The endocannabinoid system has been implicated in several neurobiological processes, including neurodegeneration, neuroprotection and neuronal plasticity. The CB1 cannabinoid receptors are abundantly expressed in the basal ganglia, the circuitry that is mostly affected in Parkinson’s Disease (PD). Some studies show variation of CB1 expression in basal ganglia in different animal models of PD, however the results are quite controversial, due to the differences in the procedures employed to induce the parkinsonism and the periods analyzed after the lesion. The present study evaluated the CB1 expression in four basal ganglia structures, namely striatum, external globus pallidus (EGP), internal globus pallidus (IGP) and substantia nigra pars reticulata (SNpr) of rats 1, 5, 10, 20, and 60 days after unilateral intrastriatal 6-hydroxydopamine injections, that causes retrograde dopaminergic degeneration. We also investigated tyrosine hydroxylase (TH), parvalbumin, calbindin and glutamic acid decarboxylase (GAD) expression to verify the status of dopaminergic and GABAergic systems. We observed a structure-specific modulation of CB1 expression at different periods after lesions. In general, there were no changes in the striatum, decreased CB1 in IGP and SNpr and increased CB1 in EGP, but this increase was not sustained over time. No changes in GAD and parvalbumin expression were observed in basal ganglia, whereas TH levels were decreased and the calbindin increased in striatum in short periods after lesion. We believe that the structure-specific variation of CB1 in basal ganglia in the 6-hydroxydopamine PD model could be related to a compensatory process involving the GABAergic transmission, which is impaired due to the lack of dopamine. Our data, therefore, suggest that the changes of CB1 and calbindin expression may represent a plasticity process in this PD model. The cannabinoid compounds are lipophilic molecules which exert their effects by binding to specific membrane receptors . Two typHigh amounts of CB1 are found in the basal ganglia , cerebellum, and hippocampus ,8. In thParkinson’s disease (PD) is a neurodegenerative disorder associated with loss of 50 to 70% of dopaminergic neurons in the SN pars compacta (SNpc), which has been extensively investigated in regard to its behavioral and molecular aspects, both in animal models and humans ,17. The Studies employing different animal models of PD, such as 6-hydroxydopamine (6-OHDA) injections in basal ganglia or intracerebroventricular in rats –29, intrTemporal variation of CB1 expression during PD progression is poorly understood. Furthermore, most studies are devoted to evaluate CB1 variation focusing on the striatum and SN, and giving minor attention to the other basal ganglia structures, such as external and internal globus pallidus . TherefSeventy-days-old male Wistar rats weighting 230-280g at the beginning of the experiments, provided by the central animal house of the Institute of Biomedical Sciences, University of São Paulo, were used in this study. The animals were housed in groups of 3–4 animals per cage, maintained at 23 °C ± 2, in a 12 h light/12 h dark cycle (lights on at 06:00 h), with free access to food and water. The experiments were carried out in accordance with the guidelines of the Brazilian College for Animal Experimentation (COBEA) and were approved by The Ethics Committee for Animal Research of the Institute of Biomedical Sciences, University of São Paulo (Protocol n° 27/68/2), which are in accordance with the procedures of national and international care of animals used in scientific research.Independent groups of rats were used to investigate the time course effects of 6-OHDA striatal injection on protein levels by immunohistochemical and immunoblotting analysis.Rats were anesthetized with 2,2,2-tribromoethanol (1 mL/100 g -250 mg/kg i.p. - Sigma®) and placed in a stereotaxic frame (David Kopf Instruments). After craniotomy, two 0.5 µl injections containing 6 µg of 6-OHDA in saline and 0.3% ascorbic acid  were made in the right striatum by way of glass micropipettes coupled to a pressure system, in the following coordinates: (1) AP: bregma, ML: -2.7, and DV: -4.5mm, (2) AP: -0.5, ML: -3.4, and DV: 4 mm . Once thPD-induced rats were deeply anesthetized with ketamine  and xylazine , and perfused through the left ventricle with phosphate-buffered saline and 4% paraformaldehyde in 0.1 M phosphate buffer . The brains were dissected out and after 4-6 hours post-fixation in paraformaldehyde solution they were transferred to a 30% sucrose PB solution to ensure cryoprotection. Coronal sections (30 µm) were then obtained on a sliding microtome and collected in PB.Sections containing substantia nigra (SN), striatum and globus pallidus (GP) regions were incubated with rabbit polyclonal antiserum anti-CB1 , a mouse monoclonal anti-tyrosine hydroxylase antibody  as an indicator of dopaminergic neurons in basal ganglia; a mouse monoclonal anti-parvalbumin antibody , and a mouse monoclonal anti-calbindin antibody , as indicators of GABAergic neurons in striatum, in PB containing 0.3% Triton X-100 plus 5% of normal goat or donkey sera. Parvalbumin is known to be present in GABAergic neurons in the basal ganglia ,35. ImmuDigital images were acquired using a Nikon E1000 microscope  and Nikon DMX1200 digital camera (Nikon Imaging Software ACT-U). Images from striatum were captured between bregmas 1.80 and -0.36; from EGP between bregmas -0.24 and -3.12; from IGP between bregmas -1.92 and -2.76; and from SNpc and SNpr between bregmas -4.56 to -6.60  . All str2 was conducted. For parvalbumin and calbindin in the striatum, positive cells were directly counted. After the quantification blind researchers to the experimental conditions (side and days after lesion) reviewed the data. The resulting indexes were always normalized to the controls.The TH expression was quantified in the striatum (6 sections) and SNpc (6-8 sections) as an indicator of the lesion extension. Parvalbumin and calbindin were quantified in the striatum (6 sections), parvalbumin in dorsolateral striatum and calbindin in ventromedial striatum. CB1 receptor expression was quantified in the striatum (6 sections), EGP (4 sections), IGP (4 sections), and SNpr (6 sections), all carried out with ImageJ (NIH/USA). For TH in SNpc, CB1 in EGP, IGP and SNpr, we measured the integrated density of immunolabeling and of the background (nonlabeled areas) from each slice. The background integrated density was deduced from the integrated density quantification of the labeled areas, to obtain a labeling index reflecting the signal-to-noise ratio. For CB1 immunostaining in the striatum the average of the integrated density of three areas of 2500 µmAt the same time points described above, the rats (n=6-8 per group) were sacriﬁced by cervical dislocation, and the SN (bregma -4.56 to -6.60 below colliculus approximately), and striatum   were rapAs described for immunohistochemistry, the TH protein levels were quantified by the immunoblotting method as an indication of the lesion extension. GAD levels were investigated in the striatum and SN to obtain indirect data on GABAergic neurons. After the quantification blind researchers to the experimental conditions (side and days after lesion) reviewed the data.Quantitative data were submitted to statistical analysis using GraphPad Prism 5 . The comparisons between control and experimental sides from each group were made using the Paired Student’s t-test. The comparisons between all groups were made using the One-Way ANOVA test, followed by Tukey’s post-test when appropriate. For all tests described, the significance value adopted was p≤0.05. All data were normalized to the controls and are expressed as means ± SEM.The analysis of TH, GAD, parvalbumin, calbindin and CB1 protein expression is summarized in In general, 6-OHDA produced a decreased TH immunoreactivity in the injured striatum and SNpc compared to control side. In the striatum, the 6-OHDA produced a gradual decrease in the TH immunoreactivity, which was more pronounced at 10 DPL, followed by a slight increase at 20 and 60 DPL . The meaIn the SNpc a gradual and progressive decrease of TH immunoreactivity was observed in immunohistochemistry  and in iThe CB1 analyses were performed only for immunohistochemistry, because a complex and distinct structure-related variation of its expression was observed and the immunoblotting analysis would not allow a detailed spatial resolution.Our studies showed that CB1 receptor was present exclusively in neuropil in all structures of the basal ganglia analyzed. In the striatum, we found higher densities of CB1 in its dorsolateral portion. In the GP, CB1 was expressed throughout the structure, and in the SN, was restricted to the SNpr. The immunolabelling for CB1 was very specific, as we had no immunostaining in our negative controls, namely the omission of primary antibody and preadsorption of the primary antibody with the corresponding control peptide for CB1 (data not shown).In the striatum, our data showed that there is no difference in the expression of CB1 in injured striatum in all DPL studied . HoweverThe CB1 expression data after the lesions are summarized in (DPL) days post-lesion, (DL) dorsolateral, (EGP) external globus pallidus, (IGP) internal globus pallidus, (SNpr) substantia nigra pars reticulata;  no change. All in relation to control side for each group.The data above were generated by comparisons between experimental and control sides for each DPL. We have also performed another statistical comparison (ANOVA) between the experimental side/control side ratios for each DPL, to evaluate the differences over time. The mean percentage of CB1 expression changes in the immunohistochemistry, as well as the p values of ANOVA between DPLs over time in the striatum, EGP, IGP, and SNpr are shown in As CB1 receptors are located in the axon terminals of GABAergic neurons in the basal ganglia , and WalCalbindin was observed mainly in cell body in the ventromedial portion. There was as an increase about 21% in the calbindin-positive cells for both 1 and 5 DPL  .Our results revealed a complex and distinct structure-specific variation of CB1 in the basal ganglia over time in 6-OHDA PD model. This variation was accompanied by an initial general TH reduction which is reversed by an increase at later periods in the striatum. No changes in the number of GABAergic neurons were observed in the striatum.Several studies have shown the participation of the cannabinoid system in different neurodegenerative diseases and suggested its neuroprotective properties –23. In tFirst, to verify a possible relationship between the CB1 expression and the progression of PD, we analyzed the TH expression as a dopaminergic lesion marker. In general, our data support the literature data regarding the dopaminergic neurons death, since we observed a marked decrease of TH expression in the injured side, both in the striatum and SN. In the SN this decrease was gradual over the time points studied, and was more pronounced in the later ones, as described by several other studies Click here for additional data file.Figure S2Semi-quantitative analysis of calbindin and parvalbumin staining in basal ganglia of PD-induced rats.The quantification is represented by percentage in the striatum of rats submitted to unilateral intrastriatal injections of 6-OHDA after 1, 5, 10, 20 and 60 days post-lesion (DPL). (A) Parvalbumin (PV) and (B) Calbidin. Comparison between the percentage of experimental sides calculated from the control side to each group (dashed line). Statistical analysis by ANOVA (one way) with Tukey post-test. *p <0.05, **p<0.01 and *** p <0.001.(TIF)Click here for additional data file.


