Benzyl isothiocyanate: maximising production in papaya tissue extracts

Abstract: Although mainly grown for its sweet flavoured fruit, papaya (Carica papaya) has also been used for pharmacological purposes for many years. The reasons for use are varied with one of the best known being its anti-fungal action. Benzyl isothiocyanate (BITC) is the constituent most often implicated in this activity. Isothiocyanates are formed when the enzyme myrosinase catalyses the hydrolysis of the non-bioactive glucosinolates. This occurs when cellular contents come into contact through chewing, cutting or during extraction processes in the laboratory. While this is common in Brassica vegetables, the glucosinolate-myrosinase system is rare in fruit, papaya being a notable exception. It contains benzyl glucosinolate (BG), the glucosinolate precursor of BITC, in significant quantities. Parameters that determine the amount of BITC formed are duration of hydrolysis, presence/absence of nitrile-specifier proteins and BG content of different cultivars and tissues. We experimented with differing BITC extraction solvents, with the intention of developing a low cost, natural anti-fungal extract based on under-utilised papaya tissues. The findings suggest that papaya seeds, particularly from quarter-ripe fruit, have the potential to produce the highest levels of BITC necessary. Furthermore, they compare well with the nitrile-specifier protein-containing garden cress seeds (Lepidium sativum). To utilise the papaya seeds as a BITC source, an organic solvent such as ethanol is required to extract the largely water-insoluble BITC from the hydrolysed papaya seed mixture.

Plasma polymer and biomolecule modification of 3-D scaffolds for tissue engineering

Plasma polymerization was used to coat a melt electrospun polycaprolactone scaffold to improve cell attachment and organization. Plasma polymerization was performed using an amine containing monomer, allylamine, which then allowed for the subsequent immobilization of biomolecules i.e. heparin and fibroblast growth factor-2. The stability of the plasma polymerized amine-coating was demonstrated by X-ray photoelectron spectroscopy analysis and imaging time-of-flight secondary ion mass spectrometry revealed that a uniform plasma amine-coating was deposited throughout the scaffold. Based upon comparison with controls it was evident that the combination scaffold aided cell ingress and the formation of distinct fibroblast and keratinocyte layers.

Natural Hendra Virus Infection in Flying-Foxes - Tissue Tropism and Risk Factors

Hendra virus (HeV) is a lethal zoonotic agent that emerged in 1994 in Australia. Pteropid bats (flying-foxes) are the natural reservoir. To date, HeV has spilled over from flying-foxes to horses on 51 known occasions, and from infected horses to close-contact humans on seven occasions. We undertook screening of archived bat tissues for HeV by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Tissues were tested from 310 bats including 295 Pteropodiformes and 15 Vespertilioniformes. HeV was detected in 20 individual flying-foxes (6.4%) from various tissues including spleen, kidney, liver, lung, placenta and blood components. Detection was significantly higher in Pteropus Alecto and Pconspicillatus, identifying species as a risk factor for infection. Further, our findings indicate that HeV has a predilection for the spleen, suggesting this organ plays an important role in HeV infection. The lack of detections in the foetal tissues of HeV-positive females suggests that vertical transmission is not a regular mode of transmission in naturally infected flying-foxes, and that placental and foetal tissues are not a major source of infection for horses. A better understanding of HeV tissue tropism will strengthen management of the risk of spillover from flying-foxes to horses and ultimately humans.

Mode of action of antitumour antibiotics: Spectrophotometric studies on the interaction of chromomycin A3 with DNA and chromatin of normal and neoplastic tissue

The binding of chromomycin A3, an antitumour antibiotic, to various DNA and chromatin isolated from mouse and rat liver, mouse fibrosarcoma and Yoshida ascites sarcoma cells was studied spectrophotometrically at 29°C in 10−2 M Tris-HCl buffer, pH 8.0, containing small amounts of MgCl2 (4.5 · 10−5−25 · 10−5 M). An isobestic point at 415 nm was observed when chromomycin A3 was gradually titrated with Image and its spectrum shifted towards higher wavelength. The rates and extent of these spectral changes were found to be dependent on the concentration of Mg2+. The change in absorbance at 440 nm was used to calculate apparent binding constant (Ka p M−1) and sites per nucleotide (n) from Scatchard plots for various DNA and chromatins. As expected, values of n for chromatin (0.06–0.10) were found to be lower than that found for corresponding DNA (0.10–0.15). Apparently no such correlation exists between binding constants (Ka p M−1 · 10−4) of DNA (6.4–11.2) and of chromatin (3.1–8.3), but Ka p M−1 of chromatin isolated from mouse fibrosarcoma and Yoshida ascites sarcoma are 1.5–3 times higher than that found for mouse and rat liver chromatin. These differences may be taken to indicate structural difference in nucleoprotein complexes caused by neoplasia. The relevance of this finding to tumour suppressive action of chromomycin A3 is discussed.

Tissue thickness calculation in ocular optical coherence tomography

Thickness measurements derived from optical coherence tomography (OCT) images of the eye are a fundamental clinical and research metric, since they provide valuable information regarding the eye’s anatomical and physiological characteristics, and can assist in the diagnosis and monitoring of numerous ocular conditions. Despite the importance of these measurements, limited attention has been given to the methods used to estimate thickness in OCT images of the eye. Most current studies employing OCT use an axial thickness metric, but there is evidence that axial thickness measures may be biased by tilt and curvature of the image. In this paper, standard axial thickness calculations are compared with a variety of alternative metrics for estimating tissue thickness. These methods were tested on a data set of wide-field chorio-retinal OCT scans (field of view (FOV) 60° x 25°) to examine their performance across a wide region of interest and to demonstrate the potential effect of curvature of the posterior segment of the eye on the thickness estimates. Similarly, the effect of image tilt was systematically examined with the same range of proposed metrics. The results demonstrate that image tilt and curvature of the posterior segment can affect axial tissue thickness calculations, while alternative metrics, which are not biased by these effects, should be considered. This study demonstrates the need to consider alternative methods to calculate tissue thickness in order to avoid measurement error due to image tilt and curvature.

