bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of VH Genes in Follicular and Diffuse Lymphoma Shows Ongoing Somatic Mutation and Multiple Isotype Transcripts in Early Disease With Changes During Disease Progression
Authors: Ottensmeier Christian H. , Thompsett Andrew R. , Zhu Delin , Wilkins Bridget S. , Sweetenham John W. , Stevenson Freda K.
Published: October 2019

Investigations of VH gene mutational patterns in B-cell tumors are often performed at an arbitrary time point of disease. To assess the effects of disease progression, tumor-derived VHgenes have been monitored from presentation through treatment and relapse in one patient with follicle center lymphoma (FCL), and two patients with primary diffuse large B-cell lymphoma (DLCL). The patient with FCL and one patient with DLCL both achieved clinical remission, although this was only partial in the FCL. However, both subsequently relapsed, and the second patient with DLCL was refractory to radiotherapy and chemotherapy. In each case, the tumor-derived VH sequence was identified, and the CDR3 “clonal signature” was used to track tumor cell sequences in subsequent biopsies. All cases showed somatic mutations, with intraclonal heterogeneity evident at presentation, and some sequences were aberrant. The VH sequences of the DLCL which responded to treatment became homogeneous at relapse. The sequences of both the FCL and the refractory DLCL remained heterogeneous. In all cases, transcripts of multiple Ig isotypes could be identified, and there was immunophenotypic evidence for expression of several Ig isotypes. The case of refractory DLCL had identifiable transcripts from IgM, IgD, IgA, IgG, and IgE, but appeared to lose the ability to produce alternative isotype transcripts and protein at the late stage of disease. These cases indicate that VH gene analysis can be used to probe tumor cell behavior in cases of lymphoma and that perturbations caused by therapy and disease progression can occur.
p53 Expression in B-Cell Chronic Lymphocytic Leukemia: A Marker of Disease Progression and Poor Prognosis
Authors: Cordone Iole , Masi Serena , Mauro Francesca Romana , Soddu Silvia , Morsilli Ornella , Valentini Tiziana , Vegna Maria Luce , Guglielmi Cesare , Mancini Francesca , Giuliacci Sonia , Sacchi Ada , Mandelli Franco , Foa Robert
Published: October 2019

We have analyzed by immunocytochemistry (ICC) the frequency of p53 protein expression in 181 cases of B-cell chronic lymphocytic leukemia (CLL) followed at a single institution to assess the relationship between p53 and the clinical and morphological features of the disease, as well as the possible involvement of this protein in the pathogenesis of the more aggressive forms of CLL. The overall frequency of p53 protein positivity in CLL was 15% (27 of 181 cases). There were no significant differences in age, sex, absolute lymphocyte count, or lymphocyte doubling time between p53-positive and -negative patients. By contrast, p53-positive patients had a significantly higher percentage of prolymphocytes (P = .002) and a significantly lower percentage of residual CD3-positive T lymphocytes (P = .0001). No correlation was found between the percentage of p53-positive cells and the percentage of cells in cycle assessed by the monoclonal antibody Ki-67. When the percentage of p53 positivity was correlated with the clinical stage of the disease, the proportion of p53-positive cases increased significantly from Binet's stage A (8 of 108; 7.4%), to stage B (12 of 49; 24.4%) and C (7 of 24; 29.2%) (P = .002). p53 positivity correlated also with the phase of the disease, showing a low expression at diagnosis (8 of 112; 7.1%) and a significantly higher expression in patients studied during the course of the disease (7 of 35; 20%) and, to a further extent, with disease progression (12 of 34; 35.3%) (P = .0001). The association of p53 protein expression with mutations in the gene was confirmed by direct sequence of the entire cDNA in 15 of the 17 ICC positive cases tested (88%). A significantly shorter treatment-free interval from diagnosis (P = .003) and a poorer response to therapy (P = .007) was observed in p53-positive compared with p53-negative patients. Overall survival from the time of diagnosis, as well as from the time of p53 protein analysis, was significantly shorter in patients with p53 protein expression (P = .03 and .0001, respectively). Moreover, in multivariate analysis, p53 expression and stage C were independently associated with a short survival. The results of this study indicate that in CLL the expression of the p53 protein, analyzed by a simple and reliable immunocytochemical method, is strongly associated with p53 gene mutations, a morphological variant (CLL with >10% prolymphocytes), advanced clinical stage, progressive disease, poor response to therapy, and short survival.
Collection, Tumor Contamination, and Engraftment Kinetics of Highly Purified Hematopoietic Progenitor Cells to Support High Dose Therapy in Multiple Myeloma
Authors: Tricot G. , Gazitt Y. , Leemhuis T. , Jagannath S. , Desikan K.R. , Siegel D. , Fassas A. , Tindle S. , Nelson J. , Juttner C. , Tsukamoto A. , Hallagan J. , Atkinson K. , Reading C. , Hoffman R. , Barlogie B.
Published: October 2019

