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Possibly the ultimate goal of bioinformatics is to be able to predict protein tertiary structure and chemical functionality from the initial amino acid sequence. Despite the best efforts of many researchers over the past two decades, a reliable modelling method has yet to be found and the folding problem continues to be a hurdle for scientists.
To surmount the economic pressures of managed care, specialists are pursuing various initiatives designed to increase revenues and market share that may put them into competition with healthcare systems. Systems contemplating collaboration with specialists to gain their loyalty may first consider employing a strategy involving physician gain sharing. It should be understood, however, that there are legal hurdles to be overcome in developing gain sharing, that there are different gain-sharing models to be used, and that there are limitations as well as benefits to such an initiative. These limitations require healthcare systems to consider other, more durable specialist integration strategies. The balance of power between a healthcare system and specialists will affect the success of whatever integration strategy is employed.
Immunotherapy of cancer is still mainly an experimental treatment. Some monoclonal antibodies have been approved for adjuvant therapy of cancer in patients, but active immunization strategies have not yet matured to this stage. The fact that vaccination against viral diseases is effective has primed high expectations for successful vaccination against cancer as well. Indeed, in some animal models, therapeutic results could be obtained against short-term established tumors, which paved the way for clinical trials. However, the first results with active immunization in cancer patients were disappointing and this led to a careful examination of current protocols and the search for more effective approaches. Evaluation of the available data suggests that cancer patients may not be comparable in their immune response to cancer vaccines with healthy persons. Furthermore, the tumor seems to be able to develop several immune-escape mechanisms, which either inactivate the specific immune cells or prevent activation of potential effector mechanisms against the tumor. Here, we review the impediments that have been identified in murine models and clinical trials for immunotherapy of cancer. It will be important to study the hurdles to come to a better understanding of the immune evasion of tumors and to achieve efficient activation of the immune system in cancer patients against the tumor. This knowledge will open new possibilities for active immunization against cancer.
The growth/no growth responses of Listeria monocytogenes inoculated at four levels (0.90, 2.58, 4.20 and 6.81 log cfu/well; 300 microl medium/well) into tryptic soy broth (TSB) were monitored at different combinations of temperature (4 to 30 degrees C), pH (3.76 to 6.44) and aw (0.888 to 0.997) for 60 days. The study was conducted in 96-well microtiter plates and growth was monitored visually and by recording the turbidity of the medium with an automated microplate reader. The growth limits of the pathogen and hence the position of the growth boundary were found to be affected by the size of the inoculum. For example, at 25 degrees C and aw 0.997 the minimum pH values that allowed growth were 4.45 and 3.94 for inoculum levels of 0.90 and 6.81 log cfu/well, respectively. Similarly, at 25 degrees C and pH 6.44 the minimum aw values where growth was observed ranged from 0.900 to 0.928 depending on the inoculum level. The results showed that at temperatures from 10 to 30 degrees C the difference in the growth limits between the tested inoculum levels was higher in environments where a single factor (pH or aw) was inhibitory than in environments where pH and aw together were inhibitory. The data were used to develop a growth/no growth interface model for each inoculum level tested, using logistic polynomial regression. The concordance indices of the models ranged from 99.8% to 99.9% and showed a good fit to the observed data in all models. This study indicates the importance of inoculum size for microbial growth initiation and provides quantitative data that show how the combinations of hurdles which prevent growth vary with inoculum size.
Since the Diabetes Control and Complications Trial demonstrated the substantial benefits of tight glycaemic control there has been renewed interest in the application of information technology (IT) based techniques for improving the day-to-day care of patients with diabetes mellitus. Computer-based educational approaches have a great deal of potential for patients use, and may offer a means of training more health-care professionals to deliver such improved care. In this article the potential role of IT in diabetes education is reviewed, focusing in particular on the application of compartmental models in both computer-based interactive simulators and educational video games. Close attention is devoted to practical applications-available today-for use by patients, their relatives, students and health-care professionals. The novel features and potential benefits of such methodologies are highlighted and some of the limitations of currently available software are discussed. The need for improved graphical user interfaces, and for further efforts to evaluate such programs and demonstrate an educational benefit from their use are identified as hurdles to their more widespread application. The review concludes with a look to the future and the type of modelling features which should be provided in the next generation of interactive diabetes simulators and educational video games.
