Molecular diagnosis in children with fractures but no extraskeletal signs of osteogenesis imperfecta.

In 26 of 94 individuals (28%) below 21 years of age who had a significant fracture history but did not have extraskeletal features of osteogenesis imperfecta (OI), we detected disease-causing mutations in OI-associated genes. INTRODUCTION: In children who have mild bone fragility but do not have extraskeletal features of OI, it can be difficult to establish a diagnosis on clinical grounds. Here, we assessed the diagnostic yield of genetic testing in this context, by sequencing a panel of genes that are associated with OI. METHODS: DNA sequence analysis was performed on 94 individuals below 21 years of age who had a significant fracture history but had white sclera and no signs of dentinogenesis imperfecta. RESULTS: Disease-causing variants were detected in 28% of individuals and affected 5 different genes. Twelve individuals had mutations in COL1A1 or COL1A2, 8 in LRP5, 4 in BMP1, and 2 in PLS3. CONCLUSIONS: DNA sequence analysis of currently known OI-associated genes identified disease-causing variants in more than a quarter of individuals with a significant fracture history but without extraskeletal manifestations of OI.

DNA sequence analysis in 598 individuals with a clinical diagnosis of osteogenesis imperfecta: diagnostic yield and mutation spectrum.

We detected disease-causing mutations in 585 of 598 individuals (98 %) with typical features of osteogenesis imperfecta (OI). In mild OI, only collagen type I encoding genes were involved. In moderate to severe OI, mutations in 12 different genes were found; 11 % of these patients had mutations in recessive genes. INTRODUCTION: OI is usually caused by mutations in COL1A1 or COL1A2, the genes encoding collagen type I alpha chains, but mutations in at least 16 other genes have also been associated with OI. It is presently unknown what proportion of individuals with clinical features of OI has a disease-causing mutation in one of these genes. METHODS: DNA sequence analysis was performed on 598 individuals from 487 families who had a typical OI phenotype. OI type I was diagnosed in 43 % of individuals, and 57 % had moderate to severe OI, defined as OI types other than type I. RESULTS: Disease-causing variants were detected in 97 % of individuals with OI type I and in 99 % of patients with moderate to severe OI. All mutations found in OI type I were dominant and exclusively affected COL1A1 or COL1A2. In moderate to severe OI, dominant mutations were found in COL1A1/COL1A2 (77 %), IFITM5 (9 %), and P4HB (0.6 %). Mutations in one of the recessive OI-associated gene were observed in 12 % of individuals with moderate to severe OI. The genes most frequently involved in recessive OI were SERPINF1 (4.0 % of individuals with moderate to severe OI) and CRTAP (2.9 %). CONCLUSIONS: DNA sequence analysis of currently known OI-associated genes identifies disease-causing variants in almost all individuals with a typical OI phenotype. About 20 % of individuals with moderate to severe OI had mutations in genes other than COL1A1/COL1A2.

Inactivation of the Odontogenic ameloblast-associated gene affects the integrity of the junctional epithelium and gingival healing.

Odontogenic ameloblast-associated (ODAM) belongs to the secretory calcium-binding phosphoprotein (SCPP) gene cluster. It is expressed by the epithelial ameloblasts during the accrued mineralisation of enamel and by cells of the junctional epithelium (JE), a specialised portion of the gingiva that plays a critical role in periodontal health. In both cases, ODAM localises at the interface between the cells and the tooth surface. It is also present among the cells of the JE, and is distinctively highly expressed in many epithelial tumours. ODAM has been proposed to be a matricellular protein implicated in the adhesion of epithelial cells to tooth surfaces, and possibly in mediating cell status. To gain further understanding of the role of ODAM, we have created an Odam knockout (KO) mouse by deleting coding exons 2-6. Inactivation of the gene was verified by Southern blot, PCR, real-time qPCR and loss of immunostaining for the protein. Young Odam KO mice showed no readily apparent phenotype. No significant differences were observed in enamel volume and density, rod-interrod organisation, and its attrition. However, in older animals, the JE presented some detachment, an increase in inflammatory infiltrate, and apical down-growth. In addition, its regeneration was delayed following a gingivectomy challenge. Our results indicate that inactivation of Odam expression has no dramatic consequence on enamel but the phenotype in older animals replicates some JE changes seen during human periodontal disease. Altogether, our results suggest that ODAM plays a role in maintaining integrity of the JE.

