17β-Estradiol Attenuates Quinolinic Acid Insult in the Rat Hippocampus

A number of studies have shown that 17-estradiol has neuroprotective properties. In this study the neuroprotective effect of 17-estradiol against quinolinic-acid-induced neuronal damage was investigated. Ovariectomized rats were separated into three groups of five animals each. Rats received daily subcutaneous injections of either olive oil or 17-estradiol in olive oil for 7 days prior to and following a single intrahippocampal injection of 1 mol quinolinic acid in 2 L phosphate-buffered saline. The brains were removed and the hippocampi either sectioned and stained for microscopic examination or used in glutamate receptor saturation binding studies. Glutamate receptor displacement binding studies were also performed using concentrations of 0.05 nM–5 M 17-estradiol or quinolinic acid. The results show that 17-estradiol protects hippocampal neurons from quinolinic-acid-induced neurodegeneration by competing with quinolinic acid to bind to the N-methyl-D-aspartate (NMDA) receptor. This would result in a decrease in intracellular free-calcium influx and resultant neuronal swelling.     

The roles of IFN-γ versus IL-17 in pathogenic effects of human Th17 cells on synovial fibroblasts

Objectives

Th17 cells, while indispensable in host defense, may play pathogenic roles in many autoimmune diseases, including rheumatoid arthritis (RA). However, the mechanisms by which human Th17 cells drive autoimmunity have not been fully defined. We assessed the potential of the human Th17 CD4 T cell subset to induce expression of cell–cell interaction molecules and inflammatory mediators by fibroblast-like synoviocytes (FLS), and the roles of IFN-γ and IL-17 in these interactions.

Methods

Th1 or Th17 cells were induced from healthy adult donor CD4 T cells and were co-cultured with FLS for 48 h with/without neutralization of IFN-γ, IL-17A, or both. Alternatively, FLS were treated only with IFN-γ or IL-17 for 48 h. FLS expression of CD40, CD54, and MHC-II, as well as IL-6 and IL-8 secretion, were assessed by surface staining followed by flow cytometry and ELISA, respectively.

Results

Both Th1 and Th17 cells secreted IL-17 as well as IFN-γ, although IFN-γ production was much greater from Th1 cells. FLS expression of CD40, CD54, and MHC-II significantly increased upon co-culture with Th1 cells, while Th17 cells increased only the percentage of FLS that were CD54+. Both T cell subsets induced IL-6 and IL-8 secretion by RA FLS. Neutralization of IL-17A did not reduce FLS expression of CD40, MHC-II, or CD54, but did inhibit IL-6 and IL-8 secretion. Although IFN-γ was a weak inducer of IL-6 secretion and significantly inhibited IL-8 secretion from FLS when used as a single stimulus, neutralization of IFN-γ inhibited the secretion of both cytokines in Th17/FLS co-cultures with RA but not OA FLS.

Conclusion

FLS cell–cell interaction molecules and soluble inflammatory mediators are differentially regulated by IFN-γ and IL-17. The effects of IFN-γ may depend in part on the particular milieu of other co-existing cytokines and its potential to induce cell–cell interactions. The potential benefit of therapeutic neutralization of either IL-17 or IFN-γ could depend on the relative proportions of these cytokines in the synovial compartment of an RA patient.     

