Prediction of heparin binding of mutated short sequences of rat thyroglobulin.

BACKGROUND: Binding of thyroglobulin (Tg) to heparin is involved in Tg transcytosis via megalin. Rat Tg (rTg) binds to heparin through an exposed carboxyl terminal region (RELPSRRLKRPLPVK, Arg2489-Lys2503) rich in positively charged residues. This region is not entirely conserved in human Tg (hTg) (Arg2489-Glu2503, REPPARALKRSLWVE), resulting in lower affinity binding. Here, we developed a score to predict to what extent secondary structure modifications affect the heparin-binding ability of rTg. METHODS: We designed eight synthetic peptides, including one with the Arg2489-Lys2503 sequence of rTg (rTgP), one with the corresponding sequence of hTg (hTgP), and six "mutant" peptides, each carrying a point mutation obtained by replacing one amino acid residue of rTgP with the corresponding residue of hTgP. Heparin binding was assessed in solid-phase assays. The Bmax and the constants of dissociation (Kd) were calculated. RESULTS: Using a no-fee online service, we obtained predictions of peptide secondary structures and developed a scoring system to estimate to what extent mutations are expected to modify rTg secondary structure. The score was designated as Probability of Secondary Structure Change (PSSC) and it significantly correlated with the BMax (R = 0.942, P < 0.001) and the Kds (R = - 0.744, P < 0.01) of heparin binding of hTgP and of the "mutant" peptides. CONCLUSIONS: The PSSC score allows predicting to what extent point mutations are likely to affect the heparin-binding ability of short sequences of proteins: in this case rTg, regardless of whether mutations affect charge of the sequence. The secondary structure of Tg is likely to play a role in heparin binding.

Bulk and Surface Electronic Structure of the Dual-Topology Semimetal Pt_{2}HgSe_{3}.

We report high-resolution angle-resolved photoemission measurements on single crystals of Pt_{2}HgSe_{3} grown by high-pressure synthesis. Our data reveal a gapped Dirac nodal line whose (001) projection separates the surface Brillouin zone in topological and trivial areas. In the nontrivial k-space range, we find surface states with multiple saddle points in the dispersion, resulting in two van Hove singularities in the surface density of states. Based on density-functional theory calculations, we identify these surface states as signatures of a topological crystalline state, which coexists with a weak topological phase.

Interleukin-17 and -22 synergy linking inflammation and EMT-dependent fibrosis in Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic inflammatory, autoimmune and systemic disorder commonly associated with dry eyes and a dry mouth. Recently, the hypothetical link between epithelial-mesenchymal transition (EMT)-dependent salivary gland (SG) fibrosis and chronic inflammatory conditions has been suggested. In this study, we present data demonstrating a negative correlation of the epithelial marker E-cadherin expression and a positive correlation of mesenchymal vimentin and collagen type I expression with increasing degrees of tissue inflammation in pSS SG specimens. In addition, as it is not clear whether dysregulated cytokines in pSS, interleukin (IL)-17 and IL-22 may also contribute to the EMT-dependent fibrosis process, the effect of IL-17 and IL-22 treatment on EMT-dependent SG fibrosis was evaluated in primary human salivary gland epithelial cells (SGEC) isolated from healthy subjects. Here we present data demonstrating that IL-17 and IL-22 can induce SGEC to undergo a morphological and phenotypical transition to a mesenchymal phenotype. In support of this, vimentin and collagen type I were up-regulated while a decreased expression of E-cadherin occurs after interleukin treatment, and co-operation between IL-17 and Il-22 was required to induce the EMT.

Binding of thyroglobulin (Tg) to the low-density lipoprotein receptor-associated protein (RAP) during the biosynthetic pathway prevents premature Tg interactions with sortilin.

