The impact of aryl hydrocarbon receptor signaling on matrix metabolism: implications for development and disease

The aryl hydrocarbon receptor (AhR) was identified as the receptor for polycyclic aromatic hydrocarbons and related compounds. However, novel data indicate that the AhR binds a variety of unrelated endogenous and exogenous compounds. Although AhR knockout mice demonstrate that this receptor has a role in normal development and physiology, the function of this receptor is still unclear. Recent evidence suggests that AhR signaling also alters the expression of genes involved in matrix metabolism, specifically the matrix metalloproteinases (MMPs). MMP expression and activity is critical to normal physiological processes that require tissue remodeling, as well as in mediating the progression of a variety of diseases. MMPs not only degrade structural proteins, but are also important mediators of cell signaling near or at the cell membrane through exposure of cryptic sites, release of growth factors, and cleavage of receptors. Therefore, AhR modulation of MMP expression and activity may be critical, not only in pathogenesis, but also in understanding the endogenous function of the AhR. In this review we will examine the data indicating a role for the AhR-signaling pathway in the regulation of matrix remodeling, and discuss potential molecular mechanisms.     

Epithelial CXCR3-B regulates chemokines bioavailability in normal, but not in Sjogren's syndrome, salivary glands

Expression of CXCR3-targeting chemokines have been demonstrated in several diseases, suggesting a critical role for CXCR3 in recruiting activated T cells to sites of immune-mediated inflammation. Sj?gren's syndrome (SS) is an autoimmune disease characterized by a mononuclear cell infiltrate of activated T cells around the duct in the salivary gland. Analysis of minor salivary gland biopsy specimens from 20 healthy subjects and 18 patients with primary SS demonstrated that CXCR3, in particular, the B form of this receptor, is constitutively expressed by human salivary gland epithelial cells. Salivary gland epithelial cell cultures demonstrated that CXCR3 participate in removing relevant amount of agonists from the supernatant of exposed cells without mediating calcium flux or chemotaxis while retaining the ability to undergo internalization. Although in normal salivary gland epithelial cells, CXCR3 behaves as a chemokine-scavenging receptor, its role in SS cells is functionally impaired. The impairment of this scavenging function might favor chemotaxis, leading to heightened immigration of CXCR3-positive T lymphocytes. These findings suggest that epithelial CXCR3 may be involved in postsecretion regulation of chemokine bioavailability. They also support a critical role for CXCR3 in the pathogenesis of SS and identify its agonists as potential therapeutic targets.     

Molecular cloning and sequencing of metallothionein in squamates: New insights into the evolution of the metallothionein genes in vertebrates

Metallothioneins are cysteine-rich, metal-binding proteins ubiquitously expressed in living organisms. In the last past years, a plethora of vertebrate metallothionein sequences have become available, but so far there has been an almost absolute lack of data about sequences of metallothionein of non-avian diapsida. In the framework of the investigations on structural and functional properties of non-mammalian metallothioneins, we have cloned and sequenced the cDNAs encoding for metallothioneins of 10 squamate reptiles, belonging to 5 different infraorders. These sequences have been used to gain insight into the evolutionary history of metallothioneins in reptiles. Phylogenetic analysis shows that reptilian metallothionein phylogeny is inconsistent with the species phylogeny. Such findings allow us to hypothesize that the identified metallothionein in each squamate species used for this study might be considered a paralogous gene derived from more events of gene duplication and losses occurred during the diversification of the squamate species. Finally, through vertebrate metallothionein comparisons and phylogenetic analysis, we also add a novel contribution to the understanding of the evolution of metallothionein genes along the major vertebrate lineages.     

