Update on immunosuppressive strategies in intestinal transplantation.

PURPOSE OF REVIEW: The intestine is the most immunologically complex solid organ allograft with the greatest risk of both rejection and graft-versus-host disease (GVHD). High levels of immunosuppression are required, further increasing morbidity. Due to low volume of transplants and few centers with experience, there is paucity of evidence-based, standardized, and effective therapeutic regimens. We herein review the most recent data about immunosuppression, focusing on novel and emerging therapies. RECENT FINDINGS: Recent data are moving the field toward increasing use of basilixumab and consideration of alemtuzumab for induction and inclusion of mammalian target of rapamycin inhibitors and antimetabolites for maintenance. For rejection, we highlight novel roles for tumor necrosis factor-α inhibition, α4ß7 integrin inhibition, microbiome modulation, desensitization protocols, and tolerance induction strategies. We also highlight emerging novel therapies for GVHD, especially the promising role of Janus kinase inhibition. SUMMARY: New insights into immune pathways associated with rejection and GVHD in intestinal allografts have led to an evolution of therapies from broad-based immunosuppression to more targeted strategies that hold promise for reducing morbidity from infection, rejection, and GVHD. These should be the focus of further study to facilitate their widespread use.

Endothelial cell-secreted MIF reduces pericyte contractility and enhances neutrophil extravasation.

Dysregulated neutrophil extravasation contributes to the pathogenesis of many inflammatory disorders. Pericytes (PCs) have been implicated in the regulation of neutrophil transmigration, and previous work demonstrates that endothelial cell (EC)-derived signals reduce PC barrier function; however, the signaling mechanisms are unknown. Here, we demonstrate a novel role for EC-derived macrophage migration inhibitory factor (MIF) in inhibiting PC contractility and facilitating neutrophil transmigration. With the use of micro-ELISAs, RNA sequencing, quantitative PCR, and flow cytometry, we found that ECs secrete MIF, and PCs upregulate CD74 in response to TNF-α. We demonstrate that EC-derived MIF decreases PC contractility on 2-dimensional silicone substrates via reduction of phosphorylated myosin light chain. With the use of an in vitro microvascular model of the human EC-PC barrier, we demonstrate that MIF decreases the PC barrier to human neutrophil transmigration by increasing intercellular PC gap formation. For the first time, an EC-specific MIF knockout mouse was used to investigate the effects of selective deletion of EC MIF. In a model of acute lung injury, selective deletion of EC MIF decreases neutrophil infiltration to the bronchoalveolar lavage and tissue and simultaneously decreases PC relaxation by increasing myosin light-chain phosphorylation. We conclude that paracrine signals from EC via MIF decrease PC contraction and enhance PC-regulated neutrophil transmigration.-Pellowe, A. S., Sauler, M., Hou, Y., Merola, J., Liu, R., Calderon, B., Lauridsen, H. M., Harris, M. R., Leng, L., Zhang, Y., Tilstam, P. V., Pober, J. S., Bucala, R., Lee, P. J., Gonzalez, A. L. Endothelial cell-secreted MIF reduces pericyte contractility and enhances neutrophil extravasation.

Prophylactic Ureteral Stenting in Laparoscopic Colectomy: Revisiting Traditional Practice.

BACKGROUND: Prophylactic placement of ureteral stents is performed during open colectomy to aid in ureteral identification and to enhance detection of injury. The effects of this practice in laparoscopic colectomy are unknown. This study compares outcomes of patients undergoing laparoscopic colectomy with and without prophylactic ureteral stenting. METHODS: A retrospective cohort study at a tertiary academic medical center was performed. The primary outcome measure was the incidence of ureteral injury. Secondary outcomes evaluated included mortality, length of stay, procedural duration, and new-onset urinary complication (hematuria, dysuria, and urinary tract infection). RESULTS: In 702 consecutive patients undergoing elective laparoscopic colectomy from 2013 to 2016, prophylactic stents were placed in 261 (37%) patients. Two ureteral injuries occurred (0.3%), both in patients who underwent ureteral stent placement (P = 0.07) and were found and repaired intraoperatively. There was no in-hospital mortality. When accounting for age-adjusted Charlson comorbidity score, procedural indication, gender, BMI, and extent of resection, no difference in hospital length of stay (P = 0.79) was noted comparing patients with and without stenting. However, stent placement prolonged operating time (P = 0.03) and increased the risk of new-onset urinary complications (P = 0.04). CONCLUSIONS: In this study, ureteral injuries only occurred in those with stent placement. Prophylactic ureteral stents in laparoscopic colectomy are associated with increased operative time and urologic morbidity. Owing to the low prevalence of ureteral injury in the elective setting and the increased risk of urinary complications, use of prophylactic ureteral stenting should be highly selective.

