Partial purification and characterization of an actin-bundling protein, band 4.9, from human erythrocytes.

Band 4.9 (a 48,000-mol-wt polypeptide) has been partially purified from human erythrocyte membranes. In solution, band 4.9 polypeptides exist as trimers with an apparent molecular weight of 145,000 and a Stokes radius of 50 A. Electron microscopy shows that the protein is a three-lobed structure with a radius slightly greater than 50 A. When gel-filtered rabbit muscle actin is polymerized in the presence of band 4.9, actin bundles are generated that are similar in appearance to those induced by "vinculin" or fimbrin. The bundles appear brittle and when they are centrifuged small pieces of filaments break off and remain in the supernatant. At low band 4.9 to actin molar ratios (1:30), band 4.9 lowers the apparent steady-state low-shear falling ball viscosity by sequestering filaments into thin bundles; at higher ratios, the bundles become thicker and obstruct the ball's movement leading to an apparent increase in steady-state viscosity. Band 4.9 increases the length of the lag phase and decreases the rate of elongation during actin polymerization as measured by high-shear Ostwald viscometry or by the increase in the fluorescence of pyrene-labeled actin. Band 4.9 does not alter the critical actin monomer concentration. We hypothesize that band 4.9, together with actin, erythrocyte tropomyosin, and spectrin, forms structures in erythroid precursor cells analogous to those formed by fimbrin, actin, tropomyosin, and TW 260/240 in epithelial brush borders. During erythroid development and enucleation, the actin filaments may depolymerize up to the membrane, leaving a membrane skeleton with short stubs of actin bundled by band 4.9 and cross-linked by spectrin.

Prognostic value of FDG-PET scan imaging in lymphoma patients undergoing autologous stem cell transplantation.

We conducted a retrospective analysis of 50 lymphoma patients (Hodgkin's disease and non-Hodgkin's lymphoma) who had an 18F-fluoro-deoxyglucose positron emission tomography (FDG-PET) scan after at least two cycles of salvage chemotherapy and before autologous stem cell transplantation (ASCT) at our institution. The patients were categorized into FDG-PET negative (N = 32) and positive (N = 18) groups. The median follow-up after ASCT was 19 months (range: 3-59). In the FDG-PET-negative group, the median progression-free survival (PFS) was 19 months (range: 2-59) with 15 (54%) patients without progression at 12 months after ASCT. The median overall survival (OS) for this group was not reached. In the FDG-PET-positive group, the median PFS was 5 months (range: 1-19) with only one (7%) patient without progression at 12 months after ASCT. The median OS was 19 months (range: 1-34). In the FDG-PET-negative group, chemotherapy-resistant patients by CT-based criteria had a comparable outcome to those with chemotherapy-sensitive disease. A positive FDG-PET scan after salvage chemotherapy and prior ASCT indicates an extremely poor chance of durable response after ASCT.

The effect of endogenous proteases on the spectrin binding proteins of human erythrocytes.

We have demonstrated that in human erythrocyte ghosts endogenous proteolytic activity is responsible for the digestion of the spectrin binding proteins (bands 2.1 to 2.6). The pH optimum, cofactor requirements and inhibitor sensitivity have been established. Our results indicate that proteolysis of bands 2.1 to 2.6 and the formation of 3', a fragment containing an active spectrin binding site, can occur through two enzymatic pathways: a cascade of consecutive proteolytic cleavages of the spectrin binding proteins inhibited by phenylmethylsulfonyl fluoride or a Ca2+-stimulated, phenylmethylsulfonyl fluoride-insensitive, EDTA-inhibited cleavage of band 2.1 to band 2.3, followed by digestion to band 3' by phenylmethylsulfonyl fluoride-inhibitable enzymes. These findings may provide the techniques necessary to prevent proteolysis of the spectrin binding proteins during purification and reconstitution experiments and provide insight into how they are formed in vivo.

Long-term event-free survivors after high-dose therapy and autologous stem-cell transplantation for low-grade follicular lymphoma.

Although follicular lymphoma (FL) is generally responsive to conventional-dose chemotherapy, improved survival in patients with this disease has been difficult to demonstrate. High-dose chemo/radiotherapy followed by autologous stem-cell transplantation (ASCT) can improve response rates, although its effects on survival remain controversial. Between 1990 and 2003, we transplanted 49 patients with low-grade FL at our institution. Twenty-two patients (45%) had undergone histologic transformation at the time of ASCT. In all, 44 patients (90%) had relapsed disease and five patients (10%) were resistant to chemotherapy at the time of transplantation. After ASCT, 30 patients (61%) were in complete remission (CR). The median overall survival (OS) has not been reached, while the median event-free survival (EFS) is 2.4 years. At a median follow-up of 5.5 years (longest 12.4 years), a plateau has been reached with 56% of patients remaining alive, and 35% event-free. ASCT was well tolerated except for two (4%) treatment-related deaths. In multivariable analysis, CR after ASCT and age less than 60 years are the best predictors of EFS and OS. ASCT is thus a safe therapeutic approach in FL, resulting in long-term EFS and OS for some patients, even with transformed disease.