Liquid Crystalline Systems for Transdermal Delivery of Celecoxib: In Vitro Drug Release and Skin Permeation Studies

Liquid crystalline systems of monoolein/water could be a promising approach for the delivery of celecoxib (CXB) to the skin because these systems can sustain drug release, improve drug penetration into the skin layers and minimize side effects. This study evaluated the potential of these systems for the delivery of CXB into the skin based on in vitro drug release and skin permeation studies. The amount of CXB that permeated into and/or was retained in the skin was assayed using an HPLC method. Polarizing light microscopy studies showed that liquid crystalline systems of monoolein/water were formed in the presence of CXB, without any changes in the mesophases. The liquid crystalline systems decreased drug release when compared to control solution. Drug release was independent of the initial water content of the systems and CXB was released from cubic phase systems, irrespective of the initial water content. The systems released the CXB following zero-order release kinetics. In vitro drug permeation studies showed that cubic phase systems allowed drug permeation and retention in the skin layers. Cubic phase systems of monoolein/water may be promising vehicles for the delivery of CXB in/through the skin because it improved CXB skin permeation compared with the control solution.KEY WORDS: celecoxib, drug delivery systems, liquid crystalline system, monoolein, skin permeation     

Bacterial Infections in Myd88-Deficient Mice

Three breeding colonies of Myd88^−/− mice had a history of significant morbidity and mortality. Although strain-specific poor reproductive performance might explain neonatal death and dystocia, mice were found dead or required euthanasia because of moribundity, distended abdomen, head tilt, and seizures. Histopathology results included bacteremia, placentitis, metritis, peritonitis with abscess formation, and suppurative meningoencephalitis. Intralesional gram-negative coccobacilli were present, often in extremely high number. Cultures of samples of the cardiac blood of a mouse and from water-bottle sipper tubes provided to some affected mice grew Pseudomonas aeruginosa. In addition, affected tissues from 2 mice and feces from a third tested PCR-positive for P. aeruginosa. Although the mice had received autoclaved reverse-osmosis–purified drinking water, we suspect that the mice were inoculated with P. aeruginosa through contaminated sipper tubes. Because of the deficiency in most of the Toll-like receptor signaling pathways, these Myd88^−/− mice were unlikely to have developed competitive innate and adaptive immune responses, resulting in bacterial infections. These clinical cases underscore the importance of understanding how genotype, phenotype and environment affect animal health. Sound husbandry and experimental practices are needed to prevent the exposure of immunodeficient mice to pathogens.Abbreviations: TLR, Toll-like receptorRodent colonies typically are managed on a ‘herd-health’ basis, with prevention, treatment, and control measures at the colony level rather than the individual level. Although sporadic cases of sick and dead mice are not unusual in large breeding colonies, unexpectedly high morbidity and mortality rates typically warrant clinical investigation and analysis of the disease triangle: the conceptual model of the interactions among the host, environment, and pathogen. Of these 3 factors, the host usually is well-characterized pertaining to its genotype, phenotype, and experimental manipulations. Similarly, the environmental conditions in a laboratory animal setting typically are controlled. However, infections with adventitious pathogens can occur and cause disease outbreak in susceptible animals. For example, poorly sanitized cages can act as fomites for pathogens that may cause clinical infections in susceptible mice.Immunodeficient mice are prone to exhibit clinical signs when infected with a pathogen. One such strain is Myd88^−/−, which lack the myeloid differentiation factor 88 (MyD88) protein, a cytoplasmic adaptor molecule essential for the signaling of IL1 and Toll-like receptor (TLR) family.^14,27 MyD88 plays a central role in innate and adaptive immune response, because it is essential for cellular responses to IL1, IL18, and many bacterial cell wall components including lipopolysaccharide, peptidoglycan, and lipopeptide.^1,10,27 Because of their phenotype, Myd88^−/− mice are commonly used for infectious disease and immunology studies.Here, we present a case series of Myd88^−/− mice that had a history of morbidity and mortality attributed to bacterial infections caused by gram-negative coccobacilli; the organism was identified to be Pseudomonas aeruginosa in one case. We underscore the importance of understanding host responses to pathogens and the provision of sound husbandry procedures to prevent infections in immunodeficient mice, such as the Myd88^−/− strain.     

