Umbilical cord blood transplantation for Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI)

Severe Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is usually fatal by early adulthood. Bone marrow transplantation is the only form of definitive enzyme replacement therapy available. A 5-year-old boy with Maroteaux-Lamy syndrome has successful recovery of bone marrow and enzymatic functions after umbilical cord blood transplant from his unaffected HLA-identical brother. Busulphan (16 mg/kg) and cyclophosphamide (200 mg/kg) were used as preparative chemotherapy with short methotrexate and long cyclosporin as prophylaxis against graft-versus-host disease (GVHD). A total of 6.08 x 10(7)/kg nucleated cells and 2.92 x 10(5)/kg CD34+ cells were transplanted with neutrophil engraftment achieved on day 26. There was no evidence of acute and chronic GVHD. Fifteen months after transplant, a normal level of N-acetylgalactosamine-4-sulphatase activity was achieved despite mixed chimerism. There was clinical improvement of hepatosplenomegaly, facial and skin features, joint mobility and resolution of suppurative middle ear effusion. He returned to school and continued to perform well in academic studies. We report here the first successful umbilical cord blood transplant as treatment of Maroteaux-Lamy syndrome.     

Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus

We sought to determine whether intramuscular injection of a recombinant adeno-associated virus (rAAV) vector expressing human factor IX (hF.IX) could direct expression of therapeutic levels of the transgene in experimental animals. High titer (10¹²–10¹³ vector genomes/ml) rAAV expressing hF.IX was prepared, purified, and injected into hindlimb muscles of C57BL/6 mice and Rag 1 mice. In the immunocompetent C57BL/6 mice, immunofluorescence staining of muscle harvested 3 months after injection demonstrated the presence of hF.IX protein, and PCR analysis of muscle DNA was positive for AAV DNA, but no hF.IX was detected in mouse plasma. Further studies showed that these mice had developed circulating antibodies to hF.IX. In follow-up experiments in Rag 1 mice, which carry a mutation in the recombinase activating gene-1 and thus lack functional B and T cells, similar results were seen on DNA analysis of muscle, but these mice also demonstrated therapeutic levels (200–350 ng/ml) of F.IX in the plasma. The time course of F.IX expression demonstrates that levels gradually increase over a period of several weeks before reaching a plateau that is stable 6 months after injection. In other experiments we demonstrate colocalization of hF.IX and collagen IV in intersitial spaces between muscle fibers. Collagen IV has recently been identified as a F.IX-binding protein; this finding explains the unusual pattern of immunofluorescent staining for F.IX shown in these experiments. Thus rAAV can be used to direct stable expression of therapeutic levels of F.IX after intramuscular injection and is a feasible strategy for treatment of patients with hemophilia B.     

Interleukin-4 and interleukin-5 map to human chromosome 5 in a region encoding growth factors and receptors and are deleted in myeloid leukemias with a del(5q)

Interleukin-4 (IL-4) is a potent mediator of growth and differentiation of cells of several hematopoietic lineages. Interleukin-5 (IL-5) is a lineage-specific hematopoietic growth factor that stimulates the production of eosinophils and eosinophil colonies from normal human bone marrow cells. By using somatic cell hybrids and in situ chromosomal hybridization, we localized the IL-4 and IL-5 genes to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted [del(5q)] in patients with myeloid disorders. By in situ hybridization, the IL-4 and IL-5 genes were found to be deleted in the 5q- chromosome of four patients with refractory anemia (RA) or therapy-related acute nonlymphocytic leukemia (t-ANLL), who had a del(5q). Thus a small segment of chromosome 5 contains IL-4, IL-5, IL- 3, and GM-CSF as well as other genes such as CD14 and EGR1. Our findings that each of these genes was deleted in the 5q- chromosome suggest that loss of function of one or more of these genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q).     

