Recent advances in tumor biology led to the realization that, in order to understand the mechanisms involved in proliferation and invasion of tumor cells, an analysis of the complex interactions that tumor cells establish with host cells of tumor microenvironment is required. The bidirectional interactions between tumor cells and components of tumor microenvironment, in particular endothelial cells, cells of monocyte/macrophage lineage and fibroblasts/myofibroblasts, play a critical role in most of the events that characterize tumor progression and metastasis. Interactions between these "reactive" normal cells and the genetically altered tumor cells, by either cell-to-cell contacts or soluble mediators, control the most aspects of tumor formation and progression. This review addresses some of the experimental evidences documenting that tumor cells may influence host cells of their own microenvironment by triggering changes that facilitate their local as well as distant dissemination. Therefore, it focuses on macrophages and fibroblasts that, upon stimulation by tumor cells, change their state towards a tumor-promoting-like phenotype. Most primary tumors can be treated successfully by surgery alone or in combination with chemotherapy, immunotherapy or radiotherapy. However, the treatment of disseminating tumors, once they have spread to secondary sites, is a much more difficult task. Effective treatment of multiple metastatic lesions by surgery or radiotherapy is usually impossible due to their distribution in vital organs. Tumor cell invasiveness and metastasis occur by a complex series of events in which malignant cells invade host tissues, penetrate into body cavities, lymphatic and/or blood circulatory systems. Subsequently they disseminate to distant sites where they invade into new surrounding tissues and proliferate to form secondary tumors -3. MetasEpidemiological and experimental evidences suggest that a wide variability exists in the metastatic spread of different human malignancies, but even cancers of the same histological type often produce quite diverse disease progression and survival outcomes for individual patients . This suThe capacity of cancers to evolve and change during their development has been termed "tumor progression" by L. Foulds 1954) . The bio54 . The Although additional interactions will almost certainly be discovered and the significance of these interactions will be elucidated, it is already at present well accepted that the reactive stroma of cancers is usually associated with increased numbers of fibroblasts, enhanced capillary density and the deposition of a new extracellular matrix that is rich in type-1-collagen and fibrin. Circulating monocytes are also recruited to the reactive stroma in response to the tumoral chemotactic factors and the wound healing-like processes occuring during tumor growth. Tumor-associated macrophages derived from differentiated monocytes and resident macrophages represent the major component of host leukocytes that infiltrate tumor tissues.This review focuses on the cancer progression towards invasiveness and metastatic spread, taking into consideration the biological role expressed by the so-called "tumor-associated macrophages" (TAM) and "cancer-associated fibroblasts" (CAF).The monocyte/macrophage lineage constitutes a large portion of tumor infiltrating host cells. They enter into the tumor mass via blood vessels throughout the life span of tumors, from early-stage nodules just beginning to vascularise to late-stage tumors that are invasive and metastatic ,32. A nuTwo major lines of evidence connect macrophages and cancer: Firstly the association of chronic inflammation, that leads to macrophage accumulation, with cancer initiation and promotion -17; secovs anti-inflammatory, immunogenic vs tolerogenic, and tissue destructive vs tissue reparative processes. In particular, we know that macrophages from healthy or inflamed tissues are capable of lysing tumor cells, presenting tumor-associated antigens to T-cells and expressing stimulatory cytokines for T- and NK-cells /CXCR4-dependent mechanism. These CAF did not show aneuploidy or in vivo tumorigenic activity [et al. showed that IL-6 secreted by CAF enhances the growth and invasiveness of estrogen receptor α-positive breast carcinoma cells through its effectors, Notch-3, Jagged-1 and carbonic anhydrase IX [By injecting tumor cell/fibroblast cell suspension into immunodeficent mice, Orimo activity . Moreoveactivity . A genomactivity . Therefodrase IX .et al. [Moreover, using an in vitro model of skin carcinogenesis, Cat et al.  demonstret al. , it was et al. .in vivo growth of prostate tumor cells, when both cell types are inoculated together. In contrast cancer-associated stromal cells co-inoculated with prostate cancer cells promote tumor growth in vivo [et al. have provided some additional evidence that prostate cancer epithelium stimulates CAF to express vimentin, α-smooth muscle actin and calponin, which is characteristic of the myofibroblast phenotype [These mesenchymal-epithelial instructive interactions are also responsible for the integrity of the prostate gland. Now, we know that alterations in the complex relationship between prostate epithelial cells and stromal cells contribute to the genomic instability that may promote the progression to a malignant state of these epithelial cells . Some ev in vivo . In Figuhenotype . Intereshenotype .et al.: tumoral IL-1, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) stimulate HGF expression in CAF, and in turn, stromal HGF leads to an invasive phenotype in carcinoma cells [et al., using a mouse model of prostate carcinoma, showed that tumor cells upregulate p53 in stromal fibroblasts, a process found to induce a selection of a subpopulation of p53 null fibroblasts. In turn, selection of a p53 null subpopulation of stromal fibroblasts contributed to the progression of carcinoma cells [It is possible that the most important feature in progression of prostate tumors is the ability of tumor cells to stimulate stromal cells to release biological agents for their growth and dissemination. Mutual interactions between carcinoma cells and CAF were reported by Nakamura ma cells . Hill etma cells .The origins of CAF were revised by Orimo and Weinberg, who suggested three major alternative hypotheses: a) genetic alteration, b) an activation without genetic alteration of normal tissue fibroblasts, and c) the activation of bone marrow-derived mesenchymal stem cells (MSC) . MSC infDespite these initial findings, additional efforts at determining the molecular mechanisms that lead to the appearance of differentiated fibroblasts and their multiple contributions in tumor progression are still urgently required.There is no tumor stroma without a tumor") and displays a substantial degree of plasticity, with the specific outcomes controlled by tumor cells themselves. Indeed, tumor cells do not only display heterogeneity and induce the expression of signaling molecules that favour their survival and invasiveness into local and distant host tissues, but also influence host stromal elements to produce relevant effectors that act as tumor promoters. Moreover, tumor cell-derived signals recruit and activate some host cells, among which monocytes/macrophages and fibroblasts are the most abundant population within the tumor microenvironment. As was discussed above, both types of cells, macrophages and fibroblasts, are involved in a intricate liaison with tumor cells, that usually leads to tumor progression and activation of the metastatic cascade. Thus, the investigation of the mechanisms that allow macrophages and fibroblasts to contribute to tumor progression, could lead to new approaches for the anti-cancer therapies that are urgently required.Tumor stroma is a specialized form of tissue composed of host cells and signals of different origin, which is associated with tumor cell growth, primarily of epithelial origin. Tumor stroma possesses unique structural features that differ from the native stroma. It is also characterised by a great degree of tumor dependency ("The authors declare that they have no competing interests.Both authors contributed to the writing, the conceptual design and the preparation of the figures of this review, and they have read and approved the final manuscript.


