Differential expression of perforin in muscle-infiltrating T cells in polymyositis and dermatomyositis.

Polymyositis (PM) and dermatomyositis (DM) are the prototypical inflammatory diseases of skeletal muscle. In PM, CD8+ T cells invade and destroy muscle fibers, whereas humoral effector mechanisms prevail in DM. We studied the expression of the cytotoxic mediator perforin in inflammatory cells in PM and DM muscle by semiquantitative PCR, immunohistochemistry and confocal laser microscopy. Similar levels of perforin mRNA were expressed in PM and DM, and abundant perforin-expressing CD3+CD8+ and CD3+ CD4+ T cells were observed in both diseases. However, there was a striking difference in the intracellular localization of perforin. In DM, perforin was distributed randomly in the cytoplasm of the inflammatory T cells. In contrast, 43% of the CD8+ T cells that contacted a muscle fiber in PM showed perforin located vectorially towards the target muscle fiber. The results suggest (a) that the random distribution of perforin in the cytoplasm of muscle-infiltrating T cells observed in DM reflects nonspecific activation, and (b) that the vectorial orientation observed only in PM reflects the specific recognition via the T cell receptor of an antigen on the muscle fiber surface, pointing to a perforin- and secretion-dependent mechanism of muscle fiber injury.     

Volatiles or TIVA: Which is the standard of care for pediatric airway procedures? A pro‐con discussion

Anesthesia for pediatric airway procedures constitutes a true art form that requires training and experience. Communication between anesthetist and surgeon to establish procedure goals is essential in determining the most appropriate anesthetic management. But does the mode of anesthesia have an impact? Traditionally, inhalational anesthesia was the most common anesthesia technique used during airway surgery. Introduction of agents used for total intravenous anesthesia (TIVA) such as propofol, short‐acting opioids, midazolam, and dexmedetomidine has driven change in practice. Ongoing debates abound as to the advantages and disadvantages of volatile‐based anesthesia versus TIVA. This pro‐con discussion examines both volatiles and TIVA, from the perspective of effectiveness, safety, cost, and environmental impact, in an endeavor to justify which technique is the best specifically for pediatric airway procedures.     

Refrigerated storage of lyophilized and rehydrated, lyophilized human red cells

Human red cells (RBCs) were collected in CPDA-1 and then freeze-dried in lyoprotective solution. The lyophilized RBCs were then stored at -20 degrees C for 7 days. At the end of the storage period, the lyophilized RBCs were rehydrated and washed in dextrose saline. The washed, reconstituted, lyophilized RBCs were resuspended in final wash solutions of ADSOL, CPDA-1, or a special additive solution containing glucose, citrate, phosphate, adenine, and mannitol, and then they were stored at 4 degrees C for an additional 7 days. The main purpose of this study was to determine whether human RBCs can be lyophilized in such a manner that normal metabolic, rheologic, and cellular properties are maintained during rehydration and subsequent storage in standard blood bank preservative solutions. Our results show that reconstituted, lyophilized RBCs maintained levels of ATP, 2,3 DPG, lactate, and cellular properties that are equal to or better than those in control nonlyophilized RBCs stored for a comparable period in CPDA-1. Reconstituted, lyophilized RBCs stored at 4 degrees C after rehydration also show better maintenance of ATP, 2,3 DPG, and lactate than do control RBCs stored in the same preservative solutions for comparable periods.     

PCR-positivity in harvested bone marrow predicts relapse after transplantation with autologous purged bone marrow in children in second remission of precursor B-cell acute leukaemia

Purging of autologous bone marrow (BM) grafts of children in second remission after a relapse of precursor B acute lymphoblastic leukaemia (ALL) in the BM has been carried out in our laboratory since 1987, initially by complement mediated cell lysis. This protocol was extended by performing an immunorosette depletion before lysis with complement. The aim of the present study was to assess by polymerase chain reaction the presence of residual leukaemic cells in the BM grafts before and after purging. The results were then correlated to clinical outcome. In 24/28 patients a PCR product was obtained by amplification of IgH and/or TcR junctional regions. BM before purging was available for analysis in 13 patients. We found that leukaemic cells could be detected in 8/13 (62%) of these grafts before purging . All these eight patients experienced a relapse, regardless of whether the purging procedure had been successful (defined as achievement of PCR-negativity) or not. In contrast, none of the five patients with PCR-negative grafts before purging relapsed ( P  = 0.0008). One patient died due to transplant-related toxicity. Of the remaining 23 patients, nine patients received a PCR-positive BM graft after purging. All these nine patients experienced a relapse as compared to 6/14 whose BM was PCR-negative after purging ( P  = 0.0072). Two of eight PCR-positive BM grafts could be purged to PCR-negativity. Thus, improvements both in treatment of leukaemia and in purging efficacy are still needed.     

