Dental defects in the primary dentition associated with hypophosphatasia from biallelic ALPL mutations.

ALPL encodes tissue-nonspecific alkaline phosphatase (TNAP), an enzyme expressed in bone, teeth, liver, and kidney. ALPL loss-of-function mutations cause hypophosphatasia (HPP), an inborn error-of-metabolism that produces skeletal and dental mineralization defects. Case reports describe widely varying dental phenotypes, making it unclear how HPP comparatively affects the three unique dental mineralized tissues: enamel, dentin, and cementum. We hypothesized that HPP affected all dental mineralized tissues and aimed to establish quantitative measurements of dental tissues in a subject with HPP. The female proband was diagnosed with HPP during childhood based on reduced alkaline phosphatase activity (ALP), mild rachitic skeletal effects, and premature primary tooth loss. The diagnosis was subsequently confirmed genetically by the presence of compound heterozygous ALPL mutations (exon 5: c.346G>A, p.A116T; exon 10: c.1077C>G, p.I359M). Dental defects in 8 prematurely exfoliated primary teeth were analyzed by high resolution micro-computed tomography (micro-CT) and histology. Similarities to the Alpl-/- mouse model of HPP were identified by additional analyses of murine dentoalveolar tissues. Primary teeth from the proband exhibited substantial remaining root structure compared to healthy control teeth. Enamel and dentin densities were not adversely affected in HPP vs. control teeth. However, analysis of discrete dentin regions revealed an approximate 10% reduction in the density of outer mantle dentin of HPP vs. control teeth. All 4 incisors and the molar lacked acellular cementum by micro-CT and histology, but surprisingly, 2 of 3 prematurely exfoliated canines exhibited apparently normal acellular cementum. Based on dentin findings in the proband's teeth, we examined dentoalveolar tissues in a mouse model of HPP, revealing that the delayed initiation of mineralization in the incisor mantle dentin was associated with a broader lack of circumpulpal dentin mineralization. This study describes a quantitative approach to measure effects of HPP on dental tissues. This approach has uncovered a previously unrecognized novel mantle dentin defect in HPP, as well as a surprising and variable cementum phenotype within the teeth from the same HPP subject.

Clodronate improves lameness in horses without changing bone turnover markers.

BACKGROUND: Clodronate is prescribed to performance horses with lameness. Despite its clinical popularity, little research has been done to understand the effects of clodronate in the horse. OBJECTIVES: Our objective was to determine if a single treatment with clodronate at the clinically approved dose altered bone remodelling, bone cell recruitment or lameness in the horse. STUDY DESIGN: Twelve university-owned equestrian team competition horses with a history of forelimb lameness due to navicular syndrome were randomised to receive either 1.4 mg/kg clodronate (CLOD n = 6) or an equivalent volume of LRS (CONT; n = 6) in a blinded manner. METHODS: Blood was evaluated weekly for 8 weeks before and after drug administration (clodronate or placebo) for bone turnover markers CTX-I and osteocalcin. Lameness evaluations were performed to assess for change in lameness 1 week before and 1, 2, 3 and 8 weeks after drug administration. Coach questionnaires were performed to assess for change in ridden performance 1, 2, 3 and 8 weeks after drug administration. Bone cell recruitment was evaluated in vitro 2 weeks before and after drug administration. RESULTS: There were no differences in in vitro bone cell recruitment from whole bone marrow or in bone turnover markers CTX-I or osteocalcin. A small but significant decrease in forelimb lameness was detected in CLOD treated horses 1 week after treatment (P = 0.005). There were no significant differences in hindlimb lameness. Coaches identified an improvement in performance significantly more often in CLOD vs. CONT (P = 0.01) at week 8. MAIN LIMITATIONS: Two CONT horses received intra-articular anti-inflammatory medication after treatment, which may have altered lameness results. CONCLUSIONS: A single dose of clodronate appears to reduce lameness without producing detectable effects on bone turnover markers. Due to the long half-life of a bisphosphonate drug, the effect of multiple doses on bone remodelling and lameness should be investigated. The Summary is available in Portuguese - see Supporting Information.

Low bone turnover and low bone density in a cohort of adults with Down syndrome.

