In utero gene therapy rescues microcephaly caused by Pqbp1-hypofunction in neural stem progenitor cells.

Human mutations in PQBP1, a molecule involved in transcription and splicing, result in a reduced but architecturally normal brain. Examination of a conditional Pqbp1-knockout (cKO) mouse with microcephaly failed to reveal either abnormal centrosomes or mitotic spindles, increased neurogenesis from the neural stem progenitor cell (NSPC) pool or increased cell death in vivo. Instead, we observed an increase in the length of the cell cycle, particularly for the M phase in NSPCs. Corresponding to the developmental expression of Pqbp1, the stem cell pool in vivo was decreased at E10 and remained at a low level during neurogenesis (E15) in Pqbp1-cKO mice. The expression profiles of NSPCs derived from the cKO mouse revealed significant changes in gene groups that control the M phase, including anaphase-promoting complex genes, via aberrant transcription and RNA splicing. Exogenous Apc4, a hub protein in the network of affected genes, recovered the cell cycle, proliferation, and cell phenotypes of NSPCs caused by Pqbp1-cKO. These data reveal a mechanism of brain size control based on the simple reduction of the NSPC pool by cell cycle time elongation. Finally, we demonstrated that in utero gene therapy for Pqbp1-cKO mice by intraperitoneal injection of the PQBP1-AAV vector at E10 successfully rescued microcephaly with preserved cortical structures and improved behavioral abnormalities in Pqbp1-cKO mice, opening a new strategy for treating this intractable developmental disorder.

Pulmonary thromboembolism as a result of ovarian vein thrombosis after laparoscopic-assisted vaginal hysterectomy for uterine myoma.

Pulmonary thromboembolism is a potentially life-threatening disorder, which can occur secondary to deep vein thrombosis. Ovarian vein thrombosis has classically been considered to be a postpartum complication and is less frequently associated with other disease processes, such as recent pelvic surgery. Herein, we report a case of pulmonary thromboembolism as a result of ovarian vein thrombosis in a 39-year-old woman after an uneventful laparoscopic-assisted vaginal hysterectomy for uterine myoma. On postoperative day 3, the patient experienced fever of unknown origin, followed by lower abdominal pain, chest discomfort and shortness of breath. A hematological examination revealed an elevated D-dimer level. Computerized tomography revealed pulmonary thromboembolism caused by left ovarian vein thrombosis. The administration of anticoagulants resolved the symptoms. In order to avoid significant morbidities and potential mortality, attention should be paid to the possibility of pulmonary thromboembolism resulting from ovarian vein thrombosis, even after minimally invasive gynecologic surgery for benign conditions.

International multicenter tool to predict the risk of four or more tumor-positive axillary lymph nodes in breast cancer patients with sentinel node macrometastases.

Recently, many centers have omitted routine axillary lymph node dissection (ALND) after metastatic sentinel node biopsy in breast cancer due to a growing body of literature. However, existing guidelines of adjuvant treatment planning are strongly based on axillary nodal stage. In this study, we aim to develop a novel international multicenter predictive tool to estimate a patient-specific risk of having four or more tumor-positive axillary lymph nodes (ALN) in patients with macrometastatic sentinel node(s) (SN). A series of 675 patients with macrometastatic SN and completion ALND from five European centers were analyzed by logistic regression analysis. A multivariate predictive model was created and validated internally by 367 additional patients and then externally by 760 additional patients from eight different centers. All statistical tests were two-sided. Prevalence of four or more tumor-positive ALN in each center's series (P = 0.010), number of metastatic SNs (P < 0.0001), number of negative SNs (P = 0.003), histological size of the primary tumor (P = 0.020), and extra-capsular extension of SN metastasis (P < 0.0001) were included in the predictive model. The model's area under the receiver operating characteristics curve was 0.766 in the internal validation and 0.774 in external validation. Our novel international multicenter-based predictive tool reliably estimates the risk of four or more axillary metastases after identifying macrometastatic SN(s) in breast cancer. Our tool performs well in internal and external validation, but needs to be further validated in each center before application to clinical use.

