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Abstract Aims/Introduction This study aimed to investigate the diagnostic potential of two simplified tests, a point‐of‐care nerve conduction device (DPNCheck™) and a coefficient of variation of R‐R intervals (CVR‐R), as an alternative to traditional nerve conduction studies for the diagnosis of diabetic polyneuropathy (DPN) in patients with diabetes. Materials and Methods Inpatients with type 1 or type 2 diabetes (n = 167) were enrolled. The study population consisted of 101 men, with a mean age of 60.8 ± 14.8 years. DPN severity was assessed using traditional nerve conduction studies, and differentiated based on Baba's classification (BC). To examine the explanatory potential of variables in DPNCheck™ and CVR‐R regarding the severity of DPN according to BC, a multiple regression analysis was carried out, followed by a receiver operating characteristic analysis. Results Based on BC, 61 participants (36.5% of the total) were categorized as having DPN severity of stage 2 or more. The multiple regression analysis yielded a predictive formula with high predictive power for DPN diagnosis (estimated severity of DPN in BC = 2.258 – 0.026 × nerve conduction velocity [m/s] – 0.594 × ln[sensory nerve action potential amplitude (μV)] + 0.528In[age(years)] – 0.178 × ln[CVR‐R], r = 0.657). The area under the curve in receiver operating characteristic analysis was 0.880. Using the optimal cutoff value for DPN with severer than stage 2, the predictive formula showed good diagnostic efficacy: sensitivity of 83.6%, specificity of 79.2%, positive predictive value of 51.7% and negative predictive value of 76.1%. Conclusions These findings suggest that DPN diagnosis using DPNCheck™ and CVR‐R could improve diagnostic efficiency and accessibility for DPN assessment in patients with diabetes.

Abstract Background Rating lymphocytes (TILs) are a prognostic marker in breast cancer and high TIL infiltration correlates with better patient outcomes. Meanwhile, parameters involving immune cells in peripheral blood have also been established as prognostic markers. High platelet-to-lymphocyte ratios (PLRs) and neutrophil-to-lymphocyte ratios (NLRs) are related to poor outcomes in breast cancer, but their mechanisms remain unknown. To date, TILs and these parameters have been examined separately. Methods We investigated the relationship between TILs and the peripheral blood markers, PLR and NLR, in the same patients, using surgical specimens from 502 patients with invasive breast carcinoma without preoperative chemotherapy. For analysis of triple-negative breast cancer (TNBC) patient outcomes, 59 patients who received preoperative chemotherapy were also examined. For immune cell profiling, multiplexed fluorescent immunohistochemistry (mfIHC) of CD3, CD4, CD8, FOXP3 and T-bet, was conducted. Results A positive correlation between PLR and TIL was observed in TNBC (P = 0.013). On mfIHC, tumors in patients with high PLR and NLR contained more CD3+CD4+FOXP3+ T-cells (P = 0.049 and 0.019, respectively), while no trend was observed in CD8+ T-cells. TNBC patients had different patterns of outcomes according to TIL and PLR, with the TIL-high/PLR-low group having the lowest rate of disease relapse and death, and the longest distant metastasis-free and overall survivals, while the TIL-low/PLR-high group had the shortest survivals. Conclusions Our data suggest that the combination of PLR with TIL assessment may enable more accurate prediction of patient outcomes with TNBC.

In a phase II trial of nivolumab in advanced thymic carcinoma (UMIN000022007), long SD (SD for more than 24 weeks) was seen in three patients and irAE (Gr2 or higher) was seen in four patients among 15 patients. Here, we report preplanned comprehensive biomarker analyses. We obtained tumor samples for immunohistochemistry, peripheral blood mononuclear cells (PBMCs), plasma and serum for pharmacokinetic analysis of nivolumab and cytokine evaluations, and whole blood for immuno pharmacogenomic (PGx) analysis. PD-L1 expression on tumor cells were not associated with therapeutic efficacy, but FOXP3 expression in tumor area and stroma, CD204 expression in stroma, and MHC class I in tumor area were all low among long SD patients. PBMC of long SD patients presented with larger number of naïve/memory cells prior to treatment, suggesting priming after nivolumab administration. Immuno-PGx analysis showed non-synonymous SNVs in ITGAX and PDCD1 had some correlation with PFS. Concentration of nivolumab in blood during the treatment was not related to PFS, with their overall trend towards decreased nivolumab concentration in patients with irAEs. Low immunogenicity of thymic carcinoma demonstrated in our study may require the activation of immune systems via a combination of immune checkpoint blockades.

