Inhibitor of apoptosis proteins as therapeutic targets in multiple myeloma.

The inhibitor of apoptosis (IAP) proteins have a critical role in the control of apoptotic machinery, and has been explored as a therapeutic target. Here, we have examined the functional importance of IAPs in multiple myeloma (MM) by using a Smac (second mitochondria-derived activator of caspases)-mimetic LCL161. We observed that LCL161 was able to potently induce apoptosis in some MM cell lines but not in others. Examining the levels of X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein 1 (cIAP1) and cellular inhibitor of apoptosis protein 2 (cIAP2) post LCL161 treatment indicated clear downregulation of both XIAP activity and cIAP1 levels in both the sensitive and less sensitive (resistant) cell lines. cIAP2, however, was not downregulated in the cell line resistant to the drug. Small interfering RNA-mediated silencing of cIAP2 significantly enhanced the effect of LCL161, indicating the importance of downregulation of all IAPs simultaneously for induction of apoptosis in MM cells. LCL161 induced marked up regulation of the Jak2/Stat3 pathway in the resistant MM cell lines. Combining LCL161 with a Jak2-specific inhibitor resulted in synergistic cell death in MM cell lines and patient cells. In addition, combining LCL161 with death-inducing ligands clearly showed that LCL161 sensitized MM cells to both Fas-ligand and TRAIL.

Small angle neutron scattering on an absolute intensity scale and the internal surface of diatom frustules from three species of differing morphologies.

The internal nanostructure of the diatoms Cyclotella meneghiniana, Seminavis robusta and Achnanthes subsessilis was investigated using small angle neutron scattering (SANS) to examine thin biosilica samples, consisting of isotropic (powder) from their isolated cell walls. The interpretation of SANS data was assisted by several other measurements. The N2 adsorption, interpreted within the Branuer-Emmet-Teller isotherm, yielded the specific surface area of the material. Fourier transform infrared (FTIR) and Raman spectroscopy indicates that the isolated material is amorphous silica with small amounts of organic cell wall materials acting as a filling material between the silica particles. A two-phase (air and amorphous silica) model was used to interpret small angle neutron scattering data. After correction for instrumental resolution, the measurements on two SANS instruments covered an extended range of scattering vectors 0.0011 nm(-1) < q < 5.6 nm(-1), giving an almost continuous SANS curve over a range of scattering vectors, q, on an absolute scale of intensity for each sample. Each of the samples gave a characteristic scattering curve where log (intensity) versus log (q) has a -4 dependence, with other features superimposed. In the high-q regime, departure from this behaviour was observed at a length-scales equivalent to the proposed unitary silica particle. The limiting Porod scattering law was used to determine the specific area per unit of volume of each sample illuminated by the neutron beam. The Porod behaviour, and divergence from this behaviour, is discussed in terms of various structural features and the proposed mechanisms for the bio-assembly of unitary silica particles in frustules.

MRK003, a γ-secretase inhibitor exhibits promising in vitro pre-clinical activity in multiple myeloma and non-Hodgkin's lymphoma.

Notch-stimulated signaling cascade results in transcriptional regulation of genes involved in cell fate decision, apoptosis and proliferation and has been implicated in various malignancies. Here, we investigated the impact of MRK003, an inhibitor of this pathway, on myeloma and lymphoma cells. We first studied the expression patterns of notch receptors and ligands on multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL) cell lines. Next, we used a γ-secretase inhibitor, MRK003 to test the importance of notch-stimulated pathways in MM and NHL disease biology. We observed expression of notch receptors and ligands on MM and NHL cell lines. MRK003 treatment induced caspase-dependent apoptosis and inhibited proliferation of MM and NHL cell lines and patient cells. Examination of signaling events after treatment showed time-dependent decrease in levels of the notch intracellular domain, Hes1 and c-Myc. MRK003 downregulated cyclin D1, Bcl-Xl and Xiap levels in NHL cells and p21, Bcl-2 and Bcl-Xl in MM cells. In addition, MRK003 caused an upregulation of pAkt, indicating crosstalk with the PI3K/Akt pathway. We evaluated MRK003 in combination with Akt1/2 kinase inhibitor and observed synergy in killing MM and NHL cell lines examined.

Sorafenib, a dual Raf kinase/vascular endothelial growth factor receptor inhibitor has significant anti-myeloma activity and synergizes with common anti-myeloma drugs.

