Recombinant immunotoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression in mice of an interleukin-2 receptor-expressing human carcinoma

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin composed of the variable domains of the monoclonal antibody anti-Tac, which binds to the p55 subunit of the interleukin-2 receptor (IL-2R), and a truncated form of Pseudomonas exotoxin (PE), which does not bind to the PE receptor (Chaudhary et al, Nature 339:394, 1989). Whereas its cytotoxic activity toward autoimmune and malignant target cells has been established, its efficacy in vivo remains unknown. To establish an animal model, we produced ATAC-4 cells by transfecting the gene encoding the low-affinity IL-2R (p55) into A431 epidermoid carcinoma cells. ATAC-4 cells contained low-affinity IL-2Rs (2 x 10(5)/cell) and formed tumors in nude mice. In tissue culture, protein synthesis in ATAC-4 cells was inhibited 50% (IC50) at 0.06 ng/mL (0.9 pmol/L) of anti-Tac(Fv)-PE40. IC50s for the derivatives anti-Tac(Fv)-PE38, which is missing PE amino acids 365-380, and anti-Tac(Fv)-PE38KDEL, which contains the same deletion plus the KDEL carboxyl terminus, were 0.04 and 0.025 ng/mL, respectively. All the agents produced complete tumor regressions in ATAC-4 tumor-bearing mice and anti-Tac(Fv)-PE38KDEL had significant antitumor activity at 1% of the LD50. The dose limiting toxicity of anti-Tac(Fv)-PE38KDEL was from hemorrhagic liver necrosis, which was observed at approximately 55% of the LD50.     

Association of metabolic syndrome with knee and hand osteoarthritis: A community-based study of women

Objective

It is unclear whether the association between osteoarthritis (OA) and metabolic syndrome (MetS) varies with the site of the affected joint and the presence of pain. Our aim was to describe the association between MetS and radiographic OA (ROA) affecting the knee or the hand in the presence or absence of concurrent joint pain.

Characterization of novel akermanite:poly‐ϵ‐caprolactone scaffolds for human adipose‐derived stem cells bone tissue engineering

In this study, three different akermanite:poly‐?‐caprolactone (PCL) composite scaffolds (wt%: 75:25, 50:50, 25:75) were characterized in terms of structure, compression strength, degradation rate and in vitro biocompatibility to human adipose‐derived stem cells (hASC). Pure ceramic scaffolds [CellCeram^TM, custom‐made, 40:60 wt%; β‐tricalcium phosphate (β‐TCP):hydroxyapatite (HA); and akermanite] and PCL scaffolds served as experimental controls. Compared to ceramic scaffolds, the authors hypothesized that optimal akermanite:PCL composites would have improved compression strength and comparable biocompatibility to hASC. Electron microscopy analysis revealed that PCL‐containing scaffolds had the highest porosity but CellCeram^TM had the greatest pore size. In general, compression strength in PCL‐containing scaffolds was greater than in ceramic scaffolds. PCL‐containing scaffolds were also more stable in culture than ceramic scaffolds. Nonetheless, mass losses after 21 days were observed in all scaffold types. Reduced hASC metabolic activity and increased cell detachment were observed after acute exposure to akermanite:PCL extracts (wt%: 75:25, 50:50). Among the PCL‐containing scaffolds, hASC cultured for 21 days on akermanite:PCL (wt%: 75:25) discs displayed the highest viability, increased expression of osteogenic markers (alkaline phosphatase and osteocalcin) and lowest IL‐6 expression. Together, the results indicate that akermanite:PCL composites may have appropriate mechanical and biocompatibility properties for use as bone tissue scaffolds. Copyright © 2012 John Wiley & Sons, Ltd.     

