MAB3412 Sigma-AldrichAnti-Cytokeratin AE1/AE3 Antibody, recognizes acidic & basic cytokeratins, clone AE1/AE3

The possibility to use a suspended tridimensional matrix as scaffolding for re-epithelialization of in vitro cutaneous wounds was investigated with the aid of a human in vitro wound healing model based on viable full thickness skin. Macroporous gelatin microcarriers, CultiSpher-S, were applied to in vitro wounds and cultured for 21 days. Tissue sections showed incorporation of wound edge keratinocytes into the microcarriers and thicker neoepidermis in wounds treated with microcarriers. Thickness of the neoepidermis was measured digitally, using immunohistochemical staining of keratins as epithelial demarcation. Air-lifting of wounds enhanced stratification in control wounds as well as wounds with CultiSpher-S. Immunohistochemical staining revealed expression of keratin 5, keratin 10, and laminin 5 in the neoepidermal component. We conclude that the CultiSpher-S microcarriers can function as tissue guiding scaffold for re-epithelialization of cutaneous wounds.

Vitamin D deficiency has been implicated in the pathogenesis of several pregnancy complications attributed to impaired or abnormal placental function, but there are few clues indicating the mechanistic role of vitamin D in their pathogenesis. To further understand the role of vitamin D receptor (VDR)-mediated activity in placental function, we used heterozygous Vdr ablated C57Bl6 mice to assess fetal growth, morphological parameters and global gene expression in Vdr null placentae. Twelve Vdr+/- dams were mated at 10-12 weeks of age with Vdr+/- males. At day 18.5 of the 19.5 day gestation in our colony, females were euthanised and placental and fetal samples were collected, weighed and subsequently genotyped as either Vdr+/+, Vdr+/- or Vdr-/-. Morphological assessment of placentae using immunohistochemistry was performed and RNA was extracted and subject to microarray analysis. This revealed 25 genes that were significantly differentially expressed between Vdr+/+ and Vdr-/- placentae. The greatest difference was a 6.47-fold change in expression of Cyp24a1 which was significantly lower in the Vdr-/- placentae (Pless than 0.01). Other differentially expressed genes in Vdr-/- placentae included those involved in RNA modification (Snord123), autophagy (Atg4b), cytoskeletal modification (Shroom4), cell signalling (Plscr1, Pex5) and mammalian target of rapamycin (mTOR) signalling (Deptor and Prr5). Interrogation of the upstream sequence of differentially expressed genes identified that many contain putative vitamin D receptor elements (VDREs). Despite the gene expression differences, this did not contribute to any differences in overall placental morphology, nor was function affected as there was no difference in fetal growth as determined by fetal weight near term. Given our dams still expressed a functional VDR gene, our results suggest that cross-talk between the maternal decidua and the placenta, as well as maternal vitamin D status, may be more important in determining pregnancy outcome than conceptus expression of VDR.

The reproductive system of chickens undergoes dynamic morphological and functional tissue remodeling during the molting period. The present study identified global gene expression profiles following oviductal tissue regression and regeneration in laying hens in which molting was induced by feeding high levels of zinc in the diet. During the molting and recrudescence processes, progressive morphological and physiological changes included regression and re-growth of reproductive organs and fluctuations in concentrations of testosterone, progesterone, estradiol and corticosterone in blood. The cDNA microarray analysis of oviductal tissues revealed the biological significance of gene expression-based modulation in oviductal tissue during its remodeling. Based on the gene expression profiles, expression patterns of selected genes such as, TF, ANGPTL3, p20K, PTN, AvBD11 and SERPINB3 exhibited similar patterns in expression with gradual decreases during regression of the oviduct and sequential increases during resurrection of the functional oviduct. Also, miR-1689* inhibited expression of Sp1, while miR-17-3p, miR-22* and miR-1764 inhibited expression of STAT1. Similarly, chicken miR-1562 and miR-138 reduced the expression of ANGPTL3 and p20K, respectively. These results suggest that these differentially regulated genes are closely correlated with the molecular mechanism(s) for development and tissue remodeling of the avian female reproductive tract, and that miRNA-mediated regulation of key genes likely contributes to remodeling of the avian reproductive tract by controlling expression of those genes post-transcriptionally. The discovered global gene profiles provide new molecular candidates responsible for regulating morphological and functional recrudescence of the avian reproductive tract, and provide novel insights into understanding the remodeling process at the genomic and epigenomic levels.