Infectivity of HBV DNA positive donations identified in look-back studies in Hyogo-Prefecture, Japan.

AIMS/OBJECTIVES: To clarify transfusion incidence of hepatitis B virus (HBV) infected blood negative for mini pool-nucleic acid amplification testing (MP-NAT). BACKGROUND: Japanese Red Cross (JRC) blood centres screen donated blood to avoid contamination with HBV. However, a low copy number of HBV may be overlooked. METHODS/MATERIALS: In Hyogo-Prefecture, JRC blood centres screened 787 695 donations for HBV from April 2005 to March 2009. Of these, 685 844 were donations from the repeat donors. To detect the donors with HBV, serological tests, MP-NAT and/or individual donation (ID)-NAT were performed. To detect the recipients with transfusion-transmitted HBV infection (TTHBI), serological analysis and/or ID-NAT were performed. RESULTS: In this study, 265 of the 685 844 repeat donations were serologically and/or MP-NAT positive for HBV. Their repository samples from the previous donation were examined in a look-back study; 13 of the 265 repository samples proved ID-NAT positive. Twelve recipients were transfused with HBV-infected blood components derived from 10 of the 13 HBV-infected donors. Only 1 of the 12 recipients was identified as TTHBI case. Seven of the 12 recipients escaped from our follow-up study and 4 recipients were negative for HBV during the observation period. CONCLUSION: On the basis of the look-back study among the repeat donors in Hyogo-Prefecture, Japan, donations with HBV-infected blood negative for MP-NAT occurred with a frequency of 13 in 685 844 donations (∼1/53 000 donations). However, more than half of the recipients transfused with HBV-infected blood negative for MP-NAT could not be followed up. It is necessary to establish a more cautious follow-up system.

Living with COVID-19: mass gatherings and minimizing risk.

During the COVID-19 pandemic, it has been important to both minimize the risk of infection and restore daily life. As a typical example, mass gathering events, such as sporting events, are gradually becoming more common, thanks to the measures taken to contain COVID-19. Some pilot studies have been launched at governments' initiative to investigate the risk of infection without measures such as face masks and physical distancing at mass gathering events, but the ethics of these studies should be carefully considered. On the other hand, it is still beneficial to implement infection control measures at mass gathering events and, in parallel, to estimate the risk of infection with measures in place, especially under a lack of vaccination progress or the spread of mutant strains possibly resistant to vaccines. To help improve compliance with measures taken by spectators and organizers and to ensure their effectiveness, we have conducted quantitative evaluations of the implementation of such measures by monitoring CO2 concentrations, assessing the proportion of people wearing face masks and analysing human flow at the event. This approach allows us to share our observations with stakeholders and participants, enabling us to protect the culture of mass gathering events, minimize the risk of infection and restore a sense of well-being in daily life.

Acute non-hemolytic transfusion reactions and HLA class I antibody: advantages of solid phase assay compared with conventional complement-dependent assay.

To evaluate the specific reactivity of HLA Class I antibodies (HLA-I Abs) in acute non-hemolytic transfusion reactions (ANHTRs) using solid phase assays (SPAs) and conventional complement-dependent lymphocyte cytotoxicity test (LCT). ANHTRs are major issues in transfusion medicine. Anti-leukocyte antibodies have been implicated as one of the causative agents of transfusion-related acute lung injury (TRALI) and febrile reaction. Antibodies to HLA Class I and/or Class II (HLA Abs) have been intensively studied using SPAs for TRALI, but not for febrile reaction. About 107 patients and 186 donors associated with ANHTRs were screened for HLA Abs by SPAs such as enzyme-linked immunosorbent assay (ELISA) and the Luminex method. When HLA-I Ab was detected, its specific reactivity was evaluated by comparing its specificity identified by the Luminex method using recombinant HLA molecules and cognate HLA antigens (Ags), as well as LCT with or without anti-human globulin (AHG). The incidences of HLA Abs were as high as 32.7% of patients' serum samples and 16% of donors' serum samples. The incidence of HLA-I Abs did not differ significantly between cases of febrile and allergic reactions. However, HLA-I Abs associated with febrile reaction showed a significantly higher rate of possessing specific reactivity to cognate HLA Ags than those associated with allergic reactions. In addition, the Luminex method enabled the detection of HLA-I Abs much earlier than AHG-LCT in serum samples from a patient with febrile reaction and platelet transfusion refractoriness (PTR). SPAs seem more useful than AHG-LCT for evaluating reactivity of antibodies in ANHTR cases.

