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Effects of the impact of a mechanical structure on the human body are of great interest in the understanding of body trauma. Experimental tests have led to first conclusions about the dangerousness of an impact observing impact forces or displacement time history with PMHS (Post Mortem human Subjects). They have allowed providing interesting data for the development and the validation of numerical biomechanical models. These models, widely used in the framework of automotive crashworthiness, have led to the development of numerical-based injury criteria and tolerance thresholds. The aim of this process is to improve the safety of mechanical structures in interaction with the body. In a military context, investigations both at experimental and numerical level are less successfully completed. For both military and civil frameworks, the literature list a number of numerical analysis trying to propose injury mechanisms, and tolerance thresholds based on biofidelic Finite Element (FE) models of different part of the human body. However the link between both frameworks is not obvious, since lots of parameters are different: great mass impacts at relatively low velocity for civil impacts (falls, automotive crashworthiness) and low mass at very high velocity for military loadings (ballistic, blast). In this study, different accident cases were investigated, and replicated with a previously developed and validated FE model of the human thorax named Hermaphrodite Universal Biomechanical YX model (HUBYX model). These previous validations included replications of standard experimental tests often used to validate models in the context of automotive industry, experimental ballistic tests in high speed dynamic impact and also numerical replication of blast loading test ensuring its biofidelity. In order to extend the use of this model in other frameworks, some real-world accidents were reconstructed, and consequences of these loadings on the FE model were explored. These various numerical replications of accident coming from different contexts raise the question about the ability of a FE model to correctly predict several kinds of trauma, from blast or ballistic impacts to falls, sports or automotive ones in a context of numerical injury mechanisms and tolerance limits investigations., (Copyright © 2015 John Wiley & Sons, Ltd.)

Despite a growing body of knowledge on the mechanisms underlying the onset and progression of cancer, treatment success rates in oncology are at best modest. Current approaches use statistical methods that fail to embrace the inherent and expansive complexity of the tumor/patient/drug interaction. Computational modeling, in particular mechanistic modeling, has the power to resolve this complexity. Using fundamental knowledge on the interactions occurring between the components of a complex biological system, large-scale in silico models with predictive capabilities can be generated. Here, we describe how mechanistic virtual patient models, based on systematic molecular characterization of patients and their diseases, have the potential to shift the theranostic paradigm for oncology, both in the fields of personalized medicine and targeted drug development. In particular, we highlight the mechanistic modeling platform ModCell™ for individualized prediction of patient responses to treatment, emphasizing modeling techniques and avenues of application., (© 2015 the author(s), publisher and licensee Libertas Academica Ltd.)

Measuring brain electrical impedance (rheoencephalography-REG) is a potential technique for noninvasive, continuous neuro-monitoring. Typically, intracranial pressure (ICP), an invasive monitoring modality, is used in brain monitoring. Our hypothesis was that both modalities would reflect cerebrovascular reactivity. In the present study we compared results of REG to results of ICP measurement. Rats were used under anesthesia ([Formula: see text]; 36 control and 59 vinpocetine infusions). REG was measured by two bipolar REG amplifiers; time constants (Tc) were 3 and 0.3 s. The vinpocetine injection caused a transient decrease in systemic arterial pressure (SAP) and a simultaneous increase in ICP and REG pulse amplitude. SAP decrease was 25%  ±  14%; ICP was 28%  ±  16%; REG pulse amplitude increase was 209%  ±  17% (Tc 3) and 107%  ±  68% (Tc 0.3). ICP increase correlated with REG pulse amplitude increase. Area under the receiver operating characteristic curve was 0.9481 for ICP-REG time constants 3 and 0.9335 for ICP-REG time constants 0.3; both with [Formula: see text]. The fact that both REG and ICP reflect cerebrovascular reactivity indicates the usefulness of REG as a potential technique for noninvasive, continuous neuro-monitoring. The Tc of REG amplifier requires optimization for continuous monitoring of pressure reactivity index.

The biological processes that keep us healthy or cause disease, as well as the mechanisms of action of possible drugs are inherently complex. In the face of this complexity, attempts at discovering new drugs to treat diseases have alternated between trial-and-error (typically on experimental systems) and grand simplification, usually based on much too little information. We now have the chance to combine these strategies through establishment of 'virtual patient' models, centred on a detailed molecular characterisation of thousands or even, in the future, millions of patients. In doing so, we lay the foundations for truly personalised therapy, as well as a far-reaching virtualisation of drug discovery and development in oncology and other areas of medicine., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Porcine Sertoli cells (pSCs) have been employed for cell therapy in pre-clinical studies for several chronic/immune diseases as they deliver molecules associated with trophic and anti-inflammatory effects. To be employed for human xenografts, pSCs products need to comply with safety and stability. To fulfill such requirements, we employed a microencapsulation technology to increase pre-transplant storage stability of specific pathogen-free pSCs (SPF-pSCs) and evaluated the in vivo long-term viability and safety of grafts., Methods: Specific pathogen free neonatal pigs underwent testis excision under sterility. pSCs were isolated, characterized by immunofluorescence (IF) and cytofluorimetric analysis (CA) and examined in terms of viability and function [namely, production of anti-müllerian hormone (AMH), inhibin B, and transforming growth factor beta-1 (TFGβ-1)]. After microencapsulation in barium alginate microcapsules (Ba-MC), long-term SPF-pSCs (Ba-MCpSCs) viability and barium concentrations were evaluated at 1, 24 throughout 40 h to establish pre-transplant storage conditions., Results: The purity of isolated pSCs was about 95% with negligible contaminating cells. Cultured pSCs monolayers, both prior to and after microencapsulation, maintained high function and full viability up to 24 h of storage. At 40 h post-encapsulation, pSCs viability decreased to 80%. Barium concentration in Ba-MCpSCs lagged below the normal maximum daily allowance and was stable for 4 months in mice with no evident side effects., Conclusions: Such results suggest that this protocol for the isolation and microencapsulation of pSCs is compatible with long-haul transportation and that Ba-MCpSCs could be potentially employable for xenotransplantation., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: To determine the optimal effective dose of sodium pyruvate in maintaining the vital signs following hemorrhagic shock (HS) in rats., Materials and Methods: Anesthetized, male Sprague-Dawley rats underwent computer-controlled HS for 30 minute followed by fluid resuscitation with either hypertonic saline, or sodium pyruvate solutions of 0.5 M, 1.0 M, 2.0 M, and 4.0 M at a rate of 5ml/kg/h (60 minute) and subsequent blood infusion (60 minute). The results were compared with sham and non- resuscitated groups. The animals were continuously monitored for mean arterial pressure, systolic and diastolic pressure, heart rate, pulse pressure, temperature, shock index and Kerdo index (KI)., Results: The Sham group remained stable throughout the experiment. Non-resuscitated HS animals did not survive for the entire experiment due to non-viable vital signs and poor shock and KI. All fluids were effective in normalizing the vital signs when shed blood was used adjunctively. Sodium pyruvate 2.0 M was most effective, and 4.0 M solution was least effective in improving the vital signs after HS., Conclusions: Future studies should be directed to use 2.0 M sodium pyruvate adjuvant for resuscitation on multiorgan failure and survival rate in HS.