Sir,KRAS-mutated tumor’ mean? Both methodological and technical issues contribute to make the answer extremely complex. Two crucial aspects deserve consideration: (i) many KRAS mutations that occur with low frequencies have not been included in the post-hoc analyses of large phase III randomised trials; (ii) mutation-enriched techniques increase the percentage of KRAS-mutated specimens by about 15%, when compared with direct sequencing  despite the inconclusive results of the post-hoc analysis of CRYSTAL trial (KRAS codon 61 mutations.Recent studies have shown that retrospective experiences are indispensable starting points to identify promising molecular tools, that need to be further evaluated in adequate trials and to be, if appropriate, introduced in clinical practice. To this end, the example of AL trial , actuallKRAS A146T mutation, although its activating power was strongly suggested by in vitro mutagenesis assays (KRAS activating alterations and was not clearly linked with lack of response to cetuximab plus irinotecan, thus raising perplexity about its negative predictive impact.On the other hand, more ambiguous findings have been reported with regard to s assays  and confKRAS alterations, including those ‘not described before in any cancer type’, a biologist's fancy or a clinical necessity? Probably neither of them. Nowadays, in daily practice, the definition of ‘KRAS-mutated tumor’ leads physicians to deny to metastatic colorectal cancer patients a class of efficacious drugs. Therefore, the relevance of rare or complex KRAS variants, of which the activating properties and actual predictive power can be hypothesised, but not clearly stated, should be evaluated with caution.Hence, in conclusion, is the comprehensive detection of all potential KRAS gene represents, instead, an inescapable investigational need, with the aim to better characterise the biology of colorectal cancer and to opportunely verify the predictive potential of KRAS rare variants.The comprehensive analysis of


In the last decade the importance of ethnicity, socio-economic and gender differences in relation to disease incidence, diagnosis, and prognosis has been realized. Differences in these areas have become a major health policy focus in the United States. Our study was undertaken to examine the demographic and clinical features of chronic myelogenous leukemia (CML) patients presenting initially at the LAC+USC Medical Center, which serves an ethnically diverse population.9/l), and significant leukocytosis (WBC >50 × 109/l) as significant adverse pretreatment prognostic factors. Using these indicators, in addition to the validated Hasford and Sokal scores, patients were stratified and analyzed via gender and ethnicity. A significantly greater proportion of women presented with significant anemia  and significant thrombocytosis  compared to men, although no differences were found in risk stratification or treatment response. MCV values for women were significantly  lower than those for men, suggesting iron deficiency anemia. Focusing on ethnicity, Hispanics as a whole had significantly lower Hasford risk stratification , and significantly greater likelihood  of achieving 3-month complete haematological remission (CHR) compared with non-Hispanics at LAC+USC Medical Center, though differences in treatment outcome were no longer significant with analysis limited to patients treated with first-line imatinib.Patients were evenly split by gender, overwhelmingly Hispanic (60.9%), and quite young  compared with previously reported CML patient populations. Previous CML studies identified significant anemia (Hgb <12 g/dl), significant thrombocytosis (platelets >450 × 10Female CML patients at LAC+USC Medical Center present with more significant adverse pre-treatment prognostic factors compared to men, but achieve comparable outcomes. Hispanic patients present with lower risk profile CML and achieve better treatment responses compared to non-Hispanic patients as a whole; these ethnic differences are no longer significant when statistical analysis is limited to patients given imatinib as first-line therapy. Our patients achieve response rates inferior to those of large-scale national studies. This constellation of findings has not been reported in previous studies, and is likely reflective of a unique patient population. In the last decade the importance of ethnicity, socio-economic and gender differences in relation to disease incidence, diagnosis, and prognosis has been realized. Differences in these areas have become a major health policy focus in the United States. Several studies have addressed ethnic differences in the behavior and mortality of prostate cancer and breast cancer and have been able to demonstrate a trend toward more aggressive disease in an African-American population when compared to a Caucasian-non Hispanic population -6. Studibcr and abl [CML is a clonal myeloproliferative malignancy caused by a specific chromosomal translocation involving chromosomes 9 and 22, giving rise to the "Philadelphia Chromosome," which fuses the two genes  and abl . There a and abl . The med and abl . The dis and abl ,16, foll and abl -23. It w and abl . Current and abl .The Los Angeles County-University of Southern California (LAC+USC) Medical Center is a large publicly-funded urban medical center in the Los Angeles serving a diverse ethnic population with a high proportion of Hispanic and minority patients. In this study we evaluated the presenting and prognostic features of patients diagnosed at LAC+USC Medical Center with CML, including the calculation of the validated Hasford  and SokaNotable features of our overall CML patient cohort at LAC+USC Medical Center are summarized in Table Significant proportions of our CML cohort present with hematological features identified in previous studies as significant adverse pre-treatment factors, such as a low hemoglobin, high platelet count, and high white blood cell count Table . For tho9/L). Proportional differences for white blood cell count data are not significant. These three characteristics were identified in previous studies as significant adverse pre-treatment prognostic factors [Notable characteristics of our CML patient population, stratified by gender, are presented in Table  factors -29. GendNotable characteristics of our CML patient population, stratified by ethnicity, are presented in Table Statistical analysis was performed to validate our calculated Hasford and Sokal scores for our CML patient cohort, as presented in Table In this current study we review several of the prognostic presentation factors of a large population of mainly Hispanic patients diagnosed and treated at LAC+USC Medical Center. An interesting observation from our study is the striking average age difference in this largely Hispanic population compared to data in the SEER registry , althougIn this study we have confirmed in our largely Hispanic patient population that women present with higher risk features, specifically a higher proportion of patients relative to men, in low hemoglobin and high platelet count categories. These factors have been demonstrated in prior studies to have an adverse effect upon outcomes -29. In oWe demonstrated lower risk stratification scores for Hispanic patients versus non-Hispanic patients combined. Hispanic patients were also more likely to achieve 3 month CHR compared to non-Hispanic patients when all patients were analysed, although these differences were no longer significant when analysis was limited to patients given imatinib as first-line treatment. These differences may more likely be due to non-Hispanic ethnicities presenting and performing more poorly than the norm, rather being attributable to superior prognostic features and outcomes for Hispanic patients. The vast majority of our non-Hispanic patients were African American or Asian. Indeed, our results may reflect a trend of non-Hispanic minority CML patients presenting and performing more poorly than national averages. We believe that socio-economic factors may play a role in explaining these differences. Additional studies are ongoing. Additionally, the disparities between risk stratification and treatment outcome in imatinib-treated patients may be attributed to the advent of imatinib as first-line therapy for CML, not yet developed at the time that the Hasford and Sokal scores were validated. However, we cannot rule out the possibility that perhaps a biological/genetic factor also contributes to this observed ethnic differences in disease presentation and behaviour, as shown previously at our institution for APL in the Hispanic patient populations .Our cohort as a whole, achieved 3-month CHR rates inferior to those examined in previous large-scale CML trials, which have yielded 3-month CHR rates of up to 86 percent -23. To oWe acknowledge that our study is limited by numerous logistical difficulties. Complete data sets were not available for all 87 patients identified in this study, limiting the number of patients for whom we could provide a full demographic profile and calculate Sokal and Hasford scores. Spleen sizes were not available for the vast majority of patients, so an average value was interpolated for the purpose of calculating Sokal and Hasford scores. Additionally, treatment response and long-term followup data was not available for numerous patients included in the pre-treatment analysis, limiting our statistical power in analysing the association between pre-treatment prognostic factors and treatment response. We were, however, able to validate our Hasford, Sokal, and treatment response data, as lower risk profiles were appropriately significantly associated with better treatment response, both when looking at all patients regardless of treatment, and when analysis was limited to patients given imatinib as first-line treatment.We conclude that female CML patients at LAC+USC Medical Center present with more significant adverse pre-treatment prognostic factors identified in previous studies compared to men, while Hispanic patients present with lower risk profile CML and achieve better treatment responses compared to our non-Hispanic patients as a whole; these ethnic differences are no longer significant when statistical analysis is limited to patients given imatinib as first-line therapy. Our cohort as a whole failed to achieve response rates seen in large-scale national studies -23. ThisWe found that women were more likely to present with significant anemia and significant thrombocytosis. An analysis of our MCV data found that values for women were significantly  lower than those for men, suggesting that the gender differences observed are likely due to iron deficiency anemia, with reactive thrombocytosis. At the same time, however, there were no significant gender-based differences in Hasford and Sokal risk stratification, or in treatment response. To our knowledge, this combination of findings has not been reported in previous studies.Surprisingly, we also found that Hispanic CML patients had lower risk stratification scores and correspondingly greater likelihood of achieving 3-month CHR compared to non-Hispanic patients as a whole when all patients are analysed regardless of treatment type; these ethnic differences in treatment response are no longer significant when analysis is limited to patients treated with imatinib as first-lined therapy. These are novel findings that have not been reported in previous studies, and may reflect a trend of our non-Hispanic minority patients presenting and performing more poorly than national norms. Although true biological differences may play a role, we believe that such differences may be attributable to the unique ethnic and socio-economic profile of our patient population at LAC+USC Medical Center, underscoring the need for further study into the significance of these factors.We began by establishing a database of all patients presenting with CML at LAC+USC Medical Center between the years 1991–2008. We identified and obtained adequate medical records for 87 CML patients. Data collected included gender, ethnicity, city and zip code of residence, date of birth, date of diagnosis, treatment, course of treatment, and any noted side effects, as well as complete blood count data at presentation (pre-treatment), comprised of hemoglobin (Hgb), hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white blood cell count (WBC), and a WBC differential. Hasford and Sokal scores were calculated for all patients as available data allowed. As spleen size was not reliably or routinely recorded in our medical records, the mean spleen size reported in the original Hasford study  was usedt test.This data was then analysed in an overall context, as well as by gender and ethnicity based stratifications, to yield our pre-treatment findings for this study. Patients who were previously diagnosed with CML at an outside institution and undergoing treatment at time of presentation to LAC+USC Medical Center were excluded from pre-treatment prognostic analysis. The associations between patient baseline demographic and clinical characteristics, and gender and ethnicity were examined using contingency tables and Fisher's exact tests. The MCV values were compared using a 2-sample 9/L and a differential of 0% immature granulocytes, a platelet count below 450 × 109/L and less than 5% basophils with a non-palpable spleen [Treatment response was determined by assessing complete hematologic response (CHR) after three months of treatment. CHR was defined as a complete blood count (CBC) with a white blood cell count below 10 × 10e spleen . A chartA contingency table and Fisher's exact were used to evaluate the associations of prognostic factors, sex, and ethnicity with CHR.p-values were 2-sided. All analyses were performed using the SAS statistical package version 9.1 .The 5% level of significance was used and This study was conducted with Institutional Review Board approval under protocol HS-06-00258, "Ethnic Differences in Hematologic Malignancies."The authors declare that they have no competing interests.JPL collected the pre-treatment data used in the study, compiled the CML patient database, participated in the study design, and drafted the manuscript. EB collected the treatment response data used in the study, and helped draft the manuscript. DM undertook background research for the study, assisted in the statement of findings, and helped to draft the manuscript. DY performed the statistical analyses utilized in the study, and helped to draft the manuscript. ASY conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Karen Edmond and Anita Zaidi highlight new approaches that could reduce the burden of neonatal sepsis worldwide. Zaman K, Roy E, Arifeen SE, Rahman M, Raqib R, et al (2008) Effectiveness of maternal influenza immunization in mothers and infants. N Engl J Med 359: 1555–1564. Recent large-scale randomised trial which conclusively demonstrated that maternal immunisation can reduce serious respiratory infections in young infants and is a feasible strategy for improving