Immunomodulatory effect of vitamin D and its potential role in the prevention and treatment of thyroid autoimmunity: a narrative review.

The main role of vitamin D is to control mineral homeostasis. However, recent studies suggested the existence of a number of extraskeletal effects. Among the latter, preclinical studies provided consistent data on the involvement of vitamin D in innate and adaptive immunity and autoimmunity. Molecular biology studies showed that both vitamin D receptor and vitamin D enzymatic complexes are expressed in a large number of cells and tissues unrelated to mineral homeostasis. In contrast, only a few randomized clinical trials in humans investigated the possible role of vitamin D in the prevention or treatment of immunological disorders. In this regard, low serum vitamin D levels have been reported in observational trials in human autoimmune disorders. The aim of the present paper was to review the potential implications of vitamin D in immune modulation, with special focus on thyroid autoimmune disorders.

Feasibility of a text-based smoking cessation intervention in rural older adults.

Text-based interventions are effective for smoking cessation, but have not been tested in rural older adults. The purpose of this study was to compare the feasibility, acceptability and preliminary efficacy of a text-based Scheduled Gradual Reduction (SGR) program to a non-SGR text messaging support condition among rural older adults. Adults over 60 years were randomized to either: (i) the SGR program (n = 20), a text-based program to reduce smoking over 4-weeks plus text-based support messages; or (ii) control (n = 20), receipt of text-based support messages only. Participants completed surveys at baseline and end of program to assess feasibility and acceptability of the intervention, and biochemically validated 7-day point prevalence cessation was assessed at end of treatment. Most participants (81%) reported reading all the messages they received. Participants found both interventions useful in quitting smoking (SGR = 57%, Control = 63%) and would recommend it to a friend (SGR = 72%, Control = 79%). Although not statically significant, the SGR group had a higher rate of biochemically validated cessation (SGR = 15%, Control = 5%, Cohen d = 0.67). Among those still smoking, the median percent reduction in cigarettes was 33.3% for both groups. Text-based cessation interventions are feasible, acceptable and can be easily disseminated to rural older adult tobacco users.

NET amyloidogenic backbone in human activated neutrophils.

Activated human neutrophils produce a fibrillar DNA network [neutrophil extracellular traps (NETs)] for entrapping and killing bacteria, fungi, protozoa and viruses. Our results suggest that the neutrophil extracellular traps show a resistant amyloidogenic backbone utilized for addressing reputed proteins and DNA against the non-self. The formation of amyloid fibrils in neutrophils is regulated by the imbalance of reactive oxygen species (ROS) in the cytoplasm. The intensity and source of the ROS signal is determinant for promoting stress-associated responses such as amyloidogenesis and closely related events: autophagy, exosome release, activation of the adrenocorticotrophin hormone/α-melanocyte-stimulating hormone (ACTH/α-MSH) loop and synthesis of specific cytokines. These interconnected responses in human activated neutrophils, that have been evaluated from a morphofunctional and quantitative viewpoint, represent primitive, but potent, innate defence mechanisms. In invertebrates, circulating phagocytic immune cells, when activated, show responses similar to those described previously for activated human neutrophils. Invertebrate cells within endoplasmic reticulum cisternae produce a fibrillar material which is then assembled into an amyloidogenic scaffold utilized to convey melanin close to the invader. These findings, in consideration to the critical role played by NET in the development of several pathologies, could explain the structural resistance of these scaffolds and could provide the basis for developing new diagnostic and therapeutic approaches in immunomediated diseases in which the innate branch of the immune system has a pivotal role.

The angiogenesis induced by HIV-1 tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells.

The HIV-1 Tat protein transactivates HIV, viral and some host cell genes. Tat can be released by infected cells and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor-1 and E selectin). Tat induces growth of Kaposi's sarcoma (KS) spindle cells and is angiogenic in vivo and in transgenic mice10-12. We previously reported that Tat is a direct angiogenic factor and noted the Tat arginine- and lysine-rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor-A (VEGF-A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (for review see ref. 13), and that Tat-induced angiogenesis is blocked by agents blocking the Flk-1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT-1, another VEGF-A tyrosine kinase receptor.

Four Ia invariant chain forms derive from a single gene by alternate splicing and alternate initiation of transcription/translation.

