Mechanotransduction of bone cells<Emphasis Type="Italic">in vitro</Emphasis>: Mechanobiology of bone tissue

Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such as fluid shear, tension or compression, can influence cells in differing ways. During dynamic loading of intact bone, fluid is pressed through the osteocyte canaliculi, and it has been demonstrated that fluid shear stress stimulates osteocytes to produce signalling molecules. It is less clear how mechanical loads act on mature osteoblasts present on the surface of cancellous or trabecular bone. Although tissue strain and fluid shear stress both cause cell deformation, these stimuli could excite different signalling pathways. This is confirmed by our experimental findings, in human bone cells, that strain applied through the substrate and fluid flow stimulate the release of signalling molecules to varying extents. Nitric oxide and prostaglandin E₂ values increased by between two- and nine-fold after treatment with pulsating fluid flow (0.6±0.3 Pa). Cyclic strain (1000 μstrain) stimulated the release of nitric oxide two-fold, but had no effect on prostaglandin E₂. Furthermore, substrate strains enhanced the bone matrix protein collagen I two-fold, whereas fluid shear caused a 50% reduction in collagen I. The relevance of these variations is discussed in relation to bone growth and remodelling. In applications such as tissue engineering, both stimuli offer possibilities for enhancing bone cell growth in vitro.     

Waardenburg syndrome: clinical differentiation between types I and II

Here we present the results of a study performed on 59 patients affected by Waardenburg syndrome (WS), 30 with the I variant, 21 having the type II, and 8 of them being isolated cases without telecanthus. These patients belong to 37 families; the main contributions and conclusions are based on the detailed study of 25 of these families, examined using standard procedures. All patients were examined as to the presence of eight cardinal signs important for the diagnosis of the condition; from each patient, from many of his/her normal relatives, and from a control sample of 300 normal individuals stratified by age and sex, 23 different craniofacial measurements were obtained. We also estimated, using our own data as well those collected from the literature, the frequencies of the cardinal signs, based on a total sample of 461 affected individuals with WSI and 121 with WSII. In order to originate discriminant functions to separate individuals affected by one of the two variants, both metric (from craniofacial measurements) as well as categoric data (based on the frequencies of the cardinal signs or symptoms) were used. Discriminant analysis based on the frequency of the eight cardinal signs can improve the separation of WSI patients without telecanthus from those presenting the variant II. We present also a Table with the conditional probabilities favoring the diagnosis of WSI for suspect subjects without telecanthus and any combination of the other seven signs/symptoms. The discriminant function based on the four ocular measurements (inner and outer intercanthal, interpupillary, and inferior lacrymal distances), on the other side, perfectly classifies patients affected by one of the variants of WS, the same taking place when the average values of the W index of all affected individuals per family are used. The discriminant function based solely in the individual W index values of patients correctly classifies 93% of WSII subjects, but only 60% of the patients with the I variant of WS.     

Subtelomeric deletions detected in patients with idiopathic mental retardation using multiplex ligation-dependent probe amplification (MLPA)

Subtelomeric rearrangements are responsible for 5% to 10% of cases of unexplained mental retardation. Despite their clinical relevance, methods to screen for these cytogenetically invisible abnormalities on a routine base are scarce. We screened patients with idiopathic mental retardation for subtelomeric aberrations using multiplex ligation-dependent probe amplification (MLPA). This recently developed technique is based on PCR amplification of ligated probes hybridized to chromosome ends. Currently, 41 telomeres can be screened in just two multiplex reactions. Four subtelomeric rearrangements (5.3%) were detected in a group of 75 patients with mild to severe mental retardation in combination with dysmorphic features and/or a familial history of mental retardation: two terminal 1p deletions, a terminal 1q deletion, and a terminal 3p deletion. Deletions could be verified by FISH and marker analysis. In one case the MLPA indicated a terminal 21q deletion due to a 3-bp deletion at the site of the probe, giving a false-positive rate of 1.3%. This study demonstrates that MLPA is a fast and reliable screening method, potentially suitable for use in routine diagnostics.     