health outcomes in mothers and young infants.Mullany LC, Darmstadt GL, Khatry SK, Katz J, LeClerq SC, et al (2006) Topical applications of chlorhexidine to the umbilical cord for prevention of omphalitis and neonatal mortality in southern Nepal: a community-based, cluster-randomised trial. Lancet 367: 910–918. The first trial to show that commonly used antiseptics could have major impacts in reducing neonatal infections and mortality in rural field-based settings in low-income countries.Boyer KM, Gotoff SP (1986) Prevention of early-onset neonatal group B streptococcal disease with selective intrapartum chemoprophylaxis. N Engl J Med 314: 1665–1669. The first trial to demonstrate conclusively that risk-based intrapartum antibiotic prophylaxis can prevent early-onset neonatal group B streptococcal disease in high-income settings.Stevens DY, Petri CR, Osborn JL, Spicar-Mihalic P, McKenzie KG, et al (2008) Enabling a microfluidic immunoassay for the developing world by integration of on-card dry reagent storage. Lab Chip 8: 2038–2045. One of a series of studies that has demonstrated that microfluidic microtechnologies can be used to create robust point-of-care diagnostic systems for low-income countries and that results can be fast, accurate, and reproducible.Bang AT, Bang RA, Baitule SB, Reddy MH, Deshmukh MD (1999) Effect of home-based neonatal care and management of sepsis on neonatal mortality: field trial in rural India Lancet 354: 1955–1961. The first study that demonstrated that home visiting from village health workers in the first days of life and the provision of parenteral antibiotics at home could substantially reduce neonatal sepsis and neonatal mortality in low-income countries.2. Indeed, new strategies that can prevent, diagnose, and treat neonates with sepsis are needed in both low- and high-income settings.Neonatal sepsis or septicaemia is a clinical syndrome characterized by systemic signs of circulatory compromise  caused by invasion of the bloodstream by bacteria in the first month of life. In the pre-antibiotic era neonatal sepsis was usually fatal. Case fatality rates in antibiotic treated infants now range between 5% and 60% with the highest rates reported from the lowest-income countries Distal risk factors for neonatal sepsis include poverty and poor environmental conditions. Proximate factors include prolonged rupture of membranes, preterm labour, maternal pyrexia, unhygienic intrapartum and postnatal care, low birth weight, and prelacteal feeding of contaminated foods and fluids Streptococcus agalactiae  is the most common cause of neonatal sepsis in many countries, though low rates are reported from many low-income countries, especially those in south Asia.Escherichia coli, Klebsiella spp., Pseudomonas spp., Acinetobacter spp.) and gram-positive cocci (such as Staphylococcus aureus and Staphylococcus epidermidis) are other important causes S. agalactiae has also led to a substantial change in the bacteria responsible for early onset neonatal sepsis; gram-negative bacilli and Staphylococcus spp. predominate in countries implementing these programs The bacteria that cause neonatal sepsis are acquired shortly before, during, and after delivery . They caTreponema pallidum, rubella virus, herpes simplex virus, cytomegalovirus, toxoplasmosis, Clostridium tetani, HIV, hepatitis B virus, and Bordetella pertussis  levels except for IgG to specific maternal antigens transferred passively across the placenta during the last trimester of pregnancy in utero requirements, including the avoidance of harmful inflammatory immune reactions The basal expression of Toll-like receptors  is similar in the neonate and adult These immunological problems are reflected in the clinical presentation of neonatal sepsis. Neonates have a rapid and fulminant progression of septicaemic disease, nonspecific clinical signs of infection, and difficult-to-interpret laboratory results including haematological and immunological biomarkers of infection and inflammation. Low birth-weight  infants have even poorer functional immunity, and are especially at risk of sepsis However, neonates do have well-functioning cationic membrane-active antimicrobial proteins and peptides (APPs) which have microbicidal properties Many older studies have demonstrated that improving maternal health and nutrition before delivery is directly associated with improved neonatal health outcomes S. agalactiae type III conjugate vaccine have demonstrated excellent placental transfer and persistence of protective levels in 2-month-old infants S. agalactiae vaccine would prevent 4% of US preterm births and 60%–70% of neonatal S. agalactiae infections Maternal immunisation is an important method of providing neonates with appropriate antibodies as soon as they are born There is strong evidence that clean delivery practices and handwashing during delivery reduces rates of neonatal sepsis in both home and health facility settings New studies from Malawi and Nepal indicate that maternal antisepsis interventions such as vaginal chlorhexidine during labour may have a significant impact on rates of neonatal mortality and sepsis in developing countries S. agalactiae from 1.7 per 1,000 live births in 1993 to 0.6 per 1,000 in 1998 Intrapartum antibiotic prophylaxis has been highly effective in reducing both early-onset neonatal bacterial and maternal sepsis in developed countries There is also strong evidence that handwashing by health care providers after delivery can reduce neonatal sepsis and infection rates, especially in hospitals In high-income settings, studies have not shown an advantage of antibiotics or antiseptics over simply keeping the umbilical cord clean There is emerging evidence that neonatal skin antisepsis preparations such as sunflower seed oil provides cheap, safe, and effective protection against nosocomial infections in hospitalized preterm neonates and infants in studies in south Asia. Application of chlorhexidine to neonatal skin has also been shown to be effective in reducing neonatal sepsis in studies from south Asia S. agalactiae and Streptococcus pneumoniae vaccines are both likely to be ineffective when given in the neonatal period Neonatal immunisation has long been considered an important method of reducing neonatal infections. However, response varies according to the antigen E. coli and other gram-negative pathogenic bacteria Breastmilk contains secretory IgA, lysozymes, white blood cells, and lactoferrin and has been shown to encourage the growth of healthy lactobacilli and reduce the growth of Neonatal micronutrient supplementation trials have focused on vitamin A supplementation. Older studies have shown significant reductions in respiratory disease in low birth-weight infants after the administration of parenteral vitamin A In high-income countries, clinical trials of immune stimulants such as granulocyte/monocyte colony stimulating factor (GM-CSF) to enhance the quantity and quality of neonatal neutrophils and monocytes appear promising but have not yet shown a significant clinical benefit Neonatal clinical sepsis syndrome identification is difficult as the clinical signs of neonatal septicaemia can be very similar to those of other life-threatening diseases such as necrotising enterocolitis, hyaline membrane disease, and perinatal asphyxia Identification of neonatal sepsis before delivery also remains challenging. A combination of maternal risk factors and clinical signs and symptoms is currently used S. epidermidis), as these organisms are both normal skin flora and pathogenic organisms in preterms and infants with indwelling blood vessel catheters Confirmation of pathogenic organisms allows targeted antibiotic therapy. However, identification of pathogenic organisms in neonates with sepsis syndrome is fraught with difficulties. Bacterial load may be low due to mothers receiving antepartum or intrapartum antibiotics and because only small amounts of blood can often be taken from newborns Automated blood culture systems have long been considered the gold standard for microbiological diagnosis. However, despite improvements in growth media and instrumentation, results of blood culture can be delayed by up to 48 hours S. agalactiae and are associated with high false positive and negative rates Antigen detection techniques allow rapid detection and identification of microorganisms without culturing. The most commonly used commercially available test is the latex agglutination assay, which is based on specific agglutination by bacterial cell wall antigens of antibody-coated latex particles. However, these tests can only detect specific organisms such as The polymerase chain reaction (PCR) has been widely used in biomedical research laboratories for pathogen identification in neonatal sepsis and in some clinical hospital laboratories. The high sensitivity of PCR allows detection of bacterial DNA even when concentrations are low The biggest problem with real time PCR testing is that the specimen must be collected with a sterile venipuncture, which may be difficult in young neonates. Neonatal capillary heel prick specimens are easier to collect but highly contaminated by skin flora. There is also high potential for contamination of enrichment media, reagents, or the sample during collection and processing Important haematological tests include microscopic examination of the blood for white cells . Advantages are that these specimens do not require sterility and a heel prick specimen can be used. However many of these indices are falsely low in a septic neonate.Biological biomarkers are human blood components that increase in response to infection. The most commonly used acute phase reactant is the C-reactive protein (CRP). However, the CRP takes 12–24 hours to increase to measurable levels; its half life is very long and it takes 5–7 days to normalize after eradication of the infectious agent. Cytokines such as IL6, IL8, TNF-α, and procalcitonin have also been extensively studied Microtechnologies, especially microfluidics, have provided the greatest recent contribution to the diagnosis of neonatal sepsis. Microfluidics is the study of the behaviour, precise control, and manipulation of fluids geometrically constrained to submillimetre (nanolitre or picolitre) channels Microfluidic technology has also allowed sample preparation and a number of different assays to be combined in small, disposable, single-use diagnostic cartridges or cards that have been called a “lab on-a-chip” or LOC  [68]. SoAs neonatal sepsis can be rapidly fatal if left untreated, highly effective antibiotic therapy must be used and delays in the provision of care must be minimised. Treatment must be effective against the causative pathogen, safe for the newborn, and feasible to deliver reliably in the hospital or community setting.Streptococcus spp., but Staphylococcus spp. can be highly resistant Klebsiella spp. E. coli resistance to ampicillin, gentamicin, and third-generation cephalosporins in hospital nurseries in both developed and developing countries is also causing increasing concern Parenteral (intravenous or intramuscular) regimens for neonatal sepsis currently recommended by national paediatric associations are a combination of penicillin/ampicillin and gentamicin, or third-generation cephalosporins  for 10–14 days. These antibiotics are safe and retain efficacy when administered at extended intervals  Streptococcus spp. and some gram-negative bacilli and has an excellent safety record. However, it has no anti-Staphylococcus coverage and resistance is emerging in gram-negative bacilli such as E. coli. New, better-absorbed oral antibiotics are also being considered. The new second-generation cephalosporins  have an excellent safety profile, a spectrum of activity similar to cotrimoxazole, and may be more effective given the high resistance of neonatal pathogens to cotrimoxazole. Ciprofloxacin also is increasingly accepted as safe in neonates and warrants further investigation for treatment of infections in newborns. However, the current cost of these agents and potential for exacerbating antimicrobial resistance may limit widespread use in developing countries Oral antibiotic therapy must be considered in settings where referral is not possible and there are no health care providers trained to give parenteral antibiotics Poor maternal-neonatal health systems, low levels of care-seeking, and lack of access to sick newborns during the first day of life, when mortality risks are highest, are also important concerns Newborn sepsis is a major cause of child mortality across the world. Industrialized countries have made remarkable progress in reducing newborn sepsis and sepsis-related mortality by providing access to hygienic skilled delivery for all women, risk-based intrapartum antibiotic prophylaxis, and high-quality intensive care for newborns that need it. Although resource constraints preclude whole-scale adoption of these strategies in developing countries, there are a number of low-cost proven interventions and promising approaches that have the potential to significantly reduce the burden of neonatal sepsis worldwide .However, practicability of implementing these new advances must be considered. Skilled attendance at delivery is increasing in low- and middle-income countries. Thus, intrapartum approaches such as risk-based antibiotic prophylaxis and improved hand washing during delivery are likely to be both cost-effective and feasible in these countries. More challenges face the implementation of diagnostic technologies. It may take many years for technologies such as the “lab-on-a-chip” to be sufficiently robust and affordable for scale-up to low-income countries. Home-based antibiotic treatment of neonatal sepsis also faces major obstacles to large-scale implementation. Concerns such as “one law for the rich and another for the poor” have already been raised. A careful assessment of the risks and benefits of new technologies and interventions is clearly needed. In low-income settings there are also difficulties with care-seeking for neonatal illnesses, and home visiting programs are needed to identify sick newborns early in life. Neonatal sepsis is also one of the most rapidly fulminating clinical diseases, and many practitioners, including experienced neonatologists, administer parenteral antibiotics rather than wait for the results of any diagnostic tests. These practitioners rightly consider that the individual patient's health is more important than the potential risks of emerging antibiotic resistance.Thus, front-line health workers and families must be partners in all research and evaluation planning. Detailed assessment of end-user attitudes and preferences using formative and qualitative research methods must be included in the development of programs to reduce morbidity and mortality from neonatal sepsis. Finally, advocacy for equitable resource allocation across and within countries must be a priority and modelling techniques to assess public health impact of neonatal sepsis interventions must be developed and used more widely.


