A high-content, in vitro cardiac fibrosis assay for high-throughput, phenotypic identification of compounds with anti-fibrotic activity.

A key feature in the pathogenesis of heart failure is cardiac fibrosis, but effective treatments that specifically target cardiac fibrosis are currently not available. A major impediment to progress has been the lack of reliable in vitro models with sufficient throughput to screen for activity against cardiac fibrosis. Here, we established cell culture conditions in micro-well format that support extracellular deposition of mature collagen from primary human cardiac fibroblasts - a hallmark of cardiac fibrosis. Based on robust biochemical characterization we developed a high-content phenotypic screening platform, that allows for high-throughput identification of compounds with activity against cardiac fibrosis. Our platform correctly identifies compounds acting on known cardiac fibrosis pathways. Moreover, it can detect anti-fibrotic activity for compounds acting on targets that have not previously been reported in in vitro cardiac fibrosis assays. Taken together, our experimental approach provides a powerful platform for high-throughput screening of anti-fibrotic compounds as well as discovery of novel targets to develop new therapeutic strategies for heart failure.

Treatment of full thickness focal cartilage lesions with a metallic resurfacing implant in a sheep animal model, 1 year evaluation.

BACKGROUND: Full depth focal cartilage lesions do not heal spontaneously and while some of these lesions are asymptomatic they might progress to osteoarthritis. Treatment for these lesions is warranted and the gold standard treatment at younger age remains biological healing by cell stimulation. In the middle-age patient the success rate of biologic treatment varies, hence the surge of non-biological alternatives. Our objective was to evaluate the efficacy and safety of a metallic implant for treatment of these lesions with respect to the long-term panarticular cartilage homeostasis. METHODS: The medial femoral condyle of 16 sheep was operated unilaterally. A metallic implant was inserted in the weight-bearing surface at an aimed height of 0.5 mm recessed. Euthanasia was performed at 6 or 12 months. Implant height and tilt was analyzed using a laser-scanning device. Damage to cartilage surfaces was evaluated macroscopically and microscopically according to the Osteoarthritis Research Society International (OARSI) recommendations. RESULTS: Thirteen sheep were available for evaluation and showed a varying degree of cartilage damage linearly increasing with age. Cartilage damage of the medial tibial plateau opposing the implant was increased compared to the non-operated knee by 1.77 units (p = 0.041; 95% CI: 0.08, 3.45) on a 0-27 unit scale. Remaining joint compartments were unaffected. Implant position averaged 0.54 recessed (95% CI: 0.41, 0.67). CONCLUSIONS: Our results showed a consistent and accurate placement of these implants at a defined zone. At this position cartilage wear of opposing and surrounding joint cartilage is limited. Thus expanded animal and human studies are motivated.

Dendritic cell-derived exosomes carry the major cat allergen Fel d 1 and induce an allergic immune response.

Exosomes are nano-sized membrane vesicles (50-120 nm), which are released from a wide variety of cells. Depending on their cellular origin, they can induce immune stimulatory-, inhibitory-, or tolerance-inducing effects. However, it is still unclear what role exosomes play during human inflammatory diseases. It has not been studied whether exosomes derived from human dendritic cells (DCs), the first cells to encounter allergens in the mucosa, can carry aeroallergens and contribute to allergic immune responses. We therefore explored whether DC-derived exosomes can present the major cat allergen Fel d 1 and whether they thereby contribute to the pathogenesis of allergic disease. Our results demonstrate that exosomes are able to present aeroallergens and thereby induce T-cell T(H)2-like cytokine production in allergic donors. Thus, these exosomes may be important immune-stimulatory factors in allergic immune responses and important targets or engineered tools in immunotherapy.

Focal knee resurfacing and effects of surgical precision on opposing cartilage. A pilot study on 12 sheep.

