Lenalidomide, rituximab (R2), and ixazomib for frontline treatment of high risk follicular and indolent non-Hodgkin lymphoma.

Lenalidomide and rituximab (R2) is an effective frontline treatment for patients with indolent B-cell non-Hodgkin lymphoma (iNHL). We investigated the safety and efficacy of addition of the proteasome inhibitor ixazomib to R2 for treatment of iNHL through a phase I/II clinical trial for high-risk patients. Twenty patients were enrolled, 18 were treated. The target dose of ixazomib 4 mg weekly was achieved during dose escalation. The most common treatment-related adverse events (AEs) were low grade gastrointestinal, rash, neuropathy, and myalgia/arthralgia. There were 33% grade 2 and 17% grade 3 infections. With median follow-up of 5.2 years, four patients discontinued treatment due to lymphoma progression. Best overall response rate (ORR) was 61.2% [55.6% CR, 5.6% PR): 22.2% had stable disease and 16.7% had disease progression. Kaplan-Meier estimates of progression free and overall survival (OS) were 73% and 87% at 36 months, respectively. R2 can safely be combined with ixazomib for treatment-naïve iNHL patients.

Heteroatom-Doped Carbon Quantum Dots and Polymer Composite as Dual-Mode Nanoprobe for Fluorometric and Colorimetric Determination of Picric Acid.

Oxygen- and nitrogen-heteroatom-doped, water-dispersible, and bright blue-fluorescent carbon dots (ON-CDs) were prepared for the selective and sensitive determination of 2,4,6-trinitrophenol (picric acid, PA). ON-CDs with 49.7% quantum yield were one-pot manufactured by the reflux method using citric acid, d-glucose, and ethylenediamine precursors. The surface morphology of ON-CDs was determined by scanning transmission electron microscopy, high-resolution transmission electron microscopy, dynamic light scattering, Raman, infrared, and X-ray photoelectron spectroscopy techniques, and their photophysical properties were estimated by fluorescence spectroscopy, UV-vis spectroscopy, fluorescence lifetime measurement, and 3D-fluorescence excitation-emission matrix analysis. ON-CDs at an average particle size of 3.0 nm had excitation/emission wavelengths of 355 and 455 nm, respectively. With the dominant inner-filter effect- and hydrogen-bonding interaction-based static fluorescence quenching phenomena supported by ground-state charge-transfer complexation (CTC), the fluorescence of ON-CDs was selectively quenched with PA in the presence of various types of explosives (i.e., 2,4,6-trinitrotoluene, tetryl, 1,3,5-trinitroperhydro-1,3,5-triazine, 1,3,5,7-tetranitro-1,3,5,7-tetraazacyclooctane, pentaerythritol tetranitrate, 3-nitro-1,2,4-triazole-5-one, and TATP-hydrolyzed H2O2). The analytical results showed that the emission intensity varied linearly with a correlation coefficient of 0.9987 over a PA concentration range from 1.0 × 10-9 to 11.0 × 10-9 M. As a result of ground-state interaction (H-bonding and CTC) of ON-CDs with PA, an orange-colored complex was formed different from the characteristic yellow color of PA in an aqueous medium, allowing naked-eye detection of PA. The detection limits for PA with ON-CDs were 12.5 × 10-12 M (12.5 pM) by emission measurement and 9.0 × 10-10 M (0.9 nM) by absorption measurement. In the presence of synthetic explosive mixtures, common soil cations/anions, and camouflage materials, PA was recovered in the range of 95.2 and 102.5%. The developed method was statistically validated against a reference liquid chromatography coupled to tandem mass spectrometry method applied to PA-contaminated soil. In addition, a poly(vinyl alcohol)-based polymer composite film {PF(ON-CDs)} was prepared by incorporating ON-CDs, enabling the smartphone-assisted fluorometric detection of PA.

Ethylenediamine-bound magnetite nanoparticles as dual function colorimetric sensor having charge transfer and nanozyme activity for TNT and tetryl detection.