Superoxide anions and endothelial cell proliferation in normoglycemia and hyperglycemia

-Oxygen free radicals are believed to play a key role in cellular proliferation, and increased concentrations of these molecules have been implicated in the pathogenesis of endothelial dysfunction in diabetes mellitus. Our aim was to study the role of superoxide anions in endothelial cell proliferation under conditions of normoglycemia and hyperglycemia. Human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) exposed to adenoviral vectors encoding CuZnSOD (AdCuZnSOD), ss-galactosidase (Adssgal), or diluent (control) were cultured in normal glucose (NG, 5.5 mmol/L) or high glucose (HG, 28 mmol/L) medium. Cell proliferation was compared by use of [(3)H]thymidine incorporation and cell count in transduced and control cells in the setting of NG and HG. Transgene expression was detected in transduced cells by X-gal staining and by Western analysis and SOD activity assay in AdCuZnSOD-transduced cells. Superoxide production was significantly (P:<0.05) decreased in AdCuZnSOD-transduced cells cultured in both NG and HG medium. In NG, AdCuZnSOD-transduced endothelial cells had decreased proliferation compared with control cells. After 48 hours in HG, superoxide levels were increased and DNA synthesis was decreased (P:<0.05) in control and Adssgal-transduced but were not affected in AdCuZnSOD-transduced cells. In addition, after 7 days in HG, cell counts were reduced (P:<0.05) in control (73+/-2.5%) and Adssgal-transduced (75+/-3.4%) but not in AdCuZnSOD-transduced cells (89+/-3.4%). These results suggest that either a deficiency or an excess of superoxide anions inhibits endothelial cell proliferation, and the inhibitory effect of increased superoxide due to hyperglycemia can be reversed by CuZnSOD overexpression.     

Induction of oral tolerance in splenocyte recipients toward pretransplant antigens ameliorates chronic graft versus host disease in a murine model

Chronic graft versus host disease (cGVHD) is a major complication that can develop after bone marrow transplantation. It involves an immune-mediated attack by transplanted donor lymphocytes, and often results in inflammatory damage of host target organs. Immune hyporesponsiveness induced by oral antigen administration has been recently shown to prevent the development of cGVHD in a murine model. The aim of this study was to evaluate whether tolerance induction in bone marrow transplant (BMT) recipients after transplantation, toward their pretransplant antigens, can alleviate preexisting cGVHD in a mouse model. cGVHD was generated by infusing 2.5 x 10(7) splenocytes from B10.D2 donor mice, to sublethally irradiated (6 Gy) BALB/c recipient mice, which differ by minor histocompatibility antigens. Transplantation resulted in cGVHD, with characteristic scleroderma-like cutaneous fibrosis, increased skin collagen content, decreased body weight, and hepatic and small bowel inflammation. Oral tolerance was induced by feeding recipient BALB/c mice with proteins extracted from BALB/c splenocytes for 11 days after B10.D2 splenocyte transplantation. Tolerance induction was evidenced by a significant reduction in mixed lymphocyte response of effector splenocytes from tolerant BALB/c mice transplanted with B10.D2 splenocytes against BALB/c target splenocytes. Oral tolerance decreased skin collagen deposits. Reduction of collagen alpha1(I) gene expression and skin collagen were shown by in situ hybridization and histochemistry, respectively. Liver and bowel biopsy specimens revealed less inflammation. Serum IL-10 levels were higher in tolerant mice than in controls, whereas IFNgamma was significantly reduced. Oral tolerance of BMT recipients toward their pretransplant antigens after splenocyte transplantation down-regulated the immune attack by transplanted cells, thus ameliorating cGVHD.     

European Myeloma Network recommendations on the evaluation and treatment of newly diagnosed patients with multiple myeloma

Multiple myeloma management has undergone profound changes in the past thanks to advances in our understanding of the disease biology and improvements in treatment and supportive care approaches. This article presents recommendations of the European Myeloma Network for newly diagnosed patients based on the GRADE system for level of evidence. All patients with symptomatic disease should undergo risk stratification to classify patients for International Staging System stage (level of evidence: 1A) and for cytogenetically defined high- versus standard-risk groups (2B). Novel-agent-based induction and up-front autologous stem cell transplantation in medically fit patients remains the standard of care (1A). Induction therapy should include a triple combination of bortezomib, with either adriamycin or thalidomide and dexamethasone (1A), or with cyclophosphamide and dexamethasone (2B). Currently, allogeneic stem cell transplantation may be considered for young patients with high-risk disease and preferably in the context of a clinical trial (2B). Thalidomide (1B) or lenalidomide (1A) maintenance increases progression-free survival and possibly overall survival (2B). Bortezomib-based regimens are a valuable consolidation option, especially for patients who failed excellent response after autologous stem cell transplantation (2A). Bortezomib-melphalan-prednisone or melphalan-prednisone-thalidomide are the standards of care for transplant-ineligible patients (1A). Melphalan-prednisone-lenalidomide with lenalidomide maintenance increases progression-free survival, but overall survival data are needed. New data from the phase III study (MM-020/IFM 07-01) of lenalidomide-low-dose dexamethasone reached its primary end point of a statistically significant improvement in progression-free survival as compared to melphalan-prednisone-thalidomide and provides further evidence for the efficacy of lenalidomide-low-dose dexamethasone in transplant-ineligible patients (2B).     

