Paul W. Naumann

Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.

Role of iron in T cell activation : TH1 clones differ from TH2 clones in their sensitivity to inhibition of DNA synthesis caused by IGG mabs against the transferrin receptor and the iron chelator deferoxamine

TH1 and TH2 helper T cell clones have been studied with respect to their sensitivity to inhibition of DNA synthesis by an IgG anti-transferrin receptor antibody (ATRA), the iron chelator deferoxamine, and the combination of the two reagents. TH1 clones are very sensitive to ATRA-mediated inhibition of DNA synthesis while TH2 clones are very resistant, but both TH1 and TH2 clones show significant down-modulation of surface transferrin receptors after ATRA exposure. TH2 clones exhibit larger chelatable iron storage pools than TH1 clones, however, and even partial chelation of TH2 cell storage iron does not fully convert a TH2 clone to the ATRA sensitivity pattern of a TH1 clone. It is therefore proposed that the greater resistance of TH2 clones to ATRA mediated inhibition derives from the combined effects of larger and less labile iron storage pools. These studies provide novel evidence indicating that nonuniform iron metabolism can exist within the T cell compartment and thus raise questions as to why such differences exist and how they can be integrated into models of the T cell activation process. These studies also suggest that the cell-mediated immune response in vivo, which is known to be sensitive to iron deficiency, may be evoked by effector cells which resemble TH1 clones insofar as iron metabolism is concerned.

Development and translational imaging of a TP53 porcine tumorigenesis model

Cancer is the second deadliest disease in the United States, necessitating improvements in tumor diagnosis and treatment. Current model systems of cancer are informative, but translating promising imaging approaches and therapies to clinical practice has been challenging. In particular, the lack of a large-animal model that accurately mimics human cancer has been a major barrier to the development of effective diagnostic tools along with surgical and therapeutic interventions. Here, we developed a genetically modified porcine model of cancer in which animals express a mutation in TP53 (which encodes p53) that is orthologous to one commonly found in humans (R175H in people, R167H in pigs). TP53(R167H/R167H) mutant pigs primarily developed lymphomas and osteogenic tumors, recapitulating the tumor types observed in mice and humans expressing orthologous TP53 mutant alleles. CT and MRI imaging data effectively detected developing tumors, which were validated by histopathological evaluation after necropsy. Molecular genetic analyses confirmed that these animals expressed the R167H mutant p53, and evaluation of tumors revealed characteristic chromosomal instability. Together, these results demonstrated that TP53(R167H/R167H) pigs represent a large-animal tumor model that replicates the human condition. Our data further suggest that this model will be uniquely suited for developing clinically relevant, noninvasive imaging approaches to facilitate earlier detection, diagnosis, and treatment of human cancers.

Effects of Anti-Transferrin Receptor Antibodies on the Growth of Neoplastic Cells

One approach to creating a state of iron deprivation in tumors is to expose them to monoclonal antibodies against the transferrin receptor (ATRAs). This paper reviews the recent history of studies with ATRAs. Both multivalent (IgM or IgA) and bivalent (IgG) ATRAs exhibit anti-tumor activity in vitro and in vivo, but IgG ATRAs appear to be most effective when used with an iron chelator such as deferoxamine or when used in pairs. Much more information is needed in order to understand: (1) how ATRAs work by themselves and in conjunction with chelators; (2) why ATRAs differ from one another in terms of their inhibitory potency; (3) whether ATRAs can be used successfully in conjunction with other anti-tumor agents, and (4) why tumors exhibit marked differences in their sensitivity to the effects of ATRAs. The toxicity of iron deprivation arising from ATRA treatment alone seems modest, but only further experimental work in vivo and in phase-1 clinical trials can determine whether the most recent observations can be converted into truly useful therapeutic tools.

