Simple Score to Predict Treatment Response to Low-Dose Tolvaptan in Patients with Heart Failure.

The recommended starting dose of Tolvaptan for heart failure (HF) is 7.5 mg/day in Japan; the recommended dose is 3.75 mg/day for older patients to avoid excessive diuresis and hypernatremia. However, low-dose Tolvaptan may delay the release of congestion in some patients. We aimed to develop a score to predict treatment responders to 3.75 mg tolvaptan.We retrospectively analyzed 106 patients with HF who initially received 3.75 mg/day of Tolvaptan in the derivation cohort (April 2013-December 2017) and 63 patients receiving 3.75 mg/day of Tolvaptan in the validation cohort (January 2018-April 2021). Treatment responders to 3.75 mg tolvaptan did not require dose escalation of Tolvaptan for congestion relief. In multivariate analysis, blood urea nitrogen (BUN) < 39 mg/dL and hematocrit > 35% were selected as variables to predict treatment responders. These were assigned 1 point each, and patients were stratified into groups with 2 points (n = 32), 1 point (n = 39), and 0 points (n = 35). The frequency of treatment responders was 82.9% in the 2-point group, 61.5% in the 1-point group, and 34.4% in the 0-point group (P < 0.05). The predictive ability of the score was acceptable with an area under the receiving operator characteristic curve (AUC) 0.726 (P < 0.05); its performance was maintained in the validation cohort (AUC 0.733, P < 0.05).A simple score using BUN and hematocrit could identify treatment responders to 3.75 mg tolvaptan, which may help determine the appropriate starting dose of Tolvaptan, balancing efficiency with safety for older patients with HF.

Development of a Portable Detection Method for Enteric Viruses from Ambient Air and Its Application to a Wastewater Treatment Plant.

The ambient air from wastewater treatment plants has been considered as a potential source of pathogenic microorganisms to cause an occupational risk for the workers of the plants. Existing detection methods for enteric viruses from the air using a liquid as the collection medium therefore require special care to handle on-site. Knowledge accumulation on airborne virus risks from wastewater has been hindered by a lack of portable and handy collection methods. Enteric viruses are prevalent at high concentrations in wastewater; thus, the surrounding air may also be a potential source of viral transmission. We developed a portable collection and detection method for enteric viruses from ambient air and applied it to an actual wastewater treatment plant in Japan. Materials of the collection medium and eluting methods were optimized for real-time polymerase chain reaction-based virus quantification. The method uses a 4 L/min active air sampler, which is capable of testing 0.7-1.6 m3 air after 3-7 h sampling with a detection limit of 102 copies/m3 air in the field. Among 16 samples collected at five to seven locations in three sampling trials (November 2007-January 2008), 56% (9/16) samples were positive for norovirus (NV) GII, with the highest concentration of 3.2 × 103 copies/m3 air observed at the sampling point near a grit chamber. Adenoviruses (4/16), NV GI (6/16), FRNA bacteriophages GIII (3/16), and enteroviruses (3/16) were also detected but at lower concentrations. The virus concentration in the air was associated with that of the wastewater at each process. The results imply that the air from the sewer pipes or treatment process is contaminated by enteric viruses and thus special attention is needed to avoid accidental ingestion of viruses via air.

Sex differences in surrogate decision-maker preferences for life-sustaining treatments of Japanese patients with heart failure.

AIMS: Patients with end-stage heart failure (HF) often require surrogate decision making for end-of-life care owing to a lack of decision-making capacity. However, the clinical characteristics of surrogate decision making for life-sustaining treatments in Japan remain to be investigated. METHODS AND RESULTS: Among 934 patients admitted to our hospital for HF from January 2004 to December 2015, we retrospectively reviewed the medical records of consecutive 106 patients who died in hospital (mean age 73 ± 13 years; male, 52.6%). During hospitalization, attending physicians conducted an average of 2.1 ± 1.4 end-of-life conversations with patients and/or their families. Only 4.7% of patients participated in the conversations and declared their preferences; surrogates made medical care decisions in 95.3% of cases. Most decisions by surrogates (98.1%) were made without the patient's advance directive. During initial end-of-life conversations, 49.4% of surrogates requested cardiopulmonary resuscitation (CPR). However, 72.0% of CPR preferences were changed to do not attempt resuscitation (DNAR) orders in the final conversation. Female surrogates were more likely to change the preference from CPR to DNAR than were male surrogates (47.1% vs. 25.0%, P = 0.023). CONCLUSIONS: Compared with male surrogates, female surrogates wavered more often in their decisions regarding life-sustaining treatments of Japanese patients with end-stage HF.

