Dentin Dysplasia in Notum Knockout Mice.

Secreted WNT proteins control cell differentiation and proliferation in many tissues, and NOTUM is a secreted enzyme that modulates WNT morphogens by removing a palmitoleoylate moiety that is essential for their activity. To better understand the role this enzyme in development, the authors produced NOTUM-deficient mice by targeted insertional disruption of the Notum gene. The authors discovered a critical role for NOTUM in dentin morphogenesis suggesting that increased WNT activity can disrupt odontoblast differentiation and orientation in both incisor and molar teeth. Although molars in Notum(-/-) mice had normal-shaped crowns and normal mantle dentin, the defective crown dentin resulted in enamel prone to fracture during mastication and made teeth more susceptible to endodontal inflammation and necrosis. The dentin dysplasia and short roots contributed to tooth hypermobility and to the spread of periodontal inflammation, which often progressed to periapical abscess formation. The additional incidental finding of renal agenesis in some Notum (-/-) mice indicated that NOTUM also has a role in kidney development, with undiagnosed bilateral renal agenesis most likely responsible for the observed decreased perinatal viability of Notum(-/-) mice. The findings support a significant role for NOTUM in modulating WNT signaling pathways that have pleiotropic effects on tooth and kidney development.

Nephronophthisis and retinal degeneration in tmem218-/- mice: a novel mouse model for Senior-Løken syndrome?

Mice deficient in TMEM218 (Tmem218(-/-) ) were generated as part of an effort to identify and validate pharmaceutically tractable targets for drug development through large-scale phenotypic screening of knockout mice. Routine diagnostics, expression analysis, histopathology, and electroretinogram analyses completed on Tmem218(-/-) mice identified a previously unknown role for TMEM218 in the development and function of the kidney and eye. The major observed phenotypes in Tmem218(-/-) mice were progressive cystic kidney disease and retinal degeneration. The renal lesions were characterized by diffuse renal cyst development with tubulointerstitial nephropathy and disruption of tubular basement membranes in essentially normal-sized kidneys. The retinal lesions were characterized by slow-onset loss of photoreceptors, which resulted in reduced electroretinogram responses. These renal and retinal lesions are most similar to those associated with nephronophthisis (NPHP) and retinitis pigmentosa in humans. At least 10% of NPHP cases present with extrarenal conditions, which most often include retinal degeneration. Senior-Løken syndrome is characterized by the concurrent development of autosomal recessive NPHP and retinitis pigmentosa. Since mutations in the known NPHP genes collectively account for only about 30% of NPHP cases, it is possible that TMEM218 could be involved in the development of similar ciliopathies in humans. In reviewing all other reported mouse models of NPHP, we suggest that Tmem218(-/-) mice could provide a useful model for elucidating the pathogenesis of cilia-associated disease in both the kidney and the retina, as well as in developing and testing novel therapeutic strategies for Senior-Løken syndrome.

Cryptogenic organizing pneumonia in Tomm5(-/-) mice.

Almost all mitochondrial proteins are encoded in the nuclear DNA and synthesized in the cytosol as pre-proteins. There is a protein translocase located in the mitochondrial outer membrane that transports mitochondrial pre-proteins into mitochondria. The central component of this translocase of the outer mitochondrial membrane (TOMM) complex is TOMM40, and TOMM5 is one of three small subunits associated with TOMM40. Translocase of outer mitochondrial membrane 5 homolog (Tomm5(-/-)) knockout mice demonstrated an unexpected lung-specific phenotype characterized by widespread intra-alveolar fibrosis. Although TOMM5-deficient mice tested normal in a very broad range of phenotyping assays, they displayed histopathological lesions in the lung that were consistent with those reported in humans with cryptogenic organizing pneumonia (COP), which is also known as bronchiolitis obliterans organizing pneumonia (BOOP). The lesions had a patchy distribution in the lung and were characterized by the presence of intraluminal fibrogenic buds consisting of fibroblasts and myofibroblasts embedded in a loose connective tissue matrix that occupied the lumina of alveoli and alveolar ducts, with preservation of underlying alveolar architecture. In addition to macrophages, which were numerous in affected and surrounding alveoli, eosinophils comprised the most common and widespread inflammatory cell. Taken together, the findings in Tomm5(-/-) mice provide yet another example of the value of histopathology as a baseline assay in high-throughput phenotyping systems.

