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Posttransplant relapse is still the major cause of treatment failure in high-risk acute leukemia. Attempts to manipulate alloreactive T cells to spare normal cells while killing leukemic cells have been unsuccessful. In HLA-haploidentical transplantation, we reported that donor-derived T regulatory cells (Tregs), coinfused with conventional T cells (Tcons), protected recipients against graft-versus-host disease (GVHD). The present phase 2 study investigated whether Treg-Tcon adoptive immunotherapy prevents posttransplant leukemia relapse. Forty-three adults with high-risk acute leukemia (acute myeloid leukemia 33; acute lymphoblastic leukemia 10) were conditioned with a total body irradiation-based regimen. Grafts included CD34(+) cells (mean 9.7 × 10(6)/kg), Tregs (mean 2.5 × 10(6)/kg), and Tcons (mean 1.1 × 10(6)/kg). No posttransplant immunosuppression was given. Ninety-five percent of patients achieved full-donor type engraftment and 15% developed ≥grade 2 acute GVHD. The probability of disease-free survival was 0.56 at a median follow-up of 46 months. The very low cumulative incidence of relapse (0.05) was significantly better than in historical controls. These results demonstrate the immunosuppressive potential of Tregs can be used to suppress GVHD without loss of the benefits of graft-versus-leukemia (GVL) activity. Humanized murine models provided insights into the mechanisms underlying separation of GVL from GVHD, suggesting the GVL effect is due to largely unopposed Tcon alloantigen recognition in bone marrow., (© 2014 by The American Society of Hematology.)

The separation of undesired product-related impurities often poses a challenge in the purification of protein therapeutic species. Product-related impurity species, which may consist of undesirable isoforms, aggregated, or misfolded variants of the desired monomeric form of the product, can be challenging to remove using preparatory scale chromatographic techniques. When using anion exchange chromatography to remove undesirable product-related impurities, the separation can be highly sensitive to relatively small changes in the chromatography operating conditions, including changes to buffer solution pH, buffer solution conductivity protein loading, and operating temperature. When performing difficult separations, slight changes to the chemical and physical properties of the anion exchange adsorbent lot may also impact the separation profile. Such lot-to-lot variability may not be readily measurable by the adsorbent manufacturer, since variability can be highly dependent on a specific protein separation. Consequently, manufacturers of chromatographic adsorbents may not be able to control adsorbent lot to lot variability tightly enough to prevent differences from occurring when performing difficult product-related separations at the preparatory scale. In such cases, it is desirable to design a chromatography step with a control strategy which accounts for adsorbent lot to lot variability in the separation performance. In order to avoid the undesired changes to process consistency and product quality, a proper adjustment of the column operating conditions can be implemented, based on the performance of each adsorbent lot or lot mixture. In this work, we describe how the adjustment of the column buffer solution composition can be used as a design space based-control strategy used to ensure consistent process performance and product quality are achieved for an anion exchange chromatography step susceptible to adsorbent lot to lot performance variability. In addition, a "use test" is described that can be employed to determine the optimal buffer solution compositions for different anion exchange adsorbent lots based on the retention volume of the therapeutic protein during a gradient elution., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The NPM1 mutation is the most frequent genetic alteration thus far identified in acute myeloid leukemia (AML). Despite progress in the clinical and biological characterization of NPM1-mutated AML, the role of NPM1 mutation in leukemogenesis in vivo has not been fully elucidated. We report a novel mouse model that conditionally expresses the most common human NPM1 mutation (type A) in the hematopoietic compartment. In Npm1-TCTG/WT;Cre(+) mice, the NPM1 mutant localized in the cytoplasm (NPMc(+)) of bone marrow (BM) cells. The mutant mice developed no AML after 1.5-year follow-up. However, NPMc(+) expression determined a significant platelet count reduction and an expansion of the megakaryocytic compartment in the BM and spleen. Serum thrombopoietin levels overlapped in mutant vs control mice, and BM cells from Npm1-TCTG/WT;Cre(+) mice formed more megakaryocytic colonies in vitro. Moreover, we demonstrated the up-regulation of microRNAs (miRNAs; miR-10a, miR-10b, and miR-20a) inhibiting megakaryocytic differentiation along with increased expression of HOXB genes. Notably, these findings mimic those of human NPM1-mutated AML, which also exhibits a similar miRNA profile and expansion of the megakaryocytic compartment. Our mouse model provides evidence that the NPM1 mutant affects megakaryocytic development, further expanding our knowledge of the role of NPM1 mutant in leukemogenesis.

