Effect of donor HSD17B13 genotype on patient survival after liver transplant: a retrospective cohort study.

Background: Several genetic variants are associated with chronic liver disease. The role of these variants in outcomes after liver transplantation (LT) is uncertain. The aim of this study was to determine if donor genotype at risk-associated variants in PNPLA3 (rs738409 C>G, p.I148M) and HSD17B13 (rs72613567 T>TA; rs80182459, p.A192Lfs∗8) influences post-LT survival. Methods: In this retrospective cohort study, data on 2346 adults who underwent first-time LT between January 1, 1999 and June 30, 2020 and who had donor DNA samples available at five large Transplant Immunology Laboratories in Texas, USA, were obtained from the United Network for Organ Sharing (UNOS). Duplicates, patients with insufficient donor DNA for genotyping, those who were <18 years of age at the time of transplant, had had a previous transplant or had missing genotype data were excluded. The primary outcomes were patient and graft survival after LT. The association between donor genotype and post-LT survival was examined using Kaplan-Meier method and multivariable-adjusted Cox proportional hazards models. Findings: Median age of LT recipients was 57 [interquartile range (IQR), 50-62] years; 837 (35.7%) were women; 1362 (58.1%) White, 713 (30.4%) Hispanic, 182 (7.8%) Black/African-American. Median follow-up time was 3.95 years. Post-LT survival was not affected by donor PNPLA3 genotype but was significantly reduced among recipients of livers with two HSD17B13 loss-of-function (LoF) variants compared to those receiving livers with no HSD17B13 LoF alleles (unadjusted one-year survival: 82.6% vs 93.9%, P < 0.0001; five-year survival: 73.1% vs 82.9%, P = 0.0017; adjusted hazard ratio [HR], 2.25; 95% CI, 1.61-3.15 after adjustment for recipient age, sex, and self-reported ethnicity). Excess mortality was restricted to those receiving steroid induction immunosuppression (crude 90-day post-LT mortality, 9.3% [95% CI, 1.9%-16.1%] vs 1.9% [95% CI, 0.9%-2.9%] in recipients of livers with two vs no HSD17B13 LoF alleles, P = 0.0012; age, sex, and ethnicity-adjusted HR, 2.85; 95% CI, 1.72-4.71, P < 0.0001). No reduction was seen among patients who did not receive steroid induction (90-day mortality 3.1% [95% CI, 0%-7.3%] vs 2% [95% CI, 0.9%-3.1%], P = 0.65; adjusted HR, 1.17; 95% CI, 0.66-2.08, P = 0.60). Interpretation: Donor HSD17B13 genotype adversely affects post-LT survival in patients receiving steroid induction. Additional studies are required to confirm this association. Funding: The National Institutes of Health and American Society of Transplant Surgeons Collaborative Scientist Grant.

Serological Response in Lung Transplant Recipients after Two Doses of SARS-CoV-2 mRNA Vaccines.

BACKGROUND: Lung-transplant (LT) recipients are at high risk for COVID-19 due to immunosuppression and respiratory tropism of SARS-CoV-2. The information on the effect of COVID-19 mRNA vaccines to elicit immunogenic responses after a two-dose (2D) regimen in LT recipients is sparse. Thus, we assessed the effect of Pfizer-BioNTech and Moderna mRNA vaccines' 2D regimen on anti-spike responses in immunocompromised LT recipients. METHODS: We utilized serum samples from LT recipients vaccinated for SARS-CoV-2 with 2D of either the Pfizer-BioNTech or Moderna vaccines and 2D-vaccinated naïve (non-transplanted and non-exposed to COVID-19) group. Antibody responses were assessed using the FDA-approved SARS-CoV-2 anti-nucleocapsid protein IgG assay (IgGNC), the SARS-CoV-2 anti-spike protein IgM assay (IgMSP), and the SARS-CoV-2 anti-spike protein IgG II assay (IgGSP). CD4+ T-cell activity was assessed as a marker of immune competence using the ImmuKnow® assay. RESULTS: About 25% (18/73) of SARS-CoV-2 uninfected-LT patients generated a positive spike-IgG response following 2D of vaccines, with 36% (9/25) in the Moderna cohort and only 19% (9/48) in the Pfizer cohort. 2D in LT patients elicited a significantly lesser median IgGSP response (1.7 AU/mL, 95% CI: 0.6-7.5 AU/mL) compared to non-transplanted, uninfected naïve subjects (14,209 AU/mL, 95% CI: 11,261-18,836 AU/mL; p < 0.0001). In LT patients, the Moderna-evoked seropositivity trend was higher than Pfizer. CONCLUSION: 2D COVID-19 vaccination elicits a dampened serological response in LT patients. Whether assessing other arms of host immunity combined with a higher vaccine dose can better capture and elicit improved immunogenicity in this immunocompromised population warrants investigation.

