Effects of centralized and onsite wastewater treatment on the occurrence of traditional and emerging contaminants in streams.

The authors conducted a survey of small streams to evaluate the effects of centralized and onsite wastewater treatment on the occurrence of selected traditional and emerging contaminants in small streams in the upper Neuse River basin, North Carolina. An undeveloped site was included to assess effects of residential land use activities on stream quality. Concentrations of nutrients and ions were higher in samples from streams in residential sites than from the stream in an undeveloped area. Overall, streams draining residential areas showed relatively small differences with respect to type of wastewater treatment. Two sites, however--one in an area of centralized wastewater treatment apparently near a suspected sewer line leak, and the second in an area of onsite wastewater treatment--showed effects of wastewater. Organic wastewater compounds were detected more frequently in samples from these two sites than from the other sites. Optical brighteners levels were correlated (r2 = .88) with the number of organic wastewater and pharmaceutical compounds detected at the residential sites and could potentially serve as a screening method to assess wastewater effects on small streams.

Coalescence kinetics in surfactant stabilized emulsions: evolution equations from direct numerical simulations.

Lattice Boltzmann simulations were used to study the coalescence kinetics in emulsions with amphiphilic surfactant, under neutrally buoyant conditions, and with a significant kinematic viscosity contrast between the phases (emulating water in oil emulsions). The 3D simulation domain was large enough (256(3) ~ 10(7) grid points) to obtain good statistics with droplet numbers ranging from a few thousand at early times to a few hundred near equilibrium. Increased surfactant contents slowed down the coalescence rate between droplets due to the Gibbs-Marangoni effect, and the coalescence was driven by a quasi-turbulent velocity field. The kinetic energy decayed at a relatively slow rate at early times, due to conversion of interfacial energy to kinetic energy in the flow during coalescence. Phenomenological, coupled differential equations for the mean droplet diameter D(t) and the number density n(d)(t) were obtained from the simulation data and from film draining theories. Local (in time) power law exponents for the growth of the mean diameter (and for the concomitant decrease of n(d)) were established in terms of the instantaneous values of the kinetic energy, coalescence probability, Gibbs elasticity, and interfacial area. The model studies indicated that true power laws for the growth of the droplet size and decrease of the number of droplets with time may not be justified, since the exponents derived using the phenomenological model were time dependent. In contrast to earlier simulation results for symmetric blends with surfactant, we found no evidence for stretched logarithmic scaling of the form D ~ [ln (ct)](α) for the morphology length, or exponential scalings associated with arrested growth, on the basis of the phenomenological model.

Cancer-associated alteration of pericentromeric heterochromatin may contribute to chromosome instability.

Many tumors exhibit elevated chromosome mis-segregation termed chromosome instability (CIN), which is likely to be a potent driver of tumor progression and drug resistance. Causes of CIN are poorly understood but probably include prior genome tetraploidization, centrosome amplification and mitotic checkpoint defects. This study identifies epigenetic alteration of the centromere as a potential contributor to the CIN phenotype. The centromere controls chromosome segregation and consists of higher-order repeat (HOR) alpha-satellite DNA packaged into two chromatin domains: the kinetochore, harboring the centromere-specific H3 variant centromere protein A (CENP-A), and the pericentromeric heterochromatin, considered important for cohesion. Perturbation of centromeric chromatin in model systems causes CIN. As cancer cells exhibit widespread chromatin changes, we hypothesized that pericentromeric chromatin structure could also be affected, contributing to CIN. Cytological and chromatin immunoprecipitation and PCR (ChIP-PCR)-based analyses of HT1080 cancer cells showed that only one of the two HORs on chromosomes 5 and 7 incorporate CENP-A, an organization conserved in all normal and cancer-derived cells examined. Contrastingly, the heterochromatin marker H3K9me3 (trimethylation of H3 lysine 9) mapped to all four HORs and ChIP-PCR showed an altered pattern of H3K9me3 in cancer cell lines and breast tumors, consistent with a reduction on the kinetochore-forming HORs. The JMJD2B demethylase is overexpressed in breast tumors with a CIN phenotype, and overexpression of exogenous JMJD2B in cultured breast epithelial cells caused loss of centromere-associated H3K9me3 and increased CIN. These findings suggest that impaired maintenance of pericentromeric heterochromatin may contribute to CIN in cancer and be a novel therapeutic target.

