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We have recently identi®ed an internal tandem duplication of the human Flt3 gene in approximately 20% of acute myeloid leukemia (AML) cases. In the present study, the wild-type and the mutant Flt3 genes were transfected into two IL-

An internal tandem duplication (ITD) of the FLT3 gene is found in nearly 20% of acute myeloid leukemia (AML) and 5% of myelodysplastic syndrome cases. Our serial studies on 51 samples with the FLT3 gene mutation indicated that the ITD was frequently (47/51) clustered in the tyrosine-rich stretch from codon 589 to 599 and rarely (3/51) in its downstream region, both of which are located within the juxtamembrane (JM) domain. One remaining sample had an insertion into the JM domain of nucleotides of unknown origin. To eludicate the biological relevance of the ITD or the insertion, we expressed various types of mutant FLT3 in Cos 7 cells. All mutant FLT3 studied were ligand-independently dimerized and their tyrosine residues were phosphorylated. The Y589 of FLT3 was essential for the phosphorylation in the wild FLT3, but a Y589F conversion did not affect the phosphorylation status of the mutant FLT3. These findings suggest that the elongation of the JM domain rather than increase of tyrosine residues causes gainof-function of FLT3. Thus, ITD is a novel modality of somatic mutation which activates its product. Since the DNA corresponding to codon 593 to 602 potentially forms a palindromic intermediate, we propose that a DNA-replication error might be associated with generating the ITD of the FLT3 gene.

RANTES (regulated on activation normal T cell expressed and secreted) is one of the natural ligands for the chemokine receptor CCR5 and potently suppresses in vitro replication of the R5 strains of HIV-1, which use CCR5 as a coreceptor. Previous studies showed that peripheral blood mononuclear cells or CD4 ؉ lymphocytes obtained from different individuals had wide variations in their ability to secrete RANTES. These findings prompted us to analyze the upstream noncoding region of the RANTES gene, which contains cis-acting elements involved in RANTES promoter activity, in 272 HIV-1-infected and 193 non-HIV-1-infected individuals in Japan. Our results showed that there were two polymorphic positions, one of which was associated with reduced CD4 ؉ lymphocyte depletion rates during untreated periods in HIV-1-infected individuals. This mutation, RANTES؊28G, occurred at an allele frequency of Ϸ17% in the non-HIV-1-infected Japanese population and exerted no inf luence on the incidence of HIV-1 infection. Functional analyses of RANTES promoter activity indicated that the RANTES؊28G mutation increases transcription of the RAN-TES gene. Taken together, these data suggest that the RANTES؊28G mutation increases RANTES expression in HIV-1-infected individuals and thus delays the progression of the HIV-1 disease.The chemokine receptor CCR5 is an essential coreceptor for the cellular entry of R5 strains (macrophage tropic͞non-syncytium-inducing strains) of HIV-1 (1-6), which predominate in the early stages of infection (7). During the course of infection, variants called X4 strains (T cell-line tropic͞ syncytium-inducing strains) emerge (1, 8-11), which use CXCR4 as a coreceptor (12). In vitro replication of R5 strains can be blocked by the ligands for CCR5, macrophage inflammatory peptide-1␣ and -1␤, and RANTES (regulated on activation normal T cell expressed and secreted; refs. 13 and 14), whereas that of X4 strains can be blocked by the CXCR4 ligands stromal cell derived factor-1␣ and -1␤ (15, 16).Mutations in HIV-1 coreceptors and their natural ligand genes have been shown to modify HIV-1 transmission and disease progression. Individuals homozygous for a 32-nt deletion in the CCR5 coding region were resistant to HIV-1 infection (17, 18), whereas heterozygosity delays disease progression (19,20). A single V-to-I substitution in the first transmembrane segment of CCR2, a minor coreceptor for dual tropic R5X4 strains (3, 5), has a significant impact on disease progression but not on HIV-1 transmission in cohorts of seroconverters (21,22). Finally, homozygosity of a single G-to-A mutation in the 3Ј noncoding region of the stromal cell derived factor-1␤ gene also showed a disease-retarding effect (23), although later studies could not confirm this effect (24,25).Among three natural CCR5 ligands, RANTES showed the highest potency to suppress in vitro replication of R5 strains of HIV-1 (13). Phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) or CD4 ϩ enriched lymphocytes obtained from different...

