Induction of Skin Allograft Chimerism by Pharmacological Mobilization of Endogenous Bone Marrow-Derived Stem Cells.

Skin substitutes including allografts remain a standard therapeutic approach to promote healing of both acute and chronic large wounds. However, none have resulted in the regrowth of lost and damaged tissues and scarless wound healing. Here, we demonstrate skin allograft chimerism and repair through the mobilization of endogenous bone marrow-derived stem and immune cells in rats and swine. We show that pharmacological mobilization of bone marrow stem cells and immune cells into the circulation promotes host repopulation of skin allografts and restoration of the skin's normal architecture without scarring and minimal contracture. When skin allografts from DA rats are transplanted into GFP transgenic Lewis recipients with a combination of AMD3100 and low-dose FK506 (AF) therapy, host-derived GFP-positive cells repopulate and/or regenerate cellular components of skin grafts including epidermis and hair follicles and the grafts become donor-host chimeric skin. Using AF combination therapy, burn wounds with skin allografts were healed by newly regenerated chimeric skin with epidermal appendages and pigmentation and without contracture in swine.

A phase I, first-in-human study to evaluate the safety and tolerability, pharmacokinetics, and pharmacodynamics of MRG-001 in healthy subjects.

Preclinical studies demonstrate that pharmacological mobilization and recruitment of endogenous bone marrow stem cells and immunoregulatory cells by a fixed-dose drug combination (MRG-001) improves wound healing, promotes tissue regeneration, and prevents allograft rejection. In this phase I, first-in-human study, three cohorts receive subcutaneous MRG-001 or placebo, every other day for 5 days. The primary outcome is safety and tolerability of MRG-001. Fourteen subjects received MRG-001 and seven received a placebo. MRG-001 is safe over the selected dose range. There are no clinically significant laboratory changes. The intermediate dose group demonstrates the most significant white blood cell, stem cell, and immunoregulatory cell mobilization. PBMC RNA sequencing and gene set enrichment analysis reveal 31 down-regulated pathways in the intermediate MRG-001 dose group compared with no changes in the placebo group. MRG-001 is safe across all dose ranges. MRG-001 may be a clinically useful therapy for immunoregulation and tissue regeneration (ClinicalTrials.gov: NCT04646603).

Evaluation of Early vs Standard Liver Transplant for Alcohol-Associated Liver Disease.

Importance: Traditionally, liver transplant (LT) for alcohol-associated liver disease (ALD) requires 6 months of abstinence. Although early LT before 6 months of abstinence has been associated with decreased mortality for decompensated ALD, this practice remains controversial and concentrated at a few centers. Objective: To define patient, allograft, and relapse-free survival in early LT for ALD, and to investigate the association between these survival outcomes and early vs standard LT. Design, Setting, and Participants: This cohort study analyzed all patients with ALD who underwent their first LT at a single academic referral center between October 1, 2012, and November 13, 2020. Patients with known pretransplant hepatocellular carcinoma, hepatitis B or C, or an alternative cause of liver failure were excluded. Follow-up period was defined as the time from LT to the most recent encounter with a transplant center or death. Exposures: The exposure of interest was early LT, which was defined as less than 180 days of pre-LT abstinence. Standard LT was defined as 180 days or more of pre-LT abstinence. Patients were separated into early LT and standard LT by time from abstinence to LT. Main Outcomes and Measures: The outcomes were patient, allograft, relapse-free, and hazardous relapse-free survival for patients who underwent early LT or standard LT. These groups were compared by log-rank testing of Kaplan-Meier estimates. Hazardous relapse was defined as binge, at-risk, or frequent drinking. Abstinence was reassessed at the most recent follow-up visit for all patients. Results: Of the 163 patients with ALD included in this study, 88 (54%) underwent early LT and 75 (46%) underwent standard LT. This cohort had a mean (SD) age at transplant of 52 (10) years and was predominantly composed of 108 male patients (66%). Recipients of early LT vs standard LT were younger (median [interquartile range (IQR)] age, 49.7 [39.0-54.2] years vs 54.6 [48.7-60.0] years; P < .001) and had a higher median (IQR) Model for End-stage Liver Disease score at listing (35.0 [29.0-39.0] vs 20.0 [13.0-26.0]; P < .001). Both recipients of early LT and standard LT had similar 1-year patient survival (94.1% [95% CI, 86.3%-97.5%] vs 95.9% [95% CI, 87.8%-98.7%]; P = .60), allograft survival (92.7% [95% CI, 84.4%-96.7%] vs 90.5% [95% CI, 81.0%-95.3%]; P = .42), relapse-free survival (80.4% [95% CI, 69.1%-88.0%] vs 83.5% [95% CI, 72.2%-90.6%]; P = .41), and hazardous relapse-free survival (85.8% [95% CI, 75.1%-92.2%] vs 89.6% [95% CI, 79.5%-94.9%]; P = .41). Conclusions and Relevance: Adherence to the 6-month rule was not associated with superior patient survival, allograft survival, or relapse-free survival among selected patients. This finding suggests that patients with ALD should not be categorically excluded from LT solely on the basis of 6 months of abstinence, but rather alternative selection criteria should be identified that are based on need and posttransplant outcomes.

