Dynamic Changes of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> Regulatory T Cells in NOD.H-2<Superscript>h4</Superscript> Mice with Iodine-Induced Autoimmune Thyroiditis

Iodine is an essential trace element for thyroid hormone synthesis and metabolism, either low or high intake may lead to thyroid disease, but the pathogenetic mechanisms by which iodine interacts with the thyroid autoimmune are poorly understood. We investigated the dynamic changes of CD4⁺CD25⁺ regulatory T cells in NOD.H-2^h4 mice with iodine-induced autoimmune thyroiditis (AIT), and explore potential immune mechanism of AIT induced by iodine. NOD.H-2^h4 mice were randomly divided into two groups, and received plain water or water containing 0.005% sodium iodide. Eight weeks after iodine provision, the incidences of thyroiditis, relative weights of thyroids, and serum thyroglobulin antibody titers in the iodine-supplied groups were significantly increased compared to the control groups (p < 0.05). The AIT mice had fewer CD4⁺CD25⁺Foxp3⁺ T cells and reduced Foxp3 mRNA expression in splenocytes compared with the controls (p < 0.01), and maintained relatively low levels during the development of thyroiditis. The changes described above aggravated gradually with the extension of iodine treatment. These data suggest that CD4⁺CD25⁺ regulatory T cells may be involved in the pathogenesis and development of AIT induced by iodine.     

The Association Between Renal Tubular Dysfunction and Zinc Level in a Chinese Population Environmentally Exposed to Cadmium

Iodine-rich herbs such as seaweed, kelp, and sea tangle were widely used to treat various types of goiter with good effect and without any adverse side effects in China. When compared with potassium iodate (PI), iodine-rich herbs had a positive effect on the recovery of goiter resulting from iodine deficiency without any obvious harmful effects. In NOD.H-2^h4 mice, an autoimmune thyroiditis-prone model, iodine excess can increase infiltration of lymphocytes and structural damage of the thyroid follicles, hence resulting in thyroiditis. Until now, there has been little research on the comparative effects of PI and iodine-rich herbs on thyroid in an autoimmune thyroiditis-prone model. This study was designed to compare the different effects of iodine-rich herbs and PI on the thyroid gland in iodine-deficient NOD.H-2^h4 mice. Excessive intake of PI cause oxidative injury in the thyroid gland and increase the risk of autoimmune thyroiditis, while iodine-rich herbs cause less oxidative injury, significantly enhancing antioxidant capacity, and inhibit the high differentiation of Th17 cells in the thyroid glands of NOD.H-2^h4 mice.     

Role of TGFbeta in development of spontaneous autoimmune thyroiditis in NOD.H-2h4 mice.

Nearly 100% of NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) and produce anti-mouse thyroglobulin autoantibodies when they receive 0.05% NaI in their drinking water beginning at 8 wk of age. Our previous studies showed that TGFbeta1 mRNA was constitutively expressed in thyroids and spleens of normal NOD.H-2h4 mice but not other strains of mice. To determine whether TGFbeta might have a role in SAT, mice were given anti-TGFbeta mAb at various times during development of SAT. Anti-TGFbeta markedly inhibited development of SAT and production of anti-mouse thyroglobulin IgG1 autoantibodies. Anti-TGFbeta was most effective in inhibiting SAT when given during the time thyroid lesions were developing, i.e., starting 4 wk after administration of NaI water. The active form of the TGFbeta1 protein was present in thyroids of mice with SAT but not in normal NOD.H-2h4 thyroids. However, thyrocytes of normal NOD.H-2h4 thyroids did express latent TGFbeta1. TGFbeta1 protein expression in the thyroid correlated with SAT severity scores, and administration of anti-TGFbeta inhibited TGFbeta1 protein expression in both the thyroid and spleen. TGFbeta1 was produced primarily by inflammatory cells and was primarily localized in areas of the thyroid containing clusters of CD4(+) T and B cells. Depletion of CD8(+) T cells had no effect on TGFbeta1 protein expression. Activation of splenic T cells was apparently not inhibited by anti-TGFbeta, because up-regulation of mRNA for cytokines and other T cell activation markers was similar for control and anti-TGFbeta-treated mice. TGFbeta1 may function by promoting migration to, or retention of, inflammatory cells in the thyroid.     