When should we intervene to control the 2009 influenza A(H1N1) pandemic?

We simulated the early phase of the 2009 influenza A(H1N1) pandemic and assessed the effectiveness of public health interventions in Japan. We show that the detection rate of border quarantine was low and the timing of the intervention was the most important factor involved in the control of the pandemic, with the maximum reduction in daily cases obtained after interventions started on day 6 or 11. Early interventions were not always effective.

DUSP22/LMW-DSP2 regulates estrogen receptor-alpha-mediated signaling through dephosphorylation of Ser-118.

In the previous study, we demonstrated the involvement of dual specificity phosphatase 22 (DUSP22/LMW-DSP2) in regulating the leukemia inhibitory factor/interleukin-6/signal transducer and activator of transcription 3-mediated signaling pathway. In this study, we show beta-estradiol (E2)-induced DUSP22 mRNA expression in estrogen receptor alpha (ERalpha)-positive breast cancer cells, whereas E2-induced phosphorylation and activation of ERalpha was suppressed by overexpression of DUSP22 but not catalytically inactive mutants. Furthermore, small-interfering RNA-mediated reduction of DUSP22 expression enhanced ERalpha-mediated transcription and endogenous gene expression. In fact, DUSP22 associated with ERalpha in vivo and both endogenous proteins interacted in ERalpha-positive breast cancer T47D cells. These results strongly suggest that DUSP22 acts as a negative regulator of the ERalpha-mediated signaling pathway.

Sentinel node biopsy in primary breast cancer: radioactive detection and metastatic disease.

AIM: To examine the relationship between the intensity of the radioactive counts and the presence of tumor metastasis in sentinel lymph nodes (SLNs) in order to correctly identify the number of SLNs to be removed. PATIENTS AND METHODS: Five hundred three breast cancer patients with successful radioisotope localization of SLNs using the combined blue dye and radioisotope method were analyzed. SLN biopsy was continued until all the blue-stained and radioactive nodes were removed. RESULTS: The mean number of harvested SLNs was 1.7+/-0.9, and the number of radioactive SLNs among the harvested nodes was 1.6+/-0.8. SLN metastasis was found in 123 of the 503 cases. The metastasis was detected in the SLN with the highest radioactive count (the hottest SLN) in 94 of the 123 cases with positive SLNs. The positive rate in the hottest SLN was 89% in 61 cases with a single radioactive SLN, and 65% in 62 cases with multiple radioactive SLNs. Of the 29 cases with positivity in other than the hottest SLNs, the metastasis was detected in the second hottest SLN in 16 cases, in the third hottest SLN in one case, in a mixture of negative radioactive SLNs and blue-dye-stained in four cases, and in the negative SLNs and positive non-SLNs (false-negative) in eight cases. Of 123 node-positive cases, 111 cases had metastasis that was detected within the first three hottest SLNs. CONCLUSIONS: These data suggest that lymph node metastasis may not always be detected in the hottest SLN. Thus, in practice, all radioactive and/or blue-dye-stained nodes should be removed for further examination.

Predictors of tumour involvement in remaining axillary lymph nodes of breast cancer patients with positive sentinel lymph node.

