The aim of this study was to provide a systematic review of studies that investigated the effectiveness of robot-assisted therapy on ankle motor and function recovery from musculoskeletal or neurologic ankle injuries.Thirteen electronic databases of articles published from January, 1980 to June, 2012 were searched using keywords ‘ankle*’, ‘robot*’, ‘rehabilitat*’ or ‘treat*’ and a free search in Google Scholar based on effects of ankle rehabilitation robots was also conducted. References listed in relevant publications were further screened. Eventually, twenty-nine articles were selected for review and they focused on effects of robot-assisted ankle rehabilitation.Twenty-nine studies met the inclusion criteria and a total of 164 patients and 24 healthy subjects participated in these trials. Ankle performance and gait function were the main outcome measures used to assess the therapeutic effects of robot-assisted ankle rehabilitation. The protocols and therapy treatments were varied, which made comparison among different studies difficult or impossible. Few comparative trials were conducted among different devices or control strategies. Moreover, the majority of study designs met levels of evidence that were no higher than American Academy for Cerebral Palsy (CP) and Developmental Medicine (AACPDM) level IV. Only one study used a Randomized Control Trial (RCT) approach with the evidence level being II.All the selected studies showed improvements in terms of ankle performance or gait function after a period of robot-assisted ankle rehabilitation training. The most effective robot-assisted intervention cannot be determined due to the lack of universal evaluation criteria for various devices and control strategies. Future research into the effects of robot-assisted ankle rehabilitation should be carried out based on universal evaluation criteria, which could determine the most effective method of intervention. It is also essential to conduct trials to analyse the differences among different devices or control strategies. Tejima, 2000 The humaTraditionally ankle injuries are rehabilitated via physiotherapy and however evidence suggests that without sufficient rehabilitation: 44% of people will have future problems,7; ambulHowever, little is known about the effects of robot-assisted therapy on ankle recovery from disabilities and the intervention most effective for a specific case. The purpose of this systematic review was to provide a comprehensive investigation and examination of published evidence on the effectiveness of robot-assisted therapy used to help human ankles recover from musculoskeletal or neurologic injuries.The literature search was limited to English-language articles  published between January 1980 and June 2012 in the following electronic databases recommended by a librarian in Auckland University: PubMed, EMBASE , MEDLINE (OvidSP), CDSR (Cochrane database of systematic reviews), Web of Science, Scopus, Compendex, IEEE Xplore, ScienceDirect, Wiley Online Library, Digital Dissertations, Academic Search Premier, SpringerLink. The electronic search terms were “‘Ankle*’ AND ‘Robot*’ AND (‘rehabilitat*’ or ‘treat*’)”. A free search in Google Scholar was also conducted and valuable references listed in relevant publications were screened, which made our search as systematic and complete as possible.The two primary reviewers  were a PHD student with expertise in mechatronics system and a senior lecturer with expertise in assistive technologies of rehabilitation, respectively. The third reviewer (SX) was a professor with expertise in medical robotics, assistive and rehabilitation robots. Two of reviewers  hold doctorates in their respective fields. The initial search was conducted in thirteen electronic databases by one primary reviewer (MZ) and the total number of articles identified was 686. To start with, one primary reviewer (MZ) assessed all the titles for eligibility using the screening criteria described below. MZ independently assessed all abstracts after the first round screening. Abstracts considered as meeting the inclusion criteria by MZ were automatically included in the full review. Otherwise, they were excluded. Studies one primary reviewer (MZ) was not sure whether to include or not were discussed by two primary reviewers. Discrepancies between the two primary reviewers were resolved through participation from the third reviewer and discussion among these three reviewers. A search in Google Scholar was conducted by one primary reviewer (MZ) based on therapeutic effects of ankle rehabilitation robots and the references of the included papers were also screened by MZ for any additional studies. These studies newly selected were also reviewed.Twenty-nine studies were included in the final review. Data extraction was then undertaken. The review concentrated on evidence based therapeutic effects of robot-assisted ankle rehabilitation, design types, levels of evidence and quality levels of the articles. The schematic overview of selection process with search results is shown in FigureRobotics was defined as: “The application of electronic, computerized control systems to mechanical devices designed to perform human functions.” The AmAll trials assessing the clinical outcomes of robot-assisted ankle rehabilitation training were included. These included participants who sustained any grade of ankle disabilities caused by musculoskeletal or neurologic injuries. Both male and female participants from athletic and non-athletic populations were included to allow the generalisation of results to different populations. Papers involving platform based ankle rehabilitation robots or wearable ankle rehabilitation robots were included.However, studies were excluded if participants underwent ankle surgeries or wore ankle prostheses. Animal based trials assessing humans and healthy subjects based trials assessing patients were also excluded. Studies focusing on the whole lower limb but not related to ankle recovery were excluded as well. Only English articles published in peer-reviewed journals or published as conference papers or abstracts were included.The data extraction form used for this study was the critical review form for quantitative studies developed by the Occupational Therapy Evidence-Based Practice Group at McMaster University, Hamilton, Ontario, Canada,20 proviThe search results are summarized in FigureA total of 29 original papers, with data from 164 patients and 24 healthy participants used as control participants in different studies, met the inclusion criteria after the eventual discussion among three reviewers. 16 of these studies focused on evaluating the therapeutic effects of platform based ankle rehabilitation devices, as shown in Table Studies meeting the inclusion criteria were quantitative. Study designs included one RCT with only 18 participants, one single case design, nine before-after designs, six single case series and six case study designs. The remNone of the studies involved outcomes classified using the health dimensions of the World Health Organization’s International Classification of Functioning, Disability, and Health. Most stIt is difficult to know how functional improvements in laboratories correlate with activities of daily life. One study showed tSeveral studies have shown that gait performance is affected by ankle muscle strength . The main effect is likely due to the alleviation of end-users’ boredom.Rehabilitation through ankle stretching was conducted in five studies,75,78,86A novel therapeutic approach (Assisted movement with enhanced sensation (AMES)) was proposed in. The effWearable robots known as exoskeleton robots or as powered orthoses are being developed in contrast to platform based rehabilitation robots. In thisThree studies,66,85 prRen, 2011 proposedAn active ankle-foot orthosis (AAFO) was developed in where thTwo studies,89 assesSeveral categories of strategies have been reported, including, assistive, challenge-based, haptic simulation, and coaching for robotic movement training following neurologic injuries. There wNot much attention has been paid to the technologies of human robot symbiosis to date because almost all robots have been designed and constructed on the assumption that the robots are physically separated from humans. In partThere is no reason to believe that a “one-size-fits-all” optimal treatment exists. In otheThe question of what is the most appropriate robot-assisted ankle rehabilitation is not evident. Hogan, 2006 demonstrAn attempt was made to ensure that all studies related with robot-assisted ankle rehabilitation with any grade of ankle disability were reviewed. In this review we assumed that all studies used different patients, but because some studies were conducted at the same place and at the same time, we cannot be certain whether only unrelated study populations were used. However, other research may exist in which robot or ankle was not identified as a key term within the article. For instance, some articles about ankle rehabilitation robots were probably described in terms of devices and lower extremity/lower limb. Only articles after 1980 were included in this study as robot-assisted ankle rehabilitation was very limited before then. We included published conference papers and abstracts as well as full peer-reviewed papers but did not include abstracts written in languages other than English or unpublished data. Some studies may therefore have been excluded on this basis, leading to potential incomplete search.Even though a range of robot-assisted ankle rehabilitation devices and control strategies are available for individuals with ankle disability, the most effective ankle rehabilitation device and control algorithm is still vague. This is due to a lack of universal evaluation criteria with effective outcome measures. Although using RCTs to assess effects of robots on ankle outcomes is expensive and time-consuming, so too are the robots designed to assist.In terms of control strategies, providing too much assistance has negative consequences and therFew studies have undergone rigorous experimental assessment with high-level evidence. Therefore, higher level trials like RCTs should be conducted aiming at assessing the therapeutic effects of robot-assisted ankle rehabilitation devices and control strategies. These trials should base on rigorous comparison with each other, and with simpler, non-robotic conventional therapy in terms of devices and control strategies. It is also necessary to set up universal evaluation criteria that should contain systematic and comprehensive outcomes to evaluate devices and control strategies, including the assessment of end-user comfort, safety and training performance. These evaluation criteria may improve the consistency of results and facilitate comparisons among ankle rehabilitation robots.RCT: Randomized Control Trial; ACC: Accident Compensation Corporation; ROM: Range Of Motion; AACPDM: American Academy for Cerebral Palsy and Developmental Medicine; VR: Virtual Reality; LE: Lower Rehabilitation; CP: Cerebral Palsy; PROM: Passive ROM; AROM: Active ROM; AMES: Assisted Movement with Enhanced Sensation; DOF: Degree Of Freedom; PPAFO: Portable Powered AFO; AFO: Ankle-Foot Orthosis; AAFO: Active AFO; IAFO: Intelligent AFO; SCI: Spinal Cord Injury; MR: Magneto-rheological; MVC: Maximum Voluntary Contraction; FIM: Functional Independence Measure; RMA: Rivermead Motor Assessment; MI: Motricity Index; SSRD: Single Subject Research Design; GRD: Group Research Design.The authors declare that they have no competing interests.MZ participated as a first reviewer in the design of the study and the data collection, data analysis and drafted the manuscript. TCD participated in the design of the review, and participated as a second reviewer in the study selection and quality assessment and helped in the data extraction and revising the manuscript. SX participated as a third reviewer to solve the discrepancies between MZ and TCD and to modify the manuscript. All authors read and approved the final manuscript.


