Proteome Analysis of Thyroid Hormone Transporter Mct8/Oatp1c1-Deficient Mice Reveals Novel Dysregulated Target Molecules Involved in Locomotor Function.

Thyroid hormone (TH) transporter MCT8 deficiency causes severe locomotor disabilities likely due to insufficient TH transport across brain barriers and, consequently, compromised neural TH action. As an established animal model for this disease, Mct8/Oatp1c1 double knockout (DKO) mice exhibit strong central TH deprivation, locomotor impairments and similar histo-morphological features as seen in MCT8 patients. The pathways that cause these neuro-motor symptoms are poorly understood. In this paper, we performed proteome analysis of brain sections comprising cortical and striatal areas of 21-day-old WT and DKO mice. We detected over 2900 proteins by liquid chromatography mass spectrometry, 67 of which were significantly different between the genotypes. The comparison of the proteomic and published RNA-sequencing data showed a significant overlap between alterations in both datasets. In line with previous observations, DKO animals exhibited decreased myelin-associated protein expression and altered protein levels of well-established neuronal TH-regulated targets. As one intriguing new candidate, we unraveled and confirmed the reduced protein and mRNA expression of Pde10a, a striatal enzyme critically involved in dopamine receptor signaling, in DKO mice. As altered PDE10A activities are linked to dystonia, reduced basal ganglia PDE10A expression may represent a key pathogenic pathway underlying human MCT8 deficiency.

The highly and perpetually upregulated thyroglobulin gene is a hallmark of functional thyrocytes.

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e., for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a unique and valuable model to study the maintenance of a high transcriptional upregulation.

Triac Treatment Prevents Neurodevelopmental and Locomotor Impairments in Thyroid Hormone Transporter Mct8/Oatp1c1 Deficient Mice.

Patients with inactive thyroid hormone (TH) transporter MCT8 display intellectual disability due to compromised central TH transport and action. As a therapeutic strategy, application of thyromimetic, MCT8-independent compounds Triac (3,5,3'-triiodothyroacetic acid), and Ditpa (3,5-diiodo-thyropropionic acid) was proposed. Here, we directly compared their thyromimetic potential in Mct8/Oatp1c1 double knock-out mice (Dko) modeling human MCT8 deficiency. Dko mice received either Triac (50 ng/g or 400 ng/g) or Ditpa (400 ng/g or 4000 ng/g) daily during the first three postnatal weeks. Saline-injected Wt and Dko mice served as controls. A second cohort of Dko mice received Triac (400 ng/g) daily between postnatal weeks 3 and 6. Thyromimetic effects were assessed at different postnatal stages by immunofluorescence, ISH, qPCR, electrophysiological recordings, and behavior tests. Triac treatment (400 ng/g) induced normalized myelination, cortical GABAergic interneuron differentiation, electrophysiological parameters, and locomotor performance only when administered during the first three postnatal weeks. Ditpa (4000 ng/g) application to Dko mice during the first three postnatal weeks resulted in normal myelination and cerebellar development but only mildly improved neuronal parameters and locomotor function. Together, Triac is highly-effective and more efficient than Ditpa in promoting CNS maturation and function in Dko mice yet needs to be initiated directly after birth for the most beneficial effects.

Hyperoxia Leads to Transient Endocrine Alterations in the Neonatal Rat During Postnatal Development.

Introduction: High oxygen concentrations have been identified as one factor contributing to the pathogenesis of the retinopathia of prematurity, chronic lung disease of the preterm infant and preterm brain injury. Preterm infants also show short- and long-term alterations of the endocrine system. If hyperoxia is one pathogenetic factor has not been investigated yet. With regard to the high prevalence of neurodevelopmental impairments in preterm infants, the hypothalamus-pituitary-thyroid (HPT) axis, the hypothalamus-pituitary-adrenal (HPA) axis and the hypothalamus-pituitary-somatotropic (HPS) axis are of special interest due to their important role in neurodevelopment. Objective: The aim of this study was to investigate the effect of hyperoxia on the endocrine system in the neonatal rat by analyzing the activities of the HPT, HPA and HPS axes, respectively. Methods: Three-days old Wistar rats were exposed to hyperoxia (oxygen 80%, 48 h). On postnatal day 5 (P5) and P11, transcript levels of thyroid-stimulating hormone (TSH), proopiomelanocortin and growth hormone (GH) were analyzed in pituitary sections by in situ hybridization. Serologic quantification of TSH and thyroxine (T4), adrenocorticotropic hormone and GH were performed by Multiplex analysis and Enzyme-linked Immunosorbent Assay. Results: At P5, significantly lower GH levels were observed in pituitaries (mRNA) and in sera of rats exposed to hyperoxia. Serum TSH was significantly elevated without changes in T4. Conclusion: This is the first study demonstrating transient endocrine alterations following hyperoxia in the neonatal rat making oxygen a possible contributor to the pathogenesis of endocrine alterations seen in preterm infants. Considering the detrimental multi-organ effects of hyperoxia on the immature organism, a rational use of therapeutic oxygen in the treatrnent of preterm infants is of utmost importance.

