Against all odds: The road to success in the development of human immune reconstitution mice.

The mouse genome has a high degree of homology with the human genome, and its physiological, biochemical, and developmental regulation mechanisms are similar to those of humans; therefore, mice are widely used as experimental animals. However, it is undeniable that interspecies differences between humans and mice can lead to experimental errors. The differences in the immune system have become an important factor limiting current immunological research. The application of immunodeficient mice provides a possible solution to these problems. By transplanting human immune cells or tissues, such as peripheral blood mononuclear cells or hematopoietic stem cells, into immunodeficient mice, a human immune system can be reconstituted in the mouse body, and the engrafted immune cells can elicit human-specific immune responses. Researchers have been actively exploring the development and differentiation conditions of host recipient animals and grafts in order to achieve better immune reconstitution. Through genetic engineering methods, immunodeficient mice can be further modified to provide a favorable developmental and differentiation microenvironment for the grafts. From initially only being able to reconstruct single T lymphocyte lineages, it is now possible to reconstruct lymphoid and myeloid cells, providing important research tools for immunology-related studies. In this review, we compare the differences in immune systems of humans and mice, describe the development history of human immune reconstitution from the perspectives of immunodeficient mice and grafts, and discuss the latest advances in enhancing the efficiency of human immune cell reconstitution, aiming to provide important references for immunological related researches.

Dclre1c-Mutation-Induced Immunocompromised Mice Are a Novel Model for Human Xenograft Research.

Severe combined immunodeficient (SCID) mice serve as a critical model for human xenotransplantation studies, yet they often suffer from low engraftment rates and susceptibility to graft-versus-host disease (GVHD). Moreover, certain SCID strains demonstrate 'immune leakage', underscoring the need for novel model development. Here, we introduce an SCID mouse model with a targeted disruption of the dclre1c gene, encoding Artemis, which is essential for V(D)J recombination and DNA repair during T cell receptor (TCR) and B cell receptor (BCR) assembly. Artemis deficiency precipitates a profound immunodeficiency syndrome, marked by radiosensitivity and compromised T and B lymphocyte functionality. Utilizing CRISPR/Cas9-mediated gene editing, we generated dclre1c-deficient mice with an NOD genetic background. These mice exhibited a radiosensitive SCID phenotype, with pronounced DNA damage and defective thymic, splenic and lymph node development, culminating in reduced T and B lymphocyte populations. Notably, both cell lines and patient-derived tumor xenografts were successfully engrafted into these mice. Furthermore, the human immune system was effectively rebuilt following peripheral blood mononuclear cells (PBMCs) transplantation. The dclre1c-knockout NOD mice described herein represent a promising addition to the armamentarium of models for xenotransplantation, offering a valuable platform for advancing human immunobiological research.

MicroRNA-29b ameliorates hepatic inflammation via suppression of STAT3 in alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) is induced by chronic excessive alcohol consumption resulting in the clinical manifestations of steatosis, inflammation, and cirrhosis. MicroRNA-29b (miR-29b) is mainly expressed in hepatic nonparenchymal cells, and its expression level varies in different diseases. In this study, we aimed to determine the role of miR-29b in a mouse model of alcohol-associated liver disease. Wild-type (WT) and miR-29b knockout (miR-29b-/-) mice were fed a Lieber-DeCarli liquid diet containing 5% alcohol for 10 days, followed by gavage of a single dose of ethanol (5 g/kg body weight). Histology, immunoblotting, and biochemical analyses were then conducted for comparison. miR-29b expression was decreased in the livers of chronic-plus-binge ethanol-fed mice. Further analysis revealed that alcohol exposure exacerbated hepatic injury by significantly increasing serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, with decreased survival rates for miR-29b-/- mice. Results from the luciferase assay indicated that miR-29b negatively regulated the signal transducer and activator of transcription 3 (STAT3). Depletion of miR-29b led to an increase in STAT3 and more noticeable inflammation in the liver, whereas overexpression of miR-29b downregulated STAT3 and proinflammatory cytokine expression in primary mouse peritoneal macrophages. Taken together, these results demonstrate a novel association between miR-29b and ALD. miR-29b plays a hepatoprotective role in alcohol-induced inflammation and liver injury by targeting STAT3.

Biological Characteristics of Severe Combined Immunodeficient Mice Produced by CRISPR/Cas9-Mediated Rag2 and IL2rg Mutation.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 is a novel and convenient gene editing system that can be used to construct genetically modified animals. Recombination activating gene 2 (Rag2) is a core component that is involved in the initiation of V(D)J recombination during T- and B-cells maturation. Separately, the interleukin-2 receptor gamma chain gene (IL2rg) encoded the protein-regulated activity of natural killer (NK) cells and shared common receptors of some cytokines. Rag2 and IL2rg mutations cause immune system disorders associated with T-, B-, and NK cell function and some cytokine activities. In the present study, 2 single-guide RNAs (sgRNAs) targeted on Rag2 and IL2rg genes were microinjected into the zygotes of BALB/c mice with Cas9 messenger RNA (mRNA) to create Rag2/IL2rg -/- double knockout mice, and the biological characteristics of the mutated mice were subsequently analyzed. The results showed that CRISPR/Cas9-induced indel mutation displaced the frameshift of Rag2 and IL2rg genes, resulting in a decrease in the number of T-, B-, and NK cells and the destruction of immune-related tissues like the thymus and spleen. Mycobacterium tuberculosis 85B antigen could not induce cellular and humoral immune response in mice. However, this aberrant immune activity compromised the growth of several tumor heterogenous grafts in the mutated mice, including orthotopic and subcutaneous transplantation tumors. Thus, Rag2/IL2rg -/- knockout mice possessed features of severe combined immunodeficiency (SCID), which is an ideal model for human xenograft.