Over half a century of converging clinical and animal research indicates that early life experiences induce enduring neuroplasticity of the HPA-axis and the developing brain. This experience-induced neuroplasticity is due to alterations in the frequency and intensity of stimulation of pups’ sensory systems  embedded in mother–infant interactions. This stimulation provides “hidden regulators” of pups’ behavioral, physiological, and neural responses that have both immediate and enduring consequences, including those involving the stress response. While variation in stimulation can produce individual differences and adaptive behaviors, pathological early life experiences can induce maladaptive behaviors, initiate a pathway to pathology, and increase risk for later-life psychopathologies, such as mood and affective disorders, suggesting that infant-attachment relationships program later-life neurobehavioral function. Recent evidence suggests that the effects of maternal presence or absence during this sensory stimulation provide a major modulatory role in neural and endocrine system responses, which have minimal impact on pups’ immediate neurobehavior but a robust impact on neurobehavioral development. This concept is reviewed here using two complementary rodent models of infant trauma within attachment: infant paired-odor-shock conditioning  and rearing with an abusive mother that converge in producing a similar behavioral phenotype in later-life including depressive-like behavior as well as disrupted HPA-axis and amygdala function. The importance of maternal social presence on pups’ immediate and enduring brain and behavior suggests unique processing of sensory stimuli in early life that could provide insight into the development of novel strategies for prevention and therapeutic interventions for trauma experienced with the abusive caregiver. Both animal and human research demonstrate that early life experiences interact with genetics to program the central nervous and endocrine systems, including the hypothalamus–pituitary–adrenal (HPA)-axis –5. InfanHere, we focus on infant experiences and the effects of early life stress and HPA-axis activation as experienced within the mother–infant dyad, as well as the pups’ attachment to the caregiver and learning about the caregiver. We review two complementary rodent models of infant trauma within attachment: infant paired-odor-shock conditioning and rearing with an abusive mother, which converge in producing a similar neurobehavioral phenotype in later-life consisting of depressive-like behavior as well as disrupted HPA-axis and amygdala function, thus enabling us to explore both the immediate and enduring effects of abusive attachment as well as role of the HPA-axis and the stress hormone corticosterone (CORT). Although infant trauma resulting from abusive attachment affects neural substrates of stress vulnerability and resilience, these can be engaged by sensory cues learned during infancy , which have the ability to normalize adult neurobehavioral dysregulation stemming from early life trauma.Attachment is a psychosocial process referring to the deep and enduring emotional bond that connects two individuals across space and time, with an individual deriving security from physical and psychological contact with the attachment figure –40. AttaThe mother–infant attachment bond is among the strongest social attachments formed by most mammals . As suchDespite the fact that childhood abuse remains a major public health concern –91, the In rats, infant-attachment occurs within a unique developmental context – the stress-hyporesponsive period (SHRP) – during which neonates show low basal plasma concentrations of CORT and reduced stress-reactivity, as indexed by limited adrenocorticotropic hormone (ACTH) and CORT responses to stressful stimuli compared to older animals, as well as low levels of corticosteroid binding globulin (CBG), which regulates glucocorticoid (GC) access into the brain , 116–123However, the SHRP during development appears to be stressor specific, since the HPA-axis is fully capable of responding to stimuli that may be considered stressful to a neonatal rat such as cold or saline injection –145. IndInfants possess a predisposition to approach the mother as well as specific sensory cues associated with her care, such as her odor and vocalizations , 162. WiIntriguingly, the sensitive-period for attachment learning in rat pups overlaps with the SHRP, suggesting that low levels of CORT and reduced HPA-axis responsiveness may contribute to the neonate’s unique neural circuitry for attachment learning. However, in order for infant-attachment to occur, the rat pup must first learn to identify the caregiver and exhibit the social behaviors necessary for survival such as orienting to and approaching the caregiver, grasping the nipple and nursing , 169. InOur lab uses infant olfactory classical conditioning in which an artificial odor  is paired with a 0.5 mA shock as a rodent model of abusive attachment. While the adult rat responds to shock with a robust CORT response, the neonatal rat does not , 189. UnNeonatal odor learning produces changes in the OB, which can be induced both naturally in the nest and experimentally in controlled learning experiments outside the nest , 204–210Infant rats (PN1–9) readily learn an odor preference to neutral odors paired with pleasant  –185, 219Experimental evidence indicates a lack of amygdala participation in the neural circuitry underlying infant paired-odor-shock conditioning during the sensitive-period, as suggested by amygdala lesions, 2-DG, and c-Fos-ir , 232, alIndeed, the natural increase of stress-induced CORT release marks the end of sensitive-period learning , 238, whIn summary, the infant learning circuit is characterized by an enhanced ability to learn odor preferences to aversive stimuli, due to a hyper-functioning LC, as well as a decreased ability to learn odor aversions that may interfere with proximity-seeking during the sensitive-period due to a hypo-functional amygdala, suggesting that the infant brain is specialized for maximizing attachment to a caregiver Figure  41, 165, 244–247Empirical evidence suggests that social support is a powerful modulator of individual differences in response to potentially stressful events in both humans and animals –253. In Yet, human parental care is disturbed under conditions of chronic stress , which cFurthermore, because maternal stimulation of pups modulates pups’ endogenous CORT, maternal care quality alters sensitive-period duration. Pups reared with a stressed mother  exhibit a precocious emergence of CORT, which is delivered through the mother’s milk , that faResponses to stressors, or conditions that threaten or are perceived to threaten physiological equilibrium, are mediated by the activation of stress-responsive neurobiological systems that help preserve allostasis, or stability through change, thereby making the stress response an essential endocrine mechanism for survival –270. StrThe effects of HPA-axis activation depend on multiple factors, including the developmental stage in which the insult occurs, number of exposures, and type of adversity , 293–297Early life adversity may lead to a maladaptive outcome to a given later environmental context. Depression is a common outcome of childhood abuse, and children with a comorbid history of depression and abuse have elevated CRF levels in the cerebrospinal fluid  as well Recently, our lab has demonstrated that both rodent models of abusive attachment  during infancy result in later-life depressive-like behavior in the Forced Swim Test (FST), a measure of behavioral despair in rodents , 329, thOur findings are in accordance with clinical and animal literature indicating that early life adversity constitutes a prime risk factor for the development of psychopathologies characterized by dysregulated HPA-axis function , 331–334An ample body of evidence suggests that the quality of infant-attachment relationships results in long-term adaptations that have the ability to program subsequent behavioral, endocrine, and neural function , 336. ReIn species requiring parental care, evolution has ensured that infants quickly learn and express robust preferences to the caregiver, regardless of the quality of care , 50. HowThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During the past several years, there has been intense research in the field of bone marrow-derived stem cell (BMSC) therapy to facilitate its translation into clinical setting. Although a lot has been accomplished, plenty of challenges lie ahead. Furthermore, there is a growing body of evidence showing that administration of BMSC-derived conditioned media (BMSC-CM) can recapitulate the beneficial effects observed after stem cell therapy. BMSCs produce a wide range of cytokines and chemokines that have, until now, shown extensive therapeutic potential. These paracrine mechanisms could be as diverse as stimulating receptor-mediated survival pathways, inducing stem cell homing and differentiation or regulating the anti-inflammatory effects in wounded areas. The current review reflects the rapid shift of interest from BMSC to BMSC-CM to alleviate many logistical and technical issues regarding cell therapy and evaluates its future potential as an effective regenerative therapy. The objective of stem cell regenerative therapy is to treat damaged organ tissues by avoiding the processes of cell death and/or inadvertent remodeled Tissue . Great oin vivo transplanted cells are exposed to local immune cells and soluble mediators, which influence the cells behavior in an unpredictable manner in the microenvironment. Thus, it is necessary to further understand the potential benefits of maximizing the paracrine effects for regenerative therapy. This review will take an in-depth look at specific mechanisms regulated by these factors and potential therapeutic applications of BMSC-CM and paracrine factors secreted by BMSCs. Indeed, understanding the therapeutic effects of regenerative therapy using BMSCs becomes more relevant when we look at the paracrine factors, which are secreted by BMSCs. For example, the frequency of stem cell engraftment and the number of newly generated cardiomyocytes or vascular cells are too insignificant to represent the remarkable cardiac functional improvement attributed to fusion or differentiation alone . In addiin vitro under specific conditions and in vivo after injection into the myocardium [BMSCs include many populations of progenitor cells: hematopoietic stem cells (HSC), mesenchymal stem cells or stromal cells (MSC), side population cells, and multipotent adult progenitor cells . BMSCs cocardium –27. ex vivo genetic modification, and multipotency [in vitro and in vivo  [in vivo and even express functional adrenergic and muscarinic receptors [in vitro proliferation and migration of endothelial cells and vascular smooth muscle cells, and enhances blood flow recovery of ischemic hindlimb [MSCs are particularly suitable for cell therapy because of easy isolation, high expansion potential giving unlimited pool of transplantable cells, low immunogenicity, amenability to ipotency , 28, 29.ipotency . Furtherin vivo  –36. Theyin vivo  –44. Moreeceptors , 46. Morhindlimb , 43, 44.hindlimb . Noiseuxhindlimb –56. The hindlimb . in vivo with little risk of malignant transformations [in vivo. However, the same group discovered that use of cytokines derived from the BMSCs did not lead to these pathological malformations. Therefore, more data describing the long-term effects of administered MSCs and their factors are needed to be established. In addition, since BMSCs can be obtained directly from adult animal models, there are no pressing ethical issues limiting their usage in clinical trials. Although the mechanisms and the therapeutic benefits have not yet been fully elucidated, general consensus states that adult stem cells, particularly BMSCs, are very safe. This is based on their usage in human studies, which have shown that they can be safely