AIMS: To characterize the various clinicopathologic features in cases of breast cancer with positive sentinel lymph nodes (SLNs), in order to determine factors that might help in predicting the involvement of the non-SLNs. METHODS: A retrospective database review was performed of 726 breast cancer patients with stage 0-II, in whom SLNs were successfully identified. One hundred eighty-five of these patients showed positive SLNs, and subsequently underwent axillary lymph node dissection (ALND). These cases were divided into two groups based on the presence or absence of metastases in the non-SLNs, i.e. positive non-SLNs (NSLN+; 81 cases) and negative non-SLNs (NSLN-; 104 cases). RESULTS: Multivariate analysis revealed that a larger size of the primary tumour (>2.0cm), presence of lymphatic invasion, larger size of the largest SLN metastasis (>2mm), and a 100% metastatic rate in the SLNs (number of positive SLNs/number of harvested SLNs) were significantly associated with NSLN+. Among the cases in which all the four factors were present, 73% (30/41) were found to have NSLN+. CONCLUSION: We found four independent predictors in relation to non-SLN metastasis. Although these factors might be useful for determining the need of additional ALND, it would seem that even the presence of all of these four factors in combination may be insufficient to safely omit ALND. Thus, until further evidence is accumulated from the results of large clinical trials, ALND would still be recommended for patients with SLN metastasis.

Impact of isolated tumor cells in sentinel lymph nodes detected by immunohistochemical staining.

AIM: Isolated tumor cells (ITCs) in lymph nodes are defined histologically as node-negative. The clinical impact of ITCs in sentinel lymph nodes (SLNs) remains unclear. We report the prognosis of breast cancer patients with ITC-positive SLNs detected by immunohistochemical staining. PATIENTS AND METHODS: One hundred and sixty-five breast cancer patients with histologically negative SLNs were seen between January 1998 and December 2000. In 69 patients, sentinel node biopsy (SNB) was immediately followed by axillary lymph node dissection, and 96 had undergone SNB alone. Permanent sections of 301 SLNs were re-examined after hematoxylin-eosin staining and cytokeratin 19 immunohistochemical staining. RESULTS: ITCs were found in 18 SLNs of 17 patients and a micrometastasis was found in one SLN of one patient. As of November 2005, only one patient with ITCs in one SLN had supraclavicular lymph node recurrence. In contrast, 18 of the 147 patients with negative SLNs had tumor recurrence. Surgical management of the axilla had no influence on recurrence-free survival in all of the patients. CONCLUSION: This study shows that breast cancer patients with ITC-positive SLNs should be clinically managed as node-negative patients.

The hypusine cascade promotes cancer progression and metastasis through the regulation of RhoA in squamous cell carcinoma.

Metastasis is a critical factor contributing to poor prognosis in cancer, but the underlying mechanisms of metastasis are still poorly understood. We established a highly metastatic cell subline (HOC313-LM) derived from an oral squamous cell carcinoma cell line (HOC313) for uncovering the mechanisms of metastasis, and identified deoxyhypusine synthase (DHPS) as a metastasis-associated gene within the specific amplification at 19p13.2-p13.13 in HOC313-LM. DHPS-mediated hypusine-modification of eukaryotic translation factor 5A facilitated the translation of RhoA, resulting in the activation of the RhoA signaling pathway and leading to not only increased cell motility, invasion and metastasis of cancer cells in vitro, but also increased tumor growth in vivo. Moreover, the use of N1-Guanyl-1,7-diaminoheptane, a DHPS inhibitor, resulted in a significant decrease in tumor formation in vivo. In patients with esophageal squamous cell carcinoma (ESCC), overexpression of DHPS in ESCC tumors was significantly associated with worse recurrence-free survival, and correlated with distant metastasis. The elucidation of these molecular mechanisms within the hypusine cascade suggests opportunities for novel therapeutic targets in SCC.

The hnRNP-Htt axis regulates necrotic cell death induced by transcriptional repression through impaired RNA splicing.