Successful treatment of primary donor-derived human herpesvirus-8 infection and hepatic Kaposi Sarcoma in an adult liver transplant recipient.

Kaposi sarcoma (KS) may rarely occur in transplant recipients through primary human herpesvirus-8 (HHV-8) infection from a seropositive donor. This report describes a patient who developed hepatic KS after receiving a split liver transplant from an HHV-8-positive donor. The recipient was treated with liposomal doxorubicin after reduction in immunosuppression led to acute cellular rejection. This treatment achieved regression of KS while preserving allograft function, demonstrating a successful therapeutic strategy for this malignancy.

Recent advances in allograft vasculopathy.

PURPOSE OF REVIEW: Despite considerable advances in controlling acute rejection, the longevity of cardiac and renal allografts remains significantly limited by chronic rejection in the form of allograft vasculopathy. This review discusses recently reported mechanistic insights of allograft vasculopathy pathogenesis as well as recent clinical evaluations of new therapeutic approaches. RECENT FINDINGS: Although adaptive immunity is the major driver of allograft vasculopathy, natural killer cells mediate vasculopathic changes in a transplanted mouse heart following treatment with donor-specific antibody (DSA). However, natural killer cells may also dampen chronic inflammatory responses by killing donor-derived tissue-resident CD4 T cells that provide help to host B cells, the source of DSA. DSA may directly contribute to vascular inflammation by inducing intracellular signaling cascades that upregulate leukocyte adhesion molecules, facilitating recruitment of neutrophils and monocytes. DSA-mediated complement activation additionally enhances endothelial alloimmunogenicity through activation of noncanonical NF-κB signaling. New clinical studies evaluating mammalian target of rapamycin and proteasome inhibitors to target these pathways have been reported. SUMMARY: Allograft vasculopathy is a disorder resulting from several innate and adaptive alloimmune responses. Mechanistic insights from preclinical studies have identified agents that are currently being investigated in clinical trials.

Attitudes toward organ donation among waitlisted transplant patients: results of a cross-sectional survey.

Organ shortage remains a major barrier to transplantation. While many efforts have focused on educating the general population regarding donation, few studies have examined knowledge regarding donation and donor registration rates among waitlisted candidates. We aimed to determine waitlisted patients' willingness to donate, elucidate attitudes surrounding organ allocation, and identify barriers to donation. A cross-sectional survey was distributed to assess demographics, knowledge regarding organ donation, and attitudes regarding the allocation process. Responses from 225 of 579 (39%) waitlisted patients were collected. Seventy-one respondents (32%) were registered donors, while 64 patients (28%) noted no interest in participating in donation. A total of 19% of respondents felt their medical treatment would change by being a donor, while 86 patients (38%) felt their condition precluded them from donation. Forty patients (18%) felt they should be prioritized on the waitlist if they agreed to donate. A minority of patients (28%) reported discussion of organ donation with their physician. Waitlisted candidates constitute a population of willing, although often unregistered, organ donors. Moreover, many endorse misconceptions regarding the allocation process and their donation eligibility. In a population for which transplantation is not always possible, education is needed regarding organ donation among waitlisted patients, as this may enhance donation rates.

Non-alcoholic fatty liver disease following liver transplantation: a clinical review.

Non-alcoholic steatohepatitis (NASH) is rapidly becoming the leading indication for liver transplantation (LT) in the United States. While post-transplantation outcomes are similar to other indications for transplant, recent evidence has suggested that reduction in risk factors for post-transplant metabolic syndrome may impose a significant survival benefit in this patient population. Cardiovascular mortality is the leading cause of death following transplantation for NASH. While pre-transplant pharmacologic and surgical approaches have been utilized to reduce cardiovascular risk factors following transplantation, the effectiveness of these treatment approaches in the post-transplant setting is poorly defined. Studies are urgently needed in the treatment of this rapidly growing population.

Novel Noninvasive Biomarkers in Liver Transplantation: A Tool on the Doorstep of Clinical Utilization.

