Inbred wild type mouse strains have distinct spontaneous morphological phenotypes.

The mouse is the most commonly used animal for modelling human disease. New approaches for generating genetically manipulated mouse models to represent human disease, as well as target the function of specific genes, has increased the importance of mice in biomedical science. For the correct interpretation of alterations in mouse phenotype the basic morphology of background mouse strains must be known. Despite on-going efforts to create publicly available baseline phenotypic data, the information concerning spontaneous lesions in wild-type mice is incomplete and scattered so far, and further studies are needed. We addressed this problem by screening haematoxylin-eosin stained sections of brain, reproductive organs, urinary bladder, kidney, thyroid, parathyroid, heart, lung, spleen, thymus, lymph nodes, adrenal glands, stomach, intestine, liver, skin and pancreas of six commonly used inbred mouse strains (C57BL6/J, C57BL6/NTac, C3HeB/FeJ, BALB/cByJ, 129P2/OlaHsd and FVB/N) for inherent spontaneous morphological lesions. Interesting spontaneous phenotypes were seen in morphology of the liver, pancreas, adrenal glands, lungs, intestines and heart. In conclusion, care should be taken when choosing the background mouse strain for genetic manipulations, since different mouse strains harbour different inherent lesions that can affect the function of targeted genes, interpretation of results and translation of results to model human disease.

[Characterization of ENU-mutant mice. Animal models for human diseases using morphological and molecular methods]. / Charakterisierung von ENU-Mausmutanten. Tiermodelle für menschliche Erkrankungen mittels morphologischer und molekularer Methoden.

Following sequencing of the human genome there are new challenges to decipher the knowledge concerning gene function and the role of gene interactions and pathways leading to disease. Mouse models have proven to be an ideal tool for this purpose. Point mutations induced by chemical mutagenesis by N-ethyl-N-nitrosourea (ENU) offer possibilities for the analysis of the phenotypic outcome of a single base pair exchange on the entire organism. The Munich ENU mouse mutagenesis project is part of the worldwide efforts to obtain mutations for each gene. The generation of new alleles or allelic series offers relevant insights into the relevance of single gene sections. Various mouse models for human diseases have been generated by a systematic large-scale genome-wide phenotyping screen in the last decade. This work illustrates how the implementation of the ENU mouse mutagenesis project with gene identification and parallel high-throughput screening is taking advantage of local cooperation with experienced phenotyping groups at the Helmholtz Zentrum München, leading to major advances in the functional analysis of the mammalian genome.

The German Mouse Clinic: a platform for systemic phenotype analysis of mouse models.

The German Mouse Clinic (GMC) is a large scale phenotyping center where mouse mutant lines are analyzed in a standardized and comprehensive way. The result is an almost complete picture of the phenotype of a mouse mutant line--a systemic view. At the GMC, expert scientists from various fields of mouse research work in close cooperation with clinicians side by side at one location. The phenotype screens comprise the following areas: allergy, behavior, clinical chemistry, cardiovascular analyses, dysmorphology, bone and cartilage, energy metabolism, eye and vision, host-pathogen interactions, immunology, lung function, molecular phenotyping, neurology, nociception, steroid metabolism, and pathology. The German Mouse Clinic is an open access platform that offers a collaboration-based phenotyping to the scientific community (www.mouseclinic.de). More than 80 mutant lines have been analyzed in a primary screen for 320 parameters, and for 95% of the mutant lines we have found new or additional phenotypes that were not associated with the mouse line before. Our data contributed to the association of mutant mouse lines to the corresponding human disease. In addition, the systemic phenotype analysis accounts for pleiotropic gene functions and refines previous phenotypic characterizations. This is an important basis for the analysis of underlying disease mechanisms. We are currently setting up a platform that will include environmental challenge tests to decipher genome-environmental interactions in the areas nutrition, exercise, air, stress and infection with different standardized experiments. This will help us to identify genetic predispositions as susceptibility factors for environmental influences.

Histopathological features of autoimmune pancreatitis.

Autoimmune pancreatitis (AIP) is a chronic fibroinflammatory disease of the pancreas characterised by lymphoplasmacytic infiltrates, interstitial fibrosis, periductal inflammation and periphlebitis. Although first described more than four decades ago, it has not gained widespread attention until the 1990s when new insights into its aetiology, clinical presentation and management were discovered. Although nowadays widely accepted as a form of chronic pancreatitis, recent evidence suggests that AIP might not be confined to the pancreas but rather be an inflammatory pancreaticobiliary disease (autoimmune pancreatocholangitis, AIPC) with possible systemic involvement and association with other autoimmune disorders. This article reviews current concepts of AIP with special focus on the histopathological features of the disease.