Fluorescence in situ hybridization mapping of translocations and deletions involving the short arm of human chromosome 12 in malignant hematologic diseases

Translocations and deletions of the short arm of chromosome 12 [t(12p) and del(12p)] are common recurring abnormalities in a broad spectrum of hematologic malignant diseases. We studied 20 patients and one cell line whose cells contained 12p13 translocations and/or 12p deletions using fluorescence in situ hybridization (FISH) with phage, plasmid, and cosmid probes that we previously mapped and ordered on 12p12-13. FISH analysis showed that the 12p13 translocation breakpoints were clustered between two cosmids, D12S133 and D12S142, in 11 of 12 patients and in one cell line. FISH analysis of 11 patients with deletions demonstrated that the deletions were interstitial rather than terminal and that the distal part of 12p12, including the GDI-D4 gene and D12S54 marker, was deleted in all 11 patients. Moreover, FISH analysis showed that cells from 3 of these patients contained both a del(12p) and a 12p13 translocation and that the affected regions of these rearrangements appeared to overlap. We identified three yeast artificial chromosome (YAC) clones that span all the 12p13 translocation breakpoints mapped between D12S133 and D12S142. They have inserts of human DNA between 1.39 and 1.67 Mb. Because the region between D12S133 and D12S142 also represents the telomeric border of the smallest commonly deleted region of 12p, we also studied patients with a del(12p) using these YACs. The smallest YAC, 964c10, was deleted in 8 of 9 patients studied. In the other patient, the YAC labeled the del(12p) chromosome more weakly than the normal chromosome 12, suggesting that a part of the YAC was deleted. Thus, most 12p13 translocation breakpoints were clustered within the sequences contained in the 1.39 Mb YAC and this YAC appears to include the telomeric border of the smallest commonly deleted region. Whether the same gene is involved in both the translocations and deletions is presently unknown.     

Mixed chimerism after bone marrow transplantation for thalassemia major

Thirty-four thalassemia patients were studied for chimerism by fluorescent in situ hybridization or variable number tandem repeats after bone marrow transplantation. Mixed chimerism was detected in 9 patients with host cells ranging from 4 to 56%. One had graft rejection and the others were transfusion independent. Mixed chimerism was common but mostly without deleterious effect.     

An inhibitory monoclonal antibody to factor X that blocks prothrombin activation but not prothrombinase enzyme assembly

A monoclonal antibody (designated alpha BFX-2b) prepared against bovine factor X inhibited factor X activity in human, bovine, porcine, rabbit, and canine plasma. In assays using purified prothrombinase components, factor Xa, factor Va, phospholipid vesicles, and calcium ion with the fluorescent active site thrombin inhibitor dansylarginyl-N-(3-ethyl-1,5- pentanediyl)amide, the antibody inhibited the conversion of prothrombin to thrombin. Antibody alpha BFX-2b also blocked prothrombinase cleavage of the macromolecular substrates prethrombin 1 and prethrombin 2 but did not inhibit factor Xa hydrolysis of the synthetic substrate benzoyl- Ile-Glu-Gly-Arg-p-nitroanilide. The antibody also prevented the inactivation of factor Xa by antithrombin III but did not prevent the inactivation by soybean trypsin inhibitor. Antibody alpha BFX-2b bound factor Xa with a stoichiometry of 1:1 and an apparent dissociation constant of 9.0 x 10(-11) mol/L as estimated from its inhibition of prothrombinase activity. Antibody alpha BFX-2b did not prevent binding of factor Xa to factor Va-phospholipid as measured by using fluorescence polarization or high-pressure liquid gel chromatography with the fluorescent Factor Xa analogue dansyl-glutamyl-glycyl-arginyl- Xa. Immunoblotting of factor X following electrophoresis on sodium dodecyl sulphate-polyacrylamide gels and transfer to nitrocellulose indicated that the antigenic determinant recognized by antibody alpha BFX-2b was found on the heavy chain of factors X and Xa. From these observations it can be concluded that antibody alpha BFX-2b recognizes a highly conserved epitope on the factor X heavy chain that is remote from the topographic sites required for prothrombinase complex assembly and substrate hydrolysis but may be located at or near a portion of the macromolecular substrate binding site.     