We determined the structural basis for the presence of electrophoretically-distinct, antigenically-related forms of invariant chains in Ia oligomers, and established the mechanisms by which they can be expressed from a single gene. S1 nuclease protection assays indicated that, in B cells, transcription of this gene initiates at a minimum of three sites. Thus, unlike previously thought, invariant chain mRNAs have heterogeneous 5' untranslated segments that may differentially affect initiation of translation. Further, restriction mapping and nucleotide sequencing of cDNAs revealed two kinds of invariant chain mRNAs differing by an internal coding segment of 192 bp. This segment represents an alternatively spliced exon, as demonstrated by nucleotide sequencing of corresponding genomic regions. The exon (exon X) encodes a cysteine-rich stretch of 64 amino acids near the COOH terminus that displays a striking and surprising homology to an internal amino acid repeat of thyroglobulin, suggesting an evolutionary mechanism of exon shuffling. Transient expression of cDNAs indicated that both types of alternatively spliced mRNAs contain two in-frame AUGs functioning as alternate start sites for translation. Thus, transfections with exon X-lacking cDNAs resulted in the expression of Mr 33,000 and 31,000 proteins, detected by immunoprecipitation with anti-invariant chain antisera, and identical by two-dimensional gel (2-D) analyses to the B cell invariant-chain forms gamma 1 (Mr 31,000), gamma 2, and gamma 3 (Mr 33,000). Similarly, exon X-containing cDNAs expressed Mr 43,000 and 41,000 proteins, also identical by 2-D migration to Ia-associated proteins. Thus, human Ia molecules contain four forms of invariant chain of closely related but nonidentical primary structure that are generated from a single gene by a complex pattern of alternate transcriptional start, exon splicing, and translational start.

Ig heavy chain variable region gene complex of lupus mice exhibits normal restriction fragment length polymorphism.

B cell hyperactivity, hypergammaglobulinemia, and autoantibody expression, the hallmarks of systemic lupus erythematosus, might be associated with structural abnormalities within the Ig heavy chain variable region (Igh-V) gene complex. The Igh-V loci from several lupus-prone mouse strains, their ancestors, and other nonautoimmune mice were therefore analyzed by restriction fragment length polymorphisms with DNA probes corresponding to seven VH gene families. These seven families comprise the majority of the known polymorphic murine VH gene repertoire, including some involved in autoantibody generation. Our study showed that the Igh-V loci from lupus and haplotype-matched nonlupus mice resulted in essentially identical restriction fragment patterns, a finding which suggests that the Igh-V gene complex does not carry a primary defect responsible for autoimmune disease.

Delineation of a defect in T cell receptor beta genes of NZW mice predisposed to autoimmunity.

In an attempt to determine whether genes involved in T cell antigen recognition are structurally abnormal and thereby promote murine systemic lupus, we analyzed the structural integrity of the D, J, and C region elements of the T cell receptor alpha and beta chain genes in all major lupus strains and several normal strains. Within the limits of restriction fragment length polymorphism analysis, all strains had an identical genomic organization, except the NZW mice, in which a deletion of the C beta 1-D beta 2-J beta 2 elements was found. Sequence analysis of NZW genomic elements containing this deletion placed its probable origin within the first exon of C beta 1, and extending to a complementary region within the first exon of C beta 2. The significance of this abnormality in the pathogenesis of systemic autoimmune disease remains to be determined.

Genetic elements used for a murine lupus anti-DNA autoantibody are closely related to those for antibodies to exogenous antigens.

The mRNAs encoding heavy and light chains of a hybridoma-derived monoclonal IgM kappa anti-DNA autoantibody from lupus-prone MRL/Mp-lpr/lpr mice (Ighj) have been transcribed into cDNA copies and molecularly cloned, and their complete nucleotide sequences have been determined. The mRNA for the heavy chain variable region, including leader peptide and 5' untranslated region, is transcribed from a heavy chain variable region (VH) gene closely related (and possibly allelic) to VH genes of the C57BL/6 (Ighb) nitrophenyl antibody family. The deduced amino acid sequence corresponding to the light chain variable region of this autoantibody shows extensive similarities with non-autoantibody molecules of the V kappa 1 group, suggesting a common variable gene origin. The joining segments, constant regions, and 3' untranslated regions of both the heavy and light chain mRNAs are nearly identical to corresponding sequences of non-autoantibodies from normal mice. Our findings suggest that this anti-DNA autoantibody originated from the same germline repertoire as antibodies to exogenous antigens.

Immunoglobulin kappa light chain variable region gene complex organization and immunoglobulin genes encoding anti-DNA autoantibodies in lupus mice.

