Evaluation of engraftment and immunological tolerance after reduced intensity conditioning in a rhesus hematopoietic stem cell gene therapy model.

Reduced intensity conditioning (RIC) is desirable for hematopoietic stem cell (HSC) targeted gene therapy; however, RIC may be insufficient for efficient engraftment and inducing immunological tolerance to transgenes. We previously established long-term gene marking in our rhesus macaque autologous HSC transplantation model following 10 Gy total body irradiation (TBI). In this study, we evaluated RIC transplantation with 4 Gy TBI in two rhesus macaques that received equal parts of CD34(+) cells transduced with green fluorescent protein (GFP)-expressing lentiviral vector and empty vector not expressing transgenes. In both animals, equivalently low gene marking between GFP and empty vectors was observed 6 months post-transplantation, even with efficient transduction of CD34(+) cells in vitro. Autologous lymphocyte infusion with GFP marking resulted in an increase of gene marking in lymphocytes in a control animal with GFP tolerance, but not in the two RIC-transplanted animals. In vitro assays revealed strong cellular and humoral immune responses to GFP protein in the two RIC-transplanted animals, but this was not observed in controls. In summary, 4 Gy TBI is insufficient to permit engraftment of genetically modified HSCs and induce immunological tolerance to transgenes. Our findings should help in the design of conditioning regimens in gene therapy trials.

Modeling tumor-host interactions of chronic lymphocytic leukemia in xenografted mice to study tumor biology and evaluate targeted therapy.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors for proliferation and survival. In particular, the B-cell receptor (BCR) and nuclear factor- κB (NF-κB) pathways are activated in the lymph node (LN) microenvironment. Thus, model systems mimicking tumor-host interactions are important tools to study CLL biology and pathogenesis. We investigated whether the recently established NOD/scid/γc(null) (NSG) mouse xenograft model can recapitulate the effects of the human microenvironment. We assessed, therefore, tumor characteristics previously defined in LN-resident CLL cells, including proliferation, and activation of the BCR and NF-κB pathways. We found that the murine spleen (SP) microenvironment supported CLL cell proliferation and activation to a similar degree than the human LN, including induction of BCR and NF-κB signaling in the xenografted cells. Next, we used this model to study ibrutinib, a Bruton's tyrosine kinase inhibitor in clinical development. Ibrutinib inhibited BCR and NF-κB signaling induced by the microenvironment, decreased proliferation, induced apoptosis and reduced the tumor burden in vivo. Thus, our data demonstrate that the SP of xenografted NSG mice can, in part, recapitulate the role of the human LN for CLL cells. In addition, we show that ibrutinib effectively disrupts tumor-host interactions essential for CLL cell proliferation and survival in vivo.

Sirolimus and post transplant Cy synergistically maintain mixed chimerism in a mismatched murine model.

Because of the toxicity associated with myeloablative conditioning, nonmyeloablative regimens are increasingly being used in vulnerable patient populations. For patients with sickle cell disease, stable mixed chimerism has proven sufficient to reverse the phenotype. Because the vast majority of patients do not have an HLA-matched sibling, a safe nonmyeloablative regimen that could be applied to the haploidentical setting would be ideal. We employed a mismatched mouse model using BALB/c donors and C57BL/6 recipients. Recipient mice were conditioned with 200 cGy TBI and sirolimus or CSA with or without post transplant Cy (PT-Cy). Our data show that when sirolimus or PT-Cy alone is given to C57BL/6 recipients, donor cells are not detected. However, when sirolimus is administered for 15 or 31 days starting 1 day before or up to 6 days after transplant with PT-Cy, all mice maintain stable mixed chimerism. In contrast, conventional therapy employing CSA with or without PT-Cy does not result in stable mixed chimerism. Lastly, mice with stable mixed chimerism after sirolimus display decreased reactivity to donor Ag both in vitro and in vivo. These data identify a novel strategy for inducing mixed chimerism for the treatment of nonmalignant hematologic diseases.