Tissue-Specific Function of Thyroid Hormone Transporters: New Insights from Mouse Models.

Thyroid hormone (TH) transporters are required for cellular transmembrane passage of TH and are thus mandatory for proper TH metabolism and action. Consequently, inactivating mutations in TH transporters such as MCT8 or OATP1C1 can cause tissue- specific changes in TH homeostasis. As the most prominent example, patients with MCT8 mutations exhibit elevated serum T3 levels, whereas their CNS appear to be in a TH deficient state. Here, we will briefly summarize recent studies of mice lacking Mct8 alone or in combination with the TH transporters Mct10 or Oatp1c1 that shed light on many aspects and pathogenic events underlying global MCT8 deficiency and also underscore the contribution of Mct10 and Oatp1c1 in tissue-specific TH transport processes. Moreover, development of conditional knock-out mice that allow a cell-specific inactivation of TH transporters in distinct tissues, disclosed cell-specific changes in TH signaling, thereby highlighting the pathophysiological significance of local control of TH action.

High prevalence of CTX-M-15-producing O25b-ST131 Escherichia coli clone in Bulgarian hospitals.

According to the European Antimicrobial Resistance Surveillance System project results, Bulgaria has become one of the European countries with dramatically increasing rates of extended-spectrum beta-lactamase (ESBL) producers. The aim of this work was to investigate the epidemiology of ESBL-producing Escherichia coli clinical isolates in Bulgaria, collected from seven clinical centers in three towns, during two study periods: 2002-2003 and 2006-2009. For 193 ESBL-producing E. coli isolates random amplified polymorphic DNA (RAPD) analyses, phylogenetic typing, and screening for O25b-ST131 isolates were carried out. Antimicrobial susceptibility, ESBL-type and transferability of resistance determinants were analyzed. Four different ESBL-types, namely TEM-139, SHV-12, CTX-M-3, and CTX-M-15 were found. CTX-M-15 dominated, being found in 88% of the isolates. RAPD-typing revealed 35 types, among which type A dominated, comprising 65% of the isolates. Sixty-eight percent of the 193 isolates belonged to the O25b-ST131 clone, to the phylogenetic group B2, mostly showed RAPD-type A (92%) and were found in all participating hospitals. O25b-ST131 isolates predominantly produced CTX-M-15 (96%), and less SHV-12 (n=3) or TEM-139 (n=2). In conclusion, this study demonstrated for the first time the country-wide dissemination of a highly resistant B2 O25b-ST131 CTX-M-15 producing E. coli clone in Bulgaria.

Pharmacologic inhibition of mTOR antagonizes the cytotoxic activity of pemetrexed in non-small cell lung cancer.

PURPOSE: Pemetrexed, an inhibitor of thymidylate synthase (TS) and additional folate-dependent enzymes, is clinically active in patients suffering from "non-squamous" non-small cell lung cancer (NSCLC). High expression of TS has been implied as biomarker predictive of resistance to pemetrexed. Against this background, we studied whether inhibition of mTOR could lower expression of TS and thus sensitize NSCLC cells to pemetrexed. METHODS AND RESULTS: Using squamous cell carcinoma and adenocarcinoma NSCLC cell lines, we observed that constitutive TS expression levels failed to correlate with sensitivity to growth inhibition or apoptosis imposed by pemetrexed in vitro. Interestingly, pemetrexed strongly induced TS RNA and protein expression in all cell lines. The allosteric "rapalogue" mTOR inhibitor everolimus suppressed constitutive, but not pemetrexed-induced TS expression. Surprisingly, cotreatment with everolimus protected NSCLC cells against pemetrexed-induced apoptosis. This resulted in increased long-term clonogenic survival of NSCLC cells treated with pemetrexed plus everolimus as compared to pemetrexed alone. No such negative interaction was observed when everolimus was combined with recombinant TRAIL, a proliferation-independent proapoptotic agent. CONCLUSIONS: Rapalogues may suppress the antitumor activity of pemetrexed by slowing cell cycle progression. This should be considered when combining pemetrexed and mTOR inhibitors in NSCLC treatment.