Standardized morphological phenotyping of mouse models of human diseases within the German Mouse Clinic.

Inbred strains are the raw material for the generation of Genetically Engineered Mice (GEM) that have become indispensable tools for cancer research, and for the identification of genes involved in human diseases. The "German Mouse Clinic" was designed to provide the scientific community with a systematic, standardized and comprehensive phenotyping of mouse models on various genetic backgrounds and generated by different methods (transgenic, knockouts, ENU mutagenesis screen and gene-trap approaches). The pathology screen within the German Mouse-Clinic was conceived to ensure a complete morphologic phenotype of mouse models, to support discovery of genes functions, and to understand how these genes influence the development of human diseases. The goal is to define disease entities that can be recognized by a pathologist and relate them to human disorders when possible. Knowing the inherent morphologic phenotype of the most frequent used mouse strains is of utmost importance for the correct interpretation of mouse models. The main challenges, which pathologist are confronted to validate mouse models for human diseases include (1) knowledge of mouse biology and of histological differences between mouse strains and humans, (2) the terminology that should be used for the classification of neoplastic lesions in GEM's, (3) to asses the usefulness of a particular GEM as model for a human disease.

Patched target Igf2 is indispensable for the formation of medulloblastoma and rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children (Dagher, R., and Helman, L. (1999) Oncologist 4, 34-44), whereas medulloblastoma, a highly malignant tumor of the cerebellum, accounts for 20% of childhood brain tumors (Goodrich, L. V., and Scott, M. P. (1998) Neuron 21, 1243-1257). Both tumors are associated with a deficiency in the tumor suppressor Patched (PTCH) in Gorlin syndrome (Gorlin, R. J. (1987) Medicine (Baltimore) 66, 98-113), and they are present in the corresponding murine models. RMS in Ptch mutant mice consistently contain elevated levels of the tumor growth-promoting insulin-like growth factor 2 (Igf2). We have investigated the mechanism of Igf2 overexpression and its significance in medulloblastoma and RMS tumorigenesis. Here we report that Igf2 is indispensable for the formation of medulloblastoma and RMS in Ptch mutants. Overexpression of Igf2 in RMS in these mice does not involve loss of imprinting, uniparental disomy, amplification of the Igf2 locus, or polyploidy. Since Igf2 is also overexpressed in non-tumor tissue deficient in Ptch, these observations suggest that Ptch regulates Igf2 levels through a transcriptional mechanism. They also identify Igf2 as a potential target for medulloblastoma and RMS.

TNF gene expression in monocytes of low and high responder individuals.

Individuals with a consistently lower immune response may be more susceptible to infection but less prone to autoimmune disease or severe sepsis. The molecular mechanisms determining the low responder status are, however, unclear. We have screened 60 male donors for tumour necrosis factor (TNF) protein levels after stimulation of monocytes with lipopolysaccharide (LPS). Among these we identified three donors each that consistently had a level of less than 20% (low responders; LR) or of more than 80% (high responders; HR) of the maximum response seen in this population. Northern blot analysis of TNF mRNA after LPS stimulation revealed lower transcript levels in LR. Half life determination after actinomycin D blockade showed a similar decay rate for LR and HR and after blockade of degradation by cycloheximide treatment mRNA levels increased but LR remained lower compared to HR. These data indicate that the lower TNF mRNA levels in LR are not due to a more rapid mRNA degradation but rather a result of lower transcription. Transcripts for interleukin 6 (IL-6) were also low in LPS-stimulated monocytes of LR. Because expression of the LPS receptor CD14 was similar in LR and HR monocytes, our data suggest that differences in signal transduction account for the LR and HR phenotype.

Interleukin-10 drives human monocytes to CD16 positive macrophages.

