Hidemi Toyoda

Dysregulation of transcriptions in primary granule constituents during myeloid proliferation and differentiation in patients with severe congenital neutropenia

We examined the expression of granule constituent genes in myeloid progenitor cells during proliferation and differentiation in patients with severe congenital neutropenia (SCN). The heterozygous mutation of the neutrophil elastase gene was identified in two of four patients. The CD34+/granulocyte‐colony stimulating factor receptor (G‐CSFR)+ cells of SCN patients showed defective responsiveness to G‐CSF in serum‐deprived culture. The CD34+/G‐CSFR+ cells expressed low levels of the granule constituent mRNAs. The transcription levels of primary granule enzyme genes in CD34+/G‐CSFR+ cells were gradually enhanced and then decreased when cells were induced toward myeloid lineage with G‐CSF in normal subjects. However, the primary up‐regulation and the following down‐regulation of these enzyme transcriptions were not clearly observed in SCN patients. No differences in expressions of the lactoferrin gene were seen between normal subjects and patients with SCN. We hypothesize that the abnormal regulation of the transcription in primary granule constituents might involve the defective proliferation and differentiation of myeloid cells in patients with SCN.

Chemical sensitization and regulation of TRAIL-induced apoptosis in a panel of B-lymphocytic leukaemia cell lines.

Summary.  Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) effectively kills tumour cells but not normal cells. We investigated TRAIL sensitivity and the TRAIL‐induced apoptosis signalling pathway in a panel of B‐lymphocytic leukaemia cell lines. Depending upon TRAIL sensitivity, leukaemia cells could be divided into three groups: highly sensitive, moderately sensitive and resistant. TRAIL receptor‐2 (DR5) plays an important role in transducing apoptosis signals. DR5 was internalized into the cytoplasm where it recruited FAS‐associated death domain protein (FADD) under TRAIL stimulation in both sensitive and resistant cells. However, the active form of caspase‐8 was recruited to FADD and only sensitive cells showed increased caspase‐8 activity upon TRAIL stimulation. The caspase‐8 specific inhibitor, Z‐IETD, impaired caspase‐8 activation and completely abrogated TRAIL‐induced apoptosis. These results suggest that TRAIL resistance in B‐lymphocytic leukaemia cells is due to negative regulation at the level of caspase‐8 activation and that caspase‐8 activation is an indispensable process in TRAIL‐induced apoptosis. However, FADD‐like interleukin‐1 β‐converting enzyme inhibitory protein (c‐FLIPL) was similarly expressed and down‐regulated after TRAIL stimulation in both sensitive and resistant cells. Interestingly, in some cell lines, TRAIL sensitivity and caspase‐8 activity was enhanced or restored with the treatment of cycloheximide (CHX). In addition, X‐linked inhibitor of apoptosis (XIAP) levels decreased significantly and rapidly following treatment with CHX. Down‐regulation of XIAP may be responsible for enhancement or restoration of TRAIL sensitivity after CHX treatment in B‐lymphocytic leukaemia cells.

Impairment of IL-12-Dependent STAT4 Nuclear Translocation in a Patient with Recurrent Mycobacterium avium Infection

We examined the immunological abnormality in a patient with recurrent Mycobacterium avium infection. T cells from the patient showed decreased ability both to produce IFN-γ and to proliferate in response to IL-12. Despite decreased expression of IL-12R β1 and β2 chains in the patient’s PHA-activated T cells, there was no difference in IL-12-induced tyrosine and serine phosphorylation of STAT4 in PHA-activated T cells between the patient and healthy subjects, suggesting that IL-12R signals are transmitted to STAT4 in the patient’s PHA-activated T cells. Using EMSA, confocal laser microscopy, and Western blotting, we demonstrated that the nuclear translocation of STAT4 in response to IL-12 is reduced in PHA-activated T cells from the patient when compared with those from healthy subjects. Leptomycin B was used to examine whether nuclear export of STAT4 is increased in the patient’s T cells. However, leptomycin B treatment did not reverse impaired IL-12-induced nuclear accumulation of STAT4. Although the exact mechanism responsible for the impaired STAT4 nuclear translocation in this patient remains unclear, the absence of mutation in the IL-12Rβ1, IL-12Rβ2, STAT4, and STAT4-binding sequence of the IFN-γ gene and preservation of STAT4 tyrosine and serine phosphorylation suggest the existence of a defective STAT4 nuclear translocation. This defect is likely responsible for the impaired STAT4 nuclear translocation in IL-12-stimulated T cells, leading to impairment of both IFN-γ production and cell proliferation. To the best of our knowledge, this is the first report of a patient with atypical mycobacterial infection associated with impairment of STAT4 nuclear translocation.

