Toshiyuki Bohgaki

Genomic instability, defective spermatogenesis, immunodeficiency, and cancer in a mouse model of the RIDDLE syndrome.

Eukaryotic cells have evolved to use complex pathways for DNA damage signaling and repair to maintain genomic integrity. RNF168 is a novel E3 ligase that functions downstream of ATM,γ-H2A.X, MDC1, and RNF8. It has been shown to ubiquitylate histone H2A and to facilitate the recruitment of other DNA damage response proteins, including 53BP1, to sites of DNA break. In addition, RNF168 mutations have been causally linked to the human RIDDLE syndrome. In this study, we report that Rnf168−/− mice are immunodeficient and exhibit increased radiosensitivity. Rnf168−/− males suffer from impaired spermatogenesis in an age-dependent manner. Interestingly, in contrast to H2a.x−/−, Mdc1−/−, and Rnf8−/− cells, transient recruitment of 53bp1 to DNA double-strand breaks was abolished in Rnf168−/− cells. Remarkably, similar to 53bp1 inactivation, but different from H2a.x deficiency, inactivation of Rnf168 impairs long-range V(D)J recombination in thymocytes and results in long insertions at the class-switch junctions of B-cells. Loss of Rnf168 increases genomic instability and synergizes with p53 inactivation in promoting tumorigenesis. Our data reveal the important physiological functions of Rnf168 and support its role in both γ-H2a.x-Mdc1-Rnf8-dependent and -independent signaling pathways of DNA double-strand breaks. These results highlight a central role for RNF168 in the hierarchical network of DNA break signaling that maintains genomic integrity and suppresses cancer development in mammals.

The efficacy of tacrolimus in patients with interstitial lung diseases complicated with polymyositis or dermatomyositis

OBJECTIVE Interstitial lung diseases (ILDs) complicated with PM or DM are frequently aggressive and refractory to treatment. Recently some reports have suggested the potential benefit of tacrolimus for severe ILD complicated with PM/DM. However, little evidence has yet shown the efficacy of tacrolimus in these settings. The aim of this study was to evaluate the efficacy of tacrolimus as a treatment for PM-/DM-related ILD. METHODS This retrospective study comprised 49 previously untreated patients diagnosed as PM-/DM-related ILD admitted to Hokkaido University Hospital from January 2000 to July 2013. These patients were treated with tacrolimus plus conventional therapy or only with conventional therapy (prednisolone, i.v. CYC and/or ciclosporin). The primary endpoint was defined as the time to relapse or death of respiratory cause or a serious adverse event. The secondary endpoint was defined as the time from the initiation of immunosuppressive treatment to relapse or death of respiratory cause. Endpoints were compared by adjusted Cox regression model by using inverse probability of treatment weighting in order to reduce the impact of these selection biases and potential confounding factors. RESULTS After adjustment, the tacrolimus group (n = 25) had significantly longer event-free survival as compared with the conventional therapy group (n = 24). The weighted hazard ratio (HR) was 0.32 (95% CI 0.14, 0.75, P = 0.008). In addition, the tacrolimus group had significantly longer disease-free survival as compared with the conventional therapy group. The weighted HR was 0.25 (95% CI 0.10, 0.66, P = 0.005). CONCLUSION The addition of tacrolimus to conventional therapy significantly improved the prognosis of patients with PM-/DM-related ILD.

DNA double-strand break signaling and human disorders

DNA double-strand breaks are among the most serious types of DNA damage and their signaling and repair is critical for all cells and organisms. The repair of both induced and programmed DNA breaks is fundamental as demonstrated by the many human syndromes, neurodegenerative diseases, immunodeficiency and cancer associated with defective repair of these DNA lesions. Homologous recombination and non-homologous end-joining pathways are the two major DNA repair pathways responsible for mediating the repair of DNA double-strand breaks. The signaling of DNA double-strand breaks is critical for cells to orchestrate the repair pathways and maintain genomic integrity. This signaling network is highly regulated and involves a growing number of proteins and elaborated posttranslational modifications including phosphorylation and ubiquitylation. Here, we highlight the recent progress in the signaling of DNA double-strand breaks, the major proteins and posttranslational modifications involved and the diseases and syndromes associated with impaired signaling of these breaks.

