Background: Cardiovascular disease remains the leading cause of death in women. Gender bias and blindness are coming into focus as relevant risk factors for patients. To date, there have been no studies that target surgeons’ potential gender bias in recognizing postoperative myocardial infarction (MI). Methods: An online clinical vignette describing a patient with postoperative MI was randomized for gender. Possible diagnoses, the next steps in management, and questions on gender bias were assessed. Results: A total of 205 surveys were analyzed. MI was recognized in 55.6% of the male case studies vs. 32.0% of the female case studies in the first question (p < 0.005). Cardiac diagnostics were initiated significantly more in male case studies (94% vs. 76%, p = 0.001). Female surgeons listed MI as the first diagnosis twice as often as male surgeons overall (43% vs. 23%, p = 0.027). Female surgeons were also more likely to mention MI across the survey at all compared to male surgeons (89% vs. 67%, p = 0.0002). Board-certified surgeons diagnosed MI by the end of the survey significantly more (88.2%) than residents (75.0%) and medical students (75.0%, p = 0.047). Conclusions: Overall, this study was able to demonstrate the presence of both gender bias and gender blindness in surgeons’ assessment of postoperative myocardial infarction symptoms with a clear disadvantage for female patients and a superior awareness for female surgeons.

Aim:: The perioperative cardiac xenograft dysfunction (PCXD), caused by ischemic crystalloid cardioplegia (Bretschneider) limited survival in a life-supporting orthotopic cardiac pig-to-baboon xenotransplantation model (oXHTx) and was here prevented by a new non-ischemic cold preservation technique. OXTx data of PCXD were compared with our previous heterotopic thoracic (htXTx) model. Another problem of the xenograft “over”growth (XOG) had to be solved with an antiproliferative therapy.

Introduction: According to the guidelines and recommendations of the ISHLT advisory board a 90-days-survival of at least 60% in a life-supporting orthotopic pig-to-baboon model (oXHTx) is necessary to begin first clinical cardiac XT studies. We started to reproduced a brilliant survival of transgenic pig hearts up to 3 years in a non-life-supporting heterotopic abdominal model ((1) Mohiuddin et al., Nat. Commun. 2016;7,11138) in our life-supporting orthotopic model with nearly the same immunosuppression (IS) dominated by a CD40mAb costimulation blockade. Another problem specific for oXHTx like the early perioperative cardiac xenograft dysfunction (PCXD, caused by ischemic crystalloid cardioplegia) and cardiac xenograft overgrowth (caused by the quick physiological pig heart growth and hypertrophy) were prevented by using a new non-ischemic cold perfusion and preservation technique and a p.o. growth inhibition by an antihypertensive and anti-proliferative drug therapy. Methods: In this group 8 GalKO/hCD46/hTM transgenic (tg) pig hearts were transplanted orthotopically in baboons. Basic IS consisting of ATG, rituximab, mycophenolate, cortisone and 50 mg/kg CD40mAb (mouse/rhesus-Ab). To prevent PCXD as an early cardiac low output and “stunning myocardium”, instead of crystalloid Bretschneider cardioplegia we used a hyperoncotic, non-ischemic 8oC cold perfusion solution with oxygenated erythrocytes and albumin. To inhibit pig xenograft overgrowth and hypertrophy, antihypertensive drugs (enalapril, metoprolol) and an intravenous mTOR inhibitor (temsirolimus) as anti-proliferative drugs were administered. Results: In contrast to a previous group with Bretschneider cardioplegia (Längin et al. Nature 2018;564:430-433 (2)) now with non-ischemic cold perfusion technique no PCXD occurred, stroke-work-index was high and catecholamines low. Two baboons with pCMV-positive donor hearts died of infections on day 14 (pancreatitis) and on day 26 (kidney failure). Using the therapies against xenograft (over)growth and hypertrophic diastolic heart failure, now all other 6 recipient baboons achieved long-term survival and 4 had to be actively terminated after 90 days with a well beating xenograft (according to the allowance of our government) and 2 experiments could be extended up to half a year (182 and 195 days). Immunologically no hyperacute or delayed rejection was observed. All baboons were in excellent physical conditions with normal parameters in hematology and clinical chemistry the whole period. Conclusion: After 25 years of intensive experimental research in oXHT of multi-tg pig hearts since 2017 a major progress and with this group the essential breakthrough occured demonstrating a consistent reproducible long-term survival up to 3 (n=4) and 6 months (n=2). This meets the prerequisite according to the ISHLT guidelines before starting a clinical phase I study with 10 patients (as bridge-to-allotransplantation or destination therapy) in the next 2 to 3 years. References: 1. (1) Mohiuddin, MM, Singh AK, Corcoran PC, Thomas Iii ML, Clark T, Lewis BG, Hoyt RF, Eckhaus M, Pierson Iii RN, Belli AJ, Wolf E, Klymiuk N, Phelps C, Reimann KA, Ayares D, Horvath KA. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft. Nat. Commun. 2016;7,11138. 2. Längin M*, Mayr T*, Reichart B, Michel S, Buchholz S, Guethoff S, Dashkevic A, Baehr A, Egerer S, Bauer A, Mihalj M, Panelli A, Issl L, Ying J, Fresch AK, Buttgereit I, Mokelke M, Radan J, Werner F, Lutzmann I, Steen S, Sjöberg T, Paskevicius A, Qiuming L, Sfriso R, Rieben R, Dahlhoff M, Kessler B, Kemter E, Klett K, Hinkel R, Kupatt C, Falkenau A, Reu S, Ellgass R, Herzog R, Binder U, Wich G, Skerra A, Ayares D, Kind A, Schönmann U, Kaup FJ, Hagl C, Wolf E, Klymiuk N, Brenner P* & Abicht JM* (* = equal). Consistent success in life-supporting porcine cardiac xenotransplantation. Nature. 2018;564:430-433.