Unfractionated peripheral blood stem cell (PBSC) grafts contain measurable quantities of myeloma cells and are therefore a potential source of relapse posttransplantation. In contrast, fluorescence-activated cell sorting (FACS)-sorted CD34+Thy1+ Lin− peripheral blood cells are substantially enriched for stem cell activity, yet contain virtually no clonal myeloma cells. A study was performed in patients with symptomatic myeloma, who had received 12 months or less of preceding standard chemotherapy, to evaluate the feasibility of large scale purification of primitive hematopoietic stem cells in order to study engraftment kinetics posttransplantation and the degree of tumor cell contamination of this cell population, based on polymerase chain reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF). A combination of elutriation and chemical lysis was used to deplete PBSC collections of monocytes, granulocytes, erythrocytes, and platelets. Subsequently, CD34+ Thy1+ Lin−progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of ≥7.2 × 105 cells/kg to support tandem transplants. After high dose melphalan (200 mg/m2) eight engrafted successfully, although granulocyte (absolute neutrophil count [ANC] >0.5 × 109/L, 16 days) and platelet recovery (platelets > 50 × 109/L, 39 days) was substantially delayed when compared with unmanipulated PBSC grafts; one patient required infusion of a reserve graft because of lack of evidence of engraftment by day +28. Three patients proceeded to a second graft with high dose melphalan and total body irradiation; two required infusion of a reserve graft and both died of infectious complications; one showed delayed, but complete, engraftment after this myeloablative regimen. Two of the nine evaluable patients attained a clinical complete remission (CR). The grafts from three patients were tested for tumor contamination and contained no detectable clonal myeloma cells. Larger quantities of purified cells may be required to resolve the problem of delayed engraftment.
Ex Vivo Culture of CD34+/Lin−/DR− Cells in Stroma-Derived Soluble Factors, Interleukin-3, and Macrophage Inflammatory Protein-1α Maintains Not Only Myeloid But Also Lymphoid Progenitors in a Novel Switch Culture Assay
Authors: Miller Jeffrey S. , McCullar Valarie , Verfaillie Catherine M.
Published: October 2019

We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1α (MIP-1α). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin−/DR−cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin−/DR− cells assayed in SNC cultures supplemented with IL-3 and MIP-1α. When CD34+/Lin−/DR− progeny from the SNC culture were plated sequentially into “NK cell progenitor switch” conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by “NK cell differentiation” conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3− NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1α directly to “NK cell differentiation” conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33− cells present after SNC cultures with IL-3 and MIP-1α, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33− population similar to fresh sorted CD34+/Lin−/DR− cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1α, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.
The Chemokine Receptor CXCR-4 Is Expressed on CD34+Hematopoietic Progenitors and Leukemic Cells and Mediates Transendothelial Migration Induced by Stromal Cell-Derived Factor-1
Authors: Möhle Robert , Bautz Frank , Rafii Shahin , Moore Malcolm A.S. , Brugger Wolfram , Kanz Lothar
Published: October 2019

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 (fusin, LESTR) are likely to be involved in the trafficking of hematopoietic progenitor and stem cells, as suggested by the reduced bone marrow hematopoiesis in SDF-1–deficient mice and the chemotactic effect of SDF-1 on CD34+ progenitor cells. Migration of leukemic cells might also depend on the expression of chemokine receptors. Therefore, we analyzed expression of CXCR-4 on mobilized normal CD34+ progenitors and leukemic cells. In addition, SDF-1–induced transendothelial migration across a bone marrow endothelial cell layer was assessed in vitro. By flow cytometry, CXCR-4 was found to be expressed in significant amounts on circulating CD34+ hematopoietic progenitor cells, including more primitive subsets (CD34+/CD38− and CD34+/Thy-1+ cells). In accordance with the immunofluorescence data, CD34+ progenitors efficiently migrated across endothelium in response to SDF-1 containing conditioned medium from the stromal cell line MS-5. Leukemic blasts (mostly CD34+) from patients with acute myeloblastic leukemia (AML) expressed variable amounts of CXCR-4, which was functionally active, as demonstrated by a positive correlation between the SDF-1–induced transendothelial migration and the cell surface density of CXCR-4 (r = 0.97). Also recombinant SDF-1β induced migration of CXCR-4–positive leukemic blasts. The effect of both conditioned medium and recombinant SDF-1 was inhibited by a CXCR-4 blocking antibody. In contrast, CD34+ leukemic cell lines (KG1, KG1a, Kasumi-1, MOLM-1) expressed low levels or were negative for CXCR-4, and did not migrate. By reverse transcriptase-polymerase chain reaction (RT-PCR), however, basal levels of CXCR-4 mRNA were also detected in all leukemic cell lines. We conclude that CXCR-4 is expressed on CD34+cells including more primitive, pluripotent progenitors, and may therefore play a role in the homing of hematopoietic stem cells. CXCR-4 expressed in variable amounts on primary AML leukemic cells is functionally active and may be involved in the trafficking of malignant hematopoietic cells.
The Structure and Function of Murine Factor V and Its Inactivation by Protein C
Authors: Yang Tony L. , Cui Jisong , Rehumtulla Alnawaz , Yang Angela , Moussalli Micheline , Kaufman Randal J. , Ginsburg David
Published: October 2019