Autoimmune disease after autologous hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) is an effective treatment for refractory autoimmune diseases. The safety and long-term outcome have been also acceptable. Infectious diseases under immune suppressive state after autologous HSCT are common transplantation related complications whereas autoimmune diseases are uncommon. Organ specific autoimmune diseases, such as immune mediated thrombocytopenia and thyroid dysfunction, are the most common after autologous HSCT. Systemic autoimmune diseases can also develop after autologous HSCT in patients with hematological disorders with genetic predisposition to autoimmune diseases. Although the mechanism of autoimmunity after HSCT is not well-known, long-term follow-up is essential in patients with autoimmune diseases treated with autologous HSCT.

RNF168 ubiquitylates 53BP1 and controls its response to DNA double-strand breaks

Significance DNA damage-repair mechanisms are highly regulated, and posttranslational modifications are one means of such regulation. p53-binding protein 1 (53BP1) is important for the response to DNA double-strand breaks (DSBs). Here we report that its functions are regulated by RING finger 168 (RNF168), a DNA DSB-signaling protein that is mutated in the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome. We show that before their localization to DNA DSBs, RNF168 interacts with 53BP1 and modifies it through the addition of a chain of ubiquitin-polypeptides. We also show that Lysine 1268 of 53BP1 is important for its ubiquitin modification by RNF168 and that loss of this modification impairs 53BP1 recruitment to sites of DNA damage and restrains its functions in the repair of DNA damage and maintenance of genomic stability. Defective signaling or repair of DNA double-strand breaks has been associated with developmental defects and human diseases. The E3 ligase RING finger 168 (RNF168), mutated in the human radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome, was shown to ubiquitylate H2A-type histones, and this ubiquitylation was proposed to facilitate the recruitment of p53-binding protein 1 (53BP1) to the sites of DNA double-strand breaks. In contrast to more upstream proteins signaling DNA double-strand breaks (e.g., RNF8), deficiency of RNF168 fully prevents both the initial recruitment to and retention of 53BP1 at sites of DNA damage; however, the mechanism for this difference has remained unclear. Here, we identify mechanisms that regulate 53BP1 recruitment to the sites of DNA double-strand breaks and provide evidence that RNF168 plays a central role in the regulation of 53BP1 functions. RNF168 mediates K63-linked ubiquitylation of 53BP1 which is required for the initial recruitment of 53BP1 to sites of DNA double-strand breaks and for its function in DNA damage repair, checkpoint activation, and genomic integrity. Our findings highlight the multistep roles of RNF168 in signaling DNA damage.

Up regulated expression of tumour necrosis factor α converting enzyme in peripheral monocytes of patients with early systemic sclerosis

Background: Systemic sclerosis (SSc) is accompanied by abnormalities in humoral and cellular immune systems. Objective: To determine the genes specifically expressed in the immune system in SSc by analysis of the gene expression profile of peripheral blood mononuclear cells (PBMC) from patients with SSc, including those treated with haematopoietic stem cell transplantation (HSCT). Additionally, to investigate the clinical significance of the up regulation of tumour necrosis factor α (TNFα) converting enzyme (TACE). Methods: PBMC from patients with SSc (n = 23) and other autoimmune diseases (systemic lupus erythematosus (SLE, n = 16), rheumatoid arthritis (RA, n = 29)), and from disease-free controls (n = 36) were examined. Complementary DNA arrays were used to evaluate gene expression of PBMC, in combination with real time quantitative polymerase chain reactions. TACE protein expression in PBMC was examined by fluorescence activated cell sorter (FACS). Results: In patients with SSc 118 genes were down regulated after HSCT. Subsequent comparative analysis of SSc without HSCT and healthy controls indicated SSc-specific up regulation for three genes: monocyte chemoattractant protein-3 (p = 0.0015), macrophage inflammatory protein 3α (p = 0.0339), and TACE (p = 0.0251). In the FACS analysis, TACE protein was mainly expressed on CD14+ monocytes both in patients with SSc and controls. TACE expression on CD14+ cells was significantly increased in patients with early SSc (p = 0.0096), but not in those with chronic SSc, SLE, or RA. TACE protein levels in SSc monocytes correlated with the intracellular CD68 levels (p = 0.0016). Conclusions: Up regulation of TACE expression was a unique profile in early SSc, and may affect the function of TNFα and other immunoregulatory molecules.

