Effect of Graft-versus-Host Disease Prophylaxis Regimens on T and B Cell Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation.

Lymphocyte reconstitution is pivotal for successful long-term outcome after allogeneic hematopoietic stem cell transplantation (HSCT), and conditioning regimen and post-transplantation immunosuppression are risk factors for prolonged immunodeficiency. Nevertheless, the effects of different immunosuppressive protocols on lymphocyte output and replicative capacity have not been investigated. Here we assessed T cell receptor excision circles (TREC), kappa-deleting recombination excision circles (KREC), and T cell telomere length (TL) as proxy markers for immune reconstitution in patients in a prospective randomized trial comparing graft-versus-host disease (GVHD) prophylaxis after transplantation (cyclosporine/methotrexate versus tacrolimus/sirolimus; n = 200). Results showed that medians of TREC, KREC, and TL were not significantly different between the prophylaxis groups at any assessment time point during follow-up (24 months), but the kinetics of TREC, KREC, and TL were significantly influenced by other transplantation-related factors. Older recipient age, the use of antithymocyte globulin before graft infusion, and use of peripheral blood stem cell grafts were associated with lower TREC levels, whereas acute GVHD transiently affected KREC levels. Patients with lymphocyte excision circle levels above the median at ≤6 months post-transplantation had reduced transplantation-related mortality and superior 5-year overall survival (P < .05). We noticed significant T cell telomere shortening in the patient population as a whole during follow-up. Our results suggest that lymphocyte reconstitution after transplantation is not altered by different immunosuppressive protocols. This study has been registered at ClinicalTrials.gov (identifier: NCT00993343).

Circulating Fibroblast Growth Factor 19 in Portal and Systemic Blood.

BACKGROUND: Bile acid homeostasis is essential and imbalance may lead to liver damage and liver failure. The bile acid induced intestinal factor fibroblast growth factor 19 (FGF19) has been identified as a key protein for mediating negative feedback inhibition of bile acid synthesis. The aim of the study was to define FGF19 and bile acid concentrations in portal and systemic blood in the fasted and postprandial state. We also addressed the question if physiological portal levels of FGF19 can be extrapolated from the concentration in systemic blood. METHODS: Portal and systemic blood was collected from 75 fasted patients undergoing liver surgery and from three organ donors before and after enteral nutrition. Serum concentration of FGF19 was determined with ELISA and bile acid concentration with gas chromatography-mass spectrometry. RESULTS: Concentration of bile acids was twice as high in portal compared to systemic blood in the fasted group and 3-5 times higher in the postprandial group. FGF19 increased after enteral nutrition but did not differ between portal and systemic blood, in either group. In addition, a strong, positive correlation between bile acids and FGF19 was found. CONCLUSION: Our results confirm that bile acids drive the postprandial increase of circulating FGF19 but a hepatic clearance of FGF19 is unlikely. We conclude that systemic concentrations of FGF19 reflect portal concentrations of FGF19.

Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells.

BACKGROUND: The use of CD19 chimeric antigen receptor (CAR) T cells to treat B-cell malignancies has proven beneficial. Several groups use serum to produce CD19 CAR T cells. Today, ready-to-use serum-free media that require no addition of serum are commercially available. Therefore, it becomes important to evaluate the production of CD19 CAR T cells with and without the addition of serum. METHODS: T cells from buffy coats were cultured in AIM-V and TexMACS (TM) supplemented with 5% human serum (A5% and TM5%, respectively), and in TM without serum. Cells were activated with OKT3 and expanded in interleukin (IL)-2. Viral transduction was performed in RetroNectin-coated plates using the spinoculation method. CD19 CAR T cells were tested for their viability, expansion, transduction efficacy, phenotype and cytotoxicity. RESULTS: CD19 CAR T cells expanded in A5% and TM5% showed significantly better viability and higher fold expansion than cells expanded in TM. TM promoted the expansion of CD8+ T cells and effector phenotype of CD19 CAR T cells. The transduction efficacy and the cytotoxic function were comparable between the different media. Higher CD107a+ cells were detected in TM and TM5%, whereas higher IL-2+ and IL-17+ cells were detected in A5%. CD19 CAR exhibited co-expression of inhibitory receptors such as TIM-3+LAG-3+ and/or TIM-3+PD-1+. CONCLUSION: Our results indicate that serum supplementation promotes better CD19 CAR T-cell expansion and viability in vitro. CD19 CAR T cells produced in TM medium showed lower CD4/CD8 ratio, which warrants further evaluation in clinical settings. Overall, the choice of culture medium impacts CD19 CAR T-cell end product.

Exogenous alpha 1-antitrypsin down-regulates SERPINA1 expression.

