Influenza Virus Affects Intestinal Microbiota and Secondary Salmonella Infection in the Gut through Type I Interferons.

Human influenza viruses replicate almost exclusively in the respiratory tract, yet infected individuals may also develop gastrointestinal symptoms, such as vomiting and diarrhea. However, the molecular mechanisms remain incompletely defined. Using an influenza mouse model, we found that influenza pulmonary infection can significantly alter the intestinal microbiota profile through a mechanism dependent on type I interferons (IFN-Is). Notably, influenza-induced IFN-Is produced in the lungs promote the depletion of obligate anaerobic bacteria and the enrichment of Proteobacteria in the gut, leading to a "dysbiotic" microenvironment. Additionally, we provide evidence that IFN-Is induced in the lungs during influenza pulmonary infection inhibit the antimicrobial and inflammatory responses in the gut during Salmonella-induced colitis, further enhancing Salmonella intestinal colonization and systemic dissemination. Thus, our studies demonstrate a systemic role for IFN-Is in regulating the host immune response in the gut during Salmonella-induced colitis and in altering the intestinal microbial balance after influenza infection.

Positive cross talk between protein kinase D and ß-catenin in intestinal epithelial cells: impact on ß-catenin nuclear localization and phosphorylation at Ser552.

Given the fundamental role of ß-catenin signaling in intestinal epithelial cell proliferation and the growth-promoting function of protein kinase D1 (PKD1) in these cells, we hypothesized that PKDs mediate cross talk with ß-catenin signaling. The results presented here provide several lines of evidence supporting this hypothesis. We found that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II (ANG II), a potent inducer of PKD activation, promoted endogenous ß-catenin nuclear localization in a time-dependent manner. A significant increase was evident within 1 h of ANG II stimulation (P< 0.01), peaked at 4 h (P< 0.001), and declined afterwards. GPCR stimulation also induced a marked increase in ß-catenin-regulated genes and phosphorylation at Ser(552) in intestinal epithelial cells. Exposure to preferential inhibitors of the PKD family (CRT006610 or kb NB 142-70) or knockdown of the isoforms of the PKD family prevented the increase in ß-catenin nuclear localization and phosphorylation at Ser(552) in response to ANG II. GPCR stimulation also induced the formation of a complex between PKD1 and ß-catenin, as shown by coimmunoprecipitation that depended on PKD1 catalytic activation, as it was abrogated by cell treatment with PKD family inhibitors. Using transgenic mice that express elevated PKD1 protein in the intestinal epithelium, we detected a marked increase in the localization of ß-catenin in the nucleus of crypt epithelial cells in the ileum of PKD1 transgenic mice, compared with nontransgenic littermates. Collectively, our results identify a novel cross talk between PKD and ß-catenin in intestinal epithelial cells, both in vitro and in vivo.

Gene therapy/bone marrow transplantation in ADA-deficient mice: roles of enzyme-replacement therapy and cytoreduction.

Gene therapy (GT) for adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) can provide significant long-term benefit when patients are given nonmyeloablative conditioning and ADA enzyme-replacement therapy (ERT) is withheld before autologous transplantation of γ-retroviral vector-transduced BM CD34+ cells. To determine the contributions of conditioning and discontinuation of ERT to the therapeutic effects, we analyzed these factors in Ada gene knockout mice (Ada(-/-)). Mice were transplanted with ADA-deficient marrow transduced with an ADA-expressing γ-retroviral vector without preconditioning or after 200 cGy or 900 cGy total-body irradiation and evaluated after 4 months. In all tissues analyzed, vector copy numbers (VCNs) were 100- to 1000-fold greater in mice receiving 900 cGy compared with 200 cGy (P < .05). In mice receiving 200 cGy, VCN was similar whether ERT was stopped or given for 1 or 4 months after GT. In unconditioned mice, there was decreased survival with and without ERT, and VCN was very low to undetectable. When recipients were conditioned with 200 cGy and received transduced lineage-depleted marrow, only recipients receiving ERT (1 or 4 months) had detectable vector sequences in thymocytes. In conclusion, cytoreduction is important for the engraftment of gene-transduced HSC, and short-term ERT after GT did not diminish the capacity of gene-corrected cells to engraft and persist.

