Permissive epigenomes endow reprogramming competence to transcriptional regulators.

Identifying molecular and cellular processes that regulate reprogramming competence of transcription factors broadens our understanding of reprogramming mechanisms. In the present study, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks including disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79/K27 methylation, but also other epigenetic pathways, catalyzed by lysine-specific histone demethylase 1A, DNA methyltransferases and histone deacetylases, allows induced pluripotent stem cell generation with almost all OCT factors. We found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors, but in fact enables dismantling of species-dependent reprogramming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these induced permissive epigenetic states, we performed an additional screen with 98 candidate genes. Thereby, we identified 25 transcriptional regulators (OTX2, SIX3, and so on) that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptual framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.

Combining Automated Organoid Workflows with Artificial Intelligence-Based Analyses: Opportunities to Build a New Generation of Interdisciplinary High-Throughput Screens for Parkinson's Disease and Beyond.

Parkinson's disease (PD) is the second most common neurodegenerative disease and primarily characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Despite decades of research and the development of various disease model systems, there is no curative treatment. This could be due to current model systems, including cell culture and animal models, not adequately recapitulating human PD etiology. More complex human disease models, including human midbrain organoids, are maturing technologies that increasingly enable the strategic incorporation of the missing components needed to model PD in vitro. The resulting organoid-based biological complexity provides new opportunities and challenges in data analysis of rich multimodal data sets. Emerging artificial intelligence (AI) capabilities can take advantage of large, broad data sets and even correlate results across disciplines. Current organoid technologies no longer lack the prerequisites for large-scale high-throughput screening (HTS) and can generate complex yet reproducible data suitable for AI-based data mining. We have recently developed a fully scalable and HTS-compatible workflow for the generation, maintenance, and analysis of three-dimensional (3D) microtissues mimicking key characteristics of the human midbrain (called "automated midbrain organoids," AMOs). AMOs build a reproducible, scalable foundation for creating next-generation 3D models of human neural disease that can fuel mechanism-agnostic phenotypic drug discovery in human in vitro PD models and beyond. Here, we explore the opportunities and challenges resulting from the convergence of organoid HTS and AI-driven data analytics and outline potential future avenues toward the discovery of novel mechanisms and drugs in PD research. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The Use of FRAX in Identifying Women Less Than 65 Years Needing Bone Mineral Density Testing.

The purpose of this study is to determine if the United States Preventive Services Task Force (USPSTF) screening guideline for osteoporosis identifies women under the age of 65 with osteoporosis needing bone mineral density (BMD) testing. If the Fracture Risk Assessment Tool (FRAX) tool fails to identify women under the age of 65 with undiagnosed osteoporosis, then diagnosis and treatment are delayed, potentially leading to increased fractures and morbidity. Another aim of this study is to characterize women under the age of 65 with osteoporosis that FRAX fails to identify and provide descriptive data on our study population. A retrospective chart review was completed between 2012 and 2018. We extracted data for 113 women ≤ 65-years with osteoporosis confirmed by BMD or fractures. Major osteoporotic fracture (MOF) risk calculation without BMD by FRAX of 9.3% or greater (high risk group) was found in 51 (45.1%) of patients. Osteoporosis by T-score < 2.5 was evident in 102 (90%) of patients. Previous osteoporotic fractures were noted in 29 (25.7%) of patients. The average age of women in the high-risk group was 58 years and 55 years in the low-risk group. The sensitivity of FRAX for identifying women with a T-score <-2.5 was 40%. The sensitivity of FRAX for identifying women with a history of fracture was 32%. The sensitivity of FRAX for identifying women with a T-score <-2.5 or identifying women with a history of fracture was 32%. These results demonstrate that the FRAX tool alone (USPSTF recommendation) fails to identify many women under the age of 65 with osteoporosis in need of BMD testing. Over half of women would not have had a BMD performed based on guidelines for screening BMD in women <65. Further study is needed to characterize women under the age of 65 with osteoporosis with a FRAX MOF risk less than 9.3%.

