Masayoshi Kibata

Relationship between serum tumor necrosis factor-α and insulin resistance in obese men with Type 2 diabetes mellitus

Abstract We analyzed serum concentrations of tumor necrosis factor-α (TNF-α) for their role in insulin resistance in 12 obese men with untreated Type 2 diabetes mellitus and in 6 age-and BMI-matched obese controls. Insulin resistance was expressed using the homeostasis model assessment (HOMA-R). Six of the patients were insulin-resistant (HOMA-R>5.0), while six were not (HOMA-R≤3.0). Serum levels of TNF-α were higher in patients with insulin resistance (4.19±0.96 pg/ml) than in patients without insulin resistance (2.52±1.64 pg/ml) and in controls (2.03±1.21 pg/ml). Fasting serum concentrations of insulin and HOMA-R were higher in patients with insulin resistance (16.2±5.0 and 6.30±1.0 IU/ml, respectively) than in patients without insulin resistance (7.3±2.2 and 2.4±0.6 IU/ml) and in controls (8.0±2.9 and 1.8±0.6 IU/ml). These data suggest that high levels of serum TNF-α in patients with insulin resistance are related to high levels of fasting insulin and HOMA-R. We conclude that TNF-α may be involved in the pathogenesis of Type 2 diabetes mellitus in obese men. The importance of this investigation is that the subjects recruited in the study are BMI matched, because human obesity is associated with an increased TNF-α mRNA expression in adipose tissue.

The Production of IL-10 by Human Regulatory T Cells Is Enhanced by IL-2 through a STAT5-Responsive Intronic Enhancer in the IL-10 Locus

STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pathology due to a reduced number of CD4+CD25+ naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element.

Bioavailability of glucosyl hesperidin in rats.

Glucosyl hesperidin (G-hesperidin) is a water-soluble derivative of hesperidin. We compared the absorption and metabolism of G-hesperidin with those of hesperidin in rats. After oral administration of G-hesperidin or hesperidin to rats, hesperetin was detected in sera hydrolyzed with β-glucuronidase, but it was not detectable in unhydrolyzed sera. Serum hesperetin was found more rapidly in rats administered G-hesperidin than in those administered hesperidin. The area under the concentration-time curve for hesperetin in the sera of rats administered G-hesperidin was approximately 3.7-fold greater than that of rats administered hesperidin. In the urine of both administration groups, hesperetin and its glucuronide were found. Urinary excretion of metabolites was higher in rats administered G-hesperidin than in those administered hesperidin. These results indicate that G-hesperidin presents the same metabolic profile as hesperidin. Moreover, it was concluded that G-hesperidin is absorbed more rapidly and efficiently than hesperidin, because of its high water solubility.

Cell-in-cell Structures Formed between Human Cancer Cell Lines and the Cytotoxic Regulatory T-cell Line HOZOT

We previously established a novel cell line, termed HOZOT, derived from umbilical cord blood mononuclear cells that is characterized as a human cytotoxic regulatory T (Treg) cell line with a FOXP3(+)CD4(+)CD8(+)CD25(+) phenotype. Here, we describe a new property of HOZOT cells: they actively penetrate into a variety of human cancer cell lines, but not into normal cell lines, and form apparent cell-in-cell structures. In the process of cell penetration, we observed that HOZOT cells adhered to target cells seemed to first insert their nuclei into the cytoplasm of target cells, distinct from the process of phagocytosis. In addition, blocking experiments showed that major histocompatibility complex class I is one of the target cell recognition molecules for HOZOT cells. Furthermore, we propose that cell-in-cell structures between HOZOT cells and target cancer cells could be one of the cytotoxic mechanisms of HOZOT cells.

