Memory-like Liver Natural Killer Cells are Responsible for Islet Destruction in Secondary Islet Transplantation.

We previously demonstrated the pivotal role of natural killer (NK) cells in islet graft loss during the early phase after intraportal syngeneic islet transplantation (IT). Liver-resident DX5- NK cells were reported to possess memory-like properties, distinguishing them from conventional DX5+ NK cells. Here, we investigated the impact of primary IT-induced liver DX5- NK cells on the engraftment of secondary-transplanted islets in mice. The culture of liver NK cells isolated from naive mice with TNF-α, IFN-γ, and IL-lß, mimicking instant blood-mediated inflammatory reaction, led to significantly increased DX5- NK cell percentage among total liver NK cells. Consistently, the prolonged expansion of DX5- CD69+ TRAIL+ CXCR3+ NK cells was observed after intraportal IT of 300 syngeneic islets (marginal mass). In most diabetic mice, 400 syngeneic islets of primary IT were sufficient to achieve normoglycaemia, whereas the same mass after secondary IT failed to induce normoglycaemia in mice that received 200 syngeneic islets during primary IT. These findings indicated that liver-resident DX5- NK cells significantly expanded even after syngeneic IT, and that these memory-like NK cells may target both originally engrafted and secondary-transplanted islets. Furthermore, anti-TNF-α treatment suppressed the expansion of liver-resident DX5- NK cells, resulting in successful islet engraftment after sequential ITs.

Quality control of hospital preparations: Establishment of a simple and rapid method for quantifying ulinastatin in vaginal suppositories.

Ulinastatin vaginal suppositories, used to prevent threatened premature delivery, are frequently used in hospitals. However, there is no established method for quantifying ulinastatin contained in suppositories. Therefore, we investigated a simple and efficient method for quantifying ulinastatin contained in suppositories. Our analytical method involved removal of the base; optimising the enzyme inhibition reaction time and enzyme reaction time; and measuring the absorbance. The modified method was reproducible, operation time was significantly shortened, and cost was reduced to approximately 1/17 of that of the previously reported method. This simple and rapid quantitative method could contribute to the improvement of quality control of ulinastatin vaginal suppositories as an extemporaneous hospital preparation.

Role of Natural Killer Cells in the Innate Immune System After Intraportal Islet Transplantation in Mice.

BACKGROUND: Both liver natural killer (NK) and NK T cells of the innate immune system play a crucial role in islet graft loss after intraportal islet transplantation, although a relationship between NK and NK T cells in islet loss has not been proven. In this study, we investigated the role of NK cells in the innate immune system in islet graft loss after intraportal islet transplantation. METHODS: To investigate the involvement of liver NK cells in islet destruction, we assessed the differences in graft survival after intraportal islet transplantation between CD1d-/- diabetic mice and NK cell-depleted CD1d-/- diabetic mice. RESULTS: The transplantation of 400 islets into the liver was sufficient to reverse hyperglycemia in wild-type diabetic mice (100%, 4/4). However, normoglycemia could not be achieved when 200 islets were transplanted (0%, 0/4). In contrast, intraportal transplantation of 200 islets in NK cell-depleted CD1d-/- diabetic mice ameliorated hyperglycemia in 71% of cases (5/7), whereas transplantation of the same number of islets in CD1d-/- diabetic mice did not (0%, 0/4). Histologic findings also confirmed that intact islets were observed in NK cell-depleted CD1d-/- diabetic mice, but were difficult to observe in CD1d-/- diabetic mice. CONCLUSIONS: The involvement of liver NK cells in the innate immune system related to islet graft loss after intraportal islet transplantation is revealed by improved graft survival and function in NK cell-depleted CD1d-/- diabetic mice. Our data reveal that regulation of NK cell activity is particularly important when insufficient islet numbers are used for transplantation.

Biomarkers for the evaluation of immunological properties during the shikoku walking pilgrimage.

