[Development and Validation of a Fully Automated, Experimental Set-Up for Ex-Vivo Burst Pressure Testing after Surgical Vessel Closure]. / Entwicklung und Validierung eines vollautomatischen, experimentellen Set-ups zur Ex-vivo-Berstungsdruckmessung nach chirurgischem Gefäßverschluss.

Background A growing number of operations are performed using minimally invasive techniques. Therefore, a lot of new requirements must be met by the staplers currently available. At the present time, the most widely used methods of minimally invasive vascular occlusion involve high-frequency energy, clips, and staplers. The most important quality parameter is burst pressure, which is measured with a variety of experimental set-ups, all of which are subject to criticism. With this study, we want to introduce a fully automated vascular burst pressure measuring system that largely mimics physiological conditions. An important feature of this set-up is the detection of very early leakage from the staple line (FAIR Leakage = First Appearance of Leakage requiring Intervention). Material and Methods Burst pressure was measured in vessel segments of porcine common carotid arteries. For vascular occlusion, we used the stapler device Micro Cutter XCHANGE® by DexteraSurgical. Prior to closure, the vessel was filled to a pressure of 80 mmHg. The pressure was increased at a defined flow rate. Burst pressure was defined as staple line leakage requiring intervention. Results and Validation 30 staple lines were examined. The average burst pressure visually determined by two independent investigators was 515.8 mmHg ± 236.3 mmHg. Maximal burst pressure was 911 mmHg, and minimal burst pressure 80 mmHg. The average burst pressure detected electronically was 511.8 mmHg ± 239.1 mmHg. Statistically, there was a highly significant correlation of visually and electronically detected burst pressures. Conclusion This is the first experimental set-up for a systematic burst pressure test that is fully automated and therefore eliminates any bias related to the investigator. The experimental set-up with a defined intravascular pressure prior to closure and the use of a liquid with blood-like viscosity enabled us to largely mimic intraoperative conditions. Since burst pressure is not defined as a complete rupture of the staple line, but as the moment of first occurrence of leakage requiring intervention, the results can be transferred into daily surgical practice.

Beyond the "B": a new concept of the surgical staple enabling miniature staplers.

BACKGROUND: Surgical staplers currently all rely on the same staple form-the "B" which necessitates a high delivery profile (12 mm). A novel "D" shape staple allows for an extremely low profile of the applicator. The acute and long-term efficacy of a D-shaped staple (Cardica, Redwood City, CA, USA) was compared to conventional B-form staples (Covidien, Norwalk, CN, USA) in an animal model for intestinal transections and anastomoses. METHODS: Jejunojejunal anastomoses (JJ) were performed via mini-laparotomy in a swine model. White & blue D- and B-shaped staples were studied in three groups (planned survival 14-84 days). Intraoperative assessment included completeness of staple line, hemostasis, and need for intervention. Postoperatively, animals were evaluated for complications. At the time of sacrifice, gross pathological and histological assessments were performed. RESULTS: Twenty-three animals had 40 anastomoses (23 "D" and 17 "B" staple anastomoses) with no intraoperative mortalities. One "D" staple application required a manual extension of the cut. Acute hemostasis was 100%. Group 1 (n = 5) compared white staples in JJs (D staple n = 5; B staple n = 5; 14-day survival = 100%). Group 2 (n = 12) compared white staples in JJs (D staple n = 12; B staple n = 6; 34-day survival = 92 %). One animal died on day 4 for a non-staple related cause. Group 3 (n = 6) compared blue staples in JJs (D staple n = 6; B staple n = 6; 84 day survival = 84%). One animal died on day 18 due to an obstruction at the B staple JJ caused by stricture. There were no other bleeding, leaks or strictures in any of the groups. Gross pathology and histology were unremarkable in all JJs. CONCLUSIONS: This study showed no difference in intraoperative performance and the chronic healing response in JJs between D- and B-shaped staples. Based on these findings, the D-shaped staple elicits a normal healing response in jejunostomies and offers the possibility of clinical use of this advance in staple design.