Tissue compression following short-term miniscleral contact lens wear

To quantify regional (nasal, superior, temporal and inferior) and location specific (corneal and scleral) tissue compression following short-term miniscleral contact lens wear.

Targeting antigen to diverse APCs inactivates memory CD8+ T cells without eliciting tissue-destructive effector function

Memory T cells develop early during the preclinical stages of autoimmune diseases and have traditionally been considered resistant to tolerance induction. As such, they may represent a potent barrier to the successful immunotherapy of established autoimmune diseases. It was recently shown that memory CD8+ T cell responses are terminated when Ag is genetically targeted to steady-state dendritic cells. However, under these conditions, inactivation of memory CD8+ T cells is slow, allowing transiently expanded memory CD8+ T cells to exert tissue-destructive effector function. In this study, we compared different Ag-targeting strategies and show, using an MHC class II promoter to drive Ag expression in a diverse range of APCs, that CD8+ memory T cells can be rapidly inactivated by MHC class II+ hematopoietic APCs through a mechanism that involves a rapid and sustained downregulation of TCR, in which the effector response of CD8+ memory cells is rapidly truncated and Ag-expressing target tissue destruction is prevented. Our data provide the first demonstration that genetically targeting Ag to a broad range of MHC class II+ APC types is a highly efficient way to terminate memory CD8+ T cell responses to prevent tissue-destructive effector function and potentially established autoimmune diseases. Copyright © 2010 by The American Association of Immunologists, Inc.

Human parvovirus B19: tissue persistence and prevalence of prototypic and new variants

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.

Growth factor-loaded microparticles for tissue engineering: The discrepancies of in vitro characterization assays

Efficient and effective growth factor (GF) delivery is an ongoing challenge for tissue regeneration therapies. The accurate quantification of complex molecules such as GFs, encapsulated in polymeric delivery devices, is equally critical and just as complex as achieving efficient delivery of active GFs. In this study, GFs relevant to bone tissue formation, vascular endothelial growth factor (VEGF) and bone morphogenetic protein 7 (BMP-7), were encapsulated, using the technique of electrospraying, into poly(lactic-co-glycolic acid) microparticles that contained poly(ethylene glycol) and trehalose to assist GF bioactivity. Typical quantification procedures, such as extraction and release assays using saline buffer, generated a significant degree of GF interactions, which impaired accurate assessment by enzyme-linked immunosorbent assay (ELISA). When both dry BMP-7 and VEGF were processed with chloroform, as is the case during the electrospraying process, reduced concentrations of the GFs were detected by ELISA; however, the biological effect on myoblast cells (C2C12) or endothelial cells (HUVECs) was unaffected. When electrosprayed particles containing BMP-7 were cultured with preosteoblasts (MC3T3-E1), significant cell differentiation into osteoblasts was observed up to 3 weeks in culture, as assessed by measuring alkaline phosphatase. In conclusion, this study showed how electrosprayed microparticles ensured efficient delivery of fully active GFs relevant to bone tissue engineering. Critically, it also highlights major discrepancies in quantifying GFs in polymeric microparticle systems when comparing ELISA with cell-based assays.

Postoperative soft tissue defects in the ankle area : The etiology and methods of reconstruction

The risk is obvious for soft tissue complications after operative treatment of the Achilles tendon, calcaneal bone or after ankle arthroplasty. Such complications after malleolar fractures are, however, seldom seen. The reason behind these complications is that the soft tissue in this region is tight and does not allow much tension to the wound area after surgery. Furthermore the area of operation may be damaged by swelling after the injury, or can be affected by peripheral vascular disease. While complications in this area are unavoidable, they can be diminished. This study attempts to highlight the possible predisposing factors leading to complications in these operations and on the other hand, to determine the solutions to solve soft tissue problems in this region. The study consists of five papers. The first article is a reprint on the soft tissue reconstruction of 25 patients after their complicated Achilles tendon surgeries were analysed. The second study reviews a series of 126 patients after having undergone an operative treatment of calcaneal bone fractures and analyses the complications and possible reasons behind them. The third part analyses a series of corrections of 35 soft tissue complications after calcaneal fracture operations. The fourth part reviews a series of 7 patients who had undergone complicated ankle arthroplasties. The last article presents a series of post operative lateral defects of the ankle treated with a less frequently used distally based peroneus brevis muscle flap and analyses the results. What can be conducted from these studies is that in general, the results after the correction of even severe soft tissue complications in the ankle region are good. For the small defects around the Achilles tendon, the local flaps are useful, but the larger defects are best treated with a free flap. We found that a long delay from trauma to surgery and a long operating time were predisposing factors that lead to soft tissue complications after operatively treated calcaneal bone fractures. The more severe the injury, the greater the risk for wound complication. Surprisingly, the long-term results after infected calcaneal osteosyntheses were acceptable and the calcaneal bone seems to tolerate chronic infections very well if the soft tissue is reconstructed successfully. Behind the complicated ankle arthroplasties, unexpectedly high number of cases experiencing arteriosclerosis of the lower extremity was found. These complications lead to ankle fusion but can be solved with a free flap if the vascularity is intact or can be reconstructed. For this reason a vascular examination of the lower extremity arteries of the patients going to ankle arthroplasty is strongly recommended. Moreover postoperative lateral malleolar wound infections which typically create lateral ankle defects can successfully be treated with a peroneus brevis muscle flap covered with a free skin graft.