Transplantation of organs and tissues  will always be associated with some risk for transmission of infectious diseases from donor to recipient. Understanding and minimizing this risk is difficult for many reasons: donor screening processes vary, screening for every infectious organism is not possible, and assessment of recipient health after transplantation to determine possibility of disease transmission is often not adequate. In May 2010, the US Food and Drug Administration held a meeting to address these challenges and establish a research agenda for minimizing these transplant transmission risks. Attendees agreed that the focus should be on standardizing donor screening, compiling disease transmissibility data, monitoring of transplant recipients’ health, and assessing effectiveness of measures to minimize disease transmission. Collaboration and sharing of perspectives, experiences, and resources of all stakeholders in the transplantation process  can improve the safety of organ and tissue transplantation. Infectious disease transmission through organ and tissue transplantation has been associated with severe complications in recipients. Determination of donor-derived infectious risk associated with organ and tissue transplantation is challenging and limited by availability and performance characteristics of current donor epidemiologic screening  and laboratory testing tools. Common methods and standards for evaluating potential donors of organs and tissues are needed to facilitate effective data collection for assessing the risk for infectious disease transmission. Research programs can use advanced microbiological technologies to define infectious risks posed by pathogens that are known to be transplant transmissible and provide insights into transmission potential of emerging infectious diseases for which transmission characteristics are unknown. Key research needs are explored. Stakeholder collaboration for surveillance and research infrastructure is required to enhance transplant safety. Multiple clusters of infection associated with allograft transplantation and poor outcomes have been described for recipients. These clusters included infection transmitted to recipients of vascularized organs or tissues such as bone, tendon, skin, or corneas. The exact risk for infection associated with organ or tissue transplantation is unknown but is related to multiple factors, including epidemiology of specific infectious exposures, tissue tropism of the organism, and transmissibility of potential pathogens through transplantation. Even for known pathogens, there are few data on the microbial characteristics that determine transmissibility. Similarly, with regard to new pathogens or pathogens that are found in new regions or populations, i.e., emerging pathogens, there are few data regarding optimal approaches to assessing risks of allograft-associated transmission.Transplantation of organs and tissues is increasing . ProspecIn an attempt to prevent donor-derived infections in transplantation, organ and tissue donors are evaluated to identify those that might be more likely to harbor transmissible pathogens. Current donor evaluation protocols rely on reviewing the potential donor’s epidemiologic and clinical history  and communicable disease test results . Donor screening methods include evaluating the donor’s medical history and physical examination results and assessing (often in the form of a questionnaire) the donor for behavioral risk factors associated with a higher prevalence of communicable diseases. Donor testing includes the donor’s microbial culture data , serologic assay results , and hepatitis C virus [HCV]), and increasingly, nucleic acid testing (NAT) results, including assays for HIV, HCV, or HBV. Although regulatory requirements and risk-benefit considerations for evaluating organ and tissue donors differ, the fundamental process for donor screening and testing, and the challenges faced in prospectively assessing the risk for donor-derived infection, are similar for organ and tissue donors. Despite the recognized need to address these challenges, there is little consensus regarding direction for improvements in donor evaluations or for identification of future epidemiologic threats posed by allograft transplantation.In May 2010, the US Food and Drug Administration held a workshop entitled Emerging Infectious Diseases: Evaluation to Implementation for Transfusion and Transplantation Safety. The goal of this meeting was to identify a research agenda to characterize the risk for transmission of donor-derived infections and inform the development of guidelines for emerging infectious diseases  is often uncertain. The incidence of recognized infectious disease transmission in organ recipients is estimated at ≈1% on the basis of limited data  have resulted in clusters of infections transmitted to organ recipients in regions where the pathogens are not endemic. Epidemiologic shifts and other disease transmission risks discussed below illustrate the need for systematic risk-based approaches to evaluating the transmissibility of pathogens through tissue and organ transplantation.Identifying potential infectious exposures is accomplished, in part, by obtaining a donor epidemiologic history. Potential organ and tissue donors may reside in, or have migrated between, geographic regions where different organisms are endemic, which confound screening and testing efforts. The changing geographic distribution of pathogens such as West Nile virus (WNV), chikungunya virus, dengue virus, The transmissibility of an organism by transplantation is generally imputed by after-the-fact recognition of the organism in the blood or tissues of the allograft donor and recipient. Detection and reporting of transmission events is incomplete. The biology of disease transmission from allografts has not been well studied, even for organisms known to be transplantation transmissible. More accurate risk assessment requires data regarding the epidemiology and transmission characteristics of a specific organism in a specific graft type.Transmission of infection is dependent on a series of factors that are organism and host dependent . These fThe pathogenesis of WNV infection illustrates the complexity of disease detection and prevention in organ transplantation. WNV is asymptomatic in 80% of immunocompetent persons infected by mosquito bites. WNV viremia in blood donors is typically detected within 1–5 days after infection, depending on whether testing is performed by using individual donation or minipool tests. Detecting WNV viremia may be complicated by low-level viremia. WNV viremia in blood donors generally clears within weeks, although viremia may persist despite the appearance of antibodies within 7–10 days after exposure  (There is variability in the performance (between specific tests and laboratories) and application (selection of a particular assay by the program performing the test) of assays used in donor testing. This variability limits the ability to compare and interpret existing testing data derived from donor populations that could, in turn, inform decisions regarding optimal assay selection. For example, some organ-procurement organizations use assays indicated for donor testing and others use diagnostic tests. Programs use antibody assays from different manufacturers (resulting in differing performance characteristics), and some use NAT routinely or only in special circumstances . There are few data regarding the clinical performance of these assays in donor populations.In the absence of routine active surveillance , donor-derived infectious disease transmission remains difficult to recognize and document. Current surveillance systems that identify transmission events are passive . Given the asymptomatic nature of some acute infections with serious, but delayed, sequelae , it is unlikely that most transmission events could be detected by passive surveillance alone. Active surveillance would increase knowledge of transmissibility and might detect emerging infectious agents within recipients of organs or tissues. Transmission rates are likely to vary among donor and recipient groups  and among organs and various tissues.,There are multiple, potentially confusing, pathways for adverse event reporting . The JoiOne deceased donor may supply organs, eyes, and numerous other tissues, which may be widely distributed over time and geographically. Some deceased donors are tested many times (by organ procurement organizations (OPOs), eye banks, and tissue banks). Collecting and sharing of donor data are challenging in the absence of linked, unique identifying information for each donor  . Effectiveness of current donor screening procedures has not been systematically evaluated. To develop data useful for assessing donor screening practices, high-quality data must be collected by using standardized protocols and assays of known performance characteristics. The nonuniformity of protocols used to screen donors among various organizations impedes critical assessment of donor evaluation protocols. One initiative to bridge this gap is the development and validation of the proposed Uniform Donor History Questionnaire for Organ, Tissue, and Eye Donors  and incomplete information about prior infectious exposures. As a result, testing donor specimens for known transmissible organisms is required to further reduce risks by excluding the infected potential donor or guiding clinical care of recipients. Given the changing epidemiology of infection, a major challenge is developing new assays for emerging infectious diseases. New assay development is typically resource-intensive and too slow to reflect rapid shifts posed by epidemic disease , but can be accomplished over time . New approaches to assay development and for evaluating such assays in organ and tissue donor populations, together with streamlined approaches to evaluating assay effectiveness in a manner appropriate to regulatory review, could enable more effective responses to threats of emerging infectious diseases.There are immediate clinical needs  and long-term research opportunities in the field of allograft transplantation. A structured approach to addressing gaps in scientific knowledge, perhaps through an overarching Department of Health and Human Services strategy, is needed to enhance the safety of organ and tissue transplantation. The data required fall into several general areas as described below and summarized in ,Baseline data for the number and types of tissue grafts distributed each year are available only through voluntary reporting to the American Association of Tissue Banks and are limited by a lack of coding and traceability after tissues are delivered to end users . Individual tissue banks rely on hospitals to return implant cards to assess use of allograft materials; compliance is voluntary and accuracy of the data is limited by incomplete return rates. Regulatory mandates require OPO and organ transplant programs to report extensive data to the OPTN on all solid-organ donors (living and deceased), candidates on the waiting lists, and recipients. However, the discard rate of donor organs on the basis of microbiologic testing are not collected by OPTN and are retained at the local OPO rather than the national level. Improved testing might change the denominator by increasing or decreasing the number of available allografts for transplantation; collection of these data is needed to guide decisions regarding optimal donor testing in organ transplantation  is unknown. Clinical data (based on cultures and serologic studies) and preclinical data from animal studies would enable more evidence-based decisions regarding donor eligibility.Processing methods vary with regard to diverse technologies and strategies used and types of tissue processed. There are few data regarding the effect of tissue processing on decreasing or eliminating infectious organisms. Similarly, the effect on the risk for transmission of infection of antimicrobial drug therapy in donors or recipients is unknown. Increasingly, some organs (notably kidneys) undergo pump perfusion before implantation, but the effect of this manipulation on disease transmission is unknown.As discussed, standardizing and validating current donor screening tools is an essential first step in collecting and analyzing data for donor risk factors and in developing refined strategies for screening and testing donors. Prospective studies comparing results of donor history questionnaires and those of microbiological testing should be performed to evaluate the effectiveness of donor-screening strategies. The availability of tissues and organs for transplantation could be increased without compromising safety.There is currently no established network or infrastructure for systematically collecting and analyzing organ and tissue donor data. Given the need for additional data to provide a scientific basis for evaluating and managing allograft donors and recipients, stakeholders must develop mechanisms to prioritize and implement joint research programs. To perform the necessary research, the allograft transplant communities need to develop systems to harmonize labeling nomenclature and data elements (product names and data that are contained on product labels), data collection methods, and mechanisms for data sharing. Elements needed to develop such a research infrastructure will include shared data repositories and software for collecting and analyzing surveillance data from donors and recipients; common epidemiologic data elements; and universal donor identifiers that link all allograft types to the original donor  and posttransplant recipient surveillance data and disease transmissibility data  and assessing the efficacy of mitigation strategies. Prioritizing the research agenda can be best driven by collaboration between government , industry , and the allograft transplant provider community . Each stakeholder has unique perspectives, experiences, and resources to share in enhancing the safety of organ and tissue transplantation and benefit the greatest number of recipients.