Occupational and environmental exposure to the heavy metal cadmium (Cd) and its inhalation from cigarette smoke are associated with emphysema. Many growth factors and extracellular matrix (ECM) cell signaling molecules are directly involved in the epithelial bronchial cell pathway. This study investigated the direct effects of Cd on the production of several ECM components in human bronchial epithelial cells (BEAS-2B) that were exposed in vitro for 48 h to sub-toxic and toxic concentrations of Cd. Gene expression of collagens, metalloproteases (MMPs), integrins, tenascin and vitronectin were quantified by RT-PCR. To study apoptosis cascade, annexin assay and cellular cytotoxicity by MTT assay were performed. We also investigated whether an imbalance in the TGFβ/TGFβ receptor (TGFβR) expression mediated Cd effects. The results showed the sub-toxic Cd dose significantly increased tenascin, vitronectin, β1 and β5 integrin gene expression. The toxic Cd dose decreased type IV and V collagen, α1, α2 and β3 integrins. Both Cd doses down-regulated type I collagen and up-regulated metalloproteases. Each Cd dose caused a different imbalance in the complex pattern of TGFβ and its receptors. No alteration in classic apoptotic marker protein expression was observed in presence of the sub-toxic dose of Cd, suggesting this metal alters ECM production without apoptotic activation. In conclusion, all these data show even sub-toxic Cd dose exposure alters the specific gene expression of several ECM components that are crucially implicated in the mechanical properties of lung parenchyma supporting the hypothesis that the mechanism underlying Cd-induced lung disease may involve downstream changes in TGFβ/TGFβR signaling., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Intravenous perfluorocarbons (PFC) have reduced the effects of decompression sickness (DCS) and improved mortality rates in animal models. However, concerns for the physiological effects of DCS combined with PFC therapy have not been examined in a balanced mixed-sex population. Thirty-two (16 male, 16 female) instrumented and sedated juvenile Yorkshire swine were exposed to 200 feet of seawater (fsw) for 31 min of hyperbaric air. Pulmonary artery pressure (PAP), cardiac output (CO), and systemic arterial pressure (SAP) were monitored before (control) and after exposure. Animals were randomized to treatment with Oxycyte (5 ml/kg; Oxygen Biotherapeutics, Inc., Morrisville, NC) vs. saline (control) with 100% oxygen administered upon DCS onset; animals were observed for 90 min. Parameters recorded and analyzed included PAP, CO, and SAP. In all animals PAP began to rise prior to cutis marmorata (CM) onset, the first sign of clinical DCS, generally peaking after CM onset. Female swine, compared with castrated males, had a more rapid onset of CM (7.30 vs. 11.46 min postsurfacing) and earlier onset to maximal PAP (6.41 vs. 9.69 min post-CM onset). Oxycyte therapy was associated with a sustained PAP elevation above controls in both sexes (33.41 vs. 25.78 mmHg). Significant pattern differences in PAP, CO, and SAP were noted between sexes and between therapeutic groups. There were no statistically significant differences in survival or paralysis between the PFC and control groups during the 48-h observation period. In conclusion, Oxycyte therapy for DCS is associated with a prolonged PAP increase in swine. These species and sex differences warrant further exploration.

Exposure to nicotine and other compounds contained in cigarette smoking affects human health. This study examined the effects of exposure to a single or multiple sub-toxic nicotine concentrations on human osteoblasts. Cell growth and expression of genes involved in bone differentiation, extracellular matrix (ECM) metabolism, and growth factor signaling pathways were investigated in nicotine-treated cells compared to untreated cells. Depending on osteoblast concentration and maturation stages, nicotine differently regulated cell growth. Real-time PCR showed regulated expressions of genes expressed by nicotine-treated osteoblasts compared to untreated cells. Among ECM genes, type I collagen was down-regulated and osteonectin was up-regulated in nicotine-treated osteoblasts; similarly, fibroblast growth factor-1 (FGF1) and fibroblast growth factor-2 (FGF2), two members of FGF signaling system, were discordantly modulated; genes involved in osteoblast maturation and differentiation such as alkaline phosphatase (ALP), runt-related transcription factor-2 (RUNX2), and bone sialoprotein (BSP) were over-expressed after drug treatment. Our results show a positive association between nicotine exposure and osteoblast phenotype and illustrate for the first time a mechanism whereby acute or chronic exposure to sub-toxic nicotine concentrations may affect bone formation through the impairment of growth factor signaling system and ECM metabolism., (© 2014 Wiley Periodicals, Inc.)

Neonatal porcine Sertoli cells (NPSC) are immune privileged cells showing innate phagocytic and antibacterial activities. NPSC have been shown capable of immunoaltering the body's response and possess lung homing capacity. These properties encourage investigation of NPSC as functional components of cell-based therapeutic protocols to treat lung infections and related complications. In this work, for the first time, NPSC were tailored to carry an antibiotic drug loaded into poly(d,l lactic) acid microparticles (MP). A loading protocol was developed, which afforded 30% drug uptake and high stability over time, with little or no effects on NPSC viability, morphology, reactive oxygen species production and DNA integrity. FSH receptor integrity, and TGFβ (transforming growth factor β) and AMH (anti-Müllerian hormone) expressions were unchanged after 1month of cryopreservation. Protein tyrosine kinase activation due to phagocytosis may have had resulted in changes in inhibin B expression. The activity of MP-loaded or NPSC alone against Pseudomonas aeruginosa was maintained throughout 1month of storage. NPSC couple an innate antibacterial activity with the capacity to embody drug loaded MP. We showed for the first time that engineered NPSC can be cryopreserved with no loss of their basic properties, thereby possibly representing a novel approach for cell-based therapeutic and drug delivery system., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Insulin resistance in type 2 diabetes mellitus (T2DM) may be due to a chronic inflammation of the visceral adipose tissue (VAT) leading to local and systemic increases in proinflammatory cytokines. Microencapsulated porcine Sertoli cells (MC-pSC), by provision of immunomodulatory and trophic factors, have been successfully used to reduce such inflammation in rodent animal models of type 1 diabetes with no complications or deleterious side effects. Herein, we have begun to investigate this novel and safe therapeutic approach in the spontaneously obese nonhuman primate with spontaneous, insulin-dependent T2DM. After MC-pSC intraperitoneal injection we have evaluated, throughout a 6-month follow-up period, daily ad libitum fed glucose levels, daily exogenous insulin supplementation, biweekly body weight measurements, periodic fasting blood glucose concentrations, glycated hemoglobin (HbA1c) levels, glucose tolerance tests (GTT), and fluorescence-activated cell sorting cytometry (FACS) assessment of peripheral blood mononuclear cells. Very preliminarily, we have observed a slight reduction in fasting (FPG) and mean nonfasting (NF) plasma glucose levels. We found minimal changes, only in 1 animal, in daily exogenous insulin requirements and HbA1c levels. Flow cytometric analysis was associated with decrease in CD8(+) cells only in 1 recipient with a reduction in mean regulatory T Cells (Treg), whereas interestingly, decrease of B lymphocytes was observed in both animals. These results may suggest that this novel MC-SC-based transplantation protocol might possibly impact the metabolic status of T2DM in higher mammals that are close to humans., (Copyright © 2014. Published by Elsevier Inc.)

