Preserving central blood volume: changes in body fluid compartments during hemodialysis.

The understanding of fluid changes during hemodialysis (HD is essential for reducing complications as well as efficacy of the procedure. Bioimpedance spectroscopy provides a non invasive method of measuring total body water (TBW), the distribution of intra (ICF) and extracellular (ECF) fluids, and their changes during HD. Segmental bioimpedance may be used to measure the same fluid shifts but from different body segments; the technique has previously been shown to com pare well with whole body measures. It is possible that fluid shifts occur differently in different body compartments during HD. Based on previous hemodynamic studies we postulated that during HD ultrafiltration (UF) the body attempts to preserve its central blood volume (cardiopulmonary circula tion plus great vessels), and thus fluid shifts would be greater from the periphery than from central compartments. To test this hypothesis, segmental bioimpedance (Xitron Technolo gies, San Diego, CA) was performed on 11 subjects undergoing HD where ECF and ICF values were obtained from the legs, arms and trunk before and after a period of UF. Blood volume change (ABV%) was also followed using an on-line optical hematocrit (Hct) sensor (Crit-Line monitor, In-Line Diagnostics, UT) where deltaBV% = deltaBV% = (1 - Hct1/Hct0) x 100 (Hct0 = baseline Hct; Hct1 = postultrafiltration Hct) The UF of 2.0 L +/- 0.79 L (M +/- SD) over 75 minutes was associated with a deltaBV% of -9.43% +/- 3.6% (M +/- SD), a significant (Student's paired t-test) reduction in total body (TB) ECF (p < 0.02), a weak correlation in reduction in TBW (p = 0.09) but not in TB ICF. The ECF reductions from the trunk, legs, and arms were all significant (minimum p < 0.02); no ICF changes from these compartments were significant. The amount of ECF reduction was greater from the legs (0.7 L +/- 0.6 L) than the arms (0.12 L +/- 0.08 L) and trunk (0.2 L +/- 0.2 L) (all M +/- SD). Multiple regression analysis showed that TB ECF changes correlated strongly with leg (r = 0.94, p < 0.001) and arm (r = 0.72, p = 0.002) ECF changes but not with trunk changes. deltaBV% correlated weakly with leg (r = 0.45, p = 0.08) and arm (r = 0.42, p = 0.10) ECF changes but not with the trunk. As the deltaBV% represents the net volume change between UF and plasma water refilling, thiss indicates that plasma water is being removed more from the peripheral compartments than from the trunk. These data suggest that plasma refilling during HD to preserve central blood volume is more dynamic from the leg ECF than from elsewhere and may, in turn, explain the frequent occurrence of leg cramps during and after hemodialysis.

Characterization of sterically stabilized cisplatin liposomes by nuclear magnetic resonance.

Extensive scientific efforts are directed towards finding new and improved platinum anticancer agents. A promising approach is the encapsulation of cisplatin in sterically stabilized, long circulating, PEGylated 100 nm liposomes. This liposomal cisplatin (STEALTH cisplatin, formerly known as SPI-77) shows excellent stability in plasma and has a longer circulation time, greater efficacy and lower toxicity than much free cisplatin. However, so far, the physicochemical characterization of STEALTH cisplatin has been limited to size distribution, drug-to-lipid ratio and stability. Information on the physical state of the drug in the liposome aqueous phases and the drug's interaction with the liposome membrane has been lacking. This study was aimed at filling this gap. We report a multinuclear NMR study in which several techniques have been used to assess the physical nature of cisplatin in liposomal formulations and if and to what extent the drug affects the liposome phospholipids. Since NMR detects only the soluble cisplatin in the liposomes and not the insoluble drug, combining NMR and atomic absorption data enables one to determine how much of the encapsulated drug is soluble in the intraliposomal aqueous phase. Our results indicate that almost all of the cisplatin remains intact during the loading process, and that the entire liposomal drug is present in a soluble form in the internal aqueous phase of the liposomes.

Effects of monofunctional platinum binding on the thermal stability and conformation of a self-complementary 22-mer.

We investigated the effect of various monofunctional platinum complexes on the thermal stability and conformation of a self-complementary 22-mer duplex oligonucleotide by means of CD and UV melting profiles. We studied several families of triamine complexes of the general formula PtA2AmCl where A2=(NH3)2 and ethylenediamine and where Am=N1-4-methyl-pyridine, N7-guanosine, and 9-ethyl-guanine. Platination by the N1-4-methyl-pyridine and 9-ethyl-guanine complexes led to a decrease in the Tm of the oligonucleotide by 2-11.5 degrees C while platination with the N7-guanosine complexes led to a rise in the melting temperature of the oligonucleotides by 4.5 degrees C. A similar inverse correlation between the two groups of platinum compounds was found in the CD spectra. In all cases, the cis isomer had a more pronounced effect on both the melting curve and the CD spectrum. The cis isomer was found to have a more destabilizing effect than its trans counterpart. This indicates that the cis geometry in fact forces a greater structural constraint on the backbone of the double helix. We have also found that the sugar of the guanosine has a significant influence on both the Tm and CD spectra; the sugar moiety contributes to the stability of the double helix, probably through the formation of hydrogen bonds.

Primary uroepithelial cultures. A model system to analyze umbrella cell barrier function.