TGF-beta promotes thyroid epithelial cell hyperplasia and fibrosis in IFN-gamma-deficient NOD.H-2h4 mice

IFN-gamma(-/-)NOD.H-2h4 mice given 0.05% NaI in their water develop severe thyroid epithelial cell (thyrocyte) hyperplasia and proliferation (TEC H/P) and fibrosis. Proliferating thyrocytes of IFN-gamma(-/-) mice with TEC H/P produce TGF-beta as demonstrated by immunohistochemical staining and in situ hybridization. Strong expression of activating phosphorylated Smad-2/3 and weak expression of inhibitory Smad-7 by proliferating thyrocytes correlate with the severity of TEC H/P. Splenocytes from IFN-gamma(-/-) mice with severe TEC H/P transfer severe TEC H/P to IFN-gamma(-/-)NOD.H-2h4.SCID mice. Mice given anti-TGF-beta had markedly reduced thyrocyte proliferation and decreased fibrosis compared with mouse Ig-treated controls, suggesting that TGF-beta plays an important role in development of TEC H/P induced by activated splenocytes. Moreover, transgenic IFN-gamma(-/-)NOD.H-2h4 mice expressing TGF-beta on thyrocytes all develop fibrosis and moderate to severe TEC H/P with accelerated kinetics, directly demonstrating a role for TGF-beta in severe TEC H/P and fibrosis.     

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot^?1, 0.40 ± 0.06 U mgprot^?1, P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot^?1, 93.0 ± 6.3 nmol mgprot^?1, P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.     

Positive selection of Tcrb-V10b+ T cells

The Tcrb-V10b⁺ T cell population has been examined with a newly established antibody, KT10b, specific for Tcrb-V10b but not Tcrb-V10a. H-2E⁺ mice have higher levels of Tcrb-V10b⁺ T cells (4.3%–11.%) than H-2E^– mice (2.2%–4.9%). This difference appears to be determined by levels of Tcrb-V10b⁺ T cells in the CD4 population. F₁ mice between H-2E⁺ and H-2E^– mice dominantly express higher levels of Tcrb-V10b⁺ T cells. [NOD (E–) x (NOD x A (E⁺))F₁] backcross mice show positive selection of Tcrb-V10b⁺ CD4⁺ T cells by H-2E. On the other hand other backcross analyses reveal positive selection of Tcrb-V10b⁺ CD8⁺ T cells by certain major histocompatibility class I molecules. Involvement of non-H-2 antigens in these positive selections remain to be determined. Address correspondence and offprint requests to: K. Tomonari.     

Fcγ receptor-mediated phagocytosis pathway was involved in phagocytosis of mIgM+ B lymphocytes from largemouth bass (Micropterus salmoides)

The potential for phagocytosis has been proven in teleost B cells, but the research on the regulatory mechanism of phagocytosis remains lacking. In this study, three largemouth bass (Micropterus salmoides) (15 ± 5 g) were injected intraperitoneally with Nocardia seriolae (10⁵ CFU/100 μl/fish) in vivo, and their spleen was collected at 72 h post-infection for mRNA-seq. After the de novo assembly of the paired-end reads, 73,622 unigenes were obtained. Gene expression profiling revealed that 2043 unigenes were differentially expressed after N. seriolae infection, comprising 1285 upregulated and 758 downregulated unigenes (q-value <0.05, log2FC > |2|) of which 181 genes were involved in phagocytosis. The Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis demonstrated that 12 differentially expressed genes (DEG) associated with phagocytosis were enriched in the Fcγ receptor-mediated phagocytosis signalling pathway. In vitro, the phagocytic ability of mIgM⁺ B lymphocytes was validated using indirect immunofluorescence assay (IIFA) and fluorescence activating cell sorter (FACS), and the phagocytosis rates of the mIgM⁺ B lymphocytes incubated with a Lyn inhibitor had decreased from 18.533 ± 6.00% to 11.610 ± 4.236% compared with the unblocked group. These results suggested that the Fcγ receptor-mediated phagocytosis signalling pathway had participated in the phagocytosis of B cells and provide further insight into the role of B cells in innate immunology.     