Analysis of dynamic surfactant mass transfer and its relationship to the transient stabilization of coalescing liquid-liquid dispersions.

In this work, experiments describing the behavior of the separation of a model liquid-liquid dispersion with various concentrations of a synthetic surfactant are presented which indicate that there is a dynamic stabilization of initially unstable emulsions that occurs when the initial surfactant concentration approaches the concentration that provides stable emulsions. A simple model is presented to suggest the mechanism for the dynamic stabilization of these emulsion systems that considers the redistribution of surfactant into the continuous phase after a coalescence event at the emulsion-bulk dispersed phase interface and the dynamic mass transport of surfactant in the continuous phase of the emulsion. The results indicate that coalescence at the interface between the emulsion layer and the bulk dispersed phase creates a local region in the vicinity of this interface where the concentration of the surfactant is much higher than the bulk surfactant concentration which could lead to a locally, dynamically stabilized emulsion at this interface. The extent to which the local excess surfactant concentration reduces the coalescence rate depends strongly on the rate of coalescence at the dense packed layer-bulk dispersed phase interface relative to the rate of surfactant diffusion through the dense packed layer and, of course, on the surfactant adsorption constant, the maximum adsorbed surfactant concentration, and the surface to volume ratio of the dispersed phase. Furthermore, the results indicate that coalescence can also act to significantly increase the local concentration of an initially very dilute surfactant in the vicinity of the interface between the emulsion layer and the bulk dispersed phase interface.

Utilization of TCD screening for primary stroke prevention in children with sickle cell disease.

BACKGROUND: In 1998, the Stroke Prevention Trial in Sickle Cell Anemia showed a >90% reduction in stroke rates after blood transfusion therapy in children with sickle cell disease (SCD) identified as high risk with transcranial Doppler ultrasonography (TCD) screening. METHODS: We studied the utilization of TCD screening in a retrospective cohort of all children with SCD within a large managed care plan from January 1993 to December 2005. Rates of first TCD screening were estimated using life table methods; predictors of TCD were evaluated using Cox proportional hazards regression. Stroke incidence rates were estimated in person-time before (pre-TCD) and after (post-TCD) first TCD. RESULTS: The average annual rate of TCD screening in 157 children with SCD was 1.8 per 100 person-years pre-1998, 5.0 from January 1, 1998, to December 31, 1999, and 11.4 after 1999. The only independent predictor of TCD screening was proximity to the vascular laboratory. The annualized stroke rate pre-TCD was 0.44 per 100 person-years, compared to 0.19 post-TCD. CONCLUSIONS: Since the Stroke Prevention Trial in Sickle Cell Anemia, the rate of transcranial Doppler ultrasonography (TCD) screening in sickle cell disease (SCD) has increased sixfold within a large health care plan. Children living farther from a vascular laboratory are less likely to be screened. Increased availability of TCD screening could improve the utilization of this effective primary stroke prevention strategy.

Flow-through pore characteristics of monolithic silicas and their impact on column performance in high-performance liquid chromatography.