Previous research suggested that soluble human recombinant thrombomodulin may reduce mortality among patients with sepsis-associated coagulopathy. OBJECTIVE To determine the effect of human recombinant thrombomodulin vs placebo on 28-day all-cause mortality among patients with sepsis-associated coagulopathy. DESIGN, SETTING, AND PARTICIPANTS The SCARLET trial was a randomized, double-blind, placebo-controlled, multinational, multicenter phase 3 study conducted in intensive care units at 159 sites in 26 countries. All adult patients admitted to one of the participating intensive care units between October 2012 and March 2018 with sepsis-associated coagulopathy and concomitant cardiovascular and/or respiratory failure, defined as an international normalized ratio greater than 1.40 without other known etiology and a platelet count in the range of 30 to 150 × 10 9 /L or a greater than 30% decrease in platelet count within 24 hours, were considered for inclusion. The final date of follow-up was February 28, 2019. INTERVENTIONS Patients with sepsis-associated coagulopathy were randomized and treated with an intravenous bolus or a 15-minute infusion of thrombomodulin (0.06 mg/kg/d [maximum, 6 mg/d]; n = 395) or matching placebo (n = 405) once daily for 6 days. MAIN OUTCOME AND MEASURES The primary end point was 28-day all-cause mortality. RESULTS Among 816 randomized patients, 800 (mean age, 60.7 years; 437 [54.6%] men) completed the study and were included in the full analysis set. In these patients, the 28-day all-cause mortality rate was not statistically significantly different between the thrombomodulin group and the placebo group (106 of 395 patients [26.8%] vs 119 of 405 patients [29.4%], respectively; P = .32). The absolute risk difference was 2.55% (95% CI, −3.68% to 8.77%). The incidence of serious major bleeding adverse events (defined as any intracranial hemorrhage; life-threatening bleeding; or bleeding event classified as serious by the investigator, with administration of at least 1440 mL [typically 6 units] of packed red blood cells over 2 consecutive days) was 23 of 396 patients (5.8%) in the thrombomodulin group and 16 of 404 (4.0%) in the placebo group. CONCLUSIONS AND RELEVANCE Among patients with sepsis-associated coagulopathy, administration of a human recombinant thrombomodulin, compared with placebo, did not significantly reduce 28-day all-cause mortality.

scribed in frame, and coded mutant FLT3 with a long JM

Congenital macrothrombocytopenia (CMTP) is a heterogeneous group of rare platelet disorders characterized by a congenital reduction of platelet counts and abnormally large platelets, for which CMTP-causing mutations are only found in approximately half the cases. We herein performed whole-exome sequencing and targeted Sanger sequencing to identify mutations that cause CMTP, in which a dominant mode of transmission had been suspected but for which no known responsible mutations have been documented. In 13 Japanese CMTP-affected pedigrees, we identified six (46%) affected by ACTN1 variants cosegregating with CMTP. In the entire cohort, ACNT1 variants accounted for 5.5% of the dominant forms of CMTP cases and represented the fourth most common cause in Japanese individuals. Individuals with ACTN1 variants presented with moderate macrothrombocytopenia with anisocytosis but were either asymptomatic or had only a modest bleeding tendency. ACTN1 encodes α-actinin-1, a member of the actin-crosslinking protein superfamily that participates in the organization of the cytoskeleton. In vitro transfection experiments in Chinese hamster ovary cells demonstrated that altered α-actinin-1 disrupted the normal actin-based cytoskeletal structure. Moreover, transduction of mouse fetal liver-derived megakaryocytes with disease-associated ACTN1 variants caused a disorganized actin-based cytoskeleton in megakaryocytes, resulting in the production of abnormally large proplatelet tips, which were reduced in number. Our findings provide an insight into the pathogenesis of CMTP.

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