BACKGROUND: The pig is generally regarded as likely to be the preferred donor animal in xenotransplantation. Although many hurdles remain to be cleared, it would be useful to be able to manipulate porcine endothelium genetically, among other reasons, to test approaches in the modulation of inflammation. However, as a nondividing cell, it is less easy to manipulate. METHODS: The authors performed in vivo and in vitro gene transfection experiments using as an adjunct the DNA-binding agent 4',6'-diamidino-2-phenylindole (DAPI), which protects DNA from degradation. RESULTS: The introduction of DAPI into a liposomal transfection system was able to increase in vitro transfection efficiency of both endothelial and vascular smooth muscle cells from the pig, even in the presence of small amounts of serum. This last observation encouraged the authors to use this system in vivo in porcine carotid arteries. In this model, the authors were also able to demonstrate a high degree of transfection efficiency using DAPI, which seemed to work by protecting DNA from degradation. CONCLUSIONS: The authors believe this technique may allow them to address many biological questions relating to intervening in vascular disease, inflammation, and immune responses in the context of transplantation and beyond.
Hazardous air pollutants, some of which have the potential for multimedia distribution, raise several hurdles for ecological risk assessment including: (1) the development of an adequate transport, fate and exposure model; and (2) the selection of exposure-response models that can accommodate multiple exposure routes for ecological receptors. To address the first issue, the EPA Office of Air Quality Planning and Standards has developed TRIM.FaTE, a mass-balance, fate, transport, and ecological exposure model that is a component of the Total Risk Integrated Methodology (TRIM) for air pollutants. In addition to abiotic transfers and transformations, TRIM.FaTE estimates the uptake of a chemical by terrestrial and aquatic organisms with time. Measures of exposure that TRIM.FaTE can provide include: (1) body burdens or tissue concentrations; (2) doses averaged over any time period; or (3) concentrations of chemicals in abiotic media. The model provides the user with the flexibility to choose the exposure-response thresholds or dose-response relationships that are best suited to data availability, routes of exposure, and the mechanism of toxicity of the chemical to an ecological receptor. One of the challenges of incorporating TRIM.FaTE into a risk assessment methodology lies in defining a streamlined model simulation scenario for initial screening-level risk assessments. These assessments may encompass multiple facilities that emit a variety of pollutants near diverse ecosystems. The information on ecological risk assessment methodology that is described is applicable to the EPA Residual Risk Program with emphasis on multimedia pollutants and the role of TRIM.FaTE.
Cationic lipids are exploited as vectors ('lipoplexes') for delivering nucleic acids, including genes, into cells for both therapeutic and cell biological purposes. However, to meet therapeutic requirements, their efficacy needs major improvement, and better defining the mechanism of entry in relation to eventual transfection efficiency could be part of such a strategy. Endocytosis is the major pathway of entry, but the relative contribution of distinct endocytic pathways, including clathrin- and caveolae-mediated endocytosis and/or macropinocytosis is as yet poorly defined. Escape of DNA/RNA from endosomal compartments is thought to represent a major obstacle. Evidence is accumulating that non-lamellar phase changes of the lipoplexes, facilitated by intracellular lipids, which allow DNA to dissociate from the vector and destabilize endosomal membranes, are instrumental in plasmid translocation into the cytosol, a prerequisite for nuclear delivery. To further clarify molecular mechanisms and to appreciate and overcome intracellular hurdles in lipoplex-mediated gene delivery, quantification of distinct steps in overall transfection and proper model systems are required.
The Republic of Pakistan is a South East Asian country with a population of over 140.7 million. Its population is fast growing and the majority (70%) live in rural areas with a feudal or tribal value system. The economy is dependent on agriculture and 35% of the population live below the poverty line. Islam is the main religion and 'mental illnesses' are stigmatized and widely perceived to have supernatural causes. The traditional healers along with psychiatric services are the main mental health service providers. The number of trained mental health professionals is small as compared to the population demands and specialist services are virtually non-existent. Lack of data on prevalence of various mental illnesses and monitory constraints are the major hurdles in the development of mental health services. A number of innovative programmes to develop indigenous models of care like the 'Community Mental Health Programme' and 'Schools Mental Health Programme' have been developed. These programmes have been found effective in reducing stigma and increase awareness of mental illness amongst the adults and children living in rural areas. Efforts by the government and mental health professionals have led to the implementation of a 'National Mental Health Policy' and 'Mental Health Act' in 2001. These aim at integrating mental health services with the existing health services, improving mental health care delivery and safeguarding the rights of mentally ill people. A favourable political will and the help of international institutions like the World Health Organization are required to achieve these aims.
