The absence of effective extracellular matrix to mimic the natural tumor microenvironment remains a significant obstacle in cancer research. Matrigel, abundant in various biological matrix components, is limited in its application due to its high cost. This has prompted researchers to explore alternative matrix substitutes. Here, we have investigated the effects of the extracellular matrix derived from pig small intestinal submucosa (ECM-SIS) in xenograft tumor modeling. Our results showed that the pig-derived ECM-SIS effectively promotes the establishment of xenograft tumor models, with a tumor formation rate comparable to that of Matrigel. Furthermore, we showed that the pig-derived ECM-SIS exhibited lower immune rejection and fewer infiltrating macrophages than Matrigel. Gene sequencing analysis demonstrated only a 0.5% difference in genes between pig-derived ECM-SIS and Matrigel during the process of tumor tissue formation. These differentially expressed genes primarily participate in cellular processes, biological regulation, and metabolic processes. These findings emphasize the potential of pig-derived ECM-SIS as a cost-effective option for tumor modeling in cancer research.

Chronic hepatitis B (CHB) infections caused by the hepatitis B virus (HBV) continue to pose a significant global public health challenge. Currently, the approved treatments for CHB are limited to interferon and nucleos(t)ide analogs, both of which have their limitations, and achieving a complete cure remains an elusive goal. Therefore, the identification of new therapeutic targets and the development of novel antiviral strategies are of utmost importance. Natural products (NPs) constitute a class of substances known for their diverse chemical structures, wide-ranging biological activities, and low toxicity profiles. They have shown promise as potential candidates for combating various diseases, with a substantial number demonstrating anti-HBV properties. This comprehensive review focuses on the current applications of NPs in the fight against HBV and provides a summary of their antiviral mechanisms, considering their impact on the viral life cycle and host hepatocytes. By offering insights into the world of anti-HBV NPs, this review aims to furnish valuable information to support the future development of antiviral drugs.

Xenotransplantation is a feasible method to treat organ failure. However, how to effectively monitor the immune rejection of xenotransplantation is a problem for physicians and researchers. This manuscript describes a simple and effective method to monitor immune rejection in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models. Circulating DNA is a potentially non-invasive biomarker for organ damage. In this study, circulating pig-specific DNA (cpsDNA) was monitored during xenograft rejection by quantitative real-time PCR (qPCR). In this protocol, porcine specific primers were designed, plasmids-containing porcine specific DNA fragments were constructed, and standard curves for quantitation were established. Species-specific primers were then used to quantify cpsDNA by qPCR in pig-to-mouse cell transplantation models and pig-to-monkey artery patch transplantation models. The value of this method suggests that it can be used as a simple, convenient, low cost, and less invasive method to monitor the immune rejection of xenotransplantation.

Monitoring for immune rejection is crucial for long-term survival of pig xenografts. Circulating DNA is a promising non-invasive biomarker for either organ injury or response to therapy. In this study, circulating pig-specific DNA (cpsDNA) was monitored during xenograft rejection. Potential targets of cpsDNA were selected by in silico analysis, and species specificity of selected primers was confirmed by PCR. Subsequently, cpsDNA as a biomarker was evaluated using a complement-dependent cytotoxicity (CDC) assay in vitro. Then, early diagnosis and response to rapamycin were assessed by an in vivo imaging model of pig-to-mouse cell transplantation. Finally, cpsDNA was monitored in a pig-to-monkey artery patch transplantation model. The results showed that (a) a method of cpsDNA quantitation was established for application in mouse and nonhuman primate models; (b) cpsDNA reflected CDC in vitro; (c) cpsDNA in vivo mirrored xenograft rejection, and correlated with xenograft loss in pig-to-mouse cell transplantation; (d) cpsDNA was significantly reduced when rapamycin was administered; and (e) dynamic cpsDNA was detectable in pig-to-monkey artery patch transplantation. In conclusion, measurement of cpsDNA could prove to be a less invasive, but more specific and sensitive low-cost biomarker enabling monitoring of xenograft rejection and the response to immunosuppressive therapy.