Diaphragm Muscle Dysfunction in Patients With Heart Failure.

BACKGROUND: Inspiratory muscle weakness is associated with the development of exercise intolerance in patients with heart failure (HF). Ultrasound assessment of the diaphragm is used to evaluate respiratory muscle function, but its application in patients with HF remains undefined. We examined the relationship of diaphragm function as assessed by ultrasonography with inspiratory muscle strength and exercise tolerance in HF. METHODS AND RESULTS: Seventy-seven patients hospitalized with HF were enrolled. Impaired diaphragm muscle function was defined as a diaphragm thickness at end-inspiration of less than the median value of 4.0 mm, which represents diaphragm muscle loss and reduced contraction. Compared with patients with preserved diaphragm muscle function, those with impaired diaphragm muscle function were older; had significantly lower vital capacity, handgrip strength, and inspiratory muscle strength as assessed by the maximum inspiratory pressure; and had a significantly shorter 6-minute walk distance (6MWD; P < .05). Although low handgrip strength was also associated with a short 6MWD, the relationship between impaired diaphragm muscle function and short 6MWD was independent from age, vital capacity, and handgrip strength. CONCLUSION: Diaphragm dysfunction as assessed by ultrasonography represents inspiratory muscle weakness and predicts exercise intolerance independently from comorbid pulmonary dysfunction and dynapenia in patients with HF.

The PFA-AMeX method achieves a good balance between the morphology of tissues and the quality of RNA content in DNA microarray analysis with laser-capture microdissection samples.

Recently, large-scale gene expression profiling is often performed using RNA extracted from unfixed frozen or formalin-fixed paraffin embedded (FFPE) samples. However, both types of samples have drawbacks in terms of the morphological preservation and RNA quality. In the present study, we investigated 30 human prostate tissues using the PFA-AMeX method (fixation using paraformaldehyde (PFA) followed by embedding in paraffin by AMeX) with a DNA microarray combined with laser-capture microdissection. Morphologically, in contrast to the case of atypical adenomatous hyperplasia, loss of basal cells in prostate adenocarcinomas was as obvious in PFA-AMeX samples as in FFPE samples. As for quality, the loss of rRNA peaks 18S and 28S on the capillary electropherograms from both FFPE and PFA-AMeX samples showed that the RNA was degraded equally during processing. However, qRT-PCR with 3' and 5' primer sets designed against human beta-actin revealed that, although RNA degradation occurred in both methods, it occurred more mildly in the PFA-AMeX samples. In conclusion, the PFA-AMeX method is good with respect to morphology and RNA quality, which makes it a promising tool for DNA microarrays combined with laser-capture microdissection, and if the appropriate RNA quality criteria are used, the capture of credible GeneChip data is well over 80% efficient, at least in human prostate specimens.

The status of donor cancer tissues affects the fate of patient-derived colorectal cancer xenografts in NOG mice.

Patient-derived xenografts (PDXs) of tumors are increasingly becoming important tools for translational research in oncology. The NOD.Cg-Prkdc(scid) Il2rg(tm1Sug)/Jic (NOG) mouse is an efficient host for PDXs. Thus as a basis for future development of methods to obtain PDXs from various disease types, we have studied the factors that affect the outcome of transplantation of human colorectal cancer in NOG mice. Of the original donor cases examined, 73% had successful engraftment. The outcome of donor-matched tissues was consistent in most cases, and was thought to show that the condition of the host did not affect engraftment. Next we analyzed the tumor aggressiveness in terms of histology grade of the original tumor and found that they were related to engraftment. Detailed histopathological examination of the transplanted tissues strongly indicated that lymphocytes engrafted with the tumor cells affect engraftment. As a factor related to transplantation of lymphocytes, we studied the human IgG concentration in the serum of tumor-bearing mice, but there was no tendency for higher concentrations to result in unsuccessful engraftment. Finally, we studied the type, density and location of T cells in the original donor tissue to determine the immune contexture and found that the unsuccessful engraftment cases tended to have an adequate or coordinated immune contexture compared to successful engraftment cases. From these results, we concluded that the aggressiveness and the T cell infiltration of the original tumor affect the outcome of transplantation in the NOG mouse.