Expression of short‐form caspase 8 correlates with decreased sensitivity to Fas‐mediated apoptosis in neuroblastoma cells

Disruption of apoptotic death signal transduction pathways may be responsible for tumor formation, progression and resistance to treatment in neuroblastoma. Caspase 8, one of the initiator caspases, plays an important role in the Fas‐Fas ligand pathway. This caspase signals through the formation of a death‐inducing signaling complex in response to Fas activation by its ligand. In this study, we evaluated the sensitivity of a series of human neuroblastoma cell lines to membrane‐bound Fas ligand induced‐cell death, as well as the expression of Fas, caspase 3 and caspase 8. Sensitivity to Fas‐mediated cell death did not correlate with the expression of Fas in neuroblastoma cells, but was directly associated with the pattern of caspase 8 protein expression. We found that the majority of neuroblastoma cell lines we evaluated lacked caspase 8 expression, and these cell lines were invariably resistant to Fas‐mediated cell death. In contrast, cell lines expressing normal caspase 8 protein were quite sensitive to Fas‐mediated cell death. More interestingly, a group of cell lines expressing a distinct short form of caspase 8 with splicing out of exon 3 consistently showed moderate sensitivity to Fas‐mediated cell death. These results indicate that the profile of caspase 8 expression is an important determinant of the response of neuroblastoma cells to Fas‐mediated cell death.

Polycythemia and Paraganglioma With a Novel Somatic HIF2A Mutation in a Male

Recently, a new syndrome of paraganglioma, somatostatinoma, and polycythemia has been discovered (known as Pacak–Zhuang syndrome). This new syndrome, with somatic HIF2A gain-of-function mutations, has never been reported in male patients. We describe a male patient with Pacak–Zhuang syndrome who carries a newly discovered HIF2A mutation. Congenital polycythemias have diverse etiologies, including germline mutations in the oxygen-sensing pathway. These include von Hippel–Lindau (Chuvash polycythemia), prolyl hydroxylase domain–containing protein-2, and hypoxia-inducible factor-2α (HIF-2α). Somatic gain-of-function mutations in the gene encoding HIF-2α were reported in patients with paraganglioma and polycythemia and have been found exclusively in female patients. Through sequencing of the HIF2A using DNA from paraganglioma in 15-year-old male patient, we identified a novel mutation of HIF2A: a heterozygous C to A substitution at base 1589 in exon 12 of HIF2A. The mutation was not found in germline DNA from leukocytes. The C1589A mutations resulted in substitution of alanine 530 in the HIF-2α protein with glutamic acid. This mutation is undoubtedly associated with increased HIF-2α activity and increased protein half-life, because it affects the vicinity of the prolyl hydroxylase target residue, proline 531. To our knowledge, this is the first report describing Pacak–Zhuang syndrome with somatic gain-of-function mutation in HIF2A in a male patient. Congenital polycythemia of unknown origin should raise suspicion for the novel disorder Pacak–Zhuang syndrome, even in male patients.

Multiple cutaneous squamous cell carcinomas in a patient with interferon γ receptor 2 (IFNγR2) deficiency

Disseminated squamous cell carcinoma (SCC) of the skin is exceedingly rare in children. SCC occurs after immunodeficiency from immunosuppression in organ transplant recipients or patients with HIV infection or leukaemia, but has not been reported in primary immunodeficiencies other than epidermodysplasia verruciformis. Interferon γ receptor 2 (IFNγR2) deficiency is an exceedingly rare primary immunodeficiency, conferring almost selective predisposition to mycobacterial diseases. A disseminated, cutaneous SCC is described that occurred in a patient homozygous for a novel frameshift deletion at positions 949 and 950 (949delTG) in the IFNGR2 gene. The patient first presented at 1 year of age with disseminated Mycobacterium avium infection, with later infections of atypical mycobacteria (Mycobacterium fortuitum and Mycobacterium porcium). At 17 years of age, the patient developed multifocal SCC lesions on the face and both hands. Histopathological examination revealed well differentiated SCC. Despite local tumour excision, multiple lesions occurred and a large SCC on the right arm required amputation. The patient died at 20 years of age of disseminated SCC. Inherited disorders of IFNγ mediated immunity may predispose patients to SCC.

