Large intragenic deletion of CDC73 (exons 4-10) in a three-generation hyperparathyroidism-jaw tumor (HPT-JT) syndrome family.

BACKGROUND: Inactivating mutations of CDC73 cause Hyperparathyroidism-Jaw Tumour syndrome (HPT-JT), Familial Isolated Hyperparathyroidism (FIHP) and sporadic parathyroid carcinoma. We conducted CDC73 mutation analysis in an HPT-JT family and confirm carrier status of the proband's daughter. METHODS: The proband had primary hyperparathyroidism (parathyroid carcinoma) and uterine leiomyomata. Her father and daughter had hyperparathyroidism (parathyroid adenoma) but no other manifestations of HPT-JT. CDC73 mutation analysis (sequencing of all 17 exons) and whole-genome copy number variation (CNV) analysis was done on leukocyte DNA of the three affecteds as well as the proband's unaffected sister. RESULTS: A novel deletion of exons 4 to 10 of CDC73 was detected by CNV analysis in the three affecteds. A novel insertion in the 5'UTR (c.-4_-11insG) that co-segregated with the deletion was identified. By in vitro assay the 5'UTR insertion was shown to significantly impair the expression of the parafibromin protein. Screening for the mutated CDC73 confirmed carrier status in the proband's daughter and the biochemistry and ultrasonography led to pre-emptive surgery and resolution of the hyperparathyroidism. CONCLUSIONS: A novel gross deletion mutation in CDC73 was identified in a three-generation HPT-JT family emphasizing the importance of including screening for large deletions in the molecular diagnostic protocol.

Surfaceome profiling reveals regulators of neural stem cell function.

The composition of cell-surface proteins changes during lineage specification, altering cellular responses to their milieu. The changes that characterize maturation of early neural stem cells (NSCs) remain poorly understood. Here we use mass spectrometry-based cell surface capture technology to profile the cell surface of early NSCs and demonstrate functional requirements for several enriched molecules. Primitive NSCs arise from embryonic stem cells upon removal of Transforming growth factor-ß signaling, while definitive NSCs arise from primitive NSCs upon Lif removal and FGF addition. In vivo aggregation assays revealed that N-cadherin upregulation is sufficient for the initial exclusion of definitive NSCs from pluripotent ectoderm, while c-kit signaling limits progeny of primitive NSCs. Furthermore, we implicate EphA4 in primitive NSC survival signaling and Erbb2 as being required for NSC proliferation. This work elucidates several key mediators of NSC function whose relevance is confirmed on forebrain-derived populations and identifies a host of other candidates that may regulate NSCs.

The adult mouse and human pancreas contain rare multipotent stem cells that express insulin.

The search for putative precursor cells within the pancreas has been the focus of extensive research. Previously, we identified rare pancreas-derived multipotent precursor (PMP) cells in the mouse with the intriguing capacity to generate progeny in the pancreatic and neural lineages. Here, we establish the embryonic pancreas as the developmental source of PMPs through lineage-labeling experiments. We also show that PMPs express insulin and can contribute to multiple pancreatic and neural cell types in vivo. In addition, we have isolated PMPs from adult human islet tissue that are also capable of extensive proliferation, self-renewal, and generation of multiple differentiated pancreatic and neural cell types. Finally, both mouse and human PMP-derived cells ameliorated diabetes in transplanted mice. These findings demonstrate that the adult mammalian pancreas contains a population of insulin(+) multipotent stem cells and suggest that these cells may provide a promising line of investigation toward potential therapeutic benefit.

Chorda tympani nerve function in children: relationship to otitis media and body mass index.

