Therapies to reduce liver fibrosis and stimulate organ regeneration are urgently needed. We conducted a first-in-human, phase 1 dose-escalation trial of autologous macrophage therapy in nine adults ...with cirrhosis and a Model for End-Stage Liver Disease (MELD) score of 10-16 (ISRCTN 10368050). Groups of three participants received a single peripheral infusion of 10
, 10
or up to 10
cells. Leukapheresis and macrophage infusion were well tolerated with no transfusion reactions, dose-limiting toxicities or macrophage activation syndrome. All participants were alive and transplant-free at one year, with only one clinical event recorded, the occurrence of minimal ascites. The primary outcomes of safety and feasibility were met. This study informs and provides a rationale for efficacy studies in cirrhosis and other fibrotic diseases.
Islet transplantation is an efficacious therapy for type 1 diabetes; however, islets from multiple donor pancreata are required, and a gradual attrition in transplant function is seen. Here, we ...manufactured human umbilical cord perivascular mesenchymal stromal cells (HUCPVCs) to Good Manufacturing Practice (GMP) standards. HUCPVCs showed a stable phenotype while undergoing rapid ex vivo expansion at passage 2 (p2) to passage 4 (p4) and produced proregenerative factors, strongly suppressing T cell responses in the resting state and in response to inflammation. Transplanting an islet equivalent (IEQ):HUCPVC ratio of 1:30 under the kidney capsule in diabetic NSG mice demonstrated the fastest return to normoglycemia by 3 days after transplant: Superior glycemic control was seen at both early (2.7 weeks) and later stages (7, 12, and 16 weeks) versus ratios of 1:0, 1:10, and 1:50, respectively. Syngeneic islet transplantation in immunocompetent mice using the clinically relevant hepatic portal route with a marginal islet mass showed that mice transplanted with an IEQ:HUCPVC ratio of 1:150 had superior glycemic control versus ratios of 1:0, 1:90, and 1:210 up to 6 weeks after transplant. Immunodeficient mice transplanted with human islets (IEQ:HUCPVC ratio of 1:150) exhibited better glycemic control for 7 weeks after transplant versus islet transplant alone, and islets transplanted via the hepatic portal vein in an allogeneic mouse model using a curative islet mass demonstrated delayed rejection of islets when cotransplanted with HUCPVCs (IEQ:HUCPVC ratio of 1:150). The immunosuppressive and proregenerative properties of HUCPVCs demonstrated long-term positive effects on graft function in vivo, indicating that they may improve long-term human islet allotransplantation outcomes.
Because of the lack of tissue available for islet transplantation, new sources of β-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the ...human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting α-like cells. Transdifferentiation or reprogramming toward insulin-secreting β-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-β1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.
IntroductionLiver cirrhosis is a growing global healthcare challenge. Cirrhosis is characterised by severe liver fibrosis, organ dysfunction and complications related to portal hypertension. There ...are no licensed antifibrotic or proregenerative medicines and liver transplantation is a scarce resource. Hepatic macrophages can promote both liver fibrogenesis and fibrosis regression. The safety and feasibility of peripheral infusion of ex vivo matured autologous monocyte-derived macrophages in patients with compensated cirrhosis has been demonstrated.Methods and analysisThe efficacy of autologous macrophage therapy, compared with standard medical care, will be investigated in a cohort of adult patients with compensated cirrhosis in a multicentre, open-label, parallel-group, phase 2, randomised controlled trial. The primary outcome is the change in Model for End-Stage Liver Disease score at 90 days. The trial will provide the first high-quality examination of the efficacy of autologous macrophage therapy in improving liver function, non-invasive fibrosis markers and other clinical outcomes in patients with compensated cirrhosis.Ethics and disseminationThe trial will be conducted according to the ethical principles of the Declaration of Helsinki 2013 and has been approved by Scotland A Research Ethics Committee (reference 15/SS/0121), National Health Service Lothian Research and Development department and the Medicine and Health Care Regulatory Agency-UK. Final results will be presented in peer-reviewed journals and at relevant conferences.Trial registration numbersISRCTN10368050 and EudraCT; reference 2015-000963-15
The neighbor of Brca1 gene (Nbr1) functions as an autophagy receptor involved in targeting ubiquitinated proteins for degradation. It also has a dual role as a scaffold protein to regulate ...growth-factor receptor and downstream signaling pathways. We show that genetic truncation of murine Nbr1 leads to an age-dependent increase in bone mass and bone mineral density through increased osteoblast differentiation and activity. At 6 mo of age, despite normal body size, homozygous mutant animals (Nbr1 tr/tr ) have ∼50% more bone than littermate controls. Truncated Nbr1 (trNbr1) co-localizes with p62, a structurally similar interacting scaffold protein, and the autophagosome marker LC3 in osteoblasts, but unlike the full-length protein, trNbr1 fails to complex with activated p38 MAPK. Nbr1 tr/tr osteoblasts and osteoclasts show increased activation of p38 MAPK, and significantly, pharmacological inhibition of the p38 MAPK pathway in vitro abrogates the increased osteoblast differentiation of Nbr1 tr/tr cells. Nbr1 truncation also leads to increased p62 protein expression. We show a role for Nbr1 in bone remodeling, where loss of function leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis.
