S100B as a new fecal biomarker of inflammatory bowel diseases.

OBJECTIVE: S100 proteins are demonstrated to exert a protective role in the gastrointestinal tract. In the present study, we investigated whether S100B protein, that is typically expressed by enteroglial cells, is detectable in feces and could be a useful noninvasive indicator of gut chronic inflammation. PATIENTS AND METHODS: This clinical prospective study included n=48 patients suffering Crohn's disease (CD) or ulcerative colitis (UC) and non IBD-controls. The clinical disease activity was evaluated using Harvey-Bradshaw or Mayo Score Index while the diagnosis of IBD was defined based on standard endoscopic and histological criteria. S100B and calprotectin were extracted and analyzed using commercial enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: Unlike calprotectin, S100B was significantly decreased in both CD and UC compared to non IBD-patients. The strongest quantitative alterations of S100B were detected concomitantly with signs of active or quiescent disease, including high/normal expression of fecal calprotectin, mucosal damage/cryptitis, mucin depletion and inflammatory infiltrate, as defined by endoscopic evaluation and histological analysis. At the onset of disease and under no Infliximab-based therapy, the lowest was detected suggesting that S100B in feces could have a potential diagnostic value for IBD. CONCLUSIONS: Testing for S100B and calprotectin could be a useful screening tool to better predict IBD activity.

In vivo imaging techniques: a new era for histochemical analysis.

In vivo imaging techniques can be integrated with classical histochemistry to create an actual histochemistry of water. In particular, Magnetic Resonance Imaging (MRI), an imaging technique primarily used as diagnostic tool in clinical/preclinical research, has excellent anatomical resolution, unlimited penetration depth and intrinsic soft tissue contrast. Thanks to the technological development, MRI is not only capable to provide morphological information but also and more interestingly functional, biophysical and molecular. In this paper we describe the main features of several advanced imaging techniques, such as MRI microscopy, Magnetic Resonance Spectroscopy, functional MRI, Diffusion Tensor Imaging and MRI with contrast agent as a useful support to classical histochemistry.

Poly-ε-caprolactone composite scaffolds for bone repair.

Synthetic biomaterials combined with cells and osteogenic factors represent a promising approach for the treatment of a number of orthopedic diseases, such as bone trauma and congenital malformations. To guarantee optimal biological properties, bone substitutes are prepared with a 3D structure and porosity grade functional to drive cell migration and proliferation, diffusion of factors, vascularization and cell waste expulsion. In this study, synthetic hydroxyapatite (HA) or rat bone extracellular matrix (BP) were examined in an effort to optimize the mechanical properties and osteogenic activity of poly-ε-caprolactone scaffolds prepared with alginate threads (PCL-AT). Using rabbit bone marrow-derived mesenchymal stem cells (rMSCs), the effects of PCL composite substrates on cell adhesion, growth and osteogenic differentiation were evaluated. Micro-CT analysis and scanning electron microscopy evidenced that porous PCL scaffolds containing HA or BP acquire a trabecular bone-like structure with interconnected pores homogenously distributed and are characterized by a pore diameter of approximately 10 µm (PCL-AT-BP) or ranging from 10 to 100 µm. Although the porosity grade of both PCL-AT-HA and PCL-AT-BP promoted optimal conditions for the cell growth of rMSCs at the early phase, the presence of BP was crucial to prolong the cell viability at the late phase. Moreover, a precocious expression of Runx2 (at 7 days) was observed in PCL-AT-BP in combination with osteogenic soluble factors suggesting that BP controls better than HA the osteogenic maturation process in bone substitutes.

In vitro evaluation of TAT-OP1 osteogenic properties and prospects for in vivo applications.

