In ovo corticosterone exposure does not influence yolk steroid hormone relative abundance or skeletal muscle development in the embryonic chicken.

In ovo corticosterone (CORT) exposure reportedly reduces growth and alters body composition traits in meat-type chickens. However, the mechanisms governing alterations in growth and body composition remain unclear but could involve myogenic stem cell commitment, and/or the presence of yolk steroid hormones. This study investigated whether in ovo CORT exposure influenced yolk steroid hormone content, as well as embryonic myogenic development in meat-type chickens. Fertile eggs were randomly divided at embryonic day (ED) 11 and administered either a control (CON; 100 µL of 10 mM PBS) or CORT solution (100 µL of 10 mM PBS containing 1 µg CORT) into the chorioallantoic membrane. Yolk samples were collected at ED 0 and ED 5. At ED 15 and hatch, embryos were humanely killed, and yolk and breast muscle (BM) samples were collected. The relative abundance of 15 steroid hormones, along with total lipid content was measured in yolk samples collected at ED 0, ED 5, ED 15, and ED 21. Muscle fiber number, cross-sectional area, and fascicle area occupied by muscle fibers were measured in BM samples collected at hatch. Relative expression of MyoD, MyoG, Pax7, PPARγ, and CEBP/ß, and the sex steroid receptors were measured in BM samples collected at hatch. The administration of CORT had a limited effect on yolk steroid hormones. In ovo CORT significantly reduced fascicle area occupied by muscle fibers and CEBP/ß expression was increased in CORT exposed birds at hatch. In addition, the quantity of yolk lipid was significantly reduced in CORT-treated birds. In conclusion, in ovo exposure to CORT does not appear to influence early muscle development through yolk steroid hormones in embryonic meat-type chickens however, the results provide a comprehensive analysis of the composition of yolk steroid hormones in ovo at different developmental time points. The findings may suggest increased mesenchymal stem cell commitment to the adipogenic lineage during differentiation and requires further investigation.

Thermoreversible hyaluronan-hydrogel and autologous nucleus pulposus cell delivery regenerates human intervertebral discs in an ex vivo, physiological organ culture model.

Numerous studies show promise for cell-based tissue engineering strategies aiming to repair painful intervertebral disc (IVD) degeneration. However, clinical translation to human IVD repair is slow. In the present study, the regenerative potential of an autologous nucleus pulposus (NP)-cell-seeded thermoresponsive hyaluronic acid hydrogel in human lumbar IVDs was assessed under physiological conditions. First, agarose-encased in vitro constructs were developed, showing greater than 90 % NP cell viability and high proteoglycan deposition within HA-pNIPAM hydrogels following 3 weeks of dynamic loading. Second, a bovine-induced IVD degeneration model was used to optimise and validate T1ρ magnetic resonance imaging (MRI) for detection of changes in proteoglycan content in isolated intact IVDs. Finally, isolated intact human lumbar IVDs were pre-scanned using the established MRI sequence. Then, IVDs were injected with HA-pNIPAM hydrogel alone or autologous NP-cell-seeded. Next, the treated IVDs were cultured under cyclic dynamic loading for 5 weeks. Post-treatment T1ρ values were significantly higher as compared to pre-treatment scans within the same IVD and region of interest. Histological evaluation of treated human IVDs showed that the implanted hydrogel alone accumulated proteoglycans, while those that contained NP cells also displayed neo-matrix-surrounded cells within the gel. The study indicated a clinical potential for repairing early degenerative human IVDs using autologous cells/hydrogel suspensions. This unique IVD culture set-up, combined with the long-term physiological culture of intact human IVDs, allowed for a more clinically relevant evaluation of human tissue repair and regeneration, which otherwise could not be replicated using the available in vitro and in vivo models.

Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications.

Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD) degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP) phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD) gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells - isolated from acute herniation tissue - produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.

Dynamic loading, matrix maintenance and cell injection therapy of human intervertebral discs cultured in a bioreactor.

Low back pain originating from intervertebral disc (IVD) degeneration affects the quality of life for millions of people, and it is a major contributor to global healthcare costs. Long-term culture of intact IVDs is necessary to develop ex vivo models of human IVD degeneration and repair, where the relationship between mechanobiology, disc matrix composition and metabolism can be better understood. A bioreactor was developed that facilitates culture of intact human IVDs in a controlled, dynamically loaded environment. Tissue integrity and cell viability was evaluated under 3 different loading conditions: low 0.1-0.3, medium 0.1-0.3 and high 0.1-1.2 MPa. Cell viability was maintained > 80 % throughout the disc at low and medium loads, whereas it dropped to approximately 70 % (NP) and 50 % (AF) under high loads. Although cell viability was affected at high loads, there was no evidence of sGAG loss, changes in newly synthesised collagen type II or chondroadherin fragmentation. Sulphated GAG content remained at a stable level of approximately 50 µg sGAG/mg tissue in all loading protocols. To evaluate the feasibility of tissue repair strategies with cell supplementation, human NP cells were transplanted into discs within a thermoreversible hyaluronan hydrogel. The discs were loaded under medium loads, and the injected cells remained largely localised to the NP region. This study demonstrates the feasibility of culturing human IVDs for 14 days under cyclic dynamic loading conditions. The system allows the determination a safe range-of-loading and presents a platform to evaluate cell therapies and help to elucidate the effect of load following cell-based therapies.

