Efficacy and safety of intravenous anti-D immunoglobulin (Rhophylac) in chronic immune thrombocytopenic purpura.

OBJECTIVES: This Phase III study examined the efficacy and safety of Rhophylac (CSL Behring AG, Bern, Switzerland), a highly pure, liquid-stable anti-D preparation, in chronic immune thrombocytopenic purpura (ITP). MATERIALS AND METHODS: Ninety-eight patients (96 adults, two adolescents) with chronic ITP and platelet counts < 30 x 10(9)/l received a single intravenous injection of 50 microg/kg bodyweight Rhophylac. RESULTS: A response (defined as an increase in platelet count by >or= 20 x 10(9)/l to >or= 30 x 10(9)/l in the first 15 days after treatment) was seen in 66% of patients. Mean time to response was 3.1 +/- 3.0 days, and mean duration of response was 19.2 +/- 1.1 days for responders. The most frequent drug-related adverse events were chills, pyrexia, an increase in bilirubin, and headache; events were mainly mild or moderate. there was no severe hemolysis or renal failure. CONCLUSION: rhophylac is well tolerated and efficacious in chronic itp.

Long-term observation of patients with anti-neutrophil cytoplasmic antibody-associated vasculitis treated with rituximab.

OBJECTIVE: Rituximab, a chimeric anti-CD20 monoclonal antibody, has been shown to be quite effective in the treatment of immune disorders resulting from autoantibodies. We prospectively studied the long-term effects of rituximab in 10 patients with anti-neutrophil cytoplasmic antibody (ANCA)-positive vasculitis refractory to conventional therapy (n=3) or in second or subsequent relapse (n=7). METHODS: The median age of patients was 53 yrs (range 38-70 yrs). Eight were classified as Wegener's granulomatosis, and two as microscopic polyangiitis. Clinical activity was assessed using the Birmingham Vasculitis Activity Score modification for Wegener's granulomatosis. Treatment consisted of intravenous infusions of rituximab given at the dose of 375 mg/m2 weekly for four consecutive weeks. RESULTS: All patients experienced a rapid clinical improvement following the administration of rituximab, with nine complete responses and one partial response at 6 months. With a median follow-up of 33.5 months (range 26-45 months), three patients have thus far relapsed. Retreatment with the monoclonal antibody at the same dose and schedule resulted in a new sustained response in all these patients. Rituximab therapy resulted in prolonged B-cell depletion. The ANCA titres decreased significantly in all patients, with eight out of 10 becoming ANCA-negative and three remaining ANCA-negative even after B-cell recovery. Infusion-related side effects were observed in one patient, but were of mild intensity and did not require discontinuation of treatment. CONCLUSIONS: Rituximab is an effective and well-tolerated treatment for patients with ANCA-associated vasculitis and should be strongly considered in severely affected patients who do not respond to standard therapy or in those in whom cytotoxic therapy bears a high risk of morbidity.

Increase in the ratio of mitochondrial Bax/Bcl-XL induces Bax activation in human leukemic K562 cell line.

The p53- and Bcl-2-negative leukemic K562 cell line showed resistant to DNA damage-induced Bax activation and apoptosis. The constitutive balanced ratio of Bax/Bcl-XL in K562 mitochondria allowed the formation of active Bax and cytochrome c release from mitochondria in the presence of a BH3-only protein, tBid, in a cell-free system. Bax transfection led to Bax undergoing a conformational change, translocation to mitochondria and homo-oligomerization but not apoptosis in the K562 cell line. After treatment with UV light, while Bcl-XL but not Bax translocated to mitochondria in K562, both Bax and Bcl-XL translocated to mitochondria in the Bax stable transfectant K/Bax cells. The increased ratio of Bax/Bcl-XL in K/Bax mitochondria led to an increased conformationally changed Bax, formation of the homo-multimer of Bax-Bax, and a reduced hetero-dimerization of Bax-Bcl-XL. Increased proportion of active Bax was accompanied with increased percentage of apoptosis. We therefore demonstrate that direct increase in the ratio of mitochondrial Bax/Bcl-XL can induce Bax activation in the p53- and Bcl-2-negative leukemic cells. Increased Bcl-XL translocation and failure in Bax translocation from cytosol to mitochondria play important roles in preventing Bax activation.

Potential mechanisms of leukemia cell resistance to TRAIL-induced apopotosis.