Functional and clinical characterization of the putative tumor suppressor WWOX in non-small cell lung cancer.

INTRODUCTION: The oxidoreductase WWOX was initially described as a putative tumor suppressor in breast cancer. Non-small cell lung cancers (NSCLCs) frequently show aberrant WWOX expression. Herein, we characterized WWOX at a functional level in preclinical NSCLC models and in primary NSCLC biopsies. METHODS: The human wild-type (wt) WWOX complementary DNA and a mutant WWOX with structurally disrupted short-chain dehydrogenase/reductase domain were conditionally expressed at physiological levels in several human NSCLC models. Resulting transgenic cell populations were analyzed with respect to clonogenic survival and apoptosis sensitivity in vitro and tumor growth in immune-deficient mice. Tissue microarrays prepared from surgically resected primary human NSCLC tumors were studied to correlate intratumoral WWOX expression with patient outcomes. RESULTS: Conditional expression of wt WWOX, but not mutant WWOX, suppressed clonogenic survival of NSCLC cells in vitro and tumor growth in vivo. In addition, preserved intratumoral WWOX expression was associated with improved outcome in a cohort of 85 patients with surgically resected NSCLC. Unexpectedly, wt WWOX failed to sensitize NSCLC cells to various apoptotic stimuli but robustly protected against apoptosis induced by inhibitors of growth factor signal transduction. CONCLUSIONS: WWOX acts as a tumor suppressor in human NSCLC models in a short-chain dehydrogenase/reductase domain-dependent manner. This activity is independent of sensitization to apoptotic cell death. WWOX expression as detected by immunohistochemistry may be a prognostic biomarker in surgically resected, early-stage NSCLC.

Prevalence of virulence genes among bulgarian nosocomial and cystic fibrosis isolates of Pseudomonas aeruginosa

The aim of this study was to evaluate the prevalence of some virulence genes among 202 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients (n=42) and non-CF in-patients (n=160) and to analyze the values according to the patient groups, infection localization and antimicrobial resistance. The following frequencies in all studied strains were established: algD (encoding GDP-mannose 6-dehydrogenase AlgD) - 91.1 percent, pilB (type IV fimbrial biogenesis protein PilB) - 23.8 percent, nan1 (neuraminidase) - 21.3 percent, lasB (elastase LasB) - 100 percent, plcH (haemolytic phospholipase C precursor) - 91.6 percent, exoS (exoenzyme S) - 62.4 percent, and exoU (exoenzyme U) - 30.2 percent. The prevalence of nan1 was significantly higher (P<0.01) in CF isolates (38.1 percent) than that in non-CF isolates (16.9 percent). The nan1-positive CF strains were cultured from 16 patients with recurrent lung exacerbations. This study revealed a statistically significant difference (P<0.01) between the portion of multidrug-resistant (MDR) nosocomial P. aeruginosa strains containing a large number (¡Ý5) of virulence genes (38.1 percent) and the respective part of non-MDR isolates (17.6 percent). Moreover, pilB, exoU and nan1 manifested a higher spread (P<0.001) among MDR than in non-MDR strains (respectively, 39.1 percent vs. 13.2 percent; 40.2 percent vs. 17.7 percent and 26.1 percent vs. 4.4 percent). In conclusion, the dissemination of nan1 in CF isolates was moderate and correlated with the lower proportion of patients with lung exacerbations. The molecular-genetic detection of this gene may be used as an indirect measure of CF pulmonary disease evolution. Simultaneous determination of virulence factors and antimicrobial resistance is the contemporary approach for examination of the microbiological aspects of infections caused by P. aeruginosa.

