Platelet sequestration and activation during GalTKO.hCD46 pig lung perfusion by human blood is primarily mediated by GPIb, GPIIb/IIIa, and von Willebrand Factor.

BACKGROUND: Here, we ask whether platelet GPIb and GPIIb/IIIa receptors modulate platelet sequestration and activation during GalTKO.hCD46 pig lung xenograft perfusion. METHODS: GalTKO.hCD46 transgenic pig lungs were perfused with heparinized fresh human blood. Results from perfusions in which αGPIb Fab (6B4, 10 mg/l blood, n = 6), αGPIIb/IIIa Fab (ReoPro, 3.5 mg/l blood, n = 6), or both drugs (n = 4) were administered to the perfusate were compared to two additional groups in which the donor pig received 1-desamino-8-d-arginine vasopressin (DDAVP), 3 µg/kg (to pre-deplete von Willebrand Factor (pVWF), the main GPIb ligand), with or without αGPIb (n = 6 each). RESULTS: Platelet sequestration was significantly delayed in αGPIb, αGPIb+DDAVP, and αGPIb+αGPIIb/IIIa groups. Median lung "survival" was significantly longer (>240 vs. 162 min reference, p = 0.016), and platelet activation (as CD62P and ßTG) were significantly inhibited, when pigs were pre-treated with DDAVP, with or without αGPIb Fab treatment. Pulmonary vascular resistance rise was not significantly attenuated in any group, and was associated with residual thromboxane and histamine elaboration. CONCLUSIONS: The GPIb-VWF and GPIIb/IIIa axes play important roles in platelet sequestration and coagulation cascade activation during GalTKO.hCD46 lung xenograft injury. GPIb blockade significantly reduces platelet activation and delays platelet sequestration in this xenolung rejection model, an effect amplified by adding αGPIIb/IIIa blockade or depletion of VWF from pig lung.

Expression of human CD46 modulates inflammation associated with GalTKO lung xenograft injury.

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose-α(1,3)-galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n = 16), and WT pig lungs (n = 16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 min compared to 120 for GalTKO (p = 0.27) and 10 for WT lungs (p < 0.001). Complement activation, platelet activation and histamine elaboration were significantly reduced during the first 2 h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120' 812 ± 230 vs. 1412 ± 1047, p = 0.02; ΔCD62P at 120' 9.8 ± 7.2 vs. 25.4 ± 18.2, p < 0.01; Δhistamine at 60' 97 ± 62 vs. 189 ± 194, p = 0.03). We conclude that, in addition to significant down-modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions.

AAV-mediated gene transfer to cardiac cells in a heterotopic rat heart transplantation model.

INTRODUCTION: Adeno-associated virus (AAV) vectors offer the possibility to transfer genes to a wide range of organ and cell types. To determine the efficiency of AAV-mediated gene transfer to cardiac cells, vectors were administered to the heart under various conditions. METHODS: In Sprague-Dawley rats, AAV vectors based on serotype 2 and coding for beta-galactosidase were injected via coronaries into hypothermic nonbeating and normothermic beating hearts before transplantation. In addition, vectors were injected intravenously or into the thigh muscle. After 28 days all animals were humanely killed and organs explanted for analysis. RESULTS: Transgenic DNA was always detectable in the liver and the heart, irrespective of the application mode. However, transgenic mRNA could not be determined in the transplanted hearts. In contrast, direct injection into the thigh muscle resulted in transgenic mRNA production and marker gene expression. After systemic application, transgenic mRNA was detected in the liver but not in the heart. CONCLUSION: The results of our study indicated that AAV-mediated gene transfer to cardiac cells is possible. However, it was impossible to detect transgenic mRNA or marker gene expression in the transplanted hearts after intracoronary perfusion or systemic injection.

Transgenic animals in experimental xenotransplantation models: orthotopic heart transplantation in the pig-to-baboon model.