Acid destabilization of a triple-helical peptide model of the macrophage scavenger receptor.

Electrostatic interactions were studied in a triple-helical peptide, (POG)3PKGQKGEKG(POG)4, which contains a lysine-rich 9 residue sequence from the collagen-like domain of the macrophage scavenger receptor (MSR). This peptide adopts a stable triple-helical conformation only when the pH is higher than 4.5, corresponding to ionization of the Glu side chain. Modeling shows Glu forms ion pairs with one of the Lys residues, stabilizing the structure. Previously studied collagen-like peptides show relatively small contributions of electrostatic interactions to stability. The large magnitude of the pH mediated structural changes seen for this peptide suggests that specific placement of charged residues in the triple-helix conformation can generate strong electrostatic interactions.

The human immune response to red blood cell antigens as revealed by repertoire cloning.

A major goal of current immunologic research is to develop specific therapeutic strategies by which the enormous diversity in immune response can be enhanced, attenuated, or eliminated, depending on the particular disease process. For nearly a century, the human immune response to red blood cell antigens has served as a paradigm for understanding the pathophysiology of autoimmune disorders and alloimmune reactions to foreign cells and tissues. Recent developments in molecular biology have facilitated the expression of immune repertoires in the form of immunoglobulin Fab fragments on the surface of filamentous bacteriophage. Such approaches have provided powerful means for producing monoclonal antibodies for research, clinical, and therapeutic applications. Our laboratory has combined these techniques with novel cell-surface selection methods to isolate extraordinarily large arrays of human antibodies to the clinically relevant red blood cell Rh(D) antigen. Our results have provided a comprehensive genetic and serologic analysis of anit-Rh(D) antibodies within single alloimmunized individuals thereby offering new insights into the development of human immune repertoires.

Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically-activated cell sorting: applications in immunohematology.

A method is described for the isolation of filamentous phage-displayed human monoclonal antibodies directed at unpurifiable cell surface-expressed molecules. To optimize the capture of antigen-specific phage and minimize the binding of irrelevant phage antibodies, a simultaneous positive and negative selection strategy is employed. Cells bearing the antigen of interest are pre-coated with magnetic beads and diluted into an excess of unmodified antigen-negative cells. Following incubation of the cell admixture with a Fab/phage library, the antigen-positive cell population is retrieved using magnetically-activated cell sorting and antigen-specific Fab/phage are eluted and propagated in bacterial culture. Utilizing this protocol with magnetically-labeled Rh(D)-positive and excess unlabeled Rh(D)-negative human red blood cells and a Fab/phage library constructed from human peripheral blood lymphocytes, dozens of unique clinically-useful gamma 1 kappa and gamma 1 lambda anti-Rh(D) antibodies were isolated from a single alloimmunized individual. This cell-surface selection method is readily adaptable for use in other systems, such as for the identification of putative tumor-specific antigens and provides a rapid (< 1 month), high-yield approach for isolating self-replicative antibody reagents directed at novel or conformationally-dependent cell-surface epitopes.

Recombinant monoclonal antibody technology.

With the development of murine hybridoma technology over a quarter century ago, the ability to produce large quantities of well-characterized monoclonal antibody preparations revolutionized diagnostic and therapeutic medicine. For many applications in transfusion medicine, however, the production of serological reagents in mice has certain biological limitations relating to the difficulty in obtaining murine monoclonal antibodies specific for many human blood group antigens. Furthermore, for therapeutic purposes, the efficacy of murine-derived immunoglobulin preparations is limited by the induction of anti-mouse immune responses. Technical difficulties inherent in human hybridoma formation have led to novel molecular approaches that facilitate the isolation and production of human antibodies without the need for B-cell transformation, tissue culture, or even immunized individuals. These technologies, referred to as 'repertoire cloning' or 'Fab/phage display', involve the rapid cloning of immunoglobulin gene segments to create immune libraries from which antibodies with desired specificities can be selected. The use of such recombinant methods in transfusion medicine is anticipated to play an important role in the development and production of renewable supplies of low-cost reagents for diagnostic and therapeutic applications.

Section 5: Structural/genetic analysis of mAbs to blood group antigens. Coordinator's report.