Cells of the monocyte/macrophage lineage are heterogenous in that some types express the CD16 molecule (Fc receptor for IgG, type III), while others are negative. We now show that culture of human blood monocytes with interleukin-10 (IL-10) will induce high levels of cell surface CD16 within 18 hr, and this goes along with increased transcript levels. After prolonged culture for 36 hr, control cultures also become CD16 positive and this induction can be prevented by anti-IL-10, but not by anti-tumor necrosis factor (TNF) antibody or isotype control. In vitro culture of blood monocytes results in a spontaneous decrease of the myelomonocytic stem cell antigen CD33, suggesting that the cells undergo maturation. IL-10 treatment will accelerate this process and result in a further reduction of cell surface CD33. These data indicate that IL-10 promotes monocyte maturation and directs these cells to develop into CD16 positive macrophages.

Retinoic acid inhibits basal and interferon-gamma-induced expression of intercellular adhesion molecule 1 in monocytic cells.

Retinoic acid (RA) and 1,25-(OH)2-vitamin D3 (1,25-D3) induced U937 cell maturation into distinct monocytic phenotypes, as demonstrated by up-regulation of CD23 by RA and CD14 by 1,25-D3. Differentiation by RA but not by 1,25-D3 was associated with reduction of basal and complete suppression of interferon-gamma (IFN-gamma)-stimulated intercellular adhesion molecule 1 (ICAM-1) expression. Induction of cyclooxygenase activity by RA and attenuation of basal ICAM-1 expression exhibited similar kinetics. Treatment with indomethacin prevented and prostaglandin E2 (PGE2), dibutyryl-cAMP, or forskolin mimicked reduction of basal ICAM-1 expression by RA, indicating that this effect of RA is mediated by PGE2 synthesis and subsequent cAMP elevation. In contrast, suppression of IFN-gamma-induced ICAM-1 expression by RA was only partly reversible by indomethacin, suggesting that inhibition of IFN-gamma stimulation was not completely due to cyclooxygenase induction. RA did not always counter-act IFN-gamma, as it cooperated with IFN-gamma in down-regulating very late activation antigen 4. Specific polymerase chain reaction and Northern blotting of ICAM-1 mRNA revealed that RA suppressed ICAM-1 induction by IFN-gamma at the transcriptional level. RA also blocked ICAM-1 induction by IFN-gamma in isolated human blood monocytes. In conclusion, inhibition of basal and stimulated ICAM-1 expression in monocytic cells may provide a mechanism for beneficial anti-inflammatory effects of retinoids.

The two soluble forms of the lipopolysaccharide receptor, CD14: characterization and release by normal human monocytes.

CD14, a glycolipid-anchored membrane glycoprotein, acts as a high affinity lipopolysaccharide receptor on leukocytes. We previously reported that the Mono-Mac-6 cell line releases two different soluble forms of CD14 (sCD14) (Labeta et al., Eur. J. Immunol. 1993. 23: 2144). Here we show that the two sCD14, which we now refer to as sCD14 alpha (low M(r)) and sCD14 beta (high M(r)), are also synthesized and released by normal human monocytes and present in normal plasma. Their mechanism of release was examined by using the Mono-Mac-6 cell line, chinese hamster ovary cell (CHO)/CD14+ transfectants and plasma from paroxysmal nocturnal hemoglobinuria (PNH) patients. It was found that: (1) sCD14 beta is released faster than sCD14 alpha and that the release of the latter is a lengthy process. (2) Monensin blocked the biosynthesis of membrane-bound CD14 (mCD14) and sCD14, additionally, a 50-kDa CD14 polypeptide accumulated in the cell lysate, suggesting that the different forms of CD14 may have a common precursor. (3) Monensin also blocked the release of sCD14 alpha from surface-labeled cells, suggesting that conversion of mCD14 to sCD14 alpha involves a mechanism of endocytosis followed by exocytosis. Interestingly, (4) sCD14 alpha and sCD14 beta were detected in PNH plasma, indicating that sCD14 alpha may also derive from an endogenous pathway. (5) Phospholipase C-released CD14 was identical in size to mCD14, thus differed from sCD14 beta by approximately 2000, indicating that release of sCD14 beta involves further processing. (6) CHO cells transfected with a CD14 cDNA coding for an eight C-terminal amino acids shorter product released an sCD14 beta-like form; thus absence of the eight C-terminal amino acids prevented mCD14 expression but not the secretion of sCD14 beta. The characterization of sCD14 alpha and sCD14 beta reported here may be useful for better understanding of variations in sCD14 levels in pathological conditions and the contribution of each sCD14 in sepsis and other, as yet unknown functions.