UNLABELLED: Increased incidence of osteoporosis in Down syndrome has been reported, but etiology is not established. We report low bone turnover markers and bone mineral density (BMD) in a cohort of people with Down syndrome without consistent clinical risk factors. Our results should guide future studies and treatments for this common problem. INTRODUCTION: To better understand the etiology for osteoporosis in Down syndrome (DS), we measured bone density by dual-energy X-ray absorptiometry (DXA) and circulating biochemical markers of bone formation and resorption in a cohort of 30 community-dwelling DS adults. METHODS: Seventeen males and 13 females followed in the University of Arkansas Down Syndrome Clinic were evaluated by DXA to estimate BMD and underwent phlebotomy to measure serum procollagen type-1 intact N-terminal propeptide (P1NP) to evaluate bone formation, and serum C-terminal peptide of type-I collagen (CTx) to evaluate bone resorption. RESULTS: Seven of 13 DS females and 12 of 17 DS males had low bone mass at one of measured sites (z≤-2.0). When data were grouped by age, males had apparent osteopenia earlier than females. The mean P1NP in the normal group was 19.2±5.2 ng/ml vs. 2.2±0.9 ng/ml in the DS group (P=0.002). Serum CTx levels in the normal group were 0.4±0.1 ng/ml vs. 0.3±0.1 ng/ml (P=0.369). CONCLUSIONS: Low BMD in adults with DS is correlated with a significant decrease in bone formation markers, compared to controls without DS, and is independent of gender. These data suggest that diminished osteoblastic bone formation and inadequate accrual of bone mass, with no significant differences in bone resorption, are responsible for the low bone mass in DS. These observations question the use of antiresorptive therapy in this population and focus attention on increasing bone mass by other interventions.

dsAAV8-mediated gene transfer and ß-cell expression of IL-4 and ß-cell growth factors are capable of reversing early-onset diabetes in NOD mice.

Type-I diabetes is a chronic disease mediated by autoimmune destruction of insulin-producing ß-cells. Although progress has been made towards improving diabetes-associated pathologies and the quality of life for those living with diabetes, no therapy has been effective at eliminating disease manifestations or reversing disease progression. Here, we examined whether double-stranded adeno-associated virus serotype 8 (dsAAV8)-mediated gene delivery to endogenous ß-cells of interleukin (IL)-4 in combination with ß-cell growth factors can reverse early-onset diabetes in NOD mice. Our results demonstrate that a single treatment with dsAAV8 vectors expressing IL-4 in combination with glucagon-like peptide-1 or hepatocyte growth factor/NK1 under the regulation of the insulin promoter enhanced ß-cell proliferation and survival in vivo, significantly delaying diabetes progression in NOD mice, and reversing disease in ∼10% of treated NOD mice. These results demonstrate the ability to reverse hyperglycemia in NOD mice with established diabetes by in vivo gene transfer to ß-cells of immunomodulatory factors and ß-cell growth factors.

Multiscale kinetic modeling of liposomal Doxorubicin delivery quantifies the role of tumor and drug-specific parameters in local delivery to tumors.

Nanoparticle encapsulation has been used as a means to manipulate the pharmacokinetic (PK) and safety profile of drugs in oncology. Using pegylated liposomal doxorubicin (PLD) vs. conventional doxorubicin as a model system, we developed and experimentally validated a multiscale computational model of liposomal drug delivery. We demonstrated that, for varying tumor transport properties, there is a regimen where liposomal and conventional doxorubicin deliver identical amounts of doxorubicin to tumor cell nuclei. In mice, typical tumor properties consistently favor improved delivery via liposomes relative to free drug. However, in humans, we predict that some tumors will have properties wherein liposomal delivery delivers the identical amount of drug to its target relative to dosing with free drug. The ability to identify tumor types and/or individual patient tumors with high degree of liposome deposition may be critical for optimizing the success of nanoparticle and liposomal anticancer therapeutics.CPT: Pharmacometrics & Systems Pharmacology (2012) 1, e15; doi:10.1038/psp.2012.16; advance online publication 21 November 2012.

Response of adult mouse uterus to early disruption of estrogen receptor-alpha signaling is influenced by Krüppel-like factor 9.