Primary human hepatocytes (PHHs) are the 'gold standard' for investigating hepatitis B virus (HBV) infection and antiviral drugs. However, poor availability, variation between batches and ethical issues regarding PHHs limit their applications. The discovery of human sodium taurocholate co‑transporting polypeptide (hNTCP) as a functional HBV receptor has enabled the development of a surrogate model to supplement the use of PHHs. In the present study, the evolutionary distance of seven species was assessed based on single‑copy homologous genes. Based on the evolutionary distance and availability, PHHs and primary rabbit hepatocytes (PRHs) were isolated and infected with hNTCP‑recombinant lentivirus, and susceptibility to HBV infection in the two cell types was tested and compared. In addition, HBV infection efficiency of hNTCP‑expressing PPHs with pooled HBV‑positive serum and purified particles was determined. The potential use of HBV‑infected hNTCP‑expressing PPHs for drug screening was assessed. The results demonstrated that pigs and rabbits are closer to humans in the divergence tree compared with mice and rats, indicating that pigs and rabbits were more likely to facilitate the HBV post‑entry lifecycle. Following hNTCP complementation and HBV infection, PPHs and Huh7D human hepatocellular carcinoma cells, but not PRHs, exhibited increased hepatitis B surface antigen and hepatitis B e‑antigen secretion, covalently closed circular DNA formation and infectious particle secretion. hNTCP‑expressing PPHs were susceptible to infection with HBV particles purified from pooled HBV‑positive sera, but were poisoned by raw HBV‑positive sera. The use of HBV‑infected hNTCP‑expressing PPHs for viral entry inhibitor screening was revealed to be applicable and reproducible. In conclusion, hNTCP‑expressing PPHs may be valuable tool for investigating HBV infection and antiviral drugs.

After hyperacute rejection in pig-to-primate xenotransplantation had been overcome by the introduction of alpha 1,3-galactosyltransferase gene-knockout (GTKO) pigs, acute and chronic antibody-mediated rejection became one of the major barriers to long-term graft survival. This was associated with exposure of non-Gal antigens to the recipient's immune system and indicated that further genetic engineering of the pigs would be necessary. We here report that Gabarapl1, a regulator of tumorigenesis, plays a role in the regulation of immunogenicity of porcine aortic endothelial cells (PAECs). Knockdown of Gabarapl1 in PAECs results in a remarkable reduction in binding of serum antibody from PAEC-immunized monkeys, associated with decreased serum cytotoxicity of pig cells. Expression of swine leukocyte antigens (SLA) II DR was downregulated by Gabarapl1 knockdown. However, suppression of expression of SLA II is associated with less reduction of antibody binding than achieved by Gabarapl1 knockdown, suggesting that other Gabarapl1-regulated xenoantigens may be more important. These findings indicate a hitherto unknown relationship between Gabarapl1 and xenoimmunogenicity, suggesting a potential new strategy to reduce rejection initiated by the presence of non-Gal antigens.

Accurate and timely diagnosis of transplant rejection is essential for the long-term survival of solid-organ transplant recipients. Biopsy is the "gold standard" for the diagnosis of rejection, but this approach is a time-consuming, expensive, and invasive process. An efficient, cheap, and noninvasive tool for monitoring of immune rejection is urgently needed. Circulating miRNA and circulating DNA can be easily amplified and quantified by PCR which could be potential biomarkers for monitoring organ survival and/or immune rejection. In this review, we briefly introduce the origins and characteristics of circulating miRNA and circulating DNA. Furthermore, we review their clinical application and potential as noninvasive biomarkers for rejection in organ transplantation. Finally, we sum up their potential as biomarkers, which may help scientists and surgeons choose the proper biomarker for routine assays.

When hyperacute rejection is avoided by deletion of Gal expression in the pig, delayed xenograft rejection (DXR) becomes a major immunologic barrier to successful xenotransplantation. This study was to investigate the potential antigens involved in DXR. We isolated primary renal microvascular endothelial cells (RMEC) and aortic endothelial cells (AEC) from a GGTA1/CMAH double-knockout (DKO) pig (and a GGTA1-KO pig) and immunized cynomolgus monkeys with both of these cells. After sensitization, monkey serum antibody binding and cytotoxicity to RMEC was significantly higher than to AEC(p < 0.05), suggesting that RMEC are more immunogenic than AEC. Transcriptome sequencing of GGTA1/CMAH DKO pigs indicated that the expression of 1,500 genes was higher in RMEC than in AEC, while expression of 896 genes was lower. Next, we selected 101 candidate genes expressed only in pig RMEC, but not in pig AEC or in monkey or human RMEC. When these genes were knocked out individually in GGTA1/CMAH DKO RMEC, 32 genes were associated with reduced antibody binding, indicating that these genes might be primary immunologic targets involved in DXR. These genes may be important candidates for deletion in producing pigs against which there is a reduced primate immune response in pig kidney xenograft.

Different cell types possess different miRNA expression profiles, and cell/tissue/organ-specific miRNAs (or profiles) indicate different diseases. Circulating miRNA is either actively secreted by living cells or passively released during cell death. Circulating cell/tissue/organ-specific miRNA may serve as a non-invasive biomarker for allo- or xeno-transplantation to monitor organ survival and immune rejection. In this review, we summarize the proof of concept that circulating organ-specific miRNAs serve as non-invasive biomarkers for a wide spectrum of clinical organ-specific manifestations such as liver-related disease, heart-related disease, kidney-related disease, and lung-related disease. Furthermore, we summarize how circulating organ-specific miRNAs may have advantages over conventional methods for monitoring immune rejection in organ transplantation. Finally, we discuss the implications and challenges of applying miRNA to monitor organ survival and immune rejection in allo- or xeno-transplantation.