cultured rmations . Pitfallrmations . Moreovermations . Anotherrmations . This laAlthough there are many benefits attributed to MSCs, it is important to know that the unique properties of MSCs do not guarantee that they will accomplish desired therapeutic outcomes or that they will evade rejection. This is the reason for the various conflicting results with regards to the mechanisms and outcomes of MSCs found in the literature. Hence, current research is highly focused on developing an efficient delivery system that can successfully transplant a sufficient number of stem cells at the desired site with higher retention and minimal biological loss or immune response. Some of our more recent work has shown that retention can be increased by four folds in the heart by using biocompatible polymeric alginate microcapsules as a delivery system, which can, in turn, help in functional recovery of acutely infarcted heart –61.Since heart disease is reaching pandemic proportions in developed and developing countries and the heart is an organ of limited regenerative capacity, much research using stem cell therapy is focused on the regeneration of the myocardium. Therefore, studies focusing on stem cell differentiation and cell fusion are often looking at the heart. In earlier studies concerning cardiac regeneration, successful transplantation of neonatal and fetal cardiomyocytes into rats with acute myocardial infarction was observed . The resOric et al. demonstrated that bone marrow could be a source of extracardiac source of progenitor cells with the ability to differentiate into cardiomyocytes and restore cardiac function , 68, 69.in vitro experiment suggesting the presence of cellular nanotubes which increased the viability of ischemic cardiomyoblasts in the presence of normal MSCs [Cell Fusion of transplanted stem cells with resident cardiomyocytes has been proven as a feasible mechanism for differentiation. Fusion of bone marrow-derived cells with purkinje neurons, hepatocytes and cardiomyocytes was reported for the first time by Alvarez-Dolado et al. . During mal MSCs . It reve+ cells were detected within the infarct border zone, in areas with viable cardiomyocytes. Although cell fusion was shown to occur by the appearance of blue cells (fusion of MSC with resident cardiomyocytes) and by the improved cardiac function, they concluded that fusion was not responsible because it happened so infrequently. Therefore, they attributed enhanced cardiac function to paracrine effects [In a separate experiment, Noiseux et al. demonstrated, using a model of acute MI in transgenic mice, that transplanted MSCs could fuse with recipient cardiomyocytes in the infarcted area . MSCs fr effects .in vivo, making it very difficult to distinguish them from background tissue leading to misinterpretation [n + 2n), or alternatively nuclear fusion will result in cells with larger nuclei 4n chromosomes going up to hexaploid (6n) DNA content. This leads to hybrid genotypes with cells failing to express proper green fluorescent phenotypes adding to the confusion of tracking differentiated cells [Despite hopeful results with experiments looking at fusion and differentiation, the mechanisms by which BMSCs work to help damaged tissue remains unclear even in seemingly successful studies. Furthermore, inconsistent results when researching stem cell regenerative capacity have been linked to improper labeling of donor cells and inability to consistently track them retation . For exaretation . Evidencretation –86. In aed cells .Since research has shown that stem cell differentiation and fusion alone cannot account for enhanced cardiac function, evidence for a nonmyogenic pathway of cardiac repair is becoming increasingly explored. The debate surrounding fusion and differentiation is of little importance now because the number of reported cardiomyocytes derived from exogenous stem cells is too low to account for the impressive enhancement of physiological function , 88. ThuMSCs such as BMSCs are known to secrete many biological factors, and under strenuous hypoxic and serum starvation, the expression of several of these factors is upregulated. This has prompted the isolation of MSC conditioned medium (MSC-CM) to be concentrated and used therapeutically. Kinnaird et al. reported the presence of cytokines as: VEGF, FGF-2, IL-6, PIGF, and MCP-1 and that intramyocardial injection of MSC-CM improved collateral blood flow recovery and limb function and reduced muscle atrophy. Because all these cytokines are known to promote angiogenesis and vascularization, the conclusion was that it was not direct cell incorporation but rather paracrine signaling that may be responsible for the effects of bone marrow cell therapy in ischemic injury.in vitro, suggesting the presence of antiapoptotic and inotropic effects [It was previously reported that stem cell transplantation into the ischemic myocardium improved cardiac function recovery and repair as early as 72 hours later, which is too early to be explained solely by MSC differentiation , 56, 79. effects . When in effects . Figure  effects .Blood vessel formation after birth proceeds primarily through 2 mechanisms: angiogenesis and arteriogenesis. Angiogenesis consists of several distinct processes which include sprouting and proliferation of pre-existing capillaries to form new networks. This process is tightly regulated by hypoxia through a number of proangiogenic factors, including VEGF, FGFs, P1GF, and hepatocyte growth factor (HGF) –94. The Under normal circumstances, myocardial tissue dies and forms scar tissue because the capillary network does not meet the demands of the hypertrophied cells. The inevitable lack of oxygen and nutrients leads to apoptosis and necrosis. Moreover, inducing angiogenesis requires the coordinated interactions between monocytes/macrophages, endothelial cells, smooth muscle cells, and pericytes , 94–99. in vitro proliferation and migration of endothelial cells and vascular smooth muscle cells and enhances in vivo collateral blood flow recovery when injected into ischemic hindlimb. Another possibility for improved function is that neovascularization by the stem cells is leading to enhanced blood supply in the peri-infarct region, thereby promoting salvage of stunned, hibernating, or otherwise susceptible cardiomyocytes [MSCs express genes encoding a broad spectrum of cytokines with angiogenic properties of some of which may be referred to in myocytes .−c-kit+) [Homing signals are extremely important for the efficacy of cell therapy because it is via these paracrine effects that the precise localization of transplanted cells is possible and can be improved. In the case of cardiac repair, stem cell therapy can be administered via intravenous, intracoronary, transmyocardial, catheter-based transendocardial with or without electromechanical voltage mapping, and finally transvenous injection into coronary veins –104. The−c-kit+) . A signiOf all techniques, the intracoronary infusion has been deemed best for treatment of acute myocardial infarction. However, strategies to augment cell function via better localization/homing is crucial if there is to be any therapeutic benefit using BMSCs for targeted regenerative therapy. Additional strategies for better homing of mesenchymal cells include inducing hypoxia for the upregulation of CXCR-4 protein receptor expression at the BMSC cell surface. CXCR-4 are chemokine protein receptors expressed in dying cells, which can be complexed with SDF-1 protein ligands , 108. Th−c-kit+) was able to give rise to new myocardium and improve left ventricle end diastolic pressure by 30% to 40% in mouse hearts with infarcts located in the free wall of the left ventricle [Orlic et al. reported that injection of BMCs . Their preliminary results suggested that much more efforts should go into further researching the benefits of conditioned media to better understand the role of individual factors that may optimize their effects. Interest in this topic started when researchers reported functional improvement of tissue before the possibility of attributing it to donor cells. Gnecchi and colleagues found significant functional improvement in the myocardium occurring in less than 72 hours after intramyocardial injection of Akt-modified MSCs in a rat model . The samβ and TNF-α factors during these optimal times suggests that they all take part in hypertrophy and proliferation of the cardiomyocytes [Recent research has also shown that there is an optimal time for conditioned media delivery since the paracrine effects are known to be time dependent. It has been demonstrated that the prohypertrophic effects on myocytes and proliferation of fibroblasts in rat myocardium were significant using media collected after 6 and 9 hours of secretion. On the other hand, medium collected at 3 and 24 hours after secretion resulted in no noticeable prohypertrophic effects. Consequently, the presence of FGF-2, IL-1, IL-6, TGF-myocytes .Similarly to stem cell therapy in myocardial regeneration, studies in the kidney provided no evidence that marrow stromal cells were incorporating into the native renal parenchyma in significant numbers or directly contributed to tubular cell repopulation . Bi and Bone marrow stromal cells also showed great potential with regards to neurological regenerative therapy. BMSCs have shown to contribute to functional recovery and tissue regeneration in rat models with spinal cord injury, cerebral ischemia, traumatic brain injury, and constructing tissue-engineered nerve grafts –130. Resin vivo. More importantly, enrichment for STRO-1 increases the expression of cardiovascular-relevant cytokines and enhanced trophic activity. STRO-1-MPC, for example, it exhibited higher expression of CXCL12 and HGF, which are crucial in endothelial tube formation and cardiac cell proliferation. Therefore, immunoselection has great potential in the field of regenerative therapy.Optimizing and maximizing the amount of secreted stem cell paracrine factors is important to assure its efficient transition in therapeutic and clinical applications. Preparation and isolation of MSC has traditionally involved plastic adherence isolation. Another method for cell isolation is by immunoselection using mesenchymal precursor cells (MPC) at higher purity. MSC involving plastic adherence (PA) isolation has been shown to be less effective than MPCs immunoselected for stromal precursor antigen-1 (STRO-1) , 133. ThEnhancing effectiveness of paracrine activity has also been attempted using heparin-binding amphiphile nanoparticles (HBPA) . HypoxicSo far, bone marrow-derived stem cells and their paracrine factors have shown all the necessary attributes for tissue regeneration, namely, homing, immunosuppression, differentiation, angiogenesis, stimulation of endogenous cells, and possible regulation of specific metabolic pathways, only to name a few. Thus, research into paracrine factors and mechanisms has shown that stem cell therapy is much more complicated and greatly enhances the potential and variety of therapeutic applications. Mediating angiogenic factors, cell proliferation, and all of the above-mentioned characteristics are crucial to stem cell therapy and provides many new approaches for therapy. Therefore, injecting MSCs into tissue and observing the extent of differentiation are to be considered superfluous unless, of course, paracrine effects are being considered. Results from experiments using only conditioned medium showed signs of remarkable promise and revealed the same regenerative capacities as studies using cell injection. However the hallmarks of transdifferentiation and cell fusion are not to be disregarded because paracrine activities will most likely make these processes more efficient. Therefore, research into using conditioned media from stem cells is crucial because it will give us a greater understanding into the most relevant and important cytokines, their optimization, and their mechanisms of action to further maximize the application and uses of regenerative stem cell therapy.


Chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR) has been established as the current standard of care for young and fit patients with chronic lymphocytic leukemia (CLL). In the early nineties of the last century, long before the advent of fludarabine or antibody-based strategies, there was realistic hope that myeloablative therapy followed by autologous stem cell transplantation (autoSCT) might be an effective and potentially curative front-line treatment option for suitable patients with CLL. Since then, several prospective trials have disenthralled this hope: although autoSCT can prolong event and progression-free survival if used as part of early front-line treatment, it does not improve overall survival, while it is associated with an increased risk of late adverse events such as secondary malignancies. In addition, autoSCT lacks the potential to overcome the negative impact of biomarkers that confer resistance to chemotherapy or early relapse. The role of autoSCT has also been explored in the context of FCR, and it was demonstrated that its effect is inferior to the currently established optimal treatment regimen. In view of ongoing attempts to improve on FCR, promising clinical activity of new substances even in relapsed/ refractory CLL patients, exciting novel cell therapy approaches and advantages in the understanding of the disease and detection of Minimal Residual Disease (MRD), autoSCT has lost its place as a standard treatment option for CLL. Chronic lymphocytic leukemia (CLL) is one of the most common lymphoid malignancies,Long before the advent of FCR and other fludarabine or monoclonal antibody-based strategies, several studies suggested that autologous stem cell transplantation (autoSCT) might represent an effective and potentially curative treatment option for suitable patients with CLL.Subsequently, prospective randomized trials were opened for enrollment, and despite being initiated in the pre-Rituximab era, the results have been eagerly awaited for. The French intergroup trial randomized patients in complete remission (CR) after mini-CHOP-and fludarabine-based therapy to autoSCT or observation. Those patients who did not achieve CR, received cytarabine-based salvage therapy followed by autoSCT or FC.A direct prospective randomized comparison between Rituximab based therapy and auto-SCT has never been undertaken, and in the view of promising new agents and cellular therapy approaches, it can be assumed that this is very unlikely to happen in the future either. However, in a recent study within this setting, 110 patients from the GCLLSG autoSCT CLL3 trial and 126 patients from the FCR arm of the GCLLSG CLL8 trial were retrospectively compared.A recently published retrospective EBMT study suggested that autoSCT might play a role in the treatment of patients with chemo-sensitive Richter's syndrome.18In addition to the lack of full curative potential and convincing advantages over conventional chemoimmunotherapy, serious long-term effects such as solid tumours and secondary haematological malignancies need to be taken into consideration after autoSCT. The 2008 WHO-classification of haematopoietic and lymphoid tissues has implemented therapy-related myeloid neoplasms (t-MNs) as a separate category, which includes therapy-related acute myeloid leukaemia (t-AML), myelodysplastic syndrome (t-MDS) and myelodysplastic/ myeloproliferative neoplasms (t-MDS/MPN).What major conclusions can be drawn from almost two decades of clinical trials on autoSCT in CLL? Firstly, autoSCT indeed has the capacity to provide prolonged disease control at least similar to modern immunochemotherapy regimens, such as FCR. However, like immunochemotherapy, autoSCT has no significant curative potential in CLL. Secondly, autoSCT does not have the potential to overcome the negative impact of biomarkers that confer resistance to chemotherapy. Therefore, patients who have gained the highest benefit from autoSCT would also most likely respond to conventional immunochemotherapy. Thirdly, autoSCT is associated with an increased risk of secondary neoplasms, which is a very serious long-term adverse event with a poor outcome.Over the past few years, CLL research and treatment have made a huge leap forward: to name a few examples, randomized clinical trials aiming to improve the potential of FCR and to optimize treatment for high-risk and older patients or those with comorbidities are ongoing, and their recruitment has for the most part been exceeding expectations. There are also several exciting new small molecule inhibitors such as the BTK-inhibitor ibrutinib or the bcl-2 inhibitor navitoclax, which show promising activity even in the relapsed/ refractory setting, and in combination with conventional chemoimmunotherapy.41


We report a case of embryonal orbital rhabdomyosarcoma (RMS) in a five year old boy. Immuno-histochemistry of incisional specimen confirmed diagnosis. Eight cycles of chemotherapy along with radiotherapy resulted in over 50% reduction in size of the mass. However, increase in size was noted subsequent to completion of therapy and exenteration was deemed prudent. Margins of the excised specimen were free from tumor cells, but after five months, the patient developed multiple metastases, including skeletal muscle involvement, and died nine months after exenteration, despite repeat chemotherapy along with radiotherapy. Orbital RMS with metastasis to skeletal muscle is a rare entity. Marked improvement in RMS survival over the past seen with the use of recent therapeutic protocols including primary radiotherapy and chemotherapy.[Rhabdomyosarcoma (RMS) is the most common primary malignancy of the orbit in children, accounting for 4% of all malignant disease in children.2 Horn anotherapy.A five-year-old boy presented to us with rapid onset of downward eccentric protrusion of right eyeball over a period of one month.  Extra ocThe orbital RMS is usually fast growing an infiltrative and often appears as an enlarging, painless mass. The initial symptoms may mimic differential diagnosis which includes inflammetry process, orbital cellulitis, metastatic neuroblastoma, chloroma, lymphangioma and ruptured dermoid cyst.In orbital RMS metastases are commonly hematogenous in origin; spread most often occurs to the lungs, liver and bones. RegionalIntergroup Rhabdomyosarcoma Study Group (IRSG) whose protocols now from the backbone of modern treatment of rhabdomyosarcoma. The progRecently excision biopsy is preferred more than incisional biopsy as it confirms diagnosis as well as it removes the tumor too, which is significantly helpful for better outcome. ExenteraThough tumor-free margins are thought to be a key element in establishing surgical cure of a patient with any form of neoplasia, which was the case following exenteration in our patient, few published reports,10 highli


Research over the past few years has provided fascinating results indicating thatbiglycan, besides being a ubiquitous structural component of the extracellular matrix(ECM), may act as a signaling molecule. Proteolytically released from the ECM, biglycanacts as a danger signal signifying tissue stress or injury. As a ligand of innate immunityreceptors and activator of the inflammasome, biglycan stimulates multifunctionalproinflammatory signaling linking the innate to the adaptive immune response. Byclustering several types of receptors on the cell surface and orchestrating theirdownstream signaling events, biglycan is capable to autonomously trigger sterileinflammation and to potentiate the inflammatory response to microbial invasion. Besidesoperating in a broad biological context, biglycan also displays tissue-specific affinitiesto certain receptors and structural components, thereby playing a crucial role in boneformation, muscle integrity, and synapse stability at the neuromuscular junction. Thisreview attempts to provide a concise summary of recent data regarding the involvement ofbiglycan in the regulation of inflammation and the musculoskeletal system, pointing outboth a signaling and a structural role for this proteoglycan. The potential of biglycan asa novel therapeutic target or agent for the treatment of inflammatory diseases andskeletal muscular dystrophies is also addressed. Biglycan is a member of the class I family of small leucine-rich proteoglycans (SLRPs) . The bigBiglycan, which is expressed ubiquitously , is syntSince the discovery of biglycan in developing bone almost 30 years ago , a multiReflecting its widespread expression and complex function, involvement of biglycan innumerous experimental and human diseases has been reported . DetailsRecent data provide evidence for a crucial role of biglycan in the regulation ofinflammation, bone growth, and muscle development and regeneration. This brief review aims tofocus on the mechanisms and consequences of biglycan interaction with cell surfacemolecules/receptors in those processes to underline the role of biglycan as a multivalent,matrix-derived signaling molecule.Research over the past few years has provided strong evidence that biglycan in its solubleform acts as danger signal bridging the innate and adaptive immune systems . FollowiEarly hints regarding the involvement of biglycan in the modulation of the inflammatoryprocess were provided in several reports. The expression of biglycan was shown to beelevated in human fibroblasts from granulation tissue, suggesting its role in thedevelopment of chronic inflammatory lesions . BiglycaThe very first indication that biglycan directly acts as a proinflammatory stimulus camefrom a study performed in murine primary peritoneal macrophages . This stBesides identifying biglycan as an ECM-derived DAMP, these studies have led to newconcepts. First, biglycan has to be present in its soluble form, because when bound to theECM, biglycan cannot act as a DAMP. Second, the magnitude of the biglycan signal can beramped up rapidly by proteolytic liberation from ECM stores without the need for de novosynthesis. Third, both infiltrating macrophages, stimulated by proinflammatory cytokinesand, at later time points, resident cells start to de novo synthesize biglycan at sites ofinjury or damage, in order to drive and shape the inflammatory response reaction overtime. Fourth, by activating both the receptor for the Gram-negative (TLR4) and theGram-positive (TLR2) bacterial response, biglycan, as a signal of tissue damage, acts asan amplifier for TLR-induced inflammation.These initial findings were confirmed by several reports describing the coincidence ofbiglycan overexpression with enhanced inflammation and severe tissue injury  in a TLRFurther studies indicated that proinflammatory effects of biglycan are mediated not onlyby its interaction