Preclinical assessment of dual CYP26[A1/B1] inhibitor, DX308, as an improved treatment for keratinization disorders.

Background: Retinoid-based therapies are commonly used in the treatment of disorders of keratinization and other skin disorders but can result in non-specific effects and adverse reactions. Use of retinoic acid metabolism blocking agents (RAMBAs) such as DX308 may address these shortcomings. Objectives: Characterize the therapeutic potential of recently discovered, CYP26-selective RAMBA, DX308. Materials and Methods: Preliminary in vitro assessment of potential off-target activity, metabolic and toxicologic profiling. Studies to assess safety and efficacy of topical treatment in correcting abnormal skin morphology in rhino mice. Extensive gene expression profiling by RNA sequencing and qPCR in 3D epidermis grown with keratinocytes (KCs) from keratinization disorders and healthy controls, to investigate modulation of retinoid biopathways. Results: In vitro, DX308 does not interact with off-target nuclear receptors or CYP450s, is not genotoxic, and is stable in skin, despite vigorous hepatic metabolism. In vivo, topical DX308 induces comedolysis and epidermal thickening without apparent adverse effects. Gene expression profiling shows potent modulation of retinoid-responsive genes by DX308 in both healthy and keratinization disorder KCs. Pathway analysis suggests DX308 may inhibit inflammatory and immune responses in KCs. Conclusions: These preliminary studies suggest that DX308 is an efficacious topical therapeutic with a favourable metabolic and safety profiles. DX308 may present an improved therapeutic alternative for the treatment of keratinization disorders and other retinoid-responsive skin ailments.

Differential gene expression analysis of ovarian cancer in a population isolate.

Gene expression products represent candidate biomarkers with the potential for early screening and therapy of patients with ovarian serous carcinoma. The present study, using patients that originate from the population isolate of South Tyrol, Italy, substantiates the feasibility of differential gene expression analysis in a genetically isolated population for the identification of potential markers of ovarian cancer. Gene expression profiles of fresh-frozen ovarian serous papillary carcinoma samples were analyzed and compared to normal ovarian control tissues using oligonucleotide microarrays complementary to 14,500 human genes. Supervised analysis of gene expression profiling data identified 225 genes that are down-regulated and 635 that are up-regulated in malignant compared to normal ovarian tissues. Class-prediction analysis identified 40 differentially expressed genes for further investigation as potential classifiers for ovarian cancer, including 20 novel candidates. Our findings provide a glimpse into the potential of population isolate genomics in oncological research.

Impaired calcineurin signaling in myeloid cells results in downregulation of pentraxin-3 and increased susceptibility to aspergillosis.

Treatment of post-transplant patients with immunosuppressive drugs targeting the calcineurin-nuclear factor of activated T cells (NFAT) pathway, including cyclosporine A or tacrolimus, is commonly associated with a higher incidence of opportunistic infections, such as Aspergillus fumigatus, which can lead to severe life-threatening conditions. A component of the A. fumigatus cell wall, ß-glucan, is recognized by dendritic cells (DCs) via the Dectin-1 receptor, triggering downstream signaling that leads to calcineurin-NFAT binding, NFAT translocation, and transcription of NFAT-regulated genes. Here, we address the question of whether calcineurin signaling in CD11c-expressing cells, such as DCs, has a specific role in the innate control of A. fumigatus. Impairment of calcineurin in CD11c-expressing cells (CD11ccrecnb1loxP) significantly increased susceptibility to systemic A. fumigatus infection and to intranasal infection in irradiated mice undergoing bone marrow transplant. Global expression profiling of bone marrow-derived DCs identified calcineurin-regulated processes in the immune response to infection, including expression of pentraxin-3, an important antifungal defense protein. These results suggest that calcineurin inhibition directly impairs important immunoprotective functions of myeloid cells, as shown by the higher susceptibility of CD11ccrecnbloxP mice in models of systemic and invasive pulmonary aspergillosis, including after allogeneic bone marrow transplantation. These findings are relevant to the clinical management of transplant patients with severe Aspergillus infections.

Intestinal CD103(+)CD11b(-) dendritic cells restrain colitis via IFN-γ-induced anti-inflammatory response in epithelial cells.