It is important to determine the immunological properties for the maintenance of health. We chose the Shikoku Walking Pilgrimage to assess the proper biomarkers for the evaluation of immunological properties. We examined whether the Shikoku Walking Pilgrimage could have a positive effect on the mental and physical health of walking participants by using several biomarkers proposed by our laboratory. Twelve non-randomized healthy male volunteers including 3 twice attendees walked the Shikoku Walking Pilgrimage distance of 58.9 km over 3 days. Plasma, serum, urine, and saliva were collected from the volunteers during the pilgrimage and at 1 week before and after it. Immunological biomarkers, including lipid metabolism, oxidative stress, immune function, and catecholamines, were measured. Additionally, mood state scores, alertness, autonomic nervous system activity, and body motion levels during sleep were assessed. A significant decrease was observed in the subjective tension-anxiety levels and in the concentrations of serum low-density lipoprotein cholesterol, plasma hydroxyoctadecadienoic acid (HODE), and urine adrenaline during the pilgrimage as compared to the values of these parameters before the participants embarked on the pilgrimage. The serum levels of brain-derived neurotrophic factor (BDNF) were significantly increased 1 week after the pilgrimage relative to those assessed previously. No significant differences in subjective fatigue and the flicker perception threshold were observed. These results suggest that the Shikoku Walking Pilgrimage can exert a positive effect on mental and physical health as particularly shown in the reduction of tensionanxiety and oxidative stress without the accompaniment of fatigue. HODE correlated significantly with typical immunological marker natural killer cell activity and immunoglobulin G. This suggests that there are promising biomarkers such as HODE, NK activity, BDNF, LDL-c, and IgG for assessing the immunological properties.

Switching from singlet-oxygen-mediated oxidation to free-radical-mediated oxidation in the pathogenesis of type 2 diabetes in model mouse.

Oxidative stress plays a key role in the development of type 2 diabetes. However, it is still unknown what kind of oxidative stress underlies the development of type 2 diabetes. We investigated hydroxyoctadecadienoic acid (HODE) isomers, which have been proposed as a biomarker for evaluating oxidative stress in vivo, during the development of diabetes in Tsumura Suzuki Obese Diabetes (TSOD) mouse, a type 2 diabetes model. It was revealed that glucose tolerance and insulin resistance index HOMA-IR in TSOD mice at 5 weeks of age were approximately normal, namely, the mice were in the prediabetic state, but these levels were significantly exacerbated from 8 weeks of age compared with those in Tsumura Suzuki Non Obesity (TSNO) mice (control). Concomitantly, the plasma levels of free-radical-mediated oxidation products, 9- and 13-(E,E)-HODE and 7ß-hydroxycholesterol, in TSOD mice were significantly higher than those in TSNO mice at 8, and 8 and 11 weeks of age, respectively. Interestingly, the plasma levels of 10- and 12-(Z,E)-HODE, which are produced specifically by singlet-oxygen-mediated oxidation, in TSOD mice were higher than those in TSNO mice only at 5 weeks of age, and not at 8, 11, and 13 weeks of age. We demonstrated that singlet-oxygen-mediated oxidation occurred in TSOD mice before development of the diabetic phenotypes, including impaired glucose tolerance and insulin resistance. These results suggest that excessive singlet-oxygen-mediated oxidation plays an important role in the pathogenesis of type 2 diabetes.

Neuroanatomical study on the tecto-suprageniculate-dorsal auditory cortex pathway in the rat.

Previous anatomical and physiological studies suggest that the superior colliculus sends integrated sensory information to the multimodal cortical areas via the thalamic suprageniculate nucleus (SG). However, the detailed distribution of rat tecto-SG axon terminals and SG neurons projecting to the multimodal cortex, as well as synaptic connections between these tectal axons and SG neurons, remains unclear. In this study, the organization of the tecto-thalamo-cortical pathway was investigated via combined injections of anterograde and retrograde tracers followed by light and electron microscopic observations. Injections of a retrograde tracer, cholera toxin B subunit (CTB), into the temporal cortex, area 2, dorsal part (Te2D), and injections of an anterograde tracer, biotinylated dextran amine (BDA), into the deep layers of the superior colliculus produced the following results: (1) Retrogradely CTB-labeled neurons were found throughout SG, predominantly in its rostral part. CTB-labeled neurons were also found in other cortical areas such as the visual cortex, the auditory cortex, the parietal association cortex, and the perirhinal cortex. (2) Anterogradely BDA-labeled axons and their terminals were also observed throughout SG. Dual visualization of BDA and CTB showed that retrogradely labeled SG neurons and anterogradely labeled tectal axon terminal boutons overlapped considerably in the rostral part of SG, and their direct synaptic contacts were also confirmed via electron microscopy. These findings suggest that multimodal information from the superior colliculus can be processed directly in SG neurons projecting to Te2D.

A cohort study of racing performance in Japanese Thoroughbred racehorses using genome information on ECA18.