A brilliant breakthrough in OI type V.

Interferon-induced transmembrane protein 5 or bone-restricted ifitm-like gene (Bril) was first identified as a bone gene in 2008, although no in vivo role was identified at that time. A role in human bone has now been demonstrated with a number of recent studies identifying a single point mutation in Bril as the causative mutation in osteogenesis imperfecta type V (OI type V). Such a discovery suggests a key role for Bril in skeletal regulation, and the completely novel nature of the gene raises the possibility of a new regulatory pathway in bone. Furthermore, the phenotype of OI type V has unique and quite divergent features compared with other forms of OI involving defects in collagen biology. Currently it appears that the underlying genetic defect in OI type V may be unrelated to collagen regulation, which also raises interesting questions about the classification of this form of OI. This review will discuss current knowledge of OI type V, the function of Bril, and the implications of this recent discovery.

Disruption of periodontal integrity induces expression of apin by epithelial cell rests of Malassez.

BACKGROUND AND OBJECTIVE: It has been suggested that epithelial cell rests of Malassez (ERM) may express enamel matrix proteins and play an important role in periodontal regeneration. Two novel proteins, apin (APIN) and amelotin (AMTN), produced by maturation-stage ameloblasts and junctional epithelium, have recently been identified. The objective of this study was to evaluate whether the ERM express APIN and AMTN under normal conditions and after periodontal challenge. MATERIAL AND METHODS: Gingivectomy and orthodontic tooth movement were carried out on the left side of the maxillae of rats. The control group included the untreated contralateral side of these animals and the maxillae of normal, untreated rats. Animals were sacrificed by intracardiac perfusion on days 3 and 5 after the experimental procedures and maxillary molars were decalcified and processed for paraffin embedding. Immunohistochemistry was used to evaluate the expression of various ameloblast products, including APIN, AMTN, ameloblastin (AMBN) and amelogenin (AMEL). RESULTS: At 3 and 5 days after periodontal challenge, ERM were more evident in the periodontal ligament along the root surface and in the root furcations. Immunodetection of APIN, but not of the other three proteins, was observed in the ERM following the disruption of periodontal integrity. No immunolabeling for APIN, AMTN, AMBN and AMEL was detected in the ERM under normal conditions. CONCLUSION: The expression of APIN at an early time-point following disruption of periodontal integrity suggests that this protein may be part of the cascade of events leading to the activation of ERM during periodontal healing and regeneration.

Psychosocial and psychosomatic diagnoses in primary care of children.

In this study in seven primary care facilities the proportion of children recognized as having behavioral, educational, or social problems was much higher than generally assumed. Although there was great variability among the facilities, at least 5% and as many as 15% of children seen in one year were diagnosed as having these problems in all but the hospital teaching facilities. The prevalence was even higher among children from poor families. The variability among facilities was much less for psychosomatic problems, which were diagnosed in 8% to 10% of the children. For both psychosocial and psychosomatic types of problems, but especially for psychosocial ones, the proportion of visits with the diagnoses was much lower than the proportion of children with them, so that these problems engendered fewer visits for their management than might have been expected from their frequency in the population. However, available evidence suggests that individuals with unresolved psychosocial problems make more than their share of visits for other diagnoses. The findings of this study have implications for the content of educational programs for primary care practitioners, for the organization of primary care practice, and for the current debate over policy concerning reimbursement and benefit packages.

Expression of the gene encoding metallothionein-3 in organs of the reproductive system.