To the Editor: More than three quarters of critically endangered species of amphibians are threatened by infectious disease; several are already extinct (Litoria castanea), which was presumed to be extinct, was rediscovered in the Southern Tablelands of New South Wales, Australia. This species of frog had not been seen for 30 years, and a chytrid fungus, Batrachochytrium dendrobatidis, was thought to be the reason  parasites forming plasmodia-like structures 10–20 μm in diameter . PlasmodLitoria booroolongensis) that had similar brain lesions and intralesional myxozoan parasites  or an infectious process might be possible causes. Staff in the zoo’s breeding program were questioned and indicated that the opportunity for introduction of a toxin was low. In addition, results for virus isolation and fungal and bacterial cultures were negative. We retrospectively reexamined histologic sections of an endangered booroolong frog (DNA was extracted from brain tissues (20 mg) by using the PureLink DNA Kit . To test for myxozoans, we used a highly Myxozoa-specific PCR to amplify the complete internal transcribed spacer of the ribosomal DNA (Cystodiscus axonis (syn. Myxidium sp. ‘brain’), as the most closely related sequence  and 1 of 5 eastern banjo frogs (Limnodynastes dumerilii).PCR produced a 973-bp amplicon with DNA from brain and liver of the yellow-spotted bell frogs and the booroolong frog. DNA from the frogs showed 100% identity with each other, as did sequences from brain and liver. A BLASTN (Cystodiscus spp. parasites of frogs are emerging and have spread widely along the eastern coast in the past 40 years; they were first detected in a frog collected in 1966 (Cystodiscus parasites in frogs endemic to Australia and in the invasive cane toad (Little information exists about the pathologic significance of myxozoan parasite in frogs and tadpoles (Frog myxozoan parasites are yet to be documented as a cause of population decline; yet, the frequent presence of these parasites in moribund animals in captivity, including the yellow-spotted bell frog, demonstrates the need to monitor parasites in endangered frog populations worldwide. On the basis of our necropsy findings in the central nervous system of 2 yellow-spotted bell frogs, we encourage other investigators to consider the potential role that myxozoan parasites may play in wild and captive populations of declining frogs worldwide.


This case report describes a 64-year-old female with longstanding rheumatoid arthritis who subsequently developed ICN with frequent episodes of sepsis requiring hospitalization and prolonged courses of antibiotics over a 4-year period. She was treated with granulocyte colony stimulating factors (G-CSF) but had a delayed, highly variable, and volatile response. She was enrolled in a clinical trial evaluating the oral investigational agent ezatiostat. Ezatiostat, a glutathione S-transferase P1-1 inhibitor, activates Jun kinase, promoting the growth and maturation of hematopoietic progenitor stem cells. She responded by the end of the first month of treatment with stabilization of her ANC (despite tapering and then stopping G-CSF), clearing of fever, and healing of areas of infection. This ANC response to ezatiostat treatment has now been sustained for over 8 months and continues. These results suggest potential roles for ezatiostat in the treatment of patients with ICN who are not responsive to G-CSF, as an oral therapy alternative, or as an adjunct to G-CSF, and further studies are warranted.Idiopathic chronic neutropenia (ICN) describes a heterogeneous group of hematologic diseases characterized by low circulating neutrophil levels often associated with recurrent fevers, chronic mucosal inflammation, and severe systemic infections. The severity and risk of complications, including serious infections, are inversely proportional to the absolute neutrophil count (ANC), with the greatest problems occurring in patients with an ANC of less than 0.5 × 10 Idiopathic chronic neutropenia (ICN) is an uncommon heterogeneous hematologic disorder characterized by persistent severe neutropenia leading to life-threatening infections . GranuloAlthough alternative treatment approaches such as administration of granulocyte/macrophage-GCF and corticosteroids have been occasionally reported, G-CSF is the generally accepted treatment for the amelioration of neutropenia in ICN. However, there is no consensus for the dose and duration of G-CSF therapy. This is mainly due to the fact that all data for idiopathic neutropenia arise from heterogeneous patient series comprising cases with diverse underlying pathogenetic mechanisms. The decision for the necessity of G-CSF administration, dose, and short- or long-term duration of treatment is individualized on the basis of infection risk and general clinical judgment rather than the ANC per se. Another important issue is prevention of osteoporosis in ICN patients. It has been shown that treatment with biophosphates significantly improves osteopenia/osteoporosis in these patients. The beneficial effect of the treatment is associated with a reduction in serum levels of IL-1β and TNF-α and, occasionally, with amelioration of neutropenia, substantiating the important role of these inflammatory cytokines in the pathophysiology of ICN .Most patients respond to daily subcutaneous administration of G-CSF; however, a subgroup of patients do not respond. ICN patients undergoing chronic G-CSF therapy often experience bone and muscle pain as well as thrombocytopenia and splenomegaly complicating their therapy.Ezatiostat is an investigational agent in development for the treatment of a variety of neoplastic and non-neoplastic hematologic disorders, including myelodysplastic syndrome (MDS), and has demonstrated significant improvement in the induction of growth and differentiation of hematologic precursor stem cells as well as an increase in apoptosis of malignant cells. Ezatiostat is an inhibitor of the enzyme glutathione S-transferase P1-1 (GSTP1-1), a negative regulator of Jun kinase (JNK). Treatment of human cells with ezatiostat leads to the activation of JNK, which promotes the growth and differentiation of hematopoietic stem cell precursors. Ezatiostat treatment has shown significant improvement in neutrophil levels in several clinical trials in MDS -15. We rA 64-year-old female with a history of rheumatoid arthritis (RA) since 1985, treated in the past with a variety of agents, including methotrexate, steroids, gold, Imuran, Enbrel, and Humira. The dose and duration of treatments are not available. The patient had borderline leukopenia and neutropenia documented as early as 2001 but developed a more progressive severe neutropenia in 2007. There was no periodicity or cyclical neutropenia. She did not have splenomegaly. Her bone marrow revealed 20-30% cellularity with mild erythroid hyperplasia and mild myeloid and megakaryocyte hypoplasia. There was non-specific lymphocytosis and no dysplasia. The maturation was orderly, with 27% erythroblasts, 1% myeloblasts, and 30% neutrophils and precursors. She experienced numerous hospitalizations for sepsis as a consequence of her neutropenia, with white blood cell counts in the 2000-3000 range and neutrophils less than 5%, hemoglobin of 12.1 gm/dL, and platelet count of 186,000. Rheumatoid factor (RF) was 67 iu, and cyclic citrullinated peptide IgG antibody (CCP-IgG) was > 250 u. Anti-nuclear antibodies varied between negative and 1:160 with a homogeneous pattern. In the 6 months prior to starting G-CSF, her clinical status deteriorated, with multiple admissions to the hospital, fevers as high as 103.8°F, non-healing perineal ulcers, and decubitus ulcers requiring treatment with broad-spectrum intravenous antibiotics. Fevers frequently recurred within 4 days following cessation of parenteral antibiotics, necessitating their resumption. She also suffered from dysphagia and underwent gastrointestinal evaluation with endoscopies, which showed only non-specific gastritis.9/L. The patient received RA treatment while on study, including fish oil, hydroxychloriquine at the start of cycle 7, and acetaminophen or ibuprofen as needed. She received ezatiostat at 2000 mg orally, daily for 21 days of a 28-day cycle, and has continued on ezatiostat therapy. Initially, G-CSF was continued along with the ezatiostat, but tapered and stopped by the end of cycle 2. Her ANC continued to respond after cessation of G-CSF for 8 months and continues.She was found to have a small clone of T-gamma lymphocytes (0.8%) in the bone marrow and peripheral blood (confirmed by PCR), but did not respond to steroid pulses or cyclosporine. She had transient moderate renal dysfunction, with a creatinine rising to 3.2 before returning to normal in 4 weeks after cessation of cyclosporine. She was then treated with PEG-filgrastim without response and thereafter with G-CSF at doses as high as 600 mcg/day for 3 months. Following an initial response to G-CSF with some wound healing, her neutropenia became intermittently more severe. Two methylprednisolone dose packs were given during an arthritis flare one month before the study observation period. She was experiencing grade 4 neutropenia and agreed to participate in a phase 2 clinical trial of ezatiostat . Her median screening absolute neutrophil count (ANC) prior to entering the 2-month observation period was 0.18 × 109/L. Figure 9/L), as well as post-ezatiostat improvement in ANC counts. Her complete blood counts have remained normal, with an ANC ranging between 1.60-3.75 × 109/L. After 11 months' participation in the study and 7 months of ezatiostat treatment, the T-gamma clone could no longer be identified by flow cytometry but persisted by PCR evaluation. RF had normalized (7 iu) but CCP-IgG remained > 250 u. She has shown remarkable clinical improvement with complete clearing of her arthritic symptoms and with an increase of her Karnofsky performance status from 50% to 90%, and she remains in complete remission from ICN. Accompanying the improvement in her ANC, she experienced a persistent infection-free period and has not required hospitalizations or antibiotic therapy since her ANC improvement. This patient has tolerated ezatiostat well, reporting only grade 1 nausea, abnormal urine and stool odor, and a metallic taste.Her post-ezatiostat treatment median ANC was 3.1 × 10Ezatiostat, a glutathione analog, has shown significant effects on growth and differentiation of human bone marrow progenitor stem cells in several preclinical models of myelopoiesis ,17. EzatWe report this unique case of a patient with longstanding ICN who had an inadequate response to G-CSF therapy and required multiple hospitalizations for recurrent infections resulting from her chronic grade 4 neutropenia. This patient received therapy with oral ezatiostat and has shown an impressive hematologic response that is durable and continues. This suggests a potential role for this agent, given its novel mechanism of action, in the treatment of patients with ICN. The oral formulation is potentially of interest for these patients with ICN, since flexibility in dosing and patient convenience in the treatment of a chronic disease such as ICN would be preferable to chronic subcutaneous injections of G-CSF.Ezatiostat, a GSTP1-1 inhibitor, activates JNK, promoting the growth and maturation of hematopoietic progenitor stem cells. This case highlights an important observation that ezatiostat produced a striking and sustained hematologic response in ANC in an ICN patient who had an inadequate response to G-CSF. Following 8 cycles of treatment with ezatiostat, she has continued to show remarkable improvement, with an ongoing complete remission from ICN without further G-CSF or need for antibiotics. Her symptomatic improvement has been dramatic. The role of ezatiostat in the suppression of her arthritic symptoms is unclear but may indicate an observation for further evaluation in patients with RA. Additionally, since ezatiostat is an oral agent, it offers ICN patients who require chronic therapy the convenience and flexibility of oral dosing. The results of this case report suggest a potential role for ezatiostat in the treatment of patients with ICN who are not adequately responsive to G-CSF or as an adjunct to G-CSF.Written informed consent was obtained from the patient when she was enrolled on this IRB-approved ezatiostat randomized phase 2 ICN trial. A copy of this informed consent, which includes permission for publication, is available to the Editors upon request. Any information that could identify our subject has been withheld.Roger Lyons received consulting research support from Amgen, Alexion, Novartis, Celgene, Incyte, and Telik, Inc. Sharon Wilks has no competing interests. Gail Brown and Shelby Young are employed by Telik, Inc.GB and SY designed the research protocol. RL treated the patient and collected the data. SW referred patient to RL and provided past medical history. GB, RL, and SY wrote the paper. All authors read and approved the final manuscript.This case report has not been previously presented.