In order to elucidate the role of the flow-through characteristics with regard to the column performance in high-performance liquid chromatography (HPLC) native and n-octadecyl bonded monolithic silica rods and columns, respectively of 100 mm length and 4.6 mm ID with mesopores in the range between 10 and 25 nm and macropores in the range between 0.7 and 6.0 microm were examined by mercury intrusion/extrusion, scanning electron microscopy, image analysis and permeability. The obtained data of the flow-through pore sizes and porosity values as well as surface-to-volume ratio of the stationary phase skeleton enabled to predict their influence to the chromatographic separation efficiency. Our data demonstrate that mercury porosimetry is a reliable technique to obtain all the characteristic parameters of the flow-through pores of silica monoliths. An important result of our examination was that the surface-to-volume ratio of monolithic silica skeletons had more significant impact to the separation process, rather than the average flow-through pore sizes. We could also show the essential differences between the particulate and monolithic stationary phases based on theoretical computation. The results, obtained from other characterization methods also indicated the structural complexity of monolithic silica samples. Permeability of columns is a generally applicable parameter to characterize all chromatographic phases no matter the chemistry or format. The correlation coefficient obtained for mercury intrusion and permeability of water was 0.998, though our investigation revealed that the surface modification is more likely influencing the obtained results. Further, the assumption of the cylindrical morphology of flow-through pores is not relevant to the investigated monolithic silica columns. These results on the morphology of the flow-through pores and of the skeletons were confirmed by the image analysis as well. Our main finding is that the flow-through pore sizes are not relevant for the estimation of the chromatographic separation efficiency of monolithic silica columns.

Demyelinating events in early multiple sclerosis have inherent severity and recovery.

BACKGROUND: It is unclear whether the severity of and recovery from the initial demyelinating event (IDE) are recapitulated in subsequent multiple sclerosis (MS) relapses. We sought to identify the factors associated with relapse severity and recovery and to evaluate whether events have inherent severity or recovery. METHODS: Patients seen at the UCSF MS Clinic within 1 year of disease onset were identified from a prospective database. Ordinal logistic regression was used to analyze predictors of three-level categorizations of event severity and recovery. RESULTS: We identified 330 patients with MS or clinically isolated syndrome; 152 had a second event and 63 had a third event. Nonwhite and younger patients were at an increased risk of more severe demyelinating events. A severe prior event predicted a substantial increase in the odds of being above any given severity cutoff for a severe subsequent event (for second event severity, odds ratio [OR] = 5.62, 95% confidence interval [CI] [2.39, 13.26], p < 0.0001; for third event severity, OR = 6.74, 95% CI [1.67, 27.18], p = 0.007). Similarly, poor recovery of the IDE predicted poor second event recovery (OR = 5.28, 95% CI [1.95, 14.25], p = 0.001), while fair or poor second event recovery predicted about a 5- or 13-fold increase in the odds of poor third event recovery. A more severe event also predicted a substantial increase in the odds of poor recovery. CONCLUSIONS: Patients with severe presentation and poor recovery at disease onset continue on a similar trajectory with subsequent demyelinating events. Whether genetic or other biologic factors are responsible for this pattern remains to be determined.

Switching first-line disease-modifying therapy after failure: impact on the course of relapsing-remitting multiple sclerosis.

