Enhancing sealing of fetal membrane defects using tissue engineered native amniotic scaffolds in the rabbit model

OBJECTIVE: The purpose of this study was to compare the efficacy of native engineered amniotic scaffolds (AS) and polyesterurethane scaffolds (DegraPol) and document wound healing response when sealing iatrogenic fetal membrane defects in the rabbit model. STUDY DESIGN: Native AS were engineered from freshly harvested membranes of 23 days' gestational age (GA; term = 31-2 d). Acellularity of AS was assessed by histology, light and scanning electron microscopy. Fetal membrane defects were created by 14 gauge-needle puncture at GA 23 days and primarily closed with AS (n = 10) or DegraPol (n = 10) or left unclosed (positive controls; n = 10). Sixty-one sacs served as negative controls. At GA 30 days a second look hysterotomy was performed to assess presence of amniotic fluid (AF) and harvest plugging sites for microscopic evaluation. RESULTS: Engineered AS had a cell-free collagenous fiber network. AF was significantly higher only in the DegraPol group (78%; P < .05) compared to the AF in positive controls (17%). Integration of plugs in the fetal membrane defect was better with AS than DegraPol, with higher reepithelialization rates (AS: 52.5% +/- 6.5%; DegraPol: 11.6% +/- 2.6%; P < .001) and proliferation indices (AS: 0.47 +/- 0.03; DegraPol: 0.28 +/- 0.04; P = .001). In both treatment groups, cell proliferation in the myometrium was increased (P < .05). CONCLUSION: Native AS seal iatrogenic fetal membrane defects better than DegraPol. Within a week, there is abundant reepithelilization and minimal local inflammation. This yields the proof of principle that engineered native, amniotic membrane scaffolds enhance fetal membrane wound healing response.

The supramolecular assemblies of voltage-dependent anion channels in the native membrane

Voltage-dependent anion channels (VDACs) are major constituents of the outer mitochondrial membrane (OMM). These primary transporters of nucleotides, ions and metabolites mediate a substantial portion of the OMM molecular traffic. To study the native supramolecular organization of the VDAC, we have isolated, characterized and imaged OMMs from potato tubers. SDS-PAGE and mass spectrometry of OMMs revealed the presence of the VDAC isoforms POM34 and POM36, as well as the translocase of the OMM complex. Tubular two-dimensional crystals of the VDAC spontaneously formed after incubation of OMMs for two to three months at 4 degrees C. Transmission electron microscopy revealed an oblique lattice and unit cells housing six circular depressions arranged in a hexagon. Atomic force microscopy of freshly isolated OMMs demonstrated (i) the existence of monomers to tetramers, hexamers and higher oligomers of the VDAC and (ii) its spatial arrangement within the oligomers in the native membrane. We discuss the importance of the observed oligomerization for modulation of the VDAC function, for the binding of hexokinase and creatine kinase to the OMM and for mitochondria-mediated apoptosis.

Hypoxia increases cytoplasmic expression of NDRG1, but is insufficient for its membrane localization in human hepatocellular carcinoma

NDRG1 is a hypoxia-inducible protein, whose modulated expression is associated with the progression of human cancers. Here, we reveal that NDRG1 is markedly upregulated in the cytoplasm and on the membrane in human hepatocellular carcinoma (HCC). We demonstrate further that hypoxic stress increases the cytoplasmic expression of NDRG1 in vitro, but does not result in its localization on the plasma membrane. However, grown within an HCC-xenograft in vivo, cells express NDRG1 in the cytoplasm and on the plasma membrane. In conclusion, hypoxia is a potent inducer of NDRG1 in HCCs, albeit requiring additional stimuli within the tumour microenvironment for its recruitment to the membrane.