Hairy cell leukemia (HCL) is a distinct clinicopathologic entity that responds well to purine analogs but is sometimes difficult to differentiate from HCL-like disorders (e.g., splenic marginal zone lymphoma and HCL variant). We recently identified the BRAF-V600E mutation as the disease-defining genetic event in HCL. In this study, we describe a new, simple, and inexpensive test for genetics-based diagnosis of HCL in whole-blood samples that detects BRAF-V600E through a sensitive allele-specific PCR qualitative assay followed by agarose-gel electrophoresis. This approach detected BRAF-V600E in all 123 leukemic HCL samples investigated containing as few as 0.1% leukemic cells. BRAF-V600E was detected at different time points during the disease course, even after therapy, pointing to its pivotal role in HCL pathogenesis and maintenance of the leukemic clone. Conversely, 115 non-HCL chronic B-cell neoplasms, including 79 HCL-like disorders, were invariably negative for BRAF-V600E. This molecular assay is a powerful tool for improving the diagnostic accuracy in HCL.

Hastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)-haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when co-infused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.

Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34(+) cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34(+) cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34(+) leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34(+) subfraction exhibiting the phenotype (CD34(+)/CD38(-)/CD123(+)/CD33(+)/CD90(-)) of leukemic stem cells. When transplanted into immunocompromised mice, CD34(+) cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34(+) fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34(-) cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Ceramic hydroxyapatite (CHT) chromatography offers unique selectivity for protein purification. However, columns composed of CHT, a crystalline form of calcium phosphate, often suffer from short column lifetimes, particularly under acidic operating conditions. In this paper, CHT was used under slightly acidic conditions (pH 6) for the production scale purification of a recombinant protein. Under these conditions, the packing quality of production scale CHT columns (45 cm diameter) degraded after 5-10 cycles of operation. This was not reproduced using a conventional scale-down chromatography model, in which a constant column bed height was maintained across scales. Thus, an alternative scale-down model was developed to better predict the lifetime of large scale CHT columns. The alternative approach, which utilized a constant column diameter-to-height aspect ratio, was able to predict column failure that approximated that of the manufacturing scale column. The alternative scale-down approach was then used to test alternate buffer formulations that significantly improved the CHT column lifetime. Screening studies, which assessed the effects of mobile phase pH and composition on the dissolution (weight loss) of CHT, were used to identify the alternative mobile phase formulations. Results from the study showed that slight changes to the existing mobile phase compositions significantly increased the column lifetime, from approximately 10 cycles to approximately 65 cycles of use, without altering the purification of the recombinant protein. The alternative scale-down model, together with relatively rapid mobile phase screening studies, provides a practical approach for predicting and optimizing the useful lifetime of CHT columns for large scale applications.

A two-dimensional model was formulated to describe the pressure-flow behavior of compressible stationary phases for protein chromatography at different temperatures and column scales. The model was based on the assumption of elastic deformation of the solid phase and steady-state Darcy flow. Using a single fitted value for the empirical modulus parameters, the model was applied to describe the pressure-flow behavior of several adsorbents packed using both fluid flow and mechanical compression. Simulations were in agreement with experimental data and accurately predicted the pressure-flow and compression behavior of three adsorbents over a range of column scales and operating temperatures. Use of the described theoretical model potentially improves the accuracy of the column scale-up process, allowing the use of limited laboratory scale data to predict column performance in large scale applications.