Differential expression of pentraxin 3 in neutrophils.

Pentraxins belong to the superfamily of conserved proteins that are characterized by a cyclic multimeric structure. Pentraxin 3 (PTX3) is a long pentraxin which can be produced by different cell types upon exposure to various inflammatory signals. Inside the neutrophil PTX3 is stored in form of granules localized in the cytoplasm. Neutrophilic granules are divided into three types: azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. PTX3 has been considered to be localized in specific (secondary) granules. Immunofluorescent analyses using confocal laser microscopic examination were performed to clarify the localization of all three groups of granules within the cytoplasm of the mature neutrophils and neutrophils stimulated with IL-8. Furthermore, PTX3 was localized in primary granules of promyelocyte cell line HL-60. As a result, we suggest that PTX3 is localized not only in specific granules, but is also partly expressed in primary and tertiary granules. After the stimulation with IL-8, irregular reticular structures called neutrophil extracellular traps (NETs) were formed, three types of granules were trapped by NETs and PTX3 showed partial colocalization with these granular components. PTX3 localized in all three types of granules in neutrophils may play important roles in host defense.

Protective effect of the long pentraxin PTX3 against histone-mediated endothelial cell cytotoxicity in sepsis.

Pentraxin 3 (PTX3), a member of the long pentraxin subfamily within the family of pentraxins, is a soluble pattern recognition molecule that functions in the innate immune system. Innate immunity affords the infected host protection against sepsis, a potentially life-threatening inflammatory response to infection. Extracellular histones are considered to be the main cause of septic death because of their cytotoxic effect on endothelial cells, which makes them a potential therapeutic target. We found that PTX3 interacted with histones to form coaggregates, which depended on polyvalent interactions and disorder in the secondary structure of PTX3. PTX3 exerted a protective effect, both in vitro and in vivo, against histone-mediated cytotoxicity toward endothelial cells. Additionally, the intraperitoneal administration of PTX3 reduced mortality in mouse models of sepsis. The amino-terminal domain of PTX3, which was required for coaggregation with histones, was sufficient to protect against cytotoxicity. Our results suggest that the host-protective effects of PTX3 in sepsis are a result of its coaggregation with histones rather than its ability to mediate pattern recognition. This long pentraxin-specific effect provides a potential basis for the treatment of sepsis directed at protecting cells from the toxic effects of extracellular histones.

Characterization of EBV-related lymphoproliferative lesions arising in donor lymphocytes of transplanted human tumor tissues in the NOG mouse.

Human tumor tissue line models established in the severely immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Sug)/Jic (NOD/Shi-scid, IL-2Rγ(null) or NOG) mouse are important tools for oncology research. During the establishment process, a lymphoproliferative lesion (LPL) that replaces the original tumor cells in the site of transplantation occurs. In the present study, we studied the impact of the LPL on the establishment process and the characteristics of the lesion, investigated the systemic distribution of the lesion in the mouse, and evaluated the potential of a simple identification method. The incidence of the lesion varied among tumor types, and the lesion was found to be the leading cause of unsuccessful establishment with gastric and colorectal cancer. The lesion consisted of a varying population of proliferating lymphoid cells that expressed CD20. The cells were positive for Epstein-Barr virus (EBV)-related antigens, and EBV DNA was detected. There was systemic distribution of the lesion within the NOG mouse, and the most consistent gross finding was splenomegaly. Additionally, identification of LPL-affected cases was possible by detecting splenomegaly in the 1st and 2nd generation mice at necropsy. From our findings the lesion was judged to arise from EBV-infected B cells originating from the donor, and monitoring splenomegaly at necropsy was thought effective as a simple method for identifying the lesion at an early stage of the establishment process.

