Jeremy Meyer

Microencapsulated human mesenchymal stem cells decrease liver fibrosis in mice

BACKGROUND & AIMS Mesenchymal stem cell (MSC) transplantation was shown to be effective for the treatment of liver fibrosis, but the mechanisms of action are not yet fully understood. We transplanted encapsulated human MSCs in two mouse models of liver fibrosis to determine the mechanisms behind the protective effect. METHODS Human bone marrow-derived MSCs were microencapsulated in novel alginate-polyethylene glycol microspheres. In vitro, we analyzed the effect of MSC-conditioned medium on the activation of hepatic stellate cells and the viability, proliferation, cytokine secretion, and differentiation capacity of encapsulated MSCs. The level of fibrosis induced by bile duct ligation (BDL) or carbon tetrachloride (CCl4) was assessed after intraperitoneal transplantation of encapsulated MSCs, encapsulated human fibroblasts, and empty microspheres. RESULTS MSC-conditioned medium inhibited hepatic stellate cell activation and release of MSC secreted anti-apoptotic (IL-6, IGFBP-2) and anti-inflammatory (IL-1Ra) cytokines. Viability, proliferation, and cytokine secretion of microencapsulated MSCs were similar to those of non-encapsulated MSCs. Within the microspheres, MSCs maintained their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. 23% (5/22) of the MSC clones were able to produce anti-inflammatory IL-1Ra in vitro. Microencapsulated MSCs significantly delayed the development of BDL- and CCl4-induced liver fibrosis. Fibroblasts had an intermediate effect against CCl4-induced fibrosis. Mice transplanted with encapsulated MSCs showed lower mRNA levels of collagen type I, whereas levels of matrix metalloproteinase 9 were significantly higher. Human IL-1Ra was detected in the serum of 36% (4/11) of the mice transplanted with microencapsulated MSCs. CONCLUSIONS MSC-derived soluble molecules are responsible for an anti-fibrotic effect in experimental liver fibrosis.

A focus on the role of platelets in liver regeneration: Do platelet-endothelial cell interactions initiate the regenerative process?

Platelets are involved in the early phases of liver regeneration. Moreover, platelet transfusion and thrombocytosis were recently shown to enhance hepatocyte proliferation. However, the precise mechanisms remain elusive. This review discusses the latest updates regarding the mechanisms by which platelets stimulate liver regeneration, focusing on their interactions with liver sinusoidal endothelial cells and on their fate within the liver. Following liver injury, platelets are recruited to and trapped within the liver, where they adhere to the endothelium. Subsequent platelet activation results in the release of platelet granules, which stimulate hepatocyte proliferation through activation of the Akt and ERK1/2 signalling pathways. Platelets activate liver sinusoidal endothelial cells, leading to the secretion of growth factors, such as interleukin-6. Finally, liver sinusoidal cells and hepatocytes can also internalize platelets, but the effects of this alternate process on liver regeneration remain to be explored. A better understanding of the mechanisms by which platelets stimulate liver regeneration could lead to improvement in post-operative organ function and allow hepatectomies of a greater extent to be performed.

Diabetogenic milieus induce specific changes in mitochondrial transcriptome and differentiation of human pancreatic islets

In pancreatic β-cells, mitochondria play a central role in coupling glucose metabolism to insulin secretion. Chronic exposure of β-cells to metabolic stresses impairs their function and potentially induces apoptosis. Little is known on mitochondrial adaptation to metabolic stresses, i.e. high glucose, fatty acids or oxidative stress; being all highlighted in the pathogenesis of type 2 diabetes. Here, human islets were exposed for 3 days to 25 mm glucose, 0.4 mm palmitate, 0.4 mm oleate and transiently to H2O2. Culture at physiological 5.6 mm glucose served as no-stress control. Expression of mitochondrion-associated genes was quantified, including the transcriptome of mitochondrial inner membrane carriers. Targets of interest were further evaluated at the protein level. Three days after acute oxidative stress, no significant alteration in β-cell function or apoptosis was detected in human islets. Palmitate specifically increased expression of the pyruvate carriers MPC1 and MPC2, whereas the glutamate carrier GC1 and the aspartate/glutamate carrier AGC1 were down-regulated by palmitate and oleate, respectively. High glucose decreased mRNA levels of key transcription factors (HNF4A, IPF1, PPARA and TFAM) and energy-sensor SIRT1. High glucose also reduced expression of 11 mtDNA-encoded respiratory chain subunits. Interestingly, transcript levels of the carriers for aspartate/glutamate AGC2, malate DIC and malate/oxaloacetate/aspartate UCP2 were increased by high glucose, a profile suggesting important mitochondrial anaplerotic/cataplerotic activities and NADPH-generating shuttles. Chronic exposure to high glucose impaired glucose-stimulated insulin secretion, decreased insulin content, promoted caspase-3 cleavage and cell death, revealing glucotoxicity. Overall, expression profile of mitochondrion-associated genes was selectively modified by glucose, delineating a glucotoxic-specific signature.