BACKGROUND: Options for non-responders to relapsing-remitting multiple sclerosis (RRMS) first-line disease-modifying therapies (DMT) are limited. We explored whether switching first-line DMT is effective. METHODS: Patients with RRMS who first received interferon-beta (IFNB) or glatiramer acetate (GA) were classified in three categories: DMT change because of suboptimal response, DMT change because of other reasons, and no DMT change during follow-up. Outcomes included annualized relapse rate (ARR) and relapse-free proportions. RESULTS: We identified 597 patients who initiated first-line DMT. For patients who did not change DMT (n = 240), pre-DMT and on-DMT median ARR were 0.50 and 0 (P < 0.0001). At 24 months, 76% (95%CI = 69-81%) of patients who did not change DMT were relapse-free. Of the 155 who switched because of suboptimal response, 101 switched to another first-line DMT. Median ARR pre-DMT, on first DMT and second DMT were: 0.50, 0.55, and 0.25 for switchers from IFNB to GA (IFNB/GA, n = 12) (pre-DMT versus first DMT: P = 0.92; first versus second DMT: P = 0.31); 0.90, 0.50, and 0 for switchers from GA to IFNB (GA/IFNB, n = 18; P = 0.19; P = 0.01); 0.50, 0.68, and 0 for switchers from an IFNB to another IFNB (IFNB/IFNB', n = 71; P = 0.34; P = 0.02). Estimated relapse-free proportion after 24 months of treatment was 42% (95%CI=15-66%) during the period on IFNB versus 53% (95%CI = 17-80%) on GA for IFNB/GA (P = 0.21); 12% (95%CI = 0-40%) on GA versus 87% (95%CI = 59-97%) on IFNB for GA/IFNB (P = 0.001); and 41% (95%CI = 29-52%) on initial IFNB versus 67% (95%CI = 53-79%) on subsequent IFNB for IFNB/IFNB' (P = 0.0001). CONCLUSIONS: Switching first-line DMT in patients with RRMS failing initial therapy may be effective in many cases.

Sulphonic acid strong cation-exchange restricted access columns in sample cleanup for profiling of endogenous peptides in multidimensional liquid chromatography. Structure and function of strong cation-exchange restricted access materials.

In this work, the pore structural parameters and size exclusion properties of LiChrospher strong cation-exchange and reverse phase restricted access materials (RAM) are analysed. The molecular weight size exclusion limit for polystyrenes was found to be about 17.7 kDa, while for standard proteins, the molecular weight size exclusion limit was higher, at approximately 25 kDa. The average pore diameter on a volume basis calculated from the pore network model changes from 8.5 nm (native LiChrospher) to 8.6 nm (diol derivative) to 8.2 nm (sulphonic acid derivative) to 6.9 nm (n-octadecyl derivative). Additional characterisations were performed on restricted access materials with nitrogen sorption at 77 K, water adsorption at 25 degrees C, intrusion-extrusion of water (in order to evaluate the hydrophobic properties of the pores of the hydrophobic RAM), and zeta potential measurements by microelectrophoresis. For peptide analysis out of the biofluids, the strong cation-exchange functionality seems to be particularly suitable mainly because of the high loadability of the strong cation-exchange restricted access material (SCX-RAM) and the fact that one can work under non-denaturing conditions to perform effective chromatographic separations. For bacitracin, the dynamic capacity of the SCX-RAM columns does not reach its maximum value in the analysed range. For lysozyme, the dynamic capacity reaches a value of 0.08 mg/ml of column volume before column is overloaded. Additionally, the proper column operating conditions that lead to the total effective working time of the RAM column to be equal to approximately 500 injections (depending on the type of sample), is comprehensively described. The SCX-RAM column was used in the same system analysing urine samples for the period of 1 month (approximately 150 injections) with run-to-run reproducibility below 5% RSD and below 10% RSD for the relative fractions.

The isoflavone genistein inhibits LPS-stimulated TNFalpha, but not IL-6 expression in monocytes from hemodialysis patients and healthy subjects.

BACKGROUND: Whole blood and peripheral blood mononuclear cells from hemodialysis (HD) patients show increased production and secretion of inflammatory cytokines. We determined the contribution of blood monocytes to the production of inflammatory cytokines in whole blood from HD patients. METHODS: Whole blood and isolated mononuclear cells from HD patients and healthy control subjects were preincubated with the isoflavone genistein and stimulated with LPS. TNFalpha, IL-6 and IL-10 formation in the whole blood was measured with ELISA and intracellular cytokine formation in CD 14-positive monocytes was determined by flow cytometry. RESULTS: Unstimulated blood levels of TNFalpha, IL-6 and IL-10 were significantly elevated in HD patients compared to controls, but intracellular monocyte content of these cytokines was identical between groups. LPS induced a robust TNFalpha response in both whole blood and monocytes, and TNFalpha formation was 2.3-fold higher in blood from HD patients compared to controls. A similar trend was observed in monocytes. Conversely, LPS stimulation increased IL-6 levels >1000-fold in whole blood, albeit without a noticeable difference between groups. Only minor increases in monocyte IL-6 content were observed. The isoflavone genistein did not inhibit IL-6 formation and did not alter basal TNFalpha levels, but genistein selectively blocked LPS-induced TNFalpha formation in whole blood and monocytes from both groups. CONCLUSION: Intracellular levels of TNFalpha, IL-6 and IL-10 in monocytes are indistinguishable between HD patients and healthy controls. However, monocytes from HD patients are selectively primed for enhanced TNFalpha secretion in response to LPS. The selective inhibition of monocyte TNFalpha production by genistein may explain the anti-inflammatory action of this phytochemical observed in vivo.

