Design of microfluidic channels to prevent negative filtration in implantable hemofiltration devices.

In order to improve the quality of life of dialysis patients, our group have been developing an implantable hemofiltration device (IHFD) composed of multiple layers of dialysis membranes and microfluidic channels. To improve the hemodialysis performance of IHFD, preventing the negative filtration, which is caused by the oncotic pressure of blood, is mandatory. In this study, we fabricated IHFDs with five different microchannel designs and experimentally investigated the performance of each device in in vitro experiment. In addition, the successful IHFD was further evaluated by ex vivo experiments with a beagle dog. The experiments verified the effectiveness of the microchannel design, which will be used for the IHFD for in vivo experiments with pigs in the future.

Cholesterol 25-hydroxylase is a metabolic switch to constrain T cell-mediated inflammation in the skin.

Interleukin-27 (IL-27) is an immunoregulatory cytokine whose essential function is to limit immune responses. We found that the gene encoding cholesterol 25-hydroxylase (Ch25h) was induced in CD4+ T cells by IL-27, enhanced by transforming growth factor­ß (TGF-ß), and antagonized by T-bet. Ch25h catalyzes cholesterol to generate 25-hydroxycholesterol (25OHC), which was subsequently released to the cellular milieu, functioning as a modulator of T cell response. Extracellular 25OHC suppressed cholesterol biosynthesis in T cells, inhibited cell growth, and induced nutrient deprivation cell death without releasing high-mobility group box 1 (HMGB1). This growth inhibitory effect was specific to actively proliferating cells with high cholesterol demand and was reversed when extracellular cholesterol was replenished. Ch25h-expressing CD4+ T cells that received IL-27 and TGF-ß signals became refractory to 25OHC-mediated growth inhibition in vitro. Nonetheless, IL-27­treated T cells negatively affected viability of bystander cells in a paracrine manner, but only if the bystander cells were in the early phases of activation. In mouse models of skin inflammation due to autoreactive T cells or chemically induced hypersensitivity, genetic deletion of Ch25h or Il27ra led to worse outcomes. Thus, Ch25h is an immunoregulatory metabolic switch induced by IL-27 and dampens excess bystander T effector expansion in tissues through its metabolite derivative, 25OHC. This study reveals regulation of cholesterol metabolism as a modality for controlling tissue inflammation and thus represents a mechanism underlying T cell immunoregulatory functions.

Heparin affects the induction of regulatory T cells independent of anti-coagulant activity and suppresses allogeneic immune responses.

Heparin is a widely used anti-coagulant that enhances anti-thrombin (AT) activity. However, heparin also suppresses immune and inflammatory responses in various rodent models and clinical trials, respectively. The mechanism by which heparin suppresses immune responses is unclear. The effect of heparin on regulatory T cells (Tregs ) in allogeneic immune responses was analysed using an acute graft-versus-host disease (aGVHD) mouse model and mixed lymphocyte reactions (MLRs). In-vitro culture systems were utilized to study the effects of heparin on Tregs . Heparin administration reduced mortality rates and increased the proportion of Tregs in the early post-transplantation period of aGVHD mice. In both murine and human MLRs, heparin increased Tregs and inhibited responder T cell proliferation. Heparin promoted functional CD4+ CD25+ forkhead box protein 3 (FoxP3)+ Treg generation from naive CD4+ T cells, increased interleukin (IL)-2 production and enhanced the activation of pre-existing Tregs with IL-2. Heparin-induced Treg increases were not associated with anti-coagulant activity through AT, but required negatively charged sulphation of heparin. Importantly, N-acetyl heparin, a chemically modified heparin without anti-coagulant activity, induced Tregs and decreased mortality in aGVHD mice. Our results indicate that heparin contributes to Treg -mediated immunosuppression through IL-2 production and suggest that heparin derivatives may be useful for immunopathological control by efficient Treg induction.

Enfold-type connecting system of artificial blood vessels for micro implantable dialysis device.

