Rie Kawano

Human Herpesvirus 6 DNA in Plasma after Allogeneic Stem Cell Transplantation: Incidence and Clinical Significance

BACKGROUND Human herpesvirus 6 (HHV-6) is increasingly recognized as an opportunistic and potentially life-threatening pathogen in recipients of allogeneic stem cell transplants (SCTs). METHODS To clarify the incidence and clinical relevance of active HHV-6 infection, serial titers of plasma HHV-6 DNA were determined for 50 allogeneic SCT recipients, using real-time polymerase chain reaction. RESULTS HHV-6 DNA was detected in plasma from 24 patients (48%). HHV-6 DNA was most frequently apparent approximately 14-27 days after transplantation. An increased risk of a positive result for HHV-6 DNA was associated with transplantation from an allelic-mismatch donor (P = .02) and administration of steroids (P = .04). Steroid use was associated with high HHV-6 DNA loads (P = .02). High HHV-6 DNA loads were correlated with delayed platelet engraftment (P = .04). Among patients who had positive results for HHV-6 DNA, the HHV-6 DNA load was higher in plasma from those who developed limbic encephalitis (n = 4) (P < .0001). CONCLUSIONS Active HHV-6 infection is not rare in SCT recipients. SCT from allelic-mismatch donors is associated with increased risk of active HHV-6 infection. Steroid therapy is associated with not only increased incidence of infection but also accelerated viral replication. Development of limbic encephalitis is associated with high HHV-6 DNA load.

Real-Time PCR Assay Compared to Nested PCR and Antigenemia Assays for Detecting Cytomegalovirus Reactivation in Adult T-Cell Leukemia-Lymphoma Patients

ABSTRACT We analyzed the efficiency of the quantitative real-time PCR assay for cytomegalovirus (CMV) reactivation in adult T-cell leukemia-lymphoma (ATL) patients and compared the results with those obtained with qualitative nested PCR and antigenemia assays. The viral load obtained by the real-time PCR assay closely paralleled the number of antigen-positive cells obtained with the antigenemia assay. Real-time PCR revealed that a large number of DNA copies could be present even in samples assessed as negative or low in antigen-positive cells (0 to 10 antigen-positive cells/50,000 cells) by antigenemia assay. CMV copy numbers did not differ between the negative and low-antigen-positive groups. When the input concentration for real-time PCR assay was 2,500 to 5,000 copies/ml, the positivity rate for the nested PCR assay was 47.3%, while the positivity rate was more than 90% at an input concentration of ≥50,000 copies/ml. Real-time PCR is more sensitive than the antigenemia and nested PCR assays. Moreover, real-time PCR was able to detect CMV reactivation earlier than the antigenemia and nested PCR assays through the use of longitudinal analysis in four ATL patients with CMV pneumonia. In longitudinal assessments, analysis of the results suggested that a cutoff level of 5,000 copies/ml might be used to initiate treatment. Real-time PCR is more suitable for monitoring CMV reactivation in ATL patients than the antigenemia and nested PCR assays. CMV viral loads of 5,000 copies/ml are proposed as the cutoff for initiating antiviral therapy in ATL patients.

Lactic Acid Bacteria Convert Human Fibroblasts to Multipotent Cells

The human gastrointestinal tract is colonized by a vast community of symbionts and commensals. Lactic acid bacteria (LAB) form a group of related, low-GC-content, gram-positive bacteria that are considered to offer a number of probiotic benefits to general health. While the role of LAB in gastrointestinal microecology has been the subject of extensive study, little is known about how commensal prokaryotic organisms directly influence eukaryotic cells. Here, we demonstrate the generation of multipotential cells from adult human dermal fibroblast cells by incorporating LAB. LAB-incorporated cell clusters are similar to embryoid bodies derived from embryonic stem cells and can differentiate into endodermal, mesodermal, and ectodermal cells in vivo and in vitro. LAB-incorporated cell clusters express a set of genes associated with multipotency, and microarray analysis indicates a remarkable increase of NANOG, a multipotency marker, and a notable decrease in HOX gene expression in LAB-incorporated cells. During the cell culture, the LAB-incorporated cell clusters stop cell division and start to express early senescence markers without cell death. Thus, LAB-incorporated cell clusters have potentially wide-ranging implications for cell generation, reprogramming, and cell-based therapy.

