Fluoxetine in Progressive Multiple Sclerosis (FLUOX-PMS) : study protocol for a randomized controlled trial

Background: Currently available disease-modifying treatments acting by modifying the immune response are ineffective in progressive multiple sclerosis (MS), which is caused by a widespread axonal degeneration. Mechanisms suspected to be involved in this widespread axonal degeneration are reduced axonal energy metabolism, axonal glutamate toxicity, and reduced cerebral blood flow. Fluoxetine might theoretically reduce axonal degeneration in MS because it stimulates energy metabolism through enhancing glycogenolysis, stimulates the production of brain-derived neurotrophic factor, and dilates cerebral arterioles. The current document presents the protocol of a clinical trial to test the hypothesis that fluoxetine slows down the progressive phase of MS. Methods/Design: The FLUOX-PMS trial is a multi-center, randomized, controlled and double-blind clinical study. A total of 120 patients with the diagnosis of either secondary or primary progressive MS will be treated either by fluoxetine (40 mg daily) or placebo for a total period of 108 weeks. The primary endpoint is the time to confirmed disease progression defined as either at least a 20% increase in the timed 25-Foot Walk or at least a 20% increase in the 9-Hole Peg Test. Secondary endpoints include the Hauser ambulation index, cognitive changes, fatigue, magnetic resonance imaging of the brain, and in a small subgroup optical coherence tomography. Discussion: The FLUOX-PMS trial will gives us information as to whether fluoxetine has neuroprotective effects in patients with progressive MS

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5) on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system

MYELOPROLIFERATIVE DISEASES Clinical, pathological and molecular features of the chronic myeloproliferative disorders: MPD 2005 and beyond

Abstract The combined use of bone marrow histopathology, biomarkers and clinical features has the potential to diagnose, stage and distinguish early and overt stages of ET, PV and idiopathic myelofibrosis, that has an important impact on prognosis and treatment of MPD patients. As the extension of the PVSG and WHO for ET, PV and agnogenic myeloid metaplasia (AMM), a new set of European clinical and pathological (ECP) criteria clearly distinct true ET from early or latent PV mimicking true ET, overt and advanced polycythemia vera (PV), and from thrombocythemia associated with prefibotic, early fibrotic stages of chronic megakaryocytic granulocytic metaplasia (CMGM) or chronic idiopathic myelofibrosis (CIMF). Cases of atypical MPD and masked PV are usually overlooked by clinicians and pathologists. Bone marrow biopsy will not differentiate between post-PV myelofibrosis versus so-called classical agnogenic myeloid metaplasia. The recent discovery of the JAK2 V617F mutation can readily explain the trilinear megakaryocytic, erythroid and granulocytic proliferation in the bone marrow, but also the etiology of the platelet-mediated microvascular thrombotic complications at increased platelet counts and red cell mass in essential thrombocythemia and polycythemia vera

The PVSG/WHO versus the Rotterdam European clinical, molecular and pathological diagnostic criteria for the classification of myeloproliferative disorders and myeloproliferative neoplasms (MPD/MPN): From Dameshek to Georgii, Vainchenker and Michiels 1950-2018

The present article extends the PVSG-WHO criteria into a simplified set of Rotterdam and European Clinical, Molecular and Pathological (RCP/ECMP) criteria to diagnose and classify the myeloproliferative neoplasms (MPNs). The crude WHO criteria still miss the masked and early stages of ET and PV. Bone marrow histology has a near to 100% sensitivity and specificity to distinguish thrombocythemia in BCR/ABL positive CML and ET, and the myelodysplastic syndromes in RARS-T and 5q-minus syndrome from BCR/ABL negative thrombocythemias in myeloproliferative disorders (MPD). The presence of JAK2V617F mutation with increased erythrocytes above 6x1012/L and hematocrit (>0.51 males and >0.48 females) is diagnostic for PV obviating the need of red cell mass measurement. About half of WHO defined ET and PMF and 95% of PV patients are JAK2V617F positive. The combination of molecular marker screening JAK2V617F, JAK2 exon 12, MPL515 and CALR mutations and bone marrow pathology is 100% sensitive and specific for the diagnosis of latent, early and classical ECMP defined MPNs. The translation of WHO defined ET, PV and PMF into ECMP criteria have include the platelet count above 350 x109/l, mutation screening and bone marrow histology as inclusion criteria for thrombocythemia in various MPNs. According to ECMP criteria, ET comprises three distinct phenotypes of true ET, ET with features of early (“forme fruste” PV), and ET with a hypercellular erythrocythemic, megakaryocytic granulocytic myeloproliferation (EMGM or masked PV). The ECMP criteria clearly differentiate early erythrocythemic, prodromal and classical PV from congenital polycythemia and idiopathic or secondary erythrocytosis. The burden of JAK2V617F mutation in heterozygous ET and in homozygous PV is of major clinical and prognostic significance. JAK2 wild type MPL515 mutated normocellular ET and MF lack PV features in blood and bone marrow. JAK2/MPL wild type hypercellular ET associated with primary megakaryocytic granulocytic myeloproliferation (PMGM) is the third distinct CALR mutated MPN. The translation of WHO into ECMP criteria for the classification of MPNs have a major impact on prognosis assessment and best choice for first line non-leukemogenic approach to postpone potential leukemogenic myelopsuppressive agents as long as possible in ET, PV and PMGM patients