Nitric oxide signaling in mechanical adaptation of bone

One of the most serious healthcare problems in the world is bone loss and fractures due to a lack of physical activity in elderly people as well as in bedridden patients or otherwise inactive youth. Crucial here are the osteocytes. Buried within our bones, these cells are believed to be the mechanosensors that stimulate bone formation in the presence of mechanical stimuli and bone resorption in the absence of such stimuli. Intercellular signaling is an important physiological phenomenon involved in maintaining homeostasis in all tissues. In bone, intercellular communication via chemical signals like NO plays a critical role in the dynamic process of bone remodeling. If bones are mechanically loaded, fluid flows through minute channels in the bone matrix, resulting in shear stress on the cell membrane that activates the osteocyte. Activated osteocytes produce signaling molecules like NO, which modulate the activity of the bone-forming osteoblasts and the bone-resorbing osteoclasts, thereby orchestrating bone adaptation to mechanical loading. In this review, we highlight current insights in the role of NO in the mechanical adaptation of bone mass and structure, with emphasis on its role in local bone gain and loss as well as in remodeling supervised by osteocytes. Since mechanical stimuli and NO production enhance bone strength and fracture resistance, these new insights may facilitate the development of novel osteoporosis treatments.     

Characterization of mutations in ATP8B1 associated with hereditary cholestasis

Progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC) are clinically distinct hereditary disorders. PFIC patients suffer from chronic cholestasis and develop liver fibrosis. BRIC patients experience intermittent attacks of cholestasis that resolve spontaneously. Mutations in ATP8B1 (previously FIC1) may result in PFIC or BRIC. We report the genomic organization of ATP8B1 and mutation analyses of 180 families with PFIC or BRIC that identified 54 distinct disease mutations, including 10 mutations predicted to disrupt splicing, 6 nonsense mutations, 11 small insertion or deletion mutations predicted to induce frameshifts, 1 large genomic deletion, 2 small inframe deletions, and 24 missense mutations. Most mutations are rare, occurring in 1-3 families, or are limited to specific populations. Many patients are compound heterozygous for 2 mutations. Mutation type or location correlates overall with clinical severity: missense mutations are more common in BRIC (58% vs. 38% in PFIC), while nonsense, frameshifting, and large deletion mutations are more common in PFIC (41% vs. 16% in BRIC). Some mutations, however, lead to a wide range of phenotypes, from PFIC to BRIC or even no clinical disease. ATP8B1 mutations were detected in 30% and 41%, respectively, of the PFIC and BRIC patients screened.     

Locally-Delivered T-Cell-Derived Cellular Vehicles Efficiently Track and Deliver Adenovirus Delta24-RGD to Infiltrating Glioma

Oncolytic adenoviral vectors are a promising alternative for the treatment of glioblastoma. Recent publications have demonstrated the advantages of shielding viral particles within cellular vehicles (CVs), which can be targeted towards the tumor microenvironment. Here, we studied T-cells, often having a natural capacity to target tumors, for their feasibility as a CV to deliver the oncolytic adenovirus, Delta24-RGD, to glioblastoma. The Jurkat T-cell line was assessed in co-culture with the glioblastoma stem cell (GSC) line, MGG8, for the optimal transfer conditions of Delta24-RGD in vitro. The effect of intraparenchymal and tail vein injections on intratumoral virus distribution and overall survival was addressed in an orthotopic glioma stem cell (GSC)-based xenograft model. Jurkat T-cells were demonstrated to facilitate the amplification and transfer of Delta24-RGD onto GSCs. Delta24-RGD dosing and incubation time were found to influence the migratory ability of T-cells towards GSCs. Injection of Delta24-RGD-loaded T-cells into the brains of GSC-bearing mice led to migration towards the tumor and dispersion of the virus within the tumor core and infiltrative zones. This occurred after injection into the ipsilateral hemisphere, as well as into the non-tumor-bearing hemisphere. We found that T-cell-mediated delivery of Delta24-RGD led to the inhibition of tumor growth compared to non-treated controls, resulting in prolonged survival (p = 0.007). Systemic administration of virus-loaded T-cells resulted in intratumoral viral delivery, albeit at low levels. Based on these findings, we conclude that T-cell-based CVs are a feasible approach to local Delta24-RGD delivery in glioblastoma, although efficient systemic targeting requires further improvement.     