Biomarkers have the potential to transform the detection, treatment, and outcomes of liver transplant complications, though their application is limited because of the lack of prospective validation. Although many genetic, proteomic, and immune markers correlating with allograft rejection and graft dysfunction have been described, evaluation of these markers in combination and validation among a broad liver transplant recipient population remain understudied. In this review, we present evidence supporting biomarker applications in 5 clinical liver transplant scenarios: (i) diagnosis of allograft rejection, (ii) prediction of allograft rejection, (iii) minimization of immunosuppression, (iv) detection of fibrosis and recurrent disease, and (v) prediction of renal recovery following liver transplantation. Current limitations for biomarker utilization and opportunities for further investigation are discussed. Accurate risk assessment, diagnosis, and evaluation of treatment responses using such noninvasive tools will pave the way for a more personalized and precise approach to management of the liver transplant patients that has profound potential to reduce morbidity and improve graft and patient longevity.

3D bioprinting of an implantable xeno-free vascularized human skin graft.

Bioengineered tissues or organs produced using matrix proteins or components derived from xenogeneic sources pose risks of allergic responses, immune rejection, or even autoimmunity. Here, we report successful xeno-free isolation, expansion, and cryopreservation of human endothelial cells (EC), fibroblasts (FBs), pericytes (PCs), and keratinocytes (KCs). We further demonstrate the bioprinting of a human skin substitute with a dermal layer containing xeno-free cultured human EC, FBs, and PCs in a xeno-free bioink containing human collagen type I and fibronectin layered in a biocompatible polyglycolic acid mesh and subsequently seeded with xeno-free human KCs to form an epidermal layer. Following implantation of such bilayered skin grafts on the dorsum of immunodeficient mice, KCs form a mature stratified epidermis with rete ridge-like structures. The ECs and PCs form human EC-lined perfused microvessels within 2 weeks after implantation, preventing graft necrosis, and eliciting further perfusion of the graft by angiogenic host microvessels. As proof-of-concept, we generated 12 individual grafts using a single donor of all four cell types. In summary, we describe the fabrication of a bioprinted vascularized bilayered skin substitute under completely xeno-free culture conditions demonstrating feasibility of a xeno-free approach to complex tissue engineering.

The Impact of COVID-19 on Organ Donation, Procurement, and Liver Transplantation in the United States.

The coronavirus disease 2019 pandemic has had an impact on all facets of our health care system, including life-saving procedures like organ transplantation. Concerns for potential exposure to the causative severe acute respiratory syndrome coronavirus type 2 have profoundly altered the process of organ donation and recovery that is vital to the execution of organ transplantation. Issues regarding adequate donor evaluation and consent, organ recovery, organ procurement organization, and donor hospital resources as well as the transplant center's acceptance of organ offers for their candidates have all required new practice paradigms. Consequently, the ability to treat patients with organ failure, in particular patients with end-stage liver disease in whom no temporizing treatments exist, and to obtain expected excellent outcomes for new liver transplant recipients has been challenged during this time. Conclusion: We summarize some of the negative effects of the current pandemic on organ recovery and liver transplantation as well as offer considerations and strategies for their mitigation that could have a lasting impact on the field even after the coronavirus disease 2019 has waned.

Inactive status is an independent predictor of liver transplant waitlist mortality and is associated with a transplant centers median meld at transplant.

BACKGROUND: Approximately 30% of patients on the liver transplant waitlist experience at least one inactive status change which makes them temporarily ineligible to receive a deceased donor transplant. We hypothesized that inactive status would be associated with higher mortality which may differ on a transplant centers' or donor service areas' (DSA) Median MELD at Transplant (MMaT). METHODS: Multi-state models were constructed (OPTN database;06/18/2013-06/08/2018) using DSA-level and transplant center-level data where MMaT were numerically ranked and categorized into tertiles. Hazards ratios were calculated between DSA and transplant center tertiles, stratified by MELD score, to determine differences in inactive to active transition probabilities. RESULTS: 7,625 (30.2% of sample registrants;25,216 total) experienced at least one inactive status change in the DSA-level cohort and 7,623 experienced at least one inactive status change in the transplant-center level cohort (30.2% of sample registrants;25,211 total). Inactive patients with MELD≤34 had a higher probability of becoming re-activated if they were waitlisted in a low or medium MMaT transplant center or DSA. Transplant rates were higher and lower re-activation probability was associated with higher mortality for the MELD 26-34 group in the high MMaT tertile. There were no significant differences in re-activation, transplant probability, or waitlist mortality for inactivated patients with MELD≥35 regardless of a DSA's or center's MMaT. CONCLUSION: This study shows that an inactive status change is independently associated with waitlist mortality. This association differs by a centers' and a DSAs' MMaT. Prioritization through care coordination to resolve issues of inactivity is fundamental to improving access.