We have investigated the genetic origin of autoantibody production in several strains of mice that spontaneously develop a systemic lupus erythematosus-like disease. Restriction fragment length polymorphism analyses of gene loci encoding kappa light chain variable regions (Igk-V) demonstrated, as shown previously for the Ig heavy chain locus, that autoantibody production and disease occur in different Igk-V haplotypes. Moreover, autoimmune mice with known genetic derivation inherited their Igk-V loci essentially unaltered from their nonautoimmune ancestors. New Zealand black lupus mice, with unknown genetic derivation, had a possibly recombinant Igk-V haplotype, composed of V kappa loci that were primarily indistinguishable from those of nonautoimmune strains from either of the two potential donor haplotypes. The heavy and light chain gene segments (variable, diversity, joining) encoding anti-DNA antibodies were diverse and often closely related, or even identical, to those found in antibodies to foreign antigens in normal mice. Only 1 of 11 sequenced variable region genes could not be assigned to existing variable region gene families; however, corresponding germline genes were present in the genome of normal mice as well. These data argue against abnormalities in the genes and mechanisms generating antibody diversity in lupus mice and suggest a remarkable genetic and structural diversity in the generation of anti-DNA binding sites.

Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA.

Heparan sulfate proteoglycans (HSPG) play a critical role in the formation of distinct fibroblast growth factor (FGF)-HS complexes, augmenting high-affinity binding and receptor activation. Perlecan, a secreted HSPG abundant in proliferating cells, is capable of inducing FGF-receptor interactions in vitro and angiogenesis in vivo. Stable and specific reduction of perlecan levels in mouse NIH 3T3 fibroblasts and human metastatic melanoma cells has been achieved by expression of antisense cDNA corresponding to the N-terminal and HS attachment domains of perlecan. Long-term perlecan downregulation is evidenced by reduced levels of perlecan mRNA and core protein as indicated by Northern blot analysis, immunoblots, and immunohistochemistry, using DNA probes and antibodies specific to mouse or human perlecan. The response of antisense perlecan-expressing cells to increasing concentrations of basic FGF (bFGF) is dramatically reduced in comparison to that in wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation. Furthermore, receptor binding and affinity labeling of antisense perlecan-transfected cells with 125I-bFGF is markedly inhibited, indicating that eliminating perlecan expression results in reduced high-affinity bFGF binding. Both the binding and mitogenic response of antisense-perlecan-expressing clones to bFGF can be rescued by exogenous heparin or perlecan. These results support the notion that perlecan is a major accessory receptor for bFGF in mouse fibroblasts and human melanomas and point to the possible use of perlecan antisense constructs as specific modulators of bFGF-mediated responses.

Structural characterization of a heterogeneous family of rat brain mRNAs.

A large heterogeneous family of RNAs derived from a single rat gene contains members that differ from each other at one or more of three positions. Their 5' ends are nested and transcription can begin at 22 or more sites covering 265 nucleotides. Many of the 5' ends are detectable only in brain RNAs, and even 5' ends common with other tissues appear with different absolute and relative abundances in brain RNA. The central portions of the RNAs are of two forms, differing only by the presence or absence of 17 nucleotides; these forms are probably produced by alternative splicing. Polyadenylation occurs at either of two sites. This complicated family of 88 RNAs encodes two novel putative proteins that differ at their C termini.

Growth factors and chemokines: a comparative functional approach between invertebrates and vertebrates.

Growth factors and cytokines control and coordinate a broad spectrum of fundamental cellular functions, and are evolutionarily conserved both in vertebrates and invertebrates. In this review, we focus our attention on the functional phylogenetic aspects of growth factors/cytokines like the Transforming Growth Factor-beta (TGF-beta), the Connective Tissue Growth Factor (CTGF), and the Vascular Endothelial Growth Factor (VEGF). We will also delve into the activites of two chemokine families, interleukin (IL)-8 (or CXCL8) and CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2). These molecules have been selected for their involvement in immune responses and wound healing processes, where they mediate and finely regulate various regeneration processes like angiogenesis or fibroplasia, not only in vertebrates, but also in invertebrates.

Cytokines and chemokines as regulators of angiogenesis in health and disease.