OBJECTIVE/HYPOTHESIS: A relationship between acute otitis media and elevated body mass index has recently been reported. Intriguingly, it was postulated that this relationship may result from altered chorda tympani nerve function impacting taste sensation and eating habits. We sought to test this directly by measuring chorda tympani nerve function in children with and without a previous history of acute otitis media and to determine the relationship to body mass index. STUDY DESIGN: Retrospective cohort study. METHODS: Institutional research ethics board approval was obtained. Study participants included 142 children (5-18 years of age) who were recruited from an otolaryngology outpatient clinic at a tertiary academic pediatric hospital between May and August 2009. Children were excluded if they were not able to communicate effectively, younger than age 5, or had developmental disabilities. Body mass index was calculated and the history of previous otologic disease carefully elicited from the caregivers. Electrogustometric threshold, a validated measure of chorda tympani function, was obtained bilaterally in each child. Children were divided into cohorts based on the number of acute otitis media episodes, and electrogustometry thresholds were compared between cohorts. RESULTS: Electrogustometric thresholds were successfully obtained in all children. There was no significant relationship between chorda tympani nerve function and history of acute otitis media. Similarly, there was no significant association between the history of otitis media and body mass index. CONCLUSION: This study did not demonstrate any effect of previous acute otitis media history on chorda tympani nerve function. Furthermore, it did not demonstrate a relationship between acute otitis media and elevated body mass index. This is counter-evidence to the previous hypothesis that increasing acute otitis media is responsible for increasing childhood obesity through alteration in chorda tympani nerve function.

Influence of previous radiation exposure on pathologic features and clinical outcome in patients with thyroid cancer.

OBJECTIVE: To determine whether previous radiation exposure to the head and neck is related to less favorable pathologic and clinical outcome in patients after surgical management of thyroid cancer. DESIGN: Retrospective chart review. SETTING: Academic teaching hospital (referral center). PATIENTS: All patients with diagnosed thyroid cancer who had been exposed to radiation before surgical treatment were retrospectively identified from the thyroid cancer database at our institution (1963-2007). One hundred twenty-five patients (95 women and 30 men) were included. Inclusion criteria included surgical treatment for thyroid cancer and a history of exposure to radiation at least 3 years before diagnosis of the disease. MAIN OUTCOME MEASURES: Pathologic features and data related to disease recurrence, distant metastasis, and survival. RESULTS: Mean (range) age at first exposure to radiation was 19.4 (1-65) years, and mean lag time to diagnosis of disease was 28.7 (3-60) years. Patients were treated surgically with either total or near-total thyroidectomy (83%) or partial or subtotal thyroidectomy (17%). Pathologic diagnoses included 111 papillary carcinomas (89%). Sixty-three percent of patients had multifocal disease, 12% had lymphovascular tumor invasion, and 26% had direct extrathyroid extension of disease. Twenty-five percent of patients had metastases to cervical lymph nodes, and 9% had distant metastases. Sixteen percent of patients experienced local recurrence of disease. At last follow-up, 86% of patients were alive and free of disease, 8% were alive with disease, 4% had died of thyroid cancer, and 2% had died of an unrelated cause. Compared with other patients with thyroid cancer, this radiation-exposed cohort was more likely to undergo total thyroidectomy, multiple operative procedures, and external radiotherapy. A higher percentage had multifocal disease, extrathyroid extension, stage IV disease, and distant metastases. At follow-up, fewer patients were free of disease, and more patients had died of thyroid disease. CONCLUSION: Patients who have been exposed to radiation have more aggressive disease and worse clinical outcome than other patients with thyroid cancer.

The importance of recognizing paradoxical vocal fold dysfunction: A case report of a 13-year-old girl presenting with stridor.

The present report details the case of a 13-year-old girl who presented to the emergency department with stridor. Treatment for presumed reactive airway disease was attempted with antibiotics, nebulized adrenaline masks and high-dose corticosteroids. Over the next month, she presented repeatedly in a similar fashion and was admitted to hospital on three separate occasions. Ultimately, she was referred to the Centre for Paediatric Voice and Laryngeal Function at The Hospital for Sick Children (Toronto, Ontario) for a speech-language pathology evaluation and direct laryngoscopy. The patient was diagnosed with paradoxical vocal fold dysfunction. After a brief treatment session with a speech-language pathologist, her stridor completely resolved and paradoxical inspiratory vocal fold adduction was no longer visualized on direct laryngoscopy. The present case highlights the fact that paradoxical vocal fold dysfunction can mimic other entities that present with stridor, and misdiagnosis can result in significant morbidity. Investigation into a patient's social history and stressors can facilitate the diagnosis, and can avoid unnecessary and potentially harmful medical and surgical interventions.

Cost-effective management of low-risk papillary thyroid carcinoma.

