Investigating the ID3/SLC22A4 as immune-related signatures in ischemic stroke.

BACKGROUND: Ischemic stroke (IS) is a fearful disease that can cause a variety of immune events. Nevertheless, precise immune-related mechanisms have yet to be systematically elucidated. This study aimed to identify immune-related signatures using machine learning and to validate them with animal experiments and single cell analysis. METHODS: In this study, we screened 24 differentially expressed genes (DEGs) while identifying immune-related signatures that may play a key role in IS development through a comprehensive strategy between least absolute shrinkage and selection operation (LASSO) regression, support vector machine (SVM) and immune-related genes. In addition, we explored immune infiltration using the CIBERSORT algorithm. Finally, we performed validation in mouse brain tissue and single cell analysis. RESULTS: We identified 24 DEGs for follow-up analysis. ID3 and SLC22A4 were finally identified as the better immune-related signatures through a comprehensive strategy among DEGs, LASSO, SVM and immune-related genes. RT-qPCR, western blot, and immunofluorescence revealed a significant decrease in ID3 and a significant increase in SLC22A4 in the middle cerebral artery occlusion group. Single cell analysis revealed that ID3 was mainly concentrated in endothelial_2 cells and SLC22A4 in astrocytes in the MCAO group. A CIBERSORT finds significantly altered levels of immune infiltration in IS patients. CONCLUSIONS: This study focused on immune-related signatures after stroke and ID3 and SLC22A4 may be new therapeutic targets to promote functional recovery after stroke. Furthermore, the association of ID3 and SLC22A4 with immune cells may be a new direction for post-stroke immunotherapy.

Re-visiting autoimmunity to sodium-iodide symporter and pendrin in thyroid disease.

OBJECTIVE: Iodide transport across thyrocytes constitutes a critical step for thyroid hormone biosynthesis, mediated mainly by the basolateral sodium-iodide-symporter (NIS (SLC5A5)) and the apical anion exchanger pendrin (PDS (SLC26A4)). Both transmembrane proteins have been described as autoantigens in thyroid disease, yet the reports on autoantibody (aAb) prevalence and diagnostic usefulness are conflicting. Reasons for the inconclusive findings may be small study groups and principle differences in the technologies used. DESIGN: We decided to re-evaluate this important issue by establishing novel non-radioactive tests using full-length antigens and comparable protocols, and analyzing a large cohort of thyroid patients (n = 323) and control samples (n = 400). METHODS: NIS and PDS were recombinantly expressed as fusion protein with firefly luciferase (Luc). Stably transfected HEK293 cells were used as reproducible source of the autoantigens. RESULTS: Recombinant NIS-Luc showed iodide transport activity, indicating successful expression and correct processing. Commercial antibodies yielded dose-dependent responses in the newly established assays. Reproducibility of assay signals from patient sera was verified with respect to linearity, stability and absence of matrix effects. Prevalence of PDS-aAb was similar in thyroid patients and controls (7.7% vs 5.0%). NIS-aAb were more prevalent in patients than controls (7.7% vs 1.8%), especially in Graves' Disease (12.3%). Neither NIS-aAb nor PDS-aAb concentrations were related to TPO-aAb or TSH-receptor-aAb concentrations, or to serum zinc or selenium status. CONCLUSIONS: Our data highlight a potential relevance of autoimmunity against NIS for thyroid disease, whereas an assessment of PDS-aAb in thyroid patients seems not to be of diagnostic value (yet).

Diversity in kinetics correlated with structure in nano body-stabilized LacY.

The structure of lactose permease, stabilized in a periplasmic open conformation by two Gly to Trp replacements (LacYww) and complexed with a nanobody directed against this conformation, provides the highest resolution structure of the symporter. The nanobody binds in a different manner than two other nanobodies made against the same mutant, which also bind to the same general region on the periplasmic side. This region of the protein may represent an immune hotspot. The CDR3 loop of the nanobody is held by hydrogen bonds in a conformation that partially blocks access to the substrate-binding site. As a result, kon and koff for galactoside binding to either LacY or the double mutant complexed with the nanobody are lower than for the other two LacY/nanobody complexes though the Kd values are similar, reflecting the fact that the nanobodies rigidify structures along the pathway. While the wild-type LacY/nanobody complex clearly stabilizes a similar 'extracellular open' conformation in solution, judged by binding kinetics, the complex with wild-type LacY did not yet crystallize, suggesting the nanobody does not bind strongly enough to shift the equilibrium to stabilize a periplasmic side-open conformation suitable for crystallization. However, the similarity of the galactoside binding kinetics for the nanobody-bound complexes with wild type LacY and with LacYWW indicates that they have similar structures, showing that the reported co-structures reliably show nanobody interactions with LacY.

