Salivary gland epithelial cell in Sjögren's syndrome: Metabolic shift and altered mitochondrial morphology toward an innate immune cell function.

Salivary gland epithelial cells (SGEC) are the main targets of the autoimmune reactivity in Sjögren's syndrome (SS). This study aimed to investigate the core proteomic differences between SS and Control- (Ct) -derived SGEC. Proteome analysis of cultured SGEC from five SS patients and four Ct was performed in a label-free quantitation format (LFQ). Electron microscopy was applied for analysis of the mitochondrial ultrastructure of SGEC in minor salivary gland sections from six SS patients and four Ct. Four hundred seventy-four proteins were identified differentially abundant in SS- compared to Ct-SGEC. After proteomic analysis, two distinct protein expression patterns were revealed. Gene ontology (GO) pathway analysis of each protein block revealed that the cluster with highly abundant proteins in SS-SGEC showed enrichment in pathways associated with membrane trafficking, exosome-mediated transport and exocytosis as well as innate immunity related mainly to neutrophil degranulation. In contrast, the low abundance protein cluster in SS-SGEC was enriched for proteins regulating the translational process of proteins related to metabolic pathways associated to mitochondria. Electron microscopy showed decreased total number of mitochondria in SS-SGEC, which appeared elongated and swollen with less and abnormal cristae compared to Ct-SGEC mitochondria. This study defines, for the first time, the core proteomic differences of SGEC between SS and Ct, substantiates the metamorphosis of SGEC into an innate immune cell and reveals that these cells are translationally shifted towards metabolism rewiring. These metabolic alterations are related mainly to mitochondria and are mirrored in situ with heavy morphological changes.

Cellular Vesicles: New Insights in Engineering Methods, Interaction with Cells and Potential for Brain Targeting.

Cellular vesicles (CVs) have been proposed as alternatives to exosomes for targeted drug delivery. CVs, prepared from human embryonic kidney 293 cells (HEK-293), C57BL/6 mouse B16F10 skin melanoma cells (B16F10), and immortalized human cerebral microvascular endothelial cells (hCMEC/D3) by liposome technology methods, were characterized for morphology, cytotoxicity, and cell uptake properties. CV brain-targeting potential was evaluated in vitro on the hCMEC/D3 blood-brain barrier (BBB) model, and in vivo/ex vivo. CV sizes were between 135 and 285 nm, and the ζ-potential was negative. The dehydration-rehydration method conferred highest calcein loading and latency to CVs compared with other methods. The increased calcein leakage from CVs when compared with liposomes indicated their poor integrity, which was increased by pegylation. The in vivo results confirmed lower liver uptake by PEG-CVs (compared with nonpegylated) proving that the calcein integrity test is useful for prediction of CV biodistribution, as used for liposomes. The cell uptake of homologous origin CVs was not always higher compared with that of non-homologous. Nevertheless, CVs from hCMEC/D3 demonstrated the highest BBB permeability (in vitro) compared with OX-26 targeted liposomes, and brain localization (in vivo). CVs from hCMEC/D3 cells grown in different media demonstrated decreased interaction with brain cells and brain localization. Significant differences in proteome of the two latter CV types were identified by proteomics, suggesting a potential methodology for identification of organotropism-determining CV components.

Calcium sensing receptor in pregnancies complicated by gestational diabetes mellitus.

