Clock gene expression in peripheral leucocytes of patients with type 2 diabetes.

AIM/HYPOTHESIS: Recent studies have demonstrated relationships between circadian clock function and the development of metabolic diseases such as type 2 diabetes. We investigated whether the peripheral circadian clock is impaired in patients with type 2 diabetes. METHODS: Peripheral leucocytes were obtained from eight patients with diabetes and six comparatively young non-diabetic volunteers at 09:00, 15:00, 21:00 and 03:00 hours (study 1) and from 12 male patients with diabetes and 14 age-matched men at 09:00 hours (study 2). Transcript levels of clock genes (CLOCK, BMAL1 [also known as ARNTL], PER1, PER2, PER3 and CRY1) were determined by real-time quantitative PCR. RESULTS: In study 1, mRNA expression patterns of BMAL1, PER1, PER2 and PER3 exhibited 24 h rhythmicity in the leucocytes of all 14 individuals. The expression levels of these mRNAs were significantly (p < 0.05) lower in patients with diabetes than in non-diabetic individuals at one or more time points. Moreover, the amplitudes of mRNA expression rhythms of PER1 and PER3 genes tended to diminish in patients with diabetes. In study 2, leucocytes obtained from patients with diabetes expressed significantly (p < 0.05) lower transcript levels of BMAL1, PER1 and PER3 compared with leucocytes from control individuals, and transcript expression was inversely correlated with HbA(1c) levels (rho = -0.47 to -0.55, p < 0.05). CONCLUSIONS/INTERPRETATION: These results suggest that rhythmic mRNA expression of clock genes is dampened in peripheral leucocytes of patients with type 2 diabetes. The impairment of the circadian clock appears to be closely associated with the pathophysiology of type 2 diabetes in humans.

Genes involved in oxidative phosphorylation are coordinately upregulated with fasting hyperglycaemia in livers of patients with type 2 diabetes.

AIMS/HYPOTHESIS: Mitochondrial oxidative phosphorylation (OXPHOS) plays an important role in the pathophysiology of type 2 diabetes. Genes involved in OXPHOS have been reported to be down-regulated in skeletal muscle from patients with type 2 diabetes; however, hepatic regulation is unknown. MATERIALS AND METHODS: We analysed expression of genes involved in OXPHOS from the livers of 14 patients with type 2 diabetes and 14 subjects with NGT using serial analysis of gene expression (SAGE) and DNA chip analysis. We evaluated the correlation between expression levels of genes involved in OXPHOS and the clinical parameters of individuals with type 2 diabetes and NGT. RESULTS: Both gene analyses showed that genes involved in OXPHOS were significantly upregulated in the type 2 diabetic liver. In the SAGE analysis, tag count comparisons of mitochondrial transcripts showed that ribosomal RNAs (rRNA) were 3.5-fold over-expressed, and mRNAs were 1.2-fold over-expressed in the type 2 diabetes library. DNA chip analysis revealed that expression of genes involved in OXPHOS, which correlated with several nuclear factors, including estrogen-related receptor-alpha or peroxisome proliferator-activated receptor-gamma, was a predictor of fasting plasma glucose levels, independently of age, BMI, insulin resistance and fasting insulin levels (p = 0.04). Surprisingly, genes involved in OXPHOS did not correlate with peroxisome proliferator-activated receptor-gamma coactivator-1alpha or nuclear respiratory factor 1. CONCLUSIONS/INTERPRETATION: Our results indicate that upregulation of genes involved in OXPHOS in the liver, which are regulated by different mechanisms from genes in the skeletal muscle, is associated with fasting hyperglycaemia in patients with type 2 diabetes.

Management of haemophilia B inhibitor patients with anaphylactic reactions to FIX concentrates.

Allergic reactions to concentrates containing factor IX (FIX) are serious complications in the treatment of haemophilia B patients with inhibitor. We have established a therapeutic protocol for such cases using an initial skin test followed by step-wise infusions of FIX concentrates under hydrocortisone cover. We have successfully treated three patients whose treatment with FIX had been suspended.

Intensification of chemotherapy using block therapies as consolidation and reinduction therapies for acute lymphoblastic leukemia during childhood.

Between April 1994 and March 1997, 143 children (age range, 1-15 years) with newly diagnosed acute lymphoblastic leukemia (ALL), except for those patients with t(9;22), were treated according to protocol-94 of the Osaka Childhood Leukemia Study Group. In this trial, the intensity of chemotherapy was enforced in the consolidation and reinduction phases by introducing AML-type block therapies consisting of concentrated administration of 4 to 6 drugs during 5 or 6 days. For patients in the higher risk groups, rotational combination chemotherapy was introduced following the early phase. A total of 124 children with B-cell precursor ALL (B-pre ALL) were classified into 3 groups, the ultrahigh-risk group (UHRG) (15 patients), the high-risk group (HRG) (61 patients), or the standard-risk group (SRG) (48 patients), based on age. leukocyte count, immunophenotype, central nervous system leukemia, response to treatment, and selected chromosomal abnormalities. The complete remission rate was 93%, and the 6-year event-free survival (EFS) rate was 79%+/-4%. EFS rates for the UHRG, HRG, and SRG groups were 67%+/-12%, 80%+/-6%, and 81%+/-6%, respectively. Nineteen patients with T-cell ALL were treated with the protocol for the UHRG. Thirteen patients (68%) attained complete remission, and the 6-year EFS rate was 55%+/-12%. Thus, intensification of chemotherapy improved the EFS rate and AML-type block therapies appeared to be effective as the consolidation and reinduction therapies for B-pre ALL.