Congenital hydrocephalus in genetically engineered mice.

There is evidence that genetic factors play a role in the complex multifactorial pathogenesis of hydrocephalus. Identification of the genes involved in the development of this neurologic disorder in animal models may elucidate factors responsible for the excessive accumulation of cerebrospinal fluid in hydrocephalic humans. The authors report here a brief summary of findings from 12 lines of genetically engineered mice that presented with autosomal recessive congenital hydrocephalus. This study illustrates the value of knockout mice in identifying genetic factors involved in the development of congenital hydrocephalus. Findings suggest that dysfunctional motile cilia represent the underlying pathogenetic mechanism in 8 of the 12 lines (Ulk4, Nme5, Nme7, Kif27, Stk36, Dpcd, Ak7, and Ak8). The likely underlying cause in the remaining 4 lines (RIKEN 4930444A02, Celsr2, Mboat7, and transgenic FZD3) was not determined, but it is possible that some of these could also have ciliary defects. For example, the cerebellar malformations observed in RIKEN 4930444A02 knockout mice show similarities to a number of developmental disorders, such as Joubert, Meckel-Gruber, and Bardet-Biedl syndromes, which involve mutations in cilia-related genes. Even though the direct relevance of mouse models to hydrocephalus in humans remains uncertain, the high prevalence of familial patterns of inheritance for congenital hydrocephalus in humans suggests that identification of genes responsible for development of hydrocephalus in mice may lead to the identification of homologous modifier genes and susceptibility alleles in humans. Also, characterization of mouse models can enhance understanding of important cell signaling and developmental pathways involved in the pathogenesis of hydrocephalus.

Amelogenesis imperfecta and other biomineralization defects in Fam20a and Fam20c null mice.

The FAM20 family of secreted proteins consists of three members (FAM20A, FAM20B, and FAM20C) recently linked to developmental disorders suggesting roles for FAM20 proteins in modulating biomineralization processes. The authors report here findings in knockout mice having null mutations affecting each of the three FAM20 proteins. Both Fam20a and Fam20c null mice survived to adulthood and showed biomineralization defects. Fam20b (-/-) embryos showed severe stunting and increased mortality at E13.5, although early lethality precluded detailed investigations. Physiologic calcification or biomineralization of extracellular matrices is a normal process in the development and functioning of various tissues (eg, bones and teeth). The lesions that developed in teeth, bones, or blood vessels after functional deletion of either Fam20a or Fam20c support a significant role for their encoded proteins in modulating biomineralization processes. Severe amelogenesis imperfecta (AI) was present in both Fam20a and Fam20c null mice. In addition, Fam20a (-/-) mice developed disseminated calcifications of muscular arteries and intrapulmonary calcifications, similar to those of fetuin-A deficient mice, although they were normocalcemic and normophosphatemic, with normal dentin and bone. Fam20a gene expression was detected in ameloblasts, odontoblasts, and the parathyroid gland, with local and systemic effects suggesting both local and/or systemic effects for FAM20A. In contrast, Fam20c (-/-) mice lacked ectopic calcifications but were severely hypophosphatemic and developed notable lesions in both dentin and bone to accompany the AI. The bone and dentin lesions, plus the marked hypophosphatemia and elevated serum alkaline phosphatase and FGF23 levels, are indicative of autosomal recessive hypophosphatemic rickets/osteomalacia in Fam20c (-/-) mice.

Situs inversus in Dpcd/Poll-/-, Nme7-/- , and Pkd1l1-/- mice.