So far, osteogenic protein 1 (OP1) is biotechnologically produced and approved for the treatment of human lumbar spine fusion and long bone non-union fractures. When combined with the TAT sequence, it has been demonstrated in vitro to be easily taken up by PC12 neuronal cells and to acquire its biological activity after intracellular refolding. In this study, TAT-OP1 was shown to be a useful strategy to efficiently drive denatured OP1 into mouse MC3T3E1 pre-osteoblasts. The correct in vitro protein refolding was verified by the activation of the BMP cascade, while the osteogenic potential of OP1 was demonstrated by increased expression of alkaline phosphatase, osteonectin and osteocalcin.

Recombinant human TAT-OP1 to enhance NGF neurogenic potential: preliminary studies on PC12 cells.

Osteogenic protein 1 (OP1), also known as bone morphogenic protein-7 (BMP7), is a multifunctional cytokine with demonstrated neurogenic potential. As the recombinant OP1 (rhOP1) was shown to provide axonal guidance cues and to prevent the reduction of dendritic growth in the injury-induced cortical cultures, it was suggested that an in vivo efficient rhOP1 delivery could enhance neurite growth and functional reconnectivity in the damaged brain. In the present work, we engineered a chimeric molecule in which rhBMP7 was fused to a protein transduction domain derived from HIV-1 TAT protein to deliver the denatured recombinant BMP7 into cells and obtain its chaperone-mediated folding, circumventing the expensive and not much efficient in vitro refolding procedures. When tested on rat PC12 cells, a widely used in vitro neurogenic differentiation model, the resulting fusion protein (rhTAT-OP1) demonstrated to enter fastly into the cells, lose HIV-TAT sequence and interact with membrane receptors activating BMP pathway by SMAD 1/5/8 phosphorylation. In comparison with nerve growth factor (NGF) and BMP7, it proved itself effective to induce the formation of more organized H and M neurofilaments. Moreover, if used in combination with NGF, it stimulated a significant (P < 0.05) and more precocious dendritic outgrowth with respect to NGF alone. These results indicate that rhTAT-OP1 fused with TAT transduction domain shows neurogenic activity and may be a promising enhancer factor in NGF-based therapies.

Involvement of rat hippocampal astrocytes in ß-amyloid-induced angiogenesis and neuroinflammation.

Although Alzheimer's disease (AD) is considered a neurodengenerative disorders, in the last few years a large amount of evidence has suggested that it is also a vascular pathology characterized by increased capillary density and expression of angiogenic factors. In AD the endothelium degenerates, promoting local neuroinflammation and activation of brain endothelium, perivascular microglia, pericytes, astrocytes. Excess tumor necrosis factor (TNF) in the cerebrospinal fluid (CSF), at a concentration of 25 times higher than in the control group, has been demonstrated in AD. Recent studies provide evidence that treatment with TNF-α antagonists may result in a rapid cognitive improvement in AD patients. In the present work we investigated the role of astrocytes in AD angiogenesis and neuroinflammation by means of conditioned media of untreated and Aß-treated rat hippocampal astrocytes (RHAs) on rat microvascular endothelial cells (RCECs). The results demonstrated that RHA media increase RCEC proliferation and capillary-like structure formation. Moreover RHAs secrete IL-1ß and, only after the Aß1-42 treatment, TNF-α promotes RCEC release of IL-1ß, IL-6 and TNF-α. The removal of IL-1ß, TNF-α and/or VEGF, a strong angiogenic inducer highly over-expressed in AD brains, by means of specific antibody-coated beads in RHA media affects RCEC release of IL-1ß, IL-6 and TNF-α. We hypothesised that astrocytes contribute to AD angiogenesis and neuroinflammation by the direct release of pro-inflammatory cytokines. The effect of an anti-inflammatory agent, such as etanercept, decreased RCEC in vitro cytokine release. This could be compared to the effect found in our experiments with antibody anti TNF-α-coated beads.

Pancreatic acellular matrix supports islet survival and function in a synthetic tubular device: in vitro and in vivo studies.

Increasing pancreatic islet survival and function is a starting point for obtaining a valuable bioartificial pancreas for the treatment of type 1 diabetes. In this context, decellularized matrices, obtained after the removal of tissue cellular part, are known to support in vitro adhesion, growth, and function of several cell types. We demonstrate that a homologous acellular pancreatic matrix is a suitable scaffold for rat islet cultures maintaining their long-term viability and function. Islets adhered to the pancreatic matrix showed a constant glucose-induced insulin release during long-term in vitro incubation, while islets cultured without a matrix or on the liver matrix showed a progressive reduction. In order to obtain implantable devices, acellular matrix/islet cultures were entrapped into poly(vinyl alcohol) (PVA)/ poly(ethylene glycol) (PEG) tubes obtained by the freezing/thawing procedure. Under this condition, an in vitro constant insulin release was detected. The devices were then implanted into diabetic rats where reduced insulin requirement was noted suggesting insulin secretory activity of islets contained in the device. Indeed, immunofluorescence confirmed the presence of insulin- and glucagon-producing cells into the explanted devices. These data show that PVA/PEG semi-permeable membrane can obtain devices that restore, at least in part, insulin secretion.

In vitro biological activity of bovine milk ribonuclease-4.

Several members of the ribonuclease superfamily possess a variety of interesting biological properties, including ribonucleolytic, angiogenic, antiproliferative, cytotoxic, embryotoxic, aspermatogenic and antitumoral activity. In this study, we report the purification from bovine milk of a protein with structural and enzymatic properties very similar to those of ribonuclease-4 (RNase-4), which is normally present in the liver and lungs, and examined its functional properties, biological activity and cytotoxic effects. RNase-4, at physiological concentrations, had a positive effect on the vitality and proliferation of human umbilical vein endothelial cells. Moreover, it induced an increase in cellular migration and the formation of in vitro capillary-like structures. We also evaluated the effect of RNase-4 in vitro on human breast, colorectal and cervical carcinoma cell lines. The protein was revealed to have a cytotoxic effect similar to that of RNase-A. We suggest that the positive effects of RNase-4 on normal cells were due to its particularly close interaction with RNase inhibitor, while good conformational stability and resistance to proteolytic degradation potentially favour ribonuclease cytotoxicity.

Middle-term expansion of hematopoietic cord blood cells with new human stromal cell line feeder-layers and different cytokine cocktails.

Cord blood (CB) is a source of hematopoietic stem cells (HSCs) and is an alternative to bone marrow for allogenic transplantation in patients with hematological disorders. The improvement of HSC in vitro expansion is one of the main challenges in cell therapy. Stromal components and soluble factors, such as cytokines, can be useful to induce in vitro cell expansion. Hence, we investigated whether feeder-layers from new stromal cell lines and different exogenous cytokine cocktails induce HSC expansion in middle-term cultures. CB HSC middle-term expansion was carried out in co-cultures on different feeder-layers exposed to three different cytokine cocktails. CB HSC expansion was also carried out in stroma-free cultures in the presence of different cytokine cocktails. Clonogenic tests were performed, and cell growth levels were evaluated. Moreover, the presence of VCAM-1 mRNA was assessed, and the mesenchymal cell-like phenotype expression was detected. All feeder-layers were able to induce a significant clonogenic growth with respect to the control culture, and all of the cytokine cocktails induced a significant increase in CB cell expansion indexes, even though no potential variation dependent on their composition was noted. The modulative effects of the different cocktails, exerted on each cell line used, was dependent on their composition. Finally, all cell lines were positive for CD73, CD117 and CD309, similar to mesenchymal stem cells present in adult bone marrow and in other human tissues, and negative for the hematopoietic markers. These data indicate that our cell lines have, not only a stromal cell-like phenotype, but also a mesenchymal cell-like phenotype, and they have the potential to support in vitro expansion of CB HSCs. Moreover, exogenous cytokines can be used in synergism with feeder-layers to improve the expansion levels of CB HSCs in preparation for their clinical use in allogenic transplantation.

Tissue-specific transcriptional initiation and activity of steroid sulfatase complementing dehydroepiandrosterone sulfate uptake and intracrine steroid activations in human adipose tissue.

