Yuji Nakamachi

miR-124a as a key regulator of proliferation and MCP-1 secretion in synoviocytes from patients with rheumatoid arthritis

MicroRNAs (miRNA) are a class of small endogenous non-coding RNAs that influence the stability and translation of messenger RNA. Synoviocytes from patients with rheumatoid arthritis (RA) were analysed for their miRNA expression profile, and it was found that miR-124a levels significantly decreased in RA synoviocytes as compared with osteoarthritis synoviocytes. Transfection of miR-124a into RA synoviocytes significantly suppressed their proliferation and arrested the cell cycle at the G1 phase. miR-124a directly binds to the 3′-untranslated region of cyclin-dependent kinase 2 (CDK-2) and monocyte chemoattractant protein 1 (MCP-1) mRNA, and the induction of miR-124a in RA synoviocytes significantly suppressed the production of the CDK-2 and MCP-1 proteins. It is proposed that miR-124a is a key miRNA in the post-transcriptional regulatory mechanisms of RA synoviocytes, and has a therapeutic potential.

MicroRNA-124 inhibits the progression of adjuvant-induced arthritis in rats

Objective MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that act as post-transcriptional regulators. We analysed the in vivo effect of miRNA-124 (miR-124, the rat analogue of human miR-124a) on adjuvant-induced arthritis (AIA) in rats. Methods AIA was induced in Lewis rats by injecting incomplete Freunds adjuvant with heat-killed Mycobacterium tuberculosis. Precursor (pre)-miR-124 was injected into the right hind ankle on day 9. Morphological changes in the ankle joint were assessed by micro-CT and histopathology. Cytokine expression was examined by western blotting and real-time RT-PCR. The effect of miR-124 on predicted target messenger RNAs (mRNAs) was examined by luciferase reporter assays. The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase staining. Results We found that miR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leucocyte infiltration and cartilage or bone destruction. Osteoclast counts and expression level of receptor activator of the nuclear factor κB ligand (RANKL), integrin β1 (ITGB1) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were reduced in AIA rats treated with pre-miR-124. Luciferase analysis showed that miR-124 directly targeted the 3′UTR of the rat NFATc1, ITGB1, specificity protein 1 and CCAAT/enhancer-binding protein α mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. Both miR-124 and miR-124a directly targeted the 3′-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts. Conclusions We found that miR-124 ameliorated AIA by suppressing critical prerequisites for arthritis development, such as RANKL and NFATc1. Thus, miR-124a is a candidate for therapeutic use for human rheumatoid arthritis.

DR negativity is a distinctive feature of M1/M2 AML cases with NPM1 mutation.

Our previous observation of a higher incidence of FLT3-ITD in DR(-) M1/M2 AML than in DR(+) M1/M2 led to an investigation of NPM1 mutation in the same samples, since DR(-) AML and AML with NPM1 mutation share such characteristics as normal karyotype, the absence of CD34, and FLT3-ITD. NPM1 mutation was found in 18 of 26 (69.2%) of DR(-) cases, but not in any of 28 DR(+) cases. FLT3-ITD was noted in 66.7% of the cases with NPM1 mutation. These findings point to DR negativity as another phenotypic feature of AML with NPM1 mutation.

SS-A/Ro52 promotes apoptosis by regulating Bcl-2 production

SS-A/Ro52 (Ro52), an autoantigen in systemic autoimmune diseases such as systemic lupus erythematosus and Sjögrens syndrome, has E3 ligase activity to ubiquitinate proteins that protect against viral infection. To investigate Ro52s role during stress, we transiently knocked it down in HeLa cells by siRo52 transfection. We found that Ro52(low) HeLa cells were significantly more resistant to apoptosis than wild-type HeLa cells when stimulated by H(2)O(2)- or diamide-induced oxidative stress, IFN-α, IFN-γ and anti-Fas antibody, etoposide, or γ-irradiation. Furthermore, Ro52-mediated apoptosis was not influenced by p53 protein level in HeLa cells. Depleting Ro52 in HeLa cells caused Bcl-2, but not other Bcl-2 family molecules, to be upregulated. Taken together, our data showed that Ro52 is a universal proapoptotic molecule, and that its proapoptotic effect does not depend on p53, but is exerted through negative regulation of the anti-apoptotic protein Bcl-2. These findings shed light on a new physiological role for Ro52 that is important to intracellular immunity.