Routine chest X-ray is not mandatory after fluoroscopy-guided totally implantable venous access device insertion

BACKGROUND The aim of this study is to determine whether systematic postoperative chest X-ray is required after totally implantable venous access port device (TIVAD) placement under fluoroscopic control. METHODS A retrospective chart review of all consecutive patients with fluoroscopy-guided TIVAD insertion from July 10, 2009 to April 16, 2012 was conducted at the Geneva University Hospitals (n = 927). Patients with an available postoperative chest X-ray were included, regardless of approach (open or percutaneous) and venous access site (subclavian, cephalic, jugular, etc.). Exclusion criteria were incomplete data and preexisting pneumothorax or hemothorax. RESULTS Eight hundred ninety-one patients were included. First-intention venous cutdown was performed in 878 patients (98.5%), with success rates of 79.4% and 88.2% when targeting the left and right cephalic veins, respectively. Percutaneous access was the chosen first-intention procedure for 12 patients (1.3%). Eight-hundred thirty-six (93.8%) insertions were performed only by the open approach and 53 (5.9%) implantations required at least one venous puncture. Two implantations were performed using previous central venous accesses. Immediate complications associated with TIVAD placement and detected on the postoperative chest X-ray consisted of 1 asymptomatic pneumothorax, 1 symptomatic hemothorax, and 2 malpositions of the catheter. One additional pneumothorax was discovered during the first night after TIVAD insertion in a patient who became symptomatic. CONCLUSIONS The very low incidence of immediate complications detected by postprocedural chest X-ray suggests that such a control is not mandatory as a routine method after fluoroscopy-guided TIVAD insertion mainly performed by venous cutdown. X-ray should be performed only in cases of clinical suspicion.

A meta-analysis of extended versus standard lymphadenectomy in patients undergoing pancreatoduodenectomy for pancreatic adenocarcinoma.

BACKGROUND Lymph node involvement in pancreatic adenocarcinoma is a key prognostic factor. Therefore, extending the number of lymph node stations excised in pancreatoduodenectomy may be beneficial to patients with pancreatic adenocarcinoma. This systematic review and meta-analysis examines the outcomes of extended versus standard lymphadenectomy in the published literature. METHODS A meta-analysis of randomized controlled trials (RCTs) comparing extended with standard lymphadenectomy in patients undergoing pancreatoduodenectomy for pancreatic adenocarcinoma was performed. Perioperative outcomes were assessed as pooled odds ratios (ORs) and weighted mean differences. Overall survival was analysed for patients with positive and negative lymph nodes. Results were reported according to the PRISMA statement. RESULTS Five RCTs were included, accounting for 724 patients. Extended lymphadenectomy was associated with greater operative time [mean difference: 63 min, 95% confidence interval (CI) 29-96; P < 0.001], increased need for blood transfusions (mean difference: 0.20, 95% CI 0.01-0.30; P = 0.030) and greater postoperative morbidity (OR 1.5, 95% CI 1.25-2.00; P = 0.030), as well as with prolonged diarrhoea after circumferential autonomic nerve dissection around major vessels (OR 12.2, 95% CI 5.3-28.5; P < 0.001). Median survival was similar across the groups in the whole cohort, as well as in subgroups of patients with, respectively, positive and negative lymph nodes. CONCLUSIONS Extended lymphadenectomy has a harmful impact on patients undergoing oncological pancreatoduodenectomy compared with standard lymphadenectomy.

Cell rearrangement in transplanted human islets

The major feature of the human pancreatic islet architecture is the organization of endocrine cells into clusters comprising central β cells and peripheral α cells surrounded by vasculature. To have an insight into the mechanisms that govern this unique islet architecture, islet cells were isolated, and reaggregation of α and β cells into islet‐like structures (pseudoislets) after culture or transplantation into mice was studied by immunohistology. The pseudoislets formed in culture displayed an unusual cell arrangement, contrasting with the transplanted pseudoislets, which exhibited a cell arrangement similar to that observed in native pancreatic islet subunits. The pattern of revascularization and the distribution of extracellular matrix around transplanted pseudoislets were alike to those observed in native pancreatic islets. This organization of transplanted pseudoislets occurred also when revascularization was abolished by treating mice with an anti‐VEGF antibody, but not when contact with extracellular matrix was prevented by encapsulation of pseudoislets within alginate hydrogel. These results indicate that the maintenance of islet cell arrangement is dependent on in vivo features such as extracellular matrix but independent of vascularization.—Lavallard, V., Armanet, M., Parnaud, G., Meyer, J., Barbieux, C., Montanari, E., Meier, R., Morel, P., Berney, T., Bosco, D. Cell rearrangement in transplanted human islets. FASEB J. 30, 748–760 (2016). www.fasebj.org

Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review

To study the biological functions of liver sinusoidal endothelial cells (LSEC) and to identify their interplay with blood or liver cells, techniques allowing for the isolation and purification of LSEC have been developed over the last decades. The objective of the present review is to summarize and to compare the efficiency of existing methods for isolating murine LSEC. Toward this end, the MEDLINE database was searched for all original articles describing LSEC isolation from rat and mouse livers. Out of the 489 publications identified, 23 reported the main steps and outcomes of the procedure and were included in our review. Here, we report and analyse the technical details of the essential steps of the techniques used for LSEC isolation. The correlations between the prevalence of some steps and the efficiency of LSEC isolation were also identified. We found that centrifugal elutriation, selective adherence and, more recently, magnetic-activated cell sorting were used for LSEC purification. Centrifugal elutriation procured high yields of pure LSEC (for rats 30–141.9 million cells for 85–98% purities; for mice 9–9.25 million cells for >95% purities), but the use of this method remained limited due to its high technical requirements. Selective adherence showed inconsistent results in terms of cell yields and purities in rats (5–100 million cells for 73.7–95% purities). In contrast, magnetic-activated cell sorting allowed for the isolation of highly pure LSEC, but overall lower cell yields were reported (for rats 10.7 million cells with 97.6% purity; for mice 0.5–9 million cells with 90–98% purities). Notably, the controversies regarding the accuracy of several phenotypic markers for LSEC should be considered and their use for both magnetic sorting and characterization remain doubtful. It appears that more effort is needed to refine and standardize the procedure for LSEC isolation, with a focus on the identification of specific antigens. Such a procedure is required to identify the molecular mechanisms regulating the function of LSEC and to improve our understanding of their role in complex cellular processes in the liver.

Impact of myeloid-derived suppressor cell on Kupffer cells from mouse livers with hepatocellular carcinoma

ABSTRACT Kupffer cells represent the first line of defense against tumor cells in the liver. Myeloid-derived suppressor cells (MDSC) have recently been observed in the liver parenchyma of tumor-bearing animals. The present study investigates the function of the MDSC subsets, and their impact on Kupffer cell phenotype and function. RIL-175 mouse hepatocellular carcinoma (HCC) cells were injected into the median liver lobe of C57BL/6 mice. Three weeks later, the median lobe hosting the tumor nodule was removed, and Kupffer cells and MDSCs were sorted from the remaining liver. Mouse livers devoid of HCC served as control. Kupffer cells expressed less co-stimulatory CD86 and MHCII and more co-inhibitory CD274 molecules in HCC-bearing livers than in control livers. Corresponding to this phenotype, Kupffer cells from HCC-bearing mice were less efficient in their function as antigen-presenting cells. Three CD11b+ cell populations were identified and sorted from HCC-bearing mice. These cells had various phenotypes with different levels of MDSC-specific surface markers (Ly6Ghigh cells, Gr1high cells, and Ly6Clow cells), and may be considered as bonafide MDSCs given their suppression of antigen-specific T cell proliferation. Primary isolated Kupffer cells in co-culture with the three MDSC subsets showed a decrease in CCL2 and IL-18 secretion, and an increase in IL-10 and IL-1β secretion, and an increased expression of CD86, CD274, and MHCII. In conclusion, these data demonstrated the existence of three MDSC subsets in HCC-bearing animals. These cells altered Kupffer cell function and may decrease the migration and activation of anticancer effector cells in the liver.

Ampullectomy for an unexpected ampullary hamartoma in a heterotaxic patient

INTRODUCTION Heterotaxy designates rare congenital disorders of organ positioning in the thoracic and abdominal cavities, which can be associated with numerous anomalies, complicating the surgical management because of the loss of conventional anatomic landmarks. PRESENTATION OF CASE A 72-year-old man was found to have asymptomatic cholestasis. Further workup included computed tomography and magnetic resonance cholangiopancreatography that revealed anomalies of lateralization of digestive organs associated with intestinal malrotation and polysplenia, and a stone-like element in the main bile duct. Endoscopic retrograde cholangiopancreatography failed to extract the lesion. Laparotomy found no stone, but a polypoid tumor with ampullary implantation. Pancreaticoduodenectomy was judged unreasonable due to the presence of macroscopic cirrhosis and a complete ampullectomy was performed. Histopathological examination revealed a hamartomatous polyp. DISCUSSION The unusual angle of the duodenoscope in a left-sided duodenum may have contributed to the improper pre-operative diagnosis. Endosonography could have recognized the tissular origin of the lesion and prompted a more detailed preoperative planning. It was fortunate that the patient ended up receiving the appropriate treatment despite the absence of an adequate pre-operative diagnosis, as the option of performing an extended resection was ruled out due to the presence of cirrhosis. CONCLUSION Although heterotaxy leads to increased technical difficulties in performing usual endoscopic and surgical procedures, it can be safely managed by experienced surgeons as illustrated by the present case. Imaging modalities have limited sensitivity in the diagnosis of small ampullary tumors. As false-negatives are likely to occur, this possibility should guide the choice of the best operation.

An optimized method for mouse liver sinusoidal endothelial cell isolation.

The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions.