Context: Due to recent advantages in cancer therapy, immune checkpoint inhibitors (ICIs) are new classes of drugs targeting programmed cell death protein 1 (PD-1) or its ligand programmed cell death protein 1-ligand 1 (PD-L1) used in many cancer therapies. Acute interstitial nephritis (AIN) is a potential and deleterious immune-related adverse events (irAE) in the kidney observed in patients receiving ICIs and the most common biopsy-proven diagnosis in patients who develop acute kidney injury (AKI). Based on previous reports, AIN in patients receiving ICIs is associated with tubular positivity for PD-L1, implicating that PD-L1 positivity reflects susceptibility to develop renal complications with these agents. It remains unclear if PD-L1 positivity is acquired specifically during ICI therapy or expressed independently in the kidney.

Methods: PD-L1 was analyzed in experimental mouse models of ischemia-reperfusion injury (IRI), folic acid-induced nephropathy (FAN), unilateral ureteral obstruction (UUO), and nephrotoxic serum nephritis (NTN) by immunostaining, SDS-PAGE, and subsequent immunoblotting. In addition, we included a total number of 87 human kidney samples (six renal biopsies with AIN related to ICI therapy, 13 nephrectomy control kidneys, and 68 ICI-naïve renal biopsies with various underlying kidney diseases to describe PD-L1 expression.

Results: We here report distinct PD-L1 expression in renal compartments in multiple murine models of kidney injury and human cases with various underlying kidney diseases, including ICI-related AIN and renal pathologies independent of ICI therapy. PD-L1 is frequently expressed in various renal pathologies independent of ICI therapy and could potentially be a pre-requisit for susceptibility to develop AKI and deleterious immune-related AIN. In addition, we provide evidence that tubular PD-L1 positivity in the kidney is associated with detection of urinary PD-L1⁺ tubular epithelial cells.

Conclusion: Our study implicates that PD-L1 is frequently expressed in various renal pathologies independent of ICI therapy and could potentially be a pre-requisit for susceptibility to develop AKI and deleterious immune-related AIN. Because non-invasive detection of PD-L1⁺ cells in corresponding urine samples correlates with intrarenal PD-L1 positivity, it is attractive to speculate that further non-invasive detection of PD-L1⁺ cells may identify patients at risk for ICI-related AIN.

Keywords: acute kidney injury; checkpoint inhibition; immune-related adverse events (irAE); inflammation; programmed cell death protein 1-ligand 1 (PD-L1).

Potato virus Y (PVY) is a serious threat to potato production due to effects on tuber yield and quality, in particular, due to induction of potato tuber necrotic ringspot disease (PTNRD), typically associated with recombinant strains of PVY. These recombinant strains have been spreading in the United States for the past several years, although the reasons for this continuing spread remained unclear. To document and assess this spread between 2011 and 2015, strain composition of PVY isolates circulating in the Columbia Basin potato production area was determined from hundreds of seed lots of various cultivars. The proportion of nonrecombinant PVY^O isolates circulating in Columbia Basin potato dropped ninefold during this period, from 63% of all PVY-positive plants in 2011 to less than 7% in 2015. This drop in PVY^O was concomitant with the rise of the recombinant PVY^N-Wi strain incidence, from less than 27% of all PVY-positive plants in 2011 to 53% in 2015. The proportion of the PVY^NTN recombinant strain, associated with PTNRD symptoms in susceptible cultivars, increased from 7% in 2011 to approximately 24% in 2015. To further address the shift in strain abundance, screenhouse experiments were conducted and revealed that three of the four most popular potato cultivars grown in the Columbia Basin exhibited strain-specific resistance against PVY^O. Reduced levels of systemic movement of PVY^O in such cultivars would favor spread of recombinant strains in the field. The negative selection against the nonrecombinant PVY^O strain is likely caused by the presence of the Ny_tbr gene identified in potato cultivars in laboratory experiments. Presence of strain-specific resistance genes in potato cultivars may represent the driving force changing PVY strain composition to predominantly recombinant strains in potato production areas.

