Meningeal B Cell Clusters Correlate with Submeningeal Pathology in a Natural Model of Multiple Sclerosis.

Multiple sclerosis (MS) is an idiopathic demyelinating disease in which meningeal inflammation correlates with accelerated disease progression. The study of meningeal inflammation in MS has been limited because of constrained access to MS brain/spinal cord specimens and the lack of experimental models recapitulating progressive MS. Unlike induced models, a spontaneously occurring model would offer a unique opportunity to understand MS immunopathogenesis and provide a compelling framework for translational research. We propose granulomatous meningoencephalomyelitis (GME) as a natural model to study neuropathological aspects of MS. GME is an idiopathic, progressive neuroinflammatory disease of young dogs with a female bias. In the GME cases examined in this study, the meninges displayed focal and disseminated leptomeningeal enhancement on magnetic resonance imaging, which correlated with heavy leptomeningeal lymphocytic infiltration. These leptomeningeal infiltrates resembled tertiary lymphoid organs containing large B cell clusters that included few proliferating Ki67+ cells, plasma cells, follicular dendritic/reticular cells, and germinal center B cell-like cells. These B cell collections were confined in a specialized network of collagen fibers associated with the expression of the lympho-organogenic chemokines CXCL13 and CCL21. Although neuroparenchymal perivascular infiltrates contained B cells, they lacked the immune signature of aggregates in the meningeal compartment. Finally, meningeal B cell accumulation correlated significantly with cortical demyelination reflecting neuropathological similarities to MS. Hence, during chronic neuroinflammation, the meningeal microenvironment sustains B cell accumulation that is accompanied by underlying neuroparenchymal injury, indicating GME as a novel, naturally occurring model to study compartmentalized neuroinflammation and the associated pathology thought to contribute to progressive MS.

Krabbe disease successfully treated via monotherapy of intrathecal gene therapy.

Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically.

AAVrh10 Gene Therapy Ameliorates Central and Peripheral Nervous System Disease in Canine Globoid Cell Leukodystrophy (Krabbe Disease).

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an inherited, neurologic disorder that results from deficiency of a lysosomal enzyme, galactosylceramidase. Most commonly, deficits of galactosylceramidase result in widespread central and peripheral nervous system demyelination and death in affected infants typically by 2 years of age. Hematopoietic stem-cell transplantation is the current standard of care in children diagnosed prior to symptom onset. However, disease correction is incomplete. Herein, the first adeno-associated virus (AAV) gene therapy experiments are presented in a naturally occurring canine model of GLD that closely recapitulates the clinical disease progression, neuropathological alterations, and biochemical abnormalities observed in human patients. Adapted from studies in twitcher mice, GLD dogs were treated by combination intravenous and intracerebroventricular injections of AAVrh10 to target both the peripheral and central nervous systems. Combination of intravenous and intracerebroventricular AAV gene therapy had a clear dose response and resulted in delayed onset of clinical signs, extended life-span, correction of biochemical defects, and attenuation of neuropathology. For the first time, therapeutic effect has been established in the canine model of GLD by targeting both peripheral and central nervous system impairments with potential clinical implications for GLD patients.

Alternatively activated macrophage-derived RELM-{alpha} is a negative regulator of type 2 inflammation in the lung.

Differentiation and recruitment of alternatively activated macrophages (AAMacs) are hallmarks of several inflammatory conditions associated with infection, allergy, diabetes, and cancer. AAMacs are defined by the expression of Arginase 1, chitinase-like molecules, and resistin-like molecule (RELM) alpha/FIZZ1; however, the influence of these molecules on the development, progression, or resolution of inflammatory diseases is unknown. We describe the generation of RELM-alpha-deficient (Retnla(-/-)) mice and use a model of T helper type 2 (Th2) cytokine-dependent lung inflammation to identify an immunoregulatory role for RELM-alpha. After challenge with Schistosoma mansoni (Sm) eggs, Retnla(-/-) mice developed exacerbated lung inflammation compared with their wild-type counterparts, characterized by excessive pulmonary vascularization, increased size of egg-induced granulomas, and elevated fibrosis. Associated with increased disease severity, Sm egg-challenged Retnla(-/-) mice exhibited elevated expression of pathogen-specific CD4(+) T cell-derived Th2 cytokines. Consistent with immunoregulatory properties, recombinant RELM-alpha could bind to macrophages and effector CD4(+) Th2 cells and inhibited Th2 cytokine production in a Bruton's tyrosine kinase-dependent manner. Additionally, Retnla(-/-) AAMacs promoted exaggerated antigen-specific Th2 cell differentiation. Collectively, these data identify a previously unrecognized role for AAMac-derived RELM-alpha in limiting the pathogenesis of Th2 cytokine-mediated pulmonary inflammation, in part through the regulation of CD4(+) T cell responses.