with TLR2/4 but also by signaling through the NLR family, pyrindomain–containing 3 (NLRP3) inflammasome . The NLRAll studies so far showed that only intact biglycan is capable of triggeringproinflammatory signaling both in macrophages and in dendritic cells . Therefolpr lupus nephritis) and in the prototypic pathogen-mediatedsystemic inflammation of LPS-induced sepsis Fig. 1). Taking all these findings into consideration, the aforementioned studies emphasize theprospect of biglycan as a therapeutic target for intervention in sterile andpathogen-mediated inflammation. Thus, further studies concerning the intricateinteractions between biglycan and innate immune receptors would most likely revealsignificant insights for the development of new drugs for the treatment ofbiglycan-mediated inflammatory diseases.A role for biglycan in the growth of bone was suspected based on the observation thatfemale patients with Turner syndrome, lacking the second X chromosome, have a shorterstature and abnormally low expression of biglycan, contrary to patients with supernumerary Xchromosomes . By the Xenopus embryosinhibits BMP4 activity and affects embryonic development. At the molecular level, biglycanbinds to BMP4 and chordin, a negative regulator of BMP4, increasing the binding efficiencybetween the aforementioned proteins, thereby blocking BMP4 activity . In addition, the response ofBMSCs to TGFβ also becomes impaired, suggesting a possible role of BMP signaling in thedevelopment of this phenotype . Indeed,activity . Besidesactivity . Moreoveactivity .However, the signaling role of biglycan in bone is not restricted to interactions withBMPs. Biglycan is also able to activate the canonical Wnt/β-catenin signaling . The actBased on findings that biglycan directly binds to WISP1 protein  and thatmdx mice, a model of Duchenne muscular dystrophy (DMD). Later,hy (DMD).mdx mouse,which has a nonsense mutation in exon 23 of the dystrophin gene  . The DGChin gene .Biglycan has been shown to bind to several components of the DGC: α-dystroglycan  as well These interactions with three distinct components of the DGC  led to tIn addition, a further study on biglycan-null mice showed that biglycan also regulates theexpression and sarcolemmal localization of other DGC components: dystrobrevin, synthrophin,and nNOS. Biglycan-null mice have a mildly dystrophic phenotype and present several defectsin the localization of DGC components. α-Dystrobrevin-1 and -2 have a selective reduction intheir localization at the sarcolemma. On the other hand, nNOS is also decreasedtranscriptionally in null mice. Different types of synthrophins are affected differently bya lack of biglycan, with the largest effect on β1-syntrophin. Remarkably, sarcolemmallocalization of these components in biglycan-null mice can be restored by the injection ofpurified biglycan core protein into muscle . Thus, bmdx mice, a therapeutic role for biglycan hasindeed been proven (mdx mice, where injection of biglycan could increase theexpression of utrophin in muscle 2.5-fold, associated with an increase in γ-sarcoglycan,β2-syntrophin, and nNOS levels at the sarcolemma. Furthermore, injection of biglycan inmdx mice ameliorated dystrophic symptoms, depending on the presence ofutrophin (In a study performed in n proven . Absenceutrophin .ImportaBesides its role in the assembly of the DGC, biglycan has also been shown to be upregulatedin regenerating skeletal muscle, although the presence of biglycan is not essential formuscle regeneration and is possibly compensated for by decorin in biglycan-null mice . ThroughResearch over the past few years has resulted in considerable progress in our understandingof the biology of biglycan. Particular attention was focused on the interaction betweenbiglycan and various cell surface binding partners, some of them being signaling receptors.The initial concept that soluble biglycan acts a signaling molecule became much more complexwhen new data showed that biglycan is capable of clustering different types of receptors onthe cell surface, thereby orchestrating their downstream signaling events.Current knowledge on biglycan in inflammation, bone development, and muscular dystrophysummarized in this review suggests that besides general effects of this proteoglycan actingas a danger signal in matrix stress or injury, other effects are strongly tissue specific.There is good evidence demonstrating the tissue-specific effects of biglycan in musculardystrophy and in bone formation. However, it is conceivable that in contrast tomuscle-specific association of biglycan with the dystrophin-glycoprotein complex, itsinteractions with BMP/TGFβ and Wnt/β-catenin pathways may play an important role not only inbone formation but also in fibrotic disorders. Thus, new studies in various tissues underphysiological and pathological conditions should expand the current knowledge.The challenges ahead lie in transferring basic research on biglycan into the clinic. Bothin sterile and pathogen-induced inflammatory conditions, the interaction of biglycan withinnate immunity receptors might represent a promising target for the development of newanti-inflammatory therapies. Possible strategies for neutralizing the proinflammatoryeffects of biglycan might include neutralizing antibodies, truncated molecules, orchemically synthesized small molecules. On the other hand, biglycan by itself could serve asa promising treatment in some skeletal muscular dystrophies. A more detailed understandingof the physicochemical and structural properties of the binding sites through which biglycaninteracts with its various binding partners will be a critical component of this effort.


SBIRT Colorado initiative to integrate SBIRT into nursing academic preparation as well as nursing practice across diverse settings. The models include: 1) in-person skills-based training; 2) rapid improvement activities; and 3) interactive, online e-learning technology called “SBIRT Substance Use NursingMentor™.” The online training uses computational linguistics, search and streaming media to teach SBIRT skills through engaging simulated interactions. The SBIRT Substance Use NursingMentor was developed as a collaborative effort between MedRespond, Peer Assistance Services, Inc., and NORC at the University of Chicago. Quantitative and qualitative data collected from nurse training participants will be described. Pilot test results from nurses who participated in the online training demonstrated an increase in knowledge, awareness, and understanding of risky drinking, illicit substance use, and related consequences. Data collected from metrics integrated into the online module launched in May 2013 will be analyzed to evaluate the use of e-learning technologies to provide skill-based SBIRT competency training. Presenters will discuss recommendations that indicate how to target and tailor training for nurses for effective SBIRT dissemination.Nurses play a critical role in integrating screening, brief intervention, and referral to treatment (SBIRT) services as standard of care in healthcare settings. Nurses are well positioned to deliver SBIRT services because of their extended patient contact and existing skill sets in health promotion, communication, and patient education. Patients consistently rate nurses in population-wide surveys to be the most trusted professional group in the United States. Data collected from SBIRT initiatives funded by the Substance Abuse and Mental Health Services Administration in the United States indicate the vast majority of SBIRT service providers are nurses, social workers, and counselors. While nurses are key to deliver SBIRT services, specific training and preparation is necessary. This presentation will review evidence and rationale that support nurse-implemented SBIRT and will describe three models employed by the


Bioremediation of hydrocarbon pollutants is advantageous owing to the cost-effectiveness of the technology and the ubiquity of hydrocarbon-degrading microorganisms in the soil. Soil microbial diversity is affected by hydrocarbon perturbation, thus selective enrichment of hydrocarbon utilizers occurs. Hydrocarbons interact with the soil matrix and soil microorganisms determining the fate of the contaminants relative to their chemical nature and microbial degradative capabilities, respectively. Provided the polluted soil has requisite values for environmental factors that influence microbial activities and there are no inhibitors of microbial metabolism, there is a good chance that there will be a viable and active population of hydrocarbon-utilizing microorganisms in the soil. Microbial methods for monitoring bioremediation of hydrocarbons include chemical, biochemical and microbiological molecular indices that measure rates of microbial activities to show that in the end the target goal of pollutant reduction to a safe and permissible level has been achieved. Enumeration and characterization of hydrocarbon degraders, use of micro titer plate-based most probable number technique, community level physiological profiling, phospholipid fatty acid analysis, 16S rRNA- and other nucleic acid-based molecular fingerprinting techniques, metagenomics, microarray analysis, respirometry and gas chromatography are some of the methods employed in bio-monitoring of hydrocarbon remediation as presented in this review. Typically, these alkanes have low aqueous solubilities. For instance, hexadecane has a water solubility of 0.9 μg l−1 and is a liquid at room temperature. Collectively, these physicochemical properties mean that mid-length aliphatic hydrocarbons are not readily volatilized or leached from soil. It is well established that interactions between hydrophobic organic contaminants may also be important in controlling their fate and behavior in the soil, involving the interactions with the mineral and organic fractions, which may invariably result in reduction in biological/chemical availability and then allow these contaminants to persist in the soil. Soil is composed of organic and inorganic components separated by pores containing water or air. The interaction between hydrocarbons and mineral surfaces  are only significant when organic matter content is <0.1%. Thus, organic matter is very important in the fate and behavior of organic contaminants. Organic matter is divided into two distinct phases: (1) soft carbon (rubbery) which is the expanded, flexible structures having humic and fulvic acids as key components with sorption described as irreversible, and (2) hard carbon (glassy) defined as rigid, condensed structures with humin, kerogen and pyrogenic carbons as commonly identified components. Sorption of hydrocarbons within the glassy region is characterized