Here we report results of non-invasive measurements of indirect markers of soft tissue healing of traumatic wounds in an observational swine study and describe the quantification of analog physiological signals. The primary purpose of the study was to measure bone healing of fractures with four different wound treatments. A second purpose was to quantify soft tissue wound healing by measuring the following indirect markers: (1) tissue oxygenation, (2) fluid content, and (3) blood flow, which were all measured by non-invasive modalities, measured with available devices. Tissue oxygenation was measured by near infrared spectroscopy; fluid content was measured by bipolar bio-impedance; and blood flow was measured by Doppler ultrasound. Immediately after comminuted femur fractures were produced in the right hind legs of thirty anesthetized female Yorkshire swine, one of four wound treatments was instilled into each wound. The four wound treatments were as follows: salmon fibrinogen/thrombin-n = 8; commercial bone filler matrix-n = 7; bovine collagen-n = 8; porcine fibrinogen/thrombin-n = 7. Fractures were stabilized with an external fixation device. Immediately following wound treatments, measurements were made of tissue oxygenation, fluid content and blood flow; these measurements were repeated weekly for 3 weeks after surgery. Analog signals of each modality were recorded on both the wounded (right) hind leg and the healthy (left) hind leg, for comparison purposes. Data were processed off-line. The mean values of 10-s periods were calculated for right-left leg comparison. ANOVA was applied for statistical analysis. Results of the bone healing studies are published separately (Rothwell et al. in J Spec Oper Med 13:7-18, 2013). For soft tissue wounds, healing did not differ significantly among the four wound treatments; however, regional oxygenation of wounds treated with salmon fibrinogen/thrombin showed slightly different time trends. Further studies are needed to establish standards for healthy wound healing and for detection of pathological alterations such as infection. Non-invasive measurement and quantification of indirect markers of soft tissue wound healing support the goals and principles of evidence-based medicine and show potential as easy to administer tools for clinicians and battlefield medical personnel to apply when procedures such as the PET scan are not available or affordable. The method we developed for storing analog physiological signals could be used for maintaining electronic health records, by incorporating vital signs such as ECG and EEG, etc.

Introduction: Two challenges of trauma triage are to identify wounded who are in danger of imminent death and to enable medics to determine if resuscitation is possible when making ?dead or alive? decisions on the battlefield. Hemorrhagic shock is the leading cause of death in combat injuries. The purpose of this study was to establish the sequence of vital sign cessation during lethal hemorrhage in swine. Our hypothesis was that brain electrical activity (electroencephalography [EEG]) and respiration are earlier indicators of imminent death than traditional modalities measured during triage, such as heart electrical activity (electrocardiography [ECG]) and blood pressure., Methods: Lethal hemorrhage was induced in anesthetized Yorkshire pigs. Vital sign modalities measured were respiration, heart electrical activity (ECG), heart sound, blood pressure (systemic arterial pressure), and brain electrical activity (EEG)., Results: The sequence of vital sign cessation was (1) respiration, (2) brain electrical activity (EEG), (3) heart sound, (4) blood pressure, and (5) heart electrical activity (ECG). Cessation of respiration occurred at approximately the same time that brain electrical activity stopped (?flatlined?) for 2 seconds and then resumed briefly before cessation; cessation of heart electrical activity occurred almost 8 minutes later., Conclusions: A 2-second EEG flatline and final respiration are useful event markers to indicate an opportunity to prevent irreversible brain damage from lethal hemorrhage. Since the 2-second EEG flatline and final respiration occur about 8 minutes before cessation of heart electrical activity (ECG), EEG and final respiration are earlier indicators of imminent death. The use of deployable noninvasive brain monitors implementing these findings can be live-saving on the battlefield as well is in civilian environments., (2013.)

Purpose: Standard treatment for decompression sickness (DCS) is recompression therapy with hyperbaric oxygen (HBO). Non-recompressive therapies are needed to address mass casualty scenarios such as a disabled submarine rescue or DCS therapy in remote environments. Intravenously delivered perfluorocarbon (PFC) emulsions improve blood oxygen content and decrease mortality in several animal models of DCS. However, the enhanced oxygen delivery of PFC emulsions may increase CNS oxygen toxicity (seizures) risk when used in conjunction with HBO. We studied seizure latency and duration in swine randomized to receive PFC or normal saline with 6 ATA of oxygen., Methods: Yorkshire swine (n = 31) were fitted with EEG electrodes and randomized to receive 5 ml/kg of the PFC Oxycyte (Oxygen Biotherapeutics Inc., Morrisville, NC) or saline intravenously 1 h before HBO. Unsedated animals were fitted with a snout mask for inhaled gas delivery, positioned inside the hyperbaric chamber, and compressed to 165 ft of sea water (6 ATA). After 2.5 min at 6 ATA, breathing gas was switched to 100 % O2 until signs of seizure were observed and EEG activity was evident. At seizure onset gas was switched back to air for 3 min, then the chamber was decompressed. After 24 h, the dive profile/oxygen exposure was repeated to ensure no secondary effects of PFC drug redistribution or emulsion metabolism., Results/conclusion: Intravenous PFC emulsion did not decrease seizure latency or increase duration on initial HBO exposure or after 24 h. This finding demonstrates the safety of PFC use in conjunction with recompression therapy to treat DCS.

Oxygen is the most widely used therapeutic strategy to prevent and treat decompression sickness (DCS). Oxygen prebreathe (OPB) eliminated DCS in 20-kg swine after rapid decompression from saturation at 60 feet of seawater (fsw). However, hyperbaric oxygen (HBO) has risks. As oxygen partial pressure increases, so do its toxic effects. Central nervous system (CNS) oxygen toxicity is the most severe side effect, manifesting as seizure. An adjunctive therapeutic is needed to extend OPB strategies to deeper depths and prevent/delay seizure onset. The Food and Drug Administration-approved anti-epileptic vigabatrin has prevented HBO-induced seizures in rats up to 132 fsw. This study aimed to confirm the rat findings in a higher animal model and determine whether acute high-dose vigabatrin evokes retinotoxicity symptoms seen with chronic use clinically in humans. Vigabatrin dose escalation studies were conducted 20-kg swine exposed to HBO at 132 or 165 fsw. The saline group had seizure latencies of 7 and 11 min at 165 and 132 fsw, respectively. Vigabatrin at 180 mg/kg significantly increased latency (13 and 27 min at 165 and 132 fsw, respectively); 250 mg/kg abolished seizure activity at all depths. Functional electroretinogram and histology of the retinas showed no signs of retinal toxicity in any of the vigabatrin=treated animals. In the 250 mg/kg group there was no evidence of CNS oxygen toxicity; however, pulmonary oxygen toxicity limited HBO exposure. Together, the findings from this study show that vigabatrin therapy is efficacious at preventing CNS oxygen toxicity in swine, and a single dose is not acutely associated with retinotoxicity.