Using 1710 Thoroughbred racehorses in Japan, a cohort study was performed to evaluate the influence of genotypes at four single nucleotide polymorphisms (SNPs) on equine chromosome 18 (ECA18), which were associated in a previous genome-wide association study for racing performance with lifetime earnings and performance rank. In males, both g.65809482T>C and g.65868604G>T were related to performance rank (P= 0.005). In females, g.65809482T>C (P = 1.76E-6), g.65868604G>T (P=6.81E-6) and g.66493737C>T (P=4.42E-5) were strongly related to performance rank and also to lifetime earnings (P < 0.05). When win-race distance (WRD) among all winning racehorses and best race distance (BRD) among elite racehorses were considered as the phenotypes, significant associations (P<0.001) were observed for all four SNPs. The favourable race distance of both elite (BRD) and novice racehorses (WRD) was also associated with genotypes in the ECA18 region, indicating the presence of a gene in this region influencing optimum race distance in Thoroughbred racehorses. Therefore, the association with performance rank is likely due to the bias in the race distances. The location of the SNPs within and proximal to the gene encoding myostatin (MSTN) strongly suggests that regulation of the MSTN gene affects racing performance. In particular, the g.65809482T>C, g.65868604G>T and g.66493737C>T SNPs, or their combinations, may be genetic diagnostic markers for racing performance indicators such as WRD and BRD.

A genome-wide association study for racing performances in Thoroughbreds clarifies a candidate region near the MSTN gene.

Using 1400 microsatellites, a genome-wide association study (GWAS) was performed to identify genomic regions associated with lifetime earnings and performance ranks, as determined by the Japan Racing Association (JRA). The minimum heritability (h(2) ) was estimated at 7-8% based on the quantitative trait model, suggesting that the racing performance is heritable. Following GWAS with microsatellites, fine mapping led to identification of three SNPs on ECA18, namely, g.65809482T>C (P=1.05E-18), g.65868604G>T (P=6.47E-17), and g.66539967A>G (P=3.35E-14) associated with these performance measures. The haplotype of these SNPs, together with a recently published nearby SNP, g.66493737C>T (P=9.06E-16) in strong linkage disequilibrium, also showed a very clear association with the performance (P<1E-05). The candidate genomic region contained eight genes annotated by ENSEMBL, including the myostatin gene (MSTN). These findings suggest the presence of a gene affecting the racing performance in Thoroughbred racehorses in this region on ECA18.

A genome-wide scan for tying-up syndrome in Japanese Thoroughbreds.

Tying-up syndrome, also known as recurrent exertional rhabdomyolysis in Thoroughbreds, is a common muscle disorder for racehorses. In this study, we performed a multipoint linkage analysis using LOKI based on the Bayesian Markov chain Monte Carlo method using 5 half-sib families (51 affected and 277 nonaffected horses in total), and a genome-wide association study (GWAS) using microsatellites (144 affected and 144 nonaffected horses) to map candidate regions for tying-up syndrome in Japanese Thoroughbreds. The linkage analysis identified one strong L-score (82.45) between the loci UCDEQ411 and COR058 (24.9-27.9 Mb) on ECA12. The GWAS identified two suggestive genomic regions on ECA12 (24.9-27.8 Mb) and ECA20 (29.3-33.5 Mb). Based on both results, the genomic region between UCDEQ411 and TKY499 (24.9-27.8 Mb) on ECA12 was the most significant and was considered as a candidate region for tying-up syndrome in Japanese Thoroughbreds.

Disseminated mucocutaneous herpes simplex virus infection in an immunocompetent woman.

Disseminated mucocutaneous herpes simplex virus (HSV) infection in an immunocompetent person is quite rare. A 19-year-old healthy Japanese woman presented with painful, umbilicated vesicles and pustules on her genital region, both nipples and on the forearm 10 days after the last sexual contact with her partner who had cold sore at that time. Tzanck test and biopsy confirmed the diagnosis of disseminated mucocutaneous HSV infection. She did not have any visceral HSV disease. Skin lesions improved after treatment with acyclovir and erythromycin for seven days. We propose that like herpes gladiatorum, HSV dissemination in this case was acquired by close body contact.

Suppressive effects of 1,4-dihydroxy-2-naphthoic acid administration on bone resorption.

