Experimental and clinical experience demonstrates that the resolution of a pathogenic challenge depends not only on the presence or absence of an immune reaction, but also on the initiation of the proper type of immune reaction. The initiation of a non-protective type of immune reaction will not only result in a lack of protection, but may also exacerbate the underlying condition. For example, in cancer, constituents of the immune system have been shown to augment tumor proliferation, angiogenesis, and metastases. This review discusses the duality of the role of the immune system in cancer, from the theories of immunosurveillance and immunostimulation to current studies, which illustrate that the immune system has both a protective role and a tumor-promoting role in neoplasia. The potential of using chemotherapy to inhibit a tumor-promoting immune reaction is also discussed. If only it were all so simple. If only there were evil people somewhere insidiously committing evil deeds, and it were necessary only to separate them from the rest of us and destroy them. But the line dividing good and evil cuts through the heart of every human being, and who is willing to destroy his own heart? Alexander Solzhenitsyn, The Gulag ArchipelagoSerratia marcescens), Coley was able to achieve a cure rate of 10% in soft-tissue sarcoma [The notion that the immune system may be manipulated into recognizing and eradicating neoplasia is not new. Heroic efforts to develop a cancer vaccine can be traced as far back as 1777 when the surgeon to the Duke of Kent injected himself with malignant tissue as a prophylaxis against development of cancer. In 1808, another attempt was made to develop a cancer vaccine by the doctor to Louis XVII who inoculated himself with breast cancer in hope of reversing a soft-tissue sarcoma, although no therapeutic effect was observed. However, it was not until 1891 that the first report of successful immunotherapy was published by William Coley, a clinician at the Memorial Sloan Kettering Cancer Institute in New York. Using heat-killed endotoxin-containing bacteria  or extracellular  nature; accordingly, the specific immune response is bifurcated into a cell-mediated branch, which offers protection against intracellular pathogens, and an antibody-mediated branch, which offers protection against extracellular pathogens. Following a challenge with gression . The abiTwo distinct theories aim to define the role of the immune system in cancer. The theory of immunosurveillance postulates that the role of the immune response in cancer is one of protection – the organism is patrolled for incipient tumor cells by the effector cells of the immune system. In contrast, the theory of immunostimulation postulates that, while in experimental systems highly immunogenic tumors may be eradicated by the immune response, the role of the immune response in spontaneous neoplasia is not one of protection, but rather of tumor promotion. This review gives a historical overview of these theories and highlights recent data supporting the validity of both immunosurveillance and immunostimulation. To explain the conflicting roles of the immune response in neoplasia it must be noted that the immune system is not a single entity, but a complex system of constituents. While these theories have historically been considered to be mutually exclusive, it is proposed that these theories describe the activation of different constituents of the immune system and hence illustrate that an appropriate immune reaction will result in protection whereas an inappropriate immune reaction will result in tumor promotion.in vivo and that the immune system is able to both recognize and protect against it [As early as 1909 Paul Ehrlich postulated that cancer occurs spontaneously ainst it . In the ainst it  introducainst it . The theThe concept of immunosurveillance initially had much intellectual appeal. First, it could explain clinical observations of spontaneous remission. Second, the most potent chemical carcinogens, dimethlybenzanthracene , urethanA number of discrepancies were noted in the theory of immunosurveillance as initially described by Thomas and Burnet, where T-cells function as the effector cells mediating immunosurveillance. First, T-cell deficient (athymic nude) mice did not develop significantly more cancers than control mice  Second, Taenia taeniaeformis will reject a large inoculum of the cestode larva yet will succumb to infection by minute inoculations [Furthermore, several studies suggest that incipient tumors may not be immunogenic. A correlation between the dose of carcinogen administrated and the immunogenicity of the subsequent tumor has been reported in mouse models -17. Sinculations . Finallyulations .Over the past two decades, data have emerged suggesting that constituents of the immune system such as natural killer (NK) cells and cytokine networks may be able to guard against cancer. Furthermore, recent studies of the incidence of neoplasia in immunosuppressed patients after organ transplantation, in contrast to earlier studies, reported that these patients are more susceptible to a wide range of cancers, including epithelial cancers ,22.in vitro [Several studies suggest that NK cells are able to protect against tumors. Beige, natural-killer-cell defective, mice have an increased incidence of spontaneous tumors  and cancin vitro -32 and tin vitro -38. NK cin vitro -43. Thesin vitro .The precise mechanism though which NK cells mediated immunosurveillance is not yet understood. It is likely that the role of NK cells, in addition to their direct cytotoxic effects, is to activate other cells of the immune system through providing cytokine support . Mature The initiation of an immune response requires cytokine support provided by T-helper cells. The nuance of cytokines produced by these cells determines the type of immune response initiated. The initiation of a cell-mediated immune response requires the support of Th1 cytokines, while the initiation of an antibody-mediated immune response requires the support of Th2 cytokines. T-helper cells that have differentiated into Th1 cells secrete IFN-γ and to a lesser extent interleukin (IL)-2 and IL-12, whereas Th2 cells secrete IL-10, IL-4 and to a lesser extent IL-5.The cell-mediated immune response is generally regarded as possessing tumor-inhibitory activities both clinically and in animal models -51. AccoFurthermore, tumors have a number of specific and non-specific ways of evading a Th1 response. Tumors secrete a number of agents, including transforming growth factor (TGF)-β, IL-10 and prostaglandin E-2, which have been shown to promote a Th2 immune response while suppressing the Th1 immune response. Indeed, it has been shown that the cytokine networks of some cancer patients are skewed toward Th2 ,65,66. TThe Th-1 cytokine IFN-γ has both direct and indirect antitumor properties. A series of experiments have demonstrated the importance of IFN-γ in eradicating incipient tumors by showing an increase in the efficacy of carcinogenesis in the absence of IFN-γ. These experiments were conducted either with neutralizing antibodies to IFN-γ or in animal models deficient in IFN-γ, the IFN-γ receptor or downstream signaling mechanisms of IFN γ, specifically, the signal transducer and activator of transcription-1 protein. In all of these models, an increase in the incidence of MCA-induced tumors and a decrease in the latency period of the tumors were observed -71. FurtIFN-γ may inhibit tumor growth by affecting proliferation, apoptosis and angiogenesis. IFN-γ has been shown to have a direct anti-proliferative effect on certain tumor models -75. It iIFN-γ may also have indirect antitumor effects by stimulating an effective antitumor immune response. In addition to influencing the Th1-Th2 cytokine balance, IFN-γ can activate cytotoxic macrophages, NK cells and NK T cells .The proof of the principle that an inappropriate type of immune response will enhance tumor growth was demonstrated as early as 1907 by Flexner and Jobling, who showed that injection of dead autologous tumor cells enhanced the growth of pre-existing tumors . In geneIn 1972, Richmond Prehn formulated the theory of immunostimulation of tumor growth . This thThis premise is also demonstrated in another study where MCA induction of tumors occurred more rapidly in irradiated thymectomized mice that had received increasing doses of lymphocytes (spleen cells) to partially restore their immune system than in mice that had either fully restored or unrestored immune systems . ComparaFurthermore, in transplantation immunology, an immune response against a graft generally promotes graft rejection. However, rabbits that received skin grafts of similar but not identical major histocompatibility complex launched an immune response that resulted not in rejection but in enhanced growth of the tissue, resulting in hyperplasia . This isCollectively, these data support the essence of the theory of immunostimulation: specifically, that spontaneous tumors may not stimulate an appropriate immune response but rather stimulate non-protective immune responses that quantitatively are not adequate for tumor eradication. These resulting "weak" immune responses are not merely non-protective but actually facilitate tumor growth. Specifically, a "weak" immune response is a state in which immunological recognition of the tumor occurs but resolution is not achieved. As this theory was developed in an era when our understanding of the composition of the immune system was limited, the exact mechanism by which a "weak" immune response stimulates tumor growth has yet to be defined in the terminology of contemporary immunology. Recent data, however, suggest numerous mechanisms by which the immune system can facilitate tumor growth and progression.Based on the observation that a tumor is in a state of chronic inflammation, Dvorak compared cancer to a wound that never heals . NumerouMast cells are leukocytes that contain inflammatory agents, such as proteases, histamine and heparin, and mediate hypersensitivity reactions. The possibility that mast cells are involved in cancer has been considered since the late 1800s. In 1877, Paul Ehrlich described a mast cell for the first time in his doctoral thesis, after identifying it using histological staining. In subsequent investigations, he and others observed that mast cells localize around tumor tissues . Interesv mice (which express a mutation in the SCF receptor) do not develop functional mast cells. In W/Wv mice, tumors do not metastasize as readily and they vascularize at a slower rate than in wild-type mice. Tumor metastasis and vascularization return to wild-type levels following restoration of mast cells [A number of studies show a correlation between mast-cell infiltration and tumor progression. For example, co-injection of mast cells with an inoculum of rat sarcoma