Tissue-engineered cartilage with inducible and tunable immunomodulatory properties

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (MSC) chondrogenesis. In this study, we combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in MSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce MSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.     

Epigenetic modulation at birth – altered DNA-methylation in white blood cells after Caesarean section

Aim:  Delivery by C-section (CS) has been associated with increased risk for allergy, diabetes and leukaemia. Whereas the underlying cause is unknown, epigenetic change of the genome has been suggested as a candidate molecular mechanism for perinatal contributions to later disease risk. We hypothesized that mode of delivery affects epigenetic activity in newborn infants.
Methods:  A total of 37 newborn infants were included. Spontaneous vaginal delivery (VD) occurred in 21, and 16 infants were delivered by elective CS. Blood was sampled from the umbilical cord and 3–5 days after birth. DNA-methylation was analyzed in leucocytes.
Results:  Infants born by CS exhibited higher DNA-methylation in leucocytes compared with that of those born by VD (p < 0.001). After VD, newborn infants exhibited stable levels of DNA-methylation, as evidenced by comparing cord blood values with those 3–5 days after birth (p = 0.55). On postnatal days 3–5, DNA-methylation had decreased in the CS group (p = 0.01) and was no longer significantly different from that of VD (p = 0.10).
Conclusion:  DNA-methylation is higher in infants delivered by CS than in infants vaginally born. Although currently unknown how gene expression is affected, or whether epigenetic differences related to mode of delivery are long-lasting, our findings open a new area of clinical research with potentially important public health implications.     

Predominance of null mutations in ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.      

Effect of delta9-tetrahydrocannabinol on quinine palatability and AM251 on sucrose and quinine palatability using the taste reactivity test

Here we provide evidence that the cannabinoid agonist, Delta9-tetrahydrocannabinol (Delta9-THC) enhances quinine palatability and the CB1 antagonist/inverse agonist, AM251, reduces sucrose and quinine palatability using the taste reactivity test, which provides a direct measure of palatability independently of appetitive behavior. In Experiment 1, rats were treated with a low dose of Delta9-THC (0.5 mg/kg) or Vehicle 30 min, 60 min, 120 min or 240 min prior to a 5-min intraoral infusion of a highly unpalatable 0.05% quinine solution. Regardless of the post-injection interval, Delta9-THC reduced rejection of quinine. The Delta9-THC-induced palatability shift was reversed by AM251. In Experiment 2, rats were injected with either AM251 (1 mg/kg) or Vehicle prior to receiving a 5-min intraoral infusion of either 32% sucrose or 0.05% quinine solution. AM251 significantly decreased sucrose-elicited hedonic reactions across both time intervals; however, AM251 did not significantly modify the rejection of 0.05% quinine solution. When the concentration of the quinine solution was reduced to 0.01% in Experiment 3, AM251 enhanced quinine aversion. Although the range of concentrations of the solutions tested in the present data is limited, our results suggest that the cannabinoid system may modulate the palatability of ingested substances regardless of the palatability of the ingested substance.     

An improved algorithm for femoropopliteal artery centerline restoration using prior knowledge of shapes and image space data

Accurate arterial centerline extraction is essential for comprehensive visualization in CT Angiography. Time consuming manual tracking is needed when automated methods fail to track centerlines through severely diseased and occluded vessels. A previously described algorithm, Partial Vector Space Projection (PVSP), which uses vessel shape information from a database to bridge occlusions of the femoropopliteal artery, has a limited accuracy in long (>100 mm) occlusions. In this article we introduce a new algorithm, Intermediate Point Detection (IPD), which uses calcifications in the occluded artery to provide additional information about the location of the centerline to facilitate improvement in PVSP performance. It identifies calcified plaque in image space to find the most useful point within the occlusion to improve the estimate from PVSP. In this algorithm candidates for calcified plaque are automatically identified on axial CT slices in a restricted region around the estimate obtained from PVSP. A modified Canny edge detector identifies the edge of the calcified plaque and a convex polygon fit is used to find the edge of the calcification bordering the wall of the vessel. The Hough transform for circles estimates the center of the vessel on the slice, which serves as a candidate intermediate point. Each candidate is characterized by two scores based on radius and relative position within the occluded segment, and a polynomial function is constructed to define a net score representing the potential benefit of using this candidate for improving the centerline. We tested our approach in 44 femoropopliteal artery occlusions of lengths up to 398 mm in 30 patients with peripheral arterial occlusive disease. Centerlines were tracked manually by four-experts, twice each, with their mean serving as the reference standard. All occlusions were first interpolated with PVSP using a database of femoropopliteal arterial shapes obtained from a total of 60 subjects. Occlusions longer than 80 mm (N = 20) were then processed with the IPD algorithm, provided calcifications were found (N = 14). We used the maximum point-wise distance of an interpolated curve from the reference standard as our error metric. The IPD algorithm significantly reduced the average error of the initial PVSP from 2.76 to 1.86 mm (p < 0.01). The error was less than the clinically desirable 3 mm (smallest radius of the femoropopliteal artery) in 13 of 14 occlusions. The IPD algorithm achieved results within the range of the human readers in 11 of 14 cases. We conclude that the additional use of sparse but specific image space information, such as calcified atherosclerotic plaque, can be used to substantially improve the performance of a previously described knowledge-based method to restore the centerlines of femoropopliteal arterial occlusions.     

