Characteristics and costs of carbapenemase-producing enterobacteria carriers (2012/2013).

OBJECTIVES: We had for aim to determine the characteristics of carbapenemase-producing enterobacteria (CPE) carriers and to assess the economic impact of isolation measures leading to loss of activity (closed beds, prolonged hospital stays) and additional personnel hours. PATIENTS AND METHODS: We conducted a retrospective study for 2years (2012/2013), in a French general hospital, focusing on CPE carriers with clinical case description. The costs were estimated by comparing the activity of concerned units (excluding the ICU) during periods with CPE carriers or contacts, during the same periods of the year (n-1), plus additional hours and rectal swabs. RESULTS: Sixteen EPC carriers were identified: 10 men and 6 women, 65±10years of age. Seven patients acquired EPC in hospital during 2 outbreaks in 2012. Four patients presented with an infection (peritonitis, catheter infection, and 2 cases of obstructive pyelonephritis) with a favorable outcome. The median length of stay was 21days [4,150]. Six patients died, 1 death was indirectly due to CPE because of inappropriate empiric antibiotic therapy. A decrease in activity was observed compared to the previous year with an estimated 547,303€ loss. The 1779 additional hours cost 63,870€, and 716 screening samples cost 30,931€. The total additional cost was estimated at 642,104€ for the institution. CONCLUSIONS: Specialized teams for CPE carriers and isolation of contact patients, required to avoid/control epidemics, have an important additional cost. An appreciation of their support is needed, as well as participation of rehabilitation units.

Tumor induction by an Lck-MyrAkt transgene is delayed by mechanisms controlling the size of the thymus.

Transgenic mice expressing MyrAkt from a proximal Lck promoter construct develop thymomas at an early age, whereas transgenic mice expressing constitutively active Lck-AktE40K develop primarily tumors of the peripheral lymphoid organs later in life. The thymus of 6- to 8-week-old MyrAkt transgenic mice is normal in size but contains fewer, larger cells than the thymus of nontransgenic control and AktE40K transgenic mice. Earlier studies had shown that cell size and cell cycle are coordinately regulated. On the basis of this finding, and our observations that the oncogenic potential of Akt correlates with its effect on cell size, we hypothesized that mechanisms aimed at maintaining the size of the thymus dissociate cell size and cell cycle regulation by blocking MyrAkt-promoted G(1) progression and that failure of these mechanisms may promote cell proliferation resulting in an enlarged neoplastic thymus. To address this hypothesis, we examined the cell cycle distribution of freshly isolated and cultured thymocytes from transgenic and nontransgenic control mice. The results showed that although neither transgene alters cell cycle distribution in situ, the MyrAkt transgene promotes G(1) progression in culture. Freshly isolated MyrAkt thymocytes express high levels of cyclins D2 and E and cdk4 but lower than normal levels of cyclin D3 and cdk2. Cultured thymocytes from MyrAkt transgenic mice, on the other hand, express high levels of cyclin D3, suggesting that the hypothesized organ size control mechanisms may down-regulate the expression of this molecule. Primary tumor cells, similar to MyrAkt thymocytes in culture, express high levels of cyclin D3. These findings support the hypothesis that tumor induction is caused by the failure of organ size control mechanisms to down-regulate cyclin D3 and to block MyrAkt-promoted G(1) progression.

Identification and expression of cosmids with an allelic variant of class I alcohol dehydrogenase in transgenic mice.

The mouse Adh1 gene exhibits tissue-specific regulation, is developmentally regulated, and is androgen regulated in kidney and adrenal tissue. To study this complex regulation phenotype a transgenic mouse approach has been used to investigate regulatory regions of the gene necessary for proper tissue expression and hormonal control. Transgenic mice have been produced with an Adh1 minigene as a reporter behind either 2.5- or 10 kb of 5'-flanking sequence [1]. Complete androgen regulation in kidney requires a region between -2.5 and -10 kb. A sequence extending to -10 kb does not confer liver expression in this minigene construct. B6.S mice express an electrophoretically variant protein resulting from a known nucleotide substitution resulting in a restriction endonuclease length polymorphism. Transgenic mice harboring B6.S cosmids can be studied for expression analysis at both protein and mRNA levels, identification of transgenic founders and inheritance studies are greatly facilitated by a PCR-restriction endonuclease cleavage approach, the entire mouse gene is used as a reporter, and the formation of heterodimeric enzyme molecules can be used to infer expression of the transgene in the proper cell types within a given tissue. Expression of a B6.S cosmid containing the entire Adh1 gene and 6 kb of 5'- and 21 kb of 3'-flanking region occurs in transgenic mice in a copy number dependent manner in a number of tissues, but expression in liver does not occur. The ability to analyze expression at the protein and mRNA levels has been confirmed using this system. Future directions will involve the use of large BAC clones modified by RARE cleavage to identify the liver specific elements necessary for expression.