Optimal conditions for lentiviral transduction of engrafting human CD34+ cells.

Cytokines are required for γ-retroviral transduction of human CD34+ cells. However, cytokines may reduce engraftment of CD34+ cells and may not be necessary for their lentiviral transduction. We sought to optimize transduction and engraftment of human CD34+ cells using lentiviral vectors. Single 24 h transduction of human CD34+ cells with human immunodeficiency virus type 1 (HIV1)-based lentiviral vectors in media containing stem cell factor (SCF), FMS-like tyrosine kinase 3 (FLT3) ligand, thrombopoietin (each 100 ng ml⁻¹) and 10% fetal bovine serum was compared with various cytokine conditions during ex vivo culture and assayed using humanized xenograft mice for 6 months after transplantation. Serum-free media improved transduction efficiency of human CD34+ cells. Interleukin-3 (20 ng ml⁻¹) had little effect on transduction efficiency or engraftment. Threefold higher cytokine mixture (each 300 ng ml⁻¹) reduced engraftment of CD34+ cells. SCF alone (100 ng ml⁻¹) proved insufficient for maintaining engraftment ability and reduced transduction efficiency. Short-term prestimulation had little effect on transduction efficiency or engraftment, yet 24 h prestimulation showed higher transduction efficiency, higher gene expression levels and lower engraftment. In summary, 24 h prestimulation followed by single 24-h lentiviral transduction in serum-free media with SCF, FLT3 ligand and thrombopoietin yields high transduction efficiency to engrafting human CD34+ cells, and is applicable in human clinical gene therapy trials.

Analysis of large-volume DNA markers and polymerase chain reaction products by capillary electrophoresis in the presence of electroosmotic flow.

We have demonstrated on-line concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large-volumes DNA samples, PEO solutions entered a capillary filled with 400 mM Tris-borate (TB) buffers by EOF and acted as sieving matrices. DNA fragments stacked between the sample zone and PEO solutions. Because sample matrixes affected PEO adsorption on the capillary wall, leading to changes in EOF, migration time, concentration, and resolving power varied with the injection length. When injecting phiX174 RF DNA-HaeIII digest prepared in 5 mM Tris-HCl buffer, pH 7.0, at 250 V/cm, peak height increased linearly as a function of injection volume up to 0.9 microl (injection time 150 s). The sensitivity improvement was 100-fold compare to that injected at 25 V/cm for 10 s (0.006 microl). When injecting 1.54 microl of GeneScan 1000 ROX, the sensitivity improvement was 265-fold. The sensitivity improvement was 40-fold when injecting 0.17 microl DNA sample containing pBR 322/HaeIII, pBR 328/BglI, and pBR 328/HinfI digests prepared in phosphate-buffered saline. This method allows the analysis of polymerase chain reaction (PCR) products amplified after 17 cycles when injecting 0.32 microl (at 30 cm height for 300 s). The total analysis time was shorter (91.6 min) than that (119.6 min) obtained from injecting PCR products after 32 cycles for 10 s.

Optimizing separation conditions for polycyclic aromatic hydrocarbons in micellar electrokinetic chromatography.

We report the separation of polycyclic aromatic hydrocarbons (PAHs) using 0.1% poly(ethylene oxide) (PEO) in micellar electrokinetic chromatography (MEKC). In the presence of PEO, adsorption of PAHs on the capillary wall was reduced, leading to better resolution and reproducibility. Effects of tetrapentylammonium iodide (TPAI), dextran sulfate (DS), methanol, and sodium lauryl sulfate (SDS) on the separation of PAHs were elucidated. In terms of resolution and speed, DS, compared to TPAI, is a better additive for separation of PAHs. When using 0.1% PEO solution containing 45% methanol, 50 mM SDS, and 0.02% DS, separation of 10 PAHs containing 2 to 5 benzene rings was accomplished in less than 12 min at 15 kV in a commercial CE system. The method has also been tested for separating seven PAHs with high quantum yields when excited at 325 nm using a He-Cd laser. Unfortunately, separation of the seven PAHs was not achieved and sensitivity diminished under the same conditions. To optimize sensitivity, resolution and speed, a stepwise technique in MEKC has been proposed. The seven PAHs were resolved in 35 min at 15 kV when separation was performed in 0.1% PEO solution containing 35 mM SDS, 40% methanol and 0.02% DS for 2 min, and subsequently in 0.1% PEO solution containing 20 mM SDS, 50% methanol, and 0.02% DS.

