A prospective, randomized, controlled trial of autologous haematopoietic stem cell transplantation for aggressive multiple sclerosis: a position paper.

BACKGROUND: Haematopoietic stem cell transplantation (HSCT) has been tried in the last 15 years as a therapeutic option in patients with poor-prognosis autoimmune disease who do not respond to conventional treatments. Worldwide, more than 600 patients with multiple sclerosis (MS) have been treated with HSCT, most of them having been recruited in small, single-centre, phase 1-2 uncontrolled trials. Clinical and magnetic resonance imaging outcomes from case series reports or Registry-based analyses suggest that a major response is achieved in most patients; quality and duration of response are better in patients transplanted during the relapsing-remitting phase than in those in the secondary progressive stage. OBJECTIVES: An interdisciplinary group of neurologists and haematologists has been formed, following two international meetings supported by the European and American Blood and Marrow Transplantation Societies, for the purpose of discussing a controlled clinical trial, to be designed within the new scenarios of evolving MS treatments. CONCLUSIONS: Objectives of the trial, patient selection, transplant technology and outcome assessment were extensively discussed. The outcome of this process is summarized in the present paper, with the goal of establishing the background and advancing the development of a prospective, randomized, controlled multicentre trial to assess the clinical efficacy of HSCT for the treatment of highly active MS.

Survival, and time to an advanced disease state or progression, of untreated patients with moderately severe multiple sclerosis in a multicenter observational database: relevance for design of a clinical trial for high dose immunosuppressive therapy with autologous hematopoietic stem cell transplantation.

Despite prolonged survival, patients with multiple sclerosis (MS) experience considerable morbidity, which adversely impacts quality of life. To assess the risk-benefit of a clinical trial of high dose immunosuppressive therapy with autologous hematopoietic stem cell transplantation for MS, we sought to determine the natural history of the disease in a comparison group of untreated patients. We identified 285 individuals with 2132 combined observation years (median: 5.6 years; 5th to 95th percentile: 1-21 years), with Expanded Disability Status Scale (EDSS) scores of 3.0-5.5 at baseline observation. Disease-related mortality was zero at five years, 5.4% at 10 years, and 22% at 15 years (40 patients contributing to the data point; 95% confidence interval: 4-32%). Risk for progression to advanced disability, defined as an EDSS score of 8, was very low for the subgroup with a baseline EDSS score of 3-3.5; however, for those with a baseline EDSS score of 4-5.5, 3% had advanced disability after two years, 5% after three years, 6% after four years, 12% after five years, and 40% after 10 years. The estimated probability of disease progression, defined as an increase in EDSS score by > or = 1.0 sustained for at least 180 days, was 5% after one year, 14% after two years, 22% after three years, 38% after five years, 57% after 10 years, and >80% after 20 years of observation. The relevance of these features to the design of the clinical trial is discussed.

Multiple sclerosis conference synopsis and discussion: cellular therapy for treatment of autoimmune diseases (October 2005).

At a conference held in October 2005, participants presented studies on high dose immunosuppression with hematopoietic cell transplant (HCT) for multiple sclerosis (MS), including neuroimmunological and magnetic resonance imaging (MRI) mechanistic approaches, clinical registry reports, and ongoing or newly-designed protocols. A discussion panel considered questions on how to define success, timing of controlled clinical trials, difficulty in patient recruitment, and future direction of high dose therapy.

Persistence of recipient plasma cells and anti-donor isohaemagglutinins in patients with delayed donor erythropoiesis after major ABO incompatible non-myeloablative haematopoietic cell transplantation.

Delayed donor erythropoiesis and pure red-cell aplasia (PRCA) complicate major-ABO mismatched non-myeloablative allogeneic stem-cell transplantation. To characterize these events, we analysed red-cell serology and chimaerism in lymphohaematopoietic lineages, including plasma cells and B cells, in 12 consecutive major-ABO incompatible transplants following cyclophosphamide/fludarabine-based conditioning. Donor erythropoiesis was delayed to more than 100 days in nine (75%) patients including six (50%) who developed PRCA. During PRCA, all patients had persistent anti-donor isohaemagglutinins and recipient plasma cells (5-42%), while myeloid and T cells were completely donor in origin. In contrast, B-cell chimaerism was frequently full-donor when significant anti-donor isohaemagglutinins persisted. Four patients with early mixed haematopoietic chimaerism and the prolonged presence of anti-donor isohaemagglutinins and recipient plasma cells developed delayed-onset (>100 days post-transplant) red cell transfusion dependence and PRCA after myeloid chimaerism converted from mixed to full donor. These findings confirm that donor-erythropoiesis is impacted by temporal disparities in donor immune-mediated eradication of recipient lymphohaematopoietic cells during major-ABO incompatibility and suggest that plasma cells are relatively resistant to graft-versus-host haematopoietic effects.

Delayed donor red cell chimerism and pure red cell aplasia following major ABO-incompatible nonmyeloablative hematopoietic stem cell transplantation.