OBJECTIVE: Sortilin, a Vps10p family member, is expressed by thyroid epithelial cells (TEC), where it binds to internalized thyroglobulin (Tg) molecules. Premature binding of Tg to sortilin during biosynthesis may cause intracellular retention of Tg. Such a premature interaction may be prevented by one or more inhibitor/s. Because both sortilin and Tg bind to the low-density lipoprotein receptor-associated protein (RAP), we investigated whether RAP serves such a function. METHODS: Immunofluorescence staining for sortilin, Tg, and RAP was performed in FRTL-5 cells. Co-immunoprecipitation experiments were performed in extracts from FRTL-5 or COS-7 cells, the former co-transfected with Tg and/or RAP and/or sortilin, or in thyroid extracts from RAP KO mice. RESULTS: Tg and sortilin did not co-localize in FRTL-5 cells following inhibition of protein synthesis, suggesting that newly synthesized, endogenous sortilin and Tg do not interact, in confirmation of which an anti-sortilin antibody did not co-precipitate Tg in FRTL-5 cells. In contrast, Tg co-localized with RAP in FRTL-5 cells. Co-immunoprecipitation of Tg with an anti-sortilin antibody in COS-7 cells transfected with sortilin and Tg was abolished when cells were co-transfected with RAP, indicating that RAP prevents binding of Tg to sortilin during biosynthesis, in confirmation of which an anti-sortilin antibody co-precipitated Tg in thyroid extracts from RAP KO mice to a greater extent than in thyroid extracts from WT mice. CONCLUSIONS: Tg does not bind prematurely to sortilin because of its interaction with RAP during protein biosynthesis. These findings add new information to the knowledge of thyroid physiology.

Toward sensitive immuno-based detection of tau protein by surface plasmon resonance coupled to carbon nanostructures as signal amplifiers.

Interest on Tau protein is fast increasing in Alzheimer's disease (AD) diagnosis. There is the urgent need of highly sensitive and specific diagnostic platforms for its quantification, also in combination with the other AD hallmarks. Up to now, SPR has been poorly exploited for tau detection by immunosensing, due to sensitivity limits at nanomolar level, whereas the clinical requirement is in the picomolar range. Molecular architectures built in a layer-by-layer fashion, biomolecules and nanostructures (metallic or not) may amplify the SPR signal and improve the limit of detection to the desired sensitivity. Mostly gold nanostructures are widely employed to this aim, but great interest is also emerging in Multi Walled Carbon Nanotubes (MWCNTs). Here MWCNTs are modified and then decorated with the secondary antibody for tau protein. Eventually we took advantage from MWCNTs-antibody conjugate to obtain a sandwich-based bioassay with the capability to increase the SPR signal of about 102 folds compared to direct detection and conventional unconjugated sandwich. With respect to these results, we hope to give a strong impulse for further investigation on studying possible roles of carbon nanotubes in optical-based biosensing.

Intracellular retention of thyroglobulin in the absence of the low-density lipoprotein receptor-associated protein (RAP) is likely due to premature binding to megalin in the biosynthetic pathway.

OBJECTIVE: The low-density lipoprotein receptor associated protein (RAP) is expressed by thyroid epithelial cells (TEC) in a TSH-dependent manner. In the thyroid RAP functions as a molecular chaperone for the thyroglobulin (Tg) endocytic receptor megalin/LRP2, which is retained intracellularly in RAP KO mice rather than being expressed on the apical membrane of TEC, its usual location. RAP binds also to Tg, which is also retained intracellularly in RAP KO mice, thereby suggesting a role of RAP in Tg secretion. Here we investigated whether Tg intracellular retention in the absence of RAP is due to premature Tg-megalin interactions during the biosynthetic pathway or to a direct action of RAP on Tg secretion. METHODS: We performed immunoprecipitation experiments in thyroid extracts from RAP KO and WT mice. In addition, we investigated Tg secretion in COS-7 cells co-transfected with human RAP (hRAP) and mouse Tg (mTg). RESULTS: An anti-megalin megalin precipitated greater amounts of Tg in thyroid extracts from RAP KO than from WT mice, suggesting increased intracellular interactions between megalin and Tg in the absence of RAP. COS-7 cells transiently transfected with hRAP, mTg or both, expressed the two proteins accordingly. RAP was found almost exclusively in cell extracts, whereas Tg was found both in extracts and media, as expected from the knowledge that RAP is ER-resident and that Tg is secreted. Regardless of whether cells were transfected with mTg alone or were co-transfected with hRAP, similar proportions of the total Tg synthesized were detected in cell extracts and media. CONCLUSIONS: The intracellular retention of Tg in the absence of RAP is likely due to its premature interaction with megalin, whereas RAP does not seem to affect Tg secretion directly.