Abnormal bone mineral content and density in people with tetrasomy 18p.

Tetrasomy 18p is a rare chromosomal abnormality, resulting from an additional iso-chromosome composed of two copies of the short arm. It is characterized by craniofacial abnormalities, neuromuscular dysfunction, and developmental delay. The Chromosome 18 Clinical Research Center has established the largest cohort of individuals with this rare genetic condition. Here, we describe a case series of 21 individuals with tetrasomy 18p who have a previously unreported clinical finding: low bone mineral density. Most individuals met criteria for low bone density despite being relatively young (mean age of 21 years). Clinicians providing care to individuals affected by Tetrasomy 18p should be aware of their increased risk for decreased bone density and pathological fractures.

Three-Dimensional Cell Culture Systems in Pediatric and Adult Brain Tumor Precision Medicine.

Primary brain tumors often possess a high intra- and intertumoral heterogeneity, which fosters insufficient treatment response for high-grade neoplasms, leading to a dismal prognosis. Recent years have seen the emergence of patient-specific three-dimensional in vitro models, including organoids. They can mimic primary parenteral tumors more closely in their histological, transcriptional, and mutational characteristics, thus approximating their intratumoral heterogeneity better. These models have been established for entities including glioblastoma and medulloblastoma. They have proven themselves to be reliable platforms for studying tumor generation, tumor-TME interactions, and prediction of patient-specific responses to establish treatment regimens and new personalized therapeutics. In this review, we outline current 3D cell culture models for adult and pediatric brain tumors, explore their current limitations, and summarize their applications in precision oncology.

Tissue clearing may alter emission and absorption properties of common fluorophores.

In recent years, 3D cell culture has been gaining a more widespread following across many fields of biology. Tissue clearing enables optical analysis of intact 3D samples and investigation of molecular and structural mechanisms by homogenizing the refractive indices of tissues to make them nearly transparent. Here, we describe and quantify that common clearing solutions including benzyl alcohol/benzyl benzoate (BABB), PEG-associated solvent system (PEGASOS), immunolabeling-enabled imaging of solvent-cleared organs (iDISCO), clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and ScaleS4 alter the emission spectra of Alexa Fluor fluorophores and fluorescent dyes. Clearing modifies not only the emitted light intensity but also alters the absorption and emission peaks, at times to several tens of nanometers. The resulting shifts depend on the interplay of solvent, fluorophore, and the presence of cells. For biological applications, this increases the risk for unexpected channel crosstalk, as filter sets are usually not optimized for altered fluorophore emission spectra in clearing solutions. This becomes especially problematic in high throughput/high content campaigns, which often rely on multiband excitation to increase acquisition speed. Consequently, researchers relying on clearing in quantitative multiband excitation experiments should crosscheck their fluorescent signal after clearing in order to inform the proper selection of filter sets and fluorophores for analysis.

Generation and Maintenance of Homogeneous Human Midbrain Organoids.

Three-dimensional cell cultures ("organoids") promise to better recapitulate native tissue physiology than traditional 2D cultures and are becoming increasingly interesting for disease modeling and compound screening efforts. While a number of protocols for the generation of neural organoids have been published, most protocols require extensive manual handling and result in heterogeneous aggregates with high sample-to-sample variation, which can hinder screening-based strategies. We have now developed a fast and efficient protocol for the generation and maintenance of highly homogeneous and reproducible midbrain organoids. The protocol is streamlined for use in fully automated workflows but can also be performed manually without the need for highly specialized equipment. It relies on the aggregation of small molecule neural precursor cells (smNPCs) in standard 96-well V-bottomed plates under static culture conditions without cumbersome matrix embedding. The result is ready-to-assay uniform 3D human midbrain organoids available in freely scalable quantities for downstream analyses in 3D cell culture. Graphic abstract: Automated midbrain organoid generation workflow and timeline.

