Preparation of three-layered porous PLA/PEG scaffold: relationship between morphology, mechanical behavior and cell permeability.

Interface tissue engineering (ITE) is used to repair or regenerate interface living tissue such as for instance bone and cartilage. This kind of tissues present natural different properties from a biological and mechanical point of view. With the aim to imitating the natural gradient occurring in the bone-cartilage tissue, several technologies and methods have been proposed over recent years in order to develop polymeric functionally graded scaffolds (FGS). In this study three-layered scaffolds with a pore size gradient were developed by melt mixing polylactic acid (PLA) and two water-soluble porogen agents: sodium chloride (NaCl) and polyethylene glycol (PEG). Pore dimensions were controlled by NaCl granulometry while PEG solvation created a micropores network within the devices. Scaffolds were characterized from a morphological and mechanical point of view in order to find a correlation between the preparation method, the pore architecture and compressive mechanical behavior. Biological tests were also performed in order to study the effect of pore size gradient on the permeation of different cell lines in co-culture. To imitate the physiological work condition, compressive tests were also performed in phosphate buffered saline (PBS) solution at 37°C. The presented preparation method permitted to prepare three-layered scaffolds with high control of porosity and pore size distribution. Furthermore mechanical behaviors were found to be strongly affected by pore architecture of tested devices as well as the permeation of osteoblast and fibroblast in-vitro.

A new method to value efficiency of enzyme blends for pancreatic tissue digestion.

Islet transplantation, since the 1990s, has been an example of human cell therapy. Nevertheless, the islet isolation procedure is not completely standardized; in fact, >50% of islet procedures do not eventuate in transplantation due both to the variability of a donor's pancreas and to the unpredictable efficiency of an enzymatic blend. The enzymes used in pancreas isolation to digest several substrates are extracted from Clostridium histolyticum. In particular, they have strong collagenolytic activity compared with vertebrate collagenases. However, several impediments persist in human islet isolation success, probably owing to the variable composition and concentration of collagenases employed during the digestion phase. For islet isolation processes, neutral proteases play important roles. However, they should be considered to be double-edged swords, contributing to tissue dissociation but, sometimes, decreasing islet yield through fragmentation, breakdown, and inactivation. Protease activities cannot be preciously adjusted in a narrow range, there is no approach to determine the optimal dosage and composition of enzymes for extraction of human islets from the pancreas. At this time, available data on commercial enzymatic activity are not sufficient to predict their efficiency for pancreas digestion; consequently, it is difficult to select enzyme batches. For these reasons, we sought to generate an innovative evaluation assay to select enzymes useful for isolation procedures of pancreatic islets.

Critical role of dipeptidyl peptidase IV in neuropeptide Y-mediated endothelial cell migration in response to wounding.

Recently, we have discovered that neuropeptide Y (NPY), a sympathetic neurotransmitter, is also present in human umbilical endothelial cells (HUVECs), and is potently chemotactic and angiogenic by acting on one or several of Y1-Y5 receptors. In HUVECs, NPY is co-localized with dipeptidyl peptidase IV (DPPIV) which cleaves Tyr(1)-Pro(2) from NPY(1-36) to form NPY(3-36) resulting in the formation of a non-Y1 receptor agonist, which remains angiogenic. Presently we studied the effects of DPPIV's blockade using monoclonal antibodies (mAbs) on migration of HUVECs in response to NPY(1-36) or NPY(3-36) following cell wounding. Both peptides caused similar dose-dependent increases in cell migration (+80% at 0.1 nM) 12 h after wounding. DPPIV mAbs, E19 and E26, significantly reduced HUVEC's migration below that of the untreated cells, and blocked responses to NPY(1-36) but not NPY(3-36). Enhanced expression of DPPIV was found in the migrating cells and in cells with their protrusions at the edge of the wound (immunostaining and Western blot). Thus, DPPIV's expression is stimulated by endothelial wounding and its enzymatic activity is required for NPY-mediated chemotaxis. Furthermore, this suggests that non-Y1 receptors activated by NPY(3-36) (Y2, Y3 and/or Y5) mediate angiogenic effects of NPY.

A novel protease-docking function of integrin at invadopodia.

