Evaluation of HMFG2 and thyroglobulin in the diagnosis of thyroid fine needle aspiration (FNA)

In order to appraise the usefulness of HMFG₂ and thyroglobulin (Tg) as specific markers for the diagnosis of thyroid disease, we studied 63 FNA smears. Cases tested included 30 benign (nine colloid goitres, six cases of Hashimoto's thyroiditis, six Hürthle cell adenomas, nine follicular adenomas) and 33 malignant lesions (nine follicular carcinomas, 12 papillary carcinomas, nine anaplastic carcinomas, three medullary carcinomas). All cases with malignant lesions except the anaplastic carcinomas were positive for HMFG₂. Immunoreactive cells to HMFG₂ were also found in 15 adenomas out of 30 benign cases. Positive Tg reaction was found in benign and malignant thyroid lesions, except six cases of Hashimoto's thyroiditis, nine anaplastic and three medullary carcinomas. The results obtained indicate that morphology paired with immunocytochemistry can usually depict a more specific profile of thyroid lesions for better evaluation of the pathology.     

Isolation and characterization of a fragment of rat thyroglobulin gene

A rat genomic library was screened for thyroglobulin gene clones with recombinant plasmids containing rat thyroglobulin complementary DNA inserts. Two identical recombinant phages were found. A map of the inserted genomic sequence established by restriction and blotting experiments, and electronic microscopy revealed that this fraction of the gene was extensively split. Exons were ≤ 200 base pair long while the introns represented 93% of the insert. A fragment subcloned in plasmid pBR 322 was shown to contain repetitive sequences when used in Southern blot experiments with rat total genomic DNA.     

Cyclooxygenase‐1 inhibition reduces amyloid pathology and improves memory deficits in a mouse model of Alzheimer's disease

Several epidemiological and preclinical studies suggest that non‐steroidal anti‐inflammatory drugs (NSAIDs), which inhibit cyclooxygenase (COX), reduce the risk of Alzheimer's disease (AD) and can lower β‐amyloid (Aβ) production and inhibit neuroinflammation. However, follow‐up clinical trials, mostly using selective cyclooxygenase (COX)‐2 inhibitors, failed to show any beneficial effect in AD patients with mild to severe cognitive deficits. Recent data indicated that COX‐1, classically viewed as the homeostatic isoform, is localized in microglia and is actively involved in brain injury induced by pro‐inflammatory stimuli including Aβ, lipopolysaccharide, and interleukins. We hypothesized that neuroinflammation is critical for disease progression and selective COX‐1 inhibition, rather than COX‐2 inhibition, can reduce neuroinflammation and AD pathology. Here, we show that treatment of 20‐month‐old triple transgenic AD (3 × Tg‐AD) mice with the COX‐1 selective inhibitor SC‐560 improved spatial learning and memory, and reduced amyloid deposits and tau hyperphosphorylation. SC‐560 also reduced glial activation and brain expression of inflammatory markers in 3 × Tg‐AD mice, and switched the activated microglia phenotype promoting their phagocytic ability. The present findings are the first to demonstrate that selective COX‐1 inhibition reduces neuroinflammation, neuropathology, and improves cognitive function in 3 × Tg‐AD mice. Thus, selective COX‐1 inhibition should be further investigated as a potential therapeutic approach for AD.     

Novel dual-reporter transgenic rodents enable cell tracking in animal models of stem cell transplantation

In the present study, we have established a novel transgenic mouse and transgenic rats with dual reporters of EGFP and ELuc. In these transgenic (Tg) rodents, both GFP fluorescent and luciferase luminescent signals were ubiquitously detected in the heart, liver, kidney and testis, while only the GFP signal was detected in the brain. This expression system is based on a P2A linked EGFP/ELuc protein allowing both signals to be generated simultaneously. Microscopy experiments, FCM, and luciferase assays showed strong expression in freshly isolated ADSCs from Tg rodents upon transplantation of Tg rat-derived ADSCs into wild-type-mice. The ELuc transgene signal was observed and traced in vivo, and EGFP positive cells could be recovered from ELuc positive tissues in engraftment sites of wild-type mice for multiple analysis. These dual reporter Tg rodents are a useful reconstituted model system of regenerative medicine and are a valuable tool to study stem cells.     