Situs inversus (SI) is a congenital condition characterized by left-right transposition of thoracic and visceral organs and associated vasculature. The usual asymmetrical positioning of organs is established early in development in a transient structure called the embryonic node. The 2-cilia hypothesis proposes that 2 kinds of primary cilia in the embryonic node determine left-right asymmetry: motile cilia that generate a leftward fluid flow, and immotile mechanosensory cilia that respond to the flow. Here, we describe 3 mouse SI models that provide support for the 2-cilia hypothesis. In addition to having SI, Dpcd/Poll(-/-) mice (for: deleted in a mouse model of primary ciliary dyskinesia) and Nme7(-/-) mice (for: nonmetastatic cells 7) had lesions consistent with deficient ciliary motility: Hydrocephalus, sinusitis, and male infertility developed in Dpcd/Poll(-/-) mice, whereas hydrocephalus and excessive nasal exudates were seen in Nme7(-/-) mice. In contrast, the absence of respiratory tract lesions, hydrocephalus, and male infertility in Pkd1l1(-/-) mice (for: polycystic kidney disease 1 like 1) suggested that dysfunction of motile cilia was not involved in the development of SI in this line. Moreover, the gene Pkd1l1 has considerable sequence similarity with Pkd1 (for: polycystic kidney disease 1), which encodes a protein (polycystin-1) that is essential for the mechanosensory function of immotile primary cilia in the kidney. The markedly reduced viability of Pkd1l1(-/-) mice is somewhat surprising given the absence of any detected abnormalities (other than SI) in surviving Pkd1l1(-/-) mice subjected to a comprehensive battery of phenotype-screening exams. However, the heart and great vessels of Pkd1l1(-/-) mice were not examined, and it is possible that the decreased viability of Pkd1l1(-/-) mice is due to undiagnosed cardiovascular defects associated with heterotaxy.

Gene trap mutagenesis.

Our ability to genetically manipulate the mouse has had a great impact on medical research over the last few decades. Mouse genetics has developed into a powerful tool for dissecting the genetic causes of human disease and identifying potential targets for pharmaceutical intervention. With the recent sequencing of the human and mouse genomes, a large number of novel genes have been identified whose function in normal and disease physiology remains largely unknown. Government-sponsored multinational efforts are underway to analyze the function of all mouse genes through mutagenesis and phenotyping, making the mouse the interpreter of the human genome. A number of technologies are available for the generation of mutant mice, including gene targeting, gene trapping and transposon, chemical or radiation-induced mutagenesis. In this chapter, we review the current status of gene trapping technology, including its applicability to conditional mutagenesis.

Activation of Hex and mEg5 by retroviral insertion may contribute to mouse B-cell leukemia.

AKXD recombinant inbred mice develop a variety of leukemias and lymphomas due to retrovirally mediated insertional activation of cellular proto-oncogenes. We describe a new retroviral insertion site that is the most frequent genetic alteration in AKXD B-cell leukemias. Multiple genes flank the site of viral insertion, but the expression of just two, Hex and mEg5, is significantly upregulated. Hex is a divergent homeobox gene that is transiently expressed in many hematopoietic lineages, suggesting an involvement in cellular differentiation. mEg5 is a member of the bim-C subfamily of kinesin related proteins that are necessary for spindle formation and stabilization during mitosis. Our data provide the first genetic evidence for the activation of these genes in leukemia, and suggest that unscheduled expression of Hex and mEg5 contributes to the development of B-cell leukemia. In addition, this work highlights the use of genomic approaches for the study of position effect mutations.

Experimental chronic Lyme borreliosis in Lewis rats.

