Intradiscal application of rhBMP-7 does not induce regeneration in a canine model of spontaneous intervertebral disc degeneration

IntroductionStrategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain.MethodsCanine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue.ResultsIn vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7.ConclusionsAn intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0625-2) contains supplementary material, which is available to authorized users.     

Regulation of perforin activation and pre‐synaptic toxicity through C‐terminal glycosylation

Perforin is a highly cytotoxic pore‐forming protein essential for immune surveillance by cytotoxic lymphocytes. Prior to delivery to target cells by exocytosis, perforin is stored in acidic secretory granules where it remains functionally inert. However, how cytotoxic lymphocytes remain protected from their own perforin prior to its export to secretory granules, particularly in the Ca²⁺‐rich endoplasmic reticulum, remains unknown. Here, we show that N‐linked glycosylation of the perforin C‐terminus at Asn549 within the endoplasmic reticulum inhibits oligomerisation of perforin monomers and thus protects the host cell from premature pore formation. Subsequent removal of this glycan occurs through proteolytic processing of the C‐terminus within secretory granules and is imperative for perforin activation prior to secretion. Despite evolutionary conservation of the C‐terminus, we found that processing is carried out by multiple proteases, which we attribute to the unstructured and exposed nature of the region. In sum, our studies reveal a post‐translational regulatory mechanism essential for maintaining perforin in an inactive state until its secretion from the inhibitory acidic environment of the secretory granule.     

Comparative analysis of dendritic cells transduced with different anti-apoptotic molecules: sensitivity to tumor-induced apoptosis

BACKGROUND: Tumors develop mechanisms to escape recognition by the immune system. It has recently been demonstrated that tumors cause apoptotic death of key immune cells, including the major antigen-presenting cells, dendritic cells (DC). Elimination of DC from the tumor environment significantly diminishes development of specific immunologic responses. We have recently demonstrated that tumor-induced DC apoptosis could be prevented by overexpression of the anti-apoptotic molecule Bcl-x(L). The aim of this study was to identify extrinsic and intrinsic tumor-induced apoptotic pathways in DC by targeting different anti-apoptotic molecules, including FLIP, XIAP/hILP, dominant-negative procaspase-9 and HSP70. METHODS: Murine bone marrow derived DC were transduced with adenoviral vectors carrying different anti-apoptotic molecules and co-incubated with tumor cells in a Transwell system. Apoptosis of DC was assessed by Annexin V and PI staining. RESULTS: We have demonstrated that adenoviral infection of DC with genes encoding different anti-apoptotic molecules exhibits different degrees of resistance to melanoma-induced apoptosis. Furthermore, we have shown that anti-apoptotic molecules other than the Bcl-2 family of proteins are able to protect DC and prevent tumor-induced apoptosis in DC. CONCLUSIONS: The results show that tumor-induced apoptosis of DC is not limited to the mitochondrial pathway of cell death and open additional possibilities for targeted molecular protection of DC longevity in cancer. Therefore, effective protection of DC from tumor-induced apoptosis may significantly improve the efficacy of DC-based therapies for cancer.     

Alternatively spliced variants of protocadherin 8 exhibit distinct patterns of expression during mouse development

Protocadherins, a subgroup of the cadherin superfamily of calcium-dependent cell adhesion molecules, are considered to play important roles in the developing embryo particularly in the central nervous system. The Protocadherin 8 (Pcdh8) gene comprises three coding exons in both human and mouse, and the exon junctions are precisely conserved between these two species. Alternative splicing of Pcdh8 RNA leads to the formation of two isoforms that differ in the length of the cytoplasmic domains. We have investigated the expression of these short and long variants of Pcdh8 during early mouse development by RT/PCR and in situ hybridization. We found that both isoforms were predominantly expressed in the nervous system, and that their expression patterns appeared to be developmentally regulated. However, the short variant had a broader pattern of expression than the long variant and was found in some non-neuronal tissues, such as paraxial mesoderm, developing somites, and in limb interdigital mesenchyme where massive programmed cell death occurs. The differential expression of two alternative cytoplasmic domain variants suggests that Pcdh8 may regulate cell adhesion in a variety of developmental processes, and that this may involve different intracellular interactions.     

Roles for alpha 1 connexin in morphogenesis of chick embryos revealed using a novel antisense approach

Gap junctional communication has been implicated in embryonic development and pattern formation. The gap junction protein, alpha 1 connexin (Cx43) is expressed in dynamic and spatially restricted patterns in the developing chick embryo and its expression correlates with many specific developmental events. High levels of expression are found in regions of budding, which leads to shaping and appears to be a necessary prelude for tissue fusions. In order to investigate the role of alpha 1 connexin in these morphogenetic events, we developed a novel method of applying unmodified antisense deoxyoligonucleotides (ODNs) to chick embryos. The use of pluronic gel to deliver antisense ODNs has allowed us to regulate the expression of alpha 1 connexin protein, both spatially and temporally. This "knockdown" results in some striking developmental defects that mimic some common congenital abnormalities, such as spina bifida, anencephaly, myeloschisis, limb malformation, cleft palate, failure of hematopoiesis, and cardiovascular deformity. The results imply a major role for alpha 1 connexin communication in the integration of signaling required for pattern formation during embryonic development. This novel antisense technique may also be widely applicable.     