BACKGROUND: Full thickness cartilage lesions (ICRS grade 3-4) and focal lesions of degenerative origin may progress to osteoarthritis (OA). Such focal lesions can be treated by metallic implants. We hypothesized that such treatment results in opposing surface cartilage damage that correlates with implant position (height) relative to the adjacent cartilage surface. This relationship was investigated using a sheep animal model. METHODS: Both medial femoral condyles of 12 sheep were operated. The implants, were inserted in the weight-bearing surface at different heights relative to the surrounding cartilage. Euthanasia was performed at 6 or 12 weeks. After retrieval, implant height was analyzed using laser scanning. Damage to the opposing tibial cartilage was evaluated macroscopically and microscopically according to the modified Mankin score. RESULTS: Twenty-two knees were available for evaluation and showed cartilage lesions ranging from severe damage (Mankin stage 11) to almost pristine conditions (Mankin stage 1). There was a strong correlation between implant height and cartilage damage. Standard deviation from the aimed implant height was 0.47 mm. CONCLUSIONS: Our results showed significant surgical imprecision and protruding implants imposed severe cartilage damage. We therefore suggest implants should be placed recessed (approx. 0.5 mm) below the surrounding cartilage in this animal model. These results encourage further studies of metallic implants yet the utmost precision regarding position is required.

Novel fibrillar structure confers adhesive property to malaria-infected erythrocytes.

Infections with the malaria parasite Plasmodium falciparum are characterized by sequestration of erythrocytes infected by mature forms of the parasite. Sequestration seems critical for the survival of the parasite, but may lead to excessive binding in the microvasculature and death of the human host. We report here that a novel electrondense fibrillar structure, containing immunoglobulins M or M and G, is found at the surface of infected erythrocytes that adhere to host cells. In cases of cerebral malaria, fibrillar strands are also seen in the microvasculature at autopsy. Our findings may explain the adhesive mechanism by which malaria-infected erythrocytes cause the vascular obstruction seen in complicated malaria infections.

Maternal plasma level of antimicrobial peptide LL37 is a major determinant factor of neonatal plasma LL37 level.

AIM: To determine cathelicidin antimicrobial peptide LL37subcellular distribution in cord neutrophils and normal plasma LL37 levels in mothers and neonates, relate them to delivery mode and relevant biochemical markers, including 25-OHvitamin D [25(OH)D] as this molecules increases cathelicidin gene expression. METHODS: A total of 115 infants were included, n = 68 with normal delivery and n = 47 with elective Caesarean section (C-section), a subset of these being 50 mother-infant pairs. Biomarkers were determined in maternal and cord blood. Subcellular peptide LL37 distribution was analysed with immunoelectron microscopy. RESULTS: Cord plasma LL37 levels were three-times higher after normal delivery compared with C-section. A highly significant correlation was observed between maternal and cord plasma LL37 levels, regardless of delivery mode. No relationship was found between LL37 and 25(OH)D levels. Neutrophils from cord blood after normal delivery contained 10-times more cytoplasmatic cathelicidin peptide compared with corresponding cells after C-section where a strict granular localization was found. CONCLUSION: These data are consistent with a placental transfer of LL37 and identifies maternal stores as the critical factor determining neonatal plasma LL37 level. An additional enhancement of neonatal cathelicidin mobilization and release is connected to normal delivery stress.

DNAJB13 is a radial spoke protein of mouse '9+2' axoneme.

It has been shown that DNAJB13, a type II heat shock protein 40, is highly expressed in the testis and is an axonemal component of mouse mature spermatozoa. By multi-tissue reverse transcription polymerase chain reaction, we found that Dnajb13 gene was expressed not only in the testis but also in several other ciliated cell-containing tissues like brain, lung and oviduct. Immunohistochemistry on mouse trachea and oviduct sections shown that DNAJB13 was present in the motile cilia of those tissues. To define further its localization in the axoneme, immunoelectron microscopy of mouse sperm flagella was performed and shown that DNAJB13 was localized to radial spokes of the axoneme. Taken together, our data indicate that DNAJB13 is a radial spoke protein of the mouse '9+2' axoneme.

Targeting of Moloney murine leukemia virus gag precursor to the site of virus budding.