A Unique Four-Hub Protein Cluster Associates to Glioblastoma Progression

Gliomas are the most frequent brain tumors. Among them, glioblastomas are malignant and largely resistant to available treatments. Histopathology is the gold standard for classification and grading of brain tumors. However, brain tumor heterogeneity is remarkable and histopathology procedures for glioma classification remain unsatisfactory for predicting disease course as well as response to treatment. Proteins that tightly associate with cancer differentiation and progression, can bear important prognostic information. Here, we describe the identification of protein clusters differentially expressed in high-grade versus low-grade gliomas. Tissue samples from 25 high-grade tumors, 10 low-grade tumors and 5 normal brain cortices were analyzed by 2D-PAGE and proteomic profiling by mass spectrometry. This led to identify 48 differentially expressed protein markers between tumors and normal samples. Protein clustering by multivariate analyses (PCA and PLS-DA) provided discrimination between pathological samples to an unprecedented extent, and revealed a unique network of deranged proteins. We discovered a novel glioblastoma control module centered on four major network hubs: Huntingtin, HNF4α, c-Myc and 14-3-3ζ. Immunohistochemistry, western blotting and unbiased proteome-wide meta-analysis revealed altered expression of this glioblastoma control module in human glioma samples as compared with normal controls. Moreover, the four-hub network was found to cross-talk with both p53 and EGFR pathways. In summary, the findings of this study indicate the existence of a unifying signaling module controlling glioblastoma pathogenesis and malignant progression, and suggest novel targets for development of diagnostic and therapeutic procedures.     

Palaeontologic and biogeochemical characterization of the Cyrtograptus lundgreni event in the black shales of eastern Mid-Sardinia, Italy

A succession of biotic and geochemical changes that occurred during the Cyrtograptus lundgreni Event (Late Wenlock) have been recorded from the 'pelagic' black-shales in the Goni section, eastern mid-Sardinia, Italy. The studied interval encompasses the Cyrtograptus rigidus to Pristiograptus dubius-Gothograptus nassa zones. The fossil association includes graptolites, chitinozoans and microplankton i.e. probable linings of agglutinated foraminifera and radiolaria capsular membranes. Analysis of the chitinozoan distribution revealed a succession of several chitinozoan associations with low species diversity and dominated by opportunistic species. Three chitinozoan faunal turnovers and three extinction events have been recorded. Two of them coincide with graptolite extinctions whereas one probably is of local significance. Disappearance of the chitinozoan and microplankton associations occurred during four consecutive graptolite zones. Geochemical data (trace elements analysis) showed significantly higher (up to c. 100%) values for Co and Cd in the sedimentary organic matter (SOM) than in the whole rock samples. Possible relationships between peaks of metal enrichment, the major faunal changes among chitinozoans, extinction events among chitinozoans and graptolites and, to a certain extent, oceanic events may be inferred. The first extinction datum is older that those occurring in Gotland, Sweden and Thüringen, Germany and is so far considered to be of local significance. The second extinction datum of Sardinia can be matched with Datum 1 of Gotland and Thüringen. A close correlation between the third extinction datum of Sardinia and Datum 2 of Thüringen and Gotland reinforces the importance of these events at global scale.     

Molecular basis underlying the biological effects elicited by extremely low-frequency magnetic field (ELF-MF) on neuroblastoma cells

