Invasive aspergillosis and endocarditis.

INTRODUCTION: Aspergillusfumigatus can cause a systemic infection called invasive aspergillosis causing pulmonary and extra-pulmonary damage. Aspergillus endocarditis (AE) is a relatively rare disease but can be life-threatening. CASE REPORTS: We report here on five cases of endocarditis due to invasive aspergillosis: a 58-year-old man receiving immunosuppressive medication following a kidney graft, a 58-year-old man undergoing chemotherapy for chronic lymphocytic leukaemia, a 55-year-old man receiving corticosteroids for IgA vasculitis, a 52-year-old HIV-infected woman under no specific treatment and a 17-year-old boy under immunosuppressive therapy for auto-immune chronic neutropenia. DISCUSSION: Aspergillus accounts for 25-30% of fungal endocarditis and 0.25% to 8.5% of all cases of infectious endocarditis. Aspergillus endocarditis results from invasion of the lung arterioles by hyphae and blood dissemination. It is associated with a very high mortality rate (42-68%). Diagnosing Aspergillus endocarditis is mainly problematic because blood cultures are almost always negative, and fever may be absent. Immunosuppression, haematological malignancies, recent cardiothoracic surgery, negative blood cultures with endocarditis and/or systemic or pulmonary emboli are predictors of AE. In the setting of endocarditis, some clinical characteristics may raise early suspicions of aspergillosis rather than a non-fungal agent: no fever, vegetations affecting the mitral valve, non-valve or aortotomy sites, aortic abscess or pseudo-aneurysm. The identification of invasive aspergillosis is based on a chest CT scan, microscopy/culture or other serological and molecular tests. The treatment of Aspergillus endocarditis requires triazole antifungal drugs, and frequently additional surgical debridement. CONCLUSION: Aspergillus endocarditis is rare but is associated with a very high mortality rate. Knowledge of its predictive factors and key clinical features can help to differentiate aspergillosis from non-fungal endocarditis and may enable improved survival rates.

Knockout mice as model systems for studying nm23/NDP kinase gene functions. Application to the nm23-M1 gene.

Mice carrying a homozygous germ-line mutation in the nm23-M1 gene that eliminates its protein expression and drives expression of beta-galactosidase by nm23-M1 promoter have been generated. nm23-M1 gene inactivation is not teratogenic and the pups can grow to adult age without apparent health problems. However, they undergo a growth retardation and knocked out females cannot feed their pups. Both effects are background dependent. Beta-galactosidase mapping of nm23-M1 promoter activation during embryogenesis shows that the nm23-M1 gene is principally expressed in epithelial layer of tissues which require inductive epithelial-mesenchymal interactions for their formation. In conclusion, invalidated mice could be interesting models to analyze the role of nm23-M1 on signal transduction pathway regulation, or cancer induction and proliferation.

Serotonin 2B receptor is required for heart development.

Several lines of evidence suggest that the serotonin (5-hydroxytryptamine, 5-HT) regulates cardiovascular functions during embryogenesis and adulthood. 5-HT binds to numerous cognate receptors to initiate its biological effects. However, none of the 5-HT receptor disruptions in mice have yet resulted in embryonic defects. Here we show that 5-HT(2B) receptor is an important regulator of cardiac development. We found that inactivation of 5-HT(2B) gene leads to embryonic and neonatal death caused by heart defects. 5-HT(2B) mutant embryos exhibit a lack of trabeculae in the heart and a specific reduction in the expression levels of a tyrosine kinase receptor, ErbB-2, leading to midgestation lethality. These in vivo data suggest that the Gq-coupled receptor 5-HT(2B) uses the signaling pathway of tyrosine kinase receptor ErbB-2 for cardiac differentiation. All surviving newborn mice display a severe ventricular hypoplasia caused by impaired proliferative capacity of myocytes. In adult mutant mice, cardiac histopathological changes including myocyte disarray and ventricular dilation were consistently observed. Our results constitute genetic evidence that 5-HT via 5-HT(2B) receptor regulates differentiation and proliferation of developing and adult heart. This mutation provides a genetic model for cardiopathy and should facilitate studies of both the pathogenesis and therapy of cardiac disorders in humans.