Multiple myeloma is characterized by increased bone marrow neovascularization driven in part by vascular endothelial growth factor (VEGF). In addition, the Ras/Raf/MEK/ERK pathway is critical for the proliferation of myeloma cells and is often upregulated. Sorafenib (Nexavar) is a novel multi-kinase inhibitor that acts predominantly through inhibition of Raf-kinase and VEGF receptor 2, offering the potential for targeting two important aspects of disease biology. In in vitro studies, sorafenib-induced cytotoxicity in MM cell lines as well as freshly isolated patient myeloma cells. It retained its activity against MM cells in co-culture with stromal cells or with interleukin-6, VEGF or IGF; conditions mimicking tumor microenvironment. Examination of cellular signaling pathways showed downregulation of Mcl1 as well as decreased phosphorylation of the STAT3 and MEK/ERK, as potential mechanisms of its anti-tumor effect. Sorafenib induces reciprocal upregulation of Akt phosphorylation; and simultaneous inhibition of downstream mTOR with rapamycin leads to synergistic effects. Sorafenib also synergizes with drugs such as proteasome inhibitors and steroids. In a human in vitro angiogenesis assay, sorafenib showed potent anti-angiogenic activity. Sorafenib, through multiple mechanisms exerts potent anti-myeloma activity and these results favor further clinical evaluation and development of novel sorafenib combinations.

ASAXS study on the formation of core-shell Ag/Au nanoparticles in glass.

Nanosized metal particles of various configurations embedded in surface regions of glass have great potential as nonlinear optical materials for photonic devices. We have prepared Ag/Au nanoparticles in core-shell configuration in soda-lime silicate glass by double-ion implantation and investigated their structural characteristics by anomalous small-angle x-ray scattering (ASAXS) and transmission electron microscopy. Measurements at x-ray energies slightly below the Au L(3) edge indicate the formation of bimetallic Ag/Au shells in some of the nanoparticles for high-dose ion implantation. An element-specific analysis of the ASAXS results allowed us not only to validate and quantify the core-shell structure, but simultaneously also the composition of the shells. Hollow nanoparticles were found for an Au-Ag implantation sequence, whereas an Ag-Au sequence generates a diluted core composition. The shift of the maximum position of optical absorption of the samples due to surface plasmon resonance of bimetallic nanoparticles, as monitored by optical spectroscopy, revealed the considerable influence of the respective particle configuration.

Simple 3D images from fossil and recent micromaterial using light microscopy.

Abstract We present a technique for extracting 3D information from small-scale fossil and Recent material and give a summary of other contemporary techniques for 3D methods of investigation. The only hardware needed for the here-presented technique is a microscope that can perform dark field and/or differential interference contrast with a mounted digital camera and a computer. Serial images are taken while the focus is successively shifted from the uppermost end of the specimen to the lowermost end, resulting in about 200 photographs. The data are then processed almost completely automatically by successive use of three freely available programs. Firstly, the stack of images is aligned by the use of CombineZM, which is used to produce a combined image with a high depth of field. Secondly, the aligned images are cropped and sharp edges extracted with the aid of ImageJ. Thirdly, although ImageJ is also capable of producing 3D representations, we preferred to process the image stack further using osirix as it has the facility to export various formats. One of the interesting export formats is a virtual Quicktime movie file (QTVR), which can be used for documentation, and stereo images can also be produced from this Quicktime VR. This method is easy to apply and can be used for documenting specimens in 3D (at least some aspects) without having to prepare them. Therefore, it is particularly useful as a safe method for documenting limited material, before using methods that may destroy the specimen of interest, or to investigate type material that cannot be treated with any preparatory technique. As light microscopes are available in most labs and free computer programs are easily accessible, this method can be readily applied.

Laser-Induced, Polarization Dependent Shape Transformation of Au/Ag Nanoparticles in Glass.

Bimetallic, initially spherical Ag/Au nanoparticles in glass prepared by ion implantation have been irradiated with intense femtosecond laser pulses at intensities still below the damage threshold of the material surface. This high-intensity laser processing produces dichroism in the irradiated region, which can be assigned to the observed anisotropic nanoparticle shapes with preferential orientation of the longer particle axis along the direction of laser polarization. In addition, the particle sizes have considerably been increased upon processing.

Magnetic field and pressure dependence of small angle neutron scattering in MnSi.

We report small angle neutron scattering of spontaneous and magnetic field aligned components of the helical spin polarization in MnSi for temperatures T down to 0.35 K, at pressures p up to 21 kbar, and magnetic field B up to 0.7 T. The parameter range of our study spans the first order transition between helical order and partial magnetic order at p{c}=14.6 kbar, which coincides with the onset of an extended regime of non-Fermi liquid resistivity. Our study suggests that MnSi above p{c} is not dominated by the remnants of the first order transition at p{c}, but that an unidentified mechanism favors stabilization of a new ground state other than helical order.

