Differential responses to parathyroid hormone-related protein (PTHrP) deficiency in the various craniofacial cartilages.

PTHrP null mutant mice exhibit skeletal abnormalities both in the craniofacial region and limbs. In the growth plate cartilage of the null mutant, a diminished number of proliferating chondrocytes and accelerated chondrocytic differentiation are observed. In order to examine the effect of PTHrP deficiency on the craniofacial morphology and highlight the differential feature of the composing cartilages, we examined the various cartilages in the craniofacial region of neonatal PTHrP deficient mice. The major part of the cartilaginous anterior cranial base appeared to be normal in the homozygous PTHrP deficient mice. However, acceleration of chondrocytic differentiation and endochondral bone formation was observed in the posterior part of the anterior cranial base and in the cranial base synchondroses. Ectopic bone formation was observed in the soft tissue-running mid-portion of the Meckel's cartilage, where the cartilage degenerates and converts to ligament in the course of normal development. The zonal structure of the mandibular condylar cartilage was scarcely affected, but the whole condyle was reduced in size. These results suggest the effect of PTHrP deficiency varies widely between the craniofacial cartilages, according to the differential features of each cartilage.     

A new functional assay for the diagnosis of X‐linked inhibitor of apoptosis (XIAP) deficiency

X‐linked inhibitor of apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and a highly variable clinical presentation. Current diagnostic screening tests such as flow cytometry for XIAP expression or lymphocyte apoptosis assays have significant limitations. Based on recent evidence that XIAP is essential for nucleotide‐binding and oligomerization domains (NOD)1/2 signalling, we evaluated the use of a simple flow cytometric assay assessing tumour necrosis factor (TNF) production of monocytes in response to NOD2 stimulation by muramyl dipeptides (L18‐MDP) for the functional diagnosis of XIAP deficiency. We investigated 12 patients with XIAP deficiency, six female carriers and relevant disease controls. Irrespective of the diverse clinical phenotype, the extent of residual protein expression or the nature of the mutation, the TNF response was severely reduced in all patients. On average, L18‐MDP induced TNF production in 25% of monocytes from healthy donors or female carriers, while fewer than 6% of monocytes responded in affected patients. Notably, the assay clearly discriminated affected patients from disease controls with other immunodeficiencies accompanied by lymphoproliferation, hypogammaglobulinaemia or inflammatory bowel disease. Functional testing of the NOD2 signalling pathway is an easy, fast and reliable assay in the diagnostic evaluation of patients with suspected XIAP deficiency.     

Altered balance between self‐reactive T helper (Th)17 cells and Th10 cells and between full‐length forkhead box protein 3 (FoxP3) and FoxP3 splice variants in Hashimoto's thyroiditis

T helper type 17 (Th17) cells play a pathogenic role in autoimmune disease, while interleukin (IL)‐10‐producing Th10 cells serve a protective role. The balance between the two subsets is regulated by the local cytokine milieu and by the relative expression of intact forkhead box protein 3 (FoxP3) compared to FoxP3Δ2, missing exon 2. Th17 and Th10 cell differentiation has usually been studied using polyclonal stimuli, and little is known about the ability of physiologically relevant self‐antigens to induce Th17 or Th10 cell differentiation in autoimmune thyroid disease. We subjected mononuclear cells from healthy donors and patients with Hashimoto's thyroiditis (HT) or Graves' disease (GD) to polyclonal stimulation, or stimulation with human thyroglobulin (TG), human thyroid peroxidase (TPO), or Esherichia coli lipopolysaccharide (LPS). TPO and LPS induced increased differentiation of naive CD4⁺CD45RA⁺CD45R0^– T cells from HT patients into Th17 cells. Th10 cell proportions were decreased in HT after polyclonal stimulation, but were comparable to those of healthy donors after antigen‐specific stimulation. Taken together, our data show that an increased Th17 : Th10 ratio was found in HT patients after stimulation with thyroid‐specific self‐antigens. We also observed an elevated baseline production of IL‐6 and transforming growth factor (TGF)‐β1 and of mRNA encoding FoxP3Δ2 rather than intact FoxP3. This may contribute to the skewing towards Th17 cell responses in HT.     