MDM2 overexpression generates a skin phenotype in both wild type and p53 null mice.

The MDM2 proto-oncogene is overexpressed in human tumours and regulates the activities of the tumour suppressors p53 and pRB. We created mice that overexpress MDM2 under the control of the CMV promoter. These mice did not display an increased tumour incidence, but rather a specific skin phenotype, characterized by desquamation and hyperkeratosis. Transgenic MDM2 was found to be overexpressed in the epidermis, a tissue that normally expresses high levels of MDM2. The phenotype appeared during the first week after birth and then lessened with age, closely following the level of expression of the transgene. MDM2 overexpression was associated with an increase in proliferation in the basal layer, thickening of the epidermis, altered expression of the differentiation markers cytokeratin CK14, CK10 and CK1, and a decrease in the size and the number of granules that contain products of differentiation. Transgenic mice on a p53 null background displayed similar although not identical changes, showing that the effects of MDM2 are to a certain degree p53 independent. The skin is a major site of MDM2 expression in mice, raising the possibility that MDM2 overexpression perturbs the normal pattern of MDM2 expression and inhibits differentiation of the epidermis.

[Generation of a trans-complementable defective recombinant provirus and loading a transgene]. / Génération d'un provirus recombinant défectif complémentable en trans et véhiculant un transgène.

This work was aimed at generating a novel system for gene transfer to tumor cell, combining the advantages of non-viral gene transfer methods with those of transfer by recombinant retroviruses. We replaced the env gene of an infectious Moloney murine leukemia provirus with the gene coding for the thymidine kinase of Herpes Simplex Virus 1 (HSV1-TK). The sole transfection of this construction allows the production of viral particles, and the encapsidation of a viral genome carrying transgene. We show that this gene is expressed at a level sufficient for conferring sensitivity to ganciclovir, a nucleoside analog that is metabolised in a toxic compound by HSV1-TK. We also show that the complementation of this recombinant defective provirus with a gene coding for a retroviral envelope, either expressed constitutively by the transduced cell, or by co-transfection, leads to the formation of infectious viral particles capable of transducing HSV1-TK into tumor cells.

Axial skeleton homeosis and forelimb malformations in Hoxd-11 mutant mice.

The Hoxd-11 gene was disrupted by homologous recombination in embryonic stem cells. We found that Hoxd-11-/- mutant mice are viable and display homeotic transformations of their sacral vertebrae, while their forelimbs present abnormalities of some metacarpals and of the first row of carpal bones. These results are discussed in the light of current models of tetrapod axial skeleton and limb patterning.

Heterologous protein expression by transimmortalized differentiated liver cell lines derived from transgenic mice (hepatomas/alpha 1 antitrypsin/ONC mouse).

A number of therapeutic plasma proteins are synthesized by human hepatocytes. Since many of these proteins undergo liver-specific post-translational modifications which are required for full biological activity, it may therefore be necessary to develop hepatocyte-based expression systems for their production. Using transgenic mice we have developed a transimmortalisation technique for the isolation of differentiated hepatic cell lines, already engineered to secrete human alpha 1 antitrypsin (alpha 1 AT), a plasma protein which is produced mainly in liver cells. This was achieved by co-expression of the mouse c-myc proto-oncogene and a genomic copy of the human alpha 1 AT gene, both under the control of the human alpha 1 AT promoter. Transgenic mice carrying this construct developed hepatomas producing human alpha 1 AT. Under defined culture conditions, cell lines secreting active alpha 1 AT were derived from these tumours. These cells maintain a differentiated hepatic phenotype and continue to secrete human alpha 1 AT for at least 40 generations.

Functional consequences of overexpressed Ia antigens in AK alpha/AK beta transgenic mice.