The traditional emphasis on developing high specificity pharmaceuticals ("magic bullets") for the treatment of Neurological and Psychiatric disorders is being challenged by emerging pathophysiology concepts that view disease states as abnormal interactions within complex networks of molecular and cellular components. So-called network pharmacology focuses on modifying the behavior of entire systems rather than individual components, a therapeutic strategy that would ideally employ single pharmacological agents capable of interacting with multiple targets ("magic shotguns"). For this approach to be successful, however, a framework for understanding pharmacological "promiscuity" - the ability of individual agents to modulate multiple molecular targets - is needed.Pharmacological promiscuity is more often the rule than the exception for drugs that target the central nervous system (CNS). We hypothesize that promiscuity is an important contributor to clinical efficacy. Modulation patterns of existing therapeutic agents may provide critical templates for future drug discovery in Neurology and Psychiatry.To demonstrate the extent of pharmacological promiscuity and develop a framework for guiding drug screening, we reviewed the ability of 170 therapeutic agents and endogenous molecules to directly modulate neurotransmitter receptors, a class of historically attractive therapeutic targets in Neurology and Psychiatry. The results are summarized in the form of 1) receptor-centric maps that illustrate the degree of promiscuity for GABA-, glycine-, serotonin-, and acetylcholine-gated ion channels, and 2) drug-centric maps that illustrated how characterization of promiscuity can guide drug development.Developing promiscuity maps of approved neuro-pharmaceuticals will provide therapeutic class-based templates against which candidate compounds can be screened. Importantly, compounds previously rejected in traditional screens due to poor specificity could be reconsidered in this framework. Further testing will require high throughput assays to systematically characterize interactions between available CNS-active drugs and surface receptors, both ionotropic and metabotropic. A common assumption underlying drug discovery is that therapeutic agents with higher specificity for their molecular targets confer better efficacy and fewer side effects. Indeed, drug discovery efforts traditionally focus on developing "magic bullets" - agents that provide the proverbial surgical strike against critical players in a disease process while minimizing collateral damage. However, there is growing interest in the possibility that drug promiscuity  may actually represent a therapeutic benefit rather than a liability. If true, then screening for "magic shotguns" - therapeutic agents that are rationally promiscuous - could be a more effective drug discovery strategy -6. This For disorders of the central nervous system (CNS), where highly complex interactions underlie normal function, drug promiscuity may be particularly relevant. Drug promiscuity is already well-recognized among certain classes of CNS-active modulators such as general anesthetics ,14, antiin vitro electrophysiology. Although indirect  and subacute/chronic  effects also likely contribute to the clinical efficacy of many CNS-active drugs, we focused only on direct functional interactions demonstrated with physiological measurements (ion flux and binding data were considered insufficient). Although the actions of these 170 modulators on the selected LGICs may or may not be functionally relevant in vivo, the degree of promiscuity illustrates the capacity of this class of protein targets to interact with diverse compounds. The potential drug discovery impact of recognizing clinically relevant promiscuity includes the idea that compound library molecules previously rejected on account of poor specificity by in vitro screening could be reconsidered in the context of "rational promiscuity".To demonstrate that CNS drugs are generally promiscuous agents and illustrate how mapping promiscuity can guide drug discovery, we collected published examples of acute, direct functional modulation of ligand-gated ion channels (LGICs) by a total of 170 pharmaceutical and endogenous molecules, as demonstrated by A, acetylcholine, glycine, and 5HT-3 receptors) by 170 compounds identified in a systematic manual search of the PubMed database between 1970 and 2008, as demonstrated by in vitro electrophysiology studies. These compounds span categories of psychiatric medications  or side effects (such as antibiotics antagonizing GABAA receptors), the physiological relevance (if any) of many of these interactions is unknown. However, the extent of promiscuity emphasizes the need for systematic characterization if the hypothesis is to be investigated for drug discovery purposes. Since the evidence for modulation was derived from a wide variety of sources not explicitly designed to test for promiscuity, meaningful comparison of the affinity and efficacy of modulation was not possible , as well as to a subset of glycine receptors in vivo [A receptors at low concentrations, act as direct agonists as higher concentrations, and open channel blockers at even higher concentrations concentrations [It is worth noting that each of these LGICs is actually comprised of multiple subtypes, each having distinct functional and pharmacological properties. Similar to a recent pharmacological interaction network based on databases of FDA-approved drugs and their targets , we haveodulation. The lev in vivo ,22. In f in vivo ,24. Modutrations -27. Diretrations -31. Locatrations .Drug promiscuity can also be evaluated from the reverse perspective, focusing instead on the ability of a single therapeutic agent to interact with multiple protein targets. The most comprehensive approach, illustrated in Figure 50 or IC50 of SSRIs for each target is approximated by the radial distance from the center of the map (based on electrophysiology data for the ion channel targets and on binding data for the metabotropic target receptors). The radius of the outer two concentric circles spans the range of estimated therapeutic CNS concentration (1-10 μM) [50 for fluoxetine enhancement of GABAA receptors was 128 μM, the enhancement was large (350%), suggesting modulation could occur at concentrations as low as 10 μM[50 0.7 μM).For drug screening purposes, however, mapping only direct interactions may be more explicitly useful. An example is shown in Figure 1-10 μM) . With th as 10 μM. FurtherUsing promiscuity maps to guide drug discovery could proceed as follows. First, a drug-centric promiscuity map akin to that shown in Figure in vivo cellular and molecular milieu involves complex interactions between cellular and molecular components. However, because these components are typically studied experimentally in isolation, it remains largely uncertain how such complexity influences signaling during normal brain function or disease states. Moreover, few studies have endeavored to explore the effect of simultaneous endogenous and pharmacological modulators acting on a receptor target, mainly because of the cumbersome combinatorial nature of studying even a subset of the possible interactions. Given the evidence for promiscuity of endogenous as well as therapeutic modulators of neurotransmitter receptors highlighted here, we propose that such information may be biologically and clinically relevant. Indeed, by aiming various high throughput functional assays toward the systematic elucidation of promiscuity maps, we anticipate that new vistas of drug discovery will be revealed. One important implication of this strategy is that one can re-investigate existing compounds that have been discarded in early preclinical studies solely on the basis of failing high specificity criteria.The Three lines of reasoning argue against the assumption that higher specificity drugs will necessarily confer increased therapeutic efficacy. First, endogenous receptors and signaling molecules are themselves multi-functional, exhibiting both ligand-centric and receptor-centric promiscuity. Thus, even theoretically high specificity agents could have unpredictable biological impact, if the intended target were involved in many functions. Second, target promiscuity may be more common than recognized for many CNS-active therapeutics,15,17,18Rational approaches to promiscuity may inform potential drug discovery strategies that consider the patterns of target modulation in relation to pharmaceutical efficacy as well as side effects. Endogenous signal transduction pathways, such as G-protein coupled receptors and receptor kinases, likely also demonstrate modulator promiscuity, and should be considered in addition to ion channel targets. Several models have been proposed to address the potential molecular basis of target-receptor promiscuity based on atomic-level interactions , as wellThe fundamental challenge of modern CNS pharmacology is to determine which aspects of drug promiscuity contribute to clinical efficacy, and which aspects are responsible for unwanted side effects. To gainThe authors declare that they have no competing interests.MTB and EJB contributed equally to conceiving the study and its design, interpretation, figure development, and drafting of the manuscript. MTB carried out the primary literature analysis. Both authors read and approved the final manuscript.Tables of modulator-target interactions. EC50, IC50, percent modulation, and references for each modulator-target interaction.Click here for file


TEM CELLS 2010; 28:1686–1702.Ineffective treatment and poor patient management continue to plague the arena of clinical oncology. The crucial issues include inadequate treatment efficacy due to ineffective targeting of cancer deposits, systemic toxicities, suboptimal cancer detection and disease monitoring. This has led to the quest for clinically relevant, innovative multifaceted solutions such as development of targeted and traceable therapies. Mesenchymal stem cells (MSCs) have the intrinsic ability to “home” to growing tumors and are hypoimmunogenic. Therefore, these can be used as (a) “Trojan Horses” to deliver gene therapy directly into the tumors and (b) carriers of nanoparticles to allow cell tracking and simultaneous cancer detection. The camouflage of MSC carriers can potentially tackle the issues of safety, vector, and/or transgene immunogenicity as well as nanoparticle clearance and toxicity. The versatility of the nanotechnology platform could allow cellular tracking using single or multimodal imaging modalities. Toward that end, noninvasive magnetic resonance imaging (MRI) is fast becoming a clinical favorite, though there is scope for improvement in its accuracy and sensitivity. In that, use of superparamagnetic iron-oxide nanoparticles (SPION) as MRI contrast enhancers may be the best option for tracking therapeutic MSC. The prospects and consequences of synergistic approaches using MSC carriers, gene therapy, and SPION in developing cancer diagnostics and therapeutics are discussed. S Approximately 25 million people live with cancer  and ∼13%Recent developments in real-time in vivo imaging technologies using image contrast enhancers offer tangible options to better guide treatment delivery and monitor outcome. Furthermore, improved treatment specificity may be achieved through gene therapy-based approaches. Using viral and nonviral vectors, genetic material can be specifically targeted to cancer cells, for example, to compensate for mutations in tumor suppressor genes, to potentiate anticancer immune responses, or to cause oncolysis . HoweverThe ability of SCs to migrate to pathological sites including wounds, ischemia, and cancer (including micrometastases) –13 underSCs from different sources have been explored for biomedical applications: embryonic SC; fetal multipotent SC; induced pluripotent SC; adult multipotent SC comprising neuronal SC (NSC), hematopoietic SC (HSC), and mesenchymal SC (MSC)  Fluoro-3-Hydroxymethylbutyl) Guanine (18F-FHBG)) has been successful in both human and animal studies for PET imaging [Given a relatively poor understanding and ability to control MSCs in vivo behavior, their application as carriers of contrast agents may not be safe . This ma imaging ; the pre imaging  or expre imaging  regulate imaging ,231, Tyr imaging  to detec imaging .Taking into account the limitations and attributes of different types of SCs, MSCs offer a feasible option in clinical oncology. New sources of MSCs are under development, including those from adipose tissue ,76,77 anOverall, it is clear that there is no single magic bullet to overcome the complexity and heterogeneity of cancer. Multifaceted approaches that exploit the best attributes of MSC biology, nanotechnology, gene-technology, and gene therapy have the potential to overcome hurdles encountered when each is used alone. However, the possibility that such multidimensional modifications may also enhance the danger of unwanted changes in MSCs functional phenotype such as gain of tumorigenic potential or loss of specific migration, cannot be ignored. A rigorous characterization of modified cells with focus toward addressing the potential regulatory issues would be crucial to achieve their speedy translation to the clinic.The authors indicate no potential conflicts of interest.


