Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells.

Hematopoietic stem and progenitor cells can differentiate into all types of blood cells. Regulatory mechanisms underlying pluripotency in progenitors, such as the ability of lymphoid progenitor cells to differentiate into T-lineage, remain unclear. We have previously reported that LIM domain only 2 (Lmo2), a bridging factor in large transcriptional complexes, is essential to retain the ability of lymphoid progenitors to differentiate into T-lineage. However, biochemical characterization of Lmo2 protein complexes in physiological hematopoietic progenitors remains obscure. Here, we identified approximately 600 Lmo2-interacting molecules in a lymphoid progenitor cell line by two-step affinity purification with LC-MS/MS analysis. Zinc finger and BTB domain containing 1 (Zbtb1) and CBFA2/RUNX1 partner transcriptional corepressor 3 (Cbfa2t3) were found to be the functionally important binding partners of Lmo2. We determined CRISPR/Cas9-mediated acute disruption of Zbtb1 or Cbfa2t3 in the lymphoid progenitor or bone marrow-derived primary hematopoietic progenitor cells causes significant defects in the initiation of T-cell development when Notch signaling is activated. Our transcriptome analysis of Zbtb1- or Cbfa2t3-deficient lymphoid progenitors revealed that Tcf7 was a common target for both factors. Additionally, ChIP-seq analysis showed that Lmo2, Zbtb1, and Cbfa2t3 cobind to the Tcf7 upstream enhancer region, which is occupied by the Notch intracellular domain/RBPJ transcriptional complex after Notch stimulation, in lymphoid progenitors. Moreover, transduction with Tcf7 restored the defect in the T-lineage potential of Zbtb1-deficient lymphoid progenitors. Thus, in lymphoid progenitors, the Lmo2/Zbtb1/Cbfa2t3 complex directly binds to the Tcf7 locus and maintains responsiveness to the Notch-mediated inductive signaling to facilitate T-lineage differentiation.

Clinical significance of 123I-BMIPP washout rate in patients with uncertain chronic heart failure.

BACKGROUND: Recently, triglyceride deposit cardiomyovasculopathy (TGCV) with defective intracellular lipolysis was found to be a disease that causes heart failure. As a diagnostic criterion for TGCV, an Iodaine-123-ß-methyl iodophenyl-pentadecanoic acid washout rate (BMIPP WOR) of < 10% is used, but its clinical significance in patients with heart failure remains to be clarified. METHODS: In 62 hospitalized patients with chronic heart failure, 123I-BMIPP myocardial single-photon emission computed tomography (SPECT) was performed predischarge state. The prevalence of TGCV was investigated. Subsequently, follow-up was conducted for ≥ 90 days (mean: 724.6 ± 392.7 days), and the association between the BMIPP WOR and cardiac events was examined, establishing all-cause mortality and admission due to heart failure as endpoints. RESULTS: Of the 62 patients, the WOR was < 10% in 41 (66.1%). Of these, 26 (41.9%) were diagnosed with definite TGCV. Furthermore, cardiac events were noted in 12 patients (19.4%). Analysis with Cox proportional hazards models showed that the BMIPP WOR < 4.5% was a significant event-predicting factor [HR 4.29, 95% CI: 1.20-16.87; p = 0.0245]. On a Kaplan-Meier curve, the WOR was 4.5%; there was a significant difference in the incidence of events (p = 0.0298). CONCLUSION: In the predischarge state of heart failure, 123I-BMIPP myocardial SPECT was performed. In approximately 40% of the patients, a diagnosis of TGCV was made. The results suggested that the BMIPP WOR is useful for predicting the prognosis of chronic heart failure patients regardless of TGCV.

Notch signaling supports the appearance of follicular helper T cells in the Peyer's patches concomitantly with the reduction of regulatory T cells.