Inappropriate early exposure of the hormone-responsive uterus to estrogenic compounds is associated with increased risk for adult reproductive diseases including endometrial cancers. While the dysregulation of estrogen receptor-alpha (ESR1) signaling is well acknowledged to mediate early events in tumor initiation, mechanisms contributing to sustained ESR1 activity later in life and leading to induction of oncogenic pathways remain poorly understood. We had shown previously that the transcription factor Krüppel-like factor 9 (KLF9) represses ESR1 expression and activity in Ishikawa endometrial glandular epithelial cells. We hypothesized that KLF9 functions as a tumor suppressor, and that loss of its expression enhances ESR1 signaling. Here, we evaluated the contribution of KLF9 to early perturbations in uterine ESR1 signaling pathways elicited by the administration of synthetic estrogen diethylstilbestrol (DES) to wild-type (WT) and Klf9 null (KO) mice on postnatal days (PNDs) 1-5. Uterine tissues collected at PND84 were subjected to histological, immunological, and molecular analyses. Compared with WT mice, KO mice demonstrated larger endometrial glands and lower endometrial gland numbers; DES exposure exacerbated these differences. Loss of KLF9 expression resulted in increased glandular ESR1 immunoreactivity with DES, without effects on serum estradiol levels. Quantitative RT-PCR analyses indicated altered expression of uterine genes commonly dysregulated in endometrial cancers (Akt1, Mmp9, Slpi, and Tgfbeta1) and of those involved in growth regulation (Fos, Myc, Tert, and Syk), with loss of Klf9, alone or in concert with DES. Our data support a molecular network between KLF9 and ESR1 in the uterus, and suggest that silencing of KLF9 may contribute to endometrial dysfunctions initiated by aberrant estrogen action.

DsAAV8-mediated expression of glucagon-like peptide-1 in pancreatic beta-cells ameliorates streptozotocin-induced diabetes.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that performs a wide array of well-characterized antidiabetic actions, including stimulation of glucose-dependent insulin secretion, upregulation of insulin gene expression and improvements in beta-cell survival. GLP-1-receptor agonists have been developed for treatment of diabetes; however, the short biological half-lives of these peptide-based therapeutics requires that frequent injections be administered to maintain sufficient circulating levels. Thus, novel methods of delivering GLP-1 remain an important avenue of active research. It has recently been demonstrated that self-complimentary, double-stranded, adeno-associated virus serotype-8 (DsAAV8) can efficiently transduce pancreatic beta-cells in vivo, resulting in long-term transgene expression. In this study, we engineered a DsAAV8 vector containing a GLP-1 transgene driven by the mouse insulin-II promoter (MIP). Biological activity of the GLP-1 produced from this transgene was assessed using a luciferase-based bioassay. DsAAV8-MIP-GLP-1 was delivered via intraperitoneal injection and beta-cell damage induced by multiple low dose streptozotocin (STZ) administration. Glucose tolerance was assessed following intraperitoneal glucose injections and beta-cell proliferation measured by PCNA expression. Expression of GLP-1 in Min6 beta-cells resulted in glucose-dependent secretion of biologically active GLP-1. Intraperitoneal delivery of DsAAV8-MIP-GLP-1 to mice led to localized GLP-1 expression in beta-cells and protection against development of diabetes induced by multiple low-dose STZ administration. This protection was associated with significant increase in beta-cell proliferation. Results from this study indicate that expression and secretion of GLP-1 from beta-cells in vivo via DsAAV8 represents a novel therapeutic strategy for treatment of diabetes.

Bone is a target for the antidiabetic compound rosiglitazone.

Rosiglitazone is an FDA-approved oral antidiabetic agent for the treatment of type 2 diabetes. This compound improves insulin sensitivity through the activation of the nuclear receptor, peroxisome proliferator-activated receptor-gamma (PPAR-gamma). In addition to sensitizing cells to insulin, the PPAR-gamma2 isoform appears to be critical for the regulation of osteoblast and adipocyte differentiation from common mesenchymal bone marrow progenitors. We have demonstrated previously that PPAR-gamma2 activated with rosiglitazone acts as a dominant inhibitor of osteoblastogenesis in murine bone marrow in vitro. Here, we show that in vivo, rosiglitazone administration results in significant bone loss. When rosiglitazone (20 microg/g body weight/d) was given to 6-month-old, nondiabetic C57BL/6 mice for 7 wk, a significant decrease in total body bone mineral density was observed. Analysis of bone microarchitecture, using micro-computed tomography, demonstrated a decrease in bone volume, trabecular width, and trabecular number and an increase in trabecular spacing. Histomorphometric analysis showed a decrease in bone formation rate, with a simultaneous increase in fat content in the bone marrow. Changes in bone morphology and structure were accompanied by changes in the expression of osteoblast- and adipocyte-specific marker genes; the expression of the osteoblast-specific genes Runx2/Cbfa1, Dlx5, and alpha1(I)collagen were decreased, whereas the expression of the adipocyte-specific fatty acid binding protein aP2, was increased. These in vivo data suggest that rosiglitazone therapy may pose a significant risk of adverse skeletal effects in humans.

Structure of problem and positive behaviors in African American youths.