Despite almost 25 years of effort, the development of a highly differentiated and functionally equivalent cell culture model of uroepithelial cells has eluded investigators. We have developed a primary cell culture model of rabbit uroepithelium that consists of an underlying cell layer that interacts with a collagen substratum, an intermediate cell layer, and an upper cell layer of large (25-100 micrometer) superficial cells. When examined at the ultrastructural level, the superficial cells formed junctional complexes and had an asymmetric unit membrane, a hallmark of terminal differentiation in bladder umbrella cells. These cultured "umbrella" cells expressed uroplakins and a 27-kDa uroepithelial specific antigen that assembled into detergent-resistant asymmetric unit membrane particles. The cultures had low diffusive permeabilities for water (2.8 x 10(-4) cm/s) and urea (3.0 x 10(-7) cm/s) and high transepithelial resistance (>8000 Omega cm2) was achieved when 1 mM CaCl2 was included in the culture medium. The cell cultures expressed an amiloride-sensitive sodium transport pathway and increases in apical membrane capacitance were observed when the cultures were osmotically stretched. The described primary rabbit cell culture model mimics many of the characteristics of uroepithelium found in vivo and should serve as a useful tool to explore normal uroepithelial function as well as dysfunction as a result of disease.

Monofunctional platinum amine complexes destabilize DNA significantly.

Both cis-[Pt(NH3)2(4-Me-Py)Cl]+ and trans-[Pt(NH3)2(4-Me-Py)Cl]+ bind DNA covalently at the N7 site of guanine residues forming mono-dentate adducts. However, like cisplatin and transplatin, only the cis isomer has anti-cancer activity, whereas the trans-isomer does not. In order to understand the molecular basis of the different activities associated with cis-[Pt(NH3)2(4-Me-Py)Cl]+ and trans-[Pt(NH3)2(4-Me-Py)Cl]+, the interactions of these two platinum compounds with the DNA heptamer CCTG*TCC:GGACAGG duplex (G* is the platinated guanine) have been examined. The reaction rate of cis-[Pt(NH3)2(4-Me-Py)Cl]+ with the single-stranded CCTGTCC is significantly faster than that of the trans isomer. The solution structure of the platinum-DNA adducts has been studied by two-dimensional NMR spectroscopy. Both the cis-platinum adducts and the trans-platinum adducts destabilize the DNA duplex significantly. The melting temperature (Tm) of the platinated heptamer duplex is estimated to be 10 degrees C lower than for the unplatinated duplex by NMR. At 2 degrees C, the base pairs located on the 5' side of the oligonucleotide, beyond the platinum lesion site, are disrupted. Over time, the platinum-DNA complex decomposes and the cis-[Pt(NH3)2(4-Me-Py)] platinum complex is gradually detached from DNA. No interstrand crosslinking is observed. The biological implications of the structural studies are discussed.

Effect of nonsteroidal anti-inflammatory drugs on the action of misoprostol in a regimen for early abortion.

Nonsteroidal anti-inflammatory drugs are frequently avoided by some investigators in protocols for medical abortion because of a concern over potential inhibition of prostaglandin action on uterine contractions. To evaluate the effects of nonsteroidal anti-inflammatory drugs on uterine cramping and resultant pregnancy expulsion, analgesic use by 449 participants with gestational age of < or = 56 days in three previously reported medical abortion trials involving methotrexate and misoprostol was reviewed. Subjects from each of the three trials were included in this analysis only if they received 50 mg/m2 methotrexate intramuscularly followed 3 or 7 days later by 800 micrograms misoprostol vaginally. Misoprostol administration was repeated if the abortion did not occur after the first misoprostol dose. A total of 416 subjects met the study criteria. For the women who took a nonsteroidal anti-inflammatory drug after the first dose of misoprostol, 132/246 (53.7%) aborted within 24 h. For those that did not take a nonsteroidal anti-inflammatory drug, 83/170 (48.8%, p = 0.38) aborted within 24 h. Similarly, 27/56 (48.2%) of the women who took a nonsteroidal anti-inflammatory drug aborted within 24 h of the second dose of misoprostol. However, only 32/145 (22.1%, p = 0.002) of the women who did not take a nonsteroidal anti-inflammatory drug aborted. Use of a nonsteroidal anti-inflammatory drug does not interfere with the action of misoprostol to induce uterine contractions and pregnancy expulsion in women receiving methotrexate and misoprostol for early abortion.

An antibody reactive with domain 4 of the platelet-derived growth factor beta receptor allows BB binding while inhibiting proliferation by impairing receptor dimerization.

A panel of murine monoclonal antibodies was generated against the extracellular domain of the human platelet-derived growth factor (PDGF) beta receptor (PDGFRbeta). These antibodies were assayed for both the ability to inhibit binding of PDGF BB to PDGFRbeta+ cells as well as the capacity to inhibit PDGF BB-mediated mitogenesis. As expected, all antibodies that could prevent PDGF BB binding also inhibited mitogenesis. However one antibody (M4TS.11), with no detectable ability to inhibit PDGF BB binding, was a potent inhibitor of proliferation induced by PDGF BB. Further characterization indicated that M4TS.11 impaired PDGFRbeta dimerization, revealing the mechanism by which it prevented PDGF BB-mediated mitogenesis. Using domain deletion mutants of the extracellular portion of PDGFRbeta, the determinant recognized by this antibody was localized to the fourth extracellular domain of PDGFRbeta, indicating that this domain, which is not involved in ligand binding, actively participates in receptor dimerization and signal transduction. The M4TS.11 antibody could also inhibit PDGF BB-mediated proliferation of responsive cells from both the baboon and the rabbit, indicating the determinant recognized by the antibody is not limited to humans and making it possible to use this antibody to evaluate the therapeutic benefit of interfering with PDGF in animal models of human disease.