The course of Lyme borreliosis in LEW/N rats inoculated intraperitoneally as infants with 10(6) Borrelia burgdorferi was followed for 360 days. Spirochetes were detected in the blood through 30 days, in the brain through 60 days, and persisted in the spleen, liver, kidneys and articular tissue through 360 days. Acute exudative arthritis, tendonitis, and bursitis were evident in multiple joints by day 30. Arthritis regressed thereafter but capsular fibrosis and lymphoplasmacytic infiltrates persisted throughout the study. Several rats developed exacerbations of acute arthritis within days 180-360, a pattern similar to that encountered in human Lyme disease. Rats had a high prevalence of nonsuppurative myocarditis and vasculitis during days 90-360. Spirochetes were visualized by microscopy in joints and other tissues during the first month of infection, but were seen only sporadically thereafter. All rats seroconverted to B. burgdorferi by day 30. IgM titers persisted and IgG titers rose progressively through day 360. Immunoblots revealed IgM reactivity to a single 41 kDa protein until 360 days, when reactivity to a 60 kDa protein emerged. IgG reactivity occurred against progressively more proteins with time, indicating continued antigenic stimulation. Chronic and recurrent arthritic lesions and myocardial involvement suggest that the rat is a reliable model for further investigation.

Comparison of minimal inhibitory and mutant prevention drug concentrations of 4 fluoroquinolones against clinical isolates of methicillin-susceptible and -resistant Staphylococcus aureus.

Staphylococcus aureus remains an important human pathogen affecting both outpatients and those hospitalized. Increasing antimicrobial resistance is global but prevalence rates are variable for different geographical areas. Fluoroquinolones have been used to treat S. aureus infections and the newer quinolones have enhanced in vitro activity against this organism. The mutant prevention concentration (MPC) defines the antimicrobial drug concentration threshold that would require an organism to simultaneously possess two mutations for growth in the presence of the drug. We tested clinical isolates of methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) S. aureus by minimum inhibitory concentration (MIC) and MPC against gatifloxacin, gemifloxacin, levofloxacin and moxifloxacin. For MSSA strains, the rank order of potency based on MIC(90) values were gemifloxacin (0.063 mg/l) = moxifloxacin (0.063 mg/l) > gatifloxacin (0.05 mg/l) = levofloxacin (0.25 mg/l) and by MPC values moxifloxacin (0.25 mg/l) > gemifloxacin (0.5 mg/l) > gatifloxacin (1 mg/l) = levofloxacin (1mg/l). For 87% of the isolates the MPC value was 0.5 mg/l for gatifloxacin. The rank order of potency based on the time the serum drug concentration exceeded the MPC(90), was as follows: moxifloxacin (>24 h) > levofloxacin (>18 h) > gatifloxacin (12 h) > gemifloxacin (9 h). Serum drug concentration remained in excess of the MPC(87) for 24 h for gatifloxacin. Both MIC(90) and MPC(90) values were higher against MRSA strains and the time above the MPC(90) was significantly shorter for all agents.

Scanning lidar with a coupled radar safety system.

A small scanning three-wavelength lidar system at NASA Langley Research Center in Hampton, Virginia, has been used since 1992 to make atmospheric measurements on stratospheric and upper tropospheric aerosols and on the evolution of aircraft exhaust plumes. Many of these measurements have been made away from the zenith, and, to reduce the hazard to air traffic produced by the laser beam, a radar safety device has been installed. The radar application is original in that the radar beam is made collinear with the laser beam by use of a dichroic mirror that transmits the laser radiation and reflects the microwaves. This mirror is inserted into the outgoing optical path prior to the radiation from both the radar and the laser passing through the independent scanning unit. Tests of the complete system show that the lidar and radar beams remain collocated as they are scanned and that the radar can be used to inhibit the laser prior to an aircraft passing through the beam.

A serologic survey for viruses and Mycoplasma pulmonis among wild house mice (Mus domesticus) in southeastern Australia.