The intracellular fragment of Notch1, a mediator of Notch signaling that is frequently detected in thymic immigrants, is critical for specifying T-cell fate in the thymus, where Delta-like 4 (Dll4) functions as a Notch ligand on the epithelium. However, as such Notch signaling has not been detected in mature T cells, how Notch signaling contributes to their response in secondary lymphoid organs has not yet been fully defined. Here, we detected the marked expression of Dll4 on the stromal cells and the active fragment of Notch1 (Notch1 intracellular domain, N1ICD) in CD4+ T cells in the follicles of Peyer's patches (PPs). In addition, N1ICD-bearing T cells were found in the T-cell zone of PPs, especially in the transcription factor Foxp3+ regulatory T (Treg) cells, with slight expression of Dll4 on the stromal cells. These fragments disappeared in Dll4-deficient conditions. It was also found that Notch1- and Notch2-deficient T cells preferentially differentiated into Treg cells in PPs, but not CXCR5+PD-1+ follicular helper T (Tfh) cells. Moreover, these phenotypes were also observed in chimeric mice reconstituted with the control and T-cell-specific Notch1/2-deficient bone marrow or Treg cells. These results demonstrated that Dll4-mediated Notch signaling in PPs is required for the efficient appearance of Tfh cells in a Treg cell-prone environment, which is common among the gut-associated lymphoid tissues, and is critical for the generation of Tfh-mediated germinal center B cells.

A near-infrared fluorescent long-chain fatty acid toward optical imaging of cardiac metabolism in living mice.

Non-invasive fatty acid (FA) metabolic imaging is crucial for the evaluation of cardiac function in the heart. Currently, single-photon emission computed tomography (SPECT) and positron emission tomography (PET) are widely employed for cardiac metabolic imaging both in pre-clinical and clinical studies. Although SPECT and PET enable highly sensitive cardiac metabolic imaging, there are several disadvantages such as the high cost of instruments and radioactive tracer synthesis. In contrast, near-infrared (NIR) optical imaging using fluorescent FAs provides a simple and useful platform for in vivo imaging of cardiac metabolism. In this work, we synthesized a NIR fluorescence labelled long-chain fatty acid (LCFA) for real-time imaging of cardiac metabolism in vivo. A NIR fluorescence labelled LCFA was designed as an analogue of ß-methyl [123I] iodophenyl-pentanedecanoic acid (123I-BMIPP), which is widely used for the diagnosis of heart diseases in clinical practice. As a NIR fluorescent label, we used an Alexa 680 fluorophore that emits over 700 nm. By conjugation of Alexa 680 to Amino-BMPP (15-(4-(3-aminopropyl)phenyl)-3-methylpentadecanoic acid), we prepared a NIR fluorescent BMIPP analogue, Alexa680-BMPP. NIR fluorescence imaging showed that Alexa680-BMPP is taken up by the mouse heart tissue after intravenous injection, showing that Alexa680-BMPP can act as a fluorescent LCFA analogue. Among Alexa680 conjugated FA analogues including short and middle chain NIR fluorescent FAs, Alexa680-BMPP was most efficiently taken up by heart tissues. For fasted and fed mice, the difference in the degree of the uptake of Alexa680-BMPP in their heart tissues was clearly observed by in vivo and ex vivo NIR fluorescence imaging. Herein, we present the synthesis of a NIR fluorescent LCFA, Alexa680-BMPP, and its capability for real-time optical imaging of cardiac metabolism in living mice.

Targeting of plasminogen activator inhibitor-1 activity promotes elimination of chronic myeloid leukemia stem cells.

Therapeutic strategies that target leukemic stem cells (LSCs) provide potential advantages in the treatment of chronic myeloid leukemia (CML). Here, we show that selective blockade of plasminogen activator inhibitor-1 (PAI-1) enhances the susceptibility of CML-LSCs to tyrosine kinase inhibitor (TKI), which facilitates the eradication of CML-LSCs and leads to sustained remission of the disease. We demonstrated for the first time that TGF-ß-PAI-1 axis was selectively augmented in CML-LSCs in the bone marrow (BM), whereby protecting CML-LSCs from TKI treatment. Furthermore, the combined administration of TKI plus a PAI-1 inhibitor, in a mouse model of CML, significantly enhanced the eradication of CML cells in the BM and prolonged the survival of CML mice. The combined therapy of imatinib and a PAI-1 inhibitor prevented the recurrence of CML-like disease in serially transplanted recipients, indicating the elimination of CML-LSCs. Interestingly, PAI-1 inhibitor treatment augmented membrane-type matrix metalloprotease-1 (MT1-MMP)-dependent motility of CML-LSCs, and the anti-CML effect of PAI-1 inhibitor was extinguished by the neutralizing antibody for MT1-MMP, underlining the mechanistic importance of MT1-MMP. Our findings provide evidence of, and a rationale for, a novel therapeutic tactic, based on the blockade of PAI-1 activity, for CML patients.