We investigated here the effect of the novel glial cell line-derived neurotrophic factor (GDNF)-family member neurturin (NTN) on transplanted fetal dopamine (DA) neurons. Three groups of rats with complete unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal DA system received intrastriatal grafts of embryonic ventral mesencephalic tissue. Following transplantation animals received repeated injections of vehicle or NTN (0.3 microg or 3.0 microg) over three weeks posttransplantation. NTN-treated animals had significantly (1.8-fold) more tyrosine hydroxylase-immunoreactive (TH-IR) neurons. Graft volume, TH-IR cell volume and overall dopaminergic host reinnervation remained unchanged. Amphetamine-induced rotation was rapidly compensated in all grafted rats. We conclude that administration of NTN may be a powerful way to increase survival of transplanted fetal DA neurons.

Netrin-1 (Ntn-1) is a multifunctional neuronal signaling molecule; however, its physiological significance, which improves the tissue-regeneration capacity of stem cells, has not been characterized. In the present study, we investigate the mechanism by which Ntn-1 promotes the proliferation of hUCB-MSCs with regard to the regeneration of injured tissues. We found that Ntn-1 induces the proliferation of hUCB-MSCs mainly via Inα6β4 coupled with c-Src. Ntn-1 induced the recruitment of NADPH oxidases and Rac1 into membrane lipid rafts to facilitate ROS production. The Inα6β4 signaling of Ntn-1 through ROS production is uniquely mediated by the activation of SP1 for cell cycle progression and the transcriptional occupancy of SP1 on the VEGF promoter. Moreover, Ntn-1 has the ability to induce the F-actin reorganization of hUCB-MSCs via the Inα6β4 signaling pathway. In an in vivo model, transplantation of hUCB-MSCs pre-treated with Ntn-1 enhanced the skin wound healing process, where relatively more angiogenesis was detected. The potential effect of Ntn-1 on angiogenesis is further verified by the mouse hindlimb ischemia model, where the pre-activation of hUCB-MSCs with Ntn-1 significantly improved vascular regeneration. These results demonstrate that Ntn-1 plays an important role in the tissue regeneration process of hUCB-MSC via the lipid raft-mediated Inα6β4 signaling pathway.

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation of AHLs leading to attenuation of virulence (quorum quenching) could pave the way for the development of new antibacterials. Penicillin V acylases (PVAs) belong to the Ntn hydrolase superfamily, together with AHL acylases. PVAs are exploited widely in the pharmaceutical industry, but their role in the natural physiology of their native microbes is not clearly understood. This report details the characterization of AHL degradation activity by homotetrameric PVAs from two gram-negative plant pathogenic bacteria, Pectobacterium atrosepticum (PaPVA) and Agrobacterium tumefaciens (AtPVA). Both the PVAs exhibited substrate specificity for degrading long-chain AHLs. Exogenous addition of these enzymes into Pseudomonas aeruginosa greatly diminished the production of elastase and pyocyanin and biofilm formation and increased the survival rate in an insect model of acute infection. Subtle structural differences in the PVA active site that regulate specificity for acyl chain length have been characterized, which could reflect the evolution of AHL-degrading acylases in relation to the environment of the bacteria that produce them and also provide strategies for enzyme engineering. The potential for using these enzymes as therapeutic agents in clinical applications and a few ideas about their possible significance in microbial physiology have also been discussed.

Data from the U.S. Geological Survey (USGS) collocated-sampler program for the National Atmospheric Deposition Program/National Trends Network (NADP/NTN) are used to estimate the overall error of NADP/NTN measurements. Absolute errors are estimated by comparison of paired measurements from collocated instruments. Spatial and temporal differences in absolute error were identified and are consistent with longitudinal distributions of NADP/NTN measurements and spatial differences in precipitation characteristics. The magnitude of error for calcium, magnesium, ammonium, nitrate, and sulfate concentrations, specific conductance, and sample volume is of minor environmental significance to data users. Data collected after a 1994 sample-handling protocol change are prone to less absolute error than data collected prior to 1994. Absolute errors are smaller during non-winter months than during winter months for selected constituents at sites where frozen precipitation is common. Minimum resolvable differences are estimated for different regions of the USA to aid spatial and temporal watershed analyses.

