Sj Ragg

Mitochondrial cytochrome c release precedes transmembrane depolarisation and caspase-3 activation during ceramide-induced apoptosis of Jurkat T cells

Whilst the role of ceramide, a second messenger of the sphingolipid family, in the initiation of receptor-mediated apoptosis is controversial, a growing body of evidence is emerging for a role of ceramide in the amplification of apoptosis via mitochondrial perturbations that culminate in the activation of execution caspases. Treatment of Jurkat T cells with the cell-permeable analog, C2-ceramide, resulted in the rapid onset of apoptosis as evidenced by Annexin V-FITC staining of externalised phosphatidylserine residues. Cells bearing this early apoptotic marker had a reduced mitochondrial transmembrane potential (ΔΨm) that was preceded by the release of cytochrome c from mitochondria. Subsequent activation of caspase-3 provides the link between these ceramide-induced mitochondrial changes and execution caspases that ultimately result in the physical destruction of the cell. Collectively these results demonstrate that ceramide signalling results in caspase-mediated apoptosis via mitochondrial cytochrome c release and are further supportive of the role of ceramide in the amplification of apoptosis.

The migration of Langerhans' cells into and out of lymph nodes draining normal, carcinogen and antigen-treated sheep skin.

Epidermal Langerhans’ cell (LC) migration to the regional lymph node and beyond into central lymph was examined in sheep following topical application of the complete chemical carcinogen 7,12‐dimethylbenz(a)anthracene(DMBA)or the contact sensitizing antigen 2,4,6‐trinitrochlorobenzene (TNCB). This was facilitated by cannulating previously constructed pseudoafferent lymphatic vessels draining the skin treated with these agents or alternatively, the efferent lymphatic vessel of the regional lymph node.

Quality controls of cryopreserved haematopoietic progenitor cells (peripheral blood, cord blood, bone marrow)

The final quality control of cryopreserved progenitor cells is a successful and persistent three lineage engraftment after transplantation. Of course, the stem cell providing institution is obliged to have a program for controlling and monitoring the manufacturing of cellular therapy products before the patients’ conditioning therapy is started. The FACT-JACIE Standards [1] and the Netcord ⁄ FACT Stan- dards prescribe that the director of the institute shall define tests and procedures for measuring and assaying cellular therapy products to ensure their safety, viability and integ- rity and shall also ensure that products meet predetermined release specifications. This requires specifications of assays and the definition of thresholds to allow release.

Association between high interleukin-6 levels and adverse outcome after autologous haemopoietic stem cell transplantation.

We studied interleukin-6 (IL-6) levels on the day of transplantation in 31 patients undergoing autologous haemopoietic stem cell transplantation (SCT) (either peripheral blood stem cell transplantation (PBSCT) or bone marrow transplantation (BMT)) for neoplastic diseases to determine if there was a relationship between IL-6 level and rate of haemopoietic recovery, length of stay in hospital, and survival. There was no apparent delay in post-transplant recovery associated with elevated IL-6 levels. However, increased values of IL-6 tended to be associated with an increased length of stay in hospital (P = 0.083). There was a highly significant adverse association between higher IL-6 levels and survival following transplantation (P = 0.0001). This association remained significant (P = 0.013) in the uniform subgroup of patients with malignant lymphoma with chemosensitive disease who had undergone BMT (that is, excluding patients who had undergone PBSCT) (n = 13). Knowledge of IL-6 levels on the day of transplant has the potential to provide valuable prognostic information in patients undergoing autologous haemopoietic SCT. Bone Marrow Transplantation (2001) 28, 929–933.

Cell cycle arrest of hematopoietic cell lines after treatment with ceramide is commonly associated with retinoblastoma activation

BACKGROUND Leukaemia cells differ from their normal counterparts in that their ability to properly regulate survival, proliferation, differentiation, and apoptosis is aberrant. Understanding the molecular mechanisms controlling cell proliferation and developing therapeutic strategies to correct nonfunctional regulatory mechanisms are emerging areas of medical research. Ceramide, a metabolite of membrane sphingomyelin hydrolysis, has recently emerged as a key regulator of cellular proliferation, differentiation, and apoptosis in leukaemia cells. METHODS Leukaemia cell lines were treated with a biologically active analogue of ceramide, C(2)-ceramide. Cell cycle status was assessed flow cytometrically using propidium iodide. Induction of apoptosis was confirmed by annexin V staining of externalised phosphatidylserine and retinoblastoma activation was determined by Western blotting. RESULTS C(2)-ceramide induced activation of retinoblastoma tumour suppressor protein, G(0)/G(1) cell cycle arrest, or apoptosis in leukaemia cell lines. In addition, these effects differed depending upon cell type, thus confirming the pleiotropic nature of the ceramide signalling pathway. Most cells studied responded to exogenous C(2)-ceramide by entering growth arrest, evidently resulting from activation of retinoblastoma protein, and by displaying some degree of apoptosis. CONCLUSIONS Taken together, these findings suggest that signalling via ceramide has novel therapeutic applications for treatment of leukaemia.