Acute mechanical injury of the human intervertebral disc: link to degeneration and pain.

Excessive mechanical loading or acute trauma to intervertebral discs (IVDs) is thought to contribute to degeneration and pain. However, the exact mechanisms by which mechanical injury initiates and promotes degeneration remain unclear. This study investigates biochemical changes and extracellular matrix disruption in whole-organ human IVD cultures following acute mechanical injury. Isolated healthy human IVDs were rapidly compressed by 5% (non-injured) or 30% (injured) of disc height. 30% strain consistently cracked cartilage endplates, confirming disc trauma. Three days post-loading, conditioned media were assessed for proteoglycan content and released cytokines. Tissue extracts were assessed for proteoglycan content and for aggrecan integrity. Conditioned media were applied to PC12 cells to evaluate if factors inducing neurite growth were released. Compared to controls, IVD injury caused significant cell death. Injury also caused significantly reduced tissue proteoglycan content with a reciprocal increase of proteoglycan content in culture media. Increased aggrecan fragmentation was observed in injured tissue due to increased matrix metalloproteinase and aggrecanase activity. Injured-IVD conditioned media contained significantly elevated interleukin (IL)-5, IL-6, IL-7, IL-8, MCP-2, GROα, and MIG, and ELISA analysis showed significantly increased nerve growth factor levels compared to non-injured media. Injured-disc media caused significant neurite sprouting in PC12 cells compared to non-injured media. Acute mechanical injury of human IVDs ex vivo initiates release of factors and enzyme activity associated with degeneration and back pain. This work provides direct evidence linking acute trauma, inflammatory factors, neo-innervation and potential degeneration and discogenic pain in vivo.

Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels.

Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications.

Mechanical injury of bovine cartilage explants induces depth-dependent, transient changes in MAP kinase activity associated with apoptosis.

OBJECTIVE: To characterize mitogen activated protein (MAP) kinase activity and chondrocyte apoptosis in an in vitro model of cartilage mechanical injury as a function of tissue depth and time post-injury. DESIGN: Mechanically injured osteochondral explants were assessed for cell viability, MAP kinase and caspase-3 activity over 15 days using immunofluorescence microscopy and Western blot. Zonal distributions of cell viability and apoptosis were quantified in the presence of specific mitogen activated protein kinase inhibitors. RESULTS: Viability rapidly decreased post-injury, most significantly in the superficial zone, with some involvement of the middle and deep zones, which correlated with increased caspase-3 activity. Transient and significant increases in extracellular-regulated protein kinase (ERK) activity were observed in middle and deep zones at 1 and 6 days post-injury, while c-Jun-amino terminal protein kinase activity increased in the deep zone at 1 and 6 days compared to uninjured controls. Changes in p38 activity were particularly pronounced, with significant increases in all three zones 30 min post-injury, but only in the middle and deep zones after 1 and 6 days. Inhibition of ERK and p38 increased chondrocyte viability which correlated with decreased apoptosis. CONCLUSIONS: Spatiotemporal patterns of MAP kinase signalling in cartilage after mechanical injury strongly correlate with changes in cell viability and chondrocyte apoptosis. Importantly, these signals may be pro-survival or pro-apoptotic depending on zonal location and time post-injury. These data yield mechanistic insights which may improve the diagnosis and treatment of cartilage injuries.

Toward a statistically relevant calibration end point for prostate seed implants.

Interstitial brachytherapy for carcinoma of the prostate is achieved through the use of a configuration of radioactive seeds placed in a manner that delivers a customized, reasonably uniform dose to the target volume. Accurate dose delivery depends on both precise seed placement and reliable seed strength in the implanted configuration. This study assumes the independence of the two issues, and quantifies the reduction in the minimum dose to the surface of the gland due only to variability in individual seed strengths. Current AAPM guidelines pertaining to the acceptable limits on seed-to-seed variability are prudent for small configurations of seeds, yet are likely to be overly stringent for applications such as prostate seed implantation. In this study we determine the reduction in the minimum peripheral dose (mPD) caused by the introduction of source strength variability, and provide statistical insight into this effect. It is concluded that the current guidelines limit the reduction in mPD to < or =0.4% relative to the prescription value, for an average configuration, due to the inclusion of strength variability. The maximum observed reduction in mPD would be < or =1.5%. This value is an order of magnitude lower than the recommendations of the AAPM Task Group 40 for the overall accuracy of brachytherapy procedures, which suggests that seed strength variability is of limited concern and that constraints on this factor should perhaps be reevaluated.

Characterization of the dose perturbation by stents as a function of X-ray beam energy.

