Takashi Machii

E2F1 and c-Myc Potentiate Apoptosis through Inhibition of NF-κB Activity that Facilitates MnSOD-Mediated ROS Elimination

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Here, we found that overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA was induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibited NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb was also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1(+/+) and E2F1(-/-) murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity.

Brain Regions Involved in Fatigue Sensation: Reduced Acetylcarnitine Uptake into the Brain

Fatigue is an indispensable sense for ordering rest. However, the neuronal and molecular mechanisms of fatigue remain unclear. Chronic fatigue syndrome (CFS) with long-lasting fatigue sensation seems to be a good model for studying these mechanisms underlying fatigue sensation. Recently, we found that most patients with CFS showed a low level of serum acetylcarnitine, which well correlated with the rating score of fatigue, and that a considerable amount of acetyl moiety of serum acetylcarnitine is taken up into the brain. Here we show by metabolite analysis of the mouse brain that an acetyl moiety taken up into the brain through acetylcarnitine is mainly utilized for the biosynthesis of glutamate. When we studied the cerebral uptake of acetylcarnitine by using [2-(11)C]acetyl-L-carnitine in 8 patients with CFS and in 8 normal age- and sex-matched controls, a significant decrease was found in several regions of the brains of the patient group, namely, in the prefrontal (Brodmanns area 9/46d) and temporal (BA21 and 41) cortices, anterior cingulate (BA24 and 33), and cerebellum. These findings suggest that the levels of biosynthesis of neurotransmitters through acetylcarnitine might be reduced in some brain regions of chronic fatigue patients and that this abnormality might be one of the keys to unveiling the mechanisms of the chronic fatigue sensation.

Possible correlation between Borna disease virus infection and Japanese patients with chronic fatigue syndrome.

Borna disease virus (BDV) is a neurotropic, as yet unclassified, non‐segmented, negative‐sense, single‐strand RNA virus. Natural infection with this virus has been reported to occur in horses and sheep. In addition, antibodies to BDV in plasma or BDV RNA in peripheral blood mononuclear cells (PBMCs) were also found in patients with neuropsychiatric diseases. We describe here the possible link between the patients with chronic fatigue syndrome (CFS) and infection with BDV.

Anti-prothrombin antibodies combined with lupus anti-coagulant activity is an essential risk factor for venous thromboembolism in patients with systemic lupus erythematosus.

Anti‐prothrombin antibodies (anti‐prothrombin) and anti‐β2‐glycoprotein I antibodies (anti‐β2‐GP I) are the most common and characterized anti‐phospholipid antibodies (aPL) detected using specific enzyme‐linked immunosorbent assay (ELISA) systems. Recently, lupus anti‐coagulant (LA) activity detected by a phospholipid‐dependent coagulation assay was reported to be associated with anti‐prothrombin and/or anti–β2‐GP I. Here we show that the co‐existence of IgG anti‐prothrombin and LA activity might be an essential risk factor for venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). We examined not only the levels of antibodies to prothrombin and anti‐β2‐GP I (both IgG and IgM isotypes) using an ELISA system, but also LA activity detected using both diluted Russells viper venom time (dRVVT) and STACLOT LA test in 124 patients with SLE. The SLE patients were divided into four groups according to the results of ELISA and LA assay results for each aPL: group A, ELISA+ and LA+ group B, ELISA+ and LA−; group C, ELISA− and LA+ group D, ELISA− and LA−. Regarding IgG anti‐prothrombin, the prevalence of VTE was significantly higher in group A (16/35 cases, 45·7%, P < 0·001, Fishers exact probability test) than in the other groups (B, 2/30, 6·7%; C, 1/22, 4·5%; D, 1/37, 2·7%). With respect to IgM anti‐prothrombin and IgG or IgM anti‐β2‐GP I, the prevalence of VTE was higher in both groups A and C than in group D, but no statistical difference in prevalence was found between groups A and C. Multivariate logistic regression analysis of risk factors for VTE confirmed that the co‐existence of IgG anti‐prothrombin and LA activity was the only significant risk factor for VTE (odds ratio, 19·13; 95% confidence intervals, 4·74–77·18).