Primary myelofibrosis is not primary anymore since the discovery of MPL515 and CALR mutations as driver causes of mono-linear megakaryocytic and dual megakaryocytic granulocytic myeloproliferation and secondary myelofibrosis

Primary myelofibrosis (PMF) is a distinct clinicopathological myeloproliferatve disease (MPD) not preceded by any other MPD ET, PV, CML,... Combined use of bone marrow histology and increased erythrocyte counts above 5.8x1012/L can replace increased red cell mass at time of presentation as the pathognomonic clue for the correct diagnosis of hetero/homozygous or homozygous mutated PV. Erythrocyte counts are in the normal range below 5.8x1012/L in heterozygous JAK2V617F mutated ET and prodromal PV but above 5.8x1012/L in heterozygous-homozygous or homozygous mutated PV. The bone marrow cellularity and morphology in pre-fibrotic ET, prodromal PV and PV carrying the JAK2V617F mutation are overlapping showing clustered increase of large mature pleomorphic megakaryocytes (M) with no increase of cellularity (<60%) in ET. The bone marrow is hypercellular (60%-80%) due to increased erythropoiesis megakaryopoiesis (EM) in prodromal and classical PV and trilinear hypercellular (80%-100% due increased megakaryopoiesis, erythropoiesis and granulopoiesis (EMG) in advanced PV and masked PV. Bone marrow cellularity ranging from normal (<60%) in ET to increased erythropoiesis (EM) in prodromal PV to hypercellular (80-100%) in advanced PV and masked PV largely depends on increasing JAK2V617F mutation load from low to high on top of other biological MPN variables like constitutional symptoms during long-term follow-up. MPL515 mutated ET is featured by an increase of clustered small and giant megakaryocytes with hyper-lobulated staghorn-like nuclei in a normal cellular bone marrow. The third entity of pronounced JAK2/MPL wild type ET associated with primary megakaryocytic granulocytic myeloproliferation (PMGM) without PV features proved to be caused by calreticulin (CALR) mutation. CALR mutated thrombocythemia is characterized by dual proliferation of megakaryocytic and granulocytic bone marrow proliferation of dense clustered large to giant immature dysmorphic megakaryocytes with bulky (bulbous) hyperchromatic nuclei, which are not seen in MPL515-mutated Thrombocythemia and JAK2V617F-Thrombocythemia, prodromal PV and classical PV.&nbsp