Basement membrane remodeling in skeletal muscles of patients with limb ischemia involves regulation of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases

BACKGROUND/AIM: Because the pericapillary basement membrane in skeletal muscles of patients with chronic critical limb ischemia (CLI) is thickened, we determined the expression patterns of genes involved in collagen metabolism, using samples from 9 CLI patients, 4 patients with acute limb ischemia and 4 healthy controls. METHODS: Gene array analysis, quantitative RT-PCR and semiquantitative grading of immunohistochemical reactivity were performed to determine mRNA/cDNA and protein concentrations. RESULTS: In CLI patients compared to controls, cDNA levels of matrix metalloproteinase (MMP)-9 and MMP-19 were higher, collagen type IV chains A1 and A2, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2 were similar and MMP-2 were lower. On the protein level, MMP-2, MMP-9, MMP-19 and TIMP-1 were more abundantly expressed. In skeletal muscles from patients with acute limb ischemia, cDNA and protein levels of MMP-9, MMP-19, collagen type IV chains, TIMP-1 and TIMP-2 were high. MMP-2 was elevated at the protein but decreased on the cDNA level. CONCLUSION: Expression of basement membrane components in skeletal muscles of CLI and acute limb ischemia patients is altered, possibly contributing to the pathogenesis of peripheral arterial disease.

Attenuation of myocardial reperfusion injury in pigs by Mirococept, a membrane-targeted complement inhibitor derived from human CR1

OBJECTIVES: Membrane-targeted application of complement inhibitors may ameliorate ischemia/reperfusion (I/R) injury by directly targeting damaged cells. We investigated whether Mirococept, a membrane-targeted, myristoylated peptidyl construct derived from complement receptor 1 (CR1) could attenuate I/R injury following acute myocardial infarction in pigs. METHODS: In a closed-chest pig model of acute myocardial infarction, Mirococept, the non-tailed derivative APT154, or vehicle was administered intracoronarily into the area at risk 5 min pre-reperfusion. Infarct size, cardiac function and inflammatory status were evaluated. RESULTS: Mirococept targeted damaged vasculature and myocardium, significantly decreasing infarct size compared to vehicle, whereas APT154 had no effect. Cardioprotection correlated with reduced serum troponin I and was paralleled by attenuated local myocardial complement deposition and tissue factor expression. Myocardial apoptosis (TUNEL-positivity) was also reduced with the use of Mirococept. Local modulation of the pro-inflammatory and pro-coagulant phenotype translated to improved left ventricular end-diastolic pressure, ejection fraction and regional wall motion post-reperfusion. CONCLUSIONS: Local modification of a pro-inflammatory and pro-coagulant environment after regional I/R injury by site-specific application of a membrane-targeted complement regulatory protein may offer novel possibilities and insights into potential treatment strategies of reperfusion-induced injury.

Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis

Invasion of non-professional phagocytes is a strategy employed by several mucosal pathogens, but has not been investigated in detail for Moraxella catarrhalis, a major cause of human respiratory tract infections. We investigated the role of outer membrane protein (OMP) UspA1 and lipooligosaccharide (LOS) in M. catarrhalis invasion into epithelial cells. An isogenic mutant of strain O35E, which lacked expression of the UspA1 adhesin, demonstrated not only severely impaired adherence (86%) to but also reduced invasion (77%) into Chang conjunctival cells in comparison with the wild-type strain. The isogenic, LOS-deficient mutant strain O35E.lpxA was attenuated in adherence (93%) and its capacity to invade was severely reduced (95%), but not abolished. Inhibition assays using sucrose and cytochalasin D, respectively, demonstrated that clathrin and actin polymerization contribute to internalization of M. catarrhalis by Chang cells. Furthermore, inhibition of UspA1-mediated binding to cell-associated fibronectin and alpha5beta1 integrin decreased invasion of M. catarrhalis strain O35E (72% and 41%, respectively). These data indicate that OMP UspA1 and LOS profoundly affect the capacity of M. catarrhalis to invade epithelial cells.

