Donor specific anti-HLA antibodies in hematopoietic stem cell transplantation. Single Center prospective evaluation and desensitization strategies employed.

BACKGROUND: In the setting of mismatched-hematopoietic stem cells transplantation, the detection of antibodies directed against donor-specific HLA allele(s) or antigen(s) (DSA) represents a barrier for engraftment. It is thus necessary to plan an immunosuppressive strategy, or to select an alternative donor. This prospective study aimed at evaluating the efficacy of our strategy for testing DSAs and the efficacy of the desensitization strategy (DS) employed between November 2017 and November 2020. MATERIALS AND METHODS: The anti-HLA antibody search was performed using the Luminex bead assays (Lifecode ID and LSA I/II-Immucor) and expressed as mean fluorescence intensity (MFI >1,000 positive). If the patient had DSAs and no alternative donors, a DS was employed with rituximab (day -15), 2 single volume plasmaphereses (PP; days -9 and -8), intravenous immunoglobulins (day -7) and infusion of HLA selected platelets, if persistent DSAs were directed against class I HLA. DS was scheduled with or without PP, according to the DSA MFI (>1,000 or <5,000) and FCXM (flow cytometry crossmatch). RESULTS: Twenty-two out of 126 patients (17.46%) showed anti-HLA antibodies, 5 of them DSAs (3.97% of total); 3 patients underwent DS obtaining engraftment. Female gender (p=0.033) and a history of previous pregnancies or miscarriages (p=0.009) showed a statistically significant impact on alloimmunization. Factors associated with a delayed neutrophil engraftment were patient's female gender (p=0.039), stem cell source (p=0.025), and a high HSCT-specific comorbidity index (p=0.028). None of the analyzed variables, including the DSA detection, influenced engraftment. CONCLUSIONS: Our study confirms the importance to test DSAs in mismatched-hematopoietic stem cells transplantation The DS used proved successful in removing DSAs. Prospective multicenter studies are needed to better define and validate consensus strategies on DSA management in HSCT.

The effects of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids on the transcription of genes regulating their uptake and metabolism by the liver: influence of cellular oxidative state.

The influence of chylomicron remnants enriched in n-6 or n-3 polyunsaturated fatty acids (PUFA) on the expression of mRNA for the low density lipoprotein receptor (LDLr), LDLr-related protein (LRP), and peroxisome proliferator activated receptor alpha (PPAR(alpha)) was investigated in normal hepatocytes and after manipulation of the cellular oxidative state by incubation with N-acetyl cysteine (NAC) or CuSO(4). In normal cells, mRNA levels for the LDLr were unaffected by incubation with chylomicron remnants, but those for the LRP and PPAR(alpha) were downregulated by remnants enriched in n-3 as compared to n-6 PUFA, suggesting that the transcription of these genes are influenced directly by the type of fatty acid delivered to the liver from the diet. Treatment with NAC or CuSO(4) was found to shift the hepatocytes into a pro-reducing or pro-oxidizing state, respectively. The abundance of mRNA for the LDLr, LRP, and PPAR(alpha) was increased after incubation with remnants enriched in n-3, but not n-6, PUFA in pro-reducing as compared to pro-oxidizing cells, and PPAR(alpha) mRNA levels were also decreased by remnants high in n-6 PUFA in the more reduced cells. These results indicate that the effects of fatty acids from the diet delivered to the liver in chylomicron remnants on the expression of hepatic genes regulating their uptake and metabolism are modulated by the redox state of the cells, and that the type of fatty acid carried by the particles also plays a part in determining the response observed.

The effects of dietary n-3 polyunsaturated fatty acids delivered in chylomicron remnants on the transcription of genes regulating synthesis and secretion of very-low-density lipoprotein by the liver: modulation by cellular oxidative state.

