Bovine explant model of degeneration of the intervertebral disc

Background: Many new treatments for degeneration of the intervertebral disc are being developed which can be delivered through a needle. These require testing in model systems before being used in human patients. Unfortunately, because of differences in anatomy, there are no ideal animal models of disc degeneration. Bovine explant model systems have many advantages but it is not possible to inject any significant volume into an intact disc. Therefore we have attempted to mimic disc degeneration in an explant bovine model via enzymatic digestion. Methods: Bovine coccygeal discs were incubated with different concentrations of the proteolytic enzymes, trypsin and papain, and maintained in culture for up to 3 weeks. A radio-opaque solution was injected to visualise cavities generated. Degenerative features were monitored histologically and biochemically (water and glycosaminoglycan content, via dimethylmethylene blue). Results and Conclusion: The central region of both papain and trypsin treated discs was macro- and microscopically fragmented, with severe loss of metachromasia. The integrity of the surrounding tissue was mostly in tact with cells in the outer annulus appearing viable. Biochemical analysis demonstrated greatly reduced glycosaminoglycan content in these compared to untreated discs. We have shown that bovine coccygeal discs, treated with proteolytic enzymes can provide a useful in vitro model system for developing and testing potential new treatments of disc degeneration, such as injectable implants or biological therapies

Tissue Microenvironments Define and Get Reinforced by Macrophage Phenotypes in Homeostasis or during Inflammation, Repair and Fibrosis

Current macrophage phenotype classifications are based on distinct in vitro culture conditions that do not adequately mirror complex tissue environments. In vivo monocyte progenitors populate all tissues for immune surveillance which supports the maintenance of homeostasis as well as regaining homeostasis after injury. Here we propose to classify macrophage phenotypes according to prototypical tissue environments, e.g. as they occur during homeostasis as well as during the different phases of (dermal) wound healing. In tissue necrosis and/or infection, damage- and/or pathogen-associated molecular patterns induce proinflammatory macrophages by Toll-like receptors or inflammasomes. Such classically activated macrophages contribute to further tissue inflammation and damage. Apoptotic cells and antiinflammatory cytokines dominate in postinflammatory tissues which induce macrophages to produce more antiinflammatory mediators. Similarly, tumor-associated macrophages also confer immunosuppression in tumor stroma. Insufficient parenchymal healing despite abundant growth factors pushes macrophages to gain a profibrotic phenotype and promote fibrocyte recruitment which both enforce tissue scarring. Ischemic scars are largely devoid of cytokines and growth factors so that fibrolytic macrophages that predominantly secrete proteases digest the excess extracellular matrix. Together, macrophages stabilize their surrounding tissue microenvironments by adapting different phenotypes as feed-forward mechanisms to maintain tissue homeostasis or regain it following injury. Furthermore, macrophage heterogeneity in healthy or injured tissues mirrors spatial and temporal differences in microenvironments during the various stages of tissue injury and repair. Copyright (C) 2012 S. Karger AG, Base

Communication Impairments in Mice Lacking Shank1: Reduced Levels of Ultrasonic Vocalizations and Scent Marking Behavior

Autism is a neurodevelopmental disorder with a strong genetic component. Core symptoms are abnormal reciprocal social interactions, qualitative impairments in communication, and repetitive and stereotyped patterns of behavior with restricted interests. Candidate genes for autism include the SHANK gene family, as mutations in SHANK2 and SHANK3 have been detected in several autistic individuals. SHANK genes code for a family of scaffolding proteins located in the postsynaptic density of excitatory synapses. To test the hypothesis that a mutation in SHANK1 contributes to the symptoms of autism, we evaluated Shank1−/− null mutant mice for behavioral phenotypes with relevance to autism, focusing on social communication. Ultrasonic vocalizations and the deposition of scent marks appear to be two major modes of mouse communication. Our findings revealed evidence for low levels of ultrasonic vocalizations and scent marks in Shank1−/− mice as compared to wildtype Shank1+/+ littermate controls. Shank1−/− pups emitted fewer vocalizations than Shank1+/+ pups when isolated from mother and littermates. In adulthood, genotype affected scent marking behavior in the presence of female urinary pheromones. Adult Shank1−/− males deposited fewer scent marks in proximity to female urine than Shank1+/+ males. Call emission in response to female urinary pheromones also differed between genotypes. Shank1+/+ mice changed their calling pattern dependent on previous female interactions, while Shank1−/− mice were unaffected, indicating a failure of Shank1−/− males to learn from a social experience. The reduced levels of ultrasonic vocalizations and scent marking behavior in Shank1−/− mice are consistent with a phenotype relevant to social communication deficits in autism.National Institute of Mental Health (U.S.) (Intramural Research Program)Simons Foundatio