ABT-737, an inhibitor of Bcl-2 family proteins, is a potent inducer of apoptosis in multiple myeloma cells.

Disruption of pathways leading to programmed cell death plays a major role in most malignancies, including multiple myeloma (MM). ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-xL, preventing the sequestration of proapoptotic molecules and shifting the cell survival/apoptosis balance toward apoptosis induction. In this study, we show that ABT-737 is cytotoxic to MM cell lines, including those resistant to conventional therapies, and primary tumor cells. Flow cytometric analysis of intracellular levels of Bcl-2 family proteins demonstrates a clear inversion of the Bax/Bcl-2 ratio leading to induction of apoptosis. Activation of the mitochondrial apoptosis pathway was indicated by mitochondrial membrane depolarization and caspase cleavage. Additionally, several signaling pathways known to be important for MM cell survival are disrupted following treatment with ABT-737. The impact of ABT-737 on survival could not be overcome by the addition of interleukin-6, vascular endothelial growth factor or insulin-like growth factor, suggesting that ABT-737 may be effective in preventing the growth and survival signals provided by the microenvironment. These data indicate that therapies targeting apoptotic pathways may be effective in MM treatment and warrant clinical evaluation of ABT-737 and similar drugs alone or in combination with other agents in the setting of MM.

Thymoglobulin targets multiple plasma cell antigens and has in vitro and in vivo activity in multiple myeloma.

Multiple myeloma is characterized by the proliferation of clonal plasma cells that have a heterogeneous expression of various cell surface markers, precluding successful use of monoclonal antibodies for therapeutic targeting of the tumor cell. Thymoglobulin (rabbit-derived polyclonal anti-thymocyte globulin), by virtue of its method of preparation, contains antibodies against several B-cell and plasma cell antigens and offers an attractive option for immunotherapy of myeloma. Here, we demonstrate potent anti-myeloma activity of the rabbit anti-thymocyte globulin preparation Thymoglobulin in vitro and in vivo in an animal model of myeloma. Thymoglobulin was able to induce dose- and time-dependent apoptosis of several myeloma cell lines, including those resistant to conventional anti-myeloma agents. Importantly, the anti-myeloma activity was preserved even when myeloma cells were grown with different cytokines demonstrating the ability to overcome microenvironment-mediated resistance. Thymoglobulin induced apoptosis of freshly isolated primary myeloma cells from patients. Using a competitive flow cytometric analysis, we were able to identify the potential antigen targets for Thymoglobulin preparation. Finally, in a plasmacytoma mouse model of myeloma, Thymoglobulin delayed the tumor growth in a dose-dependent manner providing convincing evidence for continued evaluation of this agent in the clinic in patients with myeloma, either alone or in combination with other agents.

Expression of VEGF and its receptors by myeloma cells.

Angiogenesis or new vessel formation is an essential component in the growth and progression of neoplasms and there is growing evidence of its importance in hematological malignancies including multiple myeloma (MM). Vascular endothelial growth factor (VEGF) is believed to play a role in tumor angiogenesis. We studied the expression of VEGF and its receptors (VEGFR1 or Flt-1 and VEGFR2 or Flk-1/KDR) by myeloma cell lines and plasma cells isolated from patients, using different methods. VEGF expression by the plasma cells was demonstrated by immunohistochemistry in 18 of 20 patients with MM. Enzyme-linked immunosorbent assay demonstrated VEGF secretion in all six different myeloma cell lines studied. Five patient marrow samples and seven different myeloma cell lines were then studied for VEGF mRNA expression by reverse-transcriptase polymerase chain reaction (RT-PCR), which was positive in all. We further evaluated the expression of both VEGFR1 and VEGFR2 in different myeloma cell lines and five sorted myeloma bone marrow samples by RT-PCR. All the myeloma cell lines expressed VEGFR1 and three of the cell lines expressed VEGFR2. VEGFR1 expression was detected in all and VEGFR2 in all but one of the sorted marrow samples. Increased expression of VEGF by the myeloma cells taken in the context of the suspected prognostic value of marrow angiogenesis suggests a pathogenetic role for this cytokine and presence of its receptors on myeloma cells points toward an autocrine mechanism. Demonstration of the presence of VEGFR2 in our study provides a potential biological explanation for the preclinical activity observed with VEGFR2 inhibitors.

STAT-3 activation is required for normal G-CSF-dependent proliferation and granulocytic differentiation.

