Genetic dissection of IGF1-dependent and -independent effects of permanent GH excess on postnatal growth and organ pathology of mice.

To study insulin-like growth factor 1 (IGF1)-independent effects of permanent growth hormone (GH) excess on body and organ growth and pathology in vivo, hemizygous bovine GH transgenic mice with homozygous disruption of the Igf1 gene (Igf1(-/-)/GH) were generated, and examined in comparison to Igf1(-/-), Igf1(+/-), wild-type (WT), Igf1(+/-)/GH, and GH mice. GH mice and Igf1(+/-)/GH mice showed increased serum IGF1 levels and the well-known giant-phenotype of GH transgenic mice. In contrast, the typical dwarf-phenotype of Igf1(-/-) mice was only slightly ameliorated in Igf1(-/-)/GH mice. Similar to GH mice, Igf1(-/-)/GH mice displayed hepatocellular hypertrophy, glomerulosclerosis, and reduced volumes of acidophilic cells in the pituitary gland. However, GH excess associated skin lesions of male GH mice were not observed in Igf1(-/-)/GH mice. Therefore, development of GH excess induced liver-, kidney-, and pituitary gland-alterations in GH transgenic mice is independent of IGF1 whereas GH stimulated body growth depends on IGF1.

Peri/nuclear localization of intact insulin-like growth factor binding protein-2 and a distinct carboxyl-terminal IGFBP-2 fragment in vivo.

Insulin-like growth factor binding protein-2 (IGFBP-2) as one of the most important IGFBPs has never been assessed in the intracellular compartment in vivo. Since there is evidence for novel intracellular functions of distinct IGFBPs, we investigated the presence of IGFBP-2 inside the cell. In peri/nuclear fractions of various tissues isolated from IGFBP-2 transgenic and non-transgenic mice we were able to show the presence of intact IGFBP-2. In addition, we demonstrate the presence of a highly conserved carboxyl-terminal IGFBP-2 fragment in the peri/nuclear fraction by using different peptide-induced antibodies. In pancreatic sections, confocal microscopy revealed the presence of IGFBP-2 on the nuclear surface but not within the nucleus. Our findings suggest novel functions of intact IGFBP-2 and IGFBP-2 fragments within the cell.

Mutation of the RGD sequence does not affect plasma membrane association and growth inhibitory effects of elevated IGFBP-2 in vivo.

Using insulin-like growth factor-binding protein-2 (IGFBP-2) transgenic mice (D mice) as a model of elevated IGFBP-2 expression, which is often found in unphysiological conditions, we found association of IGFBP-2 to purified plasma membranes of many organs. To determine whether the RGD (Arg-Gly-Asp) motif of IGFBP-2 mediates cell surface binding in vivo, we mutated the RGD motif of IGFBP-2 into an RGE (Arg-Gly-Glu) sequence and produced transgenic mice (E mice) which express elevated amounts of mutated IGFBP-2. Our data demonstrate that in vivo IGFBP-2 cell surface association is not dependent on the RGD motif and that mutation of this sequence does not alter growth inhibitory effects of IGFBP-2.

Contrasting obesity phenotypes uncovered by partial leptin receptor gene deletion in transgenic mice.

Non-insulin-dependent diabetes mellitus (type 2 diabetes) is known to be a polygenic and polyfactorial disorder. Here we describe the long-term examination of a transgenic mouse line showing the disruption of the leptin receptor (Lepr, Ob-R) gene caused by transgene insertion. The absence of the expression of the long isoform Ob-Rb uncovered a strong variation of the obesity and diabetes phenotype in the homozygous mutant mice of the outbred strain used. One part of the homozygous mice developed severe persistent early-onset obesity, whereas the other part developed cachexia after having shown initial obesity in the examination period up to 26 weeks p.p. The leptin-receptor-defective mice of this line might serve as a model for the investigation of genes modulating the development and mode of expression of diabetes.

Large-scale N-ethyl-N-nitrosourea mutagenesis of mice--from phenotypes to genes.

