Towards skin tolerance in vascularised composite allografts

Despite more than twenty years of clinical experience since the first successful human hand transplant in 1998, the mechanisms underlying acute rejection in such vascularised composite allografts (VCAs) remain poorly understood. To further compound the problem, the reporting of patient data, treatment protocols, and definition of VCA rejection remains in constant flux. On the other hand, chronic rejection, which was previously thought to not affect VCAs, has now been accepted and recognised as both a reality and likely eventuality for VCA patients, with no realistic exit plan beyond re-transplantation currently. Between 2015 and 2017, I served as a post-doctoral research fellow at the VCA Laboratory of the Center for Transplantation Sciences, at the Massachusetts General Hospital (MGH), a teaching affiliate of Harvard Medical School in Boston, Massachusetts, USA to work on the development of immunologic tolerance protocols to VCA in both swine and non-human primate (NHP) pre-clinical VCA models. During this time, I was also a member of the clinical VCA service at MGH where I participated in the conduction of clinical VCA trials. I have maintained research collaborations with the same group since. Data from the International Registry of Hand and Composite Tissue Transplantation (IRHCTT) have shown that more than 85% of VCA patients develop at least one episode of acute VCA rejection within the first year, compared to approximately 10% of kidney transplant patients despite the use of highly similar clinical immunosuppression regimens. These differing observations led me to hypothesise that acute rejection might develop within the dermis, due to the vicinity of the subdermal plexus and abundance of skin immune cells, before presenting clinically on the surface of VCA skin. Therefore, this thesis is based on the enclosed publications which span the gamut of clinical, basic science and translational VCA research. By targeting VCA dermis, the primary objective is successful avertion of acute rejection, with the secondary objective of allowing mixed chimerism to develop following tolerance induction through delayed donor bone marrow transplantation (DBMT) in a NHP VCA model, based on clinical trials in renal transplantation patients at MGH that successfully achieved immunosuppression withdrawal. The first two papers (#1 and #2) in this thesis provide the clinical backdrop to the most pressing problems in VCA at present – acute and chronic rejection – for which we as a field still do not have definitive solutions to. Naturally, topical immunosuppression rapidly emerged as an attractive means of addressing acute rejection in VCA given the exteriorised skin component as compared to the typical, intra-abdominal location of solid organ transplant (SOT) allografts. It was theorised that such an approach would not only allow earlier identification and treatment of acute rejection, which presumably would reduce the risk of progression to chronic rejection, but also potentially allow a reduction in the overall immunosuppressive load. In turn, the risk of systemic complications to the VCA recipient would also be lowered. The above approach was explored in NHPs in the next two papers, initially inallogenic skin grafts (#3) (i.e. neovascularised) followed by VCAs, which are primarily vascularised (#4). Concurrently, ongoing research from the VCA Laboratory at MGH using a swine model suggested that rather than transient mixed chimerism (which was sufficient for renal transplantation), VCAs would require stable mixed chimerism instead. This could potentially be achieved through the DTIP approach which was successful in NHP lung transplantation at MGH. As well, analysis of swine VCA skin correlated remarkably well with MGH’s clinical hand transplant patient following transplantation (#5). Therefore, the DTIP approach was trialed in a NHP VCA model next (#6) where chronological analysis of VCA skin both in the short- and long-term reflected our clinical observations (in Papers #1 and #2) but ultimately, tolerance was not achieved due to the persistence of acute rejection. With knowledge of results from #3 and #4, the topical approach was re-engineered for local immunosuppression delivery at the subcutaneous level through a collaboration with engineering colleagues from Rutgers University in New Jersey, USA. Subsequently, acute rejection was successfully mitigated when FK506-loaded discs were implanted subcutaneously during the VCA procedure, and mixed chimerism could be induced through DTIP using the same NHP VCA model. Further work is required however, due to the persistent, premature development of post-transplant lymphoproliferative disorder, which may be an experimental and/or NHP species-specific issue. Overall, this submission of seven papers has introduced the barriers in achieving tolerance to VCAs clinically, and described the laboratory efforts undertaken to reproduce, within ethical limits and animal welfare considerations, these immunological challenges in a robust, pre-clinical NHP VCA model. Most strikingly, the clinical observations and anticipated problems were accurately replicated in the NHP VCA studies. Potential mechanistic insight of acute rejection via VCA dermis was derived, and targeted through further studies to achieve successful proof-of-concept for the mitigation of acute rejection and ultimately, development of mixed chimerism