CD59-deficient blood cells and PIG-A gene abnormalities in Japanese patients with aplastic anaemia

Patients with aplastic anaemia (AA) frequently develop paroxysmal nocturnal haemoglobinuria (PNH) as a late complication. We investigated the frequency of the development of PNH features including a glycosyl phosphatidylinositol (GPI) anchoring defect in 73 Japanese patients with AA. A deficient expression of CD59 was found on erythrocytes and/or granulocytes in 21/73 (28.8%) of the patients. A Ham/sugar water test was positive in 13/21 patients. We also examined mutations of the PIG‐A gene in 11 patients with CD59 deficiency. A heteroduplex analysis detected PIG‐A gene abnormality in 10/11 patients tested. Nucleotide sequencing was performed in six patients and identified eight mutations including three mutations in one patient. The mutations of the PIG‐A gene were all different and included two single‐base insertions, one single‐base deletion, two two‐base deletions, and one each of eight‐base insertion and nine‐ and ten‐base deletions. All mutations but one caused frameshifts. Our findings indicate that a high proportion of Japanese patients with severe AA have a GPI‐anchoring defect and that the PIG‐A gene is mutated in the AA patients who had a GPI deficiency. We found no significant difference in the pattern of the PIG‐A gene mutation between the AA patients with a GPI deficiency and those with de novo PNH.

Risk of arterial thrombosis in patients with anticardiolipin antibodies and lupus anticoagulant

The relationship between arterial or venous thrombosis and the levels of anticardiolipin antibodies (aCL) and/or existence of lupus anticoagulant (LA) was studied. The 141 patients with systemic lupus erythematosus (SLE) were divided into four groups: aCL single positive (25 cases), LA single positive (11 cases), aCL and LA double positive (25 cases), aCL and LA double negative (80 cases). The prevalence of thrombosis was higher in aCL and LA double positive patients (21/25 cases, 84.0%, P < 0.01) than that in aCL single positive patients (4/25 cases, 16.0%), LA single positive patients (1/11 cases, 9.1%) and double negative patients (3/80 cases, 3.8%). Furthermore, in these double positive patients, all patients (10/10 cases) with a high positive level of aCL (>10 units/ml) had arterial thrombosis, whereas only 2/15 patients (13.3%) with a low positive level of aCL (3–10 units/ml) were affected. Venous thrombosis was frequently found in the low positive group (9/15 cases, 60.0%). On the contrary, none of 105 LA negative patients had arterial thrombosis and only seven (6.7%) had venous thrombosis. These findings indicate that a high aCL activity combined with a LA positive result might be a risk factor for arterial thrombosis.

Acquired activated protein C resistance is associated with the co-existence of anti-prothrombin antibodies and lupus anticoagulant activity in patients with systemic lupus erythematosus.

Summary. Venous thromboembolism (VTE) is one of the common manifestations in the anti‐phospholipid (aPL) syndrome. We examined the levels of IgG antibodies (Abs) to β2‐glycoprotein I (β2‐GP I) and prothrombin, lupus anticoagulant (LA) activity, activated protein C resistance (APC‐R), and factor V Leiden in 96 patients with systemic lupus erythematosus (SLE); 19 with VTE and 77 without VTE. Acquired APC‐R, which was not found in any patient with the factor V Leiden mutation, was present in 33 (34·4%) out of the 96 patients with SLE. The presence of acquired APC‐R was a strong risk factor for VTE. The SLE patients were divided into four groups according to the results of enzyme‐linked immunosorbent assay (ELISA) and LA activity for each aPL Abs: ELISA+, LA+; ELISA+, LA–; ELISA–, LA+; and ELISA–, LA–. A significant association was observed between APC‐R and the co‐existence of anti‐β2‐GP I Abs and LA activity or of anti‐prothrombin Abs and LA activity. There was no association between APC‐R and the presence of anti‐β2‐GP I Abs, anti‐prothrombin Abs, or LA activity alone. However, when multivariate logistical regression analysis was performed, it was clear that only the co‐existence of anti‐prothrombin and LA activity was a significant risk factor for APC‐R. These findings indicate that the co‐existence of anti‐prothrombin Abs and LA activity may be an important factor in the pathogenesis of acquired APC‐R in patients with SLE.