Twin carriers of X‐linked agammaglobulinemia (XLA) due to germline mutation in the Btk gene

We report on an X‐linked agammaglobulinemia (XLA) family in which mothers of two affected cousins were monozygotic twins. We analyzed the Btk gene of several members in three generations of the family by SSCP analysis, DNA sequencing, and RFLP analysis following polymerase chain reaction‐amplification of the individual exons. We identified a missense point mutation, G1817C (R562P), in exon 17 of the Btk gene in the affected cousins. The same mutation was also present in both mothers (twin sisters) of the cousins identifying them as carriers. However, the mutation was absent in all other relatives including the grandmother of the cousins (mother of the twin sisters). This strongly suggests that the mutation in the Btk gene had originated in one of the germ lines or in the zygote. This may be the first demonstration of a germ line (or zygotic) mutation in XLA. Am. J. Med. Genet. 90:229–232, 2000. © 2000 Wiley‐Liss, Inc.     

Physiological responses to parental separation and a strange situation are related to parental care received in juvenile Goeldi's monkeys (Callimico goeldii)

The relationship between parental care received and physiological and behavioral responses to parental separation, isolation, and reunion was investigated in seven juvenile Goeldi's monkeys living in their family groups. Physiological responses were measured noninvasively: the hypothalamic–pituitary–adrenal axis via urinary cortisol output and the autonomic nervous system via piloerection on the tail. Parent–infant aggression demonstrated high intergroup variation and predicted: (a) an increase in urinary cortisol output, r_s = 0.86, p = 0.04, and duration of piloerection, r_s = 0.71, p = 0.08, at initial separation–isolation; (b) adaptation of piloerection, r_s = −0.89, p = 0.03, to repeated separation–isolation. Juvenile Goeldi's monkeys that had received high parental aggression were more physiologically responsive to separation; they also sought more contact with their mothers at reunion, r_s = 0.93, p = 0.02. We propose that these data are consistent with the hypothesis that high emotional reactivity is related to insecure attachment to aggressive parents in this New World primate. © 1998 John Wiley & Sons, Inc. Dev Psychobiol 33: 21–31, 1998     

Frameshift mutation in the cartilage‐derived morphogenetic protein 1 (CDMP1) gene and severe acromesomelic chondrodysplasia resembling Grebe‐type chondrodysplasia

Grebe‐type chondrodysplasia exhibits a severe form of limb shortening and appendicular bone dysmorphogenesis. Here we report a family with seven males and six females who inherited the disorder in an autosomal recessive fashion. While the carrier parents did not exhibit any apparent skeletal abnormalities, all affected patients had a similar phenotype with unaffected axial and craniofacial bones. Since mutations in the cartilage‐derived morphogenetic protein 1 (CDMP1) gene have been reported in similar acromesomelic chondrodysplasias, we examined genomic DNA from affected and normal subjects for possible mutations in CDMP1. In affected subjects, an insertion of a C at nucleotide 297 of the coding sequence was discovered. This insertion produced a shift in the reading frame at amino acid residue 99, causing premature termination of the polypeptide six amino acids downstream. DNA samples from 41 control subjects did not show this mutation. The truncated CDMP1 protein in these subjects is predicted to cause a total loss of its signaling function. The present report confirms that CDMP1 plays an important role in the regulation of axial bone growth during development and suggests that its absence does not impair other developmental processes.© 2002 Wiley‐Liss, Inc.     