SARS-CoV-2 Antibody Responses Do Not Predict COVID-19 Disease Severity.

OBJECTIVES: Initial reports indicate adequate performance of some serology-based severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) assays. However, additional studies are required to facilitate interpretation of results, including how antibody levels impact immunity and disease course. METHODS: A total of 967 subjects were tested for IgG antibodies reactive to SARS-CoV-2, including 172 suspected cases of SARS-CoV-2, 656 plasma samples from healthy donors, 49 sera from patients with rheumatic disease, and 90 specimens from individuals positive for polymerase chain reaction (PCR)-based respiratory viral panel. A subgroup of SARS-CoV-2 PCR-positive cases was tested for IgM antibodies by proteome array method. RESULTS: All specificity and cross-reactivity specimens were negative for SARS-CoV-2 IgG antibodies (0/795, 0%). Positive agreement of IgG with PCR was 83% of samples confirmed to be more than 14 days from symptom onset, with less than 100% sensitivity attributable to a case with severe immunosuppression. Virus-specific IgM was positive in a higher proportion of cases less than 3 days from symptom onset. No association was observed between mild and severe disease course with respect to IgG and IgM levels. CONCLUSIONS: The studied SARS-CoV-2 IgG assay had 100% specificity and no adverse cross-reactivity. Measures of IgG and IgM antibodies did not predict disease severity in our patient population.

Allograft outcomes of treated children with kidney transplant who developed plasma cell-rich acute rejection (PCAR): A single center's experience.

INTRODUCTION: PCAR is a rare form of ACR that may compromise renal allografts. This review evaluates the outcomes of a protocol used to treat PCAR (Study group), and compares these outcomes with a matched cohort with ACR (Control group). METHODS: A retrospective analysis of 138 of pRTRs who underwent renal allograft biopsies between January 2008 and November 2016. RESULTS: Seven biopsies revealed in situ hybridization of EBER-negative PCAR (5%). Three Study group pRTRs lost their grafts within 3 months after rejection (43%). None of the Control group pRTRs lost their graft during this period. At the time of rejection, eGFR was different between the Control and Study groups (27.0 ± 19.9 mL/min per m2 vs 40.0 ± 10.6 mL/min/1.73 m2 , respectively; P < 0.05). Among Study group pRTRs with functioning allografts (n = 4), treatment resulted in an increase in eGFR from nadir levels (27.0 ± 19.9 vs 55.6 ± 18.3 mL/min/1.73 m2 , P < 0.05). In the Study group, complications included neutropenia, BK and EBV viremia, and infusion-related hypotension and hypertension. SUMMARY: (a) Graft loss in Study group while remaining high (43%) was lower than that reported in the published pediatric literature. (b) Our protocol was associated with improvement in eGFR in all surviving pRTRs within the Study group. (c) No life-threatening complications or malignancy were reported during the observation period.

Evaluation of current strategies for surveillance and management of donor-specific antibodies: Single-center study.

BACKGROUND: Although the presence of donor-specific antibodies (DSA) is known to impact lung allograft, limited data exist regarding DSA management. METHODS: We did a retrospective study at our center evaluating DSA management in adult lung transplant recipients undergoing lung transplantation between January 1, 2010 and June 30, 2014. Study follow-up was completed through October 2017. All recipients were stratified into 2 groups based on the presence or absence of DSA. Those with DSA were evaluated for the impact of treatment of DSA. The primary outcomes were postlung transplant survival and freedom from bronchiolitis obliterans syndrome (BOS), subset of chronic lung allograft dysfunction (CLAD). Simon-Makuch method was used to estimate overall survival and BOS-free survival to account for DSA as time-dependent covariate. Survival differences between the groups were analyzed using time-dependent Cox proportional hazards model. RESULTS: Sixty-four percent of 194 total subjects developed post-lung transplant DSA. Overall survival was different with worse survival in the DSA positive group that never cleared DSA (P = .002). BOS-free survival was lower, but did not reach significance in this group. Response to treatment was poor, with only 12 of 47 (25.5%) who received treatment demonstrating clearance of DSA. CONCLUSIONS: Donor-specific antibodies prevalence is high after lung transplantation. Clearance of DSA correlated with improved outcomes. Current therapeutic strategies against DSA are relatively ineffective. Multicenter collaborative studies will be required to evaluate current treatment strategies and other innovative modalities.

Complement (C1q) Binding De Novo Donor-Specific Antibodies and Cardiac-Allograft Vasculopathy in Pediatric Heart Transplant Recipients.

BACKGROUND: We hypothesized C1q binding de novo donor-specific antibody (DSA) after heart transplant (HT) is a higher risk for development of coronary artery vasculopathy (CAV) in children. METHODS: A retrospective analysis of 127 pediatric HT recipients transplanted between January 2005 and December 2014 was used to determine complement (C1q)-binding de novo DSA on the outcomes of HT and the ability of the C1q assay to predict CAV development. RESULTS: Of 127 patients, 59 (46.4%) developed de novo DSA, 37 of those had C1q+ DSA. There was no difference in baseline characteristics except patients who developed C1q+ DSA more often received a donor heart from a female compared with C1q- DSA group (P = 0.034). The DSA median fluorescent intensity (MFI) value of 7000 or greater had 80% sensitivity and 80% specificity (C statistics 0.89, P <0.05) for predicting positive C1q binding. Multivariate analyses identified C1q binding DSA as an independent risk for CAV with a hazard ratio (HR) of 3.25 (95% confidence interval [CI], 1.33-7.93; P = 0.0095). In multivariable Cox proportional hazard models, the covariates associated with graft loss included: C1q+ DSA (HR, 3.2; 95% CI, 1.34-7.86; P < 0.009), pre-HT renal insufficiency (HR, 11.3; 95% CI, 3.71-34.29; P < 0.0001), and pre-HT ventilator support (HR, 3.3; 95% CI, 1.39-7.81; P = 0.007). CONCLUSIONS: The DSA strength in MFI correlates with positive C1q-binding activity and hence functional capabilities of DSA. Close monitoring of DSA strength in MFI and function (C1q assay) may be useful for identifying pediatric HT recipient at risk for development of CAV.

Alemtuzumab (Campath-1H) therapy for refractory rejections in pediatric heart transplant recipients.

Despite substantial improvements in survival after pediatric heart transplantation, refractory rejection remains a major cause of morbidity and mortality. We have utilized ALE (Campath-1H) in six consecutive patients with refractory rejection. These rejection episodes persisted despite conventional treatment, which included intravenous methylprednisolone, rituximab, immunoglobulin G, and antithymocyte globulin. In our series, after ALE therapy, LV SF increased from 22%±5% to 33%±5% (P=.01). However, in our series, ALE therapy neither led to persistent LV function recovery nor could it prevent subsequent antibody-mediated rejection.

Management of sensitized pediatric patients prior to renal transplantation.

BACKGROUND: Data on renal allograft outcome in sensitized children are scarce. We report the clinical courses of four children who received desensitization therapy prior to renal transplantation in our institution. METHODS: Between 2009 and 2011, four pediatric patients with stage 5 chronic kidney disease received desensitization therapy due to: (1) positive donor-specific antibodies (DSA) and/or crossmatches with potential living donors, (2) more than three positive crossmatches with deceased donors or (3) high calculated panel-reactive antibody of >80 %. Desensitization with rituximab, intravenous immunoglobulin and bortezomib was performed in all patients. Induction therapy included combinations of plasmapheresis and/or alemtuzumab or anti-thymocyte globulin. Standard post-transplant medications included tacrolimus, mycophenolate mofetil and prednisolone. RESULTS: Post-transplant screening revealed DSA in three patients. Biopsy showed no evidence of rejection at 1 month in two patients, one of whom developed chronic active antibody-mediated rejection 4.5 years later. One patient developed borderline acute cellular rejection at 1 month, but the serum creatinine level was stable and DSA disappeared without treatment 1 month later, with stable long-term allograft function at 3 years. Estimated or measured glomerular filtration rate of the patients ranged between 30 and 75 ml/min/1.73 m(2) after 1 to 4.5 years. CONCLUSIONS: The four sensitized patients reported here who received desensitization therapy had successful renal transplants with a low risk of immediate post-transplant rejection. Overall, long-term allograft functions and complications from immunosuppression were encouraging.

Donor-specific HLA alloantibodies: Impact on cardiac allograft vasculopathy, rejection, and survival after pediatric heart transplantation.

BACKGROUND: There is increasing evidence that donor-specific anti-HLA antibodies (DSA) are associated with poor outcomes after cardiac transplantation in adults, but data are limited in children. The objective of this study was to examine the development and consequences of de novo DSA in pediatric recipients of heart transplants. METHODS: We analyzed 105 pediatric patients who received heart transplants at our center from January 2002 to December 2012. All patients had negative T-cell and B-cell post-transplant crossmatches. Patients underwent HLA antibody screening at 1, 2, 3, 6, and 12 months post-transplant and annually thereafter unless there was suspicion for rejection. HLA class I and II antibodies were identified using Luminex assay. Coronary angiography was performed at 1 year and annually thereafter. Acute cellular rejection, antibody-mediated rejection, and treated clinical rejections were included together as rejection events. RESULTS: Of 105 patients, 45 (43%) developed de novo DSA. DSA-positive patients had significantly higher rates of coronary artery vasculopathy (CAV) compared with DSA-negative patients (36% vs 13%). CAV-free survival at 1 year and 5 years post-transplant for DSA-negative patients was 90% and 25%, respectively, compared with 70% and 0%, respectively, for DSA-positive patients (p < 0.01). DSA-positive patients had 2.5 times more rejection events per year than DSA-negative patients. The 5-year graft survival rate was 72.4% for DSA-negative patients and 21% for DSA-positive patients (p < 0.001). CONCLUSIONS: De novo DSA has a strong negative impact on CAV, rejection, and graft survival in pediatric recipients of heart transplants.

Pancreatic islets engineered with SA-FasL protein establish robust localized tolerance by inducing regulatory T cells in mice.

Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.

Induction of tolerance to cardiac allografts using donor splenocytes engineered to display on their surface an exogenous fas ligand protein.

The critical role played by Fas ligand (FasL) in immune homeostasis renders this molecule an attractive target for immunomodulation to achieve tolerance to auto- and transplantation Ags. Immunomodulation with genetically modified cells expressing FasL was shown to induce tolerance to alloantigens. However, genetic modification of primary cells in a rapid, efficient, and clinically applicable manner proved challenging. Therefore, we tested the efficacy of donor splenocytes rapidly and efficiently engineered to display on their surface a chimeric form of FasL protein (SA-FasL) for tolerance induction to cardiac allografts. The i.p. injection of ACI rats with Wistar-Furth rat splenocytes displaying SA-FasL on their surface resulted in tolerance to donor, but not F344 third-party cardiac allografts. Tolerance was associated with apoptosis of donor reactive T effector cells and induction/expansion of CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells. Treg cells played a critical role in the observed tolerance as adoptive transfer of sorted Treg cells from long-term graft recipients into naive unmanipulated ACI rats resulted in indefinite survival of secondary Wistar-Furth grafts. Immunomodulation with allogeneic cells rapidly and efficiently engineered to display on their surface SA-FasL protein provides an effective and clinically applicable means of cell-based therapy with potential application to regenerative medicine, transplantation, and autoimmunity.

Ex vivo expansion of CD4+CD25+FoxP3+ T regulatory cells based on synergy between IL-2 and 4-1BB signaling.

Naturally occurring CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells require three distinct signals transduced via TCR, CD28, and IL-2R for their development and maintenance. These requirements served as the basis for several recently developed ex vivo expansion protocols that relied on the use of solid support-bound Abs to CD3 and CD28 in the presence of high dose IL-2. We report in this study that Treg cells up-regulate the expression of inducible costimulatory receptor 4-1BB in response to IL-2, and stimulation using this receptor via a novel form of 4-1BB ligand (4-1BBL) fused to a modified form of core streptavidin (SA-4-1BBL) was effective in expanding these cells up to 110-fold within 3 wk. Expanded cells up-regulated CD25, 4-1BB, and membranous TGF-beta, suppressed T cell proliferation, and prevented the rejection of allogeneic islets upon adoptive transfer into graft recipients. Importantly, SA-4-1BBL rendered CD4(+)CD25(-) T effector cells refractive to suppression by Treg cells. This dual function of signaling via 4-1BB, vis-à-vis Treg cell expansion and licensing T effector cells resistant to Treg cell suppression, as well as the up-regulation of 4-1BB by IL-2 may serve as important regulatory mechanisms for immune homeostasis following antigenic challenge. Stimulation using a soluble form of SA-4-1BBL represents a novel approach to expand Treg cells with potential therapeutic applications in autoimmunity and transplantation.

CD4+CD25+ T regulatory cells dominate multiple immune evasion mechanisms in early but not late phases of tumor development in a B cell lymphoma model.