There are many factors contributing to the resistance to TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. However, it is not clear whether the mechanism of resistance to TRAIL is constitutive or inductive. Therefore, the purpose of this study was to investigate the resistant mechanisms to TRAIL at different levels in the apoptotic pathway. The human T-lymphoblastic leukemic CEM cell line showed more resistant to TRAIL-induced apoptosis compared with the human chronic myeloid leukemic K562 cell line. Lower level of constitutive caspase-8 expression in the CEM cell line led to a poor response to both TRAIL-induced activation of caspase-3 and reduction in the mitochondrial membrane potential (DeltaPsim). There was no significant difference in the constitutive levels of NF-kappaB in CEM and K562 cell lines. However, CEM cells showed a faster response to TRAIL-induced NF-kappaB activation than K562 cells. TRAIL-induced regulation of Bcl-2 family of proteins included an up-regulation in Bcl-2/Bcl-XL and a down-regulation in Bax. IAPs, such as XIAP, cIAP-1, cIAP-2 and Survivin were all up-regulated during the treatment with TRAIL. In summary, our data suggest that the leukemic cells resistance to TRAIL-induced apoptosis might be due to the deficiency in the constitutive caspase-8 expression. Development of potential resistance to apoptosis by TRAIL can occur in both TRAIL-resistant and TRAIL-sensitive leukemic cells.

Major histocompatibility complex susceptibility genes and immune thrombocytopenic purpura in Caucasian adults.

Immune thrombocytopenic purpura (ITP) is a heterogeneous disorder with wide variability in response rates to treatments including corticosteroids, splenectomy and intravenous immune globulins. The nature of the underlying predisposing causes for this autoimmune disorder are not known. We have HLA typed 71 adult Caucasian patients with chronic primary ITP, and compared the data with 750 control samples. In this association study, we were not able to identify a significant immunogenetic susceptibility factor for ITP with HLA class I and class II alleles. However, it appeared that there might be an association between HLA-A2 and ITP, particularly in female patients, who are the predominantly affected group; and HLA-A2 was also present at increased frequency in patients with chronic ITP progressing to splenectomy. These findings are reviewed in the context of other similar reported HLA studies in ITP. Further studies based on larger groups of patients will be necessary to identify genetic susceptibility factors for this disease.

Liposomal daunorubicin (DaunoXome) in combination with cyclophosphamide, vincristine and prednisolone (COP-X) as salvage therapy in poor-prognosis non-Hodgkins lymphoma.

We treated 33 patients with a variant of the standard 3 weekly CHOP regime, replacing doxorubicin with liposomal daunorubicin (DaunoXome, NeXstar Pharmaceuticals) 120 mg/m2 (COP-X). Eighteen subjects had relapsed/refractory aggressive NHL and 15 had indolent NHL/CLL. Median number of courses received was 4 (1-8). Thirty-two patients were evaluable for efficacy and 26 (81%) responded. 88% of patients with aggressive NHL responded; three (18%) patients achieved complete remission (CR), 12 (70%) achieved partial remission (PR), 1 (6%) patient had stable disease (SD) and 1 (6%) patient progressed through treatment. Median duration of response for patients with aggressive NHL was 3 months. The response rate in indolent NHL/CLL was 73%. Four (27%) patients achieved CR, 7 (46%) PR and 4 (27%) SD. At two years post treatment, 55% of the patients with indolent NHL/CLL remain progression-free, although 4 patients have proceeded to consolidation therapy. Twenty-seven out of 28 (96%) patients developed neutropenia of short duration following one or more of their treatments. Twenty-three patients developed an infection at some stage during therapy (all associated with neutropenia) and required hospitalisation. There were two toxic deaths (infection) both of which occurred in patients who were neutropenic before starting COP-X. Platelet toxicity was mild in patients with normal platelet counts at the commencement of therapy. Alopecia and mucositis were mild. No clinical evidence of myocardial failure was observed. We conclude that the substitution of DaunoXome for doxorubicin in the CHOP regimen to form COP-X provides excellent efficacy against non-Hodgkin's lymphoma. Response durations were short but comparable to those reported with other regimens. COP-X was well tolerated with some suggestion of reduced non-haematological toxicity. The regimen should be considered as an alternative to CHOP with potentially less non-haematological toxicity, particularly cardiac; further studies are required to evaluate the regimen in this context.

Stem cells: progress in research and edging towards the clinical setting.

