Identification of sixty-two novel and twelve known FBN1 mutations in eighty-one unrelated probands with Marfan syndrome and other fibrillinopathies

Marfan Syndrome (MFS) is an autosomal dominant disorder of the connective tissue due to mutations of Fibrillin-1 gene (FBN1) in more than 90% of cases and Transforming Growth Factor-Beta-Receptor2 gene (TGFB2R) in a minority of cases. Genotyping is relevant for diagnosis and genotype-phenotype correlations. We describe the FBN1 genotypes and related phenotypes of 81 patients who were referred to our attention for MFS or Marfan-like phenotypes. Patients underwent multidisciplinary pertinent evaluation in the adult or paediatric setting, according to their age. The diagnosis relied on Ghent criteria. To optimise DHPLC analysis of the FBN1 gene, all coding regions of the gene were directly sequenced in 19 cases and 10 controls: heterozygous amplicons were used as true positives. DHPLC sensitivity was 100%. Then, DHPLC was used to screen 62 other cases. We identified 74 FBN1 mutations in 81 patients: 64 were novel and 17 known. Of the 81 mutations, 41 were missense (50.6%), 27, either nonsense or frameshift mutations and predicted a premature termination codon (PTC) (33%), 11 affected splice sites (13.6%), and two predicted in-frame deletions (2.5%). Most mutations (67.9%) occurred in cbEGF-like modules. Genotype was clinically relevant for early diagnosis and conclusion of the diagnostic work-up in patients with incomplete or atypical phenotypes.     

Spontaneous coronary artery dissection mimicking aortic dissection

A 51-year-old woman suffered rapidly irreversible cardiogenic shock with left hemiparesis. Transesophageal echocardiography, which represents an essential imaging tool in the emergency room, ruled out aortic dissection involving branch vessels but did not allow an in vivo diagnosis of spontaneous coronary dissection. The in vivo diagnosis of spontaneous coronary dissection is rather difficult because of the dramatic clinical presentation and selective coronary angiography requirement.     

An accessory open reading frame (orf-x) of jaagsiekte sheep retrovirus is conserved between different virus isolates

Jaagsiekte sheep retrovirus (JSRV) is the etiological agent of a contagious lung tumour of sheep known as sheep pulmonary adenomatosis (syn: ovine pulmonary carcinoma, jaagsiekte). JSRV exhibits a simple genetic organization, characteristic of the type D and type B retroviruses, with the canonical retroviral sequences gag, pro, pol and env encoding the structural proteins of the virion. An additional open reading frame (orf-x), of approximately 500 bp overlapping pol, is present in the only two complete sequences of JSRV published to date. Since very little information is available on the biology of JSRV it is important to establish if orf-x is conserved between different virus isolates. In this study we analysed the orf-x region of JSRV isolates collected from the United Kingdom, Italy, Spain and South Africa. In addition we also analysed the presence of orf-x in JSRV-related endogenous sequences (enJSRVs) present in the sheep genome. Orf-x was highly conserved in all the exogenous isolates (n=10) and in most of the endogenous sequences (n=8). Thus orf-x may be an accessory gene of JSRV and haves a biological function which might be advantageous to JSRV. Phenetic analysis conducted on the complete orf-x nucleotide sequences seems to highlight the presence of three distinct groups statistically well supported by bootstrapping: i) exogenous JSRV sequence from the UK; ii) exogenous JSRV sequences from Southern Europe and iii) the exogenous South African strain plus all the endogenous sequences analyzed and collected from Australia, Italy, UK and South Africa.     

The molecular basis of Natural Killer (NK) cell recognition and function

Natural Killer cells are likely to play an important role in the host defenses because they kill virally infected or tumor cells but spare normal self-cells. The molecular mechanism that explains why NK cells do not kill indiscriminately has recently been elucidated. It is due to several specialized receptors that recognize major histocompatibility complex (MHC) class I molecules expressed on normal cells. The lack of expression of one or more HLA class I alleles leads to NK-mediated target cell lysis. Different types of receptors specific for groups of HLA-C, HLA-B, and, very recently, HLA-A alleles have been identified. While in most instances, they function as inhibitory receptors, an activatory form of the HLA-C-specific receptors has been identified in some donors. Molecular cloning of HLA-C-, HLA-B- or HLA-A-specific receptors has revealed new members of the immunoglobulin superfamily with two or three Ig-like domains, respectively, in their extracellular portion. While the inhibitory form is characterized by a long cytoplasmic tail associated with a non-polar transmembrane portion, the activatory one has a short tail asociated with a Lys-containing transmembrane portion. Thus, these human NK receptors are different from the murine Ly49, that is a type II transmembrane protein characterized by a C-type lectin domain. A subset of activated T lymphocytes expresses NK-type class I-specific receptors. These receptors exert an inhibiting activity on T cell receptor-mediated functions and may provide an important mechanism of downregulation of T cell responses.     

