OPG, RANK and RANK ligand expression in thyroid lesions

Receptor activator of NF-kappaB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) play essential roles in bone metabolism. RANKL binds to RANK, which is expressed by osteoclasts whereas OPG acts as its decoy receptor blocking the RANK-RANKL interaction. OPG/RANK/RANKL are produced by variety of tissues including epithelial and mesenchymal cells. However, the role of RANKL/OPG in thyroid pathophysiology remains unclear. The aim of this study was to determine the expression pattern of RANK/RANKL/OPG in primary neoplastic thyroid lesions and in lymph node metastases. 27 specimens from total thyroidectomy were studied by immunohistochemistry: 9 papillary carcinomas (PC), 9 medullary carcinomas (MC), 9 macrovesicular adenomas (MA). Immunohistochemical evidence of RANKL was found in 30 % of MC, 22% of PC while RANKL has never been detected in PC. The expression of RANK is closely related to RANKL. OPG was restricted to the cytoplasm of epithelial in 1 MA and 1 MC. In contrast to pathological tissues, any expression of OPG/RANK/RANKL was detected in healthy thyroid tissue. This work reveals for the first time that OPG/RANK/RANKL are expressed in the pathological thyroid gland by follicular cells, by malignant parafollicular cells as well as in metastatic lymph node microenvironment. Thus OPG/RANK/RANKL molecular triad might play a role during pathogenesis of follicular and parafollicular tumors.     

Characterization of ligands for thyroid receptor subtypes and their interactions with co-regulators

Thyroid hormone receptors (TRs) are nuclear receptors that are activated by thyroid hormone ligands and co-regulator proteins. Two receptor subtypes, TRα and TRβ, have been suggested to play a role in numerous physiological functions. However, specificity of receptor subtype function and co-regulator interaction is unclear due to the lack of TR subtype-specific ligands. Five TR ligands were evaluated for their selectivity and interaction with the TR subtypes. A multiplex assay was used to identify co-regulator peptide interaction, and biochemical assays were used to characterize ligand-receptor specificity. In the biochemical assay, rank order ligand potencies were similar in the presence of co-activator peptides, SRC1-2 and SRC3-2, and the co-repressor peptide, NCoR1-2, with T3 and Triac potencies greater in the presence of the co-repressor. The potency of Tetrac was similar regardless of the co-regulator used while T4 and rT3 demonstrated selectivity for TRα subtype. The rank order among TR ligands at either receptor subtype in the biochemical assay correlated with the multiplex assay. These assays can be used to identify new ligands that can provide further insight into TR biology.     

Characterization and measurement of normal platelet-associated immunoglobulin G by fluorospectrophotometry

Platelet-associated immunoglobulin G of normal healthy subjects was measured and its binding characteristics studied by use of a newly developed assay which is rapid, quantitative, sensitive and inexpensive. The assay is based on fluorospectrophotometry. Fluorescein isothiocyanate was used as a fluorescent marker to label IgG. Measurement of platelet-associated IgG by this method showed that normal platelets have at least two types of binding sites for IgG of normal healthy subjects. High- and low-affinity binding sites numbering 410 ± 200 and 1800 ± 500, respectively, were identified. Specificity of binding was shown by competition between fluorescein isothiocyanate-labeled IgG and nonlabeled IgG. The effect of pH and temperature and the kinetics of binding were also investigated.     

Characterization of periostin expression in human endometrium and endometriotic lesions

Objective(s): To investigate the expression of periostin in the eutopic and ectopic endometrium of women diagnosed as endometriosis and evaluate the role of periostin in the pathogenesis of endometriosis. Study design: In this study, the expression of periostin was evaluated in the endometrial specimens from 35 women diagnosed as endometriosis and from 30 healthy women. To assess the presence and localization of periostin throughout the menstrual cycle in both eutopic and ectopic endometrium of women with endometriosis, microscopic evaluation was conducted. It was also subsequently compared with normal endometrium. Results: In the eutopic and ectopic endometrium of women with endometriosis, immunoreactivities of periostin increased compared with those of normal endometrium. We also observed a cyclic variation in the eutopic stromal periostin immunoreactivity throughout their menstrual cycle because higher H score values were observed in the proliferative phase than those in the secretory phase. Conclusion(s): These findings indicated that periostin may be involved in the pathophysiology of endometriosis.     

Identification of natural bispecific antibodies against cyclic citrullinated peptide and immunoglobulin G in rheumatoid arthritis

Background

Previous studies indicate that natural bispecific antibodies can be readily produced in vivo when the body is simultaneously stimulated with 2 distinct antigens. Patients with rheumatoid arthritis (RA) usually exhibit persistent immune responses to various autoantigens, raising the possibility that natural bispecific antibodies against 2 distinct autoantigens might exist.