Delayed donor red cell engraftment and pure red cell aplasia (PRCA) are well-recognized complications of major ABO-incompatible hematopoietic stem cell transplantation (SCT) performed by means of myeloablative conditioning. To evaluate these events following reduced-intensity nonmyeloablative SCT (NST), consecutive series of patients with major ABO incompatibility undergoing either NST (fludarabine/cyclophosphamide conditioning) or myeloablative SCT (cyclophosphamide/high-dose total body irradiation) were compared. Donor red blood cell (RBC) chimerism (initial detection of donor RBCs in peripheral blood) was markedly delayed following NST versus myeloablative SCT (median, 114 versus 40 days; P <.0001) and strongly correlated with decreasing host antidonor isohemagglutinin levels. Antidonor isohemagglutinins declined to clinically insignificant levels more slowly following NST than myeloablative SCT (median, 83 versus 44 days; P =.03). Donor RBC chimerism was delayed more than 100 days in 9 of 14 (64%) and PRCA occurred in 4 of 14 (29%) patients following NST, while neither event occurred in 12 patients following myeloablative SCT. Conversion to full donor myeloid chimerism following NST occurred significantly sooner in cases with, compared with cases without, PRCA (30 versus 98 days; P =.008). Cyclosporine withdrawal appeared to induce graft-mediated immune effects against recipient isohemagglutinin-producing cells, resulting in decreased antidonor isohemagglutinin levels and resolution of PRCA following NST. These data indicate that significantly delayed donor erythropoiesis is (1) common following major ABO-incompatible NST and (2) associated with prolonged persistence of host antidonor isohemagglutinins. The clinical manifestations of these events are affected by the degree and duration of residual host hematopoiesis.

Histologically discordant lymphomas with B-cell and T-cell components.

We describe the clinical, histologic, immunophenotypic, and genotypic features of five cases of histologically discordant lymphomas with B-cell and T-cell components. Three patients presented with B-cell lymphoma; T-cell lymphoma subsequently developed. One patient presented with T-cell lymphoma; B-cell lymphoma subsequently developed. One patient presented with synchronous B-cell and T-cell lymphomas. There were three men and two women. The median age at the initial diagnosis of lymphoma was 66 years. The mean interval between the development of the two lymphomas was 83 months. All patients died of disease. The mean survival was 96 months after the initial diagnosis of lymphoma and 14 months after the diagnosis of the histologically discordant lymphoma. Epstein-Barr virus was found in two cases--the B-cell lymphoma in the patient who presented with synchronous lymphomas, and the subsequent T-cell lymphoma in one of the patients who presented with B-cell lymphoma. Based on the results of immunophenotypic and genotypic analyses, these cases likely represent the occurrence of two distinct lymphoid neoplasms rather than histologic progression of the same neoplastic clone. Furthermore, a subset of these cases are Epstein-Barr virus-associated.

Sex cord tumor with annular tubules associated with endometriosis of the fallopian tube.

Sex cord tumor with annular tubules (SCTAT) is a distinctive ovarian sex cord-stromal tumor. The authors describe a case of SCTAT arising outside the ovaries, in the fallopian tube. The authors are aware of only one other case of extra-ovarian SCTAT, in an umbilical hernia sac in an adult woman. The case reported was an incidental finding in a hysterectomy specimen, obtained because of carcinoma in situ of the uterine cervix in a 32-year-old woman. The patient is alive and disease-free two years after surgery. The 1-cm tumor in the right fallopian tube was composed of typical ring-shaped tubules encircling hyalinized basement membrane-like material. In addition, glands and stroma of endometriosis were intimately admixed with the nests of tumor. The implications of these findings for the origin of SCTAT and ovarian sex cord structures are discussed.

Adenocarcinoid tumor of the periampullary region: a novel duodenal neoplasm presenting as biliary tract obstruction.

An adenocarcinoid tumor of the duodenum, similar to those arising in the appendix, is reported. The tumor presented as a periampullary mass causing pancreaticobiliary obstruction. The microscopic features were typical of adenocarcinoid with diffuse infiltration of mucosa, submucosa, and smooth muscle by tubuloglandular structures lined by enteroendocrine and goblet cells. Signet ring cells were also present. It is desirable to distinguish adenocarcinoids from adenocarcinomas and typical carcinoids in this setting as, by analogy with appendiceal tumors, adenocarcinoids may be expected to have an intermediate prognosis.

Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function.