Treatment of acute nonlymphocytic leukemia: use of anthracycline- cytosine arabinoside induction therapy and comparison of two maintenance regimens

Patients with acute nonlymphocytic leukemia were given remission induction therapy consisting of cytosine arabinoside and an anthracycline. Those patients who experienced complete remission received two courses of consolidation therapy and were randomized to receive maintenance therapy consisting of either daily chemotherapy with reinforcements every 3 mo or reinforcement therapy only every 6 wk. The overall complete remission rate was 66%, with 80% complete remission for previously untreated patients less than 60 yr of age who did not have a prior history of malignancy. Remission durations were the same for patients treated with both maintenance regimens. The major determinant for successful remission induction therapy was patient age, with older patients frequently succumbing to intercurrent infection. Documented leukemic cell resistance to the therapy employed was only rarely encountered. Once remission was achieved, age was no longer a determinant of patient survival, since duration of remission was independent of age. Remission durations were directly related to leukemic cell retention of cytosine arabinoside triphosphate. Hence therapy for acute nonlymphocytic leukemia can be divided into two separate areas: remission induction and remission maintenance.     

Targeted inactivation of the major positive regulatory element (HS-40) of the human alpha-globin gene locus

We have examined the role of the major positive upstream regulatory element of the human alpha-globin gene locus (HS-40) in its natural chromosomal context. Using homologous recombination, HS-40 was replaced by a neo marker gene in a mouse erythroleukemia hybrid cell line containing a single copy of human chromosome 16. In clones from which HS-40 had been deleted, human alpha-globin gene expression was severely reduced, although basal levels of alpha 1 and alpha 2-globin mRNA expression representing less than 3% of the level in control cell lines were detected. Deletion of the neo marker gene, by using FLP recombinase/FLP recombinase target system, proved that the phenotype observed was not caused by the regulatory elements of this marker gene. In the targeted clones, deletion of HS-40 apparently does not affect long-range or local chromatin structure at the alpha promoters. Therefore, these results indicate that, in the experimental system used, HS-40 behaves as a strong inducible enhancer of human alpha- globin gene expression.     

Measurement of the quality of life in rheumatic disorders using the EuroQol

The EuroQol is a validated quality of life (QOL) scale that has been used in population and clinical studies, and has been reported in patients with rheumatoid arthritis (RA). It is short, simple to complete, and might be suitable for surveys of rheumatic disease patients. The properties of this instrument were investigated in a postal survey of 1372 rheumatic disease patients, including 537 with RA, 319 with osteoarthritis (OA) and 516 with fibromyalgia. In addition, simultaneous measurements of functional disability, pain, psychological status, global severity and demographic characteristics were made. EuroQol scores (0.57) were significantly lower than VAS health state scores (0.67) and arthritis-related global severity scores (0.62). QOL was similar in RA and OA, but lower in fibromyalgia, across all instruments. The distribution of EuroQol scores had many gaps and was not continuous. EuroQol did not reflect VAS QOL scores at EuroQol levels below 0.5, and the mean score difference between the instruments below that level was 0.43. Many patients with low EuroQol scores (including some with health states that were 'worse than death') had high VAS scores. These differences appear to have arisen because disability, pain and depression questions ask about mild or moderate problems, but not both, thereby forcing scale compression in the mid ranges. In addition, the 'severe' value is so extremely abnormal that few patients endorse it. Finally, penalty scores are applied to those with at least one maximally abnormal score. The scoring properties and distributional aspects of the EuroQol indicate substantial problems in its use in rheumatic disease patients.      

Expression of a homologously recombined erythopoietin-SV40 T antigen fusion gene in mouse liver: evidence for erythropoietin production by Ito cells

We have obtained transgenic mice in which an erythropoietin-SV40 virus T antigen fusion gene is homologously recombined into the native Epo locus. This gene is expressed in a tissue-specific manner closely resembling that of the native Epo gene. Immunohistochemical detection of SV40 T antigen has been used to characterize the hepatic cell populations expressing the transgene. In mice stimulated by anaemia or hypobaric hypoxia, SV40 T antigen was demonstrated in two liver cell populations: a subset of hepatocytes and a nonparenchymal cell type. Immunohistochemical and ultrastructural characterization of these cells by light and electron microscopy showed the nonparenchymal cell type to be the Ito cells, which lie in a persinusoidal position within the space of Disse. We therefore conclude that Ito cells are the nonhepatocytic source of liver Epo production. These cells show many similarities to the Epo-producing fibroblastoid interstitial cells of the kidney.     