Methodology/Principal Findings

We identified the presence of natural bispecific antibodies against cyclic citrullinated peptide (CCP) and immunoglobulin G (IgG) in RA patients'' sera by means of a double-antigen sandwich enzyme-linked immunosorbent assay (ELISA). The spontaneous emergence of bispecific antibodies was confirmed by mixing different proportions of 1 anti-CCP-positive serum and 1 rheumatoid factor (RF)-positive serum in vitro. Among the tested samples, positive correlations were found between the presence of bispecific antibodies and both IgG4 anti-CCP antibodies and IgG4 RF (r = 0.507, p<0.001 and r = 0.249, p = 0.044, respectively), suggesting that the IgG4 subclass is associated with this phenomenon. Furthermore, bispecific antibodies were selectively generated when several anti-CCP- and RF-positive sera were mixed pairwise, indicating that factors other than the monospecific antibody titers may also contribute to the production of the natural bispecific antibodies.

Conclusions/Significance

We successfully identified the presence of natural bispecific antibodies. Our results suggest that these antibodies originate from anti-CCP and RF in the sera of RA patients. The natural occurrence of bispecific antibodies in human diseases may provide new insights for a better understanding of the diseases. Further investigations are needed to elucidate their precise generation mechanisms and explore their clinical significance in disease development and progression in a larger study population.     

Characterization of the photoproducts of protoporphyrin IX bound to human serum albumin and immunoglobulin G

Clinically useful photosensitisers (PSs) are likely bound to subcellular and molecular targets during phototherapy. Binding to a macromolecule has the potential to change the photophysical and photochemical characteristics of the PSs that are crucial for their phototoxicity and cell-killing activity. We investigated the effects of binding of a specific PS (protoporphyrin IX or PPIX) to two proteins, human serum albumin (HSA) and a commercially available immunoglobulin (IgG). These two proteins provide two different environments for PPIX. The albumin binds PPIX in hydrophobic binding sites located in subdomain IIA and IIIA, conversely IgG leaves PPIX exposed to the solvent. We show that photophysical parameters such as emission maxima and fluorescence lifetime depend on the binding site. Our results indicate that the different binding site yields very different rates of formation of photoproducts (more than three times higher for PPIX bound to HSA than to IgG) and that different mechanisms of formation may be occurring. Our characterization shows the relevance of protein binding for the photochemistry and ultimately the phototoxicity of PSs.     

Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea.

The digestion of human IgG1/K myeloma proteins with pepsin in the presence of 8 M-urea produces fragments that differ from those produced by aqueous peptic digestion, and from other characteristic immunoglobulin fragments. Fb'2, the larger urea/pepsin fragment, was previously shown to consist of the constant regions of the light chains, and the CH1 domains and hinge regions of the heavy chains. The smaller fragment, upFc, has now been characterized. After reduction, three peptides were released from fragment upFc. Amino acid sequencing, N- and C-terminal determinations and amino acid compositions have enabled these peptides to be identified as residues Ile-253 to Leu-306, residues Thr-307 to Asp-376 and residues Thr-411 to Gly-446 of the heavy chain. Fragment upFc therefore contains the entire Fc region, beginning at residue Ile-253, except for a 34-residue section from within the CH3-domain disulphide loop. Peptic digestion of IgG1/K proteins in 8M-urea therefore provides a method for isolating from gamma1 heavy chains five homogeneous peptides in good yield, which account for almost the entire constant region. Characterization of fragments Fb'2 and upFc has shown that the action of pepsin in urea is entirely different from that of aqueous pepsin. Two gamma1 heavy chains have been shown to differ in sequence at three positions from the sequence reported for protein Eu.     

Characterization of the interaction between the herpes simplex virus type I Fc receptor and immunoglobulin G

Herpes simplex virus type I (HSV-1) virions and HSV-1-infected cells bind to human immunoglobulin G (hIgG) via its Fc region. A complex of two surface glycoproteins encoded by HSV-1, gE and gI, is responsible for Fc binding. We have co-expressed soluble truncated forms of gE and gI in Chinese hamster ovary cells. Soluble gE-gI complexes can be purified from transfected cell supernatants using a purification scheme that is based upon the Fc receptor function of gE-gI. Using gel filtration and analytical ultracentrifugation, we determined that soluble gE-gI is a heterodimer composed of one molecule of gE and one molecule of gI and that gE-gI heterodimers bind hIgG with a 1:1 stoichiometry. Biosensor-based studies of the binding of wild type or mutant IgG proteins to soluble gE-gI indicate that histidine 435 at the CH2-CH3 domain interface of IgG is a critical residue for IgG binding to gE-gI. We observe many similarities between the characteristics of IgG binding by gE-gI and by rheumatoid factors and bacterial Fc receptors such as Staphylococcus aureus protein A. These observations support a model for the origin of some rheumatoid factors, in which they represent anti-idiotypic antibodies directed against antibodies to bacterial and viral Fc receptors.     