Leprosy is both a public health problem and a model for host-parasite relationship. There is much progress in many areas, and in most, hurdles are not only scientific but also social and financial. The following are goals to reach: 1) progress of growth of M. leprae in animals (to be able to breed armadillos in captivity or to substitute them with other animals); 2) to develop methods for in vitro culture of M. leprae; 3) to obtain antigens from M. leprae and characterize them immunologically; 4) to achieve techniques for objective diagnosis of infection with M. leprae and of leprosy (these techniques will most likely be serologic); 5) to develop methods to direct and manipulate immune response. This should include a better definition of immunologically active cells, their characterization by reliable methods, and an understanding of their functions. Immune control mechanisms and ways to channel them should also be understood; and 6) to have better methods of treatment including flexible adaptable schedules. It will be clear that "solving" the problem of leprosy requires "solving" the social problems in developing countries, as well as unraveling the mystery of the precise control of immune response. As I pointed out, the problems that we face are basically similar to those of autoimmune disorders and of cancer immunology. Research in autoimmune diseases or cancer might provide answers to questions posed by leprosy. It may also be that research in leprosy could supply answers to questions concerning cancer and autoimmunity.
Gene therapy has become a potential treatment for Cystic Fibrosis (CF) when the CFTR gene responsible for the disease was isolated in 1989. As premature death is still the norm, the demand is now for a dramatically improved therapy. The pulmonary manifestations of CF being life-limiting, many gene therapy studies have focused on gene transfer via various vectors into airway epithelial cells, namely in murine models of CF. These preclinical studies led to the first clinical trials, which showed that many hurdles still do exist and that more efficient vector systems need to be developed. Both a remarkable scientific effort and a close collaboration between scientists and clinicians are highly necessary in order to move from the present stage of "cautious optimism" toward the ultimate goal: a cure for CF.
Many pathogens must overcome an epithelial barrier in order to establish an infection. Unsurprisingly, such pathogens have evolved different mechanisms to overcome this obstacle, targeting specific epithelial structures or functions. These include disruption of epithelial barrier function, transcytosing from the apical to the basolateral membrane domain or inducing cell movement such as neutrophil recruitment. When studying these processes in vivo, animal models often fail to mimic the disease observed in humans and present a complex system in which many variables cannot be controlled. Therefore, in vitro transepithelial models that permit the study of a relevant biological surface have been developed, to integrate not only interactions between bacteria and epithelial cells but also, under certain conditions, to integrate a third cell type, such as neutrophils or dendritic cells. Such models are particularly useful for studying the bacteria-host relationship as it would occur in the microenvironment of the human epithelium and have enhanced our understanding of the unique strategies by which pathogenic bacteria exploit host cells to overcome the initial epithelial hurdle.
Various technologies can be used to produce genome-scale, or 'omics', data sets that provide systems-level measurements for virtually all types of cellular components in a model organism. These data yield unprecedented views of the cellular inner workings. However, this abundance of information also presents many hurdles, the main one being the extraction of discernable biological meaning from multiple omics data sets. Nevertheless, researchers are rising to the challenge by using omics data integration to address fundamental biological questions that would increase our understanding of systems as a whole.
The ability of ribozymes (i.e. RNA enzymes) to specifically recognize and subsequently catalyze the cleavage of an RNA substrate makes them attractive for the development of therapeutic tools for the inactivation of both viral RNAs and mRNAs associated with various diseases. Several applicable ribozyme models have been tested both in vitro and in a cellular environment, and have shown significant promise. However, several hurdles remain to be surpassed before we generate a useful gene-inactivation system based on a ribozyme. Among the most important requirements for further progress are a better understanding of the features that contribute to defining the substrate specificity for cleavage by a ribozyme, and the identification of the potential cleavage sites in a given target RNA. The goal of this review is to illustrate the importance of both of these factors at the RNA level in the development of any type of ribozyme based gene-therapy. This is achieved by reviewing the recent progress in both the structure-function relationships and the development of a gene-inactivation system of a model ribozyme, specifically delta ribozyme.