INTRODUCTION: Infants born from mothers with Gestational diabetes mellitus (GDM) experience several complications, including a higher rate of postnatal hypocalcemia. In this study, we investigated the association between calcium sensing receptor (CaSR) and neonatal hypocalcemia observed in GDM pregnancies. METHODS: Our study consisted of 58 pregnant women with GDM and 40 healthy women and their neonates. CaSR placental expression was evaluated with immunohistochemistry and Western Blot. Three CaSR single nucleotide polymorphisms, A986S, R990G, Q1011E, were evaluated in neonate's genomic DNA. Serum Ca, P, Mg, 25(OH)D and PTH were measured in cord blood and at 2nd day of life. RESULTS: GDM neonates had lower mean cord blood Ca levels than controls (2.47 ± 0.21 mmol/l vs 2.59 ± 0.13 mmol/l, p = 0.001) while 15.5% developed postnatal hypocalcemia. CaSR expression was lower in GDM than in healthy placentas (p < 0.001). In the GDM group, reduced CaSR immunostaining in the syncytiotrophoblast (p = 0.042) and extravillous cytotrophoblasts (p = 0.002) was associated with lower Ca cord blood levels. Moreover, the absence of the S allele of the A986S polymorphism was associated with lower serum Ca levels both at birth (AA:2.41 ± 0.23 mmol/l, AS + SS: 2.57 ± 0.12 mmol/l, p = 0.002) and at 2nd day of life (AA:2.05 ± 0.22 mmol/l, AS + SS: 2.20 ± 0.18 mmol/l, p = 0.019). CONCLUSIONS: Our results showed that CaSR is under-expressed in GDM compared with healthy placentas and this alteration may be associated with the lower Ca levels measured in cord blood of GDM infants. Placental CaSR seems to exert a local effect in fetal Ca homeostasis, which is dissociated from its contribution to the regulation of Ca homeostasis in postnatal life.

Analysis of parotid glands of primary Sjögren's syndrome patients using proteomic technology reveals altered autoantigen composition and novel antigenic targets.

Sjögren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration, destruction of the salivary and lacrimal glands and production of autoantibodies against a variety of cellular proteins. The aberrant immune response against these autoantigens may begin or extend to other proteins that are not yet defined. Several studies have shown that autoantibody production is taking place in the affected salivary glands. In the present study, using proteomic approaches, we aimed to: (a) identify new autoantigens in the salivary glands of primary SS (pSS) patients and (b) evaluate the epigenetic changes of known autoantigens. Total parotid gland extracts of pSS patients were analysed using two-dimensional gel electrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot with pSS patients' sera or purified autoantibodies and immunoprecipitation using homologous IgG. Identification of the unknown proteins was performed using mass spectrometry (MS). Immunoblot analysis on two-dimensional gels using purified anti-La/SSB antibodies revealed that pSS salivary glands contain high levels of post-translationally modified La/SSB autoantigen, while the native form of the protein is recognized faintly, in contrast to normal controls. Moreover, salivary glands of pSS patients contain post-translationally modified actin that becomes immunogenic in the microenviroment of the affected tissue. The alteration of the physicochemical properties of self-proteins could thus contribute to the break of immune tolerance against them.

Autoantibodies against aggrecan in systemic rheumatic diseases.

OBJECTIVE: This study was undertaken to investigate the presence of autoantibodies against the main cartilage proteoglycan, aggrecan, in systemic rheumatic disease sera, and to identify substructure(s) responsible for the autoimmune response. METHODS: Sera were obtained from 86 patients with various systemic rheumatic diseases, 14 with osteoarthritis (OA), 18 with cancer and 40 healthy individuals. The presence of autoantibodies against aggrecan was examined by a solid phase assay and by Western blotting, using proteoglycan aggregates treated with proteolytic enzymes. The positive bands were subjected to nanohigh performance liquid chromatography (nanoHPLC)-MS, in order to identify the aggrecan substructures involved in the autoimmune response. RESULTS: Autoantibodies against aggrecan were identified in all systemic rheumatic disease sera at a high titre, almost three times that observed in healthy controls. OA and cancer sera produced a reaction equal to that of the healthy. Western blotting analysis of aggrecan proteolytic fragments revealed the presence of a triple band, reacting with the patients' sera, of about 37 kDa, which also reacted with a polyclonal antibody against hyaluronan-binding region. NanoHPLC-MS analysis suggested that this band belonged to the G2 domain of aggrecan. CONCLUSION: At least a part of the autoimmune reaction to aggrecan, displayed by the systemic disease sera, involves the G2 domain. The significant difference observed between these sera and those from other diseases, especially cancer, may suggest a possible discriminatory role of anti-aggrecan antibodies. This may help in the differential diagnosis in complicated clinical cases. However, for this to be confirmed, studies in larger cohorts of patients should be performed.