Growth factor regulation of the promoter for calcyclin, a growth-regulated gene

The steady-state levels of calcyclin mRNA are regulated by growth factors. Using deletion mutants of the 5'-flanking region and a linked reporter (the bacterial chloroamphenicol transferase gene), we have investigated the elements of the calcyclin gene's promoter that respond to growth factors. By a transient expression assay after transfection in BALB/c/3T3 cells, we have been able to show that the serum-inducible sequences are contained in a 164-base pair fragment just upstream of the cap site. This fragment also contains an enhancer, and responds to platelet-derived growth factor as well as to serum. The sequences from -1371 to -1194 upstream of the cap site contain an element which is negatively regulated by epidermal growth factor. These findings have been confirmed in hamster cell lines in which the deletion mutants of the calcyclin promoter controlled the expression of the cDNA for human thymidine kinase. These results indicate that, like in other growth-regulated genes the activity of the calcyclin promoter is modulated by both positive and negative elements. Even more intriguing, though, is the finding that some of these negative elements may be influenced by growth factors in the environment.     

Intestinal colonization with bifidobacteria affects the expression of galectins in extraintestinal organs

This study aimed at determining the contribution of intestinal bifidobacteria to the immune system activation using widely distributed galectins as markers of immune cell homoeostasis. In human flora-associated mice, bacteria were enumerated in the gut, blood, spleen, liver and lungs, while the expression of galectin-1 (Gal-1) and galectin-3 (Gal-3) was estimated by PCR in the intestine and real-time quantitative PCR in the other organs. Gal-1 and -3 were rarely expressed in the intestine. In blood, only Gal-1 was expressed while both galectins were expressed in all other organs. A high prevalence of colonic bifidobacteria was associated with a lower expression of both pulmonary galectins, whose levels negatively correlated with bifidobacterial counts. Caecal bifidobacterial counts also negatively correlated with pulmonary Gal-3 mRNA levels. The spleen was the only organ showing an upregulation of Gal-1 expression related to its bacterial contamination. However, this upregulation was only observed when bifidobacteria were not detected in the colon. A putative mechanism explaining the reduced expression of galectins when bifidobacteria highly colonize the mouse intestine could be that, by reducing the bacterial translocation, bifidobacteria also lead to a decreased blood concentration of substances produced by intestinal bacteria.     

Glucocorticoids enhance the gamma-interferon augmentation of human monocyte immunoglobulin G Fc receptor expression

At physiologic and therapeutic concentrations, glucocorticoids decrease the number of Fc receptors for IgG (Fc gamma R) on human monocyte-like cell lines. In comparison, gamma-interferon (IFN-gamma) increases Fc gamma R expression on both human monocytes and monocyte-like cell lines. In this study, we examined the combined effects of glucocorticoids and IFN-gamma on human monocyte expression of the high affinity (72 kDa) Fc gamma R. Mononuclear cells prepared from heparinized venous blood of normal donors were treated for up to 90 hr with or without recombinant IFN-gamma and/or steroids. Monocyte Fc gamma R were measured by Scatchard analysis of the binding of human monomeric 125I-IgG1; indirect immunofluorescence plus flow cytometry, utilizing a monoclonal antibody (MoAb 32) which is specific for the high affinity Fc gamma R; and direct immunofluorescence using fluorescein isothiocyanate-labeled human monomeric IgG1 and flow cytometry quantitated using U-937 cells as a standard. Cultured monocytes incubated in the presence of both glucocorticoids and IFN-gamma for 18 hr had significantly higher (p less than 0.01) Fc gamma R levels than monocytes treated with IFN-gamma alone. The effect of combined treatment reached a plateau by 42 hr of incubation without increasing expression of other surface markers tested. Treatment with glucocorticoids alone did not consistently decrease monocyte Fc gamma R levels after either 18 or 42 hr of culture. Only glucocorticoids augmented the IFN-gamma increase in Fc gamma R; other steroids tested had no effect on IFN-gamma action. Furthermore, the effect was observed after treatment with only one type of interferon, IFN-gamma. These results describe a glucocorticoid immunoregulatory effect that may explain why combined IFN-gamma plus glucocorticoid treatment enhances mononuclear phagocyte Fc-mediated functions.     

The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54.

Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.     