Cadmium (Cd), an ubiquitous environmental metal, mainly used for industrial purposes, may be toxic at level of the reproductive system. Testis tubular-based Sertoli cells (SC), play a major role in constituting the blood-testis barrier and provide a unique microenvironment for the genesis and differentiation of germ cells. Hence SC strictly control sperm qualitative and quantitative parameters. We aimed to assess whether exposure to Cd would adversely affect superior mammal SC viability and function. We isolated and purified SC from pre-pubertal pig testes according to our method and incubated the retrieved cells with three different Cadmium chloride concentrations (5-10-15 microM). Parameters of SC function such as inhibin B and anti-Mullerian hormone (AMH) were depressed by Cd exposure, contrary to what observed in untreated controls. No impairment of the FSH receptor integrity on the SC, as assessed by 17-beta-estradiol production, upon stimulation with FSH, was observed in either 5 microM Cd-treated or untreated controls. Differences, on the contrary, were observed for higher Cd concentrations (10 and 15 mM), in terms of FSH receptor integrity, that was altered, as compared to untreated controls, in terms of lower production of 17-beta-estradiol. In addition, the apoptotic test showed a significant increase of early (ANNEXIN V-/Propidium Iodide+) (AV-/PI+) and late apoptotic cells (AV+/ PI+) in all Cd -treated SC conditions as compared to controls. In conclusion, the Cd -related toxicity on SC, clearly demonstrated by our study, even at low concentrations, is expected to damage spermatogenesis that directly is dependent upon retention of SC viability and function.

Introduction: Military servicemembers in combat operations often sustain injuries to the extremities from highspeed projectiles, resulting in bleeding and comminuted open fractures. Severe injury with bone fragmentation can result in limb amputation. Surgical treatment options include materials that promote osteogenesis and bone proliferation, such as growth hormones, stem cells, or mineralized matrix adjuncts. However, none of these are amenable to use by the first responder, nor do they address the question of hemorrhage control, which is a common problem in traumatic injuries., Hypothesis: Our hypothesis was that treatment with a fibrinogen-based protein mixture at the time of the bone injury will provide both hemostasis and a supportive environment for preservation of injured bone., Methods: A comminuted femur fracture was produced in 28 female Yorkshire swine, and one of four treatments was instilled into the wound immediately after injury. Each animal was evaluated for the following parameters: inflammation, new bone growth, osteoclast proliferation, callus formation, and femur wound cavity fill, using post-mortem computed tomography and analysis of histological sections., Results: Overall, salmon fibrinogen?thrombin and porcine fibrinogen?thrombin showed a trend for improved healing based on bone filling and calcification. However, statistically significant differences could not be established between treatment groups., Conclusions: These findings indicate that a fibrinogen?thrombin matrix may be a useful as an immediate response product to enhance fracture healing. Salmon fibrinogen?thrombin has the advantages of cost and a pathogen profile compared to mammalian fibrinogens., (2013.)

Recombinant human IGF-1 currently represents the only available treatment option for the Laron Syndrome, a rare human disorder caused by defects in the gene encoding growth hormone receptor, resulting in irreversibly retarded growth. Unfortunately, this treatment therapy, poorly impacts longitudinal growth (13% in females and 19% in males), while burdening the patients with severe side effects, including hypoglycemia, in association with the unfair chore of taking multiple daily injections that cause local intense pain. In this study, we have demonstrated that a single intraperitoneal graft of microencapsulated pig Sertoli cells, producing pig insulin-like growth factor-1, successfully promoted significant proportional growth in the Laron mouse, a unique animal model of the human Laron Syndrome. These findings indicate a novel, simply, safe and successful method for the cell therapy-based cure of the Laron Syndrome, potentially applicable to humans., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Management of pain in research swine used for studies involving painful procedures is a considerable challenge. Here we assessed whether a regional anesthesia method is effective for pain control of hindlimb injuries in pigs used for research in bone fracture healing. For this randomized controlled study, we administered regional anesthesia before an experimental femoral injury was produced. Using ultrasound guidance, we placed sterile infusion catheters near the sciatic and femoral nerves and administered local anesthetic (bupivacaine) for the first 24 h after surgery. We evaluated various behavioral and physiologic parameters to test the hypothesis that this regional anesthesia would provide superior analgesia compared with systemic analgesia alone. We also collected blood samples to evaluate serum levels of cortisol and fentanyl postoperatively. At the end of the study period, we collected sciatic and femoral nerves and surrounding soft tissues for histopathologic evaluation. Treatment pigs had lower subjective pain scores than did control animals. Control pigs had a longer time to first feed consumption and required additional analgesia earlier in the postoperative period than did treatment pigs. Ultrasound-guided regional anesthesia is a viable and effective adjunct to systemic analgesics for providing pain control in swine with experimental femoral fractures.

Centrosome morphology and number are frequently deregulated in cancer cells. Here, to identify factors that are functionally relevant for centrosome abnormalities in cancer cells, we established a protein-interaction network around 23 centrosomal and cell-cycle regulatory proteins, selecting the interacting proteins that are deregulated in cancer for further studies. One of these components, LGALS3BP, is a centriole- and basal body-associated protein with a dual role, triggering centrosome hypertrophy when overexpressed and causing accumulation of centriolar substructures when downregulated. The cancer cell line SK-BR-3 that overexpresses LGALS3BP exhibits hypertrophic centrosomes, whereas in seminoma tissues with low expression of LGALS3BP, supernumerary centriole-like structures are present. Centrosome hypertrophy is reversed by depleting LGALS3BP in cells endogenously overexpressing this protein, supporting a direct role in centrosome aberration. We propose that LGALS3BP suppresses assembly of centriolar substructures, and when depleted, causes accumulation of centriolar complexes comprising CPAP, acetylated tubulin and centrin.

Crystal micro-morphology and dimension of silica particles could be responsible for the high prevalence of silicosis as recently found among goldsmiths. In the present study we investigated two samples of silica particles with different surface sizes and shapes for their capacity to induce changes in ECM component production. In addition we investigated if their different effects could be related to cytotoxicity and apoptotic effects. Human bronchial epithelial cells were cultured with or without a sample of Silica used for casting gold jewellery, named in our experiments Silica P or a commercial sample of Silica with different physical and chemical properties, named in our experiments Silica F. After 48 h of exposure PCR analysis determined levels of several matrix components. As induction of the apoptosis cascade, annexin assay, caspase 3 activity and cellular cytoxicity by MTT assay were assayed. Silica F promoted fibronectin, MMP12, tenascin C and Integrins b5 gene expressions more than Silica P. Silica P stimulated more TGFß1 and its TGFßR1 receptor than Silica F. Cytotoxic effects were induced by the two samples of Silica. On the contrary, no alteration in classic apoptotic marker protein expression was observed in presence of either Silica F or Silica P, suggesting silica particles affect ECM production and metalloproteases through a mechanism that does not involve apoptotic activation. Different Silica micromorphology and TGFß signal pathway are linked to lung fibrotic effects but the potential role Silica in apoptotic and toxic reaction remains to be ascertained.

Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkIIβ. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells.

Skin rejection remains a major hurdle in skin reconstructive transplantation surgery. In fact, 85% of the grafted patients experience at least one episode of acute skin rejection in the first year. It has been observed that Sertoli cells (SC), when co-transplanted with allo- or xenogeneic cell/tissues, can induce graft acceptance in the absence of systemic immunosuppression. A method aimed at significantly prolonging skin allografts in rats transplanted with barium alginate-based microencapsulated xenogeneic porcine SC (SC-MCs) is described. Results demonstrated that intraperitoneal (IP) transplantation of SC-MCs with high cellular viability and function can significantly prolong allogeneic skin grafts when compared to transplantation controls receiving only empty alginate capsules (E-MCs). Lymphocytic infiltration at the skin graft site was not observed in 80% of the SC-MCs transplanted rats and these recipient animals showed a significant increased expression of T regulatory (Tregs) cells when compared to E-MCs transplantation controls. The findings of this report further substantiate the positive therapeutic effects of SC on transplantation technology mediated by Sertoli cell-induced alterations of the host's immune system and indicate new perspectives and new strategies for successful skin tissue allografts., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objectives: This article examines the challenges for health technology assessment (HTA) in the light of new developments of personalized health care, focusing on European HTA perspectives., Methods: Using the example of the Integrated Genome Research Network - Mutanom (IG Mutanom) project, with focus on personalized cancer diagnostics and treatment, we assess the scope of current HTA and examine it prospectively in the context of the translation of basic and clinical research into public health genomics and personalized health care., Results: The approaches developed within the IG-Mutanom project are based on innovative technology potentially providing targeted therapies for cancer; making translation into clinical practice requires a novel course of action, however. New models of HTA are needed that can account for the unique types of evidence inherent to individualized targeted therapies. Using constructive health technology assessment (CTA) models is an option, but further suitable models should be developed., Conclusions: Integrative, systems biology-based approaches toward personalized medicine call for novel assessment methods. The translation of their highly innovative technologies into the practice of health care requires the development of new HTA concepts.

Importance of the Field: This review analyses international studies investigating the combined genetic and environmental causes of cleft lip with or without cleft palate (CL/P) and describes successes and limitations in identifying underlying genetic and environmental factors. CL/P, the most common congenital facial malformation, is a major public health burden in terms of medical costs and emotional stress to patients and families. Because genetic and environmental factors determine risk of occurrence, CL/P has a complex, multifactor aetiology., Areas Covered in This Review: English language reports from 1980 to 2010 were searched for in Medline, PubMed, Science Citation Index, textbooks and review articles on drugs and pregnancy. Key words were diazepam or benzodiazepine(s) combined with cleft lip, cleft palate, oral malformations, prenatal exposure, GABA, gene expression and extracellular matrix., What the Reader Will Gain: This review presents an updated assessment of the mutagenic and genotoxic effects of diazepam (DZ), one of the most commonly used benzodiazepines, on CL/P occurrence., Take Home Message: Data are divergent; more studies are needed for an in-depth picture of the effects of DZ during gestation on the child's development, particularly on orofacial clefts.

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well-established non-centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin- and RNA-binding proteins. In total, we assigned novel centrosome-related functions to 24 proteins and confirmed 13 of these in human cells.

Large-scale screens in mammalian cells demand for flexible high-throughput screening platforms that allow to analyze cellular traits on a genome-wide level or to identify small-molecule inhibitors (SMIs) from complex compound libraries. In this study we developed and tested high-density cell arrays made out of polydimethylsiloxane (PDMS) that support cell growth directly on standard glass microscope objective slides. We analyzed the effect of 3 reference inhibitors (MLN-8054, VX-680, and flavopiridol) and 4 exploratory, cell permeable small-molecule kinase inhibitors (two benzothiophene-based and two 4-amino-6-arylpyrimidine-based compounds) on different cell lines, using prototype 5 × 5 and 9 × 9 array carpets. We found that high-density PDMS cell arrays support growth of a broad range of cell types, are well suited for compound screens, and are compatible with high-content screening platforms. This novel array format is of particular advantage for compound screening to identify SMIs, when a combination of flexibility with respect to culture volume, well density, and high-resolution imaging is required. In addition, we demonstrated the suitability of this format for reverse transfection and siRNA experiments.

Since occupational and environmental exposure to the heavy metal Cadmium (Cd) affects human health this study investigated the effects of exposure to a single, or multiple, sub-toxic Cd concentrations on sub-confluent and confluent human osteoblast growth and expression of specific bone differentiation markers. RT-PCR quantified gene expression of type I collagen, metalloprotease (MMP13), runt-related transcription factor-2 (RUNX2), osterix, osteocalcin, osteonectin, alkaline phosphatase, integrins and bone sialoprotein (BSP). Expression of fibroblast growth factors 1 and 2 (FGF1, FGF2), transforming growth factor-beta(3) (TGFbeta(3)) and bone morphogenetic protein-2 (BMP2) were also evaluated to determine whether Cd-related effects were mediated by an imbalance in expression. Depending on osteoblast concentration and maturation stages, Cd inhibited or stimulated cell growth, decreased type I collagen, increased MMP13, FGF1 and BMP2 gene expression and stimulated the mineralization process only in continuously exposed cultures. These results suggest that in vivo, acute or chronic exposure to sub-toxic Cd concentrations may affect bone formation differently and support the hypothesis that Cd-induced bone disorders may involve downstream changes in growth factor expression. The results are of interest in forensic and occupational medicine in establishing preventive measures to reduce professional exposure risks., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFbeta(3)) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences.

During human embryogenesis, differentiation of haematopoietic stem cells (HSCs) and their progeny is regulated spatially and temporally. In the adult, hemopoiesis is restricted to bone marrow (BM) which contains HSCs residing within the so-called 'niches'. These are microenvironments consisting of extracellular matrix (ECM) macromolecules (mainly glycosaminoglycans, proteoglycans, fibronectin and collagens) and stromal cells that act in concert to keep HSCs in quiescence or to promote their growth and differentiation, since BM stromal cells secrete specific growth factors acting on responsive stem cells. Haematopoietic precursors also secrete numerous regulatory molecules as fibroblast growth factors (FGF), interleukin 1 (IL1), and transforming growth factor-beta1 (TGFbeta1), regulating in an autocrine and/or paracrine manner the various stages of normal hematopoiesis. Although the majority of stem cells are quiescent and do nor respond to external signals, a few active stem cells responde to paracrine produced growth factors and differentiate into the more committed CD34+ haematopoietic stem cell or into a mesenchymal stem cell, which generate even more specified tissue. This review focuses on the role of both ECM molecules and growth factors that form a dynamic, interactive system crucial for lineage commitment and amplification. In this perspective, we recently described the pivotal role of ECM, FGF and TGFbeta on the GM-490 phenotype, which is a cell line established from the bone marrow of a patient with acute lymphoblastic leukemia. Our findings indicated the GM-490 cell line possess characteristics of both haematopoietic and mesenchymal precursors.