Retrovirus Moloney murine leukemia virus (M-MuLV) matures by budding at the cell surface. Central to the budding process is the myristoylated viral core protein precursor Gag which, even in the absence of all other viral components, is capable of associating with the cytoplasmic leaflet of the plasma membrane and assembling into extracellular virus-like particles. In this paper we have used heterologous, Semliki Forest virus-driven, expression of M-MuLV Gag to study the mechanism by which this protein is targeted to the cell surface. In pulse-chase experiments, BFA, monensin, and 20 degrees C block did not affect incorporation of Gag into extracellular particles thereby indicating that the secretory pathway is not involved in targeting of Gag to the cell surface. Subcellular fractionation studies demonstrated that newly synthesized Gag became rapidly and efficiently associated with membranes which had a density similar to that of plasma membrane-derived vesicles. Protease-protection studies confirmed that the Gag-containing membranes were of plasma membrane origin, since in crude cell homogenates, the bulk of newly synthesized Gag was protease-resistant as expected of a protein that binds to the cytoplasmic leaflet of the plasma membrane. Taken together these data indicate that targeting of M-MuLV Gag to the cell surface proceeds via direct insertion of the protein to the cytoplasmic side of the plasma membrane. Furthermore, since the membrane insertion reaction is highly efficient and specific, this suggests that the reaction is dependent on as-yet-unidentified cellular factors.

Nephrin is involved in podocyte maturation but not survival during glomerular development.

Nephrin, a major component of the glomerular slit diaphragm (SD), is both a structural protein as well as a signaling molecule influencing foot process (FP) formation and maintenance of podocyte integrity. Analyses of near-term embryonic kidneys showed normal cellular viability and no apoptosis in glomeruli from nephrin knockout mice. Moreover, expression and location of other SD or glomerular basement membrane components were similar in wild-type and mutant mice as was the location and levels of most podocyte-specific proteins. Transcriptional profiling showed that the lack of nephrin had minor impact on the expression of genes for FPs and SD proteins. Claudin 3, a tight-junction protein normally absent in glomeruli, was upregulated threefold in the knockout mice, suggesting a role of nephrin in claudin 3 gene expression within the glomeruli. Our results suggest that nephrin is expressed late in the process of podocyte differentiation and is a locus for the formation of SD and FP maintenance and physical integrity in vivo. Nephrin does not seem to have a primary role in cell survival but has a small impact on gene regulation during glomerular development.

The domain structure of centromeres is conserved from fission yeast to humans.

The centromeric DNA of fission yeast is arranged with a central core flanked by repeated sequences. The centromere-associated proteins, Mis6p and Cnp1p (SpCENP-A), associate exclusively with central core DNA, whereas the Swi6 protein binds the surrounding repeats. Here, electron microscopy and immunofluorescence light microscopy reveal that the central core and flanking regions occupy distinct positions within a heterochromatic domain. An "anchor" structure containing the Ndc80 protein resides between this heterochromatic domain and the spindle pole body. The organization of centromere-associated proteins in fission yeast is reminiscent of the multilayered structures of human kinetochores, indicating that such domain structure is conserved in eukaryotes.

Ultrastructural immunolocalisation of bone sialoprotein in the osteocartilagenous interface of the equine third carpal bone.

REASONS FOR PERFORMING STUDY: One of the most common causes of lameness in racehorses is osteoarthritis (OA). Pathogenesis is not clear and pathological processes of the different joint tissues interact in often progressive events. The interface between cartilage and newly synthesised bone has been shown to be particularly enriched in bone sialoprotein (BSP), a cell-binding matrix protein. OBJECTIVES: To establish whether changes in the concentration of BSP may serve as a marker for early biochemical changes of the subchondral bone. METHODS: Articular cartilage, cartilage/bone interface and subchondral bone of the proximal third carpal bone from 3 Standardbred trotters were analysed ultrastructurally for the presence of BSP in normal and degenerative areas. RESULTS: A marked increase of BSP in the cartilage/bone interface with degenerative changes of the bone and cartilage compared to the morphologically intact cartilage/bone interface was noted, but levels of the protein were distinctly lower in the distal bone. CONCLUSIONS: The results indicate that BSP has the potential to be used as a marker for changes in bone metabolism in the subchondral bone. POTENTIAL RELEVANCE: Tools to monitor early biochemical changes within the connective tissues of the joint in vivo are essential in studies of the pathogenesis of OA. These could be used to monitor and understand such changes in relation to load, exercise, training programmes, inflammation and the development of OA.

Neurokinin-A in bone and joint tissues: changes in adjuvant arthritis.