Aims/hypothesis
Type 1 diabetes complicated by hypoglycaemia is prevalent in socioeconomically deprived populations. Islet transplantation is of proven efficacy in type 1 diabetes complicated by ...hypoglycaemia, but it is not known if nationally funded programmes reach the socioeconomically deprived. Our aim was to determine: (1) socioeconomic indices in participants referred to our nationally funded programme; and (2) if metabolic outcomes in our transplant recipients were improved.
Methods
Participants referred (
n
= 106) and receiving transplants (
n
= 18; 32 infusions) were examined with respect to socioeconomic status (deprivation category score) and their ability to work and drive. In participants followed for ≥12 months after transplantation, metabolic and anthropometric measurements (
n
= 14) were recorded pre- and post-transplant (assessed ~1, ~3, ~6 and ~12 months with mixed-meal tolerance tests and 6 day continuous glucose monitoring assessments). Donor data was also examined.
Results
There was a greater prevalence of socioeconomic deprivation in referred and transplant recipients than the general population (
p
< 0.05). Of the transplant recipients, 73% were socioeconomically deprived, 88% did not hold a driver’s license and 94% had reduced ability to work (all
p
< 0.01 vs referred participants). Donors were predominantly obese and included circulatory death donors. At 12 months, 93% of participants who had received transplants had graft function, diminished frequency of hypoglycaemia (10 4–11 vs 0 0–2 hypoglycaemic episodes/week), improved awareness of hypoglycaemia (Gold score 7 5–7 vs 1 1–2) and glycaemic control (HbA
1c
: 7.9% 7.2–8.5%; 63 55–69 mmol/mol vs 7.2% 6.8–7.5%; 55 51–58 mmol/mol), diminished glycaemic lability and decreased central adiposity (all
p
< 0.05).
Conclusions/interpretation
A nationally funded islet transplant programme reaches the socioeconomically deprived and outcomes are significantly improved in this group.
Limbal stem cell deficiency (LSCD) is a disease resulting from the loss or dysfunction of epithelial stem cells, which seriously impairs sight. Autologous limbal stem cell transplantation is ...effective in unilateral or partial bilateral disease but not applicable in total bilateral disease. An allogeneic source of transplantable cells for use in total bilateral disease can be obtained from culture of donated cadaveric corneal tissue. We performed a controlled multicenter study to examine the feasibility, safety, and efficacy of allogeneic corneal epithelial stem cells in the treatment of bilateral LSCD. Patients were randomized to receive corneal epithelial stem cells cultured on amniotic membrane (AM): investigational medicinal product (IMP) or control AM only. Patients received systemic immunosuppression. Primary endpoints were safety and visual acuity, secondary endpoint was change in composite ocular surface score (OSS). Sixteen patients were treated and 13 patients completed all assessments. Safety was demonstrated and 9/13 patients had improved visual acuity scores at the end of the trial, with no significant differences between IMP and control groups. Patients in the IMP arm demonstrated significant, sustained improvement in OSS, whereas those in the control arm did not. Serum cytokine levels were measured during and after the period of immune suppression and we identified strongly elevated levels of CXCL8 in the serum of patients with aniridia, which persisted throughout the trial. This first randomized control trial of allogeneic corneal epithelial stem cells in severe bilateral LSCD demonstrates the feasibility and safety of this approach. Stem Cells Translational Medicine 2019;8:323–331
Patients with severe ocular surface disorder received transplants of amniotic membrane with (black bars) or without (gray bars) cadaveric‐donor‐derived cultured limbal stem cells. All patients received immune suppression. Only patients who received transplants containing limbal stem cells showed sustained significant improvements (reductions) in combined ocular surface scores (5 factors scored 0–3 where 0 is a normal eye score).