Multipotent mesenchymal stromal cells (MSCs) represent a rare heterogeneous subset of pluripotent stromal cells that can be isolated from many different adult tissues that exhibit the potential to give rise to cells of diverse lineages. Numerous studies have reported beneficial effects of MSCs in tissue repair and regeneration. After culture expansion and in vivo administration, MSCs home to and engraft to injured tissues and modulate the inflammatory response through synergistic downregulation of proinflammatory cytokines and upregulation of both prosurvival and antiinflammatory factors. In addition, MSCs possess remarkable immunosuppressive properties, suppressing T-cell, NK cell functions, and also modulating dentritic cell activities. Tremendous progress has been made in preclinical studies using MSCs, including the ability to use allogeneic cells, which has driven the application of MSCs toward the clinical setting. This review highlights our current understanding into the biology of MSCs with particular emphasis on the cardiovascular and renal applications, and provides a brief update on the clinical status of MSC-based therapy. In the hierarchy of ESs, cells taken from the fertilized oocyte are called tipotent . These ters Fig. . Fully dmarrow stromal stem cells or mesenchymal stem cells, and more commonly now, mesenchymal stromal cells (MSCs). MSCs are primitive cells originating from the mesodermal germ layer and were classically described to give rise to connective tissues, skeletal muscle cells, and cells of the vascular system.Adult stem cells have been described from a wide range of adult tissues, including the brain, heart, lungs, kidney, and spleen. However, the most well-characterized source for adult stem cells is still adult bone marrow. Adult bone marrow contains a heterogeneous population of cells, including hematopoietic stem cells, macrophages, erythrocytes, fibroblasts, adipocytes, and endothelial cells. In addition to these cell types, bone marrow also contains a subset of nonhematopoietic stem cells that posses a multilineage potential ,5. Thesehematopoietic stem cells. This allows MSCs to be classified as nonhematopoietic, multipotential stem cells that are capable of differentiating into both mesenchymal and nonmesenchymal cell lineages.MSCs, in the traditional view, should refer to stem cells that are also capable of producing blood cells; however, blood cells are actually derived from a distinct cell population called the More than 30 years ago, Friedenstein et al.  first replastic-adherent under standard culture conditions;positive for expression of CD105, CD73, and CD90, and absent for expression of hematopoietic cell surface markers CD34, CD45, CD11a, CD19, and HLA-DR;under specific stimulus, cells should differentiate into osteocytes, adipocytes, and chondrocytes in vitro.The true identity of MSCs has often been confused by different laboratories which employ different isolation and in vitro culture methods. These variables are responsible for the phenotype and function of resulting cell populations. Whether these conditions selectively promote the expansion of different populations of MSCs or cause similar cell populations to acquire different phenotypes is not clear. It is estimated that MSCs represent only between approximately 0.01 and 0.001% of the total nucleated cells within isolated bone marrow aspirates ,13. DespTable MSCs reside in specialized niches within various tissues, and it has been shown that bone marrow, bone, and spleen are all sites of engraftment –21. It hThe use of MSCs for therapeutic applications has been particularly hailed because of their presumed inherent ability to home to sites of inflammation following tissue injury when injected intravenously. Chapel et al.  demonstrAlthough the precise molecular mechanisms by which MSCs are able to migrate and home into sites of injury are not yet fully understood, the complex multistep process by which leukocytes migrate to peripheral sites of inflammation has been proposed as a paradigm (leukocyte adhesion cascade reviewed ). This sDuring inflammation, the recruitment of inflammatory cells requires a coordinated sequence of multistep adhesive and signaling events, including selectin-mediated rolling, cell activation by chemokines and cytokines, activation of integrins, integrin-mediated firm adhesion on endothelium, transendothelial migration, and finally the migration/invasion in the extracellular matrix involving integrin-dependent interactions and matrix-degrading proteases ,27  . These aThe inhibition of both MMP  and TIMP-1/2  through gene knockout studies have been shown to attenuate MSCs migration though the basement membranes, and it was reported that these proteins are triggered by inflammatory cytokines ,56. SteiAlthough numerous preclinical and clinical studies have demonstrated the safe and toxicity-free effects of MSC transplantation, many reports suggest that there exists a clear similarity between stem cell and cancer stem cell genetic programs . It has MSCs have received renewed interest, particularly in their use of transplantation medicine. Although traditionally the regenerative capacity of MSCs through their presumptive plasticity was seen as the driving force behind interest in MSCs, their role in modulating the immune response is now attracting greater interest. T lymphocytes (T-cells) are a major executor of the adaptive immune response, and numerous studies have demonstrated that MSCs modulate the function of T-cells. MSCs lack expression of MHC class II and most of the classical costimulatory molecules such as CD80, CD86, or CD40 . MSCs caThe traditional view that MSCs simply evaded the host immune response is not quite as simple as first proposed. MSCs have been characterized as expressing several receptors that allow them to interact with T-cells. MHC class I and several adhesion molecules, including cell adhesion molecule (VCAM), intercellular adhesion molecule one (ICAM-1), activated leukocyte cell adhesion molecule (ALCAM), lymphocyte functional antigen-3 (LFA3), and some integrins can interact with their respective ligands on T-cells . MSCs haMiesel et al.  demonstrProduction of nitric oxide (NO) by MSCs has also been implicated as a potential mechanism by which MSCs inhibit T-cell proliferation . MSCs alZhang et al.  reported2 (PGE2).The interaction between MSCs and natural killer (NK) cells may contribute to the immunomodulatory effects of MSCs. NK cells are key effector molecules of innate immunity. MSC may evade recognition by alloreactive cytotoxic T-cells (CTL) and NK cells as these were not lysed in coculture experiments . AngoulvThe inhibitory molecule programmed death one (PD-1) binding to its ligands PD-L1 and PD-L2 may also be responsible for inhibition of T-cell proliferation via cell-cell contact of MSCs, leading both to modulate the expression of cytokine receptors and activate molecules for cytokine signaling ,81. The Many kidney disorders involve both ischemic/inflammatory and immunologic injury. Therefore cell-based therapies such as those using MSCs which function through multiple mechanisms and have the potential to target the inflammatory and immunologic pathways have been considered a clinically relevant solution in contrast to pharmacologic agents that target only a single event or pathway in the pathophysiology of a given disease. The significant morbidity from long-term palliative treatments  and the ever increasing transplant waiting lists dictate a need for alternative therapy options such as cell therapy.Chronic kidney disease results in significant cell loss, accumulation of extracellular matrix proteins, and development of interstitial fibrosis . In contMorigi et al.  reported+ transgenic mice injected intravenously were reported to home to the kidney of mice with glycerol-induced ARF, but not in normal mice. These cells were shown to localize in the context of the tubular epithelial lining and expressed cytokeratin, suggesting that MSCs engrafted in the damaged kidney were able to differentiate into tubular epithelial cells. In addition, it was reported that MSCs enhanced tubular proliferation as detected by the increased number of proliferating cell nuclear antigen (PCNA) positive cells with a significant contribution of the engrafted MSCs in the regeneration of tubular epithelial cells demonstrated by the presence of a consistent number of GFPMSCs from GFPf injury . Crop etf injury  recentlyThese studies highlight the controversy that still remains about the exact mechanisms by which MSCs exert these seemingly beneficial effects. Held et al.  reportedhttp://www.who.int). In addition, cardiac transplantation is complicated by immunosuppressive therapy and is restricted by the limited supply of donor hearts. As with other degenerative diseases, the potential of cell therapy using stem cells to differentiate into viable cardiac myocytes and regenerate scar tissue is an attractive prospect, with the aim of reversing ventricular remodeling, preventing heart failure, and alleviating the need for heart transplantation.Cardiovascular disease (CVD) remains a significant cause of morbidity and mortality in the developed world. Coronary heart disease (CHD) is also the primary cause of clinical heart failure, and despite advances in medical therapy, 40% of patients with heart failure die within the first year of diagnosis . AlthougAcute myocardial infarction (AMI) is associated with the death of cardiomyocytes by apoptosis and necrosis –98. AlthIn a rat model of dilated cardiomyopathy, transplantation of MSCs led to a significant increase in capillary density (enhanced angiogenesis) and a significant inhibition of myocardial fibrosis. Both myogenic and angiogenic differentiation of MSCs were observed, in addition to characterizing the secretion of several prosurvival growth factors, including vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor, suggesting that the benefits observed were caused by a combination of differentiation and paracrine mediated effects . Jiang eThe question of the ideal route of administration remains one of the many unresolved issues facing efficient clinical application of MSCs. Clinical trials in humans aimed at improving cardiac function using stem cells have demonstrated variable but encouraging results . IntracoTable The therapeutic potential of MSCs is currently being explored in a number of phase I/II and III clinical trials, many of which have recently been completed or are underway Table . SeveralAnother small study reported that haploidentical MSCs infused in conjunction with allogeneic hematopoietic stem cell transplantation led to enhanced engraftment. In addition, all patients achieved neutrophil and platelet engraftment and 100% donor chimerism, again with the noted absence of any associated toxicity . LazarusIn another small pilot study, Gonzalo-Daganzo et al.  reportedhttp://www.osiristx.com/clinical_trials.php) is currently evaluating their proprietary adult stem cell product, Prochymal, in phase III clinical trials for three indications, including steroid refractory acute graft-versus-host disease (GvHD), newly diagnosed acute GvHD, and Crohn's disease. Prochymal are adult human MSCs derived from healthy donors. Osiris is also evaluating Prochymal in phase II clinical trials for type 1 diabetes mellitus and chronic obstructive pulmonary disease. Follow-up data from the phase II trials for the treatment of acute GvHD showed that 74% of patients experienced total clinical resolution of the disease, whereas follow-up data for the phase II trials for the treatment of Crohn's disease for patients who had failed to respond to standard treatments, such as steroids, reported a significant reduction in disease severity by day 28 with relatively low doses of Prochymal and a short treatment course. Athersys  is another industry-led company investigating the therapeutic potential of MSCs. Athersys is currently evaluating the potential of MultiStem  in several phase I clinical trials for ischemic injury  and conditions involving the immune system (autoimmune disease).In addition, significant efforts have been expended in MSC therapies from industry experts. Osiris Therapeutics Inc. , approximately 80 clinical trials are currently exploring the