OBJECTIVE: To compare the 20-year cost-effectiveness of initial hemithyroidectomy vs total thyroidectomy in the management of small papillary thyroid cancer in the low-risk patient. DESIGN: Pooled data from the published literature were used to determine key statistics for decision analysis such as rates of recurrence, rates of complications for all interventions undertaken, and rates of death. The 2005 costs were obtained from the US Department of Health and Human Services, as well as from Medicare reimbursement schedules. Future costs were discounted at 6%. SETTING: Decision analysis study. PATIENTS: Data from the published literature. MAIN OUTCOME MEASURES: A state-transition (Markov) decision model was constructed based on the most recent American Thyroid Association recommendations. A cost-effectiveness analysis was performed using fixed probability estimates and Monte Carlo microsimulation, with effectiveness defined as cause-specific mortality or recurrence-free survival. After identifying initial results, sensitivity and threshold analyses were performed to assess the strength of the recommendations. RESULTS: Initial probability estimates were determined from a review of 940 abstracts and 31 relevant studies examining outcomes in patients with low-risk thyroid cancer undergoing thyroidectomy or neck dissection. During 20 years, cost estimates (including initial surgery, follow-up, and treatment of recurrence) were between $13,896.81 and $14,241.24 for total thyroidectomy and between $15,037.58 and $15,063.75 for hemithyroidectomy. Cause-specific mortality was similar for both treatment strategies, but recurrence-free survival was higher in the total thyroidectomy group. Sensitivity and threshold analyses demonstrated that these results were sensitive to rates of recurrence and cost of follow-up but remained robust when compared with willingness to pay. CONCLUSIONS: Total thyroidectomy dominates over hemithyroidectomy as initial treatment for low-risk papillary thyroid cancer. However, in sensitivity analyses, these results varied by institution because of heterogeneity in long-term treatment outcomes. With changing protocols of management, it is possible that hemithyroidectomy will emerge as being more cost-effective. Long-term prospective trials are necessary to validate our findings.

Adhesion is prerequisite, but alone insufficient, to elicit stem cell pluripotency.

Primitive mammalian neural stem cells (NSCs), arising during the earliest stages of embryogenesis, possess pluripotency in embryo chimera assays in contrast to definitive NSCs found in the adult. We hypothesized that adhesive differences determine the association of stem cells with embryonic cells in chimera assays and hence their ability to contribute to later tissues. We show that primitive NSCs and definitive NSCs possess adhesive differences, resulting from differential cadherin expression, that lead to a double dissociation in outcomes after introduction into the early- versus midgestation embryo. Primitive NSCs are able to sort with the cells of the inner cell mass and thus contribute to early embryogenesis, in contrast to definitive NSCs, which cannot. Conversely, primitive NSCs sort away from cells of the embryonic day 9.5 telencephalon and are unable to contribute to neural tissues at midembryogenesis, in contrast to definitive NSCs, which can. Overcoming these adhesive differences by E-cadherin overexpression allows some definitive NSCs to integrate into the inner cell mass but is insufficient to allow them to contribute to later development. These adhesive differences suggest an evolving compartmentalization in multipotent NSCs during development and serve to illustrate the importance of cell-cell association for revealing cellular contribution.

Fine-needle aspiration biopsy of the thyroid: atypical cytopathological features.

OBJECTIVES: To evaluate the positive predictive value of a thyroid nodule being malignant when categorized as atypical, and to determine the prognostic implications of specific cytopathological features. DESIGN: Retrospective review of consecutive patients undergoing thyroid surgery following fine-needle aspiration biopsy (FNAB) of thyroid nodules. SETTING: Academic teaching hospital in Toronto, Ontario. PATIENTS: A total of 111 consecutive patients with atypical findings from an FNAB who underwent thyroid surgery from January 2000 to November 2005. RESULTS: Of 111 patients included in this study, 62 (56%) were diagnosed with a thyroid malignancy on final histopathological examination. The remaining 49 patients (44%) had benign disease. When comparing patients with a postoperative diagnosis of malignancy vs those with benign disease, micronucleoli (71% vs 49%; P = .01), nuclear grooves (50% vs 31%; P = .03), and powdery chromatin (37% vs 16%; P = .01) were more frequently observed in the group with cancer. The probability of malignancy was 83% if all 3 of these features were present; 32% if none of these features was present (P = .001). CONCLUSIONS: At our institution, when findings from a thyroid nodule FNAB sample were categorized as atypical, the positive predictive value of the nodule being malignant was 56%. In this series of patients, the presence of micronucleoli, nuclear grooves, and powdery chromatin increased the likelihood that an atypical specimen was representative of malignant disease. These features may help guide treatment of patients with atypical findings from a thyroid nodule FNAB sample.