The localization of neurokinin A (NK-A) in the normal ankle joint of rats was investigated by an immunoelectron microscopic technique with specific antisera to NK-A. Immunoreactivity was detected in bone matrix, myelinated nerve fiber in the periosteum, and bone marrow and synovial cells. No immunoreactivity was observed in osteoblasts, osteocytes, and osteoclasts. Using radioimmunoassay (RIA), a detectable concentration of NK-A was observed in the bone marrow, periosteum, cortical bone, and ankle of normal rats. In rats with chronic adjuvant arthritis, induced by intradermal injection of mycobacterium butyricum in paraffin oil into the base of the tail, the concentrations of NK-A using RIA in ankles and spinal cords were found to be significantly increased compared with acute or control rats. There were no significant differences between the latter two. Similarly, increased NK-A labeling was observed using immunoelectron microscopy in bone matrix and bone marrow monocyte cells of the chronic arthritic rats. These findings indicate the existence of as well as a biological role of NK-A in bone and joint tissues.

Methionine-enkephalin in bone and joint tissues.

Methionine-enkephalin (met-enk), an endogenous opiate, mimics many of the effects of morphine by binding to opiate receptors, thereby eliciting similar cellular and behavioral effects. Using biochemical and immunohistochemical techniques, several peptides have been identified in bone and joint tissues. Here we report, for the first time, the presence as well as concentration of met-enk in bone and joint tissues. Immunohistochemistry using electron and immunofluorescence microscopy showed cellular and neuronal distribution of met-enk in bone and joint tissues. The concentration of met-enk analyzed by high performance liquid chromatography electrochemical detection or radioimmunoassay was high in bone marrow, periosteum, ankle joint tissue, and cortical bone. Analysis by fast atom bombardment mass spectrometry suggested that the recovered fragment was met-enk Administration of met-enk inhibits osteoblast cell growth in culture, which is reversible by naltrexone. In arthritic rats, the concentration of met-enk was significantly decreased in ankle joints compared with controls, suggesting a role for met-enk in the pathophysiology of adjuvant arthritis.

Distribution of integrin subunits on rat metaphyseal osteoclasts and osteoblasts.

A key event in bone resorption is the attachment of osteoclasts to the bone matrix. The process apparently involves integrin plasma membrane receptors for extracellular matrix proteins. In the present study the distribution of some integrin subunits among different osteoclastic and osteoblastic domains was determined using immunocytochemistry on rat metaphyseal bone. Ultrathin cryosections were incubated with polyclonal antibodies against the alpha v beta 3 integrin and against the individual integrin subunits, alpha v, beta 1, beta 3, and beta 5. Bound antibodies were detected with 10-nm colloidal gold coated with protein A. The results show that alpha v- and beta 3-subunits are enriched at the osteoclast plasma membrane facing the bone matrix, i.e., the clear zone. Furthermore, beta 1- and beta 5-subunits were somewhat enriched at the osteoblast plasma membrane facing the bone surface. The present observations corroborate and extend our previous data indicating that the alpha v beta 3 integrin mediates tight attachment of the osteoclast to the bone matrix. Moreover, a role for integrins in the attachment and interactions of the osteoblast is suggested.

Distribution and synthesis of bone sialoprotein in metaphyseal bone of young rats show a distinctly different pattern from that of osteopontin.

Bone sialoprotein (BSP) and osteopontin (OPN) are two phosphorylated and highly glycosylated cell-binding proteins in bone. Both proteins bind to hydroxylapatite. The cell binding is mediated via an Arg-Gly-Asp (RGD) sequence and previous work indicates that both proteins can bind to the vitronectin receptor (alpha v beta 3). The present work shows that a prevailing localization of BSP in metaphyseal bone of the young rat is at the interface between calcified cartilage and bone. Thus BSP shows a conspicuous enrichment in the osteoid laid down by the invading osteoblasts immediately next to the calcified cartilage. Furthermore, the most prominent amount of BSP mRNA was detected in cells at the epiphyseal/metaphyseal border. As opposed to OPN, no prominent accumulation of BSP immunoreactivity was observed at bone surfaces that face cells. Also the synthesis OPN was most pronounced at sites very different from those of BSP. Thus, the most prominent amount of OPN mRNA was observed in cells close to the metaphyseal/diaphyseal border, where osteoclastic bone resorption is particularly active. Indeed, message was often found in cells surrounding osteoclasts without any detectable message. The distinctly different patterns of synthesis and expression of the two proteins indicate different roles in bone turnover at this stage of development. Thus, it appears that BSP has a specific role during the initial phases of bone formation at the cartilage/bone interface. On the other hand, the pattern of OPN synthesis and expression support and extend our previous data showing OPN particularly enriched at attachment sites of osteoclasts resorbing bone.