The most important tool for obtaining insight into the function of genes is the use of mutant model organisms. Homologous recombination in embryonic stem cells allows the systematic production of mouse mutants for any gene that has been cloned. Gene trap strategies have been designed to interrupt even unknown genes which are tagged by the inserted vector and can be characterised structurally and functionally. Complementary to such 'gene-driven' approaches, 'phenotype-driven' approaches are necessary to identify new genes or gene products through a search for mutants with specific defects, uncovering the function of genetic pathways in physiological and pathological processes. Mutagenesis using the alkylating agent N-ethyl-N-nitrosourea (ENU) is a powerful approach for the production of such mouse mutants. Since ENU induces mainly point mutations in premeiotic spermatogonia, this strategy allows the production of multiple alleles of a particular gene, which is pivotal for a fine tuned analysis of its function.

Overgrowth of skin in growth hormone transgenic mice depends on the presence of male gonads.

Growth hormone has been shown to possess stimulatory effects on various connective tissues. We observed that skin growth in male rat phosphoenolpyruvate carboxykinase-bovine growth hormone transgenic mice (serum growth hormone levels: 740-1940 ng per ml) is progressive with age, resulting in an "oversized coat" phenotype with a marked increase in absolute and relative skin weight and surface area, and in thickness of the dermis. Histologic changes include severe dermal fibrosis and replacement of subdermal adipose tissue by fibrous tissue. Apart from an increase in skin surface area, these changes were not noted in female transgenic mice, arguing for a specific interaction of growth hormone with male sex hormones. To clarify this point, 6 wk old male transgenic mice and control mice were castrated and compared with their noncastrated counterparts in parameters of skin growth at an age of 8 mo. The skin weight of castrated transgenic mice was smaller (p < 0.01) than that of intact transgenic mice both absolutely and relative to body weight. The relative skin weight of castrated transgenic mice was in the same range as in intact and castrated control mice. Absolute and relative skin area of castrated transgenic mice was greater (p < 0. 001 and p < 0.05) than in controls but lower than in intact transgenic mice (p < 0.001 and p < 0.05). When compared with control mice, intact transgenic mice displayed an increase (p < 0.01) in the thickness of dermis. In castrated transgenic mice the thickness of the dermis was in the same range as in control mice. Our findings demonstrate a specific interaction of growth hormone with male sex hormones resulting in a marked stimulation of skin growth.

CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.

The proper function of immune surveillance requires well-coordinated mechanisms in order to guide the patrolling immune cells through peripheral tissues and into secondary lymphoid organs. Analyzing gene-targeted mice, we identified the chemokine receptor CCR7 as an important organizer of the primary immune response. CCR7-deficient mice show severely delayed kinetics regarding the antibody response and lack contact sensitivity and delayed type hypersensitivity reactions. Due to the impaired migration of lymphocytes, these animals reveal profound morphological alterations in all secondary lymphoid organs. Upon activation, mature skin dendritic cells fail to migrate into the draining lymph nodes. Thus, in order to bring together lymphocytes and dendritic cells to form the characteristic microarchitecture of secondary lymphoid organs, CCR7 is required to rapidly initiate an adoptive immune response.

[Sex-specific analysis of bone mass in normal and growth hormone transgenic mice using dual energy x-ray absorptiometry (DXA)]. / Geschlechtsspezifische Analyse der Knochenmasse normaler und Wachstumshormon-transgener Mäuse mittels Zweienergie-Röntgen-Absorptiometrie (DXA).

The aim of the present study was the non-invasive, sex-specific measurement of bone mass in bovine growth hormone (bGH) transgenic mice and normal controls with dual energy X-ray absorptiometry (DXA). The transgenic mouse constitutes a suitable animal model to study the influence of growth hormone on the skeletal system. We analysed 28 animals, aged 12 weeks (14 transgenic, 14 controls, 7 male and 7 female, respectively), using a peripheral DXA scanner that had been adapted to the measurement of small animals. At a measurement time of 20 min, the precision (RMS average CV%) was 4.4% for bone mass (BMC), 2.5% for areal bone density (BMD), 0.86% for total body weight and 4.5% for the percentage BMC (relative to body weight). While the absolute bone mass was not significantly different between male and female animals, we found a higher percentage of the BMC relative to the total bone mass in females (+21% in controls, +31% in transgenics; p < 0.01). The absolute bone mass was higher in the transgenic animals (+71% in females and +62% in males; p < 0.01), but relative to the body weight the transgenic females yielded similar and the transgenic males lower values (-7.2%; p < 0.05). Using DXA it is possible to non-invasively determine the mass of mineralised tissue in the mouse with relatively high precision and to effectively discriminate between different groups. Although a strong influence of growth hormone on the absolute bone mass is observed, the results show that this increase is not higher than that of the total body mass.