BACKGROUND

Acute kidney injury (AKI) affects up to 60% of severely asphyxiated neonates. The diagnosis of AKI can be and is further challenged by a lack of good biomarkers. We studied the role of novel markers for AKI, neutrophil gelatinase-associated lipocalin (NGAL), interleukin-8 (IL-18), Netrin-1 (NTN-1), and sodium hydrogen exchanger isoform 3 (NHE3) on development and early diagnosis of AKI in newborns with perinatal asphyxia (PA).

METHODS

Forty-one newborns with a diagnosis of PA (15 with AKI and 26 without AKI) and 20 healthy matched controls were involved to the study. Urinary samples were obtained on postnatal days 1 and 4 for patients with PA and on postnatal day 1 for the control subjects. AKI was defined using a serum creatinine-based modification of the acute kidney injury network criteria.

RESULTS

The levels of NGAL, NTN-1, NHE3, and IL-18 on the first postnatal day urine samples were higher in patients compared to controls (p < 0.001, p <0.001, p <0.02, p <0.001, respectively). In patients with AKI, the levels of NGAL and IL-18 were higher when compared to patients without AKI (p = 0.002, p <0.001, respectively). The levels of NTN-1 and NHE3 were similar in both groups. For the samples obtained on postnatal day 4, only NGAL levels were significantly higher in patients with AKI (p = 0.004) compared to those without AKI.

CONCLUSIONS

To our knowledge, this is the largest study, which evaluated the utility of urinary biomarkers in the diagnosis of AKI in newborns with PA. First day, urine NGAL and IL-18 levels have an important diagnostic power in such patients.

In plants, RNA silencing-based antiviral defense generates viral small RNAs (sRNAs) faithfully representing the viral genomes. We employed sRNA sequencing and bioinformatics (sRNA-omics) to characterize antiviral defense and to reconstruct the full genomic sequences and their variants in the evolving viral quasispecies in cultivated solanaceous plants carrying mixed infections. In naturally infected Solanum tuberosum (potato), one case study revealed a virome comprising Potato virus Y (genus Potyvirus) and Potato virus X (genus Potexvirus), which was reconstructed by de novo-assembling separate genome-size sRNA contigs. Another case study revealed a virome comprising NTN and O strains of Potato virus Y, whose sRNAs assembled in chimeric contigs, which could be disentangled on the basis of reference genome sequences. Both viromes were stable in vegetative potato progeny. In a cross-protection trial of Solanum lycopersicum (tomato), the supposedly protective mild strain CH2 of Pepino mosaic virus (genus Potexvirus) was tested for protection against strain LP of the same virus. Reciprocal mechanical inoculations eventually resulted in co-infection of all individual plants with CH2 and LP strains, reconstructed as separate sRNA contigs. LP invasions into CH2-preinfected plants and vice versa were accompanied by alterations of consensus genome sequences in viral quasispecies, indicating a potential risk of cross-protection measures. Additionally, the study also revealed, by reconstruction from sRNAs, the presence of the mechanically nontransmissible Southern tomato virus (genus Amalgavirus) in some plants. Our in-depth analysis of sRNA sizes, 5'-nucleotide frequencies and hotspot maps revealed similarities in sRNA-generating mechanisms in potato and tomato, differential silencing responses to virome components and potential for sRNA-directed cross-targeting between viral strains which could not, however, prevent the formation of stable viromes.