Neurological outcomes in symptomatic congenital cytomegalovirus-infected infants after introduction of newborn urine screening and antiviral treatment

BACKGROUND Newborn screening for urinary cytomegalovirus (CMV) and early introduction of antiviral treatment are expected to improve neurological outcomes in symptomatic congenital CMV-infected infants. This cohort study prospectively evaluated neurological outcomes in symptomatic congenital CMV-infected infants following the introduction of hospital-based newborn urinary CMV screening and antiviral treatment. SUBJECTS/METHODS Following institutional review board approval and written informed consent from their parents, newborns were prospectively screened from 2009 to 2014 for urinary CMV-DNA by PCR within 1 week after birth at Kobe University Hospital and affiliated hospitals. CMV-positive newborns were further examined at Kobe University Hospital, and those diagnosed as symptomatic were treated with valganciclovir for 6 weeks plus immunoglobulin. Clinical neurological outcomes were evaluated at age ⩾12 months and categorized by the presence and severity of neurologic sequelae. RESULTS Urine samples of 6348 newborns were screened, with 32 (0.50%) positive for CMV. Of these, 16 were diagnosed with symptomatic infection and 12 received antiviral treatment. Four infants developed severe impairment (33%), three developed mild impairment (25%), and five developed normally (42%). CONCLUSIONS This is the first Japanese report of neurological assessments in infants with symptomatic congenital CMV infection who received early diagnosis and antiviral treatment. Urinary screening, resulting in early diagnosis and treatment, may yield better neurological outcomes in symptomatic congenital CMV-infected infants.

Troglitazone Inhibits Cell Growth and Induces Apoptosis of B-Cell Acute Lymphoblastic Leukemia Cells with t(14;18)

Peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear receptor superfamily, has been detected in several human leukemia cells. Recent studies reported that PPARγ ligands inhibit cell proliferation and induce apoptosis in both normal and malignant B-lineage cells. We investigated the expression of PPARγ and the effects of PPARγ ligands on UTree-O2, Bay91 and 380, three B-cell acute lymphoblastic leukemia (B-ALL) cell lines with t(14;18), which show a poor prognosis, accompanying c-myc abnormality. Western blot analysis identified expression of PPARγ protein and real-time PCR that of PPARγ mRNA on the three cell lines. Troglitazone (TGZ), a synthetic PPARγ ligand, inhibited cell growth in these cell lines in a dose-dependent manner, which was associated with G1 cell cycle arrest and apoptosis. We also found this effect PPARγ independent since PPARγ antagonists failed to reverse this effect. We assessed the expression of c-myc, an apoptosis-regulatory gene, since c-myc abnormality was detected in most B-ALL cells with t(14;18). TGZ was found to dose-dependently downregulate the expression of c-myc mRNA and c-myc protein in the three cell lines. These results suggest that TGZ inhibits cell growth via induction of G1 cell cycle arrest and apoptosis in these cell lines and that TGZ-induced apoptosis, at least in part, may be related to the downregulation of c-myc expression. Moreover, the downregulation of c-myc expression by TGZ may depend on a PPARγ-independent mechanism. Further studies indicate that PPARγ ligands may serve as a therapeutic agent in B-ALL with t(14;18).

Extramedullary T-lymphoid blast crisis of an ETV6/ABL1-positive myeloproliferative neoplasm with t(9;12)(q34;p13) and t(7;14)(p13;q11.2)