High incidence of cytomegalovirus, human herpesvirus-6, and Epstein–Barr virus reactivation in patients receiving cytotoxic chemotherapy for Adult T cell leukemia

The etiology of cytomegalovirus (CMV), human herpesvirus‐6 (HHV‐6), and Epstein–Barr virus (EBV) reactivation and the potential for complications following cytotoxic chemotherapy in the absence of allogeneic transplantation are not clearly understood. Patients with adult T cell leukemia (ATL) are susceptible to opportunistic infections. In this study, the incidence, kinetics and clinical significance of reactivation of CMV, HHV‐6, and EBV in ATL patients were investigated. Viral DNA in a total of 468 plasma samples from 34 patients was quantified using real‐time PCR. The probability of CMV, HHV‐6, and EBV reactivation by 100 days after the start of chemotherapy was 50.6%, 52.3%, and 21.6%, respectively. Although most CMV reactivations were self‐limited, plasma CMV DNA tended to persist or increase if the CMV DNA levels in plasma reached ≥104 copies/ml. CMV reactivation was negatively associated with survival, but the P‐value for this association was near the borderline of statistical significance (P = 0.052). One patient developed fatal interstitial pneumonia concomitant with peak CMV DNA accumulation (1.6 × 106 copies/ml plasma). Most HHV‐6 and EBV reactivations were self‐limited, and no disease resulting from HHV‐6 or EBV was confirmed. HHV‐6 and EBV reactivation were not associated with reduced survival (P = 0.35 and 0.11, respectively). These findings demonstrated that subclinical reactivation of CMV, HHV‐6, and EBV were common in ATL patients receiving chemotherapy. There were differences in the viral reactivation patterns among the three viruses. A CMV load ≥104 copies/ml plasma was indicative of subsequent exacerbation of CMV reactivation and developing serious clinical course. J. Med. Virol. 83:702–709, 2011.

Tsukushi controls the hair cycle by regulating TGF-β1 signaling.

The hair follicle contains stem/progenitor cells that supply progeny for skin development and the hair cycle. Several signaling molecules belonging to the Wnt, BMP, shh, and transforming growth factor β (TGF-β) signaling cascades are involved in the normal hair follicle cycle. However, the systemic mechanism of how these humoral factors are controlled remains largely unknown. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions extracellularly as a key coordinator of multiple signaling networks. Here, we show that TSK is expressed at the restricted areas of hair follicle during the morphogenesis and the hair cycle. Targeted disruption of the TSK gene causes the hair cycle to be delayed with low levels of TGF-β1 and phosphorylated Smad2/3 (pSmad2/3) expression. Biochemical analysis indicates that TSK directly binds to TGF-β1. Our data suggest that TSK controls the hair cycle by regulating TGF-β1 signaling.

Tsukushi is involved in the wound healing by regulating the expression of cytokines and growth factors

During the wound-healing process, macrophages, fibroblasts, and myofibroblasts play a leading role in shifting from the inflammation phase to the proliferation phase, although little is known about the cell differentiation and molecular control mechanisms underlying these processes. Previously, we reported that Tsukushi (TSK), a member of the small leucine-rich repeat proteoglycan family, functions as a key extracellular coordinator of multiple signalling networks. In this study, we investigated the contribution of TSK to wound healing. Analysis of wound tissue in heterozygous TSK-lacZ knock-in mice revealed a pattern of sequential TSK expression from macrophages to myofibroblasts. Quantitative PCR and in vitro cell induction experiments showed that TSK controls macrophage function and myofibroblast differentiation by inhibiting TGF-β1 secreted from macrophages. Our results suggest TSK facilitates wound healing by maintaining inflammatory cell quiescence.