Metallothionein-3 (MT-3) is a new MT gene-family member that inhibits survival of rat neurons cultured in presence of brain extracts. Contrary to other MT genes, which are expressed in most tissues and which are highly inducible by metals, MT-3 expression was reported to be mainly in the brain, and it failed to respond to metals in vivo. We show here that MT-3 mRNA is present in several organs other than the brain, as assayed by Northern analyses. In the rat, MT-3 mRNA was detected in the testis, prostate, epididymis, tongue, ovary, uterus, stomach, heart, and seminal vesicles. The MT-3 mRNA levels in the testis, epididymis, prostate, and tongue were 22% of those in brain, while in ovary, uterus, and stomach, they were 4% of the brain level, and they were lower still in the other organs. The MT-3 gene was not inducible by CdCl2 or lipopolysaccharide in rat testis and prostate. In the mouse and the human, relative MT-3 mRNA levels were lower than those found in the rat when compared with those present in brain. Testicular MT-3 transcript levels remained quite constant during rat postnatal development in animals aged from 6 to 43 days. In situ hybridization analyses on human testis sections showed that MT-3 mRNA was present at different levels in both the Leydig cells and the seminiferous tubules. In orchiectomized rats, prostatic MT-3 mRNA was decreased by 75%, and injections of dihydrotestosterone restored MT-3 mRNA levels to control values. Overall, these results show that MT-3 tissue-specific gene expression is broader than previously reported and provide new experimental systems to study the function and mechanism of action of the MT-3 protein.

Metallothionein induction attenuates the effects of glutathione depletors in rat hepatocytes.

Metallothionein (MT) is a protein involved in heavy metal homeostasis and detoxification. According to several studies, MT could be involved in the antioxidant defense system, in which glutathione (GSH) is an essential component. The aim of this study was to verify the implication of MT in the antioxidant defense system in isolated rat hepatocytes. For this purpose, hepatocyte cultures were exposed to treatments known to modify MT or GSH levels. Zinc (Zn) was used as an inducer of MT while diethyl maleate (DEM) and buthionine sulfoximine (BSO) were used as GSH depletors. GSH, MT, and antioxidant enzyme activities were measured under conditions of MT induction and GSH depletion. Induction of MT synthesis through an 18-hour exposure to Zn (20 microM), did not result in any significant change in GSH levels or in activities of the antioxidant enzymes, glutathione-peroxidase (GSH-Px), catalase, and superoxide dismutase (SOD). DEM caused GSH depletion in cells, whether they were exposed to Zn or not, that lasted one h; after that time, GSH rose back to basal levels. BSO also caused GSH-depletion in cells exposed or unexposed to Zn, and no recovery in GSH levels was detectable during the entire period of exposure (12 h). However, GSH depletion induced by both DEM or BSO was attenuated in Zn-treated hepatocytes. Moreover, DEM and BSO exposures led to a depletion of MT levels in Zn-treated hepatocytes, indicating a link between GSH and MT metabolism. In cells unexposed to either Zn, DEM or BSO, there was an increase in GSH-Px and SOD activities after 6 and 12 h of incubation, respectively. Under the same conditions, catalase activity was inhibited after 6 h of incubation and returned to the activity found at t = 0 after 12 h of incubation. DEM and BSO treatments had no significant effect on GSH-Px or SOD activities although they led to inhibition of catalase activity. Taken together, our data indicate that MT induction, which creates a new pool of thiol groups in the cell cytosol, can attenuate GSH depletion induced by DEM or BSO. It appears that catalase is most sensitive to oxidative stress and that MT induction can antagonize the deleterious effects of such stress on the enzyme. This study supports the view that MT is part of the hepatocyte antioxidant-defense-system.

Coping with disabilities: the perspective of young adults from different ethnic backgrounds in inner London.