Abstract Background Tumour-infiltrating lymphocyte (TIL)-high breast tumours have a high rate of pathological complete response (pCR) with neoadjuvant chemotherapy. In our routine pathological diagnoses of biopsy specimens from pCR cases, we have observed a high infiltration of plasma cells (PCs). A positive correlation of PCs with favourable patient outcome has recently been reported, but little is known about how PCs contribute to local tumour immunity. Methods We retrospectively examined biopsy specimens from 146 patients with invasive breast cancer who received neoadjuvant chemotherapy. CD138+ PC infiltration was assessed by immunohistochemistry. Multiplexed fluorescent immunohistochemistry (mfIHC) with T and B cell markers was also conducted to elucidate the profile of immune cells. Results Greater PC infiltration was observed in the pCR group (p = 0.028) and this trend was confirmed in another patient cohort. With mfIHC, we observed significantly more CD8+, T-bet+CD4+, and CD8+FOXP3+ T cells, total B cells and PCs in pCR cases. Such cases were also characterised by high expression of both PD-1 and PD-L1 on B cells and PCs. In patients with hormone receptor-negative tumours, high PC infiltration was correlated with significantly longer disease-free survival (p = 0.034). Conclusions We found that higher PC infiltration in biopsy specimens before neoadjuvant chemotherapy was associated with pCR. With mfIHC, we also revealed that the local cytotoxic immune response was clearly enhanced in pCR cases, as was the infiltration of B cells including PCs. Moreover, higher PC levels were correlated with favourable outcomes in hormone receptor-negative breast cancer patients.

Abstract Aims/Introduction Diabetic polyneuropathy (DPN) and diabetic retinopathy (DR) are traditionally regarded as microvascular complications. However, these complications may share similar neurodegenerative pathologies. Here we evaluate the correlations in the severity of DPN and changes in the thickness of neuroretinal layers to elucidate whether these complications exist at similar stages of progression. Materials and Methods A total of 43 patients with type 2 diabetes underwent a nerve conduction study (NCS), a macular optical coherence tomography, and a carotid artery ultrasound scan. Diabetic polyneuropathy was classified according to Baba’s classification using NCS. The retina was automatically segmented into four layers; ganglion cell complex (GCC), inner nuclear layer/outer plexiform layer (INL/OPL), outer nuclear layer/photoreceptor inner and outer segments, and retinal pigment epithelium (RPE). The thickness of each retinal layer was separately analyzed for the fovea and the parafovea. Results Fourteen patients were classified as having moderate to severe diabetic polyneuropathy. The thicknesses of the foveal and parafoveal INL/OPL increased in patients with diabetic polyneuropathy compared with patients without. The thickness of the parafoveal retinal pigment epithelium decreased in patients with diabetic polyneuropathy. The thinning of parafoveal ganglion cell complex and foveal and parafoveal retinal pigment epithelium were positively correlated with deterioration of nerve functions in the nerve conduction study, but the thickening of INL/OPL was positively correlated with the nerve function deterioration. The thinning of parafoveal ganglion cell complex and foveal retinal pigment epithelium were positively correlated with the thickening of the carotid intima‐media. Conclusions Depending on the progression of diabetic polyneuropathy, the ganglion cell complex and retinal pigment epithelium became thinner and the INL/OPL became thicker. These retinal changes might be noteworthy for pathological investigations and for the assessment of diabetic polyneuropathy and diabetic retinopathy.

Abstract Notch signaling has been recognized recently as a key regulator of metabolism. Here, we determined the role of Notch1 in adipogenesis in wild-type (WT) and Notch1 hetero-mutant (N1+/−) mice provided with 12-week normal or high-fat diet. Haploinsufficiency of Notch1 was associated with adipose tissue accumulation despite similar food intake. White adipose tissue (WAT) of N1+/− showed accumulation of adipogenic cells (CD34+CD68+ cells), crown-like structures, and upregulation of cell proliferation markers (cyclin D1 and Ki67). Notch1 expression in WAT reached peak levels in 8-week-old WT mice in parallel with fat accumulation, especially under HF/HS-feeding, whereas such increment was blunted in N1+/− mice. Downstream of Notch1 haploinsufficiency, over-expression of adipogenic factors PPARγ and C/EBPα was noted following down-regulation of downstream transcriptional factors of Notch signaling (Hes-1, Pref-1, and Sox9). Both pharmacological Notch signal inhibition and Notch1 knockdown enhanced adipogenesis of 3T3-L1 preadipocytes. N1+/− mice showed impaired glucose and insulin tolerance with downregulation of IRS-1 and GLUT4 in WAT after high-fat diet. Taken together, our results suggest that haploinsufficiency of Notch1 promotes fat accumulation and adipogenesis and provides a mechanistic link between Notch signaling and development of metabolic syndrome.