A reusable, low-cost, and convenient ethylenediamine (EDA)-bound magnetite nanoparticles (MNPs)-based colorimetric sensor has been developed for dual function colorimetric determination of nitroaromatic explosives such as TNT and tetryl. Colorimetric detection of analytes may occur through two independent routes: (1) nano-Fe3O4- EDA- NH2 as σ-donor may interact with the σ- and π-acceptor aromatic-poly(NO2) groups to produce a colored charge-transfer (CT) complex; (2) nano-Fe3O4-EDA-NH2 as a Fenton-type nanozyme may generate reactive species that comprise hydroxyl radicals (•OH) with H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to a blue-colored diimine (oxTMB-TMB) CT complex, where this color is bleached with TNT/tetryl because of donor-acceptor interactions between the explosive -NO2 groups and the -NH2 group of Fe3O4-EDA nanoparticles of restricted nanozyme activity. Both methods can quantify TNT well below the EPA recommended TNT residential screening level in soil, LOD being in the micromolar range. As EDA was covalently bound to MNPs, the same sensor can be separately reused six times for TNT and eight times for tetryl determination, using method (1). Common metal ions, anions, energetic materials, several camouflage materials, and soil components such as humates did not interfere with the nanosensor performance for TNT and tetryl. The combination of charge-transfer and nanozyme ability of Fe3O4- EDA-NH2 nanoparticles may bring a new approach to dual function colorimetric sensor design. To the best of our knowledge, this is the first dual function colorimetric sensor for TNT and tetryl using the same nanoparticles as sensing elements in two different detection systems involving either formation or bleaching of colored species. The proposed colorimetric sensor can determine nitroaromatic explosives in two different ways: method-1 for TNT and tetryl sensing with EDA-MNPs relies on the donor-acceptor interaction between the electron-deficient nitroaromatics and electron-rich amine groups covalently functionalized on MNPs to produce an absorbance at 512 nm. In method-2, EDA-MNPs having nanozyme activity react with H2O2 to form reactive species that can oxidize TMB to its blue-colored charge-transfer (CT) complex, where TNT and tetryl addition may partially inhibit the nanozyme activity of EDA-MNPs and cause color bleaching (decrement of 650 nm absorbance) by disrupting the CT complex formed from TMB. This is the first dual function colorimetric sensor for nitro explosives uniquely combining charge-transfer and nanozyme ability of EDA-Fe3O4 nanoparticles in the same nano-sensor.

A colorimetric probe based on 4-mercaptophenol and thioglycolic acid-functionalized gold nanoparticles for determination of phytic acid and Fe(III) ions.

As a first of its kind, we developed a highly sensitive colorimetric nanoprobe for phytic acid (PA) and Fe(III) ion detection based on 4-mercaptophenol (4MP) and thioglycolic acid (TGA)-functionalized gold nanoparticles {AuNPs@(4MP-TGA)}. AuNPs were easily derivatized by 4MP and TGA through -SH binding to gold. Fe(III) ions possibly are bound first to the phenolate groups of 4MP-AuNPs, and further coordinated several nanoparticles via the carboxylate groups of TGA-AuNPs to cause aggregation, resulting in a red-to-purple color change and a bathochromic shift in the SPR absorption band of the nanoprobe. With the addition of PA to the AuNPs@(4MP-TGA)-Fe(III) system, the aggregated particles were released due to strong complex formation between Fe(III) and PA, resulting in a restoration of the color (purple-to-red) and of the SPR band to the original 520 nm wavelength maximum. Thus, the 650-nm absorption is attenuated and the 520-nm band is enhanced upon PA-Fe(III) chelation. This means that the absorption ratio A650/A520 is an indication of Fe(III) whereas the reverse ratio A520/A650 of the PA content of complex samples. The limits of detection (LOD) of the AuNPs@(4MP-TGA) were 1.0 µM for Fe(III) ions and 0.15 µM for PA. Phytic acid extracted from bean grains was determined with the proposed probe, yielding good recoveries. In addition, common metal ions, anions, and several biomolecules did not show an adverse effect on the nanoprobe performance for ferric ions and phytate. The developed method was statistically validated against a LC-MS/MS literature method. Graphical abstract Mercaptophenolate (4MP)- and thioglycolic acid (TGA)-functionalized gold nanoparticles were prepared as nanoprobes to detect Fe(III) ions through nanoparticle aggregation accompanied by red-to-purple color shift. The same nanoprobe determined phytic acid in food through disaggregation of Fe(III)-aggregated nanoparticles by strong Fe(III)-phytate chelation and restoration of solution color from purple to red.