Plasma samples from 267 wild house mice (Mus domesticus) trapped at 14 sites in southeastern Australia were screened for antibody to 14 viruses normally associated with laboratory-reared rodents and to Mycoplasma pulmonis. Serologic prevalence was high for murine cytomegalovirus (99%, n = 94), murine coronavirus (95%), and murine rotavirus (74%). Samples from mice collected at all sites contained antibody to these viruses. The serologic prevalence was lower for mouse adenovirus, strain K87 (37%), parvovirus (33%), and reovirus type 3 (28%), with substantial site-to-site variation. Plasma from mice collected at 12 sites contained mouse adenovirus or reovirus antibody, and samples from mice at eight sites contained parvovirus antibody. Parvovirus-antibody positive mice were typically from high density populations or from low density populations that had recently declined from high density. Antibody to lymphocytic choriomeningitis virus (LCMV) and Sendai virus occurred at only three sites, and the serologic prevalence was very low (9.6% and 1.8%, respectively). All of the LCMV-positive mice were from northeastern New South Wales. The presence of this zoonotic virus in a mouse plague-prone region raises questions about human health risks resulting from cohabitation with large numbers of mice. It appeared that mouse populations at high density or declining from high density had higher prevalence of viral antibody than populations that had been at low or moderate density for some time. Thus, viral epizootics may occur among high-density populations and may be responsible for or precipitate declines in mouse density. These data raise the possibility of rodent viruses having potential as biological control agents.

Experimental Borrelia burgdorferi infection in Peromyscus leucopus.

We evaluated the susceptibility of laboratory-reared adult and infant white-footed mice (Peromyscus leucopus) to a known pathogenic isolate of Borrelia burgdorferi (N40). Two-month-old and 3-day-old Peromyscus were inoculated intradermally with 10(6) to 10(7) spirochetes. At 21 days for adults or 30 days for infants post inoculation, mice were killed, and tissues were cultured for spirochetes and examined microscopically. Based on serology and culture, adult mice became infected but did not have any gross or microscopic lesions. Mice inoculated as infants became infected, and also developed carditis and multifocal arthritis. Contact transmission between inoculated infants and their naive mothers was not observed. Age at inoculation appeared to be a critical factor in inducing Lyme borreliosis lesions in Peromyscus leucopus, as in other species.

BMP4 localization and PCNA expression during distraction osteogenesis of the porcine mandible.

This study characterized sequential molecular and cellular events in the porcine mandibular distraction osteogenesis (DO) wound. Nineteen Yucatan minipigs were divided into three treatment groups: Group A, unilateral mandibular distraction with 0 day latency, 1mm/day rate for 12 days, 24 days fixation (n=16); Group B, acute lengthening 12 mm (n=2); Group C, sham control (n=1). Group A was further divided by death date: mid-DO (n=5), end-DO (n=4), mid-fixation (n=5) and end-fixation (n=2). Groups B and C were killed on postoperative day 36, corresponding to end-fixation. Specimens were stained for proliferating cell nuclear antigen (PCNA) and bone morphogenetic protein-4 (BMP4). Cellular proliferation (PCNA) was assessed quantitatively and BMP4 staining was assessed on a semi-quantitative scale. Progenitor cell proliferation was greatest during mid-DO and decreased from end-DO through end-fixation. Proliferation in the acute lengthening group was elevated relative to sham control and comparable to end-DO. BMP4 staining intensity (localized to the periosteal cambium layer) was greatest during mid- and end-DO, decreased at mid-fixation and was undetectable at end-fixation. Progenitor cell proliferation and BMP4 expression are greatest during mid-DO and decrease progressively thereafter. At the time of death of the acute lengthening group, only increased cell proliferation was demonstrated.

Multiwavelength lidar observations of the decay phase of the stratospheric aerosol layer produced by the eruption of mount pinatubo in june 1991.

A small three-wavelength (355-, 532-, and 1064-nm) lidar system at NASA Langley Research Center in Hampton, Virginia, has been used since 1992 to make measurements on stratospheric aerosols. The data have been processed to study the decay rate of the stratospheric aerosol layer formed after the eruption of Mount Pinatubo in 1991 and its modulation, the aerosol effective radius, and the column mass loading. The stratospheric aerosol decay curves show annual and biennial cycles as well as short-term changes. At 532 nm, the decay time constant was 302 days for the period from February 1992 to August 1994 and had increased to 645 days for the period from September 1994 to December 1997. By 1996 the integrated stratospheric aerosol backscatter had fallen to levels (7.7 x 10(-5) sr(-1) at 532 nm) close to those seen in 1979 and 1989-1991. This decreasing trend was still continuing in 1997, showing no evidence for any anthropogenic contribution to the stratospheric aerosol.