application of MSCs. In addition to the application of MSCs for renal and CVD pathologies, the use of MSCs is also actively pursued in a diverse range of other conditions, including hematologic pathologies such as graft-versus-host disease (GvHD), osteogenesis imperfecta, amyotrophic lateral sclerosis (ALS), Hurler syndrome, metachromatic leukodystrophy, and Crohn's disease. In addition, MSC transplants have been investigated to improve recovery after myeloablative therapy for treatment of solid tumors. Table At the time of writing this review, according to the clinical trials Website of the United States sponsored by the National Institutes of Health (The last few years have witnessed a growing optimism by both basic scientists and clinicians for the clinical application of MSCs for many disease pathologies. Tremendous advancements have been made from significant in vitro and in vivo preclinical studies using MSCs. Although MSCs were originally heralded for their ability to contribute to tissue regeneration through engraftment and long-term survival in injured tissues via their presumed plasticity, recent findings have suggested a plethora of additional mechanisms through which MSCs exert their seemingly beneficial effects, including immunomodulation and paracrine processes. In addition, the increasing number of clinical trials demonstrating the absence of any major adverse side effects coupled with early optimistic benefits continues to drive the field of MSC therapy. However, unresolved issues such as the lack of conformity with respect to isolation and ex vivo culture-expansion protocols and the heterogeneity by which populations and subpopulations of MSCs are characterized continue to be obstacles. In addition, the conflicting data regarding the ability of MSCs to engraft and differentiate into functional cardiomyocytes or tubular epithelial cells, as well as numerous studies reporting the beneficial effects of MSCs in early time frames, suggest that the benefits are solely attributable to paracrine mediated effects. It is clear that much more work is needed and evidence from long-term studies is absolutely required to validate the nature of MSC-based therapy before the prospect of developing a genuine candidate for an “off-the-shelf” MSC biotherapeutic product is achievable.


The primary PIRSF classification unit is the homeomorphic family, whose members are both homologous (evolved from a common ancestor) and homeomorphic (sharing full-length sequence similarity and a common domain architecture). PIRSF families are curated systematically based on literature review and integrative sequence and functional analysis, including sequence and structure similarity, domain architecture, functional association, genome context, and phyletic pattern. The results of classification and expert annotation are summarized in PIRSF family reports with graphical viewers for taxonomic distribution, domain architecture, family hierarchy, and multiple alignment and phylogenetic tree. The PIRSF system provides a comprehensive resource for bioinformatics analysis and comparative studies of protein function and evolution. Domain or fold-based searches allow identification of evolutionarily related protein families sharing domains or structural folds. Functional convergence and functional divergence are revealed by the relationships between protein classification and curated family functions. The taxonomic distribution allows the identification of lineage-specific or broadly conserved protein families and can reveal horizontal gene transfer. Here we demonstrate, with illustrative examples, how to use the web-based PIRSF system as a tool for functional and evolutionary studies of protein families.The PIRSF protein classification system ( High-throughput genome projects have resulted in a rapid accumulation of predicted protein sequences. To fully realize the value of the data, scientists need to understand how these proteins function in making up a living cell. With experimentally verified information on protein function lagging far behind, computational methods are needed for reliable and large-scale functional annotation of proteins. A general approach for functional characterization of unknown proteins is to infer protein functions based on sequence similarity to annotated proteins in sequence databases. While this is a powerful approach that has led to many scientific discoveries, numerous genome annotation errors have been detected , many ofClassification of proteins is widely accepted to provide valuable clues to structure, function and evolution. Protein family classification has several advantages as a basic approach for large-scale annotation: (i) it improves the annotation of proteins that are difficult to characterize based on pair-wise alignments; (ii) it assists database maintenance by promoting family-based propagation of annotation and making annotation errors apparent; (iii) it provides an effective means to retrieve relevant biological information from vast amounts of data; and (iv) it reflects the underlying gene families, the analysis of which is essential for comparative genomics and phylogenetics.http://pir.georgetown.edu/) employs a classification-driven annotation method supported by a bioinformatics framework that provides data integration and associative analysis. This paper describes the PIRSF family classification and functional annotation approaches and illustrates how manually curated protein families can be used to support protein functional and evolutionary studies via the PIRSF web site at http://pir.georgetown.edu/pirsf/.To facilitate accurate, consistent and rich functional annotation of proteins, the Protein Information Resource  and homeomorphic (sharing full-length sequence similarity with common domain architecture). Common domain architecture is indicated by the same type, number, and order of core domains, although variation may exist for repeating domains and/or auxiliary domains. Basing classification on full-length proteins allows annotation of biological functions, biochemical activities, and sequence features that are family specific, while the domain architecture of a protein provides insight into general functional and structural properties, as well as into complex evolutionary mechanisms.Derived originally from the protein superfamily concept articulated by Margaret superfamilies and zero or more child subfamilies. The parent superfamilies connect related families and orphan proteins based on one or more common domains, which may or may not extend over the entire lengths of the proteins. The child subfamilies are homeomorphic groups that may represent functional specialization. The flexible number of parent-child levels from superfamily to subfamily reflects natural clusters of proteins with varying degrees of sequence conservation. While a protein will belong to one and only one homeomorphic family, multi-domain proteins may belong to multiple superfamilies . A domain superfamily, which consists of all proteins that contain a particular domain, is usually represented by the corresponding Pfam domain  and selected annotation from the PIR data warehouse.Automatic placement of new proteins into existing families based on BLAST and HMM results with stringent threshold values to avoid false positives. Assignments not made automatically can be added in Step 4.Computer-assisted expert analysis to define homeomorphic families based on sequence similarity, domain architecture, and taxonomic distribution. Family membership is defined, delineating full members and associate members, and selecting representative members and seed members.Hierarchies (parent superfamilies and/or child subfamilies) are created when necessary. The number of hierarchical levels varies, depending on the diversity of the protein group, evolutionary age of the subgroups and the level of functional specialization and diversity. Subfamilies are created when necessary to account for functional divergence and to provide accurate protein annotation.Expert annotation includes extensive review of relevant publications in order to assign accurate and up-to-date names and functions to the family and its members. In the absence of experimental data, functional predictions inferred from sequence and/or structural similarity, genome context, and other evidence are made whenever possible. Name, bibliography and an optional abstract are assigned to the PIRSFs.To ensure accurate and appropriate transfer of the annotations from the curated PIRSF family onto its individual member proteins, name rules and optional site rules are created.Seed members are used for the automatic generation  of family-specific hidden Markov models (HMMs), multiple sequence alignment, and neighbor-joining phylogenetic tree.The PIRSF system consists of two data sets: noncurated clusters and curated families. Currently, about a third of UniProtKB sequences are classified into over 35,000 clusters, including single-member clusters. The non-curated clusters are computationally defined using both pairwise-based parameters and cluster-based parameters. Systematic family curation is being conducted in a two-tier process to improve the quality of automated classification, with over 4,500 preliminarily curated and 3,900 fully curated families currently available. The preliminary curation provides membership and domain architecture characteristic of the family, while the full curation provides additional annotation, including family name, parent-child relationships, family description, and bibliography. Literature-based curation ensures that users are provided with high quality, accurate and up-to-date experimental data.Systematic PIRSF family curation integrates various types of information about the protein family and its members, including sequence and structure similarity, domain architecture, function, genome context, and phyletic pattern, depending upon the special properties of the protein families.It is widely known that protein function can remain conserved in related proteins across major taxonomic groups and/or when sequences have diverged so that sequence similarity is very low. This allows similarity-based predictions of functions for uncharacterized proteins and protein families, ranging from fairly obvious to those requiring elaborate sequence and structure comparisons using additional tools such as PSI-BLAST, profile searches, manual construction of sequence alignments and examination of conserved residues and motifs, structure-structure alignments, and other methods.Protein families that contain the same domains need to be considered together in order to delineate a consistent classification and to facilitate similarity-based predictions. This approach supports better understanding of higher-order relationships among PIRSF homeomorphic families and the divergence of families/superfamilies with a given domain. Furthermore, the evolutionary mobility of certain domains (resulting in domain accretion in multi-domain proteins), the rapid sequence divergence associated with reallocation of functions, and the emergence of distinct functions among relatively close members of a protein family come into focus . Proteinhttp://www.figresearch.com/) for genome context visualization to facilitate functional subsystem analysis  patterns of PIRSFs, indicating the presence or absence of corresponding proteins in completely sequenced genomes, to identify PIRSFs that occur only in given lineages or share common phylogenetic patterns. The latter sets of PIRSFs may indicate participation in the same functional system, especially if the associated pattern is unusual. Combining phylogenetic pattern and biochemical pathway information for protein families allows identification of cases where known functions have yet to be aligned with particular proteins. It may also allow for identifying alternative pathways for the same end product in different taxonomic groups .The collective use of multiple approaches often leads to functional prediction for families of uncharacterized proteins. The following example shows the annotation of several “conserved hypothetical” protein groups as the subunits of the [NiFe]-hydrogenase-3-type complex Eha, based on co-expression data, sequence conservation, genome