Abstract Aims/Introduction Diabetic polyneuropathy (DPN) develops in the early stage of diabetes. However, no common diagnostic protocol has yet been established. Here, to verify that the flicker electroretinogram using a hand‐held device can detect the early dysfunction of the peripheral nervous system in patients with diabetes, we investigated the correlation between the progression of DPN and neuroretinal dysfunction. Materials and Methods In total, 184 participants with type 1 or 2 diabetes underwent a flicker electroretinogram (ERG) using a hand‐held device RETeval™ and nerve conduction study. Participants were also evaluated for intima‐media thickness, ankle‐brachial index, toe brachial index and brachial‐ankle pulse wave velocity. Parameters of the nerve conduction study were used to diagnose the severity according to Baba’s classification. A multiple regression analysis was used to examine the associations of ERG parameters with the severity of DPN categorized by Baba’s classification. Diagnostic properties of the device in DPN were evaluated using a receiver operating characteristic curve. Results A multiple regression model to predict the severity of DPN was generated using ERG. In the model, moderate‐to‐severe DPN was effectively diagnosed (area under the receiver operating characteristic curve 0.692, sensitivity 56.5%, specificity 78.3%, positive predictive value 70.6%, negative predictive value 66.1%, positive likelihood ratio 2.60, negative likelihood ratio 0.56). In the patients without diabetic retinopathy, the implicit time and amplitude in ERG significantly correlated with the parameters of the nerve conduction study, brachial‐ankle pulse wave velocity and intima‐media thickness. Conclusions Electroretinogram parameters obtained by the hand‐held device successfully predict the severity of DPN. The device might be useful to evaluate DPN.

Introduction The COVID‐19 pandemic has been causing delay in patient arrival at hospital and starting surgery. We report a delay in a case of testicular torsion complicated by acute pneumonia during the COVID‐19 pandemic in Japan. Case presentation A 17‐year‐old Japanese boy presented to our emergency room with acute left scrotum pain and fever in January 2021. It took 2.5 h to transfer him. Physical examination and color Doppler ultrasonography revealed left testicular torsion. Chest computed tomography indicated acute pneumonia. He successfully underwent surgical detorsion 7.5 h after symptom onset, with COVID‐19 preventive measures in place. A negative polymerase chain reaction test result for COVID‐19 was revealed after surgery. Conclusion We experienced a rare case of testicular torsion complicated by acute pneumonia during the COVID‐19 pandemic. Special attention should be paid to preventing infection and surgery delay to avoid testicular loss.

Abstract Aims/Introduction A gold standard in the diagnosis of diabetic polyneuropathy (DPN) is a nerve conduction study. However, as a nerve conduction study requires expensive equipment and well‐trained technicians, it is largely avoided when diagnosing DPN in clinical settings. Here, we validated a novel diagnostic method for DPN using a point‐of‐care nerve conduction device as an alternative way of diagnosis using a standard electromyography system. Materials and Methods We used a multiple regression analysis to examine associations of nerve conduction parameters obtained from the device, DPNCheck™, with the severity of DPN categorized by the Baba classification among 375 participants with type 2 diabetes. A nerve conduction study using a conventional electromyography system was implemented to differentiate the severity in the Baba classification. The diagnostic properties of the device were evaluated using a receiver operating characteristic curve. Results A multiple regression model to predict the severity of DPN was generated using sural nerve conduction data obtained from the device as follows: the severity of DPN = 2.046 + 0.509 × ln(age [years]) − 0.033 × (nerve conduction velocity [m/s]) − 0.622 × ln(amplitude of sensory nerve action potential [µV]), r = 0.649. Using a cut‐off value of 1.3065 in the model, moderate‐to‐severe DPN was effectively diagnosed (area under the receiver operating characteristic curve 0.871, sensitivity 70.1%, specificity 87.7%, positive predictive value 83.0%, negative predictive value 77.3%, positive likelihood ratio 5.67, negative likelihood ratio 0.34). Conclusions Nerve conduction parameters in the sural nerve acquired by the handheld device successfully predict the severity of DPN.