Morphologic Observation of Mucosa‐Associated Lymphoid Tissue in the Large Intestine of Bactrian Camels (Camelus bactrianus)

The structure and distribution of the mucosa‐associated lymphoid tissue (MALT) throughout the large intestine of 10 Bactrian camels were comparatively studied by anatomical and histological methods. The results showed that Peyer's patches (PPs) were mainly located on the mucosal surfaces of the entire ileocecal orifice, the beginning of the cecum and the first third of the colon. The shape of PPs gradually changed from “scrotiform” to “faviform” along the large intestine with the scrotiform PP as the major type in the ileocecal orifice. The distribution density also gradually decreased from the ileocecal orifice to the colon. The histological observations further revealed that the MALT in the form of PPs or isolated lymphoid follicles (ILF) and lamina propria lymphocytes was mainly present in the lamina propria and submucosa from the entire ileocecal orifice, where the muscularis mucosa is usually incomplete, to the colonic forepart. In addition, lymphoid tissue was much more abundant in the lamina propria and submucosa of the ileocecal orifice as compared to the cecum and colon. Statistically, the MALT of the ileocecal orifice contained a higher number of lymphoid follicles (37.7/10 mm²) than that of the cecum, colon, or rectum (P < 0.05). The germinal centers of the lymphoid follicles were clearly visible. Together, our data suggest that the ileocecal orifice constitutes the main inductive site for the mucosal immunity in the large intestine of the Bactrian camel; and that scrotiform PPs are likely to the result of long‐term adaptation of the Bactrian camel to the harsh living environment. Anat Rec, 297:1292–1301, 2014. © 2014 Wiley Periodicals, Inc.     

Characterization of the 18 kDa translocator protein (TSPO) expression in post‐mortem normal and Alzheimer's disease brains

The 18 kDa translocator protein (TSPO) is a widely used target for microglial PET imaging radioligands, but its expression in post‐mortem normal and diseased human brain is not well described. We aimed at characterizing the TSPO expression in human control (CTRL) and Alzheimer's disease (AD) brains. Specifically, we sought to: (1) define the cell type(s) expressing TSPO; (2) compare tspo mRNA and TSPO levels between AD and CTRL brains; (3) correlate TSPO levels with quantitative neuropathological measures of reactive glia and AD neuropathological changes; and (4) investigate the effects of the TSPO rs6971 SNP on tspo mRNA and TSPO levels, glial responses and AD neuropathological changes. We performed quantitative immunohistochemistry and Western blot in post‐mortem brain samples from CTRL and AD subjects, as well as analysis of publicly available mouse and human brain RNA‐Seq datasets. We found that: (1) TSPO is expressed not just in microglia, but also in astrocytes, endothelial cells and vascular smooth muscle cells; (2) there is substantial overlap of tspo mRNA and TSPO levels between AD and CTRL subjects and in TSPO levels between temporal neocortex and white matter in both groups; (3) TSPO cortical burden does not correlate with the burden of activated microglia or reactive astrocytes, Aβ plaques or neurofibrillary tangles, or the cortical thickness; (4) the TSPO rs6971 SNP does not significantly impact tspo mRNA or TSPO levels, the magnitude of glial responses, the cortical thickness, or the burden of AD neuropathological changes. These results could inform ongoing efforts toward the development of reactive glia‐specific PET radioligands.     

Assessment of the potential pathogenicity of missense mutations identified in the GTPase‐activating protein (GAP)‐related domain of the neurofibromatosis type‐1 (NF1) gene

Neurofibromatosis type‐1 (NF1) is caused by constitutional mutations of the NF1 tumor‐suppressor gene. Although ～85% of inherited NF1 microlesions constitute truncating mutations, the remaining ～15% are missense mutations whose pathological relevance is often unclear. The GTPase‐activating protein‐related domain (GRD) of the NF1‐encoded protein, neurofibromin, serves to define its major function as a negative regulator of the Ras‐MAPK (mitogen‐activated protein kinase) signaling pathway. We have established a functional assay to assess the potential pathogenicity of 15 constitutional nonsynonymous NF1 missense mutations (11 novel and 4 previously reported but not functionally characterized) identified in the NF1‐GRD (p.R1204G, p.R1204W, p.R1276Q, p.L1301R, p.I1307V, p.T1324N, p.E1327G, p.Q1336R, p.E1356G, p.R1391G, p.V1398D, p.K1409E, p.P1412R, p.K1436Q, p.S1463F). Individual mutations were introduced into an NF1‐GRD expression vector and activated Ras was assayed by an enzyme‐linked immunosorbent assay (ELISA). Ten NF1‐GRD variants were deemed to be potentially pathogenic by virtue of significantly elevated levels of activated GTP‐bound Ras in comparison to wild‐type NF1 protein. The remaining five NF1‐GRD variants were deemed less likely to be of pathological significance as they exhibited similar levels of activated Ras to the wild‐type protein. These conclusions received broad support from both bioinformatic analysis and molecular modeling and serve to improve our understanding of NF1‐GRD structure and function. Hum Mutat 33:1687–1696, 2012. © 2012 Wiley Periodicals, Inc.     