Oncolytic measles virus therapy enhances tumor antigen-specific T-cell responses in patients with multiple myeloma.

Oncolytic virus therapy leads to immunogenic death of virus-infected tumor cells and this has been shown in preclinical models to enhance the cytotoxic T-lymphocyte response against tumor-associated antigens (TAAs), leading to killing of uninfected tumor cells. To investigate whether oncolytic virotherapy can increase immune responses to tumor antigens in human subjects, we studied T-cell responses against a panel of known myeloma TAAs using PBMC samples obtained from ten myeloma patients before and after systemic administration of an oncolytic measles virus encoding sodium iodide symporter (MV-NIS). Despite their prior exposures to multiple immunosuppressive antimyeloma treatment regimens, T-cell responses to some of the TAAs were detectable even before measles virotherapy. Measurable baseline T-cell responses against MAGE-C1 and hTERT were present. Furthermore, MV-NIS treatment significantly (P < 0.05) increased T-cell responses against MAGE-C1 and MAGE-A3. Interestingly, one patient who achieved complete remission after MV-NIS therapy had strong baseline T-cell responses both to measles virus proteins and to eight of the ten tested TAAs. Our data demonstrate that oncolytic virotherapy can function as an antigen agnostic vaccine, increasing cytotoxic T-lymphocyte responses against TAAs in patients with multiple myeloma, providing a basis for continued exploration of this modality in combination with immune checkpoint blockade.

Alternate expression of PEPT1 and PEPT2 in epidermal differentiation is required for NOD2 immune responses by bacteria-derived muramyl dipeptide.

Peptide transporters 1 and 2 (PEPT1 and PEPT2) are proton-coupled oligopeptide transporter members of the solute carrier 15 family and play a role in the cellular uptake of di/tri-peptides and peptidomimetics. Our previous work showed that PEPT2 is predominantly expressed within undifferentiated keratinocytes. Here we show that PEPT2 expression decreases as keratinocyte differentiation progresses and that PEPT1 alternately is expressed at later stages. Absolute quantification using quantitative polymerase chain reaction revealed that the expression level of PEPT1 is about 17 times greater than that of PEPT2. Immunohistochemical study of human skin provided evidence of PEPT1 in the epidermis. The uptake of glycylsarcosine into keratinocytes was significantly blocked by PEPT inhibitors, including nateglinide and glibenclamide. Moreover, we found that PEPT1 knockdown in differentiated keratinocytes significantly suppressed the influence of a bacterial-derived peptide, muramyl dipeptide (MDP), on the production of proinflammatory cytokine interleukin-8, implying that bacteria-derived oligopeptides can be transported by PEPT1 in advanced differentiated keratinocytes. Taken together, PEPT1 and PEPT2 may concertedly play an important role in MDP-NOD2 signaling in the epidermis, which provides new insight into the mechanisms of skin homeostasis against microbial pathogens.

Potassium channels in omnipause neurons.

Potassium (K+) channels are major contributors to fast and precise action potential generation. The aim of this study was to establish the immunoreactivity profile of several potassium channels in omnipause neurons (OPNs), which play a central role in premotor saccadic circuitry. To accomplish this, we histochemically examined monkey and human brainstem sections using antibodies against the voltage gated K+-channels KV1.1, KV3.1b and K+-Cl- cotransporter (KCC2). We found that OPNs of both species were positive for all three K+-antibodies and that the staining patterns were similar for both species. In individual OPNs, KV3.1b was detected on the somatic membrane and proximal dendrites, while KV1.1 was mainly confined to soma. Further, KCC2 immunoreactivity was strong in distal dendrites, but was weak in the somatic membrane. Our findings allow the speculation that the alterations in K+-channel expression in OPNs could be the underlying mechanism for several saccadic disorders through neuronal and circuit-level malfunction.