SUMMARY: The main component of the metabolic by-products of fermentation by Propionibacterium freudenreichii ET-3 is 1,4-dihydroxy-2-naphthoic acid (DHNA), which has a naphthoquinone skeleton, as in vitamin K2. This study showed that DHNA improved bone mass reduction with osteoporosis model mice caused by FK506. INTRODUCTION: Growth of the intestinal bacterium Lactobacillus bifidus is specifically facilitated by DHNA. The present study used osteoporosis model mice to investigate the effects of DHNA on bone remodeling. METHODS: FK506, an immunosuppressant, was used to prepare osteoporosis model mice. Thirty mice were divided into three groups: FK group, FK+DHNA group, and control group. In the FK group, FK506 was administered to induce bone mass reduction. In the FK-DHNA group, FK506 and DHNA were administered concurrently to observe improvements in bone mass reduction. To ascertain systemic and local effects of DHNA, we investigated systemic pathological changes in colon, kidney function and cytokine dynamics, and morphological and organic changes in bone and osteoclast dynamics as assessed by culture experiments. RESULTS: Compared to the FK group without DHNA, colon damage and kidney dysfunction were milder for FK+DHNA group, and production of inflammatory cytokines (interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha) was more suppressed. Furthermore, compared to the group without DHNA, histological analyses and radiography showed that bone resorption was suppressed for the DHNA group. Culture experiments using osteoclasts from murine bone marrow showed osteoclast suppression for the DHNA group compared to the group without DHNA. CONCLUSION: These results show that DHNA has some effects for improving bone mass reduction caused by FK506.

Herpes zoster of the nipple: rapid DNA-based diagnosis by the loop-mediated isothermal amplification method.

A 28-year-old Japanese man presented with grouped erosions and vesicles on an erythematous base affecting the right areola and the surrounding skin. A Tzanck smear from the vesicle revealed giant cells. An initial clinical diagnosis of mammary herpes simplex was considered but to explore the differential diagnosis, viral DNA was amplified by the loop-mediated isothermal amplification (LAMP) method. DNA replication was observed only in varicella zoster virus LAMP mixture, and this confirmed a diagnosis of herpes zoster. The patient was treated with 3000 mg of daily oral valacyclovir for seven days. After antiviral treatment, the lesion had healed and the pain had resolved completely.

Two-stage portal vein ligation facilitates liver regeneration in rats.

BACKGROUND/AIMS: Recent reports have demonstrated that some patients are unable to undergo scheduled liver resection after preoperative portal vein embolization due to insufficient hypertrophy of the future remnant liver. The present study examined whether two-stage portal vein ligation (PVL) increases hypertrophy of the future remnant liver compared to conventional PVL in rats. METHODS: Rats were divided into 3 groups: group A, ligation of left primary branch; group B, ligation of right and left primary branches; group C, ligation of the left primary branch, followed by 2-stage PVL 7 days postoperatively. To evaluate liver regeneration, the proliferating cell nuclear antigen labeling index (LI), mitotic index (MI) in the caudate lobe and weight ratio of caudate lobe to body weight were measured. RESULTS: The weight ratio of caudate lobe to body weight was significantly higher in group C than in groups A or B 14 days postoperatively. In groups A and B, LI and MI in the caudate lobe peaked 2 days postoperatively, then decreased to preoperative levels by 7-8 days postoperatively, but remained significantly elevated until 10-14 days postoperatively in group C. CONCLUSION: Two-stage PVL increases hypertrophy of the future remnant liver compared to conventional PVL in rats.

A pilot study to assess the efficacy of photodynamic therapy for Japanese patients with actinic keratosis in relation to lesion size and histological severity.

BACKGROUND/PURPOSE: Topical 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) is effective for actinic keratosis (AK); few studies have examined Oriental patients. The aim of this study is to assess the efficacy of PDT for the treatment of Japanese AK patients classified by lesion size and histological severity. METHODS: Thirty patients with solitary AK lesions were divided into two groups according to diameter: a small lesion group (SL), diameter < or =10 mm and a larger lesion group (LL), diameter >10 mm, and histological severity: Group I (mild and moderate) and Group II (severe). After application of 20% ALA for 4 h, exposure to an excimer-dye laser at 630 nm was performed at a dose of 50 J/cm(2) three times at an interval of 7 days. Therapeutic effects were assessed and followed for 12 months. RESULTS: In all 10 SL patients, atypical cells disappeared after PDT and did not recur for 12 months. However, for the 20 LL patients, recurrence was seen in 2 of the 14 Group I patients, while 4 of 6 Group II patients showed residual tumor cells after the first PDT session. CONCLUSION: The present study demonstrated that ALA-PDT might be useful for treatment of Japanese AK. The therapeutic outcome might depend on the lesion size and the histopathological severity.

