Thirty-two cases were graded as I or II and 15 as III or IV. The non-neoplastic tissue was examined in 40 cases, of which 22 had cirrhosis. HBsAg and anti-HCV were positive in 5/38 and 15/37 patients, respectively. The statistical analysis for possible association of immunostaining of TGF alpha and pathological features was performed through chi-square test. Results. TGF alpha was detected in 31.9% of the HCC and in 42.5% of the non-neoplastic. There was a statistically significant association between the expression of TGF alpha and cirrhosis (OR = 8.75, 95% CI = [1.93, 39.75]). The TGF alpha was detected more frequently in patients anti-HCV(+) than in those HBsAg(+). The immunoexpression of TGF alpha was not found related to tumor size or differentiation. In conclusion the TGF alpha is present in hepatocarcinogenesis in HBV negative patients. Further analysis is needed to examine the involvement of TGF alpha in the carcinogenesis associated with HCV and other possible agents. In addition, TGF alpha has an higher expression in hepatocyte regeneration and proliferation in cirrhotic livers than in HCC.

Effects of 830 and 670 nm Laser on Viability of Random Skin Flap in Rats

Objective: This study aimed to investigate the effect of 830 and 670 nm diode laser on the viability of random skin flaps in rats. Background data: Low-level laser therapy (LLLT) has been reported to be successful in stimulating the formation of new blood vessels and reducing the inflammatory process after injury. However, the efficiency of such treatment remains uncertain, and there is also some controversy regarding the efficacy of different wavelengths currently on the market. Materials and methods: Thirty Wistar rats were used and divided into three groups, with 10 rats in each. A random skin flap was raised on the dorsum of each animal. Group 1 was the control group, group 2 received 830 nm laser radiations, and group 3 was submitted to 670 nm laser radiation (power density = 0.5 mW/cm(2)). The animals underwent laser therapy with 36 J/cm(2) energy density (total energy = 2.52 J and 72 sec per session) immediately after surgery and on the 4 subsequent days. The application site of laser radiation was one point at 2.5 cm from the flap's cranial base. The percentage of skin flap necrosis area was calculated on the 7th postoperative day using the paper template method. A skin sample was collected immediately after to determine the vascular endothelial growth factor (VEGF) expression and the epidermal cell proliferation index (KiD67). Results: Statistically significant differences were found among the percentages of necrosis, with higher values observed in group 1 compared with groups 2 and 3. No statistically significant differences were found among these groups using the paper template method. Group 3 presented the highest mean number of blood vessels expressing VEGF and of cells in the proliferative phase when compared with groups 1 and 2. Conclusions: LLLT was effective in increasing random skin flap viability in rats. The 670 nm laser presented more satisfactory results than the 830 nm laser.

Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1

ADAM17, which is also known as TNF alpha-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.

Aerial organ anatomy of Smilax syphilitica (Smilacaceae)

Smilax L. in Brazil is represented by 32 taxa and it is a taxonomically difficult genus because the plants are dioecious and show wide phenotypic variation. The analysis and use of leaf anatomy characters is recognized as a frequently successful taxonomic method to distinguish between individual taxon, when floral material is absent or minute differences in flowers and foliage exist such as in Smilax. The aim of this study was to characterize the anatomical features of the aerial organs in Smilax syphilitica collected from the Atlantic Rainforest, in Santa Teresa-ES and the Smilax aff syphilitica from the Amazon Rainforest, in Manaus, Brazil. For this, a total of three samples of Smilax were collected per site. Sample leaves and stems were fixed with FAA 50, embedded in historesin, sectioned on a rotary microtome, stained and mounted in synthetic resin. Additionally, histochemical tests were performed and cuticle ornamentation was analyzed with standard scanning electron microscopy. S. syphilitica and S. aff syphilitica differed in cuticle ornamentation, epidermal cell arrangement and wall thickness, stomata type and orientation, calcium oxalate crystal type, and position of stem thorns. Leaf blades of S. syphilitica from the Amazon Rainforest have a network of rounded ridges on both sides, while in S. aff syphilitica, these ridges are parallel and the spaces between them are filled with numerous membranous platelets. Viewed from the front, the epidermal cells of S. syphilitica have sinuous walls (even more pronounced in samples from the Amazon); while in S. aff syphilitica, these cells are also sinuous but elongated in the cross-section of the blade and arranged in parallel. Stomata of S. syphilitica are paracytic, whereas in S. aff syphilitica, are both paracytic and anisocytic, and their polar axes are directed towards the mid-vein. Calcium oxalate crystals in S. syphilitica are prisms, whereas in S. aff syphilitica, crystal sand. Thorns occur in nodes and internodes in S. syphilitica but only in internodes in S. aff syphilitica. These features have proven to be of diagnostic value and may support a separation into two species, but future studies are needed to confirm that S. aff syphilitica is indeed a new taxon. Rev. Biol. Trop. 60(3): 1137-1148. Epub 2012 September 01.