Lactic acid suppresses IgE-mediated mast cell function in vitro and in vivo.

Mast cells have functional plasticity affected by their tissue microenvironment, which greatly impacts their inflammatory responses. Because lactic acid (LA) is abundant in inflamed tissues and tumors, we investigated how it affects mast cell function. Using IgE-mediated activation as a model system, we found that LA suppressed inflammatory cytokine production and degranulation in mouse peritoneal mast cells, data that were confirmed with human skin mast cells. In mouse peritoneal mast cells, LA-mediated cytokine suppression was dependent on pH- and monocarboxylic transporter-1 expression. Additionally, LA reduced IgE-induced Syk, Btk, and ERK phosphorylation, key signals eliciting inflammation. In vivo, LA injection reduced IgE-mediated hypothermia in mice undergoing passive systemic anaphylaxis. Our data suggest that LA may serve as a feedback inhibitor that limits mast cell-mediated inflammation.

Crystal structure of a LacY-nanobody complex in a periplasmic-open conformation.

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane protein, catalyzes galactoside-H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a mutant in an outward (periplasmic)-open conformation to stabilize this state of the protein. Here we describe an X-ray crystal structure of a complex between a double-Trp mutant (Gly46→Trp/Gly262→Trp) and an Nb in which free access to the sugar-binding site from the periplasmic cavity is observed. The structure confirms biochemical data indicating that the Nb binds stoichiometrically with nanomolar affinity to the periplasmic face of LacY primarily to the C-terminal six-helix bundle. The structure is novel because the pathway to the sugar-binding site is constricted and the central cavity containing the galactoside-binding site is empty. Although Phe27 narrows the periplasmic cavity, sugar is freely accessible to the binding site. Remarkably, the side chains directly involved in binding galactosides remain in the same position in the absence or presence of bound sugar.

In vivo monitoring of transfected DNA, gene expression kinetics, and cellular immune responses in mice immunized with a human NIS gene-expressing plasmid.

In assessing the effectiveness of DNA vaccines, it is important to monitor: (1) the kinetics of target gene expression in vivo; and (2) the movement of cells that become transfected with the plasmid DNA used in the immunization of a subject. In this study, we used, as a visual imaging marker, expression of the transfected human sodium/iodide symporter (hNIS) gene, which enhances intracellular radio-pertechnetate (TcO4-) accumulation. After intradermal (i.d.) and systemic injection of mice with pcDNA-hNIS and radioactive Technetium-99m (Tc-99m), respectively, whole-body images were obtained by nuclear scintigraphy. The migration of mice cells transfected with the hNIS gene was monitored over a 2-week period by gamma-radioactivity counting of isolated cell populations and was demonstrated in peripheral lymphoid tissues, especially in the draining lymph nodes (dLNs). Beginning at 24 h after DNA inoculation and continuing for the 2-week monitoring period, hNIS-expressing cells were observed specifically in the T-cell-rich zones of the paracortical area of the dLNs. Over the same time period, high levels of INF-γ-secreting CD8 T-cells were found in the dLNs of the pcDNA-hNIS immunized mice. Tumor growth was also significantly retarded in the mice that received hNIS DNA immunization followed by inoculation with CT26 colorectal adenocarcinoma cells that had been transfected with the rat NIS gene (rNIS), which is 93% homologous to the hNIS gene. In conclusion, mouse cells transfected with hNIS DNA after i.d. immunization were found to traffic to the dLNs, and hNIS gene expression in these cells continued for at least 2 weeks post immunization. Furthermore, sequential presentation of NIS DNA to T-cells by migratory antigen presenting cells could induce NIS DNA-specific Th1 immune responses and thus retard the growth of NIS-expressing tumors.

The sodium iodide symporter is unlikely to be a thyroid/breast shared antigen.