Activation of APE/Ref-1 redox activity is mediated by reactive oxygen species and PKC phosphorylation.

Reactive oxygen species (ROS) arise through normal cellular aerobic respiration, and, in combination with external sources such as ionizing radiation, cigarette tar and smoke, and particulate matter generated by combustion, can have a profound negative effect on cellular macromolecules such as DNA that may lead to a number of human pathological disorders including accelerated aging and cancer. A major end product of ROS damage to DNA is the formation of apurinic/apyrimidinic (AP) sites, which without removal are known to halt mRNA and DNA synthesis, or act as non-coding lesions resulting in the increased generation of DNA mutations. In human cells, the major enzyme in correcting the deleterious effects of AP sites in DNA is through the participation of AP endonuclease (APE), which initiates the removal of baseless sites in DNA through the catalytic scission of the phosphodiester bond 5' and adjacent to an AP site. Interestingly, APE also possesses an activity (Ref-1) that controls the redox status of a number of transcription factors including Fos and Jun. The means by which APE/Ref-1 is directed to carry out such disparate roles are unknown. The presence of a number of phosphorylation sites scattered throughout both functional domains of APE/Ref-1 however offered one possible mechanism that we reasoned could play a role in dictating how this protein responds to different stimuli. Here we show that the in vitro redox activity of APE/Ref-1 is stimulated by PKC phosphorylation. Furthermore, when human cells were exposed to the PKC activator phorbol 12-myristate 13-acetate, an increase in redox activity was observed that corresponded to an increase in the phosphorylation status of APE/Ref-1. Importantly, human cells exposed to the oxidizing agent hypochlorite, followed by methyl methanesulfanate, responded with an increase in redox activity by APE/Ref-1 that also involved an increase in PKC activity and a corresponding increase in the phosphorylation of APE/Ref-1. These results suggest that the ability of APE/Ref-1 to perform its in vivo redox function is correlated to its susceptibility to PKC phosphorylation that notably occurs in response to DNA damaging agents.

Maximization of injection volumes for DNA analysis in capillary electrophoresis.

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow.

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.

On-column preconcentration and separation of DNA fragments using polymer solutions in the presence of electroosmotic flow.

We demonstrated DNA preconcentration and separation in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large volumes of DNA samples into a capillary filled with free tris(hydroxymethyl)aminomethane (Tris)-borate (TB) buffers, PEO solutions entered the capillary by EOF and acted as sieving matrices. In contrast to conventional methods (in the absence of EOF), controlling the EOF was also useful for resolution optimization. We have found that PEO adsorption on the capillary wall was more pronounced when low ionic strength buffers were used. Thus, the EOF decreased with increasing injection length, which led to longer migration times and changes in resolution and stacking efficiency. All resolution values were higher than 1.5 when 1.0 microg/mL DNA samples were injected at 240 V/cm for 60 s (0.67 microL). In addition, as low as 0.015 microg/mL DNA samples (an about 66-fold increase in sensitivity) were detected when the injection was performed at 250 V/cm for 60 s.

Characterization of luminal paneth cell alpha-defensins in mouse small intestine. Attenuated antimicrobial activities of peptides with truncated amino termini.