Morphometric, densitometric and flow cytometric criteria for the automated classification of thyroid lesions

An automated classification of 73 thyroid lesions using a logical and mathematical approach was attempted. Densitometric, morphometric and flow cytometric parameters were used in Fisher linear discriminant functions to separate goiters or normal thyroids from adenomas and from carcinomas; the combination of this approach with binary discrimination improved the initial classification to a final efficiency of 81%. This approach, which is useful for classifying individual cells, was thus insufficient for classifying these cases. Analysis of the individual parameters showed that thyroid lesions were mainly in the near-diploid region. Two G0G1 populations were present in both benign and malignant lesions and were particularly frequent (50%) in atypical invasive follicular adenomas, probably related to the additional presence of an invasive clone. Near-triploid peaks were associated with malignancy as well as with high proliferative indexes. Nuclear and nucleolar sizes were larger in carcinomas; however, the percentage of the nucleolar area in the nucleus was greater in adenomas and nodular adenomatous goiters. A corrected staining index correlated with the nuclear size and the ploidy of abnormal cells (r = .50), being higher in malignant lesions.     

Mercaptoheterocyclic ligands grafted on a poly(ethylene vinyl alcohol) membrane for the purification of immunoglobulin G in a salt independent thiophilic chromatography

In this study, we attempted a limited combinatorial approach for designing affinity ligands based on mercaptoheterocyclic components. The template, divinyl sulfone structure (DVS), which was grafted on poly(ethylene vinyl alcohol) (PEVA) hollow fiber membrane, has served for the tethering of different heterocyclic compounds as pyridine, imidazole, purine and pyrimidine rings. Their ability to adsorb specifically IgG in a salt independent manner out of pure IgG solution, mixture of IgG/albumin and human plasma was demonstrated. Mercapto methyl imidazole (MMI) has shown the best adsorption of IgG in terms of binding capacity. No subclass discrimination was observed on all tested ligands except for mercapto methyl pyrimidine where the major IgG subclass adsorbed was IgG3. MMI gave an IgG binding capacity of 100 microg/cm2 of hollow fiber membrane surface area.     

Modeling assembly, aggregation, and chaperoning of immunoglobulin G production in insect cells

A model for immunoglobulin G (IgG) production in the baculovirus-insect cell system was developed that incorporates polypeptide synthesis, oligomer assembly, protein aggregation, and protein secretion. In addition, the capacity of a chaperone to protect heavy and light chain polypeptides from protein aggregation was considered by including in vitro chaperone-peptide binding and dissociation kinetic constants from the literature. Model predictions were then compared to experiments in which the chaperone immunoglobulin heavy chain binding protein, BiP, was coexpressed by coinfecting insect cells with BiP-containing baculovirus. The model predicted a nearly twofold increase in intracellular and secreted IgG that was similar to the behavior observed experimentally after approximately 3 days of coexpressing heterologous IgG and BiP. However, immunoglobulin aggregation was still significant in both the model simulation and experiments, so the model was then used to predict the effect of strategies for improving IgG production even further. Increasing expression of the chaperone BiP by 10-fold over current experimental levels provided a 2.5-fold increase in secreted IgG production over IgG assembly without BiP. Alternatively, the expression of BiP earlier in the baculovirus infection cycle achieved a twofold increase in protein secretion without requiring excessive BiP production. The potential effect of cochaperones on BiP activity was considered by varying the BiP binding and release constants. The utilization of lower binding and release kinetic constants led to a severalfold increase in IgG secretion because the polypeptides were protected from aggregation for greater periods. An optimized strategy for chaperone action would include the rapid peptide binding of a BiP-ATP conformation along with the slow peptide release of a BiP-ligand conformation. However, even with an optimized chaperoning system, limitations in the secretion kinetics can result in the accumulation of intracellular IgG. Thus, the entire secretory pathway must be considered when enhanced secretion of heterologous proteins is desired. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 106-116, 1997.     

Temporal and spatial regulation of expression of two galectins during kidney development of the chicken

Organogenesis and the establishment of the mature phenotype require an interplay between diverse recognition systems. Concerning protein–carbohydrate interactions, galectins are known to be involved in several extra- and intracellular functions. Due to the occurrence of two avian galectins in liver (chicken galectin-16; CG-16) and intestine (chicken galectin-14; CG-14) with different developmental regulation, the questions addressed are to what extent and where these galectins are present during chicken kidney development. Using Western blot analysis, the presence of both activities in tissue extracts was ascertained. A solid-phase assay showed peak levels at day 12 followed by a decline. A histochemical analysis was carried out in combination with routine staining. Epithelial cells of the mesonephric proximal tubules were immunoreactive in the cytoplasm for CG-14 from day 5 of incubation onwards. Additionally, epithelial cells of the metanephric collecting ducts were stained. For CG-16 a rather similar pattern of staining was seen, additional positivity in early glomerular podocytes being notable. At the electron microscopical level, a diffuse staining for CG-14 was seen in the cytoplasm, whereas immunoreactivity for CG-16 was observed mainly in mitochondria. These results demonstrate quantitative differences in the developmental regulation of the two avian galectins with obvious similarities in the cell-type pattern but with a disparate intracellular localisation profile.