BackgroundAlthough intravenous immunoglobulin (IVIG) therapy is generally safe and well tolerated, adverse reactions (ARs) do occur. The majority of these ARs are mild and transient. Risk factors for ARs associate with IVIG infusions are not well established. This study investigated possible risk factors influencing the occurrence of IVIG-associated ARs.Study Design and MethodsThis was a retrospective observational analysis of data accumulated over 5 years, including patient demographics, clinical condition, IVIG dosing regimens, number of IVIG infusions, and any ARs.ResultsARs were associated with IVIG in 4.9% of patients and 2.5% of infusions. By univariate analyses, ARs correlated with female sex, adult age, high dose IVIG, and autoimmune disease. Multivariate logistic regression identified three statistically significant of risk factors: on a per-patient basis, being female (p=0.0018), having neuromuscular disease (p=0.0002), and receiving higher doses of IVIG per patient body weight (p

Abstract Background Nivolumab is an immune checkpoint inhibitor specific to the programmed death 1 (PD-1) receptor. Nivolumab has shown clinical responses in many malignancies. Although immune-related adverse events (irAEs) associated with nivolumab are largely tolerable, severe irAEs have occurred in some patients. However, the mechanisms underlying the development of irAEs are not fully clarified. Case presentation We report 2 patients with metastatic melanoma who developed colitis, an irAEs caused by nivolumab. Both patients experienced colitis after nivolumab administration. Pathological examination of the colon showed robust infiltration of CD8+ cells and T-bet expressing CD4+ cells in both cases, indicating helper T cells (Th) 1 to be responsible for the dominant response. Additionally, we observed the serum C-reactive protein level (CRP) as well as interleukin-6 (IL-6) reflected the clinical course of irAEs clearly in the two cases. Conclusion Our two cases suggested that the development of irAEs due to nivolumab is associated with Th1 dominant response. CRP as well as IL-6 was found to be a potential biomarker for irAEs. Our findings may help to understand the mechanisms underlying irAEs caused by nivolumab and manage irAEs in clinical practice.

Abstract Aims/Introduction Although nerve conduction study (NCS) using a standard electromyography system (EMGS) is considered to be the gold standard in evaluating diabetic polyneuropathy, this examination requires expensive equipment and well‐trained technicians. We aimed to validate a point‐of‐care device, NC‐stat/DPNCheck™, that has been developed for widespread use of NCS in diabetic polyneuropathy. Materials and Methods Diabetes patients underwent two kinds of NCS: DPNCheck™ and electromyography system. Inter‐/intrarater reliability of DPNCheck™ were also determined by the intraclass correlation coefficient. Results A total of 57 patients were evaluated. The parameters of NCS between the two methods correlated well (r = 0.7734 for the sural nerve conduction velocity, r = 0.6155 for the amplitude of sural nerve action potential). The intraclass correlation coefficients were excellent (intrarater: the velocity 0.767, the amplitude 0.811; interrater: the velocity 0.974, the amplitude 0.834). Conclusions The point‐of‐care device has excellent reproducibility and good agreement with standard electromyography system. The device might be useful to evaluate diabetic polyneuropathy.