Three Dimensional Bioprinting of a Vascularized and Perfusable Skin Graft Using Human Keratinocytes, Fibroblasts, Pericytes, and Endothelial Cells.

Multilayered skin substitutes comprising allogeneic cells have been tested for the treatment of nonhealing cutaneous ulcers. However, such nonnative skin grafts fail to permanently engraft because they lack dermal vascular networks important for integration with the host tissue. In this study, we describe the fabrication of an implantable multilayered vascularized bioengineered skin graft using 3D bioprinting. The graft is formed using one bioink containing human foreskin dermal fibroblasts (FBs), human endothelial cells (ECs) derived from cord blood human endothelial colony-forming cells (HECFCs), and human placental pericytes (PCs) suspended in rat tail type I collagen to form a dermis followed by printing with a second bioink containing human foreskin keratinocytes (KCs) to form an epidermis. In vitro, KCs replicate and mature to form a multilayered barrier, while the ECs and PCs self-assemble into interconnected microvascular networks. The PCs in the dermal bioink associate with EC-lined vascular structures and appear to improve KC maturation. When these 3D printed grafts are implanted on the dorsum of immunodeficient mice, the human EC-lined structures inosculate with mouse microvessels arising from the wound bed and become perfused within 4 weeks after implantation. The presence of PCs in the printed dermis enhances the invasion of the graft by host microvessels and the formation of an epidermal rete. Impact Statement Three Dimensional printing can be used to generate multilayered vascularized human skin grafts that can potentially overcome the limitations of graft survival observed in current avascular skin substitutes. Inclusion of human pericytes in the dermal bioink appears to improve both dermal and epidermal maturation.

Progenitor-derived human endothelial cells evade alloimmunity by CRISPR/Cas9-mediated complete ablation of MHC expression.

Tissue engineering may address organ shortages currently limiting clinical transplantation. Off-the-shelf engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony-forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells can express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. CRISPR/Cas9-mediated dual ablation of ß2-microglobulin and class II transactivator (CIITA) in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.

ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes.

Complement promotes vascular inflammation in transplant organ rejection and connective tissue diseases. Here we identify ZFYVE21 as a complement-induced Rab5 effector that induces non-canonical NF-κB in endothelial cells (EC). In response to membrane attack complexes (MAC), ZFYVE21 is post-translationally stabilized on MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of cellular phosphoinositide content with miltefosine reduces ZFYVE21 induction, EC activation, and allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC and is detected in human renal and synovial tissues. Our data identifies ZFYVE21 as a Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway in complement-mediated conditions.

Transjugular intrahepatic portosystemic shunt creation for cirrhotic portal hypertension is well tolerated among patients with portal vein thrombosis.

BACKGROUND: Portal vein thrombosis (PVT) develops in cirrhotic patients because of stagnation of blood flow. Transjugular intrahepatic portosystemic shunt (TIPS) creates a low-resistance conduit that restores portal venous patency and blood flow. AIM: The effect of PVT on transplant-free survival in cirrhotic patients undergoing TIPS creation was evaluated. PATIENTS AND METHODS: A multicenter, retrospective cohort study of patients who underwent TIPS creation for cirrhotic portal hypertension was carried out. A Cox model with propensity score adjustment was developed to evaluate the effect of PVT on 90-day and 3-year transplant-free survival. A subgroup analysis examining mortality of those with superior and inferior PVT was also carried out. RESULTS: A total of 252 consecutive TIPS creations were assessed, including 65 in patients with PVT. Survival of patients with high Model for End-stage Liver Disease scores (≥18) and PVT was not statistically different compared with patients with low Model for End-stage Liver Disease scores (<18) and no PVT at 90 days (P=0.46) and 3 years (P=0.42). Those with superior PVT had improved 90-day and 3-year survival both compared with patients with a inferior PVT and those without a PVT (P<0.01, all cases). CONCLUSION: The presence of PVT does not impair the prognosis of patients following TIPS creation, particularly in patients with superior portal occlusion.

Interferon-γ converts human microvascular pericytes into negative regulators of alloimmunity through induction of indoleamine 2,3-dioxygenase 1.