tumors results in enhanced tumor growth, while pharmacologically decreasing the quantity of mast cells slows tumor growth . Furtherst cells . Furtherst cells . These sThe mechanisms by which mast cells facilitate tumor progression appear to involve the factors contained within mast-cell granules. Experimental evidence suggests that degranulation is critical in the ability of mast cells to enhance tumor growth. Inhibition of mast-cell degranulation using disodium cromoglycate impedes tumor growth . One of As is often the case in networks, activation of one component induces the activation or suppression of interacting components. Activated mast cells secrete a number of cytokines that induce the chemotactic migration of macrophages into the tumor, including IL-1, IL-6, TNF-α, IL-8, monocyte chemotactic protein-1, macrophage inflammatory protein-1α and macrophage inflammatory protein-1β ,119. TheMacrophage infiltration of tumor sites has been observed in a range of tumor types. Monocytes are recruited to the tumor by constitutive tumor-cell expression of chemoattractant cytokines. In fact, the level of expression of monocyte chemotactic protein-1, macrophage colony stimulating factor (M-CSF) and VEGF by tumor cells correlates well with the extent of macrophage infiltration ,127. WitFollowing exposure to IFN-γ or bacterial lipopolysaccharide, macrophages can exhibit direct or indirect tumor cytotoxicity. Macrophages may participate in antibody-dependent cellular cytotoxicity responses if an IgG2a antibody binds to a tumor surface antigen. In such a scenario, the macrophage would bind to the Fc portion of the antibody, releasing cytotoxic mediators such as proteinases and TNF-α. Macrophages may also mediate cytotoxicity independently of antibodies through the secretion of reactive oxygen and nitrogen species, proteinases and TNF-α. Furthermore, macrophages of this "cytotoxic" phenotype affect the cytokine profile of the microenvironment because they secrete IL-12. IL-12 favors the differentiation of naïve T-helper cells to Th1 cells, which will subsequently secrete IFN-γ and TNF-β. The role of these cytokines in the promotion of a cell-mediated immune response has already been discussed. Finally, cytotoxic macrophages produce matrix metalloproteinase-12 . Matrix In contrast to macrophages that are activated by classical mechanisms and are capable of cytotoxic activity, TAMs, for the most part, do not exhibit cytotoxic activity, are Th1 immunosuppressive and, in fact, facilitate tumor growth, vascularization and metastasis. Tumors secrete a number of cytokines including IL-4, IL-10, TGF-β, prostaglandin E-2 and VEGF. These factors modulate the macrophage phenotype, changing them from cytotoxic macrophages to suppressive macrophages -143. FurA number of studies suggest that TAMs facilitate tumor growth, vascularization and metastases. As previously stated, numerous studies have shown the presence of TAMs to be an adverse prognostic indicator. In addition, genetic evidence suggests that macrophages are important in vascularization and metastasis of experimental tumors. To assess the role of macrophages in carcinogenesis, the M-CSF deficient osteoporotic (op/op) mouse was crossed with the polyoma virus middle T transgenic mouse, which develops spontaneous mammary tumors. While no differences were observed in the early stages of tumor growth, the resulting offspring of these tumor-bearing mice had a reduced rate of progression to invasive carcinoma and contained fewer pulmonary metastases than control mice . FurtherInterestingly, TAMs are not found ubiquitously within tumor tissue. Data suggest that TAMs localize to poorly vascularized regions – that is, regions characterized by hypoxia, low pH and tissue necrosis ,148-150.One mechanism by which TAMs may augment angiogenesis is through the production of a plethora of cytokines and proteinases. As mentioned above, hypoxia induces production of VEGF. The angiogenic properties of VEGF have been extensively discussed by others . In addiFinally, TAMs have numerous indirect immunological effects. TAMs produce copious amounts of IL-10 but low amounts of IL-12 . This shin vivo neovascularization of matrigel [Chemokines are a subclass of cytokines that direct the migration of leukocytes to sites of inflammation. It has recently been observed that tumors express chemokine receptors -161. Thumatrigel . The angmatrigel ,173. Finmatrigel -177.Based on our knowledge of tumor immune evasion and immune contribution to growth of tumors, several therapeutic implications arise including: a) Use of adjuvant therapies to decrease tumor immune suppression while vaccination, b) Extracorporeal elimination of immune suppressive molecules; and c) Gene silencing of tumors to generate immunity.in vitro as well as in vivo [The armamentarium of clinically useful drugs for inhibition of tumor immunity is rapidly growing. The challenge is to identify drugs that intrinsically possess antitumor activity, while at the same time can reverse immune stimulation. One promising candidate that fits these criteria is the clinically used anti-VEGF antibody bevacizumab (Avastin). Currently approved by the FDA for treatment of advanced colon cancer patients, this antibody is believed to mediate its effects primarily by inhibition of angiogenesis . From th in vivo .Vaccination with the carboxy-terminal peptide of hCG has been shown to break tolerance to this self-molecule and induce a marginal anticancer response . CombiniAnother method of reversing tumor associated immune suppression would involve extracorporeal removal of inhibitory molecules. Although plasmapheresis techniques have been attempted with mediocre success , a novelThe introduction of RNA interference (RNAi) as a potent method of gene-specific silencing has opened new territories for gene therapy. Successful use of RNAi for immune modulation was first reported by Hill et al . SubsequThe debate on the role of the immune system in cancer has been one of the most controversial areas of science with opinions vacillating from optimistic highs to skeptic nadirs on a cyclical basis. These oscillations in opinion that are reflective of the relative progress in the immunotherapy of cancer, can be explained by the complexity of the relationship of the immune system and cancer – an interaction that can result in anti-tumor protection, tumor promotion, or in no net effect. The apparent schizoid role of the immune system in cancer may in fact be a variation on a theme borrowed from the immunology of infectious disease. That is, the mere generation of an immune response is not sufficient to attain protection against a pathogen: it is only an immune response of the proper type and the proper magnitude that will result in protection. An immune response against an agent that is not of the proper type and magnitude will be deleterious to the host.A proper type of immune response against cancer will result in protection. Evidence exists that in set conditions components of the immune system can recognize and eradicate incipient cancer cells. It is likely that some sort of immune surveillance exists in the healthy individual – though the exact mechanisms have yet to be elucidated. The genetic evidence discussed herein suggests that NK cells and IFN-γ are likely to be involved in this protection.Nevertheless, the clinical presentation of cancer suggests that neoplasia can evade these putative mechanisms of immunosurveillance. This further suggests that the ensuing immune response is not protective, or not adequately protective, to control the nascent tumor. However, this lack of a protective response is not the whole extent of the role of the immune system in cancer. Several examples have been presented in which the immune response actually facilitates tumor growth, neovascularization and progression. These observations suggest a mechanism that may explain the theory of immunostimulation, though undoubtedly, many other mechanisms may exist.This duality of the immune system in cancer arises in part because of the decentralized nature of the immune system. The non-protective immune reaction that arises resembles a bureaucratic organization in which a lack of orchestration between departments results in redundancy and counter-productivity: the individual entities that constitute the immune system react to the presence of the tumor yet they are not orchestrated to achieve the end point of tumor eradication.Secondly, in advanced tumors, the duality of the immune system in cancer may also arise from the bilateral nature of the interaction between the tumor and the immune mechanisms of the host. Not only can the immune system affect the tumor, but the tumor may also affect the immune system. The skewed cytokine milieu of a tumor, which results in the recruitment of inflammatory cells through the secretion of chemokines and growth factors and which further prevents the cytotoxic activation of these cells, is an example of how the tumor influences the immune system. This effect of the tumor on the immune system may be explained by the theory of immunoediting, which describes the iterative process of selection for cells that can evade the natural immunosurveillance mechanisms . This seAkin to the successful development of vaccines against infectious agents, the development of effective immunotherapy against cancer will require recognition of the duality of the immune response in cancer, and the stimulation of the proper type of immune response. The challenges ahead, especially in late-stage patients who have undergone immunoediting, will be to overcome the tendencies of the tumor to elicit an inappropriate immune response, one used by the tumor to promote its own growth. It may be the case that in subsets of patients the sort of immunotherapy that is desirable is not that which stimulates the immune system but that which suppresses an inappropriate, tumor promoting, immune reaction ,195. CheChemotherapy both directly and indirectly affects the immune system; however, given the multiplicity of the functions of the immune system in cancer, the net effect of this treatment modality remains to be defined. While the direct effect of chemotherapy may be myelosuppression, this may not be an undesirable consequence given that cells of the myeloid lineage, such as mast cells and macrophages, often promote the growth, vascularization and invasion of tumors. Furthermore, myelosuppression may actually stimulate a protective, Th1-type, cell mediated immune response because mast cells and macrophages secrete factors that promote a Th2 type of immune response."If only it were all so simple." If only the immune system were all good and it were necessary only to stimulate it to achieve the eradication of tumors. But the potential for good and evil is entwined within the heart of the immune reaction, and who is willing to destroy their own heart?