Pazopanib effective for bevacizumab-unresponsive epistaxis in hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) most commonly manifests with nasal mucosal telangiectasias, and vascular endothelial growth factor (VEGF) plays a significant role in this angiodysplasia. We describe a patient with HHT with epistaxis recalcitrant to several endonasal procedures and six cycles of intravenous bevacizumab, for which he was dependent on iron infusions and packed red blood cells transfusions. He then started pazopanib at 100 mg with dramatic improvements in epistaxis and normalization of hemoglobin and iron levels, without replenishment needs for 12 months. This is the first report on the efficacy of pazopanib with high selectivity for abrogating VEGF receptor-2 signaling in HHT, and needs to be explored further. Laryngoscope, 128:2234-2236, 2018.

Phase I trial of the combination of flavopiridol and imatinib mesylate in patients with Bcr-Abl+ hematological malignancies.

PURPOSE: Imatinib is an inhibitor of the Bcr-Abl tyrosine kinase; however, resistance is common. Flavopiridol, a cyclin-dependent kinase (CDK) inhibitor, down-regulates short-lived anti-apoptotic proteins via inhibition of transcription. In preclinical studies, flavopiridol synergizes with imatinib to induce apoptosis. We investigated this novel combination regimen in patients with Bcr-Abl(+) malignancies. METHODS: In a phase I dose-escalation study, imatinib was administered orally daily, and flavopiridol by 1 h intravenous infusion weekly for 3 weeks every 4 weeks. Adults with chronic myelogenous leukemia or Philadelphia chromosome-positive acute leukemia were eligible. Patients were divided into two strata based on peripheral blood and bone marrow blast counts. The primary objective was to identify the recommended phase II doses for the combination. Correlative pharmacokinetic and pharmacodynamic studies were also performed. RESULTS: A total of 21 patients received study treatment. Four dose levels were evaluated before the study was closed following the approval of the second-generation Bcr-Abl tyrosine kinase inhibitors (TKIs). Five patients responded, including four sustained responses. Four patients had stable disease. All but one responder, and all patients with stable disease had previously been treated with imatinib. One patient had a complete response sustained for 30 months. Changes in expression of phospho-Bcr/Abl, -Stat5, and Mcl-1 were monitored. No major pharmacokinetic interaction was observed. CONCLUSIONS: This is the first study to evaluate the combination of a CDK inhibitor and a TKI in humans. The combination of flavopiridol and imatinib is tolerable and produces encouraging responses, including in some patients with imatinib-resistant disease.

Sacroiliac joint dysfunction.

AIM: Sacroiliac joint dysfunction is a disorder presenting with low back and groin pain. It should be taken into consideration during the preoperative differential diagnosis of lumbar disc herniation, lumbar spinal stenosis and facet syndrome. MATERIAL AND METHODS: Four cases with sacroiliac dysfunction are presented. The clinical and radiological signs supported the evidence of sacroiliac dysfunction, and exact diagnosis was made after positive response to sacroiliac joint block. RESULTS: A percutaneous sacroiliac fixation provided pain relief in all cases. The mean VAS scores reduced from 8.2 to 2.2. CONCLUSION: It is concluded that sacroiliac joint dysfunction diagnosis requires a careful physical examination of the sacroiliac joints in all cases with low back and groin pain. The diagnosis is made based on positive response to the sacroiliac block. Sacroiliac fixation was found to be effective in carefully selected cases.

Sacroplasty: report of three cases.

AIM: Sacral stress fractures are rare fractures presenting themselves with low back and groin pain. These fractures can be treated effectively using sacroplasty. MATERIAL AND METHODS: The clinical and radiological data of three cases that underwent sacroplasty for sacral stress fractures were reviewed. The pain severity was assessed using the VAS system. The radiological investigation was performed using sacral CT and MRI. RESULTS: The sacroplasty procedure was performed in three female cases with sacral stress fractures resistant to conservative treatment. There was history of minor trauma in all cases. The diagnosis was performed using CT and MRI. The sacroplasty procedure was performed using the short-axis technique. The preoperative VAS score reduced from 8.5 to 2.3 postoperatively. CONCLUSION: It is concluded that sacroplasty is an effective and safe procedure in the treatment of the sacral stress fractures.

