CRISPR/Cas9-mediated knockin of human factor IX into swine factor IX locus effectively alleviates bleeding in hemophilia B pigs.

Hemophilia B is an X-linked recessive bleeding disorder caused by abnormalities in the coagulation factor IX gene. Without prophylactic treatment, patients experience frequent spontaneous bleeding episodes. Well-characterized animal models are valuable for determining the pathobiology of the disease and testing novel therapeutic innovations. Here, we generated a porcine model of hemophilia B using a combination of CRISPR/Cas9 and somatic cell nuclear transfer. Moreover, we tested the possibility of hemophilia B therapy by gene insertion. Frequent spontaneous joint bleeding episodes that occurred in hemophilia B pigs allowed a thorough investigation of the pathological process of hemophilic arthropathy. In contrast to the hemophilia B pigs, which showed a severe bleeding tendency and joint damage, the transgenic pigs carrying human coagulation factor IX exhibited a partial improvement of bleeding. In summary, this study not only offers a translational hemophilia B model for exploring the pathological process of hemophilic arthropathy but also provides a possibility for the permanent correction of hemophilia in the future by genome editing in situ.

Cyclodextrin Ameliorates the Progression of Atherosclerosis via Increasing High-Density Lipoprotein Cholesterol Plasma Levels and Anti-inflammatory Effects in Rabbits.

To investigate the therapeutic effects of cyclodextrin on the development of atherosclerosis in rabbits, we evaluated the effects of (2-hydroxypropyl)-ß-cyclodextrin (HPßCD) therapy on the organ coefficient, lipid profiles, inflammatory cytokines, and atherosclerotic plaques in rabbits fed a high-fat diet. Our results demonstrated that HPßCD therapy reduced plasma triglyceride levels and inflammatory cytokine levels but increased plasma high-density lipoprotein cholesterol levels. HPßCD therapy produced a significant decrease in the atherosclerotic lesion area and reduced macrophage and collagen content in the lesions. The expression levels of inflammatory genes in aortic plaques were significantly reduced by HPßCD treatment, but the expression of ATP-binding cassette (ABC) transporters A1 (ABCA1) and G1 (ABCG1) in aortic plaques and livers increased significantly. HPßCD therapy may produce additional antiatherosclerotic benefits likely via increasing high-density lipoprotein cholesterol levels.

Analyzing Differences in Hematological and Immunological Characteristics Related to Common Gene Mutations in Myelodysplastic Syndromes.

BACKGROUND: Genetic mutations play a crucial role in the development and progression of myelodysplastic syndromes (MDS), impacting the immune microenvironment and influencing the choice of treatment regimen, as well as the efficacy and prognosis of patients. The objective of this study was to examine variations in hematological and immunological characteristics associated with common gene mutations in MDS patients and establish a foundation for the precise treatment of MDS. METHODS: The hematological, immunological, and other clinical features of 71 recently diagnosed MDS patients from January 1, 2019, to July 31, 2023, were retrospectively analyzed. These patients were categorized based on their gene mutations, and the variances in hematological and immunological characteristics among distinct groups were compared. RESULTS: Hematological variances were observed among different gene mutation groups. Specifically, platelet counts in the splicing factor 3B subunit 1 (SF3B1) mutation group were notably higher compared to the wild-type group (p = 0.009). Conversely, in the additional sex combs like 1 (ASXL1) mutation groups, monocyte ratios were significantly elevated in comparison to the wild-type group (p = 0.046), and in the ten-eleven translocation 2 (TET2) mutation group, lymphocyte ratios were significantly lower (p = 0.022). Additionally, the leukocyte (p = 0.005), neutrophil ratio (p = 0.002), and lymphocyte ratio (p = 0.001) were significantly higher in the Runt-related transcription factor 1 (RUNX1) mutation group. Regarding immunological distinctions, the Natural Killer (NK) cell ratio demonstrated a significant increase in the SF3B1 mutation group (p = 0.005). Moreover, the TET2 mutation group exhibited a significantly higher Interleukin-8 (IL-8) level (p = 0.017). In contrast, the U2 small nuclear RNA auxiliary factor 1 (U2AF1) group displayed significantly lower levels of IL-1ß (p = 0.033), IL-10 (p = 0.033), and Tumour Necrosis Factor-α (TNF-α) (p = 0.009). CONCLUSION: Distinct variations exist in the immune microenvironment of MDS associated with different genetic mutations. Further studies are imperative to delve into the underlying mechanisms that drive these differences.