Immunocytochemical localization of the 70 kDa peroxisomal membrane protein in connections between peroxisomes in rat liver: support for a reticular organization of peroxisomes maintained by the cytoskeleton.

Recently it has been shown that peroxisomes interact with microtubules which affect the structure and intracellular distribution of the organelle (Schrader, M., J. K. Burkhardt, E. Baumgart, G. Lüers, H. Spring, A. Völkl, H. D. Fahimi: Interaction of microtubules with peroxisomes. Tubular and spherical peroxisomes in HepG2 cells and their alterations induced by microtubule-active drugs. Eur. J. Cell Biol. 69, 24-35 (1996)). In the present work, we have applied immunological techniques to study the organization of peroxisomes within the rat liver cell. Antibodies to a pentadecapeptide corresponding to amino acid residues 403-417 of the 70 kDa integral peroxisomal membrane protein (Kamijo, K., S. Taketani, S. Yokota, T. Osumi, T. Hashimoto: The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein super family. J. Biol. Chem. 265, 4534-4540 (1990)) were raised in rabbits and affinity purified. This antibody was found to be highly specific for peroxisomes as determined by ELISA and Western blot analysis. Immunoelectron microscopy of tissue sections from rat liver revealed that peroxisomal membranes were labeled with this antibody and, in addition, labeling was found on tubular extensions often connecting peroxisomes. Antibodies to alpha-tubulin were used to locate the microtubular system. Microtubules were often found in close connection to peroxisomes, suggesting interaction between peroxisomes and the cytoskeleton. Double-labeling experiments for the 70 kDa integral peroxisomal membrane protein and alpha-tubulin demonstrated that the tubular structures connecting peroxisomes did not colocalize with microtubules. These results suggest that peroxisomes are organized in reticular structures within rat liver cells and that the structure and localization of these reticuli may be determined by their association to the microtubular network.

Infiltrative capacity of T leukemia cell lines: a distinct functional property coupled to expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1).

Infiltrative capacity was found to distinguish separate T leukemia cell lines. Of seven T-cell lines four exhibited capacity to infiltrate Matrigel. Analysis of infiltration was performed at the single-cell level throughout the Matrigel using a depth meter. Further, we examined differences in migration capacity and metalloproteinase production between infiltrating and non-infiltrating T-cell lines. The capacity to infiltrate was not directly correlated to the capacity to adhere to the Matrigel or to migrate on/to extracellular matrix components. It is concluded that infiltration capacity does not simply reflect capacity to migrate but represents a distinct functional property. The production of metalloproteinases and their inhibitors by the separate T-cell lines was analyzed using rt PCR, biosynthetic labelling, zymography, immunoprecipitation and ELISA. All T-cell lines with capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) while non-infiltrating cell lines did not express MMP-9. Expression of MMP-1, 2, 3, 10, 14 and 17 showed no correlation to capacity to infiltrate. Analysis of infiltration in the presence of a metalloprotease inhibitor showed an increased number of cells within the gel. This enhancement of infiltration suggests that the function of MMPs and/or their inhibitors in lymphocyte infiltration is more complex than previously thought.

Porcine endothelium supports transendothelial migration of human leukocyte subpopulations: anti-porcine vascular cell adhesion molecule antibodies as species-specific blockers of transendothelial monocyte and natural killer cell migration.