B-cell malignancies in microRNA Eμ-miR-17～92 transgenic mice

miR-17∼92 is a polycistronic microRNA (miR) cluster (consisting of miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a) which frequently is overexpressed in several solid and lymphoid malignancies. Loss- and gain-of-function studies have revealed the role of miR-17∼92 in heart, lung, and B-cell development and in Myc-induced B-cell lymphomas, respectively. Recent studies indicate that overexpression of this locus leads to lymphoproliferation, but no experimental proof that dysregulation of this cluster causes B-cell lymphomas or leukemias is available. To determine whether miR-17∼92- overexpression induces lymphomagenesis/leukemogenesis, we generated a B-cell–specific transgenic mouse model with targeted overexpression of this cluster in B cells. The miR-17∼92 overexpression was driven by the Eµ-enhancer and Ig heavy-chain promoter, and a 3′ GFP tag was added to the transgene to track the miR expression. Expression analysis using Northern Blot and quantitative RT-PCR confirmed 2.5- to 25-fold overexpression of all six miRs in the transgenic mice spleens as compared with spleens from wild-type mice. Eµ-miR-17∼92 mice developed B-cell malignancy by the age of 12–18 mo with a penetrance of ∼80% (49% splenic B-cell lymphoproliferative disease, 28% lymphoma). At this stage mice exhibited severe splenomegaly with abnormal B-cell–derived white pulp expansion and enlarged lymph nodes. Interestingly, we found three classes of B-cell lymphomas/leukemias at varying grades of differentiation. These included expansion of CD19⁺ and CD5⁺ double-positive B cells similar to the aggressive form of human B-cell chronic lymphocytic leukemia, B220⁺ CD43⁺ B1-cell proliferation, and a CD19⁺ aggressive diffuse large B-cell lymphoma–like disease, as assessed by flow cytometry and histopathological analysis.MicroRNAs (miRs) are 21- to 22-nucleotide-long noncoding RNA molecules that regulate the expression of multiple cellular genes, and their dysregulation is involved in many human diseases including cancer. The MiR-17∼92 cluster frequently is up-regulated in several different malignancies including diffuse large B-cell lymphoma (DLBCL) (1) and lung cancer (2, 3). The MiR-17∼92 cluster is encoded by the chromosome 13q31 locus in humans and the 14qE4 locus in mice. This genomic region is amplified in DLBCLs and several other tumors (reviewed in ref. 4). The cluster consists of six miRs (miR-17, 18a, 19a, 20a, 19b-1, and 92a-1) and has two paralogs in the genome, miR-106a∼363 and miR-106b∼25, proposed to have arisen through series of duplication and deletion events during vertebrate evolution (5).The first suggestion for its possible role in oncogenesis came from the study in Eμ-Myc mice in which enforced expression of miR-17∼92 was shown to accelerate B-cell tumor development (6). An additional study that investigated the role of miR-17∼92 in mice by driving its overexpression under the human CD2 promoter in both B and T cells found that mice overexpressing the miR-17∼92 cluster develop autoimmunity, a lymphoproliferative disease, and die prematurely (7). No lymphomas were observed in these animals, however. Targeted deletion of this cluster, but not of its paralogs, has shown that miR-17∼92 plays an important role in B-cell development, and the KO mice die shortly after birth from lung hypoplasia and ventricular septal defects (8). Further examination of the role of individual miRs in B-cell lymphomas showed that miR-19a and miR19b are required and sufficient for the proliferative activities of the cluster (9).To understand better the role of the miR-17∼92 cluster in B-cell neoplastic progression, we generated miR-17∼92 B-cell–specific transgenic mice. These mice overexpress miR-17∼92 under the control of Ig heavy-chain promoter and Eμ enhancer and express a 3′ GFP tag to track the miRs’ expression. After screening multiple founder mice, we obtained three transgenic lines that concurrently overexpress the six miRs by about 2.5- to 25-fold in their spleens as assessed by quantitative real-time PCR of purified B cells. These mice also express GFP in their B cells which serves as a proxy for miR-17∼92 expression.These mice develop a B-cell malignancy by age of 12–18 mo with a penetrance of ∼80% (49% splenic B-cell lymphoproliferative disease, 28% lymphoma). At this stage these mice exhibit severe splenomegaly with abnormal B-cell–derived white pulp expansion and enlarged lymph nodes. Interestingly, we found three classes of B-cell lymphomas/leukemias at varying grades of differentiation. These included expansion of CD19⁺ and CD5⁺ double-positive B cells similar to the aggressive forms of human B-cell chronic lymphocytic leukemia (B-CLL), B220⁺ CD43⁺ B1-cell proliferation, and a CD19⁺ aggressive DLBCL-like disease, as assessed by flow cytometry and histopathological analysis.     