In utero haematopoietic stem cell transplantation (IUHSCT) is a non-myeloablative approach for the prenatal treatment of genetic disorders. However, in target disorders, where there is not a selective advantage for donor cells, a useful donor-cell chimerism has not been achieved. There are three possible barriers to engraftment following IUHSCT: limited space in the fetus due to host-cell competition; the large number of donor cells needed, and the immunological asset of recipient. Animal models have shown different levels of resistance to IUHSCT engraftment. In primate, goat, rat and mouse the levels of engraftment that has been achieved were low and not therapeutic. Among 46 cases of IUHSCT reported in humans, successful engraftment was obtained only in cases of X-SCID. Useful levels of chimerism has not been achieved in non-immunodeficiency diseases, and a detectable engrafment, was reported only in one case of ß-thalassemia transplanted at 12 weeks of gestation by fetal liver cells. In one α-thalassemia case, where α-globin-dependent hemoglobin production and anemia are present during fetal period, microchimerism and tolerance were suggested.To overcome the IUHSCT engraftment barriers, it is necessary to develop strategies to improve the competitive capacity of donor cells and to define the gestational age of the possible immunological “window of opportunity” in the human fetus.In utero haematopoietic stem cell transplantation (IUHSCT) is a non-myeloablative promising approach for the prenatal treatment of a variety of genetic disorders and could be an alternative option to therapeutical abortion in some congenital diseases like haematological hereditary syndromes.Observations of “naturally” occurring hematopoietic chimerismAlthough IUHSCT has been clinically successful in severe combined immunodeficiency disease (SCID), a therapeutic donnor-cell chimerism (more than 1%)The reasons for this failure are unclear and reflect a limited understanding of the engraftment barriers involved in clinical IUHSCT.There are three possible barriers to allogenic engraftment following IUHSCT : limited space in the fetus due to host-cell competition from a robust and highly proliferative fetal hematopoietic compartment; the large number of donor cells needed to successfully compete with host cells for these sites; and the immunological asset of recipient that determines the grade of the possible fetal tolerance which is conditional, depending on timing and appropiate presentation of antigenth weeks of gestationThe last barrier has been supported by successful allogenic IUHSCT in sheeps and monkeysThe goal of therapeutically relevant levels of chimerism following a single in utero transplantation has been difficult to achieve. The induction of tolerance with low levels of chimerism to facilitate postnatal cellular transplantations using minimally ablative conditioning regimens is a more realistic goal for clinical application of IUHSCT.Hematopoietic stem cells (HSC) have been isolated from fetal liverAll these cell sources were employed to perform IUHSCT; however, if postnatal cellular therapy is needed, the donor cells must be obtained from a renewable source as adult related bone marrow or mobilized peripheral bloodAn “experiment of nature” first described by Owen in 1945 is the best supporting evidence that IUHSCT might work : dizygotic cattle twins that share cross-placental circulation were born chimeric for their sibilings blood elements, and this state of “chimerism” persisted for life and was associated with specific transplantation toleranceThe first animal model was the sheep. Early gestational transplantation of allogenic, fetal liver–derived HSC into normal sheep fetuses resulted in a multilineage hematopoietic chimerismOther normal animal models have shown different levels of resistance to engraftment after in utero transplantation. In the normal primate, goat, rat and mouse (12–16) the levels of engraftment that has been achieved were low and not therapeutic: successful allogenic IUHSCT in monkeys occurred if donor and recipient fetuses are in a preimmune conditionAlmeida-Porada et al. 2000, used the in utero model of human–sheep HSC transplantation to investigate ways of improving engraftment and differentiation of donor cells after transplantationHayashi et al 2002, obtained the induction of prenatal tolerance in mice where a combined approach of IUHSCTx, performed by intraperitonel injection, followed by postnatal donor lymphocyte infusion has converted low-level mixed hematopoietic chimerism to complete donor chimerismA mouse model described by Perenteau et alHowever, further studies are needed to better characterize the timing of human fetal NK-cell maturation.The reasons for the failure in the correction most diseases and the absence of predicting tolerance in absence of immunosuppression, both in human or animal models, of IUHSCT are still unclear and reflect a limited understanding of the engraftment barriersSince Touraine et al published the first instance of intrauterine transplantation in a human fetus affected by bare lymphocyte syndrome (BLS) in 1989However, successful well-documented engraftment after in utero therapy was obtained only in cases of X-linked SCID by Touraine, Flake and WenglerUseful levels of chimerism has not been achieved in non-immunodeficiency diseases, as metachromatic leukodystrophyParticularly, a detectable engrafment, using IUHSCT, was reported only in one case of thalassemia major transplanted at 12 weeks of gestation by fetal liver cellsOnly in one α-thalassemia case, transplanted at 13 weeks of gestation with paternal CD34+ enriched cells, microchimerism and tolerance were suggestedth and the 18th weeks of gestation using cryopreserved fetal liver haematopietic stem cells and without immunodepression, was also unsuccessfulOur experience, in cases treated between the 13th and the 20th week of gestation, generated an alloimmune response as demonstrated by an high cytotoxic T cell precursor (CTLp) frequency against donor cellsParticularly, a fetus, affected by ß-thalassemia major who underwent a fetus-to-fetus transplantation between the 14We set-up a clinical protocol by which two female fetuses, 16–20 weeks old, were transplanted with circulating hematopoietic paternal stem cells after one week of low dose dexamethasone treatment with the aim to overcome the engraftment barrier by inducing a transient mild fetal immuno suppression and induce a donor-specific tolerance. During these procedures no adverse events were observed both for the mothers and the fetuses. At birth, both fetuses showed the presence of Y chromosome of donor origin but only one, at two months of age, showed the presence of paternal blood group allele A cDNA in peripheral blood and an increase of HbA concentration from 3% to 14.4%, suggesting that erithroid microchimerism has been occurred. At 5 months of age haemoglobin decreased to 6.8 g/dl and the girl started a regular blood transfusional regimenTo overcome the barriers that hinder the success of IUHSCT, it is necessary to develop strategies to improve the competitive capacity of donor cells to achieve significant engraftment, and to define the gestational age of the possible immunological “window of opportunity” in the human fetus.A significant barrier to allogeneic engraftment following IUHSCT is limited space in the fetus due to host-cell competition from a robust and highly proliferative fetal hematopoeitic compartment in circumstances where a competitive or survival advantage does not exist for donor cellsThus, strategies that selectively increase donor HSC homing and engraftment in the fetal recipient may increase both the levels and frequency of engraftment following allogeneic IUHSCT. Dipeptidyl peptidase CD26 inhibition may represent a novel approach to increasing the efficacy and success of HSC/HPC transplantationRecent in vitro studies have demonstrated that increased cleavage of stromal-derived factor 1 (SDF-1) by CD26, a chemokine involved in hematopoietic cell chemotaxis, homing, mobilization and survival, results in a loss of its chemotactic effect on primitive hematopoietic cells. Instead, its inhibition results in SDF1–induced migration In vivo, postnatal studies in a murine bone marrow transplantation (BMT) model found increased short-term bone marrow (BM) homing of enriched HSC and low-density BM donor cells after inhibition or absence of CD26 activity. Reduction of CD26 activity on low-density donor BM cells also resulted in increased long-term engraftment, competitive repopulation, secondary transplantation, and mouse survival following lethal irradiation. Therefore, CD26 inhibition may be an important adjunct to other strategies that are directed toward overcoming the barriers to fetal engraftmentOther manipulations that selectively influence expression of homing receptors and engraftment include cotransplantation with stroma. The use of stromal cotransplantation has increased short- and long-term donor cell expression in the sheep modelTherefore, the recognition of an immune barrier suggests potential immune-based strategies for application to IUHSCT that might prevent loss of engraftment.th week of gestation suggested by the presence of alloreactive T lymphocytes from the 10th week of gestation that could explain the failure of engraftment in fetuses older than this gestationth week of gestation. However, at this so early gestation it is impossible to carry out the infusion of CD34 into the umbilical cord by intravascular route or by their infusion into intraperitoneal cavity. One possible future approach could the infusion of these cells into the coelomic cavity.Some studies in miceCoelocentesis at 6–10 weeks of gestation has been successfully used to obtain fetal DNA for very early prenatal diagnosis of genetic diseasesThe injection of donor haematopoietic stem cells into this cavity could potentially induce tolerance in the fetus making successive booster stem cells infusions possible. This procedure has already been evaluated in pre-immune fetal sheep in which the injection of human stem cells through the coelomic cavity was associated with a significant level of engraftmentThe intrauterine approach was criticized by several authorities in this field who claimed that this approach did not offer any advantage over postnatal transplantation. The main arguments against intrauterine transplantation were that the fetal invasive procedure carried a certain additional risk.Conversely, promoters of intrauterine transplantation claimed that reconstitution of the immune system before birth results in reduced susceptibility to infections in the neonatal period, and to an improved psychosocial situation for the family. Other potential advantages for an intrauterine approach include cost savings and a reduced risk of GVHD in the fetal environment.Thus, a consensus has been difficult to reach and fetal transplantation has not been widely adopted, despite the fact that successful cases treated in utero had outcomes comparable with the best reported with postnatal transplantation


Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are thought to be associated with increased mortality in elderly patients. Low retinol-binding protein-4 (RBP4) is associated with a high risk of respiratory infections in the general population. Therefore, we hypothesized that low RBP4 levels are associated with an increased risk of AECOPD and can be used as a biomarker for AECOPD in elderly patients.Enzyme-linked immunosorbent assays were used to assess RBP4 levels in elderly with AECOPD within the first 24 hours after intensive care unit admission. Forty-six elderly patients with stable COPD in outpatient clinics and 50 healthy elderly persons who had physical examinations as outpatients were controls.p < 0.001). RBP4 levels were decreased by 30.6% in non-survivors compared with survivors . A higher Acute Physiology and Chronic Health Enquiry II (APACHE II) score and Simplified Acute Physiology score (SAPS II) were associated with lower RBP4 levels . RBP4 was positively correlated with creatinine and body mass index, and negatively correlated with C-reactive protein and Global Initiative for Chronic Obstructive Lung Disease stage. Multivariate logistic regression showed that RBP4 was an independent mortality predictor of AECOPD . Analysis of the area under the receiver operating characteristic (AUC) curve showed that RBP4 showed good discrimination  in predicting mortality. RBP4 improved the prognostic accuracy of mortality for the APACHE II and SAPS II scores.In AECOPD patients, RBP4 levels were lower than those in stable COPD patients and healthy controls (59.7 vs 91.2 and 113.6 mg/L, Serum RBP4 levels are significantly reduced in elderly AECOPD patients. RBP4 might be a good predictive biomarker for mortality in elderly AECOPD patients in the intensive care unit. Chronic obstructive pulmonary disease (COPD) is a condition characterized by progressive airflow limitation, which causes considerable morbidity and mortality worldwide, and results in substantial social and economic burden . PatientRecently, considerable attention has been paid to adipocytokines, biologically active factors secreted by adipose tissue as an endocrine organ . SeveralRetinol-binding protein-4 (RBP4) is a molecule found in the circulation, thought to be secreted mainly by adipose tissue and the liver, and is a specific transporter for retinol in the circulation . RBP4 haThis study aimed to identify whether RBP4 is predictive for mortality among various risk factors in elderly AECOPD patients in the ICU. We report here that lower levels of plasma RBP4 have a higher risk of mortality and can be used for predicting prognosis in elderly AECOPD patients in the ICU.1/FVC) < 70% according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines [1/FVC ratio < 0.7 and FEV1 % ≥80% predicted. Moderate COPD was defined as an FEV1/FVC ratio < 0.7 and FEV1 % 50–79% predicted. Severe COPD was defined as an FEV1/FVC ratio < 0.7 and FEV1 % 30–49% predicted. Very severe COPD was defined as an FEV1/FVC ratio < 0.7 and FEV1 % < 30% predicted.COPD was defined as dyspnea, chronic cough, or sputum production and/or a history of exposure to risk factors for the disease, and post-bronchodilator 1-second forced expiratory volume/forced vital capacity , and/or severe/worsening of hypercapnia , and/or severe/worsening of respiratory acidosis (pH < 7.25) despite supplemental oxygen and noninvasive ventilation; (4) the need for invasive mechanical ventilation; (5) hemodynamic instability and the requirement for vasopressors. Exclusion criteria included interstitial pneumonia, asthma, pulmonary tuberculosis, bronchiectasis, pulmonary fibrosis, and lung cancer according to clinical history and data [Patients with AECOPD admitted to the ICU must meet at least one of the following indications : (1) sevand data . ArteriaSystemic inflammatory response syndrome (SIRS) according to international, standardized criteria  was charAccording to the above criteria, a total of 100 patients admitted to the First and Sir Run Run Shaw Hospitals from September 2010 to February 2012 were recruited. Clinical data within the first 24 hours after ICU admission were collected. All patients were aged ≥65 years, with an ICU stay of ≥72 hours. All patients were scheduled for a follow-up visit until being discharged from the ICU (survivors) or they died in the ICU (non-survivors). To further analyze the prognostic role of RBP4 for AECOPD, we subdivided the patients according to whether they had type 2 diabetes mellitus (T2DM) or sepsis. T2DM was diagnosed according to American Diabetes Association criteria , and sepForty-six stable (no exacerbations for at least 4 weeks prior to study entry)  COPD patWritten informed consent was obtained from each participant or his or her spouse, and the study was approved by the local ethics committee .Routine blood analyses, including WBC, total bilirubin (TB), γ-glutamyl transferase, albumin (ALB), blood urea nitrogen, serum creatinine (sCr), lactic acid (LA), CRP, B-type natriuretic peptide (BNP), procalcitonin (PCT), RBP4, and blood and sputum culture were immediately performed after admission to the ICU. RBP4 serum concentrations were measured by enzyme-linked immunosorbent assay according to the manufacturer’s instructions . BNP was measured by the electrochemiluminescence technique . PCT was measured by enzyme link fluorescence assay . The homeostasis model assessment for insulin resistance (HOMA-IR) was used as a measurement of insulin resistance and was calculated as fasting plasma glucose (mmol/L) × fasting serum insulin (mU/mL)/22.5. The Acute Physiology and Chronic Health Evaluation (APACHE) II score, and Simplified Acute Physiology Score (SAPS II) were evaluated within the first 24 hours (day 1) after ICU admission.p > 0.05). Data are presented as mean and standard deviation, or median and interquartile range, as appropriate. Fisher's exact was used for comparison of the primary disease between the non-survivor group and the survivor group. For comparison of study variables between the two groups, the Student’s t-test or Mann–Whitney U test was performed. Pearson’s correlation coefficients were calculated to evaluate the relationship between serum RBP4 levels and other clinical and metabolic variables. We assessed the risk factors for ICU mortality using univariate analysis and multiple logistic regression analysis to determine independent factors and dependent factors for prognosis.For statistical analysis, measurements of RBP4, ALB, LA, PCT, and CRP were logarithmically transformed to obtain normal distributions .We compared the number of risk factors for the death probability of AECOPD. Discrimination was calculated using area under the curve (AUC) values. The AUC values were compared using a nonparametric approach. AUC analysis was also used to calculate the cut-off values, sensitivity, specificity, and overall correctness. Finally, cut-off points were calculated by calculating the best Youden index (sensitivity+specificity-1). Cumulative survival curves as a function of time were plotted using the Kaplan–Meier approach and were compared using the log rank test. A p < 0.001; Table p < 0.01). Serum RBP4 levels were different between male and female patients.The median serum concentration of RBP4 in stable COPD was 91.2 mg/L and in healthy controls it was 113.6 mg/L. AECOPD patients had significantly reduced serum RBP4 concentrations compared with stable COPD patients and healthy controls , and the mean FEV1/FVC ratio was 45% (28–63%).A total of 100 AECOPD patients meeting ICU admission standards  were incp < 0.05 or < 0.01, Table A total of 64 patients  who died were included in the non-survivor group and 36 patients  who survived were included in the survivor group. The distributions of sex, age, primary diseases , ICU days, pulmonary function, and blood gas analysis tests were not significantly different between the two groups. Premorbid diseases have not been shown to be predictive of outcome. The durations of AECOPD, APACHE II and SAPS II scores, mechanical ventilation, and ventilation time were significantly elevated in the non-survivor group compared with the survivor group (p < 0.001) , the SAPS II score (p = 0.005), LA (p = 0.032), TB (p = 0.044), duration of AECOPD (p = 0.037), and ventilation time (p = 0.001) compared with non-survivors. By contrast, WBC (p = 0.06), CRP (p = 0.08), PCT levels (p = 0.24), and FEV1 (p = 0.224) were similar in the survivor and non-survivor groups. In addition, BMI (p = 0.072), metabolic parameters on admission, including glucose (p = 0.57), and ALB levels (p = 0.33) were not different between survivors and non-survivors (Table p = 0.001).We investigated whether serum RBP4 concentrations upon ICU admission can predict clinical outcome and mortality. Interestingly, patients who died during ICU hospitalization 64/100) displayed significantly lower serum RBP4 concentrations than patients who survived     Figure c. RBP4 cp = 0.988)     levels     Figure a. RBP4 l) Figure b. RBP4 l) Figure c, but th) Figure d and ALBs Figure e. BMI hap = 0.008), the APACHE II score with the AUC was 0.75 , and the SAPS II score with the AUC was 0.77 . AUC analysis showed that RBP4 has the best discriminatory power. A combined model including RBP4 and the APACHE II score  improved the prognostic accuracy compared with the APACHE II score alone. Similarly, RBP4 tended to improve the prognostic accuracy of the SAPS II score in a combined model .To assess the prediction of RBP4 on the prognosis of mortality of AECOPD patients, we performed univariate and multivariate logistic regression, which compared the prognostic values of RBP4 and other well known risk factors, including age, BMI, the APACHE II score, the SAPS II score, glucose levels, TB levels, WBC, LA, CRP levels, and PCT levels. As shown in Table In this study, we showed that elderly AECOPD patients in the ICU had severe disease and high mortality, and RBP4 levels were lower in non-survivors than survivors. Lower RBP4 levels were accompanied by a higher mortality. For the subgroups, RBP4 serum levels were lower in patients with sepsis than in those without sepsis. RBP4 was an independent predictor of mortality of AECOPD in elderly patients in the ICU.RBP4, the circulating transporter for vitamin A, is synthesized in the liver and adipose tissue. RBP4 levels in patients with AECOPD were significantly lower than those in stable COPD and healthy subjects, as anticipated from previous studies using the same assay . RBP4 isSerum RBP4 levels were associated with nutritional status in COPD patients. Nutritional status is related to respiratory impairment and systemic inflammation in patients with AECOPD . MalnutrMultivariate logistic regression analysis showed that RBP4 was an independent predictor of mortality. In the assessment and management of critically ill patients, knowledge of prognostic factors is crucial to estimate the risk for mortality. Receiver operating curves for the prediction of mortality indicated that RBP4 with an AUC of 0.88 was higher than those of the clinical scores . Our study shows that measurement of RBP4 levels has a high prognostic use as an early and independent risk predictor for potential death to complement and thereby improve the APACHE II and SAPS II scores. Aged patients with low serum RBP4 levels caused by AECOPD might have a poor prognosis.Escherichia coli lipopolysaccharide [The possible role of low RBP4 concentrations observed in the circulation of AECOPD patients is unclear. We consider that at least five reasons may explain this finding. The first reason might involve inflammation. RBP4 levels are reduced with individual stress levels of patients, and thus mirror the stress associated with the severity and extent of illness . Tumor nccharide . We founccharide . Third, ccharide . Fourth,ccharide . Fifth, ccharide , may plaSerum RBP4 levels in AECOPD patients are correlated with liver and kidney functions, nutritional status, and disease stage, but not serum HOMA-IR, and are an independent factor of overall survival in elderly AECOPD patients. In the ICU, AECOPD patients with RBP4 deficiency have a greater risk for short-term hospital mortality, and require more intensive surveillance. In addition, we should pay more attention to the function of RBP4 as a potential determinant of prognosis.The authors declare that they have no competing interests.All authors helped to draft the manuscript and critically revise it. All authors agreed about the content of the paper and have read the manuscript and approved its submission. JQH and CYL conceived and designed the experiments. CYL and LYF collected the data. HXJ and JQH participated in statistical analysis.