Paneth cells at the base of small intestinal crypts secrete apical granules that contain antimicrobial peptides including alpha-defensins, termed cryptdins. Using an antibody specific for mouse cryptdin-1, -2, -3, and -6, immunogold-localization studies demonstrated that cryptdins are constituents of mouse Paneth cell secretory granules. Several cryptdin peptides have been purified from rinses of adult mouse small intestine by gel filtration and reverse-phase high performance liquid chromatography. Their primary structures were determined by peptide sequencing, and their antimicrobial activities were compared with those of the corresponding tissue forms. The isolated luminal cryptdins included peptides identical to the tissue forms of cryptdin-2, -4, and -6 as well as variants of cryptdin-1, -4, and -6 that have N termini truncated by one or two residues. In assays of antimicrobial activity against Staphylococcus aureus, Escherichia coli, and the defensin-sensitive Salmonella typhimurium phoP(-) mutant, full-length cryptdins had the same in vitro antibacterial activities whether isolated from tissue or from the lumen. In contrast, the N-terminal-truncated (des-Leu), (des-Leu-Arg)-cryptdin-6, and (des-Gly)-cryptdin-4 peptides were markedly less active. The microbicidal activities of recombinant cryptdin-4 and (des-Gly)-cryptdin-4 peptides against E. coli, and S. typhimurium showed that the N-terminal Gly residue or the length of the cryptdin-4 N terminus are determinants of microbicidal activity. Innate immunity in the crypt lumen may be modulated by aminopeptidase modification of alpha-defensins after peptide secretion.

Dynamic modification of the capillary wall for electrophoretic separations of small ions.

Separations of small ions were carried out under nonequilibrated conditions using capillaries treated with NaOH, HCl, or tris(hydroxymethyl)aminomethane (Tris) prior to analysis. For separations of benzoic acid isomers or acids and amines under weakly acidic conditions, capillaries flushed with 0.1 M NaOH and subsequently with running buffers prior to analysis were used. Separations of six benzoic acid isomers were accomplished in 4 min in 1 mM phosphate buffers, pH 4.01, containing 2.5 mM hydroxypropyl-beta-cyclodextrin. Without additives, the separation of biological amines and acids were also achieved in 10 min at pH 4.01. Capillaries treated with 0.1 M HCl prior to analysis were tested in separations of six phenols in 5 mM Tris solutions at pH 7.0. As a result of small electrophoretic mobilities of phenols against a small electroosmotic flow, resolution was optimized. We also found that reproducibility was improved using capillaries treated with HCl. The relative standard deviations of migration mobility of phenols were less than 1%, which were smaller than those obtained using capillaries treated with 0.1 M NaOH or Tris.

Reductions in binding and functions of D2 dopamine receptors in the rat ventral striatum during amphetamine sensitization.

Rats receiving amphetamine (5 mg/kg, i.p. once daily) for 14 continuous days develop behavioral sensitization to a subsequent amphetamine challenge (1 mg/kg) at withdrawal days 8 to 10. The present study was aimed at investigating whether there are changes in binding or functions of striatal D2 dopamine receptors in amphetamine-sensitized rats. The results indicated that the Bmax value of D2 receptors in the ventral striatum decreased 40% and 52% 7 and 10 days after amphetamine withdrawal, respectively, without changes in their binding affinities (Kd). During this withdrawal period, the D(2/3) receptor agonist-induced (a) locomotor activation (bromocriptine, 5 mg/kg, i.p. or quinpirole, 1 mg/kg, i.p.) and (b) inhibition of forskolin-enhanced adenylyl cyclase activity (bromocriptine, 50 or 150 microM) in the ventral striatum were both suppressed as compared with saline controls. The decreases in D2 receptor function were unrelated to the coupled G-proteins, since none of the G alpha i-3, G alpha o or G alpha q in the ventral striatum exhibited quantitative differences between control and amphetamine sensitized rats. Collectively, these results demonstrate that intermittent amphetamine administration for a period of 14 days leads to diminished D2 receptor expression and functions in the ventral striatum at late withdrawal periods. The decrease of D2 receptors might reflect cellular mechanisms underlying the expression of amphetamine sensitization.

Bioavailability estimation by reversed-phase liquid chromatography: high bonding density C-18 phases for modeling biopartitioning processes.