The intricate interplay between the endothelium and immune cells has been well recognized in the context of immune responses. However, the fact that this inter-relation extends well beyond immune regulation is becoming increasingly recognized, with particular regards to the influence of the immune system on the essential endothelial process of angiogenesis, where the contribution of cytokines drives the angiogenic process. As angiogenesis is an important component of numerous pathological states, among these chronic inflammatory conditions and cancer, understanding the role of cytokines and chemokines in guiding new vessel formation provides key insight into novel therapeutic modalities. Here we review the actions of principal cytokines and chemokines on the angiogenic process and discuss how both can be considered potential pharmaceutical targets or pharmaceuticals themselves for modulation of angiogenesis in chronic inflammation associated with cancer, rheumatoid arthritis and other inflammatory diseases.

Inhibition of angiogenesis-driven Kaposi's sarcoma tumor growth in nude mice by oral N-acetylcysteine.

The thiol N-acetyl-L-cysteine (NAC), an analogue and precursor of reduced glutathione, has cancer chemopreventive properties attributable to its nucleophilicity, antioxidant activity, and a variety of other mechanisms. We demonstrated recently that NAC has anti-invasive, antimetastatic, and antiangiogenic effects in in vitro and in vivo test systems. In the present study, s.c. transplantation of KS-Imm cells in (CD-1)BR nude mice resulted in the local growth of Kaposi's sarcoma, a highly vascularized human tumor. The daily administration of NAC with drinking water, initiated after the tumor mass had become established and detectable, produced a sharp inhibition of tumor growth, with regression of tumors in half of the treated mice along with a markedly prolonged median survival time. The production of vascular endothelial growth factor (VEGF) and certain proliferation markers (proliferating cell nuclear antigen and Ki-67) were significantly lower in Kaposi's sarcomas from NAC-treated mice than from control mice. Treatment of KS-Imm cells with NAC in vitro resulted in a dose-dependent inhibition of chemotaxis and invasion through inhibition of gelatinase-A (matrix metalloproteinase-2, MMP-2) activity without altering MMP-2 or MMP-9 mRNA levels. NAC also significantly inhibited VEGF production but did not affect proliferation markers in vitro. Reverse transcription-PCR analysis indicated that total VEGF mRNAs were reduced by 10 mM NAC. Taken together, these findings provide evidence that NAC, the safety of which even at high doses has been established in almost 40 years of clinical use, in addition to its chemopreventive action, has a strong antiangiogenic potential that could be exploited for preventing cancer progression as well as used in cancer adjuvant therapy.

Effects of angiostatin gene transfer on functional properties and in vivo growth of Kaposi's sarcoma cells.

Gene transfer delivery of endogenous angiogenesis inhibitors such as angiostatin would circumvent problems associated with long-term administration of proteins. Kaposi's sarcoma (KS), a highly vascular neoplasm, is an excellent model for studying tumor angiogenesis and antiangiogenic agent efficacy. We investigated the effects of angiostatin gene transfer in in vitro and in vivo models of KS-induced neovascularization and tumor growth. A eukaryotic expression plasmid and a Moloney leukemia virus-based retroviral vector for expression of murine angiostatin were generated harboring the angiostatin cDNA with cleavable leader signals under the control of either the strong cytomegalovirus promoter/enhancer or the Moloney leukemia virus long terminal repeat. Angiostatin secretion was confirmed by radioimmunoprecipitation and Western blot analysis. Supernatants of angiostatin-transfected cells inhibited endothelial cell migration in vitro. Stable gene transfer of the angiostatin cDNA by retroviral vectors in KS-IMM cells resulted in sustained angiostatin expression and delayed tumor growth in nude mice, which was associated with reduced vascularization. These findings suggest that gene therapy with angiostatin might be useful for treatment of KS and possibly other highly angiogenic tumors.

Galectin-1 suppression delineates a new strategy to inhibit myeloma-induced angiogenesis and tumoral growth in vivo.

Galectin-1 (Gal-1) is involved in tumoral angiogenesis, hypoxia and metastases. Actually the Gal-1 expression profile in multiple myeloma (MM) patients and its pathophysiological role in MM-induced angiogenesis and tumoral growth are unknown. In this study, we found that Gal-1 expression by MM cells was upregulated in hypoxic conditions and that stable knockdown of hypoxia inducible factor-1α significantly downregulated its expression. Therefore, we performed Gal-1 inhibition using lentivirus transfection of shRNA anti-Gal-1 in human myeloma cell lines (HMCLs), and showed that its suppression modified transcriptional profiles in both hypoxic and normoxic conditions. Interestingly, Gal-1 inhibition in MM cells downregulated proangiogenic genes, including MMP9 and CCL2, and upregulated the antiangiogenic ones SEMA3A and CXCL10. Consistently, Gal-1 suppression in MM cells significantly decreased their proangiogenic properties in vitro. This was confirmed in vivo, in two different mouse models injected with HMCLs transfected with anti-Gal-1 shRNA or the control vector. Gal-1 suppression in both models significantly reduced tumor burden and microvascular density as compared with the control mice. Moreover, Gal-1 suppression induced smaller lytic lesions on X-ray in the intratibial model. Overall, our data indicate that Gal-1 is a new potential therapeutic target in MM blocking angiogenesis.