Mouse embryonic stem cells have been shown to differentiate into a variety of tissues in vitro and in transplantation experiments can produce many different cell types. Multipotent stem cells in adult humans have also shown a high degree of plasticity: haemopoietic stem cells, for example, have been shown to contribute to several other tissues, such as liver. From these simple observations there has been considerable extrapolation into the use of such putative totipotent stem cells in the clinical setting, with the development of 'designer' tissue engineering, whose aim is to create large tissues or even whole organs for clinical use. In practical terms, however, there are many limitations and difficulties and clinical use has been restricted to a very few settings, eg the use of fetal cells in Parkinson's disease. Nonetheless, there is enormous potential in this area, and also in the application of embryonic or adult stem cells as carriers for gene therapy; but the limitations of such treatment, in particular the stability of manipulated cells, and the problems of ageing and Ooncogenicity, not to mention a host of ethical and regulatory issues, all need to be considered.

c-IAP1 blocks TNFalpha-mediated cytotoxicity upstream of caspase-dependent and -independent mitochondrial events in human leukemic cells.

Tumor necrosis factor-alpha (TNFalpha) mediates cytochrome c release from mitochondria, loss of mitochondrial membrane potential (DeltaPsim) and apoptosis in sensitive leukemic cells. In the present study, by using the human leukemic U937 cell line, we demonstrate that the cytochrome c release is caspase-8-dependent and can be blocked by an inhibitor of caspase-8, Z-Ile-Glu (OMe)-Thr-Asp(OMe)-fluoromethyl ketone (Z-IETD.fmk), or a pan caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD.fmk). However, TNFalpha-mediated loss of DeltaPsim was not inhibited by caspase inhibitors. The apoptotic process was blocked by either Z-IETD.fmk or Z-VAD.fmk in cells with lower DeltaPsim. U937 cells with stable transfection of the cellular inhibitor of apoptosis protein 1 (c-IAP1) are resistant to TNFalpha-induced activation of caspases, Bid cleavage, cytochrome c release and DeltaPsim collapse. In addition, both c-IAP1 and XIAP were not up-regulated upon prolonged exposure to TNFalpha. In contrast, there was a caspase-dependent cleavage of XIAP, but not c-IAP1, during treatment with TNFalpha for 7 days. These results demonstrate that c-IAP1 blocks TNFalpha signaling at a level controlling both activation of caspase-8 and a signal to cause loss of DeltaPsim. The sensitive U937 cell line failed to acquire resistance and gain a self-protecting advantage against apoptosis, upon induction of c-IAP1 expression.

Bax translocation is crucial for the sensitivity of leukaemic cells to etoposide-induced apoptosis.

Bax translocation from cytosol to mitochondria is believed to be a crucial step for triggering cytochrome c release from mitochondria. However, it is unclear whether Bax translocation is associated with Bax induction by DNA damaging agents. The induction of Bax in response to DNA damaging agents has been considered to be linked with p53. In this study, we used the p53 negative human chronic myeloid leukaemia K562 cell line. Bax up-regulation occurred at the whole cell level after DNA damage induced by etoposide. However, after incubation with etoposide, Bax failed to translocate to mitochondria and as a result, the apoptotic process was blocked. A Bax stable transfectant, the K/Bax cell line, expressed more Bax protein in the cytosol, mitochondria and nuclei. This Bax overexpression induced cytochrome c release, a reduction of cytochrome c oxidase activity and mitochondrial membrane potential (Delta(Psi)m). However, Bax-induced apoptosis was blocked downstream of mitochondria in K562 cells. The increased levels of mitochondrial Bax sensitized cells to etoposide-induced activation of caspases-2, -3 and -9 and apoptosis. However, after transient transfection with the Apaf-1 gene, K/Bax cells were sensitized to etoposide-induced caspase activation and apoptosis to a larger extent compared with Bax or Apaf-1 transfection alone. We therefore conclude that two mechanisms contribute to the resistance of K562 cells to etoposide-induced apoptosis; firstly failure of Bax targeting to mitochondria and, secondly, deficiency of Apaf-1. Uncoupling of Bax translocation from Bax induction can occur in response to etoposide-induced DNA damage.

Quantitative determination of apoptosis on leukemia cells by infrared spectroscopy.