PURPOSE: Anti-thyroid peroxidase (TPO) autoantibodies (TPOAb) seem to be protective for patients with breast cancer (BC). Thyroid and breast tissues both express the sodium iodide symporter (NIS), similarly both have a peroxidase activity, TPO and lactoperoxidase (LPO) respectively. We hypothesize a common immune response to a thyroid/breast shared antigen suggesting three putative mechanisms: (1) TPOAb react to both TPO and LPO, (2) TPO could be expressed in BC and (3) patients with TPOAb could have autoantibodies to NIS (NISAb). Previous studies excluded NISAb that block NIS activity in sera of patients with thyroid autoimmunity (TA) and/or BC. This study investigates neutral NISAb (binding without affecting function). METHODS: Clones of CHO cells stably expressing human NIS (hNIS; CHO-NIS) were isolated following transfection of hNIS in pcDNA3 vector. Expression of hNIS mRNA and surface protein was confirmed by PCR and flow cytometry respectively using a hNIS-mouse-monoclonal-antibody. CHO-NIS and controls transfected with the empty pcDNA3 vector (CHO-Empty) were incubated with 42 heat-inactivated human sera followed by an anti-human-IgG-AlexaFluor488-conjugate: 12 with BC, 11 with TA, 10 with both BC and TA and 9 with non-autoimmune thyroid diseases. The Kolmogorov-Smirnov Test was used to compare the fluorescence intensity obtained with CHO-NIS and CHO-Empty, using sera from six young males as a negative control population. RESULTS: None of the 42 sera were positive for NISAb. CONCLUSIONS: NISAb are rare and NIS is unlikely to be a common thyroid/BC shared antigen. We have recently demonstrated TPO expression in BC tissue and are currently investigating TPOAb cross-reactivity with TPO/LPO.

MicroRNA-193a-3p Reduces Intestinal Inflammation in Response to Microbiota via Down-regulation of Colonic PepT1.

Intestinal inflammation is characterized by epithelial disruption, leading to the loss of barrier function, recruitment of immune cells, and host immune responses to gut microbiota. PepT1, a di/tripeptide transporter that uptakes bacterial products, is up-regulated in inflamed colon tissue, which implies its role in bacterium-associated intestinal inflammation. Although microRNA (miRNA)-mediated gene regulation has been found to be involved in various processes of inflammatory bowel disease (IBD), the biological function of miRNAs in the pathogenesis of IBD remains to be explored. In this study we detected miRNA expression patterns in colon tissues during colitis and investigated the mechanism underlying the regulation of colonic PepT1 by miRNAs. We observed an inverse correlation between PepT1 and miR-193a-3p in inflamed colon tissues with active ulcerative colitis, and we further demonstrated that miR-193a-3p reduced PepT1 expression and activity as a target gene and subsequently suppressed the NF-κB pathway. Intracolonic delivery of miR-193a-3p significantly ameliorated dextran sodium sulfate-induced colitis, whereas the overexpression of colonic PepT1 via PepT1 3'-untranslated region mutant lentivirus vector abolished the anti-inflammatory effect of miR-193a-3p. Furthermore, antibiotic treatment eliminated the difference in the dextran sodium sulfate-induced inflammation between the presence and absence of miR-193a-3p. These findings suggest that miR-193a-3p regulation of PepT1 mediates the uptake of bacterial products and is a potent mechanism during the colonic inflammation process. Overall, we believe miR-193a-3p may be a potent regulator of colonic PepT1 for maintaining intestinal homeostasis.

EMMPRIN reduction via scFv-M6-1B9 intrabody affects α3ß1-integrin and MCT1 functions and results in suppression of progressive phenotype in the colorectal cancer cell line Caco-2.

Extracellular matrix metalloproteinase inducer (EMMPRIN) exhibits overexpression in various cancers and promotes cancer progression and metastasis via the interaction with its associated molecules. The scFv-M6-1B9 intrabody has a potential ability to reduce EMMPRIN cell surface expression. However, the subsequent effect of scFv-M6-1B9 intrabody-mediated EMMPRIN abatement on its related molecules, α3ß1-integrin, MCT1, MMP-2 and MMP-9, is undefined. Our results demonstrated that the scFv-M6-1B9 intrabody efficiently decreased α3ß1-integrin cell surface expression levels. In addition, intracellular accumulation of MCT1 and lactate were increased. These results lead to suppression of features characteristic for tumor progression, including cell migration, proliferation and invasion, in a colorectal cancer cell line (Caco-2) although there was no difference in MMP expression. Thus, EMMPRIN represents an attractive target molecule for the disruption of cancer proliferation and metastasis. An scFv-M6-1B9 intrabody-based approach could be relevant for cancer gene therapy.