Human immunodeficiency virus type 1 and influenza virus exit via different membrane microdomains

Directed release of human immunodeficiency virus type 1 (HIV-1) into the cleft of the virological synapse that can form between infected and uninfected T cells, for example, in lymph nodes, is thought to contribute to the systemic spread of this virus. In contrast, influenza virus, which causes local infections, is shed into the airways of the respiratory tract from free surfaces of epithelial cells. We now demonstrate that such differential release of HIV-1 and influenza virus is paralleled, at the subcellular level, by viral assembly at different microsegments of the plasma membrane of HeLa cells. HIV-1, but not influenza virus, buds through microdomains containing the tetraspanins CD9 and CD63. Consequently, the anti-CD9 antibody K41, which redistributes its antigen and also other tetraspanins to cell-cell adhesion sites, interferes with HIV-1 but not with influenza virus release. Altogether, these data strongly suggest that the bimodal egress of these two pathogenic viruses, like their entry into target cells, is guided by specific sets of host cell proteins.

Salivary antibodies directed against outer membrane proteins of Moraxella catarrhalis in healthy adults

Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract, but the mucosal immune response directed against surface components of this organism has not been characterized in detail. The aim of this study was to investigate the salivary immunoglobulin A (IgA) response toward outer membrane proteins (OMP) of M. catarrhalis in healthy adults, the group of individuals least likely to be colonized and thus most likely to display mucosal immunity. Unstimulated saliva samples collected from 14 healthy adult volunteers were subjected to IgA immunoblot analysis with OMP preparations of M. catarrhalis strain O35E. Immunoblot analysis revealed a consistent pattern of IgA reactivity, with the appearance of five major bands located at >250, 200, 120, 80, and 60 kDa. Eleven (79%) of 14 saliva samples elicited reactivity to all five bands. Immunoblot analysis with a set of isogenic knockout mutants lacking the expression of individual OMP was used to determine the identities of OMP giving rise to IgA bands. Human saliva was shown consistently to exhibit IgA-binding activity for oligomeric UspA2 (>250 kDa), hemagglutinin (200 kDa), monomeric UspA1 (120 kDa), transferrin-binding protein B (TbpB), monomeric UspA2, CopB, and presumably OMP CD. TbpB, oligomeric UspA2, and CopB formed a cluster of bands at about 80 kDa. These data indicate that the human salivary IgA response is directed consistently against a small number of major OMP, some of which are presently considered vaccine candidates. The functional properties of these mucosal antibodies remain to be elucidated.

Comparing oxygen transfer performance between three membrane oxygenators: effect of temperature changes during cardiopulmonary bypass

Recently, a new oxygenator (Dideco 903 [D903], Dideco, Mirandola, Italy) has been introduced to the perfusion community, and we set about testing its oxygen transfer performance and then comparing it to two other models. This evaluation was based on the comparison between oxygen transfer slope, gas phase arterial oxygen gradients, degree of blood shunting, maximum oxygen transfer, and diffusing capacity calculated for each membrane. Sixty patients were randomized into three groups of oxygenators (Dideco 703 [D703], Dideco; D903; and Quadrox, Jostra Medizintechnik AG, Hirrlingen, Germany) including 40/20 M/F of 68.6 +/- 11.3 years old, with a body weight of 71.5 +/- 12.1 kg, a body surface area (BSA) of 1.84 +/- 0.3 m(2), and a theoretical blood flow rate (index 2.4 times BSA) of 4.4 +/- 0.7 L/min. The maximum oxygen transfer (VO(2)) values were 313 mL O(2)/min (D703), 579 mL O(2)/min (D903), and 400 mL O(2)/min (Quadrox), with the D903 being the most superior (P < 0.05). Oxygen (O(2)) gradients were 320 mm Hg (D703), 235 mm Hg (D903), and 247 mm Hg (Quadrox), meaning D903 and Quadrox are more efficient versus the D703 (P < 0.05). Shunt fraction (Qs/Qt) and diffusing capacity (DmO(2)) were comparable (P = ns). Diffusing capacity values indexed to BSA (DmO(2)/m(2)) were 0.15 mL O(2)/min/mm Hg/m(2) (D703), 0.2 mL O(2)/min/mm Hg/m(2) (D903), and 0.18 mL O(2)/min/mm Hg/m(2) (Quadrox) with D903 outperforming D703 (P < 0.0005). During hypothermia (32.0 +/- 0.3 degrees C), there was a lower absolute and relative VO(2 )for all three oxygenators (P = ns). The O(2) gradients, DmO(2) and DmO(2)/m(2), were significantly lower for all oxygenators (P < 0.01). Also, Qs/Qt significantly rose for all oxygenators (P < 0.01). The oxygen transfer curve is characteristic to each oxygenator type and represents a tool to quantify oxygenator performance. Using this parameter, we demonstrated significant differences among commercially available oxygenators. However, all three oxygenators are considered to meet the oxygen needs of the patients.