In this study, we identify signaling network of necrotic cell death induced by transcriptional repression (TRIAD) by α-amanitin (AMA), the selective RNA polymerase II inhibitor, as a model of neurodegenerative cell death. We performed genetic screen of a knockdown (KD) fly library by measuring the ratio of transformation from pupa to larva (PL ratio) under TRIAD, and selected the cell death-promoting genes. Systems biology analysis of the positive genes mapped on protein-protein interaction databases predicted the signaling network of TRIAD and the core pathway including heterogeneous nuclear ribonucleoproteins (hnRNPs) and huntingtin (Htt). RNA sequencing revealed that AMA impaired transcription and RNA splicing of Htt, which is known as an endoplasmic reticulum (ER)-stabilizing molecule. The impairment in RNA splicing and PL ratio was rescued by overexpresion of hnRNP that had been also affected by transcriptional repression. Fly genetics with suppressor or expresser of Htt and hnRNP worsened or ameliorated the decreased PL ratio by AMA, respectively. Collectively, these results suggested involvement of RNA splicing and a regulatory role of the hnRNP-Htt axis in the process of the transcriptional repression-induced necrosis.

Predicting gene regulation by sigma factors in Bacillus subtilis from genome-wide data.

MOTIVATION: Sigma factors regulate the expression of genes in Bacillus subtilis at the transcriptional level. We assess the accuracy of a fold-change analysis, Bayesian networks, dynamic models and supervised learning based on coregulation in predicting gene regulation by sigma factors from gene expression data. To improve the prediction accuracy, we combine sequence information with expression data by adding their log-likelihood scores and by using a logistic regression model. We use the resulting score function to discover currently unknown gene regulations by sigma factors. RESULTS: The coregulation-based supervised learning method gave the most accurate prediction of sigma factors from expression data. We found that the logistic regression model effectively combines expression data with sequence information. In a genome-wide search, highly significant logistic regression scores were found for several genes whose transcriptional regulation is currently unknown. We provide the corresponding RNA polymerase binding sites to enable a straightforward experimental verification of these predictions.

Quantitative characterization of T-cell repertoire in allogeneic hematopoietic stem cell transplant recipients.

Allogeneic hematopoietic stem cell transplantation (HSCT) is one of curative treatment options for patients with hematologic malignancies. Although GVHD mediated by the donor's T lymphocytes remains the most challenging toxicity of allo-HSCT, graft-versus-leukemia (GVL) effect targeting leukemic cells, has an important role in affecting the overall outcome of patients with AML. Here we comprehensively characterized the TCR repertoire in patients who underwent matched donor or haplo-cord HSCT using next-generation sequencing approach. Our study defines the functional kinetics of each TCRA and TCRB clone, and changes in T-cell diversity (with identification of CDR3 sequences) and the extent of clonal expansion of certain T-cells. Using this approach, our study demonstrates that higher percentage of cord-blood cells at 30 days after transplant was correlated with higher diversity of TCR repertoire, implicating the role of cord-chimerism in enhancing immune recovery. Importantly, we found that GVHD and relapse, exclusive of each other, were correlated with lower TCR repertoire diversity and expansion of certain T-cell clones. Our results highlight novel insights into the balance between GVHD and GVL effect, suggesting that higher diversity early after transplant possibly implies lower risks of both GVHD and relapse following the HSCT transplantation.

Proliferative reaction of myelogenous leukemia cells with cytokines G-CSF, GM-CSF, M-CSF, SCF and TPO.

In recent years, many cytokines have been defined and some of them used clinically. In hematological malignancies, cytokines, including granulocyte colony-stimulating factor (G-CSF), have been widely used for leukopenia after chemotherapy. However, in acute myelogenous leukemia (AML), some leukemic cells may be induced to proliferate by these cytokines and they must be used with care. In this study, we have investigated cell reactivity and proliferation with G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CMF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF) and thrombopoietin (TPO) in cases of AML. We have also investigated the reactivity of some myeloid leukemia cell lines to TPO. G-CSF, GM-CSF, M-CSF, SCF and TPO caused proliferation of leukemic cells in 25%, 58.3%, 8.3%, 21.1% and 0% of cases, respectively. Because of this result, the use of G-CSF in AML should be regarded as potentially hazardous. TPO did not cause proliferation of leukemic cells in any case of AML, or in cell lines except MO7E, which is a megakaryocytic cell line. This result suggests that TPO might cause proliferation of some megakaryocytic leukemia cells. We cannot conclude that TPO does not cause proliferation of other AML cells, as the number of cases was small and it has been reported elsewhere that leukemia cells may proliferate when exposed to TPO in 50% of AML cases. Reactivity of AM L cells to TPO is an important factor when deciding the indications of TPO in AML and myelodysplastic syndrome.

c-fos and jun gene expression in murine precursor B lymphocytes developed in the interleukin-7-dependent bone marrow cell culture.