Objective: Although the primary purpose of the study reported here was to identify stroke risk factors among the residents of a village in eastern Hungary, the study also included a multi-faceted survey conducted in 1992-94 to ascertain the somatic, mental and socio-economic conditions of the residents. Here we report data from the survey on prevalence of mental disorders (a cross-sectional descriptive study)., Method: The screenings included the following tests administered to 535 subjects: anxiety, depression, dementia, neurosis were measured; recent medical records were compared to survey data for 330 of the same subjects., Findings: The summary of prevalence of mental disorders measured in this study was as follows: anxiety 34.7% (severe), dementia 44.68% (mild), depression 66% (mild), 15.94% (medium), 7.88% (severe), neurosis 66.73% (mild, medium, and severe). Medical records maintained by village physicians since 1960 differed from the results obtained in the present study. A treatment gap was observed between mental health treatment for neurosis, as indicated by medical records, and the diagnostic prevalence of neurosis as measured by the survey instruments: there were three times as many people diagnosed as neurotic in the survey as had been noted in village medical records. Additionally, the unique position of cerebrovascular alteration was established between the mental and somatic factors., Conclusion: The study demonstrates the successful simultaneous collection of a wide spectrum of data on somatic conditions, mental disorders, and socio-economic status of the subjects. The study showed that 1. patient-centered medical care can simultaneously address both somatic and mental factors; 2. it is possible to decrease the treatment gap in mental health; 3. there is value in systematic collection of data in order to optimize the planning of prevention, health care costs and decision making.

Unlabelled: A multi-faceted survey was conducted in 1992-94 to ascertain the somatic, mental and socio-economic conditions of the residents of a village in eastern Hungary. Here we report data on prevalence of somatic disorders from the survey., Objectives: a) To collect and compare prevalence of known cardiovascular disease, including stroke risk factors, in a specific population (a Hungarian village); b) to test a computer-based mass screening device ("Cerberus") designed to identify individuals in the test population at high risk for stroke; c) to compare results obtained with Cerberus with known stroke risk/cardiovascular disease factors and traditional medical records., Methods: A cross-sectional survey (546 subjects) was conducted in Csengersima in the early 1990s, using the Cerberus screening system, which includes: 1. a questionnaire addressing the risk factors for stroke/cardiovascular disease; 2. amplifiers to record the pulse waves of cerebral (rheoencephalography) and peripheral arteries, electrocardiogram and electroencephalogram. Additionally, subjects were measured for carotid stenosis by Doppler ultrasound and 12-lead electrocardiogram; they were also screened for blood cholesterol, glucose, and triglyceride levels., Findings: Prevalence of the following stroke risk factors was identified: overweight, 63.25%; sclerotic brain arteries by rheoencephalogram, 54.29%; heart disease, 37.92%; pathologic carotid flow, 34.24%; smoking, 30.55%; high blood cholesterol, 28.70%; hypertension, 27.83%; high triglyceride, 24.35%; abnormality of electrocardiogram, 20%; high glucose, 15.95%; symptoms of transient ischemic attack, 16.07%; alcohol abuse, 6.74%; and diabetes, 4.53%., Conclusion: The study demonstrates a possible model for primary cardiovascular disease/stroke prevention. The simple, noninvasive test uses the bioimpedance method of measurement. This method offers a standardizable, cost-effective, practical technique for mass screenings by identifying the population at high risk for cardiovascular disturbances, especially cerebrovascular disease. In this model, the rheoencephalogram can detect cerebrovascular arteriosclerosis in the susceptibility/presymptomatic phase, earlier than the Doppler ultrasound technique. The method also provides a model for storing analog physiological signals in a computer-based medical record and the first steps of turning it into an expert system also tested.

It is critical to elucidate the pathways that mediate spindle assembly and therefore ensure accurate chromosome segregation during cell division. Our studies of a unique allele of the budding yeast Ipl1/Aurora protein kinase revealed that it is required for centrosome-mediated spindle assembly in the absence of the BimC motor protein Cin8. In addition, we found that the Ase1 spindle midzone-associated protein is required for bipolar spindle assembly. The cin8 ipl1 and cin8 ase1 double mutant cells exhibit similar defects, and Ase1 overexpression completely restores spindle assembly in cin8 ipl1 strains. Consistent with the possibility that Ipl1 regulates Ase1, an ase1 mutant lacking the Ipl1 consensus phosphorylation sites cannot assemble spindles in the absence of Cin8. In addition, Ase1 phosphorylation and localization were altered in an ipl1 mutant. We therefore propose that Ipl1/Aurora and Ase1 constitute a previously unidentified spindle assembly pathway that becomes essential in the absence of Cin8.

A fusion protein based expression system was developed in the Gram-positive bacterium Bacillus subtilis to produce the soybean Bowman-Birk protease inhibitor (sBBI). The N-terminus of the mature sBBI was fused to the C-terminus of the 1st cellulose binding domain linker (CBD linker) of the BCE103 cellulase (from an alkalophilic Bacillus sp.). The strong aprE promoter was used to drive the transcription of the fusion gene and the AprE signal sequence was fused to the mature BCE103 cellulase for efficient secretion of the fusion protein into the culture medium. It was necessary to use a B. subtilis strain deficient in nine protease genes in order to reduce the proteolytic degradation of the fusion protein during growth. The fusion protein was produced in shake flasks at concentrations >1g/L. After growth, the sBBI was activated by treatment with 2-mercaptoethanol to allow the disulfide bonds to form correctly. An economical and scalable purification process was developed to purify sBBI based on acid precipitation of the fusion protein followed by acid/heat cleavage of the fusion protein at labile Asp-Pro bonds in the CBD linker. If necessary, non-native amino acids at the N- and C-termini were trimmed off using glutamyl endopeptidase I. After purification, an average of 72 mg of active sBBI were obtained from 1L of culture broth representing an overall yield of 21% based on the amount of sBBI activated before purification.

Extracellular matrix (ECM) molecules and growth factors, such as fibroblast growth factor (FGF), play a crucial role in Alzheimer's disease (AD). The purpose of this investigation was to determine whether phenotypic alterations in ECM production are present in non-neuronal AD cells associated with different FGF expression and response. Synthesis of glycosaminoglycans (GAG) and collagen were measured in skin fibroblasts from patients with familial, sporadic AD (FAD and SAD respectively), and from age-matched controls by radiolabeled precursors. Proteoglycans (PG), metalloprotease (MMP)-1, and FGF gene expressions were measured by reverse transcription-polymerase chain reaction. The results showed different ECM neosynthesis and mRNA levels in the two AD fibroblast populations. FAD accumulated more collagen and secreted less GAG than SAD. Biglycan PG was upregulated in FAD while betaglycan, syndecan, and decorin were markedly downregulated in SAD fibroblasts. We found a significant decrease of MMP1, more marked in FAD than in SAD fibroblasts. Constitutive FGF expression was greatly reduced in both pathological conditions (SAD>FAD). Moreover, an inverse high affinity/low affinity FGF receptor ratio between SAD and FAD fibroblasts was observed. FGF treatment differently modulated ECM molecule production and gene expression in the two cell populations. These observations in association with the changes in FGF gene expression and in the FGF receptor number, suggest that cellular mechanisms downstream from FGF receptor binding are involved in the two different forms of AD.

