Targeted Resequencing of 29 Candidate Genes and Mouse Expression Studies Implicate ZIC3 and FOXF1 in Human VATER/VACTERL Association

The VATER/VACTERL association describes the combination of congenital anomalies including vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. As mutations in ciliary genes were observed in diseases related to VATER/VACTERL, we performed targeted resequencing of 25 ciliary candidate genes as well as disease‐associated genes (FOXF1, HOXD13, PTEN, ZIC3) in 123 patients with VATER/VACTERL or VATER/VACTERL‐like phenotype. We detected no biallelic mutation in any of the 25 ciliary candidate genes; however, identified an identical, probably disease‐causing ZIC3 missense mutation (p.Gly17Cys) in four patients and a FOXF1 de novo mutation (p.Gly220Cys) in a further patient. In situ hybridization analyses in mouse embryos between E9.5 and E14.5 revealed Zic3 expression in limb and prevertebral structures, and Foxf1 expression in esophageal, tracheal, vertebral, anal, and genital tubercle tissues, hence VATER/VACTERL organ systems. These data provide strong evidence that mutations in ZIC3 or FOXF1 contribute to VATER/VACTERL.     

Circulating concentrations of IGF-I and IGFBP-3 are not predictive of incidence or clinical behavior of pediatric osteosarcoma

BACKGROUND: Preclinical studies suggest a role of insulin-like growth factor-1 (IGF-1) in the proliferation of osteosarcoma cells in vivo. The purpose of this study is to address the relationship between serum levels of IGF-1 and its binding protein (IGFBP-3), and the clinical behavior and outcome of osteosarcoma in children, and to compare those levels present in osteosarcoma patients with a normal population. PROCEDURE: Serum IGF-1 and IGFBP-3 levels were determined by ELISA in 37 patients with osteosarcoma treated on the same treatment regimen (OS-91 protocol), and who had available serum samples from diagnosis. IGF-1 and IGFBP-3 levels were compared with those previously established in the normal population, matched for age and gender, and were correlated with the presence of metastatic disease, histologic response to preoperative chemotherapy, and event-free survival. RESULTS: In osteosarcoma patients the median IGF-1 level was 275 ng/ml (range, 105-613) and the median IGFBP-3 level was 3.4 mg/L (range, 2.3-5.1). IGF-1 levels differed from those in the normal population (P = 0.029); although we anticipated higher IGF-1 levels than normal children, 68% of observed standardized scores were less than 0. Furthermore, IGF-1 or IGFBP-3 levels failed to correlate with the presence of metastatic disease (P = 0.12 and P = 0.12, respectively), histologic response (Rosen-Huvos grades 3/4 vs. grades 1/2) (P = 0.95 and P = 0.71, respectively), or event-free survival (P = 0.52 and P = 0.41, respectively). There was a strong association observed between IGF-1 and IGFBP-3 levels (P < 0.001). CONCLUSIONS: In this retrospective study of 37 patients, we found that circulating levels of IGF-1 and IGFBP-3 are not predictive of the development or clinical characteristics of pediatric osteosarcoma. However, further studies on a larger patient population should be performed in order to investigate this relationship.     

Effect of the group I metabotropic glutamate agonist DHPG on the visual cortex

Metabotropic glutamate receptors have a variety of effects in visual cortex that depend on the age of the animal, the layer of the cortex, and the group of the receptor. Here we describe these effects for group I receptors, using both in vivo and in vitro preparations. The metabotropic group I glutamate receptor agonist 3,5 dihydroxyphenylglycine (DHPG) potentiates the responses to N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) in slices of rat visual cortex. It also increases, initially, the visual response in the cat visual cortex. Both these effects are largest at 3-4 wk of age and decline to insignificance by 10 wk of age. Both are also largest in lower layers of cortex, which explains why the facilitatory effects found with the general metabotropic glutamate agonist 1S,3R aminocyclopentane-1,3-dicarboxylic acid (ACPD) are observed only in lower layers. Prolonged application of DHPG in the cat visual cortex, after the initial excitatory effect, produces depression. We also found that DHPG facilitates the NMDA response in fast-spiking cells, which are inhibitory, providing a partial explanation for this. Thus there are multiple effects of group I metabotropic glutamate receptors, which vary with layer and age in visual cortex.     