Pulmonary veno-occlusive disease following allogeneic peripheral blood stem cell transplantation for chronic myeloid leukaemia

A 49-year-old Japanese woman was admitted to our hospital on 23 July 1998 because of leucocytosis noted at a medical check. Her leucocyte and platelet counts were 14Æ1 · 10 ⁄ l and 637 · 10 ⁄ l respectively. She was diagnosed as having chronic myelogenous leukaemia (CML) based on the presence of a Philadelphia chromosome (Ph), and a BCR–ABL fusion gene was demonstrated. She was initially treated with interferon-a but became depressed. After 6 months of interferon-a, allogeneic peripheral blood stem cell transplantation (PBSCT) was carried out. Her human leucocyte antigen (HLA)-matched sister was treated with 300 lg of granulocyte colony-stimulating factor (G-CSF) for 6 d, and 119Æ25 · 10 of CD34-positive cells (2Æ93 · 10 ⁄ kg of recipient body weight) were obtained. After high-dose cyclophosphamide (60 mg ⁄ kg ⁄ d for 2 d) and total body irradiation (2 Gy twice daily for 3 d), PBSCT was performed on 18 March 1999. She received a short course of methotrexate and daily cyclosporin A to suppress acute graft-versus-host disease. Four days after PBSCT, 300 lg of G-CSF was administered, and her leucocyte and platelet counts improved. Her blood type changed to that of her sister and the BCR–ABL gene was no longer demonstrated by reverse transcriptase polymerase chain reaction. On 2 September, she complained of dyspnoea after walking. Her blood gas analysis showed severe hypoxia (PaO2 56Æ1 torr, SaO2 90%, PaCO2 32Æ0 torr). A computerized tomography scan of her thorax suggested decreased blood flow in both lower lobes of her lungs. Pulmonary blood perfusion scintigraphy confirmed this (left). Pulmonary digital subtraction angiography showed no thrombus-associated obstruction, but a reduction of blood flow in this area (right). A diagnosis of pulmonary veno-occlusive disease (PVOD) was made, and she was treated with pulse steroid therapy. Although her hypoxia improved transiently, she developed pneumonia and died of acute renal failure 1 month later. An autopsy showed severe pneumonia but the pulmonary vessels were normal. Although PVOD is very rare, it should be recognized as an important reversible complication of stem cell transplantation.

The E3 ligase PIRH2 polyubiquitylates CHK2 and regulates its turnover

The serine threonine kinase checkpoint kinase 2 (CHK2) is a DNA damage checkpoint protein important for the ATM-p53 signaling pathway. In addition to its phosphorylation, CHK2 is also ubiquitylated, and both post-translational modifications are important for its function. However, although the mechanisms that regulate CHK2 phosphorylation are well established, those that control its ubiquitylation are not fully understood. In this study, we demonstrate that the ubiquitin E3 ligase PIRH2 (p53-induced protein with a RING (Really Interesting New Gene)-H2 domain) interacts with CHK2 and mediates its polyubiquitylation and proteasomal degradation. We show that the deubiquitylating enzyme USP28 forms a complex with PIRH2 and CHK2 and antagonizes PIRH2-mediated polyubiquitylation and proteasomal degradation of CHK2. We also provide evidence that CHK2 ubiquitylation by PIRH2 is dependent on its phosphorylation status. Cells deficient in Pirh2 displayed accumulation of Chk2 and enhanced hyperactivation of G1/S and G2/M cell-cycle checkpoints. This hyperactivation was, however, no longer observed in Pirh2−/−Chk2−/− cells, providing evidence for the importance of Chk2 regulation by Pirh2. These findings indicate that PIRH2 has central roles in the ubiquitylation of Chk2 and its turnover and in the regulation of its function.