6-O-[(2-Hydroxyethyl)poly(2-oxyethyl)]chitosan ("glycolchitosan") was oxidatively cleaved with nitrous acid and then partly acetylated with acetic anhydride, reacted with bromoacetyl-N-hydroxysuccinimide, and reacted further with acetic anhydride. Conditions were selected, including fractionation by size-exclusion chromatography, so that the resulting "Chitin Leash" had an estimated, average molecular weight of 10,000 (dextran standards), corresponding to a length of approximately 40 sugar residues. It possessed 0.9 terminal aldehyde and 2.6 random (presumably) side-chain bromoacetyl reactive groups per chain (average values). As a model system, the Chitin Leash was used to crosslink staphylococcal nuclease (SNase) to ribonuclease A (RNase) with retention of 75 and 78%, respectively, of the starting enzyme activities. For this coupling, the Nase was first converted to a sulfhydryl SNase derivative which retained 74% of the activity of starting enzyme. The yields in this synthesis were: 13% Chitin Leash from glycolchitosan, 24% Chitin Leash-RNase from Chitin Leash and 45% SNase-Chitin Leash-RNase from the latter conjugate. The ratio of SNase to RNase in this conjugate was 1.0:0.94. In a second preparation, in which [14C]acetic anhydride was used, a longer reaction time was employed for the coupling of Chitin Leash to RNase. This gave a 1.0:1.8:0.95 molar ratio of Nase: [14C]Chitin Leash: RNase, revealing multiple attachment of the [14C]Chitin Leash to RNase. The activity of the RNase in the final conjugate was 20%. The latter conjugate was approximately 70% hydrolyzed by diaminooctyl-succinyl-lysozyme, disconnecting the two enzymes while not affecting their activities.

A method has been developed in which the DNA of leukocytes (as the buffy coat from blood) is isolated in the form of its constituent deoxynucleotides. The steps in this method are as follows: (1) lyse the leukocytes with sodium dodecyl sulfate (SDS) and enzymatically digest the proteins and RNA, (2) remove the SDS on a non-polar adsorbent (Bio-Beads SM-4) and then trap the DNA on a quaternary amine silica cartridge, (3) wash the column with 1 M NaCl-buffer, (4) digest the DNA on the column with staphylococcal nuclease and (5) elute the digested DNA with 0.5 M NaCl-buffer and digest it further with bovine spleen phosphodiesterase II to deoxynucleotide-3'-monophosphates. From a 40-microliters sample of butty coat was obtained 126 +/- 14 micrograms (two experiments, eight sample total) of deoxynucleotides. Reversed-phase high-performance liquid chromatography, which removed the added enzymes, showed only peaks for deoxynucleotides. For comparison, the amount of deoxynucleotides obtained from the leukocytes by an automated phenol extraction procedure was 101 +/- 5.4 micrograms (one experiment in triplicate).

Both the active ester and maleimide moieties of the cross-linking reagent, N-[(gamma-maleimidobutyryl)oxy]succinimide (GMBS), were found to react with the primary amino groups on ribonuclease (RNase). This largely inactivated RNase towards a polymeric (but not monomeric) substrate. Citraconylating the RNase first, so that essentially only a single primary amino group remained to react with GMBS, overcame this problem. The subsequent maleimido-citraconyl-RNase was used to prepare a 1:1.1 M conjugate of anti-T-2 toxin Fab' and RNase (Fab'-RNase) in a 76% yield. The conjugate was used to detect as little as 0.1 microgram of T-2 toxin based on the ability of T-2 toxin to specifically displace Fab'-RNase complexed to a T-2 agarose affinity gel.

Fluorescence post-labeling of nucleotides is a potentially useful technique for the detection of trace amounts of damaged DNA (DNA adducts). Towards this goal, we have studied a derivatization procedure starting with the four major 5'-deoxynucleotides as model compounds. The 5'-phosphate group was first labeled with ethylenediamine via a phosphorimidazolide intermediate in methanol with an organic-soluble carbodiimide. The resulting ethylenediaminephosphoramidate products were reacted in turn with fluorescein isothiocyanate. The reaction sequence has been characterised at all stages by high-performance liquid chromatography.