The main goal of the therapy with purified human plasma alpha1-antitrypsin (A1AT) is to increase A1AT levels and to prevent lungs from elastolytic activity in patients with PiZZ (Glu342Lys) A1AT deficiency-related emphysema. Potential hepatic gains of this therapy are unknown. Herein, we investigated the effect of A1AT therapy on SERPINA1 (gene encoding A1AT) expression. The expression of SERPINA1 was determined in A1AT or A1AT plus Oncostatin M (OSM) treated primary human hepatocytes isolated from liver tissues from A1AT deficient patients and control liver tissues. In addition, SERPINA1 mRNA was assessed in lung tissues from PiZZ emphysema patients with and without A1AT therapy, and in adherent human peripheral blood mononuclear cells (PBMC) isolated from healthy PiMM donors. In a dose-dependent manner purified A1AT lowered SERPINA1 expression in hepatocytes. This latter effect was more prominent in hepatocytes stimulated with OSM. Although it did not reach statistical significance (P = 0.0539)-analysis of lung tissues showed lower SERPINA1 expression in PiZZ emphysema patients receiving augmentation therapy relative to those without therapy. Finally, exogenously added purified A1AT (1mg/ml) reduced SERPINA1 expression in naïve as well as in lipopolysaccharide (LPS)-stimulated human adherent PBMCs. Exogenous A1AT protein reduces its own endogenous expression. Hence, augmentation with native M-A1AT protein and a parallel reduction in expression of dysfunctional mutant Z-A1AT may be beneficial for PiZZ liver, and this motivates further studies.

Addition of Dexamethasone Alters the Bile Acid Composition by Inducing CYP8B1 in Primary Cultures of Human Hepatocytes.

BACKGROUND: Primary human hepatocytes offer the best human in vitro model for studies on human liver cell metabolism. Investigators use a variety of different media supplements and matrix biocoatings and the type of culture system used may influence the outcome. OBJECTIVES: To optimize in vitro conditions for primary human hepatocytes with regard to bile acid synthesis. METHODS: Human hepatocytes were isolated and cultured on collagen type I or EHS matrigel in cell media with or without dexamethasone. The glucocorticoid receptor (GR) antagonist RU486 was used to elucidate the involvement of GR. RESULTS: Hepatocytes cultured on EHS matrigel produced more bile acids and expressed higher levels of cholesterol 7α-hydroxylase (CYP7A1) than cells cultured on rat tail collagen. Supplementation with dexamethasone increased the formation of cholic acid (CA) and decreased chenodeoxycholic acid formation. In line with these results, the mRNA expression of sterol 12α-hydroxylase (CYP8B1) increased following dexamethasone treatment. Surprisingly, the mRNA expression of CYP7A1 and CYP27A1 was not increased to the same extent. By using the GR antagonist RU486, we concluded that CYP8B1 induction is mediated via a GR-independent pathway. An altered expression of retinoid-related orphan receptor (ROR) α and ROR α target gene Glucose-6-phosphatase (G6Pase) suggests that ROR α signaling may regulate CYP8B1 expression. CONCLUSION: Primary human hepatocytes have an increased bile acid synthesis rate when cultured on matrigel as compared to collagen. Exposure to glucocorticoid hormones stimulates the expression of CYP8B1, leading to an increased formation of CA and alteration of the bile acid composition. The effect is most likely mediated through a GR-independent pathway, possibly through ROR α.

Strategies for short-term storage of hepatocytes for repeated clinical infusions.

Hepatocyte transplantation is an upcoming treatment for patients with metabolic liver diseases. Repeated cell infusions over 1-2 days improve clinical outcome. Isolated hepatocytes are usually cold stored in preservation solutions between repeated infusions. However, during cold storage isolated hepatocytes undergo cell death. We investigated if tissue preservation and repeated isolations are better than storage of isolated hepatocytes when cold preserving human hepatocytes. Liver tissue obtained from liver surgery or organ donors was divided into two pieces. Hepatocytes were isolated by collagenase digestion. Hepatocytes were analyzed directly after isolation (fresh) or after storage for 48 h at 4°C in University of Wisconsin solution (UW cells). Liver tissue from the same donor was stored at 4°C in UW and hepatocytes were isolated after 48 h (UW tissue cells). Hepatocyte viability and function was evaluated by trypan blue exclusion, plating efficiency, ammonia metabolism, CYP 1A1/2, 2C9, 3A7, and 3A4 activities, phase II conjugation, and apoptosis evaluation by TUNEL assay and caspase-3/7 activities. Hepatocytes stored in UW showed a significantly lower viability compared to fresh cells or hepatocytes isolated from tissue stored for 48 h (54% vs. 71% vs. 79%). Plating efficiency was significantly decreased for cells stored in UW (40%) compared to fresh and UW tissue cells (63% vs. 55%). No significant differences between UW cells and UW tissue cells could be shown for CYP activities or ammonia metabolism. Hepatocytes stored in UW showed a strong increase in TUNEL-positive cells, whereas TUNEL staining in cold-stored liver tissue and hepatocytes isolated after 48 h was unchanged. This observation was confirmed by increased caspase-3/7 activities in UW cells. Although preservation of isolated hepatocytes in UW maintains function, cold storage of liver tissue and repeated hepatocyte isolations is superior to cold storage of isolated hepatocytes in preserving hepatocyte viability and function.