MicroRNAs (miRNAs) are small RNAs of 21–25 nucleotides that specifically regulate cellular gene expression at the post-transcriptional level. miRNAs are derived from the maturation by cellular RNases III of imperfect stem loop structures of ~ 70 nucleotides. Evidence for hundreds of miRNAs and their corresponding targets has been reported in the literature for plants, insects, invertebrate animals, and mammals. While not all of these miRNA/target pairs have been functionally verified, some clearly serve roles in regulating normal development and physiology. Recently, it has been queried whether the genome of human viruses like their cellular counterpart also encode miRNA. To date, there has been only one report pertaining to this question. The Epstein-Barr virus (EBV) has been shown to encode five miRNAs. Here, we extend the analysis of miRNA-encoding potential to the human immunodeficiency virus (HIV). Using computer-directed analyses, we found that HIV putatively encodes five candidate pre-miRNAs. We then matched deduced mature miRNA sequences from these 5 pre-miRNAs against a database of 3' untranslated sequences (UTR) from the human genome. These searches revealed a large number of cellular transcripts that could potentially be targeted by these viral miRNA (vmiRNA) sequences. We propose that HIV has evolved to use vmiRNAs as a means to regulate cellular milieu for its benefit. Caenorhabditis elegans as regulators of temporal control of post-embryonic development [Initially discovered in elopment ,2, miRNAelopment ,4. To daelopment . Structuelopment . Accordielopment . Mechanielopment -10.r et al. identified virus-encoded miRNA sequences in Epstein-Barr virus (EBV) infected cells [Recently, in addition to plants, insects, invertebrate animals, and mammals, Pfeffeed cells . They retrans-activation responsive RNA) and RRE (Rev-responsive element) [StemEd [gag, near the gag-pol frameshift, in the nef gene, and in the 3'LTR [We wondered if HIV-1 maintains RNA structures that resemble pre-miRNAs. As a proof-of-principle, we examined pre-miRNA structures in one specific example of HIV-1, the genome of the pNL4-3 molecular clone. Because HIV has well-described stem-loops such as TAR (element) , one mig [StemEd , we uncohe 3'LTR ,15. The The 5 HIV-1 encoded pre-miRNA candidates can in principle yield 10 mature vmiRNAs. To ask, whether these putative vmiRNAs, if expressed in infected cells, could be used by HIV-1 to modulate host cell gene expression profiles (i.e. suppress the translatability of cellular mRNAs), we checked each vmiRNA sequence against a 3'UTR database for human genes. Because the exact rules governing suppression of translation based on miRNA complementarity to 3' UTR remain unclear, we collected all "hits" that had 6 or fewer mismatches with an additional constraint that the 5'-most nucleotide of the vmiRNA cannot be mis-matched with the target sequence. Surprisingly, based on the above criteria, a very large number of cellular targets were found Figure . On averHere, we introduce the concept that the HIV genome could reasonably encode 5 candidate pre-miRNAs. We further suggest that a large number of cellular transcripts could potentially be targeted if these 5 pre-miRNAs were processed into 10 predicted mature vmiRNAs Figure . StudiesThe authors declare that they have no competing interests.YB and KTJ conceived of the ideas and wrote the paper. SYL did the computation work for the study. MLY participated in the discussion of the data.


The objectives of this report are to review the assessment of patient-focused outcomes in pediatric orthopedic surgery, to describe a framework for identifying appropriate sets of measures, and to illustrate an application of the framework to a challenging orthopedic problem.A detailed framework of study design and measurement factors is described. The factors are important for selecting appropriate instruments to measure health status and health-related quality of life (HRQL) in a particular context. A study to evaluate treatment alternatives for patients with neurofibromatosis type 1 and congenital tibial dysplasia (NF1-CTD) provides a rich illustration of the application of the framework. The application involves great variability in the instrument selection factors. Furthermore, these patients and their supportive caregivers face numerous complex health challenges with long-term implications for HRQL.Detailed summaries of important generic preference-based multi-attribute measurement systems, pediatric health profile instruments, and pediatric orthopedic-specific instruments are presented. Age-appropriate generic and specific measures are identified for study of NF1-CTD patients. Selected measures include the Activities Scale for Children, Gillette Functional Assessment Questionnaire Walking Scale, Health Utilities Index, and Pediatric Inventory of Quality of Life.Reliable and valid measures for application to pediatric orthopedics are available. There are important differences among measures. The selected measures complement each other. The framework in this report provides a guide for selecting appropriate measures. Application of appropriate sets of measures will enhance the ability to describe the morbidity of pediatric orthopedic patients and to assess the effectiveness of alternative clinical interventions. The framework for measurement of health status and HRQL from a patient perspective has relevance to many other areas of orthopedic practice. Assessments of patient-focused health status and health-related quality of life (HRQL) are being recognized increasingly by clinicians, patient advocates, regulatory authorities, administrators and policy makers as primary measures of the need, efficacy, effectiveness and efficiency associated with health care services. These types of measures are commonly reported in the published literature of many health care disciplines although there are few published reports concerning orthopedic surgery. However, the orthopedic community is becoming interested in using evidence reported by patients and patients' family members to determine what is best for patients.Patient-focused evidence (including patient-reported outcomes, PROs) refers to results from functional health status (FHS) and HRQL studies of orthopedic outcomes with measurements obtained from the perspective of patients or their non-clinician caregivers (e.g., parents of patients too young to self-report). The field of orthopedics is concerned with all musculo-skeletal problems from the top to the bottom of the human body. This broad anatomical range is associated with a wide range of functional problems. Some orthopedic problems are highly focused in regards to anatomy and associated with highly effective therapies. However, other orthopedic problems are of a diverse nature, the etiology is often poorly understood, and effective treatment strategies are sometimes elusive.The breadth and depth of some particularly challenging orthopedic problems has stimulated an interest in patient-based perspectives of their own FHS and HRQL. These types of measures will be referred to as "health measures" for the purposes of this paper. Health measures can be used to assess the burden of morbidity associated with orthopedic diagnoses, and to assess the effectiveness and efficiency of therapies. The diversity of issues also led the American Academy of Orthopedic Surgeons and the Pediatric Orthopedic Society of North America to develop the Pediatric Outcomes Data Collection Instrument (PODCI), and led other researchers to develop function-specific instruments such as the Activities Scale for Kids (ASK). However, there are a large variety of potentially appropriate health measures and there are limits to the number of these measures that can be used in any given study. The most appropriate set of health measures should be selected on the basis of underlying study objectives and design criteria .a priori specification of the comprehensive measure of health and a credible assessment viewpoint for the purposes of primary analysis of study results. Secondary study objectives may involve use of data collected from other health measures or from other assessment perspectives.Objectives and designs vary greatly across orthopedic studies. In general, orthopedic procedures and programs are undertaken to improve the health of patients. The health of patients can be measured in different ways. Conventional clinical or physiological measures are generally very useful for diagnostic and therapeutic purposes. Conventional clinical measures may, however, provide an incomplete assessment of health. For example, clinical examination and gait measures have been used to describe the quality and quantity of limitations in walking ability associated with hip flexion contracture but these types of assessments provide little information about the importance of this contracture relative to that of other serious health problems. In addition, various measures may provide conflicting results: some indicating improvement while others suggest decline in patients' health. Therefore, in the interest of scientific rigor, protocols to assess the overall effectiveness of therapy should include FHS and HRQL measures are important for a variety of reasons that complement conventional clinical measures . FHS mea"Health-related quality of life is the value assigned to duration of life as modified by the impairments, functional states, perceptions, and social opportunities that are influenced by disease, injury, treatment or policy" .It is generally accepted that a goal of therapy is to make patients feel better  and thisA series of studies from a randomized controlled clinical trial (RCT) and prospective cohort study of elective total hip arthroplasty are illustrative of these purposes. These examples, while not from a pediatric setting, are orthopedic and demonstrate how data can be used for a variety of purposes. In the RCT, Rorabeck and colleagues  undertooThere are many factors to consider when designing studies using patient-focused health measures for orthopedic patients. Among the most important factors are the study objectives, types of patients, the dimensions and FHS constructs associated with the health problem under study, the appropriateness of the questionnaire for the age range of the patients, the viewpoint of the assessors answering the questionnaire, the method of collecting questionnaire information (self-completion or interviewer-administration), the period of time respondents are asked to consider when answering the questionnaire , and measurement properties of assessment tools. Measurement properties include content and construct validity, test-retest and inter-rater reliability, responsiveness, and practical limitations of data collection. It takes many years, and often decades, for credible and relevant evidence to be accumulated about the measurement properties of instruments. Instruments with well-established measurement properties should be used whenever possible. Two prime issues for assessments of children are the developmental stage of the subjects and the most appropriate respondent for questionnaires.The methods section of the paper presents a framework of important factors for consideration in designing studies to assess FHS and HRQL for orthopedic patients. The results were generated by applying the framework in the context of developing a proposal for a comprehensive international study of patients with neurofibromatosis type 1 (NF1) and congenital tibial dysplasia (CTD). The NF1-CTD protocol provides a rich illustration because it encompasses great variability in the instrument selection factors, and these patients with their supportive caregivers face numerous complex health status challenges with long-term implications for HRQL. Furthermore, there is little information in the published