Introduction

Acute cellular rejection (ACR) remains a common complication causing significant morbidity after liver transplantation.^1,2 Recent studies suggest that the proportion of non-HLA mismatched SNPs, referring to all SNPs outside the HLA region, also known as major histocompatibility complex (MHC), was associated with increased rejection risk after kidney transplantation.^3-6 This is currently unexplored in liver transplantation.

Aims & Methods

We aimed to investigate whether donor-recipient non-HLA genetic mismatch is associated with increased incidence of biopsy-proven grade 2/3 ACR, hereafter referred to solely as ACR, within 1-year after liver transplantation. We conducted an international multicenter case-control genome-wide association study of donor and liver transplant recipient pairs in three independent cohorts, totaling 1846 pairs. To assess the burden of donor-recipient genetic incompatibility, we calculated sum scores for single nucleotide polymorphism (SNP) mismatch based on all non-HLA functional SNPs. Additionally, we performed an analysis specifically including only SNPs coding for transmembrane or secreted proteins, which are more likely to affect the immune system. We analyzed the association between the log-normalized non-HLA mismatch scores and ACR in a multivariable cox regression model per cohort, followed by a weighted meta-analysis.

Results

During the first-year post-transplantation, 90 of 689 (13%), 161 of 720 (22%) and 48 of 437 (11%) recipients experienced ACR in cohorts 1-3, respectively. After correcting for potential confounders using multivariable cox regression analysis, our weighted meta-analyses showed that higher mismatch in functional non-HLA SNPs was associated with increased incidence of 1-year ACR (HR 5.31; 95%CI 1.39-20.08; P=0.015). Moreover, we also confirmed the effect of mismatch in the subset of SNPs coding only for transmembrane or secreted proteins on 1-year ACR (HR 6.69; 95%CI 1.82-24.78; P=0.004).

Conclusion

In this multi-center genome-wide meta-analysis we show that genetic non-HLA incompatibility between donors and recipients is an important independent predictor of ACR after liver transplantation. Donor-recipient mismatch of functional non-HLA SNPs overall and, especially, of SNPs coding for transmembrane or secreted proteins correlated with ACR developed within the first year after liver transplantation. Identifying high-risk immunological burden between donor-recipient pairs may potentially prevent early graft rejection and help future efforts to personalize immuno-suppressive medication. This is important as immune suppressive medication causes significant morbidity and mortality.

References

1. Burra P. The adolescent and liver transplantation. J Hepatol. 2012; doi:10.1016/j.jhep.2011.07.032
2. Choudhary NS, Saigal S, Bansal RK, et al. Acute and Chronic Rejection After Liver Transplantation: What A Clinician Needs to Know. J Clin Exp Hepatol. 2017; doi:10.1016/j.jceh.2017.10.003
3. Reindl-Schwaighofer R, Heinzel A, Kainz A, et al. Contribution of non-HLA incompatibility between donor and recipient to kidney allograft survival: genome-wide analysis in a prospective cohort. Lancet. 2019; doi:10.1016/S0140-6736(18)32473-5
4. Pineda S, Sigdel TK, Chen J, et al. Novel non-histocompatibility antigen mismatched variants improve the ability to predict antibody-mediated rejection risk in kidney transplant. Front Immunol. 2017; doi:10.3389/fimmu.2017.01687
5. Steers NJ, Li Y, Drace Z, et al. Genomic Mismatch at LIMS1 Locus and Kidney Allograft Rejection. N Engl J Med. 2019; 380(20):1918-1928. doi: 10.1056/NEJMoa1803731. PMID: 31091373; PMCID: PMC6589355.
6. Markkinen S, Helanterä I, Lauronen J, et al. Mismatches in Gene Deletions and Kidney-related Proteins as Candidates for Histocompatibility Factors in Kidney Transplantation. Kidney Int Rep. 2022; 7(11):2484-2494. doi: 10.1016/j.ekir.2022.08.032. PMID: 36531875; PMCID:

Introduction

Currently, there is no approved medication for the treatment of Metabolic Associated Fatty Liver Disease (MASLD). However, numerous clinical trials have examined the effect of antihyperglycemic agents on MASLD in type 2 diabetic (T2D) patients, given the close association between T2D and this hepatopathy, both of which are closely linked to insulin resistance. Our aim is to evaluate and compare the therapeutic effects of different hypoglycemic agents in patients with MASLD and T2D.

Aims & Methods

This is a prospective cross-sectional study involving 200 T2D patients over a 6-month period. The presence of hepatic steatosis was confirmed by abdominal ultrasound and/or a Fatty Liver Index (FLI) score greater than 60 in 122 patients. Hepatic fibrosis was assessed using FibroScan in all patients with MASLD. Two comparative studies were conducted to identify the therapeutic effects of hypoglycemic medications on hepatic steatosis and significant fibrosis, defined by a LSM value ≥ 8 kPa.