Rheological and biological properties of a hydrogel support for cells intended for intervertebral disc repair

<p>Abstract</p> <p>Background</p> <p>Cell-based approaches towards restoration of prolapsed or degenerated intervertebral discs are hampered by a lack of measures for safe administration and placement of cell suspensions within a treated disc. In order to overcome these risks, a serum albumin-based hydrogel has been developed that polymerizes after injection and anchors the administered cell suspension within the tissue.</p> <p>Methods</p> <p>A hydrogel composed of chemically activated albumin crosslinked by polyethylene glycol spacers was produced. The visco-elastic gel properties were determined by rheological measurement. Human intervertebral disc cells were cultured <it>in vitro </it>and <it>in vivo </it>in the hydrogel and their phenotype was tested by reverse-transcriptase polymerase chain reaction. Matrix production and deposition was monitored by immuno-histology and by biochemical analysis of collagen and glycosaminoglycan deposition. Species specific <it>in situ </it>hybridization was performed to discriminate between cells of human and murine origin in xenotransplants.</p> <p>Results</p> <p>The reproducibility of the gel formation process could be demonstrated. The visco-elastic properties were not influenced by storage of gel components. <it>In vitro </it>and <it>in vivo </it>(subcutaneous implants in mice) evidence is presented for cellular differentiation and matrix deposition within the hydrogel for human intervertebral disc cells even for donor cells that have been expanded in primary monolayer culture, stored in liquid nitrogen and re-activated in secondary monolayer culture. Upon injection into the animals, gels formed spheres that lasted for the duration of the experiments (14 days). The expression of cartilage- and disc-specific mRNAs was maintained in hydrogels <it>in vitro </it>and <it>in vivo</it>, demonstrating the maintenance of a stable specific cellular phenotype, compared to monolayer cells. Significantly higher levels of hyaluronan synthase isozymes-2 and -3 mRNA suggest cell functionalities towards those needed for the support of the regeneration of the intervertebral disc. Moreover, mouse implanted hydrogels accumulated 5 times more glycosaminoglycans and 50 times more collagen than the <it>in vitro </it>cultured gels, the latter instead releasing equivalent quantities of glycosaminoglycans and collagen into the culture medium. Matrix deposition could be specified by immunohistology for collagen types I and II, and aggrecan and was found only in areas where predominantly cells of human origin were detected by species specific <it>in situ </it>hybridization.</p> <p>Conclusions</p> <p>The data demonstrate that the hydrogels form stable implants capable to contain a specifically functional cell population within a physiological environment.</p

A semiparametric Bayesian proportional hazards model for interval censored data with frailty effects

<p>Abstract</p> <p>Background</p> <p>Multivariate analysis of interval censored event data based on classical likelihood methods is notoriously cumbersome. Likelihood inference for models which additionally include random effects are not available at all. Developed algorithms bear problems for practical users like: matrix inversion, slow convergence, no assessment of statistical uncertainty.</p> <p>Methods</p> <p>MCMC procedures combined with imputation are used to implement hierarchical models for interval censored data within a Bayesian framework.</p> <p>Results</p> <p>Two examples from clinical practice demonstrate the handling of clustered interval censored event times as well as multilayer random effects for inter-institutional quality assessment. The software developed is called survBayes and is freely available at CRAN.</p> <p>Conclusion</p> <p>The proposed software supports the solution of complex analyses in many fields of clinical epidemiology as well as health services research.</p

Worldwide distribution of NAT2 diversity: Implications for NAT2 evolutionary history

