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). (C) 2019 American Society for Blood and Marrow Transplantation

Lipid content in the liver.

<p>(A) Lathosterol content in the liver from animals treated for 10 weeks, n=10 per group. (B) Lathosterol content in the liver from animals treated for 25 weeks, n=7 in controls, n=9 in the other groups. (C) Free cholesterol and cholesteryl ester content in liver from animals treated for 10 weeks, n=10 per group. (D) Free cholesterol and cholesteryl ester content in liver from animals treated for 25 weeks, n=7 in controls, n=9 in the other groups. (E) Triglyceride content in liver from animals treated for 10 weeks, n=10 per group. (F) Triglyceride content in liver from animals treated for 25 weeks, n=7 in controls, n=9 in the other groups. All data are corrected for protein content and show mean ± SEM.</p

Serum profiling of granulocyte and monocyte differentiation factors and of chemokines and cytokines.

<p>Granulocytes and monocytes differentiation factors, cytokines and chemokines measured in sera, n=5 per group. Data show mean ± SEM for the control group. Panel 2A presents the data for granulocyte-macrophage colony-stimulating factor (GM-CSF). Panel 2B presents the data for monocyte chemoattractant protein 1 (MCP-1), interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF) alpha and interferon γ.</p

Fecal excretion.

<p>(A) Fecal excretion of neutral sterols from animals treated for 10 weeks, feces was collected group wise (pooled) for 48h, n=10 per group. (B) Fecal excretion of bile acids from animals treated for 10 weeks, feces was collected group wise (pooled) for 48h, n=10 per group.</p

Cholesterol content in aorta.

<p>(A) Free cholesterol and cholesteryl ester content in the aorta from mice treated for 10 weeks, n=10 in control and KB group, n= 9 in Atorvastatin group and combination group. (B) Free cholesterol and cholesteryl ester content in the aorta from mice treated for 25 weeks, n=6 in controls, n=7 in KB group, n= 8 in Atorvastatin group and combination group. Values in (A) and (B) are corrected for protein levels and show mean ± SEM. (C) Hematoxylin/eosin (1,3) and oil red staining (2,4) of aorta from one control animal treated for 25 weeks (1, 2) and one animal treated with KB3495 for 25 weeks (3, 4). pl indicates plaque formation, sv indicates semilunar valves. Scalebar in 2 represents 300 μm and is valid for 1-4. </p

Lipid content in the skin.

<p>(A) Free cholesterol and cholesteryl ester content in the skin, data are corrected for protein levels and show mean ± SEM. n=7 in controls, n=9 in KB group, n= 9 in atorvastatin group, n=8 combination group. (B) Histological staining of skin with the specific macrophage Mac-3 marker. ED indicates epidermis, D indicates dermis. 1: control, 2: KB3495, 3: atorvastatin, 4: combination of KB3495 and atorvastatin.</p

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

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

Total bile acid concentration in gallbladder bile collected from control mice or humanized mice, with or without injection of FGF19.

<p>Statistics were performed on logtransformed data inorder to stabilize variances.</p><p>prior to one-way ANOVA followed by post-hoc analysis according to the least.</p><p>significance differance (LSD) test. In humanized mice the average bile acid levels was significantly lower after injection of FGF19, p = 0.001 and also in wild type mice bile acid levels was lower after injection of FGF19, p = 0.01. The overall significance of the experiment was p = 0.0048.</p

Lipoproteins in mouse serum.

<p>A, Serum cholesterol lipoprotein profiles measured by size exclusion chromatography of wild type mice, human, high (90%) and low (45%) levels of repopulation in humanized FRG mice. Panel B showing percentage of different lipoprotein fractions, as well as ratio of LDL/HDL in wild type mice, human controls, repopulated FRG mice and FRG controls. C, Western blot analysis of human (h) and mouse (m) Apolipoprotein E in serum samples of human and mouse control samples, 1–6. Humanized FRG with different levels of repopulation are shown in lane 7–9.</p