Expression analysis by reverse transcriptase (RT)-PCR indicates that human adipose tissue is not likely to perform de novo synthesis of steroid hormones from cholesterol because the mRNAs of cytochromes P450scc and P450c17, and of the steroidogenic-related proteins, steroidogenic acute regulatory protein and steroidogenic factor 1, were not detected. Instead, our data support an intracrine role of adipose tissue, in which adrenal dehydroepiandrosterone sulfate (DHEA-S), the most abundant circulating androgen in man, is selectively uptaken, desulfated, and converted into bioactive androgens and estrogens. Three organic anion-transporting polypeptides-B, -D, and -E, presumably involved in DHEA-S transmembrane transport, were demonstrated at the mRNA level. While sulfotransferase expression was not found, the occurrence of steroid sulfatase (STS), converting DHEA-S to DHEA, was established at the mRNA, protein and catalytic activity levels. The 5'-rapid amplification of cDNA ends analysis showed that STS transcription in adipose tissue is regulated by the use of two promoters which differ from the prevalent placental one. The adipose transcripts contain a distinct untranslated first exon, 0a or 0b, followed by a common partially translated exon 1b, and nine other exons that are also shared by the main placental transcript. The presence of an upstream open reading frame in the new transcript variants could lead to an N-terminal divergence restricted to the cleavable signal peptide and thus not interfering with the catalytic activity of the mature STS protein. The adipose transcripts are also present in the placenta as minor isoforms. Western blotting revealed the characteristic approximately 64 kDa band of STS in both the placenta and adipose tissue. The specific enzymatic activity of STS in adipocytes was 118 pmol/10(6) cells per hour, about 50-100 times lower than in the placenta. A similar rate of [3H] DHEA-S uptake plus desulfation was measured in preadipocytes and adipocytes, equivalent to 40-45 pmol/10(6) cells per hour. Thus, an excessive accumulation of fat may out-compete other peripheral organs that are also dependent on intracrine DHEA-S utilization, especially when the adrenal production is low or declining with aging.

Effects of some vanadyl coordination compounds on the in vitro insulin release from rat pancreatic islets.

Many lines of evidence indicate that vanadium inorganic salts possess insulin-mimetic and insulinotropic properties. However, they are poorly absorbed, so high oral doses are required to achieve effective plasma concentrations with possible undesirable toxic side-effects ensuing. Various organically-chelated vanadium compounds have been synthesized that are more potent than inorganic vanadium salts in their insulin-like effects due to their greater bioavailability. Unfortunately, little is known about the possible insulin secretagogue action of organic vanadyl coordination compounds. Hence, we investigated the effect of [VO(metformin)2]H2O, [VO(salicylidene-ethylenedimmine)2] and [VO(pyrrolidine-N-dithiocarbamate)2](VODTC) on insulin release from isolated rat pancreatic islets, and compared it to that of vanadyl sulfate (VOSO4). Of the three coordination compounds, only VODTC was found to exert insulin secretagogue action. VODTC, within concentrations ranging from 0.1 to 1.0 mM, enhanced both basal and glucose (11 mM)-stimulated insulin release. The effect involves calcium channels, since it was not appreciable in Ca2+-free medium. The stimulating action of VODTC required the presence of the whole metal-chelator complex inasmuch as the chelator DTC alone was ineffective. VOSO4 was unable to bring about any significant rise in insulin release from isolated islets. Taken together, our findings indicate that VODTC may be considered a potential elective pharmaceutical tool in the therapy of diabetes, especially of type 2, through its concomitant stimulatory effect on insulin secretion and insulin-mimetic action.

Experimental abdominal wall defect repaired with acellular matrix.

