Pancreas-derived mesenchymal stromal cells share immune response-modulating and angiogenic potential with bone marrow mesenchymal stromal cells and can be grown to therapeutic scale under Good Manufacturing Practice conditions.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs). METHODS: MSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs. RESULTS: MSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions. CONCLUSIONS: LPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs.

Development, functional characterization and validation of methodology for GMP-compliant manufacture of phagocytic macrophages: A novel cellular therapeutic for liver cirrhosis.

BACKGROUND AIMS: Autologous macrophage therapy represents a potentially significant therapeutic advance for the treatment of severe progressive liver cirrhosis. Administration of macrophages has been shown to reduce inflammation and drive fibrotic scar breakdown and tissue repair in relevant models. This therapeutic approach is being assessed for safety and feasibility in a first-in-human trial (MAcrophages Therapy for liver CirrHosis [MATCH] trial). METHODS: We outline the development and validation phases of GMP production. This includes use of the CliniMACS Prodigy cell sorting system to isolate CD14+ cells; optimizing macrophage culture conditions, assessing cellular identity, product purity, functional capability and determining the stability of the final cell product. RESULTS: The GMP-compliant macrophage products have a high level of purity and viability, and have a consistent phenotypic profile, expressing high levels of mature macrophage markers 25F9 and CD206 and low levels of CCR2. The macrophages demonstrate effective phagocytic capacity, are constitutively oriented to an anti-inflammatory profile and remain responsive to cytokine and TLR stimulation. The process validation shows that the cell product in excipient is remarkably robust, consistently passing the viability and phenotypic release criteria up to 48 hours after harvest. CONCLUSIONS: This is the first report of validation of a large-scale, fully Good Manufacturing Practice-compliant, autologous macrophage cell therapy product for the potential treatment of cirrhosis. Phenotypic and functional assays confirm that these cells remain functionally viable for up to 48 h, allowing significant flexibility in administration to patients.

Scotblood 2015: Improving and delivering blood products, novel cellular therapies, and celebrating patients and donor engagement within transfusion services.

Blood Transfusion Services are striving to continually improve the efficacy and quality of their blood products whilst also simultaneously diversifying into novel cellular products. For this to be successful the relationships between the various arms of the organisation must be strong and interlinked. As new technologies impact on the products that blood transfusion services supply it should be noted that the interaction between the service and its donor base is also affected by advancing technologies. Social media has fundamentally altered the way in which the public can access information and news, as such blood services must engage and interact appropriately with these new forms of media. As a reflection of these challenges the Scotblood 2015 programme was focussed on service and product improvement, donor engagement and people centred transfusion. This commentary comprises summaries of the presentations, based in part on the abstracts provided by the speakers.

Islet transplantation from a nationally funded UK centre reaches socially deprived groups and improves metabolic outcomes.

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 (HbA1c: 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.

Phenotypic and functional characterization of macrophages with therapeutic potential generated from human cirrhotic monocytes in a cohort study.

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-ß, 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 × 10(8) and 2.5 ± 0.56 × 10(8), respectively. The mean yield of mature macrophages cultured was also not significantly different between cirrhotic patients and controls (0.9 × 10(8) ± 0.38 × 10(8), with more than 90% viability and 0.65 × 10(8) ± 0.16 × 10(8), 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.

Innovative research, improving quality, and celebrating patient successes and the role of donors within transfusion services.

A modern successful Blood service is reliant on numerous elements to continually improve. With the constant increments in regulatory and quality management legislation the backbone to the service requires the implementation and maintenance of a modern Quality management system. It also relies on successful relationships between the various arms of the organisation in driving the service forward, and finally it is dependent on a relationship between its donor base and the staff. It is vital that those involved with transfusion appreciate the impact that they are having on the donors and patients, and it is important to appreciate and engage with donors without whom there would be no transfusion or transplantation. As a reflection of this the Scotblood 2014 programme was focused on service improvement and people centred transfusion. This commentary comprises summaries of the presentations, based in part on the abstracts provided by the speakers.

Use of the BacT/alert system for rapid detection of microbial contamination in a pilot study using pancreatic islet cell products.