BACKGROUND: In cases where hyperacute rejection has been prevented, pig to primate organ transplantation results in a delayed rejection mediated by graft-infiltrating leukocytes. The migration of human leukocytes across porcine endothelium is poorly characterized, but may offer targets for species-specific antirejection therapy. METHODS: Transwell tissue culture inserts with endothelial cells growing on polycarbonate filters were used to characterize the migration of peripheral blood monocuclear cells and purified leukocyte subpopulations across pig and human endothelial cells and cell lines. Endothelial cell morphology was evaluated by scanning and transmission electron microscopy, and the contribution of different adhesion receptor pairs to transendothelial migration was evaluated by antibody blocking experiments. RESULTS: There were no evident quantitative or qualitative differences in the capacity of human and porcine endothelium to support transendothelial migration of human leukocytes [T, B, and natural killer (NK) cells, monocytes, and neutrophils]. Monocytes and large granular CD3+ lymphocytes migrated most efficiently across the endothelium. Antiporcine vascular cell adhesion molecule-1 antibodies blocked transendothelial migration of human monocytes and NK cells across tumor necrosis factor-alpha stimulated pig endothelium by at least 60%. Anti-CD18 antibodies had no effect on the migration of human NK cells across pig endothelium, whereas they partly blocked migration of NK cells across human endothelium and migration of monocytes across porcine endothelium. Interleukin-2 stimulated, but not unstimulated, T and NK cells were cytotoxic to porcine endothelium. CONCLUSIONS: Porcine endothelium supports transendothelial migration of human leukocyte subpopulations as efficiently as human endothelium. Incompatibilities in some adhesion receptor pairs may be compensated for by other adhesion receptor pairs, as exemplified by human NK cells whose migration across human, but not pig, endothelium was blocked by anti-CD18 antibodies. Antiporcine vascular cell adhesion molecule-1 antibodies may be used as species-specific blockers of transendothelial NK cell and monocyte migration, and as such may prove to be useful inhibitors of cellular organ xenograft rejection.

Somatostatin immunoreactivity in bone and joint tissues.

Using immunoelectron microscopy we have investigated the presence of somatostatin in normal bone and joint tissues. We observed somatostatin labeling in the myelinated nerve fibers of the periosteum, the bone marrow cells and in the mature bone matrix but only slightly in the synovial cells. Quantification of somatostatin in bone tissue by radioimmunoassay showed highest levels in bone marrow followed by periosteum and cortical bone. These findings suggest a role for somatostatin in bone and joint physiology.

Improved survival of macroencapsulated islets of Langerhans by preimplantation of the immunoisolating device: a morphometric study.

Encapsulation of cells in a semipermeable membrane may in the future provide an opportunity to treat a variety of endocrine and neurological disorders, without the need for lifelong immunosuppression. The physiological conditions in the device are crucial factors for graft survival. Previously, we have shown that the exchange across the immunoisolating membrane and the microcirculation around the TheraCyte device increase around 3 months after implantation. The aim of this study was to determine whether preimplantation of the TheraCyte device would improve the survival of a later transplanted islet graft. A TheraCyte device was implanted SC on one side of the back of a nondiabetic SD rat. After 3 months, 1500 islets isolated from SD rats were transplanted via the device port. At the same time, another device, loaded with the same number of islets, was implanted on the other side of the back. Both devices were explanted 2 weeks after islet transplantation (i.e., 3.5 months and 0.5 month after device implantation, respectively). Six pairs of devices were evaluated by morphometery. The volume densities of viable islets were 0.22 +/- 0.04 in the preimplanted device vs. 0.06 +/- 0.03 in the nonpreimplanted one (p < 0.05). The corresponding volume densities of fibrosis and necrosis were 0.64 +/- 0.13 vs. 0.85 +/- 0.08 (p < 0.05) and 0.11 +/- 0.14 vs. 0.09 +/- 0.07 (ns), respectively. When the absolute volumes (mm3) were calculated, preimplanted devices contained 1.1 +/- 0.7 endocrine cells while nonpreimplanted ones contained 0.4 +/- 0.2 (p < 0.05). The percentages of insulin- positive beta-cells in the preimplanted versus nonpreimplanted device were 80 +/- 5% and 67 +/- 6%, respectively (p < 0.01). The corresponding volumes of fibrotic tissue were 3.0 +/- 1.8 vs. 5.2 +/- 1.2 (p < 0.05), while the amount of necrotic tissue did not differ significantly (0.42 +/- 0.5 vs. 0.50 +/- 0.3). Preimplantation of the TheraCyte device seems to improve the survival of an encapsulated islet graft and reduce fibroblast outgrowth in the device.