Prolonged survival in mice with advanced tumors treated with syngeneic or allogeneic intra-bone marrow–bone marrow transplantation plus fetal thymus transplantation

Thymic function decreases in line with tumor progression in patients with cancer, resulting in immunodeficiency and a poor prognosis. In the present study, we attempted to restore thymic function by BALB/c (H-2^d) syngeneic (Syn), or B6 (H-2^b) allogeneic (Allo) bone marrow transplantation (BMT) using intra-bone marrow–bone marrow transplantation (IBM–BMT) plus Syn-, Allo- or C3H (H-2^k) 3rd-party fetal thymus transplantation (TT). Although the BALB/c mice with advanced tumors (Meth-A sarcoma; H-2^d, >4 cm²) treated with either Syn- or Allo-BMT alone showed a slight improvement in survival compared with non-treated controls, the mice treated with BMT + TT showed a longer survival. The mice treated with Allo-BMT + Allo-TT or 3rd-party TT showed the longest survival. Interestingly, although there was no difference in main tumor size among the BMT groups, lung metastasis was significantly inhibited by Allo-BMT + Allo-TT or 3rd-party TT. Numbers of CD4⁺ and CD8⁺ T cells, Con A response, and IFN-γ production increased significantly, whereas number of Gr-1⁺/CD11b⁺ myeloid suppressor cells and the percentage of FoxP3⁺ cells in CD4⁺ T cells significantly decreased in these mice. Furthermore, there was a positive correlation between survival days and the number of T cells or T cell function, while there was a negative correlation between survival days and lung metastasis, the number of Gr-1⁺/CD11b⁺ cells, or the percentage of FoxP3⁺ cells. These results suggest that BMT + TT, particularly Allo-BMT + Allo-TT or 3rd-party TT, is most effective in prolonging survival as a result of the restoration of T cell function in hosts with advanced tumors.     

Iodine and IFN-gamma synergistically enhance intercellular adhesion molecule 1 expression on NOD.H2h4 mouse thyrocytes

NOD.H2(h4) mice spontaneously develop autoimmune lymphocytic thyroiditis that mimics human Hashimoto's thyroiditis, a disease where iodine, IFN-gamma, and adhesion molecules have all been implicated in the pathogenesis. To study how iodine and IFN-gamma modulate the expression of ICAM-1, we analyzed NOD.H2(h4) thyrocytes in baseline conditions (day 0) and at several time points following supplementation of iodine in the drinking water. On day 0, a small percentage ( approximately 10%) of thyrocytes constitutively expressed ICAM-1. The expression gradually increased to 13, 25, and 41% on days 7, 14 and 28, respectively, returning to baseline (9%) on day 35. The initial ICAM-1 kinetics was paralleled by thyroidal infiltration of CD45(+) hemopoietic cells, which increased from an average of 4% on day 0 to an average of 13, 21, and 24% on days 14, 28, and 35, respectively. To distinguish whether the observed ICAM-1 increase was a direct effect of iodine or a consequence of the immune infiltrate, we treated mouse primary thyrocyte cultures with 0.01 mM sodium iodine and showed a 3-fold increased ICAM-1 expression. To assess interaction between IFN-gamma and iodine, we analyzed CD45 and ICAM-1expression on thyrocytes from NOD.H2(h4) wild-type and NOD.H2(h4) thyr-IFN-gamma transgenic littermates. Strikingly, IFN-gamma interacted synergistically with iodine to enhance ICAM-1 expression on thyrocytes. These findings suggest that iodine and IFN-gamma cooperate to promote thyroidal expression of ICAM-1 in this mouse model of thyroiditis, highlighting the complex interplay present in the pathogenesis of Hashimoto's thyroiditis.     

