Proteome of Personalized Tissue-Engineered Veins.

Vascular diseases are the largest cause of death globally and impose a major global burden on healthcare. The gold standard for treating vascular diseases is the transplantation of autologous veins, if applicable. Alternative treatments still suffer from shortcomings, including low patency, lack of growth potential, the need for repeated intervention, and a substantial risk of developing infections. The use of a vascular ECM scaffold reconditioned with the patient's own cells has shown successful results in preclinical and clinical studies. In this study, we have compared the proteomes of personalized tissue-engineered veins of humans and pigs. By applying tandem mass tag (TMT) labeling LC/MS-MS, we have investigated the proteome of decellularized (DC) veins from humans and pigs and reconditioned (RC) DC veins produced through perfusion with the patient's whole blood in STEEN solution, applying the same technology as used in the preclinical studies. The results revealed high similarity between the proteomes of human and pig DC and RC veins, including the ECM texture after decellularization and reconditioning. In addition, functional enrichment analysis showed similarities in signaling pathways and biological processes involved in the immune system response. Furthermore, the classification of proteins involved in immune response activity that were detected in human and pig RC veins revealed proteins that evoke immunogenic responses, which may lead to graft rejection, thrombosis, and inflammation. However, the results from this study imply the initiation of wound healing rather than an immunogenic response, as both systems share the same processes, and no immunogenic response was reported in the preclinical and clinical studies. Finally, our study assessed the application of STEEN solution in tissue engineering and identified proteins that may be useful for the prediction of successful transplantations.

Repair of Long Nerve Defects with a New Decellularized Nerve Graft in Rats and in Sheep.

Decellularized nerve allografts (DC) are an alternative to autografts (AG) for repairing severe peripheral nerve injuries. We have assessed a new DC provided by VERIGRAFT. The decellularization procedure completely removed cellularity while preserving the extracellular matrix. We first assessed the DC in a 15 mm gap in the sciatic nerve of rats, showing slightly delayed but effective regeneration. Then, we assayed the DC in a 70 mm gap in the peroneal nerve of sheep compared with AG. Evaluation of nerve regeneration and functional recovery was performed by clinical, electrophysiology and ultrasound tests. No significant differences were found in functional recovery between groups of sheep. Histology showed a preserved fascicular structure in the AG while in the DC grafts regenerated axons were grouped in small units. In conclusion, the DC was permissive for axonal regeneration and allowed to repair a 70 mm long gap in the sheep nerve.

Resolving the taxonomic conundrum in Graphoderus of the east Palearctic with a key to all species (Coleoptera, Dytiscidae).

The Holarctic diving beetle genus Graphoderus (Dytiscinae, Aciliini) contains relatively few and well-known species but these may still be difficult to identify based on external characters. A taxonomic problem in the eastern Palearctic was discovered that relates to the Palearctic Graphoderus zonatus (Hoppe, 1795) and the Nearctic Graphoderus perplexus Sharp, 1882. Based on qualitative and quantitative characters, especially on male genitalia which have been poorly studied in the past, it is shown that eastern Palearctic specimens identified by previous authors as either of the two species in fact belongs to a third species. The synonymized name Graphoderus elatus Sharp, 1882, is reinstated as a valid species (stat. n.) and a lectotype is designated from the mixed syntype series. The male genitalia of all known Graphoderus species have been examined and an illustrated identification key to the genus is provided. The three species in the complex of focus, Graphoderus elatus, Graphoderus zonatus and Graphoderus perplexus are found to have allopatric distributions; Graphoderus perplexus in the Nearctic region, Graphoderus zonatus in the west Palearctic region and eastwards to the Yenisei-Angara river and Graphoderus elatus east of the Yenisei-Angara river. All previous records of either Graphoderus zonatus or Graphoderus perplexus in the east Palearctic, east of the Yenisei-Angara river turned out to be misidentified Graphoderus elatus. This conclusion also brings with it that dimorphic females, thought only to be present in the single subspecies Graphoderus zonatus verrucifer (CR Sahlberg, 1824), proved to be present also in a second species, Graphoderus elatus. The dimorphic female forms is either with dorsally smooth elytra and pronotum or conspicuously granulated elytra and wrinkly pronotum. As has been shown in Graphoderus zonatus verrucifer there is a correlation between the occurrence of granulate female forms in a population and an increase in the number of adhesive discs on pro- and mesotarsus in males within Graphoderus elatus.