At the Islet Isolation Laboratory of the Scottish National Blood Transfusion Service, manual sterility testing data show that contamination rates are 57.7% for pancreas transport fluid, 4.3% for postpurification islet samples, and 0% for pretransplant islet samples. This pilot study presents the BacT/Alert System as an alternative to manual testing to provide more rapid and sensitive sterility results for islet cell products.

Optimal use of blood and innovative approaches to stem cells, regenerative medicine and donor recruitment.

The annual scientific meeting of the Scotblood National Blood Transfusion Service, (SNBTS), continues to enjoy success. Scotblood 2013 focused on the contemporary issues affecting the various essential areas of blood transfusion and transfusion medicine. Presentations ranged from the challenges of recruiting young donors, forecasting future blood demand and celebrating the success of the better blood transfusion program. The meeting also discussed potential future developments in regenerative medicine particularly the potential of mesenchymal stromal cells and discussion of the ongoing Bloodpharma project, the ultimate aim of developing cultured red blood cells. This commentary comprises summaries of the presentations, based in part on the abstracts provided by the speakers. The Scotblood Conference began with the welcoming introduction by SNBTS Director Mrs. Mary Morgan, during which she updated the ongoing developments within SNBTS over the last year. Mrs. Morgan described how SNBTS met the challenges and obstacles that have been prevalent in all Blood Transfusion Services, whilst also meeting the transfusion needs of the people of Scotland. Mrs. Morgan then introduced the keynote speaker Dr. Aileen Keel CBE, Deputy Chief Medical Officer of Scotland. Dr. Keel's presentation was entitled "Twenty years in the Scottish Government-edited highlights" in which she described the various challenges that have presented themselves to her throughout her career. Dr. Keel highlighted how the various risks in the blood transfusion field (from HCV, HIV through to nvCJD) have arisen and then reduced to miniscule levels through hard work and perseverance. The highlights of the conference are summarised below.

Scotblood 2012: transfusion/transplant medicine in the 2nd decade of the 21st century.

Transfusion medicine is a technology-based discipline, undergoing continual changes for improvement. It requires staff at all levels to be continually educated and trained in appropriate multidisciplinary skills, in line with the rapid developments in all areas of transfusion practice: from blood/organ collection, through processing and storage to the more advanced cellular and hospital-based transfusion/transplantation therapies. Whilst the majority of the challenges to improve hospital and general transfusion practice can be overcome through team work, education, timely objectives and perseverance, it is important to envisage opportunities for implementing digital technologies to reduce all of the applicable hazards associated with transfusion. These can vary widely from new and emerging pathogens to limitations of supply due to growing demographic changes in populations. In the first decade of 21st century we have already witnessed unprecedented advances in haematopoietic stem cell transplantation to minimise the toxicities of graft versus host disease (GvHD), and in cell therapy to explore immunotherapy against cancer and other malignant disorders. Today there are 1000 genome project hapmaps that only the extreme cost of their implementation to routine practices may limit. Transfusion medicine, like all disciplines of medicine, nevertheless, will face difficult choices between increasing healthcare technology and increasing worldwide health. Drs. Colligan and McGowan, the new lead organisers of this wonderful yearly educational programme have agreed to follow the previous organisers' strategy to make a summary report of their meeting to become available, through TRASCI to broader interested groups, with the sprit that "sharing is caring". The main highlights of the 2012 conference were: targeting transfusion practices in hospital, a continuing journey; emerging infections and the potential causes and possible remedial actions; building for the future; the challenging issues of donor recruitment/retention; and finally; the application of Information Technology as a decision making tool, utilising clinical audit monitoring to evaluate good practice. This year's conference also coincided with the retirement of Martin Bruce OBE, after his 41years distinguished career, who gave the most delightful and humorous talk of a" life time of learning" which delighted all the participants. Finally, 2012 also marked the retirements of the previous lead Scotblood organisers Prof. Robin Fraser and Dr. Hagop Bessos after over thirty years service to SNBTS, and to whom we would like to dedicate this meeting report and wish them a happy and healthy retirement. This commentary comprises summaries of the presentations, based in part on the abstracts provided by the speakers.