Immunosuppressive effects of surgery assessed by flow cytometry in nonhuman primates after nephrectomy.

Despite previous studies suggesting that surgery cause immune suppression, the underlying biologic mechanisms have not been studied using advanced immune function assays. Unilateral nephrectomy was performed in nonhuman primates. Blood was collected before surgery and at different time-points through 14 days after surgery. Lymphocyte proliferation (expression of proliferating cell nuclear antigen in cells in S/G(2)M-phase), production of intracellular cytokines [interleukin (IL)-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha] and expression of surface-activation antigens (CD25, CD71) on T-lymphocytes were assessed in whole blood using flow cytometry. Results were compared with nonoperated control animals. The procedure caused a decrease of 25% in absolute lymphocyte count on postoperative day 3. Inhibition of lymphocyte proliferation was maximal on postoperative day 1 (55% normalized to preoperative values) and was detectable until postoperative day 7, when it was 25%. Expression of T-cell activation antigens was decreased during the first postoperative week with a maximum on postoperative day 1 for CD71 (29%) and on postoperative day 3 for CD25 (49%). Intracellular production of cytokines by T cells was decreased only on postoperative day 1 (50% for IL-2, 29% for IFN-gamma and 22% for TNF-alpha). Immune functions returned to presurgery values by day 14. A major surgical procedure severely inhibits lymphocyte proliferation and various T-cell functions up to 1 week postoperatively.

The effect of soluble complement receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts.

BACKGROUND: In pig-to-nonhuman primate solid organ xenotransplantation using organs from donors transgenic for human decay-accelerating factor (hDAF), the main type of rejection is antibody-mediated (acute humoral xenograft rejection, AHXR). This occurs despite the complement-regulatory function of the transgene, neutralization of natural antibodies to Galalpha1-3Gal (Gal) using soluble glycoconjugates, and chronic immunosuppression. As complement components play a major role in graft destruction after antibody binding, we evaluated the efficacy of chronic complement inhibition by soluble complement receptor type 1 (TP10). METHODS: Life-supporting hDAF-transgenic kidney transplantation was performed in cynomolgus monkeys, using cyclophosphamide induction, and maintenance immunosuppression with cyclosporin A, mycophenolate sodium, and tapering steroids. Rejection was treated with bolus steroid injections: if not successful animals were terminated. Three groups were studied: in group 1 (n=4) GAS914 (a soluble glycoconjugate comprising Gal on a poly-L-lysine backbone) was added before and after transplantation; group 2 (n=2) received GAS914 as in group 1 and in addition TP10 before and after transplantation; in group 3 (n=4) GAS914 was only given before transplantation and TP10 as in group 2. Monitoring included the regular assessment of anti-porcine antibodies, complement activity (soluble C5b-9), therapeutic drug monitoring, and graft histology. RESULTS: Survival in group 1 was 6, 12, 31 and 37 days, respectively, and in all four cases graft histology showed AHXR. The two animals in groups 2 survived 3 and 15 days, respectively, and similarly showed AHXR in graft histology. In group 3 two animals showed AHXR (10 and 37 days survival, respectively), and two others did not show AHXR (20 and 32 days survival, respectively). The diagnosis AHXR included the deposition of complement activation products in the graft, which were present at lower intensity in animals treated with TP10. In all animals GAS914 effectively neutralized circulating anti-Gal antibody. Antibodies were detectable in the circulation of all animals using porcine erythrocytes in a hemolytic assay, although at lower levels than before transplantation. Soluble C5b-9 was not detectable in the circulation of animals receiving TP10, and circulating TP10 concentrations in these animals were in a presumed pharmacologically active range. CONCLUSIONS: The inclusion of TP10 in the immunosuppressive protocol does not clearly lead to improved xenograft survival. Despite effective neutralization of anti-Gal antibodies and effective inhibition of systemic complement activity, AHXR was apparent in four of six animals under chronic TP10 treatment, including deposits of complement activation products in the graft. Apparently, effective systemic complement inhibition by TP10 in combination with local complement regulation by the hDAF transgene product does not necessarily result in effective inhibition of complement activation at locations in the xenograft upon binding of anti-porcine antibodies to the grafted endothelium.