Aortic insufficiency associated with Impella that required surgical intervention upon implantation of the durable left ventricular assist device.

The Impella is an axial-flow percutaneous ventricular assist device for cardiogenic shock. In this report, we describe two patients who developed aortic insufficiency (AI) associated with Impella and required surgical intervention upon implantation of the durable left ventricular assist device (LVAD). Both patients presented with cardiogenic shock and underwent insertion of Impella 5.0 as a bridge to decision. The cardiac function in these patients did not improve and obtaining approval for heart transplantation required time. They were managed with Impella for 91 and 98 days, respectively. In both cases, moderate AI that was not present before Impella insertion was observed when the Impella was removed. Therefore, we performed aortic valve closure to control the AI during durable LVAD implantation. In patients with durable LVAD implantation, AI may occur and progress after the operation in several cases. Aortic valve surgery is often performed to prevent deterioration of AI, especially in patients with AI before the surgery. Hence, AI is an important complication following Impella device implantation as a bridge to decision. Careful observation of AI is essential when the Impella is removed as the evaluation of AI by echocardiogram during Impella management is cumbersome because of device-generated artifacts.

Newly developed selective immunoinactivation assay revealed reduction in adipose triglyceride lipase activity in peripheral leucocytes from patients with idiopathic triglyceride deposit cardiomyovasculopathy.

Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare and newly identified disease among patients requiring cardiac transplantation. TGCV is characterized by cardiomyocyte steatosis and triglyceride (TG)-deposit atherosclerosis, resulting from the abnormal intracellular metabolism of TG. TGCV is classified into primary and idiopathic types. Primary TGCV carries ultra-rare genetic mutations in the adipose triglyceride lipase (ATGL), a rate-liming enzyme that hydrolyzes intracellular TG in adipose and non-adipose tissues. Idiopathic TGCV, first identified among autopsied individuals with diabetes mellitus (DM) with severe heart diseases, shows no ATGL mutations and its causes and underlying mechanisms are still unknown. TGCV is difficult to diagnose in daily clinics, thereby demanding feasible diagnostic procedures. We aimed to develop an assay to measure ATGL activity using peripheral leucocytes. Human his6-ATGL was expressed in COS1 cells, purified to homogeneity, and used to raise a polyclonal antibody neutralizing TG-hydrolyzing activity of ATGL. We developed a selective immunoinactivation assay (SIIA) for the quantitation of ATGL activity in cell lysates of leucocytes by the antibody neutralizing ATGL activities. ATGL activity was measured in 13 idiopathic TGCV patients, with two patients with primary TGCV as the negative control. Healthy (non-DM) and DM controls without heart diseases were also subjected. The developed SIIA assay revealed significant reduction in ATGL activity in leucocytes from patients with idiopathic TGCV who did not carry ATGL mutations as compared with non-DM and DM controls. Thus, ATGL in leucocytes may be an important biomarker for the diagnosis of TGCV and our assay may provide insights into pathophysiology and elucidate the underlying mechanism of TGCV and related disorders.

Effect of lipid metabolism on male fertility.

Cholesterol and lipid homeostasis is important for male fecundity. However, the plasma total cholesterol level does not reflect sperm concentration and motility. Adipose tissue in mammals is the main tissue contributing to the dynamic equilibrium of lipid synthesis and catabolism. However, recent studies suggested that local lipolysis has an important role in male fertility. If so, which plasma lipid metabolism parameter reflects sperm concentration and motility? Leptin receptor-deficient db/db mice show hyperphagia, hyperglycemia, hypercholesterolemia, hypertriglyceridemia, morbid obesity and are reported to be infertile. Impairment of spermatogenesis and sperm motility in db/db mice was observed in our experiments. Medium-chain triglycerides (MCTs) are more quickly metabolized as fuel compared to long-chain triglycerides (LCTs). If the LCTs are replaced with MCTs in the diet, even in hyperphagia, does it affect spermatogenesis and sperm motility? In this study, we investigated the effect of a MCT replacement diet on sperm parameters using db/db mice. Six weeks of MCT replacement diet improved not only spermatogenesis but also the maturation processes in the epididymis for sperm to acquire the ability to move forward. Plasma chylomicron and large VLDL levels showed positive correlation with total and motile sperm concentrations. The MCT replacement diet could be an effective treatment for idiopathic non-obstructive oligozoospermia or asthenozoospermia men with low levels of chylomicron and large VLDL.