17β-Estradiol Prevents Early-Stage Atherosclerosis in Estrogen Receptor-Alpha Deficient Female Mice

Estrogen is atheroprotective and a high-affinity ligand for both known estrogen receptors, ERα and ERβ. However, the role of the ERα in early-stage atherosclerosis has not been directly investigated and is incompletely understood. ERα-deficient (ERα−/−) and wild-type (ERα+/+) female mice consuming an atherogenic diet were studied concurrent with estrogen replacement to distinguish the actions of 17β-estradiol (E₂) from those of ERα on the development of early atherosclerotic lesions. Mice were ovariectomized and implanted with subcutaneous slow-release pellets designed to deliver 6 or 8 μg/day of exogenous 17β-estradiol (E₂) for a period of up to 4 months. Ovariectomized mice (OVX) with placebo pellets (E₂-deficient controls) were compared to mice with endogenous E₂ (intact ovaries) and exogenous E₂. Aortas were analyzed for lesion area, number, and distribution. Lipid and hormone levels were also determined. Compared to OVX, early lesion development was significantly (p < 0.001) attenuated by E₂ with 55–64% reduction in lesion area by endogenous E₂ and >90% reduction by exogenous E₂. Compared to OVX, a decline in lesion number (2- to 4-fold) and lesser predilection (~4-fold) of lesion formation in the proximal aorta also occurred with E₂. Lesion size, development, number, and distribution inversely correlated with circulating plasma E₂ levels. However, atheroprotection was independent of ERα status, and E₂ athero-protection in both genotypes was not explained by changes in plasma lipid levels (total cholesterol, triglyceride, and high-density lipoprotein cholesterol). The ERα is not essential for endogenous/exogenous E₂-mediated protection against early-stage atherosclerosis. These observations have potentially significant implications for understanding the molecular and cellular mechanisms and timing of estrogen action in different estrogen receptor (ER) deletion murine models of atherosclerosis, as well as implications to human studies of ER polymorphisms and lipid metabolism. Our findings may contribute to future improved clinical decision-making concerning the use of hormone therapy.     

17β-Estradiol administration attenuates seawater aspiration-induced acute lung injury in rats

There is very little evidence on the value of administering estrogen in cases of seawater drowning which can induce acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Therefore, this study aimed to investigate whether 17β-estradiol (E2) treatment can attenuate seawater aspiration-induced ALI in rats. In the experiment, ALI was induced by endotracheal instillation of seawater (4 mL/kg) and the rats were then given intraperitoneal injection of E2 (5 mg/kg) 20 min after seawater instillation. Finally, the changes of arterial blood gases which contained hydrogen ion concentration (pH), arterial oxygen tension (PaO₂) and arterial carbon dioxide tension (PaCO₂) were measured and the measurement of extravascular lung water (EVLW) was observed. The pulmonary histological changes were evaluated by hematoxylin–eosin stain. The expression of aquaporins (AQPs) 1, AQP5, and estrogen receptor-β (ERβ) was measured by western blotting and immunohistochemical methods. The results showed that compared with normal saline water, seawater aspiration induced more serious ALI in rats which was markedly alleviated by E2 treatment. Meanwhile, the ERβ in lung tissues was activated after E2 administration. The seawater aspiration group also presented with severe pulmonary edema which was paralleled with over expressed AQP1 and AQP5. However, the up-regulation of AQP1 and AQP5 was suppressed by the administration of E2, resulting in an attenuation of lung edema. In conclusion, E2 treatment could effectively attenuate seawater aspiration-induced acute lung injury in rats by the down-regulation of AQP1 and AQP5.     

Evidence Against Significant 19-nor-Deoxycorticosterone Production in Patients with 17α-Hydroxylase Deficiency

Tetrahydro-19-nor-deoxycorticosterone (3α, 21-dihydroxy-19-nor-5β-pregnan-20-one, a presumed metabolite of 19-nor-DOC was sought in the conjugated steroid fractions of urine from patients with 17α-hydroxylase deficiency syndrome. The reference material was prepared by microbial reduction (Clostridium paraputrificum) of 19-nor-DOC. Urinary steroid fractions of appropriate polarity were examined by high resolution gas chromatography and gas chromatography/mass spectrometry (GC/MS) but no tetrahydro-19-nor-DOC was found. The high selectivity of the GC/MS technique ensured that the excretion of this compound could not exceed about 1% of the excretion of tetrahydroDOC. Assuming that tetrahydro-19-nor-DOC is a major metabolite of 19-nor-DOC, it can be stated that 19-nor-DOC production is not a significant feature of 17α-hydroxylase deficiency. This assumption may not be valid if 19-nor-DOC is formed from DOC in the kidney and is excreted unmetabolized soon after synthesis.     

