Enzymatic mineralization of gellan gum hydrogel for bone tissue‐engineering applications and its enhancement by polydopamine

Interest is growing in the use of hydrogels as bone tissue‐engineering (TE) scaffolds due to advantages such as injectability and ease of incorporation of active substances such as enzymes. Hydrogels consisting of gellan gum (GG), an inexpensive calcium‐crosslinkable polysaccharide, have been applied in cartilage TE. To improve GG suitability as a material for bone TE, alkaline phosphatase (ALP), an enzyme involved in mineralization of bone by cleaving phosphate from organic phosphate, was incorporated into GG hydrogels to induce mineralization with calcium phosphate (CaP). Incorporated ALP induced formation of apatite‐like material on the submicron scale within GG gels, as shown by FTIR, SEM, EDS, XRD, ICP‐OES, TGA and von Kossa staining. Increasing ALP concentration increased amounts of CaP as well as stiffness. Mineralized GG was able to withstand sterilization by autoclaving, although stiffness decreased. In addition, mineralizability and stiffness of GG was enhanced by the incorporation of polydopamine (PDA). Furthermore, mineralization of GG led to enhanced attachment and vitality of cells in vitro while cytocompatibility of the mineralized gels was comparable to one of the most commonly used bone substitute materials. The results proved that ALP‐mediated enzymatic mineralization of GG could be enhanced by functionalization with PDA. Copyright © 2012 John Wiley & Sons, Ltd.     

Interleukin-4 stimulates expression of urokinase-type-plasminogen activator in cultured human foreskin microvascular endothelial cells

The effect of interleukin-4 (IL-4) on the fibrinolytic system of human microvascular and macrovascular endothelial cells in culture was studied. Only foreskin microvascular endothelial cells (EC) responded to IL-4 treatment with a dose- and time-dependent increase in urokinase- type plasminogen activator (u-PA) (control: 3.0 +/- 0.8 ng/10(5) cells/24 h; 200 U/mL IL-4: 6.7 +/- 0.8 ng/10(5) cells/24 h), whereas human macrovascular EC remained unaffected. A maximum effect was achieved with 200 U/mL IL-4. Little u-PA activity was detected in the conditioned media of human foreskin microvascular EC (HFMEC) treated without and with IL-4 before plasmin treatment (control: 0.03 +/- 0.003 IU/10(5) cells/20 h; 200 U/mL IL-4: 0.09 +/- 0.007 IU/10(5) cells/20 h). These values increased to 0.18 +/- 0.02 IU/10(5) cells/20 h and 0.53 +/- 0.04 IU/10(5) cells/20 h, respectively, after plasmin treatment, indicating that u-PA is released by HFMEC predominantly in its inactive precursor form single-chain u-PA (scu-PA). u-PA activity increased also in the cell lysates of HFMEC up to 2.5-fold after IL-4 treatment. Plasminogen activator inhibitor type-1 (PAI-1) levels produced by HFMEC remained unaffected by IL-4, whereas tissue-type plasminogen activator (t-PA) levels were slightly decreased when HFMEC were treated with IL-4. These findings were also reflected in the specific mRNA levels as determined by Northern blotting. u-PA-specific mRNA increased significantly in HFMEC in the presence of IL-4, whereas t-PA mRNA and PAI-1-specific mRNA in HFMEC and u-PA specific mRNA in human saphenous vein EC (HSVEC) remained unaffected by IL-4 treatment. Our findings suggest a role for IL-4 in the process of angiogenesis, in addition to its known proliferative effect on human microvascular EC, by increasing the fibrinolytic potential of such EC, thereby facilitating extracellular proteolysis and cell migration.     

Specific antibody-blocking activities in antilymphocyte globulin as correlates of efficacy for the treatment of aplastic anemia

Horse anti-human thoracic duct lymphocyte globulin (ATDLG) has been used successfully for the treatment of severe aplastic anemia, although not all lots have comparable efficacy. We have characterized the antibody specificities contained in one lot of Swiss ATDLG found to provide a response rate of 69% and another lot that provided only a 31% response rate. Antibody specificities were analyzed quantitatively by competitive inhibition assays with the use of a panel of fluorescein- conjugated murine monoclonal antibodies that recognize T cell antigens, common leukocyte antigens, and "la-like" antigens. Although there was wide variation in the amounts of individual antibody specificities within each lot, the effective lot of ATDLG contained an average of 2 1/2 times as much of each antibody specificity as the less effective lot. There were only two antibody specificities that differed remarkably from this pattern; and these deviations did not appear sufficient to account for the variation in ATDLG efficacy. It is possible that antibody specificities other than those tested were responsible for therapeutic efficacy. Alternatively, the data suggest that it might be possible to achieve improved results for the treatment of severe aplastic anemia with higher doses of less effective lots of ATDLG.     