With the blooming of oil and gas industries, oily saline wastewater treatment becomes a viable option to resolve the oily water disposal issue and to provide a source of water for beneficial use. Reverse osmosis (RO) has been touted as a promising technology for oily saline wastewater treatment. However, one great challenge of RO membrane is fouling phenomena, which is caused by the presence of hydrocarbon contents in the oily saline wastewater. This study focuses on the fabrication of antifouling RO membrane for accomplishing simultaneous separation of salt and oil. Thin film nanocomposite (TFN) RO membrane was formed by the layer by layer (LbL) assembly of positively charged TNS (pTNS) and negatively charged TNS (nTNS) on the surface of thin film composite (TFC) membrane. The unique features, rendered by hydrophilic TNS bilayer assembled on TFC membrane in the formation of a hydration layer to enhance the fouling resistance by high concentration oily saline water while maintaining the salt rejection, were discussed in this study. The characterization findings revealed that the surface properties of membrane were improved in terms of surface hydrophilicity, surface roughness, and polyamide(PA) cross-linking. The TFC RO membrane coated with 2-bilayer of TNS achieved >99% and >98% for oil and salt rejection, respectively. During the long-term study, the 2TNS-PA TFN membrane outperformed the pristine TFC membrane by exhibiting high permeability and much lower fouling propensity for low to high concentration of oily saline water concentration (1000 ppm, 5000 ppm and 10,000 ppm) over a 960 min operation. Meanwhile, the average permeability of uncoated TFC membrane could only be recovered by 95.7%, 89.1% and 82.9% for 1000 ppm, 5000 ppm and 10,000 ppm of the oily saline feedwater, respectively. The 2TNS-PA TFN membrane achieved almost 100% flux recovery for three cycles by hydraulic washing.

Keywords: antifouling; layer by layer assembly; oily saline wastewater; reverse osmosis; thin film composite.

Optimal oxidative stress (OS) is important throughout pregnancy; however, an increased OS may alter placental angiogenesis culminating in an imbalanced of angiogenic growth mediators (AGMs). Suboptimal Health Status (SHS), a physical state between health and disease, may be associated with increased OS and unbalanced AGMs. In this study, we explored the association between SHS, biomarkers of OS (BOS) and AGMs among normotensive pregnant women (NTN-PW) in a Ghanaian Suboptimal Health Cohort Study (GHOACS). This comparative GHOACS recruited 593 NTN-PW from the Komfo Anokye Teaching Hospital, Ghana. SHS was measured using a Suboptimal Health Status Questionnaire-25 (SHSQ-25). Along with the subjective SHS measure, objective BOS: 8-hydroxy-2-deoxyguanosine (8-OHdG), 8-epiprostaglandinF2 alpha (8-epi-PGF2α), total antioxidant capacity (TAC), and AGMs: vascular endothelial growth factor-A (VEGF-A), soluble fms-like tyrosine kinase receptor 1 (sFlt-1), placental growth factor (PIGF) and soluble endoglin (sEng) were evaluated. Compared to optimal health NTN-PW, levels of PlGF, VEGF-A and TAC were significantly (p < 0.05) reduced and negatively associated with SHS whilst sEng, sFlt-1, 8-epiPGF2α, 8-OHdG, and combined ratios of sFlt-1/PlGF, 8-epiPGF2α/PlGF, 8-OHdG/PlGF, and sEng/PlGF were significantly increased and positively associated with SHS. The first quartile for PIGF (2.79-fold) and VEGF-A (5.35-fold), and the fourth quartile for sEng (4.31-fold), sFlt-1 (1.84-fold), 8-epiPGF2α (2.23-fold), 8-OHdG (1.90-fold) and urinary 8-OHdG (1.95-fold) were independently associated with SHS (p < 0.05). SHS is associated with increased OS and unbalanced AGMs. Early identification of SHS-related OS and unbalanced AGMs may inform clinicians of the need for therapeutic options.

Ca(2+) is one of the most universal and versatile signalling molecules and is involved in almost every aspect of cellular processes. Accumulating evidence suggests that Ca(2+) serves as a messenger in many growth and developmental processes and in plant responses to biotic and abiotic stresses. Numerous signals have been shown to induce transient elevation of cytoplasmic [Ca(2+)](cyt) in plants. The calcium free ions were detected cytochemically in Solanum tuberosum cv. Rywal tissues as a hypersensitive response (HR) from 10h to 5 days after a Potato virus Y (PVY) infection. Calcium was detected in vivo by its reaction with Alizarin S Red, producing an intense red staining in contact with calcium free ions. Calcium was found in the necrotic area of the epidermal and mesophyll cells 3 days after the PVY infection (when morphological symptoms on potato leaves appeared). Calcium ions were detected cytochemically in HR also by its reaction with potassium pyroantimonate. Inoculation with PVY(NTN) and also PVY(N) Wi induced a rapid hypersensitive response during which highly localised increased accumulation of electron-dense deposits of calcium pyroantimonate were detected. Calcium deposition was observed in necrotic and non-necrotic areas, starting from 10h after PVY infection. The deposits were present along ER cisternae, chloroplasts and mitochondria envelopes connected with PVY particles. The precipitates of calcium antimonate were detected near the nuclear envelope, inside karyolymph and along tracheary elements, especially when virus particles were present inside.