Invadopodia are membrane extensions of aggressive tumor cells that function in the activation of membrane-bound proteases occurring during tumor cell invasion. We explore a novel and provocative activity of integrins in docking proteases to sites of invasion, termed invadopodia. In the absence of collagen, alpha(3)beta(1) integrin and the gelatinolytic enzyme, seprase, exist as nonassociating membrane proteins. Type I collagen substratum induces the association of alpha(3)beta(1) integrin with seprase as a complex on invadopodia. The results show that alpha(3)beta(1) integrin is a docking protein for seprase to form functional invadopodia. In addition, alpha(5)beta(1) integrin may participate in the adhesion process necessary for invadopodial formation. Thus, alpha(3)beta(1) and alpha(5)beta(1) integrins play major organizational roles in the adhesion and formation of invadopodia, promoting invasive cell behavior.

Temporal-spatial expression of two Paracentrotus lividus cell surface proteins.

The temporal expression of two cell surface proteins, called BEP1 and BEP4, during Paracentrosus lividus embryonic development was studied. These proteins are found in both monomeric and dimeric forms in egg and embryos and we have established that their specific form is related to their being in the cytoplasm or on the cell surface. The spatial distribution of BEP1 and BEP4 proteins in eggs and embryos was established by whole mount immunohistochemistry. These proteins are located in the animal part of unfertilized and fertilized eggs; thereafter they are much less represented in structures derived from the vegetal cells of the embryo such as the micromeres of the 16 cell stage, the primary mesenchyme of blastula and the gut of gastrula. At the prism stage BEP1 and BEP4 proteins are present to some ectodermal parts and thereafter, at the pluteus stage, to the oral region.

Molecular cloning of seprase: a serine integral membrane protease from human melanoma.

Seprase is a homodimeric 170 kDa integral membrane gelatinase whose expression correlates with the invasiveness of the human melanoma cell line LOX. Here, we report the molecular cloning of a cDNA that encodes the 97 kDa subunit of seprase. Its deduced amino acid sequence predicts a type II integral membrane protein with a cytoplasmic tail of 6 amino acids, followed by a transmembrane domain of 20 amino acids and an extracellular domain of 734 amino acids. The carboxyl terminus contains a putative catalytic region (approximately 200 amino acids) which is homologous (68% identity) to that of the nonclassical serine protease dipeptidyl peptidase IV (DPPIV). The conserved serine protease motif G-X-S-X-G is present as G-W-S-Y-G. However, sequence analysis of seprase cDNA from LOX and other cell lines strongly suggests that seprase and human fibroblast activation protein alpha (FAP alpha) are products of the same gene. We propose that seprase/FAP alpha and DPPIV represent a new subfamily of serine integral membrane proteases (SIMP).

Identification and characterization of a constitutive HSP75 in sea urchin embryos.

An antiserum against a hsp of the 70-kDa family was prepared, by means of a fusion protein, which was able to detect a constitutive 75-kDa hsc in the sea urchin P. lividus. This hsc was present both during oogenesis and at all developmental stages. A two-dimensional electrophoresis has revealed four isolectric forms of this 75-kDa hsc. The amino acid sequence of the fragment used to prepare the anti-hsp70 antibodies revealed a 43% identity with the corresponding part of sea urchin sperm receptor, and in mature eggs a brighter immunofluorescence was seen all around the cell cortex where the receptor for sea urchin sperm is localized. In oocytes the hsp75 was localized in the cytoplasms but not in the nuclei. In the embryos a higher hsp75 concentration was found in the portion facing the lumen of the cells which invaginate at gastrulation.

Cell adhesion-dependent regulation of cell growth during sea urchin development.

In the sea urchin embryo there are at least two cell adhesion molecules related to mammalian cadherins, one of them, similar to E-cadherin, is expressed in embryos at very early developmental stages, the second appears at the blastula stage (G. Ghersi et al. Mech. Dev. 41, 47-55 (1993)). We show here that when sea urchin embryos are treated with monovalent fragments of antibodies directed against the extracellular domain of these molecules, the decompaction of the embryo is accompanied by a sharp reduction of the rate of cell division. Treatment of the embryos with Fab fragments inhibits thymidine incorporation, but does not affect thymidine uptake or amino acid incorporation. After the first day of development treated embryos have 10 times less blastomeres than normal; later, however, they resume development and give eventually rise to normal-looking plutei. Analysis of putative second messengers shows that treatment of the embryos with anti-cadherin Fabs leads to a decreased tyrosine phosphorylation of the two cadherins and of two cadherin-associated proteins and to a doubling of the intracellular concentration of cAMP. These results are discussed in view of the importance of cell adhesion signals for cell growth control.

