Although amyloid ␤ (A␤) oligomers are presumed to cause synaptic and cognitive dysfunction in Alzheimer's disease (AD), their contribution to other pathological features of AD remains unclear. To address the latter, we generated APP transgenic mice expressing the E693⌬ mutation, which causes AD by enhanced A␤ oligomerization without fibrillization. The mice displayed age-dependent accumulation of intraneuronal A␤ oligomers from 8 months but no extracellular amyloid deposits even at 24 months. Hippocampal synaptic plasticity and memory were impaired at 8 months, at which time the presynaptic marker synaptophysin began to decrease. Furthermore, we detected abnormal tau phosphorylation from 8 months, microglial activation from 12 months, astrocyte activation from 18 months, and neuronal loss at 24 months. These findings suggest that A␤ oligomers cause not only synaptic alteration but also other features of AD pathology and that these mice are a useful model of A␤ oligomer-induced pathology in the absence of amyloid plaques.

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The online version of this article contains a supplementary appendix. BackgroundThe diagnosis of myelodysplastic syndromes is not always straightforward when patients lack specific diagnostic markers, such as blast excess, karyotype abnormality, and ringed sideroblasts. Design and MethodsWe designed a flow cytometry protocol applicable in many laboratories and verified its diagnostic utility in patients without those diagnostic markers. The cardinal parameters, analyzable from one cell aliquot, were myeloblasts (%), B-cell progenitors (%), myeloblast CD45 expression, and channel number of side scatter where the maximum number of granulocytes occurs. The adjunctive parameters were CD11b, CD15, and CD56 expression (%) on myeloblasts. Marrow samples from 106 control patients with cytopenia and 134 low-grade myelodysplastic syndromes patients, including 81 lacking both ringed sideroblasts and cytogenetic aberrations, were prospectively analyzed in Japan and Italy. ResultsData outside the predetermined reference range in 2 or more parameters (multiple abnormalities) were common in myelodysplastic syndromes patients. In those lacking ringed sideroblasts and cytogenetic aberrations, multiple abnormalities were observed in 8/26 Japanese (30.8%) and 37/55 Italians (67.3%) when the cardinal parameters alone were considered, and in 17/26 Japanese (65.4%) and 42/47 Italians (89.4%) when all parameters were taken into account. Multiple abnormalities were rare in controls. When data from all parameters were used, the diagnostic sensitivities were 65% and 89%, specificities were 98% and 90%, and likelihood ratios were 28.1 and 8.5 for the Japanese and Italian cohorts, respectively. ConclusionsThis protocol can be used in the diagnostic work-up of low-grade myelodysplastic syndromes patients who lack specific diagnostic markers, although further improvement in diagnostic power is desirable.Key words: myelodysplastic syndromes, flow cytometry, diagnosis.Citation: Ogata K, Della Porta MG, Malcovati L, Picone C, Yokose N, Matsuda A, Yamashita T, Tamura H, Tsukada J, and Dan K. Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study. Haematologica 2009;94:1066-1074. doi:10.3324/haematol.2009 This is an open-access paper. Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study

During disease progression in myelodysplastic syndromes (MDS), clonal blasts gain a more aggressive nature, whereas nonclonal immune cells become less efficient via an unknown mechanism. Using MDS cell lines and patient samples, we showed that the expression of an immunoinhibitory molecule, B7-H1 (CD274), was induced by interferon-␥ (IFN␥) and tumor necrosis factor-␣ (TNF␣) on MDS blasts. This induction was associated with the activation of nuclear factor-B (NF-B) and nearly completely blocked by an NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC). B7-H1 ؉ MDS blasts had greater intrinsic proliferative capacity than B7-H1 ؊ MDS blasts when examined in various assays. Furthermore, B7-H1 ؉ blasts suppressed T-cell proliferation and induced T-cell apoptosis in allogeneic cocultures. When fresh bone marrow samples from patients were examined, blasts from high-risk MDS patients expressed B7-H1 molecules more often compared with those from low-risk MDS patients. Moreover, MDS T cells often overexpressed programmed cell death 1 (PD-1) molecules that transmit an inhibitory signal from B7-H1 molecules. Taken IntroductionB7-H1 (CD274), which was identified by us as a costimulatory molecule, plays a crucial role in T-cell regulation in various immune responses. 1,2 B7-H1 molecules deliver a costimulatory signal through an unknown receptor on naive T cells. [1][2][3] They also deliver an inhibitory signal to activated T cells through programmed cell death 1 (PD-1) molecules, 4 which are a type I transmembrane protein belonging to the CD28 receptor family and were originally identified in T cells undergoing apoptosis. 5 B7-H1 expression is detected not only on antigenpresenting cells but also on activated T cells and some tumor cells (ie, renal cell, colon, breast, and lung carcinoma, and Hodgkin lymphoma). [6][7][8][9][10] Rodent data suggest that B7-H1 molecules on tumor cells deliver negative signals through PD-1 and other receptors on tumorspecific cytotoxic T lymphocytes and inhibit antitumor immune responses. 11,12 Consistent with those data, it was reported that in patients with renal cell carcinoma and breast cancer, patients whose tumor cells expressed B7-H1 had a poor prognosis. 9,13 In a mouse leukemia model in which mice were immunized with irradiated DA1-3b leukemia cells and then challenged with live DA1-3b cells, only leukemia cells expressing high levels of B7-H1 survived for a long period. Moreover, these cells gained tolerance to specific cytotoxic T lymphocytemediated killing. 14 Therefore, B7-H1 molecules on leukemia cells may be associated with immune evasion in this model.Myelodysplastic syndromes (MDS) are clonal hematologic stem cell disorders characterized by cytopenias, excessive apoptosis of hematopoietic cells, and a high risk of progression to acute myeloid leukemia (AML). In MDS, various immune abnormalities, including lymphopenia and T-cell dysfunction, have been reported, 15-17 although data on B7-related molecules, in particular B7-H1, are lacking. With disease progression, that is, with i...