Allosteric modulators of NR2B-containing NMDA receptors: molecular mechanisms and therapeutic potential

N-methyl-D-aspartate receptors (NMDARs) are ion channels gated by glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). They are widespread in the CNS and are involved in numerous physiological and pathological processes including synaptic plasticity, chronic pain and psychosis. Aberrant NMDAR activity also plays an important role in the neuronal loss associated with ischaemic insults and major degenerative disorders including Parkinson''s and Alzheimer''s disease. Agents that target and alter NMDAR function may, thus, have therapeutic benefit. Interestingly, NMDARs are endowed with multiple extracellular regulatory sites that recognize ions or small molecule ligands, some of which are likely to regulate receptor function in vivo. These allosteric sites, which differ from agonist-binding and channel-permeation sites, provide means to modulate, either positively or negatively, NMDAR activity. The present review focuses on allosteric modulation of NMDARs containing the NR2B subunit. Indeed, the NR2B subunit confers a particularly rich pharmacology with distinct recognition sites for exogenous and endogenous allosteric ligands. Moreover, NR2B-containing receptors, compared with other NMDAR subtypes, appear to contribute preferentially to pathological processes linked to overexcitation of glutamatergic pathways. The actions of extracellular H⁺, Mg²⁺, Zn²⁺, of polyamines and neurosteroids, and of the synthetic compounds ifenprodil and derivatives (‘prodils’) are presented. Particular emphasis is put upon the structural determinants and molecular mechanisms that underlie the effects exerted by these agents. A better understanding of how NR2B-containing NMDARs (and NMDARs in general) operate and how they can be modulated should help define new strategies to counteract the deleterious effects of dysregulated NMDAR activity.     

Treatment outcome after low intensity chemotherapy [CVP] in children and adolescents with early stage nodular lymphocyte predominant Hodgkin's lymphoma - an Anglo-French collaborative report

PurposeTo examine whether three cycles of a low-intensity chemotherapy consisting of cyclophosphamide [500 mg/m² – day 1], vinblastine [6 mg/m² – days 1 and 8] and prednisolone [40 mg/m² – days 1–7] (CVP) is safe and therapeutically effective in children and adolescents with early stage nodular lymphocyte predominant Hodgkin lymphoma [nLPHL].Patients and methodsFifty-five children and adolescents with early stage nLPHL [median age 13 years, range 4–17 years] diagnosed between June 2005 and October 2010 in the UK and France are the subjects of this report. Staging investigations included conventional cross sectional as well as 18 fluro-deoxyglucose [FDG] PET imaging. Histology was confirmed as nLPHL by an expert pathology panel.ResultsOf the 45 patients, who received CVP as first line treatment, 36 [80%, 95% Confidence Interval [CI]: (68; 92)] either achieved a complete remission [CR] or CR unconfirmed [CRu], the remaining nine patients achieved a partial response. All nine subsequently achieved CR with salvage chemotherapy [n = 7] or radiotherapy [n = 2]. Ten patients received CVP at relapse after primary treatment that consisted of surgery alone and all achieved CR. To date, only three patients have relapsed after CVP chemotherapy and all had received CVP as first line treatment at initial diagnosis. The 40-month freedom from treatment failure and overall survival for the entire cohort were 75.4% (SE ± 6%) and 100%, respectively. No significant early toxicity was observed.ConclusionsOur results show that CVP is an effective chemotherapy regimen in children and adolescents with early stage nLPHL that is well tolerated with minimal acute toxicity.     

Frontline treatment of localized osteosarcoma without methotrexate: results of the St. Jude Children's Research Hospital OS99 trial

BACKGROUND:

The standard treatment of osteosarcoma includes cisplatin and high‐dose methotrexate (HDMTX); both agents exert significant toxicity, and HDMTX requires complex pharmacokinetic monitoring and leucovorin rescue. In the previous OS91 trial, the treatment of localized disease with carboplatin, ifosfamide, doxorubicin, and HDMTX yielded outcomes comparable to those of cisplatin‐based regimens and caused less toxicity. To build on this experience, the authors conducted a multi‐institutional trial (OS99) that evaluated the efficacy of carboplatin, ifosfamide, and doxorubicin without HDMTX in patients with newly diagnosed, localized, resectable osteosarcoma.