Fluorescence-based Single-cell Analysis of Whole-mount-stained and Cleared Microtissues and Organoids for High Throughput Screening.

Three-dimensional (3D) cell culture, especially in the form of organ-like microtissues ("organoids"), has emerged as a novel tool potentially mimicking human tissue biology more closely than standard two-dimensional culture. Typically, tissue sectioning is the standard method for immunohistochemical analysis. However, it removes cells from their native niche and can result in the loss of 3D context during analyses. Automated workflows require parallel processing and analysis of hundreds to thousands of samples, and sectioning is mechanically complex, time-intensive, and thus less suited for automated workflows. Here, we present a simple protocol for combined whole-mount immunostaining, tissue-clearing, and optical analysis of large-scale (approx. 1 mm) 3D tissues with single-cell level resolution. While the protocol can be performed manually, it was specifically designed to be compatible with high-throughput applications and automated liquid handling systems. This approach is freely scalable and allows parallel automated processing of large sample numbers in standard labware. We have successfully applied the protocol to human mid- and forebrain organoids, but, in principle, the workflow is suitable for a variety of 3D tissue samples to facilitate the phenotypic discovery of cellular behaviors in 3D cell culture-based high-throughput screens. Graphic abstract: Automatable organoid clearing and high-content analysis workflow and timeline.

Bilateral atypical fractures of the femur: Ten years AFTER ten years of bisphosphonate therapy.

BACKGROUND: Atypical femur fracture (AFF) is a clinically important complication of bisphosphonate (BP) use in the treatment of osteoporosis. The benefits of long-term BP therapy in preventing osteoporotic fractures have been shown to outweigh the risks of treatment. Discontinuation of BPs or "drug holidays" have been implemented as a strategy to reduce the risk of rare complications such as AFF. CASE REPORT: We present the case of a 70-year-old postmenopausal woman who suffered bilateral AFF ten years after discontinuation of BP treatment. Management of this patient included fixation of the complete AFF with an intramedullary rod. A single dose of denosumab was administered prior to referral to endocrinology and seemed to contribute to callus formation. Denosumab was discontinued to prevent progression of the contralateral incomplete AFF. Teriparatide was indicated for the treatment of this patient's osteoporosis and also led to the resolution of the incomplete AFF. CONCLUSION: Patients receiving long-term BP therapy should be periodically reevaluated in order to maximize the benefit and minimize the risk of treatment. Current research supports the implementation of drug holidays to decrease the risk of AFF; however, this case report confirms the need for continued monitoring after discontinuation of BP therapy. Additionally, our review of current literature highlights the need for more specific research regarding duration of BP treatment and drug holidays.

Cell-Type-Specific High Throughput Toxicity Testing in Human Midbrain Organoids.

Toxicity testing is a crucial step in the development and approval of chemical compounds for human contact and consumption. However, existing model systems often fall short in their prediction of human toxicity in vivo because they may not sufficiently recapitulate human physiology. The complexity of three-dimensional (3D) human organ-like cell culture systems ("organoids") can generate potentially more relevant models of human physiology and disease, including toxicity predictions. However, so far, the inherent biological heterogeneity and cumbersome generation and analysis of organoids has rendered efficient, unbiased, high throughput evaluation of toxic effects in these systems challenging. Recent advances in both standardization and quantitative fluorescent imaging enabled us to dissect the toxicities of compound exposure to separate cellular subpopulations within human organoids at the single-cell level in a framework that is compatible with high throughput approaches. Screening a library of 84 compounds in standardized human automated midbrain organoids (AMOs) generated from two independent cell lines correctly recognized known nigrostriatal toxicants. This approach further identified the flame retardant 3,3',5,5'-tetrabromobisphenol A (TBBPA) as a selective toxicant for dopaminergic neurons in the context of human midbrain-like tissues for the first time. Results were verified with high reproducibility in more detailed dose-response experiments. Further, we demonstrate higher sensitivity in 3D AMOs than in 2D cultures to the known neurotoxic effects of the pesticide lindane. Overall, the automated nature of our workflow is freely scalable and demonstrates the feasibility of quantitatively assessing cell-type-specific toxicity in human organoids in vitro.