Aim: A growing number of mutations mapped in the receptor gene for fibroblast growth factor have been implicated in several cranial development disorders including the Apert and Crouzon syndromes. The present paper investigated cellular mechanisms underlying Apert phenotype, by analyzing the effects of FGF2 in primary cultures of Apert periosteal fibroblasts carrying the FGFR2 Pro253Arg mutation., Results: FGF2 administration significantly decreased extracellular matrix production in mutant cells by stimulating degradative enzymatic activities. Gene expression analysis revealed that decorin and biglycan, two proteoglycans involved in collagen fibrillogenesis, were more expressed in mutant cells and down-regulated by FGF2. FGF2 receptor binding showed little differences in high affinity receptor counts between mutant and wild-type cells, while we showed for the first time that low affinity receptors are significantly fewer in mutant cells. Differences were found in Crouzon syndrome, where both high and low affinity receptor counts were up-regulated., Conclusions: The different mutation and low affinity receptor regulation in mutant receptors support the hypothesis that the impact on the activity of the ligand-receptor complex could allow distinct modes of FGF2 activation in Apert and Crouzon syndromes, which interfere with the FGFR2 signalling cascade.

The small dimension and particle shape of silica in gypsum used to prepare moulds for lost wax casting might be responsible for the high prevalence of silicosis in gold jewellery. To test this hypothesis, human pulmonary epithelial cell (BEAS-2B) cultures were exposed to two samples of silica with different crystal micro-morphologies: Silica Powder (Silica P) which is used in casting gold jewellery, and no powder Silica (Silica F). Extracellular matrix (ECM) production was evaluated using radio-labelled precursors and quantified by RT-PCR analysis. Expression of basic fibroblast growth factor (FGF2) and its receptor (FGFR2) was also evaluated. The results demonstrated Silica P particles had a very fine lamellar crystalline structure while Silica F was characterized by larger rounded crystals. Silica P stimulated collagen production significantly more than Silica F and downregulated laminin and metalloprotease expression. Both silica samples down-regulated FGF2 but only Silica F enhanced FGF2 receptor expression. In conclusion each Silica sample promoted a profibrotic lung microenvironment in a different manner and also elicited different FGF2 signalling pathways. The data confirm that different micromorphology of Silica particles affects the fibrogenic potential and the molecular mechanisms of dust pathogenicity.

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.

The spindle assembly checkpoint guards the fidelity of chromosome segregation. It requires the close cooperation of cell cycle regulatory proteins and cytoskeletal elements to sense spindle integrity. The role of the centrosome, the organizing center of the microtubule cytoskeleton, in the spindle checkpoint is unclear. We found that the molecular requirements for a functional spindle checkpoint included components of the large gamma-tubulin ring complex (gamma-TuRC). However, their localization at the centrosome and centrosome integrity were not essential for this function. Thus, the spindle checkpoint can be activated at the level of microtubule nucleation.

During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-beta (TGF-beta), retinoic acid (RA), and gamma-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-beta binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA--which, at pharmacologic doses, induces cleft palate in newborns of many species--were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-beta3 mRNA expression and TGF-beta receptor number were higher and RA receptor-alpha (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-beta3 mRNA expression but reduced the number of TGF-beta receptors. TGF-beta receptor type I mRNA expression was decreased, TGF-beta receptor type II was increased, and TGF-beta receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-beta signaling systems could be involved in human cleft palate fibroblast phenotype.

Immunological features of GM-490 cells, a new blood cell line from a patient with acute lymphoblastic leukemia, included lack of CD34, CD38, CD45, CD14, HLA-DR, and lymphoid and myeloid markers and expression of CD29, CD36, CD44, CD54, CD71, CD105, and CD133. Molecular analysis indicated CD45 gene expression was absent but CD34 mRNA was present. GM-490 cells constitutively produced fibronectin (FN), type III and traces of type I collagen, collagenases, glycosaminoglycans (GAG) and biglycan and betaglycan proteoglycans (PG) as well as FGF2 and TGFbeta1. When FGF2 and/or TGFbeta1 were added to cells in vitro, they stimulated cell proliferation and differently modulated matrix production and growth factor receptor expression. Reverse transcription-polymerase chain reaction (RT-PCR) detection of transcripts encoding for osteocalcin and RUNX2 suggests GM-490 cells differentiate towards the osteoblast pathway. GM-490 cells expressed the low affinity nerve growth factor receptor (p75LNGFR), a somatic stem cell marker that is not detected in hematopoietic cells, leading to the hypothesis that GM-490 has mesenchymal stem cell properties. The reciprocal modulating effects of FGF2 and TGFbeta1 on each other's receptors make the GM-490 cell line a new model for investigating the relationship between these growth factors and their receptors in autocrine loops which are believed to sustain the malignant clone in hematological diseases., (Copyright 2005 Wiley-Liss, Inc.)

Classical protocols for the isolation of centrosomes from higher eukaryotic cells are based on enrichment of cell organelles by density gradient centrifugation. Various successful protocols have been described that isolate centrosomes from mammalian tissue culture cells, tissue, clam oocytes, Drosophila, and yeast, to mention only some of the more frequently used sources. The material produced is subsequently used in various assays. These include functional tests such as the microtubule nucleation assay, electron microscopic study of centrosome morphology, and antigen localization; the organelles may also be used for the generation of antibodies. Furthermore, centrosomal preparations have been used for the characterization of their protein composition. The method described here focuses on the isolation of centrosomes from the syncytial stages of the early Drosophila embryo. This is a particularly attractive system because these organelles are not bounded by cellular membranes. Moreover, the abundance of pericentriolar material of these centrosomes produces excellent total protein yields.

Background: Recently, a wide variety of bandages have been formulated to attempt to improve the effectiveness of emergency intervention in situations of uncontrolled bleeding. The best of these dressings contain a mixture of human thrombin and fibrinogen. The presence of human components in these bandages, although effective, increases the cost of the dressing and raises questions of availability of raw materials and transmission of pathogens. The purpose of this study was to investigate the efficacy of dressings composed of salmon thrombin and fibrinogen in a swine aortotomy model., Methods: A 4.4-mm aortotomy was produced in the abdominal aorta of 19 anesthetized, splenectomized swine. The United States Army standard field gauze was applied to 8 animals, and the salmon thrombin-fibrin dressing (SFD) was applied to 11 animals. Survival, blood loss, and other parameters were measured over a 60-minute period., Results: All 11 animals that received the SFD survived the aortotomy injury, and bleeding stopped within 7.5 +/- 1.5 min. Seven of 8 animals in the control group were killed when bleeding continued and blood pressures decreased to the cutoff values as outlined in the animal protocol. Bleeding was significantly less in the SFD group compared with the gauze group (241 +/- 65.3 vs. 932.7 +/- 142.4 mL)., Conclusion: Fibrin dressing using salmon-derived thrombin and fibrinogen is effective in controlling severe, uncontrolled bleeding. This dressing may offer an alternative to dressings composed of human coagulation proteins.