Effects of Lithium on Age-related Decline in Mitochondrial Turnover and Function in Caenorhabditis elegans

Aging has been associated with the accumulation of damages in molecules and organelles in cells, particularly mitochondria. The rate of damage accumulation is closely tied to the turnover of the affected cellular components. Perturbing mitochondrial turnover has been shown to significantly affect the rate of deterioration of mitochondrial function with age and to alter lifespan of model organisms. In this study, we investigated the effects of upregulating autophagy using lithium in Caenorhabditis elegans. We found that lithium treatment increased both the lifespan and healthspan of C. elegans without any significant change in the mortality rate and oxidative damages to proteins. The increase in healthspan was accompanied by improved mitochondrial energetic function. In contrast, mitochondrial DNA copy number decreased faster with age under lithium. To better understand the interactions among mitochondrial turnover, damage, and function, we created a mathematical model that described the dynamics of functional and dysfunctional mitochondria population. The combined analysis of model and experimental observations showed how preferential (selective) autophagy of dysfunctional mitochondria could lead to better mitochondrial functionality with age, despite a lower population size. However, the results of model analysis suggest that the benefit of increasing autophagy for mitochondrial function is expected to diminish at higher levels of upregulation due to a shrinking mitochondrial populatio

Novel cement curing technique by using controlled release of carbon dioxide coupled with nanosilica

Nanotechnology has attracted a lot of interest in the modification of building materials involving nanoparticles. Among the nanoparticles available, the incorporation of nano-silica draws intense attention due to the similarity of its chemical composition with cement and its pozzolanic properties. In this work, the potential capability to utilise CO2 in improving cement composites properties through carbonation acceleration mechanism was explored. In this study, various type of nano silica was used as a CO2 carrier and incorporated into cement mortar design with different amount of carbonated silica loading, ranging from 0.55 wt% to 2.42 wt% and cured in water and ambient air condition. The aim of this study is to examine the effects on the compressive strength of nano-silica impregnated with CO2 and incorporated into cement mortar. From the results, it was found that at 1.89% silica loading, the hydrophilic silica mortar (HSAM) samples can achieve the highest compressive strength of 34.1 MPa at 7 days and 40.7 MPa at 28 days, with a percentage gain of +38.06% and +17.29% respectively as compared to blank samples. However, the incorporation of silica for more than 1.89 wt% resulted in a negative effect on the compressive strength gain of HSAM samples. By the incorporation of 2.42 wt%, the samples showed a significant drop in compressive strength of −21.46% at 7 days and −17.29% at 28 days. The results proved that nano-silica coupled with CO2 can accelerate curing of cement mortar by means of carbonation

Abstract: Analysis of acute skin rejection in non-human primate models of face and hand allotransplantation

