Potential therapeutic roles of stem cells in ischemia-reperfusion injury.

Ischemia-reperfusion injury (I/RI), produced by an initial interruption of organ blood flow and its subsequent restoration, contributes significantly to the pathophysiologies of stroke, myocardial infarction, renal I/RI, intestinal I/RI and liver I/RI, which are major causes of disability (including transplant failure) and even mortality. While the restoration of blood flow is required to restore oxygen and nutrient requirements, reperfusion often triggers local and systemic inflammatory responses and subsequently elevate the ischemic insult where the duration of ischemia determines the magnitude of I/RI damage. I/RI increases vascular leakage, changes transcriptional and cell death programs, drives leukocyte entrapment and inflammation and oxidative stress in tissues. Therapeutic approaches which reduce complications associated with I/RI are desperately needed to address the clinical and economic burden created by I/RI. Stem cells (SC) represent ubiquitous and uncommitted cell populations with the ability to self-renew and differentiate into one or more developmental 'fates'. Like immune cells, stem cells can home to and penetrate I/R-injured tissues, where they can differentiate into target tissues and induce trophic paracrine signaling which suppress injury and maintain tissue functions perturbed by ischemia-reperfusion. This review article summarizes the present use and possible protective mechanisms underlying stem cell protection in diverse forms of ischemia-reperfusion.

Antibacterial and antibiofouling clay nanotube-silicone composite.

INTRODUCTION: Invasive medical devices are used in treating millions of patients each day. Bacterial adherence to their surface is an early step in biofilm formation that may lead to infection, health complications, longer hospital stays, and death. Prevention of bacterial adherence and biofilm development continues to be a major healthcare challenge. Accordingly, there is a pressing need to improve the anti-microbial properties of medical devices. MATERIALS AND METHODS: Polydimethylsiloxane (PDMS) was doped with halloysite nanotubes (HNTs), and the PDMS-HNT composite surfaces were coated with PDMS-b-polyethylene oxide (PEO) and antibacterials. The composite material properties were examined using SEM, energy dispersive spectroscopy, water contact angle measurements, tensile testing, UV-Vis spectroscopy, and thermal gravimetric analysis. The antibacterial potential of the PDMS-HNT composites was compared to commercial urinary catheters using cultures of E. coli and S. aureus. Fibrinogen adsorption studies were also performed on the PDMS-HNT-PEO composites. RESULTS: HNT addition increased drug load during solvent swelling without reducing material strength. The hydrophilic properties provided by PEO were maintained after HNT addition, and the composites displayed protein-repelling properties. Additionally, composites showed superiority over commercial catheters at inhibiting bacterial growth. CONCLUSION: PDMS-HNT composites showed superiority regarding their efficacy at inhibiting bacterial growth, in comparison to commercial antibacterial catheters. Our data suggest that PDMS-HNT composites have potential as a coating material for anti-bacterial invasive devices and in the prevention of institutional-acquired infections.

Immunodetection of a type III sodium-dependent phosphate cotransporter in tissues and OK cells.

Polyclonal antibodies were raised in rabbits against a 14-amino acid portion of the gibbon ape leukemia virus human membrane receptor Glvr-1. This epitope also contained seven amino acids common to the receptor for the amphotropic murine retrovirus Ram-1. Antibody specificity and molecular size of Glvr-1/Ram-1-related proteins were assayed by Western blot. Using a standard Laemmli buffer system, under reducing conditions, a single band of approximately 85 kDa (designated p85) was immunodetected in membranes prepared from opossum kidney (OK) cells and in brain membranes from rat, rabbit and hamster. In mouse brain, p85 as well as a protein of 70-72 kDa were immunodetected. This protein was also present in several other mouse tissues. Limited proteolysis of p85 and the 70-72kDa-protein from mouse yielded similar peptide fragments, suggesting that both proteins are related. Fragments of the same molecular masses were also detected in OK cell membranes following proteolysis, showing that p85 in both models (mouse brain and OK cell) share a similar sequence. p85 is not N-glycosylated since an assay using endoglycosidase F/N-glycosidase F did not alter the electrophoretic mobility of p85. We also observed that regulation of phosphate transport by incubating OK cells without any phosphate or by PTH treatment occurs without any changes in the amount of p85. In conclusion, these data demonstrate for the first time a Western blot detection of a type III phosphate transporter using polyclonal antibodies. They also suggest that, conversely to type I and type II phosphate transporters which are localized in the kidney, this third type of transporter is ubiquitous and probably absorbs the readily available phosphate from interstitial fluid for normal cellular functions in many species and tissues, serving as a housekeeping Na+/Pi cotransport system. This is also the first report showing that p85 is not regulated in the same manner as type II phosphate transporters.