Downstream activation of NF-κB in the EDA-A1/EDAR signalling in Sjögren's syndrome and its regulation by the ubiquitin-editing enzyme A20.

Sjögren's syndrome (SS) is an autoimmune disease and the second most common chronic systemic rheumatic disorder. Prevalence of primary SS in the general population has been estimated to be approximately 1-3%, whereas secondary SS has been observed in 10-20% of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE) and scleroderma. Despite this, its exact aetiology and pathogenesis are largely unexplored. Nuclear factor-kappa B (NF-κB) signalling mechanisms provide central controls in SS, but how these pathways intersect the pathological features of this disease is unclear. The ubiquitin-editing enzyme A20 (tumour necrosis factor-α-induced protein 3, TNFAIP3) serves as a critical inhibitor on NF-κB signalling. In humans, polymorphisms in the A20 gene or a deregulated expression of A20 are often associated with several inflammatory disorders, including SS. Because A20 controls the ectodysplasin-A1 (EDA-A1)/ectodysplasin receptor (EDAR) signalling negatively, and the deletion of A20 results in excessive EDA1-induced NF-κB signalling, this work investigates the expression levels of EDA-A1 and EDAR in SS human salivary glands epithelial cells (SGEC) and evaluates the hypothesis that SS SGEC-specific deregulation of A20 results in excessive EDA1-induced NF-κB signalling in SS. Our approach, which combines the use of siRNA-mediated gene silencing and quantitative pathway analysis, was used to elucidate the role of the A20 target gene in intracellular EDA-A1/EDAR/NF-κB pathway in SS SGEC, holding significant promise for compound selection in drug discovery.

Determination of pharmaceutical and illicit drugs in oral fluid by ultra-high performance liquid chromatography-tandem mass spectrometry.

A simple and extremely fast procedure for the quantitative determination in oral fluid samples of 44 substances, including the most common drugs of abuse and several pharmaceutical drugs, was developed and fully validated. Preliminary sample treatment was limited to protein precipitation. The resulting acetonitrile solution was directly injected into an ultra-high performance liquid chromatograph (UHPLC) equipped with a C18 column (100mm×2.1mm, 1.7µm). The mobile phase eluted with linear gradient (water/formic acid 5mM: acetonitrile/formic acid 5mM; v:v) from 98:2 to 0:100 in 5.0min, followed by isocratic elution at 100% B for 1.0min. The flow rate was 0.6mL/min and the total run time was 9.0min including re-equilibration at the initial conditions. The analytes were revealed by a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The method proved to be simple, accurate, rapid and highly sensitive, allowing the simultaneous detection of all compounds. The ease of sample treatment, together with the wide range of detectable substances, all with remarkable analytical sensitivity, make this procedure ideal for the screening of large populations in several forensic and clinical contexts, whenever oral fluid sampling has to be preferred to blood sampling, as for example in short retrospective investigations.

Possible role of oral ibandronate administration in Osteonecrosis of the Jaw: a case report.

We describe a case of Osteonecrosis of the Jaw (ONJ) that developed in a 65-year-old Caucasian woman with osteopenia and other risk factors who was receiving low doses of oral bisphosphonate therapy (ibandronate, 150 mg monthly). Computed tomography (CT), panoramic radiographs (OPT), 99mTc-Sn-MDP scintigraphy, and magnetic resonance imaging (MRI) were performed to study the diseased area; cytological examination also revealed the presence of suppurative material around the area of exposed bone. A diagnosis of bisphosphonate-related osteonecrosis of the jaw complicated by osteomyelitis was made. The patient was prescribed a drug protocol consisting of metronidazole 250 mg 2 times daily, chlorhexidine mouthwashes 3 times daily and chewing exercises for two months. Ibandronate was stopped and replaced with strontium ranelate. The symptoms improved and the patient is still under close follow-up. Assessment of the benefits versus risks is particularly necessary in patients with several risk factors to ascertain their eligibility for treatment with antiresorptive drugs and when this is not possible to choose alternative medications.