<p>Abstract</p> <p>Background</p> <p>The N-acetyltransferase 2 (<it>NAT2</it>) gene plays a crucial role in the metabolism of many drugs and xenobiotics. As it represents a likely target of population-specific selection pressures, we fully sequenced the <it>NAT2 </it>coding region in 97 Mandenka individuals from Senegal, and compared these sequences to extant data on other African populations. The Mandenka data were further included in a worldwide dataset composed of 41 published population samples (6,727 individuals) from four continental regions that were adequately genotyped for all common <it>NAT2 </it>variants so as to provide further insights into the worldwide haplotype diversity and population structure at <it>NAT2</it>.</p> <p>Results</p> <p>The sequencing analysis of the <it>NAT2 </it>gene in the Mandenka sample revealed twelve polymorphic sites in the coding exon (two of which are newly identified mutations, C345T and C638T), defining 16 haplotypes. High diversity and no molecular signal of departure from neutrality were observed in this West African sample. On the basis of the worldwide genotyping survey dataset, we found a strong genetic structure differentiating East Asians from both Europeans and sub-Saharan Africans. This pattern could result from region- or population-specific selective pressures acting at this locus, as further suggested in the HapMap data by extremely high values of <it>F</it>_STfor a few SNPs positions in the <it>NAT2 </it>coding exon (T341C, C481T and A803G) in comparison to the empirical distribution of <it>F</it>_STvalues accross the whole 400-kb region of the <it>NAT </it>gene family.</p> <p>Conclusion</p> <p>Patterns of sequence variation at <it>NAT2 </it>are consistent with selective neutrality in all sub-Saharan African populations investigated, whereas the high level of population differentiation between Europeans and East Asians inferred from SNPs could suggest population-specific selective pressures acting at this locus, probably caused by differences in diet or exposure to other environmental signals.</p

Craniectomy for Malignant Cerebral Infarction: Prevalence and Outcomes in US Hospitals

Randomized trials have demonstrated the efficacy of craniectomy for the treatment of malignant cerebral edema following ischemic stroke. We sought to determine the prevalence and outcomes related to this by using a national database.Patient discharges with ischemic stroke as the primary diagnosis undergoing craniectomy were queried from the US Nationwide Inpatient Sample from 1999 to 2008. A subpopulation of patients was identified that underwent thrombolysis. Two primary end points were examined: in-hospital mortality and discharge to home/routine care. To facilitate interpretations, adjusted prevalence was calculated from the overall prevalence and two age-specific logistic regression models. The predictive margin was then generated using a multivariate logistic regression model to estimate the probability of in-hospital mortality after adjustment for admission type, admission source, length of stay, total hospital charges, chronic comorbidities, and medical complications.After excluding 71,996 patients with the diagnosis of intracranial hemorrhage and posterior intracranial circulation occlusion, we identified 4,248,955 adult hospitalizations with ischemic stroke as a primary diagnosis. The estimated rates of hospitalizations in craniectomy per 10,000 hospitalizations with ischemic stroke increased from 3.9 in 1999-2000 to 14.46 in 2007-2008 (p for linear trend<0.001). Patients 60+ years of age had in-hospital mortality of 44% while the 18-59 year old group was found to be 24% (p = 0.14). Outcomes were comparable if recombinant tissue plasminogen activator had been administered.Craniectomy is being increasingly performed for malignant cerebral edema following large territory cerebral ischemia. We suspect that the increase in the annual incidence of DC for malignant cerebral edema is directly related to the expanding collection of evidence in randomized trials that the operation is efficacious when performed in the correct patient population. In hospital mortality is high for all patients undergoing this procedure

Interferon-γ Regulates the Proliferation and Differentiation of Mesenchymal Stem Cells via Activation of Indoleamine 2,3 Dioxygenase (IDO)

The kynurenine pathway (KP) of tryptophan metabolism is linked to antimicrobial activity and modulation of immune responses but its role in stem cell biology is unknown. We show that human and mouse mesenchymal and neural stem cells (MSCs and NSCs) express the complete KP, including indoleamine 2,3 dioxygenase 1 (IDO) and IDO2, that it is highly regulated by type I (IFN-β) and II interferons (IFN-γ), and that its transcriptional modulation depends on the type of interferon, cell type and species. IFN-γ inhibited proliferation and altered human and mouse MSC neural, adipocytic and osteocytic differentiation via the activation of IDO. A functional KP present in MSCs, NSCs and perhaps other stem cell types offers novel therapeutic opportunities for optimisation of stem cell proliferation and differentiation

Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges

Lumbar discectomy is a very effective therapy for neurological decompression in patients suffering from sciatica due to hernia nuclei pulposus. However, high recurrence rates and persisting post-operative low back pain in these patients require serious attention. In the past decade, tissue engineering strategies have been developed mainly targeted to the regeneration of the nucleus pulposus (NP) of the intervertebral disc. Accompanying techniques that deal with the damaged annulus fibrous are now increasingly recognised as mandatory in order to prevent re-herniation to increase the potential of NP repair and to confine NP replacement therapies. In the current review, the requirements, achievements and challenges in this quickly emerging field of research are discussed