In this Letter, Mayuko Kurome and Valeri Zakhartchenko have been added to the author list (affiliated with Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany). The author list and 'Author contributions' section have been corrected online; see accompanying Amendment.

Objectives Life-supporting orthotopic cardiac xenotransplantations (OHXTx) in a pig-to-baboon model with GalT-KO/hCD46/hTM transgenic donor pigs were performed with a compatible, non-toxic immunosuppression (IS) based on CD40mAb co-stimulation blockade in group G1 and in a group G2 with a new pasylated, non-thrombogenic CD40L-Ab. Chemical procedure of PASylation prolongs plasma half-life by 170-fold to several days. Primary aims were to realize a constant preclinical long-term survival in a life-supporting cardiac xenotransplantation model and to prevent perioperative cardiac xenograft dysfunction (PCXD) as a kind of cardiac low output. Methods OHXTx according to the technique of Lower and Shumway were performed in 11 baboons with transgenic pig hearts. The new IS consisted of a recombinant mouse-rhesus chimeric CD40 (clone 2C10R4) antibody in G1 (n=7) or a PASylated Fab-CD40L antibody (XL-protein/Wacker-Chemie) in G2 (n=4) combined with ATG, rituximab, MMF (no tacrolimus or cyclosporine!) and steroids. Results Survival in G1 with CD40mAb were 3, 1, 30, 1, 1** and 27 day(s) with 2 cases of PCXD. Using CD40L-Ab in G2 PCXD was observed in 1 case and the recipients survived 1, 18 and 40 days. Ischemic time of the hearts ranged from 112-128 min. Primary cause of death mostly were renal and hepatic failure, one died of a neurological deficit**, another after hematothorax, but no hyperacute or delayed xenograft rejection occurred. After several weeks a pig donor organ (over)growth was found. All long-term surviving baboons were in good general conditions until to the last days. Mean survival in G1 (CD40Ab) was 10.5+/-4.90 days and in G2 (Fab-CD40L) 19.7+/-7.84 days and without PCXD and brain damage** 20+/-6.55 days in G1 and 29+/-7.78 days in G2. In a preliminary modified subgroup (unpublished data und matter of a patent) one baboon with CD40L-Ab survived 50 days and another baboon (B67 with CD40Ab) reached and was successfully terminated at the endpoint of study on day 90 with no signs of rejection of the xenograft. Conclusion Co-stimulation blockade with CD40L antibody tended to prolong survival time after orthotopic cardiac xenotransplantation, but with so far incomplete groups statistically not significant. In total survival in this important life-supporting orthotopic model was significantly prolonged with a less toxic IS and better quality of life. This is an important milestone and progress on the way to a long-term survival of 2 -3 months, which is necessary for first clinical cardiac xenotransplantations. This aim was now first time worldwide reached with our last 90-days surviving baboon B67 in the last subgroup and must be replicated another 5 times before starting a clinical study.

