Flexible and elastic porous poly(trimethylene carbonate) structures for use in vascular tissue engineering.

Biocompatible and elastic porous tubular structures based on poly(1,3-trimethylene carbonate), PTMC, were developed as scaffolds for tissue engineering of small-diameter blood vessels. High-molecular-weight PTMC (M(n) = 4.37 x 10(5)) was cross-linked by gamma-irradiation in an inert nitrogen atmosphere. The resulting networks (50-70% gel content) were elastic and creep resistant. The PTMC materials were highly biocompatible as determined by cell adhesion and proliferation studies using various relevant cell types (human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) and mesenchymal stem cells (MSCs)). Dimensionally stable tubular scaffolds with an interconnected pore network were prepared by particulate leaching. Different cross-linked porous PTMC specimens with average pore sizes ranging between 55 and 116 microm, and porosities ranging from 59% to 83% were prepared. These scaffolds were highly compliant and flexible, with high elongations at break. Furthermore, their resistance to creep was excellent and under cyclic loading conditions (20 deformation cycles to 30% elongation) no permanent deformation occurred. Seeding of SMCs into the wall of the tubular structures was done by carefully perfusing cell suspensions with syringes from the lumen through the wall. The cells were then cultured for 7 days. Upon proliferation of the SMCs, the formed blood vessel constructs had excellent mechanical properties. Their radial tensile strengths had increased from 0.23 to 0.78 MPa, which is close to those of natural blood vessels.

Collision tumor of the stomach: a case of an adenocarcinoma and a gastrointestinal stromal tumor.

A collision tumor of the stomach is a rare event. We report the case of a collision tumor of the stomach consisting of an adenocarcinoma and a gastrointestinal stromal tumor (GIST). This is, to our knowledge, the second report in the literature of such a case. A 71-year-old man with abdominal discomfort underwent an esophagogastroduodenoscopy which revealed a tumor of the oesophagogastric junction. A total gastrectomy was performed. Histologic examination showed a mixed tumor consisting of a primary adenocarcinoma and multiple nodules of GIST. The adenocarcinoma showed both diffuse and intestinal growth, angio-invasion and metastasis to lymph nodes. The GIST tumor cells were strongly immunoreactive to CD117 and CD34. Based on mitotic index, size and cytonuclear details, the biological behavior of this GIST tumor was supposed to be benign. This case reports the rare finding of a collision tumor consisting of an adenocarcinoma and a GIST with an unknown etiology.

[Distinguishing nodal naevus from melanoma metastases in the sentinel node in patients with melanoma]. / Een nodale naevus in de schildwachtklier bij patiënten met melanoom: onderscheid met melanoommetastase.

OBJECTIVE: Establishing the frequency of nodal naevi in lymph-node dissections from patients with a melanoma who have undergone a sentinel-node procedure and/or regional node dissection and distinguishing naevi from melanoma metastases. DESIGN: Retrospective and descriptive. METHODS: Patients with a nodal naevus in the sentinel node were selected from a database containing clinical and pathological data on all 65 patients who underwent a sentinel-node procedure for melanoma at our hospital between 1998 and 2001. Also data from the pathology department on the case frequency and the nodal frequency of nodal naevi in the total number of patients with melanoma in whom a sentinel-node procedure and/or therapeutic node dissection had been carried out during the same period, were examined. RESULTS: In 5 patients a nodal naevus was found in the sentinel node. The case frequency was 6.2% and the nodal frequency 0.65%. Distinction from melanoma metastases was made by the use of H&E colouring, localization, architectural and morphological features of the melanocyte cell clusters in the lymph node and sometimes after consultation with the National Melanoma Panel. Immunohistochemical markers provided supplementary information only. CONCLUSION: Nodal naevi in lymph nodes were not uncommon in people with melanoma and can be distinguished from the micrometastases from melanoma.

The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes.

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface. In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.

Glomerular extracellular matrix components and integrins.