One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells.

Human hepatocytes display substantial functional inter-individual variation regarding drug metabolizing functions. In order to investigate if this diversity is mirrored in hepatocytes derived from different human pluripotent stem cell (hPSC) lines, we evaluated 25 hPSC lines originating from 24 different donors for hepatic differentiation and functionality. Homogenous hepatocyte cultures could be derived from all hPSC lines using one standardized differentiation procedure. To the best of our knowledge this is the first report of a standardized hepatic differentiation procedure that is generally applicable across a large panel of hPSC lines without any adaptations to individual lines. Importantly, with regard to functional aspects, such as Cytochrome P450 activities, we observed that hepatocytes derived from different hPSC lines displayed inter-individual variation characteristic for primary hepatocytes obtained from different donors, while these activities were highly reproducible between repeated experiments using the same line. Taken together, these data demonstrate the emerging possibility to compile panels of hPSC-derived hepatocytes of particular phenotypes/genotypes relevant for drug metabolism and toxicity studies. Moreover, these findings are of significance for applications within the regenerative medicine field, since our stringent differentiation procedure allows the derivation of homogenous hepatocyte cultures from multiple donors which is a prerequisite for the realization of future personalized stem cell based therapies.

Genetic variants of GSK3B are associated with biomarkers for Alzheimer's disease and cognitive function.

BACKGROUND: Glycogen synthase kinase 3 beta (GSK3B) is the major kinase phosphorylating tau protein. Hyperphosphorylated tau is one of the hallmarks of Alzheimer's disease (AD). Despite extensive research, the role of GSK3B in AD pathogenesis is not fully understood. OBJECTIVE: To evaluate possible associations between gene variants of GSK3B and risk of AD. METHODS: Twelve GSK3B tag single-nucleotide polymorphisms (SNPs), together with the previously AD-associated rs334558, were analyzed in 583 AD patients and 673 controls. Analyses on single marker and haplotype levels were done to relate to risk of AD, Mini-Mental State Examination (MMSE) scores, and cerebrospinal fluid (CSF) biomarker levels of total tau (T-tau), hyperphosphorylated tau (P-tau181), and amyloid-ß (Aß42). RESULTS: After correction for multiple testing, we found a number of associations of gene variants with CSF biomarker levels and cognitive function in the AD patients. Firstly, rs334558 was associated with elevated T-tau levels (pc = 0.04). Next, rs1154597 showed association with reduced Aß42 levels (pc = 0.007). Lastly, rs3107669 was associated with lower MMSE scores (pc = 0.03). In addition, one more SNP was nominally significantly associated with reduced Aß42 levels and another was associated with reduced MMSE. CONCLUSION: We found GSK3B gene variants associated with cognitive function and CSF biomarkers T-tau and Aß42. To our knowledge, this is the first time GSK3B has been associated with cognitive function or CSF biomarkers reflecting neuronal degeneration (T-tau) and brain amyloid load (Aß42). The regulation of GSK3B needs to be investigated further, to fully understand how these GSK3B gene variants are involved in AD pathogenesis.

Identification of novel biomarkers for doxorubicin-induced toxicity in human cardiomyocytes derived from pluripotent stem cells.