Since catechol estrogens are potent competitive inhibitors of catechol-O-methyl transferase (COMT), it has been suggested that they may prolong the half-life of catecholamines which in turn can cause hypertension. Thus, experiments were carried out to study the effect of catechol estrogens on blood pressure in the male rat following chronic administration. Results demonstrate that 2-hydroxyesterone (2,3-dihydroxyestra-1,3,5(10)-trien-17-one) and 2-hydroxy-estradiol (estra-1,3,5(10)-triene-2,3,17 beta-triol) even when administered in high doses do not alter blood pressure.

A radioimmunoassay method for urinary catechol estrogens is described; The specific nature of the antisera allows direct analyses of acid hydrolyzed urine. A LH-20 Sephadex column chromatography can be employed for individual determinations of 2-hydroxyestrone and 2-hydroxyestradiol. The excretion of catechol estrogens during menstrual cycles ranged from 14.48 to 50.15 microgram per 24 hours, whereas, during the last trimester of pregnancies, the values ranged from 129.30 to 1758. 20 microgram per 24 hours.

A monoclonal antibody for T-2 toxin is converted to a Fab'-fluorescein derivative. The latter is specifically complexed onto a T-2 agarose gel. Fifteen successive doses of T-2 ranging from 1 to 50 ng are then repetitively and linearly detected using a column packed with a small volume (0.2 ml) of this gel without recharging with Fab'-fluorescein. For these assays the effluent from the column is monitored with a spectrofluorometer.

Transamination conjugates of cytidine-3'-phosphate and polycytidylic acid (poly C) with diaminopropane, diaminohexane and diaminooctane (DAO) are formed both at 25 degrees C and 60 degrees C. The extent of reaction and formation of side products, with intermittent hydrolysis to mononucleotides in the case of aminoalkyl-poly C, is monitored by reversed-phase high-performance liquid chromatography. Both Dns-DAO-poly C and succinyl-lysozyme-DAO-poly C covalent conjugates are then prepared and similarly characterized, including separation on a size-exclusion diol high-performance liquid chromatography column. The retention of the latter on a wide-pore reversed-phase column seems to be controlled by the protein moiety.

A stable T-2 hydrazide gel is prepared by activating T-2 toxin with tresyl chloride followed by coupling to agarose-adipic acid hydrazide. Utilized as an affinity chromatography column, this T-2 hydrazide gel purifies a monoclonal antibody for T-2 in high yield directly from ascites fluid. Specific antibody trapped on the column is eluted either with excess T-2 or at pH 11.6. Much less successful are two other T-2 affinity columns that were prepared and evaluated: T-2 bovine serum albumin Affi-Gel 15 and T-2 hexylamine Sepharose.

DNA was subjected to bisulfite-catalyzed transamination at the N4 sites of its cytosine residues with 1,8-diaminooctane (DAO). The product, DNA-DAO, was non-specifically degraded with a cloned staphylococcal nuclease (Nase). The products from the Nase digestion were determined by high-performance liquid chromatography (HPLC) to define the extent of reaction with DAO. Mostly, nucleoside 3'-monophosphates were obtained, along with four Nase-resistant dinucleotides: TpdGp, dApdGp, TpdCp-DAO and dApdCp-DAO. The addition of spleen phosphodiesterase II gave a faster hydrolysis and left no dinucleotides. A mixture of Nase, snake venom phosphodiesterase I and alkaline phosphatase gave a fast hydrolysis as well but two dinucleotides, apparently TpdC-DAO and dApdC-DAO, persisted. Further modification of the diaminooctyl side chains with fluorescein isothiocyanate or biotin N-hydroxysuccinimide ester was similarly investigated. Interestingly, derivatization of the DAO side chain with biotin eliminates the resistance of TpdCp-DAO and dApdCp-DAO to Nase digestion. This work provides some guidelines for using Nase, alone or with other nucleases, along with HPLC, for characterizing alkyldiamine DNA products, and should be similarly useful for studying other modifications of DNA.