Abstract Background Transient CD4+ T cell depletion led to the proliferation of tumor-specific CD8+ T cells in the draining lymph node and increased infiltration of PD-1+CD8+ T cells into the tumor, which resulted in strong anti-tumor effects in tumor-bearing mice. This is a first-in-human study of IT1208, a defucosylated humanized anti-CD4 monoclonal antibody, engineered to exert potent antibody-dependent cellular cytotoxicity. Methods Patients with advanced solid tumors were treated with intravenous IT1208 at doses of 0.1 or 1.0 mg/kg. The first patient in each cohort received a single administration, and the other patients received two administrations of IT1208 on days 1 and 8. Results Eleven patients were enrolled in the 0.1 mg/kg (n = 4) and 1.0 mg/kg cohorts (n = 7). Grade 1 or 2 infusion-related reactions was observed in all patients. Decreased CD4+ T cells in peripheral blood due to IT1208 were observed in all patients and especially in those receiving two administrations of 1.0 mg/kg. CD8+ T cells increased on day 29 compared with baseline in most patients, resulting in remarkably decreased CD4/8 ratios. One microsatellite-stable colon cancer patient achieved durable partial response showing increased infiltration of both CD4+ and CD8+ T cells into tumors after IT1208 administration. Moreover, transcriptomic profiling of the liver metastasis of the patient revealed upregulation of the expression of interferon-stimulated genes, T cell activation-related genes, and antigen presentation-related genes after IT1208 administration. Two additional patients with gastric or esophageal cancer achieved stable disease lasting at least 3 months. Conclusions IT1208 monotherapy successfully depleted CD4+ T cells with a manageable safety profile and encouraging preliminary efficacy signals, which warrants further investigations, especially in combination with immune checkpoint inhibitors.

Although Vaccinium virgatum Aiton leaves and stems inhibit adult T-cell leukemia (ATL) cells, leaves and stems can differ between individual plants and by time and location. In this study, leaf and stem components were profiled in the same individual plant using direct-injection electron ionization-mass spectrometry (DI-EI-MS) metabolomics, with the aims of analyzing the anti-ATL activity, and quantifying proanthocyanidins (PACs). Leaves, stems, and leaf/stem mixtures showed distinct and characteristic spectra. Anti-ATL activity was stronger in stems than leaves, and the PAC content was higher in stems than leaves. These data were subjected to bivariate analysis to identify the factor (m/z) responsible for the inhibitory effect of ATL based on the highest coefficient of determination (R2). The results of this DI-EI-MS metabolomics analysis suggest that among PACs contained in V. virgatum stems and leaves, the fragment ion at m/z 149 contributes significantly to anti-ATL activity.

Macrophages play an indispensable role in both innate and acquired immunity, while the persistence of activated macrophages can sometimes be harmful to the host, resulting in multi-organ damage. Macrophages develop from monocytes in the circulation. However, little is known about the organ affinity of macrophages in the normal state. Using in vivo imaging with XenoLight DiR®, we observed that macrophages showed strong affinity for the liver, spleen and lung, and weak affinity for the gut and bone marrow, but little or no affinity for the kidney and skin. We also found that administered macrophages were still alive 168 hours after injection. On the other hand, treatment with clodronate liposomes, which are readily taken up by macrophages via phagocytosis, strongly reduced the number of macrophages in the liver, spleen and lung.

We previously reported that rabbit-eye blueberry (Vaccinium virgatum Aiton) leaves exhibit multiple functions. In this study, we evaluated whether V. virgatum stems can also be used as functional materials similar to leaves and clarified the major constituents and their biological activity (antioxidant activity and anti–adult T cell leukemia (ATL) activity). Water extracts of V. virgatum stems were separated into 19 fractions using a Diaion HP-20 open column. Sugars and organic acids were detected in the highly water-soluble fractions. Polyphenols and proanthocyanidin were detected in the hydrous methanol-soluble fractions. In biological activity evaluations, a difference in antioxidant activity was observed in the water-containing methanol-eluted fractions, and fractions exhibiting anti-ATL activity differed depending on cell type. These results suggest that blueberry stems, like leaves, are rich in polyphenols and exhibit antioxidant activity and inhibit ATL cell growth. In the future, aerial parts of blueberries, including stems and leaves, could be used as functional materials and/or medicinal resources.

Blueberry (Vaccinium virgatum Aiton; Kinisato 35 Gou) leaves have recently attracted increasing attention as a useful material for the prevention of lifestyle diseases. Here, we examined the effects of the hot water extract of blueberry leaves (BLEx) on lipogenesis and uric acid production in 3T3-L1 adipocytes. The results showed that BLEx suppressed lipid accumulation and the mRNA expression of differentiation markers in 3T3-L1 adipocytes. A fractionation study showed that the highly polymerized proanthocyanidin-rich fraction was responsible for this effect. Upon maturation to adipocytes, 3T3-L1 cells produced uric acid and tumor necrosis factor-α, and hypoxia stimulated the production of uric acid and xanthine oxidoreductase activity. BLEx suppressed the production of uric acid under these conditions. Although BLEx inhibited the enzymatic activity of xanthine oxidase, this activity was observed in several fractions containing catechin, epicatechin, chlorogenic acid, rutin, and low molecular weight proanthocyanidins. Taken together, these results indicate that BLEx contains various compounds with the ability to suppress lipid accumulation and uric acid production in adipocytes.