Doxorubicin is a chemotherapeutic agent indicated for the treatment of a variety of cancer types, including leukaemia, lymphomas, and many solid tumours. The use of doxorubicin is, however, associated with severe cardiotoxicity, often resulting in early discontinuation of the treatment. Importantly, the toxic symptoms can occur several years after the termination of the doxorubicin administration. In this study, the toxic effects of doxorubicin exposure have been investigated in cardiomyocytes derived from human embryonic stem cells (hESC). The cells were exposed to different concentrations of doxorubicin for up to 2 days, followed by a 12 day recovery period. Notably, the cell morphology was altered during drug treatment and the cells showed a reduced contractile ability, most prominent at the highest concentration of doxorubicin at the later time points. A general cytotoxic response measured as Lactate dehydrogenase leakage was observed after 2 days' exposure compared to the vehicle control, but this response was absent during the recovery period. A similar dose-dependant pattern was observed for the release of cardiac specific troponin T (cTnT) after 1 day and 2 days of treatment with doxorubicin. Global transcriptional profiles in the cells revealed clusters of genes that were differentially expressed during doxorubicin exposure, a pattern that in some cases was sustained even throughout the recovery period, suggesting that these genes could be used as sensitive biomarkers for doxorubicin-induced toxicity in human cardiomyocytes. The results from this study show that cTnT release can be used as a measurement of acute cardiotoxicity due to doxorubicin. However, for the late onset of doxorubicin-induced cardiomyopathy, cTnT release might not be the most optimal biomarker. As an alternative, some of the genes that we identified as differentially expressed after doxorubicin exposure could serve as more relevant biomarkers, and may also help to explain the cellular mechanisms behind the late onset apoptosis associated with doxorubicin-induced cardiomyopathy.

Complement gene single nucleotide polymorphisms and biomarker endophenotypes of Alzheimer's disease.

The complement system has been implicated in both physiological synapse elimination and Alzheimer's disease (AD). Here, we investigated associations between four single nucleotide polymorphisms (SNPs) in complement genes and cerebrospinal fluid (CSF) biomarkers for AD in 452 neurochemically or neuropathologically verified AD cases and 678 cognitively normal controls. None of the SNPs associated with risk of AD but there were potential associations of rs9332739 in the C2 gene and rs4151667 in the complement factor B gene with CSF tau levels (p = 0.023) and Mini-Mental State Examination scores (p = 0.012), both of which may be considered markers of disease intensity/severity.

Tumour-loaded α-type 1-polarized dendritic cells from patients with chronic lymphocytic leukaemia produce a superior NK-, NKT- and CD8+ T cell-attracting chemokine profile.

Tumour-loaded dendritic cells (DCs) from patients with chronic lymphocytic leukaemia (CLL) matured using an α-type 1-polarized DC cocktail (IL-1ß/TNF-α/IFN-α/IFN-γ/poly-I:C;αDC1) were recently shown to induce more functional CD8(+) T cells against autologous tumour cells in vitro than DCs matured with the 'standard' cocktail (IL-1ß/TNF-α/IL-6/PGE(2) ;PGE(2) DCs). However, the ability of vaccine DCs to induce a type 1-polarized immune response in vivo probably relies on additional features, including their ability to induce a CXCR3-dependent recruitment of NK cells into vaccine-draining lymph nodes. Moreover, their guiding of rare tumour-specific CD8(+) T cells to sites of DC-CD4(+) T cell interactions by secretion of CCL3 and CCL4 is needed. We therefore analysed the chemokine profile and the lymphocyte-attracting ability in vitro of monocyte-derived PGE(2) DCs and αDC1s from patients with CLL. αDC1s produced much higher levels of CXCR3 ligands (CXCL9/CXCL10/CXCL11) than PGE(2) DCs. Functional studies further demonstrated that αDC1s were superior recruiters of both NK and NKT cells. Moreover, αDC1s produced higher levels of CCL3/CCL4 upon CD40 ligation. These findings suggest that functional αDC1s, derived from patients with CLL, produce a desirable NK-, NKT- and CD8(+) T cell-attracting chemokine profile which may favour a guided and Th1-deviated priming of CD8(+) T cells, supporting the idea that αDC1-based vaccines have a higher immunotherapeutic potential than PGE(2) DCs.