by irreversible sequestration . Aliphatic hydrocarbons can strongly partition into organic matter and diffuse into the three-dimensional structure of organic matter. Hydrocarbons may be sequestered within the soil through sorption to organic matter and mineral fractions and or diffuse into the three-dimensional structure of the soil  involves the enzymes that have a strict requirement for molecular oxygen, i.e., monooxygenases (mixed function oxidases) or dioxygenases. The most common pathway depends on the action of monooxygenase enzymes and is specific for n-alkanes  to CO2 and H2O pyrene as the sole source of energy, although transformations through co-metabolic activities have been reported  procedure   than other bacteria with or without plasmid. Bogan et al.  in petroleum hydrocarbon-contaminated Alpine soils. They used this method to quantify CO2 produced during a 24-h period by titration. Their results correlated positively with the microbiological data obtained attributing the hydrocarbon attenuation observed to microbial activities rather than abiotic losses. In the same vein, Siciliano et al.  fingerprinting of total soil bacterial DNA and PCR-amplification of the catechol-2,3-dioxygenase (C23O) genes. Amouric et al.  tetrazolium-based redox dye technology. The method uses 96-well microplates/ecoplates containing 31 or 95 different carbon sources (Torsvik and Øvreås http://www.biolog.com). The results from the CLPP analysis can be further used to describe the community’s functional diversity using diversity indices like the Shannon–Weaver index or statistical tools like the principal component analysis (PCA)  present in an environment with respect to the number of defined substrates that can be oxidized by the autochthonous bacteria found there Maila . BasicalTotal phospholipids fatty acids extracted from environmental samples have been used to study microbial community structures and metabolic states and to compare similarities and differences among soil microbial communities  in soil samples containing high concentrations of diesel that received NPK fertilizer. However, PLFA analyses are not without limitations as fatty acid composition can be influenced by temperature and nutrition  to characterize microbial communities in PAHs and polychlorinated biphenyl (PCB) contaminated soil. According to GenBank identification using BLAST, the bacteria Sphingomonas adhaesiva, Sphingomonas terrae, Sphingopyxis witflariensis, Sphingomonas sp., Methylocystis sp., Pseudomonas sp., Pseudomonas marginalis, Acidocella sp. and Acidiphilium facilis were among the dominant sequences in the PAH and PCB-polluted soils. Phylogenetic analysis with dendograms constructed using CLUST software also revealed diversity of aromatic hydrocarbon degradative genes that clustered the genera Burkholderia, Sphingomonas, Pseudomonas, Bacillus, Methylobacterium, Klebsiella and Rhodococcus as well as Vibrio together, thus describing the production of catechol 2,3 dioxygenase and naphthalene dioxygenase. By supplementing phylogenetic-based community dynamics tools with analysis of microbial community composition and diversity using specific catabolic gene markers, clearer information can be obtained on the behavior of the organisms that are specifically responsible for degrading hydrocarbons, free from variation in members of the community that do not directly participate in hydrocarbon degradation.Microbial diversity and dynamics in environments such as soil undergoing bioremediation have been assessed by the analysis of conserved marker genes such as 16S rRNA genes. In addition, large databases of reference sequences, such as Greengenes, SILVA or Ribosomal Database Project II (RDP II) (Simon and Daniel The clear-cut means for evaluating bioremediation of hydrocarbon-contaminated soils is a direct measurement of hydrocarbon dissipation or the residuum. However, the success of a bioremediation project can often be estimated by measuring the biodegradability of the contaminants. Because biodegradation is a microbial-driven process, it can be indirectly assessed by measuring the microbial numbers, biomass and/or activity. Methods that are used include enumeration of hydrocarbon degraders using any hydrocarbon as a selective pressure, measurement of community metabolic activities by respirometry or Biolog system, degradative enzyme-based assays, metagenomic/nucleic acid-based techniques and phospholipid fatty acid analysis. Proper application of these strategies and techniques assists the investigators to monitor population dynamics during the course of bioremediation.


With the exponential increase in iPSC research in recent years, human iPSCs have been successfully derived with different technologies and from various cell types. Although there remain a great deal to learn about patient-specific iPSC safety, the reprogramming mechanisms, better ways to direct a specific reprogramming, ideal cell source for cellular grafts, and the mechanisms by which transplanted stem cells lead to an enhanced functional recovery and structural reorganization, the discovery of the therapeutic potential of iPSCs offers new opportunities for the treatment of incurable neurologic diseases. However, iPSC-based therapeutic strategies need to be thoroughly evaluated in preclinical animal models of neurological diseases before they can be applied in a clinical setting.Many human neurological diseases are not currently curable and result in devastating neurologic sequelae. The increasing availability of induced pluripotent stem cells (iPSCs) derived from adult human somatic cells provides new prospects for cellreplacement strategies and disease-related basic research in a broad spectrum of human neurologic diseases. Patient-specific iPSC-based modeling of neurogenetic and neurodegenerative diseases is an emerging efficient tool for  Human neurological diseases including stroke, neurodegenerative disorders, neurotrauma, multiple sclerosis (MS), and neurodevelopmental disorders are caused by a loss of neurons and glial cells in the brain or spinal cord. They usually cause morbidity and mortality as well as increase social and economic burdens of patients and their caregivers . Stroke Stem cells are characterized by the ability to renew themselves  through mitotic cell division and differentiate into a diverse range of specialized cell types . These cPluripotent stem cells can give rise to every cell of an organism except extraembryonic tissues, such as placenta . ESCs caThe third type is multipotent stem cells that only generate specific lineages of cells. Neural stem cells (NSCs) are multipotent stem cells which are derived from neural tissues . These cThe potential applications of stem cell therapies for treating neurological disorders are enormous. Many laboratories are focusing on stem cell treatments for CNS diseases, including SCI, stroke, ALS, PD, MS, and epilepsy –16. http://clinicaltrials.gov/). However, ethical concerns, immune rejection of grafted stem cells, and tumor formation limit the use of human ESCs.  Finally, clinical trial on stem cell therapy for treatment of neurological disorders was started. Autologous bone marrow stem cells and mesenchymal stem cells are used for treatment of amyotrophic lateral sclerosis. In addition, Geron Corp has started clinical trial using hESC-derived oligodendrocyte progenitor cells for spinal cord injury , SRY-box containing gene 2 (SOX2), cytoplasmic Myc protein (c-MYC), and Krueppel-like factor 4 (KLF4) . SpecifiGeneration of iPCS from somatic cells is a complicated process that is affected by many factors such as the source of the initial cell type (type of the somatic cell used for reprogramming), the particular cocktail of factors used for reprogramming, as well as the methods for delivery of reprogramming factors and culture conditions see . β cells [ In addition to fibroblasts, iPSCs have now been generated from a large variety of somatic cell populations. Cell types that have been used for iPSCs derivation include keratinocytes , pancreaβ cells , neural β cells , mature β cells , melanocβ cells  hepatocyβ cells , amniotiβ cells , 40, andβ cells , 42. HowThe inherent differences among the cell types that have been used for reprogramming may affect the efficiency of iPSC generation as well as the quality of the generated iPSCs. For instance, iPSCs derived from mouse embryonic fibroblasts or hepatocytes exhibit a lower tendency to form teratomas when compared to iPSCs derived from mouse tail-tip fibroblasts . Dependiβ (TGF β) inhibitors or high level of Oct4 [Reprogramming factors, such as OCT4, SOX2, KLF4, c-MYC, Nanog, and LIN28, have putative roles in ESC development. OCT4 seems to be the essential reprogramming factor for most initial cell types . Sox2 sy of Oct4 , 46, 47. of Oct4 . Much of of Oct4 . Instead of Oct4 .As many of the current reprogramming factors tend to endow iPSCs with tumor formation capacity, it is necessary to find new reprogramming factors that lack tumorigenicity. Specific chemicals, such BIX-01294 (BIX), which is a G9a histone methyltransferase (G9a HMTase) inhibitor  5-aza-2′Retroviral and lentiviral vectors have been widely used for the delivery of reprogramming factors. Critical steps in iPSC generation include that reprogramming factors can be efficiently overexpressed in the somatic cell type that is chosen for reprogramming and that expression of the exogenously applied reprogramming factors is silenced once the cells have been transformed to a pluripotent stage. Retroviruses efficiently infect only proliferating cells, but expression of reprogramming factors is silenced in the ESC stage. Lentiviral vectors on the other hand infect dividing as well as nondividing cells. Yet, a major disadvantage of lentiviral vectors is that reprogramming factors are not efficiently silenced once the cells reach pluripotency. Major disadvantages, which both vector systems have in common, is that they contain oncogenic transcription factors and that they randomly integrate into the genome of infected cells. Adenoviral vectors, which do not integrate into the infected cells genome have also been used to deliver reprogramming factors . Yet, th New strategies have been suggested to generate safe and less tumorigenic iPSCs by using nonviral methods or by omitting the oncogenic factors c-MYC and KLF4 , 48. TheViral free methods have been explored to deliver reprogramming factors by transfecting cells with standard transfection methods, such as liposomes. However, these approaches have a limited efficiency that may be overcome through the recent development of polycistronic vectors. OriP/EBNA1 vectors derived from the Epstein-Barr virus were used to transfect human somatic cells