Membrane topography of the renal phosphate carrier NaPi-2: limited proteolysis studies.

The rat sodium/phosphate cotransporter NaPi-2 is a 70 kDa polypeptide (p70) for which eight transmembrane segments have been predicted. We have shown that p70 exists predominantly as p45 and p40 fragments which are linked by disulfide bonds. In this work, the p40 fragment, corresponding to the C-terminus of NaPi-2, was purified from renal brush-border membranes using non-reducing and then reducing column electrophoresis followed by enzymatic deglycosylation and SDS-PAGE. The N-terminal sequence obtained for this fragment, VEAIG, indicates that the formation of p45 and p40 arises from the cleavage of p70 between arginine-319 and valine-320. In order to determine the membrane topography of NaPi-2, brush-border membrane vesicles were digested with various proteases and the transporter-derived proteolytic peptides were subsequently identified by Western blotting using N- and C-terminal-directed antibodies. Our results lead us to propose an alternative topographical model in which p45 and p40 possess three transmembrane domains each and indicate that the processing site of p70 for the generation of p45 and p40 is localized in a large protein core facing the extracellular milieu. This localization of the cleavage site indicated that NaPi-2 could either be processed intracellularly by vesicular proteases or extracellularly by secretory proteases or by brush-border membrane ectoenzymes.

Phosphate deprivation induces overexpression of two proteins related to the rat renal phosphate cotransporter NaPi-2.

Polyclonal antibodies were raised in rabbits against the C-terminal portion of the rat renal brush-border membrane sodium/phosphate cotransporter NaPi-2. Antibody specificity and molecular sizes of proteins related to NaPi-2 were assayed by Western blot analysis. Proteins of 40 and 70-75 kDa (p40 and p70) were immunodetected in rat and mouse brush-border membranes and proteins of 72 and 82 kDa were detected in rabbit. The absence or presence of beta-EtSH in the samples before electrophoresis greatly influenced the immunodetection profile of the rat proteins. Since the 40 kDa protein (p40) can only be detected under reducing conditions, it probably originates from reduction of disulfide bonds in p70. Tryptic cleavage of p40 and p70 revealed identical protein fragments showing the close structural identity of those proteins. Both proteins were more abundant in the outer cortex portion of the rat kidney than in the juxtamedullary portion. Furthermore, rats fed a low-phosphate diet for 24 h showed a 20- and 14-fold increase in the amount of p40 and p70, respectively, compared to control rats, showing that the adaptation to P(i) deprivation by increasing renal phosphate reabsorption is not only the result of overproduction of p70, as previously shown, but is also due to the novel p40 which most probably derives from p70.

Successful use of indwelling cuffed femoral vein catheters in ambulatory hemodialysis patients.