To investigate the role of signal transducer and activator of transcription (STAT) proteins in granulocyte colony-stimulating factor (G-CSF)-regulated biological responses, we generated transgenic mice with a targeted mutation of their G-CSF receptor (termed d715F) that abolishes G-CSF-dependent STAT-3 activation and attenuates STAT-5 activation. Homozygous mutant mice are severely neutropenic with an accumulation of immature myeloid precursors in their bone marrow. G-CSF-induced proliferation and granulocytic differentiation of hematopoietic progenitors is severely impaired. Expression of a constitutively active form of STAT-3 in d715F progenitors nearly completely rescued these defects. Conversely, expression of a dominant-negative form of STAT-3 in wild-type progenitors results in impaired G-CSF-induced proliferation and differentiation. These data suggest that STAT-3 activation by the G-CSFR is critical for the transduction of normal proliferative signals and contributes to differentiative signals.

T cell activation induces human osteoclast formation via receptor activator of nuclear factor kappaB ligand-dependent and -independent mechanisms.

In unstimulated conditions, osteoclast (OC) formation is regulated by stromal cell production of the key osteoclastogenic factors receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the mechanisms of accelerated osteoclastogenesis and bone loss characteristic of inflammatory conditions are poorly understood but appear to involve T cells. In addition, the mechanism by which OCs arise spontaneously in cultures of peripheral blood mononuclear cells in the absence of stromal cells or added cytokines remains unclear. Using a stromal cell free human osteoclast generating system, we investigated the ability of activated T cells to support osteoclastogenesis. We show that when activated by phytohemagglutinin-P (PHA), T cells (both CD4+ and CD8+) stimulate human OC formation in vitro. Although both soluble M-CSF and RANKL were detected in activated T cell supernatants, the presence of M-CSF was not essential for macrophage survival or RANKL-dependent osteoclast formation, suggesting that other soluble T cell-derived factors were capable of substituting for this cytokine. We also found that saturating concentrations of osteoprotegerin (OPG) failed to neutralize 30% of the observed OC formation and that T cell conditioned medium (CM) could superinduce osteoclastogenesis in cultures of purified monocytes maximally stimulated by RANKL and M-CSF. Together, these data suggest that activated T cells support osteoclastogenesis via RANKL-dependent and -independent mechanisms. Although not relevant for T cell-induced osteoclastogenesis, secretion of soluble M-CSF is a previously undescribed property of activated T cells.

B lymphocytes inhibit human osteoclastogenesis by secretion of TGFbeta.

The role of B lymphocytes in osteoclast (OC) formation is controversial, because both stimulatory and inhibitory effects of B-lineage cells on osteoclastogenesis and life span have been reported. In this study, we have investigated the effects of mature B cells on human osteoclastogenesis using cultures of peripheral blood stem cells (PBSC), a system that generates functional OCs in the absence of stromal cells. We report that B cells inhibit the formation of OCs and shorten the life span of mature OCs by secreting transforming growth factor beta (TGFbeta), a factor that induces apoptosis in these cells. The antiosteoclastogenic effects of B cells are abolished by addition of anti-TGFbeta antibody to osteoclast cultures and mimicked by treatment of B cell-deprived PBSC cultures with recombinant TGFbeta, thus confirming TGFbeta as the B cell produced antiosteoclastogenic activity. Thus, the ability of B cells to downregulate osteoclastogenesis by secretion of the apoptotic cytokine TGFbeta provides new insights into the ability of immune cells to regulate OC formation under basal and inflammatory conditions.

Allogeneic peripheral blood stem cell transplantation following CD34+ enrichment by density gradient separation.

GVHD is a significant cause of morbidity and mortality following allogeneic peripheral blood stem cell transplantation (AlloPBSC). CD34+ cell selection could reduce GVHD by negative selection of T cells. In an attempt to reduce the T cell content of alloPBSC we carried out a trial in which 11 patients with hematologic malignancies received alloPBSC from HLA-matched siblings following density gradient separation using an isotonic colloidal silica solution (BDS 60; Dendreon Corporation). Cyclosporine and methylprednisone were used for GVHD prophylaxis. The mean yield of CD34+ cells was 69 +/- 15.6% with a purity of 2.9 +/- 1.7%. The mean number of CD3+ cells infused was 1.0 +/- 1.2 x 107/kg, representing a 1.3 log depletion. A high risk of acute GVHD was observed: grade II-IV in 7/11 (64%) and grade III-IV GVHD in 5/11 (45%) patients. Nine of the 11 (82%) patients died with a median survival of 68 days. Cytokine expression in PBSC was compared pre and post processing. Interferon-gamma was detected only following density gradient separation while IL-8 expression increased 3- to 6-fold post processing. Therefore, processing with this device may augment production of pro-inflammatory cytokines. Bone Marrow Transplantation (2000) 25, 1223-1228.