Genotype-Phenotype Features of Germline Variants of the TMEM127 Pheochromocytoma Susceptibility Gene: A 10-Year Update.

PURPOSE: This work aimed to evaluate genotype-phenotype associations in individuals carrying germline variants of transmembrane protein 127 gene (TMEM127), a poorly known gene that confers susceptibility to pheochromocytoma (PHEO) and paraganglioma (PGL). DESIGN: Data were collected from a registry of probands with TMEM127 variants, published reports, and public databases. MAIN OUTCOME ANALYSIS: Clinical, genetic, and functional associations were determined. RESULTS: The cohort comprised 110 index patients (111 variants) with a mean age of 45 years (range, 21-84 years). Females were predominant (76 vs 34, P < .001). Most patients had PHEO (n = 94; 85.5%), although PGL (n = 10; 9%) and renal cell carcinoma (RCC, n = 6; 5.4%) were also detected, either alone or in combination with PHEO. One-third of the cases had multiple tumors, and known family history was reported in 15.4%. Metastatic PHEO/PGL was rare (2.8%). Epinephrine alone, or combined with norepinephrine, accounted for 82% of the catecholamine profiles of PHEO/PGLs. Most variants (n = 63) occurred only once and 13 were recurrent (2-12 times). Although nontruncating variants were less frequent than truncating changes overall, they were predominant in non-PHEO clinical presentations (36% PHEO-only vs 69% other, P < .001) and clustered disproportionately within transmembrane regions (P < .01), underscoring the relevance of these domains for TMEM127 function. Integration of clinical and previous experimental data supported classification of variants into 4 groups based on mutation type, localization, and predicted disruption. CONCLUSIONS: Patients with TMEM127 variants often resemble sporadic nonmetastatic PHEOs. PGL and RCC may also co-occur, although their causal link requires further evaluation. We propose a new classification to predict variant pathogenicity and assist with carrier surveillance.

A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids.

Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates ('organoids') or 3D structures with less physiological relevance ('spheroids'). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates. The resulting organoids possess a highly homogeneous morphology, size, global gene expression, cellular composition, and structure. They present significant features of the human midbrain and display spontaneous aggregate-wide synchronized neural activity. By automating the entire workflow from generation to analysis, we enhance the intra- and inter-batch reproducibility as demonstrated via RNA sequencing and quantitative whole mount high-content imaging. This allows assessing drug effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow.

In 1907, the American zoologist Ross Granville Harrison developed the first technique to artificially grow animal cells outside the body in a liquid medium. Cells are still grown in much the same way in modern laboratories: a single layer of cells is placed in a warm incubator with nutrient-rich broth. These cell layers are often used to test new drugs, but they cannot recapitulate the complexity of a real organ made from multiple cell types within a living, breathing human body. Growing three-dimensional miniature organs or 'organoids' that behave in a similar way to real organs is the next step towards creating better platforms for drug screening, but there are several difficulties inherent to this process. For one thing, it is hard to recreate the multitude of cell types that make up an organ. For another, the cells that do grow often fail to connect and communicate with each other in biologically realistic ways. It is also tough to grow a large number of organoids that all behave in the same way, making it hard to know whether a particular drug works or whether it is just being tested on a 'good' organoid. Renner et al. have been able to overcome these issues by using robotic technology to create thousands of identical, mid-brain organoids from human cells in the lab. The robots perform a series of precisely controlled tasks ­ including dispensing the initial cells into wells, feeding organoids as they grow and testing them at different stages of development. These mini-brains, which are the size of the head of a pin, mimic the part of the brain where Parkinson's disease first manifests. They can be used to test new drugs for Parkinson's, and to better understand the biology of the brain. Perhaps more importantly, other types of organoids can be created using the same technique to model diseases that affect other areas of the brain, or other organs altogether. For example, Renner et al. also generated forebrain organoids using an automated approach for both generation and analysis. This research, which shows that organoids can be grown and tested in a fully automated, reproducible and scalable way, creates a platform to quickly, cheaply and easily test thousands of drugs for Parkinson's and other difficult-to-treat diseases in a human setting. This approach has the potential to reduce research waste by increasing the chances that a drug that works in the lab will also ultimately work in a patient; and reduce animal experiments, as drugs that do not work in human tissues will not proceed to animal testing.