There have been many attempts to estimate biological activity with either 1-octanol/water partition coefficients or chromatographic retention parameters. Bulk phases may not be appropriate, however, for modeling a partitioning process in an interphase such as biological membranes. Chromatographic stationary phases can be argued as having structure similar to a membrane because of chain organization; however, the density of the grafted stationary-phase chains in commercially available stationary phases is much too low to provide a suitable model. We have previously developed a new scheme for derivatizing silica surfaces that produces stationary phases of significantly higher chain density than traditional methods. Investigation of the molecular mechanism and thermodynamics of solute partitioning into the different phases has shown that densely bonded reversed-phase stationary phases mimic partitioning to a biomembrane better than does bulk-phase octanol. Here we report chromatographic retention for pesticides, PAHs, and barbiturates using a C-18 column with high alkyl chain density, and in all cases, correlations of log k'w with bioavailability are equivalent to or better than correlations of bioavailability with the octanol/water partition coefficient.

Treatment of failing glaucoma filtering cystic blebs with tissue plasminogen activator (tPA).

Tissue plasminogen activator (tPA) is a clot-specific fibrinolytic agent shown to be effective in decreasing postsurgical fibrin formation and enhancing filtration after trabeculectomy. To evaluate the efficacy of tPA on blebs showing clinical evidence of failure, a retrospective analysis was performed on 20 eyes of 20 glaucoma patients with failing or failed cystic filtering bleb treated with tPA intrableb and/or intracameral injection. The average pre-tPA intraocular pressure (IOP) was 18.0 +/- 7.2 mmHg and post-tPA was 12.9 +/- 6.2 mmHg. Fifteen of 20 eyes (75%) were able to maintain their IOP at or below target level, in the follow-up period of 3 to 7 months. Eight of the 15 eyes with successful outcome were free of glaucoma medication. Patients experienced no pain or irritation during and after the injection of tPA. No adverse effects on visual acuity or major complications were encountered. Transient mild complications related to tPA injections were subconjunctival hemorrhage (2 eyes), hyphema (1 eye) and hypotony (6 eyes). The revision of failing cystic bleb by tPA intrableb and/or intracameral injection may be considered as a useful adjunct with low risk in management of failing cystic blebs. A well designed clinical trial would provide needed information pertaining to the nature of cystic bleb formation as well as the efficacy, safety and clinical implication on the tPA treatment.

Mouse Paneth cell defensins: primary structures and antibacterial activities of numerous cryptdin isoforms.

Cryptdins are antimicrobial peptides of the defensin family that are produced by intestinal Paneth cells. mRNAs encoding 17 cryptdin isoforms have been characterized from a cDNA library generated from a single jejunal crypt. Six cryptdin cDNAs correspond to known peptides, and the remainder predict 11 novel Paneth cell defensins. Most cryptdin cDNAs have > or = 93% nucleotide sequence identity overall, except for cryptdin 4 and 5 cDNAs, whose respective mature peptide-encoding regions are only 74 and 78% identical to that of cryptdin 1. Cryptdin cDNAs differ at a small number of nucleotide positions: frequent substitutions were found in codons 38 and 52 of the propiece and in codons 68, 73, 76, 87, and 89 of the deduced peptides; cDNA clones with changes in codons 74, 83, and 88 were found, but there were fewer of these. The antimicrobial activities of cryptdins 1 to 6 were tested against Escherichia coli ML35 in two assays. In an agar diffusion assay, the potencies of cryptdins 1 to 3, 5, and 6 were approximately equivalent to that of rabbit neutrophil defensin NP-1 but cryptdin 4 was 30 times more active than NP-1. In a bactericidal assay system, cryptdins 1 and 3 to 6 were equally active at 10 micrograms/ml but cryptdin 2 and rabbit NP-1 were not active at this concentration. Since cryptdins 2 and 3 differ only at residue 10 (Thr and Lys, respectively), this amino acid appears to function in bactericidal interaction with E. coli. The demonstration that Paneth cells express a diverse population of microbicidal defensins further implicates cryptdins in restricting colonization or invasion of small intestinal epithelium by bacteria.