Histopathological characteristics of human non-tumor thyroid tissues in a long-term model of adenomatous goiter xenografts in the NOD/Shi-scid, IL-2Rγ(null) mouse.

There is a growing need for modeling the human thyroid to link data obtained from animals to humans because of its sensitivity to radiation exposure and endocrine disruption chemicals. In a scid mouse model produced by transplanting human thyroid tissues, leakiness and thymic lymphoma that occurs spontaneously in the scid mouse can complicate the interpretation of experimental results. Considering that the NOD.Cg-Prkdc(scid)Il2rg(tm1Sug)/Jic mouse (NOD/Shi-scid, IL-2Rγ(null) or NOG mouse) may be a better host because this strain has low incidence of leakiness and thymic lymphoma, we have evaluated the potential of a model that allows long-term observation of non-tumor human thyroid tissues in this mouse. We transplanted tissues of human adenomatous goiter into NOG mice and examined the tissues histopathologically. The morphology of human adenomatous goiter tissues was maintained from 24 to 44 weeks after transplantation in NOG mice with no noted differences between donor-matched tissues or the weeks after transplantation. The tissues expressed thyroglobulin protein and mRNA as well as thyroperoxidase. Endothelial cells originating from human were found in the transplanted tissues and were thought to be a characteristic of this model. The intactness of the tissues before transplantation was found to affect the rate of tissue engraftment. From the present results we have concluded that transplanted thyroid tissues in NOG mice maintain the histopathological characteristics of their origin for long terms. Therefore this model was thought feasible for toxicity evaluation.

LGR5-positive colon cancer stem cells interconvert with drug-resistant LGR5-negative cells and are capable of tumor reconstitution.

The cancer stem cell (CSC) concept has been proposed as an attractive theory to explain cancer development, and CSCs themselves have been considered as targets for the development of diagnostics and therapeutics. However, many unanswered questions concerning the existence of slow cycling/quiescent, drug-resistant CSCs remain. Here we report the establishment of colon cancer CSC lines, interconversion of the CSCs between a proliferating and a drug-resistant state, and reconstitution of tumor hierarchy from the CSCs. Stable cell lines having CSC properties were established from human colon cancer after serial passages in NOD/Shi-scid, IL-2Rγ(null) (NOG) mice and subsequent adherent cell culture of these tumors. By generating specific antibodies against LGR5, we demonstrated that these cells expressed LGR5 and underwent self-renewal using symmetrical divisions. Upon exposure to irinotecan, the LGR5(+) cells transitioned into an LGR5(-) drug-resistant state. The LGR5(-) cells converted to an LGR5(+) state in the absence of the drug. DNA microarray analysis and immunohistochemistry demonstrated that HLA-DMA was specifically expressed in drug-resistant LGR5(-) cells, and epiregulin was expressed in both LGR5(+) and drug-resistant LGR5(-) cells. Both cells sustained tumor initiating activity in NOG mice, giving rise to a tumor tissue hierarchy. In addition, anti-epiregulin antibody was found to be efficacious in a metastatic model. Both LGR5(+) and LGR5(-) cells were detected in the tumor tissues of colon cancer patients. The results provide new biological insights into drug resistance of CSCs and new therapeutic options for cancer treatment.

The proteomic profile of circulating pentraxin 3 (PTX3) complex in sepsis demonstrates the interaction with azurocidin 1 and other components of neutrophil extracellular traps.