Follicle-forming cat thyroid cell lines synthesizing extracellular matrix and basal membrane components: a new tool for the study of thyroidal morphogenesis

Interactions between follicular epithelial cells and extracellular matrix (ECM) are supposed to play an important role in the development and maintenance of thyroid tissue architecture. In the present study we have therefore investigated the synthesis of ECM components by a feline thyroid cell line which is able to form follicle-like structures in vitro, and also in v-ras-transfected and control-transfected sublines. Transfections were performed by lipofection with pZSR (viral Harvey ras gene; neo) and pSV2-neo (control, neo only) plasmids. We have adapted a semisolid culture system composed exclusively of polymerized alginate and therefore devoid of ECM components. Feline cells embedded in alginate gels as single cells and cultured for up to 90 days formed cell clusters within 10 days. Follicle-like structures were formed in the original cell lines and also in the v-ras- and control-transfected cells. Differences in proliferation rates were observed, the v-ras-transfected cells growing up to two to three times faster than the non-transfected cells. Immunostaining was done using rabbit first antibodies directed against mouse collagen IV, human fibronectin, laminin (tumor Engelbreth-Holm-Swarm laminin), perlecan and other ECM components. For comparison, immunostaining was also performed on cryosections of nodular goiters of six hyperthyroid cats. The cell lines and their transfected clones stained strongly positive for collagen IV and fibronectin, and positively but less strongly for laminin and perlecan. The cat goiter tissue stained positively for collagen IV, laminin, perlecan, and fibronectin, and positive staining for S-laminin (containing the beta2-chain) was seen in blood vessel walls in this tissue. In conclusion, cat cell lines grow three-dimensionally in alginate beads over several weeks, they form follicle-like structures and express the same ECM components as the native cat goiter tissue. Transfection with v-ras does increase proliferation rate, but does not fundamentally alter formation of follicle-like structures and ECM expression. Alginate gel culture is a promising new tool for the study of follicular morphogenesis, polarity, the expression pattern of ECM components and of the interaction between thyrocytes and ECM. It avoids interference caused by gels composed of ECM components.

Meprins, membrane-bound and secreted astacin metalloproteinases

The astacins are a subfamily of the metzincin superfamily of metalloproteinases. The first to be characterized was the crayfish enzyme astacin. To date more than 200 members of this family have been identified in species ranging from bacteria to humans. Astacins are involved in developmental morphogenesis, matrix assembly, tissue differentiation and digestion. Family members include the procollagen C-proteinase (BMP1, bone morphogenetic protein 1), tolloid and mammalian tolloid-like, HMP (Hydra vulgaris metalloproteinase), sea urchin BP10 (blastula protein) and SPAN (Strongylocentrotus purpuratus astacin), the 'hatching' subfamily comprising alveolin, ovastacin, LCE, HCE ('low' and 'high' choriolytic enzymes), nephrosin (from carp head kidney), UVS.2 from frog, and the meprins. In the human and mouse genomes, there are six astacin family genes (two meprins, three BMP1/tolloid-like, one ovastacin), but in Caenorhabditis elegans there are 40. Meprins are the only astacin proteinases that function on the membrane and extracellularly by virtue of the fact that they can be membrane-bound or secreted. They are unique in their domain structure and covalent subunit dimerization, oligomerization propensities, and expression patterns. They are normally highly regulated at the transcriptional and post-translational levels, localize to specific membranes or extracellular spaces, and can hydrolyse biologically active peptides, cytokines, extracellular matrix (ECM) proteins and cell-surface proteins. The in vivo substrates of meprins are unknown, but the abundant expression of these proteinases in the epithelial cells of the intestine, kidney and skin provide clues to their functions.