We report the creation and characterization of several transgenic mouse lines that carry genes coding for the Ak alpha or Ak beta MHC class II (or Ia) molecules. In all these lines, the transgenes are expressed at the RNA and protein level with correct tissue and cell type specificity. Crosses between certain of them yield progeny displaying very high surface levels of class II protein--roughly five times the normal amount--allowing us to evaluate the consequences of quantitative variation in Ia molecule density on the organization and function of the immune system. The effects appear rather limited: we detect subtle changes in thymic lymphocyte subpopulations, as well as an enhanced Ag presentation capacity in vitro. Yet, in vivo responses are largely unaffected, and Ia overexpression to such levels does not provoke lymphoproliferation, immunodeficiency, or autoimmunity.

Compartmentalization of MHC class II gene expression in transgenic mice.

A set of transgenic mouse lines carrying Ek alpha genes with promoter region deletions was created in an attempt to compartmentalize MHC class II gene expression. Fine immunohistological analyses established that one transgenic line is essentially devoid of E complex in the thymic cortex, another displays almost no E in the thymic medulla or on peripheral macrophages, and two lines display no E on greater than 98% of B cells. We have assayed these mice for immune function: E-dependent tolerance, antigen presentation, T cell priming, and antibody response. Certain of the findings are difficult to reconcile with currently popular hypotheses, e.g., tolerance induction to E molecules in the virtual absence of E complex in the thymic medulla and efficient antibody responses to E-restricted antigens when almost all B cells are E-.

Monocistronic translation of alfalfa mosaic virus RNAs.

The four alfalfa mosaic virus RNAs (respectively 24 S, 20 S, 17 S and 12 S) have been used separately as messengers in two in vitro protein synthesizing systems: wheat germ and rabbit reticulocyte lysate. In both systems a polypeptide corresponding to the translation of the entire length of the RNA can be found for RNAs 24 S, 20 S and 12 S, but not for 17 S RNA, the translation product of which is only 35,000 daltons. The number of initiation sites has been determined for each RNA by analyzing the initiation peptides synthesized in the presence of spasomycin and show that there is only one initiation or binding site perRNA. We thus conclude that each AMV RNA behaves as a monocistronic messenger in in vitro translating systems.

Isolation and biochemical properties of toxic tryptic peptides of ricinotoxin from Ricinus communis seeds.

Tryptic hydrolysis conditions of ricinotoxin were studied in order to produce not only digestion of this glycoprotein but also still toxic tryptic peptides. No hydrolysis was obtained without prior denaturation. The best conditions of denaturation were obtained with 0.2 M guanidine hydrochloride and, to a lower extent, by heat treatment at 90 degrees C during 6 minutes. The hydrolysates were fractionated on Sephadex G100 column. In each case highly toxic peptide fractions were obtained which showed, like native ricinotoxin, a strong inhibitory action on the in vitro protein synthesis ina cell-free eukaryotic system but were without any action on a prokaryotic cell-free system.

Messenger RNA translation in the presence of homologous and heterologous tRNA.

The effect of tRNA distribution in the cell on the rate of specific protein synthesis has been investigated. A tRNA-dependent cell-free protein-synthesizing system derived from Krebs-II ascites cells has been worked out. In this system it is possible to synthesize specific proteins (egg-white proteins or globin) by the translation of oviduct or reticulocyte messenger RNA in the presence of tRNA from homologous or heterologous tissues. The rate of translation of a give messenger RNA is optimal in the presence of tRNA from the homologous tissue. The lower level of protein synthesis obtained with an excess of heterologous tRNA can be overcome by adding the homologous tRNA. Nevertheless homologous isoaccepting species partly purified by a reversed-phase chromatography or a benzoylated DEAE-cellulose column cannot fully restore the optimal synthesis, eliminating the hypothesis according to which a particular tRNA species is involved in this phenomenon. The correct distribution of tRNA is necessary for optimal translation of a messenger RNA.