Quantitative trait locus on chromosome 1q influences bone loss in young Mexican American adults.

Bone loss occurs as early as the third decade and its cumulative effect throughout adulthood may impact risk for osteoporosis in later life, however, the genes and environmental factors influencing early bone loss are largely unknown. We investigated the role of genes in the change in bone mineral density (BMD) in participants in the San Antonio Family Osteoporosis Study. BMD change in 327 Mexican Americans (ages 25-45 years) from 32 extended pedigrees was calculated from DXA measurements at baseline and follow-up (3.5 to 8.9 years later). Family-based likelihood methods were used to estimate heritability (h(2)) and perform autosome-wide linkage analysis for BMD change of the proximal femur and forearm and to estimate heritability for BMD change of lumbar spine. BMD change was significantly heritable for total hip, ultradistal radius, and 33% radius (h(2) = 0.34, 0.34, and 0.27, respectively; p < 0.03 for all), modestly heritable for femoral neck (h(2) = 0.22; p = 0.06) and not heritable for spine BMD. Covariates associated with BMD change included age, sex, baseline BMD, menopause, body mass index, and interim BMI change, and accounted for 6% to 24% of phenotype variation. A significant quantitative trait locus (LOD = 3.6) for femoral neck BMD change was observed on chromosome 1q23. In conclusion, we observed that change in BMD in young adults is heritable and performed one of the first linkage studies for BMD change. Linkage to chromosome 1q23 suggests that this region may harbor one or more genes involved in regulating early BMD change of the femoral neck.

The Convergence of Stem Cell Technologies and Phenotypic Drug Discovery.

Recent advances in induced pluripotent stem cell technologies and phenotypic screening shape the future of bioactive small-molecule discovery. In this review we analyze the impact of small-molecule phenotypic screens on drug discovery as well as on the investigation of human development and disease biology. We further examine the role of 3D spheroid/organoid structures, microfluidic systems, and miniaturized on-a-chip systems for future discovery strategies. In highlighting representative examples, we analyze how recent achievements can translate into future therapies. Finally, we discuss remaining challenges that need to be overcome for the adaptation of the next generation of screening approaches.

Chronic Myeloid Leukemia Associated Hypercalcemia: A Case Report and Literature Review.

BACKGROUND Hypercalcemia associated with chronic myeloid leukemia (CML) is an ominous sign. Although rare, several cases have been reported and multiple pathophysiologic mechanisms have been independently proposed. We present a patient case and a literature review of the clinical presentation and mechanisms of CML-associated hypercalcemia. CASE REPORT A 58-year-old male with a past medical history of CML diagnosed six years earlier, presented to the emergency department with one week of acute confusion, disorientation, polyuria, and polydipsia. On physical examination, we observed tachycardia, altered mental status, and dehydration. Blood analysis revealed leukocytosis, thrombocytosis, and marked hypercalcemia (18.6 mg/dL). His chest CT scan showed diffuse lytic lesions and bone destruction concerning for diffuse bone marrow involvement. The patient was diagnosed with hypercalcemia in the context of a CML blast phase. Treatment with hydration, calcitonin, and zoledronic acid lead to control of his symptoms and normalization of his serum calcium levels. After discharged, the patient was maintained on palliative treatment and zoledronic acid management without new episodes of hypercalcemia. However, eight months later, the patient died. CONCLUSIONS Evidence from the literature demonstrates a highly variable clinical presentation of CML-associated hypercalcemia, commonly occurring during an accelerated or a blast phase, and associated with poor survival. Multiple mechanisms could be involved and are not exclusive of each other. Better understanding of the pathophysiologic mechanisms involved in CML-associated hypercalcemia could lead to improvement in clinical and laboratory evaluation of these patients and be the foundation for the development of better management strategies and possibly target-directed therapy to positively improve prognosis.

