Role of CCL19/21 and its possible signaling through CXCR3 in development of metallophilic macrophages in the mouse thymus

We have already shown that metallophilic macrophages, which represent an important component in the thymus physiology, are lacking in lymphotoxin-β receptor-deficient mice. However, further molecular requirements for the development and correct tissue positioning of these cells are unknown. To this end, we studied a panel of mice deficient in different chemokine ligand or receptor genes. In contrast to normal mice, which have these cells localized in the thymic cortico-medullary zone (CMZ) as a distinct row positioned between the cortex and medulla, in plt/plt (paucity of lymph node T cells) mice lacking the functional CCL19/CCL21 chemokines, metallophilic macrophages are not present in the thymic tissue. Interestingly, in contrast to the CCL19/21-deficient thymus, metallophilic macrophages are present in the CCR7-deficient thymus. However, these cells are not appropriately located in the CMZ, but are mostly crowded in central parts of thymic medulla. The double staining revealed that these metallophilic macrophages are CCR7-negative and CXCR3-positive. In the CXCL13-deficient thymus the number, morphology and localization of metallophilic macrophages are normal. Thus, our study shows that CCL19/21 and its possible signaling through CXCR3 are required for the development of thymic metallophilic macrophages, whereas the CXCL13–CXCR5 signaling is not necessary.     

Engineered cardiac tissues

Cardiac tissue engineering offers the promise of creating functional tissue replacements for use in the failing heart or for in vitro drug screening. The last decade has seen a great deal of progress in this field with new advances in interdisciplinary areas such as developmental biology, genetic engineering, biomaterials, polymer science, bioreactor engineering, and stem cell biology. We review here a selection of the most recent advances in cardiac tissue engineering, including the classical cell-scaffold approaches, advanced bioreactor designs, cell sheet engineering, whole organ decellularization, stem cell-based approaches, and topographical control of tissue organization and function. We also discuss current challenges in the field, such as maturation of stem cell-derived cardiac patches and vascularization.     

The mechanism of tail-anchored protein insertion into the ER membrane

Tail-anchored (TA) proteins access the secretory pathway via posttranslational insertion of their C-terminal transmembrane domain into the endoplasmic reticulum (ER). Get3 is an ATPase that delivers TA proteins to the ER by interacting with the Get1-Get2 transmembrane complex, but how Get3's nucleotide cycle drives TA protein insertion remains unclear. Here, we establish that nucleotide binding to Get3 promotes Get3-TA protein complex formation by recruiting Get3 to a chaperone that hands over TA proteins to Get3. Biochemical reconstitution and mutagenesis reveal that the Get1-Get2 complex comprises the minimal TA protein insertion machinery with functionally critical cytosolic regions. By engineering a soluble heterodimer of Get1-Get2 cytosolic domains, we uncover the mechanism of TA protein release from Get3: Get2 tethers Get3-TA protein complexes into proximity with the ATPase-dependent, substrate-releasing activity of Get1. Lastly, we show that ATP enhances Get3 dissociation from the membrane, thus freeing Get1-Get2 for new rounds of substrate insertion.     

Galectin‐3 deficiency protects pancreatic islet cells from cytokine‐triggered apoptosis in vitro

Beta cell apoptosis is a hallmark of diabetes. Since we have previously shown that galectin‐3 deficient (LGALS3^?/?) mice are relatively resistant to diabetes induction, the aim of this study was to examine whether beta cell apoptosis depends on the presence of galectin‐3 and to delineate the underlying mechanism. Deficiency of galectin‐3, either hereditary or induced through application of chemical inhibitors, β‐lactose or TD139, supported survival and function of islet beta cells compromised by TNF‐α + IFN‐γ + IL‐1β stimulus. Similarly, inhibition of galectin‐3 by β‐lactose or TD139 reduced cytokine‐triggered apoptosis of beta cells, leading to conclusion that endogenous galectin‐3 propagates beta apoptosis in the presence of an inflammatory milieu. Exploring apoptosis‐related molecules expression in primary islet cells before and after treatment with cytokines we found that galectin‐3 ablation affected the expression of major components of mitochondrial apoptotic pathway, such as BAX, caspase‐9, Apaf, SMAC, caspase‐3, and AIF. In contrast, anti‐apoptotic molecules Bcl‐2 and Bcl‐XL were up‐regulated in LGALS3^?/? islet cells when compared to wild‐type (WT) counterparts (C57BL/6), resulting in increased ratio of anti‐apoptotic versus pro‐apoptotic molecules. However, Fas‐triggered apoptotic pathway as well as extracellular signal‐regulated kinase 1/2 (ERK1/2) was not influenced by LGALS‐3 deletion. All together, these results point to an important role of endogenous galectin‐3 in beta cell apoptosis in the inflammatory milieu that occurs during diabetes pathogenesis and implicates impairment of mitochondrial apoptotic pathway as a key event in protection from beta cell apoptosis in the absence of galectin‐3. J. Cell. Physiol. 228: 1568–1576, 2013. © 2012 Wiley Periodicals, Inc.     