Evidence for the epithelial to mesenchymal transition in biliary atresia fibrosis.

The epithelial to mesenchymal transition has recently been implicated as a source of fibrogenic myofibroblasts in organ fibrosis, particularly in the kidney. There is as yet minimal evidence for the epithelial to mesenchymal transition in the liver. We hypothesized that this process in biliary epithelial cells plays an important role in biliary fibrosis and might be found in patients with especially rapid forms, such as is seen in biliary atresia. We therefore obtained liver tissue from patients with biliary atresia as well as a variety of other pediatric and adult liver diseases. Tissues were immunostained with antibodies against the biliary epithelial cell marker CK19 as well as with antibodies against proteins characteristically expressed by cells undergoing the epithelial to mesenchymal transition, including fibroblast-specific protein 1, the collagen chaperone heat shock protein 47, the intermediate filament protein vimentin, and the transcription factor Snail. The degree of colocalization was quantified using a multispectral imaging system. We observed significant colocalization between CK19 and other markers of the epithelial to mesenchymal transition in biliary atresia as well as other liver diseases associated with significant bile ductular proliferation, including primary biliary cirrhosis. There was minimal colocalization seen in healthy adult and pediatric livers, or in livers not also demonstrating bile ductular proliferation. Multispectral imaging confirmed significant colocalization of the different markers in biliary atresia. In conclusion, we present significant histologic evidence suggesting that the epithelial to mesenchymal transition occurs in human liver fibrosis, particularly in diseases such as biliary atresia and primary biliary cirrhosis with prominent bile ductular proliferation.

RELMbeta/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract.

Gastrointestinal (GI) nematode infections are an important public health and economic concern. Experimental studies have shown that resistance to infection requires CD4(+) T helper type 2 (Th2) cytokine responses characterized by the production of IL-4 and IL-13. However, despite >30 years of research, it is unclear how the immune system mediates the expulsion of worms from the GI tract. Here, we demonstrate that a recently described intestinal goblet cell-specific protein, RELMbeta/FIZZ2, is induced after exposure to three phylogenetically distinct GI nematode pathogens. Maximal expression of RELMbeta was coincident with the production of Th2 cytokines and host protective immunity, whereas production of the Th1 cytokine, IFN-gamma, inhibited RELMbeta expression and led to chronic infection. Furthermore, whereas induction of RELMbeta was equivalent in nematode-infected wild-type and IL-4-deficient mice, IL-4 receptor-deficient mice showed minimal RELMbeta induction and developed persistent infections, demonstrating a direct role for IL-13 in optimal expression of RELMbeta. Finally, we show that RELMbeta binds to components of the nematode chemosensory apparatus and inhibits chemotaxic function of a parasitic nematode in vitro. Together, these results suggest that intestinal goblet cell-derived RELMbeta may be a novel Th2 cytokine-induced immune-effector molecule in resistance to GI nematode infection.

Homozygous loss of menin is well tolerated in liver, a tissue not affected in MEN1.