A phase 2 trial of immunotherapy with mitumprotimut-T (Id-KLH) and GM-CSF following rituximab in follicular B-cell lymphoma.

We evaluated the efficacy and safety of patient-specific immunotherapy with mitumprotimut-T idiotype keyhole limpet hemocyanin and granulocyte-monocyte colony-stimulating factor (GM-CSF) following rituximab in patients with follicular B-cell lymphoma. Patients with previously untreated or relapsed/refractory CD20+ follicular lymphoma received 4 weekly infusions of rituximab and those with a complete response (CR), partial response (PR), or stable disease received mitumprotimut-T and GM-CSF injections subcutaneously. Courses were given monthly for 6 doses, every 2 months for 6 doses, and then every 3 months until disease progression. Computed tomography scans were obtained every 3 to 6 months and reviewed centrally. The primary endpoint was event-free survival (EFS). Among 103 patients treated with rituximab, 92 (54 relapsed/refractory and 38 previously untreated) received mitumprotimut-T/GM-CSF; median age was 53 years, 91% had stage III to IV disease, and 59% had failed earlier therapy. The premitumprotimut-T objective response rate was 47% (2 CRs, 41 PRs). During the mitumprotimut-T treatment phase, 16 patients converted to CR resulting in an overall objective response rate of 60% (18 CRs, 37 PRs). Median EFS was 15.2, 20.8, and 13.5 months for all, treatment-naive, and relapsed/refractory disease patients, respectively. Anti-Id cellular immune responses were detected in 13 of 18 (72%) patients and humoral immune responses in 17 of 83 (20%) patients. Adverse events were usually mild-to-moderate. The most common adverse event was injection site reactions. Mitumprotimut-T/GM-CSF-induced anti-Id cellular immune responses in most patients. The occurrence of late CRs and favorable EFS suggested a clinical benefit of active immunotherapy and led to a placebo-controlled phase 3 trial.

Phase I trial of fludarabine, bortezomib and rituximab for relapsed and refractory indolent and mantle cell non-Hodgkin lymphoma.

Based on the hypothesis that bortezomib may potentiate fludarabine activity by inhibiting DNA repair, we designed a phase I trial using this combination with rituximab in patients with relapsed and refractory indolent and mantle cell non-Hodgkin lymphoma. Twenty-four patients were enrolled. Non-Hodgkin lymphoma subtypes included 12 patients with follicular lymphoma, four with marginal zone lymphoma, three with lymphoplasmacytic lymphoma, three with mantle cell lymphoma and two with small lymphocytic/chronic lymphocytic leukaemia. Fludarabine and bortezomib were escalated in cohorts of three patients. Rituximab was added to the maximum tolerated dose of fludarabine and bortezomib and added significant dose-limiting myelosuppression. The maximum tolerated dose was fludarabine 25 mg/m(2) on days 1-3, bortezomib 1.3 mg/m(2) on days 1, 4, 8, 11, with rituximab 375 mg/m(2) on day 1 administered every 21 d. Clinical responses were observed in 11 patients, five of whom were refractory to their most recent treatment regimen. Six additional patients had stable disease for a median of 10 months (range 4-30+). Cumulative myelosuppression and neuropathy was observed. The combination of fludarabine, bortezomib, and rituximab appears to be an active regimen with manageable toxicity for relapsed NHL.

SDF-1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction.