A randomized controlled clinical trial to determine the optimum duration of G-CSF priming prior to BM stem cell harvesting.

BACKGROUND Harvesting of hemopoietic stem cells (HSC) from G-CSF-primed BM for autologous transplantation is an alternative to collection of unprimed BM or G-CSF-primed peripheral blood (PB). However, the optimum number of days of G-CSF administration for this purpose is unknown. We set out to determine whether cell yields could be optimized by varying the number of days of G-CSF administration prior to BM stem cell harvesting. METHODS We conducted a randomized controlled single-center trial of 6 days (the standard) vs. 4 days of G-CSF administration and compared yields of total nucleated cells (TNC), CD34(+) HSC and CFU-GM cells per kilogram patient body weight. Statistical analysis was by Students t-test. RESULTS Twenty-four patients were enrolled; 13 received 6 days and 11 received 4 days of G-CSF administration. Analysis of the first harvest aspirate showed higher proportions of CD34(+) HSC (P=0.02) and CFU-GM (P=0.03) in the 4-day group. For the 6-day and 4-day groups, respectively, the median yield of TNC/kg was 6.5 x 10(8) and 5.4 x 10(8) (P=0.28), of CD34(+) cells/kg 0.56 x 10(6) and 0.98 x 10(6) (P=0.04) and of CFU-GM cells/kg 1.66 x 10(5) and 1.55 x 10(5) (P=0.75). DISCUSSION These results suggest that by 6 days the HSC-stimulating effect of G-CSF has passed its peak and that 4 days should be adopted as the standard for G-CSF priming prior to BM stem cell harvesting for autologous transplantation.

Dendritic Cells Migrating from Carcinogen-Treated Skin Have Reduced Antigen-Presenting Function

Chemical carcinogens have a recognised ability to reduce the status of cutaneous immunity. Antigen presentation through carcinogen-treated skin results in immune suppression rather than activation of effector mechanisms (reviewed by 1). Topical application of the complete chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) leads not only to the development of skin tumours but also induces a rapid depletion of Langeihans cells (LC) from the epidermis - up to 50% of the LC are depleted from the epidermis in the 3–4 days following DMBA application2.

An audit of patients with mature T‐cell non‐Hodgkin lymphoma by transplant status in Tasmania

This retrospective audit of patients diagnosed with mature T‐cell lymphoma across a 10‐year period provides contemporary information on the outcomes and treatment patterns of an Australian cohort. Forty‐two patients diagnosed with mature T‐cell lymphoma were identified from the Tasmanian Cancer Registry and analysed using medical records and simple statistical analysis. The demographics and outcomes of patients in this cohort were comparable to large international studies with treatment patterns in line with the best available evidence.

Use of 'rainy day' autologous haemopoietic stem cells: a single-institution experience over 10 years.

High‐dose chemotherapy and autologous haematopoietic stem cell transplantation is an important therapeutic modality in the treatment of many haematological malignancies. Generally, stem cells are collected close to the time of the transplant, but an alternative is to collect and cryopreserve cells at an early stage of the illness so they are available for later use (‘rainy day harvesting’). Although this practice has been commonplace in Australia, there is little evidence to document eventual use of cells collected in this manner.

Use of ‘rainy day’ haematopoietic stem cells over 10 years

Aim: High dose therapy and autologous haematopoietic stem cell (AHSC) transplantation is an important treatment for many haematological malignancies. AHSCs can be harvested during remission or consolidation treatment for subsequent transplantation if required (‘rainy day’ collection). Although the practice is widespread, evidence base is minimal. We investigated the eventual transplantation of ‘rainy day’ collections, delay to transplantation and patient outcomes. Methods: This was a retrospective audit of practice between 1/1/2001 and 31/12/2010. ‘Rainy day’ harvest was defined as collection of AHSCs where transplantation was not anticipated in the next 6 months. Results: 532 collections were performed in 474 patients for haematological indications. 342 (71%) patients had rainy day AHSCs collected. Disease indications included non-Hodgkin lymphoma (NHL; n = 205, 60%), multiple myeloma (MM; 45, 13%), Hodgkin lymphoma (HL; 38, 11%), acute myeloid leukaemia (AML; 20, 6%), chronic myelogenous leukaemia (CML; 5, 1%), acute lymphoblastic leukaemia (ALL; 6, 2%), other myeloproliferative neoplasms (21, 6%), chronic lymphocytic leukaemia (CLL; 3, 1%). Overall transplantation rate was 14%. For MM collections 27% were transplanted, compared to 16% for HL and 14% for NHL. Only 5% of collections for AML were transplanted and there were no transplants for CML. The median delay to transplantation was 18 months (range 6.3–112); 81% of patients transplanted engrafted within specified limits. Of the 296 patients who had ‘rainy day’ AHSC collections that have not been transplanted, 255 (85%) are still alive Discussion: ‘Rainy day’ AHSC collection is resource intensive. We found high rates of eventual usage for certain indications (MM, HL) and low rates for others (AML, CML). Findings from this study may help to inform guidelines for ‘rainy day’ AHSC collection.