Does thyroid peroxidase provide an antigenic link between thyroid autoimmunity and breast cancer?

Women with breast cancer (BC) and antithyroid peroxidase (TPO) autoantibodies (TPOAb) have a better prognosis than women lacking TPOAb. Sera from women with TPOAb displayed immunoreactivity to BC tissue by immunofluorescence that was not apparent in women without TPOAb. We hypothesize a BC/thyroid shared antigen that provides a target for humoral or cell-mediated immune activity; candidates include the sodium/iodide symporter (expressed in thyroid and BC), cross-reacting epitopes in TPO and lactoperoxidase (LPO) or TPO itself. As the association is with TPOAb, we investigated TPO expression in BC, breast peritumoral tissue (PT), other tissues (tumoral and not) and thyroid as positive control. Transcripts for known and novel TPO isoforms were detected in BC (n = 8) and PT (n = 8) but at approximately 10(4) -fold lower than in thyroid while in non-BC tumors (n = 5) they were at the limit of detection. TPO was expressed also in adipose tissue (n = 17), 10(3) -fold lower than in thyroid. Full length TPO (Mr 105-110 kDa) was detected in Western blots in the majority of examined tissues; preabsorption of the TPO antibody with recombinant TPO (but not LPO) reduced the signal, indicating specificity. The same occurred with some lower molecular weight bands, which could correspond to smaller TPO transcript isoforms, present in all samples. In conclusion, TPO is weakly expressed in BC and other tissues; this could partly explain the high frequency and protective role of TPOAb in BC patients. Further studies will investigate tissue specificity, function and immunogenicity of the novel TPO variants (some BC-specific) identified.

Soy extracts suppressed iodine uptake and stimulated the production of autoimmunogen in rat thyrocytes.

Soy consumption is associated with thyroid disorders such as hypothyroidism, goiter, and autoimmune thyroid disease (ATD) as well as increased iodine requirement in certain cases. However, the anti-thyroid component(s) in soy are yet to be identified and the molecular mechanism(s) involved remain unclear. This study examined the effects of soy isoflavones (ISF) on iodide uptake and expression of thyroglobulin (Tg) and sodium/iodide symporter (NIS) in thyrocytes. Fischer rat thyroid cells (FRTL) were treated with Novasoy (a soy alcohol extract containing 30% ISF) or major ISF aglycones or glycosides for 24 h. Iodide uptake was measured by a colorimetric assay. The protein level of Tg and NIS was measured by Western blotting. Cytotoxicity of tested compounds was determined by the MTT cell proliferation assay. Iodide uptake in FRTL cells was dose-dependently suppressed by Novasoy added into the cell culture (10, 25, or 50 µg/mL, P < 0.05). However, neither the major ISF aglycones nor glycosides alone or in combination had similar effects. Novasoy (up to 200 µg/mL) had no cytotoxic effect. Novasoy (1, 10, and 50 µg/mL) and genistein (1 and 10 µM) markedly increased the protein content of a 40 kDa Tg fragment (P40, a known autoimmunogen) and non-glycosylated NIS in the FRTL cells (P < 0.05). Overall, this study demonstrated that the alcohol soluble component(s) other than the major ISF in soy remarkably inhibited iodide uptake in the FRTL cells. Soy ISF, particularly genistein, induced the production of P40, which might be responsible for the higher incidence of ATD reported in soy infant formula-fed children.

Anti-natrium/iodide symporter antibodies and other anti-thyroid antibodies in children with Turner's syndrome.