Prevalence of virulence genes among bulgarian nosocomial and cystic fibrosis isolates of pseudomonas aeruginosa.

The aim of this study was to evaluate the prevalence of some virulence genes among 202 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients (n=42) and non-CF in-patients (n=160) and to analyze the values according to the patient groups, infection localization and antimicrobial resistance. The following frequencies in all studied strains were established: algD (encoding GDP-mannose 6-dehydrogenase AlgD) - 91.1%, pilB (type IV fimbrial biogenesis protein PilB) - 23.8%, nan1 (neuraminidase) - 21.3%, lasB (elastase LasB) - 100%, plcH (haemolytic phospholipase C precursor) - 91.6%, exoS (exoenzyme S) - 62.4%, and exoU (exoenzyme U) - 30.2%. The prevalence of nan1 was significantly higher (P<0.01) in CF isolates (38.1%) than that in non-CF isolates (16.9%). The nan1-positive CF strains were cultured from 16 patients with recurrent lung exacerbations. This study revealed a statistically significant difference (P<0.01) between the portion of multidrug-resistant (MDR) nosocomial P. aeruginosa strains containing a large number (≥5) of virulence genes (38.1%) and the respective part of non-MDR isolates (17.6%). Moreover, pilB, exoU and nan1 manifested a higher spread (P<0.001) among MDR than in non-MDR strains (respectively, 39.1% vs. 13.2%; 40.2% vs. 17.7% and 26.1% vs. 4.4%). In conclusion, the dissemination of nan1 in CF isolates was moderate and correlated with the lower proportion of patients with lung exacerbations. The molecular-genetic detection of this gene may be used as an indirect measure of CF pulmonary disease evolution. Simultaneous determination of virulence factors and antimicrobial resistance is the contemporary approach for examination of the microbiological aspects of infections caused by P. aeruginosa.

New variant of CTX-M-type extended-spectrum beta-lactamases, CTX-M-71, with a Gly238Cys substitution in a Klebsiella pneumoniae isolate from Bulgaria.

A single Klebsiella pneumoniae strain isolated in a Bulgarian hospital was found to produce CTX-M-71, a new CTX-M variant characterized by one amino acid substitution from glycine to cysteine at position 238 in comparison to CTX-M-15. This exchange decreased the hydrolytic activity of the beta-lactamase for cefotaxime, ceftazidime, and cefepime.

A novel molecular mechanism of primary resistance to FLT3-kinase inhibitors in AML.

Currently, FLT3 tyrosine kinase inhibitors (TKIs) are emerging as the most promising drug therapy to overcome the dismal prognosis of acute myelogenous leukemia (AML) patients harboring internal tandem duplications (ITDs) of FLT3. However, up-front drug resistance occurs in approximately 30% of patients, and molecular mechanisms of resistance are poorly understood. Here, we have uncovered a novel mechanism of primary resistance to FLT3 TKIs in AML: an FLT3 receptor harboring a nonjuxtamembrane ITD atypically integrating into the beta-2 sheet of the first kinase domain (FLT3_ITD627E) induces dramatic up-regulation of the anti-apoptotic myeloid cell leukemia 1 protein (MCL-1). Using RNA interference technology, deregulated MCL-1 protein expression was shown to play a major role in conferring the resistance phenotype of 32D_ITD627E cells. Enhanced and sustained binding of the adaptor protein GRB-2 to the FLT3_ITD627E receptor is involved in MCL-1 up-regulation and is independent from TKI (PKC412)-induced inhibition of the receptor kinase. Thus, we describe a new mechanism of primary resistance to TKIs, which operates by reprogramming local and distant signal transduction events of the FLT3 tyrosine kinase. The data presented suggest that particular ITDs of FLT3 may be associated with rewired signaling and differential responsiveness to TKIs.

Identification of a novel type of ITD mutations located in nonjuxtamembrane domains of the FLT3 tyrosine kinase receptor.