Pig organs are at risk for hyperacute and acute vascular rejection mediated by anti-pig antibodies, mainly binding to the Galalpha(1,3)Gal epitope. Acute cellular rejection is characterized by progressive infiltration of mononuclear cells. There is an ongoing search for immunosuppressive regimens that provide adequate protection against all patterns of xenograft rejection, but have no severe impact on the condition of xenograft recipients. Herein orthotopic heart transplantations were performed from hDAF or hCD46 piglets to nonsplenectomized baboons. Basic immunosuppression consisted of tacrolimus, sirolimus, GAS914, steroids, and ATG. Group 1 received basic immunosuppression. Group 2 was additionally treated with rituximab and group 3 with half-dose cyclophosphamide. Group 4 received cyclophosphamide and an anti-HLA-DR antibody. Three baboons received GAS914 and TPC. Monitoring included the regular assessment of anti-porcine antibodies, blood counts, therapeutic drug monitoring, and graft histology. Two grafts failed due to technical mistakes. In group 1, baboons died after 1 and 9 days. In group 2, maximum survival was 30 hours. In group 3, baboons lived 20 hours, 25 days, and 14 days. Group 4 survival times were 9.5 hours, 5.5 hours, 4 days, 34 hours, and 3 days. An increase of non-Galalpha(1,3)Gal antibodies was observed. Depositions of immunoglobulins and complement revealed a humoral rejection process. No cellular infiltration could be observed. In conclusion, suppressing cellular rejection with half-dose cyclophosphamide together with tacrolimus and sirolimus produced longer graft survival with a good general condition. Prevention of acute xenograft rejection further needs inhibition of non-Galalpha(1,3)Gal cytotoxicity by sufficient depression of B-cell activation.

Ergonomic and physiotherapeutic interventions for treating work-related complaints of the arm, neck or shoulder in adults.

BACKGROUND: Conservative interventions such as physiotherapy and ergonomic adjustments (such as keyboard adjustments or ergonomic advice) play a major role in the treatment of most work-related complaints of the arm, neck or shoulder (CANS). OBJECTIVES: This systematic review aims to determine whether conservative interventions have a significant impact on outcomes for work-related CANS in adults. SEARCH STRATEGY: We searched the Cochrane Bone, Joint and Muscle Trauma Group Specialised Register (March 2005) and Cochrane Rehabilitation and Related Therapies Field Specialised Register (March 2005), the Cochrane Controlled Trials Register (The Cochrane Library, Issue 1, 2005), PubMed, EMBASE, CINAHL, AMED and reference lists of articles. The date of the last search was March 2005. No language restrictions were applied. SELECTION CRITERIA: We included randomised and non-randomised controlled trials studying conservative interventions (e.g. exercises, relaxation, physical applications, biofeedback, myofeedback and work-place adjustments) for adults suffering CANS. DATA COLLECTION AND ANALYSIS: Two authors independently selected trials from the search yield, assessed the methodological quality using the Delphi list, and extracted relevant data. We pooled data or, in the event of clinical heterogeneity or lack of data, we used a rating system to assess levels of evidence. MAIN RESULTS: For this update we included six additional studies; 21 trials in total. Seventeen trials included people with chronic non-specific neck or shoulder complaints, or non-specific upper extremity disorders. Over 25 interventions were evaluated; five main subgroups of interventions could be determined: exercises, manual therapy, massage, ergonomics, and energised splint. Overall, the quality of the studies was poor. In 14 studies a form of exercise was evaluated, and contrary to the previous review we now found limited evidence about the effectiveness of exercises when compared to massage and conflicting evidence when exercises are compared to no treatment. In this update there is limited evidence for adding breaks during computer work; massage as add-on treatment on manual therapy, manual therapy as add-on treatment on exercises; and some keyboard designs when compared to other keyboards or placebo in participants with carpal tunnel syndrome. AUTHORS' CONCLUSIONS: There is limited evidence for the effectiveness of keyboards with an alternative force-displacement of the keys or an alternative geometry, and limited evidence for the effectiveness of exercises compared to massage; breaks during computer work compared to no breaks; massage as an add-on treatment to manual therapy; and manual therapy as an add-on treatment to exercises.

Fluorescent microspheres reveal different regional blood flow in hyperacutely rejected nontransgenic and hDAF pig hearts.