Pulmonary sarcoidosis is associated with high‐level inducible co‐stimulator (ICOS) expression on lung regulatory T cells – possible implications for the ICOS/ICOS‐ligand axis in disease course and resolution

Sarcoidosis is a granulomatous inflammatory disorder of unknown aetiology. The increased frequency of activated lung CD4⁺ T cells with a T helper type 1 (Th1) cytokine profile in sarcoidosis patients is accompanied by a reduced proportion and/or impaired function of regulatory T cells (T_regs). Here we evaluated the expression of the inducible co‐stimulator (ICOS) on lung and blood CD4⁺ T cell subsets in sarcoidosis patients with different prognosis, by flow cytometry. Samples from the deep airways were obtained by bronchoalveolar lavage (BAL). We show that T_regs from the inflamed lung of sarcoidosis patients were characterized by a unique ICOS^high phenotype. High‐level ICOS expression was restricted to T_regs from the inflamed lung and was absent in blood T_regs of sarcoidosis patients as well as in lung and blood T_regs of healthy volunteers. In addition, lung T_regs exhibited increased ICOS expression compared to sarcoid‐specific lung effector T cells. Strikingly, ICOS expression on T_regs was in particularly high in the lungs of Löfgren's syndrome (LS) patients who present with acute disease which often resolves spontaneously. Moreover, blood monocytes from LS patients revealed increased ICOS‐L levels compared to healthy donors. Sarcoidosis was associated with a shift towards a non‐classical monocyte phenotype and the ICOS‐L^high phenotype was restricted to this particular monocyte subset. We propose a potential implication of the ICOS/ICOS‐L immune‐regulatory axis in disease activity and resolution and suggest to evaluate further the suitability of ICOS as biomarker for the prognosis of sarcoidosis.     

Pathogenic presenilin 1 mutations (P436S & I143F) in early‐onset Alzheimer's disease in the UK

Familial Alzheimer's disease (AD) is an autosomal dominant disorder characterized by memory impairment and multiple cognitive deficits which occurs in mid to late life. Early onset AD has been associated with mutations in three genes, of which presenilin 1 (PS1) mutations are the most frequent. We sequenced the open reading frame from genomic DNA of a series of 21 early onset AD (AD3) UK families in which there were at least two affected individuals in two or more generations with a diagnosis of probable or definite AD. We found PS1 mutations in six of these families with no sequence variation in the remaining 15. The six families contained between them five different mutations of which two, I143F and P436S, have not been found elsewhere. I143F shows incomplete penetration within the affected family. P436S is the most carboxy‐terminal presenilin 1 mutation reported to date. © 1999 Wiley‐Liss, Inc.     

Deficiency of FK506‐binding protein (FKBP) 51 alters sleep architecture and recovery sleep responses to stress in mice