Sjögren's syndrome pathological neovascularization is regulated by VEGF-A-stimulated TACE-dependent crosstalk between VEGFR2 and NF-κB.

We explore the involvement of tumor necrosis factor α (TNF-α)-converting enzyme (TACE) in vascular endothelial growth factor (VEGF) and its receptor 2 (VEGFR2) (VEGF-A/VEGFR2)-mediated angiogenesis in Sjögren's syndrome (SS), one of the most common autoimmune rheumatic diseases. To test the hypothesis that SS autoantibodies (Abs) regulate VEGF-A/VEGFR2 expression by a TACE-dependent nuclear factor-κB (NF-κB) activation pathway, their effects on the expression and activation of the VEGF-A/TACE/VEGFR2/NF-κB pathway were determined in human salivary gland epithelial cells (SGEC). An enhanced angiogenesis in SS salivary gland biopsies was observed, associated with an increased VEGF-A expression and activation of VEGF-A/VEGFR2 signaling. Human cytokine array analysis of the pro-inflammatory and pro-angiogenic protein response in SGEC treated with SS Abs revealed an overexpression of multiple pro-angiogenic factors. TACE RNA knockdown, the use of anti-VEGF-A monoclonal antibody and the inhibition of NF-κB activity significantly abrogated the release of pro-angiogenic factors, demonstrating that VEGF-A/TACE/VEGFR2/NF-κB axis dysfunction may be contributory to pathogenesis and exacerbation of this autoimmune condition.

Absence of primary hypothyroidism and goiter in Slc26a4 (-/-) mice fed on a low iodine diet.

BACKGROUND: Mutations in the SLC26A4 gene, coding for the anion transporter pendrin, are responsible for Pendred syndrome, characterized by congenital sensorineural deafness and dyshormonogenic goiter. The physiological role of pendrin in the thyroid is still unclear and the lack of a thyroid phenotype in some patients with SLC26A4 mutations and in Slc26a4 (-/-) mice indicate the existence of environmental or individual modifiers able to compensate for pendrin inactivation in the thyroid. Since pendrin can transport iodide in vitro, variations in iodide supply have been claimed to account for the thyroid phenotype associated with pendrin defects. AIM: The Slc26a4 (-/-) mouse model was used to test the hypothesis that iodide supply may influence the penetrance and expressivity of SLC26A4 mutations. MATERIALS AND METHODS: Slc26a4 (-/-) and (+/+) mice were fed up to 6 months on a standard or low iodine diet and were evaluated for thyroid structural abnormalities or biochemical hypothyroidism. RESULTS: A 27-fold iodide restriction induced similar modifications in thyroid histology, but no differences in thyroid size, T4 or TSH levels were observed between between Slc26a4 (-/-) and (+/+) mice, either in standard conditions and during iodine restriction. CONCLUSIONS: Iodide restriction is not able to induce a thyroid phenotype in Slc26a4 (-/-) mice. These experimental data, together with those coming from a review of familial Pendred cases leaving in regions either with low or sufficient iodide supply, support the idea that the expression of thyroid phenotype in Pendred syndrome is more powerfully influenced by individual factors than by dietary iodide.

Binding, uptake, and degradation of internalized thyroglobulin in cultured thyroid and non-thyroid cells.