Pentraxin 3 (PTX3), a long pentraxin subfamily member in the pentraxin family, plays an important role in innate immunity as a soluble pattern recognition receptor. Plasma PTX3 is elevated in sepsis (~200 ng/ml) and correlates with mortality. The roles of PTX3 in sepsis, however, are not well understood. To investigate the ligands of PTX3 in sepsis, we performed a targeted proteomic study of circulating PTX3 complexes using magnetic bead-based immunopurification and shotgun proteomics for label-free relative quantitation via spectral counting. From septic patient fluids, we successfully identified 104 candidate proteins, including the known PTX3-interacting proteins involved in complement activation, pathogen opsonization, inflammation regulation, and extracellular matrix deposition. Notably, the proteomic profile additionally showed that PTX3 formed a complex with some of the components of neutrophil extracellular traps. Subsequent biochemical analyses revealed a direct interaction of bactericidal proteins azurocidin 1 (AZU1) and myeloperoxidase with PTX3. AZU1 exhibited high affinity binding (K(D) = 22 ± 7.6 nm) to full-length PTX3 in a calcium ion-dependent manner and bound specifically to an oligomer of the PTX3 N-terminal domain. Immunohistochemistry with a specific monoclonal antibody generated against AZU1 revealed a partial co-localization of AZU1 with PTX3 in neutrophil extracellular traps. The association of circulating PTX3 with components of the neutrophil extracellular traps in sepsis suggests a role for PTX3 in host defense and as a potential diagnostic target.

Right- and left-sided carcinoid heart disease without intracardiac shunting.

We present a case of a 54-year-old man with carcinoid heart disease and mitral valve involvement. He had hepatic carcinoid with an extremely elevated urinary excretion of 5-hydroxyindole acetic acid and was referred to our division for shortness of breath and leg edema. Transthoracic echocardiography showed the thickened and retracted tricuspid valve leaflets and severe tricuspid regurgitation. Moderate mitral regurgitation was observed, with the involvement of mitral valve leaflets. A patent foramen ovale was not detected on transesophageal echocardiography. The extremely high concentration of circulating serotonin and/or other vasoactive substances may have contributed to left- as well as right-sided carcinoid heart disease.

Fenofibrate enhances neovascularization in a murine ischemic hindlimb model.

Fenofibrate have been illustrated to stimulate nitric oxide (NO) pathway, which plays pivotal roles in neovascularization. Here, we evaluated the effect of fenofibrate on neovascularization using a murine ischemic hindlimb model. C57BL/6J mice were treated with fenofibrate and/or NG-nitro-l-arginine methyl ester hydrochloride (l-NAME) for 28 days after ischemia operation. We exploited a concentration of L-NAME that did not affect blood pressure levels but suppress NO activity. Limb blood perfusion and capillary density in ischemic limb, serum NO levels, and aortic NOS activity were significantly increased by fenofibrate treatment when compared with the untreatment group. And, these effects were abolished by coadministration of L-NAME. Fenofibrate treatment significantly lowered serum triglyceride levels. Cotreatment of L-NAME did not inhibit serum triglyceride level, lowering effect of fenofibrate. These results suggested that the lowering in serum triglyceride levels is not involved in the improvement of neovascularization. In an in vitro experiment, fenofibrate stimulated NOS activity in human umbilical vein endothelial cells. Also, fenofibrate stimulated in vitro angiogenesis, and this effect was abolished by coincubation with L-NAME. In conclusions, fenofibrate enhanced neovascularization in a murine hindlimb ischemia model. The mechanism is most likely through activation of NO pathway in endothelial cells.

Spontaneous thymic lymphomas in the non-obese diabetic/Shi-scid, IL-2R gamma (null) mouse.

The NOD/Shi-scid, IL-2Rgamma(null) (NOG) mouse is a severely immunodeficient mouse used for the engraftment of human tissues and cells. In this study, 2406 mice (8-62 weeks old, 503 males and 1903 females) were subcutaneously engrafted with human tissues. In 16 mice (12-26 weeks old, 1 male and 15 females), a mass was seen in the anteroventralis of the thorax on gross examination with an incidence of 0.7%. Histologically, the masses were composed of sheets of lymphoblastic cells. A 'starry sky' pattern was observed with numerous mitoses. Immunohistochemically the lymphoblastic cells were positive for Thy 1. The lymphoblastic cells were also seen in the spleen, lung, liver, kidney and heart. The gross and histopathological findings led to the diagnosis of spontaneous thymic lymphoma in NOG mice.

First detection of genotype 3 hepatitis E virus RNA in river water in Cambodia.

