Gary P. Swain

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

ABSTRACT 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.

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.

Cell proliferation in the lamprey central nervous system

After spinal cord transection, axons regenerate both in larval and adult lampreys. It is not known to what degree cells proliferate, even in the uninjured animal. Therefore, we have determined the prevalence of mitosis in the lamprey central nervous system (CNS). Bromodeoxyuridine (BrdU) was injected and incorporated for 4 hours into 2‐ to 5‐year‐old larvae, animals undergoing metamorphosis, and young adults. Labeled cells were counted in the rhombencephalon (where most supraspinal projecting neurons are located) and spinal cord. A mitotic index (MI) was calculated as the percentage of nuclei that were labeled. There was a seasonal variation in mitotic activity, with higher MIs occurring in summer. Within the summer, there was an additional transient spike in mitosis, especially in the rhombencephalon. There was no correlation between age and MI within the range of developmental stages examined. Baseline MIs in the rhombencephalon and spinal cord were approximately 0.15% and 0.20%, respectively. In most animals, the highest mitotic rates in both the rhombencephalon and spinal cord were seen in the ependyma, but many labeled cells were found in nonependymal regions as well. During the summer spike, almost all of the additional mitosis in the rhombencephalon was in the ependyma, but this finding was not true in the spinal cord. Many BrdU‐labeled cells in the spinal cord and rhombencephalon were also stained by monoclonal antibodies specific for lamprey glial keratin but were never labeled by anti‐neurofilament antibodies. These results suggest that (1) neurogenesis is uncommon in the lamprey CNS; (2) during most of the year, baseline gliogenesis occurs mainly in the ependyma with substantial contribution by nonependymal areas. During the summer, a spike of mitotic activity occurs in the ependyma of the rhombencephalon and throughout the spinal cord. J. Comp. Neurol. 469:298–310, 2004.

A Method for in Situ Hybridization in Wholemounted Lamprey Brain: Neurofilament Expression in Larvae and Adults

Nonisotopic in situ hybridization (NISH) using both cDNA and cRNA probes is rapidly gaining favor over autoradiographic methods. Typically, either biotinylated or digoxigenin-labeled probes are used to detect mRNAs in sectioned tissue or in cultured cells. With a few exceptions, most applications of NISH in wholemount preparations have been limited to Drosophila embryos. A protocol developed for NISH in whole adult Drosophila CNS was extended to wholemounted larval and adult lamprey brain preparations. Digoxigenin-labeled RNA probes were transcribed from cloned fragments of a lamprey neurofilament (NF180) cDNA. Hybridization with these probes, and comparisons with Nissl-stained wholemounts and wholemounts retrogradely labeled by injections of tracer into the spinal cord, demonstrated that NF180 mRNA was expressed in only a subset of neurons in the lamprey CNS. These included primarily neurons with long axons that project out of the brainstem, e.g., reticulospinal neurons and cranial motor neurons. Metamorphosis from the larval to the adult form was accompanied by an increase in the number of neurons expressing NF180 and in the apparent level of NF expression as judged by the intensity of labeling. For example, in the oculomotor and trochlear nuclei, expression of NF180 was seen in postmetamorphic young adult lampreys but not in larvae. In the trigeminal motor nucleus, both the number of neurons expressing NF180 and the intensity of the hybridization labeling increased with metamorphosis. The ability to do NISH in lamprey brain wholemounts eliminates the need for serial reconstructions and thus facilitates the study of selected gene expression during metamorphosis and regeneration.

A novel colonic repressor element regulates intestinal gene expression by interacting with Cux/CDP.

ABSTRACT Intestinal gene regulation involves mechanisms that direct temporal expression along the vertical and horizontal axes of the alimentary tract. Sucrase-isomaltase (SI), the product of an enterocyte-specific gene, exhibits a complex pattern of expression. Generation of transgenic mice with a mutated SI transgene showed involvement of an overlapping CDP (CCAAT displacement protein)-GATA element in colonic repression of SI throughout postnatal intestinal development. We define this element as CRESIP (colon-repressive element of the SI promoter). Cux/CDP interacts with SI and represses SI promoter activity in a CRESIP-dependent manner. Cux/CDP homozygous mutant mice displayed increased expression of SI mRNA during early postnatal development. Our results demonstrate that an intestinal gene can be repressed in the distal gut and identify Cux/CDP as a regulator of this repression during development.