Antibodies against the Na/I symporter (anti-NIS ab) have been found in adult patients with autoimmune thyroid diseases. As easily available for the immune system, NIS can play a role in the initial stage of autoimmune thyroid diseases. Children with Turner's syndrome (TS) being at high risk of autoimmune thyroid disease development seem a valuable group for the investigation of the early autoimmune process. The aim of the study was to investigate the presence of anti-NIS ab and its potential clinical significance in TS children. Fifty four girls with TS were examined (age 11.9 ± 2.46 years), and 23 healthy girls with normal thyroid function, free of autoimmune diseases. Anti-NIS antibodies were measured by the in-house ELISA method and the Western blotting. Sera considered positive for anti-NIS ab were used for the iodide uptake bioassay using COS7 cells stably transfected with hNIS. In all patients the thyroid function, antithyroid antibodies presence and thyroid ultrasonography were evaluated. In 20% of the patients a subclinical hypothyroidism was diagnosed and 70.4% had antithyroid antibodies (anti-TPO - 64.8% and Anti-Tg - 24%). Anti-NISab were present in 14.8% girls with TS and in none of the control group. Their presence was unrelated to other antithyroid antibodies titre or patients' age. A positive correlation between the anti-NIS ab presence and the hypothyroidism was found (p < 0.04). Anti-NIS ab-positive sera did not suppress iodine uptake. In conclusion, anti-NIS antibodies were present in 14.8% of children with TS and they were related to the presence of hypothyroidism.

K+-Cl- cotransporter-2 KCC2 in chicken cardiomyocytes.

Using antibodies prepared against a unique region (exon 22-24) of rat K(+)-Cl(-) cotransporter-2 (KCC2), we confirmed that the ~140-kDa KCC2 protein is exclusively expressed in rat brain, but in chicken, we observed strong reactivity not only with the ~140-kDa KCC2 protein in brain but also a slightly larger ~145-kDa protein in heart. In silico analysis showed that while exon 22 of KCC2 is unique to this isoform in therian mammals, it is retained in KCC2's closest paralog, KCC4, of lower vertebrates, including chicken. To eliminate potential cross-reactivity with chicken KCC4, the antibodies were preadsorbed with blocking peptides prepared over the only two regions showing significant sequence identity to chicken KCC4. This completely eliminated antibody recognition of exogenously expressed chicken KCC4 but not of the ~145-kDa protein in chicken heart, indicating that chicken heart expresses KCC2. Real-time PCR confirmed robust KCC2 transcript expression in both chicken brain and heart. Chicken heart expressed predominantly the longer KCC2a splice variant consistent with the larger ~145-kDa protein in chicken heart. Immunofluorescence microscopy revealed prominent plasma membrane KCC2 labeling in chicken ventricular cardiomyocytes. We hypothesize that KCC2 is an important Cl(-) extrusion pathway in avian cardiomyocytes that counters channel-mediated Cl(-) loading during high heart rates with ß-adrenergic stimulation. While KCC2 is absent from mammalian cardiomyocytes, understanding the role that the other KCC isoforms play in Cl(-) homeostasis of these cells represents a nascent area of research.

KCC2a expression in a human fetal lens epithelial cell line.

The fetal human lens epithelial cell (LEC) line (FHL124) possesses all four K(+)Cl(-) (KCC) cotransporter isoforms, KCC1-4, despite KCC2 being typically considered a neuronal isoform. Since at least two spliced variants, KCC2a and KCC2b, are co-expressed in cells of the central nervous system, this study sought to define the KCC2 expression profile in FHL124 cells. KCC2a, but not KCC2b transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Proteins of molecular weights ranging from 95 to 135 kDa were found by Western blotting using non-variant specific anti-KCC2 antibodies directed against two different regions of the KCC2 proteins, and by biotinylation suggesting membrane expression. Immunofluorescence revealed membrane and punctate cytoplasmic staining for KCC2. Low levels of cytosolic αA and αB crystallines, and neuron-specific enolase were also detected contrasting with the strong membrane immunofluorescence staining for the Na/K ATPase α1 subunit. Since the lack of neuron-specific expression of the KCC2b variant in non-neuronal tissues has been proposed under control of a neuron-restrictive silencing element in the KCC2 gene, we hypothesize that this control may be lifted for the KCC2a variant in the FHL124 epithelial cell culture, a non-neuronal tissue of ectodermal origin.