Upregulation of TGF-beta1 expression may be necessary but is not sufficient for excessive scarring.

Transforming growth factor beta 1 (TGF-beta1) upregulation has been implicated in hypertrophic scars and keloids, but it is unclear if it is the cause or an effect of excessive scar formation. In this study, we overexpressed TGF-beta1 in fibroblasts and characterized its role. Normal human dermal fibroblasts were genetically modified to overexpress TGF-beta1 as the wild-type latent molecule or as a mutant constitutively active molecule. TGF-beta1 secretion was measured, as were the effects of TGF-beta1 upregulation on cell proliferation, expression of smooth muscle cell alpha actin (SMC alpha-actin) and ability to contract collagen lattices. Fibroblasts were implanted intradermally into athymic mice and tissue formation was analyzed over time by histology and immunostaining. Gene-modified fibroblasts secreted approximately 20 times the TGF-beta1 released by control cells, but only cells expressing mutant TGF-beta1 secreted it in the active form. Fibroblasts expressing the active TGF-beta1 gene had increased levels of SMC alpha-actin and enhanced ability to contract a collagen lattice. After intradermal injection into athymic mice, only fibroblasts expressing active TGF-beta1 formed "keloid-like" nodules containing collagen, which persisted longer than implants of the other cell types. We conclude that upregulation of TGF-beta1 by fibroblasts may be necessary, but is not sufficient for excessive scarring. Needed are other signals to activate TGF-beta1 and prolong cell persistence.

Neurite bridging across micropatterned grooves.

After injury, regenerating axons must navigate complex, three-dimensional (3D) microenvironments. Topographic guidance of neurite outgrowth has been demonstrated in vitro with culture substrates that contain micropatterned features on the nanometer-micron scale. In this study we report the ability of microfabricated biomaterials to support neurite extension across micropatterned grooves with feature sizes on the order of tens of microns, sizes relevant to the design of biomaterials and tissue engineering scaffolds. Neonatal rat dorsal root ganglion (DRG) neurons were cultured on grooved substrates of poly(dimethyl siloxane) coated with poly-L-lysine and laminin. Here we describe an unusual capability of a subpopulation of DRG neurons to extend neurites that spanned across the grooves, with no underlying solid support. Multiple parameters influenced the formation of bridging neurites, with the highest numbers of bridges observed under the following experimental conditions: cell density of 125,000 cells per sample, groove depth of 50 microm, groove width of 30 microm, and plateau width of 200 microm. Bridges were formed as neurites extended from a neuron in a groove, contacted adjacent plateaus, pulled the neuron up to become suspended over the groove, and the soma translocated to the plateau. These studies are of interest to understanding cytoskeletal dynamics and designing biomaterials for 3D axon guidance.

Effects of alendronate on bone mineral density in men with prostate cancer treated with androgen deprivation therapy.

Bone mineral density (BMD) is low in men with prostate cancer treated with androgen deprivation therapy (ADT). Intravenous bisphosphonates have been shown to prevent the bone loss, however, the effectiveness of oral bisphosphonates have not been studied in this population. In this retrospective cohort study, we examine the effect of alendronate on BMD in men with prostate cancer receiving ADT. We reviewed the charts of patients receiving ADT referred from the VA Urology Clinic for BMD measurements. Forty seven patients had follow up BMD measurements (17.6+8.3 months). Twenty-two men (47%) were also receiving alendronate 70 mg every week. There was a statistically significant difference (p<0.05) in the percent change of BMD per year at the spine (-1.29+/-0.7% vs. +1.41+/-0.7%), total hip (-0.94+/-0.6% vs. +0.97+/-0.5%), femoral neck (-2.17+/-0.7% vs. +0.32+/-0.6%) and trochanter (-2.01+/-0.7% vs. +0.79+/-0.8%) in the patients not treated compared to those treated with alendronate. In the four other measured sites at the radius (proximal, mid, ultra distal and total), there were no statistically significant differences (p>0.05). These findings confirm that bone loss occurs in men receiving ADT at all sites measured. The use of alendronate prevents bone loss at the spine and hip, but does not seem to have the same protective effect at the radius.