This paper reports the connecting mechanism for the artificial blood vessels along with the recent development of the micro implantable dialysis device. Our group has been studying the micro implantable dialysis device, which will drastically improve the quality of life of dialysis patients. We expect to replace the device every couple of years, which will involve surgery. In order to simplify the surgery to reduce the load to the patients, we develop a connector for the artificial vessels, which allows the exchange of the device by low invasive surgery. The connector needs to be designed not to induce blood coagulation. We designed a connecting mechanism that enfolds the artificial vessels to allow blood to contact only to the surface of the artificial vessels. In order to verify effectiveness of the proposed connecting mechanism, we investigated the connector surfaces with SEM after blood circulation tests. Then, we evaluated blood coagulation capacity of the connecting system as well as the set of the connecting system and the micro dialysis device with respect to the activated partial thromboplastin time (APTT). No remarkable increase of blood coagulation at the connecting point was observed after 72 hours of blood circulation tests. Short-term experiments for 120 minutes to evaluate APTT showed a small decrease of APTT, which needs to be further investigated in a longer-term experiments.

Does the Age of Donor Kidneys Affect Nocturnal Polyuria in Patients With Successful Real Transplantation?

BACKGROUND: We investigated whether the age of donor kidneys influences the incidence of nocturnal polyuria in patients with successful renal transplantation (RTX). METHODS: Eighty-five patients (45 men and 40 women) undergoing RTX (median age, 47 years) were included in this study. Twenty-four-hour bladder diaries were kept for 3 days, and nocturnal polyuria was defined as a nocturnal polyuria index (nocturnal urine volume/24-hour urine volume) of >0.33. Risk factors for nocturnal polyuria were analyzed in patients with RTX by means of the Mann-Whitney U test, χ2 test, and a logistic regression analysis. RESULTS: End-stage renal disease (ESRD) developed from diabetes mellitus in 16 patients (19%). Sixty-five patients (76%) received pre-transplant dialysis, with a median duration of 5 years. The median serum creatinine level and body mass index at the most recent visit were 1.2 mg/dL and 21.2 kg/m2, respectively. On the basis of the 24-hour bladder diaries, nocturnal polyuria was identified in 48 patients (56%). A logistic regression analysis revealed that diabetes mellitus as the original disease for ESRD was the only risk factor for nocturnal polyuria (odds ratio, 8.95; 95% confidence interval, 2.01-65.3; P = .0028). The age of donor kidneys at examination did not affect the incidence of nocturnal polyuria (P = .9402). CONCLUSIONS: Nocturnal polyuria was not uncommon in patients with successful RTX. Diabetes mellitus as the original disease for ESRD was the only risk factor for nocturnal polyuria, whereas the age of donor kidneys at examination did not affect the incidence of nocturnal polyuria. Thus, nocturnal polyuria is caused by recipient factors but not donor factors.

Evaluation of biofouling for implantable micro dialysis system.

Implantable artificial kidney can drastically improve the quality of life of the renal disease patients. In previous study, our group has developed a multi-layered micro dialysis device which is composed of micro stainless steel channels and nano-porous polyethersulfone (PES) membranes. The device conducts hemofiltration without dialysis fluids, which is advantageous in miniaturization. We investigated the water-permeability of the PES membrane through in vivo experiments using rat model of renal disease for 5 hours and verified the effectiveness of the device. We investigated the permeability of PES membrane via in vitro experiments for 24 days. Biofouling on the PES membrane was found and caused deterioration of dialysis performance of the membrane. In this research, we investigated the biofouling such as thrombus, coagulation and protein adhesion on the sidewall of the micro fluidic channels. We investigated the micro fluidic channel geometry and surface condition associated with the processing methods. Conducting in vitro experiment for 7 days, biofouling was found to be mainly caused by the surface conditions. The mirror surface formed by electrolytic etching could substantially prevent biofouling.

Complications after intraoral vertical ramus osteotomy: relationship to the shape of the osteotomy line.

Intraoral vertical ramus osteotomy (IVRO) is used widely to correct mandibular prognathism. However, several disadvantages of this procedure have been reported, such as condylar luxation and bony interference at the osteotomy site. The aim of this study was to survey the incidence of complications (condylar luxation and bony interference) based on the shape of the osteotomy line. One hundred and eighty-five rami in 118 patients with jaw deformities, which were treated with IVRO, were examined retrospectively. The shape of the osteotomy line and the postoperative complications were examined on panoramic radiographs. Osteotomy lines were classified into three types: vertical, C-shaped, and oblique. Of the 185 osteotomy sites, 98 were vertical, 37 C-shaped, and 50 oblique. Condylar luxation was found in six rami (3.2%); four had undergone vertical osteotomy and two had undergone C-shaped osteotomy. Bony interference occurred in seven rami (3.8%), all with vertical type osteotomy lines. Most complications occurred in the vertical type cases and no complications were found in oblique type cases. Condylar luxation was found mainly in unilateral IVRO cases and bony interference was found in bilateral IVRO cases. These results suggest that the oblique type of osteotomy line has the advantage of avoiding complications.