Stem cell transplantation at the time of acute myocardial infarction (AMI) improves cardiac function. Whether the improved cardiac function results from regeneration of cardiac myocytes, modulation of remodeling, or preservation of injured tissue through paracrine mechanisms is actively debated. Because no specific stem cell population has been shown to be optimal, we investigated whether the benefit of stem cell transplantation could be attributed to a trophic effect on injured myocardium. Mesenchymal stem cells secrete SDF-1 and the interaction of SDF-1 with its receptor, CXCR4, increases survival of progenitor cells. Therefore, we compared the effects of MSC and MSC engineered to overexpress SDF-1 on cardiac function after AMI. Tail vein infusion of syngeneic MSC and MSC:SDF-1 1 day after AMI in the Lewis rat led to improved cardiac function by echocardiography by 70.7% and 238.8%, respectively, compared with saline controls 5 wk later. The beneficial effects of MSC and MSC:SDF-1 transplantation were mediated primarily through preservation, not regeneration of cardiac myocytes within the infarct zone. The direct effect of SDF-1 on cardiac myocytes was due to the observation that, between 24 and 48 h after AMI, SDF-1-expressing MSC increased cardiac myocyte survival, vascular density (18.2+/-4.0 vs. 7.6+/-2.3 vessels/mm2, P<0.01; SDF-1:MSC vs. MSC), and cardiac myosin-positive area (MSC: 49.5%; mSC:SDF-1: 162.1%) within the infarct zone. There was no evidence of cardiac regeneration by the infused MSC or endogenous cardiac stem cells based on lack of evidence for cardiac myocytes being derived from replicating cells. These results indicate that stem cell transplantation may have significant beneficial effects on injured organ function independent of tissue regeneration and identify SDF-1:CXCR4 binding as a novel target for myocardial preservation.

Peritransplant use of ultraviolet-B irradiation (UV-B) therapy is detrimental to allogeneic stem cell transplantation outcome.

Whole-body UV-B phototherapy has been used for the treatment of graft-versus-host disease (GVHD) of the skin and has systemic immunosuppressive and tolerogenic effects. We hypothesized that whole-body UV-B therapy would improve donor engraftment and decrease the incidence and severity of GVHD that is associated with decreased intensity allogeneic hematopoietic stem cell transplantation. This study tested the feasibility of using UV-B phototherapy that was initiated before grafting and continued until engraftment to determine its effect on transplantation outcome. Eight patients (median age, 55.5 years; range, 32-65 years) with hematologic malignancies were included. Allogeneic peripheral blood stem cells were obtained from matched related (n=5) or matched unrelated (n=3) donors. Conditioning regimen was fludarabine 30 mg/m2 intravenously for 5 days, cyclophosphamide 1 g/m2/d intravenously for 2 days, and equine antithymocyte globulin 30 mg/kg/d for 2 days. GVHD prophylaxis included cyclosporine, methylprednisolone, and escalating doses of narrowband UV-B (311 nm) according to skin tolerance, 3 days a week, from 10 days before to 28 days after transplantation. The conditioning regimen and the UV-B therapy were well tolerated. Two patients received all 14 prescribed UV-B treatments (cumulative doses of 2000 and 3260 mJ/cm2, respectively) and 6 patients received 8 to 13 treatments with a cumulative dose range of 528-3465 mJ/cm2. There was a rapid decrease in epidermal CD1a+ cells by day of transplantation. Myeloid engraftment was rapid. One patient had secondary engraftment failure at 3 months and another had mixed chimerism at day 100. Seven of 8 patients developed severe acute GVHD (grade III, n=5; grade IV, n=2). Six had skin involvement, 5 had gastrointestinal involvement, and 1 had liver involvement. Four patients died (2 from sepsis, 1 from acute GVHD, and 1 from chronic GVHD). Four patients are alive (130-287 days), 3 with extensive chronic GVHD. We conclude that extended peritransplant UV-B therapy at the standard minimally erythemogenic dose is detrimental to the outcome of allogeneic stem cell transplantation. It is unclear how UV-B at this immunsuppressive dose might have altered skin and systemic cytokine and immune cell compositions in the host and increased GVHD- and treatment-related mortalities. Different UV-B dose and schedules should be further explored. However, although other phototherapeutic modalities may be effective against GVHD, extended UV-B therapy should not be used during early phases of decreased conditioning allogeneic transplantation.

A perspective for the selection of surgical approaches in patients with upper thoracic and cervicothoracic junction instabilities.