Long-chain fatty acid triglyceride (TG) metabolism disorder impairs male fertility: a study using adipose triglyceride lipase deficient mice.

STUDY QUESTION: Does the deletion of adipose triglyceride lipase (Atgl) gene impair male fertility? SUMMARY ANSWER: The deletion of Atgl gene impaired male fertility but the effect was partially reversed by a low long-chain triglyceride (TG) diet. WHAT IS KNOWN ALREADY: ATGL specifically hydrolyses long-chain fatty acid TG to diacylglycerol and a high level of expression of ATGL in testes has been reported. However, the role of ATGL in male fertility is unknown. STUDY DESIGN, SIZE, DURATION: To investigate the effect of deletion of Atgl gene on male fertility, cauda epididymides and testes were collected from wild-type, heterozygous and homozygous Atgl-deficient mice at 10 weeks of age and epididymal sperm analysis and histological analysis of the testes were performed. To investigate whether a medium-chain triglycerides (MCTs) replacement diet mitigated the impaired male fertility by deletion of Atgl gene, homozygous Atgl-deficient mice were fed a MCT replacement diet, or a standard diet including long-chain triglycerides (LCTs) in a control group, for 6 weeks from 5 weeks of age (n = 22). The systematic and local effects of the MCT replacement diet on spermatogenesis and sperm maturation in the epididymis were analyzed at 10 weeks of age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Hematoxylin and eosin staining in paraffin-embedded sections of testes and Oil Red O staining in frozen sections of testes were performed. The epididymal sperm concentrations were analyzed. Statistical analyses were performed using the Student's t-test or Mann-Whitney U test with Shapiro-Wilk Normality test. MAIN RESULTS AND THE ROLE OF CHANCE: Although heterozygous mice were fertile and showed a similar number of epididymal total and motile sperm concentrations to wild-type mice, the deletion of Atgl gene in homozygous mice led to accumulation of TG deposits in testes and impaired spermatogenesis. The deletion of Atgl gene also impaired the sperm maturation process required for sperm to acquire the ability to move forward in the epididymis. The MCT replacement diet for 6 weeks increased the plasma level of non-esterified fatty acid (NEFA) (1.5-fold, P = 0.005), but not the plasma total cholesterol (T-Cho) and TG levels. In testes, the MCT replacement diet decreased the number of Oil Red O stain positive vacuoles (-40%, P < 0.001) and increased testis tissue weight (1.1-fold, P = 0.012), total sperm concentration (1.5-fold, P = 0.011) and motile sperm concentration (2.1-fold, P < 0.001) compared to the control group. However, there was no significant change in the sperm survival rate between the two groups. LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: One previous study reported that Atgl-deficient male mice were fertile. In most studies heterozygous Atgl(+/-) mice were used to generate homozygous Atgl-deficient Atgl(-/-) mice. Although the same gene targeting mice were used in this study and the formation of vaginal plugs were observed after mating with Atgl(-/-) male mice, there were no pregnant wild-type mice observed after mating with Atgl(-/-) male mice. WIDER IMPLICATIONS OF THE FINDINGS: Local TG metabolism in the male reproductive system could affect spermatogenesis and sperm motility in men. The MCT replacement diet could be an effective therapy for idiopathic non-obstructive oligozoospermia or asthenozoospermia in men with low levels of serum NEFA. STUDY FUNDING AND COMPETING INTEREST(S): This study was supported in part by the Japan Society for the Promotion of Science JSPS KAKENHI Grant (Nos. JP24249080, JP25462557, JP16K11086). The authors declare no conflict of interest.

Hepatic aberrant glycosylation by N-acetylglucosaminyltransferase V accelerates HDL assembly.