Embryonic cortical neural stem cells migrate ventrally and persist as postnatal striatal stem cells.

Embryonic cortical neural stem cells apparently have a transient existence, as they do not persist in the adult cortex. We sought to determine the fate of embryonic cortical stem cells by following Emx1(IREScre); LacZ/EGFP double-transgenic murine cells from midgestation into adulthood. Lineage tracing in combination with direct cell labeling and time-lapse video microscopy demonstrated that Emx1-lineage embryonic cortical stem cells migrate ventrally into the striatal germinal zone (GZ) perinatally and intermingle with striatal stem cells. Upon integration into the striatal GZ, cortical stem cells down-regulate Emx1 and up-regulate Dlx2, which is a homeobox gene characteristic of the developing striatum and striatal neural stem cells. This demonstrates the existence of a novel dorsal-to-ventral migration of neural stem cells in the perinatal forebrain.

Intrinsic differences distinguish transiently neurogenic progenitors from neural stem cells in the early postnatal brain.

Recent reports of stem cell plasticity have led to the suggestion that there are few intrinsic differences between precursor cells, and that environment dictates fundamental cellular properties such as differentiation potential. This suggestion has been buoyed by other work suggesting that apparent in vivo differences between neural precursor cells are lost when placed in a culture environment. We sought to further test this hypothesis by comparing neural precursors present in various neural tissues during the early postnatal period. Precursors from three postnatal actively neurogenic regions and three postneurogenic regions (cerebral cortex, lateral striatum, and optic nerve) were assayed at postnatal day 1, day 10, and adulthood, and compared to well-characterized ventricular subependymal neural stem cells. In contrast to stem cells that remain multipotential throughout life, the progenitor cells become restricted in a time- and region-dependent manner to an exclusively glial-producing phenotype, a phenomenon that occurs both in vitro and in vivo. Transcription factors associated with neural precursor identity are expressed regardless of brain region of origin or time in vitro. Environmental coculture manipulations are only able to rescue neurogenesis in olfactory bulb precursors but not other restricted progenitors. Thus, in contrast to the views that the in vitro environment has a homogenizing effect on distinct neural precursors, our data suggest that robust intrinsic differences with respect to self-renewal and continued neuron production exist between neural precursors from different brain regions. These differences are evident in vitro and in vivo.

Clonal identification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.

The clonal isolation of putative adult pancreatic precursors has been an elusive goal of researchers seeking to develop cell replacement strategies for diabetes. We report the clonal identification of multipotent precursor cells from the adult mouse pancreas. The application of a serum-free, colony-forming assay to pancreatic cells enabled the identification of precursors from pancreatic islet and ductal populations. These cells proliferate in vitro to form clonal colonies that coexpress neural and pancreatic precursor markers. Upon differentiation, individual clonal colonies produce distinct populations of neurons and glial cells, pancreatic endocrine beta-, alpha- and delta-cells, and pancreatic exocrine and stellate cells. Moreover, the newly generated beta-like cells demonstrate glucose-dependent Ca(2+) responsiveness and insulin release. Pancreas colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. These cells represent a previously unidentified adult intrinsic pancreatic precursor population and are a promising candidate for cell-based therapeutic strategies.

Primitive neural stem cells from the mammalian epiblast differentiate to definitive neural stem cells under the control of Notch signaling.

Basic fibroblast growth factor (FGF2)-responsive definitive neural stem cells first appear in embryonic day 8.5 (E8.5) mouse embryos, but not in earlier embryos, although neural tissue exists at E7.5. Here, we demonstrate that leukemia inhibitory factor-dependent (but not FGF2-dependent) sphere-forming cells are present in the earlier (E5.5-E7.5) mouse embryo. The resultant clonal sphere cells possess self-renewal capacity and neural multipotentiality, cardinal features of the neural stem cell. However, they also retain some nonneural properties, suggesting that they are the in vivo cells' equivalent of the primitive neural stem cells that form in vitro from embryonic stem cells. The generation of the in vivo primitive neural stem cell was independent of Notch signaling, but the activation of the Notch pathway was important for the transition from the primitive to full definitive neural stem cell properties and for the maintenance of the definitive neural stem cell state.