Hyperacute rejection of hDAF-transgenic pig organ xenografts in cynomolgus monkeys: influence of pre-existing anti-pig antibodies and prevention by the alpha GAL glycoconjugate GAS914.

BACKGROUND: Our introductory pig-to-cynomolgus monkey heart or kidney transplantation using organs from pigs transgenic for human decay-accelerating factor (hDAF), showed a high incidence of hyperacute rejection (HAR), which was ascribed to extraordinary high levels of anti-pig antibodies. We evaluated the efficacy of GAS914, a Gal alpha 1-3Gal trisaccharide linked to a poly-l-lysine backbone, in inhibition of HAR. METHODS: hDAF transgenic heterotopic heart (n = 15) or life-supporting kidney (n = 8) transplantation included induction with cyclophosphamide or anti-thymocyte globulin, and maintenance with cyclosporine or tacrolimus, steroids and mycophenolate sodium/mofetil. Four doses of GAS914 were given before transplantation. Rejection was confirmed by graft histology, and anti-pig antibody levels were determined in various assays. RESULTS: Four of six heart transplants without GAS914 treatment showed HAR. Nine subsequent transplants with GAS914 pre-treatment, did not show HAR (chi-square, P < 0.05). Two of four kidney transplants without GAS914 treatment ended with HAR. Four subsequent transplants with GAS914 did not show HAR. Animals with HAR showed extremely high antibody levels. Samples just before transplantation showed significantly higher antibody levels in recipients presenting with HAR. In all assays antibody levels were significantly lowered by GAS914 pre-treatment. CONCLUSIONS: HAR of hDAF solid organs could be ascribed to high levels of anti-pig antibodies. It is hypothesized that the hDAF transgene shows a threshold in efficacy, above which an overwhelming attack by antibodies and complement activation cannot be modulated to prevent HAR. HAR does not occur when animals with lower levels are used, or when antibodies are effectively depleted from the circulation by GAS914 treatment.

Treatment with humanized monoclonal antibodies against CD80 and CD86 combined with sirolimus prolongs renal allograft survival in cynomolgus monkeys.

BACKGROUND: Co-stimulatory blockade has been shown to prolong allograft survival in different transplant models. We investigated the effect of combining humanized anti-CD80 and anti-CD86 monoclonal antibodies (mAb) with sirolimus in cynomolgus monkey renal transplant recipients. METHODS: After renal transplantation, groups of four animals were treated daily with sirolimus, sirolimus and nine weekly doses of mAb, two weekly doses of mAb, or sirolimus and two weekly doses of mAb. RESULTS: Survival was significantly better in monkeys treated with the combination of sirolimus and mAb when compared with treatment with either agent alone (P=0.0067 by log-rank analysis). When combined with sirolimus, nine weekly doses of mAb did not result in an additional survival benefit compared with only two mAb doses (P=0.74). None of the treatment regimens used in this study resulted in development of transplantation tolerance. CONCLUSIONS: Sirolimus can be successfully combined with humanized mAb against CD80 and CD86. Induction with a short course of mAb is effective in prolonging allograft survival in combination with sirolimus.

Treatment by mycophenolate mofetil of advanced graft vascular disease in non-human primate recipients of orthotopic aortic allografts.

Failure to control chronic graft dysfunction [e.g. graft vascular disease (GVD)] is the primary cause of immunologic graft failure. This is the first study of mycophenolate mofetil (MMF) for the treatment of GVD in non-human primate recipients of aortic allografts. Abdominal aortic allografts were exchanged between mixed leukocyte reaction (MLR) -mismatched, blood-group-compatible cynomolgus monkeys. Six control recipients were untreated. Individualized treatment with frequent dose adjustments of MMF insured that treatment was close to the maximum tolerated dose (mean 99.2 mg/kg/day). Immune-mediated injury proceeded unhindered until day 45, after which MMF treatment began. Changes in intimal volume (IV) were quantified by intravascular ultrasound (IVUS) and compared to histology on day 105. Serial IVUS measurements of IV (mm(3)) in controls showed progressive GVD. In four out of six animals, MMF was well tolerated, thus enabling optimum treatment; in all these animals, IV was significantly less than in the control animals (p = 0.02). In the two remaining animals, high doses were not tolerated; at day 105, there was no significant difference in IV between them and the controls. We found a significant correlation between the mean MMF tolerated dose and the inhibition of progression of IV (r = -0.88, p = 0.015). When high MMF doses were tolerated, MMF slowed progression of GVD.