Knock-out mouse for Canavan disease: a model for gene transfer to the central nervous system.

BACKGROUND: Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by deficiency of aspartoacylase (ASPA) and increased levels of N-acetylaspartic acid (NAA) in brain and body fluids, severe mental retardation and early death. Gene therapy has been attempted in a number of children with CD. The lack of an animal model has been a limiting factor in developing vectors for the treatment of CD. This paper reports the successful creation of a knock-out mouse for Canavan disease that can be used for gene transfer. METHODS: Genomic library lambda knock-out shuttle (lambdaKOS) was screened and a specific pKOS/Aspa clone was isolated and used to create a plasmid with 10 base pair (bp) deletion of exon four of the murine aspa. Following linearization, the plasmid was electroporated to ES cells. Correctly targeted ES clones were identified following positive and negative selection and confirmed by Southern analysis. Chimeras were generated by injection of ES cells to blastocysts. Germ line transmission was achieved by the birth of heterozygous mice as confirmed by Southern analysis. RESULTS: Heterozygous mice born following these experiments have no overt phenotype. The homozygous mice display neurological impairment, macrocephaly, generalized white matter disease, deficient ASPA activity and high levels of NAA in urine. Magnetic resonance imaging (MRI) and spectroscopy (MRS) of the brain of the homozygous mice show white matter changes characteristic of Canavan disease and elevated NAA levels. CONCLUSION: The newly created ASPA deficient mouse establishes an important animal model of Canavan disease. This model should be useful for developing gene transfer vectors to treat Canavan disease. Vectors for the central nervous system (CNS) and modulation of NAA levels in the brain should further add to the understanding of the pathophysiology of Canavan disease. Data generated from this animal model will be useful for developing strategies for gene therapy in other neurodegenerative diseases.

TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway.

Tpl2 knockout mice produce low levels of TNF-alpha when exposed to lipopolysaccharide (LPS) and they are resistant to LPS/D-Galactosamine-induced pathology. LPS stimulation of peritoneal macrophages from these mice did not activate MEK1, ERK1, and ERK2 but did activate JNK, p38 MAPK, and NF-kappaB. The block in ERK1 and ERK2 activation was causally linked to the defect in TNF-alpha induction by experiments showing that normal murine macrophages treated with the MEK inhibitor PD98059 exhibit a similar defect. Deletion of the AU-rich motif in the TNF-alpha mRNA minimized the effect of Tpl2 inactivation on the induction of TNF-alpha. Subcellular fractionation of LPS-stimulated macrophages revealed that LPS signals transduced by Tpl2 specifically promote the transport of TNF-alpha mRNA from the nucleus to the cytoplasm.

Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation.

Provirus insertion in the last intron of the Tpl-2 gene in retrovirus-induced rat T-cell lymphomas results in the enhanced expression of a carboxy-terminally truncated Tpl-2 kinase. Here we show that the truncated protein exhibits an approximately sevenfold higher catalytic activity and is two- to threefold more efficient in activating the MAPK and SAPK pathways relative to the wild-type protein. The truncated Tpl-2 protein and a GST fusion of the Tpl-2 carboxy-terminal tail interact when coexpressed in Sf9 cells. Their interaction down-regulates the kinase activity of the truncated protein suggesting that tail-directed intramolecular interactions regulate the Tpl-2 kinase. Tpl-2 transgenic mice expressing the wild-type protein from the proximal Lck promoter fail to show a biological phenotype, whereas mice expressing the truncated protein develop large-cell lymphoblastic lymphomas of T-cell origin. These results show that Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation.

Interspecific backcrosses provide an important new tool for centromere mapping of mouse chromosomes.