Results

Patients were predominantly female (73.2%) with a median age of 59.76 ± 10 years. The duration of T2D, although longer in cases of steatosis, was not statistically significant. Glycemic control, assessed by fasting blood glucose and HbA1c levels, was less optimal in patients with MASLD, with an average HbA1c level of 8.98%, compared to patients without hepatic steatosis, who had an average HbA1c level of 8.74% (p=0.002). A similar trend was observed with fasting blood glucose levels (81.96% vs 73.07%), but it did not reach statistical significance.

Degenerative complications, regardless of their type, were comparable between the two groups.

The prescription of biguanides was associated with a reduced risk of hepatic steatosis (p=0.05), while insulin use was frequently observed in patients with steatosis, with a statistically significant correlation for long-acting human insulin and rapid-acting analogs (33.61% vs 19.23% in the absence of steatosis, p=0.02) and (4.92% vs 0% in the absence of steatosis, p=0.05) respectively. No other therapeutic modality, including different classes of oral antidiabetic agents, was correlated with the risk of steatosis.

The prescription of sulfonylureas and rapid-acting insulin analogs was associated with a higher risk of significant hepatic fibrosis, with p-values of 0.05 and 0.008 respectively. In contrast, the use of long-acting human insulin was less frequent in patients with significant fibrosis, with a statistical correlation of 0.03. No other treatment modality, including various types of oral antidiabetic agents, was associated with the risk of fibrosis.

Conclusion

The use of biguanides appears to reduce the risk of hepatic steatosis, while insulin therapy, particularly rapid-acting analogs, is associated with an increased risk of hepatic steatosis and fibrosis. These observations emphasize the importance of an individualized therapeutic approach to optimize the management of MASLD in type 2 diabetic patients. Further studies are needed to validate these associations

References

Tang W, Xu Q, Hong T, Tong G, Feng W, Shen S, et al. Comparative efficacy of anti‐diabetic agents on nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus: a systematic review and meta‐analysis of randomized and non‐randomized studies. Diabetes Metabolism Res. févr 2016;32(2):200‑16.

Yan J, Yao B, Kuang H, Yang X, Huang Q, Hong T, et al. Liraglutide, Sitagliptin, and Insulin Glargine Added to Metformin: The Effect on Body Weight and Intrahepatic Lipid in Patients With Type 2 Diabetes Mellitus and Nonalcoholic Fatty Liver Disease. Hepatology. juin 2019;69(6):2414‑26.

Introduction

The treatments for liver fibrosis are still in need so far. As the crucial initiators, activated hepatic stellate cells (aHSCs) undergo a series of epigenetic changes. The study intends to explore transcription factors (TFs) that facilitate HSCs activation and provide novel therapeutic targets for liver fibrosis.

Aims & Methods

Three distinct HSCs activation models were established in present study. Firstly, in vitro activation involved isolating primary HSCs from normal Sprague-Dawley rats and culturing them for two days (n=5) and seven days (n=5), namely natural activation model. Secondly, in vivo activation entailed isolating primary HSCs from bile duct ligated rats (n=4) and normal controls (n=4), followed by culturing until the next day. Lastly, transforming growth factor-β (TGF-β, 2.5ng/ml) (n=10) or control buffer (n=10) were added to the human HSCs cell line LX2 and cultured for 24 hours. Subsequently, assay for transposase-accessible chromatin using sequencing (ATAC-seq) was conducted on the abovementioned cells, along with motif enrichment and footprint analysis. Pathway enrichment analysis was performed on differentially accessible genes (DAGs). RNA sequencing (RNA-seq) was employed to analyze gene expression profiles of primary HSCs in the natural activation model (n=4). The integration of ATAC-seq and RNA-seq further identified potential key TFs. RT-qPCR and differential expression analysis of public datasets was utilized to confirm the dysregulation of TFs in primary HSCs, fibrotic liver and fibrotic model of induced pluripotent stem cells. Finally, enrichment at enhancers and promoters of these genes was validated by H3K27Ac and H3K4me3 chromatin immunoprecipitation followed by sequencing (ChIP-seq) of human HSCs.

