Functional evaluation of hTM, CD46 and HLA-E transgenes in vitro and in pig leg xenoperfusion experiments

Background Besides α1,3 galactosyltransferase (Gal) gene knockout several transgene combinations to prevent pig-to-human xenograft rejection are being investigated. hCD46/HLA-E double transgenic pigs were tested for prevention of xenograft rejection in an ex vivo pig-to-human xenoperfusion model. In addition, expression of human thrombomodulin (hTM-) on wild-type and/or multi-transgenic (GalTKO/hCD46) background was evaluated to overcome pig-to-human coagulation incompatibility. Methods hCD46/HLA-E double transgenic as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human blood and autologous blood, respectively. Blood samples were analyzed for production of porcine and/or human inflammatory cytokines. Biopsy samples were examined for deposition of complement proteins as well as E-selectin and VCAM-1 expression. Serial blood cell counts were performed to analyze changes in human blood cell populations. In vitro, PAEC were analyzed for ASGR1 mediated human platelet phagocytosis. In addition, a biochemical assay was performed using hTM-only and multi-transgenic (GalTKO/hCD46/hTM) pig aortic endothelial cells (PAEC) to evaluate the ability of hTM to generate activated protein C (APC). Subsequently, the anti-coagulant properties of hTM were tested in a microcarrier based coagulation assay with PAEC and human whole blood. Results No hyperacute rejection was seen in the ex vivo perfusion model. Extremity perfusions lasted for up to 12 h without increase of vascular resistance and had to be terminated due to continuous small blood losses. Plasma levels of porcine IL1β (P < 0.0001), and IL-8 (P = 0.019) as well as human C3a, C5a and soluble C5b-9 were significantly (P < 0.05–<0.0001) lower in blood perfused through hCD46/HLA-E transgenic as compared to wild-type limbs. C3b/c, C4b/c, and C6 deposition as well as E-selectin and VCAM-1 expression were significantly (P < 0.0001) higher in tissue of wild-type as compared to transgenic limbs. Preliminary immunofluorescence staining results showed that the expression of hCD46/HLA-E is associated with a reduction of NK cell tissue infiltration (P < 0.05). A rapid decrease of platelets was observed in all xenoperfusions. In vitro findings showed that PAEC express ASGR1 and suggest that this molecule is involved in human platelet phagocytosis. In vitro, we found that the amount of APC in the supernatant of hTM transgenic cells increased significantly (P < 0.0001) with protein C concentration in a dose-dependent manner as compared to control PAEC lacking hTM, where the turnover of the protein C remained at the basal level for all of the examined concentration. In further experiments, hTM also showed the ability to prevent blood coagulation by three- to four-fold increased (P < 0.001) clotting time as compared to wild-type PAEC. The formation of TAT complexes was significantly lower when hTM-transgenic cells (P < 0.0001) were used as compared to wild-type cells. Conclusions Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since the terminal pathway of complement, endothelial cell activation, inflammatory cytokine production and NK-cell tissue infiltration were all down-regulated. We also found ASGR1 expression on the vascular endothelium of pigs, and this molecule may thus be involved in binding and phagocytosis of human platelets during pig-to-human xenotransplantation. In addition, use of the hTM transgene has the potential to overcome coagulation incompatibilities in pig-to-human xenotransplantation.

Radiation dose reduction in computer assisted navigation for functional endoscopic sinus surgery--cadaver head experiments and clinical implementation

Computed tomography based navigation for endoscopic sinus surgery is inflationary used despite of major public concern about iatrogenic radiation induced cancer risk. Studies on dose reduction for CAS-CT are almost nonexistent. We validate the use of radiation dose reduced CAS-CT for clinically applied surface registration.

Serial in vivo intravascular ultrasound-based echogenicity changes of everolimus-eluting bioresorbable vascular scaffold during the first 12 months after implantation insights from the ABSORB B trial

This study sought to investigate quantitative and homogeneity differential echogenicity changes of the ABSORB scaffold (1.1) during the first year after implantation.

Serial analysis of the malapposed and uncovered struts of the new generation of everolimus-eluting bioresorbable scaffold with optical coherence tomography

The aim of this study is to assess the serial changes in strut apposition and coverage of the bioresorbable vascular scaffolds (BVS) and to relate this with the presence of intraluminal masses at 6 months with optical coherence tomography (OCT).

Reporting Experiments in Homeopathic Basic Research—Description of the Checklist Development

The objective of this study was to develop a criteria catalogue serving as a guideline for authors to improve quality of reporting experiments in basic research in homeopathy. A Delphi Process was initiated including three rounds of adjusting and phrasing plus two consensus conferences. European researchers who published experimental work within the last 5 years were involved. A checklist for authors provide a catalogue with 23 criteria. The “Introduction” should focus on underlying hypotheses, the homeopathic principle investigated and state if experiments are exploratory or confirmatory. “Materials and methods” should comprise information on object of investigation, experimental setup, parameters, intervention and statistical methods. A more detailed description on the homeopathic substances, for example, manufacture, dilution method, starting point of dilution is required. A further result of the Delphi process is to raise scientists' awareness of reporting blinding, allocation, replication, quality control and system performance controls. The part “Results” should provide the exact number of treated units per setting which were included in each analysis and state missing samples and drop outs. Results presented in tables and figures are as important as appropriate measures of effect size, uncertainty and probability. “Discussion” in a report should depict more than a general interpretation of results in the context of current evidence but also limitations and an appraisal of aptitude for the chosen experimental model. Authors of homeopathic basic research publications are encouraged to apply our checklist when preparing their manuscripts. Feedback is encouraged on applicability, strength and limitations of the list to enable future revisions.

Steerable intravitreal inserts for drug delivery: in vitro and ex vivo mobility experiments

The progress of wet age-related macular degeneration can now be controlled by intravitreal drug injection. This approach requires repeated injections, which could be avoided by delivering the drug to the retina. Intraocular implants are a promising solution for drug delivery near the retina. Currently, their accurate placement is challenging, and they can only be removed after a vitrectomy. In this paper, we introduce an approach for minimally invasive retinal drug delivery using magnetic intraocular inserts. We briefly discuss the electromagnetic-control system for magnetic implants and then focus on evaluating their ability to move in the vitreous humor. The mobility of magnetic intraocular implants is estimated in vitro with synthesized vitreous humors, and ex vivo with experiments on cadaver porcine eyes. Preliminary results show that with such magnetic implants a vitrectomy can be avoided.

Accordion-optimized DEPT experiments

In this contribution, a pulse sequence is described for recording accordion-optimized DEPT experiments. The proposed ACCORDEPT experiment detects a wide range of one-bond coupling constants using accordion optimization. As a proof of concept, this strategy has been applied to a mesogen containing a large range of one-bond (1)J(CH) coupling constants associated with the various structural elements. The ACCORDEPT experiment afforded significant enhancements for the resonances with the larger 1JCH couplings, similar SNR for aliphatic resonances, but reduced SNR for aliphatic resonances as compared with the standard DEPT experiment. In addition, the ACCORDEPT is straightforward to implement, does not require any supplementary calibration procedures and can be used under automated conditions without difficulty by inexperienced users. Copyright (C) 2010 John Wiley & Sons, Ltd.