Many therapeutic strategies aimed at relieving symptoms of Parkinson's disease (PD) are currently used for treatment of this disease. With a hallmark of progressive degeneration of dopaminergic neurons, the absence of properly operational dopaminergic circuitry becomes a therapeutic target. Following diagnosis, dopamine replacement can be given in the form of L-DOPA (L-3,4-dihydroxyphenylalanine). Even though it is recognized as standard of care, this treatment strategy does not prevent the affected neurons from degenerating. Therefore, studies have been performed using gene therapy (GT) to make dopamine (DA) available from within the brain using an artificial DA circuitry. One approach is to administer a GT aimed at delivering the key enzymes for DA synthesis using a lentiviral vector system (Palfi et al., 2014). A similar approach has been investigated with adeno-associated virus (AAV) expressing aromatic L-amino acid decarboxylase, tyrosine hydroxylase, and GTP-cyclohydrolase I (Bankiewicz et al., 2000), which are downregulated in PD. Another GT approach to mitigate symptoms of PD used AAV-mediated delivery of GAD-67 (glutamate decarboxylase) (Kaplitt et al., 2007). This approach mimics the inhibitory effect of DA neurons on their targets, in reducing motor abnormalities. Finally, disease modifying strategies have been undertaken using neurotrophic factors such as neurturin (NTN) (Marks et al., 2008; Bartus et al., 2013a) or are ongoing with the closely related Glial cell line-derived neurotrophic factor. Those approaches are aiming at rescuing the degenerating neurons. All of the above mentioned strategies have their own merits, but also some disadvantages. So far, none of clinical applied GT studies has resulted in significant clinical benefit, although some clinical studies are ongoing and results are expected over the next few years.

The main function of lysosomal proteins is to degrade cellular macromolecules. We purified a novel lysosomal protein to homogeneity from bovine kidneys. By gene annotation, this protein is defined as a bovine phospholipase B-like protein 1 (bPLBD1) and, to better understand its biological function, we solved its structure at 1.9 Å resolution. We showed that bPLBD1 has uniform noncomplex-type N-glycosylation and that it localized to the lysosome. The first step in lysosomal protein transport, the initiation of mannose-6-phosphorylation by a N-acetylglucosamine-1-phosphotransferase, requires recognition of at least two distinct lysines on the protein surface. We identified candidate lysines by analyzing the structural and sequentially conserved N-glycosylation sites and lysines in bPLBD1 and in the homologous mouse PLBD2. Our model suggests that N408 is the primarily phosphorylated glycan, and K358 a key residue for N-acetylglucosamine-1-phosphotransferase recognition. Two other lysines, K334 and K342, provide the required second site for N-acetylglucosamine-1-phosphotransferase recognition. bPLBD1 is an N-terminal nucleophile (Ntn) hydrolase. By comparison with other Ntn-hydrolases, we conclude that the acyl moiety of PLBD1 substrate must be small to fit the putative binding pocket, whereas the space for the rest of the substrate is a large open cleft. Finally, as all the known substrates of Ntn-hydrolases have amide bonds, we suggest that bPLBD1 may be an amidase or peptidase instead of lipase, explaining the difficulty in finding a good substrate for any members of the PLBD family.

A reverse transcription polymerase chain reaction (RT-PCR) protocol used for the detection of potato viruses in dormant tubers and leaves and in an aphid vector is described. Problems in plant sample preparation from different hosts, uneven distribution or low concentration of viruses and the presence of PCR inhibitors in plant extracts are discussed and various ways to eliminate their effect are described. Using Potyviridae viruses, it has been shown that RT-PCR in various modified forms can be used to differentiate viruses at the level of family, genus, species, strains and their subtypes or serotypes. The specificity of primer pairs and PCR modifications has been used to separate closely related potato viruses A and PVY strains (PVY(O), PVY(N) and PVY(NTN)) from a known mixture.