literature about the comprehensive health status and HRQL of patients with NF1-CTD. The rationale and approach to studying FHS and HRQL from a patient perspective have relevance to many other areas of orthopedics.This section identifies specific study design and measurement instrument factors that should be considered in selecting measures for use in studies, and presents some background information for illustrating the application of these factors.Study design factors are specified in detailed protocols that clearly define the study objectives and conditions. Well-defined study objectives identify the study subjects, and type and number of measurements required. Study conditions describe practical issues including data collection sites, and budgets for time and resources.A highly focused, single objective study may require relatively few instruments while a broad-based, multi-faceted study will require numerous measures. For example, a RCT of efficacy for an experimental technique to achieve union of bone compared to a conventional technique might specify patients' reports from a single well-established walking ability scale as the primary outcome measure. However, an economic evaluation of the cost-effectiveness and cost-utility of the experimental technique would require at least two instruments: the single well-established walking ability scale to estimate effectiveness for the numerator in the cost-effectiveness ratio; and a utility-based scale of overall HRQL to estimate quality-adjusted life years for the numerator in the cost-utility ratio.The age of study subjects affects the choice of measurement instruments and the method of collecting data. Each instrument is valid for a specific age range and the age range is quite small for many pediatric instruments. For example, developmental changes during childhood make it difficult for single instruments to be valid from infancy through adolescence. Many instruments are valid for one or more of the following age categories: infants, pre-school children, primary-school children, adolescents, young / middle-age adults, seniors and elderly. Inability to read well (e.g., young children or populations with high illiteracy rates), or see well (e.g., many elderly populations), or concentrate well (e.g., people on certain strong medications) inhibits use of self-complete questions. Well-designed interviewer-administered questionnaires pose clear, short, well-focused questions with readily understood and easily remembered response options (e.g., yes or no response options).It is becoming widely accepted in many disciplines that studies of health care programs and technologies should include measures of patients' perceptions of their FHS and HRQL . MeasureThe locations and cultural characteristics of the study population will determine the language and sometimes the choice of assessors. Relatively few instruments have been carefully translated and culturally-adapted to facilitate use in a large variety of communities. Some cultures accept a variety of assessment viewpoints  while other cultures recognize only a physician viewpoint for health assessments.The literature suggests that there may be important effects due to mode of data collection  and, theFHS questionnaires should have well-specified assessment periods to help ensure that the subjects and researchers know the period of time covered by the responses. Assessment recall period refers to the period of time the assessor is asked to consider when answering the questionnaire. The period should match the objectives of the study. For instance, if a new surgical technique is thought to reduce the peri-operative burden of morbidity then frequent assessments with a brief recall period (24 hours) might be suitable. Standard assessment periods include the past 24 hours, the past 1 week, the past 2 weeks and the past 4 weeks. Short assessment periods should be used in studies of patients whose FHS varies over time and in studies involving serial assessments to ensure that there is no overlap in assessment times. Relatively long recall assessment durations may be used when it can be assumed patients' health status is fairly stable. For example, a four-week recall assessment period was used to measure the HRQL of patients in a randomized clinical trial and economic evaluation of two alternative treatment strategies for patients with knee osteoarthritis ,16.There are limits to the number and type of measures that respondents can be expected to complete without getting tired or frustrated, and/or that the study budget can afford in regards to both data collection and analysis. The evidence about the limits of respondent burden is sparse. All else being equal, studies involving serial assessments should expect to collect fewer measurements per assessment than single-assessment cross-sectional surveys. To maximize efficiency, instruments should be selected to provide complementary rather than overlapping information.There are numerous definitions of FHS and HRQL. For the purposes of the paper, a FHS measure is defined as being descriptive in terms of functional ability and HRQL is defined as involving some form of valuation of that health status.One published taxonomy  suggestsEach multi-attribute system includes a descriptive classification scheme to describe and assess health status, and a preference-based valuation system. HRQL scores for health states defined by multi-attribute systems are calculated from models fitted from directly measured preference measurements (see below). Multi-attribute systems provide descriptive information about comprehensive health status, and interval-scale preference scores of overall HRQL from a community perspective on a scale where 0.00 is the score for being dead and 1.00 is the score for being in perfect health. Several multi-attribute systems define negative scores of overall HRQL to represent preferences for states considered worse than being dead. A few systems include single-attribute preference-based scales of morbidity. Single-attribute morbidity scales are defined such that the least desirable level within an attribute  has a score of 0.00 (blind) and the most desirable level has a score of 1.00. The community perspective is most widely recommended for technology assessment and reference case economic evaluation analyses -29. InteA valid measure is "sound and sufficient" . There aA measure is reliable if it is sound and dependable . ReliabiResponsiveness is also referred to as sensitivity to change. It is an important feature for determining a measure's ability to detect effects of treatments or natural changes over time (e.g., due to the aging process). Husted and colleagues reviewed the literature and defined two major types of responsiveness: internal and external responsiveness . InternaCeiling and floor effects are undesirable properties that reduce the validity, reliability, and responsiveness of measures. A ceiling effect may occur when a large proportion of measurement observations are close to the upper bound of the measurement scale. A ceiling effect results in a positively skewed distribution of measurements, limited ability of the measure to discriminate among subjects at the upper end of the scale, and attenuated responsiveness to improvements in health in longitudinal studies. A floor effect may occur when a large proportion of measurement observations are close to the lower bound of the measurement scale. Floor effects create a negatively skewed distribution of measurements, limited ability of the measure to discriminate among subjects at the lower end of the scale, and decreased responsiveness to decrements in health in longitudinal studies. Many generic and specific measures of HRQL may be subject to ceiling effect problems in that they may not be able to describe patients or subjects with above average  health. Some measures are subject to floor effect problems. Some are subject to both. Typically floor effect problems are more serious in clinical studies (which often involve patients with disabilities) and ceiling effect problems may be more problematic in general population studies.The limits of respondent burden depend upon many factors including the number of questions presented, how the questions are presented, the complexity of questions, the sophistication of respondents, and the respondents' interest in the questions. In general, the allowable length of questionnaire is shorter for mail and phone administration than for face-to-face interviewer administration . One setApplications of FHS and HRQL measures are greatly facilitated by expert advice, detailed instructions and other services designed to support users of a measure. Supporting documentation is usually protected by copyright and should not be used without written permission of the original developers. Documentation obtained from third-party sources should be considered suspect because it is frequently invalid. Licensing fees are used to fund high quality, readily accessible service centers. Permission to use copyright materials is typically granted one study at a time. Support services may also include consultation about the most appropriate versions of questionnaires for use in a specific study. Application packages may include data collection instruments such as questionnaires, procedure manuals, coding algorithms and scoring systems, as well as background information about the conceptual and measurement properties of the instrument.Recently there has been interest in using measures of FHS and HRQL to evaluate treatment alternatives for NF1-CTD. NF1 is one of the most common genetic disorders in childhood . It is eCTD is rare in the general population, approximately 1 per 140,000 . It has Conventional clinical measures of CTD include the Crawford classification system . These mSurveys of the published literature, experts in the fields, web sites and other sources of information were conducted to determine the dimensions of health that are affected by NF1-CTD, the types of FHS and HRQL measures that have been used, which measures should be considered as potentially useful for studies of NF1-CTD, the measurement properties of potentially useful measures, and the relative merits of various measures. A review of the on-line Quality of Life Instruments Database (QOLID) developed by Dr. Marcello Tamburini and the MAPI Research Institute , and corThere are five important research objectives of an NF1-CTD study that provide a context for applying the framework described in the Methods section:1) to document long-term health outcomes associated with the disease and its treatment;2) to measure the burden of disease and treatment during active therapy;3) to investigate the hypothesis that improved HRQL is associated with initial amputation compared with multiple limb-saving procedures;4) to determine relationships of FHS and HRQL with conventional clinical variables used in diagnosis and management; and5) to assess the measurement properties  of selected FHS and HRQL measures in NF1-CTD patients.These detailed objectives require the identification and assessment of leading FHS and HRQL measures for use in both cross-sectional and prospective longitudinal surveys.The prevalence of NF1-CTD is relatively low. Patients will need to be recruited from numerous clinical centers in North America to generate precise estimates of FHS and HRQL. Questionnaires should be available in at least 3 major languages: English, French and Spanish. The survey population ranges in age from newborn into adulthood and linking results across the study objectives requires that at least some of the assessment tools be in common across the age range of study patients. To avoid potential confounding effects, data collection techniques should be consistent across subjects and measures.The patient-focus will be represented by collecting