Stem and progenitor cells: the premature desertion of rigorous definitions.

A current disturbing trend in stem cell biology is the abandonment of rigorous definitions of stem and progenitor cells in favor of more ambiguous, all-encompassing concepts. However, recent studies suggest that there are consistent, functional differences in the biology of these two cell types. Admittedly, it can be difficult to harmonize the in vivo and in vitro functional differences between stem and progenitor cells. Nonetheless, these distinctions between cell types should be emphasized rather than ignored, as they can be used to test specific hypotheses in neural stem cell biology.

In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord.

Stem cells isolated from the fourth ventricle and spinal cord form neurospheres in vitro in response to basic fibroblast growth factor (FGF2)+heparin (H) or epidermal growth factor (EGF)+FGF2 together. To determine whether these growth factor conditions are sufficient to induce stem cells within the fourth ventricle and spinal cord to proliferate and expand their progeny in vivo, we infused EGF and FGF2, alone or together, with or without H, into the fourth ventricle for 6 days via osmotic minipumps. Animals were injected with bromodeoxyuridine (BrdU) on days 4, 5 and 6 of infusion in order to label cells proliferating in response to the growth factors. Infusions of EGF+FGF2+H into the fourth ventricle resulted in the largest proliferative effect, a 10.8-fold increase in the number of BrdU+ cells around the fourth ventricle, and a 33.5-fold increase in the number of BrdU+ cells around the central canal of the spinal cord, as compared to vehicle infused controls. The majority of the cells were nestin+ after 6 days of infusion. Seven weeks post-infusion, 22 and 30% of the number of BrdU+ cells induced to proliferate after 6 days of EGF+FGF2+H infusions were still detected around the fourth ventricle and central canal of the spinal cord, respectively. Analysis of the fates of the remaining cells showed that a small percentage of BrdU+ cells around the fourth ventricle and in the white matter of the spinal cord differentiated into astrocytes and oligodendrocytes. BrdU+ neurons were not found in the brainstem or in the grey matter of the cervical spinal cord 7 weeks post-infusion. These results show that endogenous stem cells and progenitors around the fourth ventricle and central canal of the spinal cord proliferate in response to exogenously applied growth factors, but unlike in the lateral ventricle where they generate some new neurons, they only produce new astrocytes and oligodendrocytes at 7 weeks post-infusion.

Adult rodent neurogenic regions: the ventricular subependyma contains neural stem cells, but the dentate gyrus contains restricted progenitors.

Neurogenesis persists in two adult brain regions: the ventricular subependyma and the subgranular cell layer in the hippocampal dentate gyrus (DG). Previous work in many laboratories has shown explicitly that multipotential, self-renewing stem cells in the subependyma are the source of newly generated migrating neurons that traverse the rostral migratory stream and incorporate into the olfactory bulb as interneurons. These stem cells have been specifically isolated from the subependyma, and their properties of self-renewal and multipotentiality have been demonstrated in vitro. In contrast, it is a widely held assumption that the "hippocampal" stem cells that can be isolated in vitro from adult hippocampus reside in the neurogenic subgranular layer and represent the source of new granule cell neurons, but this has never been tested directly. Primary cell isolates derived from the precise microdissection of adult rodent neurogenic regions were compared using two very different commonly used culture methods: a clonal colony-forming (neurosphere) assay and a monolayer culture system. Importantly, both of these culture methods generated the same conclusion: stem cells can be isolated from hippocampus-adjacent regions of subependyma, but the adult DG proper does not contain a population of resident neural stem cells. Indeed, although the lateral ventricle and other ventricular subependymal regions directly adjacent to the hippocampus contain neural stem cells that exhibit long-term self-renewal and multipotentiality, separate neuronal and glial progenitors with limited self-renewal capacity are present in the adult DG, suggesting that neuron-specific progenitors and not multipotential stem cells are the source of newly generated DG neurons throughout adulthood.