Immunological Reconstitution after Autologous Hematopoietic Stem Cell Transplantation in Patients with Systemic Sclerosis: Relationship Between Clinical Benefits and Intensity of Immunosuppression

Objective. To analyze the relationship between clinical benefits and immunological changes in patients with systemic sclerosis (SSc) treated with autologous hematopoietic stem cell transplantation (HSCT). Methods. Ten patients with SSc were treated with high-dose cyclophosphamide followed by highly purified CD34+ cells (n = 5) or unpurified grafts (n = 5). Two groups of patients were retrospectively constituted based on their clinical response (good responders, n = 7; and poor responders, n = 3). As well as clinical findings, immunological reconstitution through autologous HSCT was assessed by fluorescence-activated cell sorter analysis, quantification of signal joint T cell receptor rearrangement excision circles (sjTREC), reflecting the thymic function, and foxp3, a key gene of regulatory T cells, mRNA levels. Results. Patients’ clinical and immunological findings were similar between good and poor responders, or CD34-purified and unpurified groups at inclusion. The sjTREC values were significantly suppressed at 3 months after autologous HSCT in good responders compared with poor responders (p = 0.0152). Reconstitution of CD4+CD45RO− naive T cells was delayed in good responders compared with poor responders. The phenotype of other lymphocytes, cytokine production in T cells, and foxp3 gene expression levels after autologous HSCT did not correlate with clinical response in good or poor responders. Clinical and immunological findings after autologous HSCT were similar between CD34-purified and unpurified groups. Conclusion. Our results suggest that immunosuppression intensity, sufficient to induce transient suppression of thymic function, is attributable to the feasible clinical response in patients with SSc treated with autologous HSCT. Appropriate monitoring of sjTREC values may predict clinical benefits in transplanted SSc patients after autologous HSCT.

An independent validation of the Global Anti-Phospholipid Syndrome Score in a Japanese cohort of patients with autoimmune diseases

Sir, We previously proposed a quantitative score defined as ‘antiphospholipid score (aPL-S)’, in which, by testing multiple antiphospholipid antibodies (aPL), thrombotic risk may be evaluated in antiphospholipid syndrome (APS) patients. This score is validated in our cohort, as well as cohorts in other institution, as a useful quantitative index for diagnosing APS and predicting thrombosis in autoimmune diseases. Recently, Sciascia et al. broadened this idea by taking into account the aPL profile with conventional cardiovascular risks, and developed and validated the ‘Global Anti-Phospholipid Syndrome Score’ (GAPSS) as a marker of APS manifestations. In order to validate the GAPSS independently, we applied it to a cohort of 282 consecutive patients who attended the Hokkaido University Hospital rheumatology clinic from January 2002 to December 2003. There were 41 APS (17 primary APS) patients, 88 systemic lupus erythematosus (SLE) without APS, 50 rheumatoid arthritis, 16 Sjogren’s syndrome, 21 systemic sclerosis, 10 polymyositis/dermatomyositis and 56 other autoimmune diseases. Overall, thrombosis and/or pregnancy loss (APS manifestations) were observed in 43 patients (38 arterial thrombosis, 24 venous thrombosis and 11 pregnancy loss). Higher values of GAPSS were observed in patients who had experienced one or more of the APS manifestations compared with the patients without APS manifestations (Figure 1). When clinical subgroups were analyzed, patients with a history of arterial and/or venous thrombosis showed higher GAPSS compared with patients without APS manifestations. Patients with a history of pregnancy morbidity failed to show a significant difference in GAPSS compared with patients without APS manifestations (Figure 1). The smaller number of patients with history of pregnancy loss in our cohort was likely to be a primary factor in the insignificance of GAPSS in evaluating risks of pregnancy loss. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR), and area under the curve (AUC) of the receiver operating characteristic (ROC) curve at various levels of GAPSS cut-off are shown in Table 1. In our cohort, maximum AUC of ROC curve was at the cut-off level of GAPSS 6, lower than the cut-off level of GAPSS 10 in the original cohort in the UK. The reason for differences in adequate cut-off levels of ROC curve in GAPSS between two cohorts may be attributed to their different characteristic backgrounds. Our cohort consists of patients with various autoimmune diseases, while the original UK cohort comprised patients with APS and/or SLE. In our cohort, non-APS patients who scored GAPSS> 10 were all SLE patients (6/282). The high GAPSS in SLE patients was mainly due to the high incidence of ‘aPL carriers’. The adequate cut-off value of GAPSS may differ among patients with and without SLE. We demonstrated that aPL profile with conventional cardiovascular risks can be successfully quantified by GAPSS in an independent cohort of patients with autoimmune diseases. GAPSS correlated with a history of APS manifestations, particularly with thrombosis, suggesting that it is a suitable quantitative marker for APS. However, one should consider the appropriate cut-off to be adapted to different kinds of cohorts by reviewing their basic characteristics.