Abstract Chronic stress is closely linked to the metabolic syndrome, diabetes, hyperuricemia and thromboembolism, but the mechanisms remain elusive. We reported recently that stress targets visceral adipose tissue (VAT), inducing lipolysis, low-grade inflammation with production of inflammatory adipokines, metabolic derangements such as insulin resistance, and prothrombotic state. In the present study, we hypothesized the involvement of VAT xanthine oxidoreductase (XOR), a source of reactive oxygen species (ROS) and uric acid (UA) in the above processes. Restraint stress in mice resulted in upregulation of XOR and xanthine oxidase activity, accumulation of ROS in VAT as well as liver and intestine, increase in serum UA levels, upregulation of NADPH oxidase subunits and downregulation of antioxidant enzymes. Immunohistochemistry and RT-PCR analysis also showed that restraint stress induced VAT monocyte accumulation and proinflammatory adipokine production, resulting in reduced insulin sensitivity and induction of plasminogen activator inhibitor-1 and tissue factor in VAT. Treatment with febuxostat, a potent XO inhibitor, suppressed stress-induced ROS production and VAT inflammation, resulting in improvement of serum UA levels, insulin sensitivity, and prothrombotic tendency. Our results suggest that stress perturbs glucose and UA metabolism, and promotes prothrombotic status, and that XO inhibition by febuxostat might be a potential therapy for stress-related disorders.

Immune checkpoint inhibitors have now become a standard therapy for malignant melanoma. However, as immunotherapies are effective in only a limited number of patients, biomarker development remains one of the most important clinical challenges. Biomarkers predicting clinical benefit facilitate appropriate selection of individualized treatments for patients and maximize clinical benefits. Many biomarkers derived from tumors and peripheral blood components have recently been reported, mainly in retrospective settings. This review summarizes the recent findings of biomarker studies for predicting the clinical benefits of immunotherapies in melanoma patients. Taking into account the complex interactions between the immune system and various cancers, it would be difficult for only one biomarker to predict clinical benefits in all patients. Many efforts to discover candidate biomarkers are currently ongoing. In the future, verification, by means of a prospective study, may allow some of these candidates to be combined into a scoring system based on bioinformatics technology.

Purpose We tested a new head-mounted display (HMD) system for surgery on the upper urinary tract. Surgical Technique Four women and one man with abnormal findings in the renal pelvis on computed tomography and magnetic resonance imaging underwent surgery using this new system. A high definition HMD (Sony, Tokyo, Japan) is connected to a flexible ureteroscope (Olympus, Tokyo, Japan) and the images from the ureteroscope are delivered simultaneously to various participants wearing HMDs. Furthermore, various information in addition to that available through the endoscope, such as the narrow band image, the fluoroscope, input from a video camera mounted on the lead surgeon’s HMD and the vital monitors can be viewed on each HMD. Results Median operative duration and anesthesia time were 53 and 111 minutes, respectively. The ureteroscopic procedures were successfully performed in all cases. There were no notable negative outcomes or incidents (Clavien-Dindo grade ≥1). Conclusion The HMD system offers simultaneous, high-quality magnified imagery in front of the eyes, regardless of head position, to those participating in the endoscopic procedures. This affordable display system also provides various forms of information related to examinations and operations while allowing direct vision and navigated vision.

To obtain compounds that inhibit the interaction of von Willebrand factor (vWF) and glycoprotein (GP) Ib, a novel binding assay was established. The binding of fixed platelets to vWF-R497 mutant was quantified by a solid phase assay. In this assay, fixed platelets bound to the vWF-R497 mutant, carrying the deletion of Glu497-Tyr508 and the missense mutation of Arg545 to Ala, without binding modulators such as ristocetin. The Kd value of the binding was 2.8 nM, which was consistent with the result from liquid binding assay. The binding was inhibited by aurin tricarboxylic acid (ATA) and an anti GPIb antibody, AK2. Using this binding assay, we screened our library compounds and obtained D74-3736. This compound also inhibited ristocetin-induced platelet aggregation in the human platelet-rich plasma. Keywords:: biotinylation of fixed platelets, binding assay of von Willebrand factor (vWF) to glycoprotein Ib (GPIb)

