Sixty-one metabolites were identified in the CSF from glioma patients using targeted, quantitative and non-targeted, semi-quantitative analysis. The citric and isocitric acid levels were significantly higher in the glioblastoma (GBM) samples than in the grades I–II and grade III glioma samples. In addition, the lactic and 2-aminopimelic acid levels were relatively higher in the GBM samples than in the grades I–II glioma samples. The CSF levels of the citric, isocitric, and lactic acids were significantly higher in grade I–III gliomas with mutant IDH than in those with wild-type IDH. The tumor location and enhancement obtained using MRI did not significantly affect the metabolite profiles. Higher CSF levels of lactic acid were statistically associated with a poorer prognosis in grades III–IV malignant gliomas. Our study suggests that the metabolomic analysis of CSF from glioma patients may be useful for predicting the glioma grade, metabolic state, and prognosis of gliomas.Metabolomics has recently undergone rapid development; however, metabolomic analysis in cerebrospinal fluid (CSF) is not a common practice. We analyzed the metabolite profiles of preoperative CSF samples from 32 patients with histologically confirmed glioma using gas chromatography/mass spectrometry (GC/MS). We assessed how alterations in the metabolite levels were related to the World Health Organization (WHO) tumor grades, tumor location, gadolinium enhancement on magnetic resonance imaging (MRI), and the The online version of this article (doi:10.1007/s11060-013-1090-x) contains supplementary material, which is available to authorized users. Cerebrospinal fluid (CSF) has various functions, such as protecting the brain, transporting biological substances, and excreting toxic and waste substances. CSF is in direct contact with the extracellular fluid of the brain. Although the CSF composition reflects that of the blood plasma, active transport from the blood and secretions from the brain contribute to the CSF composition. Therefore, the CSF composition can indicate biological brain processes, and CSF analysis is indispensable for diagnosing and understanding central nervous system (CNS) disorders –4. A cheisocitrate dehydrogenase (IDH) genes have been identified in gliomas [+-dependent dehydrogenases and convert isocitrate into α-ketoglutarate. Previous reports have indicated that these mutations are frequently observed in astrocytic and oligodendroglial tumors of grades II and III [IDH mutations may alter global cellular metabolism [Gliomas, the most common primary CNS tumors, are classified as grades I to IV based on the histopathological and clinical criteria established by the WHO . Grade I gliomas , 14. Bot and III . Becausetabolism .IDH mutation. Our study indicates that a metabolomic analysis of CSF from glioma patients may be useful for predicting the malignancy grade and IDH mutation status.Metabolomics has recently undergone rapid development. Metabolomics includes the analysis of metabolites from biofluids or tissues using nuclear magnetic resonance (NMR) or mass spectrometry (MS)-based approaches, including liquid chromatography/mass spectrometry (LC/MS) or gas chromatography/mass spectrometry (GC/MS). To date, the global metabolic profiling of human biofluids, such as urine and sera, has been used to visualize the distinct metabolic profiles of patients with cancer and gastroenterological disease . FurtherOur study included 32 patients with intracranial glial tumors, and the information concerning the participants is summarized in Table 2O:CHCl3 = 2.5:1:1) containing 10 μl of 1.0 mg/ml 2-isopropylmalic acid dissolved in distilled water as an internal standard. The solution was shaken for 30 min at 37 °C and centrifuged. A total of 250 μl of the resultant supernatant was transferred to a fresh tube, and 200 μl of distilled water was added. After mixing, the solution was centrifuged, and 250 μl of the resultant supernatant was transferred to a tube and lyophilized using a freeze dryer. For oximation, 40 μl of 20 mg/ml methoxyamine hydrochloride dissolved in pyridine was mixed with the lyophilized sample and shaken at 1,200 rpm for 90 min at 30 °C. Next, 20 μl of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was added for the derivatization step, and the mixture was mixed at 1,200 rpm for 30 min at 37 °C. The mixture was then centrifuged, and the resultant supernatant was subjected to GC/MS measurements. To assess the technical