OBJECTIVE: To reach the upper thoracic vertebrae, a number of extensive approaches have been proposed. The purpose of this study is to provide a clear perspective for the selection of surgical approaches in patients who undergo vertebral body resection, reconstruction, and stabilization for upper thoracic and cervicothoracic junction instabilities. METHODS: Seventeen patients with upper thoracic or cervicothoracic junction (C7-T6) instability underwent surgery between January 1999 and May 2004. All patients presented with pain and/or neurological deficits. The causes of instabilities were 10 traumas and 7 pathological fractures. The approach chosen was primarily dictated by 3 factors including (1) type of injury, (2) level of lesion, and (3) time of admission. Ventral surgical approach was performed to all pathological and traumatic fractures causing anterior spinal cord compression. Level of lesion determined the selection of the type of ventral surgical approach, namely, supramanubrial, transmanubrial, or lateral transthoracic. On the other hand, combined (anterior and posterior) approach was performed to all late admitted trauma patients. RESULTS: Twelve anterior, 2 combined (anterior and posterior), and 3 posterior approaches were performed in this study. Anterior approaches included 3 transmanubrial, 5 upper lateral transthoracic, and 4 supramanubrial cervical dissection procedures for decompression, fusion, and plate-screw fixation depending on the levels of the lesion. The mean follow-up period was 18 months, ranging from 10 to 58 months. Nonunion or instrument-related complications were not observed. The postoperative neurological conditions were statistically significantly better than the preoperative ones (P = .003). CONCLUSION: Consideration of the type of injury, level of lesion, and time of admission can provide a perspective for the selection of side of surgical approach for this transitional part of the spinal column. This study also suggests that supramanubrial cervical approach achieves sufficient exposure up to T2, transmanubrial approach for T3, and lateral transthoracic approach below T3.

The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro but does not decrease apoptosis under serum deprivation.

BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) can be used for myocardial repair following myocardial infarction. Increased expression of stromal cell-derived factor-1 (SDF-1) by an ischemic myocardium attracts CXCR4+ stem cells toward it. CXCR4, the receptor for SDF-1, is important in the migration, homing, and survival of hematopoietic stem cells. Although low levels of CXCR4 expression were found in minor subpopulations of cultured MSCs, most MSCs do not express CXCR4. To optimize the migration and survival of human MSCs, we expressed the CXCR4 gene in these cells using retroviral transduction. MATERIALS AND METHODS: We isolated and cultured MSCs from healthy volunteers and transduced them with a retroviral vector containing either CXCR4 and green fluorescent protein (GFP; CXCR4/GFP vector) or GFP alone (control vector). Flow cytometry confirmed successful transduction and GFP and CXCR4 expression. We used a transwell migration system to study MSC migration to SDF-1. We used Annexin V and propidium iodide stains to assess cell survival before and after the survival challenge. RESULTS: Flow cytometry showed that, on average, 83.4+/-17.7% of transduced MSCs expressed CXCR4. Compared with control MSCs, MSCs transduced with CXCR4 showed significantly more migration toward SDF-1, threefold greater at 3 h and more than fivefold greater at 6 h. Mesenchymal stem cells transduced with CXCR4 showed no significant difference in survival under normal to serum-deprived growth conditions. CONCLUSION: Mesenchymal stem cells can be efficiently transduced to express CXCR4, and transduced MSCs migrate rapidly toward SDF-1. CXCR4 expression does not render survival advantage to MSCs under serum-deprived conditions.

Randomised comparison of two B-cell purging protocols for patients with B-cell non-Hodgkin lymphoma: in vivo purging with rituximab versus ex vivo purging with CliniMACS CD34 cell enrichment device.