In April 1993, national reforms of the method of offering community health and social care have come into effect in the U.K. A cornerstone of the reforms will be the appointment of care managers by local authorities to oversee needs assessment and care of vulnerable people. A survey was undertaken of 65 young people with physical disabilities living in inner south east London, an area of deprivation and ethnic diversity. The aim was to ascertain the perceptions of young adults in terms of access to services, lifestyle and future plans. The information was intended to inform joint work by the local health and social services departments in their own needs assessment in implementing the Community Care Act. The majority of those interviewed were living with informal carers, usually their mother. Most people had complex disabilities and were wheelchair users, but it did not follow that those with greater need were being supported more by either statutory or voluntary sectors. In fact, people were losing what contact they once had, particularly with social services. Access to respite and organised social outlets was strictly limited, particularly for people with complex disabilities. Although over half the interviewees had taken formal examinations, only five were currently employed, and several had lost their job recently. Few had received advice about careers or independent living and almost half of those interviewed did not know where to go for family planning advice. Over 40% of interviewees were from ethnic minorities, and lived in close family networks where coping skills were similar to that of the white British group.(ABSTRACT TRUNCATED AT 250 WORDS)

Successful transfusion in the presence of anti-K4 (anti-Kpb).

A 71-year-old group A, DC female, with anti-K4(-Kpb), -E, and -S was admitted for her second coronary artery surgery. Four units of autologous red blood cells (1BCs) were transfused perioperatively, and four units of homologous K:-4, E-, S- RBCs were transfused over the next 24 hours. Ten units of fresh frozen plasma and 28 units of platelets were also transfused. Continued bleeding necessitated calling donors from other states in AUStralia and from the International Panel of Donors of Rare Type, Bristol, UK, maintained by the World Health Organization. Four units of group 0, K:4, E-, S- KHCs were transfused in the next 24 hours while the K:-4 blood was in transit. No immediate signs of a transfusion reaction were noted. Blood pressure, temperature, bilirubin, and urinary output were normal, and an increase in hemoglobin was observed. A positive direct antiglobulin test was evident 3 days posttransfusion of the incompatible units and was still present 8 days later. Anti-A and anti-K4 were eluted from the patient's RBCs. The titer of anti-K4 increased from 8 preoperatively to 2,048 19 days posttransfusion of K:4 RBCs. Subsequent transfusions were not required, and the patient was discharged 3 weeks after the operation without further incident.

Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector.

Gene transfer using viral vectors offers the potential for the sustained expression of proteins in specific target tissues. However, in the case of calcified tissues, in vivo delivery remains problematic because of limited accessibility. The aim of this study was to test the efficiency of lentiviral vectors (LVs) on osteogenic cells in vitro, and determine the feasibility of directly transducing resident bone cells in vivo. LVs encoding for green fluorescent protein (GFP) and ameloblastin (AMBN), a protein associated with mineralization not reported in bone, were generated. The transduction efficiency of the LVs was evaluated using the MC3T3 cell line and primary calvaria-derived osteogenic cells. For in vivo delivery, the LVs were infused using osmotic minipumps through holes created in the bone of the rat hemimandible and tibia. The production of GFP and AMBN in vitro and in vivo was monitored using fluorescence microscopy. Both transgenes were expressed in MC3T3 and primary osteogenic cells. In vivo, GFP was detected at the infusion site and fibroblast-like cells, osteoblasts, osteocytes and osteoclasts expressed AMBN. Our data demonstrate, for the first time, that primary osteogenic cells are efficiently transduced with LVs and that their infusion is advantageous for locally delivering DNA to bone cells.

Metallothionein in physiological and physiopathological processes.