CD18-dependent and L-selectin-dependent neutrophil emigration is diminished in neonatal rabbits

Human neonatal neutrophils manifest decreases in mobility, adherence, and emigration compared with adult neutrophils that may contribute to the increased susceptibility of neonates to infection. In a developmental rabbit model, we show a reduced ability of neutrophils from 1-day-old rabbit pups to emigrate to inflamed peritoneium (3.7 +/- 0.35 x 10(6) neutrophils/mL peritoneal exudate) compared with 14-day- old (8.5 +/- 0.7 x 10(6)/mL) and adult rabbits (9.4 +/- 1.4 x 10(6) mL, P < .05) despite significantly increased blood neutrophil counts. Because the reductions in functional Mac-1 (CD11b/CD18) as well as the amount of surface L-selectin are hypothesized to be primarily responsible for the differences in human neonatal neutrophil mobility, we examined CD11b/CD18 and L-selectin in our model. Using flow cytometric analysis we found that similar to human neonates, neutrophils from 1-day-old rabbit pups had 57% of adult rabbit levels of L-selectin and, in contrast with adults, failed to show significant decreases in L-selectin after chemotactic stimulation. In addition, neutrophils from 1-day-old pups compared with adults showed a significantly diminished capacity to upregulate CD11b/CD18 after chemotactic stimulation in vitro, or after emigration to the inflamed peritoneum. Systemic administration of anti-L-selectin monoclonal antibody (MoAb) resulted in significant reduction in peritoneal neutrophils in adult (47%, P < .05) and 14-day-old rabbits (47%, P < .05), but was without effect in 1-day-old rabbits. Administration of anti-CD18 MoAb resulted in significant reduction in peritoneal neutrophil accumulation in all age groups though less in 1 day and 14 day (58% and 65%, respectively) than in adults (91%, P < .05). Only in the 14-day-old rabbits was there an additive effect of anti-L-selectin and anti-CD18 MoAbs compared with anti-CD18 alone (84% v 65%, P < .05). The findings in this in vivo rabbit model support the hypothesis that the previously described in vitro defects in human neonatal L-selectin and CD11b/CD18 may be major contributors to human neonatal inflammatory deficits.     

Inhibitory effect of icariin on Ti-induced inflammatory osteoclastogenesis

Background

Wear particle-induced periprosthetic osteolysis that results in aseptic loosening is the most common cause of long-term failure after total joint replacement.

Materials and methods

Icariin (ICA), a flavonoid isolated from Epimedium pubescens, inhibits osteoclast formation, but its effects on wear particle-induced inflammatory osteoclastogenesis remains unclear. We investigated the role of ICA in the regulation of osteoclast differentiation in a murine macrophage cell line (RAW264.7), which is stimulated by titanium (Ti) particles and the receptor activator of NF-κB ligand.

Results

ICA effectively inhibited osteoclast formation and bone resorption in the differentiation medium. ICA (10⁻⁷ mol/L) significantly reduced the number of tartrate-resistant acid phosphatase-positive cells compared with the control, and significantly reduced the percentage of the surface covered by resorption lacunae. Quantitative real-time polymerase chain reaction analysis showed that ICA inhibited messenger RNA expression for the receptor activator of nuclear factor-κB, cathepsin K, tartrate-resistant acid phosphatase-positive, and matrix metalloproteinase-9 in RAW264.7 cells stimulated by Ti particles and receptor activator of NF-κB ligand. ICA also reduced pro-inflammatory cytokine expression of interleukin-1β and tumor necrosis factor-α in RAW264.7 cells cultured with Ti particles. In addition, incubation with cholecystokinin-8 showed that ICA had no toxic effects on RAW264.7 cells.

Conclusions

ICA possibly elicited inhibitory effects on inflammatory osteoclastogenesis induced by Ti particles, indicating that ICA may be useful for the prevention and treatment of wear particle-induced osteolysis.