Protein synthesis inhibitor omacetaxine is effective against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the sixth most common and the fourth most deadly cancer worldwide. The development cost of new therapeutics is a major limitation in patient outcomes. Importantly, there is a paucity of preclinical HCC models in which to test new small molecules. Herein, we implemented potentially novel patient-derived organoid (PDO) and patient-derived xenografts (PDX) strategies for high-throughput drug screening. Omacetaxine, an FDA-approved drug for chronic myelogenous leukemia (CML), was found to be a top effective small molecule in HCC PDOs. Next, omacetaxine was tested against a larger cohort of 40 human HCC PDOs. Serial dilution experiments demonstrated that omacetaxine is effective at low (nanomolar) concentrations. Mechanistic studies established that omacetaxine inhibits global protein synthesis, with a disproportionate effect on short-half-life proteins. High-throughput expression screening identified molecular targets for omacetaxine, including key oncogenes, such as PLK1. In conclusion, by using an innovative strategy, we report - for the first time to our knowledge - the effectiveness of omacetaxine in HCC. In addition, we elucidate key mechanisms of omacetaxine action. Finally, we provide a proof-of-principle basis for future studies applying drug screening PDOs sequenced with candidate validation in PDX models. Clinical trials could be considered to evaluate omacetaxine in patients with HCC.

What's the score? A comparison of deceased donor kidney scoring systems and correlation with graft outcome.

BACKGROUND: A number of deceased donor kidney scoring systems have been developed to predict post-transplant graft failure. However, studies comparing the predictive ability of these scoring systems to each other are lacking. METHODS: We used single-center histopathologic and UNOS data from 140 marginal deceased donor kidneys and transplant recipients to compare the predictive accuracy of the Maryland Aggregate Pathology Index (MAPI), Kidney Donor Risk Index (KDRI), Remuzzi, and Nyberg scoring systems for 2-year graft survival using time-dependent receiver operating curves and Kaplan-Meier analysis. RESULTS: MAPI had the highest predictive accuracy (area under curve [AUC] = 0.81) compared to KDRI (AUC = 0.45), Remuzzi (AUC = 0.59), and Nyberg (AUC = 0.63) for 2-year graft survival. Furthermore, when analyzing each score according to its pre-defined risk strata, MAPI was the only scoring system for which 2-year graft survival was significantly different across strata (84.3% for low risk, 56.5% for intermediate risk, and 50% for high risk, P < .001). Additionally, MAPI was the only risk score significantly associated with 2-year graft survival (hazard ratio per point: 1.12, 95% confidence interval [CI]: 1.01-1.23, P = .03). CONCLUSIONS: In a single-center cohort of biopsied marginal kidneys used for transplantation, MAPI had the best predictive ability of these four scoring systems. When biopsy data are available for kidneys considered for transplantation, the MAPI score may provide additional information that could be used to better identify kidneys likely to have longer graft survival.

Stem Cell Mobilization Is Lifesaving in a Large Animal Preclinical Model of Acute Liver Failure.

INTRODUCTION: Acute liver failure (ALF) affects 2000 Americans each year with no treatment options other than liver transplantation. We showed previously that mobilization of endogenous stem cells is protective against ALF in rodents. The objective of this study was to assess whether stem cell mobilizing drugs are lifesaving in a large animal preclinical model of ALF, to assess readiness for a clinical trial. METHODS: Male Yorkshire pigs (14-18 kg) were divided into 2 groups, control (n = 6) and treatment (n = 6). All pigs received an intravenous bolus of the hepatotoxin D-galactosamine (0.5 g/kg) via central line and were followed up until death or day 28. Treated animals received simultaneous intramuscular injection of plerixafor (1 mg/kg) and G-CSF (2 µg/kg) at baseline, 24 and 48 hours after toxin infusion to mobilize endogenous stem cells, as previously described. Control animals received saline. RESULTS: All control animals (6/6) succumbed to liver failure within 91 hours, confirmed by clinical, biochemical, and histopathological evidence of ALF. In the treatment group (5/6) animals survived indefinitely despite comparable biochemical changes during the first 48 hours (P = 0.003). White blood cell count increased by a mean of 4× in the treated group at the peak of mobilization (P = 0.0004). CONCLUSIONS: Stem cell mobilizing drugs were lifesaving in a preclinical large animal model of ALF. Since no therapeutic options other than liver transplantation are currently available for critically ill patients with ALF, a multicenter clinical trial is warranted.