Cell fusion contributes to the rescue of apoptotic cardiomyocytes by bone marrow cells

Cardiomyocyte apoptosis is an important contributor to the progressive cardiac dysfunction that culminates in congestive heart failure. Bone marrow cells (BMCs) restore cardiac function following ischaemia, and transplanted BMCs have been reported to fuse with cells of diverse tissues. We previously demonstrated that the myogenic conversion of bone marrow stromal cells increased nearly twofold when the cells were co‐cultured with apoptotic (TNF‐α treated) cardiomyocytes. We therefore hypothesized that cell fusion may be a major mechanism by which BMCs rescue cardiomyocytes from apoptosis. We induced cellular apoptosis in neonatal rat cardiomyocytes by treatment with hydrogen peroxide (H₂O₂). The TUNEL assay demonstrated an increase in apoptosis from 4.5 ± 1.3% in non‐treated cells to 19.0 ± 4.4% (P < 0.05) in treated cells. We subsequently co‐cultured the apoptotic cardiomyocytes with BMCs and assessed cell fusion using flow cytometry. Fusion was rare in the non‐treated control cardiomyocytes (0.3%), whereas H₂O₂ treatment led to significantly higher fusion rates than the control group (P < 0.05), with the highest rate of 7.9 ± 0.3% occurring at 25 μM H₂O₂. We found an inverse correlation between cell fusion and completion of cardiomyocyte apoptosis (R² = 0.9863). An in vivo mouse model provided evidence of cell fusion in the infarcted myocardium following the injection of BMCs. The percentage of cells undergoing fusion was significantly higher in mice injected with BMCs following infarction (8.8 ± 1.3%) compared to mice that did not undergo infarction (4.6 ± 0.6%, P < 0.05). Enhancing cell fusion may be one method to preserve cardiomyocytes following myocardial infarction, and this new approach may provide a novel target for cardiac regenerative therapies.     

APC Activation Restores Functional CD4+CD25+ Regulatory T Cells in NOD Mice that Can Prevent Diabetes Development

Background

Defects in APC and regulatory cells are associated with diabetes development in NOD mice. We have shown previously that NOD APC are not effective at stimulating CD4⁺CD25⁺ regulatory cell function in vitro. We hypothesize that failure of NOD APC to properly activate CD4⁺CD25⁺ regulatory cells in vivo could compromise their ability to control pathogenic cells, and activation of NOD APC could restore this defect, thereby preventing disease.

Methodology/Principal Findings

To test these hypotheses, we used the well-documented ability of complete Freund''s adjuvant (CFA), an APC activator, to prevent disease in NOD mice. Phenotype and function of CD4⁺CD25⁺ regulatory cells from untreated and CFA-treated NOD mice were determined by FACS, and in vitro and in vivo assays. APC from these mice were also evaluated for their ability to activate regulatory cells in vitro. We have found that sick NOD CD4⁺CD25⁺ cells expressed Foxp3 at the same percentages, but decreased levels per cell, compared to young NOD or non-NOD controls. Treatment with CFA increased Foxp3 expression in NOD cells, and also increased the percentages of CD4⁺CD25⁺Foxp3⁺ cells infiltrating the pancreas compared to untreated NOD mice. Moreover, CD4⁺CD25⁺ cells from pancreatic LN of CFA-treated, but not untreated, NOD mice transferred protection from diabetes. Finally, APC isolated from CFA-treated mice increased Foxp3 and granzyme B expression as well as regulatory function by NOD CD4⁺CD25⁺ cells in vitro compared to APC from untreated NOD mice.

Conclusions/Significance

These data suggest that regulatory T cell function and ability to control pathogenic cells can be enhanced in NOD mice by activating NOD APC.     