Natural killer cells affect the natural course, drug resistance, and prognosis of multiple myeloma.

Multiple myeloma (MM), a stage-developed plasma cell malignancy, evolves from monoclonal gammopathy of undetermined significance (MGUS) or smoldering MM (SMM). Emerging therapies including immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies, chimeric antigen-T/natural killer (NK) cells, bispecific T-cell engagers, selective inhibitors of nuclear export, and small-molecule targeted therapy have considerably improved patient survival. However, MM remains incurable owing to inevitable drug resistance and post-relapse rapid progression. NK cells with germline-encoded receptors are involved in the natural evolution of MGUS/SMM to active MM. NK cells actively recognize aberrant plasma cells undergoing malignant transformation but are yet to proliferate during the elimination phase, a process that has not been revealed in the immune editing theory. They are potential effector cells that have been neglected in the therapeutic process. Herein, we characterized changes in NK cells regarding disease evolution and elucidated its role in the early clinical monitoring of MM. Additionally, we systematically explored dynamic changes in NK cells from treated patients who are in remission or relapse to explore future combination therapy strategies to overcome drug resistance.

A novel method for simultaneously measuring boronophenylalanine uptake in brain tumor cells and number of cells using inductively coupled plasma atomic emission spectroscopy.

Boron neutron capture therapy (BNCT) combines neutron irradiation with boron compounds that are selectively uptaken by tumor cells. Boronophenylalanine (BPA) is a boron compound used to treat malignant brain tumors. The determination of boron concentration in cells is of great relevance to the field of BNCT. This study was designed to develop a novel method for simultaneously measuring the uptake of BPA by U87 and U251 cells (two brain tumor cell lines) and number of cells using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The results revealed a linear correlation between phosphorus intensity and the numbers of U87 and U251 cells, with correlation coefficients (R2) of 0.9995 and 0.9994, respectively. High accuracy and reliability of phosphorus concentration standard curve were also found. Using this new method, we found that BPA had no significant effect on phosphorus concentration in either U87 or U251 cells. However, BPA increased the boron concentration in U87 and U251 cells in a concentration-dependent manner, with the boron concentration in U87 cells being higher than that in U251 cells. In both U87 and U251 cells, boron was mainly distributed in the cytoplasm and nucleus, accounting for 85% and 13% of the total boron uptake by U87 cells and 86% and 11% of the total boron uptake by U251 cells, respectively. In the U87 and U251 cell-derived xenograft (CDX) animal model, tumor exhibited higher boron concentration values than blood, heart, liver, lung, and brain, with a tumor/blood ratio of 2.87 for U87 cells and 3.11 for U251 cells, respectively. These results suggest that the phosphorus concentration in U87 and U251 cells can represent the number of cells and BPA is easily uptaken by tumor cells as well as in tumor tissue.

Early Diagnosis of Colorectal Cancer Based on Bisulfite-free Site-specific Methylation Identification PCR Strategy: High-Sensitivity, Accuracy, and Primary Medical Accessibility.

Due to its decade-long progression, colorectal cancer (CRC) is most suitable for population screening to achieve a significant reduction in its incidence and mortality. DNA methylation has emerged as a potential marker for the early detection of CRC. However, the current mainstream methylation detection method represented by bisulfite conversion has issues such as tedious operation, DNA damage, and unsatisfactory sensitivity. Herein, a new high-performance CRC screening tool based on the promising specific terminal-mediated polymerase chain reaction (STEM-PCR) strategy is developed. CRC-related methylation-specific candidate CpG sites are first prescreened through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases using self-developed bioinformatics. Next, 9 homebrew colorectal cancer DNA methylated STEM‒PCR assays (ColoC-mSTEM) with high sensitivity (0.1%) and high specificity are established to identify candidate sites. The clinical diagnostic performance of these selected methylation sites is confirmed and validated by a case-control study. The optimized diagnostic model has an overall sensitivity of 94.8% and a specificity of 95.0% for detecting early-stage CRC. Taken together, ColoC-mSTEM, based on a single methylation-specific site, is a promising diagnostic approach for the early detection of CRC which is perfectly suitable for the screening needs of CRC in primary healthcare institutions.

