Alternatively spliced, truncated GCSF receptor promotes leukemogenic properties and sensitivity to JAK inhibition.

Granulocyte colony-stimulating factor (GCSF) drives the production of myeloid progenitor and precursor cells toward neutrophils via the GCSF receptor (GCSFR, gene name CSF3R). Children with severe congenital neutropenia chronically receive pharmacologic doses of GCSF, and ∼30% will develop myelodysplasia/acute myeloid leukemia (AML) associated with GCSFR truncation mutations. In addition to mutations, multiple isoforms of CSF3R have also been reported. We found elevated expression of the alternatively spliced isoform, class IV CSF3R in adult myelodysplastic syndrome/AML patients. Aside from its association with monosomy 7 and higher rates of relapse in pediatric AML patients, little is known about the biology of the class IV isoform. We found developmental regulation of CSF3R isoforms with the class IV expression more representative of a progenitor cell stage. Striking differences were found in phosphoprotein signaling involving Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and cell cycle gene expression. Enhanced proliferation by class IV GCSFR was associated with diminished STAT3 and STAT5 activation, yet showed sensitivity to JAK2 inhibitors. Alterations in the C-terminal domain of the GCSFR result in leukemic properties of enhanced growth, impaired differentiation and resistance to apoptosis, suggesting that they can behave as oncogenic drivers, sensitive to JAK2 inhibition.

Epidemiology of sarcoptic mange in free-ranging raccoon dogs (Nyctereutes procyonoides) in Yokohama, Japan.

Free-ranging raccoon dogs (Nyctereutes procyonoides) from Nogeyama Zoological Gardens, Kanazawa Zoological Gardens, and Yokohama Zoological Gardens frequently rescued dogs having Sarcoptes scabiei infestation. However, the epidemiology of S. scabiei infestation has not yet been elucidated. In the present study, we investigated the epidemiology of S. scabiei infestation in raccoon dogs and its influence on the population of masked palm civets in Yokohama, Japan. We examined records of raccoon dog rescue between 1981 and 2010 and classified the dogs into the following 4 categories on the basis of the reason for rescue: dogs with S. scabiei infestation, scabies-infested dogs involved in car accidents, uninfested dogs involved in car accidents, and other reasons for rescue. We found that the number of dogs rescued due to car accidents and other reasons increased from 1989 onwards, and an S. scabiei outbreak was recorded since 1993. The infestation spread from the southern to the northern regions of Yokohama. The total number of raccoon dogs rescued annually peaked in 1995 and declined thereafter. The number of masked palm civets (Paguma larvata) rescued gradually increased with a decline in the number of raccoon dogs rescued. In the present study, we revealed the epidemiology of S. scabiei infestation in the raccoon dog. The outbreak might be induced by the increased population density, and the infestation spread immediately from the southern to the northern regions of Yokohama since 1993. Further, the population of masked palm civets may have increased due to the decrease in the population of the raccoon dog.

Loss of SHIP-1 protein expression in high-risk myelodysplastic syndromes is associated with miR-210 and miR-155.

The myelodysplastic syndromes (MDSs) comprise a group of disorders characterized by multistage progression from cytopenias to acute myeloid leukemia (AML). They display exaggerated apoptosis in early stages, but lose this behavior during evolution to AML. The molecular basis for loss of apoptosis is unknown. To investigate this critical event, we analyzed phosphatidylinositol (PI) 3'kinase signaling, implicated as a critical pathway of cell survival control in epithelial and hematological malignancies. PI 3'kinase activates Akt through its production of 3' phosphoinositides. In turn, the phosphoinositides are dephosphorylated by two lipid phosphatases, PTEN and SHIP-1, in myeloid cells. We studied primary MDS-enriched bone marrow cells and bone marrow sections by western blotting, immunohistochemistry, immunocytochemistry and quantitative PCR for components of the SHIP/PTEN/PI 3'kinase signaling circuit. We reported constitutively activated Akt, variable levels of PTEN and uniformly decreased SHIP-1 expression in MDS progenitor cells. Overexpression of SHIP-1, but not the phosphatase-deficient form, inhibited myeloid leukemic growth. Levels of microRNA (miR)-210 and miR-155 transcripts, which target SHIP-1, were increased in CD34(+) MDS cells compared with their normal counterparts. Direct binding of miR-210 to the 3' untranslated region of SHIP-1 was confirmed by luciferase reporter assay. Transfection of a myeloid cell line with miR-210 resulted in loss of SHIP-1 protein expression. These data suggest that miR-155 and miR-210/SHIP-1/Akt pathways could serve as clinical biomarkers for disease progression, and that miR-155 and miR-210 might serve as novel therapeutic targets in MDS.

RNAi-mediated silencing of p190Bcr-Abl inactivates Stat5 and cooperates with imatinib mesylate and 17-allylamino-17-demetoxygeldanamycin in selective killing of p190Bcr-Abl-expressing leukemia cells.

The 190 kD (p190) and 210 kD (p210) Bcr-Abl proteins are responsible for the pathophysiology of Philadelphia chromosome (Ph)(+) leukemia. We applied RNA interference (RNAi) to specific killing of p190(+) cells, and determined the optimal sequences for gene silencing in the BCR, junctional and ABL regions of p190, respectively. Then, p190(+) and p210(+) cells were infected with lentiviral vectors encoding these shRNAs, resulting in efficient killing of p190(+) cells, while p210(+) cells were only sensitive to shBCR and shABL. In p190-transformed Ba/F3 cells, silencing of p190 specifically inhibited tyrosine phospohorylation of Stat5 prior to their death, but did not affect phosphorylation of Jak2, Akt or MEK1/2. In contrast, downregulation of p190 by their treatment with 17-allylamino-17-demetoxygeldanamycin (17-AAG) was associated with reduced protein levels of Jak2, Akt and MEK1/2. shRNA targeting p190 collaborated additively with imatinib and 17-AAG in growth inhibition of Ba/F3-p190wt and imatinib-resistant Ba/F3-p190Y253 H cells. Collectively, RNAi-mediated silencing of p190 is a promising option both for delineating signal transduction and for therapeutic application in 190(+) leukemia.

Shedding of herpes simplex virus type 1 into saliva in patients with orofacial fracture.

Shedding of herpes simplex virus type 1 (HSV-1) into saliva was studied in 83 patients with orofacial fractures. Infectious virus was isolated from 14 of 83 patients (16.8%) with no detectable herpetic lesion during hospitalization. Of the 83 patients, 44 (53%) had HSV-1 specific antibody. Virus shedding into saliva was observed only in the patients with antibody to HSV-1; thus, the frequency of virus shedding patients among those with antibody was 31% (14 of 44 patients). The frequency was obviously higher than that of a healthy population. The period of HSV-1 shedding had a mean of 3.7 days with range of 1 to 8 days, which is also significantly longer than that of a healthy population. These results strongly suggest that both treatment and operation lead effectively to reactivation of latently infected HSV-1.