Classic features of hyperacute rejection show differential severity in the inner compared to the outer myocardium. In the present study, regional blood flow (RBF) measured by fluorescent microspheres served as a marker of the extent of hyperacute rejection. Using a working heart model, hearts of nontransgenic and hDAF transgenic pigs were perfused with human blood. Additionally, hDAF transgenic pig hearts were perfused with human blood containing GAS914 or the GPIIb/IIIa inhibitor tirofiban. Injections of fluorescent microspheres into the donor heart were performed in situ and during perfusion. Reference arterial blood samples were collected from the inferior aorta and the afterload line. Perfusion was terminated before hyperacutely rejected hearts failed to pump against the afterload column. RBF was determined in tissue samples of standardized areas of the left atrium and ventricle. Each specimen was divided into subepicardial and subendocardial tissue samples. Fluorescence intensity was measured using an automated luminescence spectrometer. At the end of perfusion with human blood, hyperacutely rejected nontransgenic pig hearts showed a higher RBF in the subendocardium. In hDAF-transgenic pig hearts perfused with unmodified human blood the subendocardial/subepicardial blood flow ratio changed in favor of the subepicardium. This ratio was not further improved by GAS914. In contrast, tirofiban was able to assimilate subepicardial and subendocardial blood flow. In conclusion, RBF of hyperacutely rejected pig hearts was inhomogeneous. Inhibition of complement activation improved the reduced subepicardial RBF, but depletion of antibodies had no positive effect. The ability of tirofiban to further increase subepicardial RBF affirms thrombosis of subepicardial veins as the defining characteristic of hyperacute rejection.

Ergonomic and physiotherapeutic interventions for treating upper extremity work related disorders in adults.

BACKGROUND: Conservative interventions such as physiotherapy and ergonomic adjustments play a major part in the treatment of most work-related musculoskeletal disorders (WRMD). OBJECTIVES: The objective of this systematic review is to determine whether conservative interventions have a significant impact on short and long-term outcomes for upper extremity WRMD in adults. SEARCH STRATEGY: We searched the Cochrane Musculoskeletal Injuries Group specialised register (January 2002) and Cochrane Rehabilitation and Related Therapies Field specialised register (January 2002), the Cochrane Controlled Trials Register (The Cochrane Library Issue 3, 2001), PubMed (1966 to November 2001), EMBASE (1988 to November 2001), and CINAHL (1982 to November 2001). We also searched the Physiotherapy Index (1988 to November 2001) and reference lists of articles. No language restrictions were applied. SELECTION CRITERIA: Only randomised controlled trials and concurrent controlled trials studying conservative interventions for adults suffering from upper extremity WRMD were included. Conservative interventions may include exercises, relaxation, physical applications, biofeedback, myofeedback and work place adjustments. DATA COLLECTION AND ANALYSIS: Two reviewers independently selected the trials from the search yield and assessed the clinical relevance and methodological quality using the Delphi list. In the event of clinical heterogeneity or lack of data we used a rating system to assess levels of evidence. MAIN RESULTS: We included 15 trials involving 925 people. Twelve trials included people with chronic non-specific neck or shoulder complaints, or non-specific upper extremity disorders. Over 20 interventions were evaluated; seven main subgroups of interventions could be determined: exercises, manual therapy, massage, ergonomics, multidisciplinary treatment, energised splint and individual treatment versus group therapy. Overall, the quality of the studies appeared to be poor. In 10 studies a form of exercise was evaluated, and there is limited evidence about the effectiveness of exercises only when compared to no treatment. Concerning manual therapy (1 study), massage (4 studies), multidisciplinary treatment (1 study) and energised splint (1 study) no conclusions can be drawn. Limited evidence is found concerning the effectiveness of specific keyboards for patients with carpal tunnel syndrome. REVIEWER'S CONCLUSIONS: This review shows limited evidence for the effectiveness of keyboards with an alternative force-displacement of the keys or an alternative geometry, and limited evidence for the effectiveness of individual exercises. The benefit of expensive ergonomic interventions (such as new chairs, new desks etc) in the workplace is not clearly demonstrated.

Pulmonary function and intradermal tests in workers exposed to soft-paper dust.

A survey was carried out in the processing department of a paper mill, consisting of environmental and personal monitoring, a short questionnaire concerning chronic non-specific lung disease (CNSLD), spirometry, and intradermal tests. Spirometry was performed on Monday, Wednesday, and Friday. Exposures to paper dust regularly exceeded the Dutch Maximal Allowable Concentrations (MACs) for total and respirable dust. There were indications of a qualitatively different exposure to molds inside the mill in comparison with the outdoor air. A comparison between a group of workers exposed to paper dust and a control group showed significantly more persons with positive late and delayed intradermal test reactions in the exposed population. The analysis of the spirometric measurements after correction for differences between the groups in age, height, and smoking habits gave evidence of the existence of lower FEV1, MMEF, MEF50, and MEF25 among exposed workers with a positive immediate intradermal reaction on Friday. The same workers showed a decline in pulmonary function over the week compared with the controls. These changes suggest an obstructive airway reaction with an immunologic mechanism to dust exposure in a paper mill.