Thyroid hormone release requires degradation of thyroglobulin (Tg) by thyroid epithelial cells, which occurs mainly in the lysosomal pathway following Tg endocytosis. Non-specific fluid-phase endocytosis is thought to be the main route of Tg uptake leading to degradation, whereas receptor- mediated endocytosis is believed to lead to post-endocytic pathways other than degradation. To gain more insights into these issues, we investigated handling of Tg by various cell types. Tg bound similarly to thyroid (FRTL-5, FRT) and non-thyroid (COS-7, IRPT) cells, indicating the presence of membrane-binding sites, presumably receptors, in both cell types. Tg was internalized and degraded by all cells and degradation paralleled uptake, with the exception of FRTL- 5 cells, in which a lower proportion of Tg was degraded, suggesting that in FRTL-5 cells mechanisms that target Tg to the various post-endocytic pathways (either receptors or postreceptorial factors) are differently represented. Immunoelectronmicroscopy showed a common path of endocytosis in FRTL-5, COS-7, and IRPT cells, namely the formation of pseudopods engulfing Tg, followed by internalization and accumulation of Tg in cytoplasmic vesicles and lysosomes. The fastest rate was observed in COS-7 cells, probably reflecting a lower impact of endocytic receptors. Our findings suggest that Tg uptake and degradation are not thyroid-specific, that Tg binding sites exist in different cell types, and that uptake and/or degradation are differently regulated in differentiated thyroid cells, presumably because of a different impact of endocytic receptors or post-endocytic mechanisms, which are probably responsible for the regulation of hormone release.

Quercetin decreases proliferation of orbital fibroblasts and their release of hyaluronic acid.

BACKGROUND: Inhibition of fibroblast (FB) proliferation and hyaluronic acid (HA) production may be a therapeutic approach to Graves' ophthalmopathy (GO). The flavonoid quercetin has a wide range of activities, including reduction of FB growth. AIM: To investigate the effects of quercetin in orbital FB from GO patients and control subjects. METHODS: Primary cultures of orbital FB were treated with quercetin or with its glycosides rutin and quercitrin. Cell proliferation, necrosis, apoptosis, HA production, and cell cycle were measured. RESULTS: Beginning at a 30 µM concentration, quercetin, but not rutin and quercitrin, reduced cell proliferation, with no difference between GO and control FB. The effect of quercetin on proliferation was due to necrosis and cell cycle blockade, whereas apoptosis was unaffected. Quercetin reduced HA in the cell media, with no difference between GO and control FB. CONCLUSIONS: Quercetin reduces cell proliferation and HA release in orbital FB. Whether these initial findings have any potential for the use of quercetin in the clinical practice remains to be established.

Autoimmune pancreatitis: a challenging diagnostic puzzle for clinicians.

Autoimmune pancreatitis is a form of pancreatitis with autoimmune stigmata that may present as either focal or diffuse gland involvement. In focal forms, autoimmune pancreatitis shares demographic, clinical, biochemical and imaging features with pancreatic cancer. Since autoimmune pancreatitis is a benign disease and steroid therapy can rapidly resolve symptoms, improve radiological findings and avoid unnecessary surgery, the current clinical challenge is how to differentiate autoimmune pancreatitis from pancreatic neoplasia. Even though definitive diagnosis of the disease is difficult, several diagnostic criteria have been proposed and progress has been made in imaging studies. The management of this unique form of pancreatitis should, therefore, be handled in centres with knowledge of all aspects of the disease. This article briefly reviews clinical aspects of autoimmune pancreatitis with a focus on its diagnostic imaging and management.

Anti-Ro/SSA autoantibody-mediated regulation of extracellular matrix fibulins in human epithelial cells of the salivary gland.

OBJECTIVES: The fibulins are a family of extracellular matrix (ECM) molecules that regulate the organ shape along with other growth factors and stromal cells and have recently been shown to be involved in a variety of cellular functions including proliferation, migration, differentiation, and survival. Important changes in acinar and ductal morphology and function, together with pronounced ECM remodelling, are detectable in the labial salivary glands (LSGs) of patients with Sjögren's syndrome (SS). Here we report the in vitro expression of the recently identified ECM proteins fibulin-6 and fibulin-7 by human salivary gland epithelial cells (SGECs). The ability of anti-Ro/SSA autoantibodies (Abs) to modulate fibulin-6 and fibulin-7 expression was investigated. METHODS: Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR were used to analyse fibulin-6 and fibulin-7 mRNA expression. Confocal microscopy and fluorescence-activated cell sorting (FACS) were used to study expression of the proteins in primary human SGEC cultures, established from biopsies of minor LSGs, in both untreated control cells and anti-Ro/SSA Abs-treated cells. RESULTS: The methods used show the expression of fibulin-6 and fibulin-7 in SGECs. Treatment of cells with anti-Ro/SSA Abs results in a down-regulation of fibulin-6 mRNA expression whereas no significant differences were observed in fibulin-7 expression between untreated and treated cells. CONCLUSION: Dysregulation of fibulin expression in SGECs by anti-Ro/SSA Abs may contribute to disorganization of the ECM environment and thus cause injury to the salivary gland architecture and functionality observed in SS.