Feline leukaemia virus: protective immunity is mediated by virus-specific cytotoxic T lymphocytes

Feline leukaemia virus (FeLV) nucleic acid vaccination of domestic cats affords protection against viraemia and the development of latency without inducing antiviral antibodies.1 To determine the contribution of cell-mediated immunity to the control of virus replication and clearance from the host, FeLV-specific cytotoxic T lymphocyte (CTL) responses were compared in vaccine-protected, transiently viraemic, and persistently viraemic cats. Vaccinal immunity was associated with the detection of higher levels of virus-specific effector CTL in the peripheral blood and lymphoid organs to FeLV Gag/Pro and Env antigens than those observed in unvaccinated control, persistently viraemic cats (P<0.001). Likewise, higher levels of virus-specific CTLs were also observed in transiently viraemic cats which recovered following exposure to FeLV. In cats that controlled their infection, recognition of Gag/Pro antigens was significantly higher than the recognition of Env antigens. This is the first report highlighting the very significant role that virus-specific CTL have in determining the outcome of FeLV infection in either vaccinated cats or cats recovering naturally from FeLV exposure.     

A new animal model of binge eating: key synergistic role of past caloric restriction and stress

Dieting and stress are important in the etiology and maintenance of eating disorders, and dieting strongly predicts stress-induced overeating in humans. We hypothesized that caloric restriction and stress interact in a unique manner to promote binge eating. To test this hypothesis, a group of young female rats were cycled through a restriction period (4 days of 66% of control food intake) followed by 6 days of free feeding prior to being stressed by acute foot shock. After three of these cycles, the food intake of rats exposed only to restriction (R), or only to stress (S), did not differ from controls. However, R+S rats that were restricted and refed, despite normal body weight and food intake after free feeding, engaged in a powerful bout of hyperphagia when stressed (Experiment 1). The R + S effect was replicated in an older group of rats (Experiment 2). The hyperphagia was characteristically binge-like, it constituted a 40% selective increase in highly palatable (HP) food (P < .001) over a discrete period of time (within 24 h post-stress), and reflected feeding for reward (higher HP:chow ratio) over metabolic need as occurred after restriction (higher chow:HP ratio). Subsequent experiments revealed that binge eating did not occur if only chow was available (Experiment 3) or if restriction-refeeding (R-R) did not proximally precede stress (Experiment 4). Experiment 5 revealed that a history of R-R cycles followed by only one stress episode was sufficient to increase intake to 53% above controls as early as 2 h after stress (P < .001). This animal model of binge eating should facilitate investigations into the neurochemical changes induced by dieting and environmental stress to produce disordered eating and provide a preclinical tool to test preventive strategies and treatments more relevant to bulimia nervosa, multiple cases of binge eating disorder (BED) and binge-purge type anorexia nervosa.     

Referrals for bereavement counselling in primary care: a qualitative study

The growth in the provision of counselling services in British primary care offers an opportunity for general practitioners (GPs) to refer patients to counsellors following bereavement. This study explores the factors that influence GPs referral decisions. Qualitative interviews were conducted with 50 GPs from two cities in southern UK. The study found that GPs draw on notions of abnormal bereavement in making referral decisions. Indicators of bereavement problems related to: the nature of the death; level of social support; and reaction to the death. GPs views about the types of patients likely to benefit from counselling were further criteria employed in referral decisions. The study indicated that consideration of these factors may discriminate against certain types of patients being referred. Further education in the range of psychological theories of bereavement may assist GPs in understanding their bereaved patients' experiences and in developing their skills in recognising abnormal reactions and making appropriate referrals.     

Identification of estrogen-responsive genes involved in breast cancer metastases to the bone

Bone metastasis is the most common metastasis in breast cancer patients. Clinical observations propose strong association between estrogen receptor (ER)-positive tumors and the development of bone metastases. We hypothesized of biologically diverse sets of hormone-dependent tumors predisposed to bone metastases and of possible role of ER-signaling pathways in the development and progression of bone metastases. We developed a novel in vitro estrogen (E2)-responsive model system, in which breast cancer cells and bone cells express high levels of either ERα or ERβ. Using co-culture approach and gene array technology we identified E2-responsive genes involved in the interaction between cancer cells and bone cells. We detected 13 genes that were altered solely by ERα and 11 genes that were regulated solely by ERβ in cancer cells. Only 5 genes were modified by both ERα and ERβ. Interestingly, the majority of genes in bone cells were altered through ERβ. Two genes, namely MacMarcks and Muc-1, whose changes in expressions in cancer cells in response to E2 were highly significant, were selected for immunohistochemical analysis using tissue microarrays of 59 infiltrating ductal carcinomas. Our results indicated that both MacMarcks and Muc-1 were expressed at high frequency in ER-positive tumors. The correlation between ERα- and ERβ-status of hormone-dependent tumors with combined expression of these two markers might suggest a more aggressive tumor phenotype associated with bone metastases. Further analysis of tissues with clinicopathological characteristics and known bone metastatic disease will indicate potential prognostic values of these and other markers in the development of bone metastases in a subgroup of “bad” hormone-dependent breast cancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.