An acquired CSF3R mutation in an adult chronic idiopathic neutropenia patient who developed acute myeloid leukaemia

Granulocyte colony-stimulating factor (G-CSF, CSF3) and its receptor (G-CSFR, CSF3R) play an essential role in the regulation of granulopoiesis. Recently, it was shown that point mutations in the gene encoding CSF3R (CSF3R) were detected in 20% of severe congenital neutropenia (SCN) patients. In particular, a truncation mutation in the cytoplasmic domain of CSF3R has been identified in some SCN patients who eventually developed myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML), and it was thought that this mutation was strongly related to leukemogenesis (Hunter & Avalos, 2000). On the other hand, chronic idiopathic neutropenia (CIN) is characterized by a low incidence of developing AML, and usually has a benign outcome. However, various point mutations in CSF3R have also been reported in a few cases of CIN patients who developed AML (Forbes et al, 2002; Papadaki et al, 2004). There has been some controversy about the association between the pathogenesis of CIN and CSF3R point mutations, as well as about the existence of a subset of CIN patients with a tendency to develop AML (Papadaki et al, 2002). We identified a point mutation in the cytoplasmic domain of CSF3R in a patient with a history of acquired unexplained neutropenia who developed AML. We herein discuss the association between CSF3R mutations and the development of MDS and AML. A 55-year-old male was admitted to our hospital in September 1998 because of neutropenia. At presentation, he had a low peripheral blood neutrophil count 0Æ077 · 10/l), normal haemoglobin level and platelet count (260 · 10/l), was negative for antineutrophil antibodies, and had a normal bone marrow karyotype, with hypocellularity and maturation arrest of precursor neutrophils, and no signs suggestive of myelodysplasia. He was diagnosed to have non-immune CIN on the basis of the diagnostic criteria (Papadaki et al, 2001). He suffered frequent and severe infectious episodes whilst receiving intermittent recombinant human G-CSF therapy. In 2003, the patient was readmitted because of the appearance of blast cells in his peripheral blood. Bone marrow aspiration demonstrated 52Æ7% infiltration with blast cells that were positive for CD7, CD19, CD13, CD33, and CD34 on a flow cytometric analysis. The cytogenetic analysis showed 46, XY, add (7) (q22), add (14) (q32). He was diagnosed to have AML and underwent allogeneic stem cell transplantation from a human leucocyte antigen (HLA)-matched related donor, but he died of idiopathic pulmonary syndrome. The Ethics Committee of Oita University Faculty of Medicine approved the use of patient-derived cells and the protocols used for this study. Reverse transcription polymerase chain reaction and a nucleotide sequence analysis were carried out using the bone marrow mononuclear cells from the patient by conventional methods. A point mutation in the CSF3R cDNA from this patient was identified. The mutation occurred at nucleotide 2396 and was a C-to-T transition, changing the CAG codon at position 743 (Gln 743) to a TAG stop codon (National Center for Biotechnology Information GenBank accession No.M59818) (Fig 1). This mutation is predicted to result in a protein product with a 120-amino acid C-terminal truncation. However, this mutation was not detected in the CSF3R cDNA during the first bone marrow examination when the patient was initially diagnosed with CIN. Further investigations revealed no mutations in the transmembrane domain or the extracellular region of the CSF3R. CSF3R mutations are exclusively detected in patients with SCN, primarily those who develop secondary MDS or AML, and these mutations play an important role in leukemogenesis. These SCN cases show similar types of molecular defects that introduce premature stop codons leading to a truncation of the CSF3R. A previous report demonstrated that most patients with MDS/ AML secondary to SCN harboured nonsense mutations at nucleotides 2384, 2390, 2396, 2412, 2425, and 2429 (Germeshausen et al, 2007). Although CSF3R mutations in a few de novo AML and MDS cases have been reported, the breakpoint was variable, involving extracellular or transmembrane domains. The relationship with leukemogenesis has also remained unclear. Link et al (2007) reported that no CSF3R mutations located between nucleotide 2384 and 2429 were detected in cases of de novo AML, suggesting that CSF3R mutations are rare in de novo AML. In addition, although mutations of tyrosine kinase genes were common in de novo AML, no mutations in these genes were detected in the SCN patients (Link et al, 2007). It is therefore thought that the leukemogenic mechanism differs between de novo AML, and AML with SCN. In addition, while monitoring the Severe Chronic Neutropenia International Registry, it was discovered that CIN patients do not show an increased propensity to develop MDS or AML, despite longterm G-CSF treatment (Dale et al, 2003). In the present case, the mutation occurred at nucleotide 2396, and we thought that it played an important role in the clinical pathogenesis of the disease, because the mutation was not detected when the patient had CIN without AML, but it was detected after he was diagnosed with AML that developed from CIN. Carapeti et al (1997) reported a similar point mutation (nucleotide 2390) in a case that developed AML from MDS. However, we could find no other case reports. The present case is therefore only the second case with a truncated Correspondence