To identify and describe hepatitis E virus (HEV) strains in aquatic environments in Cambodia, we collected two river-water samples from upstream and downstream sites along the Siem Reap River and examined them for HEV using a virus concentration method followed by TaqMan reverse transcription PCR and genetic analysis of the PCR product. Genotype 3 HEV strain was detected from the river-water sample collected from the river downstream, indicating that genotype 3 HEV strains are circulating in this region. To our knowledge, this is the first report demonstrating HEV contamination in aquatic environments in Cambodia.

The expression profile of glypican-3 and its relation to macrophage population in human hepatocellular carcinoma.

BACKGROUND: Glypican-3 (GPC3) is frequently upregulated in hepatocellular carcinoma (HCC). Analysis of GPC3-deficient mice implies GPC3 involvement in macrophage-lineage cells. AIM: In this study, we first assessed the association of GPC3 expression with the macrophage population in liver tissues from 30 HCC patients using immunohistochemistry. METHODS: The GPC3 expression was categorized into three patterns - one with GPC3-negative staining and two with GPC3-positive staining (one with unclear membrane staining and one with clear membrane staining, designated GPC3+/C). The number of macrophages that were stained with resident macrophage (rMvarphi) or pan-macrophage (pMvarphi) markers was counted for each GPC3 expression pattern. RESULTS: GPC3 immunoreactivity was observed in 76.7% of the HCC specimens. No significant differences were observed in the number of rMvarphi marker-positive cells among the three expression patterns. In contrast, the GPC3+/C pattern showed a significantly higher number of pMvarphi-positive cells compared with the other two patterns, most of which tended to take on the morphology of migrating macrophages. A second experiment conducted to compare macrophage infiltration between the xenograft tissues of a GPC3-transfected HCC cell line and its parent GPC3-nonexpressing cell line revealed that the increase in macrophages was stimulated by membrane expression of GPC3. CONCLUSION: The observations suggest that the increased macrophages in the GPC3+/C pattern are likely to be recruited macrophages, not resident macrophages, and that the expression of GPC3 in the membrane is involved in macrophage recruitment.

Bacterial contamination of raw vegetables, vegetable-related water and river water in Ho Chi Minh City, Vietnam.

The study attempts to identify the potential routes of bacterial infection via consumption of raw vegetables, drinking water and vegetable-related water in Ho Chi Minh City (HCMC). Vegetables in the markets and restaurants had higher total coliforms and E. coli counts than the vegetables at the vegetable cultivation fields. In search of the potential contamination sources, it was found that vegetables are washed in nearby canals after harvesting. Those canals are contaminated with human and animal excreta, which in turn may contaminate the vegetables. At the markets, although the tap water was found to be free of microbes, contaminated and non-contaminated vegetables are mixed and washed in the same bowl, which may bring about further spreading of infectious bacteria. The results of this study suggested that an integrated countermeasure that incorporates reducing microbial contamination of canals, raising the awareness of microbial infection among the local farmers and wholesalers, and providing enough clean water to the food markets should be implemented to reduce the incidence of food-borne illness in HCMC.

Establishment and characterization of in vivo human tumor models in the NOD/SCID/gamma(c)(null) mouse.

Immunodeficient mice are widely used for xenografts of human cells and tissue. The purpose of the present study was to investigate the characteristics of xenograft human tumor models using engraftment of various non-hematopoietic tumors in the NOD/SCID/gamma(c) (null) mouse. For tumor models, human solid tumor tissues were serially passaged three or more times to establish tissue lines. A total of 326 fresh tumor specimens, mainly gastrointestinal and female genital tissue, were engrafted with 54 established tissue lines. The types of tissue lines varied and included tumor tissue of both epithelial and mesenchymal origin. In some cases the original surgical specimen was replaced with large mononuclear cells. In the established tumor tissue lines, differentiation and tumor structure were similar to that of the original surgical specimen. The interstitium of the xenograft tissue in the tissue lines was relatively well preserved although slightly decreased and replaced by host tissue. These results indicate that human solid tumors can be successfully engrafted into the NOD/SCID/gamma(c) (null) mouse and that tissue lines with the characteristics of the original tumors can be established. Investigators in the field of tumor research will benefit from the availability of tissue lines that allow the establishment of more relevant in vivo human tissue models.