Sequencing of the human genome in combination with computational annotation has provided tremendous data on predicted genes. However, for most of them, no corresponding cDNAs are available yet. Furthermore, even where cDNA clones were obtained, gene transcripts often have many different splice variants that are not covered by current gene collections. For direct synthesis of cDNA clones corresponding to predicted genes, new splice variants, or any other gene of interest, we established optimal PCR conditions for the direct amplification of exons from genomic DNA, which require a specific Taq aptamer. PCR products comprising differently tagged exons were concatenated by overlap extension into full-length cDNAs. To prove the effectiveness of the approach, the 1900-bp full-length open reading frame of the human mitochondrial aldehyde dehydrogenase (ALDH2) gene was synthesized in a two-step reaction comprising all 13 exons. Thus, our conditions are of general value for in vitro synthesis of cDNAs and alternative splice variants from genomic DNA.

In repetitive measurements of flow-mediated dilatation (FMD), the duration of the interval between measurements remains controversial. In this pilot study, we conducted three sequential measurements of low-flow-mediated constriction (L-FMC), FMD and flow-mediated total dilation (FMTD; L-FMC+ FMD) at baseline and intervals of 15 and 60 min in 30 healthy males. FMD15, L-FMC15, and FMTD15 were significantly lower than the respective first measurements, but all indices showed full recovery at 60 min in all subjects. The baseline diameter was slightly increased at 15 min and restored at 60 min, but the maximum diameter, and the baseline and reactive flow velocity unchanged. We examined the relationship between recovery rate of FMTD at 15 min (FMTD-R) and cardio-ankle vascular index (CAVI). Univariate analysis showed moderate correlation between FMTD-R, and CAVI and L-FMC0. Patients were divided according to FMTD-R value; the low-FMTD-R group [below the median value (-26.2%)] included a significantly higher proportion of smokers and higher CAVI values than the high-FMTD-R group. The reproducibility of FMTD and FMTD-R was evaluated in another group of 25 healthy subjects. The range of variation across measurements was 1.1% for FMTD and 4.6% for FMTD-R; with intraclass correlation coefficients of 0.93 and 0.95, respectively. The present study demonstrated blunted recovery of FMD within 15 min, suggesting the need for selection of a more adequate interval between measurements to avoid underestimation of FMD in subsequent measurements. The findings demonstrated the reproducibility of FMTD-R and FMTD measurements, and that FMTD-R might be involved in arterial stiffness and early vascular impairment in the healthy subjects.

Macrophage infiltration of skin GVHD lesions correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear and GVHD with many macrophages is a therapeutic challenge. Here, we characterize the macrophages involved in GVHD and report that dexamethasone palmitate (DP), a liposteroid, can ameliorate such GVHD by inhibiting macrophage functions. We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD. DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro. DP treatment on day 7 and day 14 decreased macrophage number, and attenuated GVHD score and subsequent mortality in a murine model. This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.

A strong causal link exists between psychological stress and insulin resistance as well with hypertension. Meanwhile, stress-related responses play critical roles in glucose metabolism in hypertensive patients. As clinical trials suggest that angiotensin-receptor blocker delays the onset of diabetes in hypertensive patients, we investigated the effects of irbesartan on stress-induced adipose tissue inflammation and insulin resistance. C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle, 3 and 10 mg/kg/day irbesartan. The plasma concentrations of lipid and proinflammatory cytokines [Monocyte Chemoattractant Protein-1 (MCP-1), tumor necrosis factor-α, and interleukin-6] were assessed with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was observed with CD11b-positive cell counts and mRNA expressions of CD68 and F4/80 using immunohistochemistry and RT-PCR methods respectively. The mRNA levels of angiotensinogen, proinflammatory cytokines shown above, and adiponectin in WAT were also assessed with RT-PCR method. Glucose metabolism was assessed by glucose tolerance tests (GTTs) and insulin tolerance tests, and mRNA expression of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in WAT. Restraint stress increased monocyte accumulation, plasma free fatty acids, expression of angiotensinogen and proinflammatory cytokines including MCP-1, and reduced adiponectin. Irbesartan reduced stress-induced monocyte accumulation in WAT in a dose dependent manner. Irbesartan treatment also suppressed induction of adipose angiotensinogen and proinflammatory cytokines in WAT and blood, and reversed changes in adiponectin expression. Notably, irbesartan suppressed stress-induced reduction in adipose tissue weight and free fatty acid release, and improved insulin tolerance with restoration of IRS-1 and GLUT4 mRNA expressions in WAT. The results indicate that irbesartan improves stress-induced adipose tissue inflammation and insulin resistance. Our results suggests that irbesartan treatment exerts additive benefits for glucose metabolism in hypertensive patients with mental stress.