We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has approximately 0.3 mol Pi/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol Pi/mol 18,000-D light chain. MLCP, which was partially purified, selectively removes phosphate from the 18,000-D light chain but not from the heavy chain of Dictyostelium myosin. Phosphatase-treated Dictyostelium myosin has less than or equal to 0.01 mol Pi/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to greater than or equal to 0.96 mol Pi/mol 18,000-D light chain by incubation with MLCK and ATP. We found myosin thick filament assembly to be independent of the extent of 18,000-D light-chain phosphorylation when measured as a function of ionic strength. However, actin-activated Mg2+-ATPase activity of Dictyostelium myosin was found to be directly related to the extent of phosphorylation of the 18,000-D light chain. MLCK-treated myosin moved in an in vitro motility assay (Sheetz, M. P., and J. A. Spudich, 1983, Nature (Lond.), 305:31-35) at approximately 1.4 micron/s whereas phosphatase-treated myosin moved only slowly or not at all. The effects of phosphatase treatment on the movement were fully reversed by subsequent treatment with MLCK.

Cross-linking of actin filament networks by self-association and actin-binding macromolecules.

We have used low shear falling ball viscometry to measure the effects of actin purity, solution conditions, and cross-linking macromolecules on the formation of actin filament networks. Removal of minor contaminants from conventional muscle actin (Spudich, J. A., and Watt, S. (1971) J. Biol. Chem. 246, 4866-4871) by gel filtration (MacLean-Fletcher, S. and Pollard, T.D. (1980c) Biochem. Biophys. Res. Commun. 96, 18-27), greatly promotes the interaction of the filaments. Purified actin filaments form a gel (apparent viscosity greater than 12,000 cp) at approximately 2 mg/ml whereas approximately 12 mg/ml of conventional actin filaments have a viscosity of less than 400 cp. The apparent viscosity of the filaments depends on pH and the concentration of monovalent and divalent cations. The viscosity of purified action filaments is more sensitive to such variables when compared to that of conventional actin filaments. Together these experiments suggest that actin filament self-associations contribute to the stabilization of actin gels. A number of basic macromolecules, including aldolase, histones, lysozyme, polylysine, and RNase A can bind to and crosslink conventional actin filaments to form a gel. Since it is unlikely that all of these molecules are bound to actin in vivo, experimental approaches in addition to viscometry and sedimentation must be applied to prove that a given protein functions as an actin cross-linking protein in the cell.

The interaction of actin filaments with microtubules and microtubule-associated proteins.

Purified actin and microtubule proteins polymerized together form a gel, while mixtures of actin with tubulin polymers lacking microtubule-associated proteins (MAPs) have low viscosities close to the sum of the viscosities of the constituents. Mixtures of actin and MAPs also have high viscosities. Our interpretation of these observations was that there is interaction of actin filaments and microtubules which is mediated by MAPs (Griffith, L. M., and Pollard, T. D. (1978) J. Cell Biol. 78, 958-965). We report here further evidence for this interaction. 1) Actin filaments and microtubules can form gels at physiological ionic strength providing the anion is glutamate rather than chloride. Both glutamate and chloride inhibit actin-MAPs interaction, but this is compensated for in glutamate where the microtubules are longer than in chloride. 2) The low shear viscosity of mixtures of isolated MAPs and actin filaments is enhanced by acidic pH and inhibited by high ionic strength. 3) MAPs can be fractionated to yield four different fractions with actin cross-linking activity: a subset of high molecular weight MAPs, purified "MAP-2" and two different fractions of tau polypeptides. 4) We have reconstituted a gel from actin, purified tubulin, and whole MAPs, but have not yet been successful with actin, purified tubulin, and any single purified MAP.

Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins.

We have used low shear viscometry and electron microscopy to study the interaction between pure actin filaments and microtubules. Mixtures of microtubules having microtubule-associated proteins (MAPs) with actin filament have very high viscosities compared with the viscosities of the separate components. MAPs themselves also cause a large increase in the viscosity of actin filaments. In contrast, mixtures of actin filaments with tubulin polymers lacking MAPs have low viscosities, close to the sum of the viscosities of the separate components. Our interpretation of these observations is that there is an interaction between actin filaments and microtubules which requires MAPs. This interaction is inhibited by ATP and some related compounds. Electron micrographs of thin sections through mixtures of actin and microtubules show numerous close associations between the two polymers which may be responsible for their high viscosity.

Diurnal periodicity of uridine uptake by Hymenolepis diminuta.

A diurnal pattern in the uptake of uridine was displayed by the rat cestode Hymenolepis diminuta. No periodicity in the uptake of uracil was observed over a 48-hr period. A high level of uridine uptake occurred at 6 PM. when 10-day-old worms were in a posterior location in the intestine of rats maintained on a 6 PM.-6 AM. dark cycle-feeding regime, while low levels of uptake were correlated with an anteriad location at 6 AM. The lowest levels of uridine uptake were recorded at noon. Coincubation with thymine caused a stimulation of uridine uptake at midnight, 6 AM., and noon when uridine's transport rate in the absence of thymine was low. Stimulation was not demonstrable when uridine's transport rate was at its highest at 6 PM. Preincubation with uridine did not alter the diurnal uridine uptake pattern. This diurnal phenomenon is an important consideration essential to future studies on transport in parasitic and other organisms.