Novel mechanisms of suppressor activity exhibited by cytotoxic regulatory T cell lines, HOZOT

OBJECTIVE Regulatory T (Treg) cells, which play a central role in maintaining immune tolerance, can be grouped into different subtypes, such as naturally occurring Treg, type-1 T regulatory, and Th3. The suppressor activities of Treg cells are mediated through several molecular mechanisms, including immunosuppressive cytokines, cell surface molecules, and cytolytic molecules. In a previous report, we described a novel regulatory human T-cell line (termed HOZOT). The line was established by cocultivating human umbilical cord blood cells with mouse stromal cells in the absence of exogenous cytokines. In this study, we investigated the mechanism of HOZOTs suppressor activity. MATERIALS AND METHODS Suppressor activity of HOZOT was evaluated in vitro by assessing their inhibition of allogeneic mixed lymphocyte reaction, in which CD4+CD25(-) responder T cells were stimulated by dendritic cells (DCs). Responder T cells as well as DCs were prepared from umbilical cord blood using magnetic-activated cell sorting separation system. DNA microarray analysis was performed to search for specific molecules involved in HOZOTs suppressor mechanisms. RESULTS We confirmed that suppressing effects were observed in all three subpopulations of CD4/CD8 phenotype. We ruled out possible involvement of HOZOTs cytotoxic activity as well as participation of surface molecules, including cytotoxic T-lymphocyte-associated protein-4, programmed death-1, and glucocorticoid-inducible tumor necrosis factor receptor in suppressor. The supernatant obtained from HOZOT and DC coculture revealed mixed lymphocyte reaction inhibitory activity, indicating the presence of a soluble factor, which mediates suppressor function. Blocking experiments demonstrated that interleukin-10 and transforming growth factor-beta were not responsible factors. CONCLUSIONS HOZOT exerted suppressor activity in the absence of cell contact mechanisms, which are distinct from those of naturally occurring Treg, type-1 T regulatory, and Th3.

SERUM α-TOCOPHEROL, COENZYME Q, AND THIOBARBITURIC ACID-REACTIVE SUBSTANCE IN ACUTE MYOCARDIAL DAMAGE AND STROKE

We reported earlier that serum vitamin E level correlates inversely with the serum lipid peroxide level,’ which in lapan is routinely determined by Yagi’s fluorometric method‘ and designated as the thiobarbituric acid (TBA) value. We have also investigated the behaviors of serum TBA and vitamin E in patients with acute myocardial infarction (AMI) and stroke (CVA).3 Vitamin E dropped rapidly and TBA increased within one to two days from the outbreak of vascular accidents. Oral or cutaneous administration of vitamin E to 54 stroke patients in the acute phase successfully prevented the elevation of TBA. and this w a s d u e to the increase of serum vitamin E. The findings led to our assumption that vitamin E migrated to the lesions and was utilized as an extinguisher against the lipid peroxidation process. Coenzyme Q1,, is known to be a member of the electron transport system in mitochondria and is receiving attention as a therapeutic agent for cerebral, coronary, or renal artery ischemias in acute stages, as its antioxidative activity is being more and more elucidated. In this paper, the authors report the results of their observations on the behavior of coenzyme Qlu in comparison with that of vitamin E in the acute vascular accidents AM1 and CVA. Further, we conducted observations of these factors using, as a model of AMI. rats wherein myocardial necrosis was induced by injection of a large dosage of isoproterenol.

Interleukin-8 and RANTES are signature cytokines made by HOZOT, a new type of regulatory T cells

Distinct cytokine production profiles define the effector functions of both helper and regulatory T cells. Recently, we established novel cytotoxic regulatory T (Treg) cell lines, HOZOT, which have been characterized as IL-10-producing T cells. In this study, we further characterized HOZOT by performing comprehensive analyses of cytokines produced by HOZOTs in order to identify a signature cytokine profile. Using DNA microarrays, we compared the gene expression profiles of HOZOT-4, a representative HOZOT cell line, under three different conditions. Seven genes, including IL-8, IL-10, IL-13, MIP-1alpha, and MIP-1beta, were identified as inducible cytokines when stimulated with stromal cells or anti-CD3/CD28 antibodies. Twelve genes, including IL-2, IL-3, IL-4, IL-22, CCL1, and lymphotactin, were categorized as antibody stimulation-responsive but stromal cell-non-responsive. Three genes, IL-32, RANTES, and CCL23, were constitutively expressed irrespective of stimulation condition. Among these cytokines, we focused on two chemokines, IL-8 and RANTES for further studies, and found that only HOZOT produced both of them at considerable levels whereas other T cell subsets, including Tregs and helper T cells, did not. Kinetic and inhibition experiments revealed contrasting properties for the two chemokines. IL-8 was induced only after stimulation, whereas RANTES mRNA and protein accumulated to high levels even before stimulation. Interestingly, IL-8 mRNA was induced by cycloheximide treatment and RANTES showed reduced mRNA but increased protein expression by antibody stimulation. As a whole, the unique cytokine signature profile consisting of Th1, Th2, and cytolytic T cell cytokines as well as Treg cytokines reflect the multifunctional nature of HOZOT. In particular, the dual production of IL-8 and RANTES by distinct mechanisms is a hallmark of HOZOT.