A crosstalk between commensals, gut immune cells, and colonic epithelia is required for a proper function of intestinal mucosal barrier. Here we investigated the importance of two distinct intestinal dendritic cell (DC) subsets in controlling intestinal inflammation. We show that Clec9A-diphtheria toxin receptor (DTR) mice after depletion of CD103(+)CD11b(-) DCs developed severe, low-dose dextran sodium sulfate (DSS)-induced colitis, whereas the lack of CD103(+)CD11b(+) DCs in Clec4a4-DTR mice did not exacerbate intestinal inflammation. The CD103(+)CD11b(-) DC subset has gained a functional specialization that able them to repress inflammation via several epithelial interferon-γ (IFN-γ)-induced proteins. Among others, we identified that epithelial IDO1 and interleukin-18-binding protein (IL-18bp) were strongly modulated by CD103(+)CD11b(-) DCs. Through its preferential property to express IL-12 and IL-15, this particular DC subset can induce lymphocytes in colonic lamina propria and in epithelia to secrete IFN-γ that then can trigger a reversible early anti-inflammatory response in intestinal epithelial cells.

Studies of the murine DDB1 and DDB2 genes.

Human DDB (Damaged DNA Binding protein) is a heterodimer of 48 and 127kDa subunits whose activity is absent from cell strains derived from a subset of Xeroderma Pigmentosum (XP) complementation group E individuals (Ddb(-)) [Keeney, S., Wein, H., and Linn, S., (1992). Mut. Res. 273, 49-56]. Whereas in vivo DNA repair appears to be compromised in both Ddb(-) and Ddb(+) XPE cells, DDB activity is not necessary for nucleotide excision repair (NER) in vitro. In this study, the presence of a specific UV-damaged DNA binding activity in mouse cell-free extracts that is comparable to the activity observed in HeLa cells was demonstrated. The mouse DDB2 cDNA, coding for DDB p48 subunit, was cloned and the partial genomic structure of DDB2 was obtained. A search of current databases revealed amino acid sequences of mouse and Drosophila predicted p127 homologues, but not of a Drosophila p48 homologue. The alignment of these higher eukaryotic p127 sequences uncovered the presence of three highly conserved domains in the p127 polypeptides which we hypothesize could function in DNA binding, transcription-transactivation, and protein-protein interaction, respectively.

Mutation producing alternative splicing of exon 26 in the COL1A2 gene causes type IV osteogenesis imperfecta with intrafamilial clinical variability.

We have characterized a familial form of osteogenesis imperfecta (OI). Following the identification by ultrasound of short limbs and multiple fractures in a fetus at 25 weeks of gestation, the family was referred with a provisional diagnosis of severe OI. We detected subtle clinical and radiological signs of OI in the father and in the paternal grandmother of the proposita, who had never received a diagnosis of OI. Linkage analysis indicated COL1A2 as the disease locus. Heteroduplex analysis of reverse transcription-polymerase chain reaction (RT-PCR) amplification products of pro alpha2(I) mRNA from an affected member and subsequent sequencing of the candidate region demonstrated the presence of normal transcripts and a minority of transcripts lacking exon 26 (54 bp) of COL1A2. Sequencing of PCR-amplified genomic DNA identified an A --> G transition in the moderately conserved +3 position of the IVS 26 donor splice site. The mutant pre-mRNA molecules were alternatively spliced, yielding both full-length and deleted transcripts that represented less than 30% of the total pro alpha2(I) mRNA. The biochemical data on type I collagen synthesized by dermal fibroblasts showed intracellular retention of the mutant protein; failure to detect the shortened alpha2(I) chains either in the medium or in the cell layer may be the consequence of their instability at physiological temperature. These observations justified the mild resulting phenotype.

[The association between liver cirrhosis and bacterial endocarditis. Description of a typical case]. / L'associazione tra cirrosi epatica ed endocardite batterica. Descrizione di un caso tipico.

A typical case of an association of cirrhosis of the liver and bacterial endocarditis involving only the aortic valve is described. The essential role of echocardiography in diagnosis and the ability of the technique to supply useful information for correct therapy is emphasised. Finally, evidence that, in spite of all negative blood cultures, Escherichia Coli via the urinary ways may be considered the aetiological agent is presented.

Type I interferon and Toll-like receptor expression characterizes inflammatory myopathies.

OBJECTIVES: Juvenile dermatomyositis (JDM), adult dermatomyositis, and polymyositis (PM) are idiopathic inflammatory myopathies (IIMs) characterized by muscle infiltration and specific muscle fiber alterations. They are thought to have an autoimmune etiology, but triggering factors, and how immunologic attack induces muscle weakness, remain unknown. Recent evidence suggests a key role for type I interferon (IFN)-mediated innate immunity in dermatomyositis, which we explored in JDM, dermatomyositis, and PM by gene expression profiling, and other methods. METHODS: Ten IIM and 5 control muscle biopsies were assessed for expression of approximately 16,000 genes by microarray; 37 additional IIM, 10 dystrophinopathic, and 14 nonmyopathic control muscles were studied for type I IFN-dependent genes, and Toll-like receptor (TLR) expression by immunochemistry and PCR. RESULTS: Type I IFN-dependent transcripts were significantly upregulated in IIM muscles compared to controls; in JDM the most expressed were ISG15 (408-fold), IFIT3 (261-fold), MX1 (99-fold), and IRF7 (37-fold). IFN-ß (but not IFN-α) transcripts were upregulated in PM as well as dermatomyositis/JDM. TLR3 was upregulated particularly in JDM, being localized on vascular endothelial cells, muscle infiltrating cells (mainly myeloid dendritic cells), and regenerating myofibers; TLR7 and TLR9 proteins were present in IIM (prominently in PM), mainly on cell infiltrates, particularly plasma cells, and on some injured myofibers. CONCLUSIONS: IFN-ß and type I IFN-induced molecules are involved in PM as well as JDM/dermatomyositis. Endosomal TLRs (effectors of innate immunity) are also involved (but differently) in the 3 conditions, further suggesting viral involvement, although TLR activation could be secondary to tissue damage.

Chronic myocarditis leading to a right ventricular cardiomyopathy. Case report.

A case of dilated cardiomyopathy arose after a spontaneous abortion at the second month of pregnancy is reported. Whereas clinical, hemodynamic and gross features pointed to a diagnosis of right ventricular cardiomyopathy, histologic findings of inflammatory infiltration, myocyte degeneration and interstitial fibrosis of the atrial and ventricular walls allowed to a correct diagnosis of chronic myocarditis.

Intrafamilial variable expressivity of osteogenesis imperfecta due to mosaicism for a lethal G382R substitution in the COL1A1 gene.

Fibroblasts from a 23 week old fetus affected with lethal (type II) osteogenesis imperfecta (OI) produced normal and abnormal type I procollagen molecules. The abnormal molecules were shown to contain pro alpha 1(I) chains in which the glycine at position 382 of the triple helical domain was substituted by arginine, as the result of a G-to-C transversion at nucleotide 1797 of the pro alpha (I) coding sequence. Also fibroblasts from the apparently normal father produced abnormal type I collagen but the overmodified alpha 1(I) chains tended to disappear with increasing passage number. We determined that the mutant allele accounted for approximately 36% of the COL1A1 alleles in the father's skin fibroblasts. Upon careful clinical reexamination, the man appeared to be very mildly affected with OI. The most plausible explanation for such a phenotypic variation is that the father is a mosaic for a mutation that is lethal in the heterozygous son. This finding confirms previous observations that somatic mosaicism for new dominant mutations is responsible for extreme intrafamilial variability and poses some caveats in genetic counselling.

[Complete sub-His AV block caused by carotid sinus massage. Possible direct vagal effect on the His-Purkinje system]. / Blocco AV totale sottohissiano indotto dal massaggio del seno carotideo. Possibile effetto vagale diretto sul sistema His-Purkinje.

A 64 year old male patient, with frequent syncopes, underwent an electro-physiologic study. A complete left bundle branch block and a first degree His-Purkinje system atrioventricular block (AH = 100 msec, HV = 60 msec) were present in basal condition. Left carotid sinus massage caused extreme sinus bradycardia (with PP intervals as long as 3000 msec), without AH and HV interval changes. Right carotid sinus massage caused a His-Purkinje atrioventricular block. Ventricular asystole of 4800 msec occurred while the sinus cycle varied between 1440 and 590 msec. Since His-Purkinje atrioventricular block is induced by the right carotid sinus massage with PP intervals even shorter than the basal cycles and since the block was not reproduced at PP intervals longer than 1440 msecs, a direct vagal effect on the His-Purkinje system may be suggested, rather than a bradycardia dependent phase 4 block.

Four new cases of lethal osteogenesis imperfecta due to glycine substitutions in COL1A1 and genes. Mutations in brief no. 152. Online.