Consultant post prospects for Specialist Registrars (SpRs) in all the medical specialties have been analysed using the databases of the Royal College of Physicians and the Joint Committee on Higher Medical Training (JCHMT). A 'bulge' in the numbers of SpRs obtaining Certificates of Completion of Specialist Training (CCSTs) is anticipated over the next 2-3 years, but this effect will be alleviated by the increasing trend to spend extra years in research or flexible training. There are profound differences between the specialties in the ratios of SpRs holding National Training Numbers (NTNs) to consultants, and in the proportions of female SpRs, and these factors will markedly influence consultant post prospects. The outlook within most specialties is favourable, but this is critically dependent on the rate of consultant expansion over the next few years.

Microarrays are one of the new emerging methods in plant virology currently being developed by various laboratories. In this study, a new approach is described on the detection of plant viruses using short synthetic single-stranded oligomers (40 nt) instead of PCR products as capture probes. A microchip detecting potato viruses, PVA, PVS, PVM, PVX, PVY and PLRV, in both single and mixed infections was developed and tested. The chip was also designed to distinguish between the main strains of PVY and PVS. Results of initial tests with PVY(NTN) and PVY(O) strains using several different probes for one virus are presented. Possibilities and advantages of the new oligonucleotide-based microarray approach for plant viral diagnosis are discussed.

Netrins are a family of matrix-binding proteins that function as guidance signals. Netrin-4 displays pathologic roles in tumorigenesis and neovascularization. To answer the question whether netrin-4 acts either pro- or anti-angiogenic, angiogenesis in the retina was assessed in Ntn-4(-/-) mice with oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV), mimicking hypoxia-mediated neovascularization and inflammatory mediated angiogenesis. The basement membrane protein netrin-4 was found to be localised to mature retinal blood vessels. Netrin-4, but not netrin-1 mRNA expression, increased in response to relative hypoxia and recovered to normal levels at the end of blood vessel formation. No changes in the retina were found in normoxic Ntn-4(-/-) mice. In OIR, Ntn-4(-/-) mice initially displayed larger avascular areas which recovered faster to revascularization. Ganzfeld electroretinography showed faster recovery of retinal function in Ntn-4(-/-) mice. Expression of netrin receptors, Unc5H2 (Unc-5 homolog B, C. elegans) and DCC (deleted in colorectal carcinoma), was found in Müller cells and astrocytes. Laser-induced neovascularization in Nnt-4(-/-) mice did not differ to that in the controls. Our results indicate a role for netrin-4 as an angiogenesis modulating factor in O2-dependent vascular homeostasis while being less important during normal retinal developmental angiogenesis or during inflammatory neovascularization.

The identification of endogenous neurotrophic factors and their receptors in human spinal cord is important not only to understand development, but also in the consideration of possible future therapies for neurodegenerative disorders and trauma. Using in situ hybridization, the expression of glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN), persephin (PSP), GFRalpha-1, GFRalpha-2, GFRalpha-3 and RET mRNA in human fetal spinal cord was studied. Strong GDNF mRNA hybridization signal, presumably restricted to Clarke's nucleus, was detected in the thoracic spinal cord. mRNA encoding GFRalpha-1 was expressed in the entire spinal cord gray matter with particularly high expression in the ventral horn. GFRbeta-1 was also expressed more weakly in dorsal root ganglia. NTN and persephin mRNA were not detected in either the fetal spinal cord or the dorsal root ganglia. mRNA coding for GFRalpha-2, however, was found in most cells of the spinal cord gray matter. A strong expression of GFRalpha-3 mRNA was detected in dorsal root ganglia cells and Schwann cells. The transducing receptor RET was expressed strongly in motorneurons and dorsal root ganglion neurons. We conclude that basic features concerning the role of the GDNF family of ligands and their receptors revealed in rodents applies to humans.