Differing Sensitivity of Non-Hematopoietic Human Tumors to Synergistic Anti-Transferrin Receptor Monoclonal Antibodies and Deferoxamine in vitro

We tested non-hematopoietic human tumors for in vitro sensitivity to either a pair of synergistic IgG antitransferrin (Tf) receptor monoclonal antibodies (MAbs), deferoxamine (DFO) or the combination thereof. With an equimolar mixture of the two MAbs (A27.15, E2.3), two prostate tumors showed similar degrees of maximal growth inhibition (PC-3: 35%, DU 145: 38%), two breast tumors showed more variability (MDA-MB-231: 26%, SK-BR-3: 52%) and two neuroblastomas showed the most variability (SK-N-SH: 4%, SK-N-MC: 76%). When the MAbs were applied together with DFO, the D50 for DFO was reduced for all tumors (PC-3: 2.5x, DU 145: 3.7x; MDA-MB-231: 2.9x, SK-BR-3: 1.9x, and SK-N-SH: 2.6x, SK-N-MC: 7.0x). Sensitivity to MAbs was more closely correlated with the relative decrease in Tf receptor density resulting from antibody exposure than with initial receptor density. The degree of reduction of D50 for DFO resulting from the joint application with the MAbs was, however, most closely related to the growth rate of the tumors. Since some non-hematopoietic tumors exhibit sensitivity to the effects of a synergistic pair of IgG anti-Tf receptor MAbs and DFO, it appears that further preclinical studies with such tumors, especially those with higher Tf densities, would be of interest.

Genetically modified species in research: opportunities and challenges for the histology core laboratory

Abstract Translational research using animal models has traditionally involved genetically modified rodents; however, there is increasing use of other novel genetically engineered species. As histology laboratories interface with researchers studying these novel species there will be many situations in which protocols will need to be adapted to the species, model and research goals. This paper gives examples of protocol adaptations to meet research needs and addresses common considerations that should be addressed for all research tissues submitted to the histotechnology laboratory. Positioning the histotechnologist, as well as the investigator, to meet the challenges associated with novel research models will help maximize research efficacy and quality.

Increased susceptibility to otitis media in a Splunc1-deficient mouse model

ABSTRACT Otitis media (inflammation of the middle ear) is one of the most common diseases of early childhood. Susceptibility to otitis is influenced by a number of factors, including the actions of innate immune molecules secreted by the epithelia lining the nasopharynx, middle ear and Eustachian tube. The SPLUNC1 (short palate, lung, nasal epithelial clone 1) protein is a highly abundant secretory product of the mammalian nasal, oral and respiratory mucosa that is thought to play a multifunctional role in host defense. In this study we investigated Splunc1 expression in the ear of the mouse, and examined whether this protein contributes to overall host defense in the middle ear and/or Eustachian tube. We found that Splunc1 is highly expressed in both the surface epithelium and in submucosal glands in these regions in wild-type mice. In mice lacking Splunc1, we noted histologically an increased frequency of otitis media, characterized by the accumulation of leukocytes (neutrophils with scattered macrophages), proteinaceous fluid and mucus in the middle ear lumens. Furthermore, many of these mice had extensive remodeling of the middle ear wall, suggesting a chronic course of disease. From these observations, we conclude that loss of Splunc1 predisposes mice to the development of otitis media. The Splunc1−/− mouse model should help investigators to better understand both the biological role of Splunc1 as well as host defense mechanisms in the middle ear. Summary: We document expression of the innate immune factor Splunc1 in the murine middle ear and Eustachian tube, and describe spontaneous development of otitis media in mice lacking functional Splunc1.