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples.

Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning.

Analysis and parametric sensitivity of the behavior of overshoots in the concentration of a charged adsorbate in the adsorbed phase of charged adsorbent particles: practical implications for separations of charged solutes.

In this work, an analysis of the parametric sensitivity of the overshoot in the concentration of the adsorbate in the adsorbed phase, which occurs under certain conditions during an ion-exchange adsorption process, is presented and used to suggest practical implications of the concentration overshoot phenomenon on operational policies and configurations of chromatographic columns and finite bath adsorption systems. The results presented in this work demonstrate and explain how the development of an overshoot in the concentration of the adsorbate in the adsorbed phase could be enhanced or suppressed by (i) varying the diffusion coefficient, D3, of the adsorbate relative to the diffusion coefficients, D1 and D2, of the cations and anions, respectively, of the background/buffer electrolyte, (ii) altering the initial surface charge density, delta0, of the charged adsorbent particles, (iii) varying the Debye length, lambda, and (iv) changing the initial concentration, Cd3(0), of the adsorbate in the bulk liquid of the finite bath. The influence of the pH and ionic strength, Iinfinity, of the liquid solution on the development of an overshoot in the concentration of the adsorbate in the adsorbed phase is also presented and discussed through the relationships of these parameters to delta0 and lambda, respectively. Furthermore, a detailed explanation of the effects of each parameter on the interplay between the diffusive and electrophoretic molar fluxes, as well as on the structure and functioning of the electrical double layer, which are responsible for the concentration overshoot phenomenon, is presented.

Expressions for evaluating the possibility of slip at the liquid-solid interface in open tube capillary electrochromatography.

In this work, expressions are constructed and solved that describe the velocity field of electroosmotic flow (EOF) in open tube capillary electrochromatography (CEC) systems when the possibility of having unequal tangential velocities at the liquid-solid interface is considered and a slip condition is employed as a boundary condition for the velocity of the EOF at the capillary wall. The coupled equations of hydrodynamics (momentum balance equation) and electrostatics (Poisson equation) are solved numerically in order to obtain the distribution of the velocity field as well as the value of the volumetric flow rate in the open tube. Also, expressions for the velocity field and the volumetric flow rate of the EOF are presented that are valid for certain electrolytic systems and for certain parameter values for which analytical solutions to the momentum balance and Poisson equations could be obtained. The results presented in this work indicate that having slip in the velocity of the EOF at the wall of the capillary could (i) substantially increase the electroosmotic velocity in the plug-flow region of the radial domain of the open capillary tube and (ii) increase the portion of the radial domain of the open capillary tube where the velocity of the EOF has a plug-flow profile, which in turn could increase the average velocity and volumetric flow rate of the EOF in the open capillary tube. Furthermore, the modeling approach and the results presented in this work indicate a method for experimentally evaluating the possibility of having slip in the velocity of the EOF at the capillary wall.

Novel general expressions that describe the behavior of the height equivalent of a theoretical plate in chromatographic systems involving electrically-driven and pressure-driven flows.