The porcine cytomegalovirus (PCMV) is a herpesvirus that may pose a risk for xenotransplantation using pig cells, tissues, or organs. Here, three orthotopic pig heart transplantations into baboons were studied. To detect PCMV, a real-time PCR and a Western blot assay based on four PCMV protein sequences, including two tegument proteins, were used. The transmission of PCMV from the donor pig to the recipient baboon was found in two cases, despite PCMV not being detected in the blood of the donor pigs by real-time PCR. Although it was not in the blood, PCMV was detected in different organs of the donor pigs, and in sibling animals. Immunohistochemistry using an antiserum that is specific for PCMV detected virus protein-expressing cells in all of the organs of the recipient baboon, most likely representing disseminated pig cells. Therefore, for the first time, the distribution of PCMV in organs of the donor pigs and the recipient baboons was described. In addition, baboon cytomegalovirus (BaCMV) was found activated in the recipient, and a screening for hepatitis E virus (HEV) and porcine lymphotropic herpesviruses (PLHV) was performed. For the first time, a cross-reactivity between antibodies directed against PCMV and BaCMV was found.

Objectives: In our orthotopic cardiac xenotransplantation (OHXTx) pig-to-baboon model with GalT-KO/hCD46/hTM transgenic donor pigs we used a non-toxic immunosuppression (IS) based on CD40mAb co-stimulation blockade in group G1 and in a group G2 a new PAsylated, nonthrombogenic CD40L-Ab. The procedure of PASylation prolongs plasma half-life to several days. Primary aims were to achieve a constant preclinical long-term survival in a life-supporting cardiac xenotransplantation model and to prevent perioperative cardiac xenograft dysfunction (PCXD).

Genetically engineered pigs are a promising source for islet cell transplantation in type 1 diabetes, but the strong human anti-pig immune response prevents its successful clinical application. Here we studied the efficacy of neonatal porcine islet-like cell clusters (NPICCs) overexpressing LEA29Y, a high-affinity variant of the T cell co-stimulation inhibitor CTLA-4Ig, to engraft and restore normoglycemia after transplantation into streptozotocin-diabetic NOD-SCID IL2rγ −/− (NSG) mice stably reconstituted with a human immune system. Transplantation of INS LEA29Y expressing NPICCs resulted in development of normal glucose tolerance (70.4%) and long-term maintenance of normoglycemia without administration of immunosuppressive drugs. All animals transplanted with wild-type NPICCs remained diabetic. Immunohistological examinations revealed a strong peri- and intragraft infiltration of wild-type NPICCs with human CD45 + immune cells consisting of predominantly CD4 + and CD8 + lymphocytes and some CD68 + macrophages and FoxP3 + regulatory T cells. Significantly less infiltrating lymphocytes and only few macrophages were observed in animals transplanted with INS LEA29Y transgenic NPICCs. This is the first study providing evidence that beta cell-specific LEA29Y expression is effective for NPICC engraftment in the presence of a humanized immune system and it has a long-lasting protective effect on inhibition of human anti-pig xenoimmunity. Our findings may have important implications for the development of a low-toxic protocol for porcine islet transplantation in patients with type 1 diabetes.