Presence of different collagens and collagen mRNAs during embryogenesis and in adult tissues of the sea urchin Paracentrotus lividus.

The presence of four different collagen genes had been previously described in the sea urchin genome and four different cDNAs had been cloned and sequenced. Two of them code for 140 and 300 KDaltons proteins, belonging to the fibrillar collagens, and the other two families code for two type IV collagens with a molecular weight of about 210 KDaltons. In this paper immunological evidence is provided for the presence in the developing P. lividus sea urchin embryo of at least seven major collagen proteins. Western blot analyses, carried out by means of specific polyclonal antibodies, show a series of collagenase sensitive bands, with molecular weights ranging from 55 to 200 KDaltons, which are present from eggs to plutei. Northern blot analyses show the presence of the previously described 6 and 9 Kb RNA bands from oocytes till plutei; in the later stages two other collagen RNAs are detected. The presence of two sets of genes coding for the 6 Kb mRNAs, differentially expressed during development, is also discussed. Immunofluorescence histological analyses show the location of collagen in gonads, oocytes, eggs, embryos and adult tissues.

Spatial distribution of two maternal messengers in Paracentrotus lividus during oogenesis and embryogenesis.

We demonstrated that two mRNAs that are synthesized during the vitellogenic period of oogenesis and that code for cell surface proteins are asymmetrically distributed in the unfertilized egg of Paracentrotus lividus. At fertilization, these RNAs rapidly localize in the cortical zone at the animal pole of the egg. They are then detected in the mesomeres and the macromeres, but not in the micromeres, and thereafter are found in the ectoderm but not in the vegetal plate, mesenchyme cells, or early intestine. They disappear in late gastrula. The proteins synthesized by these mRNAs show the same territorial location during the period examined here, which included the unfertilized egg and the 16-blastomere stage. These conclusions were reached on the basis of in situ hybridization and immunostaining experiments, as well as Northern and Western blot analyses of isolated blastomeres. The possible significance of this asymmetric distribution of these two mRNAs and proteins in the establishment of the animal/vegetal axis is discussed.

Identification of a protein encoded by a mouse simple repeated sequence.

In order to study the possibility that a mouse repeated 'simple sequence', containing an ORF, could encode a protein, we inserted a fragment of a cDNA clone into the expression vector pEX31C. The fragment containing the short sequence 'CAGAGAGG' was expressed as MS2 polymerase fusion protein in Escherichia coli. This fusion protein (H5fp) was injected subcutaneously into rabbit and the corresponding polyclonal antibodies generated. Western blot analysis of proteins extracted from different mouse tissues established that anti H5fp antibodies recognized, in vivo, an antigen of 28 KDa. Immunolocalization with anti H5fp antibodies showed the presence of the related antigen in the cytoplasm of the examined sections. An hypothetical secondary structure for the protein was predicted by the Chou and Fasman method.

Human breast carcinoma cells cultured in the presence of serum shed membrane vesicles rich in gelatinolytic activities.

Human breast carcinoma cell lines, 8701-BC and MCF-7, in culture shed membrane vesicles with similar morphology. Vesicles shed in the presence of serum were rich in gelatinolytic activities, but not those obtained in the absence of serum. Zymographic analyses of the vesicles from 8701-BC and MCF-7, using gelatin as substrate, showed three predominant activities at 68-kDa, 97-kDa, and above 200-kDa. The ratio of the three activities was similar in the vesicles recovered from the two cell lines, but the vesicles from 8701-BC cells contained greater amounts of activities than those from MCF-7 cells. Optimal pH and sensitivity to protease inhibitors suggest that all gelatinolytic activities detected in vesicles are metalloproteinases. Treatment of the vesicles extracts with 4-aminophenylmercuric acetate and comparison with the purified enzyme indicate that 97-kDa gelatinase is the precursor of matrix metalloproteinase-9 (gelatinase B). These results support the early hypothesis that vesicle shedding from the plasma membrane may participate in metastatic cascade of cancer cells.