Early acute rejection of human allografts is mediated by circulating alloreactive host effector memory T cells (TEM). TEM infiltration typically occurs across graft postcapillary venules and involves sequential interactions with graft-derived endothelial cells (ECs) and pericytes (PCs). While the role of ECs in allograft rejection has been extensively studied, contributions of PCs to this process are largely unknown. This study aimed to characterize the effects and mechanisms of interactions between human PCs and allogeneic TEM. We report that unstimulated PCs, like ECs, can directly present alloantigen to TEM, but while IFN-γ-activated ECs (γ-ECs) show increased ability to stimulate alloreactive T cells, IFN-γ-activated PCs (γ-PCs) instead suppress TEM proliferation but not cytokine production or signaling. RNA sequencing analysis of PCs, γ-PCs, ECs, and γ-ECs reveal induction of indoleamine 2,3-dioxygenase 1 (IDO1) in γ-PCs to significantly higher levels than in γ-ECs that correlates with tryptophan depletion in vitro. Consistently, shRNA knockdown of IDO1 markedly reduces γ-PC-mediated immunoregulatory effects. Furthermore, human PCs express IDO1 in a skin allograft rejection humanized mouse model and in human renal allografts with acute T cell-mediated rejection. We conclude that immunosuppressive properties of human PCs are not intrinsic but instead result from IFN-γ-induced IDO1-mediated tryptophan depletion.

Antigen Presentation by Vascular Cells.

Antigen presentation by cells of the vessel wall may initiate rapid and localized memory immune responses in peripheral tissues. Peptide antigens displayed on major histocompatibility complex (MHC) molecules on the surface of endothelial cells (ECs) can be recognized by T cell receptors on circulating effector memory T cells (TEM), triggering both transendothelial migration and activation. The array of co-stimulatory receptors, adhesion molecules, and cytokines expressed by ECs serves to modulate T cell activation responses. While the effects of these interactions vary among species, vascular beds, and vascular segments within the same tissue, they are capable of triggering allograft rejection without direct involvement of professional antigen-presenting cells and may play a similar role in host defense against infections and in autoimmunity. Once across the endothelium, extravasating TEM then contact mural cells of the vessel wall, including pericytes or vascular smooth muscle cells, which may also present antigens and provide signals that further regulate T cell responses. Collectively, these interactions provide an unexplored opportunity in which targeting of vascular cells can be used to modulate immune responses. In organ transplantation, targeting ECs with siRNA to reduce expression of MHC molecules may additionally mitigate perioperative injuries by preformed alloantibodies, further reducing the risk of graft rejection. Similarly, genetic manipulation of vascular cells to minimize antigen-dependent responses can be used to increase perfusion of tissue engineered organs without triggering rejection.

Can residents detect errors in technique while observing central line insertions?

BACKGROUND: Procedural teaching and the ability to detect and correct errors are important components of surgical education. This study evaluates whether review of an instructional video will improve residents' ability to detect errors. We hypothesized that clinical experience and confidence do not correlate with ability to detect errors. METHODS: Participants were randomized to 2 groups: the study group viewed an instructional video demonstrating correct technique, whereas the control group did not view the instructional video. Forty general surgery residents described errors in technique during an ultrasound-guided right internal jugular vein catheterization pre and post randomization. RESULTS: Participants who viewed the video improved their error identification rate by 72.6% (P < .001). No correlation between postgraduate year or confidence in error detection and the actual ability to detect errors was noted (r = .17 and r = .14 respectively). CONCLUSIONS: Experience and seniority may not be sufficient to detect procedural errors during central line insertion. Instructional videos improve error recognition.

A p190BRhoGAP mutation and prolonged RhoB activation in fatal systemic capillary leak syndrome.

We describe a fatal case of pediatric systemic capillary leak (Clarkson's disease) associated with a point mutation in p190BRhoGAP. Dermal microvascular endothelial cells (ECs) isolated from this patient form monolayers with similar levels and distribution of junctional proteins and transendothelial electrical resistance compared with normal human dermal microvascular ECs. However, patient-derived ECs demonstrate a greater increase in permeability and impaired recovery of barrier function in response to tumor necrosis factor (TNF) compared with normal donor EC cultures. TNF transiently activates RhoB in ECs coincident with developing leak, and inactivation of RhoB correlates with barrier recovery. The mutation in p190BRhoGAP impairs RhoB inactivation, and the mutant phenotype of patient-derived ECs is replicated by siRNA knockdown of p190BRhoGAP in normal ECs. These data suggest a previously unknown function for p190BRhoGAP in control of capillary EC barrier function that may also be important in acquired systemic capillary leak associated with critical illness in humans.