METHODS:

Treatment was comprised of 12 cycles of chemotherapy administered over 35 weeks: 3 cycles of carboplatin (dose targeted to area under the concentration‐time curve of 8 mg/mL × min on Day 1) and ifosfamide (at a dose of 2.65 g/m² daily ×3 days) and 1 cycle of doxorubicin (at a dose of 25 mg/m² daily ×3 days) before surgical resection, followed by 2 additional cycles of the combination of carboplatin and ifosfamide and 3 cycles each of doxorubicin (25 mg/m² daily ×2 days) combined with ifosfamide or carboplatin.

RESULTS:

A total of 72 eligible patients (median age, 13.4 years) were enrolled between May 1999 and May 2006. Forty of the 66 (60.6%) evaluable patients had good histologic responses (>90% tumor necrosis) to preoperative chemotherapy. The estimated 5‐year event‐free survival rate was 66.7% ± 7.0% for the OS99 trial compared with 66.0% ± 6.8% for the OS91 trial (P = .98). The estimated 5‐year survival rate was 78.9% ± 6.3% for the OS99 trial and 74.5% ± 6.3% for the OS91 trial (P = .40).

CONCLUSIONS:

The regimen used in the OS99 trial was found to produce outcomes comparable to those of cisplatin‐containing or HDMTX‐containing regimens. This therapy offers a good alternative for patients, particularly those who demonstrate an intolerance of HDMTX, and for institutions that cannot provide pharmacokinetic monitoring for MTX. Cancer 2011. © 2011 American Cancer Society.     

Fractionation of Spatial Memory in GRM2/3 (mGlu2/mGlu3) Double Knockout Mice Reveals a Role for Group II Metabotropic Glutamate Receptors at the Interface Between Arousal and Cognition

Group II metabotropic glutamate receptors (mGluR2 and mGluR3, encoded by GRM2 and GRM3) are implicated in hippocampal function and cognition, and in the pathophysiology and treatment of schizophrenia and other psychiatric disorders. However, pharmacological and behavioral studies with group II mGluR agonists and antagonists have produced complex results. Here, we studied hippocampus-dependent memory in GRM2/3 double knockout (GRM2/3^−/−) mice in an iterative sequence of experiments. We found that they were impaired on appetitively motivated spatial reference and working memory tasks, and on a spatial novelty preference task that relies on animals'' exploratory drive, but were unimpaired on aversively motivated spatial memory paradigms. GRM2/3^−/− mice also performed normally on an appetitively motivated, non-spatial, visual discrimination task. These results likely reflect an interaction between GRM2/3 genotype and the arousal-inducing properties of the experimental paradigm. The deficit seen on appetitive and exploratory spatial memory tasks may be absent in aversive tasks because the latter induce higher levels of arousal, which rescue spatial learning. Consistent with an altered arousal–cognition relationship in GRM2/3^−/− mice, injection stress worsened appetitively motivated, spatial working memory in wild-types, but enhanced performance in GRM2/3^−/− mice. GRM2/3^−/− mice were also hypoactive in response to amphetamine. This fractionation of hippocampus-dependent memory depending on the appetitive-aversive context is to our knowledge unique, and suggests a role for group II mGluRs at the interface of arousal and cognition. These arousal-dependent effects may explain apparently conflicting data from previous studies, and have translational relevance for the involvement of these receptors in schizophrenia and other disorders.     

Use of SPME-HS-GC-MS for the analysis of herbal products containing synthetic cannabinoids

The increasing prevalence and use of herbal mixtures containing synthetic cannabinoids presents a growing public health concern and legal challenge for society. In contrast to the plant-derived cannabinoids in medical marijuana and other cannabinoid-based therapeutics, the commonly encountered synthetic cannabinoids in these mendaciously labeled products constitute a structurally diverse set of compounds of relatively unknown pharmacology and toxicology. Indeed, the use of these substances has been associated with an alarming number of hospitalizations and emergency room visits. Moreover, there are already several hundred known cannabinoid agonist compounds that could potentially be used for illicit purposes, posing an additional challenge for public health professionals and law enforcement efforts, which often require the detection and identification of the active ingredients for effective treatment or prosecution. A solid-phase microextraction headspace gas chromatography-mass spectrometry method is shown here to allow for rapid and reliable detection and structural identification of many of the synthetic cannabinoid compounds that are currently or could potentially be used in herbal smoking mixtures. This approach provides accelerated analysis and results that distinguish between structural analogs within several classes of cannabinoid compounds, including positional isomers. The analytical results confirm the continued manufacture and distribution of herbal materials with synthetic cannabinoids and provide insight into the manipulation of these products to avoid legal constraints and prosecution.