Fatal graft-versus-host disease following blood transfusion in Hodgkin's disease documented by HLA typing

Fatal graft-versus-host disease (GVHD) developed in a patient with Hodgkin's disease treated with combined chemotherapy and radiotherapy following the transfusion of 2 U of packed red blood cells. Clinical features of the GVHD included the development of exfoliative dermatitis, progressive hepatic dysfunction, aplastic anemia, and finally progressive fatal pneumonia. GVHD was documented by skin biopsy and chimerism by HLA typing. The HLA phenotype of the patient's skin fibroblasts [A3, Bw44 (w4)/A2, B15 (w4)] was appropriate for parental haplotypes and probably represented her true HLA phenotype. Lymphocytes from the patient (peripheral blood and lymph node biopsy) were of a different HLA phenotype (A3; Bw35, w38, w4, w6; Cw4), which was inappropriate for parental HLA haplotypes but identical to the HLA phenotype of one of the blood donors. The HLA-DR typing of the patient's family and of the blood donor demonstrated that the patient and the donor probably were HLA-DR identical (DRw5/DRw6), although no B lymphocytes could be obtained from the patient for direct DR typing. We are currently irradiating all blood products administered to patients with Hodgkin's disease receiving intensive treatment. Further observations will be necessary to determine whether transfusions to other cancer patients with immunodeficiency states should be restricted to irradiated blood products.     

Identification of novel K562 membrane proteins that adhere to bone marrow fibroblasts

Adhesion of myeloid leukemia cells to the bone marrow (BM) microenvironment is mediated in part by Beta 1 and Beta 2 integrins. Cells of the erythroleukemia line K562, derived from a patient with chronic myeloid leukemia, bind to BM fibroblasts (BMFs) but the adhesion cannot be accounted for by integrins or other known adhesion proteins including CD44 or members of the Ig or selectin families. Membrane fragments from K562 cells were radioiodinated and allowed to adhere to BMF monolayers. Adherent proteins were solubilized together with the fibroblasts, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Four adherent proteins were consistently observed. Two of these, with reduced molecular weights of 52 kD and 35 to 37 kD, were prominent. Addition of soluble thrombospondin and heparin but not fibronectin inhibited binding of K562 membrane proteins to adherent BMFs and immobilized thrombospondin- and heparin-bound K562 proteins. The 52-kD protein has a multimeric structure nonreduced and has characteristics of a glycoprotein. Its adhesion to fibroblasts is divalent cation and temperature sensitive. The 35- to 37-kD protein, whose function is divalent cation but not temperature sensitive, is phosphoinositol- linked and has characteristics identical to an adherent 35- to 37-kD protein identified on murine progenitor cells. Membrane preparations from two cases of acute myeloid leukemia showed an adherent 35- to 37- kD protein and in one case an adherent 52-kD protein without other adherent bands. A K562 subclone with reduced adherence to BMFs showed reduced amounts of adherent 52-kD and 35- to 37-kD proteins. These proteins may be responsible for the adhesion of malignant and normal hematopoietic progenitor cells to the BM microenvironment.     

Interaction of hemoglobin E and pyrimidine 5' nucleotidase deficiency

A Bangladeshi family is described in which the genes for both hemoglobin E (Hb E) and pyrimidine 5' nucleotidase deficiency are segregating. An individual homozygous for both these conditions has a severe hemolytic anemia, whereas family members who are homozygous for Hb E are asymptomatic and those homozygous for pyrimidine 5' nucleotidase deficiency have the mild hemolytic anemia that is characteristic of this disorder. Globin-chain synthesis experiments have shown that the mechanism underlying the interaction between these two genotypes is a marked decrease in the stability of Hb E in pyrimidine 5' nucleotidase-deficient red blood cells (RBCs). It has also been found that in the enzyme-deficient RBCs in which Hb E is highly unstable, free alpha-chains, though not beta E-chains, acoumulate on the membrane. In view of the increasing evidence that the hemolysis associated with pyrimidine 5' nucleotidase deficiency results not only from an increase in the level of erythrocyte pyrimidines, but also from inhibition of the hexose monophosphate shunt activity in young erythrocytes, it is likely that the marked instability of Hb E in the enzyme-deficient cells results from oxidant damage acting on a mildly unstable Hb variant. These observations may have important implications for the better understanding of the pathophysiology of Hb E/beta-thalassemia, globally the commonest important form of thalassemia.