Modulation of osteopontin in proteinuria-induced renal interstitial fibrosis

Proteinuria is associated with macrophage-dependent interstitial fibrosis (IF). Osteopontin (OPN), a macrophage chemoattractant, may be involved in the transition of proteinuria to IF but protective properties have also been reported. To elucidate whether OPN may be involved in the proteinuria-induced cascade of tubulointerstitial damage, renal expression of OPN was studied during the development of proteinuria-induced renal damage and during anti-proteinuric intervention with ACE inhibition (ACEi). First, the temporal relationships between proteinuria, interstitial OPN induction, and IF in adriamycin nephrosis (AN), a model of chronic proteinuria-induced renal damage, were studied. Second, the effect of anti-proteinuric treatment on OPN expression was investigated. The time course of OPN induction and markers of renal damage was studied in rats with unilateral AN at 6-week intervals until week 30. In a second study, a renal biopsy was taken 6 weeks after induction of bilateral AN; subsequently, rats were treated with ACEi until termination (week 12). In unilateral AN, proteinuria developed gradually and stabilized at week 10. In proteinuric kidneys, OPN expression was induced from week 12 onwards. Simultaneously, a progressive increase in interstitial macrophages, alpha-smooth muscle actin (alpha-SMA), collagen type III, and focal glomerulosclerosis (FGS) was observed. In bilateral AN, ACEi reduced proteinuria and OPN protein and stabilized fibrosis. In untreated animals, OPN mRNA increased, with stable OPN protein and fibrosis and increased FGS. Thus, in AN, development of proteinuria is followed by up-regulation of OPN along with markers of renal damage. The up-regulation of OPN is reversible by anti-proteinuric treatment without a corresponding reduction in fibrosis. Whereas these data are consistent with a role for OPN in the cascade of transition from proteinuria to fibrosis, intervention with ACEi showed that reduction of OPN does not attenuate established fibrosis.     

Association of DJ-1 and parkin mediated by pathogenic DJ-1 mutations and oxidative stress

The identification of rare monogenic forms of Parkinson's disease (PD) has provided tremendous insight into the molecular pathogenesis of this disorder. Heritable mutations in alpha-synuclein, parkin, DJ-1 and PINK1 cause familial forms of PD. In the more common sporadic form of PD, oxidative stress and derangements in mitochondrial complex-I function are considered to play a prominent role in disease pathogenesis. However, the relationship of DJ-1 with other PD-linked genes and oxidative stress has not been explored. Here, we show that pathogenic mutant forms of DJ-1 specifically but differentially associate with parkin, an E3 ubiquitin ligase. Chemical cross-linking shows that pathogenic DJ-1 mutants exhibit impairments in homo-dimer formation, suggesting that parkin may bind to monomeric DJ-1. Parkin fails to specifically ubiquitinate and enhance the degradation of L166P and M26I mutant DJ-1, but instead promotes their stability in cultured cells. The interaction of parkin with L166P DJ-1 may involve a larger protein complex that contains CHIP and Hsp70, perhaps accounting for the lack of parkin-mediated ubiquitination. Oxidative stress also promotes an interaction between DJ-1 and parkin, but this does not result in the ubiquitination or degradation of DJ-1. Parkin-mediated alterations in DJ-1 protein stability may be pathogenically relevant as DJ-1 levels are dramatically increased in the detergent-insoluble fraction from sporadic PD/DLB brains, but are reduced in the insoluble fraction from parkin-linked autosomal recessive juvenile-onset PD brains. These data potentially link DJ-1 and parkin in a common molecular pathway at multiple levels that may have important implications for understanding the pathogenesis of inherited and sporadic PD.     

Long-term in vivo biostability of poly(dimethylsiloxane)/poly(hexamethylene oxide) mixed macrodiol-based polyurethane elastomers