FK506‐binding protein 51 (FKBP51) is a co‐chaperone of the glucocorticoid receptor, functionally linked to its activity via an ultra‐short negative feedback loop. Thus, FKBP51 plays an important regulatory role in the hypothalamic–pituitary–adrenocortical (HPA) axis necessary for stress adaptation and recovery. Previous investigations illustrated that HPA functionality is influenced by polymorphisms in the gene encoding FKBP51, which are associated with both increased protein levels and depressive episodes. Because FKBP51 is a key molecule in stress responses, we hypothesized that its deletion impacts sleep. To study FKBP51‐involved changes in sleep, polysomnograms of FKBP51 knockout (KO) mice and wild‐type (WT) littermates were compared at baseline and in the recovery phase after 6‐h sleep deprivation (SD) and 1‐h restraint stress (RS). Using another set of animals, the 24‐h profiles of hippocampal free corticosterone levels were also determined. The most dominant effect of FKBP51 deletion appeared as increased nocturnal wake, where the bout length was significantly extended while non‐rapid eye movement sleep (NREMS) and rapid eye movement sleep were rather suppressed. After both SD and RS, FKBP51KO mice exhibited less recovery or rebound sleep than WTs, although slow‐wave activity during NREMS was higher in KOs, particularly after SD. Sleep compositions of KOs were nearly opposite to sleep profiles observed in human depression. This might result from lower levels of free corticosterone in FKBP51KO mice, confirming reduced HPA reactivity. The results indicate that an FKBP51 deletion yields a pro‐resilience sleep phenotype. FKBP51 could therefore be a therapeutic target for stress‐induced mood and sleep disorders.     

Surfactant protein D (SP‐D) deficiency is attenuated in humanised mice expressing the Met(11)Thr short nucleotide polymorphism of SP‐D: implications for surfactant metabolism in the lung

Surfactant protein D (SP-D) is part of the innate immune system involved in lung homeostasis. SP-D knockout mice show accumulations of foamy alveolar macrophages, alveolar lipoproteinosis and pulmonary emphysema. Three single nucleotide polymorphisms (SNPs) have been described in the coding sequence of the human SP-D gene SFTPD. Clinical studies showed that the SNP SFTPD with a nucleotide change from A to C resulting in a Met to Thr substitution at position 11 in the protein (Met(11)Thr), is relevant. This study set out to create a humanised mouse model of the Met(11)Thr SNP. Transgenic mice lines expressing either Met(11) or Thr(11) SP-D under the control of the ubiquitously expressed pROSA26 promoter in C57Bl/6 SP-D deficient mice (DKO) was created. Both Met(11) (142 ± 52 ng mL⁻¹) and Thr(11) (228 ± 76 ng mL⁻¹) mice lines expressed human SP-D at almost similar levels. According to the literature this was within the range of SP-D levels found in wildtype (WT) mice (253 ± 22 ng mL⁻¹). Met(11) or Thr(11) SP-D in serum from transgenic mice bound maltose in a calcium-dependent manner, and binding was inhibited in the presence of EDTA or maltose. Bronchoalveolar lavage showed for both transgenic mice lines complementation of the DKO phenotype by restoring cell counts, phospholipid levels and protein content back to WT levels. Cytospins of BAL pellet cells showed a resemblance to WT but both mice lines showed some foamy alveolar macrophages. The stereological analysis showed for none of the mice lines a complete abrogation of emphysematous alterations. However, both Met(11) and Thr(11) mice lines were partially reverted back to a WT phenotype when compared with DKO mice, indicating important effects on surfactant metabolism in vivo.     

A novel luminescence‐based method for the detection of functionally active antibodies to muscarinic acetylcholine receptors of the M3 type (mAchR3) in patients' sera

In different bioassays, functional antibodies reacting with the human muscarinic acetylcholine receptor M3(mAchR3) have been detected in sera from patients with Sjögren's syndrome (SS), and there is strong evidence that those antibodies may have pathogenetic relevance. However, depending on the method of detection, their prevalence varied. Furthermore, those bioassays are difficult to standardize. We report on the development and optimization of a novel test system based on a luminometric method to determine downstream signalling of mAchR3 which produces specific and reproducible results. Chinese hamster ovarian (CHO) cells were transfected with plasmids encoding mAchR3 and a green fluorescence protein (GFP)/aequorin fusion protein. Incubation of cells with carbachol resulted in an increase in intracellular [Ca²⁺], which was detected by measuring light emission with a luminometer, and the effect of incubation with patients' immunoglobulins (Ig) was evaluated. Optimal cell density, Ig preparation and time of incubation with patients' sera were determined. Sera from patients with primary Sjögren's syndrome (pSS; n = 40), systemic sclerosis (SSc; n = 47), myasthenia gravis (MG; n = 133) and 50 blood donors were analysed. Optimal assay conditions were obtained with a cell density of 100 000 cells/ml, isolation of Ig by ammonium sulphate precipitation and short‐term incubation. Based on this highly reliable assay, 50% of the pSS patients had antibodies which inhibited carbachol‐induced activation of mAchR3; none of the SSc patients, 6% of the patients with MG and 12% of the blood donors had antibodies which reacted with the mAchR3. This method facilitates the determination of functional anti‐mAchR3 antibodies in patients' sera, confirmed their high prevalence in pSS patients and may, therefore, help to analyse their pathogenetic and clinical relevance in more detail.     