HOZOTs, novel human regulatory T-cell lines, exhibit helper or suppressor activities depending on dendritic cell or anti-CD3 stimulation

OBJECTIVE HOZOT cell lines (HOZOTs) are a new type of regulatory T cells established from human umbilical cord blood without using cytokines. In addition to their unique FOXP3(+)CD4(+)CD8(+)CD25(+) phenotype, HOZOTs are bifunctional and can exert either suppressor or cytotoxic activities. To further characterize HOZOTs, we cocultured HOZOTs with responder T cells under different stimulation conditions and found another function of HOZOTs. MATERIALS AND METHODS Naïve CD4(+)T cells as responder cells were stimulated with dendritic cells or plate bound anti-CD3 antibody. As effector cells, HOZOTs were added to this culture and proliferation of the responder cells were monitored by (3)H-thymidine incorporation or carboxyfluorescein succinimidyl ester dilution method. To investigate the molecular mechanisms, antibodies specific for interleukin (IL)-2/IL-2R or cell surface molecules were used for blocking experiments. RESULTS The proliferation of naïve CD4(+)T cells was suppressed by one HOZOT line, HOZOT-4, when the responder cells were stimulated with dendritic cells. However, responder cell proliferation was augmented by HOZOT-4 when these cells were stimulated with anti-CD3 antibody. This opposing function to responder cells was unique to HOZOTs because naturally occurring regulatory T cells suppressed proliferation of both dendritic cell- and anti-CD3-antibody-stimulated cells. IL-2 was not involved in the mechanism of the helper activity of HOZOT-4 as blocking antibodies for IL-2 and IL-2R did not abrogate the helper activity. Moreover, this helper activity could not be reduced by blocking costimulatory pathways such as CD28/B7, CD4/human leukocyte antigen-DR, and intercellular adhesion molecule-1/lymphocyte function-associated antigen-1. CONCLUSION We demonstrated a new function of HOZOTs as helper T cells in addition to suppressor and cytotoxic activities, characterizing HOZOTs as multifunctional T cells.

Free radicals in the cerebrospinal fluid are associated with neurological disorders including mitochondrial encephalomyopathy

The free radical levels in the cerebrospinal fluid of 8 patients with neurological diseases and 9 undergoing lumbar anesthesia for surgery were measured. The ascorbate free radical level 10 min after lumbar puncture showed a positive correlation with the hydroxyl radical level. In the patient with mitochondrial encephalomyopathy, the levels of hydroxyl and ascorbate free radicals increased upon discontinuation of treatment and decreased upon its resumption, and the ascorbate free radical levels without therapy fell after lumbar puncture. The free radical levels in the cerebrospinal fluid may reflect the degree of oxidative stress in the central nervous system.

Re-induction of complete remission with a new synthetic retinoid, Am-80, for relapse of acute promyelocytic leukaemia previously treated with all-trans retinoic acid

Two patients with relapsed acute promyelocytic leukaemia previously treated with all‐trans retinoic acid (ATRA), were treated with a new synthetic retinoid, Am‐80. In both patients pancytopenia gradually resolved without an increase in leukaemic cells, and differentiation of leukaemic cells was observed morphologically in bone marrow. Without the use of anti‐leukaemic agents, both cases achieved complete remission (CR) on days 52 and 38 of treatment, respectively. On the day of CR, PML gene rearrangement and the t(15;17) translocation disappeared, though PML‐RAR α chimaeric messenger RNA was still detected by reverse transcriptase polymerase chain reaction. Both patients then received conventional chemotherapy for consolidation of CR. These clinical experiences suggest that Am‐80 may be an active agent for APL patients who have relapsed from ATRA‐induced remission.