Most tumor suppressor genes show a widespread pattern of expression, yet individuals with germline, heterozygous loss of function of such genes develop tumors in a restricted set of tissues. This paradox has generated a multitude of speculative hypotheses. The gene for multiple endocrine neoplasia type I (MEN1) encodes a ubiquitously expressed tumor suppressor of unknown function called menin. Humans and mice with germline, heterozygous loss-of-function mutations in the MEN1 gene almost always develop at least one endocrine tumor by late adulthood, and examination of those tumors invariably reveals loss of the wild-type allele. To investigate the paradox of tissue-specific tumor phenotype in MEN1, mice homozygous for an Men1 gene with exons 3-8 flanked by loxP sites were bred to transgenic mice expressing cre from the albumin promoter. This strategy allowed us to generate mice with homozygous deletion of the Men1 gene in liver, a tissue not normally predisposed to developing tumors in humans or mice with heterozygous MEN1 loss-of-function mutations. Livers that were completely null for menin expression appeared entirely normal and remained tumor free until late adulthood. These results argue against certain hypotheses previously proposed for the tissue specificity of tumor suppressor genes and provide insights to the mechanism of tissue specificity in MEN1.

Of mice and MEN1: Insulinomas in a conditional mouse knockout.

Patients with multiple endocrine neoplasia type 1 (MEN1) develop multiple endocrine tumors, primarily affecting the parathyroid, pituitary, and endocrine pancreas, due to the inactivation of the MEN1 gene. A conditional mouse model was developed to evaluate the loss of the mouse homolog, Men1, in the pancreatic beta cell. Men1 in these mice contains exons 3 to 8 flanked by loxP sites, such that, when the mice are crossed to transgenic mice expressing cre from the rat insulin promoter (RIP-cre), exons 3 to 8 are deleted in beta cells. By 60 weeks of age, >80% of mice homozygous for the floxed Men1 gene and expressing RIP-cre develop multiple pancreatic islet adenomas. The formation of adenomas results in elevated serum insulin levels and decreased blood glucose levels. The delay in tumor appearance, even with early loss of both copies of Men1, implies that additional somatic events are required for adenoma formation in beta cells. Comparative genomic hybridization of beta cell tumor DNA from these mice reveals duplication of chromosome 11, potentially revealing regions of interest with respect to tumorigenesis.

Anatomic and functional characteristics of the rat ileal pouch.

BACKGROUND: The definitive operation for chronic ulcerative colitis (UC) and familial adenomatous polyposis is total proctocolectomy with ileal pouch-anal anastomosis (IPAA). Mild inflammation (pouchitis) is omnipresent in pouches and becomes severe in 50% of UC patients with IPAA. The etiology of pouchitis is likely due to combined genetic, microbial, and immunologic factors. Epithelial cell exposure to surgical trauma and/or to changes in intestinal bacterial composition may account for the inflammatory infiltrate. Progress in understanding pouchitis is restricted by the lack of suitable animal models. METHODS: An ileal pouch-rectal anastomosis [IPRA] in rats was developed to reproduce a model of human IPAA and clinical, gross and histologic criteria were determined. RESULTS: Many shared features with human ileal pouch were observed. CONCLUSION: IPRA is an important in vivo model to study mechanisms of repair, defense and immunity that may contribute to pouchitis.

Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice.

BACKGROUND & AIMS: Intestinal-type gastric cancer is often preceded by intestinal metaplasia in humans. The genetic events responsible for the transdifferentiation that occurs in intestinal metaplasia are not well understood. Cdx2, a transcription factor whose expression is normally limited to the intestine, has been detected in gastric intestinal metaplasia. Cdx2 induces differentiation of intestinal epithelial cells in vitro; therefore, we sought to establish whether a causal relationship exists between Cdx2 activation and intestinal metaplasia. METHODS: Cdx2 expression was directed to the gastric mucosa in transgenic mice using cis-regulatory elements of Foxa3 (Hnf3gamma). Transgenic mice were analyzed for histologic and gene expression changes. RESULTS: Histologic examination of the gastric mucosa of the Foxa3/Cdx2 mice revealed the presence of alcian blue-positive intestinal-type goblet cells, a hallmark of intestinal metaplasia. In addition, Cdx2 induced the expression of intestine-specific genes. CONCLUSIONS: Gastric expression of Cdx2 alone was sufficient to induce intestinal metaplasia in mice. These mice represent a powerful tool to investigate the molecular mechanisms that promote intestinal metaplasia. Moreover, as gastric cancer in humans is often preceded by intestinal metaplasia, the phenotype described here strongly suggests involvement of Cdx2 in the initiation of the process leading to intestinal neoplasia of the gastric mucosa.