It has become apparent that extracellular matrix components and their cellular receptors, the integrins, are important regulators of glomerular development and function. In this rapidly evolving field we studied the production of extracellular matrix components and integrins by rat glomerular visceral epithelial and mesangial cells, using molecular probes and antibodies that have recently become available. Special attention was paid to laminin isoforms and to splice variants of the integrin subunits alpha 3 and alpha 6. Results were compared to the in vivo expression in human fetal, newborn and adult kidneys. The mesangial cells were found to produce laminin-1, nidogen and two as yet unidentified laminin isoforms with putative alpha chains of about 395 (alpha x) and of 375 kDa (alpha y), tentatively described before as bovine kidney laminin. Furthermore, they expressed the integrins alpha 1 beta 1, alpha 2 beta 1, alpha 3A beta 1, alpha 5 beta 1, alpha v beta 3, alpha v beta 5, and small amounts of alpha 6A beta 1 and alpha 6B beta 1. The glomerular visceral epithelial cells produced the two new laminin isoforms mentioned above, laminin-5, but no laminin-1 or nidogen. The integrins alpha 2 beta 1, alpha 3A beta 1, alpha 6A beta 4, alpha 6B beta 4 and the integrin subunit alpha v were found to be expressed. We show that during nephrogenesis, the laminin alpha 1 chain disappears and is replaced by another alpha chain, possibly one of the two as yet unidentified alpha chains mentioned above. The laminin beta 1 chain is replaced by the beta 2 chain somewhat later in glomerular development. In general, the integrins found to be expressed in glomeruli of adult kidney were consistent with those found in cultured glomerular visceral epithelial and mesangial cells. No splice variant switch of the integrin alpha 3 or alpha 6 subunits could be demonstrated during nephrogenesis. Our results suggest an important role for the mesangial cell in providing nidogen as a crucial component of the supramolecular structure of the glomerular basement membrane. Furthermore our results indicate that laminin alpha x beta 2 gamma 1 and alpha y beta 2 gamma 1 isoforms are important in the glomerulus of adult kidney and that the integrin alpha 3A beta 1 is the main integrin receptor for laminin isoforms on glomerular visceral epithelial and mesangial cells, both in vitro and in vivo.

The A and B variants of the alpha 3 integrin subunit: tissue distribution and functional characterization.

The alpha subunits of the laminin-binding integrins alpha 3 beta 1, alpha 6 beta 1, and alpha 7 beta 1 have homologous sequences and are similar in structure. Two cytoplasmic variants, A and B, have been identified for each of these alpha subunits, although the alpha 3B splice variant has been detected only at the mRNA level. We prepared a panel of mouse monoclonal antibodies specific for the A and B variants of the alpha 3 subunit to study their tissue distribution. Four monoclonal antibodies react with alpha 3A, one of which recognizes only the nonphosphorylated form; of the three anti-alpha 3B antibodies, one cross-reacts with alpha 6B. Reverse transcriptase-PCR analysis of various human tissues revealed the presence of alpha 3B mRNA in brain, heart, and skeletal muscle. Moreover, the alpha 3B protein was detected by immunoblotting in brain and heart tissue but not in skeletal muscle. In contrast, alpha 3A mRNA and protein were present in all tissues studied. Thus, the expression of alpha 3B in adult tissues is more restricted than that of alpha 3A. Immunohistochemical studies showed that in brain tissue, both variants are exclusively expressed on small blood-vessel endothelium, whereas in heart tissue their distribution patterns differ markedly. Although alpha 3A is strongly expressed on vascular smooth muscle cells, alpha 3B is detected only on endothelial cells of veins. Expression of the two variant forms of alpha 3 in K562 cells revealed that the ligand-binding specificities of alpha 3A beta 1 and alpha 3B beta 1 are identical: both bind human laminin-2 and -4, laminin-5, and laminins isolated from bovine kidney, but not bovine laminin-2 and -4, mouse laminin-1, or human fibronectin. In addition, adhesion mediated by both integrins is induced to the same extent by phorbol 12-myristate 13-acetate. The alpha 3A, but not the alpha 3B subunit, is phosphorylated; and phosphorylation of alpha 3A increases after phorbol 12-myristate 13-acetate stimulation. Thus, we found no differences between the adhesion functions of the A and B variants of alpha 3.

Differential binding of Haemophilus influenzae to human tissues by fimbriae.

The hypothesis was investigated that tissue tropism of Haemophilus influenzae during colonisation and infection is associated with the ability of fimbriate bacteria to bind to the organs and cell types involved. H. influenzae type b with fimbriae (strain 770235f+) bound to several cell types, including ciliated columnar epithelial cells, pneumocytes, ependymal cells, glial cells, connective tissue fibroblasts, synovial cells, antigen-presenting cells, lymphocytes, erythrocytes and endothelial cells. Binding of H. influenzae to kidney, liver and conjunctiva cells was poor. Fimbriae-specific monoclonal antibody (MAb 6HE8) inhibited this binding. Some binding to endothelial cells and macrophages was also observed with non-fimbriate strains. This binding was not inhibited by MAb 6HE8. We conclude that in-vitro binding of fimbriate H. influenzae is mainly to those tissues and cells where H. influenzae is found during colonisation and infection. The data suggest that a shift to the non-fimbriate form is required for bacteria in the bloodstream to escape clearance mechanisms mediated by blood cells.