Neighbor of Brca1 gene (Nbr1) functions as a negative regulator of postnatal osteoblastic bone formation and p38 MAPK activity.

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 approximately 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.

Study protocol: a multicentre, open-label, parallel-group, phase 2, randomised controlled trial of autologous macrophage therapy for liver cirrhosis (MATCH).

INTRODUCTION: Liver 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 ANALYSIS: The 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 DISSEMINATION: The 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 NUMBERS: ISRCTN10368050 and EudraCT; reference 2015-000963-15.

Human umbilical cord perivascular cells improve human pancreatic islet transplant function by increasing vascularization.

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.

Binding Immunoglobulin Protein (BIP) Inhibits TNF-α-Induced Osteoclast Differentiation and Systemic Bone Loss in an Erosive Arthritis Model.

OBJECTIVE: The association between inflammation and dysregulated bone remodeling is apparent in rheumatoid arthritis and is recapitulated in the human tumor necrosis factor transgenic (hTNFtg) mouse model. We investigated whether extracellular binding immunoglobulin protein (BiP) would protect the hTNFtg mouse from both inflammatory arthritis as well as extensive systemic bone loss and whether BiP had direct antiosteoclast properties in vitro. METHODS: hTNFtg mice received a single intraperitoneal administration of BiP at onset of arthritis. Clinical disease parameters were measured weekly. Bone analysis was performed by microcomputed tomography and histomorphometry. Mouse bone marrow macrophage and human peripheral blood monocyte precursors were used to study the direct effect of BiP on osteoclast differentiation and function in vitro. Monocyte and osteoclast signaling was analyzed by Western blotting, flow cytometry, and imaging flow cytometry. RESULTS: BiP-treated mice showed reduced inflammation and cartilage destruction, and histomorphometric analysis revealed a decrease in osteoclast number with protection from systemic bone loss. Abrogation of osteoclast function was also observed in an ex vivo murine calvarial model. BiP inhibited differentiation of osteoclast precursors and prevented bone resorption by mature osteoclasts in vitro. BiP also induced downregulation of CD115/c-Fms and Receptor Activator of NF-κB (RANK) messenger RNA and protein, causing reduced phosphorylation of the p38 mitogen-activated protein kinases, extracellular signal-regulated kinases 1/2 and p38, with suppression of essential osteoclast transcription factors, c-Fos and NFATc1. BiP directly inhibited TNF-α- or Receptor Activator of NF-κB Ligand (RANKL)-induced NF-κB nuclear translocation in THP-1 monocytic cells and preosteoclasts by the canonical and noncanonical pathways. CONCLUSION: BiP combines an anti-inflammatory function with antiosteoclast activity, which establishes it as a potential novel therapeutic for inflammatory disorders associated with bone loss.

Allogeneic Ex Vivo Expanded Corneal Epithelial Stem Cell Transplantation: A Randomized Controlled Clinical Trial.

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.

Safety profile of autologous macrophage therapy for liver cirrhosis.

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 107, 108 or up to 109 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.

Good Manufacturing Practice (GMP) Translation of Advanced Cellular Therapeutics: Lessons for the Manufacture of Erythrocytes as Medicinal Products.

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 × 1012). 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.

Granulocyte colony-stimulating factor and autologous CD133-positive stem-cell therapy in liver cirrhosis (REALISTIC): an open-label, randomised, controlled phase 2 trial.