PURPOSE: External beam irradiation of coronary arteries has been shown to be detrimental in an animal model for the prevention of neointimal hyperplasia in the presence of stents when orthovoltage x-ray beams are used. The present study investigated the effect of beam energy on the dose distribution in the wall of the artery in the presence of stents. MATERIALS AND METHODS: We used 250-kVp x-rays and 6-MV x-rays to irradiate a stent placed in a homogeneous phantom. Radiochromic film densitometry and Monte Carlo calculations were used to measure and to simulate the dose distribution in the proximity of the stent. RESULT: External beam irradiation not only failed to prevent neointimal hyperplasia, but actually accentuated the neointimal response to a prompt mechanical injury in the artery. The photoelectric effect, which dominates low-energy x-ray interactions, produces recoil electrons in the stent, which enhance the dose surrounding the intima. The photoelectrons generated in nickel and iron have an extremely short range in normal tissue, approximately 0.1 mm. Initial estimates of orthovoltage x-ray interactions with the stent indicate a dose enhancement in the orthovoltage range by a factor of 2-6 due to the rise in the photoelectric cross section in this energy range depending on the elemental composition of the stent. Film densitometry verifies this dose enhancement. The Monte Carlo calculation yields a dose enhancement and the dose fall-off with distance from the stent when irradiated with orthovoltage x-rays. Conversely when the tissue and stent are irradiated with megavoltage x-rays, the dose enhancement in this region is a factor of 1.15 in close proximity to the stent and 1.0 at distances greater than 0.1 mm. The 6-MV photon interactions in tissue and Ni/Ti are predominantly through Compton scattering. The Compton effect is dependent on the electron density in the medium, in contrast to the atomic number, which is more relevant for photoelectric absorption. The dose estimates for megavoltage x-rays adjacent to the stent are complicated by the lack of charged particle equilibrium. CONCLUSIONS: There is a limited but definite increase in the dose delivery to the arterial wall when stents are irradiated with orthovoltage x-ray energies. This increase may explain the negative response in other studies. The presence of the stent does perturb the character and magnitude of the dose in the normal arterial wall as a function of beam quality.

Quality assurance in linac-based stereotactic radiosurgery and radiotherapy.

Stereotactic Radiosurgery demands extraordinary attention to quality assurance issues. This is related to the high accuracy needed to perform a successful procedure, accuracy demanded by the proximity of the target lesion to neighboring fragile and eloquent structures in the head and large doses delivered. The nature of the linac-based radiosurgery procedure is that of a series of steps, each linked together and requiring quality control, for if one step is faulty the final result will be equally faulty. The salient points associated with the quality assurance of each step are laid out in this article. Implementation of a linac-based radiosurgery program in an institution must be well thought out and must be a team effort, involving expertise in medical physics, radiological imaging, radiation oncology, and specially trained radiation therapists in order to be successful and safe.

Clients' representations of their therapists over the course of psychotherapy.

Using a cross-sectional design, the forms, functional themes, and corresponding affects of clients' (n = 88) representations of their therapists were compared across three distinct time phases of therapy: up to one year, between one and three years, and more than three years. Results indicated that clients in the beginning phase of therapy were less likely to employ representations in the service of continuing the therapeutic dialogue in-between sessions, and less likely to use representations to relieve the pain associated with missing the therapist during separations than clients who have been in therapy for more than one year. Clients in all three phases of therapy most commonly felt comforted and accepted when evoking therapist representations in between sessions.

Lung carcinoma in a patient with Lucite sphere plombage thoracoplasty.

A patient with a history of pulmonary tuberculosis was treated in 1949 with Lucite sphere plombage thoracoplasty. She subsequently developed squamous cell carcinoma of the lung despite having no history of exposure to known carcinogens associated with the development of squamous cell carcinoma. The patient's lung carcinoma developed adjacent to the plombage space. Lung carcinoma has not previously been reported in association with Lucite sphere plombage.

[Cyclosporin A in the treatment of pemphigus foliaceus and pemphigus erythematosus]. / Cyclosporin A in der Behandlung von Pemphigus foliaceus und Pemphigus erythematosus.

We present the cases of two female patients with pemphigus foliaceus and pemphigus erythematosus, respectively, who did not respond to long-term azathioprine and prednisone treatment in high dosages. In both patients the skin condition improved nearly completely after therapy with cyclosporine A (3.5-6.5 mg/kg per day) in combination with prednisone (7.5-10 mg every 2 days). We did not observe any side-effects during 1 year of this therapy.

Spectrum of clinical disease in pulmonary infection with Mycobacterium avium-intracellulare.

Cases of infection with Mycobacterium avium-intracellulare reported in Milwaukee, Wisconsin, have become more common in recent years, and their incidence--50 cases per year--is now similar to that of tuberculosis. Cases usually occur in middle-aged men with underlying lung diseases, but variations in age, sex, presentation, and severity of disease are wide. Several cases that illustrate pathogenesis and spectrum of disease, from primary, to chronic-active, to healing stages, are presented. The disease tends to run an indolent course in most cases, but extensive disease and unfavorable early course indicate poor prognosis. At a 10-year review, mortality had reached sizable numbers in this aging population with frequent serious underlying problems. Only a small number of these deaths can be attributed directly to mycobacterial disease.