PURPOSE: The diversion of ambulances away from their intended emergency departments (EDs) in the United States has become commonplace and may compromise patient care. Although ambulance diversion resulting from ED overcrowding has been well described in the literature, little is known about how to reduce the incidence of ambulance diversion on a regional level. We describe the development, implementation, and impact of a region-wide program to reduce ambulance diversion. BASIC PROCEDURES: This study was undertaken in the greater Sacramento, California region from January 2001 to December 2003. This comprehensive ambulance diversion reduction program was implemented May 15, 2002, with analysis of data for this 3-year time frame. The data for this study were obtained from 17 hospitals with ambulance diversion hours being the main outcome measure for this study. FINDINGS: The greater Sacramento region had 23785 hours of ambulance diversion in 2001. In 2003, there were 7143 ambulance diversion hours. Comparing the 17-month period before implementation of this program with the 19-month period after implementation, the difference in the means of these two groups was -1428 hours per month (95% confidence interval, -1252 to -1597), a 74% decrease in ambulance diversion hours. Notably, this reduction occurred despite overall increases in ED census (6.5%), hospital admissions from the ED (8.8%), EMS arrivals to the ED (17.1%), inpatient hospital census (7.4%), and overall Sacramento population (5.7%). CONCLUSIONS: Our results demonstrate a sizeable reduction of ambulance diversion in a large urban region after the successful implementation of a comprehensive ambulance diversion reduction program. The description of this effort may serve as a model for other regions across the country that do not have an organized approach in place for ambulance diversion, although boarding of admitted patients will still be a major hurdle to effective reduction of ambulance diversion.
Hypertension is a debilitating disease with significant socioeconomic and emotional impact. Despite recent success in the development of traditional pharmacotherapy for the management of hypertension, the incidence of this disease is on the rise and has reached epidemic proportions by all estimates. This has led many to conclude that traditional pharmacotherapy has reached an intellectual plateau, and novel approaches for the treatment and control of hypertension must be explored. We have begun to investigate the possibility of treating and/or curing hypertension by using genetic means. In this review, we will provide evidence in favor of targeting of the renin-angiotensin system by antisense gene therapy as an effective strategy for the lifelong prevention of hypertension in the spontaneously hypertensive rat model. In addition, we will discuss the properties of an ideal vector for the systemic delivery of genes and the potential experimental hurdles that must be overcome to take this innovative approach to the next level of evaluation.
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Plasmid-mediated gene therapy can restore dystrophin expression in skeletal muscle in the mdx mouse, a model of Duchenne muscular dystrophy. However, sufficient long-term expression and distribution of dystrophin remain a hurdle for translating this technology into a viable treatment for Duchenne muscular dystrophy. To improve plasmid-mediated gene therapy for muscle diseases, we studied the effects of targeted plasmid integration using a phage integrase (phiC31) that can mediate the integration of suitably modified plasmids into the mammalian genome. Using a luciferase expression plasmid, we monitored plasmid gene expression noninvasively in living mice by bioluminescence imaging. Coinjection of an integrase plasmid resulted in 5- to 10-fold higher levels of sustained luciferase expression. Likewise, plasmid-mediated dystrophin expression in mdx muscle was enhanced by integration. Using a combination of dystrophin and luciferase plasmids, we analyzed the functional benefit of dystrophin expression in the dystrophic muscle. The expression of dystrophin slowed the loss of luciferase expression associated with muscle degeneration, and that protection was enhanced by targeted integration of the dystrophin plasmid. In the presence of integrase, dystrophin expression was distributed along a much greater length of individual fibers, and this was associated with increased protection against degenerative changes. These data demonstrate the importance of both the level and distribution of dystrophin expression to achieve therapeutic efficacy, and that the efficacy can be enhanced by targeted plasmid integration.