In the surgical repair of congenital abdominal-wall defects (AWD), the ready availability of a non-immunogenic and non-prosthetic biomaterial that could guide the regeneration of normal tissue is a fascinating possibility. Biomaterials are already in use, but in our experience, an acellular matrix (ACM) can stimulate exact regeneration of the absent tissue. We explored the possibility of using an ACM to repair a muscular AWD in an animal model. Male New Zealand white rabbits (3-4 kg, n = 18) were anesthetized and the abdominal wall was shaved and scrubbed; a vertical incision was made in the left lower quadrant and a large patch of external-oblique muscle was resected (3 x 3 cm). The animals underwent reconstruction with homologous diaphragm acellular matrix (HDAM) grafts that were previously prepared using a detergent enzymatic method. The patches were evaluated histologically at 9 (n = 6), 40 (n = 6), and 90 (n = 6) days post-surgery in each group; moreover, 90 days post-surgery an electromyogram (EMG) (n = 6) of the implanted matrix was recorded. Histologic analysis demonstrated that the HDAM supported fibroblast migration, deposition of newly-formed collagen, and neovascularization. No signs of necrosis, or evidence of skeletal-muscle-cell ingrowth were detected. The EMG revealed minimum muscular electrophysiologic activity, probably due to muscle underlying the patch. The HDAM we employed was thus not able to produce reconstruction of the skeletal muscle, and was progressively remodeled into fibrous tissue. Since the ultimate reason for failure of muscle regeneration is a lack of myogenesis, future studies will use ACMs preconditioned by various regulators of myoblast proliferation and differentiation.

Essential amino acids increase the growth and alkaline phosphatase activity in osteoblasts cultured in vitro.

An inadequate protein intake seems to be involved in the pathogenesis of osteoporosis. Moreover, protein from animal sources appears to protect against hip fracture, while protein from vegetable sources, which present low levels of essential amino acids, has no effect. In this preliminary work, the growth, the alkaline phosphatase activity and the collagen synthesis were evaluated in osteoblast cultures obtained from calvaria of newborn Sprague-Dawley rats and incubated with lysine, threonine, methionine, triptophan and arginine. Our results have shown that the essential amino acids can modulate the growth and the differentiation of osteoblasts cultured in vitro, confirming the relationship between osteoporotic hip fracture and inadequate protein intake. The compounds have mainly enhanced cell growth and alkaline phosphatase activity, and, to a lower degree, collagen synthesis. In summary, the essential amino acids can stimulate bone formation and could represents useful agents for the prevention and therapy of osteoporosis.

Prostaglandin F2 alpha can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro.

The effects of PGF2 alpha on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF2 alpha at concentrations equal to or lower than 10(-6) M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10(-5) M PGF2 alpha induced a decrease in cell growth under all culturing conditions. PGF2 alpha did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF2 alpha induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF2 alpha and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE2.

Short bowel syndrome: experimental approach to increase intestinal surface in rats by gastric homologous acellular matrix.

BACKGROUND/PURPOSE: In this preliminary work the authors used homologous acellular matrix obtained by the gastric wall to increase the small bowel surface in Sprague-Downey rats; through this experimental model the authors verified that homologous acellular matrix can support cell migration and the reconstruction of the intestinal wall. METHODS: A tract of about 2 cm of tubular gastric acellular matrix was inserted with bilateral anastomosis in an isolated ileal loop, which was located in endoabdominal position through a short subcutaneous tunnel. Twelve animals were analyzed at each of the time-points ranging from 1 to 6 weeks after surgery. RESULTS: Histologic evaluation showed that the implanted matrix can be reintegrated in the normal small bowel in a period ranging between 3 and 6 weeks from surgery. The implanted matrix was organized with 4 different tonacae from the third week after the surgery, without interruption at the site of the anastomosis. CONCLUSIONS: To date, the authors do not have a demonstration of the function of the ileal loop reconstructed with this technique; based on these results the authors are engaged in an experimental trial of restoration of intestinal viability with the ileal prosthesis after 3 weeks to study its function.

Production of epidermal growth factor in human prostatic cells cultured in vitro.