The association of 21 problem and positive behaviors was explored in a sample of 283 inner-city, African American youths of ages 8 through 12. Data reduction yielded 4 factors, 3 representing different types or levels of problem behavior, labeled Interpersonal-Minor Deviance, School Problems, and Covert-High Deviance, and 1 representing positive behaviors. The 3 problem behavior factors, although not the positive behavior factor, were significantly correlated with an underlying second-order general deviance factor. The problem behavior clusters identified differed by the settings in which they occur as well as their inherent magnitude of deviance. Discriminant validity analyses confirmed that deviance was not a unitary phenomenon. Limitations as well as other implications of the data are discussed.

Comparison of oral ritodrine and magnesium gluconate for ambulatory tocolysis.

Magnesium sulfate has been administered intravenously to arrest preterm labor but the oral form of this drug cannot be used for continual tocolysis. This trial involved the administration of oral magnesium gluconate to determine its effectiveness compared with that of ritodrine hydrochloride in 50 patients whose labor had been arrested by parenteral therapy. Group A (n = 25) received 1 gm of oral magnesium gluconate every 2 to 4 hours for tocolysis and group B (n = 25) received 10 mg of ritodrine every 2 to 4 hours. The number of patients who progressed to 37 weeks' gestation was similar (group A, 21 versus group B, 19) and the time gained in utero was not different (group A, 6.4 weeks versus group B, 5.9 weeks). There was a trend toward more side effects with the use of ritodrine (40%) compared with magnesium gluconate (16%), but the numbers were too small to reveal a significant difference. These data suggest that magnesium gluconate used as an oral tocolytic is as effective as a beta-agonist in patients whose labor is arrested initially with intravenous therapy.

Tocolysis with oral magnesium.

Seventeen patients in whom uterine activity responded favorably to parenteral magnesium sulfate were given oral magnesium gluconate for continued tocolysis. The mean serum magnesium level before therapy was 1.44 +/- 0.22 mg/100 ml, whereas 2 hours after initiation of oral magnesium it was 2.16 +/- 0.32 mg/100 ml (p less than 0.05). One patient had nausea without vomiting or diarrhea. These data suggest that magnesium ingested orally can raise the serum magnesium level significantly.

Gap junction modulation in rat uterus. I. Effects of estrogens on myometrial and serosal cells.

The ability of estradiol benzoate (E2 B) and diethylstilbestrol (DES) to affect the formation and internalization of gap junctions was examined in several uterine cell types from hypophysectomized rats. Both myometrial and serosal cells respond to daily administration of E2 B or DES by increasing gap junction membrane in a dose-dependent fashion. The myometrial cell response arises from a zero base with gap junctions detected within 24 h of a single injection of 500 micrograms E2 B, while five daily injections of 5 micrograms E2 B were required to induce their formation at the lower dosage. Uterine serosal cells exhibit small macular gap junctions 60 days posthypophysectomy with E2 B stimulation leading to an increase in the number of macular gap junctions and the induction of annular gap junctions. Myometrial cell gap junctions were modulated by L-thyroxine and progesterone when administered in combination with E2 B but were without effect when administered alone. Combination of indomethacin with E2 B injections antagonized E2 B-stimulated junction growth in both myometrial and serosal cells, however, only serosal cells responded to exogenous prostaglandin (PG) injections, with PGE1 increasing and PGF2 alpha decreasing the number of serosal cell gap junctions. These studies support the assumption that the induction of gap junctions in uterine myometrium is hormone dependent.

The cytosine arabinoside (Ara-C) syndrome.

Four patients with non-Hodgkin lymphoma and two with acute lymphocytic leukemia (ages 4 and 4 months to 16 years 6 months) exhibited a unique reaction to intravenously administered cytosine arabinoside (Ara-C) given alone as a part of the previously reported LSA2-L2 treatment protocol. The syndrome was characterized by fever, myalgia, bone pain, and occasionally by chest pain, maculopapular rash, and conjunctivitis. Each of the eleven episodes of this syndrome occurred within 6-12 hours of drug infusion and always abated after cessation of Ara-C. Prior to the reaction, patients had been on therapy for an average of 13.5 months during which they were exposed to 2298-5387 mg/m2 (mean of 3200 mg/m2) of Ara-C. The high incidence of this syndrome (50% of our patients on the LSA2-L2 regimen and 33% of those receiving Ara-C) has not been previously reported. Considering the prolonged exposure to Ara-C and our inability to document infections in the patients or pyrogens contaminating the drug lots, the most likely explanation for this syndrome is a hypersensitivity reaction to Ara-C. Prevention of these symptoms with corticosteroids supports this contention and provides a reasonable alternative to discontinuing Ara-C.