Factor V (FV) is a central regulator of hemostasis, serving both as a critical cofactor for the prothrombinase activity of factor Xa and the target for proteolytic inactivation by the anticoagulant, activated protein C (APC). To examine the evolutionary conservation of FV procoagulant activity and functional inactivation by APC, we cloned and sequenced the coding region of murine FV cDNA and generated recombinant wild-type and mutant murine FV proteins. The murine FV cDNA encodes a 2,183-amino acid protein. Sequence comparison shows that the A1-A3 and C1-C2 domains of FV are highly conserved, demonstrating greater than 84% sequence identity between murine and human, and 60% overall amino acid identity among human, bovine, and murine FV sequences. In contrast, only 35% identity among all three species is observed for the poorly conserved B domain. The arginines at all thrombin cleavage sites and the R305 and R504 APC cleavage sites (corresponding to amino acid residues R306 and R506 in human FV) are invariant in all three species. Point mutants were generated to substitute glutamine at R305, R504, or both (R305/R504). Wild-type and all three mutant FV recombinant proteins show equivalent FV procoagulant activity. Single mutations at R305 or R504 result in partial resistance of FV to APC inactivation, whereas recombinant murine FV carrying both mutations (R305Q/R504Q) is nearly completely APC resistant. Thus, the structure and function of FV and its interaction with APC are highly conserved across mammalian species.
Adeno-Associated Viral Vector-Mediated Gene Transfer of Human Blood Coagulation Factor IX Into Mouse Liver
Authors: Nakai Hiroyuki , Herzog Roland W. , Hagstrom J. Nathan , Walter Johannes , Kung Szu-Hao , Yang Edmund Y. , Tai Shing Jen , Iwaki Yuichi , Kurtzman Gary J. , Fisher Krishna J. , Colosi Peter , Couto Linda B. , High Katherine A.
Published: October 2019

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (1012 to 1013 particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1α promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 × 1010 particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1α-F.IX of 2.7 × 1011particles resulted in plasma levels of 700 to 3,200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1α-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.
Parathyroid Hormone-Related Protein-Induced Hypercalcemia in SCID Mice Engrafted With Adult T-Cell Leukemia Cells
Authors: Takaori-Kondo Akifumi , Imada Kazunori , Yamamoto Itsuo , Kunitomi Akane , Numata Yasuharu , Sawada Hitoshi , Uchiyama Takashi
Published: October 2019

Parathyroid hormone-related protein (PTHrP) is considered to be one of the main causes of hypercalcemia associated with adult T-cell leukemia (ATL). To clarify the role of PTHrP and bone remodeling in the development of hypercalcemia in ATL, we examined the SCID mouse model of ATL that has previously been shown to mimic the disease in humans. Using this model, we found clear elevations in serum levels of calcium and C-terminal PTHrP (C-PTHrP). PTHrP mRNA was highly expressed in ATL cells proliferating in vivo. After the development of hypercalcemia, ATL mice were killed and bone histomorphometric analysis was performed. Bone volume was clearly decreased in the ATL mice. In comparison to control SCID mice, bone formation indices were very low in the ATL mice. Surprisingly, no significant difference was detected between the ATL mice and the control SCID mice in eroded surface/bone surface (ES/BS), a parameter of bone resorption. To our knowledge, the model presented here is the first animal model of ATL with humoral hypercalcemia. This is in contrast to previously reported, well-characterized animal models of human solid tumors associated with humoral hypercalcemia of malignancy (HHM). Furthermore, this model not only provides us with the opportunity to study the mechanisms underlying development of elevated calcium levels in ATL, but also allows us to test new therapeutic agents designed to treat hypercalcemia.
The Therapeutic Potential of Ribozymes
Authors: James Helen A. , Gibson Ian
Published: October 2019