The yin-yang of immunity: Immune dysregulation in myelodysplastic syndrome with different risk stratification.

Myelodysplastic syndrome (MDS) is a heterogeneous group of myeloid clonal diseases with diverse clinical courses, and immune dysregulation plays an important role in the pathogenesis of MDS. However, immune dysregulation is complex and heterogeneous in the development of MDS. Lower-risk MDS (LR-MDS) is mainly characterized by immune hyperfunction and increased apoptosis, and the immunosuppressive therapy shows a good response. Instead, higher-risk MDS (HR-MDS) is characterized by immune suppression and immune escape, and the immune activation therapy may improve the survival of HR-MDS. Furthermore, the immune dysregulation of some MDS changes dynamically which is characterized by the coexistence and mutual transformation of immune hyperfunction and immune suppression. Taken together, the authors think that the immune dysregulation in MDS with different risk stratification can be summarized by an advanced philosophical thought "Yin-Yang theory" in ancient China, meaning that the opposing forces may actually be interdependent and interconvertible. Clarifying the mechanism of immune dysregulation in MDS with different risk stratification can provide the new basis for diagnosis and clinical treatment. This review focuses on the manifestations and roles of immune dysregulation in the different risk MDS, and summarizes the latest progress of immunotherapy in MDS.

Evaluation of Pharmacokinetics of Boronophenylalanine and Its Uptakes in Gastric Cancer.

Boron neutron capture therapy (BNCT), a cellular-level particle radiation therapy, combines boron compounds selectively delivered to tumor tissue with neutron irradiation. Boronophenylalanine (BPA) is a boron compound widely used in malignant melanoma, malignant brain tumors, and recurrent head and neck cancer. However, neither basic nor clinical research was reported for the treatment of gastric cancer using BPA. Selective distribution of boron in tumors rather than that in blood or normal tissue prior to neutron irradiation is required for the successful treatment of BNCT. This study evaluated the pharmacokinetics and safety of 10B-labeled BPA (10B-BPA, abbreviated as BPA) and its uptakes in gastric cancer. Pharmacokinetics and safety were evaluated in Sprague-Dawley (SD) rats intravenously injected with BPA. The uptakes of boron in gastric cancer cell line MKN45 and in cell-derived xenografts (CDX) and patient-derived xenografts (PDX) animal models were measured. The results showed that the boron concentration in the blood of rats decreased fast in the first 30 min followed by a steady decrease following the observation time, having a half-life of 44.11 ± 8.90 min and an AUC-last of 815.05 ± 62.09 min×µg/ml. The distribution of boron in different tissues (heart, liver, lung, stomach, and small intestine) of rats revealed a similar pattern in blood except for that in the brain, kidney, and bladder. In MKN45 cells, boron concentration increased in a time- and concentration-dependent manner. In both CDX and PDX animal models, the boron is preferentially distributed in tumor tissue rather than in blood or normal tissues. In addition, BPA had no significant adverse effects in rats. Taken together, the results suggested that BPA revealed a fast decrease in boron concentration in rats and is more likely to distribute in tumor cells and tissue.

Aggressive natural killer cell leukemia with skin manifestation associated with hemophagocytic lymphohistiocytosis: A case report.