Three hemodialysis patients with multiple upper extremity vascular access complications and central vein stenosis were treated for as long as 3 months using an indwelling femoral vein catheter having a buried felt cuff in its subcutaneous tunnel. Four catheters were placed in these three patients. In one case, initial failure due to poor flow and clotting occurred using a straight catheter with its tunnel crossing the inguinal ligament and exiting caudally on the anterior thigh. Otherwise, each patient had successful placement of a 180-degree, curved catheter that exited the femoral vein in the usual fashion but had a subcutaneous tunnel and skin exit pointing cephalad in the inferior portion of the right lower quadrant. The three successful devices functioned immediately after placement, having acceptable outflow pressures and recirculation values. One of three catheters was removed 3 weeks after placement when persisting infection was thought to reside on the device. No other bleeding, thromboembolic, or infectious complications occurred in these patients. In conclusion, short-term indwelling femoral vein access may be feasible in ambulatory hemodialysis patients with previous access difficulties that complicate temporary dialysis treatment.

Successful use of cuffed central venous hemodialysis catheters inserted percutaneously.

Although endogenous fistulae and grafts are preferred for permanent hemodialysis access, central venous catheters are often required for varying intervals when creating permanent access is not feasible. The prospective experience with 118 catheters in over a 3.5-yr period is reported; 93 (79%) were placed by percutaneous techniques, and 25 (21%) were placed by operative techniques. Seventy seven catheters (65%) were placed in the subclavian vein, 36 (31%) were placed in the internal jugular vein (usually right side), and 5 (4%) were placed in the femoral vein. Early postplacement complications were infrequent. Catheter function at last local follow-up ranged from several days to nearly 2 yr, averaging approximately 3 mo, even though many patients returned to their referring centers with a functioning catheter after only a short follow-up. Actuarial survival for percutaneously placed catheters was approximately 60% at 6 mo and 30% at 12 mo. Catheter failure occurred in 36% of cases, equally divided between malfunction (thrombosis refractory to fibrinolysis, extrusion, kinking, or related event) and infection with septicemia requiring removal. Such failure was not more frequent after percutaneous placement than after operative placement. Failure due to mechanical malfunction, but not that due to infection, tended to be less frequent among catheters placed in the internal jugular vein than among catheters placed in the subclavian vein. Finally, infection with septicemia involved 22% of all catheters and occurred at an average cumulated rate of approximately one infection per patient-year. Coagulase-positive staphylococcus was the most common organism isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of disulphide bonds in the renal sodium/phosphate co-transporter NaPi-2.

The rat renal brush border membrane sodium/phosphate co-transporter NaPi-2 was analysed in Western blots with polyclonal antibodies raised against its N-terminal and C-terminal segments. Under reducing conditions, proteins of 45-49 and 70-90 kDa (p45 and p70) were detected with N-terminal antibodies, and proteins of 40 and 70-90 kDa (p40 and p70) were detected with C-terminal antibodies. p40 and p45 apparently result from a post-translational cleavage of NaPi-2 but remain linked through one or more disulphide bonds. Glycosidase digestion showed that both polypeptides are glycosylated; the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2. In the absence of reducing agents, both N-terminal and C-terminal antibodies detected p70 and a protein of 180 kDa (p180), suggesting the presence of p70 dimers. Much higher concentrations of beta-mercaptoethanol were required to produce a given effect in intact membrane vesicles than in solubilized proteins, indicating that the affected disulphide bonds are not exposed at the surface of the co-transporter. Phosphate transport activity decreased with increasing concentrations of reducing agents [beta-mercaptoethanol, dithiothreitol and tris-(2-carboxyethyl)phosphine] and was linearly correlated with the amount of p180 detected. The target sizes estimated from the radiation-induced loss of intensity of p40, p70 and p180 were all approx. 190 kDa, suggesting that NaPi-2 exists as an oligomeric protein in which the subunits are sufficiently close to one another to allow substantial energy transfer between the monomers. When protein samples were pretreated with beta-mercaptoethanol [2.5% and 5% (v/v) to optimize the detection of p40 and p70] before irradiation, target sizes estimated from the radiation-induced loss of intensity of p40 and p70 were 74 and 92 kDa respectively, showing the presence of disulphide bridges in the molecular structure of NaPi-2.