A non-destructive technique for 3-D microstructural phenotypic characterisation of bones in genetically altered mice: preliminary data in growth hormone transgenic animals and normal controls.

A non-destructive, three-dimensional technique for microstructural phenotypic characterisation of skeletal elements in genetically altered mice is presented. Preliminary data in bovine growth-hormone transgenic animals and control littermates are shown. The technique is based on microcomputed tomography (microCT) and digital postprocessing and allows for a differential quantitative analysis of the cortical and trabecular bone compartments in the axial and peripheral skeleton. The distal femora and the first lumbar vertebral bodies of six animals were CT scanned in the axial plane with an isotropic resolution of 20 microm. The periostal surface and the marrow spaces were segmented fully automatically, and the trabecular and cortical compartments were separated interactively. After 3-D reconstruction, various regions of interest (diaphyseal, metaphyseal and epiphyseal) were selected for the analysis. The femora and vertebrae of the transgenic animals showed obvious differences in size, shape, and trabecular arrangement compared with the control animals. The total bone mass was increased by a factor of two to three, but the trabecular bone was increased much more (up to 12 times) than the cortical bone. The transgenic animals showed an increased ratio of trabecular vs cortical bone (0.90 to 1.27 vs 0.14 to 0.36 in the femoral diaphysis) and an elevated trabecular bone volume fraction (49% to 73% vs 18% to 43% in the femoral metaphysis). The mean 3-D cortical thickness was similar in the normal and transgenic animals (values between 93 microm and 232 microm in the dia- and metaphyses), but the minimal cortical thickness was lower in the transgenic animals (22 to 31 microm vs 54 microm to 110 microm in the diaphysis). The technique presented is suitable for phenotypic characterisation of bone structure in genetically altered mice.

In vivo and in vitro characterization of two camelpoxvirus isolates with decreased virulence.

Two camelpoxvirus (CPV) strains isolated from camels with generalized skin-disease were serially passaged on Vero cells. Various phenotypic properties were investigated in vitro and in vivo and compared with those of the corresponding wildtype strains. In many aspects no differences were observed. However, in a mouse model both passaged strains proved to be highly attenuated. In addition, both strains failed to replicate in a cell line derived from camel skin cells. Comparison of physical maps established for enzymes HindIII and Xhol revealed deletions accounting for a total of 22 kbp in one attenuated strain. In the second strain only minor alterations were noted.

Characterization of camelpoxvirus isolates from Africa and Asia.

Five orthopoxvirus isolates of camels from different geographic regions of Africa and Asia were analysed with respect to their biological and genomic attributes. The behaviour of the isolates in various cell cultures, the type of pock lesions on the chorioallantoic membrane of embryonated chicken eggs, and the respective ceiling temperatures were determined. Additionally, physical maps for restriction endonucleases HindIII and XhoI were established. The data obtained from biological assays and DNA analyses demonstrated minor differences between the five isolates. However, these findings confirm previous reports suggesting that orthopoxviruses of camels constitute a separate species within the genus Orthopoxvirus.

[The effectiveness of immunization with vaccinia virus type "MVA" against an infection with cowpox virus type "OPV 85" in rabbits]. / Die Wirksamkeit einer Immunisierung mit Vaccinia-Virus Stamm "MVA" gegen eine Infektion mit Kuhpocken-Virus Stamm "OPV 85" beim Kaninchen.

The immunological efficacy of vacciniavirus "MVA" was tested against a dermal and intradermal infection with cowpoxvirus "OPV 85" in rabbits: A single vaccination with "MVA" provided only insufficient immunity, a revaccination induced good immunity. Intramuscular immunizations protected better than subcutaneous applications. Immunized rabbits showed "revaccination reactions" after infection with "OPV 85" indicating a cellular immunity. After immunization with "MVA" and after infection with cowpoxvirus "OPV 85" all rabbits developed N- and ELISA-antibodies. HAI-antibodies were not found after immunization, but indicated a multiplication of cowpoxvirus after challenge. Vacciniavirus "MVA" is suggested for immunization of man and animal against possible infections with cowpoxvirus and cowpoxlike viruses.