Ribozymes are catalytic RNA molecules that recognize their target RNA in a highly sequence-specific manner. They can therefore be used to inhibit deleterious gene expression (by cleavage of the target mRNA) or even repair mutant cellular RNAs. Targets such as the mRNAs of oncogenes (resulting from base mutations or chromosome translocations, eg, ras or bcr-abl) and viral genomes and transcripts (human immunodeficiency virus–type 1 [HIV-1]) are ideal targets for such sequence-specific agents. The aim of this review is therefore to introduce the different classes of ribozymes, highlighting some of the chemistry of the reactions they catalyze, to address the specific inhibition of genes by ribozymes, the problems yet to be resolved, and how new developments in the field give hope to the future for ribozymes in the therapeutic field.
Atherogenic Lipoproteins Support Assembly of the Prothrombinase Complex and Thrombin Generation: Modulation by Oxidation and Vitamin E
Authors: Rota Simin , McWilliam Nicola A. , Baglin Trevor P. , Byrne Christopher D.
Published: October 2019

The importance of lipoproteins in the etiology of atherosclerosis is well established. Evidence is now accumulating to implicate thrombin in the pathogenesis of atherosclerosis. We have investigated whether atherogenic lipoproteins can support thrombin generation. In the absence of platelets or endothelial cells, both very low-density lipoprotein (VLDL) and oxidized low-density lipoprotein (LDL) support assembly of the prothrombinase complex and generation of thrombin. Thrombin generation (per μg of apolipoprotein) supported by VLDL was 19.4-fold greater than that supported by high-density lipoprotein (HDL), P < .00001, and 11.7-fold greater than that supported by LDL, P < .00001. Oxidation of LDL increased lipoprotein-supported thrombin generation 12-fold compared to unmodified LDL, P < .0001. We have shown that the phenomenon of lipoprotein-supported thrombin generation is mediated predominantly by specific phospholipids and is enhanced by oxidation of these phospholipids. The addition of vitamin E (α-tocopherol) markedly reduced the increase in thrombin generation observed after oxidation of LDL (822 ± 57 v 138 ± 47 nmol/L;P < .0001). These effects suggest that lipoproteins are important in the production of thrombin and that vitamin E may confer protection from the detrimental effects of lipoprotein oxidation by limiting thrombin formation. These results suggest that atherogenic lipoproteins are linked to the development of atherosclerosis in part by their capacity to support thrombin generation.
Interferon-α Resistance in a Cutaneous T-Cell Lymphoma Cell Line Is Associated With Lack of STAT1 Expression
Authors: Sun Wenn H. , Pabon Carlos , Alsayed Yazan , Huang Paul P. , Jandeska Sara , Uddin Shahab , Platanias Leonidas C. , Rosen Steven T.
Published: October 2019

Interferon-alpha (IFNα) mediates its biological effects through activation of the JAK-STAT signaling pathway and it has been shown to be one of most effective therapeutic agents for a number of hematological malignancies, including cutaneous T-cell lymphoma (CTCL). Nevertheless, its efficacy is limited by the development of clinical resistance but the reasons for resistance in CTCL are unknown. Here, we report the development of an IFNα-resistant CTCL cell line (HUT78R), characterized by its ability to proliferate in high concentration of recombinant IFNα, which can be used as a model system to study IFN resistance. The levels of IFN receptor expression and binding affinity were found to be comparable between the parental sensitive (HUT78S) and resistant (HUT78R) cells. However, IFNα stimulation failed to induce interferon-stimulated gene factor 3 (ISGF3) complex formation in HUT78R cells. In addition, the expression of the IFN-inducible 2-5 OAS gene was significantly reduced in HUT78R cells, suggesting the presence of a defect in the Jak-STAT signaling pathway. Our results showed that the IFNα-activated form of a latent transcriptional factor STAT1 was not found in HUT78R cells, whereas activated STAT2 and STAT3 were clearly detectable. By Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, we found that HUT78R cells do not express any STAT1 protein or mRNA, suggesting the possibility of a null mutation in the STAT1 gene. Resistance to the growth inhibitory effect of IFNα in CTCL cells may result from lack of STAT1 expression.
Fetal Hemoglobin in Starvation Ketosis of Young Women
Authors: Peters Achim , Rohloff Dagmar , Kohlmann Thomas , Renner Florian , Jantschek Günther , Kerner Wolfgang , Lorenz Fehm Horst
Published: October 2019

