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).

Mitochondrial membrane potential and delayed graft function following kidney transplantation.

Delayed graft function (DGF) complicates 20%-40% of deceased-donor kidney transplants and is associated with increased length of stay and subsequent allograft failure. Accurate prediction of DGF risk for a particular allograft could influence organ allocation, patient counseling, and postoperative planning. Mitochondrial dysfunction, a reported surrogate of tissue health in ischemia-perfusion injury, might also be a surrogate for tissue health after organ transplantation. To understand the potential of mitochondrial membrane potential (MMP) in clinical decision-making, we analyzed whether lower MMP, a measure of mitochondrial dysfunction, was associated with DGF. In a prospective, single-center proof-of-concept study, we measured pretransplant MMP in 28 deceased donor kidneys and analyzed the association between MMP and DGF. We used hybrid registry-augmented regression to adjust for donor and recipient characteristics, minimizing overfitting by leveraging Scientific Registry of Transplant Recipients data. The range of MMP levels was 964-28 333 units. Low-MMP kidneys (MMP<4000) were more likely from female donors (75% vs 10%, P = .002) and donation after cardiac death donors (75% vs 12%, P = .004). For every 10% decrease in MMP levels, there were 38% higher odds of DGF (adjusted odds ratio = 1.08 1.381.78 , P = .01). In summary, MMP might be a promising pretransplant surrogate for tissue health in kidney transplantation and, after further validation, could improve clinical decision-making through its independent association with DGF.

Pharmacological Mobilization of Endogenous Bone Marrow Stem Cells Promotes Liver Regeneration after Extensive Liver Resection in Rats.

Rapid regeneration of the remnant liver is critical for preventing liver failure and promoting recovery after extensive liver resection. Numerous studies have demonstrated the involvement of bone marrow-derived stem cells in liver regeneration and the potential benefits of bone marrow stem cell therapy. To avoid the preparation of stem cells, we proposed in this study to mobilize endogenous bone marrow stem cells pharmacologically with a combination of AMD3100 (A), an antagonist of CXCR4 and low-dose FK506 (F). Here we show that AF combination therapy significantly increased lineage negative (Lin-) CD34+ and Lin-CD133+ stem cells in peripheral blood and enhanced recruitment of CD133+ cells into the remnant liver in a rat model of 85% partial hepatectomy. Recruiting CD133+ stem cells in the remnant liver was associated with increased proliferation of hepatic oval cells and paralleled the increased SDF-1, CXCR4 and HGF expression. Importantly, AF combination therapy increased the number of Ki67 positive hepatocytes and BrdU incorporation in the remnant liver and improved serum levels of albumin. Our results demonstrate that pharmacological mobilization of endogenous bone marrow stem cells with AF combination therapy can enhance endogenous stem cell mobilization to promote liver regeneration and improve liver function after extensive hepatectomy.

Arteriosclerosis in kidneys from healthy live donors: comparison of wedge and needle core perioperative biopsies.

CONTEXT: Although risks associated with live kidney donation are low, there are few pathologic studies of kidneys from live donors, and possible risk factors for development of hypertension or renal insufficiency remain unknown. There are many studies of histopathologic changes in deceased donor kidneys and how these changes affect subsequent graft function; most are based on wedge rather than needle core biopsies. OBJECTIVE: To examine the frequency and severity of arterial fibrointimal thickening and other pathologic lesions in kidneys from healthy live donors and compare wedge and needle core biopsies as methods for evaluating these changes. DESIGN: For 36 of 332 live donor renal transplantations performed from January 2004 through November 2006, a wedge biopsy of the transplanted kidney was done prior to and/or after implantation, and a needle core biopsy was done postimplantation or during the ensuing 7 days. For these 36 allografts, we compared pathologic features of the wedge and core perioperative biopsies. RESULTS: Findings on core and wedge biopsies were similar, except for arterial fibrointimal thickening. Moderate thickening (Banff cv2) was present on 13 core biopsies, and mild thickening (cv1) was present on another 10; by contrast, no wedge biopsies showed cv2 lesions, and only 8 showed cv1. Arterial thickening on core but not wedge biopsies correlated significantly with increasing patient age. CONCLUSIONS: The findings indicate that needle core biopsies are superior to wedge biopsies for evaluating vascular changes in donor kidneys, and they suggest a need for studies correlating such changes with long-term outcomes of live donors, particularly older donors.

Excellent outcomes after transplantation of deceased donor kidneys with high terminal creatinine and mild pathologic lesions.

BACKGROUND: Outcomes after kidney transplantation using deceased donors with high terminal creatinine are not well described but potentially represent an underutilized source of renal allografts. Utility of renal biopsy of these kidneys is similarly not well established. METHODS: To better understand the posttransplant function of kidneys from donors with high terminal creatinine, we reviewed our database of almost 500 cadaveric kidney transplants. We compared the 65 nonexpanded criteria donors with a final donor creatinine > or = 2.0 mg/dl (range 2.0-4.9 mg/dl) with kidneys procured from donors with terminal creatinine of <1.5. Biopsy results were correlated with graft function. RESULTS: Kidneys from deceased donors with high terminal creatinine performed as well as kidneys from donors with a normal terminal creatinine with respect to primary non-function, acute rejection, 6-year graft and patient survival, and function over the first 48 months. High creatinine kidneys with moderate or severe lesions on biopsy demonstrated poorer function at 6 months and 1 year as compared to those with mild or no histological lesions. CONCLUSIONS: Under select conditions, kidneys from donors with high terminal creatinine can be used safely with excellent results.