with episomes . HoweverOther nonviral delivery methods including RNA and protein transfection have also been tried for iPS generation. Recently, Warren et al. could show that human somatic cells converted to iPSCs by using synthetic mRNAs . This syA major problem in iPSC generation for therapeutical us is that xenogenic products are used at multiple steps in current protocols for iPSC generation and maintenance. For instance, fetal bovine serum (FBS) containing media processed with animal-derived enzymes  are used to maintain primary cultures of the human somatic cells that are to be reprogrammed. Xenogenic contamination might also occur when viruses are used to transduce somatic cells with reprogramming factors. Feeder cell layers of mitotically inactivated mouse embryonic fibroblasts plated on gelatin of animal origin and culture media containing serum substitutes, such as knockout serum replacement (KO-SR), are used for the growth and selection of reprogrammed iPSC colonies and their maintenance. Therefore, xeno-free alternatives to those products have been tested for the derivation and maintenance of human embryonic stem cell (hESC) lines , 71. RecFor all these reasons, strict quality control procedures related to the culture of iPSCs are crucial, especially if the generated iPSCs are intended to be delivered to human subjects. Another common problem in cell culture work is mycoplasma contamination, and contamination tests should be performed on iPSCs and all other cells that were used during their derivation. This control is particularly critical because mycoplasma infection has been shown to dramatically change stem cell viability and function. Another problem is the tendency of cells in culture to become genetically instable, and karyotype analysis on iPSCs should be performed, particularly after extended serial passaging .in vitro while a more stringent test would comprise an examination of their teratoma formation potential in vivo [Patient-specific iPSCs should validated and subjected to rigorous quality controls before deemed pluripotent. Therefore, a test for pluripotency should be performed by testing for the presence of pluripotency markers in human iPSCs that will be used in cell theraoy. These markers include cytoplasmic alkaline phosphatase and cell surface markers such as the stage-specific embryonic antigens (SSEA) SSEA-3, SSEA-4 and the tumor recognition antigens (TRA) TRA-1–60 and TRA-1–81. Immunocytochemistry and flow cytometry can be routinely used to assay the expression of these pluripotency markers. Pluripotent iPSCs and hESCs also endogenously express the nuclear transcription factors OCT4, SOX2, and NANOG and endogenous expression of these factors must be distinguished from expression of the exogenously introduced factors that might have been delivered during the reprogramming procedure. Oligonucleotide primers specific for either the endogenous or exogenous factors can be designed and used in RT-PCR in order to solve this problem. In addition, gene expression profiling can also be used to test for the presence of pluripotency markers. Most of the characteristic genes, which are associated with the pluripotent state, have been identified by microarray studies using hESCs. Therefore, the expression of these pluripotency markers can be analyzed by performing multiple standard quantitative RT-PCR, specialized RT-PCR arrays, or by using microarray platforms. Important for cell replacement therapy, the differentiation potential of iPSCs and hESCs should also be tested. A simple method to check for pluripotency of iPSCs and hESCs is to test for their ability to form embryoid bodies (EBs)  in vivo .in vivo, and proper timing of exposure to these factors can give rise to well-defined neuronal populations. The use of iPSCs for the treatment of neurological disorders requires that iPSCs can differentiate into the relevant neuronal subtypes that should be replaced or repaired by the therapy. The extended knowledge of neural development has provided a good opportunity to generate neural cells from iPSCs, and neurons of different parts of the neural tube have been successfully generated, including spinal motoneurons , midbraiin vitro differentiation of neural cells from human iPSCs using the embryoid body formation method. The earliest recognizable cell type in the neural lineage is the neural ectoderm. The differentiation of human iPSCs including patient-specific iPSCs into neural ectoderm cells [βIII-TUBULIN-positive neurons as well as GFAP-positive astrocytes [Several groups have reported rm cells  has beenrm cells , increasrm cells , 84. Yamtrocytes , 48.IPSCs and ESCs share major properties such as self-renewal and pluripotency; that is, they are capable of producing cells of all kind of tissues and organs , 85. IPSThe generation of human iPSCs offers new approaches to model and cure human diseases. In 2008, Park et al., for the first time, created patient- as well as disease-specific iPSCs from skin fibroblasts of patients that suffered from a variety of genetic diseases, including adenosine deaminase deficiency-related severe combined immunodeficiency, Gaucher disease type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington's the disease (HD), juvenile-onset, type 1 diabetes mellitus, Down syndrome (DS)/trisomy 21, and the carrier state of Lesch-Nyhan syndrome . Similarin vivo remains to be identified, patient-specific iPSCs represent a novel and promising resource to study the mechanisms of SMA. Further functional studies, including electrophysiological experiments and coculture studies with motor neurons derived from SMA-iPSCs and muscle fibers, are needed. It has been proposed that more iPSC clones from other patients and control cases will reduce the concern that the observed phenotype could be a consequence of intrinsic iPSC variability [Certain monogenic neurological diseases are characterized by young childhood-onset, such as spinal muscular atrophy (SMA) and familial dysautonomia (FD). These diseases are autosomal recessive and are caused by the loss of function of a single specific gene, and rapid disease progression occurs within the first years of life. SMA is a group of autosomal recessive diseases caused by loss or mutations in the survival motor neuron (SMN) genes . SMA typiability , 91. Lee and coworkers recently established iPSCs from another fatal neurodegenerative disease, FD, that results from an aberrant splicing of the IkB kinase complex-associated protein (IKBKAP) . FD, alsKu et al. have reported the derivation of patient-specific iPSCs from skin fibroblasts of two Friedreich's ataxia patients by transcription factor reprogramming . FriedrePatient-specific iPSCs have also been derived from diseases in which genomic imprinting is affected and which are accompanied by neurological symptoms , 98. AngTwo research groups have recently established iPSCs from patients with Down's syndrome (DS), a frequent genetic cause of mental retardation in humans, occurring in 1 out of 700 births , 99. ThiIn addition to early-onset genetic disorders, patient-specific iPSCs have also been obtained from patients with late-onset neurodegenerative diseases including HD, PD, and ALS. Interestingly, fibroblasts obtained from elderly patients could be reprogrammed with similar efficiency as those obtained from younger patients , 95. Parin vitro differentiation protocol, the authors differentiated motor neurons from embryoid bodies formed by patient-specific iPSCs. Within the same cultures glial cells could also be identified. Genotypic analysis revealed a L144F polymorphism in SOD1 gene both in patient-specific iPSCs and iPSC-derived motor neurons [In contrast to HD, the vast majority of ALS and PD cases are sporadic. Yet, the study of rare familial forms of these neurodegenerative diseases can also provide valuable information about the pathogenesis of age-related sporadic forms of ALS and PD as both familial and sporadic cases share common phenotypic traits, such as the involvement and loss of specific neuron subpopulations. ALS is a progressive fatal neurodegenerative disease affecting upper and lower motor neurons. In 2008, Dimos et al. generated patient-specific iPSCs derived from skin fibroblasts of two elderly sisters with ALS-associated mutations in the gene encoding superoxide dismutase (SOD1) . One of  neurons . However neurons , 82. The neurons .in vitro and in defined cell populations is an important advancement and would make it possible to understand cellular mechanisms of the neurodegenerative diseases [in vitro [In neurology, most of the current knowledge about disease-related neuronal phenotypes is gathered from postmortem studies because obtaining live brain tissue is limited. This creates a problem for understanding disease progression and development, because postmortem samples only represent the end-stage of the disease. In addition, aspects of the pathology that are observed in these samples could be secondary and not faithfully reflect the exact disease phenotype on a cellular level . Therefodiseases . Reprogrin vitro , 82. Natin vitro . Many stIncomplete disease modeling using iPSCs has first been reported in autosomal recessive neurodegenerative diseases by Ebert et al. it showed that SMA-iPSCs-derived neural cells undergo selective neuronal death, which could be reversed by adding the compounds known to raise the production of SMN protein . Howeverα-synuclein (SNCA), PARKIN, and DJ-1 have been associated with familial PD cases. It would be interesting to see whether iPSCs or their derivatives generated from familial PD cases could exhibit specific disease genotypes and phenotypes in vitro. Subsequently, iPSCs have also been established from the patients with late-onset neurodegenerative diseases. Dimos et al. successfully directed differentiation of iPSCs which were generated from cells of two familial ALS patients. The iPSCs were able to differentiate into motor neurons expressing appropriate motor neuron markers such as Hb9 and ISLET but no disease phenotype was observed . The genAnother potential impact of human iPSC research is the use of patient-specific cells for the process of drug discovery. in vitro provides an invaluable tool for testing candidate neurotherapeutic agents in patient-specific iPSC-based disease models [The ability to analyze drug responses e models .The drug discovery process is time consuming and costly because of high attrition rates and protracted research and development cycles. Many candidate drugs that have significant effect on animal models fail to show significant benefits in clinical trials . Tests bin vitro using patient-specific iPSCs has been demostrated. A study by Ebert et al. provides a proof-of-principle