Hemoglobin North Shore: a variant hemoglobin associated with the phenotype of beta-thalassemia

Hemoglobin (Hb) North Shore (beta 134 val leads to glu) is a mutant hemoglobin that is associated with the phenotype of mild heterozygous beta-thalassemia. Heterozygotes are characterized low normal hemoglobin levels or mild anemia, microcytosis, increased HbA2, and 34%-38% Hb North Shore. The mechanism of the anemia and microcytosis associated with Hb North Shore was explored by studies of hemolysate thermal instability, peripheral blood globin biosynthesis, and whole blood oxygen affinity. Hb North Shore was mildly heat unstable in comparison to normal adult hemolysate. Pulse labeling of reticulocytes with 3H- leucine showed an alpha/beta ratio of 1.35 (normal 1.0). The beta North Shore/alpha ratio was 0.22-0.27, which was less than expected on the basis of gene dosage and less than that seen for most beta-chain variants. The beta A/alpha ratio was 0.50, as would be expected. The beta North Shore/alpha ratio was 0.26 after a 15-min pulse and did not decrease during 120 min of chase. These findings suggest that suboptimal synthesis rather than posttranslational degradation is responsible for the thalassemic phenotype associated with this variant hemoglobin. These observations parallel the findings in heterozygous HbE. It is not presently known whether the thalassemia phenotype is conferred by the structural mutation itself or by another mutation cis to the beta North Shore gene.     

Structural mechanisms of DREAM complex assembly and regulation

The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52–p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence.     

CHARGE syndrome: a review of the immunological aspects

CHARGE syndrome is caused by a dominant variant in the CHD7 gene. Multiple organ systems can be affected because of haploinsufficiency of CHD7 during embryonic development. CHARGE syndrome shares many clinical features with the 22q11.2 deletion syndrome. Immunological abnormalities have been described, but are generally given little attention in studies on CHARGE syndrome. However, structured information on immunological abnormalities in CHARGE patients is necessary to develop optimal guidelines for diagnosis, treatment and follow-up in these patients. Here, we provide an overview of the current literature on immunological abnormalities in CHARGE syndrome. We also explore immunological abnormalities in comparable multiple congenital anomaly syndromes to identify common immunological phenotypes and genetic pathways that might regulate the immune system. Finally, we aim to identify gaps in our knowledge on the immunological aspects in CHARGE syndrome that need further study.     

Evaluation of a CD5-specific immunotoxin for treatment of acute graft- versus-host disease after allogeneic marrow transplantation

Acute graft-versus-host disease (GVHD) is most often treated with high dose glucocorticoids, but less than half of patients have durable overall improvement. Previous phase I and phase II studies suggested that treatment with a CD5-specific immunotoxin (XomaZyme-CD5 Plus) could ameliorate symptoms of GVHD. In a randomized, double-blind trial, we compared XomaZyme-CD5 Plus and glucocorticoids versus placebo and glucocorticoids as initial therapy for 243 patients who developed acute GVHD after allogeneic marrow transplantation. The study drug (XomaZyme. CD5-Plus or an identical appearing placebo) was administered at a dose of 0.1 mg/kg body weight on each of 14 consecutive days. All patients were treated concomitantly with a standard regimen of methylprednisolone. At the time of entry on study, 94% of patients had a rash, 56% had hyperbilirubinemia, 61% had diarrhea, and 84% had nausea and vomiting. At 3, 4, and 5 weeks after starting treatment, symptom severity was less in the CD5 group than in the placebo group. At 4 weeks, 40% of patients assigned to the CD5 group had complete response compared with 25% of those assigned to the control group (P = .019). At 6 weeks, 44% of patients assigned to the CD5 group had complete response as compared with 38% in the placebo group (P = .36). Clinical extensive chronic GVHD developed in 65% of patients in the CD5 group compared with 72% in the control group (P = .35). Survival at 1 year after treatment was 49% in the CD5 group and 45% in the control group (P = .68). Side effects required close monitoring and appropriate adjustment of treatment. The combined administration of a CD5-specific immunotoxin and glucocorticoids controls GVHD manifestations more effectively than treatment with glucocorticoids alone during the first 5 weeks after starting treatment. Use of this immunotoxin does not result in any long-term clinical benefit for patients with acute GVHD.     

The role of HLA-DPB1 disparity in the development of acute graft-versus- host disease following unrelated donor marrow transplantation

The role of HLA-DPB1 disparity in the development of acute graft-versus- host disease (GVHD) following unrelated donor (URD) marrow transplantation is unknown. We studied 129 patients who underwent marrow transplantation from HLA-A, -B, -DRB, and -DQB matched URDs to determine whether matching for HLA-DPB1 alleles significantly decreased the risk of developing acute GVHD. HLA-DPB1 alleles were determined by sequence-specific oligonucleotide hybridization and by the number of patient DPB1 alleles not shared by the donor scored. The Kaplan-Meier probability of developing grades II to IV acute GVHD was determined for patients incompatible for zero (group A), one (group B), or two (group C) DPB1 alleles. Of the 129 pairs, there was no recipient DPB1 incompatibility in 28 (22%), one DPB1 mismatch in 72 (56%), and two DPB1 mismatches in 29 (22%). The probability of grades II to IV acute GVHD was 0.69 (0.50, 0.86) for group A, 0.83 (0.73, 0.91) for group B, and 0.72 (0.56, 0.87) for group C (P = .63). These results indicate that matching patients and unrelated donors for HLA-A, -B, -DRB, and - DQB does not predict for matching at DPB1. However, recipient incompatibility for DPB1 alleles does not detectably influence the risk of acute GVHD. Therefore, HLA-DP disparity should not be used as an exclusion criterion for donor selection in unrelated marrow transplantation.