Urinary calcium excretion in primary hyperparathyroidism: relationship to 25-hydroxyvitamin d status.

OBJECTIVE: To determine the prevalence of vitamin D deficiency in patients with primary hyperparathyroidism (PHPT) and evaluate the relationship between urinary calcium excretion and serum 25-hydroxyvitamin D (25-OH-D) levels in patients with PHPT. METHODS: We present a case report and a review of the medical records of patients with PHPT. Of 75 patients with PHPT substantiated by hypercalcemia and increased levels of intact parathyroid hormone (iPTH), 35 were identified with laboratory evaluation of vitamin D levels and 24-hour urinary calcium excretion. These study subjects were stratified as 25-OH-D deficient, insufficient, or replete (on the basis of serum values of <15, 15 to 25, or >25 ng/mL, respectively). Total 24-hour urinary calcium excretion and the fractional excretion of calcium (FECa) were analyzed as a function of 25-OH-D status. RESULTS: Of the 35 study subjects, 14 (40%) and 13 (37%) had 25-OH-D deficiency or insufficiency, respectively. Those patients with a 25-OH-D level <15 ng/mL had higher serum iPTH concentrations as well as lower urinary calcium excretion and FECa. No significant correlations were found, however, between 25-OH-D status and iPTH concentrations (r = -0.21; P = 0.23), total 24-hour urinary calcium excretion (r = 0.07; P = 0.7), or FECa (r = 0.04; P = 0.8). CONCLUSION: Vitamin D deficiency (25-OH-D levels <15 ng/mL) was common in our population of patients with PHPT. Urinary calcium excretion was not significantly altered by 25-OH-D deficiency in patients with newly recognized PHPT. Measurements of total urinary calcium excretion and FECa can be reliably used to rule out familial benign hypocalciuric hypercalcemia in the initial evaluation of PHPT, regardless of 25-OH-D status. Determining 25-OH-D concentrations best assesses the vitamin D status.

Sequence analysis of measles virus nucleocapsid transcripts in patients with Paget's disease.

It has been debated for almost 30 years whether Paget's disease of bone results from paramyxoviral infection of osteoclasts (OCs). Paramyxoviral-like nuclear inclusions are found in OCs from patients with Paget's disease, and measles virus (MV) or canine distemper virus (CDV) messenger RNA (mRNA) transcripts have been detected by in situ hybridization in bone cells from pagetic lesions. Furthermore, immunocytochemical studies have shown the presence of several paramyxoviral species in OCs from patients with Paget's disease. However, others have been unable to detect paramyxoviral transcripts in bone samples from patients with Paget's disease or marrow cultures from involved sites of patients with Paget's disease. Furthermore, no one has been able to isolate an infectious virus from pagetic bone samples or marrow cells from patients with Paget's disease, and a full-length viral gene has not been sequenced from pagetic samples. In this study, we have obtained the full-length sequence for the MV nucleocapsid (MVNP) gene in bone marrow from an involved site from a patient with Paget's disease and more than 700 base pairs (bps) of MVNP sequence in 3 other patients with Paget's disease. These sequences were undetectable in four normal marrow samples studied simultaneously. The sequences from the patients contained multiple mutations that differed from the Edmonston strain MVNP gene. These findings are consistent with the presence of a chronic MV infection in affected sites from these patients with Paget's disease.