Negative cross-talk between calcium-sensing receptor and ß-catenin signaling systems in colonic epithelium.

Here, we examined the role of the extracellular Ca(2+)-sensing receptor (CaSR) in the control of colonic epithelial cell proliferation in vivo and changes in ß-catenin triggered by CaSR stimulation in human colonic epithelial cells in vitro. The in vivo studies, using a novel Casr intestinal-specific knock-out mouse, indicate that the genetic ablation of the Casr leads to hyperproliferation of colonic epithelial cells, expansion of the proliferative zone, changes in crypt structure, and enhanced ß-catenin nuclear localization. The in vitro results indicate that stimulation of the CaSR, by Ca(2+) or by the calcimimetic R-568, produced a striking and time-dependent decrease in the phosphorylation of ß-catenin at Ser-552 and Ser-675, two amino acid residues that promote ß-catenin transcriptional activity. The reduced phosphorylation of ß-catenin coincided with a decline in its nuclear localization and a marked redistribution to the plasma membrane. Furthermore, CaSR stimulation promoted a down-regulation of ß-catenin-mediated transcriptional activation. These studies demonstrate that signaling pathways emanating from the CaSR control colonic epithelial cell proliferation in vivo and suggest that the mechanism involves regulation of ß-catenin phosphorylation.

Long-term survival of the juvenile lethal arginase-deficient mouse with AAV gene therapy.

Arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia. Human patients suffer from neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation. In a murine model, onset of the phenotypic abnormality is heralded by weight loss beginning around day 15 with death occurring typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. The goal of this study was to address the development of a gene therapy approach for arginase deficiency beginning in the neonatal period. Lifespan extension, body weight, circulating amino acids and ammonia levels were examined as outcome parameters after gene therapy with an adeno-associated viral vector expressing arginase was administered to mice on the second day of life (DOL). One-hundred percent of untreated arginase-deficient mice died by DOL 24, whereas 89% of the adeno-associated virus (AAV)-treated arginase deficient mice have survived for >8 months. While animals at 8 months demonstrate elevated glutamine levels, ammonia is less than three times that of controls and arginine levels are normal. These studies are the first to demonstrate that AAV-based therapy for arginase deficiency is effective and supports the development of gene therapy for this and the other urea cycle disorders.

Protein kinase D1 mediates stimulation of DNA synthesis and proliferation in intestinal epithelial IEC-18 cells and in mouse intestinal crypts.

We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p < 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p < 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.

Rapid protein kinase D1 signaling promotes migration of intestinal epithelial cells.

We have examined the role of protein kinase D1 (PKD1) signaling in intestinal epithelial cell migration. Wounding monolayer cultures of intestinal epithelial cell line IEC-18 or IEC-6 induced rapid PKD1 activation in the cells immediately adjacent to the wound edge, as judged by immunofluorescence microscopy with an antibody that detects the phosphorylated state of PKD1 at Ser(916), an autophosphorylation site. An increase in PKD1 phosphorylation at Ser(916) was evident as early as 45 s after wounding, reached a maximum after 3 min, and persisted for ≥15 min. PKD1 autophosphorylation at Ser(916) was prevented by the PKD family inhibitors kb NB 142-70 and CRT0066101. A kb NB 142-70-sensitive increase in PKD autophosphorylation was also elicited by wounding IEC-6 cells. Using in vitro kinase assays after PKD1 immunoprecipitation, we corroborated that wounding IEC-18 cells induced rapid PKD1 catalytic activation. Further results indicate that PKD1 signaling is required to promote migration of intestinal epithelial cells into the denuded area of the wound. Specifically, treatment with kb NB 142-70 or small interfering RNAs targeting PKD1 markedly reduced wound-induced migration in IEC-18 cells. To test whether PKD1 promotes migration of intestinal epithelial cells in vivo, we used transgenic mice that express elevated PKD1 protein in the small intestinal epithelium. Enterocyte migration was markedly increased in the PKD1 transgenic mice. These results demonstrate that PKD1 activation is one of the early events initiated by wounding a monolayer of intestinal epithelial cells and indicate that PKD1 signaling promotes the migration of these cells in vitro and in vivo.