Introduction: The incidence of acute rejection (AR) of the skin within the first year after hand or face transplantation is approximately 85% and up to 56% of patients experience multiple episodes1. Significant immunosuppression is required to prevent allograft loss, and recent studies suggest that repeated AR episodes can lead to VCA dysfunction and loss2. The mechanisms underlying variability in AR presentation remain poorly defined however. Materials and Methods: 8 cynomolgus monkeys received either an orthotopic hand (n=2) or heterotopic face VCA (n=6) from MHC-mismatched donors following induction with anti-thymocyte globulin. Post-operatively, triple immunosuppression – tacrolimus, mycophenolate mofetil, methylprednisolone – was maintained for up to 120 days before bone marrow transplantation (BMT) was performed. Protocol biopsies of VCA skin were performed at 30-day intervals for histopathology and flow cytometric analysis of resident skin leukocyte populations; VCA-resident cells were differentiated by H38 status (mouse antihuman HLA class I monoclonal antibody that cross reacts with cynomolgus monkeys) for donor or recipient derivation. Clinical AR was treated with steroids and further biopsies were taken for histologic confirmation; corresponding anti-donor responses were evaluated by mixed lymphocyte reaction (MLR) and allo-antibody formation. Results: Up to three episodes of AR (from POD 14, Banff I to II) developed while recipient animals were maintained on triple immunosuppression. Corresponding flow cytometric analyses demonstrate > 80% of skin-resident T lymphocytes (CD4+, CD8+) within VCA dermis were of recipient origin, suggesting rapid immigration of various lineages into the VCA. These observations coincided with the first episode of AR in fully mismatched recipients but haplomatched animals remained rejection-free. All but one episode of AR were successfully treated. No allo-antibodies were detected and anti-donor responses by MLR were comparable to that against third-party. Following BMT, mixed chimerism was detected and enabled immunosuppression withdrawal. However, this was transient and once lost, clinical AR developed and nearly 100% of both dermal and epidermal lymphocytes were recipient-derived. Conclusion: We report a clinically-relevant model for studying AR in VCA. Our results suggest that further understanding of the relative importance of MHC differences in transplant pairs may lead to differences in outcomes for VCA recipients maintained under standard immunosuppression. Immunosuppression-free tolerance of non-hematopoietic antigens in composite tissues can be achieved, but require additional strategies to achieve stable, rather than transient mixed chimerism following BMT

The association between post-stroke depression, aphasia, and physical independence in stroke patients at 3-month follow-up

Objective: Few studies have examined the association between post-stroke depression (PSD), aphasia, and physical independence in Chinese patients. This study investigated the above association in stroke patients in China at 3-month follow-up. Methods: Altogether 270 patients within 14 days after ischemic stroke were recruited and followed up at 3 months. PSD, aphasia, and physical functional status were measured using the Stroke Aphasia Depression Questionnaire (SADQ), Western Aphasia Battery (WAB), and modified Rankin Scale (mRS), respectively. Patients with mRS total score \u3e2 were considered as having “physical dependence.” Results: Out of 248 patients at 3-month follow up, 119 (48%) were rated as having physical dependence. Multiple logistic regression analyses revealed that female (p = 0.04; OR = 2.2; 95% CI: 1.0–5.1), more severe stroke at admission (p \u3c 0.01; OR = 1.4; 95% CI: 1.3–1.5), and more severe PSD at 3 months (p = 0.01; OR = 1.05; 95% CI: 1.01–1.1) were independently associated with physical dependence at 3 months. Conclusions: Greater PSD and stroke severity were independently associated with physical dependence at 3months after stroke. Aphasia was also associated with physical dependence but the relationship was not significant. Early and effective depression screening, treatment and stroke rehabilitation appear to be important to improve the physical outcome and reduce the burden of stroke survivors

CatNorth: An Improved Gaia DR3 Quasar Candidate Catalog with Pan-STARRS1 and CatWISE

A complete and pure sample of quasars with accurate redshifts is crucial for quasar studies and cosmology. In this paper, we present CatNorth, an improved Gaia DR3 quasar candidate catalog with more than 1.5 million sources in the 3$\pi$ sky built with data from Gaia, Pan-STARRS1, and CatWISE2020. The XGBoost algorithm is used to reclassify the original Gaia DR3 quasar candidates as stars, galaxies, and quasars. To construct training/validation datasets for the classification, we carefully built two different master stellar samples in addition to the spectroscopic galaxy and quasar samples. An ensemble classification model is obtained by averaging two XGBoost classifiers trained with different master stellar samples. Using a probability threshold of $p_{\mathrm{QSO\_mean}}>0.95$ in our ensemble classification model and an additional cut on the logarithmic probability density of zero proper motion, we retrieved 1,545,514 reliable quasar candidates from the parent Gaia DR3 quasar candidate catalog. We provide photometric redshifts for all candidates with an ensemble regression model. For a subset of 89,100 candidates, accurate spectroscopic redshifts are estimated with the Convolutional Neural Network from the Gaia BP/RP spectra. The CatNorth catalog has a high purity of > 90% while maintaining high completeness, which is an ideal sample to understand the quasar population and its statistical properties. The CatNorth catalog is used as the main source of input catalog for the LAMOST phase III quasar survey, which is expected to build a highly complete sample of bright quasars with $i < 19.5$.Comment: 24 pages, 13 figures, submitted to AAS journals. Table 4 (The CatNorth quasar candidate catalog) is available at https://nadc.china-vo.org/res/r101313

