Engineering of biologically active living heart valve leaflets using human umbilical cord-derived progenitor cells

This study demonstrates the engineering of biologically active heart valve leaflets using prenatally available human umbilical cord-derived progenitor cells as the only cell source. Wharton's Jelly-derived cells and umbilical cord blood-derived endothelial progenitor cells were subsequently seeded on biodegradable scaffolds and cultured in a biomimetic system under biochemical or mechanical stimulation or both. Depending on the stimulation, leaflets showed mature layered tissue formation with functional endothelia and extracellular matrix production comparable with that of native tissues. This demonstrates the feasibility of heart valve leaflet fabrication from prenatal umbilical cord-derived progenitor cells as a further step in overcoming the lack of living autologous replacements with growth and regeneration potential for the repair of congenital malformation.     

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFα, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFα and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.     

Immune reactivity of candidate reference materials

Immune reactivity is a key issue in the evaluation of the quality of recombinant allergens as potential reference materials. Within the frame of the CREATE project, the immune reactivity of the natural and recombinant versions of the major allergens of birch pollen (Bet v 1), grass pollen (Phl p 1 and 5), olive pollen (Ole e 1), and house dust mite (Der p 1 and 2, and Der f 1 and 2) was analysed. The IgE binding capacity of the allergens was studied by direct RAST and RAST inhibition, and their biological activity by basophil histamine release, using sera of allergic patients selected across Europe. For birch pollen, rBet v 1 is an excellent mimic of the natural allergen. For grass pollen, rPhl p 1 showed a significant lower IgE reactivity and was not considered a suitable candidate, whereas rPhl p 5a exhibited an immune reactivity closer to that of its natural counterpart. For olive, rOle e 1 had a lower IgE binding capacity in RAST but a higher biological activity in histamine release. For house dust mite, recombinant group 1 allergens were significantly less potent than their natural counterparts, but recombinant group 2 allergens were close mimics of their natural homologues.     

Dependence of cross-bridge kinetics on myosin light chain isoforms in rabbit and rat skeletal muscle fibres

Cross-bridge kinetics underlying stretch-induced force transients was studied in fibres with different myosin light chain (MLC) isoforms from skeletal muscles of rabbit and rat. The force transients were induced by stepwise stretches (< 0.3% of fibre length) applied on maximally Ca²⁺-activated skinned fibres. Fast fibre types IIB, IID (or IIX) and IIA and the slow fibre type I containing the myosin heavy chain isoforms MHC-IIb, MHC-IId (or MHC-IIx), MHC-IIa and MHC-I, respectively, were investigated. The MLC isoform content varied within fibre types. Fast fibre types contained the fast regulatory MLC isoform MLC2f and different proportions of the fast alkali MLC isoforms MLC1f and MLC3f. Type I fibres contained the slow regulatory MLC isoform MLC2s and the slow alkali MLC isoform MLC1s. Slow MLC isoforms were also present in several type IIA fibres. The kinetics of force transients differed by a factor of about 30 between fibre types (order from fastest to slowest kinetics: IIB > IID > IIA ≫ I). The kinetics of the force transients was not dependent on the relative content of MLC1f and MLC3f. Type IIA fibres containing fast and slow MLC isoforms were about 1.2 times slower than type IIA fibres containing only fast MLC isoforms. We conclude that while the cross-bridge kinetics is mainly determined by the MHC isoforms present, it is affected by fast and slow MLC isoforms but not by the relative content of MLC1f and MLC3f. Thus, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the cross-bridge kinetics.     

Influence of various cell-detachment solutions on endothelial cells after catheter abrasion for prosthesis colonization prior to implantation

We (1) evaluated the effectiveness of different media on the detachment of endothelial cells (ECs) from a catheter brush (Cragg thrombolytic catheter system) that was previously used for endothelial cell abrasion in 10 cm human umbilical vein (HUV) segments; (2) tested the practicability of endovascular catheter brush abrasion followed by EC detachment from the catheter brush, in vitro culture of harvested ECs, and finally endothelialization of a prosthesis; and (3) analyzed the defect created by the catheter brush in HUV segments after endovascular catheter abrasion. Best results in detachment of ECs from the catheter brush were obtained with a mixture of phosphate-buffered saline + 1% human albumin. EC vitality was time-dependent in the collected HUV segments postdelivery. Harvested EC viability decreased from (26.28 +/- 5.76)% (0-3 h postdelivery) to (17.29 +/- 4.56)% (after 4-8 h). ECs were easily cultured ex vivo within 2-3 weeks; seeded on nitinol stents, they grew to confluency and formed a monolayer on the stent surface (determined by scanning electron microscopy - SEM). Histological and SEM analysis of HUV segments that had undergone previous catheter brush abrasion revealed slight disruption of the intima but intact subintimal layers. Our findings indicate an advantageous method of capturing and culturing primary ECs for gene therapy or for the analysis and diagnosis of certain blood vessel diseases, especially in cases in which endovascular intervention is performed anyway. Moreover, and of high relevance to the biomaterial field, theoretically the procedure could be used to endothelialze a prosthesis ex vivo for implantation into the patient from whom the ECs were harvested, to reduce the inherent thrombogenicity of the prosthesis.     

Lymphatic tissue fibrosis is associated with reduced numbers of naive CD4+ T cells in human immunodeficiency virus type 1 infection

The organized structure of lymphatic tissues (LTs) constitutes a microenvironment referred to as a niche that plays a critical role in immune system homeostasis by promoting cellular interactions and providing access to cytokines and growth factors on which cells are dependent for survival, proliferation, and differentiation. In chronic human immunodeficiency virus type 1 (HIV-1) infection, immune activation and inflammation result in collagen deposition and disruption of this LT niche. We have previously shown that these fibrotic changes correlate with a reduction in the size of the total population of CD4+ T cells. We now show that this reduction is most substantial within the na?ve CD4+ T-cell population and is in proportion to the extent of LT collagen deposition in HIV-1 infection. Thus, the previously documented depletion of na?ve CD4+ T cells in LTs in HIV-1 infection may be a consequence not only of a decreased supply of thymic emigrants or chronic immune activation but also of the decreased ability of those cells to survive in a scarred LT niche. We speculate that LT collagen deposition might therefore limit repopulation of na?ve CD4+ T cells with highly active antiretroviral therapy, and thus, additional treatments directed to limiting or reversing inflammatory damage to the LT niche could potentially improve immune reconstitution.     

Herpes virus entry mediator synergizes with Toll-like receptor mediated neutrophil inflammatory responses

In microbial infections polymorphnuclear neutrophils (PMN) constitute a major part of the innate host defence, based upon their ability to rapidly accumulate in inflamed tissues and clear the site of infection from microbial pathogens by their potent effector mechanisms. The recently described transmembrane receptor herpes virus entry mediator (HVEM) is a member of the tumour necrosis factor receptor super family and is expressed on many haematopoietic cells, including T cells, B cells, natural killer cells, monocytes and PMN. Interaction of HVEM with the natural ligand LIGHT on T cells has a costimulatory effect, and increases the bactericidal activity of PMN. To further characterize the function of HVEM on PMN, we evaluated the effect of receptor ligation on human PMN effector functions using an agonistic monoclonal antibody. Here we demonstrate that activation of HVEM causes activation of neutrophil effector functions, including respiratory burst, degranulation and release of interleukin-8 in synergy with ligands for Toll-like receptors or GM-CSF. In addition, stimulation via HVEM enhanced neutrophil phagocytic activity of complement opsonized, but not of non-opsonized, particles. In conclusion, these results indicate a new, as yet unknown, participation of HVEM in the innate immune response and points to a new link between innate and adaptive immunity.     

Elevated serum 8-oxo-dG in hemodialysis patients: a marker of systemic inflammation?

Does inflammation, as assessed by high sensitivity C-reactive protein (hs-CRP), in patients with end-stage renal disease (ESRD) tightly associate with increased serum levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8- oxo-dG)? Increased oxidative stress and inflammation have both been highlighted among several nontraditional risk factors for cardiovascular disease, which is the main cause of mortality in ESRD patients. In contrast to oxidative stress effects on proteins and lipids, DNA base damage has not been well demonstrated in ESRD. Two groups of hemodialysis patients were studied, one group with persistent inflammation (n = 13, with constant elevation of CRP > 10 mg/L for 6 months) and one group of noninflamed patients (n = 19, with constant CRP < 10 mg/L for 6 months). Serum 8-oxo-dG was significantly elevated in persistent inflammation in comparison to noninflamed patients. At an individual level, a significant correlation was found between serum 8-oxo-dG and hsCRP. Extracellular 8-oxo-dG leads to intracellular oxidative damage on the nucleotide pool, thus providing a sensitive marker for inflammatory response. Serum levels of 8-oxo-dG, in combination with other inflammatory markers, serve as useful diagnostic tools for identification of patients in risk for inflammatory complications.     

Tsetse fly saliva accelerates the onset of Trypanosoma brucei infection in a mouse model associated with a reduced host inflammatory response

Tsetse flies (Glossina sp.) are the vectors that transmit African trypanosomes, protozoan parasites that cause human sleeping sickness and veterinary infections in the African continent. These blood-feeding dipteran insects deposit saliva at the feeding site that enables the blood-feeding process. Here we demonstrate that tsetse fly saliva also accelerates the onset of a Trypanosoma brucei infection. This effect was associated with a reduced inflammatory reaction at the site of infection initiation (reflected by a decrease of interleukin-6 [IL-6] and IL-12 mRNA) as well as lower serum concentrations of the trypanocidal cytokine tumor necrosis factor. Variant-specific surface glycoprotein-specific antibody isotypes immunoglobulin M (IgM) and IgG2a, implicated in trypanosome clearance, were not suppressed. We propose that tsetse fly saliva accelerates the onset of trypanosome infection by inhibiting local and systemic inflammatory responses involved in parasite control.     

Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFalpha, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFalpha and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.