The multipurpose nature of MT that we have presented in this review has drawn attention from many different fields of research: biochemistry, molecular biology, toxicology, pharmacology, etc. In recent years, considerable advances have been made concerning the regulation of MT genes by metals. Little, however, is known at the molecular level about the mechanisms of MT induction by nonmetallic inducers such as growth factors. This is of particular interest since MT is highly expressed during liver regeneration, an event orchestrated by a series of growth stimulators and inhibitors. The significance of the nuclear distribution of MT in growing cells and what controls its translocation are questions that remain unanswered at the present time. The possibility that MT could participate in a DNA synthesis-related process through donation or abstraction of Zn to and from transcription factors has been inferred from in vitro studies. Such transfer mechanisms, however, have yet to be confirmed in vivo. Overexpression of MT is often accompanied by increased resistance towards a variety of alkylating agents and chemotherapeutic drugs. The mechanisms by which MT protects cells against these agents may depend on their distinct mode of toxic action. For some, MT cysteines can be the target of the direct attack from the parent compound. For others such as N-methyl-N-nitroso compounds, MT cysteines may serve as a sink for the reactive oxygen species now known to be derived from their metabolism. In either case, a primary consequence of such interactions is the release of the metals initially bound to MT. Therefore, the metal composition of MT appears to be an important factor to consider in determining the overall effect of MT in the resistance process.

Cadmium-2-acetylaminofluorene interaction in isolated rat hepatocytes.

Cadmium (Cd) is a non-essential, highly toxic heavy metal and a ubiquitous environmental contaminant. Evidence exists that Cd can affect parameters which are of great importance in the response towards xenobiotics. However, there is a lack of information about the mechanisms that take place at the cellular and molecular levels upon dual exposure to Cd and other toxins. The purpose of the present work was therefore to examine the biochemical interactions between Cd and a well-known genotoxic hepatocarcinogen, 2-acetylaminofluorene (AAF) in isolated rat hepatocytes. The cells were incubated for 10 hr with a sub-cytotoxic concentration (0.22 microM) of 109Cd. This was followed by a 10 hr exposure to 1 microM [3H]AAF. Cellular distribution of Cd and 3H was determined. Sephadex G-75 elution profiles of the cytosol showed that Cd was almost entirely associated with the intermediate molecular weight (IMW) fractions containing metallothionein (MT) ( > 80%), and with high molecular weight proteins. In parallel, the highest proportion of 3H was found in the low molecular weight components. Further analysis of IMW fractions by DEAE A-25 anion-exchange chromatography revealed that, in addition to Cd, there was some 3H which coeluted along with MT-I and MT-II isoforms, but preferentially with MT-I. Moreover, Cd pretreatment caused a 1.6-fold increase in MT level, as measured by the silver-saturation assay. Under these conditions, there was a 17% lower binding of 3H to the DNA. This reduced binding was neither accompanied by diminished AAF uptake nor by inhibition of cytochrome P-450 activity. Taken together, these results suggest that Cd exposure has a protective effect against the genotoxicity of AAF. MT, whose synthesis is induced, could play a role in the Cd-AAF interaction through scavenging of reactive metabolites.

Induction of metallothionein gene expression by epidermal growth factor and its inhibition by transforming growth factor-beta and dexamethasone in rat hepatocytes.

Metallothionein (MT) is a small cysteine-rich protein thought to be mainly involved in metal regulation and detoxification. The implication of MT in cell growth and differentiation has also been suggested. This latter hypothesis was further investigated in adult rat hepatocytes induced to proliferate by epidermal growth factor (EGF). Exposure of hepatocytes to EGF resulted in significant increases (approximately twofold) in MT protein and MT-1 messenger RNA (mRNA) levels, which were maximal after 48 hours. As revealed by nuclear run-on analysis, these changes were the result of transcriptional activation. Increases of MT occurred concomitantly with stimulation of DNA synthesis (48 hours). Addition of ZnSO4 or dexamethasone (Dex) was also effective at inducing MT protein (approximately 3.6 to 3.3 times) and mRNA. Combined addition of Zn and EGF produced an additive increase in MT protein and MT-1 mRNA levels. When both Dex and EGF were present together, the EGF-induced MT protein and mRNA expression was lost, whereas it had only minor inhibitory effects on DNA synthesis. Transforming growth factor beta (TGF-beta), a known antagonist of EGF on hepatocytes, blocked the EGF-induced MT accumulation and stimulation of DNA synthesis. In addition, under the same conditions, the EGF-induced c-fos mRNA accumulation was blocked by Dex whereas TGF-beta had no effect. These results show that growth factors believed to play a role in liver regeneration can also modulate MT gene expression in vitro. This modulation does not strictly parallel that of DNA synthesis. The possibility that c-fos stimulation may play a role in MT induction by EGF cannot be ruled out.