Mosaic variegated aneuploidy with multiple congenital abnormalities: homozygosity for total premature chromatid separation trait.

Separation of chromatids of all mitotic chromosomes, here called total premature chromatid separation (total PCS), was observed in 67 to 87.5% of repeated cultures of peripheral blood lymphocytes from two unrelated infants. Also noted was a variety of mosaic aneuploidies, especially trisomies, double trisomies, and monosomies, to be called mosaic variegated aneuploidy. The infants both showed severe pre- and postnatal growth retardation, profound developmental retardation, uncontrollable seizures, severe microcephaly, hypoplasia of the brain, Dandy-Walker anomaly, abnormal facial appearance, and bilateral cataract. Patient 1, a girl, in addition had a cleft palate, multiple renal cysts, and Wilms tumor of the left kidney. Whereas patient 2, a boy, had ambiguous external genitalia. They both died within 2 years of age. In the two families of the infants, their parents and three other members showed 2.5 to 47% lymphocytes with total PCS but without mosaic variegated aneuploidy or phenotypic abnormalities. Another 10 relatives studied showed 0 to 1% cells with total PCS and so were judged negative for the total PCS trait. It was deduced that the total PCS trait in the two families was transmitted in an autosomal-dominant fashion, and the two affected infants were homozygous for the trait.

Effect of recombinant human granulocyte-colony stimulation factor (rhG-CSF) on immune system in pediatric patients with aplastic anemia.

To determine the effect of recombinant human granulocyte-colony stimulation factor (rhG-CSF) on the immune system, serum immunoglobulins, lymphocyte subsets, and serum cytokines were analyzed in eight pediatric patients with aplastic anemia (AA) during 8-week rhG-CSF therapy. The rhG-CSF was administered either subcutaneously (200 micrograms/m2 x 4 weeks, followed by 400 micrograms/m2 x 4 weeks) or intravenously (400 micrograms/m2 x 4 weeks, followed by 800 micrograms/m2 x 4 weeks). In response to rhG-CSF therapy, neutrophil counts exceeded the pretreatment counts by twofold during the first week except for one case that did not attain twofold increase until day 41. While serum IgG and IgA were not affected, serum IgM was elevated during treatment in six of the eight cases to more than 1.2-fold basal levels (P < 0.04); however, there was no increase in serum interleukin (IL)-6 and interferon-gamma levels. On the other hand, CD56 positive NK cells significantly dropped from 7.7% to 4.5% (P < 0.02). These results indicate that systemic administration of rhG-CSF affects not only the neutrophil count, but also serum IgM levels and the natural killer cell population in patients with AA.

Heterogeneous origin of established non-T,non-B cell lines from two adolescent patients with acute lymphoblastic leukemia.

Three leukemic cell lines were established from the bone marrow cells of two adolescents with non-T,non-B acute lymphoblastic leukemia (ALL) at relapse. Two cell lines from a 14-year-old girl and one from an 11-year-old boy were designated as KH-3A, KH-3B and KH-4, respectively. Leukemic cells started to grow attached to the bone marrow stromal (BMS) cells. KH-3A was positive for OKIa1 and positive at low percentages for B1, Leu-1 and J5 antigens; KH-3B reacted with OKIa1 and J5. Except for OKIa1, these two cell lines showed no surface marker change after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. On TPA treatment, clones (KH-3A-2 and KH-3A-3) isolated from KH-3A in agarose showed the induction of differentiation into T and B cell lineage. KH-4 was positive for OKIa1 and positive at low percentages for B1 and J5, and showed a strong reaction with OKIa1, B1 and J5 after TPA treatment. T cell receptor (TCR) beta-chain gene and immunoglobulin gene (JH and C mu) rearrangements were found in KH-3A, KH-3B, and sublines isolated from KH-3 (KH-3A-2 and -3) simultaneously. These findings indicate that BMS cells are useful for the establishment of leukemic cell lines and that some common ALL (cALL) cell populations may be heterogeneous.

Four cases of t(4;11) acute leukemia and its myelomonocytic nature in infants.

Four cases of acute infantile leukemia with translocation (4;11) (q21;q23) are reported. Although leukemia with this chromosomal abnormality has been classified as L2 acute lymphoblastic leukemia by the FAB classification, two of our cases appeared to be of myelomonocyte origin as demonstrated by cytochemical, immunologic, and electron microscopic studies and differentiation induction by 12-tetradecanoyl-phorbol-13-acetate and methylformamide. This chromosomal change is associated with poor prognosis.

Neutrophilic polymorphonuclear leucocytes in obese children. Studies by Sudan Black B and electron microscope.

In 378 obese and 35 control children, a patho-histometric study was performed regarding the relation of the morphology of neutrophilic polymorphonuclear leucocytes(PMNL) to obesity. Sudan Black B staining studies were undertaken to compare the change in PMNL in obese and nonobese childhood groups. The size and density of the cytoplasm of the PMNL were greater in the obese group. The average diameter of PMNL was 14.2 +/- 0.1 micra in the obese group and 13.6 +/- 0.2 in the control group. The electron microscopic feature of PMNL in the obese group included an increase in the number of the specific granules with diameter of 0.1 to 0.2 micron. These PMNL in obesity may be correlated with changes in the serum or hepatic lipid levels. An elevation of cholesterol, triglyceride, and beta-lipoprotein in the serum was found in obese children. The hepatic function was abnormal only in advanced degree of obesity.