Removal of Noninfected Arteriovenous Fistulae after Kidney Transplantation is a Safe and Beneficial Management Strategy for Unused Dialysis Access.

BACKGROUND: Renal transplant recipients often maintain their hemodialysis access in the event of future allograft failure. Patients may develop complications related to the unused dialysis access, and it also limits vein availability for phlebotomy. Accordingly, a change in the current paradigm may be warranted. This study evaluates the indications for, and safety of, arteriovenous fistula (AVF) removal in patients after successful renal transplantation. METHODS: All patients who underwent AVF excision at a single institution from 2006 to 2016 were retrospectively reviewed. Within that cohort, those undergoing removal after renal transplantation were included for analysis. Baseline patient characteristics, including renal function at the time of removal, reason for excision, and age of the AVF, were examined. The primary outcome was the need for dialysis after AVF removal. RESULTS: A total of 114 patients, of which 36 (31.6%) were recipients of renal transplants, underwent fistula removal during the study period. Within the transplant cohort, the median fistula age at the time of excision was 1,903 days (interquartile range: 556-3,394 days). The most common indications for excision included aneurysmal degeneration (n = 9, 25%), pain (n = 6, 16.7%), upper extremity steal syndrome (n = 5, 13.9%), thrombosis (n = 5, 13.9%), high cardiac output heart failure (n = 4, 11%), and extremity swelling secondary to venous hypertension (n = 2, 5.6%). Most patients (30, 83.3%) had intact graft function. Average creatinine and eGFR at the time of excision in these patients were 1.6 mg/dL and 52.3 mL/min/m2, respectively. Two of these 30 patients (6.7%), who had creatinine values of 2.0 and 9.7 mg/dL, went on to require dialysis following excision. The remaining 28 have maintained normal renal function with improvement in their preoperative symptomatology. Two patients (5.6%) experienced postoperative complications-a hematoma requiring evacuation and a superficial wound infection requiring oral antibiotics. CONCLUSIONS: Removal of symptomatic, unused AVFs can be performed safely in renal transplant recipients. Considering the morbidity associated with large AVFs (including high output cardiac failure), the current paradigm of maintaining asymptomatic hemodialysis access in patients with normally functioning renal transplants should be reconsidered.

Disappearance of GFP-positive hepatocytes transplanted into the liver of syngeneic wild-type rats pretreated with retrorsine.

BACKGROUND AND AIM: Green fluorescent protein (GFP) is a widely used molecular tag to trace transplanted cells in rodent liver injury models. The differing results from various previously reported studies using GFP could be attributed to the immunogenicity of GFP. METHODS: Hepatocytes were obtained from GFP-expressing transgenic (Tg) Lewis rats and were transplanted into the livers of wild-type Lewis rats after they had undergone a partial hepatectomy. The proliferation of endogenous hepatocytes in recipient rats was inhibited by pretreatment with retrorsine to enhance the proliferation of the transplanted hepatocytes. Transplantation of wild-type hepatocytes into GFP-Tg rat liver was also performed for comparison. RESULTS: All biopsy specimens taken seven days after transplantation showed engraftment of transplanted hepatocytes, with the numbers of transplanted hepatocytes increasing until day 14. GFP-positive hepatocytes in wild-type rat livers were decreased by day 28 and could not be detected on day 42, whereas the number of wild-type hepatocytes steadily increased in GFP-Tg rat liver. Histological examination showed degenerative change of GFP-positive hepatocytes and the accumulation of infiltrating cells on day 28. PCR analysis for the GFP transgene suggested that transplanted hepatocytes were eliminated rather than being retained along with the loss of GFP expression. Both modification of the immunological response using tacrolimus and bone marrow transplantation prolonged the survival of GFP-positive hepatocytes. In contrast, host immunization with GFP-positive hepatocytes led to complete loss of GFP-positive hepatocytes by day 14. CONCLUSION: GFP-positive hepatocytes isolated from GFP-Tg Lewis rats did not survive long term in the livers of retrorsine-pretreated wild-type Lewis rats. The mechanism underlying this phenomenon most likely involves an immunological reaction against GFP. The influence of GFP immunogenicity on cell transplantation models should be considered in planning in vivo experiments using GFP and in interpreting their results.