BACKGROUND: Aggressive natural killer cell leukemia (ANKL) is a rare natural killer cell neoplasm characterized by systemic infiltration of Epstein-Barr virus and rapidly progressive clinical course. ANKL can be accompanied with hemophagocytic lymphohistiocytosis (HLH). Here, we report a case of ANKL with rare skin lesions as an earlier manifestation, accompanied with HLH, and review the literature in terms of etiology, clinical manifestation, diagnosis and treatment. CASE SUMMARY: A 30-year-old woman from Northwest China presented with the clinical characteristics of jaundice, fever, erythema, splenomegaly, progressive hemocytopenia, liver failure, quantities of abnormal cells in bone marrow, and associated HLH. The immunophenotypes of abnormal cells were positive for CD2, cCD3, CD7, CD56, CD38 and negative for sCD3, CD8 and CD117. The diagnosis of ANKL complicated with HLH was confirmed. Following the initial diagnosis and supplementary treatment, the patient received chemotherapy with VDLP regimen (vincristine, daunorubicin, L-asparaginase and prednisone). However, the patient had severe adverse reactions and complication such as severe hematochezia, neutropenia, and multiple organ dysfunction syndrome, and died a few days later. CONCLUSION: This is the first reported case of ANKL with rare skin lesions as an earlier manifestation and associated with HLH.

Abnormality of hepatic triglyceride metabolism in ApcMin/+ mice with colon cancer cachexia.

AIMS: Colorectal cancer syndrome has been one of the greatest concerns in the world. Although several epidemiological studies have shown that hepatic low lipoprotein lipase (LPL) mRNA expression may be associated with dyslipidemia and tumor progression, it is still not known whether the liver plays an essential role in hyperlipidemia of ApcMin/+ mice. MAIN METHODS: We measured the expression of metabolic enzymes that involved fatty acid uptake, de novo lipogenesis (DNL), ß-oxidation and investigated hepatic triglyceride production in the liver of wild-type and ApcMin/+ mice. KEY FINDINGS: We found that hepatic fatty acid uptake and DNL decreased, but there was no significant difference in fatty acid ß-oxidation. Interestingly, the production of hepatic very low-density lipoprotein-triglyceride (VLDL-TG) decreased at 20 weeks of age, but marked steatosis was observed in the livers of the ApcMin/+ mouse. To further explore hypertriglyceridemia, we assessed the function of hepatic glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) for the first time. GPIHBP1 is governed by the transcription factor octamer-binding transcription factor-1 (Oct-1) which are involved in the nuclear factor-κB (NF-κB) signaling pathway in the liver of ApcMin/+ mice. Importantly, it was also confirmed that sn50 (100 µg/mL, an inhibitor of the NF-κB) reversed the tumor necrosis factor α (TNFα)-induced Oct-1 and GPIHBP1 reduction in HepG2 cells. SIGNIFICANCE: Altogether, these findings highlighted a novel role of GPIHBP1 that might be responsible for hypertriglyceridemia in ApcMin/+ mice. Hypertriglyceridemia in these mice may be associated with their hepatic lipid metabolism development.

Abnormality of intestinal cholesterol absorption in ApcMin/+ mice with colon cancer cachexia.

Colorectal cancer syndrome has been one of the greatest concerns in the world, particularly in developed countries. Several epidemiological studies have shown that dyslipidemia may be associated with the progression of intestinal cachexia, but there is little research on the function of the small intestine, which is involved in blood lipid metabolism, in dyslipidemia. In the present study, we aimed to explore the function of intestinal cholesterol absorption in the ApcMin/+ mouse model using an intestinal lipid absorption test. We found that both triglyceride (TG) and total cholesterol (TC) uptake were inhibited in the intestine of ApcMin/+ mice with age and the intestinal peroxisome proliferator-activated receptor α (PPARα) downregulated the processes of ß-oxidation, oxidative stress response, and cholesterol absorption in APC-deficient mice. In addition, reduced expression levels of farnesoid X receptor (FXR) and apical sodium-dependent bile acid transporter (ASBT) indicated that bile acid metabolism might be associated with intestinal cholesterol absorption in ApcMin/+ mice. Thus, our data suggested that the intestine plays an essential role in cholesterol uptake and that bile acid metabolism seems to cause a decrease in intestinal cholesterol uptake in ApcMin/+ mice.