Quantitation of apoptotic cell death in vivo has become an important issue for patients with acute leukemia. We describe herein a new analytical method, based on infrared (IR) spectroscopy, to estimate the percentage of apoptotic leukemic cells in two different cell lines (CEM and K562), induced with etoposide (VP-16). As the percentage of apoptosis increases, the protein structure shifts from dominantly beta-sheet to unordered (random coil), the overall lipid content increases and the amount of detectable DNA decreases. These changes can be directly related to the percentage of apoptosis as determined by two standard reference methods: flow cytometry and DNA ladder formation. The correlation between the significant IR spectral changes and the percentage of apoptotic leukemia cells in the two cell lines was optimal up to 24 h following etoposide treatment (r = 0.99 for CEM cells and r = 0.96 for K562 cells). Furthermore, IR spectroscopy is able to detect apoptotic changes in these cells already after 4 h treatment with VP-16, compared to flow cytometry which needs 6 h to observe significant changes. Our study suggests that IR spectroscopy may have potential clinical utility for the early, fast and reagent free assessment of chemotherapeutic efficacy in patients with leukemia.

Apaf-1 protein deficiency confers resistance to cytochrome c-dependent apoptosis in human leukemic cells.

The human leukemia cell lines K562, CEM, CEM/VLB(100), human leukemic blasts, and the bladder cancer J82 cell line have different sensitivities to UV light-induced apoptosis. It is reported that resistance to UV light-induced apoptosis occurs at a point in the apoptotic pathway upstream of caspase-3 but downstream of mitochondrial cytochrome c release. It is demonstrated that the block is due to deficiency of Apaf-1, a critical member of the apoptosome. Sensitivity to apoptosis was independent of caspase-9b or XIAP (inhibitors of apoptosis proteins) expression or levels of procaspase-9. Transfection of Apaf-1 conferred sensitivity to apoptosis in resistant cells. Apaf-1 deficiency may constitute a significant mode of resistance to apoptosis in human leukemia.

Vigam-S, a solvent/detergent-treated intravenous immunoglobulin, in idiopathic thrombocytopenic purpura.

The efficacy of Vigam-S, a highly purified intravenous immunoglobulin, was investigated by an open, noncomparative study in 20 adults with chronic idiopathic thrombocytopenic purpura (ITP). Fifteen patients responded to the initial 3-day infusion of 0.4 g kg(-1) day(-1) by exhibiting an incremental increase in platelet count of >or= 30 x 10(9) L(-1), in eight of whom platelet count normalized (> 150 x 10(9) L(-1)). The peak platelet count for responders on day 4 was 163 x 10(9) L(-1) (baseline = 18 x 10(9) L(-1)). No benefit was derived from an extra 2 days infusion in nonresponders. Further treatment (either a single 0.8 g kg(-1) dose or another 3-day infusion) given to responders when platelet counts fell below 30 x 10(9) L(-1) was effective on eight of 14 occasions. Increases in total serum IgG concentration (to a mean peak of 25.3 g L(-1)) were not correlated with platelet response. There was no evidence of seroconversion to virus markers, or of alteration in renal function, following Vigam-S infusion. Most adverse events were mild and transient; however, three patients had severe headache and vomiting (possible aseptic meningitis syndrome) and one had marked superficial thrombophlebitis. Therefore Vigam-S provides effective and well tolerated therapy in the management of adults with ITP although individual patient response remains difficult to predict.

Trail-induced apoptosis in Type I leukemic cells is not enhanced by overexpression of bax.

We have previously shown that Bax translocation was crucial in TNFalpha or etoposide-induced apoptosis. Overexpression of Bax sensitized chronic myeloid leukemic K562 cells to etoposide-induced apoptosis. Treatment with TNF-related apoptosis-inducing ligand (TRAIL) induces a loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondria, activation of caspases-8, -9, and -3, and cleavage of Bid in the K562 cell line. Bax failed to sensitize K562 cells to TRAIL-induced apoptosis. TRAIL did not induce Bax expression and/or translocation from cytosol to mitochondria in the K562 cell line. However, 100 microM Z-VAD.fmk, a pan caspase inhibitor, completely blocked TRAIL-initiated mitochondrial alterations and cleavages of caspases and Bid. We propose that TRAIL-induced apoptosis in K562 cells is via Type I apoptotic signal pathway. Bax translocation is not essential for TRAIL-induced cytochrome c release and DeltaPsim collapse in the Type I cells.

8-Cl-adenosine mediated cytotoxicity and sensitization of T-lymphoblastic leukemia cells to TNFalpha-induced apoptosis is via inactivation of NF-kappaB.

