Determination of carazolol residues in porcine tissue by radioreceptor assay

A radioreceptor assay was developed for the determination of the p-blocker carazolol in porcine muscle and kidney. The method involves a simple alkaline extraction procedure using diethyl ether followed by a competitive assay between carazolol residues and [H-3]-dihydroalprenolol ([H-3]-DHA) using solubilised beta2-adrenoceptors isolated from a transfected cell line. The limit of detection (LOD) was determined using 20 reference blank samples of pig kidney and pig muscle. LODs for muscle (0.93 mug kg(-1)) and kidney (1.47 mug kg(-1)) were well below their respective European community maximum residue limits, (MRLs 5 and 25 mug kg(-1), respectively). The assay was used to investigate if carazolol residues persisted in pig tissues for up to 30 h post-intramuscular injection at the recommended dose rate (10 mug carazolol/kg body weight). The highest mean +/- S.D. concentrations were detected at 1 h post-injection in kidney (10.84 +/- 1.3 mug kg(-1)) and muscle (3.59 +/- 0.2 mug kg(-1)) which were less than the respective MRLs. It is concluded that this method offers a robust and rapid alternative to other methods for the screening of carazolol residues in pig meat. (C) 2002 Elsevier Science B.V. All rights reserved.

Autoradiographic and scintillation analysis of 5-aminolevulinic acid permeation through epithelialised tissue: Implications for topical photodynamic therapy of superficial gynaecological neoplasias

Purpose. Aminolevulinic acid (5-ALA) diffusion through both keratinised and non-keratinised tissue, used as a model tissue substrates, was evaluated, together with the depth of permeation and the concentration achieved following delivery from bioadhesive patch and proprietary cream formulations. Materials and Methods. Moisture-activated, bioadhesive patches loaded with 5-ALA at concentrations of 19.0, 38.0 and 50.0 mg cm(-2) and an o/w cream (20% w/w 5-ALA) were radiolabelled with C14 5-ALA and applied to excised human vaginal tissue and porcine skin. After 1, 2 and 4 h, tissue was sectioned in two orientations and the 5-ALA concentration at specific depths determined using autoradiography and liquid scintillation counting (LSC). Results. The stratum corneum was a significant barrier to 5-ALA permeation, with concentrations in tissue dependent on application time and drug loading. 5-ALA was detected at 6 mm using autoradiography after 2 h, with LSC showing phototoxic concentrations at 2.375 mm after 4 h of application. Inclusion of oleic acid and dimethyl sulphoxide in bioadhesive patches increased 5-ALA significantly in neonate porcine tissue, but only for patches cast from blends containing 5% w/w oleic acid. Conclusions. The bioadhesive patch described delivered 5-ALA to depths of at least 2.5 mm in tissue types indicative of vulval skin, suggesting that photodynamic therapy of deep vulval intraepithelial neoplasia is feasible using this means of bioadhesive 5-ALA delivery.

A comparison of human and porcine acellular dermal tissues in the subcutaneous space of a rat model

The study compared the host response to a human and a porcine acellular dermal tissue implanted in the subcutaneous space of a rat model. The human and porcine acellular grafts were surgically implanted in the subcutaneous tissue of rats (5 rats/group) and the materials were evaluated at 7, 15, 30, 60 and 180 postoperative days (PO). The histological immune response was quantified using a digital image analysis system, which evaluated the number of vessels present in the implants and in the surrounding soft tissue, the area of inflammatory cell infiltration in the grafts, the width of the capsular formation present around the tissues and the area of implants absorbed. The data were submitted to statistical analysis. Light microscopy showed mononuclear cellular infiltration, the presence of a capsular formation surrounding the grafts and the presence of vacuolar structures (optically empty spaces) inside the implants. The image analysis comparing both materials showed significant inflammatory cells in the human graft at 15 and 30 PO, thicker capsular formation in the porcine tissue at 60 PO, increased number of vessels inside the implants and in the surrounding tissues in the porcine graft and a similar absorption pattern in both materials at 180 PO. The histological findings showed that both tissues were well-tolerated when implanted in the subcutaneous tissue of rats, allowing us to consider the porcine acellular dermal graft as a provisional alternative material for reconstructive plastic surgery. Copyright © 2005 Taylor & Francis LLC.

Bystander-induced differentiation: a major response to targeted irradiation of a urothelial explant model.

A ureter primary explant technique, using porcine tissue sections was developed to study bystander effects under in vivo like conditions where dividing and differentiated cells are present. Targeted irradiations of ureter tissue fragments were performed with the Gray Cancer Institute charged particle microbeam at a single location (2 microm precision) with 10 3He2+ particles (5 MeV; LET 70 keV/microm). After irradiation the ureter tissue section was incubated for 7 days allowing explant outgrowth to be formed. Differentiation was estimated using antibodies to Uroplakin III, a specific marker of terminal urothelial differentiation. Even although only a single region of the tissue section was targeted, thousands of additional cells were found to undergo bystander-induced differentiation in the explant outgrowth. This resulted in an overall increase in the fraction of differentiated cells from 63.5+/-5.4% to 76.6+/-5.6%. These changes are much greater than that observed for the induction of damage in this model. One interpretation of these results is that in the tissue environment, differentiation is a much more significant response to targeted irradiation and potentially a protective mechanism.

Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA-E double transgenic pig forelimbs with human blood.

BACKGROUND Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model. METHODS α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples. RESULTS No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions. CONCLUSION Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection.

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400 mg kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Influence of porcine genotype on the abundance of thyroid hormones and leptin in sow milk and its impact on growth, metabolism and expression of key adipose tissue genes in offspring

Neonatal mortality is greater in commercial porcine genotypes, compared with the ancient Meishan breed that rapidly lay down adipose tissue; this may be related to hormones, such as triiodothyronine (T3) or leptin. Leptin is present in maternal milk; however, the extent to which this supply provides the neonate with leptin is unknown, but may play a role in growth and development. We investigated whether thyroid hormones and leptin concentrations in maternal milk differed between genotypes; and whether this influenced piglet concentrations or expression of genes involved in adipose tissue regulation. Eight Meishan and six commercial sows were entered into the study and milk samples from the day of parturition to day 4 postpartum was taken daily. The median birth weight piglet in each litter had a daily venous blood sample taken and was euthanised on day 4. Gene expressions of IGF-I, IGF-binding protein 3 (IGFBP-3), peroxisome proliferators activated receptor (PPAR) and glucocorticoid receptor (GR) were measured in adipose tissue using real-time PCR. T3 was increased in Meishan milk, but not in piglet plasma. Milk thyroxine was similar between breeds but commercial piglet levels were significantly higher. Leptin was higher in commercial sow milk throughout the study. Milk leptin was strongly correlated to plasma leptin during the first postnatal days and also to organ and body weight in Meishan piglets that also had significantly higher expression of GR, but not IGF-I, IGFBP-3 or PPAR. In conclusion, we have found a significant disparity in the provision of thyroid hormones in Meishan and commercial sow’s milk. These changes are not always translated to plasma concentrations of hormone in the piglet. Leptin appears to have a stronger role in growth and development in the Meishan genotype compared with commercial; along with the increased GR expression, this may also represent a potential mechanism behind the rapid accumulation of adipose tissue in Meishan piglets.

Acellular dermal tissue study: an ultrastructural evaluation of human and porcine derived tissues in a rat model

The purpose of this study was to evaluate the host response of a human and a porcine derived acellular dermal tissue (ADT) implanted in the subcutaneous tissue of a rat model. Two subcutaneous pockets were surgically created along the dorsal midline of 25 rats (5 rats/group). The human ADT was placed superiorly and the porcine ADT, inferiorly. The animals were sacrificed at 07, 15, 30, 60 and 180 postoperative days (PO) and the ADTs and surrounding soft tissues were assessed for ultrastructural evaluation by transmission electron microscopy. The ultrastructural findings were similar in both materials. Normal collagen and elastic fibers bundles were observed during all experimental moments, as well as macrophages presenting cytoplasmic enlargements digesting cellular portions after 15 PO. From 30 until 180 PO, vacuolar structures filled with an amorphous, electron-transparent substance, were present inside and outside the fibroblasts. Both human and porcine ADT showed similar pattern of ultrastructural response when implanted in the subcutaneous tissue of rats. The porcine ADT appears as a good alternative to be used as a biomaterial.

Differential response of porcine osteoblasts and chondrocytes in cell or tissue culture after 5-aminolevulinic acid-based photodynamic therapy

OBJECTIVE: Outcome in osteochondral allografting is limited by the immunological incompatibility of the grafted tissue. Based on a resistance of chondrocytes to photodynamic therapy in cell culture it is proposed that 5-aminolevulinic acid-based photodynamic therapy (5-ALA-PDT) might be used to inactivate bone while maintaining viability of chondrocytes and thus immunomodulate bone selectively. METHODS: Chondrocytes and osteoblasts from porcine humeral heads were either isolated (cell culture) or treated in situ (tissue culture). To quantify cytotoxic effects of 5-ALA-PDT (0-20J/cm(2), 100mW/cm(2)) an (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide) (MTT)-assay was used in cell culture and in situ hybridization in tissue culture to assess metabolic active cells (functional osteoblasts: colalpha(1)(I) mRNA, functional chondrocytes: colalpha(1)(II) mRNA). RESULTS: In cell culture, survival after 5-ALA-PDT was significantly higher for chondrocytes (5J/cm(2): 87+/-12% compared to untreated cells) than for osteoblasts (5J/cm(2): 12+/-11%). In tissue culture, the percentage of functional chondrocytes in cartilage showed a decrease after 5-ALA-PDT (direct fixation: 92+/-2%, 20J/cm(2): 35+/-15%; P<0.0001). A significant decrease in the percentage of bone surfaces covered by functional osteoblasts was observed in freshly harvested (31+/-3%) compared to untreated tissues maintained in culture (11+/-4%, P<0.0001), with no further decrease after 5-ALA-PDT. CONCLUSION: Chondrocytes were more resistant to 5-ALA-PDT than osteoblasts in cell culture, while in tissue culture a loss of functional chondrocytes was observed after 5-ALA-PDT. Since osteoblasts - but not chondrocytes - were sensitive to the tissue culture conditions, devitalized bone with functional cartilage might already be achieved by applying specific tissue culture conditions even without 5-ALA-PDT.