Simple Resistance Exercise helps Patients with Non-alcoholic Fatty Liver Disease.

To date, only limited evidence has supported the notion that resistance exercise positively impacts non-alcoholic fatty liver disease. We evaluated the effects of resistance exercise on the metabolic parameters of non-alcoholic fatty liver disease (NAFLD) in 53 patients who were assigned to either a group that performed push-ups and squats 3 times weekly for 12 weeks (exercise group; n=31) or a group that did not (control; n=22). Patients in the control group proceeded with regular physical activities under a restricted diet throughout the study. The effects of the exercise were compared between the 2 groups after 12 weeks. Fat-free mass and muscle mass significantly increased, whereas hepatic steatosis grade, mean insulin and ferritin levels, and the homeostasis model assessment-estimated insulin resistance index were significantly decreased in the exercise group. Compliance with the resistance exercise program did not significantly correlate with patient background characteristics such as age, sex, BMI and metabolic complications. These findings show that resistance exercise comprising squats and push-ups helps to improve the characteristics of metabolic syndrome in patients with non-alcoholic fatty liver disease.

Genetic diversity of Mycobacterium tuberculosis isolates from foreign-born and Japan-born residents in Tokyo.

Sequences of the full genomes of 259 clinical isolates of Mycobacterium tuberculosis, obtained from foreign-born and Japan-born patients in Tokyo, Japan, were determined, and a phylogenetic tree constructed by concatenated single-nucleotide polymorphism (SNP) sequences. The 259 isolates were clustered into four clades: Lineage 2 (East Asian or "Beijing" genotype; n = 182, 70.3%), Lineage 4 (Euro-American, n = 46, 17.8%), Lineage 1 (Indo-Oceanic, n = 23, 8.9%), and Lineage 3 (East African-Indian, n = 8, 3.1%). Of the 259, 36 (13.9%) were resistant to at least one drug. There was no multi-drug-resistant isolate. Drug resistance was greater for the strains in Lineage 2 than the non-Lineage 2. The proportion of Lineage 2 isolates was significantly smaller in foreign-born (n = 43/91, 47.3%) than in Japan-born (n = 139/168, 82.7%) patients, whereas the proportion of Lineage 1 isolates was significantly larger in foreign-born (n = 19/91, 20.9%) than in Japan-born (n = 4/168, 2.4%) patients. We also found eight SNPs specific to the typical Beijing sub-genotype in Lineage 2, including 4 non-synonymous SNPs. Of the 259 isolates, 244 had strain-specific SNP(s) and small (1-30-bp) insertions and deletions (indels). The numbers of strain-specific SNPs and indels per isolate were significantly larger from foreign-born (median 89, range 0-520) than from Japan-born (median 23, range 0-415) (p 3.66E-15) patients. These results suggested that M. tuberculosis isolates from foreign-born patients had more genetic diversity than those from Japan-born patients.

Development of implantable hemodialysis system using PES membranes with high water-permeability.

This paper presents development of high water-permeable dialysis membranes. We proposed the system that does not use dialysis fluid for the implantable micro dialysis treatment and development of such membranes is crucial. We developed micro dialysis system composed by nanoporous membranes and microfluidic channels in our prior work. The membranes were made of nanoporous polyethersulfone (PES), which was not water-permeable. By not using dialysate, our device can be simplified because the pumps and storage tanks for the dialysis fluid are not necessary. This treatment is termed as hemofiltration. We measured the water permeability of PES membrane with respect to the concentrations of the PES, the additives, and the solvents in the casting solution. We could find the membranes with sufficiently high water permeability through in vitro experiments using a syringe pomp and whole cow blood, and the membrane had enough mechanical strength. We conducted experiments with multi-layered device in in vitro and in vivo using rats, where the system was connected to the vein and artery. We successfully collected the filtrate beyond target line, which was set by a medical doctor, without any leakage of blood from the device. The results verified that the filtration device can be scaled-up by increasing a number of the layer. We connected the device to a rat for 5h. It was verified the device maintained almost constant water permeability beyond our target line.