Dear Editor, Chromosomal translocations involving ETV6 at 12p13 and ABL1 at 9q34 are very rare, but are very rare but recurrent aberrations in hematological malignancies including BCR / ABL1-negative chronic myeloid leukemia (CML), myeloproliferative neoplasm (MPN), and acute leukemia [1, 2]. The resultant ETV6/ABL1 fusion protein is thought to play a crucial role in leukemic transformation by the constitutive activation of tyrosine kinase [1]. Similar to BCR /ABL1-positive CML, it has been reported that several cases of ETV6 /ABL1 -positive CML/MPN presented with blast crisis (BC) in lymph nodes as well as bone marrow [3–7]. Here, we describe the first case of extramedullary T-lymphoid BC of ETV6 /ABL1 -positive MPN. A 31-year-old man was admitted because of generalized lymphadenopathy and leukocytosis. Peripheral blood values were hemoglobin 113 g/L, platelets 438×10/L, and leukocytes 50.7×10/L with 20 % myelocytes, 17 % metamyelocytes, 10 % band forms, 32 % segmented neutrophils, and 11 % eosinophils. Bone marrow was hypercellular with myeloid hyperplasia and prominent eosinophilia, and compatible with MPN (Fig. 1a–c). Pathological examination of inguinal lymph nodes was consistent with T-lymphoblastic lymphoma (T-LBL) (Fig. 1d). Immunohistochemistry showed that lymphoma cells were positive for CD3, CD1a, and TdT (Fig. 1e). The patient was treated by dasatinib with hyper-CVAD protocol followed by myeloablative allogeneic stem cell transplantation (SCT). However, he died of idiopathic pneumonia syndrome, 11 months after initial diagnosis. G banding and spectral karyotyping of bone marrow cells on admission showed 46,XY,der(9)t(9;12)(q34;p13), del(12)(p13)[1]/46,sl,t(7;14)(p13;q11.2)[18]/47,sdl1,+19[1] (Fig. 2a). The karyotype of lymph node cells was similar as fol lows: 46,XY,t (7 ;14)(p13;q11.2) ,der(9) t (9 ;12) (q34;p13),del(12)(p13)[4]. Fluorescence in situ hybridization (FISH) on metaphase spreads of bone marrow cells detected the ETV6 /ABL1 fusion signal on the der(9)t(9;12)(q34;p13) (Fig. 2b). FISH on interphase nuclei of lymph node cells also confirmed the ETV6 /ABL1 fusion signals (Fig. 2c). Reverse transcription-polymerase chain reaction (RTPCR) demonstrated two types of ETV6 /ABL1 fusion transcripts (types B and A) only in bone marrow cells of the patient (Fig. 2d) [2]. Southern blot analyses of lymph node cells showed rearrangement of TRD@ Jδ1, but germline configuration of IGH@ JH, TRB@ Cβ1, and TRG@ Jγ (data not shown). We have detected a rare translocation der(9)t(9;12) (q34;p13) resulting in an ETV6 /ABL1 fusion gene. We confirmed an expression of the fusion transcripts in K. Yamamoto (*) :K. Yakushijin :Y. Miyata :Y. Sanada : A. Okamura :H. Matsuoka :H. Minami Division of Medical Oncology/Hematology, Department of Medicine, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan e-mail: kyamamo@med.kobe-u.ac.jp

Low total IgM values and high cytomegalovirus loads in the blood of newborns with symptomatic congenital cytomegalovirus infection

Abstract Aims: Neurological outcomes differ considerably between symptomatic and asymptomatic infants with congenital cytomegalovirus (CMV) infection. Our objective was to characterize laboratory markers in symptomatic newborns in comparison with asymptomatic newborns with congenital CMV infection. Methods: Ten newborns with symptomatic and 13 newborns with asymptomatic congenital CMV infection were included in this 3-year prospective cohort study. Total immunoglobulin M (IgM), CMV-IgM, CMV antigenemia, and CMV-DNA in blood and urine were measured and their positive rates and quantitative values compared between the symptomatic and asymptomatic groups. Results: Fifty percent of newborns in the symptomatic group were positive based on total IgM; this was significantly lower than in the asymptomatic group (100%). Quantitative total IgM values were significantly lower, and there were significantly more copies of CMV-DNA in the blood of symptomatic newborns than in asymptomatic newborns (median values for total IgM: 14 vs. 43 mg/dL and blood CMV-DNA: 3.2×102 vs. 3.5×101 copies/106 white blood cells). CMV-IgM, CMV antigenemia, and urine CMV-DNA did not differ significantly between groups. Conclusion: Low total IgM values and high blood CMV loads were associated with the presence of symptoms in newborns with congenital CMV infection.

Expression of the novel NUP98/PSIP1 fusion transcripts in myelodysplastic syndrome with t(9;11)(p22;p15).

Objectives:  The t(9;11)(p22;p15) is a very rare but recurrent translocation in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) blast crisis. The translocation results in a fusion gene between NUP98 at 11p15 and PSIP1 encoding two transcriptional coactivators, p52 and p75, at 9p22. Here, we describe the first case of myelodysplastic syndrome (MDS) with t(9;11)(p22;p15).

Epidermolysis bullosa simplex with mottled pigmentation with noncicatricial alopecia: identification of a recurrent p.P25L mutation in KRT5 in four affected family members.

Abstract Epidermolysis bullosa simplex with mottled pigmentation (EBS-MP) is a rare subtype of EBS that is characterized by blistering, mottled pigmentation of the trunk and limbs, punctate hyperkeratosis of the palms and soles, and dystrophic nails.(1) EBS-MP is caused by a mutation in the KRT5 or KRT14 gene encoding keratin 5 (K5) and keratin 14 (K14), respectively.(1, 2) Dermatopathia pigmentosa reticularis (DPR), caused by a KRT14 mutation, is characterized by reticulate pigmentation, noncicatricial alopecia, onychodystrophia and loss of dermatoglyphics.(3) Here, we report cases of EBS-MP with noncicatricial alopecia, a clinical feature of DPR, with a recurrent p.P25L mutation in KRT5 in four Japanese family members. This article is protected by copyright. All rights reserved.