BACKGROUND: Results of small-scale studies have suggested that stem-cell therapy is safe and effective in patients with liver cirrhosis, but no adequately powered randomised controlled trials have been done. We assessed the safety and efficacy of granulocyte colony-stimulating factor (G-CSF) and haemopoietic stem-cell infusions in patients with liver cirrhosis. METHODS: This multicentre, open-label, randomised, controlled phase 2 trial was done in three UK hospitals and recruited patients with compensated liver cirrhosis and MELD scores of 11·0-15·5. Patients were randomly assigned (1:1:1) to receive standard care (control), treatment with subcutaneous G-CSF (lenograstim) 15 µg/kg for 5 days, or treatment with G-CSF for 5 days followed by leukapheresis and intravenous infusion of three doses of CD133-positive haemopoietic stem cells (0·2 × 106 cells per kg per infusion). Randomisation was done by Cancer Research UK Clinical Trials Unit staff with a minimisation algorithm that stratified by trial site and cause of liver disease. The coprimary outcomes were improvement in severity of liver disease (change in MELD) at 3 months and the trend of change in MELD score over time. Analyses were done in the modified intention-to-treat population, which included all patients who received at least one day of treatment. Safety was assessed on the basis of the treatment received. This trial was registered at Current Controlled Trials on Nov 18, 2009; ISRCTN, number 91288089; and the European Clinical Trials Database, number 2009-010335-41. FINDINGS: Between May 18, 2010, and Feb 26, 2015, 27 patients were randomly assigned to the standard care, 26 to the G-CSF group, and 28 to the G-CSF plus stem-cell infusion group. Median change in MELD from day 0 to 90 was -0·5 (IQR -1·5 to 1·1) in the standard care group, -0·5 (-1·7 to 0·5) in the G-CSF group, and -0·5 (-1·3 to 1·0) in the G-CSF plus stem-cell infusion group. We found no evidence of differences between the treatment groups and control group in the trends of MELD change over time (p=0·55 for the G-CSF group vs standard care and p=0·75 for the G-CSF plus stem-cell infusion group vs standard care). Serious adverse events were more frequent the in G-CSF and stem-cell infusion group (12 [43%] patients) than in the G-CSF (three [11%] patients) and standard care (three [12%] patients) groups. The most common serious adverse events were ascites (two patients in the G-CSF group and two patients in the G-CSF plus stem-cell infusion group, one of whom was admitted to hospital with ascites twice), sepsis (four patients in the G-CSF plus stem-cell infusion group), and encephalopathy (three patients in the G-CSF plus stem-cell infusion group, one of whom was admitted to hospital with encephalopathy twice). Three patients died, including one in the standard care group (variceal bleed) and two in the G-CSF and stem-cell infusion group (one myocardial infarction and one progressive liver disease). INTERPRETATION: G-CSF with or without haemopoietic stem-cell infusion did not improve liver dysfunction or fibrosis and might be associated with increased frequency of adverse events compared with standard care. FUNDING: National Institute of Health Research, The Sir Jules Thorn Charitable Trust.

How do we use ambulatory measurement of blood pressure in the management of hypertension?

We have explored the use and interpretation of ambulatory blood pressure monitoring (ABPM) among clinicians at an Edinburgh Cardiovascular Risk Clinic and among a group of international experts in blood pressure monitoring. Locally, we were able to demonstrate major discrepancies in management advice between doctors and nurses. Although all of the international experts used ABPM regularly, they did not agree on thresholds levels for treatment or target BP. This gives food for thought as we consider how to advise internists and primary care physicians on the use and interpretation of ABPM.

Generation of Functional Beta-Like Cells from Human Exocrine Pancreas.

Transcription factor mediated lineage reprogramming of human pancreatic exocrine tissue could conceivably provide an unlimited supply of islets for transplantation in the treatment of diabetes. Exocrine tissue can be efficiently reprogrammed to islet-like cells using a cocktail of transcription factors: Pdx1, Ngn3, MafA and Pax4 in combination with growth factors. We show here that overexpression of exogenous Pax4 in combination with suppression of the endogenous transcription factor ARX considerably enhances the production of functional insulin-secreting ß-like cells with concomitant suppression of α-cells. The efficiency was further increased by culture on laminin-coated plates in media containing low glucose concentrations. Immunocytochemistry revealed that reprogrammed cultures were composed of ~45% islet-like clusters comprising >80% monohormonal insulin+ cells. The resultant ß-like cells expressed insulin protein levels at ~15-30% of that in adult human islets, efficiently processed proinsulin and packaged insulin into secretory granules, exhibited glucose responsive insulin secretion, and had an immediate and prolonged effect in normalising blood glucose levels upon transplantation into diabetic mice. We estimate that approximately 3 billion of these cells would have an immediate therapeutic effect following engraftment in type 1 diabetes patients and that one pancreas would provide sufficient tissue for numerous transplants.