Using scanning electron microscopy, microscopic structures have been identified in fresh cerebrospinal fluid (CSF) in patients with schizophrenia and bipolar disorder, but only rarely in control subjects. However, it has not been determined whether these microscopic particles represent state or trait markers, i.e. if their presence is related to clinical manifestations of the disease or if they also can be found in as yet asymptomatic individuals with a genetic liability. This question can be addressed by studying twins discordant or concordant for schizophrenia or bipolar disorder.We investigated microscopic structures in CSF in 102 individuals: 21 monozygotic and 16 dizygotic twins affected or not affected with schizophrenia, schizoaffective disorder or bipolar disorder and in 65 healthy singleton controls. A first and a second fraction of CSF was freshly applied on filters and examined by scanning electron microscopy technique. Spherical particles with lipid appearance averaging between 0.1 to 8.0 µm in diameter were detected in the center of the filter as well as located in the margins of larger aggregates binding in a viscous state. Structures were found in 12 of 17 probands, 5 of 12 healthy co-twins and 3 of 73 healthy controls. Thus, a positive microscopic finding significantly increased the likelihood of belonging to the proband group  and the co-twin-group . Age, sex, history of alcohol abuse or anxiety syndrome, somatic disorder and markers of acute inflammatory activity did not account for group differences; nor did exposure to psychotropic medication.Presence of microscopic particles in CSF may possibly reflect trait dependent genetic or environmental vulnerability in patients with schizophrenia, schizoaffective disorder or bipolar disorder. Identifying biomarkers for psychiatric disorders would be an important milestone and a step towards developing tools for improved diagnostic procedures, identification of high-risk individuals, and personalized psychiatric treatment. Cerebrospinal fluid (CSF) is an especially suitable substrate to use in the quest for biomarkers in psychiatric disorder Schizophrenia is presumably a neurodevelopmental disorder characterized by hallucinations, delusions, and cognitive deficits. Bipolar disorder is an affective disorder that presents with episodic mood swings from high periods or mania to low periods of depression, but is also often accompanied by cognitive deficits and psychotic symptoms. Schizophrenia and bipolar disorder have a symptom overlap and in line with this reasoning, epidemiological and genetic studies have suggested a partly shared genetic liability between bipolar disorder and schizophrenia In 2002, micrometer-sized structures were identified in CSF from patients with schizophrenia using a scanning electron microscopic (SEM) technique. In 20 of 22 patients with schizophrenia but in only 2 of 38 non-schizophrenic controls micrometer-sized spherical particles were found in CSF It is still unknown whether the microscopic patterns identified in the CSF are present in vulnerable but as yet asymptomatic individuals. If this were the case, SEM analysis of CSF could be used to identify individuals with increased risk for the disorders under study The aim of the present study was to investigate the incidence of microscopic particles in two fractions of fresh cerebrospinal fluid in twin subjects discordant or concordant for schizophrenia or bipolar disorder, unaffected twin pairs, and healthy individuals without major psychiatric disorders. A secondary aim was to examine a large cohort of healthy controls using identical CSF sampling techniques that were used in the study of probands with bipolar disorder The ethical review board of Karolinska Institutet, Stockholm, Sweden (#2007/779) approved the study of the twins, which was performed in compliance with the Helsinki Declaration. The collection of the healthy singleton subjects was part of an ongoing study approved by the Regional Ethics Committee in Stockholm (#2009/1221-32) and conducted in accordance with the latest Helsinki Protocol. All enrolled patients and control subjects consented orally and in writing to participate in the study. There were no adverse effects in the twins or the singletons, which may be explained by fixed routine and that the same neurologist performed the lumbar punctures.Nineteen twin pairs were recruited from a nationwide cohort of Swedish-born, same-sex twins with schizophrenia, bipolar disorder and healthy control-pairs ascertained through the Swedish Twin Registry. In total, 38 twins were included in the study where one or both twins in a pair had an ongoing or past history of bipolar disorder, schizophrenia, or schizoaffective disorder. In four of the included twin-pairs none was affected by bipolar disorder, schizophrenia or schizoaffective disorder. The sibling of one of the 38 recruited twins did not agree to participate in the lumbar puncture. None of the participants was hospitalized at the time of examination. All 37 twins and 65 healthy controls, who received a small remuneration for their participation, consented orally and in writing to take part in the study. The regional ethical review board of Karolinska Institutet, Stockholm, Sweden, approved the study, which was performed in compliance with the Helsinki Declaration.The zygosity of the twins, mono- or dizygotic, was validated using a robust panel of 47 highly multiplexed single nucleotide polymorphisms (SNPs) that provide reliable and high quality data on a range of different DNA templates All twin individuals were interviewed with The Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I) www.scb.se) from the National Population Register in the Stockholm area. The participants were matched on sex and age with patients with bipolar disorder enrolled in a prospective study (the St. Göran bipolar project) as previously described Healthy volunteers were selected randomly for the study by Statistics Sweden , using a Swedish modified version of the Affective Disorder Evaluation assessment tool 9/L . Height and weight were recorded on the same day as blood and CSF sampling for calculation of the body mass index (BMI) as a heuristic proxy for body fat of the participants.The blood and CSF investigations for the twins lasted from March 2008 to September 2011 and for the controls from November 2009 to December 2010. Blood samples were collected with the subjects in a fasting state at 08 h under sterile conditions before the lumbar puncture. The integrity of the blood-CSF barrier of the twin and control samples was assessed by the albumin ratio, expressed as CSF albumin (mg/L)/serum albumin (g/L). Blood-CSF barrier dysfunction was defined as an albumin ratio >6.8 in individuals ≤44 years and >10.2 in individuals ≥45 years according to the reference limits presented by Blennow and coworkers The same neurologist performed the 102 lumbar punctures with all subjects in a sitting position. Sixteen twin pairs were examined on the same day and the remaining two pairs within the same month. One of two types of fine disposable needles was used; either Becton Dickinson (BD) 22 GA 3.00 IN, 0.70×75 mm (Quincke needle used in 26 twins and 17 control subjects) or BD Whitaker Needle 25 GA 3.50 IN, 0.50×90 mm (Sprotte needle used in 12 twins and 48 control subjects). The skin in the lumbar region was washed with sterile cotton swabs and chlorhexidine 5 mg/mL (Fresenius Kabi) before puncture. The needles were inserted in vertebral interspace L3 to L4, or L4 to L5, and the very first 12 drops of CSF, approximately 0.6 mL, were collected in a sterile test tube for microscopic examination. The following 12 mL of CSF were allowed to drip spontaneously, or by suction six times using a 2 mL syringe due to slow flow, in a second test tube, which was gently inverted 10 times to secure homogeneous mixing of the components to avoid gradient effects. An aliquot of 0.6 mL of the mixed 12 mL was transferred into a third sterile test tube for immediate filtration and gold coating before microscopic examination. The first and the second fraction of CSF were handled in a similar way for SEM. The last mL of CSF for the control subjects was sent within 30 min after CSF withdrawal to the hospital laboratory for visual inspection and cell counting.Of each 0.6 mL fresh CSF fractions, 200 µL were pipetted and dripped onto the surface of a polycarbonate filter  with 0.6 µm pores. The polycarbonate filter was specially prepared by GP Plastic AB  and supplied by Sempore AB . The filter was fitted to an airtight device designed with flow channels that allows CSF to stream to the center of the filter when vacuum suction is applied from below. This design does not allow particles with sizes larger than 0.6 µm to drip through the filter. The remaining structures in the CSF were thus concentrated in the center of the filter. Once the filter was dried it was coated in a JEOL JFC-1200 Fine Coater  during two minutes with ionized gold to a thickness of 40 Å.The 204 filters in the present study were analyzed in a SEM microscope . The total area of each filter with a diameter of one cm was examined in the microscope and rated by an experienced researcher (RN). The peripheral area outside the center was mostly free of structures. To standardize the procedure, SEM images of the central areas of the filters were enlarged 50, 500 and 2000 times and saved for further reference. A second researcher (LW) rated the same images with similar results. The microscopic quantity of the morphological structures on each filter was rated in the following four categories: 0 = no, 1 = few, 2 = several and 3 = many structures  as descr9/L .Blood samples were collected under sterile conditions before the lumbar puncture. The integrity of the blood-CSF barrier of the twin and control samples was assessed by the albumin ratio, expressed as CSF albumin (mg/L)/serum albumin (g/L). Blood-CSF barrier dysfunction was defined as an albumin ratio >6.8 in individuals ≤44 years and >10.2 in individuals ≥45 years according to the reference limits presented by Blennow and coworkers The results of the clinical assessment of the 37 twins are presented in Descriptive statistics are expressed as means (standard deviations) or medians  for continuous variables and frequencies (percentages) for categorical variables. Group differences in our data were examined using the exact conditional logistic regression model  Structures of the CSF were examined on two filters from each participant, one from the first and one from the second sample of fresh CSF. Spherical particles were noted with the appearance of small lipid bodies averaging between 0.1 to 8.0 µm. Filters with a negative finding did not present any structures . ParticlDemographic data are presented in A series of analyses were conducted in order to determine whether a history of alcohol abuse or anxiety syndrome, somatic disorder, smoking, blood-CSF-barrier dysfunction , BMI, type of needle used in the lumbar puncture procedure, acute infection or inflammation and use of psychotropic medications could account for the above associations. A previous or ongoing history of alcohol abuse or dependency strongly predicted the risk of being a proband . However, when we tested the effect of a positive CSF finding among the probands and controls with no history of alcohol abuse or dependency (probands n = 10 and controls n = 72), microscopic particles in the CSF continued to be associated with proband status . Anxiety syndrome, somatic disorder, smoking and type of needle at lumbar puncture (Quincke vs. Sprotte needle) did not predict the risk of being a proband or a co-twin. Including high vs. low levels of albumin ratio in the model did not change the significance of the associations between CSF particles and proband or co-twin status. When individuals (n = 7) with a high albumin ratio - indicating a defect blood-CSF-barrier - were removed from the analysis, the association between proband/co-twin status and a positive CSF finding remained significant.Since acute or chronic infection may be a cause of the structures in CSF, we investigated measures of acute and chronic infection or inflammation. HS-CRP and WBC were used as indicators of acute infection. Excluding subjects from the analysis with HS-CRP or WBC above the reference limit did not change the significance of the associations of CSF particles with the proband or co-twin status. The effect of a previous infection, chronic rheumatic disorder, or gastro-inflammatory disorder were included separately in the model but did not affect the association between proband or co-twin status and microscopic particles.The effect of psychotropic medication on the microscopic CSF findings was tested in the proband group, since the non-proband subjects had not received psychotropic medications with the exception of three co-twins and two control-twins treated with antidepressants at the time of lumbar puncture. Neither “any psychotropic medication” , nor any of the separately tested psychotropic medications  had a significant effect on the presence of CSF structures. The effect of antidepressants was tested on the individuals with a diagnosis of schizophrenia, schizoaffective disorder, bipolar disorder, and depression .Six individuals, five twins and one healthy singleton were prescribed statins (cholesterol-lowering drugs) of whom three displayed microscopic CSF particles.To explore a possible genetic contribution to the CSF results, we tested the effect of zygosity in the co-twin group. There was a clear tendency for a positive CSF finding to be more frequent in the MZ co-twins (57%) than in the DZ co-twins (20%), but this difference did not reach significance in this limited sample of twins .Detailed analyses were performed in the sample to investigate the association between particles in the CSF and the separate diagnostic groups divided into schizophrenia or schizoaffective disorder and bipolar disorder type I or type II. Microscopic particles were strongly associated to schizophrenia and schizoaffective disorder  and their co-twins . Moreover particles were associated to bipolar disorder type I and II , but not to their respective co-twins .In the group of patients with bipolar disorder type I, all subjects had experienced at least one psychotic episode. We therefore analyzed the association between particles and patients with psychotic features including probands with schizophrenia, schizoaffective disorder and bipolar disorder type 1, removing the patients and co-twins with bipolar disorder type II. The association continued to be significant in probands  and co-twins .The association between CSF-particles and group-status when excluding the subjects with a history of lifetime depression in the control-group increased in the proband group  as well as in co-twins .We performed a separate analysis to compensate for non-independency within twin-pairs by removing either twin 1 or twin 2 from the disease concordant pairs in the proband group and from the healthy twin-pairs in the control-group. When we tested the effect in probands (n = 14) and co-twins (n = 12) compared to controls (n = 69) particles in CSF continued to be associated with proband status  and co-twin status . We got the same results when removing twin 1 as when removing twin 2.9/L), an increased level of HS-CRP (16.9 mg/L) and protein-like aggregates were observed in his CSF sample (9/L), a slightly increased level of HS-CRP (3.5 mg/L) and displayed spherical particles in his CSF  may help to answer the question of whether structures of the CSF precede a manifest disease state and may serve as a trait marker. Further work is needed to explore the origin and the composition of the anatomical structures in both the first and second CSF fractions.The main conclusion from this study is the strong statistical support for structures in CSF identified with the SEM technique in patients with bipolar disorder or schizophrenia. The results indicate that the CSF structures likely originate from trait-dependent factors.Table S1Detailed information on the characteristics of 102 individuals included in the study.(DOC)Click here for additional data file.


ASPM  gene has been proposed as a major determinant of cerebral cortical size among primates, including humans. Yet the specific functions of ASPM and its connection to human intelligence remain controversial. This debate is limited in part by a taxonomic focus on Old World monkeys and apes. Here we expand the comparative context of ASPM sequence analyses with a study of New World monkeys, a radiation of primates in which enlarged brain size has evolved in parallel in spider monkeys (genus Ateles) and capuchins (genus Cebus). The primate community of Costa Rica is perhaps a model system because it allows for independent pairwise comparisons of smaller- and larger-brained species within two taxonomic families. Accordingly, we analyzed the complete sequence of exon 18 of ASPM in Ateles geoffroyi, Alouatta palliata, Cebus capucinus, and Saimiri oerstedii. As the analysis of multiple species in a genus improves phylogenetic reconstruction, we also analyzed eleven published sequences from other New World monkeys. Our exon-wide, lineage-specific analysis of eleven genera and the ratio of rates of nonsynonymous to synonymous substitutions (dN/dS) on ASPM revealed no detectable evidence for positive selection in the lineages leading to Ateles or Cebus, as indicated by dN/dS ratios of <1.0 . Our results suggest that a multitude of interacting genes have driven the evolution of larger brains among primates, with different genes involved in this process in different encephalized lineages, or at least with evidence for positive selection not readily apparent for the same genes in all lineages. The primate community of Costa Rica may serve as a model system for future studies that aim to elucidate the molecular mechanisms underlying cognitive capacity and cortical size.The  ASPM  gene has been proposed as a major determinant of cerebral cortical size among primates A disproportionately large cerebral cortex is a hallmark of human evolution. It facilitates increased information processing and thus our perceived high level of intelligence and rapid rate of cultural innovation; for instance, our capacity for tool-making, social intelligence, and language ASPM gene encodes a 10,434-bp-long coding sequence, in 28 exons, and spans 65 kb of genomic DNA. Exon 3 spans approximately 1500 bp while exon 18 spans approximately 4700 bp. The remaining exons of the ASPM gene span less than 200 bp each. The ASPM gene encodes a protein that is widely conserved between primates. The ASPM protein contains four distinguishable regions: a putative N-terminal microtubule-binding domain, a calponin-homology domain, an IQ repeat domain containing multiple IQ repeats , and a C-terminal region The Calmodulin binding to IQ motifs induces a conformational change in proteins that regulate the binding of actin to the aminoterminal CH domains. It has been proposed that changes in ASPM induce changes in the orientation of the mitotic spindle of neuroblasts, which induces symmetric mitosis generating two progenitor cells; as opposed to one progenitor cell and one postmitotic neuron, typical of asymmetric mitosis. The additional rounds of symmetric duplication cause an exponential expansion of the progenitor pool. Control of this proliferative symmetry can cause dramatic alterations in cerebral cortical size, and so changes in ASPM could regulate cortical size by making subtle changes in spindle orientation ASPMASPM is expressed in numerous proliferating tissues outside the cerebral cortex, suggesting it has functions apart from neuroblast replication. Other authors linked ASPM to more general mechanisms such as ciliary function and spermatogenesis rather than neural development, further confusing the functional significance of ASPM during human evolution ASPM was linked to recent and ongoing selective sweeps among populations of modern humans ASPM genetic variants and human intelligence have been refuted ASPM function, but do little to resolve why ASPM evinces a purported signature of positive selection in certain primate lineages.Such claims have fueled further research on the biology and function of ASPM and brain size evolution have been based on Old World monkeys, apes, and humans ASPM variation and brain size across primates using codon-specific maximum likelihood tests of selection. Such an approach aimed to detect mutations in specific sites that might have impacted the function of the ASPM protein in specific lineages in ways that might have positively affected the fitness of an individual that carried the mutation (such that