These data show that 8-Cl-cAMP is cytotoxic to the lymphoblastic leukemia cell line CEM and its vinblastine selected multidrug resistant derivative, CEM/VLB100 although PKA was not involved in these effects. The cytotoxic effects of 8-Cl-cAMP was abrogated by cotreatment with either ADA or IBMX which indicated a degradation form of 8-Cl-cAMP was needed for this cytotoxicity. CEM and CEM/VLB100 cells displayed a notable sensitivity to 8-Cl-adenosine-induced growth inhibition and apoptosis. 8-Cl-adenosine increased the cytosolic levels of IkappaBalpha which prevented NF-kappaB nuclear translocation. 8-Cl-adenosine also prevented TNFalpha-induced IkB decay and NF-kappaB activation in CEM and CEM/VLB100 cells.

Apaf-1XL is an inactive isoform compared with Apaf-1L.

Apaf-1 plays a crucial role in the cytochrome c/dATP-dependent activation of caspase-9 and -3. We found that the human myeloid leukemic K562 cells were more resistant to cytochrome c-induced activation of caspase-9 and -3 in a cell-free system compared with the human T-lymphoblastic subclone CEM/VLB(100) cells. Apaf-1 cDNA sequencing revealed an additional insert of 11 aa between the CARD and CED-4 (ATPase) domains in K562 cells, which was identical to the sequence of Apaf-1XL. Immunoprecipitation of Apaf-1 with caspase-9 after a cell-free reaction demonstrated that Apaf-1XL in the K562 cell line showed a lower binding ability to caspase-9 compared with Apaf-1L protein. The resistance of K562 cells to cytochrome c-dependent apoptosis may be partly due to this Apaf-1XL form. These results suggest that the additional insert between CARD and CED-4 domains might affect Apaf-1 recruitment of caspase-9 during apoptosis.

Hydrogen peroxide and tumour necrosis factor-alpha induce NF-kappaB-DNA binding in primary human T lymphocytes in addition to T cell lines.

Reactive oxygen intermediates (ROIs), such as hydrogen peroxide (H2O2), have been implicated as second messengers in the activation of NF-kappaB by a variety of stimuli, including tumour necrosis factor-alpha (TNF-alpha). The aim of the present study was to examine the effects of ROIs on NF-kappaB activation in primary human CD3+ T lymphocytes and human peripheral blood mononuclear cells (PBMCs). For comparison purposes, Jurkat T cells (subclones JR and JE6.1) were also investigated. Cells were incubated in the presence of either H2O2 or TNF-alpha and nuclear proteins were extracted. NF-kappaB binding was assessed by electrophoretic mobility shift assays (EMSAs). The concentration of H2O2 required to activate NF-kappaB in human primary CD3+ T lymphocytes was as low as 1 microM. In contrast, much higher concentrations of H2O2 were required to activate NF-kappaB in PBMCs and in the JR subclone of Jurkat T cells. H2O2-induced NF-kappaB activation was not observed in the JE6.1 subclone of Jurkat T cells. NF-kappaB was activated by TNF-alpha in all four cell types tested. In PBMCs and Jurkat T cells (subclones JR and JE6.1), this activation could be inhibited by pre-treatment with the antioxidants, pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC). Our results support a role for ROIs in NF-kappaB-DNA binding in human primary T lymphocytes.

Generation of reactive oxygen species is not involved in idarubicin-induced apoptosis in human leukaemic cells.

The anthracycline antibiotic idarubicin (IDA) induces double-stranded DNA breaks, the generation of reactive oxygen species (ROS) and apoptosis in human leukaemic cells. It is unclear whether the generation of ROS is associated with the apoptotic process. Using the T-lymphoblastic leukaemic CEM cell line, we found that IDA-induced DNA breaks were correlated with final cell death. The reduction in mitochondrial membrane potential (Deltapsim) and the generation of ROS occurred simultaneously with IDA-induced activation of caspase-9 and caspase-3. Inhibition of caspases by a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) completely blocked IDA-induced reduction of Deltapsim, apoptosis and final cell death. Interestingly, ROS generation was significantly enhanced by Z-VAD-fmk. ROS generation was neither caspase dependent nor part of the apoptotic process. IDA-mediated reduction in Deltapsim is caspase dependent and is not a consequence of the generation of ROS. These results indicate that IDA-induced generation of ROS and apoptosis are separate events. Inhibition of caspases facilitates IDA-mediated generation of ROS.