Akhirin regulates the proliferation and differentiation of neural stem cells in intact and injured mouse spinal cord

Although the central nervous system is considered a comparatively static tissue with limited cell turnover, cells with stem cell properties have been isolated from most neural tissues. The spinal cord ependymal cells show neural stem cell potential in vitro and in vivo in injured spinal cord. However, very little is known regarding the ependymal niche in the mouse spinal cord. We previously reported that a secreted factor, chick Akhirin, is expressed in the ciliary marginal zone of the eye, where it works as a heterophilic cell‐adhesion molecule. Here, we describe a new crucial function for mouse Akhirin (M‐AKH) in regulating the proliferation and differentiation of progenitors in the mouse spinal cord. During embryonic spinal cord development, M‐AKH is transiently expressed in the central canal ependymal cells, which possess latent neural stem cell properties. Targeted inactivation of the AKH gene in mice causes a reduction in the size of the spinal cord and decreases BrdU incorporation in the spinal cord. Remarkably, the expression patterns of ependymal niche molecules in AKH knockout (AKH−/−) mice are different from those of AKH+/+, both in vitro and in vivo. Furthermore, we provide evidence that AKH expression in the central canal is rapidly upregulated in the injured spinal cord. Taken together, these results indicate that M‐AKH plays a crucial role in mouse spinal cord formation by regulating the ependymal niche in the central canal.

A New Amino Acid Substitution at G150S in Lanosterol 14-α Demethylase (Erg11 protein) in Multi-azole-resistant Trichosporon asahii

The mechanisms of azole resistance in Trichosporon asahii have not yet been fully clarified. We previously showed that T. asahii has the ERG11 gene, coding lanosterol 14-α-demethylase (Erg11 protein; Erg11p), which is the primary target of azoles. A single amino acid substitution at G453R in Erg11p was found to induce changes in the affinity of this enzyme for azoles, especially fluconazole, in vitro. In the present study, we investigated the DNA sequences of the ERG11 gene using six different strains of clinically isolated T. asahii that were highly resistant to multi-azoles, including fluconazole, itraconazole, and voriconazole. All of the T. asahii strains had a point mutation (G448A) that caused a single amino acid substitution at G150S in Erg11p. This amino acid is highly conserved among major fungal pathogens. We identified a new point mutation in the ERG11 gene that is common to clinically isolated azole-resistant T. asahii strains, suggesting that this mutation is associated with the multi-azole resistance of T. asahii.

Cadherin-7 enhances Sonic Hedgehog signalling by preventing Gli3 repressor formation during neural tube patterning

Sonic Hedgehog (Shh) is a ventrally enriched morphogen controlling dorsoventral patterning of the neural tube. In the dorsal spinal cord, Gli3 protein bound to suppressor-of-fused (Sufu) is converted into Gli3 repressor (Gli3R), which inhibits Shh-target genes. Activation of Shh signalling prevents Gli3R formation, promoting neural tube ventralization. We show that cadherin-7 (Cdh7) expression in the intermediate spinal cord region is required to delimit the boundary between the ventral and the dorsal spinal cord. We demonstrate that Cdh7 functions as a receptor for Shh and enhances Shh signalling. Binding of Shh to Cdh7 promotes its aggregation on the cell membrane and association of Cdh7 with Gli3 and Sufu. These interactions prevent Gli3R formation and cause Gli3 protein degradation. We propose that Shh can act through Cdh7 to limit intracellular movement of Gli3 protein and production of Gli3R, thus eliciting more efficient activation of Gli-dependent signalling.