Synovial Sarcoma Complicating Total Knee Arthroplasty.

Synovial sarcoma is a relatively common periarticular soft tissue malignancy, occurring mostly in the extremities of younger patients. Occasionally, diversity in its clinical features may lead to misdiagnosis and inappropriate management. The authors report herein a unique case of a patient who underwent a primary total knee arthroplasty to treat osteoarthritis. During the operation, a mass was discovered but was attributed to synovitis. Biopsy revealed a rare intra-articular synovial sarcoma. The patient underwent reoperation with wide excision and endoprosthesis placement and is disease-free at the 8-year follow-up.

Collection of blood stem cells from patients with sickle cell anemia.

Prior to initiating gene therapy trials for sickle cell disease (SCD), methods to collect sufficient numbers of hematopoietic stem and progenitor cells will need to be developed. Bone marrow harvest entails significant morbidity that could be severe in patients with SCD. In addition, an ability to perform repeated stem cell collections so that several transfers of genetically modified cells could be attempted would be advantageous. In other settings, apheresis collection of mobilized blood stem cells has become the preferred source of stem cells for transplantation. Unfortunately, patients with SCD do not tolerate granulocyte-colony stimulating factor and therefore cannot be mobilized using these conventional methods. In this pilot study, we investigated whether withdrawal of hydroxyurea therapy results in an increase in circulating numbers of CD34+ cells and hematopoietic progenitors. In addition, we performed leukapheresis in one patient with severe SCD in an attempt to determine whether blood stem cell collection can be performed safely in patients who would be candidates for SC gene therapy trials. Our results highlight some of the potential difficulties in initiating gene therapy clinical trials for sickle cell disease.

Spontaneous switch from Agamma- to beta-globin promoter activity in a stable transfected dual reporter vector.

Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for (A)gamma- and beta-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as 1-2 years from transfection. The transfected GM979 cells rapidly (within 1 month) evolved into a stable population which expresses constant levels of reporters for more than a year of continuous bulk culture. No silencing of the inserted construct was observed over time. In contrast, after 1 month, the reporter activity (both from (A)gamma- and beta-promoter) expressed per cell increased over time. The analysis of the Luciferase contained in single cell clones indicated that the higher reporter activity was due to increased gene expression per cell rather than to clonal selection of the most expressing clones. Since the activity driven by the beta-promoter increased 10-fold more than that driven by the (A)gamma one, the ratio between (A)gamma-driven/((A)gamma-driven + beta-driven) reporter activity in the cells decreased after 1 month and became similar to the gamma/(gamma + beta) globin mRNA ratio expressed by adult erythroid cells. Moreover, although both cells from early and late bulk culture responded to incubation with butyric acid, a known inducer of fetal globin gene expression, by increasing the reporter activity driven by the (A)gamma-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the beta-promoter. These results suggest that the rapid changes in activity driven by the (A)gamma- and beta-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the (A)gamma- and beta-promoter during the fetal to adult hemoglobin switch and confirm GM979 cells stably transfected with the dual reporter construct as a reliable assay for automated screening of chemical inducers of fetal globin gene activation.

The histone deacetylase inhibitor, trichostatin A, reactivates the developmentally silenced gamma globin expression in somatic cell hybrids and induces gamma gene expression in adult BFUe cultures.

Somatic cell hybrids that have undergone globin gene switching and developmental silencing of gamma globin expression were treated with the histone deacetylase inhibitor trichostatin A (TSA). Culture of the post-switch hybrids in the presence of TSA reactivated gamma globin expression and concommitantly downregulated beta globin expression, as determined by both mRNA quantitation and immunofluorescent quantitation of gamma globin expressing cells. In contrast, similar treatment of pre-switch hybrids, which were expressing predominantly gamma globin and only small levels of beta globin, had no effect on the relative gamma or beta globin gene expression. In addition, trichostatin A induced gamma gene expression in adult BFUe cultures in a maturation-independent fashion. The results provide direct evidence that inhibition of HDAC activity can alter expression from the human beta globin locus in the adult stage of development.