The heavy and light chain immunoglobulin variable region nucleotide sequences for 219 mAbs to human red blood cells were collected from workshop participants, published reports, and Genbank. Information regarding antigen specificity, species of origin, method of cloning, and other relevant serological properties was correlated with the sequence data. Immunoglobulin sequences were analyzed to determine the heavy- and light-chain immunoglobulin genes used and the overall extent of somatic mutation from germline configuration. Approximately 50% of the sequences encoded antibodies with Rh(D) specificity with the remaining sequences encoding mAbs to other Rh-related antigens, antigens of the ABO, MNS, and Kell blood group systems, and several others. Surprisingly, no sequence data were available for mAbs with specificity for a number of common Rh antigens, common Kell antigens, or antigens of the Lewis, Kidd, or Duffy blood group systems. The majority of mAbs were of human origin but included a significant number of macaque mAbs, murine mAbs, and a small number of synthetically-designed recombinant antibodies. Both cellular (EBV-transformation, cell fusion) and molecular (phage display) approaches were used for antibody cloning. Analysis of certain groups of sequences demonstrated patterns of immunoglobulin gene restriction, repertoire shift, and somatic mutation. Analysis of other mAbs demonstrated the value of antibody sequence data for the design and production of novel reagents useful in blood group serology.

Second auto-SCT is safe and effective salvage therapy for relapsed multiple myeloma.

Therapeutic options for patients with multiple myeloma whose disease has relapsed after a prior auto-SCT include novel biologic therapies, traditional chemotherapy or a second transplant, with no clear standard of care. Few published studies address the safety and efficacy of a second auto-SCT for relapsed disease. We reviewed the Abramson Cancer Center experience with salvage auto-SCT for relapsed multiple myeloma. Forty-one patients had received a salvage auto-SCT at our institution; the median time between transplants was 37 months (range 3-91). The overall response rate in assessable patients was 55%, and treatment-related mortality was 7%. With a median follow-up time of 15 months, the median PFS was 8.5 months and the median overall survival (OS) was 20.7 months. In a multivariate analysis of OS, independent prognostic factors were >or=5 prior lines of therapy and time to progression after initial auto-SCT of

Isolation of an IgG anti-B from a human Fab-phage display library.

BACKGROUND: ABO incompatibility is a common cause for mild hemolysis in the newborn, ranging from 1 in 30 to 1 in 150 births. Fortunately, hemolysis requiring transfusion is rare and restricted to blood group O mothers, because blood group A and B individuals make poor IgG anti-B and anti-A responses. No human IgG ABO antibody sequences have been reported, in part because of the difficulty in obtaining human IgG hybridomas. Phage-display technology may be able to circumvent these difficulties, but its application to carbohydrate antigens is poorly studied. STUDY DESIGN AND METHODS: A human IgG1 phage-display Fab library was constructed from splenocytes derived from a nonhyperimmunized blood group O person, and panned against group B RBCs. RESULTS: After five rounds of panning, essentially all phage bound to group B RBCs. Nucleotide sequence analysis of a single monoclonal IgG1lambda phage, FB5.7, revealed a highly mutated VH4 family heavy chain, and a nearly germline VL7 family lambda light chain. The Fab agglutinated group B, but not group A, random-donor RBCs. However, group B ELISA reactivity could be inhibited by soluble B-trisaccharide, soluble A-trisaccharide, galactose, and N-acetyl galactosamine. Similarly, galactose and N-acetyl galactosamine were able to inhibit group B RBC agglutination. CONCLUSION: FB5.7 is the first human IgG ABO MoAb described. Alhough it behaves serologically like a group B-specific antibody, it demonstrates interaction with both the A and B epitopes. Phage-display technology can be used to better define the relationship between antibody genotype and phenotype in anti-carbohydrate responses in nonhyperimmunized hosts, and thus to improve our understanding of the composition of the antibody repertoire.

Genetic and immunological properties of phage-displayed human anti-Rh(D) antibodies: implications for Rh(D) epitope topology.

Understanding anti-Rh(D) antibodies on a molecular level would facilitate the genetic analysis of the human immune response to Rh(D), lead to the design of therapeutically useful reagents that modulate antibody binding, and provide relevant information regarding the structural organization of Rh(D) epitopes. Previously, we described a Fab/phage display-based method for producing a large array of anti-Rh(D) antibodies from the peripheral blood lymphocytes of a single alloimmunized donor. In the current study, we present a detailed analysis of 83 randomly selected clones. Sequence analysis showed the presence of 28 unique gamma1 heavy chain and 41 unique light chain gene segments. These paired to produce 53 unique Fabs that had specificity for at least half of the major Rh(D) epitopes. Surprisingly, despite this diversity, only 4 closely related heavy chain germline genes were used (VH3-30, VH3-30.3, VH3-33, and VH3-21). Similarly, nearly all Vkappa light chains (15/18) were derived from one germline gene (DPK9). lambda light chains showed a more diverse VL gene usage, but all (23/23) used the identical Jlambda2 gene. Several Fabs that differed in epitope specificity used identical heavy chains but different light chains. In particular, 2 such clones differed by only 3 residues, which resulted in a change from epD2 to epD3 specificity. These results suggest a model in which footprints of anti-Rh(D) antibodies are essentially identical to one another, and Rh(D) epitopes, as classically defined by panels of Rh(D) variant cells, are not discrete entities. Furthermore, these data imply that the epitope specificity of an anti-Rh(D) antibody can change during the course of somatic mutation. From a clinical perspective, this process, which we term epitope migration, has significance for the design of agents that modulate antibody production and for the creation of mimetics that block antibody binding in the settings of transfusion reactions and hemolytic disease of the newborn.