Generation of tissue constructs for cardiovascular regenerative medicine: From cell procurement to scaffold design

The ability of the human body to naturally recover from coronary heart disease is limited because cardiac cells are terminally differentiated, have low proliferation rates, and low turn-over rates. Cardiovascular tissue engineering offers the potential for production of cardiac tissue ex vivo, but is currently limited by several challenges: (i) Tissue engineering constructs require pure populations of seed cells, (ii) Fabrication of 3-D geometrical structures with features of the same length scales that exist in native tissue is non-trivial, and (iii) Cells require stimulation from the appropriate biological, electrical and mechanical factors. In this review, we summarize the current state of microfluidic techniques for enrichment of subpopulations of cells required for cardiovascular tissue engineering, which offer unique advantages over traditional plating and FACS/MACS-based enrichment. We then summarize modern techniques for producing tissue engineering scaffolds that mimic native cardiac tissue.     

Millipedes vs. pathogens: Defensive secretions of some julids (Diplopoda: Julida) as potential antimicrobial agents

In the light of growing interest in discovering new sources of natural antimicrobial agents, we extracted and tested the efficiency of julid defensive secretions in comparison with commercially used antibiotic and antimycotics. This work involved chemical characterization of the defensive secretions of Cylindroiulus boleti (C. L. Koch, 1847), Megaphyllum bosniense (Verhoeff, 1897) and M. unilineatum (C. L. Koch, 1838), as well as in vitro evaluation of their antimicrobial activity against 11 bacteria, one yeast and eight filamentous fungi. Compounds of the analysed defensive secretions included p‐benzoquinones, alkyl esters of fatty acids and ketones. Ketones were recorded for the first time in the order Julida, and they were detected in secretions of both Megaphyllum species. All three analysed defensive secretions showed antibacterial and antifungal potential against all of the tested pathogens. Staphylococcus aureus proved to be the most sensitive bacterial strain to all analysed secretions. Also, defensive secretion of M. unilineatum showed significant antibacterial potential against Bacillus subtilis. The most resistant bacterial strains in this study were Escherichia coli, Erwinia persicina and Pseudomonas syringae pv. tomato. Analysed defensive secretions achieved the strongest antifungal activity against Aspergillus parasiticus (secretions of all three millipede species), Penicillium griseofulvum (secretions of C. boleti and M. bosniense) and Cladosporium oxysporum (secretion of M. unilineatum). The most resistant mycromycetes were A. niger (to all tested defensive secretions), A. flavus (to secretion of M. unilineatum) and P. lanosum (to secretions of C. boleti and M. bosniense). Our results showed a generally lower level of activity compared to antibiotic and a significantly higher level compared to antimycotics. The results of this study elucidate and open opportunities for further research in the field of millipede chemical ecology.     

Chemical Composition,Antimicrobial and Cytotoxic Activity of Heracleum verticillatum Pančić and H. ternatum Velen. (Apiaceae) Essential Oils

In this work, the chemical composition, antimicrobial and cytotoxic activity of Heracleum verticillatum Pan?i? and H. ternatum Velen . root, leaf, and fruit essential oils were investigated. The composition was analyzed by GC and GC/MS. Heracleum verticillatum and H. ternatum root oils were dominated by monoterpenes, mostly β‐pinene (23.5% and 47.3%, respectively). Heracleum verticillatum leaf oil was characterized by monoterpenes, mainly limonene (20.3%), and sesquiterpenes, mostly (E)‐caryophyllene (19.1%), while H. ternatum leaf oil by the high percentage of phenylpropanoids, with (Z)‐isoelemicin (35.1%) being dominant constituent. Both fruit oils contained the majority of aliphatic esters, mostly octyl acetate (42.3% in H. verticillatum oil and 49.0% in H. ternatum oil). The antimicrobial activity of the oils was determined by microdilution method against eight bacterial and eight fungal strains. The strongest effect was exhibited by H. verticillatum root oil, particularly against Staphylococcus aureus, Salmonella typhimurium (MICs = 0.14 mg/ml, MBCs = 0.28 mg/ml), and Trichoderma viride (MIC = 0.05 mg/ml, MFC = 0.11 mg/ml). Cytotoxic effect was determined by MTT test against malignant HeLa, LS174, and A549 cells (IC₅₀ = 5.9 – 146.0 μg/ml), and against normal MRC‐5 cells (IC₅₀ > 120.1 μg/ml). The best effect was exhibited by H. verticillatum root oil on A549 cells (IC₅₀ = 5.9 μg/ml), and H. ternatum root oil against LS174 cells (IC₅₀ = 6.7 μg/ml).     

The dynamic co-evolution of memory and regulatory CD4+ T cells in the periphery

Whereas memory T cells are required to maintain immunity, regulatory T cells have to keep the immune system in check to prevent excessive inflammation and/or autoimmunity. Both cell types must be present during the lifetime of the organism. However, it is not clear whether both subsets are regulated in tandem or independently of each other, especially because thymic involution severely restricts the production of T-cell populations during ageing. In this Opinion article, we discuss recent evidence in both mice and humans that supports the hypothesis that some CD4(+)CD25(+)FOXP3(+)regulatory T cells can differentiate from rapidly proliferating memory T cells in the periphery.     

Altered Topographic Distribution and Enhanced Neuronal Expression of Adenosine-Metabolizing Enzymes in Rat Hippocampus and Cortex from Early to late Adulthood

Neurochemical Research - The present study demonstrates altered topographic distribution and enhanced neuronal expression of major adenosine-metabolizing enzymes, i.e. ecto-5?-nucleotidase...