Ligand and cytokine dependence of the immunosuppressive pathway of tryptophan catabolism in plasmacytoid dendritic cells

Murine plasmacytoid dendritic cells (pDCs) have been credited with a unique ability to express indoleamine 2,3-dioxygenase (IDO) function and mediate immunosuppression in specific settings; yet, the conditions of spontaneous versus induced activity have remained unclear. We have used maneuvers known to up-regulate IDO in different cell types and have examined the relative efficacy and mechanisms of the induced activity in splenic pDCs, namely, after specific receptor engagement by CTLA-4-Ig, CD200-Ig or CD28-Ig, the latter in combination with silenced expression of the suppressor of cytokine signaling 3 (SOCS3) gene. We found that pDCs (CD11c+ mPDCA-1+ 120G8+) do not express IDO and are not tolerogenic under basal conditions. B7-1 engagement by CTLA-4-Ig, CD200R1 engagement by CD200-Ig and B7-1/B7-2 engagement by CD28-Ig in SOCS3-deficient pDCs were each capable of initiating IDO-dependent tolerance via different mechanisms. IFN-gamma was the major cytokine responsible for CTLA-4-Ig effects, and type I IFNs for those of CD200-Ig. Immunosuppression by CD28-Ig in the absence of SOCS3 required IFN-gamma induction and IFN-like actions of IL-6. Therefore, although pDCs do not mediate IDO-dependent tolerance constitutively, multiple ligands and cytokines will contribute to the expression of a tolerogenic phenotype by pDCs in the mouse.     

An ELISA serum assay for autoantibodies to HSP70 in immune-mediated hearing loss

A Western blot to detect anti-HSP70 autoantibodies has been reported to be of diagnostic value for immune-mediated hearing loss patients. While setting up this Western blot in our lab, we detected two main problems. First, some patients were positive for antibodies to a 70-kDa protein when tested against a whole cell lysate, but negative if the antigen used was purified HSP70. Second, if high amounts of purified HSP70 were loaded on the gel, both patients and healthy controls were positive. We have developed and optimized an ELISA as an alternative to the Western blot. This assay is more appropriate to identify positive and negative individuals because it is semi-quantitative. The ELISA is also more sensitive, requiring very low concentrations of the antigen and thus minimizing false positives. Finally, we demonstrated that immune-mediated hearing loss patients recognize mainly the native form of HSP70, a fact that potentially leads to false negatives when a denaturing Western blot assay is used for diagnosis. To test the diagnostic value of the ELISA, we performed a blind test with 70 hearing loss patients, as well as 30 healthy controls. A sensitivity of 84% and a specificity of 93% were obtained, superior to what has been reported so far for the Western blot.     

Identification of a 7S globulin as a novel coconut allergen.

BACKGROUND: Coconut (Cocos nucifera) is a monocotyledonous plant of the Arecaceae family. Allergy to coconut is infrequent, with only 5 cases reported so far in the medical literature. OBJECTIVE: To identify coconut allergens in 2 patients allergic to this food. METHODS: We describe 2 patients allergic to coconut: an adult pollen-allergic patient monosensitized to coconut who presented with severe oropharyngeal symptoms and a child with a previous allergy to walnut, not allergic to pollen, who developed anaphylaxis on coconut ingestion. Both patients had positive skin prick test results and serum specific IgE (CAP) to coconut. IgE sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting was performed to identify the allergens involved, and a strong IgE binding band detected in both patients was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). Stability to pepsin digestion of the coconut extract and its cross-reactivity with tree nuts were studied. RESULTS: An immunoblot showed an almost identical profile of IgE binding proteins in the coconut extract in both patients who reacted strongly to a band of approximately 29 kDa. The peptide analysis by MALDI-TOF MS of this band obtained the sequence GHGKREDPEKR. The protein with the highest correlation with this peptide was found to be a 7S globulin from Elaeis guineensis, another oil palm species also belonging to the Arecaceae family. The 29-kDa band was digested by pepsin in less than 1 minute. Cross-reactivity among coconut, walnut, and hazelnut was demonstrated by CAP inhibition in patient 2. CONCLUSION: We have identified a 7S storage protein as a novel coconut allergen.