Prediction of Reactivity to Noninherited Maternal Antigen in MHC-Mismatched, Minor Histocompatibility Antigen-Matched Stem Cell Transplantation in a Mouse Model

The immunologic effects of developmental exposure to noninherited maternal Ags (NIMAs) are quite variable. Both tolerizing influence and inducing alloreaction have been observed on clinical transplantation. The role of minor histocompatibility Ags (MiHAs) in NIMA effects is unknown. MiHA is either matched or mismatched in NIMA-mismatched transplantation because a donor of the transplantation is usually limited to a family member. To exclude the participation of MiHA in a NIMA effect for MHC (H-2) is clinically relevant because mismatched MiHA may induce severe alloreaction. The aim of this study is to understand the mechanism of NIMA effects in MHC-mismatched, MiHA-matched hematopoietic stem cell transplantation. Although all offsprings are exposed to the maternal Ags, the NIMA effect for the H-2 Ag was not evident. However, they exhibit two distinct reactivities, low and high responder, to NIMA in utero and during nursing depending on the degree of maternal microchimerism. Low responders survived longer with less graft-versus-host disease. These reactivities were correlated with Foxp3 expression of peripheral blood CD4+CD25+ cells after graft-versus-host disease induction and the number of IFN-γ–producing cells stimulated with NIMA pretransplantation. These observations are clinically relevant and suggest that it is possible to predict the immunological tolerance to NIMA.

Prognostic significance of CD155 mRNA expression in soft tissue sarcomas

CD155 was initially identified as a receptor for poliovirus. Several studies have demonstrated that CD155 overexpression in cancer cells is significant in their migration, invasion, proliferation and metastasis. The objective of the present study was to investigate the correlation between CD155 expression and the clinical aggressiveness of soft tissue tumors. The CD155 expression levels in 43 surgically-resected soft tissue tumors were evaluated using the quantitative real-time polymerase chain reaction (PCR). The clinicopathogical factors affecting the expression levels of CD155 mRNA were investigated and the association between the expression levels of CD155 and patient prognosis was identified. The CD155 expression level was not correlated with the patient gender, site of the primary tumor, tumor depth, tumor size or presence of distant metastasis at presentation, but was correlated with patient age (Fisher’s exact test). The local recurrence-free survival rate for patients with a high CD155 expression level was observed to be significantly poorer compared with that of patients with low CD155 expression levels (P=0.0401). Moreover, a multivariate analysis indicated that a high CD155 expression level was an independent adverse prognostic factor for local recurrence-free survival (hazard ratio, 6.369; P=0.0328). The present study therefore suggests that the expression level of CD155 is a useful marker for predicting the local recurrence of soft tissue tumors.

Elevated Von Willebrand factor propeptide for the diagnosis of thrombotic microangiopathy and for predicting a poor outcome.

Thrombotic microangiopathy (TMA) is associated with vascular endothelial cell injury and is sometimes linked with poor outcome. Von Willebrand factor (VWF) propeptide (VWFpp) is considered to be a marker of vascular endothelial cell injury. The plasma levels of VWF, VWFpp, and thrombomodulin (TM) were evaluated for their use in the diagnosis of TMA in 75 patients with TMA. There were 30 TMA patients with marked decreases in ADAMTS13 (TMA/ADAMTS13) and 45 without the decrease (TMA/other). The plasma levels of TM, VWF, and VWFpp values were significantly high in patients with TMA, especially TMA/other group. The plasma levels of TM and VWFpp were significantly high in non-survivor with TMA. In the TMA/other group, the plasma levels of VWFpp were negatively correlated with ADAMTS13 activity. The plasma levels of TM correlated with the renal function, but the plasma levels of VWFpp did not. A ROC analysis indicated that VWFpp and TM were useful markers for the prediction of a poor outcome. These findings suggest that VWFpp is an useful marker for the diagnosis of TMA and for the prediction of poor outcome.

Recurrent inflammatory aortic aneurysms in chronic mucocutaneous candidiasis with a gain-of-function STAT1 mutation

a Department of Cardiology and Nephrology, Mie University Graduate School of Medicine, Tsu, Japan b Department of Pediatrics, Mie University Graduate School of Medicine, Tsu, Japan c Department of Thoracic and Cardiovascular Surgery, Mie University Graduate School of Medicine, Tsu, Japan d Department of Hematology and Oncology, Mie University Graduate School of Medicine, Tsu, Japan e Department of Pathology and Matrix Biology, Mie University Graduate School of Medicine, Tsu, Japan f Department of Cell Transplantation, Mie University Graduate School of Medicine, Tsu, Japan g Mie University Research Center for Matrix Biology, Tsu, Japan