Tumors use a complex set of direct and indirect mechanisms to evade the immune system. Naturally arising CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells have been implicated recently in tumor immune escape mechanism, but the relative contribution of these cells to overall tumor progression compared with other immune evasion mechanisms remains to be elucidated. Using the A20 B cell lymphoma as a transplantable tumor model, we demonstrate that this tumor employs multiple direct (expression of immunoinhibitory molecule PD-L1, IDO, and IL-10, and lack of expression of CD80 costimulatory molecule) and indirect (down-regulation of APC function and induction of Treg cells) immune evasion mechanisms. Importantly, Treg cells served as the dominant immune escape mechanism early in tumor progression because the physical elimination of these cells before tumor challenge resulted in tumor-free survival in 70% of mice, whereas their depletion in animals with established tumors had no therapeutic effect. Therefore, our data suggest that Treg cells may serve as an important therapeutic target for patients with early stages of cancer and that more vigorous combinatorial approaches simultaneously targeting multiple immune evasion as well as immunosurveillance mechanisms for the generation of a productive immune response against tumor may be required for effective immunotherapy in patients with advanced disease.

Senescent fibroblasts resist apoptosis by downregulating caspase-3.

In replicative senescence, cells undergo permanent exit from cell cycle traverse; this is traditionally thought to occur at the end of a culture's in vitro life span, after serial passaging. In general, the checkpoint for replicative senescence is found at the G(1)/S border, controlled by the modulation of a battery of proteins, typified by gaining inhibitors of cell cycle traverse, such as cyclin-dependent kinases or RB hyperphosphorylation, and losing pro-proliferation gene expressions such as c-fos, c-myc, and a cadre of proliferation-dependent kinases. Here, we present evidence that replicatively senescent fibroblasts are resistant to apoptotic death, associated with a lack of key enzyme activities, caspase-3 being the chief executioner. This observation, coupled with our earlier report that senescent fibroblasts maintain persistently high levels of pro-survival factor Bcl-2, suggests that the molecular signaling program present in fibroblasts at the end of their in vitro life span may not only cater to the state of permanent exit from cell cycle traverse, but also dictate an inability to commit cellular suicide. Future experiments will reveal whether replicatively senescent fibroblasts that can neither proliferate nor die contribute to organismic aging, and whether their accumulation over time in tissue becomes detrimental to the normal aging process.

Designer microarrays: from soup to nuts.

The recognition that multigene mechanisms control the pathways determining the aging process renders gene screening a necessary skill for biogerontologists. In the past few years, this task has become much more accessible, with the advent of DNA chip technology. Most commercially available microarrays are designed with prefixed templates of genes of general interest, allowing investigators little freedom of choice in attempting to focus gene screening on a particular thematic pathway of interest. This report describes our "designer microarray" approach as a next generation of DNA chips, allowing individual investigators to engage in gene screening with a user friendly, do-it-yourself approach, from designing the probe templates to data mining. The end result is the ability to use microarrays as a platform for versatile gene discovery.

Establishing lymphoblastoid cell lines from frozen blood of extremely old individuals.

In population studies involving peripheral blood samples from nonagenarian and centenarian donors, the amount of biological material available for research is restricted, as only a few millilitres of blood can be obtained from extremely old donors without significantly compromising their health. Here we describe a protocol to immortalize small amounts of total frozen blood from extremely old donors (90+) using the Epstein-Barr virus, despite the low level of circulating B cells present in nonagenarian and centenarian blood samples. This methodology provides a unique way to maximize resources of biological material available for research.

Alterations in TNF- and IL-related gene expression in space-flown WI38 human fibroblasts.

Spaceflight, just like aging, causes profound changes in musculoskeletal parameters, which result in decreased bone density and muscular weakness. As these conditions decrease our ability to conduct long-term manned space missions, and increase bone frailty in the elderly, the identification of genes responsible for the apparition of these physiological changes will be of great benefit. Thus, we developed and implemented a new microarray approach to investigate the changes in normal WI38 human fibroblast gene expression that arise as a consequence of space flight. Using our microarray, we identified changes in the level of expression of 10 genes, belonging to either the tumor necrosis factor- (TNF) or interleukin- (IL) related gene families in fibroblasts when WI38 cells exposed to microgravity during the STS-93 Space Shuttle mission were compared with ground controls. The genes included two ligands from the TNF superfamily, TWEAK and TNFSF15; two TNF receptor-associated proteins, NSMAF and PTPN13; three TNF-inducible genes, ABC50, PTX3, and SCYA13; TNF-alpha converting enzyme, IL-1 receptor antagonist, and IL-15 receptor alpha chain. Most of these are involved in either the regulation of bone density, and as such the development of spaceflight osteopenia, or in the development of proinflammatory status.