Expression and characterization of recombinant anti-Rh(D) antibodies on filamentous phage: a model system for isolating human red blood cell antibodies by repertoire cloning.

The production of human anti-red blood cell (RBC) Igs in vitro from immunized individuals would greatly facilitate the genetic analysis of the human immune response to RBC antigens and also provide useful serologic reagents. Technical difficulties inherent in human B-cell immortalization have led to the development of molecular approaches that bypass the need for cell transformation. By cloning human Ig gene segments into bacterial expression vectors, libraries are created of filamentous phage particles displaying Fab fragments on their surfaces. Libraries have been screened with purified, soluble antigen and selected clones genetically manipulated in Escherichia coli to produce soluble Fab fragments. Our goal has been to adapt this technique to the study of RBC autoantibodies and alloantibodies that have specificities against unpurifiable membrane-bound antigens. To test the feasibility of this approach, two sets of phage were created, one set expressing a human anti-Rh(D) Ig and the other expressing a human antitetanus toxoid Ig. After verifying the presence of functional phage-displayed Fabs through biochemical, flow cytometric, and electron microscopic analyses, a model library was constructed comprising one anti-Rh(D)-expressing phage per 10(4) antitetanus toxoid-expressing phage. A method was developed for screening the library with intact Rh(D)-positive RBCs. After four rounds of panning, anti-Rh(D) specificity was enriched more than 10,000-fold to a final frequency of approximately 100%. Plasmid DNA derived from anti-Rh(D) phage was used to produce milligram quantities of soluble recombinant anti-Rh(D) Fabs purified by nitrogen cavitation and nickel-chelation affinity chromatography. The authenticity of the Fabs was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, which showed bands with molecular weights of approximately 50 kD and 26 kD under nonreducing and reducing conditions, respectively. Binding of recombinant anti-Rh(D) Fabs to Rh(D)-positive RBCs was demonstrated by flow cytometry and by an agglutination assay. Our results suggest that repertoire cloning by cell-surface enrichment may have broad application to the study of the human immune response to erythroid antigens in addition to membrane-bound antigens present on other hematopoietic cells.

Inappropriate testing for diarrheal diseases in the hospital.

To assess the degree to which routine stool cultures, ova and parasite examinations, and Clostridium difficile toxin assays may be inappropriately ordered on hospitalized patients, we conducted a retrospective study to determine the relative yield of these tests on specimens collected from outpatients and inpatients as a function of time after admission. During a 3-year period, only 1 of 191 positive stool cultures and none of the 90 ova and parasite examinations with positive results were from the group of patients who had stool specimens submitted after 3 days of hospitalization. Analysis of laboratory work load for a 1-year period showed that specimens from this patient group contributed nearly 50% of the more than 3000 specimens received each year. In contrast, approximately 25% (range, 17% to 33%) of samples, regardless of admission status, were positive for C difficile toxin. Eliminating routine stool cultures and ova and parasite examinations on hospitalized patients would significantly reduce hospital and patient costs without altering patient care. Nationwide, such a policy might achieve a cost savings of +20 to +30 million per year.

Direct NMR measurement of folding kinetics of a trimeric peptide.

Direct NMR measurements of the folding kinetics are performed on a collagen-like triple helical peptide. The triple helical peptide was designed to model a biologically important region of collagen and has the sequence (POG)3ITGARGLAG(POG)4. Triple helical peptides were synthesized with specifically labeled 15N amino acid residues in key positions, and the kinetics of folding of the individual residues were monitored directly by measuring the loss of monomer intensity and the increase in trimer intensity. The residues at the terminal ends and central region could be followed independently and quantitated directly. Residues located at the terminal ends have rates and kinetics of folding that are distinct from residues in the central region of the peptide. This allows the monitoring of different steps in the folding mechanism and the postulation of the existence of a kinetic intermediate. The NMR data are consistent with a mechanism of association/nucleation and propagation. Hereditary connective tissue diseases are associated with mutations that result in abnormal folding of collagen, and the NMR folding experiments on a collagen-like peptide provide a basis for characterizing the molecular defect in folding mutations.