Hashimoto's thyroiditis in Melkersson-Rosenthal syndrome patient: casual association or related diseases?

Melkersson-Rosenthal syndrome (MRS) is a rare disorder of unknown etiology. MRS is classically defined as a triad of recurrent orofacial edema, relapsing paralysis of the facial nerve, and fissured tongue. The authors present the case of a 52-year-old woman with orofacial swelling and facial pain attacks. The patient reported to suffer of hypothyroidism and laboratory findings disclosed free triiodothyronine (FT(3)), free thyroxine (FT(4)), and thyrotropin (TSH) altered. Endocrinological consult led to the diagnosis of Hashimoto's thyroiditis. Antithyroper-oxidase antibodies (anti-TPO) were highly elevated and thyroid function tests had evidenced a clinically significant hypothyroidism. A link between MRS and immunological disorders such as sarcoidosis, Crohn's disease, unilateral anterior uveitis and multiple sclerosis was documented. The literature did not report any association between Hashimoto's thyroiditis and Melkersson-Rosenthal syndrome. The presence of the anti-TPO antibodies in the case reported here could suggest a possible correlation between immunological alteration characteristic of autoimmune thyroiditis and MRS.

Kidney abnormalities in low density lipoprotein receptor associated protein knockout mice.

Mice lacking the LDL receptor associated protein (RAP) have a severe defect of thyroglobulin secretion into the colloid, associated with moderately increased serum TSH levels and histological features of early goiter. RAP is expressed also in renal proximal tubule cells, where it functions as a molecular chaperone for the endocytic receptor megalin, which is responsible for reabsorption of low molecular weight proteins from the glomerular filtrate. Here we investigated whether the thyroid phenotype in RAP knockout (KO) mice is associated with kidney alterations. By immunohistochemistry, we found that in RAP KO mice megalin expression on the apical membrane of renal proximal tubule cells was markedly reduced, with intracellular retention of the receptor. The reduced expression of megalin was associated with its impaired function. Thus, urinary protein concentrations and urinary protein excretion in 24 h were higher in RAP KO than in wild-type mice. Coomassie staining of urine samples revealed an increased intensity of low molecular mass bands in the urine of RAP KO mice, indicating that they had low molecular weight proteinuria. Therefore, we concluded that disruption of the RAP gene determines not only thyroid abnormalities, but also a severe defect of megalin expression and function in the kidney.

Sequencing of the entire coding region of the receptor associated protein (RAP) in patients with primary hypothyroidism of unknown origin.

The LDL receptor-associated protein (RAP) is involved in secretion of thyroglobulin (Tg) from the thyrocyte to the colloid. Disruption of the RAP gene in mice results in a reduced Tg content within the colloid, leading to subclinical hypothyroidism and histological alterations resembling early goiter. Here we studied the entire coding sequence of RAP in genomic DNA samples from 18 patients with primary hypothyroidism not due to thyroid autoimmunity or dysgenesis. The control group included 21 subjects with no evidence of thyroid alterations. Eleven different polymorphisms with amino-acid substitution and 4 different missense polymorphisms without amino-acid substitution were found in various regions of the RAP gene. Only one polymorphism in exone 7 (V311M) was observed exclusively in patients, but it had been previously reported in normal subjects as well. The remaining polymorphisms were found either both in patients and controls or only in controls and had not been previously reported. The frequency of the various polymorphisms did not differ significantly between patients and controls. Based on these findings, we conclude that alterations of the RAP gene are not a common cause of hypothyroidism, although it cannot be excluded that other, rarer alterations with a pathogenic effect exist, and that they should be investigated in a larger number of patients.