measurements from all patients old enough to provide self-assessments, and from parents acting as proxy assessors for all children and adolescents. Self-complete questionnaires requiring minimal supervision should be used to eliminate the need for interviewers at each clinical center, to facilitate use of mail-out surveys, and to avoid potential "interviewer" effects. The number and type of measures per assessment, and the number of serial assessments per patient, should be sufficient to address all the study objectives within the limits of study resources and assessor burden. Measures of morbidity associated with NF1-CTD should be comparable with data on norms from surveys of general populations and other patient groups, and be useful for assessing the effectiveness and efficiency of health care services.The HRQL measure should be comprehensive and preference-based, to facilitate a broad variety of comparisons. A pediatric health profile measure and other specific measures will be selected to complement the selected preference-based HRQL measure. FHS measures may be focused on one or more of the following: the population of interest (e.g., pediatrics); the major underlying disease (e.g., NF1); the major human function of most interest ; the medically-defined health problem of most interest ; the medical speciality most involved with treatment of the health problem (e.g., pediatric orthopedics).There are six major generic preference-based HRQL utility systems , presentThe major population-specific health profiles include the Child Health Questionnaire (CHQ), Pediatric Inventory of Quality of Life (PedsQL), Pediatric Evaluation of Disability Inventory (PEI) and TNO-AZL Pre-School Children Quality of Life questionnaire (TAPQOL). The PEI is limited to children age 0.5 – 7 years of age and requires a structured parent interview or clinician observation . TAPQOL The major pediatric disease-/function-/specialty-specific instruments include the PODCI , ASK, Gillette Functional Assessment Questionnaire Walking Scale , and Wee-FIM. Wee-FIM , a populA summary of the major pediatric generic health profiles appears in Tables Table The choice of existing measures is based on a process of elimination considering the relative strengths of each instrument and the complementarities among measures. Neither the SF-36 nor the EQ-5D is valid for use in adolescent patients with orthopedic problems . A revieIn summary, there are few measures available for assessing subjects less than 5 years of age and even fewer for subjects less than 2 years of age. Most relevant measures are available in self-complete format only. Preliminary recommendations for the NF1-CTD study were that PedsQL be used as the generic health profile, HUI be the multi-attribute preference-based measure of HRQL utility scores for children age 5 years and older and that CHSCS-PS be the measure for children age 2 through 4 years, ASK be the measure of activity limitation, FAQ walking scale be the measure of walking ability, and that a small feasibility study of these instruments be completed with a convenience sample.A pilot feasibility study surveyed 8 NF1 patients using HUI and FAQ walking scale measures. Questionnaires were completed by 6 NF1 patients and 3 parents. The combined HUI and FAQ walking scale questions took respondents an average of 13 minutes (range, 9–20 minutes) to complete. The patients were 11 to 50+ years old and had health problems ranging from mild to severe. One patient with tibial dysplasia and 2 patients with scoliosis were included.HUI data were collected from 5 patients, 2 parents and both the patient and parents in one case. Health problems were reported in 7 of the 8 HUI3 attributes . The attributes associated with the most morbidity, as assessed using HUI3 single-attribute utility scores , were paFAQ walking scale data were collected from 4 patients (1 of the 5 survey patients did not answer the question), 2 parents and both the patient and parents in one case. Five patients were reported to be at Level 10 , one patient to be at Level 8 , and one patient at Level 6 .In summary, the feasibility study showed that the HUI and FAQ walking scale questions were acceptable to patients' families and that results, especially for HUI, reflected the large variability in HRQL of the sample of patients.No single measure will provide sufficient data to address all the important study objectives. A set of measures is required. The set of measures should provide complementary data of health status and preference-based scores of HRQL. Redundancy in measurement is reduced, and efficiency of measurement is increased, by selecting the most comprehensive generic measures and then supplementing these measures with the most appropriate set of specific measures.It is recommended that HUI be selected as the comprehensive generic measure for ten reasons:a) it includes both generic health profile and preference-based scoring systems;b) the preference-based scoring systems are well-validated;c) it is the most comprehensive, compact and efficient of these types of systems;d) it includes many of the most important domains in the context of NF1-CTD;e) it is applicable for all people age 5 years and older;f) well-developed data collection questionnaires are available to match the study design criteria;g) HUI results facilitate integrating effects of morbidity and mortality, and cost-utility economic evaluations;h) it has been used successfully in a variety of studies of musculoskeletal problems;i) population norm data are available; andj) a closely-related health status system, the CHSCS-PS, is available to assess children 2 through 4 years of age.The HUI will provide a broad set of measures for comparisons with other populations and for estimating HRQL on a general scale such that dead = 0.00 and perfect health = 1.00. As a generic measure, HUI also has the ability to capture side effects and the effects of co-morbidities. However, these broad measures may not be responsive to small but important changes in health status. Therefore, HUI should be complemented by a set of instruments focused on pediatric, orthopedic and walking issues.The PedsQL 4.0, a pediatric generic health profile, should also be included in the set of measures because:a) it includes domains, social and school function, which complement HUI and CHSCS-PS domains;b) it is appropriate for children ages 2 through 18 years;c) it is not overly burdensome in terms of data collection;d) patient and parent assessment questionnaires are available; ande) it can be interviewer-administered to facilitate data collection by telephone, if necessary.Two specific measures should also be part of the set of instrumentation: the ASK and the FAQ walking scale. ASK is an orthopedic-specific instrument which has been shown to cover the most important domains in the context of musculoskeletal disorders, including the impact of limb lengthening surgery experienced by many children with tibial dysplasia. ASK is also attractive because it provides overall summary scores for both performance and capability measures, and is only moderately burdensome to complete. Walking ability is one of the most important aspects of health that is frequently compromised in NF1-CTD patients, and the FAQ walking scale is the most complete scale of functional walking ability currently available and it only requires asking one question.CHQ is not recommended because it does not add much to the set of recommended measures and it is burdensome to complete. PODCI is not recommended because it is very burdensome to complete, it has been reported to have major problems with "missing data", and the system for collapsing questionnaire responses into summary scores is not well validated.Children ages 2 to 5 years of age should be assessed by their parents using three questionnaires: the CHSCS-PS (12 questions), the PedsQL (23 questions); and the FAQ walking scale (1 question). It is expected that all three of these questionnaires can be completed in an average of 15 minutes.Children and adolescents ages 5 to 17 years of age should be assessed by their parents using four questionnaires: the HUI (15 questions), the PedsQL (23 questions), the FAQ walking scale (1 question), and the ASK (30 questions). These four questionnaires are expected to be completed in an average of 20 to 30 minutes.Children and adolescents older than 11 years should provide self-assessments using four questionnaires: the HUI (15 questions), the PedsQL (23 questions), the FAQ walking scale (1 question), and the ASK (30 questions). On average, it is expected that respondents will complete all four questionnaires in 20 to 30 minutes.This paper highlights reasons why patient-focused measures of FHS and HRQL should be considered important tools in the field of orthopedic surgery. It has also noted that there is increasing competition for scarce health-care resources, that allocation decisions about these resources are being informed by evidence based on patient-focused health measures, and that these measures are being under-utilized by the orthopedic surgery community.The orthopedic community faces numerous obstacles in utilizing FHS and HRQL measures. One major obstacle is that the multitude of existing measures makes it difficult to decide which measures may be appropriate for a specific application. A second obstacle is that most of the information about FHS and HRQL measures is not reported in the orthopedic literature. A third obstacle is that usually no one measure can capture all the important aspects associated with a specific orthopedic issue. The framework outlined in the paper provides guidance for selecting appropriate FHS and HRQL measures. The framework guides orthopedic investigators to combine their basic study criteria, including objectives and clinical context, with key criteria for FHS and HRQL measures from the published literature.The results in this paper identify some major sources of information about health measures, identify some of the most widely used measures of FHS and HRQL, and provide summaries of key characteristics for selected measures in three major taxonomical classes: generic preference-based multi-attribute systems; generic pediatric health profile systems; and orthopedic-specific systems. It is clear that there are many important differences among measures both within and across taxonomical classes. All measures are not equal. There are sound factors for making judgements about which measures are most appropriate for a given application. A process of appraisal and elimination was used to select one measure from each taxonomical class for inclusion in the NF1-CTD study illustrative example, and a pilot study of the most readily available selected measures confirmed the feasibility of their use in a small sample of NF1-CTD patients.The paper shows that a set of relevant, valid, reliable, responsive and practical patient-focused health measures for use in an orthopedic study can be readily identified and selected from the published literature and information available on the worldwide web. We encourage orthopedic researchers to use the framework to identify and select appropriate patient-focused health measures in their future studies.®) instrumentation and provides methodological advice on the use of HUI.W. Furlong and D. Feeny have a proprietary interest in Health Utilities Inc. which distributes copyright Health Utilities Index (HUIAll authors were involved in critical review of drafts for intellectual content and have given final approval to the version submitted for publication. W Furlong was responsible for much of the overall design, acquisition of data, and initial drafting. R Barr and D Feeny made important contributions to the analysis and interpretation of results. S Yandow conceived the idea of presenting the information in a published manuscript.