Vascular image in autoimmune pancreatitis by contrast-enhanced color-Doppler endoscopic ultrasonography: Comparison with pancreatic cancer.

INTRODUCTION: In autoimmune pancreatitis (AIP), veins of various sizes are highly affected by obliterative phlebitis without damage to arteries, in contrast, the involvement of both arteries and veins is observed in the mass of pancreatic cancer. A vascular image without blooming artifact in the pancreas is clearly observed in the directional eFLOW (Prosound α10, Aloca Co., Tokyo, Japan) color mode using contrast-enhanced color-Doppler endoscopic ultrasonography (CC-EUS) despite perfusion of the contrast media. AIMS AND METHODS: The aim of this study was to compare the vascular structure of AIP with that of pancreatic cancer using CC-EUS. We evaluated the perfusion image and the vascular image of the mass in AIP patients (11) with an increase in serum IgG4 levels (477.3 ± 314.2 IU/mL) and in pancreatic cancer patients (11) with elevated serum CA19-9 levels (49839.0 ± 80061.6 mg/dl), on CC-EUS. Perfusion images were obtained at 20-30 s after injection of a contrast agent, Sonazoid (GE Healthcare AG, Oslo, Norway), by extended pure harmonic detection mode and were assessed as to homogeneity or heterogeneity (containing partial low echoic areas or multiple spotty low echoic areas) enhancement. The vascular image was assessed in the directional eFLOW color mode despite perfusion of the contrast media (40-50 s after injection of Sonazoid) as to the presence of a dendritic vessel network or only a few feeder vessels. The parameters for imaging were as follows: Mechanical index, 0.22-0.24; transmission frequency, 5.0 MHz; and receiving frequency, 5.0 MHz. The Chi-square test or Fisher's exact test was used for comparison of categorical data of the two groups when appropriate. This study was approved by the institutional review board of Sendai City Medical Center. All subjects gave informed consent. RESULTS: A homogenous pattern in perfusion imaging was seen in 73% of patients with AIP (8/11) and 55% of those with pancreatic cancer (6/11). The rates were not significantly different between the two groups (P = 0.33). In the other patients with a heterogenous pattern, multiple spotty low echoic areas were seen in 33% (1/3) and 80% (4/5) in each group, respectively. A dendritic vascular pattern in the eFLOW color mode was seen in 82% (9/11) of patients with AIP, but was not seen in any of patients with pancreatic cancer. The other patients with AIP (18%) and all patients with pancreatic cancer showed only a few feeder vessels in the mass on CC-EUS. CONCLUSION: The eFLOW color mode using Sonazoid may be useful for evaluating the vascular structure of AIP for differential diagnosis from pancreatic cancer.

A novel method for designing and fabricating custom-made artificial bones.

Artificial bones are useful for tissue augmentation in patients with facial deformities or defects. Custom-made artificial bones, produced by mirroring the bone structure on the healthy side using computer-aided design, have been used. This method is simple, but has limited ability to recreate detailed structures. The authors have invented a new method for designing artificial bones, better customized for the needs of individual patients. Based on CT data, three-dimensional (3D) simulation models were prepared using an inkjet printer using plaster. The operators applied a special radiopaque paraffin wax to the models to create target structures. The wax contained a contrast medium to render it radiopaque. The concentration was adjusted to achieve easy manipulation and consistently good-quality images. After the radiopaque wax was applied, the 3D simulation models were reexamined by CT, and data on the target structures were obtained. Artificial bones were fabricated by the inkjet printer based on these data. Although this new technique for designing artificial bones is slightly more complex than the conventional methods, and the status of soft tissue should also be considered for an optimal aesthetic outcome, the results suggest that this method better meets the requirements of individual patients.

Anti-apoptotic function of Xbp1 as an IL-3 signaling molecule in hematopoietic cells.

