Successful xenografting of cryopreserved primary pancreatic cancers

In order to assess the suitability of cryopreserved neoplastic tissues for xenografting into nude (nu/nu) mice, we compared the take rate in 28 samples of pancreatic ductal carcinoma. Eleven fresh samples were implanted in nu/nu mice, and 17 were frozen in cryopreserving solution and implanted at a later time. All samples were examined for the presence of neoplastic tissue in cryostat sections. A total of 15 tumors grew in the animals; five from the freshly implanted samples and ten from those cryopreserved. Ten xenografted tumors were characterized for alterations in p53, K-ras, and p16 genes, which were found in six, eight, and nine cases, respectively. Our results demonstrate that the take rate for xenografting is comparable between cryopreserved and fresh tissue samples. The procedure allows for the exchange of tumor material between institutions and permits the establishment of centralized facilities for the storage of an array of different primary tumor samples suitable for the production of in vivo models of cancers.     

Experimental model for studying the effects of 2-ethylhexyl-phthalate and dialysate on connective tissue

In order to have a model for studying the possible implications of 2-ethylhexyl-phthalate and dialysate on connective tissue, we evaluated their direct effects on the air pouch lining tissue and on fibroblast cultures. Air pouches were formed on the backs of 60 ten-week-old Wistar rats by subcutaneous injections of 10 ml sterile air. On the tenth day 2 ml sterile air, or 2 ml 5 microg/L or 2 ml 10 microg/L 2-ethylhexyl-phthalate in olive oil, or 2 ml olive oil alone, or 2 ml 5 mg/ml or 12 mg/ml lyophilized dialysate were injected into the air pouches. After sampling at seven or twenty-one days, the rats were killed. The biochemical data showed an increase in sulphated glycosaminoglycans with 2-ethylhexyl-phthalate and dialysate. Electron microscopy findings revealed cellular alterations such as vacuolation and cell remnants with 2-ethylhexyl-phthalate, while the cells of the air pouches treated with dialysate showed regular organelles with increased and dilated cisternae of rough endoplasmic reticulum. Moreover, an increase in collagen fibres surrounding the damaged zones was noticed in 2-ethylhexyl-phthalate and dialysate treated rats. The glycosaminoglycan modifications and collagen fibre increase seem to suggest that the morphological changes, with the features of fibrosis, could be the result of 2-ethylhexyl-phthalate and dialysate action on connective tissue. Moreover, the air pouch technique can be considered a good model for studying the direct effects of 2-ethylhexyl-phthalate and other substances, such as uremic toxins, on connective tissue.     

Immunohistochemical evidence for epinephrine-containing retinal amacrine cells

The enzyme for the synthesis of epinephrine, phenylethanolamine-N-methyltransferase, has been localized, by an indirect immunofluorescent staining method, to a subpopulation of amacrine cells in the rat retina. The immunoreactive cells are located primarily in the inner nuclear layer and send a single process to the inner plexiform layer. Most of the immunoreactivity is found in the center of the inner plexiform layer. A small percentage of immunoreactive cell bodies were found in the inner plexiform layer and occasionally cells were observed in the ganglion cell layer. These epinephrine-containing amacrine cells are morphologically distinct from the dopamine-containing amacrine cells previously described by formaldehyde fluorescence and we speculate from reports in the literature that epinephrine-containing amacrine cells may play a role in modulating the activity of dopamine-containing amacrine cells.     

Fascin,an actin-bundling protein,modulates colonic epithelial cell invasiveness and differentiation in vitro

In epithelial tissue, cell-matrix and cell-cell adhesive interactions have important roles in the normal organization and stabilization of the cell layer. The malignant conversion of epithelial cells involves alterations in the expression and function of these adhesion systems that enable a switch to a migratory phenotype in tumor invasion and metastasis. Fascin is an actin-crosslinking protein that is found in the core actin bundles of cell-surface spikes and projections that are implicated in cell motility. We demonstrate that fascin is not detectable in normal colonic epithelium, but is dramatically up-regulated in colorectal adenocarcinoma. To test the hypothesis that fascin could participate in tumor invasive behavior, we developed a cell culture model to examine the effect of fascin expression on the adhesive interactions, invasiveness, and differentiation of colonic epithelial cells. We report marked effects on the organization of cell-surface protrusions, actin cytoskeleton, and focal adhesions in the absence of alterations in the protein levels of the major components of these structures. These effects correlate with alterations in cell movements on two-dimensional matrix, and increased invasiveness in three-dimensional matrix. The cells also show increased proliferation and decreased capacity for normal glandular differentiation in collagen gels. We propose that up-regulation of fascin, by promoting the formation of protrusive, actin-based, cell-motility structures, could be a significant component in the acquisition of invasive phenotype in colonic carcinoma.     