Ketones can reactivate the production of fetal hemoglobin (HbF) in vitro and in vivo. A reactivation of HbF by ketones, which are generated during starvation, remains largely speculative. Therefore, we investigated HbF in 31 women with anorexia nervosa or bulimia, using both of these as models of intermittent starvation ketosis. For comparison, we also studied 42 female control subjects matched for age. β-Hydroxybutyrate levels were higher in patients than in controls (460 ± 90 v 110 ± 20 μmol/L; P < .0001). We correlated β-hydroxybutyrate, metabolic, and hematologic parameters with HbF. HbF was measured with high pressure liquid chromatography. The data were analyzed with logistic regression analysis. An elevated HbF fraction (>0.87%) was observed four times as often in patients than in controls (29%v 7%, P = .01). After adjustment for age, we found HbF elevations associated with β-hydroxybutyrate levels (P= .005). No other correlations between the various metabolic/hematologic parameters and HbF were significant. In conclusion, β-hydroxybutyrate generated in starvation is associated with increased levels of HbF. Thus, unrestrained lipolysis can produce β-hydroxybutyrate in sufficient quantities to induce a clinically measurable amount of HbF. These findings suggest that intermittent ketosis might also explain some increases of HbF in type 1 diabetes and pregnancy.
Developmental Expression of Mouse Erythrocyte Protein 4.2 mRNA: Evidence for Specific Expression in Erythroid Cells
Authors: Zhu Lingyun , Kahwash Samir B. , Chang Long-Sheng
Published: October 2019

Erythrocyte protein 4.2 (P4.2) is an important component of the erythrocyte membrane skeletal network with an undefined biologic function. Presently, very little is known about the expression of the P4.2 gene during mouse embryonic development and in adult animals. By using the Northern blot and in situ hybridization techniques, we have examined the spatial and temporal expression of the P4.2 gene during mouse development. We show that expression of the mouse P4.2 gene is temporally regulated during embryogenesis and that the P4.2 mRNA expression pattern coincides with the timing of erythropoietic activity in hematopoietic organs. P4.2 transcripts are first detected in embryos on day 7.5 of gestation and are localized exclusively in primitive erythroid cells of yolk sac origin. These erythroid cells remain to be the only source for P4.2 expression until the switch of the hematopoietic producing site to fetal liver. In mid- and late-gestation periods, P4.2 mRNA expression is restricted to the erythroid cells in fetal liver and to circulating erythrocytes. Around and after birth, the site for P4.2 expression is switched from liver to spleen and bone marrow, and P4.2 transcripts are only detected in cells of the erythroid lineage. These results provide the evidence for specific P4.2 expression in erythroid cells. In addition, the timing and pattern of expression of the P4.2 gene suggest the specific regulation of the P4.2 gene.
Mortality in a Cohort of Men Expressing the Glucose-6-Phosphate Dehydrogenase Deficiency
Authors: Cocco Pierluigi , Todde Pierfelice , Fornera Susanna , Bonaria Manca Maria , Manca Pierina , Rosa Sias Ana
Published: October 2019

The objective of this study was to test the hypothesis of a lower mortality from cancer and cardiovascular diseases among men expressing glucose-6-phosphate dehydrogenase (G6PD) deficiency. We designed a mortality study based on death certificates from January 1, 1982 through December 31, 1992 in a cohort of G6PD-deficient men. Cohort members were 1,756 men, identified as expressing the G6PD-deficient phenotype during a 1981 population screening of the G6PD polymorphism. The setting was the island of Sardinia, Italy. Outcome measures were cause-specific standardized mortality ratios (SMRs), which were computed as 100 times the observed/expected ratio, with the general Sardinian male population as the reference. Deaths from all causes were significantly less than expected due to decreased SMRs for ischemic heart disease (SMR, 28; 95% confidence interval [CI], 10 to 62), cerebrovascular disease (SMR, 22; 95% CI, 6 to 55), and liver cirrhosis (SMR, 12; 95% CI, 0 to 66), which explained 95.6% of the deficit in total mortality. All cancer mortality was close to the expectation, with a significant increase in the SMR for non-Hodgkin's lymphoma (SMR, 545; 95% CI, 147 to 1,395). A decrease in mortality from cardiovascular diseases was one of the study hypotheses, based on an earlier human report and experimental evidence. However, selection bias is also a likely explanation. Further analytic studies are warranted to confirm whether subjects expressing the G6PD-deficient phenotype are protected against ischemic heart disease and cerebrovascular disease. This cohort study is consistent with more recent case-control studies in rejecting the hypothesis of a decreased cancer risk among G6PD-deficient subjects. The observed increase in mortality from non-Hodgkin's lymphoma and decrease in mortality from liver cirrhosis were not previously reported.
Expression of the Lung Resistance Protein Predicts Poor Outcome in De Novo Acute Myeloid Leukemia
Authors: Filipits Martin , Pohl Gudrun , Stranzl Thomas , Suchomel Ralf W. , Scheper Rik J. , Jäger Ulrich , Geissler Klaus , Lechner Klaus , Pirker Robert
Published: October 2019