Neurturin (NTN) is a recently identified homologue of glial-cell-line-derived neurotrophic factor. Both factors promote the survival of dopaminergic (DA) neurons. We investigated the biological activity of mammalian-cell-produced NTN versus purified Escherichia coli-produced NTN. Baby hamster kidney cells were engineered to stably secrete mature human NTN. Mammalian-cell-derived NTN enhanced the activity of embryonic DA neurons in vitro, with greater potency (maximum effect achieved in the picogram range) than purified E. coli-produced NTN. Cell-based delivery of NTN (less than 10 ng/day) was also shown to be biologically active in vivo. These results suggest that mammalian-cell-derived NTN, synthesized de novo and delivered in small quantities to the parenchyma at the target site, may be as active as much larger quantities of purified, E. coli-produced NTN, delivered by other means.

Implantable ventricular assist devices have been regarded as a promising instrument in the clinical treatment of patients with severe heart failures. In this article, a three-dimensional model of the Kyoto-NTN magnetically suspended centrifugal blood pump was generated and a computational fluid dynamics solution of the inner flow field of the pump including the static pressure distributions, velocity profiles, and the shear stress distributions of the blood was presented. The results revealed that reverse flow generally occurred in the impeller blade channels during the operation of the pump, due to the imbalance of the flow and the pressure gradient generated in the blade channels. The flow pattern at the exit of the blade channels was varying with its angular positions in the pump. The reverse flow at the exit of the impeller blade channels was found to be closely related with the static pressure distribution in the volute passage. Higher pressure in the volute caused severe backflow from the volute into the blade channels. To clarify the effects of a moving impeller on the blood, shear stresses of the blood in the pump were investigated according to the simulation results. The studies indicated that at the beginning of the splitter plate and the cutwater, the highest shear stress exceeded 700 Pa. At other regions such as the inlet and outlet of the impeller blade channels and some regions in the volute passage, shear stresses were found to be about 200 Pa. These areas are believed to have a high possibility of rendering blood trauma.

Reduction of the negative charge of the glomerular capillary wall alters its charge- and size-selective properties. To investigate the effect of alteration in glomerular charge properties on antibody localization, we prepared cationic and anionic fractions of antibodies to subepithelial and glomerular basement membrane (GBM) antigens, and compared their deposition in normal rats and rats treated with protamine sulfate or aminonucleoside of puromycin to reduce capillary wall charge. IgG antibodies were eluted from kidneys of rats with active Heymann's nephritis (AICN), passive Heymann's nephritis (PHN), or anti-GBM nephritis (NTN), separated into cationic and anionic fractions, and radiolabeled with iodine 125 or iodine 131. Relative antibody content of each fraction was determined by incubation with an excess of glomerular antigen. Varying amounts of cationic and anionic IgG eluted from kidneys of rats with AICN or PHN were injected into 24 normal or protamine sulfate-treated rats. Glomerular binding of all antibodies was highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 4 hours was 1.08 +/- 0.07 for AICN eluate and 0.37 +/- 0.04 for PHN eluate. The ratios were not significantly different in animals pretreated with protamine sulfate (1.15 +/- 0.06 and 0.44 +/- 0.06, respectively; P greater than 0.05). Varying amounts of cationic and anionic IgG eluted from kidneys of rats with NTN were injected into 10 normal rats and four rats treated with aminonucleoside of puromycin. Glomerular binding of antibody was again highly correlated with IgG delivery to the kidney. The ratio of cationic to anionic antibody deposited in the glomeruli of normal rats after 1 hour was 1.03 +/- 0.06, and was not significantly altered in rats treated with aminonucleoside of puromycin (1.05 +/- 0.03, P greater than 0.5). Proteinuria in PHN rats was also unaffected by treatment with protamine sulfate for 5 days (controls: 68 +/- 21 mg/day; protamine sulfate-treated: 65 +/- 14 mg/day; n = 25, P greater than 0.08). These results demonstrate that treatment to reduce glomerular polyanion does not significantly alter the ratio of cationic to anionic antibodies to fixed glomerular antigens that deposit in the glomerulus, or reduce proteinuria caused by deposition of antibody to a fixed subepithelial antigen.