An epidemic of tuberculosis in an elementary school, Sanremo, Italy, 1993.

In late 1992, three cases of tuberculosis were identified in school children attending a small elementary school in Sanremo, Italy. In order to identify further cases and determine the source, an epidemiologic investigation was undertaken. Tine test and X rays were performed on all students and school personnel. A total of 80% of the 59 students in the school had positive tine test reactions, as did 100% of the 12 teachers. All but one of the positive students had converted since last tested, as had the nine teachers who had been previously negative. The source of the outbreak was a teacher who had been in direct classroom contact with two of the five classes and had worked closely with the rest in building of a Christmas creche for the school. This outbreak suggests that increasing attention should be paid to school as potential foci for the spread of tuberculosis and that greater attention be paid to teacher screening, particularly in areas of higher tuberculosis prevalence.

Differential expression and function of cadherin-like proteins in the sea urchin embryo.

Cadherins are Ca(+2)-dependent cell surface proteins involved in the specification of the adhesive properties of cells. They are supposed to play a critical role in morphogenesis and pattern formation. In this paper we show that in the sea urchin embryo there are at least two different cadherins of relative molecular masses 140 and 125 kDa. The 140 kDa cadherin is already present in the fertilized egg and is the sea urchin equivalent of E-cadherin. The 125 kDa cadherin, which can be detected using a broad-spectrum anti-cadherin antibody, appears only at later stages of development. In later embryos these two molecules are distributed differently: E-cadherin is present predominantly in the invaginating endoderm of the gastrula while the 125 kDa protein is present on the cell surface of most epithelia. Consistently with the observed differences in expression and in distribution, antibodies directed against these two cadherins differently perturb sea urchin development. For example, when these antibodies are added to early gastrulas only the antibodies against the 125 kDa component can induce a complete disaggregation of the ectoderm, while anti E-cadherin antibodies induce an abnormal development of the endoderm while the embryo maintains its basic integrity. These results are discussed in view of the need for multiple adhesion receptors during pattern formation and embryogenesis.

Characterization of bep1 and bep4 antigens involved in cell interactions during Paracentrotus lividus development.

We have identified and partially characterised two antigens, extracted with 3% butanol, from Paracentrotus lividus embryos dissociated at the blastula stage, and encoded by the cDNA clones previously described as bep1 and bep4 (bep-butanol extracted proteins). The cDNA fragments containing the specific central portions of bep1 and bep4 were expressed as MS2 polymerase fusion proteins in Escherichia coli. These two fusion proteins, called 1C1 (bep1) and 4A1 (bep4), were injected subcutaneously into rabbits and the corresponding polyclonal antibodies generated. Western blot analysis of proteins, extracted with 3% butanol, from sea urchin embryos at the blastula stage (b.e.p.), established that both antibodies recognize two 33 KDa proteins. Reducing and non-reducing electrophoretic conditions show that both antibodies against bep1 and bep4 related proteins react also with a protein band of a molecular weight 66 KDa, indicating that these two antigens probably exist as dimers. Immunolocalization with anti 1C1 and 4A1 antibodies shows the presence of the related antigens also on the cell surface. Fab fragments of the polyclonal antibodies against 1C1 and 4A1 inhibited reaggregation of sea urchin embryonic cells, dissociated from blastula stage embryos. This prevention of reaggregation indicates that these proteins probably play a role in cell interaction during sea urchin embryonic development.

An acid extract from dissociation medium of sea urchin embryos, induces mesenchyme differentiation.

When material extracted by 1 M acetic acid from the dissociation medium of sea urchin embryos is added at low concentrations to isolated primary mesenchyme cells, it induces skeletogenesis. The same material added to dissociated blastula cells, or to embryos at the blastula stage, stimulates skeleton formation and pigment cell differentiation. On dissociated cells, it also increases cell reaggregation, thymidine incorporation and survival. On embryos, it induces exogastrulation and appearance of extraembryonic pigment cells. The activity of the extract is resistant to raised temperatures and partially to tryptic digestion but is abolished by trypsin treatment followed by heating. The active fraction does not readily filter through Amicon XM-50 and is retarded by column chromatography on Bio-Gel P-60.