IMPORTANCEAn unmet need remains for safe and efficacious treatments for Duchenne muscular dystrophy (DMD). To date, there are limited agents available that address the underlying cause of the disease.OBJECTIVE To evaluate the safety, tolerability, and efficacy of viltolarsen, a novel antisense oligonucleotide, in participants with DMD amenable to exon 53 skipping. DESIGN, SETTING, AND PARTICIPANTSThis phase 2 study was a 4-week randomized clinical trial for safety followed by a 20-week open-label treatment period of patients aged 4 to 9 years with DMD amenable to exon 53 skipping. To enroll 16 participants, with 8 participants in each of the 2 dose cohorts, 17 participants were screened.

Both mislocalization of TDP-43 and downregulation of RNA-editing enzyme ADAR2 colocalize in the motor neurons of amyotrophic lateral sclerosis patients, but how they are linked is not clear. Here we demonstrate that activation of calpain, a Ca 2 þ -dependent cysteine protease, by upregulation of Ca 2 þ -permeable AMPA receptors generates carboxyterminal-cleaved TDP-43 fragments and causes mislocalization of TDP-43 in the motor neurons expressing glutamine/arginine site-unedited GluA2 of conditional ADAR2 knockout (AR2) mice that mimic the amyotrophic lateral sclerosis pathology. These abnormalities are inhibited in the AR2res mice that express Ca 2 þ -impermeable AMPA receptors in the absence of ADAR2 and in the calpastatin transgenic mice, but are exaggerated in the calpastatin knockout mice. Additional demonstration of calpain-dependent TDP43 fragments in the spinal cord and brain of amyotrophic lateral sclerosis patients, and high vulnerability of amyotrophic lateral sclerosis-linked mutant TDP43 to cleavage by calpain support the crucial role of the calpain-dependent cleavage of TDP43 in the amyotrophic lateral sclerosis pathology.

GluR2 is a subunit of the AMPA receptor, and the adenosine for the Q/R site of its pre-mRNA is converted to inosine (A-to-I conversion) by the enzyme called adenosine deaminase acting on RNA 2 (ADAR2). Failure of A-to-I conversion at this site affects multiple AMPA receptor properties, including the Ca 2ϩ permeability of the receptor-coupled ion channel, thereby inducing fatal epilepsy in mice (Brusa et al., 1995; Feldmeyer et al., 1999). In addition, inefficient GluR2 Q/R site editing is a disease-specific molecular dysfunction found in the motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients (Kawahara et al., 2004). Here, we generated genetically modified mice (designated as AR2) in which the ADAR2 gene was conditionally targeted in motor neurons using the Cre/loxP system. These AR2 mice showed a decline in motor function commensurate with the slow death of ADAR2-deficient motor neurons in the spinal cord and cranial motor nerve nuclei. Notably, neurons in nuclei of oculomotor nerves, which often escape degeneration in ALS, were not decreased in number despite a significant decrease in GluR2 Q/R site editing. All cellular and phenotypic changes in AR2 mice were prevented when the mice carried endogenous GluR2 alleles engineered to express edited GluR2 without ADAR2 activity (Higuchi et al., 2000). Thus, loss of ADAR2 activity causes AMPA receptor-mediated death of motor neurons.

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