Application of the Trivex system in the treatment of primary severe superficial varicose veins of the lower extremity.

PURPOSE: The aim of this study was to evaluate the clinical effects of the Trivex system in the treatment of primary severe superficial varicose veins of the lower extremity and compare Trivex to the point-form-stripping combined with foam sclerotherapy (FS). METHODS: A total of 64 patients (35 females, 29 males; mean age, 57 ±â€¯11 years [range, 29-79 years]) with primary severe superficial varicose veins of the lower extremity involving 64 legs were included between October 2015 and July 2019. The maximum diameter of the vein branches was >20 mm, which appeared to be cystic dilatation and forms large-scale in the crus or the thigh. All patients underwent high ligation and endovenous laser ablation or stripping of the trunk under general anesthesia. The surgical time, pain/phlebitis, number of incisions, amount of bleeding, recurrence of varicose vein, incidence of surgical site infections (SSIs), satisfaction score, and improvement in clinical symptoms were evaluated respectively with the patients in two groups: Group A, with patients who underwent treatment with the Trivex system, and Group B, patients who underwent treatment by point-form-stripping combined with FS. RESULTS: All procedures were performed successfully. The average operative time in Group A was 56 ±â€¯11 min, whereas that of Group B was 90 ±â€¯33 min, which was a significant difference (p < 0.05). Group A patients felt little pain after surgery, whereas in Group B the level of pain peaked on postoperative day 30, mostly due to thrombophlebitis after FS. There was no recurrence of varicose vein was observed in any patient, however, there were some residual effects in Group B, including the amount of bleeding volume, in-hospital stays, pain/phlebitis, and number of incisions (P < 0.05). There were no significant differences with respect to SSIs, improvement in clinical symptoms, and satisfaction scores observed (p>0.05). CONCLUSIONS: This study shows that patients benefited from both treatment options. However, primary severe superficial varicose veins of the lower extremity treated with the Trivex system suffered less pain with fewer incisions than severe branches treated with the point-form-stripping combined with foam sclerotherapy (FS). In summary, the Trivex system is a suitable treatment prior to point-form-stripping combined with foam sclerotherapy (FS) for those who demand a high level of appearance, and especially for young patients, the Trivex system is recommended.

Anti-proliferation of breast cancer cells with itraconazole: Hedgehog pathway inhibition induces apoptosis and autophagic cell death.

Itraconazole is a common antifungal which may have promise for treating various human cancers. We report that itraconazole was cytotoxic to MCF-7 and SKBR-3 breast cancer cell lines via apoptosis by altering mitochondria membrane potential, reducing BCL-2 expression and elevating caspase-3 activity. Itraconazole also induced autophagic cell death via LC3-II expression upregulation, P62/SQSTM1 degradation, autophagosome formation and increases in autophagic puncta. Itraconazole treatment inhibited hedgehog pathway key molecular expression, such as SHH and Gli1, resulting in promotion of apoptosis and autophagy. The anti-proliferation effect of itraconazole-induced apoptosis and autophagy via hedgehog pathway inhibition was confirmed with Gli1 inhibitor GANT61 and SHH siRNA, GANT61 and SHH siRNA synergistically enhanced cytotoxicity induced by itraconazole. A human xenograft nude mouse model corroborated the anti-breast cancer activity as evidenced by reduced tumor size, and increased tumor tissue apoptosis and autophagy. Thus, itraconazole has a potent anti-breast cancer activity that may be improved when combined with hedgehog pathway inhibitors.

Cabazitaxel-induced autophagy via the PI3K/Akt/mTOR pathway contributes to A549 cell death.

Cabazitaxel has been used to treat castration-resistant prostate cancer since its approval by the US Food and Drug Administration in 2010. However, whether cabazitaxel may inhibit the proliferation of other tissue­derived cancer cells, and its underlying mechanism, remains unknown. In the present study, the A549 lung adenocarcinoma cancer cell line was exposed to cabazitaxel, in order to investigate its cytotoxic effect and determine the underlying mechanism. The results demonstrated that cabazitaxel was able to induce autophagy in A549 cells, as evidenced by the formation of autophagosomes, upregulated LC3­II expression and increased LC3 puncta. Cabazitaxel­induced autophagy had a cytotoxic effect on A549 cells, as evidenced by the induction of cell death and cell cycle arrest at G2/M phase, which was independent of the apoptotic pathway. Furthermore, transfection with Beclin1 small interfering RNA and treatment with the autophagy inhibitor 3­methyladenine protected cells from cabazitaxel­induced cell death, thus confirming that cabazitaxel­induced autophagy contributed to A549 cell death. In addition, cabazitaxel targeted the phosphoinositide 3­kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway to induce autophagy, as indicated by reduced phosphorylation of Akt and mTOR. In conclusion, the present study demonstrated that cabazitaxel exerts a cytotoxic effect on A549 cells by acting on the PI3K/Akt/mTOR pathway to promote autophagic cell death. This result supports the potential use of cabazitaxel as a chemotherapeutic agent for the treatment of lung cancer.