the mutation would have swept to fixation rapidly).In general, ties between ASPM and an increase in neonatal brain size across 21 anthropoid genera based on a fraction (2.3%) of the codon sites predicted to present a significant increase in non-synonymous substitutions over synonymous substitutions. Accordingly, Montgomery et al. ASPM, and suggested that it might be responsible for independent increases in brain size in all major clades of primates, including New World monkeys.Ali and Meier ASPM evolution within primates. However, if positive selection of ASPM is related to an ancestral increase in brain size, a comparative analysis requires the inclusion of multiple related species within genera, which allows for proper reconstruction of the ancestral states of ASPM sequences. Here we expand on these earlier findings by incorporating platyrrhine genera from all previous studies and adding four species from the primate community of Costa Rica, for a total of 16 species. Our analysis not only allows for a more complete treatment of the families Atelidae and Cebidae, but it also highlights the practical value of focusing on communities as model systems for studying the evolution of cerebral cortical size.Montgomery et al.’s Alouatta palliata) and the black-handed spider monkey (Ateles geoffroyi). The two species are comparable in body size, yet the brain of A. geoffroyi is nearly twofold larger (Cebus capucinus) and the squirrel monkey (Saimiri oerstedii). The relative brain size of Cebus is among the largest of any nonhuman primate and 10–20% larger than that of Saimiri  Ateles, the selective advantages associated with remembering the location and availability of such patchy resources has been tied to the evolution of its relatively large brain Ateles are reported to exceed those of squirrel monkeys and macaques under experimental conditions The tropical forests of Costa Rica are highly dynamic fluctuating systems that require complex foraging strategies to meet nutritional needs over all phases of an annual cycle. Despite similar body sizes, Cebus and Saimiri also share similar body sizes and morphologies, and often forage sympatrically on the same substrates Cebus and Saimiri differ considerably in the extent to which they acquire resources manually Saimiri tends to glean invertebrates from exposed surfaces, Cebus spends ca. 50% of its day searching for and extracting embedded foods Cebus manufactures the greatest variety of tools and uses them more often than all nonhuman primates excepting chimpanzees Cebus, the cognitive demands and selective advantages associated with conceptualizing unseen foods and the use of tools to extract them was possibly a major contributing factor to the evolution of its large brain Ateles and Cebus, i.e. the need to remember scarce resources or imagine and extract concealed ones, it is worth testing whether there has been convergent adaptive evolution in these lineages of the same genes believed to be responsible for human cortex size and cognitive ability. More specifically, the pair-wise coexistence of large- and small-brained atelid and cebid primates in Costa Rica provides a model system for testing molecular hypothesis focused on the evolution of brain size. In this study we compared the complete sequence of exon 18 in ASPM across eleven platyrrhine genera, including multiple species of large-brained Ateles and Cebus, and small-brained Alouatta and Saimiri, and studied patterns of natural selection on this gene coding sequence using both an exon-wide lineage-specific analysis similar to Evans et al. ASPM gene and has been shown to code for most of the critically functioning region of the ASPM protein; the Cadmodulin-binding domain, consisting of repetitive IQ domains. Early dN/dS analyses of anthropoid primates indicated different rates of evolution along the ASPM protein, where rapidly evolving residues were mainly concentrated in the IQ repeat domain, coded in its majority by Exon 18, making it the prime region to consider in studies of natural selection Given the ecological and cognitive challenges faced by ASPM was reported to be under positive selection in the lineages leading to humans and other primates with relatively increased cortex size. In particular, a human-chimpanzee comparison revealed repeated selection on multiple sites along the ASPM gene Ateles and Cebus, which evolved increased cortical sizes independently within their taxonomic families. In contrast to their shared increased cortical sizes, amino acid replacements specific to humans, Cebus and Ateles found in exon 18 of the ASPM protein do not overlap, underlying the independent evolution of this region in these species of interest (ASPM gene resulted in dN/dS ratios of 0.6502 for the Ateles lineage and 0.4268 for the Cebus lineage (N/dS) on ASPM thus revealed no detectable evidence for positive selection. It should be noted that the only branch to present a positive dN/dS ratio, was that of Alouatta palliata , and was not significantly different from a model of neutral evolution (p = 0.235).Previously, interest . Lineage lineage . Such va 3.0377, , howeverN/dS to vary across codon positions as well as across lineages, and then recording the differences in the distribution of positively selected changes between “foreground” branches (Cebus and Ateles branches) and the rest of the phylogenetic tree. Our results from this test also failed to reject a model of neutral evolution in the Ateles and Cebus lineages (p = 0.491). The discrepancy in results may be due to the addition of an extra species within the genus Ateles, allowing for better phylogenetic reconstruction.Although our exon-wide lineage-specific results within the Atelidae stand in contrast with those of Ali and Meier N/dS value of 0.479 Finally, following the methodology of Montgomery et al. ASPM in the Atelidae, and no evidence of strong positive selection in any other lineage of New World monkeys. We also found no evidence of gradual positive selection over the platyrrhine clade, despite evidence for independent cerebral enlargement in the lineages leading to Ateles and Cebus. This finding is somewhat contradictory to Montgomery et al.’s ASPM into their analysis, so it is possible that our results are consistent with positive selection occurring on exon 3. Because changes in exon 18 are most likely to affect neural cell duplication, we expected positive selection associated with brain size increase to occur in this region. It is possible that positive selection of ASPM is associated with processes other than brain development, at least in New World monkeys, thus explaining the inconsistencies between our results and previously published results.In contrast to the results of Ali and Meier ASPM exon 18, even if such changes did in fact partly account for the large differences in brain anatomy among New World monkeys. It is also plausible that changes in other ASPM exons that we did not study, or in ASPM regulatory regions, may harbor evidence of positive selection in these taxa. Of course, brain development and higher cognitive skills are almost certainly affected by a multitude of interacting genes, such that convergent phenotypic features may be achieved through different modifications on separate lineages.We note that it is also possible that our tests had insufficient power to detect evidence of positive selection on any specific changes in ASPM with species under similar selective pressures, one might be able to provide insight into human neurogenetics. Species such as Cebus and Ateles offer independent examples of encephalization and relatively increased intelligence among primates.The primate community of Costa Rica, featuring coexisting small- and large-brained species with remarkable behavioral diversity, has the potential to be an instructive model system for testing hypotheses relevant to human evolution. By contrasting candidate genes such as We affirm that our research methods were approved by all relevant government agencies and the Chancellor’s Animal Research Committee of the University of California, Santa Cruz . UC-Santa Cruz was the institutional affiliation of the corresponding author at the time of the research. Samples were collected with the approval of Costa Rica’s Ministerio de Ambiente y Energía (permit no. 069-2001 OFAU). Buccal swabs containing DNA were exported from Costa Rica (Ministerio de Agricultura y Ganaderia permiso fitosanitario no. 331) and imported into the USA (CITES permit no. 06US130146/9).Alouatta palliata, Ateles geoffroyi, Cebus capucinus, Saimiri oerstedii) in the Pacific region of Costa Rica (Ministerio de Ambiente y Energía permit no. 069-2001 OFAU). Buccal swabs containing DNA were exported from Costa Rica (Ministerio de Agricultura y Ganaderia permiso fitosanitario no. 331) and imported into the USA (CITES permit no. 06US130146/9). The DNA was extracted using a standard Qiagen DNeasy blood and tissue extraction kit. This protocol was reviewed and approved by the Chancellor’s Animal Research Committee of the University of California, Santa Cruz .One of the authors (G.A.G.-E.) tranquilized wild individuals ASPM. Primers were designed specifically for exon 18 . All new sequences were deposited into GenBank with the following accession numbers: Alouatta palliata [GQ221757], Ateles geoffroyi [GQ221758], Cebus capucinus [GQ221759], Saimiri oerstedii [GQ221760]. Sequences for the remaining species were acquired from GenBank: Saimiri boliviensis [AY485419]; Aotus sp. ; Lagothrix lagotricha ; Cebus albifrons [HQ540103]; Alouatta caraya [HQ540104]; Leontopithecus chrysomelas [HQ540105]; Callicebus moloch [HQ540106]; Pithecia pithecia [HQ540107; Comparative exon 18 sequence for N/dS ratios were estimated in PAML using model 1. In this model, ω is the average dN/dS within lineages with independent ω’s between lineages. In the absence of synonymous substitutions, the dN/dS ratio cannot be calculated; as a result, some of our branches lack a dN/dS ratio (S = 0), PAML is unable to properly estimate dN/dS. In these cases dN was reported individually. To test if the high value of dN/dS in Alouatta palliata is significantly different from neutrality, a specific model in which ω evolves at a different rate for this branch was tested against a neutral model using a likelihood ratio test.Sequence alignment was performed in Bioedit dS ratio . BecauseAteles and Cebus branches were selected as foreground branches based on behavioral and morphological observations. The cut-off for statistical significance was assigned at the 0.05 level of probability by testing twice the difference of the log-likelihood (2Δl) output from PAML compared against a chi-square distribution (d.f. = 1).As suggested by Ali and Meier Saguinus labiatus, Callithrix jacchus and Leontopithecus chrysomelas from our sample. The removal of this family led to no differences in results for the remaining species.Following the recommendations of Montgomery et al. Table S1ASPM Exon 18 primers and Invitrogen TOPO cloning vector primers.(XLS)Click here for additional data file.File S1Abstract (Spanish).(DOC)Click here for additional data file.