Mesangial cells are glomerular cells of stromal origin. During immune complex mediated crescentic glomerulonephritis (Crgn), infiltrating and proliferating pro-inflammatory macrophages lead to crescent formation. Here we have hypothesised that mesangial cells, given their mesenchymal stromal origin, show similar immunomodulatory properties as mesenchymal stem cells (MSCs), by regulating macrophage function associated with glomerular crescent formation. We show that rat mesangial cells suppress conA-stimulated splenocyte proliferation in vitro, as previously shown for MSCs. We then investigated mesangial cell-macrophage interaction by using mesangial cells isolated from nephrotoxic nephritis (NTN)-susceptible Wistar Kyoto (WKY) and NTN-resistant Lewis (LEW) rats. We first determined the mesangial cell transcriptome in WKY and LEW rats and showed that this is under marked genetic control. Supernatant transfer results show that WKY mesangial cells shift bone marrow derived macrophage (BMDM) phenotype to M1 or M2 according to the genetic background (WKY or LEW) of the BMDMs. Interestingly, these effects were different when compared to those of MSCs suggesting that mesangial cells can have unique immunomodulatory effects in the kidney. These results demonstrate the importance of the genetic background in the immunosuppressive effects of cells of stromal origin and specifically of mesangial cell-macrophage interactions in the pathophysiology of crescentic glomerulonephritis.

In hypertensive adults (HTN), cardiovascular risk increases disproportionately during environmental cold exposure. Despite ample evidence of dysregulated sympathetic control of the peripheral vasculature in hypertension, no studies have examined integrated neurovascular function during cold stress in HTN. The findings of the present study show that whole-body cold stress elicits greater increases in sympathetic outflow directed to the cutaneous vasculature and, correspondingly, greater reductions in skin blood flow in HTN. We further demonstrate an important role for non-adrenergic sympathetic co-transmitters in mediating the vasoconstrictor response to cold stress in hypertension. In the context of thermoregulation and the maintenance of core temperature, sympathetically-mediated control of the cutaneous vasculature is not only preserved, but also exaggerated in hypertension. Given the increasing prevalence of hypertension, clarifying the mechanistic underpinnings of hypertension-induced alterations in neurovascular function during cold exposure is clinically relevant.

Despite ample evidence of dysregulated sympathetic control of the peripheral vasculature in hypertension, no studies have examined integrated neurovascular function during cold stress in hypertensive adults (HTN). We hypothesized that (i) whole-body cooling would elicit greater cutaneous vasoconstriction and greater increases in skin sympathetic nervous system activity (SSNA) in HTN (n = 14; 56 ± 2 years) compared to age-matched normotensive adults (NTN; n = 14; 55 ± 2 years) and (ii) augmented reflex vasoconstriction in HTN would be mediated by an increase in cutaneous vascular adrenergic sensitivity and a greater contribution of non-adrenergic sympathetic co-transmitters. SSNA (peroneal microneurography) and red cell flux (laser Doppler flowmetry; dorsum of foot) were measured during whole-body cooling (water-perfused suit). Sympathetic adrenergic- and non-adrenergic-dependent contributions to reflex cutaneous vasoconstriction and vascular adrenergic sensitivity were assessed pharmacologically using intradermal microdialysis. Cooling elicited greater increases in SSNA (NTN: +64 ± 13%baseline vs. HTN: +194 ± 26%baseline ; P < 0.01) and greater reductions in skin blood flow (NTN: -16 ± 2%baseline vs. HTN: -28 ± 3%baseline ; P < 0.01) in HTN compared to NTN, reflecting an increased response range for sympathetic reflex control of cutaneous vasoconstriction in HTN. Norepinephrine dose-response curves showed no HTN-related difference in cutaneous adrenergic sensitivity (logEC50 ; NTN: -7.4 ± 0.3 log M vs. HTN: -7.5 ± 0.3 log M; P = 0.84); however, non-adrenergic sympathetic co-transmitters mediated a significant portion of the vasoconstrictor response to cold stress in HTN. Collectively, these findings indicate that hypertension increases the peripheral cutaneous vasoconstrictor response to cold via greater increases in skin sympathetic outflow coupled with an increased reliance on non-adrenergic neurotransmitters.