The long-term biostability of a novel thermoplastic polyurethane elastomer (Elast-Eon 2 80A) synthesized using poly(hexamethylene oxide) (PHMO) and poly(dimethylsiloxane) (PDMS) macrodiols has been studied using an in vivo ovine model. The material's biostability was compared with that of three commercially available control materials, Pellethane 2363-80A, Pellethane 2363-55D and Bionate 55D, after subcutaneous implantation of strained compression moulded flat sheet dumbbells in sheep for periods ranging from 3 to 24 months. Scanning electron microscopy, attenuated total reflectance-Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy were used to assess changes in the surface chemical structure and morphology of the materials. Gel permeation chromatography, differential scanning calorimetry and tensile testing were used to examine changes in bulk characteristics of the materials. The results showed that the biostability of the soft flexible PDMS-based test polyurethane was significantly better than the control material of similar softness, Pellethane 80A, and as good as or better than both of the harder commercially available negative control polyurethanes, Pellethane 55D and Bionate 55D. Changes observed in the surface of the Pellethane materials were consistent with oxidation of the aliphatic polyether soft segment and hydrolysis of the urethane bonds joining hard to soft segment with degradation in Pellethane 80A significantly more severe than that observed in Pellethane 55D. Very minor changes were seen on the surfaces of the Elast-Eon 2 80A and Bionate 55D materials. There was a general trend of molecular weight decreasing with time across all polymers and the molecular weights of all materials decreased at a similar relative rate. The polydispersity ratio, Mw/Mn, increased with time for all materials. Tensile tests indicated that UTS increased in Elast-Eon 2 80A and Bionate 55D following implantation under strained conditions. However, ultimate strain decreased and elastic modulus increased in the explanted specimens of all three materials when compared with their unimplanted unstrained counterparts. The results indicate that a soft, flexible PDMS-based polyurethane synthesized using 20% PHMO and 80% PDMS macrodiols has excellent long-term biostability compared with commercially available polyurethanes.     

Renal cell carcinoma with mixed features of papillary and clear cell cytomorphology: a fluorescent in situ hybridization study

We performed fluorescent in situ hybridization (FISH) to investigate the numeric change of chromosomes 7, 17, and Y and loss of chromosome 3p in “papillary renal cell carcinomas (RCC) with extensive clear cell changes (CCC).” Consecutive cases of RCC over a 12-year period were reviewed to identify “papillary RCC with extensive CCC.” Immunostaining for cytokeratin 7 and alpha-methylacyl-CoA racemase (AMACR) and FISH for chromosomes 7, 17, Y, and 3p were applied. Of the total of 521 RCC retrieved, there were 49 RCC with papillary architecture and clear cell areas that could be divided into: Group 1 (12 cases), typical clear cell RCC with focal areas of papillary formation; Group 2 (28 cases), focal typical papillary RCC having papillary architecture with extensive CCC; and Group 3 (nine cases), RCC with an admixture of eosinophilic/clear cytoplasm and solid/papillary architecture. Group 1 showed negative immunoreactivity for CK7 and AMACR and absence of numeric chromosomal gain or loss of chromosomes 7/17 and Y. Groups 2 and 3 showed variable reactivity for CK7 and AMACR. Tumors in group 2 and five in group 3 showed trisomies of chromosomes 7 and/or 17 with or without loss of chromosome Y. Loss of small arm 3p was observed in groups 1 and 3 but not in group 2 tumors. In conclusion, papillary RCC may show phenotypical CCC mimicking clear cell RCC. In a small number of cases with mixed histopathological features, FISH is helpful in subtyping RCC.     

Characterization of the antibody response against Plasmodium falciparum erythrocyte membrane protein 1 in human volunteers

The immune response against the Plasmodium falciparum variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a key component of clinical immunity against falciparum malaria. In this study, we used sera from human volunteers who had been infected with the P. falciparum 3D7 strain to investigate the development, specificity, and dynamics of anti-PfEMP1 antibodies measured against six different strain 3D7 Duffy binding-like domain 1α (DBL1α) fusion proteins. We observed that a parasitemia of 20 to 200 infected erythrocytes per μl was required to trigger an antibody response to DBL1α and that antibodies against one DBL1α variant cross-react with other DBL1α variants. Both serum and purified immunoglobulin Gs (IgGs) were able to agglutinate infected erythrocytes, and purified anti-DBL1α IgGs bound to the live infected red blood cell surface in a punctate surface pattern, confirming that the IgGs recognize native PfEMP1. Analysis of sera from tourists naturally infected with P. falciparum suggests that the anti-PfEMP1 antibodies often persisted for more than 100 days after a single infection. These results help to further our understanding of the development of acquired immunity to P. falciparum infections.     