Iodine concentration of milk in a dose–response study with dairy cows and implications for consumer iodine intake

Most feed is poor in iodine and iodine supplementation of cow's diets must guarantee milk iodine concentrations for humans that contribute to prevention of the deficiency and minimize the risk of exceeding an upper limit of iodine intake. Five Holstein cows were fed four iodine doses (via Ca(ΙO₃)₂·6H₂O). In four sequential 14-d periods, doses of 0.2 (basal diet), 1.3, 5.1, and 10.1 mg iodine kg^?1 diet dry matter (DM) were administered. Samples of milk were collected during each period; blood was also sampled from each cow for each iodine dosage. In an 18-d depletion period, a non-supplemented diet was provided. Iodine was determined by inductively coupled plasma-mass spectrometry. The iodine content of milk and serum reflected the iodine dosages in feed significantly. The levels for the four doses tested in milk were 101±32, 343±109, 1215±222, and 2762±852 μg iodine kg^?1. The total amount of iodine in milk per day was 30–40% of ingested supplemental iodine. Omitting additional iodine resulted in a short-term reduction of serum and milk iodine following an exponential decay function. The iodine supplementation of 0.5–1.5 mg kg^?1 diet DM represents the requirement of the cow, resulting in 100–300 μg iodine L^?1 milk, which optimally contributes to human supply. The maximum dietary levels of former and present EU legislations (10 and 5 mg iodine kg^?1 cow feed) increase the risk of iodine excess in humans.     

Modulation of p38 kinase by DUSP4 is important in regulating cardiovascular function under oxidative stress

Over-activation of p38 is implicated in many cardiovascular diseases (CVDs), including myocardial infarction, hypertrophy, heart failure, and ischemic heart disease. Numerous therapeutic interventions for CVDs have been directed toward the inhibition of the p38 mitogen-activated protein kinase activation that contributes to the detrimental effect after ischemia/reperfusion (I/R) injuries. However, the efficacy of these treatments is far from ideal, as they lack specificity and are associated with high toxicity. Previously, we demonstrated that N-acetyl cysteine (NAC) pretreatment up-regulates DUSP4 expression in endothelial cells, regulating p38 and ERK1/2 activities, and thus providing a protective effect against oxidative stress. Here, endothelial cells under hypoxia/reoxygenation (H/R) insult and isolated heart I/R injury were used to investigate the role of DUSP4 in the modulation of the p38 pathway. In rat endothelial cells, DUSP4 is time-dependently degraded by H/R (0.25±0.07-fold change of control after 2 h H/R). Its degradation is closely associated with hyperphosphorylation of p38 (2.1±0.36-fold change) and cell apoptosis, as indicated by the increase in cells immunopositive for cleaved caspase-3 (12.59±3.38%) or TUNEL labeling (29.46±3.75%). The inhibition of p38 kinase activity with 20 µM SB203580 during H/R prevents H/R-induced apoptosis, assessed via TUNEL (12.99±1.89%). Conversely, DUSP4 gene silencing in endothelial cells augments their sensitivity to H/R-induced apoptosis (45.81±5.23%). This sensitivity is diminished via the inhibition of p38 activity (total apoptotic cells drop to 17.47±1.45%). Interestingly, DUSP4 gene silencing contributes to the increase in superoxide generation from cells. Isolated Langendorff-perfused mouse hearts were subjected to global I/R injury. DUSP4^-/- hearts had significantly larger infarct size than WT. The increase in I/R-induced infarct in DUSP4^-/- mice significantly correlates with reduced functional recovery (assessed by RPP%, LVDP%, HR%, and dP/dt_max) as well as lower CF% and a higher initial LVEDP. From immunoblotting analysis, it is evident that p38 is significantly overactivated in DUSP4^-/- mice after I/R injury. The activation of cleaved caspase-3 is seen in both WT and DUSP4^-/- I/R hearts. Infusion of a p38 inhibitor prior to ischemia and during the reperfusion improves both WT and DUSP4^-/- cardiac function. Therefore, the identification of p38 kinase modulation by DUSP4 provides a novel therapeutic target for oxidant-induced diseases, especially myocardial infarction.     

Effector and naturally occurring regulatory T cells display no abnormalities in activation induced cell death in NOD mice

Background

Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff) and regulatory T cells (Treg) to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice.

Principal Findings

Both effector (CD25⁻, FoxP3⁻) and suppressor (CD25⁺, FoxP3⁺) CD4⁺ T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP tranegenes. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL) in both strains. The effector and suppressor CD4⁺ subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4⁺CD25⁻ T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice.

Conclusion

These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis.     

TheH-2 class II genes and the susceptibility to the development of pulmonary adenoma in mice

To determine the locus in theH-2 complex that affects susceptibility to the development of pulmonary adenomas in mice,H-2 congenic and recombinant strains of mice with A/Wy, BALB/c, C3H, and B10 backgrounds were subjected to treatment with urethane. The average number and the incidence of adenoma foci were recorded five months after the treatment. InH-2 congenic strains on the A/Wy background, the average number of adenoma foci per mouse was significantly higher in mice of the A/Wy, A/J, and A-Tla ^b (H-2 ^a ) strains than in A.BY (H-2 ^b ) mice. In BALB/c and C3H congenic strains, the strains carrying theH-2 ^k haplotype were more susceptible than those carrying theH-2 ^b haplotype. InH-2 congenic strains on the B 10 background, the average number and incidence of foci was also higher in haplotypesa, h2, k, andj than in haplotypesb, s, f, d, r, h4, i3, i5, and4. The average numbers of adenoma foci in (A/J × A.BY)F₁ (H-2 ^a /H-2 ^b ) and (B10 × B10.A)F₁ (H-2 ^b /H-2 ^a ) were intermediate between the numbers in the parental strains. In [B10.A (4R) × B10.A (3R)]F₁ (H-2 ^h4 /H-2 ⁱ³ ) and [B10.A (4R) × B10.A (5R)]F₁ (H-2 ^h4 /H-2 ⁱ⁵ ), the numbers of adenoma foci were higher than in resistant parental recombinants. These patterns of response to urethane matched the patterns of the immune response to lactate dehydrogenase-B (LDH-B) and immunoglobulin gamma 2a (IgG2a) proteins. These differences between mice in their susceptibility to the development of pulmonary adenomas is probably due to the polymorphism of the class II genes in theH-2 complex.     

Effects of Hypoxia,Glucose Deprivation and Recovery on the Expression of Nucleoside Transporters and Adenosine Uptake in Primary Culture of Rat Cortical Astrocytes

The aim of this study was to explore effects of hypoxia, glucose deprivation (HGD) and recovery on expression and activities of equilibrative nucleoside transporters (rENT) and concentrative nucleoside transporters (rCNT) in rat astrocytes in primary culture. Amounts of cellular ATP in the control group (CG, 5% CO₂ in air, medium containing 7 mM d-glucose, 1 mM Na⁺-pyruvate, 1 h), HGD group (2% O₂/5% CO₂ in N₂, pyruvate-free medium containing 1.5 mM d-glucose and 10 mM 2-deoxy-d-glucose, 1 h) and recovery group (RG, HGD for 1 h, followed by 1 h exposure to the same conditions as the CG) were (nmol/mg protein, n = 4) 18 ± 1.6, 4.9 ± 0.6 and 10.1 ± 0.8, respectively. Extracellular adenosine concentrations increased from (nM, n = 3) 42 ± 4 in the CG, to 99 ± 8 in the HGD group and 86 ± 3 in the RG. Real-time PCR and immunoblotting revealed that in the HGD group and RG, the amounts of rENT1 mRNA and protein were reduced to 40 and 50%, when compared to the CG, respectively. Astrocyte cultures took up [³H]adenosine by concentrative and equilibrative transport processes; however, rENT1-mediated uptake was absent in the RG and cultures from the RG took up significantly less [³H]adenosine by equilibrative mechanisms than cultures from the CG.     

Eriodictyol regulated ferroptosis,mitochondrial dysfunction,and cell viability via Nrf2/HO-1/NQO1 signaling pathway in ovarian cancer cells

This study aimed to investigate the antitumor effect and the underlying molecular mechanism of eriodictyol on ovarian cancer cells. CaoV3 and A2780 were exposed to eriodictyol at different concentrations of 0−800 μM. Cell apoptosis and viability were determined by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay and Cell Counting Kit-8 (CCK-8) assay, respectively. Mitochondrial membrane potential was evaluated by flow cytometers with a JC-1 detection kit. Fe²⁺ content was evaluated using an iron assay kit. The section of tumor tissues was observed using hematoxylin-eosin (H&E) staining and nuclear factor erythroid 2-related factor 2 (Nrf2) expression was detected by immunohistochemistry (IHC) staining. Eriodictyol suppressed cell viability and induced cell apoptosis of CaoV3 and A2780 cells. Half maximal inhibitory concentration (IC₅₀) value of CaoV3 at 24 and 48 h was (229.74 ± 5.13) μM and (38.44 ± 4.68) μM, and IC₅₀ value of A2780 at 24 and 48 h was (248.32 ± 2.54) μM and (64.28 ± 3.19) μM. Fe²⁺ content and reactive oxygen species production were increased and protein levels of SLC7A11 and GPX4 were decreased by eriodictyol. Besides, eriodictyol reduced the ratio of JC-1 fluorescence ratio, glutathione and malondialdehyde contents but elevated Cytochrome C level. Nrf2 phosphorylation were obviously downregulated by eriodictyol. Finally, eriodictyol suppressed tumor growth, aggravated mitochondrial dysfunction and downregulated Nrf2 expression in tumor tissue in mice. Eriodictyol regulated ferroptosis, mitochondrial dysfunction and cell viability via Nrf2/HO-1/NQO1 signaling pathway in ovarian cancer.     

H-2D end confers dominant protection from IL-10-mediated acceleration of autoimmune diabetes in the nonobese diabetic mouse.

BALB/c mice that express IL-10 as a transgene in their pancreatic beta cells (Ins-IL-10 mice) do not develop diabetes, even after crossing to nonobese diabetic (NOD) mice ((Ins-IL-10 x NOD)F(1) mice). However, backcross of F(1) mice to NOD mice (NOD.Ins-IL-10 mice) results in N2 and N3 generations that develop accelerated diabetes. In this study, we found that NOD.Ins-IL-10 mice that expressed BALB/c-derived MHC molecules (NOD.Ins-IL-10(H-2(g7/d)) mice) were protected from diabetes. This protection associated with peri-islet infiltration and preserved beta cell function. Moreover, expression of I-A(d) and I-E(d) MHC class II molecules of BALB/c origin was not responsible for protection, but NOD.Ins-IL-10 mice that expressed BALB/c MHC class I D(d) molecules (NOD.Ins-IL-10(H-2(g7/d)) mice) did not develop diabetes. To directly test the possibility of a protective role of H-2D(d) in the development of accelerated diabetes, we generated transgenic mice expressing D(d) under the control of the MHC class I promoter. We found that double transgenic NOD.Ins-IL-10-D(d) mice developed accelerated diabetes in a fashion similar to NOD.Ins-IL-10 mice that were D(d) negative. Microsatellite analysis of H-2D(d)-linked loci confirmed association between BALB/c-derived alleles and protection of NOD.Ins-IL-10(H-2(g7/d)) mice. These results suggest a control of H-2D(d)-linked gene(s) on IL-10-mediated acceleration of autoimmune diabetes and dominant protection of the D(d) region in NOD.Ins-IL-10 mice.     

Determination of iodine in human milk and infant formulas

The aim of this study was to develop a method to determine iodine in human milk and infant formulas using ICP-MS. The milk samples were digested using an alkaline digestion (5% NH₃, 45 W, 2 min and 30 s), and the method was validated using a certified reference material (CRM) BCR CRM151. On the other hand the milk was separated in three fractions, whey, fat and caseins using ultracentrifugation (15 min, 4 °C, 50,000 rpm) and the iodine was determined in the different fractions. About 27 samples of different infant formulas and 14 samples of human milk have been studied. In the human milk the values found were between 144±93.2 μg kg⁻¹, whereas in the infant formulas the values were 53.3±19.5. For both types of samples the bigger amount of iodine is in the whey fraction, between 80% and 90%, whereas in the fat there is about a 2% of the total iodine and in the casein fraction the levels are between 5% and 10% depending on the type of sample.