Results

For ATAC-seq, primary HSCs exhibited higher chromatin accessibility after activation compared with the controls. After peak annotation, the up-regulated peaks were mainly located in the promoter region in all comparison groups, uncovering the dynamic transcriptional regulation during HSC activation. In the motif enrichment analysis, 150 aHSCs related TFs and 54 quiescent HSCs (qHSCs) related TFs were identified in the in vitro activation model. The three models shared 12 aHSCs related TFs, namely Runx-aml, Runx1, Runx2, Runx, Ets:Runx, Atf2, Atf4, Atf7, c-Jun-Cre, Creb5, Smad3 and CArG; and 19 qHSCs related TFs, including Foxf1, Foxo3, Foxk1, Foxp1, Hoxa1, Hoxa2, Stat3, Hnf4a, Hnf6, Elk1, Elk4, Cux1, Crx, Duxbl, Vdr, Tr4, Ebf2, Gata2, Gata6. Footprint analysis validated the alteration of accessibility in Atf2, Atf4, Atf7, Creb5, Hnf6 and Gata2. Actin filament organization, Ras protein signal transduction and intrinsic apoptotic signal pathways were enriched in DAGs. Integration of ATAC-seq and RNA-seq showed that gene expression was significantly correlated with chromatin accessibility, among which Runx2, Creb5, and Smad3 were significantly upregulated in aHSCs at transcription level. The dysregulation of these three genes was verified in primary HSCs, fibrotic liver and fibrotic model of human induced pluripotent stem cells. Finally, enrichment at active enhancers and promoters of these TFs was validated by H3K27Ac and H3K4me3 ChIP-seq of human HSCs, revealing the crucial role of genes in the transcription.

Conclusion

We generated chromatin accessibility profiles of different HSCs activation models, highlighting the significance of Runx2, Creb5, and Smad3 as key TFs mediating HSCs activation. These findings proposed these TFs as promising targets for future liver fibrosis treatment.

Disclosure

The author declare no competing interests and financial relationships with commercial interests.

Introduction

The relationship between disruptions in the intestinal microbiome, psychological well-being, and modern lifestyle factors in individuals with prevalent non-communicable diseases, such as atherosclerosis, has been partially elucidated but remains inadequately investigated. This raises the inquiry into the feasibility of diagnosing, preventing, and treating atherosclerosis through the measurement and adjustment of disease markers associated with intestinal microbiome, psychological state, and lifestyle indicators.

Aims & Methods

This study aims to broaden the spectrum of these markers to a degree adequate for establishing models to diagnose atherosclerosis within the alternative categories of "no atherosclerosis," "asymptomatic atherosclerosis," and "symptomatic atherosclerosis," as well as by the level of associated health risks (non-significant, significant, and high). Our cohort study data were employed to fulfill this goal.

Results

We identified microbial markers from 955 gut microbiome species and other markers from 52 indicators of psychological state and lifestyle among participants. Marker properties were assessed based on their ability to distinguish between "no atherosclerosis," "asymptomatic atherosclerosis," and "symptomatic atherosclerosis" groups, as well as "not significant risk," "significant risk," and "high risk" groups. We measured their correlation with each participant's group membership using the p-level parameter to prioritize markers, resulting in a rating for each. Eleven microbial species (including Oxalobacter aliiformigenes, Lactobacillus acidophilus, and Bifidobacterium longum) and six other indicators (including "feeling of slowness" and "duration of physical activity") showed the highest scores. Cluster-type models based on these markers accurately predict group membership, with predictive accuracies ranging from 93.7% to 97.1%. This study provides novel insights into atherosclerosis-associated markers, laying a foundation for future research in this field.

Conclusion

The inclusion of microbial, psychological state, and lifestyle indicators has significantly enhanced the existing markers for atherosclerosis. These new markers have facilitated the development of models that, for the first time, enable the diagnosis of atherosclerosis within the categories of "no atherosclerosis," "asymptomatic atherosclerosis," and "symptomatic atherosclerosis," as well as by the associated health risks (non-significant, significant, and high). In addition to microbial markers, these models also incorporate an individual's psychological state and lifestyle indicators.

References

1. Meleshko T. V., Pallah O. V., Rukavchuk R. O., Yusko L. S., & Boyko N. V. (2020). Edible Fruits Extracts Affect Intestinal Microbiota Isolated from Patients with Noncommunicable Diseases Associated With Chronic Inflammation. Biotechnologia Acta, 13 (5), 87-100.
2. Toth, S., Boyko, N., & Sihotsky, V. (2021). Hypothesis of “stroke-stop” formula: a tool for risk index determination in development of acute cerebrovascular disease in asymptomatic individuals with carotid stenosis.
3. Bubnov, R. V., Spivak, M. Y., Lazarenko, L. M., Bomba, A., & Boyko, N. V. (2015). Probiotics and immunity: provisional role for personalized diets and disease prevention. EPMA Journal, 6, 1-11.
4. Kopolovets, I., Berek, P., Stefanic, P., Lotnyk, D., Mucha, R., Hertelyova, Z., ... & Sihotsky, V. (2020). Relevance of Lp-PLA2 blood concentration for morphological structure of atherosclerotic plaque: a shift from reactive to predictive, preventive and personalized medicine to predict stroke in patients with asymptomatic internal carotid artery stenosis.