We investigated the feasibility, safety and efficacy of two B-cell purging methods in patients with CD20+ non-Hodgkin lymphoma (NHL) receiving autologous stsem cell transplantation. Myeloid and immune recoveries between the methods were compared. Twenty-seven patients were randomised to either in vivo purging with rituximab or ex vivo purging by CD34+ cell selection. Both purging methods were efficient at eliminating B-cells in infusates. When compared with in vivo purging, ex vivo purging was associated with CD34+ cell loss and delayed median neutrophil (10 d vs. 11 d) and platelet (12.5 d vs. 17 d) count recoveries. Lymphocyte recovery was similar in both groups, but immunoglobulin recovery was delayed after in vivo purging. Late-infectious complications were few in both arms. At a median follow-up of 27 months, 2-year probabilities of event-free survival (EFS) rates were 81% for in vivo purging and 76% for ex vivo purging (P = 0.66). When compared with 53 unpurged patients, all 27 purged patients had improved 3-year probabilities of overall survival (89% vs. 70%, P = 0.014) and a trend for improved EFS (78% vs. 57%, P = 0.075). In conclusion, although both purging methods were feasible and safe, rituximab purging was superior as it did not impair CD34+ cell mobilisation and was associated with faster myeloid recovery. Further studies are needed to determine whether rituximab purging is more effective than the use of unpurged autografts.

Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive T cells.

Human bone marrow-derived mesenchymal cells (MSCs) are precursors of nonhematopoietic mesenchymal cells of the bone marrow microenvironment. MSCs were shown to inhibit alloreactive T lymphocytes, but the mechanism and mediators of this effect are not fully understood. Here we describe a novel interaction between blood monocytes and bone marrow-derived, culture-expanded MSCs, which results in inhibition of T-lymphocyte activation. We found that CD14+ monocytes from blood activate MSCs to secrete inhibitory molecules that lead to inhibition of alloreactive T cells. This cellular communication is not contact-dependent, but rather is mediated by soluble factors that include interleukin (IL)-1beta. MSC-mediated inhibition of alloreactive T lymphocytes is associated with downregulation of activation markers CD25, CD38, and CD69 detected both in CD4+ and CD8+ T lymphocytes. The cytokines secreted by MSCs that mediate T-cell inhibition include transforming growth factor-beta1, but not IL-10. The interaction between blood monocytes and the MSCs represents a unique immune regulatory paradigm that can potentially be exploited in clinic.

N-benzoylstaurosporine (PKC412) inhibits Akt kinase inducing apoptosis in multiple myeloma cells.

Multiple myeloma is a clonal malignancy of plasma cells that invariably progresses to a chemoresistant state. The PI3K/Akt pathway mediates signals downstream of several growth factors involved in myeloma pathogenesis, and constitutive activation of Akt was observed in myeloma cells. We now report that a staurosporine derivative, N-benzoylated staurosporine or PKC412, induces cell death in myeloma cell lines (RPMI8226S, U266, MM1S and MM1R) with loss of mitochondrial membrane potential Delta psi m, caspase 3 and PARP cleavage. ZVAD.fmk, but not interleukin-6, rescued these cells from PKC412 effects. Upstream of the mitochondria, PKC412 inhibited Bad phosphorylation and attenuated Akt kinase activity by suppressing its phosphorylation on serine residue in its activation loop. Reduced phosphorylation of downstream Akt substrates GSK3 alpha/beta and FKHR was also noted. Stable transfection of 8226S cells with constitutively active Akt (8226S-myAkt) partially protected against PKC412 cytotoxicity. Primary myeloma cells isolated from refractory myeloma patients (n=4), were equally sensitive to PKC412 treatment. More importantly, PKC412 did not affect CFU-GM or BFU-E colony formation. In summary, our results demonstrate that PKC412 suppresses Akt kinase activation and induces apoptosis in myeloma cell lines, as well as primary resistant cells. PKC412 is an appropriate candidate for novel treatment protocols for multiple myeloma.

Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients.