Novel general expressions are constructed and presented that describe the behavior of the height equivalent of a theoretical plate (plate height), H, as a function of the linear velocity, Vx, along the axis, x, of the column and the kinetic parameters that characterize the mass transfer and adsorption mechanisms in chromatographic columns. Open tube capillaries as well as columns packed with either non-porous or porous particles are studied. The porous particles could have unimodal or bimodal pore-size distributions and intraparticle convective fluid flow and pore diffusion are considered. The expressions for the plate height, H, presented in this work could be applicable to high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) systems, and could be used together with experimental plate height, H, versus linear velocity, Vx, data to determine the values of the parameters that characterize intraparticle convective fluid flow and pore diffusion. Furthermore, chromatographic systems under unretained as well as under retained conditions are examined. The experimental values of the plate height, H, versus the linear velocity, Vx, for a CEC system involving charged porous silica C8 particles and an uncharged analyte are compared with the theoretical results for the plate height, H, obtained from the expressions presented in this work. The agreement between theory and experiment is good, and the results indicate that the magnitude of the intraparticle electroosmotic flow (EOF) in the pores of the particles is substantial while the pore diffusion coefficient was of small magnitude. But the overall intraparticle mass transfer resistance in these particles was low because of the significant contribution of the intraparticle EOF. Simulation results are also presented (i) for a hybrid HPLC-CEC system, and (ii) for different CEC systems involving open capillaries as well as packed columns having non-porous or porous particles. The analysis of the results indicates (a) the reasons for the superior performance exhibited by the hybrid HPLC-CEC system over the performance obtained when the system is operated only in the HPLC mode, and (b) the operational configuration and the properties that the structure of the porous particles would have to have in CEC systems involving uncharged or charged analytes under unretained or retained conditions in order to obtain high CEC efficiency (low values of the plate height, H).

Chromosome engineering: prospects for gene therapy.

Recent advances in chromosome engineering and the potential for downstream applications in gene therapy were presented at the Artificial Chromosome Session of Genome Medicine: Gene Therapy for the Millennium in Rome, Italy in September 2001. This session concentrated primarily on the structure and function of human centromeres and the ongoing challenge of equipping human artificial chromosomes (HACs) with centromeres to ensure their mitotic stability. Advances in the 'bottom up' construction of HACs included the transfer into HT1080 cells of circular PACs containing alpha satellite DNA, and the correction of HPRT deficiency in cells using HACs. Advances in the 'top down' construction of HACs using telomere associated chromosome fragmentation in DT40 cells included the formation of HACs that are less than a megabase in size and transfer of HACs through the mouse germline. Significant progress has also been made in the use of human minichromosomes for stable trans-gene expression. While many obstacles remain towards the use of HACs for gene therapy, this session provided an optimistic outlook for future success.

The interplay of diffusional and electrophoretic transport mechanisms of charged solutes in the liquid film surrounding charged nonporous adsorbent particles employed in finite bath adsorption systems.