ABSTRACT Purpose: To determine the predictive factors for postoperative urinary incontinence (UI) following holmium laser enucleation of the prostate (HoLEP) during the initial learning period. Patients and Methods: We evaluated 127 patients with benign prostatic hyperplasia who underwent HoLEP between January 2011 and December 2013. We recorded clinical variables, including blood loss, serum prostate-specific antigen levels, and the presence or absence of UI. Blood loss was estimated as a decline in postoperative hemoglobin levels. The predictive factors for postoperative UI were determined using a multivariable logistic regression analysis. Results: Postoperative UI occurred in 31 patients (24.4%), but it cured in 29 patients (93.5%) after a mean duration of 12 weeks. Enucleation time >100 min (p=0.043) and blood loss >2.5g/dL (p=0.032) were identified as significant and independent risk factors for postoperative UI. Conclusions: Longer enucleation time and increased blood loss were independent predictors of postoperative UI in patients who underwent HoLEP during the initial learning period. Surgeons in training should take care to perform speedy enucleation maneuver with hemostasis.

Background/Aim: The prognostic impact of the administration of antibiotics and proton pump inhibitors (PPIs) in immune checkpoint inhibitor (ICI) therapy for advanced cancer has recently been documented. However, how these drugs affect the outcomes of first-line ICI combination therapy for advanced renal cell carcinoma (RCC) remains unclear. Patients and Methods: We retrospectively evaluated the data of 128 patients with RCC who received first-line ICI combination therapy. The patients were grouped according to their history of antibiotics and PPIs use one month before the initiation of ICI combination therapy. Progression-free survival (PFS), overall survival (OS), and objective response rate (ORR) after ICI combination therapy were compared between patients treated with and without antibiotics or PPIs. Results: Of the 128 patients, 30 (23%) and 44 (34%) received antibiotics and PPIs, respectively. Patients treated with antibiotics exhibited shorter PFS and OS compared to those who did not receive antibiotics (median PFS: 4.9 vs. 16.1 months, p<0.0001; OS: 20.8 vs. 49.0 months, p=0.0034). Multivariate analyses showed that antibiotic administration was an independent predictor of shorter PFS (hazard ratio: 2.54: p=0.0002) and OS (hazard ratio: 2.56: p=0.0067) after adjusting for other covariates. In contrast, there were no significant differences in either PFS or OS between patients who received PPIs and those who did not. (PFS: p=0.828; OS: p=0.105). Conclusion: Antibiotics administration before ICI combination therapy was negatively associated with outcomes of first-line ICI combination therapy for advanced RCC. Therefore, careful monitoring is required for potentially high-risk patients undergoing ICI combination therapy. [ABSTRACT FROM AUTHOR]

In 2022, we conducted a survey to assess how institutions were handling regenerative medical products. The survey was distributed to 203 facilities, including university hospitals, national and public hospitals, Japan Marrow Donor Program-certified hospitals, children's hospitals, and perinatal medical centers. A total of 140 facilities responded. The current status of in-hospital adoption of regenerative medical products is as follows: among 11 types of human cell/tissue products, Temcell® HS had the high usage rate of 80.7%, while Kymriah® (Tisagenlecleucel) had a usage rate of 28.6%. Of the three types of gene therapy product, Zolgensma® (Onasemnogene abeparvovec-xioi) had the highest usage rate at 18.6%. Regarding transfusion departments, while these were involved in the apheresis and management of regenerative medical products, they had was no involvement with Nepic® or Ocural®. It should be noted that these products are specified as designated-regenerative medical products, and the storage of administration records within the institution is therefore mandated, similarly as for blood products. Some of these products involve the participation of specialized departments, but the majority were under management of the respective medical department and pharmacy division. This survey highlighted challenges in the management and handling of regenerative medical products within the hospital from the perspective of the transfusion department. [ABSTRACT FROM AUTHOR]