The influence of chylomicron remnants enriched in n-3 or n-6 polyunsaturated fatty acids (PUFA) (derived from fish or corn oil, respectively) on the expression of mRNA for four genes involved in the regulation of the synthesis, assembly, and secretion of very-low-density lipoprotein (VLDL) in the liver was investigated in normal rat hepatocytes and after manipulation of the cellular oxidative state by incubation with N-acetyl cysteine (NAC) or CuSO(4). The four genes investigated were those encoding apolipoprotein B (apoB), the microsomal triacylglycerol transfer protein (MTP), and the enzymes acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2), which play a role in the regulation of triacylglycerol and cholesteryl ester synthesis, respectively. mRNA levels for apoB, MTP, and DGAT were unaffected by either fish or corn oil chylomicron remnants, but the amount of ACAT2 mRNA was significantly reduced after incubation of the hepatocytes with fish oil remnants as compared with corn oil remnants or without remnants. These findings indicate that the delivery of dietary n-3 PUFA to hepatocytes in chylomicron remnants downregulates the expression of mRNA for ACAT2, and this may play a role in their inhibition of VLDL secretion. However, when the cells were shifted into a pro-oxidizing or pro-reducing state by pretreatment with CuSO(4) (1 mM) or NAC (5 mM) for 24 hr, levels of mRNA for MTP were increased by about 2- or 4-fold, respectively, by fish oil remnants, whereas corn oil remnants had no significant effect. Fish oil remnants also caused a smaller increase in apoB mRNA in comparison with corn oil remnants in NAC-treated cells (+38%). These changes would be expected to lead to increased VLDL secretion rather than the decrease associated with dietary n-3 PUFA in normal conditions. These findings suggest that relatively minor changes in cellular redox levels can have a major influence on important liver functions such as VLDL synthesis and secretion.

Snail controls differentiation of hepatocytes by repressing HNF4alpha expression.

Epithelial-to-mesenchymal transition (EMT) is a coordinated process, occurring both during morphogenesis and tumor progression, that allows epithelial cells to dissociate from initial contacts and migrate to secondary sites. The transcriptional repressors of the Snail family induce EMT in different epithelial cell lines and their expression is strictly correlated with EMT during the development and progression of carcinomas. We have previously shown that EMT in hepatocytes correlates with the downregulation of hepatic differentiation key factors HNFs (hepatocyte nuclear factors), and in particular of HNF4alpha. Here, we demonstrate that Snail overexpression is sufficient (i) to induce EMT in hepatocytes with conversion of morphology, downregulation of several epithelial adhesion molecules, reduction of proliferation and induction of matrix metalloproteinase 2 expression and, (ii) most relevantly, to repress the transcription of the HNF4alpha gene through a direct binding to its promoter. These finding demonstrate that Snail is at the crossroads of the regulation of EMT in hepatocytes by a dual control of epithelial morphogenesis and differentiation.

Steatosis and intrahepatic lymphocyte recruitment in hepatitis C virus transgenic mice.

To assess the effects of constitutive hepatitis C virus (HCV) gene expression on liver, transgenic mice carrying the entire HCV open reading frame inserted in the alpha1 antitrypsin (A1AT) gene were generated. Expression of A1AT/HCV mRNA was found to be mainly limited to perivascular areas of the liver as indicated by in situ hybridization analysis. HCV core protein was detected in Western blots of liver extracts, whereas the expression of E2, NS3 and NS5 proteins was revealed by immunostaining of liver samples using HCV-specific antisera. Histological analysis of HCV transgenic mice showed that these animals develop extensive steatosis, but very little necrosis of liver tissue. Moreover, a consistent T cell infiltrate and a slight hepatocyte proliferation were observed. Phenotypic analysis of cells infiltrating the liver indicated that recruitment and/or expansion of residing CD8(+), NK, NKT and gammadelta T cells occurred in transgenic animals. Among these cells, a large fraction of CD8(+) T lymphocytes released mainly IL-10 and, to a lesser extent, IFN-gamma upon mitogenic stimulation in vitro. Furthermore, both intrahepatic lymphocytes and splenocytes did not produce cytokines in response to HCV antigens. Thus, these data indicate that constitutive expression of HCV proteins may be responsible for intrahepatic lymphocyte recruitment in absence of viral antigen recognition. This response is likely to be driven by virus-induced cellular factors and may play a significant role in the immunopathology of chronic HCV infection and liver disease.