Pharmacological mobilization of endogenous stem cells significantly promotes skin regeneration after full-thickness excision: the synergistic activity of AMD3100 and tacrolimus.

Stem cell therapy has shown promise in treating a variety of pathologies including skin wounds, but practical applications remain elusive. Here, we demonstrate that endogenous stem cell mobilization produced by AMD3100 and low-dose tacrolimus is able to reduce by 25% the time of complete healing of full-thickness wounds created by surgical excision. Equally important, healing was accompanied by reduced scar formation and regeneration of hair follicles. Searching for mechanisms, we found that AMD3100 combined with low-dose tacrolimus mobilized increased number of lineage-negative c-Kit+, CD34+, and CD133+ stem cells. Low-dose tacrolimus also increased the number of SDF-1-bearing macrophages in the wound sites amplifying the "pull" of mobilized stem cells into the wound. Lineage tracing demonstrated the critical role of CD133 stem cells in enhanced capillary and hair follicle neogenesis, contributing to more rapid and perfect healing. Our findings offer a significant therapeutic approach to wound healing and tissue regeneration.

Stem cells in acute liver failure.

Patients with acute liver failure are a particularly challenging group, with unique difficulties faced in treatment decisions. Life-saving therapy is available, but organ shortage, delays in transplantation, and complications in management result in a high mortality in this group of patients even after transplant. Any pharmacologic intervention that improved outcomes in this population of critically ill patients would be of great benefit. Based on available evidence, different scenarios of participation of HSCs in liver recovery are conceivable. Encouraging HSCs to differentiate into hepatocytes or supply paracrine and cellular level support to accelerate ongoing local repair mechanisms and assist a failing liver with inadequate mass and functional capacity might be directed to occur effectively in humans. Evidence within small animal models of liver injury and observations within the human population suggest that this might also be encouraged. The use of pharmacologic agents to mobilize hematopoietic stem cells is well established and effectively used in a different population of patients. As such, extending the use of these drugs, such as plerixafor, to the human population has a sound basis. However, there is a need for clarification of the mechanisms by which these cells exert their effect as well as which specific population of cells is involved in the regenerative process. To be clinically relevant in scenarios of acute liver failure, stem cell mobilizing strategies would have to impact survival when administered well after injury. Applications in other settings may also prove useful. Limits to liver resection exist where the size of the future liver remnant governs the extent of resection possible. Preexisting functional impairment may be restrictive, and strategies involving stem cells may assist the future liver remnant in both normal and functionally impaired livers. Benefit has already been reported from treatment with G-CSF in other injured tissues, including the injured myocardium and acutely injured kidney. However, as yet no clinical trial exists to assess the effects of stem cell mobilization in humans with acute liver failure. The familiarity in the use of and success demonstrated in the clinical and experimental use of plerixafor and G-CSF make exploration of hematopoietic stem cells as therapy in patients with acute liver failure appealing.

Chronic pancreatitis in a patient with malnutrition due to anorexia nervosa.

CONTEXT: Both acute and chronic pancreatitis are associated with eating disorders, including malnutrition found in anorexia, bulimia, and major depression. CASE REPORT: We report a case of a female patient suffering from severe malnutrition and anorexia with repeated attacks of pancreatic pain and an enlarging cystic lesion in the pancreatic head. Due to a progressively enlarging lesion on CT, a pancreaticoduodenectomy was performed. Histology demonstrated chronic pancreatitis. CONCLUSIONS: The pathogenesis of chronic pancreatitis remains to be well defined. There is evidence that an imbalance between oxidative stress and antioxidant capacity results in pancreatic inflammation and activation of periacinar myofibroblasts. It has been demonstrated that protein energy malnutrition is associated with increased levels of proinflammatory cytokines as well as pancreatic acinar cell damage and ductal disruption. Furthermore, it has been shown that protein energy malnutrition including anorexia nervosa is associated with a depleted antioxidant status. Thus there is a possible pathogenic basis for severe malnutrition leading to chronic pancreatitis. Our patient underwent surgery based on the presumption that she had a symptomatic cystic neoplasm. Chronic pancreatitis was demonstrated. Patients presenting with malnutrition and recurrent epigastric pain should be investigated for pancreatic pathology and the possibility of pancreatitis and the presence of pseudocysts entertained.