for the use of iPSC in drug screening by using the SMA patient-specific iPSCs or their derivatives [The feasibility of exploring the therapeutic action of candidate drugs for human neurological diseases ivatives . In thisivatives . The plaivatives . Althougivatives . These rin vitro systems such as iPSC-based assays can actually faithfully predict the toxicity of drugs in a complex in vivo setting [Possible toxic effects of candidate drugs are often missed in rodent cells and animal models in general due to specific interactions with human biological processes that cannot be accurately recapitulated in these assay systems . Human i setting .As summarized above, the recent advances in deriving iPSCs from patients offer new and exciting possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation in neurological diseases. There are two main strategies for using iPSCs to treat neurological disorders. The first is to produce new neurons to replace those lost during disease progression. The second is to produce glial cells that could protect neurons from ongoing degeneration by expressing and secreting critical humoral factors, for example, growth factors. Park et al. showed that growth factor-expressing glial cells can contribute to motor neuron protection in ALS mice. This study underlines the important fact that (micro-) environmental conditions should also be considered in cell-based therapies and might include the delivery of glial cell types along with neurons. in vitro [in vivo experimental transplantation studies suggest that engrafted neurospheres derived from human iPSCs are able to differentiate to various types of neural cells such as neurons, astrocytes, and oligodendrocytes in the central nervous system of embryonic mice [As summarized before, iPSCs derived from patients with chronic neurological diseases can be successfully differentiated into different types of neural cells, neuronal subtypes, or neural cell precursors in vitro , 95, 115nic mice . Upon trnic mice . IPSCs oFetal tissue transplantation for PD has given way to cell replacement therapy for neurodegenerative diseases . PD is m Despite advances in medical and surgical care, current clinical therapies for SCI are limited. SCI can affect relatively young people who often become a huge burden to themselves and society due to high medical costs throughout their lives. Rationales for therapeutic use of stem cells for SCI include replacement of damaged neurons and glial cells, secretion of trophic factors, regulation of gliosis and scar formation, and enhancement of axon regeneration . However Furthermore, transplanted cells also contributed to endogenous remyelination, induced the axonal regeneration and axonal outgrowth of host serotonergic fibers, and promoted the recovery of locomotor function. In contrast to this, the transplantation of iPSC-derived neurospheres sorted as unsafe exhibited teratoma formation and even had detrimental effects on the functional recovery following SCI . The theAcute stroke is the second or third most common cause of death and the primary cause of morbidity and long-term neurological disability worldwide. The only FDA-approved treatment for stroke is the use of the thrombolytic agent, tissue-plasminogen activator. This therapy is very limited due to a narrow therapeutic time window and the risk of hemorrhagic complications. The regeneration of the brain after damage is active even weeks after stroke occurs, which might provide a rational for cell-replacement therapy . Transpl Moreover, transplanted undifferentiated iPSCs formed tridermal tumorigenesis in ischemic mouse brain, suggesting a facilitating and promoting effect of the postischemic microenvironment for the development of tumors. IPSCs grafted into ischemic brains formed teratoma with higher probability and larger volume as compared to those formed in intact brain tissue. The expression of matrix metalloproteinase-9 and phosphorylated vascular endothelial growth factor receptor2 are significantly increased in iPSC-derived tumors in the ischemic mouse brain . The effAs outlined above, the major limitation of iPSC-based therapies is its tumorigenic potential. Current differentiation methods are not efficient enough to transform all iPSCs, and undifferentiated cells remain in the preparation , 123. ThMost patient-specific iPSCs have been generated by lentiviruses or retroviruses containing reprogramming factors, which both lead to genomic integration of the transgene and which may not get silenced efficiently at the pluripotent stage. Moreover, reactivation of the silenced transgenes can cause undesirable side effects in reprogrammed cells. Thus, avoiding genomic integration may be a crucial step in the derivation of safe iPSCs for cell replacement therapy and can be achieved by improving transgene-free or nonviral methods , 125. Several groups put considerable effort in working out nonviral delivery methods, such as using chemicals that can improve efficiency in order to derive iPSCs , 127. OnFor regenerative medicine, human iPSCs and hESCs are promising cell sources. They have the common ability to differentiate into all three germ layers; however, one recent study revealed that heterogeneity in gene expression levels is much greater among hiPSCs than among hESCs. It is also suggested that human iPSCs occupy an alternate and less stable pluripotent state. Moreover, human iPSCs display slower growth kinetics and impaired directed differentiation when compared to hESCs .in vivo function. In addition, the variability that has long been observed between different human ESC lines can be even more dramatic in human iPSCs because of their diverse origins and the different modes of derivation [in vivo reprogramming has also been reported [According to recent studies, many mouse iPSCs are also prone to epigenetic abnormalities and sustain a transient epigenetic memory of their donor cells , 131. Thrivation . Thus, vrivation , 134. Inreported . Thus, aAnother challenge of iPSC technology is that the process of well-characterized and carefully validated iPSC line generation is a time-consuming process. The lag between the initiation of reprogramming and cell delivery to a patient might render the process inappropriate for therapeutic applications, for instance in acute organ failure or injury .In addition to the difficulty of isolating the optimal cell type that has the best potential for transplantation, poor survival rates, limited self-renewal, and limited homing/migration after transplantation should be taken into consideration to improve iPSCs based-approaches , 137.IPSCs cell-based therapies are still in their infancy. Therefore, numerous hurdles must be overcome before they can be used in clinical applications. in vitro within a few days of cell culture or whether the exposure to different types of environmental factors or genetic stress to the cells is necessary for revealing the desired disease phenotype. One study related to PD showed that iPSCs derived from sporadic PD patients do not display apparent abnormalities when compared with wildtype neurons [in vitro would be crucial.So far, iPSCs technology has been used to investigate monogenetic neurodegenerative diseases and some promising data have been published providing a new way to understand the pathology of the diseases. However, multifactorial neurodegenerative disease modeling has not been performed yet. One of the questions concerning iPSC-based disease modelling is whether late-onset diseases like AD and PD can be efficiently recapitulated  neurons . Most of neurons , 138. Re neurons , 140. Th neurons –143. Non neurons , 145. ThDespite a large body of current research, iPSC technology is still in its infancy. There are many limitations that have to be solved before clinical trials for treatment of neurological disorders can be performed. For monogenic diseases, which require gene targeting to repair mutant alleles, new targeting strategies need to be developed concomitantly . A recen IPSCs technology needs to be standardized in order to create medically relevant cells. For the translation of iPSCs into therapeutics and human clinical applications, current good manufacturing practices (cGMP) will also be necessary . The sci So far, most of the iPSCs lines have been exposed to animal products either in direct or indirect ways, which could make these cells improper for transplantation. Therefore, cGMP standards have to construct standardized animal-free methods for the derivation of iPSC lines . Human iAn alternative to the feeder cell-free culture method has been established by using autologous skin fibroblasts derived from the same patient, which provides an appropriate source of feeder cells , 149. AdNonviral and transgene-free reprogramming methods have been significantly improved, yet the cancer-causing potential of safe iPSCs still needs to be evaluated in animal models before clinical applications in humans. Besides small animals such as mice and rats, iPSC therapies need to be validated in large animal models which are anatomically and physiologically more relevant to humans. IPSCs have been generated from monkeys –153 and The safety and efficacy of cell-based therapies for neurodegenerative diseases also depend on the method of cell administration. One study has suggested that intranasally administered cells could pass through the blood-brain barrier , 157. ThSince the first generation of human iPSCs, a large number of patient-specific iPSC lines have been developed. Recently, iPSC studies have exploded and the potential application of these cells in establishing disease models, drug screening, and cell transplantation-based therapy have been widely recognized. IPSCs have distinct advantages over hESC such as the lack of ethical restrictions and immune rejection of the graft. In addition to their direct therapeutic use in cell replacement therapy, disease modeling is most likely the most important aspect of iPSC technology. IPSCs are the patient's own cells and therefore the best possible source for cell transplantation treatment provided that their genetic program can be changed for the treatment of neurodegenerative diseases. Human iPSCs hold great promise for the treatment of incurable neurodevelopmental and neurodegenerative diseases. Furthermore, iPSC-based cell therapy may contribute to neurorepair in the chronic phase of acute CNS injuries such as stroke and neurotrauma. However, iPSC technology still faces specific difficulties such as low efficiency and high variability. Finally, iPSC-based therapeutic strategies need to be evaluated in preclinical animal models of neurological diseases before clinical trials.The authors declare no competing financial interests.