Tuning of innate immunity and polarized responses by decoy receptors

After the identification of the interleukin (IL)-1 type II receptor as the prototype, decoy receptors have been identified for a number of members of the IL-1/IL-18, TNF, IL-10 and IL-13 receptor families. Moreover, the silent receptor D6 is a promiscuous decoy and scavenger receptor of inflammatory chemokines. The IL-1 decoy receptor is regulated by pro- and anti-inflammatory signals and its levels may serve as a readout of the activation of anti-inflammatory pathways, for instance by glucocorticoid hormones. Decoy receptors represent a strategy to tune inflammatory and polarized adaptive responses.     

Diltiazem impairs maturation and functions of human dendritic cells

The aim of this study was to define the effects of diltiazem, a calcium antagonist drug used in cardiology and in clinical transplantation, on the differentiation and maturation of human dendritic cells (DC). Herein, we demonstrate that diltiazem, in association with granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), induces monocytes to differentiate into cells with many of the characteristic of DC. However, diltiazem-induced DC express high levels of mannose receptor and Fc gamma RII and, consequently, manifest a higher endocytic activity compared with GM-CSF+IL-4-induced DC. Importantly, diltiazem-induced DCs have an impaired responsiveness to lipopolysaccharide and CD40 ligand because they produce decreased levels of IL-12 and reveal a reduced ability to stimulate alloreactive T-cell responses as well as in inducing interferon-gamma producing Th1 cells. These effects may contribute to a decreased DC-dependent T-cell activation and may help to explain the immunoregulatory function of diltiazem and its effectiveness in preventing transplant rejection.     

Prostaglandin E2 in gastric mucosa of children with Helicobacter pylori gastritis: relation to thickness of mucus gel layer.

AIMS--To evaluate the changes in mucus gel layer thickness and prostaglandin E2 (PGE2) content caused by Helicobacter pylori infection in the antral mucosa of children: to assess whether decreased mucus gel thickness is related to PGE2 production. METHODS--Antral biopsy specimens were taken at endoscopy from 153 children. H pylori gastritis was evident in 45 and normal mucosa in 59. The other 49 children were studied one month after antibiotic treatment that eradicated the infection in 37 of them had been stopped. One antral specimen was immersed in ice-cold saline, put under an inverse microscope with an eyepiece graticule. Mucus gel thickness was measured and then the processed for histological examination; another specimen was weighed and processed for in vitro prostanoid generation. RESULTS--Mucus gel layer thickness was significantly decreased in children with H pylori gastritis (90 (SD) 29) microns v 120 (58) microns in controls, p < 0.01) but returned to control values after H pylori had been eradicated. PGE2 generation was significantly increased in children with H pylori gastritis (1022 (811) ng/g v 641 (473) ng/g in controls, p < 0.01). One month after treatment PGE2 generation significantly decreased in children without infection (880 (534), p < 0.01), but was still high where infection persisted. A significant inverse correlation was found between PGE2 generation and mucus gel layer thickness (p < 0.05). CONCLUSIONS--These data suggest that H pylori damages the mucus gel layer, and that the gastric mucosa increases generation of PGE2 in response to back diffusion of acid and pepsin.     

Distribution of acid alpha-naphthyl acetate esterase among human T lymphocyte subsets

Human T lymphocyte subsets, identified by means of OKT3, 4 and 8 monoclonal antibodies, were isolated by a fluorescence activated cell sorter (FACS IV) and analyzed for distribution of alpha-naphthyl acetate esterase (ANAE) activity. As compared to OKT8⁺ lymphocytes a higher proportion of OKT4⁺ lymphocytes was ANAE-positive exibiting a spot or dot-like pattern in the cytoplasm. OKT8 and 4 positive subsets showed a similar ANAE distribution in diffuse granular form. Although OKT4 and OKT8 populations presented a different ANAE dot-like reactivity, this marker did not allow as clear a distinction between them as that reported for T_G and T_M lymphocytes.