Genetic profile of insertion mutations in mouse leukemias and lymphomas.

Murine leukemia retroviruses (MuLVs) cause leukemia and lymphoma in susceptible strains of mice as a result of insertional mutation of cellular proto-oncogenes or tumor suppressor genes. Using a novel approach to amplify and sequence viral insertion sites, we have sequenced >200 viral insertion sites from which we identify >35 genes altered by viral insertion in four AKXD mouse strains. The class of genes most frequently altered are transcription factors, however, insertions are found near genes involved in signal transduction, cell cycle control, DNA repair, cell division, hematopoietic differentiation, and near many ESTs and novel loci. Many of these mutations identify genes that have not been implicated in cancer. By isolating nearly all the somatic viral insertion mutations contributing to disease in these strains we show that each AKXD strain displays a unique mutation profile, suggesting strain-specific susceptibility to mutations in particular genetic pathways.

Humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus.

BACKGROUND AND OBJECTIVES: Two serotypes of autonomously replicating parvoviruses infect laboratory mice. Genome regions coding for the nonstructural proteins of minute virus of mice [MVM] and mouse parvovirus [MPV] are almost identical, whereas capsid-coding sequences are divergent. We addressed these questions: Does humoral immunity confer protection from acute infection after challenge with homotypic or heterotypic parvovirus, and if it confers protection against acute MPV infection, does it also protect against persistent MPV infection? METHODS: Infant mice without maternal antibody or antibody to MVM or MPV and young adult mice given normal mouse serum or antibody to MVM or MPV were challenged with homotypic or heterotypic virus. In situ hybridization with target tissues was the indicator of infection. RESULTS: Humoral immunity failed to confer protection against acute heterotypic parvovirus infection. In passive transfer studies, MPV DNA was observed occasionally in lymph nodes, intestine, or the spleen of MPV-challenged mice given homotypic antibody and kept for 6 or 28 days. Variable proportions of mice given MPV antibody and homotypic challenge had viral DNA in lymphoid tissues 56 days after virus inoculation. CONCLUSION: A mouse or colony that has sustained infection with MVM or MPV is probably fully susceptible to infection with the heterotypic virus.

Enzyme-linked immunosorbent assay for detection of antibody to lymphocytic choriomeningitis virus in mouse sera, with recombinant nucleoprotein as antigen.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibody to lymphocytic choriomeningitis virus (LCMV) in mouse sera. This assay is based on recombinant LCMV nucleoprotein generated in a baculovirus system. Sera from experimentally and naturally infected as well as noninfected mice were tested, and the results were compared with those obtained from an established immunofluorescence assay (IFA) that uses infected cells as antigen. An excellent correlation was found; the ELISA specificity and sensitivity were calculated to be 100 and 95% respectively. Unlike the IFA, this ELISA does not require the handling of infective virus. It eliminates the need to work with a zoonotic agent in the laboratory while allowing effective screening of laboratory mouse populations for LCMV antibody.

Characterization of a novel alpha-mannosidosis-causing mutation and its use in leukocyte genotyping after bone marrow transplantation.

Alpha-Mannosidosis is a lysosomal storage disorder caused by deficiency of lysosomal alpha-mannosidase (LAMAN). Major symptoms include mental retardation, skeletal changes and recurrent infections. Recently, a successful bone marrow transplantation (BMT) in an alpha-mannosidosis patient was reported. Here we show that this patient was homozygous for a novel mutation, a 1-bp insertion (1197-1198insA) in exon 9 of the LAMAN gene. By using this mutation as a marker, we demonstrate that 1 year post-BMT, the LAMAN genotype of the patient's leukocytes was identical to that of the donor. This method of genotyping blood cells is a fast and accurate way to monitor the colonization of donor bone marrow cells.