BackgroundThe perioperative phase of preclinical cardiac xenotransplantations significantly affects the experimental outcome. Moderate or even severe hemodynamic and respiratory impairment occurs frequently in baboons after receiving a cardiac transplant. The perioperative management of such postoperative instability is very demanding, especially in the experimental setting. We compared perioperative changes of hemodynamic and laboratory findings during orthotopic and heterotopic thoracic cardiac xenotransplantations and describe our monitoring, treatment and intensive care.MethodsTwenty-eight pig-to-baboon cardiac xenotransplantations were performed using either the orthotopic (oHTx, n=5) or heterotopic thoracic (htHTx; n=23) technique. In both techniques, cardioplegia and an intraoperative cardiopulmonary bypass (CPB) were required. Preoperatively, intensive care (eg, transfusions, catecholamine therapy) was provided and fast extubation was targeted. A central venous catheter, afemoral arterial thermodilution catheter, a telemetric pressure transmitter and transthoracic echocardiography were used to monitor the animal. Baboon jackets with a tethering system were used to continuously apply medication postoperatively and permit blood sampling, also after extubation of the animal and transfer into the cage. Perioperative survival, hemodynamics, catecholamine doses, respiratory function and weaning from respirator were compared. Perioperative organ damage was evaluated based on laboratory findings 12hours after transplantation.ResultsRecipients could be weaned from CPB in the 20 htHTx and all five oHTx experiments, and three htHTx procedures were terminated during the operation. The time of cardiopulmonary bypass was significantly lower in the heterotopic group (oHTx median 171 [157-193] minutes; htHTx median 144 [100-190] minutes; P=.02). In 17 htHTx procedures, no inotropics were used, whereas epinephrine had to be administered in four of the five oHTx experiments; the mean time of catecholamine support was longer in the oHTx group (oHTx 972348minutes vs htHTx 111 +/- 92minutes; P<.01). After htHTx, weaning off the respirator was possible in 19 of 20 cases (one died due to pneumothorax). After oHTx, three of the five baboons could be weaned off the respirator; in these cases, the arterial saturation was higher compared with the extubated baboons after htHTx (oHTx 99 +/- 1% vs htHTx 91 +/- 4%, P=.01). Intraoperative blood loss was similar between the two groups, and hemostasis was impaired after all procedures, but relevant postoperative bleeding never occurred.ConclusionIntensive intra- and postoperative monitoring and care is required in both transplantation techniques as a requirement for successful weaning from CPB and respirator. After htHTx, the animals needed less catecholamines and were hemodynamically more stable. Even though pulmonary function was often impaired after htHTx, weaning from the respirator and extubation was more successful in this group.

Aim: To achieve a constant preclinical long-term survival in a life-supporting cardiac xenotransplantation model (pig-to-baboon), a nontoxic immunosuppression based on CD40/CD40L co-stimulation blockade was used and an improvement of primary graft function with non-ischemic myocardial preservation in a group G2.

Background: The complement system plays a crucial role in acute xenogeneic reactions after cardiac transplantation. We used an ex vivo perfusion model to investigate the -effect of Cp40, a compstatin analog and potent inhibitor of complement at the level of C3.Methods: Fifteen wild-type pig hearts were explanted, cardiopleged, and reperfused ex vivo after 150 minutes of cold ischemia. Hearts were challenged in a biventricular working heart mode to evaluate cardiac perfusion and function. In the treatment group (n= 5), the complement cascade was blocked at the level of C3 using Cp40, using diluted human blood. Untreated human and porcine blood was used for controls.Results: Throughout the perfusion, C3 activation was inhibited when Cp40 was used (mean of all time points: 1.11 +/- 0.34% vs 3.12 +/- 0.48% control activation; P<. 01). Compared to xenoperfused controls, the cardiac index improved significantly in the treated group (6.5 +/- 4.2 vs 3.5 +/- 4.8 mL/min/g; P=. 03, 180 minutes perfusion), while the concentration of lactate dehydrogenase as a maker for cell degradation was reduced in the perfusate (583 +/- 187 U/mL vs 2108 +/- 1145 U/mL, P=. 02). Histological examination revealed less hemorrhage and edema, and immunohistochemistry confirmed less complement fragment deposition than in untreated xenoperfused controls.Conclusions: Cp40 efficiently prevents C3 activation of the complement system, resulting in reduced cell damage and preserved function in wild--type porcine hearts xenoperfused ex vivo. We suggest that this compstatin analog, which blocks all main pathways of complement activation, could be a beneficial perioperative treatment in preclinical and in future clinical xenotransplantation.