The thyroid receptor modulator KB3495 reduces atherosclerosis independently of total cholesterol in the circulation in ApoE deficient mice.

BACKGROUND: Thyroid hormones (TH) regulate cholesterol metabolism but their use as lipid-lowering drugs is restricted due to negative cardiac effects. TH mimetic compounds modulating TH receptor ß (THRß) have been designed as potential drugs, reducing serum cholesterol levels while avoiding apparent deleterious cardiac effects. OBJECTIVE: Using ApoE deficient mice, we examined whether KB3495, a TH mimetic compound, reduces atherosclerosis and if there is a synergistic effect with atorvastatin. The effect of KB3495 was investigated after 10 and 25 weeks. RESULTS: KB3495 treatment reduced atherosclerotic plaque formation in aorta and decreased the cholesteryl ester (CE) content by 57%. Treatment with KB3495 was also associated with a reduction of macrophage content in the atherosclerotic plaques and reduced serum levels of IL-1ß, TNFalpha, IL-6, Interferon γ, MCP-1 and M-CSF. Serum lipoprotein analysis showed no change in total cholesterol levels in ApoB-containing lipoproteins. KB3495 alone increased fecal BA excretion by 90%. The excretion of neutral sterols increased in all groups, with the largest increase in the combination group (350%). After 25 weeks, the animals treated with KB3495 showed 50% lower CE levels in the skin and even further reductions were observed in the combination group where the CE levels were reduced by almost 95% as compared to controls. CONCLUSION: KB3495 treatment reduced atherosclerosis independently of total cholesterol levels in ApoB-containing lipoproteins likely by stimulation of sterol excretion from the body and by inhibition of the inflammatory response.

Mice with chimeric livers are an improved model for human lipoprotein metabolism.

OBJECTIVE: Rodents are poor model for human hyperlipidemias because total cholesterol and low density lipoprotein levels are very low on a normal diet. Lipoprotein metabolism is primarily regulated by hepatocytes and we therefore assessed whether chimeric mice extensively repopulated with human cells can model human lipid and bile acid metabolism. DESIGN: FRG [ F ah(-/-) R ag2(-/-)Il2r g (-/-)]) mice were repopulated with primary human hepatocytes. Serum lipoprotein lipid composition and distribution (VLDL, LDL, and HDL) was analyzed by size exclusion chromatography. Bile was analyzed by LC-MS or by GC-MS. RNA expression levels were measured by quantitative RT-PCR. RESULTS: Chimeric mice displayed increased LDL and VLDL fractions and a lower HDL fraction compared to wild type, thus significantly shifting the ratio of LDL/HDL towards a human profile. Bile acid analysis revealed a human-like pattern with high amounts of cholic acid and deoxycholic acid (DCA). Control mice had only taurine-conjugated bile acids as expcted, but highly repopulated mice had glycine-conjugated cholic acid as found in human bile. RNA levels of human genes involved in bile acid synthesis including CYP7A1, and CYP27A1 were significantly upregulated as compared to human control liver. However, administration of recombinant hFGF19 restored human CYP7A1 levels to normal. CONCLUSION: Humanized-liver mice showed a typical human lipoprotein profile with LDL as the predominant lipoprotein fraction even on a normal diet. The bile acid profile confirmed presence of an intact enterohepatic circulation. Although bile acid synthesis was deregulated in this model, this could be fully normalized by FGF19 administration. Taken together these data indicate that chimeric FRG-mice are a useful new model for human lipoprotein and bile-acid metabolism.

Comparison of culture media for bile Acid transport studies in primary human hepatocytes.

BACKGROUND: Primary human hepatocytes are a useful in vitro model system to examine hepatic biochemical pathways, liver disorders and/or pharmacotherapies. This system can also be used for transport studies to investigate uptake and excretion of bile acids. Proper modeling of hepatic function requires careful attention to media components, and culture substrates and conditions. OBJECTIVES: To examine the effects of different culture media and conditions on bile acid transport in cultured human hepatocytes. METHODS AND RESULTS: Hepatocytes cultured in Williams' medium E showed an increase in both uptake and excretion of taurocholate compared to cells cultured in Dulbecco's Modified Eagle Medium (DMEM). Supplementation of DMEM with glutathione or ascorbic acid did not compensate for the lower transport. The difference can be explained by lower mRNA expression of the transporter proteins sodium taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP; ABCB11) when cultured in DMEM. Hepatocytes cultured in DMEM also display fewer and smaller bile canaliculi. Following extended time in culture supplementation of Williams' medium E with dexamethasone increased the expression of NTCP and BSEP. CONCLUSION: Williams' medium E is superior to DMEM for transport studies in primary human hepatocytes. Supplementation with dexamethasone increase mRNA levels of NTCP and BSEP.