Low circulating levels of gastrin-17 in patients with Barrett＇s esophagus

AIM: To examine whether the fasting levels of serum gastrin-17 (G-17) are lower in Barrett's esophagus (BE) patients than in non-Barrett controls. METHODS: Nineteen patients with BE (presenting with a tubular segment ≥2 cm long in lower esophagus and intestinal metaplasia of incomplete type ("specialized columnar epithelium") in endoscopic biopsies from the tubular segment below the squamocolumnar junction were collected prospectively from outpatients referred to diagnostic gastroscopy. The controls comprised 199 prospectively collected dyspeptic outpatients without BE or any endoscopically visible lesions in the upper GI tract. Fasting levels of serum G-17 (G-17fast) were assayed with an EIA test using a Mab highly specific to amidated G-17. None of the patients and controls received therapy with PPIs or other antisecretory agents. RESULTS: The mean and median levels of G-17fast in serum were significantly lower (P= 0.001) in BE patients than in controls. The positive likelihood ratios (LR+) of low G-17fast to predict BE in the whole study population at G-17fast levels <0.5, <1, or <1.5 pmol/L were 3.5, 3.0, and 2.8, respectively. Among patients and controls with healthy stomach mucosa, the LR+ were 5.6, 3.8, and 2.6, respectively. In the whole study population, serum G-17 was below 2 pmol/L in 15 of 19 BE patients (79%). The corresponding prevalence was 66 of 199 (33%) in controls (P<0.001). The G-17fast was 5 pmol/L or more in only one of the 19 BE patients (5%). In controls, 76 of the 199 patients (38%) had such high serum G-17fast levels (P<0.01). CONCLUSION: Serum levels of G-17fast tend to be lower in native patients with BE than in healthy controls.     

17β-Estradiol Protects Against Quinolinic Acid-Induced Lipid Peroxidation in the Rat Brain

The neurotoxin quinolinic acid has been identified as a causative agent in Huntington's disease and is a metabolite of the tryptophan pathway in the brain. In the present study, the in vivo and in vitro effect of 17-estradiol on lipid peroxidation induced by quinolinic acid was investigated. For the in vivo experiments ovariectomized female rats were administered with 100 g 17-estradiol daily for seven days prior to and seven days following the intrahippocampal injection of 1 mol quinolinic acid. The level of lipid peroxidation in brain homogenate was investigated using the thiobarbituric acid test. The in vitro experiments were performed in brain homogenates of ovariectomized female rats. The homogenate was treated with quinolinic acid alone or in combination with 17-estradiol. Quinolinic acid increased lipid peroxidation in a dose dependent manner in vitro, while homogenate co-treated with 17-estradiol showed a significant reduction in lipid peroxidation. 17-estradiol was also shown to be protective against quinolinic acid in vivo. These results could explain the neuroprotective effect of 17-estradiol.     

Physiological and biochemical effects of 17β estradiol in aging female rat brain

Aging in females and males is considered as the end of natural protection against age related diseases like osteoporosis, coronary heart disease, diabetes, Alzheimer's disease and Parkinson's disease. These changes increase during menopausal condition in females when the level of estradiol is decreased. The objective of this study was to observe the changes in activities of monoamine oxidase, glucose transporter-4 levels, membrane fluidity, lipid peroxidation levels and lipofuscin accumulation occurring in brains of female rats of 3 months (young), 12 months (adult) and 24 months (old) age groups, and to see whether these changes are restored to normal levels after exogenous administration of estradiol (0.1 μg/g body weight for 1 month). The results obtained in the present work revealed that normal aging was associated with significant increases in the activity of monoamine oxidase, lipid peroxidation levels and lipofuscin accumulation in the brains of aging female rats, and a decrease in glucose transporter-4 level and membrane fluidity. Our data showed that estradiol treatment significantly decreased monoamine oxidase activity, lipid peroxidation and lipofuscin accumulation in brain regions of aging rats, and a reversal of glucose transporter-4 levels and membrane fluidity was achieved, therefore it can be concluded from the present findings that estradiol's beneficial effects seemed to arise from its antilipofuscin, antioxidant and antilipidperoxidative effects, implying an overall anti-aging action. The results of this study will be useful for pharmacological modification of the aging process and applying new strategies for control of age related disorders.     