Fine specificity and subclasses of IgG anti-actin autoantibodies differ in health and disease.

Current opinions suggest that autoantibodies occurring in autoimmune diseases are generated by B-cells which primarily produce polyspecific natural autoantibodies, through either polyclonal activation or specific antigen selection of these B-cells. In this study, we compared the immunological properties (polyspecificity, fine specificity and IgG subclasses) between natural anti-actin antibodies (N-AAA) and disease-associated AAA (D-AAA). IgG AAA from sera of healthy donors, patients with autoimmune hepatitis type 1 (AIH-1) and patients with primary biliary cirrhosis (PBC) were affinity-purified on actin immunoadsorbent and tested initially for polyspecificity against various cytoskeleton proteins by enzyme-linked immunosorbent assay (ELISA). Fine specificity was studied by Western blotting using proteolytic peptides of actin and by ELISA using synthetic 12 mer peptides, spanning the 221-377 aa sequence of actin. Results showed that both N-AAA and D-AAA are polyspecific. Nevertheless, D-AAA from both diseases showed a specific reactivity pattern as compared to N-AAA, against the 16 kDa C-terminal (229-377 aa) proteolytic peptide of actin and more specifically against the P36 synthetic peptide (351-362 aa). Quantitation of AAA IgG subclasses revealed that IgG1 and IgG3 were specifically increased in D-AAA from AIH-1 and PBC, respectively, as compared to N-AAA. We conclude that D-AAA are differentiated from N-AAA in terms of fine specificity and IgG subclasses, probably through specific antigen selection of B-cells primarily producing N-AAA.

C-MYC and IGF-II mRNA-binding protein (CRD-BP/IMP-1) in benign and malignant mesenchymal tumors.

Mouse coding region determinant-binding (mCRD-BP) and human IGF-II mRNA-binding 1 (hIMP-1) proteins are orthologous mRNA-binding proteins that recognize c-myc and IGF-II mRNA, respectively, and regulate their expression posttranscriptionally. Here, we confirm that human CRD-BP/IMP-1 binds to c-myc mRNA and that it is predominantly expressed in fetal tissues. Moreover, hCRD-BP/IMP-1 expression was detected in cell lines of neoplastic origin and in selected primary tumors. In a series of 33 malignant and 10 benign mesenchymal tumors, 73% and 40%, respectively, were found to express hCRD-BP/IMP-1. In particular, expression was significant in 14 Ewing's sarcomas, all of which were positive. The data suggest that hCRD-BP/IMP-1 plays a role in abnormal cell proliferation in mesenchymal tumors.

Expression profiling across many samples via manifold-assisted mRNA processing.

Analysis of mRNA provides a condensed view of gene structure, and quantitative analyses can reveal induction of physiological or pathological gene expression programs. One of the main hurdles for routine mRNA analyses is the need to prepare large sets of samples in a rapid and standardized manner. We describe here a procedure for mRNA isolation and cDNA synthesis using manifold devices, consisting of a set of prongs that project into individual reaction wells. The prongs have a high binding capacity for the polyA-tails of mRNA and the captured mRNA is directly used to synthesize cDNA on the supports, followed by amplification. The convenience and reproducibility of the procedure allows profiling of gene expression over time, by comparing many different samples. Using the device mRNA was simultaneously isolated and accurately measured from up to 96 different samples of anywhere between 10 and 200 000 cells. The amounts of a leukemia-specific transcript could be measured when the malignant cells represented

Seven-color time-resolved fluorescence hybridization analysis of human papilloma virus types.

Identification of human papilloma virus (HPV) types is important in order to determine the risk of cervical carcinoma in women. This requires a technique to probe individual samples for multiple virus specificities. Here we describe simultaneous multicolor analysis of amplification products for any of seven amplified HPV types 16, 18, 31, 33, 35, 39, and 45, associated with cancer of the cervix. A seminested polymerase chain reaction was performed in a single tube using a biotinylated inner primer. Sets of amplification products, immobilized on a 96-pronged manifold solid support, were rendered single stranded and probed with a mix of seven type-specific, differentially labeled oligonucleotides. These probes contained 10 or 20 lanthanide chelates at the 5' ends with seven distinct combinations of europium, terbium, and samarium ions. The seven viral strains were correctly identified by time-resolved fluorescence measurement of the specifically hybridized probes. Using this assay format, simultaneous detection of any of seven or even more target variants is possible.