In acute myeloid leukemia (AML), internal tandem duplications (ITDs) of the juxtamembrane (JM) of FLT3 have been shown to play a crucial role in driving proliferation and survival of the leukemic clone. Here, we report the identification of FLT3_ITD mutations located in non-JM domains of the FLT3-receptor. This novel type of FLT3_ITD mutation was found in 216 of 753 (28.7%) of unselected FLT3_ITD-positive AML cases. An FLT3 receptor harbouring a prototypic non-JM ITD (FLT3_ITD627E) mediated constitutive phosphorylation of FLT3 and of STAT5, suggesting that non-JM ITDs confer constitutive activation of the receptor. FLT3_ITD627E induced transformation of hematopoietic 32D cells and led to a lethal myeloproliferative disease in a syngeneic mouse model. Our results indicate that a significant proportion of activating FLT3_ITD mutations is not confined to the JM domain of FLT3. Further studies are warranted to define the biologic and clinical characteristics of non-JM ITDs.

The kinase inhibitor LS104 induces apoptosis, enhances cytotoxic effects of chemotherapeutic drugs and is targeting the receptor tyrosine kinase FLT3 in acute myeloid leukemia.

Activating mutations of FLT3 are found in approximately one-third of acute myeloid leukemia (AML)-cases and are considered to represent an attractive therapeutic target. In this study, we report that the hydroxystyryl-acrylonitrile compound LS104 inhibits proliferation and induces potent cytotoxic effects in FLT3 expressing leukemic cells in vitro. Immunoblot and phosphoprotein-FACS analysis demonstrated inhibiton of phosphorylation of FLT3-ITD and of its downstream targets. In pharmacokinetic studies, a rapid and dose dependent cellular uptake of LS104 lasting up to 11h could be demonstrated. Combination of LS104 with chemotherapeutic agents markedly enhanced cytotoxic effects. Recently, a phase I clinical trial investigating LS104 in refractory/relapsed hematologic malignancies has been initiated.

LS104, a non-ATP-competitive small-molecule inhibitor of JAK2, is potently inducing apoptosis in JAK2V617F-positive cells.

The activating JAK2V617F mutation has been described in the majority of patients with BCR-ABL-negative myeloproliferative disorders (MPD). In this report, we characterize the small-molecule LS104 as a novel non-ATP-competitive JAK2 inhibitor: Treatment of JAK2V617F-positive cells with LS104 resulted in dose-dependent induction of apoptosis and inhibition of JAK2 autophosphorylation and of downstream targets. Activation of these targets by JAK2 was confirmed in experiments using small interfering RNA. LS104 inhibited JAK2 kinase activity in vitro. This effect was not reversible using elevated ATP concentrations, whereas variation of the kinase substrate peptide led to modulation of the IC50 value for LS104. In line with these data, combination treatment using LS104 plus an ATP-competitive JAK2 inhibitor (JAK inhibitor I) led to synergistically increased apoptosis in JAK2V617F-positive cells. Furthermore, LS104 strongly inhibited cytokine-independent growth of endogenous erythroid colonies isolated from patients with JAK2V617F-positive MPD in vitro, whereas there was no significant effect on growth of myeloid colonies obtained from normal controls. Based on these data, we have recently started a phase I clinical trial of LS104 for patients with JAK2V617F-positive MPDs. To the best of our knowledge, this is the first report on a non-ATP-competitive kinase inhibitor being tested in a clinical trial.

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in Bulgarian hospitals.

The aim of the study was to describe the emergence, the spread, and the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Bulgaria. Over eight years (1996-2003), 442 ESBL-screen-positive isolates were collected in nine medical institutions in four Bulgarian towns. Class A ESBLs of the SHV, TEM, and CTX-M groups were identified in seven species. SHV-type enzymes persisted during the whole study period, TEM-ESBLs appeared first in 1999, and CTX-M-types appeared first in 2001. The rate of CTX-M enzyme producers increased rapidly between 2001 and 2003, while the rate of SHV producers decreased. Six different ESBL-types were identified, namely, SHV-2, -5, and -12, CTX-M-3 and -15, and a new TEM-3-like variant (TEM-139). The most widespread enzymes were SHV-12, CTX-M-15, and CTX-M-3 found in seven centers. TEM-139 was identified mainly in one center. A trend for strains harboring more than one ESBL gene, for example, CTX-M + SHV, was observed since 2002. Plasmid fingerprinting and random amplified polymorphic DNA analysis typing revealed wide dissemination of identical plasmids among different bacterial species and hospitals, as well as clonal spread of ESBL producers. Our data contribute to clarify the dynamics in the prevalence of ESBLs in Bulgaria and demonstrate the importance of molecular procedures for their analysis.