Innovations in allogeneic stem-cell transplantation.

Allogeneic bone marrow transplantation (BMT) is associated with prolonged periods of neutropenia and thrombocytopenia, which can lead to severe infections and bleeding complications. Transplantation-related side effects might be ameliorated by use of cytokine-mobilized peripheral blood progenitor cells (PBPC) Instead of bone marrow. We have studied PBPC mobilization and transplantation in more than 150 patients with high-risk hematologic malignancies. Normal donors can be sufficiently mobilized with granulocyte colony-stimulating factor (G-CSF), with 91% of G-CSF-stimulated normal donors producing more than 2 x 10(6) CD34+ cells/kg by a single apheresis. The combination of G-CSF plus granulocyte-macrophage colony-stimulating factor (GM-CSF) was more effective than mobilization with G-CSF alone. A clear relationship was seen between numbers of resting CD34+ cells premobilization and numbers of PBPC collected by apheresis, indicating that resting CD34+ cells might be used to predict mobilization results and identify donors who could benefit from more effective mobilization regimens. Transplantation of G-CSF-mobilized PBPC was associated with a more rapid engraftment than that observed for BMT. While engraftment was safe and acute graft-versus-host disease (aGvHD) rates were not increased over BMT, chronic GvHD rates were higher after PBPC transplantation. An additional PBPC infusion on day +3 resulted in a further shortening of neutropenia and thrombocytopenia. Incorporation of these innovative approaches with "minimal" conditioning regimens has resulted in near-complete elimination of fever, neutropenia, thrombocytopenia, and the need for antibiotics and RBC and platelet transfusions after allogeneic transplantation.

Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene expression.

Investigation of the molecular basis of megakaryocyte (MK) and platelet biology has been limited by an inadequate source of genetically manipulable cells exhibiting physiologic MK and platelet functions. We hypothesized that ex vivo cultured MKs would exhibit agonist inducible glycoprotein (GP) IIb-IIIa activation characteristic of blood platelets and that these cultured MKs would be capable of transgene expression. Microscopic and flow cytometric analyses confirmed that human hematopoietic stem cells cultured in the presence of pegylated recombinant human MK growth and development factor (PEG-rHuMGDF) differentiated into morphologic and phenotypic MKs over 2 weeks. Cultured MKs expressed functional GPIIb-IIIa receptors as assessed by agonist inducible soluble fibrinogen and PAC1 binding. The specificity and kinetics of fibrinogen binding to MK GPIIb-IIIa receptors were similar to those described for blood platelets. The reversibility and internalization of ligands bound to MK GPIIb-IIIa also shared similarities with those observed in platelets. Cultured MKs were transduced with an adenoviral vector encoding green fluorescence protein (GFP) or beta-galactosidase (beta-gal). Efficiency of gene transfer increased with increasing multiplicities of infection and incubation time, with 45% of MKs expressing GFP 72 hours after viral infection. Transduced MKs remained capable of agonist induced GPIIb-IIIa activation. Thus, ex vivo cultured MKs (1) express agonist responsive GPIIb-IIIa receptors, (2) are capable of expressing transgenes, and (3) may prove useful for investigation of the molecular basis of MK differentiation and GPIIb-IIIa function.

A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)-deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8-stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR-deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR-deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR-deficient PMNs was significantly impaired, suggesting a defect in beta2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR-deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness.

[Positive and negative symptoms in chronic schizophrenic patients under maintenance therapy with flupenthixol decanoate for a twelve month perioid]. / Positiv- und Negativsymptomatik bei chronisch schizophrenen Patienten unter Erhaltungstherapie mit Flupentixol-Decanoat im 12-Monats-Verlauf.

62 chronic schizophrenics, who after a psychotic exacerbation were stabilized again, were randomized on 3 different maintenance dosages of flupentixol-decanoate and observed for 12 months. 15 (24.2%) patients had a relapse independently of the dose. 34 of the 47 patients without relapse had complete documentations of the follow-up. After 3 to 6 months there was an obvious and constant clinical improvement as assessed by various test inventories. The BPRS-scores for psychopathology, negative and positive symptoms improved significantly between 14 to 18%. There was also significant reduction of negative symptoms assessed by SANS (22%) as well in the severity of illness (CGI) and an amelioration of psychosocial functioning (GAS, Strauss-Carpenter). At the end of trial 26.4% of the patients had mild involuntary movements (AIMS), 23.5% were on antiparkinson-medication. It is concluded that there could be an indication for flupentixol-decanoate in the long term maintenance therapy especially of chronic schizophrenics with negative symptoms and problems of compliance under oral medication.