Sirolimus (rapamycin) monotherapy prevents graft vascular disease in nonhuman primate recipients of orthotopic aortic allografts.

BACKGROUND: Delayed treatment with sirolimus (SRL) halts progression of graft vascular disease (GVD) in nonhuman primate (NHP) aortic allograft recipients. In this study, we investigated whether SRL monotherapy prevents the development of GVD. METHODS AND RESULTS: Pairs of 3-cm infrarenal aortic segments were exchanged between mixed lymphocyte reaction-mismatched, blood group-compatible NHPs (n=12). Six NHPs were untreated controls, and 6 were treated orally with SRL starting on the day of transplantation. Follow-up was 105 days. SRL doses were adjusted individually by assessing SRL blood concentrations, immune function, and clinical status. The severity of GVD was determined every 3 weeks by intravascular ultrasound, which quantified intimal area (IA) and intimal volume (IV) for the middle 1-cm graft segments. The mean+/-SEM SRL plasma levels were 14.5+/-9 ng/mL. In grafts from treated NHPs, IA and IV values on days 63, 84, and 105 were significantly lower than for controls (P<0.05 to P<0.001). On day 105, in the grafts from SRL-treated NHPs compared with grafts from controls, values (mean+/-SEM) were IA, 2.9+/-0.9 versus 5.5+/-0.7 mm2, P<0.001 and IV, 29.6+/-4.6 versus 55.2+/-2.8 mm3, P<0.001; IA and IV values for grafts from SRL-treated NHPs did not increase significantly between days 21 and 105. CONCLUSIONS: We show that SRL monotherapy prevented GVD in NHP aortic allograft recipients, suggesting the value of SRL for controlling GVD in clinical transplantation.

A life-supporting technique of renal allotransplantation in Macaca fascicularis to evaluate novel immunosuppressive drugs in nonhuman primates.

BACKGROUND: The use of nonhuman primates in preclinical transplantation studies is becoming more common. This report details complete procedures developed for a successful life-supporting kidney allotransplantation program using the cynomolgus monkey (Macaca fascicularis). MATERIALS AND METHODS: All transplants were performed in wild-caught, ABO-matched, MLR-mismatched adult males. Transplant procedures were staggered: an animal first served as a donor, was allowed to recover for 4 weeks, and was subsequently used as a recipient. The kidney was flushed in situ while the aorta was briefly cross-clamped. The graft was implanted heterotopically end-to-side in the right iliac fossa using microsurgical techniques. An ureteroneocystostomy was constructed; a telemetry probe was inserted into the aorta, and the remaining native kidney was removed. RESULTS: Sixty-two transplants were performed in 6.9 +/- 0.1 kg animals. Operating times in the donor and recipient were 126 +/- 3 and 166 +/- 5 min, respectively. The cold ischemia time was 55 +/- 1 min. There were no intraoperative deaths. Postoperative complications were observed in six (9.7%) monkeys and consisted of one early renal arterial thrombosis and five ureteral complications (two of which were successfully repaired). Transplants were ultimately successful in 93.6% (58/62) of cases. Immediate kidney function was satisfactory, with a mean serum creatinine of 1.7 +/- 0.2 mg/dL and a mean urine output of 140 +/- 15 ml on postoperative day 7. CONCLUSION: Life-supporting kidney transplantation in cynomolgus macaques is a demanding operation that requires great attention to details. Precise surgical techniques, telemetric monitoring, ultrasound surveillance, and aggressive, early, postoperative fluid resuscitation produced a 94% success rate.