Double deficiency in IL-17 and IFN-γ signalling significantly suppresses the development of diabetes in the NOD mouse

Aims/hypothesis

T helper type (Th) 17 cells have been shown to play important roles in mouse models of several autoimmune diseases that have been classified as Th1 diseases. In the NOD mouse, the relevance of Th1 and Th17 is controversial, because single-cytokine-deficient NOD mice develop diabetes similarly to wild-type NOD mice.

Methods

We studied the impact of IL-17/IFN-γ receptor double deficiency in NOD mice on the development of insulitis/diabetes compared with IL-17 single-deficient mice and wild-type mice by monitoring diabetes-related phenotypes. The lymphocyte phenotypes were determined by flow cytometric analysis.

Results

IL-17 single-deficient NOD mice showed delayed onset of diabetes and reduced severity of insulitis, but the cumulative incidence of longstanding diabetes in the IL-17-deficient mice was similar to that in wild-type mice. The IL-17/IFN-γ receptor double-deficient NOD mice showed an apparent decline in longstanding diabetes onset, but not in insulitis compared with that in the IL-17 single-deficient mice. We also found that double-deficient NOD mice had a severe lymphopenic phenotype and preferential increase in regulatory T cells among CD4⁺ T cells compared with the IL-17 single-deficient mice and wild-type NOD mice. An adoptive transfer study with CD4⁺CD25^? T cells from young non-diabetic IL-17 single-deficient NOD mice, but not those from older mice, showed significantly delayed disease onset in immune-deficient hosts compared with the corresponding wild-type mice.

Conclusions/interpretation

These results indicate that IL-17/Th17 participates in the development of insulitis and that both IL-17 and IFN-γ signalling may synergistically contribute to the development of diabetes in NOD mice.     

The miR-17～92 cluster collaborates with the Sonic Hedgehog pathway in medulloblastoma

Medulloblastomas (MBs) are the most common brain tumors in children. Some are thought to originate from cerebellar granule neuron progenitors (GNPs) that fail to undergo normal cell cycle exit and differentiation. Because microRNAs regulate numerous aspects of cellular physiology and development, we reasoned that alterations in miRNA expression might contribute to MB. We tested this hypothesis using 2 spontaneous mouse MB models with specific initiating mutations, Ink4c−/−; Ptch1+/− and Ink4c−/−; p53−/−. We found that 26 miRNAs showed increased expression and 24 miRNAs showed decreased expression in proliferating mouse GNPs and MBs relative to mature mouse cerebellum, regardless of genotype. Among the 26 overexpressed miRNAs, 9 were encoded by the miR-17∼92 cluster family, a group of microRNAs implicated as oncogenes in several tumor types. Analysis of human MBs demonstrated that 3 miR-17∼92 cluster miRNAs (miR-92, miR-19a, and miR-20) were also overexpressed in human MBs with a constitutively activated Sonic Hedgehog (SHH) signaling pathway, but not in other forms of the disease. To test whether the miR-17∼92 cluster could promote MB formation, we enforced expression of these miRNAs in GNPs isolated from cerebella of postnatal (P) day P6 Ink4c−/−; Ptch1+/− mice. These, but not similarly engineered cells from Ink4c−/−; p53−/− mice, formed MBs in orthotopic transplants with complete penetrance. Interestingly, orthotopic mouse tumors ectopically expressing miR-17∼92 lost expression of the wild-type Ptch1 allele. Our findings suggest a functional collaboration between the miR-17∼92 cluster and the SHH signaling pathway in the development of MBs in mouse and man.