Mesenchymal stem cells (MSCs) are found in a variety of tissues, including human bone marrow; secrete hematopoietic cytokines; support hematopoietic progenitors in vitro; and possess potent immunosuppressive properties. We hypothesized that cotransplantation of culture-expanded MSCs and hematopoietic stem cells (HSCs) from HLA-identical sibling donors after myeloablative therapy could facilitate engraftment and lessen graft-versus-host disease (GVHD); however, the safety and feasibility of this approach needed to be established. In an open-label, multicenter trial, we coadministered culture-expanded MSCs with HLA-identical sibling-matched HSCs in hematologic malignancy patients. Patients received either bone marrow or peripheral blood stem cells as the HSC source. Patients received 1 of 4 study-specified transplant conditioning regimens and methotrexate (days 1, 3, and 6) and cyclosporine as GVHD prophylaxis. On day 0, patients were given culture-expanded MSCs intravenously (1.0-5.0 x 10(6)/kg) 4 hours before infusion of either bone marrow or peripheral blood stem cells. Forty-six patients (median age, 44.5 years; range, 19-61 years) received MSCs and HLA-matched sibling allografts. MSC infusions were well tolerated, without any infusion-related adverse events. The median times to neutrophil (absolute neutrophil count > or = 0.500 x 10(9)/L) and platelet (platelet count > or = 20 x 10(9)/L) engraftment were 14.0 days (range, 11.0-26.0 days) and 20 days (range, 15.0-36.0 days), respectively. Grade II to IV acute GVHD was observed in 13 (28%) of 46 patients. Chronic GVHD was observed in 22 (61%) of 36 patients who survived at least 90 days; it was extensive in 8 patients. Eleven patients (24%) experienced relapse at a median time to progression of 213.5 days (range, 14-688 days). The probability of patients attaining disease- or progression-free survival at 2 years after MSC infusion was 53%. Cotransplantation of HLA-identical sibling culture-expanded MSCs with an HLA-identical sibling HSC transplant is feasible and seems to be safe, without immediate infusional or late MSC-associated toxicities. The optimal MSC dose and frequency of administration to prevent or treat GVHD during allogeneic HSC transplantation should be evaluated further in phase II clinical trials.

High-dose carmustine, etoposide, and cisplatin for autologous stem cell transplantation with or without involved-field radiation for relapsed/refractory lymphoma: an effective regimen with low morbidity and mortality.

Over a 10-year period (January 1993 to October 2002), 101 relapsed or refractory non-Hodgkin lymphoma patients were treated at our center with high-dose chemotherapy and autologous transplantation. The median patient age was 54 years (range, 25-70 years). Thirty-two patients had indolent (low-grade), 42 had aggressive (intermediate-grade), and 27 had very aggressive (high-grade) non-Hodgkin lymphoma. Thirty-six patients had primary refractory disease, 20 had a chemoresistant relapse, 35 patients had a chemosensitive relapse, and 10 patients were "initial high risk" patients. The median number of prior chemotherapy regimens was 2 (range, 1-5). The preparative regimen (BEP) was bischloroethylnitrosourea (BCNU) 600 mg/m 2 , etoposide 2400 mg/m 2 , and Platinol (cisplatin) 200 mg/m 2 given intravenously over 5 days. Within 3 weeks before transplantation, 70 patients received involved-field radiotherapy (IFR) 20 Gy to sites of currently active (>2 cm) or prior bulky (>5 cm) disease. Most patients (n = 93) received mobilized peripheral blood stem cells (median CD34 + cell dose, 6.7 x 10 6 /kg). Median neutrophil (>500/microL) and platelet (>20 000/microL, untransfused) recoveries were 11 days (range, 7-19 days) and 14 days (range, 7-36 days), respectively. At a median follow-up of 41 months (range, 4 to 118 months) for survivors, Kaplan-Meier 5-year probabilities of overall survival (OS) and disease-free survival (DFS) were 58.6% and 51.1%, respectively. Four patients (4%) died within 30 days of stem cell infusion (1 pulmonary embolism, 2 septicemias with multiorgan failure, and 1 progressive lymphoma). Two patients (2%) developed interstitial pneumonitis most likely secondary to high-dose BCNU. Three cases (3%) of secondary acute myelogenous leukemia occurred. On multivariate analysis, age (<60 or > or =60 years), histologic grade (low versus intermediate or high), the use of IFR, and chemotherapy response at baseline did not affect OS or DFS. Of 70 patients given IFR, 27 relapsed: 10 (37%) within and 17 (63%) outside the radiation field. The use of IFR did not affect either OS or DFS, probably because IFR was offered to patients with bulky or chemoresistant disease. BEP with or without IFR is a highly effective and well-tolerated regimen in the relapsed/refractory lymphoma setting. It has low morbidity and transplant-related mortality and a low incidence (3%) of posttransplantation malignancy.