[Infections with original cowpox virus and cowpox-like agents in humans and animals: a literature review]. / Infektionen mit originärem Kuhpockenvirus und kuhpockenähnlichen Erregern bei Mensch und Tier: Eine Literaturübersicht.

In an evaluation of literature the biological, physical-chemical and antigenic characteristics of cowpoxviruses and cowpox-like agents are presented, the according diseases following a natural and experimental infection are described and their epizootiological and epidemiological aspects discussed.

A serological survey of the prevalence of Aujeszky's disease antibodies in Thailand using enzyme-linked immunosorbent assays (ELISA), serum neutralization (SN) and latexagglutination tests (LT).

The presence of Aujeszky's disease (AD) antibodies in eluates of whole blood on filter paper and corresponding sera from Thai pigs was determined by ELISA, SNT and LT. From a total of 800 samples tested by ELISA, 26% of the sera and 18% of the eluates showed positive results. From 640 samples tested by SNT and chosen because they gave negative, suspicious, or weakly positive results by ELISA, 22% were positive. A total of 182 suspicious samples were also tested by LT, and among them 63 (35%) were clearly positive. The investigation demonstrated that the older the animal, the greater the probability that antibodies would be found. Owner surveys tended to state that few animals had been vaccinated. This coupled with the high frequency of antibodies detected, indicates that AD-infection among Thailand's swine population is a common problem.

[Experimental studies of in vitro cultivation of the cells of Kärtner honeybees (Apis mellifera carnica Pollmann, 1879)]. / Experimentelle Untersuchungen zur in vitro-Kultivierung von Zellen der Kärntner Honigbiene (Apis mellifera carnica Pollmann, 1879).

A synopsis about published methods and results on experiments to cultivate bee cells in vitro is given. Experimental investigations were performed with haemocytes of larvae of the L-5 stage using many different media and methods for the preparation of primary tissue culture. Monolayers could be prepared and a high rate of reproduction has been achieved, although subpassages could not be obtained. Haemocytes could be kept alive up to 27 days by using BML-TC/7A medium according to Gardiner and Stockdale, modified by Skatulla (pers. communic., 1987). Further experiments are necessary in order to study the suitability of bee cells to detect specific pathogens and toxic substances.

[Epizoologic studies of the detection of antibodies against Aujeszky's disease virus in sera and blood eluates of swine from Thailand using ELISA ("Enzygnost," Behring), serum neutralization test and "Aujeszky Latex Kit" (Iffa Merieux)]. / Epizootiologische Untersuchungen zum Nachweis von Antikörpern gegen das Virus der Aujeszkyschen Krankheit in Seren und Bluteluaten thailändischer Schweine mittels ELISA ("Enzygnost", Behring), Serum-Neutralisations-Test und "Aujeszky-Latex-Kit" (Iffa Merieux).

The results of three tests for Aujeszky's disease were analysed and compared. The presence of Aujeszky's antibodies was determined by "Enzyme-linked-Immunosorbent-Assays" (ELISA, "Enzygnost"), Behring company, Marburg; "Serum-Neutralization-Tests" (SNT); and "Latex Agglutination-Tests" (LT, "Aujeszky-Latex-Kit"), Iffa Merieux company, Laupheim. Whole blood and sera samples were taken from 805 swine from 26 of Thailand's provinces. These samples were analysed to determine if eluates of whole blood on filter paper were as effective as corresponding sera samples in determining the presence of Aujeszky's disease antibodies. From a total of 805 samples, 26% of the serum and 18% of the blood eluate samples showed a positive result when tested by the ELISA method. Clearly, testing whole blood eluates provides results which are inferior to results from sera samples. Therefore the ELISA whole blood eluates test can only be recommended with reservations. Further testing was done on 645 serum samples using SNT. Samples tested were those which gave negative, suspicious, or weakly positive results when tested by ELISA. Using SNT, 23% of these showed a positive result. Many serum and blood eluate samples were also tested by LT. Most of these test samples were chosen because they were deemed suspicious. Suspicious samples were defined as those which had deviant test results. According to these results the sensitivity of LT was between the sensitivity of SNT and ELISA. Owner survey responses tended to state that few animals had been vaccinated. This coupled with the frequency of antibody occurrence proves the high rate of infection among Thailand's swine population.