PTEN tumor suppressor regulates p53 protein levels and activity through phosphatase-dependent and -independent mechanisms.

We show in this study that PTEN regulates p53 protein levels and transcriptional activity through both phosphatase-dependent and -independent mechanisms. The onset of tumor development in p53(+/-);Pten(+/-) mice is similar to p53(-/-) animals, and p53 protein levels are dramatically reduced in Pten(-/-) cells and tissues. Reintroducing wild-type or phosphatase-dead PTEN mutants leads to a significant increase in p53 stability. PTEN also physically associates with endogenous p53. Finally, PTEN regulates the transcriptional activity of p53 by modulating its DNA binding activity. This study provides a novel mechanism by which the loss of PTEN can functionally control "two" hits in the course of tumor development by concurrently modulating p53 activity.

Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer.

The murine Pten prostate cancer model described in this study recapitulates the disease progression seen in humans: initiation of prostate cancer with prostatic intraepithelial neoplasia (PIN), followed by progression to invasive adenocarcinoma, and subsequent metastasis with defined kinetics. Furthermore, while Pten null prostate cancers regress after androgen ablation, they are capable of proliferating in the absence of androgen. Global assessment of molecular changes caused by homozygous Pten deletion identified key genes known to be relevant to human prostate cancer, including those "signature" genes associated with human cancer metastasis. This murine prostate cancer model provides a unique tool for both exploring the molecular mechanism underlying prostate cancer and for development of new targeted therapies.

Dysfunction of ouabain-induced cardiac contractility in mice with heart-specific ablation of Na,K-ATPase beta1-subunit.

Na,K-ATPase is composed of two essential alpha- and beta-subunits, both of which have multiple isoforms. Evidence indicates that the Na,K-ATPase enzymatic activity as well as its alpha(1), alpha(3) and beta(1) isoforms are reduced in the failing human heart. The catalytic alpha-subunit is the receptor for cardiac glycosides such as digitalis, used for the treatment of congestive heart failure. The role of the Na,K-ATPase beta(1)-subunit (Na,K-beta(1)) in cardiac function is not known. We used Cre/loxP technology to inactivate the Na,K-beta(1) gene exclusively in the ventricular cardiomyocytes. Animals with homozygous Na,K-beta(1) gene excision were born at the expected Mendelian ratio, grew into adulthood, and appeared to be healthy until 10 months of age. At 13-14 months, these mice had 13% higher heart/body weight ratios, and reduced contractility as revealed by echocardiography compared to their wild-type (WT) littermates. Pressure overload by transverse aortic constriction (TAC) in younger mice, resulted in compensated hypertrophy in WT mice, but decompensation in the Na,K-beta(1) KO mice. The young KO survivors of TAC exhibited decreased contractile function and mimicked the effects of the Na,K-beta(1) KO in older mice. Further, we show that intact hearts of Na,K-beta(1) KO anesthetized mice as well as isolated cardiomyocytes were insensitive to ouabain-induced positive inotropy. This insensitivity was associated with a reduction in NCX1, one of the proteins involved in regulating cardiac contractility. In conclusion, our results demonstrate that Na,K-beta(1) plays an essential role in regulating cardiac contractility and that its loss is associated with significant pathophysiology of the heart.

Disruption of arginase II alters prostate tumor formation in TRAMP mice.