We analyzed the expression of c-fos and jun family gene in murine pre-B cells developed in the interleukin-7-(IL-7) dependent bone marrow cell culture. The c-fos gene was expressed in the pre-B cells. The c-fos RNA became undetectable after IgM+ B cells were developed in the culture. The c-fos expression in the pre-B cells was IL-7-dependent. However, the c-fos RNA was not induced when the pre-B cells cultured without IL-7 for 6 h were restimulated with IL-7. The expression of c-jun RNA was slightly induced in the restimulated pre-B cells. The junB and junD RNA were the steady state level in the cells. These gene products formed AP-1 molecule in the pre-B cells. These results suggest that the AP-1 plays a role in the IL-7-dependent pre-B cell development.

Long-term molecular remission induced by donor lymphocyte infusions for recurrent acute myeloblastic leukemia after allogeneic bone marrow transplantation.

A case of acute myelogenous leukemia with a t(8;21) translocation relapsed 5 months after allogeneic bone marrow transplantation (allo-BMT). After chemotherapy-induced hematologic remission, the patient received donor lymphocyte infusions (DLI); 4.9 x 108/kg T cells were infused. After DLI, she achieved molecular CR for the first time after allo-BMT, which lasted for 40 months. However, she suffered from grade III acute GVHD of the skin and the liver. Hepatic GVHD was sustained and resulted in fatal outcome. The case demonstrates that DLI is a double-edged sword. Further study is necessary before DLI can be considered to be a beneficial therapy for acute leukemia. Bone Marrow Transplantation (2000) 26, 809-810.

Coagulation abnormalities and thrombotic microangiopathy following bone marrow transplantation from HLA-matched unrelated donors in patients with hematological malignancies.

Thrombotic microangiopathy is a well-known heterogeneous disorder that occurs as a complication of allogenic bone marrow transplantation (BMT). We evaluated 12 consecutive patients receiving HLA-matched unrelated BMT and 12 consecutive recipients of HLA-identical related BMT for the development of bone marrow transplantation-associated thrombotic microangiopathy (BMT-TM) on days 30 and 60 following BMT. A diagnosis was made in four of 12 (33.3%) unrelated compared to none of 12 (0%) HLA-identical cases. Levels of serum lactic dehydrogenase (LDH), fibrin degraded products (FDP) and de novo thrombocytopenia were elevated in eight of 12 patients (66.7%) receiving unrelated donor BMT, and none of the patients receiving related donor BMT. Our findings suggest clinical or subclinical microangiopathic changes may occur frequently in unrelated donor BMT. FDP elevation is possibly an important marker of microangiopathic changes as early complications of BMT.

Epstein-Barr virus-negative high grade B cell lymphoma of donor origin developing 19 months after unrelated allogeneic bone marrow transplantation.

A 22-year-old man, in first complete remission of acute myelogenous leukemia, developed a high grade B cell lymphoma 19 months after an allogeneic bone marrow transplant (allo-BMT) from an HLA-identical unrelated donor. Biopsy of a cervical lymph node revealed a lymphoma that was negative for Epstein-Barr virus-encoded small nuclear RNAs (EBERs) in situ hybridization. Genotypic analyses identified the lymphoma to be of donor origin, and there was no evidence of the Epstein-Barr virus (EBV) DNA in the lymphoma by Southern blot analysis. The lymphoma went into complete remission, following four courses of combination chemotherapy, but relapsed after a month and the patient died of congestive heart failure. The patient was thought to be persistently immunosuppressed 11 months after cessation of immunosuppressants, and the lymphoma was thought to be induced by one or more factors other than EBV.