Glycosylation is involved in various pathophysiological conditions. N-Acetylglucosaminyltransferase V (GnT-V), catalyzing ß1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in cancer and the immune system. Recent findings indicate that aberrant N-glycan structure can modify lipid metabolism. In this study, we investigated the effects of aberrant glycosylation by GnT-V on high-density lipoprotein cholesterol (HDL) assembly. We used GnT-V transgenic (Tg) mice and GnT-V Hep3B cell (human hepatoma cell line) transfectants. The study also included 96 patients who underwent medical health check-ups. Total serum cholesterol levels, particularly HDL-cholesterol (HDL-C) levels, were significantly increased in Tg vs. wild-type (WT) mice. Hepatic expression of apolipoprotein AI (ApoAI) and ATP-binding cassette subfamily A member 1 (ABCA1), two important factors in HDL assembly, were higher in Tg mice compared with WT mice. ApoAI and ABCA1 were also significantly elevated in GnT-V transfectants compared with mock-transfected cells. Moreover, ApoAI protein in the cultured media of GnT-V transfectants was significantly increased. Finally, we found a strong correlation between serum GnT-V activity and HDL-C concentration in human subjects. Multivariate logistic analyses demonstrated that GnT-V activity was an independent and significant determinant for serum HDL-C levels even adjusted with age and gender differences. Further analyses represented that serum GnT-V activity had strong correlation especially with the large-size HDL particle concentration. These findings indicate that enhanced hepatic GnT-V activity accelerated HDL assembly and could be a novel mechanism for HDL synthesis.

Delta-like 4-mediated Notch signaling is required for early T-cell development in a three-dimensional thymic structure.

Delta-like 4 (Dll4)-mediated Notch signaling is critical for specifying T-cell fate, but how Dll4-mediated Notch signaling actually contributes to T-cell development in the thymus remains unclear. To explore this mechanism in the thymic three-dimensional structure, we performed fetal thymus organ culture using Dll4-deficient mice. DN1a/b+DN2mt cells, which had not yet committed to either the αß T or γδ T/NK cell lineage, did not differentiate into the αß T-cell lineage in Dll4-deficient thymus despite the lack of cell fate conversion into other lineages. However, DN3 cells efficiently differentiated into a later developmental stage of αß T cells, the double-positive (DP) stage, although the proliferation was significantly impaired during the differentiation process. These findings suggest that the requirement for Notch signaling differs between the earliest and pre-TCR-bearing precursors and that continued Notch signaling is required for proper differentiation with active proliferation of αß T lineage cells. Furthermore, we showed that Notch signaling increased the c-Myc expression in DN3 cells in the thymus and that its overexpression rescued the proliferation and differentiation of DN3 cells in the Dll4-null thymus. Therefore, c-Myc plays a central role in the transition from stage DN3 to DP as a downstream target of Notch signaling.

Anti-fibrotic effects of a novel small compound on the regulation of cytokine production in a mouse model of colorectal fibrosis.

Intestinal fibrotic stricture is a major complication of inflammatory bowel disease. Despite its clinical importance, anti-fibrotic therapy has not been implemented. Transforming growth factor-ß (TGF-ß) is considered to be a major factor contributing to tissue fibrosis. We have previously shown that the administration of a small compound, HSc025, which promotes the nuclear translocation of YB-1 as a downstream effector of IFN-γ and antagonizes TGF-ß/Smad signaling, improves fibrosis in several murine tissues. In this study, we evaluated the anti-fibrotic effect of HSc025 on colorectal fibrosis in TNBS-induced murine chronic colitis. Daily oral administration of HSc025 (3, 15 and 75 mg/kg) suppressed collagen production and decreased the severity of colorectal fibrosis in a dose-dependent manner. In addition, the local production of TGF-ß was decreased after HSc025 treatment, whereas that of IL-13 and TNF-α was not affected. HSc025 administration maintained the level of IFN-γ production, even at a late stage when IFN-γ production was lost without the drug treatment. These results demonstrate that HSc025 could be a therapeutic candidate for intestinal fibrosis in inflammatory bowel disease that acts by altering the local production of cytokines, as well as by directly suppressing collagen production.

Selective evaluation of high density lipoprotein from mouse small intestine by an in situ perfusion technique.