BACKGROUND: Arginase II (AII) is involved in the polyamine synthetic pathway, and elevated levels of expression have been found in a high proportion of prostate cancer samples and patients. However, the biological function of arginase II in prostate cancer still remains to be elucidated. In this study, we utilized the TRAMP mouse prostate cancer model to better understand the contribution of AII on tumor development. METHODS: AII expression was determined in prostates from TRAMP mice at 23 weeks of age by real-time RT-PCR and Western blot analysis. Additionally, AII expression was disrupted in the TRAMP model by crossbreeding arginase II knockout (AII KO) mice with TRAMP mice in order to generate the TRAMP/AII KO line. In each group, genito-urinary (GU) tract weights were determined and a pathological evaluation of the tumors was completed. RESULTS: AII expression was only detectable in those mice without the presence of macroscopic tumors; it was also absent in the TRAMP-C2 cell line, which is characteristic of an advanced prostate tumor. Assessment of the GU weights revealed larger average GU weights in the TRAMP/AII KO mice compared to TRAMP mice. Additionally, a greater percentage of more advanced pathology was found in the TRAMP/AII KO group compared to the TRAMP cohort. CONCLUSIONS: Based on these results, AII deficiency in the TRAMP model seems to accelerate prostate tumor progression, leading to an overall more advanced cancer stage in these mice. These findings support the possibility that prostatic arginase II could be a potentially useful marker of disease progression.

Lentiviral vectors with CMV or MHCII promoters administered in vivo: immune reactivity versus persistence of expression.

Lentiviral vectors (LVs) are potential tools for genetic vaccination. To improve the safety of LV vaccines, we evaluated the selectivity, bio-distribution, persistence of expression, and immune potency of vesicular stomatitis virus G (VSV-G)-pseudotyped vectors transcriptionally targeted to antigen presenting cells (APCs) through a major histocompatibility complex class II (MHCII) promoter. Control vectors contained the ubiquitous cytomegalovirus (CMV) promoter. Bio-distribution studies after intravenous injections of LVs expressing green fluorescent protein (GFP) or luciferase were conducted by a combination of flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (RT-Q-PCR) and whole-body bioluminescence analyses. GFP-expressing vectors showed selective expression in MHCII(+) cells of spleen and LV-CMV-GFP administration produced noticeable spleen inflammation, whereas LV-MHCII-GFP did not. Long-term optical imaging analyses of C57BL/6 mice injected with LV-CMV-LUC showed diminishing luciferase expression in the liver and spleen over time. Vaccination/boost with LV-CMV expressing the melanoma antigen tyrosinase-related protein 2 (TRP2) yielded dose-dependent antigen-specific CD8(+) T-cell reactivity and high protection against B16 melanoma challenge. Unexpectedly, administration of LVs containing the MHCII promoter resulted in persistence of luciferase expression and viral integration in MHCII(+) splenocytes and virtually no CD8(+) T-cell responses against TRP2. These studies reveal that APC transduction by LVs could lead to immune reactivity (LV-CMV) or persistence of transgene expression (LV-MHCII), providing a relevant paradigm for vaccination and gene replacement approaches.

Genetic background controls tumor development in PTEN-deficient mice.

PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. Germ line mutations of PTEN have been detected in three rare autosomal-dominant disorders. However, identical mutations in the PTEN gene may lead to different symptoms that have traditionally been described as different disorders, such as Cowden disease, Lhermitte-Duclos disease, and Bannayan-Zonana syndromes. This lack of genotype-phenotype correlation prompted us to directly test the possible effects of genetic background or modifier genes on PTEN-controlled tumorigenesis using genetically engineered mouse models. In this study, we generated two animal models in which either exon 5 (Pten(Delta5)) or promoter to exon 3 (Pten(-)) of the murine Pten gene were deleted and compared phenotypes associated with individual mutations on two genetic backgrounds. We found that the onset and spectrum of tumor formation depend significantly on the genetic background but less on the type of mutation generated. Our results suggest that PTEN plays a critical role in cancer development, and genetic background may influence the onset, the spectrum, and the progression of tumorigenesis caused by Pten mutation.