Detecting Genes with Ligases

The combination of synthetic oligonucleotide probes and DNA ligases is central to several recently developed genetic assays. Among the advantages of ligase-mediated gene detection is that ligation of probe pairs provides highly specific detection of unique DNA sequences in genomic samples. The technique also allows for convenient distinction between sequence variants, since mismatched bases at the junction of the probe pair prevent ligation. Moreover, the circumstance that two probes are joined into one molecule can be exploited for detection in several ways, for instance by observing the change in probe size upon ligation. Alternatively, a detectable function on one probe can be demonstrated to become linked to a retrievable function on another one through ligation. Ligation products can also be recruited as templates for subsequent ligation reactions in powerful amplification schemes. So-called padlock probes lock to their targets by encircling them, remaining in place even after denaturing washes. Here, we will describe two ligase-mediated assays: one that serves to monitor the presence of common sequence variants in amplified samples of genomic DNA and another that is suitable to detect localized gene sequences.

Padlock probes: circularizing oligonucleotides for localized DNA detection.

Nucleotide sequence information derived from DNA segments of the human and other genomes is accumulating rapidly. However, it frequently proves difficult to use such short DNA segments to identify clones in genomic libraries or fragments in blots of the whole genome or for in situ analysis of chromosomes. Oligonucleotide probes, consisting of two target-complementary segments, connected by a linker sequence, were designed. Upon recognition of the specific nucleic acid molecule the ends of the probes were joined through the action of a ligase, creating circular DNA molecules catenated to the target sequence. These probes thus provide highly specific detection with minimal background.

Solid-phase synthesis of chelate-labelled oligonucleotides: application in triple-color ligase-mediated gene analysis.

Oligonucleotides labelled with detectable groups are essential tools in gene detection. We describe here the synthesis of pyrimidine deoxynucleotide-building blocks, modified at their C-5 position with a protected form of a strongly chelating agent. These reagents can be used to introduce multiple metal ions into oligodeoxynucleotides during standard oligonucleotide synthesis. The chelating functions form strongly fluorescent complexes with europium ions, characterized by a wide separation between the excitation and emission spectra. Moreover, the long decay time of the fluorescence permits sensitive time-resolved fluorescence measurements. The chelates also have the stability required to function in triple-color assays involving europium, samarium, and terbium ions. We demonstrate the application of these reagents for ligase-based gene analysis reactions.

Dual-color detection of DNA sequence variants by ligase-mediated analysis.

Genetic screening for sequence variants associated with disease is assuming increasing importance in clinical medicine as well as in research. We describe an efficient method for such analyses, comprising a combination of practical features: (1) Amplified DNA samples are analyzed for their ability to serve as templates in standardized allele-specific ligation reactions between oligonucleotide probes; (2) Two allele-specific probes, differentially labeled with either of two lanthanide labels, compete for ligation to a third oligonucleotide (the signal from the two labeled probes can thus be directly compared in a sensitive time-resolved fluorescence detection reaction); and (3) Large sets of analyses are processed in parallel using a 96-pin capture manifold, serving to reduce pipetting steps and the risk of contamination. We present here the basis of the technique and its application to the screening for two common mutations causing cystic fibrosis and alpha 1-antiytrypsin deficiency.

A manifold support for molecular genetic reactions.

Large numbers of samples can be efficiently processed through sequential reaction steps using a 96-pronged support that projects into individual microtiter wells. The support was constructed by creating a porous surface on a disposable polystyrene manifold, with avidin coupled to this greatly expanded surface. The shape and high binding capacity of the device allow the parallel transfer of large sets of reaction intermediates between different binding or enzymatic processing steps. We have applied the support to increase the efficiency of preparative and analytical molecular genetic reactions. The support also reduce the risks of sample mix-up and contamination in applications such as PCR and DNA sequencing.