The small intestine (SI) is the second-greatest source of HDL in mice. However, the selective evaluation of SI-derived HDL (SI-HDL) has been difficult because even the origin of HDL obtained in vivo from the intestinal lymph duct of anesthetized rodents is doubtful. To shed light on this question, we have developed a novel in situ perfusion technique using surgically isolated mouse SI, with which the possible filtration of plasma HDL into the SI lymph duct can be prevented. With the developed method, we studied the characteristics of and mechanism for the production and regulation of SI-HDL. Nascent HDL particles were detected in SI lymph perfusates in WT mice, but not in ABCA1 KO mice. SI-HDL had a high protein content and was smaller than plasma HDL. SI-HDL was rich in TG and apo AIV compared with HDL in liver perfusates. SI-HDL was increased by high-fat diets and reduced in apo E KO mice. In conclusion, with our in situ perfusion model that enables the selective evaluation of SI-HDL, we demonstrated that ABCA1 plays an important role in intestinal HDL production, and SI-HDL is small, dense, rich in apo AIV, and regulated by nutritional and genetic factors.

Genetic mutations in adipose triglyceride lipase and myocardial up-regulation of peroxisome proliferated activated receptor-γ in patients with triglyceride deposit cardiomyovasculopathy.

Adipose triglyceride lipase (ATGL, also known as PNPLA2) is an essential molecule for hydrolysis of intracellular triglyceride (TG). Genetic ATGL deficiency is a rare multi-systemic neutral lipid storage disease. Information regarding its clinical profile and pathophysiology, particularly for cardiac involvement, is still very limited. A previous middle-aged ATGL-deficient patient in our institute (Case 1) with severe heart failure required cardiac transplantation (CTx) and exhibited a novel phenotype, "Triglyceride deposit cardiomyovasculopathy (TGCV)". Here, we tried to elucidate molecular mechanism underlying TGCV. The subjects were two cases with TGCV, including our second case who was a 33-year-old male patient (Case 2) with congestive heart failure requiring CTx. Case 2 was homozygous for a point mutation in the 5' splice donor site of intron 5 in the ATGL, which results in at least two types of mRNAs due to splicing defects. The myocardium of both patients (Cases 1 and 2) showed up-regulation of peroxisome proliferated activated receptors (PPARs), key transcription factors for metabolism of long chain fatty acids (LCFAs), which was in contrast to these molecules' lower expression in ATGL-targeted mice. We investigated the intracellular metabolism of LCFAs under human ATGL-deficient conditions using patients' passaged skin fibroblasts as a model. ATGL-deficient cells showed higher uptake and abnormal intracellular transport of LCFA, resulting in massive TG accumulation. We used these findings from cardiac specimens and cell-biological experiments to construct a hypothetical model to clarify the pathophysiology of the human disorder. In patients with TGCV, even when hydrolysis of intracellular TG is defective, the marked up-regulation of PPARγ and related genes may lead to increased uptake of LCFAs, the substrates for TG synthesis. This potentially vicious cycle of LCFAs could explain the massive accumulation of TG and severe clinical course for this rare disease.

Coronary triglyceride deposition in contemporary advanced diabetics.

It is of importance to clarify pathophysiology of diabetic heart diseases such as heart failure and coronary artery disease. We reported a novel clinical phenotype called triglyceride deposit cardiomyovasculopathy (TGCV), showing aberrant TG accumulation in both coronary arteries and myocardium, in a cardiac transplant recipient. Here, we examined autopsied diabetics for TG deposition in cardiovasculature. Consecutive series of hearts from advanced diabetes mellitus (DM) subjects (DM group: DMG, n = 20) and those from age- and sex-matched non-diabetic controls (non DM group: NDMG, n = 20) were examined. The diagnostic criteria of 'advanced DM' was made based on 2014 Clinical Practice Recommendations proposed by the American Diabetes Association. The mean duration of DM was 15.8 years. All DMG suffered from heart diseases including coronary artery diseases and 14 subjects had multi-vessel disease. Tissue TG contents were measured biochemically. Coronary arterial TG contents was significantly higher in DMG compared with NDMG. Spatial distribution of TG in transverse sections of coronary arteries showed TG deposition mainly in smooth muscle cells by Imaging Mass Spectrometry. Abundant TG deposition in coronary artery might be associated with advanced DM.

Practice recommendation for measuring washout rates in 123I-BMIPP fatty acid images.

The purpose of this practice recommendation is to specifically identify the critical steps involved in performing and interpreting 123I-ß-methyl-iodophenyl-pentadecanoic acid (BMIPP) single-photon emission computed tomography (SPECT) and measurement of washout rate (WR) from the heart. This document will cover backgrounds, patient preparation, testing procedure, visual image interpretation, quantitation methods using planar and SPECT studies, and reporting of WR. The pitfall and some tips for the calculation of 123I-BMIPP WR are also included. The targets of global and regional WR calculation include ischemic heart disease, cardiomyopathy, heart failure, and triglyceride deposit cardiomyovasculopathy, an emerging rare heart disease.