The effect of gas plasma modification on platelet and contact phase activation processes

Medical-grade polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), polyetherurethane (PEU) and ultrahigh molecular weight polyethylene (UHMWPE) were plasma treated with O2, Ar, N2 and NH3. Their surface properties were characterised using X-ray photoelectron spectroscopy (XPS), static secondary ion mass spectroscopy (SSIMS), atomic force microscopy (AFM) and dynamic contact angle (DCA) analysis. Platelet adhesion, aggregation, activation and release of microparticles were determined after contact with whole blood in a cone and plate viscometer. Activation of the coagulation system was quantified in a static environment using a partial thromboplastin time (PTT) assay. The chemical compositions of the untreated surfaces were found to be very similar to those of the bulk material except for PEU, whose surface was comprised almost entirely of soft ether segments. For all materials, the different plasma treatments resulted in moderate etching with the incorporation of functional groups and removal of side groups: defluorination, dehydrogenation, cleavage of methyl side groups and soft segments for PTFE, UHMWPE, PDMS and PEU, respectively. Consequently, plasma treatment resulted in increased wettability in all cases. Blood contact with the virgin materials resulted in activation of platelets and the clotting cascade. Plasma treatment resulted in a significant reduction in platelet adhesion for all materials and all treatments. In the case of PTFE and PEU, the activation status of these cells was also reduced. Plasma treatment of all materials reduced fluid-phase CD62P expression. Platelet aggregate size correlated well with degree of aggregate formation, but many treatments increased the degree of aggregation, as was the case for microparticle shedding. There was no correlation between CD62P expression, aggregate formation and platelet microparticle (PMP) shedding. It is concluded that despite incorporation of the same chemical groups, the pattern of response to blood in vitro is not the same across different polymers.     

Changes in central artery blood pressure and wave reflection during a cold pressor test in young adults

The relative contribution of sympathetic nervous system (SNS)-induced increase in peripheral vascular resistance on central artery blood pressure (BP) and aortic wave reflection (augmentation index; AIx) is not completely understood. Central BP and wave reflection characteristics were measured using radial artery applanation tonometry before, during a 3-min cold pressor test (CPT), and 90 and 180-s post-CPT in 15 young, healthy adults (25 +/- 1 years). The CPT resulted in a greater magnitude of change in the estimated aortic systolic (31 vs. 23%, P < 0.05) and pulse (31 vs. 13%, P < 0.05) BP compared with the change in brachial artery BP. Additionally, the CPT resulted in an increased mean arterial pressure (MAP) (P < 0.05) and AIx (10 +/- 2 vs. 26 +/- 2%, P < 0.05). The change in MAP during the CPT was correlated to the change in AIx (r = 0.73, P < 0.01) and inversely related to roundtrip duration of the reflected wave to the periphery and back (r = -0.57, P < 0.05). The present study suggests that cold pressor testing results in a significant increase in arterial wave reflection intensity, possibly due to an increased MAP. However, the greater increase in systolic and pulse BP in the central compared with the peripheral circulation suggests that increased central artery wave reflection intensity contributes to increased left ventricular myocardial oxygen demand during CPT-induced hypertension.     

Characterization of chromosome structures of Falconinae (Falconidae, Falconiformes, Aves) by chromosome painting and delineation of chromosome rearrangements during their differentiation

Karyotypes of most bird species are characterized by around 2n = 80 chromosomes, comprising 7–10 pairs of large- and medium-sized macrochromosomes including sex chromosomes and numerous morphologically indistinguishable microchromosomes. The Falconinae of the Falconiformes has a different karyotype from the typical avian karyotype in low chromosome numbers, little size difference between macrochromosomes and a smaller number of microchromosomes. To characterize chromosome structures of Falconinae and to delineate the chromosome rearrangements that occurred in this subfamily, we conducted comparative chromosome painting with chicken chromosomes 1–9 and Z probes and microchromosome-specific probes, and chromosome mapping of the 18S–28S rRNA genes and telomeric (TTAGGG) _n sequences for common kestrel (Falco tinnunculus) (2n = 52), peregrine falcon (Falco peregrinus) (2n = 50) and merlin (Falco columbarius) (2n = 40). F. tinnunculus had the highest number of chromosomes and was considered to retain the ancestral karyotype of Falconinae; one and six centric fusions might have occurred in macrochromosomes of F. peregrinus and F. columbarius, respectively. Tandem fusions of microchromosomes to macrochromosomes and between microchromosomes were also frequently observed, and chromosomal locations of the rRNA genes ranged from two to seven pairs of chromosomes. These karyotypic features of Falconinae were relatively different from those of Accipitridae, indicating that the drastic chromosome rearrangements occurred independently in the lineages of Accipitridae and Falconinae.