A model that describes the diffusive and electrophoretic mass transport of the cation and anion species of a buffer electrolyte and of a charged adsorbate in the liquid film surrounding nonporous adsorbent particles in a finite bath adsorption system, in which adsorption of the charged adsorbate onto the charged surface of the nonporous particles occurs, is constructed and solved. The dynamic behavior of the mechanisms of this model explicitly demonstrates (a) the interplay between the diffusive and electrophoretic molar fluxes of the charged adsorbate and of the species of the buffer electrolyte in the liquid film surrounding the nonporous adsorbent particles, (b) the significant effect that the functioning of the electrical double layer has on the transport of the charged species and on the adsorption of the charged adsorbate, and (c) the substantial effect that the dynamic behavior of the surface charge density has on the functioning of the electrical double layer. It is found that at equilibrium, the value of the concentration of the charged adsorbate in the fluid layer adjacent to the surface of the adsorbent particles is significantly greater than the value of the concentration of the adsorbate in the finite bath, while, of course, the net molar flux of the charged adsorbate in the liquid film is equal to zero at equilibrium. This result is very different than that obtained from the conventional model that is currently used to describe the transport of a charged adsorbate in the liquid film for systems involving the adsorption of a charged adsorbate onto the charged surface of nonporous adsorbent particles; the conventional model (i) does not consider the existence of an electrical double layer, (ii) assumes that the transport of the charged adsorbate occurs only by diffusion in the liquid film, and (iii) causes at equilibrium the value of the charged adsorbate in the liquid layer adjacent to the surface of the particles to become equal to the value of the concentration of the charged adsorbate in the liquid of the finite bath. Furthermore, it was found that a maximum can occur in the dynamic behavior of the concentration of the adsorbate in the adsorbed phase when the value of the free molecular diffusion coefficient of the adsorbate is relatively large, because the increased magnitude of the synergistic interplay between the diffusive and electrophoretic molar fluxes of the adsorbate in the liquid film allows the adsorbate to accumulate (to be entrapped) in the liquid layer adjacent to the surface of the adsorbent particles faster than the concentrations of the electrolyte species, whose net molar fluxes are significantly hindered due to their opposing diffusive and electrophoretic molar fluxes, can adjust to account for the change in the surface charge density of the particles that arises from the adsorption of the charged adsorbate. The results presented in this work also have significant implications in finite bath adsorption systems involving the adsorption of a charged adsorbate onto the surface of the pores of charged porous adsorbent particles, because the diffusion and the electrophoretic migration of the charged solutes (cations, anions, and charged adsorbate) in the pores of the adsorbent particles will depend on the dynamic concentration profiles of the charged solutes in the liquid film surrounding the charged porous adsorbent particles. The results of the present work are also used to illustrate how the functioning of the electrical double layer could contribute to the development of inner radial humps (concentration rings) in the concentration of the adsorbate in the adsorbed phase of charged porous adsorbent particles.

Homing and engraftment defects in ex vivo expanded murine hematopoietic cells are associated with downregulation of beta1 integrin.

OBJECTIVE: Hematopoietic progenitors generated by ex vivo expansion "home" less efficiently to the bone marrow (BM) after intravenous transplantation than fresh cells. To explore the underlying cause of this transplantation defect, we examined the homing and engraftment properties in vivo of fresh and cultured marrow cells differing in beta1 integrin expression. MATERIALS AND METHODS: Fresh murine BM cells, or the expanded progeny of enriched Sca-1(+) c-kit(+)Lin(-) stem cells, were fractionated into beta1(-/lo) and beta1(+) subpopulations by cell sorting. These populations were assayed for their content of in vitro colony-forming cells (CFCs), cells able to provide radioprotection, and early and long-term multilineage hematopoietic reconstitution following transplantation into myeloablated recipients. These endpoints were correlated with the homing properties of beta1(-/lo) and beta1(+) cells that were labeled with 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) and tracked to hematopoietic organs 24 hours after injection into lethally irradiated mice. RESULTS: Most normal stem and progenitor cells express high levels of beta1 integrin. In contrast, most clonogenic cells generated in vitro are beta1(-/lo). Consequently, expanded beta1(-/lo) progenitors failed to provide radioprotection or repopulate the hematopoietic system following intravenous transplantation. Defective engraftment of expanded cells was associated with reduced homing of beta1(-/lo) cells to the bone marrow. CONCLUSION: Downregulation of beta1 integrin on primitive hematopoietic cells during ex vivo expansion reduces their homing efficiency and negatively impacts hematopoietic reconstitution in vivo. Strategies directed at preserving beta1 integrin expression during culture may improve the clinical utility of expanded hematopoietic cells.

Nuclear factor-kappaB is constitutively activated in primitive human acute myelogenous leukemia cells.