The Epidermal Growth Factor (EGF) plays an important role in the regulation of in vitro growth of prostate cells inducing a strong mitogenic effect. Nevertheless in our previous study we observed that the treatment of human hypertrophic prostate cell line U285 with exogenous EGF produces a restricted effect on the cellular growth rate. This phenomenon could be due to the capacity of the cells to produce EGF. In this study we aimed to verify this hypothesis by evaluating the presence of mRNA of EGF and EGF receptor (EGF-R) and of their translation products in U285 cells, before and after the treatment with suramin and exogenous EGF. Moreover we studied the effects exerted by these substances on the proliferative rate of the cells U285 after different treatment protocols. The presence in the cells of mRNA for EGF and EGF-R and of their translation products was demonstrated by means of reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical methods respectively. The modification of growth rate induced by these drugs was studied by FRAME Cytotoxicity Test. The operative modalities adopted to carry out these growth assays tended to 1) focus the effects of suramin in relation to in vitro cellular growth phase; 2) verify the reversibility of its effects; 3) ascertain if it was possible to antagonize the action of suramin by adding exogenous EGF. The results obtained from the RT-PCR showed the presence, in the control cells and in the treated ones, of mRNA coding for EGF and EGF-R. The immunocytochemical analysis indicated that 20% of the control cells are EGF positive, and 83% are EGF-R positive, confirming the results obtained with RT-PCR. Moreover, these stainings showed that the treatment with EGF does not significantly modify the percentage of cells marked by the anti-EGF antibody, while treatments with suramin and suramin plus EGF double this percentage. None of the treatments modifies the percentage of EGF-R positive cells. The growth assays showed that the exposition to highest doses of suramin in the first 24 h of cultures causes a decrease (p < 0.05) of the cellular proliferation during the following 48 h and 72 h and that these effects are irreversible. Moreover, a contemporaneous exposition of the cells to EGF and suramin at seeding strengthens the cytotoxic action of the last drug. To sum up, the demonstration of the presence in the U285 cells of mRNA coding for EGF and EGF-R and of the corresponding proteins, confirms the hypothesis that these cells can produce EGF. Moreover, the cytotoxicity experiments allowed a focusing of the role of the endogenous EGF in the regulation of the U285 cells proliferation and confirmed the importance of biological events that take place in U285 cells during the first 24 h of culture.

Experimental defect in rabbit urethra repaired with acellular aortic matrix.

Urethral reconstruction following failed hypospadias repair or post-traumatic chronic stricture requires adequate amounts of tissue. Many surgical techniques utilizing different types of biological tissues have been attempted: (a) vascularized skin flaps from the prepuce, scrotum or penile shaft; (b) full-thickness free skin grafts; (c) vesical or buccal mucosa grafts; (d) ureter; artery; vein and appendix tissue. More recently, biodegradable polymers have also been used as delivery vehicles of urothelial cells in animals. It has been demonstrated that the implant of an acellular tissue matrix in the bladder can guide the regeneration of urothelium, blood vessels, smooth muscle and nerves. The aim of this study was to create an experimental model of urethral defect, and then repair it by implanting homologous acellular aortic grafts as urethral substitutes. An acellular matrix was obtained by detergent enzymatic treatment of rabbit thoracic aorta. The growth of urethral epithelium was verified in vitro, and homologous acellular vessels were then implanted in rabbits, bridging a previous surgical urethral defect. The outcome of reconstructive surgery was evaluated histologically at 10 days, 3 weeks, 3 and 12 months. As the time after surgery increased, the neourothelium became less thick, signs of inflammatory response disappeared, and the orientation of collagen fibrils and smooth muscle fascicles resembled that of a normal urethra. The implants displayed abundant vascularization, and the luminal surface started to become irregular. Acellular blood vessels may represent a promising approach to urethral defect therapy for different reasons: (a) unlimited availability, (b) readily obtainable in different lengths and gauges, (c) the potential for being organized as tissue bank, and (d) that just one simple surgical procedure is needed. Nevertheless, before this technique can be applied in humans, it must be tested in more species and animals.