Rat hepatocytes with elevated metallothionein expression are resistant to N-methyl-N'-nitro-N-nitrosoguanidine cytotoxicity.

Metallothionein (MT) is a small cysteine-rich metal-binding protein involved in Zn and Cu homeostasis as well as in heavy metal detoxication. It is also believed that when MT is overexpressed, it can confer resistance against alkylating agents. However, the mechanisms involved are still poorly understood. The purpose of the present work was to investigate whether metal treatment, which induces MT synthesis, could protect isolated rat hepatocytes against the cytotoxic effects of the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Exposure to 12.5 microM ZnSO4 for 18 hr raised MT levels approximately 15-fold (as measured by the 109Cd-heme assay). When these cells were exposed to increasing concentrations of MNNG, a significant reduction in cell death (as measured by lactate dehydrogenase leakage into extracellular medium) was observed (LC50 = 468 +/- 20 microM vs 362 +/- 13 microM for control cells). On the other hand, Zn pretreatment was not accompanied by resistance against MMS toxicity. In addition, the synthesis of graded amounts of MT, achieved by incubation with various concentrations of Zn or Cu, led to a high correlation between MT levels and the extent of hepatocyte survival. Cd (another MT inducer) failed to protect hepatocytes from MNNG cytotoxicity. Time-course studies also revealed a good correlation between the onset of MT induction by Zn (> 3 hr) and that of protection against MNNG (> 3 hr). The stability of MT in the presence of MNNG was studied by incubating 109Cd-labeled MT with MNNG and by analyzing the mixture using Sephadex G-75 Chromatography. Direct interaction of MNNG with rabbit liver (Cd,Zn)-MT was demonstrated by the release of 109Cd bound to MT. Similar results were obtained with 109Cd-exposed hepatocytes, 109Cd being redistributed from MT to high-molecular-weight proteins after incubation with MNNG. None of the metals used to induce MT modulated glutathione (GSH) because it remained at control levels after 18 hr. However, within 15 min of incubation, MNNG had completely depleted GSH in both control and Zn-pretreated hepatocytes equally. This was followed by a marked decline in MT levels. Taken together, these results suggest that Zn- and Cu-induced tolerance against killing by MNNG appears to be related to the accumulation of MT. The mechanism of protection might reside in the antioxidant properties of MT and on its ability to scavenge electrophilic species.

A national evaluation of school breakfast clubs: where does economics fit in?

STUDY OBJECTIVE: To describe the economics of UK school breakfast clubs, to estimate costs resulting from clubs and to investigate relationships between costs and outcomes. DESIGN: A postal survey of schools with a 1-year follow-up, a cluster randomized controlled trial, case studies, semi-structured interviews with parents and a secondary econometric analysis. SETTING: England, the UK. MAIN RESULTS: Key economic differences were identified between clubs based in primary schools and those based in secondary schools in terms of both funding levels and cost structures. However, funding levels were not a significant determinant of the observed outcomes in either type of school. CONCLUSIONS: For formal economic evaluation to succeed during implementation of a new initiative, a clearer understanding of relevant outcomes and the distinction between short- and long-term outcomes and potential individual, institutional and societal benefits are required from an early stage.

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1.

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn(2+)-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1-MRE and the MEP-1-MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn(2+) ions but not by Cd(2+), UV irradiation or PMA, and occurred on ice as well as at 21 degrees C. In control and Zn(2+)-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn(2+) or a preincubation at 37 degrees C. However, recombinant MTF-1 produced in vitro required Zn(2+) activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.