Neurogenesis in the lamprey central nervous system following spinal cord transection

After spinal cord transection, lampreys recover functionally and axons regenerate. It is not known whether this is accompanied by neurogenesis. Previous studies suggested a baseline level of nonneuronal cell proliferation in the spinal cord and rhombencephalon (where most supraspinal projecting neurons are located). To determine whether cell proliferation increases after injury and whether this includes neurogenesis, larval lampreys were spinally transected and injected with 5‐bromo‐2&prime‐deoxyuridine (BrdU) at 0–3 weeks posttransection. Labeled cells were counted in the lesion site, within 0.5 mm rostral and caudal to the lesion, and in the rhombencephalon. One group of animals was processed in the winter and a second group was processed in the summer. The number of labeled cells was greater in winter than in summer. The lesion site had the most BrdU labeling at all times, correlating with an increase in the number of cells. In the adjacent spinal cord, the percentage of BrdU labeling was higher in the ependymal than in nonependymal regions. This was also true in the rhombencephalon but only in summer. In winter, BrdU labeling was seen primarily in the subventricular and peripheral zones. Some BrdU‐labeled cells were also double labeled by antibodies to glial‐specific (antikeratin) as well as neuron‐specific (anti‐Hu) antigens, indicating that both gliogenesis and neurogenesis occurred after spinal cord transection. However, the new neurons were restricted to the ependymal zone, were never labeled by antineurofilament antibodies, and never migrated away from the ependyma even at 5 weeks after BrdU injection. They would appear to be cerebrospinal fluid‐contacting neurons. J. Comp. Neurol. 522:1316–1332, 2014.

Developmental Increases in Expression of Neurofilament mRNA Selectively in Projection Neurons of the Lamprey CNS

Neurofilaments of the sea lamprey are unique in being homopolymers of a single subunit (NF‐180). Digoxigenin‐labeled RNA probes complementary to NF‐180 were used to determine the distribution and timing of expression of neurofilament message in the brain and spinal cord of the lamprey. In the brainstem, detection of NF‐180 mRNA was restricted to neurons with axons projecting to the spinal cord or the periphery. The majority of brainstem neurons, whose axons project locally, did not express NF‐180 within the detection limits of this technique. NF‐180‐positive neurons included cells with a wide range of axon diameters, suggesting neurofilament mRNA expression was linked to axon length rather than caliber. To further evaluate this hypothesis, expression was studied in animals of different developmental stages between larvae and adults. In younger (shorter) larvae, the large Mauthner and rhombencephalic Müller cells did not express NF‐180 mRNA, even though their axons are among the largest caliber in the animal and extend the entire length of the spinal cord. In contrast, many other reticulospinal neurons, whose axons are smaller in diameter than those of the Müller and Mauthner cells, expressed NF‐180 message throughout larval development. Furthermore, neurons of the cranial motor nuclei did not express NF‐180 until later developmental stages and the extraocular motor neurons did not label until metamorphosis. Therefore, while detectable neurofilament mRNA expression in the lamprey is restricted to neurons with long axons, its expression in this population of neurons appears to be developmentally regulated by factors still not determined. It is postulated that need for NF message is determined by a balance between the volume of axon to be filled and the rate of turnover of NF in that axon.

Intraoperative near-infrared fluorescence imaging targeting folate receptors identifies lung cancer in a large-animal model

Complete tumor resection is the most important predictor of patient survival with non–small cell lung cancer. Methods for intraoperative margin assessment after lung cancer excision are lacking. This study evaluated near‐infrared (NIR) intraoperative imaging with a folate‐targeted molecular contrast agent (OTL0038) for the localization of primary lung adenocarcinomas, lymph node sampling, and margin assessment.

Cerebrospinal Fluid Calbindin D Concentration as a Biomarker of Cerebellar Disease Progression in Niemann-Pick Type C1 Disease.

Niemann-Pick type C (NPC) 1 disease is a rare, inherited, neurodegenerative disease. Clear evidence of the therapeutic efficacy of 2-hydroxypropyl-β-cyclodextrin (HPβCD) in animal models resulted in the initiation of a phase I/IIa clinical trial in 2013 and a phase IIb/III trial in 2015. With clinical trials ongoing, validation of a biomarker to track disease progression and serve as a supporting outcome measure of therapeutic efficacy has become compulsory. In this study, we evaluated calcium-binding protein calbindin D-28K (calbindin) concentrations in the cerebrospinal fluid (CSF) as a biomarker of NPC1 disease. In the naturally occurring feline model, CSF calbindin was significantly elevated at 3 weeks of age, prior to the onset of cerebellar dysfunction, and steadily increased to >10-fold over normal at end-stage disease. Biweekly intrathecal administration of HPβCD initiated prior to the onset of neurologic dysfunction completely normalized CSF calbindin in NPC1 cats at all time points analyzed when followed up to 78 weeks of age. Initiation of HPβCD after the onset of clinical signs (16 weeks of age) resulted in a delayed reduction of calbindin levels in the CSF. Evaluation of CSF from patients with NPC1 revealed that calbindin concentrations were significantly elevated compared with CSF samples collected from unaffected patients. Off-label treatment of patients with NPC1 with miglustat, an inhibitor of glycosphingolipid biosynthesis, significantly decreased CSF calbindin compared with pretreatment concentrations. These data suggest that the CSF calbindin concentration is a sensitive biomarker of NPC1 disease that could be instrumental as an outcome measure of therapeutic efficacy in ongoing clinical trials.

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.