Development of the post-natal growth plate requires intraflagellar transport proteins

In the post-natal growth plate, chondrocytes are arranged in columns parallel to the long axis of the bone. Chondrocytes divide perpendicular to this axis and then move into position one on top of another in a process called "rotation" that maintains columnar organization. Primary cilia are non-motile microtubule base appendages extending from the surface of almost all vertebrate cells. Primary cilia were described on chondrocytes almost 40 years ago but the function of these structures in cartilage biology is not known. Intraflagellar transport (IFT) is the process by which primary cilia are generated and maintained. This study tested the hypothesis that IFT plays an important role in post-natal skeletal development. Kif3a, a subunit of the Kinesin II motor complex, that is required for intraflagellar transport and the formation of cilia, was deleted in mouse chondrocytes via Col2a-Cre-mediated recombination. Disruption of IFT resulted in subsequent depletion of cilia and post-natal dwarfism due to premature loss of the growth plate likely a result of reduced proliferation and accelerated hypertrophic differentiation of chondrocytes. Cell shape and columnar orientation in the growth plate were also disrupted suggesting a defect in the process of rotation. Alterations in chondrocyte rotation were accompanied by disruption of the actin cytoskeleton and alterations in the localization of activated FAK to focal adhesion-like structures on chondrocytes. This is the first report indicating a role for IFT and primary cilia in the development of the post-natal growth plate. The results suggest a model in which IFT/cilia act to maintain the columnar organization of the growth plate via the process of chondrocyte rotation.     

Altered glycosylation of acetylcholinesterase in APP (SW) Tg2576 transgenic mice occurs prior to amyloid plaque deposition

Previous studies have shown that a minor glycoform of acetylcholinesterase (AChE) is increased in Alzheimer's disease brain and cerebrospinal fluid. This glycoform can be distinguished from other AChE species by its lack of binding to concanavalin A (Con A). In this study, the temporal relationship between AChE glycosylation and Abeta deposition was examined in Tg2576 mice. There was a significant (p < 0.05) difference in AChE glycosylation in Tg2576 mice compared with age-matched background strain control mice at 4 months of age. This difference in glycosylation was also observed in 8- and 12-month-old Tg2576 mice. In contrast, Abeta plaques were only seen in the Tg2576 mice at 12 months of age, and were not detected at 4 and 8 months of age. Soluble human-sequence Abeta was detected as early as 4 months of age in the transgenic mice. The altered AChE glycosylation was due to an increase in a minor AChE isoform, which did not bind Con A, similar to that previously observed to be increased in Alzheimer's disease brain and cerebrospinal fluid. The results demonstrate that in transgenic mice altered AChE glycosylation is associated with very early events in the development of AD-like pathology. The study supports the possibility that glycosylation may also be a useful biomarker of AD.     

Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4⁺ T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.     

ER stress responses in the absence of apoptosome: A comparative study in CASP9 proficient vs deficient mouse embryonic fibroblasts

Cells respond to endoplasmic reticulum (ER) stress through the unfolded protein response (UPR), autophagy and cell death. In this study we utilized casp9^+/+ and casp9^−/− MEFs to determine the effect of inhibition of mitochondrial apoptosis pathway on ER stress-induced-cell death, UPR and autophagy. We observed prolonged activation of UPR and autophagy in casp9^−/− cells as compared with casp9^+/+ MEFs, which displayed transient activation of both pathways. Furthermore we showed that while casp9^−/− MEFs were resistant to ER stress, prolonged exposure led to the activation of a non-canonical, caspase-mediated mode of cell death.     

A novel heterozygous mutation of the AIRE gene in a patient with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED)

Background

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) is an autosomal recessive disease due to mutations of the autoimmune regulator (AIRE) gene. Typical manifestations include candidiasis, Addison's disease, and hypoparathyroidism. Type 1 diabetes, alopecia, vitiligo, ectodermal dystrophy, celiac disease and other intestinal dysfunctions, chronic atrophic gastritis, chronic active hepatitis, autoimmune thyroid disorders, pernicious anemia and premature ovarian failure are other rare associated diseases although other conditions have been associated with APECED.

Case presentation

What follows is the clinical, endocrinological and molecular data of a female APECED patient coming from Lithuania. The patient was affected by chronic mucocutaneous candidiasis, hypoparathyroidism and pre-clinical Addison's disease. Using direct sequencing of all the 14 exons of the AIRE gene in the patient's DNA, we identified in exon 6 the known mutation c.769 C>T (p.Arg257X) in compound heterozygosity with the newly discovered mutation c.1214delC (p.Pro405fs) in exon 10. The novel mutation results in a frameshift that is predicted to alter the sequence of the protein starting from amino acid 405 as well as to cause its premature truncation, therefore a non-functional Aire protein.

Conclusions

A novel mutation has been described in a patient with APECED with classical clinical components, found in compound heterozygosity with the c.769 C>T variation. Expanded epidemiological investigations based on AIRE gene sequencing are necessary to verify the relevancy of the novel mutation to APECED etiopathogenesis in the Lithuanian population and to prove its diagnostic efficacy in association with clinical and immunological findings.