Immuno-PET in Inflammatory Bowel Disease: Imaging CD4-Positive T Cells in a Murine Model of Colitis.

Inflammatory bowel diseases (IBDs) in humans are characterized in part by aberrant CD4-positive (CD4+) T-cell responses. Currently, identification of foci of inflammation within the gut requires invasive procedures such as colonoscopy and biopsy. Molecular imaging with antibody fragment probes could be used to noninvasively monitor cell subsets causing intestinal inflammation. Here, GK1.5 cys-diabody (cDb), an antimouse CD4 antibody fragment derived from the GK1.5 hybridoma, was used as a PET probe for CD4+ T cells in the dextran sulfate sodium (DSS) mouse model of IBD. Methods: The DSS mouse model of IBD was validated by assessing changes in CD4+ T cells in the spleen and mesenteric lymph nodes (MLNs) using flow cytometry. Furthermore, CD4+ T cell infiltration in the colons of colitic mice was evaluated using immunohistochemistry. 89Zr-labeled GK1.5 cDb was used to image distribution of CD4+ T cells in the abdominal region and lymphoid organs of mice with DSS-induced colitis. Region-of-interest analysis was performed on specific regions of the gut to quantify probe uptake. Colons, ceca, and MLNs were removed and imaged ex vivo by PET. Imaging results were confirmed by ex vivo biodistribution analysis. Results: An increased number of CD4+ T cells in the colons of colitic mice was confirmed by anti-CD4 immunohistochemistry. Increased uptake of 89Zr-maleimide-deferoxamine (malDFO)-GK1.5 cDb in the distal colon of colitic mice was visible in vivo in PET scans, and region-of-interest analysis of the distal colon confirmed increased activity in DSS mice. MLNs from colitic mice were enlarged and visible in PET images. Ex vivo scans and biodistribution confirmed higher uptake in DSS-treated colons (DSS, 1.8 ± 0.40; control, 0.45 ± 0.12 percentage injected dose [%ID] per organ, respectively), ceca (DSS, 1.1 ± 0.38; control, 0.35 ± 0.09 %ID per organ), and MLNs (DSS, 1.1 ± 0.58; control, 0.37 ± 0.25 %ID per organ). Conclusion:89Zr-malDFO-GK1.5 cDb detected CD4+ T cells in the colons, ceca, and MLNs of colitic mice and may prove useful for further investigations of CD4+ T cells in preclinical models of IBD, with potential to guide development of antibody-based imaging in human IBD.

Mouse model for human arginase deficiency.

Deficiency of liver arginase (AI) causes hyperargininemia (OMIM 207800), a disorder characterized by progressive mental impairment, growth retardation, and spasticity and punctuated by sometimes fatal episodes of hyperammonemia. We constructed a knockout mouse strain carrying a nonfunctional AI gene by homologous recombination. Arginase AI knockout mice completely lacked liver arginase (AI) activity, exhibited severe symptoms of hyperammonemia, and died between postnatal days 10 and 14. During hyperammonemic crisis, plasma ammonia levels of these mice increased >10-fold compared to those for normal animals. Livers of AI-deficient animals showed hepatocyte abnormalities, including cell swelling and inclusions. Plasma amino acid analysis showed the mean arginine level in knockouts to be approximately fourfold greater than that for the wild type and threefold greater than that for heterozygotes; the mean proline level was approximately one-third and the ornithine level was one-half of the proline and ornithine levels, respectively, for wild-type or heterozygote mice--understandable biochemical consequences of arginase deficiency. Glutamic acid, citrulline, and histidine levels were about 1.5-fold higher than those seen in the phenotypically normal animals. Concentrations of the branched-chain amino acids valine, isoleucine, and leucine were 0.4 to 0.5 times the concentrations seen in phenotypically normal animals. In summary, the AI-deficient mouse duplicates several pathobiological aspects of the human condition and should prove to be a useful model for further study of the disease mechanism(s) and to explore treatment options, such as pharmaceutical administration of sodium phenylbutyrate and/or ornithine and development of gene therapy protocols.