Human acute myelogenous leukemia (AML) is thought to arise from a rare population of malignant stem cells. Cells of this nature, herein referred to as leukemic stem cells (LSCs), have been documented for nearly all AML subtypes and appear to fulfill the criteria for stem cells in that they are self-renewing and give rise to the cells found in many leukemic populations. Because these cells are likely to be critical for the genesis and perpetuation of leukemic disease, the present studies sought to characterize unique molecular properties of the LSC population, with particular emphasis on the transcription factor, nuclear factor-kappaB (NF-kappaB). Previous experiments have shown that unstimulated human CD34(+) progenitor cells do not express NF-kappaB. In contrast, primary AML CD34(+) cells display readily detectable NF-kappaB activity as assessed by electrophoretic mobility shift assay and gene expression studies. Furthermore, detailed analyses of enriched AML stem cells (CD34(+)/CD38(-)/CD123(+)) indicate that NF-kappaB is also active in the LSC population. Given the expression of NF-kappaB in leukemic, but not normal primitive cells, the hypothesis that inhibition of NF-kappaB might induce leukemia-specific apoptosis was tested by treating primary cells with the proteasome inhibitor MG-132, a well-known inhibitor of NF-kappaB. Leukemic CD34(+)/CD38(-) cells displayed a rapid induction of cell death in response to MG-132, whereas normal CD34(+)/CD38(-) cells showed little if any effect. Taken together, these data indicate that primitive AML cells aberrantly express NF-kappaB and that the presence of this factor may provide unique opportunities to preferentially ablate LSCs.

Stable gene expression from a mammalian artificial chromosome.

We have investigated the potential of PAC-based vectors as a route to the incorporation of a gene in a mammalian artificial chromosome (MAC). Previously we demonstrated that a PAC (PAC7c5) containing alpha-satellite DNA generated mitotically stable MACs in human cells. To determine whether a functional HPRT gene could be assembled in a MAC, PAC7c5 was co-transfected with a second PAC containing a 140 kb human HPRT gene into HPRT-deficient HT1080 cells. Lines were isolated containing a MAC hybridizing with both alpha-satellite and HPRT probes. The MACs segregated efficiently, associated with kinetochore proteins and stably expressed HPRT message after 60 days without selection. Complementation of the parental HPRT deficiency was confirmed phenotypically by growth on HAT selection. These results suggest that MACs could be further developed for delivering a range of genomic copies of genes into cells and that stable transgene expression can be achieved.

Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver.

The rate of reconstitution following hematopoietic stem cell (HSC) transplantation differs widely depending on the tissue source of the cells infused. To test the hypothesis that variability in engraftment kinetics is related to differences in the efficiency with which intravenously transplanted HSCs "home" to the bone marrow (BM), the homing properties of murine fetal liver (FL), adult BM, and mobilized peripheral blood (MPB) cells were compared. Lethally irradiated mice transplanted with 2 x 10(6) FL, BM, or MPB cells exhibited sequentially slower recovery of circulating leukocytes and platelets that correlates with the progressively lower frequency of colony-forming cells (CFCs) in these tissues. However, differences in the rate and degree of early and long-term reconstitution were maintained even after infusing equal numbers of CFCs derived from FL, BM, and MPB. To compare the homing of progenitors from these tissues, cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. Three hours later, PKH26(+) cells were reisolated from the BM and spleen by fluorescence-activated cell sorting and assayed for in vitro CFCs. Despite the higher level of very late antigen (VLA)-2, VLA-4, and VLA-5 on Sca-1(+)c-kit(+) cells from FL compared to BM, 10-fold fewer FL CFCs homed to hematopoietic organs than those from BM. MPB cells homed slightly better, but still less efficiently than BM cells. Therefore, clonogenic cells from different tissues exhibit striking variations in homing efficiency that does not necessarily correlate with engraftment kinetics. Homing is likely counterbalanced by intrinsic differences in proliferative potential that ultimately determine the rate of hematopoietic reconstitution.