Caenorhabditis elegans was previously shown to be a relevant model organism for several late endosomal/lysosomal membrane proteins associated with LSDs. The aim of this study was to identify and characterize C. elegans orthologs to both human lysosomal luminal proteins α-GAL and α-NAGA.Human α-galactosidase A (α-GAL) and α-N-acetylgalactosaminidase (α-NAGA) are presumed to share a common ancestor. Deficiencies of these enzymes cause two well-characterized human lysosomal storage disorders (LSD) – Fabry (α-GAL deficiency) and Schindler (α-NAGA deficiency) diseases. C. elegans gene (R07B7.11) with homology to both human genes  – gana-1. We cloned and sequenced the complete gana-1 cDNA and elucidated the gene organization.BlastP searches for orthologs of human α-GAL and α-NAGA revealed a single Phylogenetic analyses and homology modeling of GANA-1 based on the 3D structure of chicken α-NAGA, rice α-GAL and human α-GAL suggest a close evolutionary relationship of GANA-1 to both human α-GAL and α-NAGA.C. elegans mixed culture homogenates. However, α-GAL activity on an artificial substrate was completely inhibited by the α-NAGA inhibitor, N-acetyl-D-galactosamine.Both α-GAL and α-NAGA enzymatic activities were detected in ::GFP fusion protein expressed from a transgene, containing the complete gana-1 coding region and 3 kb of its hypothetical promoter, was not detectable under the standard laboratory conditions. The GFP signal was observed solely in a vesicular compartment of coelomocytes of the animals treated with Concanamycin A (CON A) or NH4Cl, agents that increase the pH of the cellular acidic compartment.A GANA-1Immunofluorescence detection of the fusion protein using polyclonal anti-GFP antibody showed a broader and coarsely granular cytoplasmic expression pattern in body wall muscle cells, intestinal cells, and a vesicular compartment of coelomocytes.gana-1 by RNA interference resulted in a decrease of both α-GAL and α-NAGA activities measured in mixed stage culture homogenates but did not cause any obvious phenotype.Inhibition of C. elegans ortholog of both human α-GAL and α-NAGA proteins. Phylogenetic, homology modeling, biochemical and GFP expression analyses support the hypothesis that GANA-1 has dual enzymatic activity and is localized in an acidic cellular compartment.GANA-1 is a single  Humans have two enzymes with α-galactosidase activity and an acidic pH optimum, α-N-acetylgalactosaminidase (α-NAGA)  and α-galactosidase A (α-GAL). Hereditary deficiency of each of the hydrolases causes a distinct lysosomal storage disorder in humans, Schindler and Fabry diseases, respectively ,2.Early studies suggested that both human enzymes were glycoforms with similar substrate specifities. Purified enzymes had similar physical properties, including subunit molecular mass ~46 kDa), homodimeric structure, and amino acid sequences. However, additional studies showed kinetic, structural, and immunologic differences proving that α-GAL and α-NAGA were products of two different genes ,4. The t6 kDa, ho8 barrel structure, a domain fold observed commonly in glycosidases. Domain II has eight antiparallel β strands, packed into two β sheets in a β sandwich fold containing a Greek key motif [Both enzymes belong to the glycoside hydrolase family 27 clan D . GlycosiThe physiological importance of both enzymes is evidenced by the severe presentation of α-NAGA and α-GAL deficiencies in humans ,2.m [Our recent study on degradation of blood group A and B glycolipids in Fabry cells indicated a high residual activity in Fabry cells toward natural substrate glycolipid B-6-2  althoughm ,12. Its m .C. elegans α-GAL and α-NAGA ortholog (gana-1) to both human genes. We present evidence suggesting that this gene has indeed evolved from the α-GAL/α-NAGA ancestral gene before the duplication event which resulted in separate α-NAGA and α-GAL genes in higher metazoans. We further performed structural analysis of the GANA-1 3D model acquired by homology modeling. We determined the spatial and temporal expression of the gene in transgenic worms using a translational reporter and examined the effect of RNA interference (RNAi) as a first step in the possible use of C. elegans as a model organism for Schindler and Fabry diseases.We investigated the phylogenesis of a single C. elegans genome [gana-1 (R07B7.11) that has sequence similar to human genes encoding α-GAL and α-NAGA. Similar results were obtained from searching the available C. briggsae genome sequence [gana-1 gene consists of 5 exons and 4 introns and is annotated as an ortholog of human α-NAGA. Several EST clones for this open reading frame (ORF) have been reported and open-reading-frame sequence tag (OST) is present in the Worfdb database [The complete s genome ,15 contadatabase . Availabgana-1 and the two predicted downstream genes R07B7.12 and R07B7.13. Although this region has not been reported as an operon, the physical distances between this cluster of three genes are suggestive of an operon [gana-1 cDNA.We verified the gene structure by sequencing the PCR products from genomic DNA and cDNA Figure . The anan operon -19. No an operon . We noteAnopheles gambiae GAL (46%), the lowest similarity was observed with Streptomyces avertimilis GAL (22%). The results of our phylogenetic analysis are in accordance with generally accepted evolutionary concepts. The analysis identified four main clades: animal NAGAs, animal GALs, plant/lower organisms GALs and the clade containing sequences of Drosophila melanogaster, Anopheles gambiae and Caenorhabditis elegans. The branch including C. elegans is positioned between higher animal GALs and NAGAs and plant/lower organisms GALs. This position in the tree infers the evolutionary ancestrality of gana-1 gene to both animal GALs and animal NAGAs. However, this conclusion is not in complete agreement with the presence of pairs of genes in Drosophila and Anopheles genomes annotated as α-GALs and α-NAGAs. The presence of these genes in the Caenorhabditis/Drosophila/Anopheles branch (and not in the GAL and NAGA clades) could be due to low divergence of these sequences from a common ancestral gene or to independent gene duplication in the Drosophila/Anopheles ancestral organism. It is also important to note that the phylogenetic analysis by maximum parsimony algorithm placed the Caenorhabditis/Drosophila/Anopheles branch into the neighborhood of the animal NAGAs branch [We aligned GANA-1 protein sequence with other melibiase family members Figure  and conss branch . In thisC. elegans. In this case the enzyme found in C. elegans would probably be the ortholog of human α-NAGA and the lost gene would likely be the ortholog of human α-GAL. The likelihood of these two hypotheses depends on functional divergence of duplicated gene products and their dispensability for organism's metabolic pathways [In our opinion, the phylogenetic analysis provides evidence that the GANA-1 evolved from a common ancestor of α-GAL and α-NAGA enzymes. However, the topology of the tree could also be explained by a loss of the second gene during the evolution of pathways .8 barrel structure which represents a common motif in many glycoside hydrolases. Less conserved is domain II that has a β domain with β sandwich structure containing a Greek key motif. The active site pocket of GANA-1 is formed by the same twelve amino acids (W31, D76, D77, Y118, C126, K152, D154, C156, S186, A189, Y190 and R211) , found between GANA-1 backbone atoms and the chicken α-NAGA template , was 0.5) Figure  as in chResidues forming the "N-acetyl recognition loop" in the chicken α-NAGA  (S172, AAccording to Garman  the key Standard bacteria/nematode separation protocol previously used by other authors ,23 whileC. elegans N2 strain using 4-methylumbelliferyl (MU) substrates. The α-NAGA activity was dominant over the α-GAL activity. The activity of α-NAGA measured at 37°C was 430 nmol.mg-1.h-1 with MU-α-N-acetylgalactosaminide compared to the activity of α-GAL with MU-α-galactopyranoside of 43 nmol.mg-1.h-1 (about 10% of that of α-NAGA).We found both α-GAL and α-NAGA enzymatic activities in the homogenates from In the assay of α-GAL, the degradation of the MU-α-galactopyranoside was inhibited up to 95% in the presence of N-acetyl-D-galactosamine , whereas in the presence of D-galactose  the degradation of the same substrate was inhibited up to 75%. In the assay of α-NAGA, the degradation of the MU-α-N-acetylgalactosaminide was inhibited up to 97% by D-GalNAc and up to 90% by D-Gal. No inhibition of α-NAGA and α-GAL activity by D-glucose was observed.C. elegans has only one enzyme with both α-GAL and α-NAGA activities. Nevertheless, these experiments were not conducted with the pure enzyme and thus do not provide absolute proof of this hypothesis.According to published observations in human enzymes, D-GalNAc has no inhibitory effect on α-GAL activity. On the other hand, human α-NAGA activity is inhibited by both, D-GalNAc and D-Gal . The modgana-1, employing combination of microinjection and feeding approaches, did not reveal any abnormal morphological phenotypes. Nevertheless, measurement of α-GAL and α-NAGA activities in four different experiments showed a simultaneous decrease of both enzymatic activities in RNAi-treated worms  under the control of a 3 kb region of the gana-1 hypothetical promoter. The presence of the gana-1::GFP transgene in the worms was confirmed on the level of genomic DNA, cDNA and protein. However, no GFP signal was observed by fluorescence microscopy under the standard laboratory conditions. As Western blotting showed the presence of fusion protein of the expected size (data not shown), we assumed that the absence of the GFP signal was caused by a pH-dependent quenching of GFP fluorescence, which has neutral to alkaline optimum (pH 5.5–12) [To study the expression