Atherogenic diet causes lethal ileo-ceco-colitis in cyclooxygenase-2 deficient mice.

Cyclooxygenases (COX) regulate a variety of inflammatory diseases, including inflammatory bowel disease (IBD). While the pathological effects of COX-1 inhibition by NSAIDs on intestinal ulceration are well established, the role of COX-2 on intestinal inflammation remains under investigation. In this paper, we report a protective role for COX-2 against diet-mediated intestinal inflammation in mice. COX-2(-/-) mice fed an atherogenic diet or diet containing cholate, but not chow or fat alone, had a high mortality whereas COX-1(-/-) mice and wild-type mice were unaffected by the dietary changes. Histological analysis identified the cause of death in COX-2(-/-) mice due to severe intestinal inflammation that was surprisingly limited to the ileo-ceco-colic junction. COX-2 expression is induced in the cecum of wild-type mice fed an atherogenic diet. Our findings show that COX-2 plays an anti-inflammatory role at the ileo-ceco-colic junction in mice, and the pathology of diet-mediated intestinal inflammation in COX-2(-/-) mice offers an excellent model system to elucidate the molecular mechanisms of intestinal inflammation.

Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee.

The Pathological Classification of Prostate Lesions in Genetically Engineered Mice (GEM) is the result of a directive from the National Cancer Institute Mouse Models of Human Cancer Consortium Prostate Steering Committee to provide a hierarchical taxonomy of disorders of the mouse prostate to facilitate classification of existing and newly created mouse models and the translation to human prostate pathology. The proposed Bar Harbor Classification system is the culmination of three meetings and workshops attended by various members of the Prostate Pathology Committee of the Mouse Models of Human Cancer Consortium. A 2-day Pathology Workshop was held at The Jackson Laboratory in Bar Harbor, Maine, in October 2001, in which study sets of 93 slides from 22 GEM models were provided to individual panel members. The comparison of mouse and human prostate anatomy and disease demonstrates significant differences and considerable similarities that bear on the interpretation of the origin and natural history of their diseases. The recommended classification of mouse prostate pathology is hierarchical, and includes developmental, inflammatory, benign proliferative, and neoplastic disorders. Among the neoplastic disorders, preinvasive, microinvasive, and poorly differentiated neoplasms received the most attention. Specific criteria were recommended and will be discussed. Transitions between neoplastic states were of particular concern. Preinvasive neoplasias of the mouse prostate were recognized as focal, atypical, and progressive lesions. These lesions were designated as mouse prostatic intraepithelial neoplasia (mPIN). Some atypical lesions were identified in mouse models without evidence of progression to malignancy. The panel recommended that mPIN lesions not be given histological grades, but that mPIN be further classified as to the absence or presence of documented associated progression to invasive carcinoma. Criteria for recognizing microinvasion, for classification of invasive gland-forming adenocarcinomas, and for characterizing poorly differentiated tumors, including neuroendocrine carcinomas, were developed and are discussed. The uniform application of defined terminology is essential for correlating results between different laboratories and models. It is recommended that investigators use the Bar Harbor Classification system when characterizing new GEM models or when conducting experimental interventions that may alter the phenotype or natural history of lesion progression in existing models.

Classification of proliferative pulmonary lesions of the mouse: recommendations of the mouse models of human cancers consortium.