Differential Dimerization of Variants Linked to Enhanced S‐Cone Sensitivity Syndrome (ESCS) Located in the NR2E3 Ligand‐Binding Domain

NR2E3 encodes the photoreceptor‐specific nuclear hormone receptor that acts as a repressor of cone‐specific gene expression in rod photoreceptors, and as an activator of several rod‐specific genes. Recessive variants located in the ligand‐binding domain (LBD) of NR2E3 cause enhanced short wavelength sensitive‐ (S‐) cone syndrome (ESCS), a retinal degeneration characterized by an excess of S‐cones and non‐functional rods. We analyzed the dimerization properties of NR2E3 and the effect of disease‐causing LBD missense variants by bioluminescence resonance energy transfer (BRET²) protein interaction assays. Homodimerization was not affected in presence of p.A256V, p.R039G, p.R311Q, and p.R334G variants, but abolished in presence of p.L263P, p.L336P, p.L353V, p.R385P, and p.M407K variants. Homology modeling predicted structural changes induced by NR2E3 LBD variants. NR2E3 LBD variants did not affect interaction with CRX, but with NRL and rev‐erbα/NR1D1. CRX and NRL heterodimerized more efficiently together, than did either with NR2E3. NR2E3 did not heterodimerize with TLX/NR2E1 and RXRα/NR2C1. The identification of a new compound heterozygous patient with detectable rod function, who expressed solely the p.A256V variant protein, suggests a correlation between LBD variants able to form functional NR2E3 dimers and atypical mild forms of ESCS with residual rod function.     

B‐cell responses to B‐cell activation factor of the TNF family (BAFF) are impaired in the absence of PI3K delta

B‐cell activating factor of the TNF family (BAFF) is critical for the survival and maturation of B cells. The molecular mechanisms by which BAFF regulates the survival of developing B cells are becoming better understood. Recent evidence has begun to emerge demonstrating a role for the PI3K/Akt signalling pathway in response to BAFF. However, the importance of the PI3K family for BAFF‐signalling and the effects of loss of PI3K function on BAFF responses are still unknown. We therefore investigated the BAFF‐mediated responses of B cells deficient for the PI3K catalytic subunit P110δ. We find that the loss of P110δ impairs the BAFF‐mediated survival of cultured B cells demonstrating a direct role for this member of the PI3K family in regulating the survival of B cells in response to BAFF. P110δ was required for the growth of B cells in response to BAFF and was critical for the upregulation of the receptor for BAFF following BCR crosslinking. Our findings reveal an important role for p110δ in regulating B‐cell responses to BAFF.     

A dystroglycan mutation (p.Cys667Phe) associated to muscle‐eye‐brain disease with multicystic leucodystrophy results in ER‐retention of the mutant protein

