Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they ...maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic.
Hibernating American black bears have significantly different clotting parameters than their summer active counterparts, affording them protection against venous thromboembolism during prolonged ...periods of immobility. We sought to evaluate if significant differences exist between the expression of microRNAs in the plasma of hibernating black bears compared with their summer active counterparts, potentially contributing to differences in hemostasis during hibernation.
MicroRNA sequencing was assessed in plasma from 21 American black bears in summer active (n = 11) and hibernating states (n = 10), and microRNA signatures during hibernating and active state were established using both bear and human genome. MicroRNA targets were predicted using messenger RNA (mRNA) transcripts from black bear kidney cells. In vitro studies were performed to confirm the relationship between identified microRNAs and mRNA expression, using artificial microRNA and human liver cells.
Using the bear genome, we identified 15 microRNAs differentially expressed in the plasma of hibernating black bears. Of these microRNAs, three were significantly downregulated (miR-141-3p, miR-200a-3p, and miR-200c-3p), were predicted to target SERPINC1, the gene for antithrombin, and demonstrated regulatory control of the gene mRNA expression in cell studies.
Our findings suggest that the hibernating black bears’ ability to maintain hemostasis and achieve protection from venous thromboembolism during prolonged periods of immobility may be due to changes in microRNA signatures and possible upregulation of antithrombin expression.
•Hibernating American black bears appear to possess an innate protection against venous thromboembolism during prolonged periods of immobility.•Using microRNA sequenced from the plasma of 21 American black bears in summer active (n = 11) and hibernating states (n = 10), we identified three differentially expressed microRNAs (miR-141-3p, miR-200a-3p, and, miR-200c-3p) that target SERPINC1, the gene for antithrombin.•In vitro studies using human hepatocytes (HUH7.5) confirmed the regulatory control of these microRNAs on SERPINC1, with an average of 35% gene silencing with the introduction of artificial microRNAs.
Large B cell lymphomas can comprise numerous CD14+ cells in the tumor stroma, which raises the question of whether monocytes can support B cell survival and proliferation. We show that the coculture ...of monocytes with B cells from peripheral blood or from diffuse large B cell lymphoma enabled prolonged B cell survival. Under these conditions, diffuse large lymphoma B cells proliferated, and addition of B cell‐activating factor of the TNF family (BAFF) and IL‐2 enhanced cell division. Monocytes and dendritic cells (DC) had similar antiapoptotic activity on healthy B cells but displayed differences with respect to B cell proliferation. Monocytes and cord blood‐derived CD14+ cells promoted B cell proliferation in the presence of an anti‐CD40 stimulus, whereas DC supported B cell proliferation when activated through the BCR. DC and CD14+ cells were able to induce plasmocyte differentiation. When B cells were activated via the BCR or CD40, they released the leukocyte attractant CCL5, and this chemokine is one of the main chemokines expressed in diffuse large B cell lymphoma. The data support the notion that large B cell lymphoma recruit monocytes via CCL5 to support B cell survival and proliferation.