of  5.5–12) .To study the tissue and intracellular distribution of the fusion protein, we resorted to immunofluorescence detection of the transgene product. Immunofluorescence detection of GFP fusion protein showed a specific and coarsely granular cytoplasmic pattern of fusion protein expression. This transgene product was limited to body wall muscle cells (30% of population) Figure  or found4Cl and concanamycin A (CON A) experiments . The GFP signal intensity was dependent on the time of incubation and the concentration of the alkalizing agent used. The first visible GFP signal was observed after 8 hour incubation in 100 mM NH4Cl and within 2 hours of incubation in 50 nM CON A. Lower concentrations of both NH4Cl and CON A did not result in visible GFP signal even after prolonged incubation of up to 24 hours. The reappearance of the GFP signal after treatment of the worms with compounds increasing the acidic compartment pH indirectly confirms lysosomal localization of the fusion protein. The GFP signal in coelomocytes had the same coarsely granular pattern as that observed after immunostaining.To confirm that the absence of the GFP signal was due to the quenching of fluorescence by low pH in the acidic cellular compartment, we used two agents specifically alkalizing acidic cellular compartment ,34 to enLimited access of alkalizing agents to the tissues can explain the differences between the results of immunofluorescence and alkalization studies.gana-1 is the only ortholog of human α-NAGA and α-GAL in C. elegans. Based on phylogenetic and homology modeling analyses we speculate that GANA-1 most probably developed from a hypothetical ancestral gene before the duplication event which gave rise to separate α-NAGA and α-GAL genes.Our findings showed that gana-1 gene product has both α-NAGA and α-GAL activities as detected in C. elegans homogenates. Importantly, both activities in the worm were inhibited by D-galactose and N-acetyl-D-galactosamine, which is a specific inhibitor of human α-NAGA and does not inhibit α-GAL.We also speculate that The GANA-1::GFP fusion protein had a pattern of distribution that is compatible with lysosomal subcellular localization. The lysosomal localization of the fusion protein was also supported by pH sensitive fluorescence of GFP that was detectable only after alkalization of the acidic cellular compartment.gana-1 yielded no abnormal morphological phenotypes, most likely because it did not provide sufficient knockdown of enzymatic activities, necessary for development of lysosomal storage as observed in human pathology states. Nevertheless, gana-1 RNAi resulted in a partial decrease of both enzymatic activities supporting the notion that this gene encodes both of them.Not suprisingly, RNAi of gana-1 may provide more insight into the function of gana-1 and efforts are underway to isolate such alleles. Deletion alleles of lysosomal hydrolases may serve as valuable models of human lysosomal storage disorders.It is possible that a deletion allele of Caenorhabditis elegans [The wild type Bristol N2 strain was used for all experiments and was handled under standard laboratory conditions as described previously . Standar elegans  and has Wormbase (2002–2004 versions and freeze versions ,38,39) dtrans splicing; the 1142 bp fragment of the 3' end of cDNA was amplified with T7 primer and a gene specific primer (5'CTTAAGTTTGGAATTTATGAA). The dominant PCR products were cloned with TOPO TA cloning kit (Invitrogen) into the pCR 2.1 vector. Positive clones were sequenced using the Li-Cor automated fluorescent sequencer and sequences were aligned with R07B7 reference cosmid sequence in the AlignIR software (Li-Cor) to evaluate splicing boundaries and overall gene organization.Total RNA was isolated from mixed stages of N2 cultures  and reveConfirmed or predicted amino acid sequences of melibiase family members  represenThe 3D model of GANA-1 is based on the X-ray structure of chicken α-NAGA, rice α-GAL and human α-GAL (PDB codes 1ktcA, 1uas and 1r47, respectively) ,51. The gana-1 gene, including 3 kb of its 5'upstream sequence, was amplified from N2 genomic DNA through a nested PCR reaction using DyNAzyme EXT™ PCR system (Finnzymes) and two pairs of primers: the external pair (5'GTGAGAGTGGGGAGATAGAA and 5'TCAATTTGCTTGAGGTACATA) and the internal primers, with overhangs containing SphI and SalI restriction sites respectively (5'ACATGCATGCAACTTTCACAGGAACACAAC and 5'CGACGTCGACAATTGAACTCTATTGGTTCTCAA). The amplified DNA fragment (4709 bp) was cloned using TOPO-XL cloning kit (Invitrogen) into the pCR-XL-TOPO vector. The SphI and SalI gana-1 restriction fragment was recloned into the GFP reporter vector pPD95.67 (supplied by A. Fire, Stanford University). The in-frame nature of the insert was confirmed by sequencing. The green fluorescent protein (GFP) fusion construct pJH3 (50 ng/μl) and pRF4 plasmid (50 ng/μl) used as the dominant marker were co-injected into the gonads of young adult N2 worms. Transgenic animals were screened for GFP signal. Nikon Eclipse E800 with C1 confocal module and 488 nm and 543 nm lasers and differential interference contrast (DIC) optics was used for specimen examination. EZ-C1 software (Nikon) was used for picture analysis and 3D rendering  [4Cl method, animals were suspended in 0, 10, 25, 50, 75 and 100 mM aqueous solutions of NH4Cl. Small aliquots of worms were examined after 30 min, 2, 4, 6, 8 and 24 hours. For CON A, animals were suspended in 0, 10, 20, 50, 100, 150, 200 nM solutions of CON A in aqueous media. Small aliquots of worms were examined after 1, 3, 6 and 24 hours.Mixed stage pJH3 and N2 (control) cultures were harvested from NGM OP50 plates and washed with water. Worms were pelleted by centrifugation (max. 1000 RPM, 2 min.) between the washes. Worms were treated with either one of two agents (NH– CON A) ,34, thatMicroscopical examination was performed as described above.3, 11.1 mM glucose, 1.6 mM CaCl2·H2O, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4·7H2O, pH 7.4). The reduction/digestion step was performed twice. Pellets were washed 3 times with 1 × PBS and stored for further processing in AbA buffer (1 × PBS, 0.1% Triton X-100, 1% BSA, 0.05% NaN3). AbA buffer was used for antibody dilution. Primary antibody ) was diluted 1:500. Secondary antibody (goat anti-rabbit IgG Alexa Fluor 488 labeled (Molecular Probes)) was diluted 1:1000. Both incubations were performed overnight at room temperature, with AbB buffer (1 × PBS, 0.1% Triton X-100, 0.1% BSA, 0.05% NaN3) washes in between.The fixation and immunofluorescence staining procedures were based on the approaches of Nonet et al. . Mixed sNuclei were counterstained with SYTOX orange (Molecular Probes) and the microscopic evaluation was performed as described above.Mixed stage pJH3 and N2 cultures were harvested from NGM OP50 plates. Worms were homogenized by sonication and the concentration of protein was measured by the Hartree method . The progana-1 cDNA was cloned into pCRII-TOPO vector (Invitrogen) and L4440 double promoter vector for microinjection and feeding RNAi respectively. In-vitro transcription employing T7 and SP6 RNA polymerases (Promega) was used to generate antisense single stranded RNA molecules, which were annealed to generate double stranded RNA (dsRNA). dsRNA was microinjected into N2 worms which were fed on HT115 [E. coli strain carrying L4440 plasmid with gana-1 insert. The F1 and early F2 progeny was screened for morphological phenotypes. N2 worms microinjected with water and fed on HT115 E. coli transformed with L4440 vector without insert were used as a control. 5–7 worms were microinjected both with dsRNA and water in each of 4 separate experiments, single worm progeny reaching 110–150 individuals.The PCR product containing the entire on HT115 E. coli sPrior to all activity measurements worms were washed from culture plates and repeatedly (6 times) washed and centrifuged in M9 buffer and finally pelleted by centrifugation. 4-methylumbelliferyl (MU)-α-D-N-acetylgalactosaminide (1 mM), 4-MU-α-D-galactopyranoside and 4-MU-β-D-glucopyranoside in theMcIlvaine buffer (0.1 M citrate/0.2 M phosphate buffer at acidic pH) were used as enzyme substrates. Reaction mixtures (sample and enzyme substrate) were incubated at 37°C and reactions were stopped by 600 μl of 0.2 M glycine/ NaOH buffer (pH 10.6) ,57. FluoJH carried out molecular, RNAi, expression and biochemical analyses and wrote the first draft of the manuscript. JS participated in the design of all experiments and participated on the bioinformatic, molecular, RNAi and expression analyses and wrote the final version of the manuscript. RD performed phylogenetic analyses including homology modeling. HP participated on the biochemical analyses. MK and JL participated on the coordination of the project. MH conceived the project and provided fundraising. All authors read and approved the final version of the manuscript.Structure of GANA-1 dimer. The color of the backbone represents differences of amino acids between GANA-1 and chicken NAGA. Blue color represents identical residues and orange stands for non-conservative changes. The colors from cyan to green represent different degrees of conservation. The surface of one monomer unit at the interface area is rendered with colors representing electrostatic potential. N-acetyl-D-galactosamine (inhibitor) is placed in the active site pocket of both monomer units . K257 arrowhead depicts predicted dimerisation residue.Click here for fileImmunofluorescence detection of GANA-1::GFP in muscle cells. 3D volume rendered and animated image corresponding to Figure 5AClick here for fileImmunofluorescence detection of GANA-1::GFP in muscle cells and coelomocytes. 3D volume rendered and animated image corresponding to Figure 5BClick here for file