Extremely low-frequency magnetic fields (ELF-MFs) may affect human health because of the possible associations with leukemia but also with cancer, cardiovascular, and neurological disorders. In the present work, human SH-SY5Y neuroblastoma cells were exposed to a 50 Hz, 1 mT sinusoidal ELF-MF at three different times, that is, 5 days (T5), 10 days (T10), and 15 days (T15) and then the effects of ELF-MF on proteome expression and biological behavior were investigated. Through comparative analysis between treated and control samples, we analyzed the proteome changes induced by ELF-MF exposure. Nine new proteins resolved in sample after a 15-day treatment were involved in a cellular defense mechanism and/or in cellular organization and proliferation such as peroxiredoxin isoenzymes (2, 3, and 6), 3-mercaptopyruvate sulfurtransferase, actin cytoplasmatic 2, t-complex protein subunit beta, ropporin-1A, and profilin-2 and spindlin-1. Our results indicated that ELF-MFs exposure altered the proliferative status and other important cell biology-related parameters, such as cell growth pattern, and cytoskeletal organization. These findings support our hypothesis that ELF radiation could trigger a shift toward a more invasive phenotype.     

IgM-linked SerpinB3 and SerpinB4 in sera of patients with chronic liver disease

Background

Epidemiological studies indicate that a growing number of cirrhotic patients will develop hepatocellular carcinoma (HCC) in the next decade. Recent findings have demonstrated that Squamous cell carcinoma antigen 1 (SCCA1) and 2 (SCCA2) isoforms, now classified as serpinB3 and serpinB4, are over-expressed in HCC, but not in normal liver. As reported, high levels of circulating SCCA-IgM immunocomplexes in patients with cirrhosis are significantly associated with HCC development.

Aim

To ascertain whether IgM-linked SCCA isoforms circulate in patients with chronic liver disease, compared to total SCCA-IgM levels.

Methodology and Findings

79 patients with chronic liver disease were studied, including 17 patients with chronic hepatitis, 36 patients with cirrhosis and 26 with HCC. 28 blood donors were used as control. Monoclonal antibodies against serpinB3 and serpinB4 were used as catcher antibodies to set up specific ELISA assays, while total SCCA-IgM immunocomplexes were detected by commercially available ELISA assay. Overall, the results revealed a better diagnostic sensitivity of total SCCA-IgM assay, compared to both serpinB3 and serpinB4 IgM-linked assays. SerpinB4-IgM median values obtained with SCC103 antibody were moderately higher in patients with cirrhosis than in those with HCC, median values: 0.168 (IQR 0.140–0.427) vs. 0.140 (IQR 0.140–0.278), (p = 0.177). A trend toward decreasing serpinB4-IgM/serpinB3-IgM median ratio was observed in patients with advanced liver disease, being 1.08 in patients with HCC, 1.10 in patients with cirrhosis and 1.40 in patients with chronic hepatitis (p = 0.079).

Conclusions

IgM-linked SCCA isoforms in serum of patients with chronic liver diseases were quantified for the first time. Although the number of patients was limited, this preliminary study reveals that the relative balance of the two serpin isoforms is altered in HCC and it is characterized by a lower serpinB4-IgM/serpinB3-IgM ratio, determined by lower serpinB4 levels.     

Gold nanoparticles capped by a GC-containing peptide functionalized with an RGD motif for integrin targeting

Gold nanoparticles were obtained by reduction of a tetrachloroaurate aqueous solution in the presence of a RGD-(GC)(2) peptide as stabilizer. As comparison, the behavior of the (GC)(2) peptide has been studied. The (GC)(2) and RGD-(GC)(2) peptides were prepared ad hoc by Fmoc synthesis. The colloidal systems have been characterized by UV-visible, TGA, ATR-FTIR, mono and bidimensional NMR techniques, confocal and transmission (TEM) microscopy, ζ-potential, and light scattering measurements. The efficient cellular uptake of Au-RGD-(GC)(2) and Au-(GC)(2) stabilized gold nanoparticles into U87 cells (human glioblastoma cells) were investigated by confocal microscopy and compared with the behavior of (GC)(2) capped gold nanoparticles. A quantitative determination of the nanoparticles taken up has been carried out by measuring the pixel brightness of the images, a measure that highlighted the importance of the RGD termination of the peptide. Insight in the cellular uptake mechanism was investigated by TEM microscopy. Various important evidences indicated the selective uptake of RGD-(GC)(2) gold nanoparticles into the nucleus.