Glycosylation contributes to variability in expression of murine cytomegalovirus m157 and enhances stability of interaction with the NK-cell receptor Ly49H

NK cell‐mediated resistance to murine cytomegalovirus (MCMV) is controlled by allelic Ly49 receptors, including activating Ly49H (C57BL/6 strain) and inhibitory Ly49I (129 strain), which specifically recognize MCMV m157, a glycosylphosphatidylinositol‐linked protein with homology to MHC class I. Although the Ly49 receptors retain significant homology to classic carbohydrate‐binding lectins, the role of glycosylation in ligand binding is unclear. Herein, we show that m157 is expressed in multiple, differentially N‐glycosylated isoforms in m157‐transduced or MCMV‐infected cells. We used site‐directed mutagenesis to express single and combinatorial asparagine (N)‐to‐glutamine (Q) mutations at N178, N187, N213, and N267 in myeloid and fibroblast cell lines. Progressive loss of N‐linked glycans led to a significant reduction of total cellular m157 abundance, although all variably glycosylated m157 isoforms were expressed at the cell surface and retained the capacity to activate Ly49HB6 and Ly49I129 reporter cells and Ly49H+ NK cells. However, the complete lack of N‐linked glycans on m157 destabilized the m157‐Ly49H interaction and prevented physical transfer of m157 to Ly49H‐expressing cells. Thus, glycosylation on m157 enhances expression and binding to Ly49H, factors that may impact the interaction between NK cells and MCMV in vivo where receptor–ligand interactions are more limiting.

NIAM-Deficient Mice Are Predisposed to the Development of Proliferative Lesions including B-Cell Lymphomas

Nuclear Interactor of ARF and Mdm2 (NIAM, gene designation Tbrg1) is a largely unstudied inhibitor of cell proliferation that helps maintain chromosomal stability. It is a novel activator of the ARF-Mdm2-Tip60-p53 tumor suppressor pathway as well as other undefined pathways important for genome maintenance. To examine its predicted role as a tumor suppressor, we generated NIAM mutant (NIAMm/m) mice homozygous for a β-galactosidase expressing gene-trap cassette in the endogenous gene. The mutant mice expressed significantly lower levels of NIAM protein in tissues compared to wild-type animals. Fifty percent of aged NIAM deficient mice (14 to 21 months) developed proliferative lesions, including a uterine hemangioma, pulmonary papillary adenoma, and a Harderian gland adenoma. No age-matched wild-type or NIAM+/m heterozygous animals developed lesions. In the spleen, NIAMm/m mice had prominent white pulp expansion which correlated with enhanced increased reactive lymphoid hyperplasia and evidence of systemic inflammation. Notably, 17% of NIAM mutant mice had splenic white pulp features indicating early B-cell lymphoma. This correlated with selective expansion of marginal zone B cells in the spleens of younger, tumor-free NIAM-deficient mice. Unexpectedly, basal p53 expression and activity was largely unaffected by NIAM loss in isolated splenic B cells. In sum, NIAM down-regulation in vivo results in a significant predisposition to developing benign tumors or early stage cancers. These mice represent an outstanding platform for dissecting NIAMs role in tumorigenesis and various anti-cancer pathways, including p53 signaling.

A method for histopathological study of the multifocal nature of spinal cord lesions in murine experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a well-established mouse model for multiple sclerosis and is characterized by infiltration of mononuclear cells and demyelination within the central nervous system along with the clinical symptoms of paralysis. EAE is a multifocal and random disease, which sometimes makes histopathologic analysis of lesions difficult as it may not be possible to predict where lesions will occur, especially when evaluating cross sections of spinal cord. Consequently, lesions may be easily missed due to limited sampling in traditional approaches. To evaluate the entire length of the spinal cord while maintaining anatomic integrity, we have developed a method to section the cord within the decalcified spinal column, which allows for the study of the multifocal nature of this disease and also minimizes handling artifact. HE and Luxol fast blue staining of these spinal cord sections revealed a paucity of lesions in some areas, while others showed marked inflammation and demyelination. The percentage of spinal cord affected by EAE was evaluated at four separate areas of longitudinally sectioned cord and it varied greatly within each animal. Immunohistochemical staining of in situ spinal cords which had undergone decalcification was successful for key immuno-markers used in EAE research including CD3 for T cells, B220 for B cells and F4/80 for murine macrophages. This method will allow investigators to look at the entire spinal cord on a single slide and evaluate the spinal cord with and without classic EAE lesions.