Inhibition of Cullin-RING E3 ubiquitin ligase 7 by simian virus 40 large T antigen

Simian virus 40 (SV40) large tumor antigen (LT) triggers oncogenic transformation by inhibition of key tumor suppressor proteins, including p53 and members of the retinoblastoma family. In addition, SV40 transformation requires binding of LT to Cullin 7 (CUL7), a core component of Cullin-RING E3 ubiquitin ligase 7 (CRL7). However, the pathomechanistic effects of LT–CUL7 interaction are mostly unknown. Here we report both in vitro and in vivo experimental evidence that SV40 LT suppresses the ubiquitin ligase function of CRL7. We show that SV40 LT, but not a CUL7 binding-deficient mutant (LT^Δ69–83), impaired 26S proteasome-dependent proteolysis of the CRL7 target protein insulin receptor substrate 1 (IRS1), a component of the insulin and insulin-like growth factor 1 signaling pathway. SV40 LT expression resulted in the accumulation and prolonged half-life of IRS1. In vitro, purified SV40 LT reduced CRL7-dependent IRS1 ubiquitination in a concentration-dependent manner. Expression of SV40 LT, or depletion of CUL7 by RNA interference, resulted in the enhanced activation of IRS1 downstream signaling pathways phosphatidylinositol-3-kinase/AKT and Erk mitogen-activated pathway kinase, as well as up-regulation of the downstream target gene c-fos. Finally, SV40 LT-positive carcinoma of carcinoembryonic antigen 424/SV40 LT transgenic mice displayed elevated IRS1 protein levels and activation of downstream signaling. Taken together, these data suggest that SV40 LT protects IRS1 from CRL7-mediated degradation, thereby sustaining high levels of promitogenic IRS1 downstream signaling pathways.Studies with simian virus 40 (SV40), a member of the Polyomaviridae family of tumor viruses, have led to fundamental insights into molecular processes of cell transformation and oncogenesis (1, 2). SV40 encodes the large tumor antigen (LT) with the potential to transform cells in culture and induce tumors in rodents. The tumorigenic features of SV40 have been attributed to binding and deactivation of key tumor suppressor proteins of the host cell including p53 and members of the retinoblastoma (pRB) family (1–3). In addition, SV40 LT was shown to be physically associated with Cullin 7 (CUL7; also named p185 or p193) (4, 5) as well as insulin receptor substrate 1 (IRS1) (6). It has been proposed that the association of SV40 LT with either CUL7 or IRS1 is critical to SV40 oncogenic transformation (7–9). However, the functional effect of LT interaction with CUL7/IRS1 and their pathophysiological interrelation remains mostly unknown.CUL7 is a scaffold protein responsible for assembling the multisubunit Cullin-RING E3 ubiquitin ligase 7 (CRL7) that consists of the RING-finger protein ROC1 and the Skp1-Fbw8 substrate-targeting subunit (10, 11). Genetic studies documented a pivotal growth-regulatory role of CRL7. Both cul7 (12) and fbw8 (13) null mice exhibit intrauterine growth retardation. In addition, CUL7 germ-line mutations were linked to 3-M syndrome, a hereditary disorder characterized by pre- and postnatal growth retardation in humans (14, 15), as well as Yakut dwarfism syndrome (16). DeCaprio and colleagues mapped the CUL7 interaction domain on SV40 LT to residues 69–83 and demonstrated that the CUL7 binding-deficient deletion mutant (LT^Δ69–83) lost its transformation potential despite maintaining its ability to bind and inactivate p53 and pRB members (8, 9). This suggested that CUL7 may act as a tumor suppressor and that constraining growth-inhibitory functions of CRL7 may be critical to SV40 transformation.We previously identified IRS1, a component of the insulin and insulin-like growth factor 1 (IGF1) signaling pathway, as a proteolytic target of CRL7 (17). Binding of insulin or IGF1 to its receptor induces tyrosine phosphorylation of IRS1 and subsequent activation of phosphatidylinositol-3-kinase (PI3K)/AKT and Erk mitogen-activated pathway kinase (MAPK) pathways (18). It was shown that CRL7-induced degradation of IRS1 is part of a negative feedback loop via mechanistic target of rapamycin complex 1 (mTORC1) to restrain IRS1 downstream signaling (17, 19). A more recent study suggested an mTORC2-dependent feedback inhibition of IRS1 by direct phosphorylation of Fbw8, resulting in enhanced stability of this F-box protein that promotes IRS1 degradation (20). Collectively, these studies have implicated roles for CRL7 in regulating both mTORC1 and mTORC2 signaling. Based on the above observations, we investigated whether SV40 LT impacts on CRL7 feedback regulation of IRS1 signaling in addition to its effects on p53 and pRB members.