Cytokine signaling is critical for proliferation, survival and differentiation of hematopoietic cell, and interleukin-3 (IL-3) is required for maintenance of many hematopoietic cell lines, such as BaF3. We have isolated apoptosis-resistant clones of BaF3 using retroviral insertional mutagenesis and the Xbp1 locus was identified as a retroviral integration site. Expression and splicing of the Xbp1 transcript was conserved in the resistant clone but was promptly disappeared on IL-3 withdrawal in parental BaF3. IL-3 stimulation of BaF3 cells enhanced Xbp1 promoter activity and induced phosphorylation of the endoplasmic reticulum stress sensor protein IRE1, resulting in the increase in Xbp1S that activates unfolded protein response. When downstream signaling from IL-3 was blocked by LY294002 and/or dn-Stat5, Xbp1 expression was downregulated and IRE1 phosphorylation was suppressed. Inhibition of IL-3 signaling as well as knockdown of Xbp1-induced apoptosis in BaF3 cells. In contrast, constitutive expression of Xbp1S protected BaF3 from apoptosis during IL-3 depletion. However, cell cycle arrest at the G1 stage was observed in BaF3 and myeloid differentiation was induced in IL-3-dependent 32Dcl3 cells. Expression of apoptosis-, cell cycle- and differentiation-related genes was modulated by Xbp1S expression. These results indicate that the proper transcriptional and splicing regulation of Xbp1 by IL-3 signaling is important in homeostasis of hematopoietic cells.

Mapping quantitative trait loci for proteinuria-induced renal collagen deposition.

The progression of chronic kidney disease is a complex process influenced by genetic factors. Proteinuria is a predictor of functional deterioration and an accelerator of disease progression through renal parenchymal damage and interstitial fibrosis. To determine genetic components that might mediate renal fibrosis due to proteinuria, we mapped loci influencing the phenotype of two mouse strains differing in proteinuria-induced renal type I collagen (COLI) deposition. Collagen I deposition in 129S1/svImJ and C57BL/6J mice differs significantly among tested strains. We backcrossed 120 hemi-nephrectomized (129S1/svImJ x C57BL/6J) F1 x 129S1/svImJ backcrossed mice loaded with bovine serum albumin giving rise to proteinuria and renal COLI deposition. Quantitative trait loci (QTL) mapping was performed and our analysis identified one suggestive linkage for renal COLI deposition peaking at 87 cM near D2Mit224 (logarithm of odds: 2.41) on Chr 2. In silico analysis uncovered nine candidate genes. Hence, although more studies are needed, these QTL provide an initial cue to subsequent gene discovery, which might help unravel the genetics of renal fibrosis.

Expression and role of connexins in the rat renal vasculature.

Gap junctions are present in the juxtaglomerular apparatus enabling intercellular communication. Our study determined the location of different connexin subtypes within the juxtaglomerular apparatus of the rat, and the role of these subtypes in renal hemodynamics through the use of specific mimetic peptides. Immunohistochemical analysis showed connexins 37 and 40 expression in the endothelial and renin-secreting cells of the afferent arteriole, while connexin 40 was also found in extra- and intraglomerular mesangial cells. In contrast, connexin 43 was weakly expressed in endothelial cells of the afferent arteriole and within the glomerulus. Intra-renal infusion of the peptides (GAP) reported to block specific gap junctions ((Cx37,43)GAP27 or (Cx40)GAP27), elevated blood pressure, plasma renin activity, and angiotensin II levels, while decreasing renal plasma flow without a significant change in the glomerular filtration rate. Subsequent restoration of blood pressure reduced both renal plasma flow and glomerular filtration rate. In contrast, (Cx43)GAP26 reduced glomerular filtration rate without alterations in blood pressure, renal plasma flow, plasma renin activity, or angiotensin II levels. Hence, connexins 37 and 40 are expressed in the rat juxtaglomerular apparatus and these proteins control, in part, the renin-angiotensin system and renal autoregulation.

Vascular compliance is secured under angiotensin inhibition in non-diabetic chronic kidney diseases.

Cardiovascular diseases constitute major cause of death in chronic kidney diseases (CKDs). We examined the effects of angiotensin inhibition either with angiotensin-converting enzyme inhibitor or with angiotensin receptor blocker on patient prognosis and heart-ankle pulse wave velocity (haPWV) in CKDs. Randomized controlled study was performed on 102 patients with non-diabetic CKDs. Patients were divided into two groups with or without angiotensin inhibition, and followed until death, creatinine clearance was halved or starting renal replacement therapy, whichever occurred first. For 4 years, haPWV was assessed repeatedly in the surviving patients. While both groups showed well blood pressure control throughout 4 years (129+/-1 to 131+/-2/71+/-1 to 73+/-2 mm Hg), renal prognosis was better in angiotensin inhibition group (P<0.05). In addition, angiotensin inhibition reduced cardiovascular and renal death (P<0.05). Age, sex, heart rate, systolic blood pressure and proteinuria were correlated to haPWV (R(2)=0.76, P<0.0001). Although haPWV was similar between two groups at the start of the study (1098+/-31 vs 1094+/-37 cm/s), it was higher in patients without angiotensin inhibition than that with angiotensin inhibition 4 years later (1034+/-38 cm/s (n=28) vs 1242+/-37 cm/s (n=23), P<0.01). The present results provided the evidence that angiotensin inhibition arrested a time-dependent elevation of haPWV in non-diabetic CKDs, conferring organ protection. Furthermore, our data indicated that angiotensin inhibition improved patient prognosis in non-diabetic chronic kidney diseases with mild-to-moderate renal dysfunction.

Attenuated radial augmentation index is associated with successful long-term antihypertensive treatment.

Pulse wave analysis was performed in apparently normal volunteers (n=164) and in essentially hypertensive patients without cardiovascular complications (n=171) using a newly developed non-invasive pulse wave measurement device (HEM-9010AI). Our results suggest that early wave reflections measured by radial augmentation index (AIr) are enhanced in volunteers with systolic blood pressure (SBP) >or= 160 mm Hg compared with the volunteers with their SBP<160 mmHg (98+/-18 vs 88+/-12, P<0.05). Furthermore, AIr is lower in hypertensive patients with long-term antihypertensive treatment than in those with short-term treatment (84+/-10 vs 89+/-13, P<0.01).

Alpha2-antiplasmin is involved in the production of transforming growth factor beta1 and fibrosis.

BACKGROUND: Fibrotic disease occurs in most tissues. Transforming growth factor (TGF)-beta is the major inducer of fibrosis. The fibrinolytic system is considered to play an important role in the degradation of extracellular matrices. However, the detailed mechanism of how this system affects fibrosis remains unclear. METHODS AND RESULTS: We examined experimental fibrosis in mice with a deficiency of alpha(2)-antiplasmin (alpha2AP), which is a potent and specific plasmin inhibitor. We found that the lack of alpha2AP attenuated bleomycin-induced TGF-beta(1) synthesis and fibrosis. In addition, the production of TGF-beta(1) from the explanted fibroblasts of alpha2AP(-/-) mice decreased dramatically as compared to that in wild-type mice. Moreover, we found that alpha2AP specifically induces the production of TGF-beta(1) in fibroblasts. CONCLUSION: The lack of alpha2AP attenuated TGF-beta(1) synthesis, thereby resulting in attenuated fibrosis. This is the first report to describe the crucial role that alpha2AP plays in TGF-beta(1) synthesis during the process of fibrosis. Our results provide new insights into the role of alpha2AP in fibrosis.

Arginine-specific gingipains from Porphyromonas gingivalis deprive protective functions of secretory leucocyte protease inhibitor in periodontal tissue.

Chronic periodontitis is correlated with Porphyromonas gingivalis infection. In this study, we found that the expression of secretory leucocyte protease inhibitor (SLPI), an endogenous inhibitor for neutrophil-derived proteases, was reduced in gingival tissues with chronic periodontitis associated with P. gingivalis infection. The addition of vesicles of P. gingivalis decreased the amount of SLPI in the media of primary human gingival keratinocytes compared to untreated cultures. We therefore investigated how arginine-specific gingipains (Rgps) affect the functions of SLPI, because Rgps are the major virulence factors in the vesicles and cleave a wide range of in-host proteins. We found that Rgps digest SLPI in vitro, suppressing the release of SLPI. Rgps proteolysis of SLPI disrupted SLPI functions, which normally suppresses neutrophil elastase and neutralizes pro-inflammatory effects of bacterial cell wall compounds in cultured human gingival fibroblasts. The protease inhibitory action of SLPI was not exerted towards Rgps. These results suggest that Rgps reduce the protective effects of SLPI on neutrophil proteases and bacterial proinflammatory compounds, by which disease in gingival tissue may be accelerated at the sites with P. gingivalis infection.