context and phyletic profile.eha) operon encodes a putative multisubunit membrane-bound [NiFe]-hydrogenase Eha in Methanobacterium thermoautotrophicum  from Escherichia coli, and CO-induced hydrogenase (Coo) from Rhodospirillum rubrum, all of which form a distinct group of multisub-unit membrane-bound [NiFe]-hydrogenases and together are called hydrogenase-3-type hydrogenases.The energy-converting hydrogenase A  and in Methanobacterium thermoformicicum (not a complete genome). Genome context of the corresponding genes in these organisms is also conserved, with the exception of the EhaI (PIRSF036537), which occurs only in M. thermoautotrophicum and M. thermoformicicum, and EhaK (PIRSF036538), which is missing in M. kandleri (they are replaced in the operons by unrelated membrane proteins). Based on these data, the corresponding eleven protein families were annotated as subunits of the multisubunit membrane-bound [NiFe]-hydrogenase Eha . They are conserved only in four complete genomes of closely related Euryarchaeota , with summaries organized in several sections: (i) general information: PIRSF number and general statistics , as well as additional annotation for curated families, such as family name, bibliography, family description, representative and seed members, and domain architecture; (ii) membership: lists of all members separated by major kingdoms and members from model organisms; (iii) function, structure, and family relationship: enzyme classification , structure hierarchy , “preliminary” , or “uncurated”  meaning fully curated with optional description and bibliography, partially curated with membership and domain architecture, or automatically classified and not yet curated, respectively. The curated families, each with a unique ID and family name, are labeled with an evidence tag of “validated” to indicate those containing at least one member with experimentally-validated function, “predicted” for families whose functions are inferred computationally based on sequence similarity and/or functional associative analysis, or “tentative” to indicate cases where experimental evidence is not decisive. PIRSF protein family reports provide supporting evidence for both experimentally validated and computationally predicted annotations.The classification and expert annotation results are presented in PIRSF family reports  PIR taxonomy tree browser from the “Taxonomy Range” field, which displays the taxonomy distribution of all family members and the phylogentic pattern of members in complete genomes; (ii) PIR interactive alignment and tree viewer from the “Alignment and Tree” field, which displays ClustalW multiple alignment and neighbor-joining tree, together with a protein annotation table, all dynamically generated from seed members of curated families; and (iii) PIR DAG browser from the “PIRSF Hierarchy” field, which displays the PIRSF family hierarchy with Pfam domain superfamilies and protein membership in a network structure.More than 20 PIRSF fields are searchable, including database unique identifiers  and annotations . For example, one can identify all PIRSFs sharing one or more Pfam domains based on Pfam ID or name, or identify all PIRSFs in a SCOP fold superfamily based on SCOP fold name. The search results are returned in a summary table, listing each PIRSF with its ID, name, domain architecture and other major attributes, which can be tailored using different display options. The PIRSFs in the result summary table can be selected for further browsing and analysis, including multiple sequence alignment, taxonomy distribution and domain display. For PIRSF classification of a query protein, the “PIRSF Scan” program allows one to identify best-matched PIRSF families based on HMMER match  against functional convergence and functional divergence. There are different approaches for comprehensive studies of protein evolutionary and functional relationships. Functional convergence was investigated by Protein classification facilitates systematic sequence and functional analysis of groups of proteins, allowing one to draw conclusions about protein evolution. In particular, PIRSF families that are fully curated based on integrative sequence and functional analysis and literature review allow the user to proceed directly to advanced comparative studies, for example, to detect Many PIRSF families cover broad taxonomic ranges and can be used to infer function of unknown proteins based on characterized homologs across different taxonomic groups as shown in the example of the PIRSF025009 family  and its two subfamilies—PIRSF50023 8  and PIRSF500239 . As shown in the DAG browser view of the PIRSF025009 family hierarchy , both suAn individual protein domain may fulfill the same biochemical role in various physiological systems or in different taxonomic groups; on the other hand, the combination of multiple domains may confer functional specialization unique to each particular system. The PIRSF classification system provides a convenient overview and comparison of protein families that share one or more Pfam domains. The domain architecture of curated families is represented in order from N- to C- terminus and may include functional Pfam domains that are not detected in all members using the default Pfam threshold values.PF000702 and “PIRSF name” Validated (The relationship between domain architecture and function can be illustrated by the various types of response regulators that share the CheY-like phosphoacceptor domain (PF00072) and are involved in signal transduction by two-component signaling systems. These response regulators usually consist of an N-terminal CheY-like receiver domain and a C-terminal output  domain . In addialidated  and the alidated .As revealed by the domain architecture and functional annotations of these PIRSF families, the presence of a unique output domain in response regulator families often signifies their involvement in a distinct regulatory pathway. For example, members of PIRSF006198 contain the LytTR DNA-binding domain and control the genes involved in biosynthesis of extracellular polysaccharides and bacteriocins, and in expression of exoproteins, including toxins, fimbriation, and quorum sensing . B-type In the PIRSF classification system, subfamilies are created to reflect functional specialization in homeo-morphic families. In many cases, functional divergence observed in the subfamilies are in fact completely new biological functions, as illustrated in the example of PIRSF000886 . The family includes both prokaryotic and eukaryotic proteins that share the metal binding motif found in many other phosphoesterases. The PIRSF000886 family members, however, do not have the conserved catalytic His residue in the GNH[DE] portion of the common consensus pattern DXH(X25)GDXXD(X25)GNH[DE] . As showMembers of this subfamily are involved in vesicle-mediated transport . While members of these four families are under the same Pfam domain family, PIRSF classification view reflects evolutionary subgroups within this Pfam group and allows functional distinction among them. For example, it separates proteins with cobaltochelatase/ferrochelatase activity (PIRSF004877) from those with bifunctional chelatase/precorrin isomerase activity (PIRSF036559).Combined information on curated PIRSF families, domain architecture and SCOP fold superfamily often reveals interesting information, such as functional convergence of evolutionarily unrelated proteins, as illustrated in the In addition, the ability to display customizable columns adds to the integrative view of the families. This is illustrated by the display of domain architecture column along with the SCOP Superfamily column, which reveals that PF01903 and another Pfam domain, PF06180  share the same SCOP fold superfamily (chelatase), indicating that these proteins may have arisen from a common ancestor .The analysis and examination of highly curated PIRSF reports  further provides a more complete picture of the cobaltochelatase enzymes and the relationship of the class II chelatases to other chelatases, as summarized below. Tetrapyrrole biosynthetic chelatases fall into three classes, which are evolutionary unrelated based on sequence and structures . MembersClass I chelatases require three subunits for activity and utilize ATP. Aerobic cobalt chelatase consists of three subunits, CobT (PIRSF031715), CobN  and CobS (PIRSF037030) . Class I2+and Co2+ and can, therefore, function in both siroheme and cobalamin biosynthesis. In many organisms, precorrin-2 oxidase/ferrochelatase is fused with the uroporphyrin-III C-methyltransferase to form a two-domain siroheme synthase (PIRSF036426). PIRSF004999 represents a stand-alone form of precorrin-2 oxidase/ferrochelatase (eg yeast Met8p), which corresponds to the N-terminal domain of siroheme synthase. As with the class II chelatases, these proteins are homodimers and do not require ATP for activity. However, they share no structural similarity with the class II chelatases, and likely have arisen by the acquisition of a chelatase function within a dehydrogenase catalytic framework  and Met8p (PIRSF004999), which are multifunctional proteins, acting as dehydrogenases and chelatases . They arramework . The cobTaxonomic analysis of related PIRSF families can reveal potential horizontal gene transfer that may have functional implications, as shown by the example of nematode chorismate mutase (PIRSF036575). Chorismate mutase (CM) (EC 5.4.99.5) catalyzes the reaction at the branch point of the biosynthetic pathway leading to the three aromatic amino acids, phenylalanine, tryptophan and tyrosine. It is part of the shikimate pathway, which is present only in bacteria, archaea, fungi, and plants. The taxonomic browser view of the nine chorismate mutase-related PIRSF families , howeverSequence similarity analysis shows that the nematode CM is most closely related to periplasmic CM (PIRSF026640). Periplasmic CM is a subclass of the AroQ class CM, and is twice the size of cytoplasmic AroQ protein due to a unique C-terminal domain of unknown function . Members of the periplasmic CM family may be involved in pathogenicity, as most members are pathogenic bacteria. This opens a possibility that chorismate mutases may have been acquired in plant-pathogenic nematodes as a result of horizontal gene transfer from bacteria with the conservation of pathogenicity-related function.PIRSF classification, which considers both full-length similarity and domain architecture, discriminates between single- and multi-domain proteins, showing functional differences associated with the presence or absence of one or more domains. Furthermore, specific biological functions  can seldom be inferred solely from the generic functions of the constituent domains, and proteins with different biological functions may have similar domain organization. Therefore, full-length protein functional annotation, based on homeomorphic protein families  and subfamilies (sharing the same function), is also important for providing the high-quality functional annotation.The PIRSF classification provides family-based annotation for individual protein members. This annotation method has advantages over traditional “genome-by-genome” or “protein-by-protein” annotation, especially when coupled with the PIR name rules and site rules for accurate and consistent transfer of annotations from the corresponding PIRSF families and subfamilies .Coupling protein family classification and data integration allows associative studies of protein sequence, function, and structure. Domain-based or structural classification-based searches allow identification of protein families sharing domains or structural fold classes. Functional convergence and functional divergence are revealed by the relationships between protein family classification and curated family names and functions. With the underlying taxonomic information, protein families that occur in given lineages can be identified. The systematic approach for protein family curation using integrative data leads to novel predictions and functional inferences for uncharacterized “hypothetical” proteins, and to detection and correction of genome annotation errors. Such studies may serve as a basis for further analysis of protein functional evolution.