Centromere mapping of mouse chromosomes has been problematic due to a paucity of appropriate markers. As a result, the mapping of centromeres has most often relied on the use of Robertsonian chromosomes to mark chromosome ends. Many Robertsonian translocations have been shown to suppress recombination in pericentric regions; therefore, centromere mapping data generated by using Robertsonian chromosomes must be interpreted with caution. We have utilized a new tool for centromere mapping that is applicable to all mouse chromosomes (except the Y chromosome) and that potentially overcomes the inherent limitations of using Robertsonian translocations. Briefly, an interspecific backcross mapping panel was constructed from crosses of C57BL/6Ros and Mus spretus mice. The centromere of each chromosome was subsequently typed by in situ hybridization, using a major satellite probe that uniformly labels C57BL/6Ros centromeres but hybridizes only weakly to M. spretus centromeres. Genetic markers that were already known to map in the proximal region of each of the mouse chromosomes were then typed by segregation analyses of restriction fragment length polymorphisms. These studies have made it possible to align the interspecific genetic map of each of the mouse autosomes and the X chromosome with respect to the centromere. They also provide a basis for comparison with centromere mapping data generated previously by other means.

An interspecific linkage map of mouse chromosome 15 positioned with respect to the centromere.

We have used an interspecific backcross between C57BL/6J and Mus spretus to derive a molecular genetic linkage map of chromosome 15 that includes 25 molecular markers and spans 93% of the estimated length of chromosome 15. Using a second interspecific backcross that was analyzed with a centromere-specific marker, we were also able to position our map with respect to the chromosome 15 centromere. This map provides molecular access to many discrete regions on chromosome 15, thus providing a framework for establishing relationships between cloned DNA markers and known mouse mutations and for identifying homologous genes in mice and humans that may be involved in disease.

A molecular genetic linkage map of mouse chromosome 18 reveals extensive linkage conservation with human chromosomes 5 and 18.

An interspecific backcross between C57BL/6J and Mus spretus was used to generate a molecular genetic linkage map of mouse chromosome 18 that includes 23 molecular markers and spans approximately 86% of the estimated length of the chromosome. The Apc, Camk2a, D18Fcr1, D18Fcr2, D18Leh1, D18Leh2, Dcc, Emb-rs3, Fgfa, Fim-2/Csfmr, Gnal, Grl-1, Grp, Hk-1rs1, Ii, Kns, Lmnb, Mbp, Mcc, Mtv-38, Palb, Pdgfrb, and Tpl-2 genes were mapped relative to each other in one interspecific backcross. A second interspecific backcross and a centromere-specific DNA satellite probe were used to determine the distance of the most proximal chromosome 18 marker to the centromere. The interspecific map extends the known regions of linkage homology between mouse chromosome 18 and human chromosomes 5 and 18 and identifies a new homology segment with human chromosome 10p. It also provides molecular access to many regions of mouse chromosome 18 for the first time.

Transgenic mice carrying a murine amylase 2.2/SV40 T antigen fusion gene develop pancreatic acinar cell and stomach carcinomas.

The mouse pancreatic amylase Amy-2.2 gene was fused to the structural gene for SV40 T antigen, and 51 independent transgenic founder mice carrying the fusion gene were generated. The majority of the founders and 100% of their offspring in the derived transgenic lines developed pancreatic acinar cell carcinomas and stomach carcinomas. Transgenic animals also had a high incidence of metastatic carcinomas in other tissues. The development of stomach carcinomas was unexpected because the Amy-2.2 promoter was not previously known to be expressed in stomach. Northern blot analyses and ribonuclease protection assays showed that Amy-2.2 is expressed in stomach, at approximately 0.05% of the level in pancreas. Expression of the fusion gene in stomach, therefore, appears to represent a previously unrecognized activity of the Amy-2.2 promoter. Examination of young transgenic mice demonstrated that preneoplastic lesions were present in pancreas and stomach before the development of neoplastic lesions in either tissue, consistent with the notion that stomach neoplasms are primary neoplasms and not metastases from the pancreas. Ribonuclease protection assays demonstrated that properly initiated large T and small t antigen transcripts were present in pancreas and stomach during tumorigenesis. T antigen protein was also detected in pancreas and stomach by immunohistochemistry. A time course for tumorigenesis was established for several transgenic mouse lines in which distinct types of lesions appeared at predictable times. This study provides the basis for future analysis of the role of SV40 T antigen in the progression and maintenance of pancreatic and stomach carcinomas.

An interspecific backcross linkage map of the proximal half of mouse chromosome 14.

We have generated a 30-cM molecular genetic linkage map of the proximal half of mouse chromosome 14 by interspecific backcross analysis. Loci that were mapped in this study include Bmp-1, Ctla-1, Hap, hr, Plau, Psp-2, Rib-1, and Tcra. A region of homology between mouse chromosome 14 and human chromosome 10 was identified by the localization of Plau to chromosome 14. This interspecific backcross map will be valuable for establishing linkage relationships of additional loci to mouse chromosome 14.