Antimicrobial Activity of Essential Oils against Streptococcus mutans and their Antiproliferative Effects

This study aimed to evaluate the activity of essential oils (EOs) against Streptococcus mutans biofilm by chemically characterizing their fractions responsible for biological and antiproliferative activity. Twenty EO were obtained by hydrodistillation and submitted to the antimicrobial assay (minimum inhibitory (MIC) and bactericidal (MBC) concentrations) against S. mutans UA159. Thin-layer chromatography and gas chromatography/mass spectrometry were used for phytochemical analyses. EOs were selected according to predetermined criteria and fractionated using dry column; the resulting fractions were assessed by MIC and MBC, selected as active fractions, and evaluated against S. mutans biofilm. Biofilms formed were examined using scanning electron microscopy. Selected EOs and their selected active fractions were evaluated for their antiproliferative activity against keratinocytes and seven human tumor cell lines. MIC and MBC values obtained for EO and their active fractions showed strong antimicrobial activity. Chemical analyses mainly showed the presence of terpenes. The selected active fractions inhibited S. mutans biofilm formation (P < 0.05) did not affect glycolytic pH drop and were inactive against keratinocytes, normal cell line. In conclusion, EO showed activity at low concentrations, and their selected active fractions were also effective against biofilm formed by S. mutans and human tumor cell lines.

Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo

(Anatomical analysis of peach palm (Bactris gasipaes) leaves cultivated in vitro, ex vitro and in vivo). The present work characterized and compared the anatomical structures of the leaves of Bactris gasipaes (Arecaceae) plants grown under different cultivation conditions (in vitro, ex vitro and in vivo) with the goal of identifying the origins of the difficulties encountered in acclimatizing micro-plants. The Quant program was used to determine leaf tissue thicknesses and areas, and histochemical tests were performed on leaf sections and analyzed using light microscopy. Stomatal and trichome densities were determined using the epidermal impression method and by scanning electronic microscopy. Our results indicated that there were no discernible alterations of the anatomical characteristics of the leaves of micro-plants cultivated under differing conditions and that the thickening of the mesophyll and the vascular fibers indicated adaptive responses to ex vitro conditions. As such, the observed difficulties in acclimatizing peach palm micro-plants to ex vitro conditions cannot be attributed to plant anatomical characteristics acquired during in vitro cultivation.

Identification of Novel Cellular Transcription Factors that Regulate Early Promoters of Human Papillomavirus Types 18 and 16

Background. The long control region (LCR) of human papillomavirus (HPV) regulates early gene transcription by interaction with several viral and cellular transcription factors (TFs). Methods. To identify novel TFs that could influence early expression of HPV type 18 (HPV-18) and HPV type 16 (HPV-16), a high-throughput transfection array was used. Results. Among the 704 TFs tested, 28 activated and 36 inhibited the LCR of HPV-18 by more than 2-fold. For validation, C33 cells were cotransfected with increasing amounts of selected TF expression plasmids in addition to LCR-luciferase vectors of different molecular variants of HPV-18 and HPV-16. Among the TFs identified, only GATA3, FOXA1, and MYC have putative binding sites within the LCR sequence, as indicated using the TRANSFAC database. Furthermore, we demonstrated FOXA1 and MYC in vivo binding to the LCR of both HPV types using chromatin immunoprecipitation assay. Conclusions. We identified new TFs implicated in the regulation of the LCR of HPV-18 and HPV-16. Many of these factors are mutated in cancer or are putative cancer biomarkers and could potentially be involved in the regulation of HPV early gene expression.

Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.