Identification of a mutation associated with fatal Foal Immunodeficiency Syndrome in the Fell and Dales pony.

The Fell and Dales are rare native UK pony breeds at risk due to falling numbers, in-breeding, and inherited disease. Specifically, the lethal Mendelian recessive disease Foal Immunodeficiency Syndrome (FIS), which manifests as B-lymphocyte immunodeficiency and progressive anemia, is a substantial threat. A significant percentage (∼10%) of the Fell ponies born each year dies from FIS, compromising the long-term survival of this breed. Moreover, the likely spread of FIS into other breeds is of major concern. Indeed, FIS was identified in the Dales pony, a related breed, during the course of this work. Using a stepwise approach comprising linkage and homozygosity mapping followed by haplotype analysis, we mapped the mutation using 14 FIS-affected, 17 obligate carriers, and 10 adults of unknown carrier status to a ∼1 Mb region (29.8 - 30.8 Mb) on chromosome (ECA) 26. A subsequent genome-wide association study identified two SNPs on ECA26 that showed genome-wide significance after Bonferroni correction for multiple testing: BIEC2-692674 at 29.804 Mb and BIEC2-693138 at 32.19 Mb. The associated region spanned 2.6 Mb from ∼29.6 Mb to 32.2 Mb on ECA26. Re-sequencing of this region identified a mutation in the sodium/myo-inositol cotransporter gene (SLC5A3); this causes a P446L substitution in the protein. This gene plays a crucial role in the regulatory response to osmotic stress that is essential in many tissues including lymphoid tissues and during early embryonic development. We propose that the amino acid substitution we identify here alters the function of SLC5A3, leading to erythropoiesis failure and compromise of the immune system. FIS is of significant biological interest as it is unique and is caused by a gene not previously associated with a mammalian disease. Having identified the associated gene, we are now able to eradicate FIS from equine populations by informed selective breeding.

Thyroperoxidase, thyroglobulin, Na(+)/I(-) symporter, pendrin in thyroid autoimmunity.

The autoimmune thyroid diseases (AITD), Graves' disease (GD) and Hashimoto's thyroiditis (HT) are most common endocrine disorders in humans. Both disorders are characterized by lymphocytic infiltration of the thyroid gland and the production of autoantibodies (aAb) against proteins that are thyroid-specific or expressed predominantly in the thyroid. The three main autoantigens are thyroperoxidase (TPO), thyroglobulin (Tg), and thyrotropin hormone receptor. Recently, the thyroidal iodide transporters Na+/I- symporter (NIS) and pendrin have also been identified as novel antigens in AITD. TPO-aAb and Tg-aAb are hallmarks of AITD, whereas the pathological and clinical relevance of NIS and pendrin aAb are still uncertain. To gain a greater understanding of the pathogenic mechanism(s) of autoimmune thyroid diseases at the molecular level, further characterisation of the autoantigens is required in order to shed light on why and how these molecules are seen by the immune system. This review summarizes current knowledge regarding the patho-physiological function and immunogenic response to the proteins TPO, Tg, NIS, and pendrin.

Immunohistochemical analysis of MCT1 and CD147 in equine skeletal muscle fibres.

Monocarboxylate transporter 1 (MCT1) and its ancillary protein CD147 facilitate efflux of lactate from the muscle. Expression of MCT1 and CD147 were studied with immunohistochemistry in type I, IIA, IIAB and IIB fibres of equine gluteal muscle. Staining intensity of MCT1 in the cytoplasm as well as in the membranes of fibre types decreased in the order I=IIA>IIAB>IIB and correlated with the oxidative capacity. Capillaries were pronounced in the MCT1 staining. CD147 antibody stained plasma membranes of all fibre types evenly, whereas the staining in the cytoplasm followed that of MCT1. In the middle gluteal muscle the expression of MCT1 follows the oxidative capacity of muscle fibres, but the expression of CD147 in sarcolemma does not vary among fibre types. The use of horse specific MCT1 and CD147 antibodies can in future studies help to evaluate lactate efflux from different muscle fibre types.