Etretinate enhances the susceptibility of human skin squamous cell carcinoma cells to 5-aminolaevulic acid-based photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolaevulinic acid (5-ALA) is a noninvasive and effective treatment for superficial skin cancers. Etretinate, a derivate of vitamin A, with the chemical formula ethyl(2E,4E,6E,8E)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-2,4,6,8-nona-tetraenoate, has been reported to have antitumour effects and to regulate the proliferation and differentiation of skin cancers. OBJECTIVE: In order to develop more efficient PDT, we investigated whether etretinate enhanced the cytotoxic action of ALA-based PDT against human squamous cell carcinoma cell line, HSC-5. METHOD: The in vitro cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic cells were detected by double-staining with fluorescent annexin V and propidium iodide. Intracellular protoporphyrin IX (PpIX) converted from exogenous ALA was measured by a fluorescence meter. RESULTS: HSC-5 cells pretreated with a nontoxic concentration of etretinate became more susceptible to the cytotoxic action of ALA-based PDT. Etretinate-pretreated cells underwent apoptosis in response to ALA-based PDT. Etretinate pretreatment resulted in enhanced accumulation of ALA-dependent intracellular PpIX. CONCLUSIONS: The results suggest that etretinate enhances the susceptibility of HSC-5 cells to ALA-based PDT via the intracellular increase of ALA-dependent PpIX. Etretinate might be useful for improvement of ALA-based PDT.

Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly.

Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription.

Effect of tyrosine kinase inhibition by imatinib mesylate on mast cell tumors in dogs.

BACKGROUND: Imatinib mesylate is a small molecule targeted at dysregulated protein-tyrosine kinase. Mutation of c-kit exon 11, which induces constitutive phosphorylation of KIT, is one of the mechanisms for the development or progression of mast cell tumor (MCT) in dogs. The purpose of this study was to examine the therapeutic potential of imatinib mesylate in canine MCT. HYPOTHESIS: Imatinib mesylate has activity against MCT in dogs, and response to treatment can be correlated to presence of mutation within exon 11 of c-kit. ANIMALS: Twenty-one dogs with MCT with gross tumor burden and median tumor size of 7.2 cm (range, 1.0-25.3 cm) before treatment. METHODS: Tumors were analyzed for mutation of c-kit exon 11. Imatinib mesylate was administered PO to the dogs at a dose of 10 mg/kg daily for 1-9 weeks. RESULTS: Ten of 21 dogs (48%) had some beneficial response to imatinib mesylate treatment within 14 days of treatment initiation. All 5 dogs with a demonstrable c-kit mutation in exon 11 responded to the drug (1 complete remission, 4 partial remission). CONCLUSIONS AND CLINICAL IMPORTANCE: Imatinib mesylate has clinical activity against MCT in dogs. Response could not be predicted based on presence of absence of a mutation in exon 11 of c-kit.

Circadian clock molecules CLOCK and CRYs modulate fibrinolytic activity by regulating the PAI-1 gene expression.

Disruptions of circadian rhythms are associated with the development of many disorders. However, whether a disruption of the circadian clock can cause anomalies of the hemostatic balance remains unknown. The present study examines coagulation and fibrinolytic activities in circadian clock mutants, a homozygous Clock mutant and Cry1/Cry2 double knockout (Cry1/2-deficient) mice. The euglobulin clot lysis time (ELT) showed circadian variations that peaked at 21:00 (early night) in wild-type mice, suggesting that fibrinolytic activity is lowest at this time. The ELT was continuously reduced in Clock mutants, while the ELT was significantly increased and did not differ between day and night (9:00 and 21:00) in Cry1/2-deficient mice. The prothrombin time (PT) and activated partial prothrombin time (APTT) were constant in all genotypes. To identify which factors cause the loss of ELT rhythm, we measured fibrinolytic parameters in Clock mutant and Cry1/2-deficient mice. The robust circadian fluctuation of plasma plasminogen activator inhibitor 1 (PAI-1) that peaked at early night was damped to trough levels in Clock mutant mice. On the other hand, PAI-1 levels in Cry1/2-deficient mice remained equivalent to the peak levels of those in wild-type mice at both 9:00 and 21:00. Circadian changes in plasma PAI-1 levels seemed to be regulated at the level of gene expression, because the plasma PAI-1 levels in Clock mutant and Cry1/2-deficient mice were closely correlated with the level of PAI-1 mRNA transcript in these mice. Plasma plasminogen and hepatic mRNA levels were not rhythmic in wild-type mice, and continuously higher in Clock mutant than in wild-type or Cry1/2-deficient mice. In contrast, the activity and mRNA levels of tissue type plasminogen activator (t-PA), plasma levels and mRNA levels of plasminogen, and plasma levels of alpha2 plasmin inhibitor (alpha2PI) in all genotypes were constant throughout the day. Coagulation parameters such as factor VII, factor X, prothrombin and fibrinogen remained constant throughout the day, and were not affected by clock gene mutations. These results suggest that circadian clock molecules play an important role in hemostatic balance by regulating the fibrinolytic systems.