Rapid advances in generating new mouse genetic models for lung neoplasia provide a continuous challenge for pathologists and investigators. Frequently, phenotypes of new models either have no precedents or are arbitrarily attributed according to incongruent human and mouse classifications. Thus, comparative characterization and validation of novel models can be difficult. To address these issues, a series of discussions was initiated by a panel of human, veterinary, and experimental pathologists during the Mouse Models of Human Cancers Consortium (NIH/National Cancer Institute) workshop on mouse models of lung cancer held in Boston on June 20-22, 2001. The panel performed a comparative evaluation of 78 cases of mouse and human lung proliferative lesions, and recommended development of a new practical classification scheme that would (a) allow easier comparison between human and mouse lung neoplasms, (b) accommodate newly emerging mouse neoplasms, and (c) address the interpretation of benign and preinvasive lesions of the mouse lung. Subsequent discussions with additional experts in pulmonary pathology resulted in the current proposal of a new classification. It is anticipated that this classification, as well as the complementary digital atlas of virtual histological slides, will help investigators and pathologists in their characterization of new mouse models, as well as stimulate further research aimed at a better understanding of proliferative lesions of the lung.

Uropathic observations in mice expressing a constitutively active point mutation in the 5-HT3A receptor subunit.

Mutant mice with a hypersensitive serotonin (5-HT)3A receptor were generated through targeted exon replacement. A valine to serine mutation (V13'S) in the channel-lining M2 domain of the 5-HT3A receptor subunit rendered the 5-HT3 receptor 70-fold more sensitive to serotonin and produced constitutive activity when combined with the 5-HT3B subunit. Mice homozygous for the mutant allele (5-HT3Avs/vs) had decreased levels of 5-HT3A mRNA. Measurements on sympathetic ganglion cells in these mice showed that whole-cell serotonin responses were reduced, and that the remaining 5-HT3 receptors were hypersensitive. Male 5-HT3Avs/vs mice died at 2-3 months of age, and heterozygous (5-HT3Avs/+) males and homozygous mutant females died at 4-6 months of age from an obstructive uropathy. Both male and female 5-HT3A mutant mice had urinary bladder mucosal and smooth muscle hyperplasia and hypertrophy, whereas male mutant mice had additional prostatic smooth muscle and urethral hyperplasia. 5-HT3A mutant mice had marked voiding dysfunction characterized by a loss of micturition contractions with overflow incontinence. Detrusor strips from 5-HT3Avs/vs mice failed to contract to neurogenic stimulation, despite overall normal responses to a cholinergic agonist, suggestive of altered neuronal signaling in mutant mouse bladders. Consistent with this hypothesis, decreased nerve fiber immunoreactivity was observed in the urinary bladders of 5-HT3Avs/vs compared with 5-HT3A wild-type (5-HT3A+/+) mice. These data suggest that persistent activation of the hypersensitive and constitutively active 5-HT3A receptor in vivo may lead to excitotoxic neuronal cell death and functional changes in the urinary bladder, resulting in bladder hyperdistension, urinary retention, and overflow incontinence.

Monitoring antiproliferative responses to kinase inhibitor therapy in mice with 3'-deoxy-3'-18F-fluorothymidine PET.

UNLABELLED: The aim of this study was to evaluate, whether PET with (18)F-FDG and 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) may be used to monitor noninvasively the antiproliferative effects of tyrosine kinase inhibitors. METHODS: Using a high-resolution small animal scanner, we measured the effect of the ErbB-selective kinase inhibitor PKI-166 on the (18)F-FDG and (18)F-FLT uptake of ErbB1-overexpressing A431 xenograft tumors. RESULTS: Treatment with PKI-166 markedly lowered tumor (18)F-FLT uptake within 48 h of drug exposure; within 1 wk (18)F-FLT uptake decreased by 79%. (18)F-FLT uptake by the xenografts significantly correlated with the tumor proliferation index as determined by proliferating cell nuclear antigen staining (r = 0.71). Changes in (18)F-FLT uptake did not reflect inhibition of ErbB kinase activity itself but, rather, the effects of kinase inhibition on tumor cell proliferation. Tumor (18)F-FDG uptake generally paralleled the changes seen for (18)F-FLT. However, the baseline signal was significantly lower than that for (18)F-FLT. CONCLUSION: These results indicate that (18)F-FLT PET provides noninvasive, quantitative, and repeatable measurements of tumor cell proliferation during treatment with ErbB kinase inhibitors and provide a rationale for the use this technology in clinical trials of kinase inhibitors.