Natural killer cell-derived large granular lymphocyte lymphoma of lung developed in a patient with hypersensitivity to mosquito bites and reactivated Epstein-Barr virus infection

A 17‐year‐old female developed natural killer (NK) cell‐derived large granular lymphocyte (LGL) lymphoma of the lung. She had a past history of hypersensitivity to mosquito bites (HMB). After an eight‐year chronic, active Epstein‐Barr virus (EBV) infection, she developed multiple lung lesions and pleural effusion. In the effusion, 60% of the cells were LGL. They were CD2+, 3−, 16+, 56+, 57+, 45RO+/RA + weak, and possessed strong NK activity. No rearrangement of T‐cell–receptor genes was detected. From all these results, a diagnosis of NK‐LGL lymphoma of the lung was made. EB virus DNA was detected in cells infiltrating the pleural effusion. The clonality of the LGLs was determined by Southern blot hybridization with the terminal repeat sequence of EB virus as a probe, and by chromosomal abnormalities. The patient died from respiratory failure. Necropsy of the lung revealed diffuse lymphoma composed of polymorphic cells with typical angiocentric lesions. Reportedly, lymphomas of NK lineage show predominantly extranodal involvement, and primary lung lesions are rare. In the pleural effusion of the present case, abnormally high levels of soluble Fas ligand, interleukin‐10 and interferon γ were detected. This hypercytokinemia, reflecting the microenvironment of lymphoma cells, may play a role in the progression of the lymphoma and organ injury in the lung. Am. J. Hematol. 59:309–315, 1998.

Increased frequency of somatic mutations at glycophorin A loci in patients with aplastic anaemia, myelodysplastic syndrome and paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH), aplastic anaemia (AA) and myelodysplastic syndrome (MDS) are haemopoietic stem cell disorders. These disorders have some features in common, and a percentage of cases progress to acute leukaemia. We speculated that changes in gene stability are involved in the pathogenesis of these haemopoietic stem cell disorders. Therefore we investigated in vivo mutation frequencies in these disorders by erythrocyte glycophorin A (GPA) mutation assay. The assay enumerates NO or NN variant cells in 106 erythrocytes of the MN type using a flowcytometric technique. Patients undergoing chemotherapy known to be at risk of hypermutageneity were also studied. Events exceeding the 95th percentile of healthy donors (≧ 32 and 34 events, respectively for NO and NN variants) were defined as abnormal. Abnormal events in the NO variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, two out of seven patients with MDS, and four out of nine patients with PNH. Abnormal events in the NN variants were found in three out of seven patients undergoing chemotherapy, two out of nine patients with AA, one out of seven patients with MDS, and two out of nine patients with PNH. These results suggest that not only PIG‐A, but also other genes including the GPA gene, are hypermutable in haemopoietic stem cell disorders, and that mutagenic pressure and/or gene instability can contribute to the pathogenesis of these disorders.

Borna Disease Virus Infection in Two Family Clusters of Patients with Chronic Fatigue Syndrome

A high rate of Borna disease virus (BDV) infection has been demonstrated in patients with chronic fatigue syndrome (CFS). Herein, we focused on BDV infection in two family clusters of patients with CFS: a father, mother, two sons and one daughter (family #1); and a father, mother, two daughters and one son (family #2). All members, except for the elder son in family #1 and the father and son in family #2, were diagnosed with CFS. The results supported that all the family members with CFS were infected with BDV, as evidenced by the presence of antibodies to viral p40, p24 and/or gp18 and BDV p24 RNA in peripheral blood mononuclear cells. The healthy members, except for the father of family #2 who was positive for antibody to p24, were all negative by both assays. Follow‐up studies in family #1 continued to reveal BDV antibodies and BDV RNA, except in the mother, who lost the RNA upon slight recovery from the disease.