Axillorenal arteriovenous graft: a new approach for dialysis access.

As the population requiring hemodialysis grows, it becomes increasingly common to encounter patients with limited options for vascular access. Because inability to secure vascular access is a life-threatening problem, it is important to consider all possible options in each patient. We report a new arteriovenous grafting procedure in which the left renal vein is used for outflow in a patient with multiple venous occlusions. Patency of the graft continues 18 months after placement. This graft carries acceptable morbidity, and can be revised. Consideration of this graft is appropriate in selected patients.

Locally derived cytokines and upregulation of MHC class II genes in allografts.

BACKGROUND: In vitro, various cytokines can modulate the level of expression of major histocompatibility complex (MHC) Class II antigens. Major histocompatibility complex Class II hyperexpression occurs in many immunologic disorders in vivo, but the cytokines that affect this are difficult to analyze because they are produced in small amounts, they act locally, and their mRNAs have short half-lives. METHODS: We studied the expression of cytokines known to up-regulate MHC Class II genes in heart allografts in mice from B10.BR donors to B10.D2 recipients by reverse transcription of mRNA and polymerase chain reaction amplification. The I-Abeta(k) gene expression was also studied in the same fully MHC incompatible strain combination. RESULTS: Messenger RNA for interferon (INF)-gamma, interleukin (IL)-4, and tumor necrosis factor (TNF)-alpha, known inducers of MHC Class II expression in vitro, could be detected in allografts either 24 hours before or simultaneously with massive induction of graft specific I-Abeta mRNA. Interleukin-6 mRNA could be detected as early as 1 day after grafting. CONCLUSION: These data suggest that known cytokine up-regulators of MHC Class II genes, i.e., IFN-gamma, IL-4, and TNF-alpha may contribute to the upregulation of graft-specific MHC Class II antigens during an allograft reaction. Also, IL-6 expression in allografts may result from the stress of the grafting procedure, as it is evident very early after grafting.

Breath biomarkers for detection of human liver diseases: preliminary study.

Chronic liver disease is initially occult, has multiple aetiologies, involves complex diagnostic questions, and requires follow-up because progression is likely. Blood tests and biopsies are generally used, but have disadvantages. We have developed a new test for liver disease based on abnormal concentrations of metabolic products detected in exhaled breath. This test can be used, in conjunction with other clinically accepted diagnostic protocols, to detect and classify chronic liver diseases. Samples of breath collected from spontaneously breathing human subjects (86 patients presenting with 13 liver diseases and 109 subjects with normal liver function) were concentrated cryogenically and analysed by wide-bore capillary gas chromatography using various detectors. The concentrations of various molecules in exhaled breath were examined for potential use as biomarkers of liver function. Subjects with chronic liver diseases could be differentiated from those with normal liver function by comparing levels of breath carbonyl sulphide, carbon disulphide and isoprene; these differences were confirmed and correlated by comparing the levels with standard clinical blood markers of liver damage. The presence of chronic liver failure can thus be detected with sensitivity and specificity by quantifying sulphur-containing compounds arising from the abnormal metabolism associated with liver disease. The breath test we have developed appears to distinguish between hepatocellular and biliary tract aetiologies, and allows staging for severity. This approach may provide the clinician with a simple, non-invasive technique for use in the screening of large populations and follow-up for patients with chronic liver disease.

Expression of fetal isoforms of actin after transplantation injury.

Trauma and injury to transplanted organs in the early post-transplant period are significant factors that affect long-term graft survival. Fetal isoforms of actin are integral members of the immediate early gene family and are expressed in response to free radical injury. We therefore studied actin gene expression in heart transplantation to determine if reperfusion injury activates fetal isoforms of actins. Heterotopic cardiac transplantations were performed in mice. mRNA was extracted from allo- and isografted hearts as well as from normal hearts and spleen. Northern hybridization with actin cDNA to alpha and beta/gamma actin mRNA was performed and analyzed by densitometry. The beta/gamma actin gene expression in the transplanted hearts was found to be significantly elevated within 48 h after transplantation. Analysis of beta/gamma actin gene expression in isografts substantiates the possibility of de novo increase in actin expression. Our studies demonstrate for the first time that fetal isoforms of actin are induced in the allograft heart after transplantation.

A case of severe benign intrahepatic cholestasis treated with liver transplantation.

A male patient with benign recurrent cholestasis since age 2.5 yr developed unremitting cholestasis with incapacitating pruritus and hepatic fibrosis by age 21. He was tried on numerous medical therapies for pruritus with transient or no relief. He responded only temporarily to biweekly plasmapheresis, which was carried out for 4 yr. He underwent orthotopic liver transplantation at age 25 with immediate resolution of his pruritus. At age 30 he is a happy, asymptomatic, fully employed professional.