Complaints of unrefreshing sleep are a prominent component of chronic fatigue syndrome (CFS); yet, polysomnographic studies have not consistently documented sleep abnormalities in CFS patients. We conducted this study to determine whether alterations in objective sleep characteristics are associated with subjective measures of poor sleep quality in persons with CFS.We examined the relationship between perceived sleep quality and polysomnographic measures of nighttime and daytime sleep in 35 people with CFS and 40 non-fatigued control subjects, identified from the general population of Wichita, Kansas and defined by empiric criteria. Perceived sleep quality and daytime sleepiness were assessed using clinical sleep questionnaires. Objective sleep characteristics were assessed by nocturnal polysomnography and daytime multiple sleep latency testing.Participants with CFS reported unrefreshing sleep and problems sleeping during the preceding month significantly more often than did non-fatigued controls. Participants with CFS also rated their quality of sleep during the overnight sleep study as significantly worse than did control subjects. Control subjects reported significantly longer sleep onset latency than latency to fall asleep as measured by PSG and MSLT. There were no significant differences in sleep pathology or architecture between subjects with CFS and control subjects.People with CFS reported sleep problems significantly more often than control subjects. Yet, when measured these parameters and sleep architecture did not differ between the two subject groups. A unique finding requiring further study is that control, but not CFS subjects, significantly over reported sleep latency suggesting CFS subjects may have an increased appreciation of sleep behaviour that may contribute to their perception of sleep problems. Chronic fatigue syndrome (CFS) is a complex illness defined by unexplained persistent or relapsing fatigue for ≥ 6 months that is not attributable to exertion and is not improved by rest. The fatigue must be accompanied by at least 4 of 8 defining symptoms  and the illness must cause substantial functional impairment . Nearly Further, polysomnographic studies have not consistently documented sleep abnormalities in people with CFS ,12. ThesIn the present study, we evaluated the relationship between subjective and objective measures of sleep alterations in persons with CFS and non-fatigued controls. As detailed previously , we condThis study adhered to U.S. Department of Health and Human Services human experimentation guidelines and received Institutional Review Board approval from the CDC and collaborating institutions. All participants gave informed consent.Between January and July 2003, we conducted a 2-day in-hospital study of adults identified with CFS from the general population of Wichita . The in-th percentile of published US population on the physical function (≤ 70), or role physical (≤ 50), or social function (≤ 75), or role emotional (≤ 66.7) subscales of the SF-36. Finally, subjects reporting ≥ 4 symptoms and scoring ≥ 25 on the Symptom Inventory Case Definition Subscale were considered to have substantial accompanying symptoms.We classified participants as having CFS at the time of the study based on the empirical application  of the 1primary hypnotics , narcotic analgesics , anti-depressants , anti-anxiety , anti-histamines , decongestants , anti-convulsants , anti-sleep phase disorder (melatonin), blood pressure controlling , anti-psychotics , stimulants , peristaltic stimulants (metoclopramide), and muscle relaxants (cyclobenzaprine).To assess whether medical conditions exclusionary for CFS  had developed since the surveillance study, participants provided a standardized past medical history and a listing of current medications, underwent a standardized physical examination, and provided blood and urine for routine analysis. Medications that affect sleep were considered 'sleep medications' for the purpose of analysis and include: To identify psychiatric conditions exclusionary for CFS , licensed and specifically trained psychiatric interviewers administered the Diagnostic Interview Schedule for Axis I psychiatric disorders.We classified participants meeting the 3 criteria  for CFS and in whom no exclusionary medical (including sleep) or psychiatric conditions were identified as having CFS. Participants whose scores were in the normal range on all of the above mentioned instruments and who had no exclusionary medical or psychiatric conditions identified were classified as non-fatigued. Persons with exclusionary medical or psychiatric conditions were not included in the analysis.Sleep studies were conducted in a 4-bed clinical research unit at Wesley Medical Center, Wichita, Kansas . These sDaytime sleepiness was measured with the MSLT, which has demonstrated objective sensitivity to the effects of sleep deprivation, sleep fragmentation, sleep restriction, insufficient sleep hypersomnia, and in disease states such as sleep apnea and narcolepsy ,25. MultMeasures of sleep architecture and diagnoses of primary sleep disorders were based upon data from MSLT and the second nocturnal polysomnography . Clinical outcomes of polysomnographic assessment and MSLT included obstructive sleep apnea, periodic limb movements, narcolepsy, insufficient sleep syndrome, primary/secondary insomnia, delayed sleep phase syndrome, bruxism, central sleep apnea, and upper airway resistance syndrome.total sleep time (TST) (in min), sleep efficiency (% of time spent in bed asleep), the percentage of TST spent in non-REM (NREM) and REM sleep, latency to sleep onset (in min) to three consecutive epochs of sleep, and REM latency, defined as the time between the first epoch of any stage of sleep and the first epoch of REM-sleep. Brief arousals were scored following criteria set forth by the American Academy of Sleep Medicine, and the number of arousals expressed as a rate per hour of sleep adjusted for TST. Periodic leg movements both with and without accompanying arousals, were scored according to conventional criteria Besides the theoretical issues addressed above, the present study is not without practical limitations. First, due to stringent selection criteria, our sample size was small, especially considering the number of variables examined. This circumstance limited the power to detect more subtle differences in responses to sleep questionnaire items between groups. Both CFS subjects and controls showed moderately impaired sleep quality  and polysomnography is not an optimal measure of insomnia. Further studies with larger sample sizes are clearly warranted. Second, while sleep-altering medications were frequently used by both CFS subjects and controls, their use was more common among CFS subjects. Prescribed medications in CFS subjects may in turn have influenced CFS subjects' reports of sleep quality. Many published studies of sleep in persons with CFS do not consider medication use. Our attempt to statistically adjust for differences in use of medications that affect sleep as a binary measure (use/non-use) might be inadequate to completely control for the confounding effect of medication use. However, our small sample size precluded conducting a stratified analysis among cases and controls who did and did not use medications that alter sleep or whether they medications induced or inhibited sleep. Finally, the mean duration of illness among CFS cases in this population was 7.3 years . Thus, fThese findings suggest that alterations in standard objective sleep parameters do not explain the etiology of symptoms of unrefreshing sleep and presence of sleep problems reported by persons with CFS who do not have readily diagnosable sleep disorders. Further studies examining the causes of apparent altered sleep-state perception may be helpful in understanding CFS.The author(s) declare that they have no competing interests.WCR and CH were principal investigators of the study. MM, CH, MJD, and WCR wrote the manuscript. All authors contributed to the manuscript. WCR, CH, ERU, LSY, JFJ, and MJD designed the study protocol and supervised data collection during the study. MJD designed sleep protocols, trained sleep technicians, and supervised sleep studies. MJD, CH, WCR, JFJ supervised the conduct of clinical studies and data collection. MM and MJD conducted the statistical analyses with assistance from BMG.The pre-publication history for this paper can be accessed here: