Bacterial Contamination of Tissue Allografts – Experiences of the Donor Tissue Bank of Victoria

The aim of this study is to report the experience of the Donor Tissue Bank of Victoria with bacteria isolated from musculoskeletal, skin and cardiac allografts retrieved from cadaveric donors. The results of all quality control samples for bacterial culture, taken during retrieval and processing of allografts at the DTBV for a 12 month period, were extracted and analysed. It was found that 15.7% of skin, 15.1% of heart valves and 5.8% of musculoskeletal samples had positive culture results. The number and types of organisms isolated varied with tissue type. The most commonly isolated organisms were Staphylococcus species (including S. aureus). The identity of the isolate and the number of positive specimens from the same donor were considerations in the decision concerning the suitability of tissue for subsequent implantation.     

The PAXgene? Tissue System Preserves Phosphoproteins in Human Tissue Specimens and Enables Comprehensive Protein Biomarker Research

Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE) tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assay (ELISA) to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology.     

Tissue expansion: dividend or loan?

Epidermal mitotic activity during tissue expansion has been assessed in the guinea pig using tritiated thymidine and other confirmatory techniques. Implant inflation results in a threefold elevation of epidermal mitotic activity within 24 hours, followed by a gradual return to normal baseline over 2 to 5 days. Implant deflation, conversely, causes a transient decrease in epidermal mitotic activity. Neither of these phenomena has been previously described. It confirms previous animal studies which illustrate the highly responsive nature of the epidermis to physical stimuli and supports the view that tissue expansion is a highly useful manipulation of normal physiologic processes.     

‘Saviour Siblings’? The Distinction between PGD with HLA Tissue Typing and Preimplantation HLA Tissue Typing

One of the more controversial uses of preimplantation genetic diagnosis (PGD) involves selecting embryos with a specific tissue type so that the child to be born can act as a donor to an existing sibling who requires a haematopoietic stem cell transplant. PGD with HLA tissue typing is used to select embryos that are free of a familial genetic disease and that are also a tissue match for an existing sibling who requires a transplant. Preimplantation HLA tissue typing occurs when parents select embryos that are not at risk of a familial genetic disease to be a match for an existing sibling who requires a transplant. In Victoria, Australia, applications to use PGD with HLA tissue typing are reviewed by the Infertility Treatment Authority on a case by case basis. Preimplantation HLA tissue typing is prohibited prima facie because the embryo to be tested would not be at risk for a genetic abnormality or disease. Arguments for or against the use of PGD/HLA tissue typing are based on several key issues including the commodification and welfare of the donor child. This essay aims to show that that the same arguments apply to both PGD with HLA tissue typing and Preimplantation HLA tissue typing, and that the policy distinction between the two procedures is therefore ethically inconsistent.     

Evaluation of Serum and Tissue Levels of α-Tocopherol

This study was designed to test the effect of supplementation of several antioxidants, including α-tocopherol, on the clinical reduction of premalignant oral lesions. Samples of oral mucosa and serum were taken from baseline to 9 months of supplementation from patients with premalignant oral lesions and analyzed for α-tocopherol by HPLC. Statistical increases in both serum and tissue α-tocopherol were found after supplementation. There was no statistical relationship between α-tocopherol and β-carotene levels.     

Plant dehydrins — Tissue location, structure and function

Dehydrins (DHNs) are part of a large group of highly hydrophilic proteins known as LEA (Late Embryogenesis Abundant). They were originally identified as group II of the LEA proteins. The distinctive feature of all DHNs is a conserved, lysine-rich 15-amino acid domain, EKKGIMDKIKEKLPG, named the K-segment. It is usually present near the C-terminus. Other typical dehydrin features are: a track of Ser residues (the S-segment); a consensus motif, T/VDEYGNP (the Y-segment), located near the N-terminus; and less conserved regions, usually rich in polar amino acids (the Φ-segments). They do not display a well-defined secondary structure. The number and order of the Y-, S-and K-segments define different DHN sub-classes: Y_nSK_n, Y_nKn, SK_n, K_n and K_nS. Dehydrins are distributed in a wide range of organisms including the higher plants, algae, yeast and cyanobacteria. They accumulate late in embryogenesis, and in nearly all the vegetative tissues during normal growth conditions and in response to stress leading to cellular dehydration (e.g. drought, low temperature and salinity). DHNs are localized in different cell compartments, such as the cytosol, nucleus, mitochondria, vacuole, and the vicinity of the plasma membrane; however, they are primarily localized to the cytoplasm and nucleus. The precise function of dehydrins has not been established yet, but in vitro experiments revealed that some DHNs (YSK_n-type) bind to lipid vesicles that contain acidic phospholipids, and others (K_nS) were shown to bind metals and have the ability to scavenge hydroxyl radicals [Asghar, R. et al. Protoplasma 177 (1994) 87–94], protect lipid membranes against peroxidation or display cryoprotective activity towards freezing-sensitive enzymes. The SK_n-and K-type seem to be directly involved in cold acclimation processes. The main question arising from the in vitro findings is whether each DHN structural type could possess a specific function and tissue distribution. Much recent in vitro data clearly indicates that dehydrins belonging to different subclasses exhibit distinct functions. An erratum to this article is available at .     

Tissue transglutaminase in tumour progression: friend or foe?

Summary. Basic biological processes in which tissue transglutaminase (TG2, tTG) is thought to be important including apoptosis, cell adhesion and migration, ECM homeostasis and angiogenesis are key stages in the multistage tumour progression cascade. Studies undertaken with primary tumours and experimental models suggest that TG2 expression and activity in the tumour body and surrounding matrix generally decreases with tumour progression, favouring matrix destabilisation, but supporting angiogenesis and tumour invasion. In contrast, in the secondary metastatic tumour TG2 is often highly expressed whereby its potential roles in cell survival both at the intra- and extracellular level become important. In the following review the underlying molecular basis for the selection of these different phenotypes in tumour types and the anomaly for the requirement of TG2 is discussed in relation to the complex events of tumour progression.     

Tissue differential expression of lycopene β-cyclase gene in papaya

Carotene pigments in flowers and fruits are distinct features related to fitness advantages such as attracting insects for pollination and birds for seed dispersal. In papaya, the flesh color of the fruit is considered a quality trait that correlates with nutritional value and is linked to shelf-life of the fruit. To elucidate the carotenoid biosynthesis pathway in papaya, we took a candidate gene approach to clone the lycopene β-cyclase gene, LCY-B. A papaya LCY-B ortholog, cpLCY-B, was successfully identified from both cDNA and bacterial artificial chromosome （BAC） libraries and complete genomic sequence was obtained from the positive BAC including the promoter region. This cpLCY-B shared 80% amino acid identity with citrus LCY-B. However, full genomic sequences from both yellow- and red-fleshed papaya were identical. Quantitative real-time PCR （qPCR） revealed similar levels of expression at six different maturing stages of fruits for both yellow- and red-fleshed genotypes. Further expression analyses of cpLCY-B showed that its expression levels were seven- and three-fold higher in leaves and, respectively, flowers than in fruits, suggesting that cpLCY-B is down-regulated during the fruit ripening process.     

Quantitation of γH2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks¹. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)². Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy². Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer³. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo^4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.Download video file.^{(284M, mp4)}     

Tissue migration by parasitic helminths - an immunoevasive strategy?

Migration through host tissues has major costs for parasitic helminths in terms of energy expenditure, risks of attrition and the need to adapt to varying physicochemical environments. Nevertheless, such migratory phases seem to confer a specific survival advantage. One reason for this might be the avoidance of specific host immune-defence mechanisms designed to protect against threats at mucosal surfaces.     

Tissue trauma: the underlying cause of overtraining syndrome?

An athlete who trains intensely, yet consistently underperforms, is considered to be suffering from overtraining syndrome (OTS). OTS is a complex state that involves a large variety of signs and symptoms. Symptoms include changes in mood or behaviour, decreases or increases in concentration of different blood molecules, and alterations in immune function. Although several hypotheses have been proposed, each only explains a selective aspect of OTS. Presently, the sole agreement is that OTS is associated with excessive training and insufficient rest and recovery. The hypothesis proposed in this paper suggests that excessive training/competing causes repetitive tissue trauma, either to muscle and/or connective tissue and/or to bony structures, and that this results in chronic inflammation. It is further proposed that traumatized tissue synthesizes a group of inflammatory molecules, cytokines. Cytokines have been shown to coordinate the different systems of the body to promote recovery. Suggestions are made to detect, prevent, and rehabilitate the overtrained athlete.     

Tissue culture of the desert plant Anastatica hiërochuntica

Summary Callus derived from the winter annual desert plant Anastatica hiërochuntica was grown on different media, Murashige and Skoog (1962) medium giving the best results. Large amounts of lignified xylem elements were formed resulting in an extremely hard tissue. The growth responses to different auxins, cytokinins and abscisic acid were investigated. When salts (high Na⁺, Ca²⁺ and Cl^--contents) as they can be found in aqueous extracts of desert soils from a natural A. hiëerochuntica habitat were added to Abou-Mandour (1977) or MS-media, growth of callus was inhibited drastically. In the presence of abscisic acid, however, original growth was completely restored. In salt free control media on the other hand, ABA proved to be inhibitory. Drought stress caused a decrease of both cytokinins and indoleacetic acid in the callus while ABA levels were increased, but by far not as distinct as in intact plants. Proline level was not affected by stress.Abbreviations ABA abscisic acid - AM Abou-Mandour-medium - BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DHZR dihydroxyzeatinriboside - DW dry weight - ELISA enzyme linked immuno sorbent assay - FW freshweight - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IPA isopentenyladenosine - Kin kinetin - MS Murashige and Skoog-medium     

β3-Adrenergic Receptor Stimulation Induces E-Selectin-mediated Adipose Tissue Inflammation

Inflammation induced by wound healing or infection activates local vascular endothelial cells to mediate leukocyte rolling, adhesion, and extravasation by up-regulation of leukocyte adhesion molecules such as E-selectin and P-selectin. Obesity-associated adipose tissue inflammation has been suggested to cause insulin resistance, but weight loss and lipolysis also promote adipose tissue immune responses. While leukocyte-endothelial interactions are required for obesity-induced inflammation of adipose tissue, it is not known whether lipolysis-induced inflammation requires activation of endothelial cells. Here, we show that β₃-adrenergic receptor stimulation by CL 316,243 promotes adipose tissue neutrophil infiltration in wild type and P-selectin-null mice but not in E-selectin-null mice. Increased expression of adipose tissue cytokines IL-1β, CCL2, and TNF-α in response to CL 316,243 administration is also dependent upon E-selectin but not P-selectin. In contrast, fasting increases adipose-resident macrophages but not neutrophils, and does not activate adipose-resident endothelium. Thus, two models of lipolysis-induced inflammation induce distinct immune cell populations within adipose tissue and exhibit distinct dependences on endothelial activation. Importantly, our results indicate that β₃-adrenergic stimulation acts through up-regulation of E-selectin in adipose tissue endothelial cells to induce neutrophil infiltration.     

Tissue engineered nerve constructs:where do we stand?

Driven by enormous clinical need, interest in peripheral nerve regeneration has become a prime focus of research and area of growth within the field of tissue engineering. While using autologous donor nerves for bridging peripheral defects remains today's gold standard, it remains associated with high donor site morbidity and lack of full recovery. This dictates research towards the development of biomimetic constructs as alternatives. Based on current concepts, this review summarizes various approaches including different extracellular matrices, scaffolds, and growth factors that have been shown to promote migration and proliferation of Schwann cells. Since neither of these concepts in isolation is enough, although each is gaining increased interest to promote nerve regeneration, various combinations will need to be identified to strike a harmonious balance. Additional factors that must be incorporated into tissue engineered nerve constructs are also unknown and warrant further research efforts. It seems that future directions may allow us to determine the "missing link".     

Rivermouth Alteration of Agricultural Impacts on Consumer Tissue δ15N

Terrestrial agricultural activities strongly influence riverine nitrogen (N) dynamics, which is reflected in the δ¹⁵N of riverine consumer tissues. However, processes within aquatic ecosystems also influence consumer tissue δ¹⁵N. As aquatic processes become more important terrestrial inputs may become a weaker predictor of consumer tissue δ¹⁵N. In a previous study, this terrestrial-consumer tissue δ¹⁵N connection was very strong at river sites, but was disrupted by processes occurring in rivermouths (the ‘rivermouth effect’). This suggested that watershed indicators of N loading might be accurate in riverine settings, but could be inaccurate when considering N loading to the nearshore of large lakes and oceans. In this study, the rivermouth effect was examined on twenty-five sites spread across the Laurentian Great Lakes. Relationships between agriculture and consumer tissue δ¹⁵N occurred in both upstream rivers and at the outlets where rivermouths connect to the nearshore zone, but agriculture explained less variation and had a weaker effect at the outlet. These results suggest that rivermouths may sometimes be significant sources or sinks of N, which would cause N loading estimates to the nearshore zone that are typically made at discharge gages further upstream to be inaccurate. Identifying definitively the controls over the rivermouth effect on N loading (and other nutrients) will require integration of biogeochemical and hydrologic models.     

Enhanced Human Tissue Microdialysis Using Hydroxypropyl-?-Cyclodextrin as Molecular Carrier

Microdialysis sampling of lipophilic molecules in human tissues is challenging because protein binding and adhesion to the membrane limit recovery. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) forms complexes with hydrophobic molecules thereby improving microdialysis recovery of lipophilic molecules in vitro and in rodents. We tested the approach in human subjects. First, we determined HP-ß-CD influences on metabolite stability, delivery, and recovery in vitro. Then, we evaluated HP-ß-CD as microdialysis perfusion fluid supplement in 20 healthy volunteers. We placed 20 kDa microdialysis catheters in subcutaneous abdominal adipose tissue and in the vastus lateralis muscle. We perfused catheters with lactate free Ringer solution with or without 10% HP-ß-CD at flow rates of 0.3–2.0 µl/min. We assessed tissue metabolites, ultrafiltration effects, and blood flow. In both tissues, metabolite concentrations with Ringer+HP-ß-CD perfusate were equal or higher compared to Ringer alone. Addition of HP-ß-CD increased dialysate volume by 10%. Adverse local or systemic reactions to HP-ß-CD did not occur and analytical methods were not disturbed. HP-ß-CD addition allowed to measure interstitial anandamide concentrations, a highly lipophilic endogenous molecule. Our findings suggest that HP-ß-CD is a suitable supplement in clinical microdialysis to enhance recovery of lipophilic molecules from human interstitial fluid.     

Tissue engineering and regenerative medicine –where do we stand?

Tissue Engineering (TE) in the context of Regenerative Medicine (RM) has been hailed for many years as one of the most important topics in medicine in the twenty-first century. While the first clinically relevant TE efforts were mainly concerned with the generation of bioengineered skin substitutes, subsequently TE applications have been continuously extended to a wide variety of tissues and organs. The advent of either embryonic or mesenchymal adult stem-cell technology has fostered many of the efforts to combine this promising tool with TE approaches and has merged the field into the term Regenerative Medicine. As a typical example in translational medicine, the discovery of a new type of cells called Telocytes that have been described in many organs and have been detected by electron microscopy opens another gate to RM. Besides cell-therapy strategies, the application of gene therapy combined with TE has been investigated to generate tissues and organs. The vascularization of constructs plays a crucial role besides the matrix and cell substitutes. Therefore, novel in vivo models of vascularization have evolved allowing axial vascularization with subsequent transplantation of constructs. This article is intended to give an overview over some of the most recent developments and possible applications in RM through the perspective of TE achievements and cellular research. The synthesis of TE with innovative methods of molecular biology and stem-cell technology appears to be very promising.