The solid-state catalytic isotope exchange of hydrogen in dalargin

A [³H]Dalargin preparation with a molar radioactivity of 52 Ci/mmol was obtained by the high temperature solid-state catalytic isotope exchange (HSCIE) of tritium for hydrogen at 150°C. This tritium-labeled peptide was shown to completely retain its biological activity in the test of binding to opioid receptors from rat brain. The dissociation constant of the Dalargin-opioid receptor complex was found to be 4.3 nM. The dependences of the chemical yield and the molar radioactivity on the reaction time and temperature of HSCIE were determined. The activation energy of the HSCIE reaction for the peptide was calculated to be 32 kcal/mol. The amino acid analysis showed that tritium is distributed between all the amino acid residues of [³H]Dalargin at the HSCIE reaction, with the temperature growth significantly increasing the total tritium incorporation and, especially, enhancing the radioactivity incorporation into aromatic residues.     

Stable carbon and oxygen isotopes in human tooth enamel: Identifying breastfeeding and weaning in prehistory. Am. J. Phys. Anthropol. 106: 1–18.

ERRATUM: Wright LE and Schwarcz HP (1998) Stable Carbon and Oxygen Isotopes in Human Tooth Enamel: Identifying Breastfeeding and Weaning in Prehistory. Am. J. Phys. Anthropol. 106: 1–18. Isotopic ratios were incorrectly printed as percentages (%) rather than units permil (‰). Wherever “breast feeding” and “breast fed” occur, the words should be combined into “breastfeeding” and “breastfed” respectively. The correct information on isotopic ratios is the following: p. 1, Abstract: all % signs should be “‰”; p. 2, column two, last line: “… δ¹³C values¹, have been …”; p. 2, Footnote 1 should read: “Isotope ratios of carbon and oxygen are expressed in δ notation as follows, δ = [(R_sample/R_standard) − 1] × 1000, where R = ¹³C/¹²C for δ¹³C, and R = ¹⁸O/¹⁶O for δ¹⁸O, and are in units permil, ‰.”; p. 3, column two: all % signs should be “‰”, except for the 23rd line from the bottom, which reads: “… provided 95% of water intake by all infants …”; p. 5: all % signs should be “‰”; p. 6, column one: 8th and 9th line from bottom should be “… mean deviations were 0.029‰ for δ¹³C and 0.024‰ for δ¹⁸O …”; p. 6, column one: 2nd line from bottom should be “… only 0.208‰ for δ¹³C and only 0.091‰ for δ¹⁸O …”; p. 6, column two: top line: “… of 0.5‰ in δ¹³C and 0.2‰ in δ¹⁸O …”; p. 8, 9 and 10: all % signs should be “‰”; p. 11, column two: 7th line from top: “… the lipids are 4-6‰ lighter …”; p. 12, 13 and 14: all % signs should be “‰.”     

Antiproliferative activity of long chain acylated esters of quercetin-3-O-glucoside in hepatocellular carcinoma HepG2 cells

Despite their strong role in human health, poor bioavailability of flavonoids limits their biological effects in vivo. Enzymatically catalyzed acylation of fatty acids to flavonoids is one of the approaches of increasing cellular permeability and hence, biological activities. In this study, six long chain fatty acid esters of quercetin-3-O-glucoside (Q3G) acylated enzymatically and were used for determining their antiproliferative action in hepatocellular carcinoma cells (HepG2) in comparison to precursor compounds and two chemotherapy drugs (Sorafenib and Cisplatin). Fatty acid esters of Q3G showed significant inhibition of HepG2 cell proliferation by 85 to 90% after 6 h and 24 h of treatment, respectively. The cell death due to these novel compounds was associated with cell-cycle arrest in S-phase and apoptosis observed by DNA fragmentation, fluorescent microscopy and elevated caspase-3 activity and strong DNA topoisomerase II inhibition. Interestingly, Q3G esters showed significantly low toxicity to normal liver cells than Sorafenib (P < 0.05), a chemotherapy drug for hepatocellular carcinoma. Among all, oleic acid ester of Q3G displayed the greatest antiproliferation action and a high potential as an anti-cancer therapeutic. Overall, the results of the study suggest strong antiproliferative action of these novel food-derived compounds in treatment of cancer.