HIV-tat protein is a heparin-binding angiogenic growth factor.

Transgenic animal studies have linked the expression of the HIV-1 tat gene to the appearance of Kaposi's sarcoma (KS)-like lesions. We have recently shown that recombinant tat is angiogenic in vivo, and that tat angiogenic response is enhanced by heparin. Also in the rabbit cornea model, recombinant HIV-1 tat alone is poorly angiogenic, but gives a good response when combined with heparin. Like many angiogenic growth factors, tat has a basic domain similar to that of several heparin binding angiogenic factors, including FGF, VEGF and HGF, suggesting that this region is important in endothelial cell activation. We show that tat binds heparin sepharose with a high affinity, similar to bFGF. Binding of tat to the cell surface is also modulated by heparin. Biological activities of tat, such as induction of endothelial cell growth, migration and invasion in vitro are all enhanced by low concentrations and inhibited by high concentrations of heparin, as has been shown for other heparin-binding angiogenic factors. Heparan sulfate is also effective, whereas the unsulfated polysaccharide K5 does not enhance tat activity. Furthermore, a peptide encompassing the tat basic domain is able to induce growth and migration of endothelial cells, while an adjacent peptide is not. Our data indicate that the tat basic domain plays a key role in its vascular cell activation properties, and strongly suggest that extracellular HIV-tat is essentially a 'new' heparin-binding angiogenic factor.

RB1 oncosuppressor gene over-expression inhibits tumor progression and induces melanogenesis in metastatic melanoma cells.

The retinoblastoma gene (RB1) is frequently deleted or mutated in many tumor types and in all cases of retinoblastoma. Apart from its role in regulation of the cell cycle, the RB1 gene product (p110RB1) appears to be involved in control of differentiation. Malignant metastatic cells show many properties of poorly differentiated cells, and are highly invasive in vitro and in vivo. We have transfected the human RB1 cDNA in an expression vector under the control of the beta-actin promoter into B16F10 murine melanoma cells. These cells highly overexpress RB1 mRNA and the p110RB1 product, show reduced growth rate and increased melanogenesis in vitro. Vector control transfectants showed no alteration of invasiveness. The p110RB1 over-expressing cells also had a reduced capacity to migrate and invade through an artificial basement membrane, key characteristics of metastatic cells. When injected into nude mice, the p110RB1 over-expressing cells showed reduced tumor growth and reduced metastatic potential. The few metastasis observed were predominantly melanotic. These data indicate that RB1 gene expression is involved in melanoma cell differentiation and plays a role in downregulation of migration, invasion and metastatic potential of these cells.

Altered steady-state mRNA levels of basement membrane proteins in diabetic mouse kidneys and thromboxane synthase inhibition.

We examined steady-state levels of mRNA encoding type IV collagen, B1 chain of laminin, and the basement membrane heparan sulfate proteoglycan in the kidney cortex of a mouse model (KKAy) of non-insulin-dependent diabetes. mRNAs encoding laminin B1 and the proteoglycan were unchanged in kidneys taken from diabetic mice with demonstrable basement membrane thickening. mRNA levels for type IV collagen, in contrast, were significantly elevated (2-fold) in diabetic mice concurrent with but not preceding morphologically thickened basement membranes. There was a negative correlation between a ratio of proteoglycan/type IV collagen and levels of albuminuria in the diabetic mice. No correlation was noted with laminin. We also examined the effects of inhibiting the synthesis of thromboxane, a potent vasoconstrictor, on the steady-state levels of type IV collagen in the diabetic mice. Inhibition of thromboxane stopped the progression of albuminuria and prevented an increase in type IV collagen mRNA levels. We conclude that basement membrane thickening in diabetes, a hallmark of diabetic nephropathy, is partly a consequence of an unbalanced increase in the production of type IV collagen. The relative decrease in proteoglycan production may contribute to chronic albuminuria.