The 110-kD lung resistance protein (LRP) is overexpressed in P-glycoprotein–negative multidrug-resistant cell lines and most likely involved in the multidrug resistance (MDR) of these cell lines. To determine the clinical significance of LRP, we have studied LRP expression of leukemic blasts and its association with clinical outcome in patients with de novo acute myeloid leukemia (AML). LRP expression of leukemic blasts obtained from peripheral blood or bone marrow of previously untreated patients (n = 86) was determined by immunocytochemistry by means of monoclonal antibody LRP-56. LRP expression at diagnosis was detected in 31 (36%) patients. LRP expression was independent of age and sex of the patients, French-American-British subtype, cytogenetic abnormalities, and lactate dehydrogenase levels, but correlated with white blood cell count (P = .01). Eighty-two patients received standard induction chemotherapy that included cytarabine and MDR drugs (daunorubicin in most patients, additional etoposide in the majority of patients). The complete remission rate of induction chemotherapy was 72% (95% confidence interval [CI] = 61% to 82%) for the total study population. The complete remission rate was 81% (95% CI = 67% to 91%) for patients without LRP expression but only 55% (95% CI = 36% to 74%) for patients with LRP expression (P = .01). Overall survival and disease-free survival were estimated according to Kaplan-Meier in 82 and 59 patients, respectively. Overall survival was significantly longer in patients without LRP expression than in patients with LRP expression. At a median follow-up of 16 months, median overall survival was 17 months (95% CI = 12 to 38 months) for LRP-negative patients but only 8 months (95% CI = 4 to 12 months) for -positive patients (P = .006). Disease-free survival was 9 months (95% CI = 7 to 11 months) for LRP-negative patients and 6 months (95% CI = 5 to 8 months) for -positive patients (P = .078). Outcome was best in patients lacking both LRP and P-glycoprotein expression. In conclusion, LRP predicts for poor outcome and thus theLRP gene appears to be another clinically relevant drug resistance gene in AML.
Retroviral-Mediated Gene Transfer of the Leukocyte Integrin CD18 Into Peripheral Blood CD34+ Cells Derived From a Patient With Leukocyte Adhesion Deficiency Type 1
Authors: Bauer Thomas R. , Schwartz Barbara R. , Conrad Liles W. , Ochs Hans D. , Hickstein Dennis D.
Published: October 2019

Leukocyte adhesion deficiency or LAD is a congenital immunodeficiency disease characterized by recurrent bacterial infections in which the leukocytes from affected children fail to adhere to endothelial cells and migrate to the site of infection due to heterogeneous defects in the leukocyte integrin CD18 subunit. To assess the feasibility of human gene therapy of LAD, we transduced granulocyte colony-stimulating factor (G-CSF)-mobilized, CD34+peripheral blood stem cells derived from a patient with the severe form of LAD using supernatant from the retroviral vector PG13/LgCD18. The highest transduction frequencies (31%) were found after exposure of the cells to retroviral vector on a substrate of recombinant fibronectin fragment CH-296 in the presence of growth factors interleukin-3 (IL-3), IL-6, and stem cell factor. When the phenotype of the transduced cells was monitored by fluorescence-activated cell sorting following in vitro differentiation with growth factors G-CSF and granulocyte-macrophage CSF (GM-CSF), CD11a surface expression was detected immediately after transduction. CD11b and CD11c were expressed at low levels immediately following transduction, but increased over 3 weeks in culture. Adhesion of the transduced cells was nearly double that of nontransduced cells in a cell adhesion assay using human umbilical vein endothelial cells. Transduced cells also demonstrated the ability to undergo a respiratory burst in response to opsonized zymosan, a CD11/CD18-dependent ligand. These experiments show that retrovirus-mediated gene transfer of the CD18 subunit complements the defect in LAD CD34+ cells resulting in CD11/CD18 surface expression, and that the differentiated myelomonocytic cells derived from the transduced LAD CD34+ cells display CD11/CD18-mediated adhesion function. These results indicate that ex vivo gene transfer of CD18 into LAD CD34+ cells, followed by re-infusion of the transduced cells, may represent a therapeutic approach to LAD.
Type 2M von Willebrand Disease: F606I and I662F Mutations in the Glycoprotein Ib Binding Domain Selectively Impair Ristocetin- but not Botrocetin-Mediated Binding of von Willebrand Factor to Platelets
Authors: Hillery Cheryl A. , Mancuso David J. , Evan Sadler J. , Ponder Jay W. , Jozwiak Mary A. , Christopherson Pamela A. , Cox Gill Joan , Paul Scott J. , Montgomery Robert R.
Published: October 2019

von Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T → A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A → T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.
Natural Killer Cell–Mediated Eradication of Neuroblastoma Metastases to Bone Marrow by Targeted Interleukin-2 Therapy
Authors: Lode Holger N. , Xiang Rong , Dreier Torsten , Varki Nissi M. , Gillies Stephen D. , Reisfeld Ralph A.
Published: October 2019

Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody–IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell–dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell–deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell–stimulating agents, such as poly I:C or recombinant mouse interferon-γ. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.
Genetic Polymorphism in MDR-1: A Tool for Examining Allelic Expression in Normal Cells, Unselected and Drug-Selected Cell Lines, and Human Tumors
Authors: Mickley Lyn A. , Lee Jong-Seok , Weng Zheng , Zhan Zhirong , Alvarez Manuel , Wilson Wyndham , Bates Susan E. , Fojo Tito
Published: October 2019

By using RNase protection analysis, residues 2677 and 2995 ofMDR-1 were identified as sites of genetic polymorphism. Through use of oligonucleotide hybridization, the genomic content and expression of individual MDR-1 alleles were examined in normal tissues, unselected and drug selected cell lines, and malignant lymphomas. In normal tissues, unselected cell lines, and untreated malignant lymphoma samples, expression of MDR-1 from both alleles was similar. In contrast, in drug selected cell lines, and in relapsed malignant lymphoma samples, expression of one allele was found in a large percentage of samples. To understand how expression of one allele occurs, two multidrug resistant sublines were isolated by exposing a Burkitt lymphoma cell line to increasing concentrations of vincristine. The resistant sublines expressed only one allele and had a hybrid MDR-1 gene composed of non–MDR-1 sequences proximal to MDR-1. Previous studies showing hybridMDR-1 genes after rearrangements provided a potential explanation for activation and expression of one MDR-1 allele. We conclude that oligonucleotide hybridization can be used as a sensitive tool to examine relative allelic expression of MDR-1,and can identify abnormal expression from a single allele. Acquired drug resistance in vitro and in patients is often associated with expression of a single MDR-1 allele, and this can be a marker of a hybrid MDR-1 gene.
Origin of the Hodgkin/Reed-Sternberg Cells in Chronic Lymphocytic Leukemia With “Hodgkin's Transformation”
Authors: Ohno Toshiyuki , Smir Bassam N. , Weisenburger Dennis D. , Gascoyne Randy D. , Hinrichs Steven D. , Chan Wing C.
Published: October 2019

A lymphoma with the characteristic features of Hodgkin's disease (HD) occasionally develops in patients with B-cell chronic lymphocytic leukemia (CLL), and has been called Richter's syndrome with HD features. In such cases, large tumor cells have the morphological and immunophenotypic features of classical Hodgkin and Reed-Sternberg (H-RS) cells. However, it is not known whether the H-RS cells arise from transformation of the underlying CLL cells or from a different pathological process. We report herein a study of the clonal relationship between the CLL cells and the H-RS cells in three cases of Richter's syndrome with HD features by using a single cell assay. We isolated single CLL cells and H-RS cells from immunostained tissue sections by micromanipulation. The immunoglobulin heavy chain gene (IgH) complementarity determining region (CDR) III of each cell was amplified by the polymerase chain reaction (PCR). The products were then compared by gel electrophoresis and nucleotide sequencing. The IgH CDRIII sequences from the H-RS cells were identical to those from the CLL cells in two cases. In one case, the clonal relationship between the two types of cells could not be determined because PCR products could not be obtained from any of the H-RS cells. This study shows that the H-RS cells and the CLL cells belong to the same clonal population in some cases of Richter's syndrome with HD features. Furthermore, our findings indicate that mature B cells can undergo transformation to cells with the features of H-RS cells, in association with a cellular background typical of HD. This study also supports recent findings suggesting that the H-RS cells in classical HD are derived from transformed B cells.
Purging of Mammary Carcinoma Cells During Ex Vivo Culture of CD34+ Hematopoietic Progenitor Cells With Recombinant Immunotoxins
Authors: Spyridonidis A. , Schmidt M. , Bernhardt W. , Papadimitriou A. , Azemar M. , Wels W. , Groner B. , Henschler R.
Published: October 2019

Tumor cells have been found in autologous hematopoietic cell transplants used after high-dose chemotherapy. To specifically eliminate contaminating mammary tumor cells during ex vivo expansion of CD34+ hematopoietic progenitor cells, we used recombinant immunotoxins (ITs) directed against cell-surface antigens expressed on mammary carcinoma cells. ITs were expressed from fusion cDNAs combining a single-chain antibody fragment (scFv) directed against the Erb-B2 or epidermal growth factor (EGF) receptors with a truncatedPseudomonas exotoxin A fragment devoid of its cell-binding domain. CD34+ hematopoietic progenitor cells did not express Erb-B2 and EGF receptors as detected by Western blotting. Ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors in the presence of stem-cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (Epo) was not affected when ITs were added to the cultures. In contrast, MDA-MB 453 and MCF-7 mammary carcinoma cells were depleted in a dose- and time-dependent manner by more than 3 log in coculture with CD34+ cells over a period of 7 days in the presence of 100 to 1,000 ng/mL of anti–Erb-B2 IT. This included elimination of the subpopulations with regrowth potential. Similarly, addition of either anti–Erb-B2 or anti-EGF receptor ITs to primary breast cancer cells isolated from patients with metastatic disease resulted in elimination of cytokeratin-positive cells in seven of seven samples. ITs are highly efficient and convenient to use for the depletion of mammary tumor cells during ex vivo expansion of hematopoietic progenitor-cell autografts.
Characterization and Use of an Antibody Detecting the CBFβ-SMMHC Fusion Protein in inv(16)/t(16;16)-Associated Acute Myeloid Leukemias
Authors: Viswanatha David S. , Chen I.-Ming , Liu Pu Paul , Slovak Marilyn L. , Rankin Cathy , Head David R. , Willman Cheryl L.
Published: October 2019

The inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFβ-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFβ-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFβ-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR–negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFβ-SMMHC fusion proteins. Molecular characterization of one RT-PCR–positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, −0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR–positive AML cases with inv(16) or t(16;16) could be rapidly identified. This study demonstrates the use of this antibody as an investigational tool in inv(16)/t(16;16) AML and suggests that the development of such reagents may have potential clinical diagnostic use.
A Common Genetic Polymorphism (46 C to T Substitution) in the 5′-Untranslated Region of the Coagulation Factor XII Gene Is Associated With Low Translation Efficiency and Decrease in Plasma Factor XII Level
Authors: Kanaji Taisuke , Okamura Takashi , Osaki Koichi , Kuroiwa Mika , Shimoda Kazuya , Hamasaki Naotaka , Niho Yoshiyuki
Published: October 2019

We studied the Hga I polymorphism (46 C/T) in the 5′-untranslated region of the coagulation factor XII (FXII) gene corresponding to four bases upstream from the ATG translation initiation codon. By using allele-specific restriction analysis with restriction endonuclease Hga I, the allele frequency of 46C/T was estimated to be 0.27/0.73 in Orientals (allele number =152), and conversely, 0.8/0.2 in Caucasians (allele number =40). Because it has been reported that plasma levels of FXII were lower in Orientals than in Caucasians, we investigated the relationship between this polymorphism and plasma levels of FXII. As a result, there were significant differences in plasma FXII levels between these three allele types: C/C,170±38% (178±27%); C/T, 141±29% (123±34%); and T/T, 82±19% (61±11%) [FXII activity (FXII antigen levels)]. In heterozygotes of 46 C/T both alleles were equally transcribed in hepatocytes, as determined by reverse transcription polymerase chain reaction (RT-PCR), suggesting little influence of the polymorphism at the level of transcription or on the stability of mRNA. In in vitro transcription/translation analysis, less FXII was produced from cDNA containing 46 T than from that containing 46 C. Therefore, it is highly likely that the 46 T polymorphism in the FXII gene decreased the translation efficiency and led to low plasma levels of FXII activity and antigen, probably due to the creation of another ATG codon and/or impairment of the consensus sequence for the translation initiation scanning model.
Antibody to Granulocyte-Macrophage Colony-Stimulating Factor Is a Dominant Anti-Cytokine Activity in Human IgG Preparations
Authors: Svenson Morten , Hansen Morten Bagge , Ross Christian , Diamant Marcus , Rieneck Klaus , Nielsen Henrik , Bendtzen Klaus
Published: October 2019

Pharmaceutical preparations of normal human immunoglobulin (IgG) are known to contain high-avidity and neutralizing antibodies (Ab) to the cytokines interleukin (IL)-1α, IL-6, and interferon (IFN)α. To test for other cytokine Ab, 23 batches of IgG were tested for saturable binding to eight 125I-labeled recombinant cytokines. All batches bound granulocyte-macrophage colony-stimulating factor (GM-CSF) with high avidity (Kav ≈ 10 pmol/L) and capacities of up to 5 μmol GM-CSF/mol IgG. Only 1 of 15 batches bound IL-5, also with high avidity, whereas 13 of 15 batches bound to IL-10 but with lower capacities and avidities. None of the IgG preparations bound IL-1 receptor antagonist (IL-1ra), IL-2, IL-3, IL-4, or G-CSF. Cross-binding and absorption analyses revealed identical or slightly stronger binding of recombinant GM-CSF, IL-5, and IL-10 than their native counterparts. GM-CSF–IgG complexes did not bind to cellular GM-CSF receptors, but Fc-dependent binding occurred to blood polymorphonuclear cells. Increased binding of GM-CSF to patient sera correlated positively with the binding capacities of infused IgG preparations. Patient and normal sera did not interfere with the binding of Ab to GM-CSF. From these and previous experiments, we conclude that pools of normal human IgG contain variable amounts of specific and high-avidity Ab to some cytokines, and that Ab to GM-CSF constitute a dominant anti-cytokine activity in these preparations. These Ab are available for reactionin vivo following IgG therapy.