Introduction: We used a non-toxic immunosuppression based on CD40/CD40L co-stimulation blockade in group G1 and additionally to improve primary xenograft function in a group G2 a new non-ischemic myocardial preservation technique to achieve a constant preclinical long-term survival in a life-supporting cardiac xenotransplantation model (pig-to-baboon). Methods: According to the technique of Lower and Shumway 9 orthotopic (OHXTx) heart transplantations were performed in baboons with genetically-modified GalKO/hCD46/hTM transgenic pig hearts. The immunosuppression (IS) consisted of ATG, rituximab, MMF, tapered down cortisone and CD40 antibody or PASylated Fab-CD40L. Because a “perioperative cardiac xenograft dysfunction” (PCXD) played a critical role, cardioplegia with crystalloid solution (Bretschneider solution, 50 ml/kg; G1: n = 5) was replaced in the last 4 cases of G2 by Steen´s “non-ischemic preservation technique”: This 8°C cold myocardial perfusion solution consists of a modified Krebs-Henseleit solution with albumin and 10% erythrocytes, hormones and vasoactive agents (Steen et al, 2016*). Under pressure, flow and temperature control heart is constantly perfused during explantation and storage time, intermittently during implantation, using an independent portable heart-lung-machine. Results: Ischemic time ranged from 112–128 min. Survival in G1 with Bretschneider solution were 3, 1, 30, 1 and 1 day(s). Using non-ischemic preservation technique in G2 PCXD was not observed and the recipients survived 18, 1**, 27 and 40 days. Baboons mostly died of respiratory problems, renal and hepatic failure, one was sacrificed due to a major p.o. neurological deficit**, another died after hematothorax, but no hyperacute or delayed xenograft rejection was found. A problem after 3 weeks was a donor organ (over)growth. All long-term surviving baboons (especially in G2) were in good general conditions. Conclusion: With conventional hypothermic, ischemic heart preservation techniques, perioperative cardiac xenograft dysfunction plays a detrimental role after orthotopic xHTx. This was prevented with the Steen’s “non-ischemic preservation technique, an important step on the way to a long-term survival of 2–3 months as an important prerequisite for cardiac xenotransplantation in a clinical use. References: 1. * Steen S, Paskevicius A, Liao Q, Sjöberg T. Safe orthotopic transplantation of 24-hour preserved heart harvested 24 hours after brain death. Scand Cardiovasc J 2016;50:193-200

OBJECTIVESThe incidence of cardiovascular morbidity and mortality in premenopausal women is comparatively low, but increases sharply after menopause. The principal aim of this study was to determine whether women with ascending aortic disease (AAD) have a different reproductive history from that of an age-matched control group.METHODSIn this retrospective study, women who had undergone ascending aortic aneurysm (AscAA) repair between 2000 and 2010 were asked to complete a questionnaire concerning risk factors and reproductive history. Data from 142 women with AAD were evaluated, and a subgroup (n = 64) with AscAA ≥5 cm was analyzed and compared to an age-matched random control group without known aortic diseases.RESULTSAlmost all women were menopausal at the time of the questionnaire (98.4% vs. 90.6%, AscAA ≥5 cm subcohort vs. control, p = 0.12) and all subjects presented with a comparable age of menarche (13.7 ± 2.6 years vs. 14.2 ± 1.8 years, AscAA ≥5 cm subcohort vs. control, log-rank 0.04, p = 0.84). However, mean menopausal age was significantly lower in the case subcohort than in controls (48.1 ± 4.8 years vs. 50.6 ± 5.8 years, AscAA ≥5 cm subcohort vs. control, log-rank 8.35, p = 0.004), and reproductive life span was correspondingly shorter (34.2 ± 5.2 years vs. 36.2 ± 5.7 years, p = 0.04). Furthermore, hypertension was more prevalent in women with AscAA ≥5 cm compared to controls (89.1% vs. 61.9%, AscAA ≥5 cm subcohort vs. control, p < 0.001).CONCLUSIONWomen who experience menopause at an earlier age than the regional mean could profit from screening for cardiovascular disease in general and particularly for AAD. Screening would enable early aneurysm detection and could, therefore, reduce morbidity and mortality.

BACKGROUND:After transplantation of pig hearts into baboons, a particularly high increase of liver parameters was observed in 1 animal. To evaluate whether porcine hepatitis E virus (HEV) was involved in the pathological changes, the donor pig and the recipient baboon were screened for the presence of HEV.MATERIAL AND METHODS:Screening for HEV was performed using highly sensitive and specific PCR methods as well as immunological screening for HEV-specific antibodies.RESULTS:HEV was not detected in the donor pig or the baboon recipient. At necropsy, histopathological examination of liver sections showed acute coagulative necrosis of hepatocytes and hemorrhage, but minimal inflammatory cell activity.CONCLUSIONS:The liver failure observed in the recipient animal was not due to transmission of porcine HEV. Liver failure could have been caused by the onset of cardiac failure related to delayed transplant rejection.