A C-terminal fragment of the mitochondrial gene for 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI. Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates.COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%), with low degrees of pairwise haplotype divergence within populations (0–1%).Herein we present experimental evidence that the mitochondrial 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI, especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.We strongly advocate the use of  The use of short DNA sequences for the standardized identification of organisms has recently gained attention under the terms DNA barcoding or DNA taxonomy -3. AmongCOI or COXI) [Although not necessarily under the specific concepts of DNA barcoding and DNA taxonomy, the diagnosis and even definition of taxa by their DNA sequences are realities in many fields and organism groups, such as prokaryotes, fungi, and soil invertebrates ,6. To usor COXI) ,11. Thisor COXI) ,4,7,11.A genetic marker suitable for DNA barcoding needs to meet a number of criteria . First, 16S) in DNA barcoding of amphibians. As a contribution to the discussion about suitable standard markers we provide experimental data on comparative amplification success of 16S and COI in amphibians, empirical data on conservedness of priming sites, and an example from the 16S-based identification of amphibian larval stages.Here we explore the performance of a fragment of the 16S ribosomal RNA gene  that did not amplify for COI at all .We performed independent amplification experiments with one set of  primers ,7 focusiCOI. The sequences of some species were largely consistent with the primers: Xenopus had two mutations only at each of the priming regions. However, other sequences were strongly different, with up to seven mutations, all at third codon positions. No particular pattern was recognizable for any major group that would facilitate designing COI primers specific for frogs, salamanders or caecilians. Interestingly the variability among the amphibian sequences available was as large as or larger than among the complete set of vertebrates at many nucleotide positions of COI priming sites . This indicates that cases in which haplotypes of a species are more similar to those of another species than to those of other conspecific individuals or populations, are rare in these frogs. Sharing of identical haplotypes among individuals belonging to different species, in our dataset, was limited to three closely related species pairs of low genetic divergences: Boophis doulioti and B. tephraeomystax, B. goudoti and B. cf.periegetes, Mantella aurantiaca and M. crocea. Depending on the taxonomic scheme employed, our complete data set contains 200–300 species of Madagascan frogs. Hence, haplotype sharing was demonstrated in 2–3% of the total number of species only.Phylogenetic and phenetic analyses (Bayesian and Neighbor-joining) of these and many additional sequences (to be published elsewhere) mostly grouped sequences of those specimens from Ranomafana and Andasibe that a priori had been considered to be conspecific . In only 5 cases (2%) conspecific adults collected from a different site than the tadpoles yielded higher BLAST scores although adult sequences from the same site were in the database.To explore the reliability of tadpole identification using the DNA barcoding has great appeal as a universally applicable tool for identification of species and variants of organisms, possibly even in automated handheld devices . HoweverMantidactylus boulengeri, specimens from Andasibe and Ranomafana have similar advertisement calls and  similar morphologies, but their 16S haplotypes were not a monophyletic group (unpublished data). This species belongs to a group of direct-developing frogs that, like the Neotropical Eleutherodactylus [Mantidactylus blommersae from these two sites are conspecific, yet the Ranomafana haplotypes are closer to those of the clearly distinct species M. domerguei. The species pairs where haplotype sharing has been observed (see Results) all appear to be allopatrically to parapatrically distributed and show no or only low differences in advertisement calls, indicating that occasional hybridization along contact zones may be possible [e.g., [Haplotype sharing due to incomplete lineage sorting or introgression is also known in amphibians  althoughdactylus  may be ce [e.g., ]. HaplotAlthough our data show that DNA barcoding in mantellids is a largely valid approach when both reference and test sequences come from the same site, the occurrence of non-monophyletic and highly divergent haplotypes within species characterizes these and other amphibians as a challenging group for this technique. Certainly, DNA barcoding is unable to provide a fully reliable species identification in amphibians, especially if reference sequences do not cover the entire genetic variability and geographic distribution of a species. However, the same is true for any other morphological or bioacoustic identification method. Case studies are needed to estimate more precisely the margin of error of molecular identification of amphibian species. For many approaches, such as the molecular survey of the trade in frog legs for human consumption , the err18S and 28S), proposed as standard marker [Phenomena of haplotype sharing or non-monophyletic conspecific haplotypes will affect any DNA barcoding approach that uses mitochondrial genes, and are also to be expected with nuclear genes [e.g., ]. Nevertd marker , has a hCOI primers may enable amplification of a 5' terminal fragment from representatives of most animal phyla due to their robustness [As a consequence, despite the need of consensus on markers for universal applicability of DNA barcoding, the use of different genes in different groups of organisms seems reasonable. It has been hypothesized that universal bustness . The sucbustness ,4,7.16S primers commonly used in amphibians to COI primers that had been developed for other vertebrates [COI from amphibians. However, our results from mantellid frogs .A further optimization of terflies ,7. Howev16S sequences is complicated by the prevalence of insertions and deletions, and this gene is less variable than COI [COI to assign vertebrate sequences to the level of classes and orders.Alignment of than COI . Neverth16S gene is a highly conserved mitochondrial marker but mutations are common in some variable regions, corresponding to loops in the ribosomal RNA structure. In amphibians, where many species are relatively old entities [16S is sufficiently variable to unambiguously identify most species.The entities , this encytochrome b. This gene can easily be amplified in salamanders and archaeobatrachian frogs using primers that anneal with adjacent tRNA genes. However, neobatrachian frogs (the wide majority of amphibian species) are characterized by rearrangements of the mitochondrial genome [cytochrome b in these species borders directly to the control region. Although cytochrome b primers are available that work in many neobatrachians [COI primers tested here.A further mitochondrial gene that has been widely used in amphibian phylogenetic and phylogeographic studies is l genome ,26, and rachians ,28, they16S primer pair used here can be considered as truly universal not only for amphibians but even for vertebrates. This is also reflected by the high number of amphibian 16S sequences in Genbank (2620 hits for 16S vs. 483 hits for COI, as of September 2004). Moreover, the 16S and 12S rRNA genes have been selected as standard markers for phylogeny reconstruction in amphibians [16S as additional standard DNA barcoding marker for vertebrates, especially for but not limited to applications that involve pooled samples.In contrast, the phibians , which wphibians  is envisTo test for universality of primers and cycling conditions, we performed parallel experiments in three different laboratories (Berkeley, Cologne, Konstanz) using the same primers but different biochemical products and thermocyclers, and slightly different protocols.16S [The selected primers for 16S  amplify COI we tested (1) three primers designed for birds [COI that had performed well in mammals and fishes were kindly made available by P. D. N. Hebert  and these primers yielded similar results (not shown).For or birds  that ampor birds  that ampCOI primers were determined using a gradient thermocycler and were found to be 49–50°C; the 16S annealing temperature was 55°C. Successfully amplified fragments were sequenced using various automated sequencers and deposited in Genbank. Accession numbers for the complete data set of adult mantellid sequences used for the assessment of intra- and interspecific divergences .Nucleotide variability was scored using the software DNAsp  at COI a16S sequences of a large sample of Madagascan frogs were used to build a database in Bioedit [ Bioedit . Tadpole Bioedit  as impleCOI and 16S in assigning taxa to inclusive major clades was tested based on gene fragments homologous to those amplified by the primers used herein (see above), extracted from the complete mitochondrial sequences of 68 vertebrate taxa. Sequences were aligned in Sequence Navigator (Applied Biosystems) by a Clustal algorithm with a gap penalty of 50, a gap extend penalty of 10 and a setting of the ktup parameter at 2. PAUP* [The performance of 2. PAUP*  was usedMV designed the study and drafted the manuscript. MT performed parts of the PCR experiments and carried out the molecular identifications of tadpoles. AVDM and DRV performed part of the PCR experiments. YC provided results on 16S differentiation among Madagascan frogs. All authors read and approved the final manuscript.Summary of results of amplification experiments, and detailed data of inter- and intraspecific divergences in mantellid frogs.Click here for file