Laboratory diagnosis and molecular classification of von Willebrand disease

A complete set of laboratory investigations, including bleeding time, PFA-100 closure times, factor VIII (FVIII) coagulant activity (FVIII:C), von Willebrand factor (VWF) ristocetin cofactor (VWF:RCo), collagen binding (VWF:CB), antigen (VWF:Ag) and propeptide (VWFpp), ristocetin-induced platelet aggregation (RIPA), multimeric analysis of VWF and the response of FVIII:C and VWF parameters to desmopressin (DDAVP), is necessary to fully diagnose all variants of von Willebrand disease (VWD) and to discriminate between type 1 and type 2 and between severe VWD type 1 and type 3. The response to DDAVP of VWF parameters is normal in pseudo VWD (mild VWF deficiency due to blood group O), in mild VWD type 1 and in carriers of recessive severe VWD type 1 and 3. The response to DDAVP is rather good but restricted followed by increased clearance in dominant type 1/2E, good but transient in mild type 2A group II, good for VWF:CB, with only poor response for VWF:RCo in 2M and 2U, poor in 2A group I, 2B, 2C and 2D, and very poor or non-responsive in severe recessive VWD type 1 and 3. Homozygosity or double heterozygosity for nonsense (null) mutations in the VWF gene result in recessive VWD type 3. The combination of a nonsense and missense mutation or of two missense mutations (homozygous or double heterozygous) may cause recessive severe VWD type 1. Recessive VWD type 2A subtype IIC (2C) is caused by homozygous or double heterozygous gene defects in the D1-D2 domain. Homozygosity or double heterozygosity for a FVIII binding defect of the VWF is the cause of recessive VWD type 2N (Normandy) characterized by low FVIII:C, mild or moderate VWF deficiency and normal VWF multimers. Dominant VWD type 1/2E is a mixed quantitative and qualitative multimerization defect caused by a heterozygous cysteine mutation in the D3 domain resulting in abnormal multimerization with a secretion and clearance defect of VWF not due to increased proteolysis. Dominant VWD type 1 Vicenza is a qualitative defect with normal secretion but rapid clearance with equally low levels of FVIII:C, VWF:Ag, VWF:RCo, VWF:CB and the presence of unusually large VWF multimers in plasma due to a specific mutation (R1205H) in the D3 domain. Dominant VWD type 2M and 2U are caused by loss-of-function mutations in the A1 domain resulting in quantitative/qualitative deficiencies with a selectively decreased platelet-dependent function with decreased VWF:RCo but normal VWF:CB, a relative decrease in large VWF multimers and the presence but relative loss of large VWF multimers. VWD type 2A and 2B show loss of large VWF multimers due to increased proteolysis. Dominant type 2A is caused by heterozygous missense mutations in the A2 domain. VWD type 2B is due to gain-of-function mutations in the A1 domain and differs from 2A by a normal VWF multimeric pattern in platelets and increased RIPA. DDAVP response curves and VWFpp/Ag ratios contribute to the diagnostic differentiation of VWD type 1 and 2. Rapid clearance of VWF after DDAVP with increased VWFpp/Ag ratios >10 appears to be diagnostic for VWD Vicenza. VWD type 1/2E due to the mutations in the D3 domain uniformly show increased VWFpp/Ag ratios ranging from 3.2 to 4.69 indicating clearance of the VWF/FVIII complex. Normal VWFpp/Ag ratios in mild VWD type 1 with mutations in the D1-D2 and the D4-B-C domains reflect a synthesis/secretion defect.status: publishe

The European Clinical, Molecular, and Pathological (ECMP) Criteria and the 2007/2008 Revisions of the World Health Organization for the Diagnosis, Classification, and Staging of Prefibrotic Myeloproliferative Neoplasms Carrying the JAK2V617F Mutation

OBJECTIVE: The prefibrotic stages of JAK2V617F essential thrombocythemia (ET) and JAK2V617F polycythemia vera (PV) can easily be diagnosed clinically without use of bone marrow biopsy histology. We assessed the 2008 WHO and European Clinical, Molecular, and Pathological (ECMP) criteria for the diagnosis of myeloproliferative neoplasms (MPNs). METHODS: Studied patients included 6 JAK2V617F-mutated ET and 4 PV patients during long-term follow-up in view of critical analysis of the literature. The bone marrow biopsy histology diagnosis without use of clinical data was PV in 7 (of which 3 were cases of ET with features of early prodromal PV) and classical PV in 4. RESULTS: The ECMP criteria distinguish 3 sequential phenotypes (1, 2, or 3) of JAK2V617F-mutated ET: normocellular ET-1; ET-2, with clinical and bone marrow features of PV (prodromal PV), and ET-3, with hypercellular dysmorphic megakaryocytic and granulocytic myeloproliferation (ET.MGM). The 3 patients with ET-2 or prodromal PV developed slow-onset PV after a follow-up of about 10 years. Bone marrow biopsy histology differentiates MPNs of various molecular etiologies from all variants of primary or secondary erythrocytoses and thrombocytoses with sensitivity and specificity of near 100%. CONCLUSION: Normocellular ET (WHO-ET), prodromal PV, and classical PV show overlapping bone marrow biopsy histology features with similar pleomorphic clustered megakaryocytes in the prefibrotic stages of JAK2V617F mutated MPN. Erythrocytes are below 6x1012/L in normocellular ET and prodromal PV, and are consistently above 6x1012/L in classical PV and at the time of transition from prodromal PV into classical PV. Red cell count at a cut-off level of 6x1012/L separates ET from PV and obviates the need for red cell mass measurement when bone marrow histology and JAK2V617F mutation screening are included in the diagnostic work-up of MPNs