Perinatal lethal osteogenesis imperfecta is the result of heterozygous mutations of the COL1A1 and COL1A2 genes. Here we describe the molecular defects responsible for four case of lethal OI. Two glycine substitutions within the COL1A1 gene (G478S, G994D) and two glycine substitutions within the COLIA2 gene (G319V, G697C) were identified. The mutation sites were localized in proalpha2(I) and proalpha2(I)mRNA molecules, respectively, by chemical cleavage of mismatch in hereteroduplex nucleic acids. Subsequent reverse transcription PCR amplification, cloning and sequencing, led to mutation identification. The aminoacid substitutions were due to two G-->A transitions in COL1A1(cases 1,2), to a G-->T transversion in COL1A2 (case 3), and to two contiguous point mutations in COL1A2 (case 4). All five nucleotide changes appeared to be fresh mutations. COLIA1(accession number Z74615) and COL1A2(accession number Z74616) wild type coding sequences (cDNA) were deduced from the EMBL DNA sequence database. The mutations described here can also be found in the human type I collagen mutation database at the web site:http://www.le.ac.uk/genetics/collagen.

Gly85 to Val substitution in pro alpha 1(I) chain causes mild osteogenesis imperfecta and introduces a susceptibility to protease digestion.

In this paper we describe a mild moderate form of osteogenesis imperfecta caused by a point mutation in COL1A1 which converted glycine 85 to valine. The valine substitution introduced into the triple-helical domain of type-I collagen a conformational perturbation causing susceptibility to digestive proteases. In fact, SDS/PAGE of pepsin-treated collagen showed the presence of a faint band, migrating between alpha 1(I) and alpha 2(I), both in the medium and in the cell layer. On trypsin digestion the band, a shortened form of alpha 1(I), had a melting temperature of 39.5 degrees C. If the triple-helical collagen was obtained after trypsin or chymotrypsin digestion of procollagen, two shortened bands were identified; the enzymes cleaved about 40% of the trimers. The mutant procollagen was normally secreted and processed in the extracellular matrix at a normal rate. When native type-I collagen was formed after dextran-sulfate incubation, only chains of normal length were found, suggesting that the fibroblast proteases did not recognize the alteration introduced by the mutation. The effects of glycine 85 to valine substitution are compared with those produced by a previously described arginine substitution of the same residue (Deak et al., 1991).

Severe (type III) osteogenesis imperfecta due to glycine substitutions in the central domain of the collagen triple helix.

The molecular defects responsible for three cases of severe (type III) osteogenesis imperfecta (OI) were investigated. The mutation sites were localized in pro alpha 1(I) and pro alpha 2(I) mRNA molecules, respectively, by chemical cleavage of mismatch in heteroduplex nucleic acids. Mutation identification was achieved by reverse transcription-polymerase chain reaction-DNA amplification, followed by cloning and sequencing. Two unrelated patients were demonstrated to bear the same G-A transition at nucleotide 2418 of the pro alpha 1(I) coding region, leading to G589S substitution and resulting in very similar clinical manifestations. In the latter patient, a G-T transversion at nucleotide 2166 was found in one pro alpha 2(I) allele, which caused a G586V substitution and again severe OI. Presumably all three mutations occurred de novo in the probands, since they were not found in their parents' DNA. The biochemical findings on type I collagen were very similar in all the probands: the mutations here described had little destabilizing effects on triple helix formation, secretion and stability. The half-life of the collagen incorporated into the insoluble matrix was comparable with that of controls. These mutations are localized in the gap zone of the fibrils where mineral nucleation occurs. This fact suggests that they probably do not exert destabilizing effects on the individual collagen molecules, but rather on the mineralization process, once the defective molecules are incorporated into the fibrils, hence causing severe phenotypes.

[Right cardiac echinococcosis with coronary compression. Description of a clinical case]. / Echinococcosi cardiaca destra con compressione coronarica. Descrizione di un caso clinico.

An outstanding case of cardiac echinococcosis is described. Clinical symptomatology was characterized by typical nitroglycerin-responsive crisis of angina pectoris associated to an electrocardiographic pattern of negative T wave on anterolateral and inferior leads. The diagnosis was suspected on the basis of thoracic computerized tomography and coronary arteriography. The anatomical localization was mainly pericardial but the right ventricle was involved too. Left anterior descending artery was dislocated and squeezed.

Allelic frequencies of FBN1 gene polymorphisms and genetic analysis of italian families with Marfan syndrome.

The fibrillin gene (FBN1) is the disease locus for Marfan syndrome. This disorder shows a high degree of clinical and allelic heterogeneity. Direct mutation screening has proven difficult and inefficient and at present cannot be utilized for routine analysis. In familial cases linkage analysis represents a useful tool for molecular diagnosis. We have determined the allelic frequencies of 5 polymorphic markers within the FBN1 locus in the Italian population and have successfully employed them for prenatal diagnosis and resolution of clinically equivocal cases.