Multiparameter flow cytometry is a robust and reliable method for determining tumour DNA content applicable to formalin-fixed paraffin-embedded (FFPE) tissue. This study examined the clinical and pathological associations of DNA content in primary breast cancer using an improved multiparametric technique.The FFPE tissue from 201 primary breast cancers was examined and the cancers categorised according to their DNA content using multiparametric flow cytometry incorporating differential labelling of stromal and tumour cell populations. Mathematical modelling software (ModFit 3.2.1) was used to calculate the DNA index (DI) and percentage S-phase fraction (SPF%) for each tumour. Independent associations with clinical and pathological parameters were sought using backward stepwise Binary Logistic Regression (BLR) and Cox's Regression (CR) analysis.Tumours were grouped into four categories based on the DI of the tumour cell population. Low DI tumours (DI=0.76-1.14) associated with progesterone receptor-positive status (P=0.012, BLR), intermediate DI (DI=1.18-1.79) associated with p53 mutant tumours (P=0.001, BLR), high DI (DI1.80) tumours with human epidermal growth factor receptor 2 (HER2)-positive status (P=0.004, BLR) and 'multiploid tumours' (two or more tumour DNA peaks) did not show any significant associations. Tumours with high SPF% (10%) independently associated with poor overall survival (P=0.027, CR).Multiparametric flow analysis of FFPE tissue can accurately assess tumour DNA content. Tumour sub-populations associated with biomarkers of prognosis or likely response to therapy. The alterations in DNA content present the potential for greater understanding of the mechanisms underlying clinically significant biomarker changes in primary breast cancer.

Calretinin is an intracellular calcium-binding EF-hand protein of the calmodulin superfamily. It plays a role in diverse cellular functions, including message targeting and intracellular calcium signaling. It is expressed in the mesothelium, mast cells, some neural cells, and fat cells, among others. Because of its relative specificity for mesothelial neoplasms, calretinin is widely used as one of the primary immunohistochemical markers for malignant mesothelioma and in differentiating it from adenocarcinoma. On the basis of our sporadic observation on calretinin immunoreactivity in desmoid fibromatosis, we systematically evaluated calretinin, keratin cocktail (AE1/AE3), and WT1 immunoreactivity in 268 fibroblastic/myofibroblastic neoplasms. Calretinin was observed in 75% (44/58) of desmoid fibromatosis, 50% (21/42) of proliferative fasciitis, 23% (8/35) of nodular fasciitis, 33% (13/40) of benign fibrous histiocytoma, 35% (22/62) of malignant fibrous histiocytoma, and 13% (4/31) of solitary fibrous tumors but not in normal connective tissue fibroblasts at various sites. Keratin AE1/AE3 immunoreactivity was also commonly (6/13) present in the large ganglion-like cells of proliferative fasciitis and sometimes in nodular fasciitis (3/35), solitary fibrous tumor (3/27), and malignant fibrous histiocytoma (9/62). Nuclear immunoreactivity for WT1 or keratin 5 positivity was not detected in myofibroblastic tumors. On the basis of these observations, it can be concluded that calretinin and focal keratin immunoreactivity is fairly common in benign and malignant fibroblastic and myofibroblastic lesions. Calretinin-positive and keratin-positive spindle cells in desmoid and nodular fasciitis or calretinin-positive ganglion-like cells in proliferative fasciitis should not be confused with elements of epithelioid or sarcomatoid mesothelioma. These diagnostic pitfalls can be avoided with careful observation of morphology, quantitative differences in keratin expression, and use of additional immunohistochemical markers such keratin 5 and WT1 to verify true epithelial and mesothelial differentiation typical of mesothelioma.