Heterocyclic derivatives of all-trans retinoic acid: in vitro effects on fibroblast/keratinocyte growth and differentiation, and in vivo effects on guinea-pig skin.

We investigated the biological effects of five all-trans retinoic acid derivatives, bearing heterocyclic ring systems in the side chain. Growth assays performed on submerged human fibroblast and keratinocyte cultures revealed that (E)4-[2-(5-terbuthyl-thiophen-2-yl)propenyl]benzoic acid (compound 5) is the best compound among the studied derivatives for it exhibits a weaker antiproliferative activity and induces, like all-trans retinoic acid does, a significant increase in fibroblast and keratinocyte growth. The morphological and morphometrical analyses of submerged human fibroblast cultures and human epidermis reconstructed in vitro showed that the compound 5 behaves similarly to all-trans retinoic acid: it induces a decrease in all the cell parameters of submerged fibroblast cultures, and modulates the differentiation of keratinocytes in in vitro reconstructed epidermis. Compound 5 induces thickening of epidermis in vivo, one of the most remarkable pharmacological effects of retinoids on skin, but compared to all-trans retinoic acid, it induces a weaker irritation on guinea-pig skin in terms of both erythema and scaling. Compound 5 could then represent a promising candidate for the treatment of certain dermatological diseases.

Effects of prostaglandin E2 on growth, morphology, morphometry and keratin pattern of bovine corneal epithelial cells cultured in vitro.

In this work the relationship between the proliferation of bovine corneal epithelial cells and PGE2 has been studied. Our data indicate that PGE2 plays an important role in the growth of corneal epithelial cells. Actually, epithelial cells cultured on a keratocyte feeder-layer and exposed to indomethacin, a cyclooxygenase inhibitor, have shown a decrease in growth rate at drug concentrations which otherwise did not induce a reduction in the viability of the keratocytes as well as in epithelial cells in separate cultures. This effect has been reversed by an exogenous PGE2 addition to the culture media. Moreover, significant increases have been found in the growth of epithelial cells cultured in the presence of keratocytes, with basal medium and with conditioning medium after adding exogenous PGE2 at concentrations equal to or lower than 10(-6) M. Significant decreases in the dimensions of the corneal epithelial cells have been found only when PGE2 has been added to basal and to conditioning medium, suggesting that the autacoid maintains cell dimension and morphology. The appearance of keratins with high molecular weight (54 and 57 kDa) coupled with the tendency to stratification of the cells cultivated with media supplemented with PGE2, indicates that the autacoid could favour cell differentiation. The action of PGE2 on the corneal epithelial cells does not seem to be influenced by the presence of the fibroblasts and their products, since PGE2 has induced increases in cell growth and morphological variations, independent of cultural conditions and therefore also only in the presence of basal medium.

Bovine corneal stroma and epithelium reconstructed in vitro: characterisation and response to surfactants.

In order to define safety profiles and proper handling procedures for new industrial products, it is essential to determine their potential for ocular irritation. The Draize test is normally employed but it involves using rabbits. There is today a great need for all researchers to limit the use of animals for laboratory experiments and to encourage the development and adoption of alternative in vitro methods to evaluate the potential toxicity of new products. This study proposes a three-dimensional model of bovine corneal stroma and epithelium that is not only easy to reproduce but may also be used in the toxicological field as an alternative to animal experimentation. The data presented here show that this model allows the growth of epithelium similar in features to in vivo epithelium. Basal cells are cube-shaped, whereas superficial areas are horizontally longer; desmosomes and 64 kDa keratin, as a marker for differentiation of corneal epithelial cells, are both expressed; the basal lamina is synthesised also. The 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) assay was carried out on the model to evaluate the toxicity of some surfactants: benzalkonium chloride, Triton X-100, sodium dodecylsulphate and Tween 20. Since the in vitro data fit very well the results of the Draize test in vivo as reported in the literature, the three-dimensional culture may be used to predict the potential cytotoxicity of surfactants.