Apert and Crouzon syndromes are well known craniostenosis. In the last 10 years several studies were performed to provide a better understanding of the etiology and pathogenesis of these diseases. Both have an autosomal dominant mode of transmission, and a mutation in the gene encoding for the fibroblast growth factor receptor 2 (FGFR2) is the cause in most patients. However, the fact that the same mutation can produce a wide range of phenotypic expression makes the mechanism of anomalous development more complex. The extracellular matrix (ECM) is composed of proteins, glycosaminoglycans, and cytokines that are secreted in an autocrine and paracrine manner and are able to modify the ECM. Fibroblast growth factors are complexed with heparan sulfate, a component of the ECM, before binding the FGFR2. Data exist about different expressions of cytokines and ECM macromolecule in craniostenosis-derived fibroblasts and osteoblasts. Changes in ECM composition could explain the altered osteogenic process and account for pathologic variations in cranial development in addition to the FGFR2 mutations.

Unregulated fibroblast growth factor 2 (FGF2) signaling caused by mutations in the fibroblast growth factor receptor (FGFR2) leads to human craniosynostosis such as the Apert syndrome. In an in vitro control model of calvarial osteoblasts from Apert patients carrying the FGFR2 P253R mutation, we studied the changes in cellular phenotype and evaluated the effects of FGF2. Compared with wild-type controls, osteocalcin mRNA was down-regulated in Apert osteoblasts, Runt-related transcription factor-2 (RUNX2) mRNA was differentially spliced, and FGF2 secretion was greater. Total protein synthesis, fibronectin and type I collagen secretion were up-regulated, while protease and glycosidase activities and matrix metalloproteinase-13 (MMP-13) transcription were decreased, suggesting an altered ECM turnover. Adding FGF2 increased protease and glycosidase activities and down-regulated fibronectin and type I collagen secretion in Apert osteoblasts. High affinity FGF2 receptors were up-regulated in Apert osteoblasts and analysis of signal transduction showed elevated levels of Grb2 tyrosine phosphorylation and the Grb2-p85 beta association, which FGF2 stimulation strongly reduced. All together these findings suggest increased constitutive receptor activity in Apert mutant osteoblasts and an autocrine loop involving the FGF2 pathway in modulation of Apert osteoblast behavior., (2004 Wiley-Liss, Inc.)

Chromosome alignment on the mitotic spindle is monitored by the spindle checkpoint. We identify Sgo1, a protein involved in meiotic chromosome cohesion, as a spindle checkpoint component. Budding yeast cells with mutations in SGO1 respond normally to microtubule depolymerization but not to lack of tension at the kinetochore, and they have difficulty attaching sister chromatids to opposite poles of the spindle. Sgo1 is thus required for sensing tension between sister chromatids during mitosis, and its degradation when they separate may prevent cell cycle arrest and chromosome loss in anaphase, a time when sister chromatids are no longer under tension.

Intravenous administration of liposomes, including Doxil, can cause severe life-threatening hemodynamic changes in pigs. The reaction is due to complement activation, and it is characterized by massive pulmonary hypertension, systemic hypotension, and severe cardiac abnormalities including falling cardiac output, tachy-or bradycardia with arrhythmia. There were no data suggesting the involvement of cerebrovascular changes in this reaction; however, clinical observations allowed this hypothesis. Here we measured the accompanying changes during liposome infusion by monitoring pulsatile electrical impedance (rheoencephalogram- REG) on the skull (n=24 pigs, 57 trials, 19 types of liposomes). A transient but significant decrease of REG pulse amplitudes followed the injection of liposomes (78.43% in the total sample, and 91.66% in the Doxil subgroup; P=0.003, n=12), indicating the involvement of cerebrovascular reaction during liposome infusion.

To establish the role of posttranslational modification in modulating the immune response to collagen, recombinant human type II collagen (rCII) was produced using a yeast expression system (rCII(pic)) and a baculovirus expression system (rCII(bac)). The biosynthesis of CII requires extensive posttranslational modification including the hydroxylation of prolyl and lysyl residues and glycosylation of selected hydroxylysyl residues. Amino acid analyses indicated that the rCII(bac) was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue-derived CII, whereas rCII(pic) was adequately hydroxylated at prolyl residues but unhydroxylated at lysyl residues and had no glycosylation. When DBA/1 mice were immunized with rCII, rCII(pic) induced a lower incidence of arthritis than tissue-derived CII, whereas rCII(bac) induced an intermediate level of arthritis. The severity of the arthritis was significantly lower in mice immunized with rCII(pic) compared with mice immunized with tissue-derived CII, whereas that of rCII(bac) was intermediate. These data indicate that the degree of lysine hydroxylation and glycosylation plays a role in the induction of arthritis. The recombinant collagens were then compared with tissue-derived CII when given as i.v. or oral tolerogens to suppress arthritis. Both recombinant collagens were less potent than tissue-derived CII, and this decrease in arthritis was associated with a decrease in Ab response to CII. These data suggest that the degree of glysosylation affects the immune response to CII, so that underglycosylated CII is less effective in the induction of arthritis and in its ability to suppress collagen-induced arthritis.

The tools of recombinant protein expression are now being used to provide recombinant sources of both collagen and gelatin. The primary focus of this review is to discuss alternatives to bovine collagen for biomedical applications. Several recombinant systems have been developed for production of human sequence collagens. Mammalian and insect cells were initially used, but were thought to be too costly for commercial production. Yeast have been engineered to express high levels of type I homotrimer and heterotrimer and type II and type III collagen. Co-expression of collagen genes and cDNAs encoding the subunits of prolyl hydroxylase has lead to the synthesis of completely hydroxylated, thermostable collagens. Human types I and III collagen homotrimers have been expressed in transgenic tobacco plants, while transgenic mice have been engineered to produce full-length type I procollagen homotrimer as well as a alpha2 (I) homotrimeric mini-collagen. Most recently, a transgenic silkworm system was used to produce a fusion protein containing a collagenous sequence. Each of these transgenic systems holds great promise for the cost-effective large-scale production of recombinant human collagens. As seen in other recombinant expression systems, transgenic silkworms, tobacco, and mice lack sufficient endogenous prolyl hydroxylase activity to produce fully hydroxylated collagen. In mice and tobacco, this was overcome by over-expression of prolyl hydroxylase, analogous to what has been done in yeast and insect cell culture. In addition to recombinant alternatives to bovine collagen, other sources such as fish and sponge collagen are discussed briefly. Recombinant gelatin has been expressed in Pichia pastoris and Hansenula polymorpha in both non-hydroxylated and hydroxylated forms. Pichia was shown to be a highly productive system for gelatin production. The recombinant gelatins produced in yeast are of defined molecular weight and physio-chemical properties and represent a new biomaterial not previously available from animal sources. Genetic engineering has made great progress in the areas of recombinant collagen and gelatin expression, and there are now several alternatives to bovine material that offer an enhanced safety profile, greater reproducibility and quality, and the ability of these materials to be tailored to enhance product performance.