Adoptive immunotherapy with Epstein–Barr virus (EBV)-specific T cells is an effective treatment for relapsed or refractory EBV-induced post-transplant lymphoproliferative disorders (PTLD) with ...overall survival rates of up to 69%. EBV-specific T cells have been conventionally made by repeated stimulation with EBV-transformed lymphoblastoid cell lines (LCL), which act as antigen-presenting cells. However, this process is expensive, takes many months, and has practical risks associated with live virus. We have developed a peptide-based, virus-free, serum-free closed system to manufacture a bank of virus-specific T cells (VST) for clinical use. We compared these with standard LCL-derived VST using comprehensive characterization and potency assays to determine differences that might influence clinical benefits. Multi-parameter flow cytometry revealed that peptide-derived VST had an expanded central memory population and less exhaustion marker expression than LCL-derived VST. A quantitative HLA-matched allogeneic cytotoxicity assay demonstrated similar specific killing of EBV-infected targets, though peptide-derived EBV T cells had a significantly higher expression of antiviral cytokines and degranulation markers after antigen recall. High-throughput T cell receptor-beta (TCRβ) sequencing demonstrated oligoclonal repertoires, with more matches to known EBV-binding complementary determining region 3 (CDR3) sequences in peptide-derived EBV T cells. Peptide-derived products showed broader and enhanced specificities to EBV nuclear antigens (EBNAs) in both CD8 and CD4 compartments, which may improve the targeting of highly expressed latency antigens in PTLD. Importantly, peptide-based isolation and expansion allows rapid manufacture and significantly increased product yield over conventional LCL-based approaches.
Blood transfusion is a mainstay of modern medical practice. In many parts of the world the use of this life-saving therapy is hampered by issues of supply and the potential for transfusion ...transmitted infections. Accordingly, there are many studies seeking to find an alternative to donated red blood cells (RBCs) for transfusion, including large-scale production from adult and pluripotent stem cells, or erythroid cell lines. Translating basic studies, using any cell lineage, into protocols that are suitable for the generation of cellular therapies requires a wide range of biological and regulatory procedures to be put in place. Additionally, there are specific challenges for the production of RBCs caused by the number of cells needed for a single dose (approx. 1-2 × 10
). In this chapter, we will review critical areas in the development and good manufacturing practice (GMP) translation of cellular therapeutics through to early phase clinical trials and how this learning can be applied to in vitro RBC therapies.
Abstract Background aims Macrophages have complex roles in the liver. The aim of this study was to compare profiles of human monocyte-derived macrophages between controls and cirrhotic patients, to ...determine whether chronic inflammation affects precursor number or the phenotype, with the eventual aim to develop a cell therapy for cirrhosis. Methods Infusion of human macrophages in a murine liver fibrosis model demonstrated a decrease in markers of liver injury (alanine transaminase, bilirubin, aspartate transaminase) and fibrosis (transforming growth factor-β, α-smooth muscle actin, phosphatidylserine receptor) and an increase in markers of liver regeneration (matrix metalloproteinases MMP-9, MMP-12 and TNF-related weak inducer of apoptosis). CD14+ monocytes were then isolated from controls. Monocytes were matured into macrophages for 7 days using a Good Manufacturing Practice–compatible technique. Results There was no significant difference between the mean number of CD14+ monocytes isolated from cirrhotic patients (n = 9) and controls (n = 10); 2.8 ± SEM 0.54 × 108 and 2.5 ± 0.56 × 108 , respectively. The mean yield of mature macrophages cultured was also not significantly different between cirrhotic patients and controls (0.9 × 108 ± 0.38 × 108 , with more than 90% viability and 0.65 × 108 ± 0.16 × 108 , respectively. Maturation to macrophages resulted in up-regulation of a number of genes (MMP-9, CCL2, interleukin IL-10 and TNF-related weak inducer of apoptosis). A cytokine and chemokine polymerase chain reaction array, comparing the control and cirrhotic macrophages, revealed no statistically significant differences. Conclusions Macrophages can be differentiated from cirrhotic patients' apheresis-derived CD14 monocytes and develop the same pro-resolution phenotype as control macrophages, indicating their suitability for clinical therapy.
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