A large-scale in vitro 3D tumor model was generated to evaluate gene delivery procedures in vivo. This 3D tumor model consists of a "tissue-like" spheroid that provides a micro-environment supportive of melanoma proliferation, allowing cells to behave similarly to cells in vivo. This functional spheroid measures approximately 1 cm in diameter and can be used to effectively evaluate plasmid transfection when testing various electroporation (EP) electrode applicators. In this study, we identified EP conditions that efficiently transfect green fluorescent protein (GFP) and interleukin 15 (IL-15) plasmids into tumor cells residing in the 3D construct. We found that plasmids delivered using a 6-plate electrode applying 6 pulses with nominal electric field strength of 500 V/cm and pulse-length of 20 ms produced significant increase of GFP (7.3-fold) and IL-15 (3.0-fold) expression compared to controls. This in vitro 3D model demonstrates the predictability of cellular response toward delivery techniques, limits the numbers of animals employed for transfection studies, and may facilitate future developments of clinical trials for cancer therapies in vivo.

We report the first case, to our knowledge, of a possible primary, signet-ring cell melanoma of the gastroesophageal junction. The mass was initially diagnosed as an invasive, poorly differentiated carcinoma; however, on further review and immunohistochemical workup, the diagnosis of signet-ring cell melanoma was made. The lesion consisted of oval to round epithelioid cells undermining the gastric mucosa and infiltrating the muscularis mucosae. Tumor cells demonstrated abundant cytoplasm and eccentrically located nuclei, many with signet-ring cell morphology. The tumor cells were negative for mucin and pancytokeratin, strongly positive for S100 protein and Melan-A, and focally but strongly positive for human melanoma black-45. Diagnostic imaging failed to prove another site of melanoma, and no history of melanoma or cutaneous lesion was reported by the patient. Therefore, it was determined this was likely a primary lesion. We review the literature and previously reported cases of this rare histologic variant of melanoma.

We use both large and small animal models in our pre-clinical evaluation of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy. Here, we report the use of a large animal model to assess three non-viral GTAs: 25 kDa-branched polyethyleneimine (PEI), the cationic liposome (GL67A) and compacted DNA nanoparticle formulated with polyethylene glycol-substituted lysine 30-mer. GTAs complexed with plasmids expressing human cystic fibrosis transmembrane conductance regulator (CFTR) complementary DNA were administered to the sheep lung (n=8 per group) by aerosol. All GTAs gave evidence of gene transfer and expression 1 day after treatment. Vector-derived mRNA was expressed in lung tissues, including epithelial cell-enriched bronchial brushing samples, with median group values reaching 1-10% of endogenous CFTR mRNA levels. GL67A gave the highest levels of expression. Human CFTR protein was detected in small airway epithelial cells in some animals treated with GL67A (two out of eight) and PEI (one out of eight). Bronchoalveolar lavage neutrophilia, lung histology and elevated serum haptoglobin levels indicated that gene delivery was associated with mild local and systemic inflammation. Our conclusion was that GL67A was the best non-viral GTA currently available for aerosol delivery to the sheep lung, led to the selection of GL67A as our lead GTA for clinical trials in CF patients.

Matrix-based therapy restoring the cell microenvironment is a new approach in regenerative medicine successfully treating human chronic pathologies by using a heparan sulfate mimetic (ReGeneraTing agents [RGTA]). Periodontitis are inflammatory diseases destroying the tooth-supporting tissues with no satisfactory therapy. We studied in vivo RGTA ability to fully restore the tooth-supporting tissues. After periodontitis induction, hamsters were treated with RGTA (1.5 mg kg(-1) w(-1)) or saline. Bone loss was evaluated and immunohistochemical labeling of molecules expressed during cementum development was performed. RGTA treatment restored alveolar bone and the attachment apparatus where fibers were inserted in acellular decorin-negative cementum. RGTA treatment increased the epithelial rests of Malassez, previously depleted by periodontitis. Bone morphogenetic protein (BMP) expressions were compartmentalized: BMP-3 was strongly expressed by epithelial rests of Malassez; BMP-7 was expressed by cells lying on the cementum and BMP-2 by osteoprogenitors around bone formation sites but not at the root-bone interface. Cells near the cementum and bone expressed the ALK2 receptor. This is the first evidence that reconstructing the extracellular matrix scaffold with a heparan sulfate mimetic regenerated the root interface despite the persistence of the bacteria responsible for the disease The improved cellular microenvironment led to the sequential recruitment of cell populations involved in attachment apparatus regeneration.