DNA Diagnostics of Hereditary Hearing Loss: A Targeted Resequencing Approach Combined with a Mutation Classification System

Although there are nearly 100 different causative genes identified for nonsyndromic hearing loss (NSHL), Sanger sequencing‐based DNA diagnostics usually only analyses three, namely, GJB2, SLC26A4, and OTOF. As this is seen as inadequate, there is a need for high‐throughput diagnostic methods to detect disease‐causing variations, including single‐nucleotide variations (SNVs), insertions/deletions (Indels), and copy‐number variations (CNVs). In this study, a targeted resequencing panel for hearing loss was developed including 79 genes for NSHL and selected forms of syndromic hearing loss. One‐hundred thirty one presumed autosomal‐recessive NSHL (arNSHL) patients of Western‐European ethnicity were analyzed for SNVs, Indels, and CNVs. In addition, we established a straightforward variant classification system to deal with the large number of variants encountered. We estimate that combining prescreening of GJB2 with our panel leads to a diagnosis in 25%–30% of patients. Our data show that after GJB2, the most commonly mutated genes in a Western‐European population are TMC1, MYO15A, and MYO7A (3.1%). CNV analysis resulted in the identification of causative variants in two patients in OTOA and STRC. One of the major challenges for diagnostic gene panels is assigning pathogenicity for variants. A collaborative database collecting all identified variants from multiple centers could be a valuable resource for hearing loss diagnostics.     

DNA methylation testing for endometrial cancer detection in urine,cervicovaginal self-samples and cervical scrapes

Endometrial cancer incidence is rising and current diagnostics often require invasive biopsy procedures. DNA methylation marker analysis of minimally- and non-invasive sample types could provide an easy-to-apply and patient-friendly alternative to determine cancer risk. Here, we compared the performance of DNA methylation markers to detect endometrial cancer in urine, cervicovaginal self-samples and clinician-taken cervical scrapes. Paired samples were collected from 103 patients diagnosed with stage I to IV endometrial cancer. Urine and self-samples were collected at home. All samples were tested for nine DNA methylation markers using quantitative methylation-specific PCR. Methylation levels measured in endometrial cancer patients were compared to unpaired samples of 317 healthy controls. Diagnostic performances were evaluated by univariable and multivariable logistic regression analysis, followed by leave-one-out cross-validation. Each methylation marker showed significantly higher methylation levels in all sample types of endometrial cancer patients compared to healthy controls (P < .01). Optimal three-marker combinations demonstrated excellent diagnostic performances with area under the receiver operating curve values of 0.95 (95% CI: 0.92-0.98), 0.94 (0.90-0.97) and 0.97 (0.96-0.99), for endometrial cancer detection in urine, self-samples and scrapes, respectively. Sensitivities ranged from 89% to 93% at specificities of 90% to 92%. Virtually equal performances were obtained after cross-validation and excellent diagnostic performances were maintained for stage I endometrial cancer detection. Our study shows the value of methylation analysis in patient-friendly sample types for endometrial cancer detection of all stages. This approach has great potential to screen patient populations at risk for endometrial cancer.     

Challenging Behaviours in Dementia: a Project at Hornsby/Ku-Ring-Gai Hospital

Abstract: This paper introduces a two year study at Hornsby/Ku-Ring-Gai Hospital which aims to shed more light on the nature of behaviour problems in dementia, and on methods of dealing with them. Rather than trialing a standard therapeutic technique across a range of behaviours, the project attempts to reflect what is often a complex clinical situation not amenable to standard answers. A series of interventions is being instituted in a controlled trial on the assumption that each case is idiosyncratic both in aetiology and other variables which affect treatment choice. Data are collected on quantitative and qualitative aspects of each case, including presumed causes of the behaviour. Outcome is assessed two and five months after the intervention using measures of patient behaviour, and of carer distress, coping and perception of the problem.