The mononuclear phagocyte system of human lymphoid tissue comprises macrophages and dendritic cells (DCs). The heterogeneity of the non-DC mononuclear phagocyte population in human lymphoid tissue ...has been little addressed. Here, we studied the expression of 2 monocyte-derived markers, CD14 and CD169 (sialoadhesin), in reactive human lymphoid tissue as well as in a series of 51 B-cell lymphomas by immunohistochemistry on paraffin-embedded tissue. We confirmed that lymph node sinusoidal monocyte-derived cells were the only population staining for CD169. Although most sinusoidal histiocytes also expressed CD14, monocyte-derived cells with phagocytosis such as erythrophagocytosis, anthracosis, or tingible bodies macrophage lacked CD14 and CD169. Among B-cell lymphomas, splenic marginal zone lymphoma was the only one associated with an expansion of the CD14
+CD169
+ cells in the cords. With respect to nodal B-cell lymphomas, CD14
+ cells were rare among B-chronic lymphocytic leukemia, follicular lymphoma (FL), mantle cell lymphoma (MCL). However, strikingly, we found a strong expansion of CD14
+CD169
− cells in numerous diffuse large B-cell lymphomas (DLBCLs), except in cases associated with numerous mitoses, apoptotic bodies, and tingible bodies macrophages. When cultivated in granulocyte/macrophage colony stimulating factor/interleukin 4, DLBCL purified CD14
+ cells differentiate into plasmacytoid cells, expressing DC-specific intercellular adhesion molecule 3–grabbing nonintegrin, suggesting dendritic cell differentiation potential. Our observation fits well with the lymph node and host response cluster signatures described in the gene profiling signatures of DLBCL. However, the role of this CD14
+ population that may constitute a microenvironment-related marker of this subgroup of DLBCL remains to be determined.
Immature dendritic cells (DCs) reside in interstitial tissues (int-DC) or in the epidermis, where they capture antigen and, thereafter, mature and migrate to draining lymph nodes (LNs), where they ...present processed antigen to T cells. We have identified int-DCs that express both TRANCE (tumor necrosis factor–related activation-induced cytokine) and RANK (receptor activator of NF-κB) and have generated these cells from CD34+ human progenitor cells using macrophage colony-stimulating factor (M-CSF). These CD34+-derived int-DCs, which are related to macrophages, are long-lived, but addition of soluble RANK leads to significant reduction of cell viability and Bcl-2 expression. This suggests that constitutive TRANCE-RANK interaction is responsible for CD34+-derived int-DC longevity. Conversely, CD1a+ DCs express only RANK and are short-lived. However, they can be rescued from cell death either by recombinant soluble TRANCE or by CD34+-derived int-DCs. CD34+-derived int-DCs mature in response to lipopolysaccharide (LPS) plus CD40 ligand (L) and become capable of CCL21/CCL19-mediated chemotaxis and naive T-cell activation. Upon maturation, they lose TRANCE, making them, like CD1a+DCs, dependent on exogenous TRANCE for survival. These findings provide evidence that TRANCE and RANK play important roles in the homeostasis of DCs.
The predominantly Afrotropical genus Charaxes is represented by 31 known species outside of Africa (excluding subgenus Polyura Billberg). We explored the biogeographic history of the genus using ...every known non-African species, with several African species as outgroup taxa. A phylogenetic hypothesis is proposed, based on molecular characters of the mitochondrial genes cytochrome oxidase subunit I (COI) and NADH dehydrogenase 5 (ND5), and the nuclear wingless gene. Phylogenetic analyses based on maximum parsimony and Bayesian inference of the combined dataset implies that the Indo-Pacific Charaxes form a monophyletic assemblage, with the exception of Charaxes solon Fabricius. Eight major lineages are recognized in the Indo-Pacific, here designated the solon (+African), elwesi, harmodius, amycus, mars, eurialus, latona, nitebis, and bernardus clades. Species group relationships are concordant with morphology and, based on the phylogeny, we present the first systematic appraisal and classification of all non-African species. A biogeographical analysis reveals that, after the genus originated in Africa, the evolutionary history of Charaxes in the Indo-Pacific, in particular Wallacea, may be correlated with the inferred geological and climatic history of the region. We propose that Wallacea was the area of origin of all Charaxes (excluding C. solon) occurring to the east of Wallace's 1863 Line. The earliest Indo-Pacific lineages appear to have diverged subsequent to the initial fragmentation of a palaeo-continent approximately 13 million years ago. Further diversification in Indo-Pacific Charaxes appears primarily related to climatic changes during the Pliocene and possibly as recently as the Pleistocene. Although both dispersal and vicariance have played important roles in the evolution of the genus within the region, the latter has been particularly responsible for diversification of Charaxes in Wallacea.
PDF