Dystroglycan (DG) is a cell adhesion complex composed by two subunits, the highly glycosylated α‐DG and the transmembrane β‐DG. In skeletal muscle, DG is involved in dystroglycanopathies, a group of heterogeneous muscular dystrophies characterized by a reduced glycosylation of α‐DG. The genes mutated in secondary dystroglycanopathies are involved in the synthesis of O‐mannosyl glycans and in the O‐mannosylation pathway of α‐DG. Mutations in the DG gene (DAG1), causing primary dystroglycanopathies, destabilize the α‐DG core protein influencing its binding to modifying enzymes. Recently, a homozygous mutation (p.Cys699Phe) hitting the β‐DG ectodomain has been identified in a patient affected by muscle‐eye‐brain disease with multicystic leucodystrophy, suggesting that other mechanisms than hypoglycosylation of α‐DG could be implicated in dystroglycanopathies. Herein, we have characterized the DG murine mutant counterpart by transfection in cellular systems and high‐resolution microscopy. We observed that the mutation alters the DG processing leading to retention of its uncleaved precursor in the endoplasmic reticulum. Accordingly, small‐angle X‐ray scattering data, corroborated by biochemical and biophysical experiments, revealed that the mutation provokes an alteration in the β‐DG ectodomain overall folding, resulting in disulfide‐associated oligomerization. Our data provide the first evidence of a novel intracellular mechanism, featuring an anomalous endoplasmic reticulum‐retention, underlying dystroglycanopathy.     

Endoplasmic reticulum stress causes autophagy and apoptosis leading to cellular redistribution of the autoantigens Ro/Sjögren's syndrome‐related antigen A (SSA) and La/SSB in salivary gland epithelial cells

The aim of this study was to examine the levels of endoplasmic reticulum (ER) stress in minor salivary glands, to investigate the interplay between ER stress-induced autophagy and apoptosis in human salivary gland (HSG) cells and to test the effect of ER stress-induced apoptosis on the cellular redistribution of the two major Sjögren’s syndrome (SS) autoantigens Ro/Sjögren’s syndrome-related antigen A (SSA) and La/Sjögren’s syndrome-related antigen B (SSB). Minor salivary gland biopsies from SS patients and sicca controls were examined by immunohistochemistry for the expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP) as an indicator of unfolded protein response (UPR). HSG cells were treated with thapsigargin (TG) and cell viability, autophagy and apoptosis were assessed. Immunoblot was applied to detect the conversion of LC3I to LC3II and the protein levels of GRP78/BiP and X-box binding protein-1 (XBP-1). Apoptosis was evaluated by a single-stranded DNA enzyme-linked immunosorbent assay (ELISA). Ro/SSA and La/SSB localization was visualized using immunofluorescence. GRP78/BiP was expressed by acinar and ductal epithelial cells in salivary glands of patients and sicca controls. TG treatment induced autophagy, as indicated by enhanced protein expression of LC3II. The protein levels of UPR marker XBP-1 were increased after TG treatment, while GRP78/BiP levels were decreased. TG treatment resulted in induction of HSG apoptosis. Ro/SSA and La/SSB autoantigens were localized predominantly to the cytoplasm in resting cells, while they were redistributed to cell membrane and blebs in the apoptotic cells. In conclusion, ER stress is activated in minor salivary gland epithelial cells from SS patients and controls. ER stress-induced apoptosis in HSG cells leads to cell surface and apoptotic blebs relocalization of Ro/SSA and La/SSB autoantigens.     

Factors related to genetic testing in adults at risk for Huntington disease: the prospective Huntington at‐risk observational study (PHAROS)

Huntington disease (HD) is a late onset ultimately fatal neurodegenerative disorder caused by a cytosine–adenine–guanine ( CAG) triplet repeat expansion in the Huntingtin gene which was discovered in 1993. The PHAROS study is a unique observational study of 1001 individuals at risk for HD who had not been previously tested for HD and who had no plans to do so. In this cohort, 104 (10%) individuals changed their minds and chose to be tested during the course of the study but outside of the study protocol. Baseline behavioral scores, especially apathy, were more strongly associated with later genetic testing than motor and chorea scores, particularly among subjects with expanded CAG repeat length. In the CAG expanded group, those choosing to be tested were older and had more chorea and higher scores on the behavioral section of the unified Huntington's disease rating scale at baseline than those not choosing to be tested. Following genetic testing, 56% of subjects with CAG < 37 had less depression when compared to prior to testing, but depression generally stayed the same or increased for 64% of subjects in the expanded group. This finding suggests that approaches to testing must continue to be cautious, with appropriate medical, psychological and social support.