Plasminogen activator inhibitor-1 promotes immune evasion in tumors by facilitating the expression of programmed cell death-ligand 1.

Background: Increased levels of plasminogen activator inhibitor-1 (PAI-1) in tumors have been found to correlate with poor clinical outcomes in patients with cancer. Although abundant data support the involvement of PAI-1 in cancer progression, whether PAI-1 contributes to tumor immune surveillance remains unclear. The purposes of this study are to determine whether PAI-1 regulates the expression of immune checkpoint molecules to suppresses the immune response to cancer and demonstrate the potential of PAI-1 inhibition for cancer therapy. Methods: The effects of PAI-1 on the expression of the immune checkpoint molecule programmed cell death ligand 1 (PD-L1) were investigated in several human and murine tumor cell lines. In addition, we generated tumor-bearing mice and evaluated the effects of a PAI-1 inhibitor on tumor progression or on the tumor infiltration of cells involved in tumor immunity either alone or in combination with immune checkpoint inhibitors. Results: PAI-1 induces PD-L1 expression through the JAK/STAT signaling pathway in several types of tumor cells and surrounding cells. Blockade of PAI-1 impedes PD-L1 induction in tumor cells, significantly reducing the abundance of immunosuppressive cells at the tumor site and increasing cytotoxic T-cell infiltration, ultimately leading to tumor regression. The anti-tumor effect elicited by the PAI-1 inhibitor is abolished in immunodeficient mice, suggesting that PAI-1 blockade induces tumor regression by stimulating the immune system. Moreover, combining a PAI-1 inhibitor with an immune checkpoint inhibitor significantly increases tumor regression. Conclusions: PAI-1 protects tumors from immune surveillance by increasing PD-L1 expression; hence, therapeutic PAI-1 blockade may prove valuable in treating malignant tumors.

Genetic and Functional Analyses of Patients with Marked Hypo-High-Density Lipoprotein Cholesterolemia.

AIM: This study aimed to analyze two cases of marked hypo-high-density lipoprotein (HDL) cholesterolemia to identify mutations in ATP-binding cassette transporter A1 (ABCA1) and elucidate the molecular mechanism by which these novel pathological mutations contribute to hypo-HDL cholesterolemia in Tangier disease. METHODS: Wild type and mutant expression plasmids containing a FLAG tag inserted at the C-terminus of the human ABCA1 gene were generated and transfected into HEK293T cells. ABCA1 protein expression and cholesterol efflux were evaluated via Western blotting and efflux assay. The difference in the rate of change in protein expression was evaluated when proteolytic and protein-producing systems were inhibited. RESULTS: In case 1, a 20-year-old woman presented with a chief complaint of gait disturbance. Her HDL-C level was only 6.2 mg/dL. Tangier disease was suspected because of muscle weakness, decreased nerve conduction velocity, and splenomegaly. Whole-exome analysis showed compound heterozygosity for a W484* nonsense mutation and S1343I missense mutation, which confirmed Tangier disease. Cholesterol efflux decreased by a mixture of W484* and S1343I mutations. The S1343I mutation decreased the protein production rate but increased the degradation rate, decreasing the protein levels. This patient also had Krabbe disease. The endogenous ABCA1 protein level of macrophage cell decreased by knocking down its internal galactocerebrosidase.Case 2, a 51-year-old woman who underwent tonsillectomy presented with peripheral neuropathy, corneal opacity, and HDL-C of 3.4 mg/dL. Whole-exome analysis revealed compound heterozygosity for R579* and R1572* nonsense mutations, which confirmed Tangier disease. CONCLUSION: Case 1 is a new ABCA1 mutation with complex pathogenicity, namely, a W484*/S1343I compound heterozygote with marked hypo-HDL cholesterolemia. Analyses of the compound heterozygous mutations indicated that decreases in ABCA1 protein levels and cholesterol efflux activity caused by the novel S1343I mutation combined with loss of W484* protein activity could lead to marked hypo-HDL cholesterolemia. Galactocerebrosidase dysfunction could also be a potential confounding factor for ABCA1 protein function.