Relationship between the magnitude of intraocular pressure during an episode of acute elevation and retinal damage four weeks later in rats

PURPOSE: To determine relationship between the magnitude of intraocular pressure (IOP) during a fixed-duration episode of acute elevation and the loss of retinal function and structure 4 weeks later in rats. METHODS: Unilateral elevation of IOP (105 minutes) was achieved manometrically in adult Brown Norway rats (9 groups; n = 4 to 8 each, 10-100 mm Hg and sham control). Full-field ERGs were recorded simultaneously from treated and control eyes 4 weeks after IOP elevation. Scotopic ERG stimuli were white flashes (-6.04 to 2.72 log cd.s.m(-2)). Photopic ERGs were recorded (1.22 to 2.72 log cd.s.m(-2)) after 15 min of light adaptation (150 cd/m(2)). Relative amplitude (treated/control, %) of ERG components versus IOP was described with a cummulative normal function. Retinal ganglion cell (RGC) layer density was determined post mortem by histology. RESULTS: All ERG components failed to recover completely normal amplitudes by 4 weeks after the insult if IOP was 70 mmHg or greater during the episode. There was no ERG recovery at all if IOP was 100 mmHg. Outer retinal (photoreceptor) function demonstrated the least sensitivity to prior acute IOP elevation. ERG components reflecting inner retinal function were correlated with post mortem RGC layer density. CONCLUSIONS: Retinal function recovers after IOP normalization, such that it requires a level of acute IOP elevation approximately 10 mmHg higher to cause a pattern of permanent dysfunction similar to that observed during the acute event. There is a 'threshold' for permanent retinal functional loss in the rat at an IOP between 60 and 70 mmHg if sustained for 105 minutes or more

Calcium Intake and Risk of Colorectal Cancer According to Tumor-infiltrating T Cells

Calcium intake has been associated with a lower risk of colorectal cancer. Calcium signaling may enhance T-cell proliferation and differentiation, and contribute to T-cell–mediated antitumor immunity. In this prospective cohort study, we investigated the association between calcium intake and colorectal cancer risk according to tumor immunity status to provide additional insights into the role of calcium in colorectal carcinogenesis. The densities of tumor-infiltrating T-cell subsets [CD3+, CD8+, CD45RO (PTPRC)+, or FOXP3+ cell] were assessed using IHC and computer-assisted image analysis in 736 cancer cases that developed among 136,249 individuals in two cohorts. HRs and 95% confidence intervals (CI) were calculated using Cox proportional hazards regression. Total calcium intake was associated with a multivariable HR of 0.55 (comparing ≥1,200 vs. <600 mg/day; 95% CI, 0.36–0.84; Ptrend = 0.002) for CD8+ T-cell–low but not for CD8+ T-cell–high tumors (HR = 1.02; 95% CI, 0.67–1.55; Ptrend = 0.47). Similarly, the corresponding HRs (95% CIs) for calcium for low versus high T-cell–infiltrated tumors were 0.63 (0.42–0.94; Ptrend = 0.01) and 0.89 (0.58–1.35; Ptrend = 0.20) for CD3+; 0.58 (0.39–0.87; Ptrend = 0.006) and 1.04 (0.69–1.58; Ptrend = 0.54) for CD45RO+; and 0.56 (0.36–0.85; Ptrend = 0.006) and 1.10 (0.72–1.67; Ptrend = 0.47) for FOXP3+, although the differences by subtypes defined by T-cell density were not statistically significant. These potential differential associations generally appeared consistent regardless of sex, source of calcium intake, tumor location, and tumor microsatellite instability status. Our findings suggest a possible role of calcium in cancer immunoprevention via modulation of T-cell function