variation in the metabolomic experiments, each of the samples was extracted, derivatized, and measured in six replicates. Then, the average of the data set per sample was calculated.The CSF samples were centrifuged, and the supernatants were transferred to fresh tubes and stored at −80 °C until use. Fifty microliters of CSF were mixed with 250 μl of a solvent mixture . The peak detection and alignment were performed using MetAlign software . The resulting data were analyzed using in-house analytical software (AI output).The GC/MS analysis was conducted according to the method described by Tsugawa et al. . This anWe constructed a three-dimensional matrix using the sample names (observations), metabolite numbers (variable indices), and normalized peak intensities (variables). A principal component analysis (PCA) was performed using commercially available SIMCA-P + Software version 12.0.1 .IDH mutations, forward and reverse primers were designed to amplify exon 4 (codon R132) of the IDH1 gene and exon 4 (codon R172) of the IDH2 gene. The polymerase chain reaction products were sequenced using the primers and the BigDye Terminator v3.1 Cycle Sequencing kit .Genomic DNA was isolated from paraffin blocks of glioma tissue using the DNeasy FFPE kit , according to the manufacturer’s instructions. To detect the U test. The statistical significance among three groups was determined using the Steel–Dwass test. Multiple tests were controlled by false discovery rate (FDR), the expected proportion of false positive results that is declared significant [P < 0.05 was considered to be statistically significant. We divided the patients into two groups: the “higher (larger) group” had levels higher than the median, and the “lower  group” had levels lower than the median. Statistical analysis was performed using the SPSS, version 12.0, software package. FDR analysis was performed using the R, Q-value package (version 2.15.2) .The statistical significance between two groups was determined using the Mann–Whitney nificant . Q < 0.1p = 0.0125, Q = 0.1002); 1.83-fold, vs. grade III ) and isocitric acid ; 1.83-fold, vs. grade III ) , and fumaric acid was significantly decreased in GBMs compared with anaplastic astrocytomas . . The concentration of downstream molecules of isocitric acid in the TCA cycle, i.e., succinic acid, fumaric acid, and malic acid, were relatively lower in GBMs than in grades I-II or III gliomas (Supplementary Table S1). None of the amino acids or their derivatives manifested significant differences among each grade of gliomas.CSF samples from 32 glioma patients were subjected to metabolite level measurements with GC/MS Table . A totalp = 0.033) and 2-aminopimelic acid  were higher in GBMs than in grades I–II gliomas . The levels of lactic acid , which produces lactic acid from pyruvate, in all glioma samples analyzed. Overall, the LDHA was predominantly expressed in tumor cells rather than in vascular tissues, as shown in the Supplementary Fig. S3. Notably, the tumor cells near the necrotic tissues markedly expressed LDHA . From these findings, tumor cells, particularly hypoxic tumor cells, appear to predominantly produce and release lactic acid. The comparison of the LDHA expression level of GBMs and other gliomas demonstrated that LDHA expression is stronger in GBMs than in other gliomas . This finding is consistent with the results demonstrated for the CSF lactic acid levels.Because 1H-MRS studies were performed for 9 patients in this study, we compared the CSF metabolite levels measured by GC/MS with those of tumors measured by 1H-MRS. The MRI and 1H-MRS images of 3 patients are indicated in Supplementary Fig. S1 . Conversely, in low-grade gliomas alone, the CSF lactic acid levels were significantly higher in the group of proximally located gliomas than in distally located gliomas (p = 0.028). Additionally, because gadolinium enhancement of the tumor on MRI is known to involve blood–brain barrier (BBB) disruption, we determined whether there were alterations in the metabolite levels in MRI-enhanced tumors . Next, we classified the high-grade gliomas (grades III + IV) into a larger tumor size group and a smaller tumor size group. We also compared the CSF metabolite levels between these groups; however, there were no significant differences in the metabolites between the larger group and the smaller group.To examine the differences in the metabolite levels related to tumor location, we divided the gliomas into two groups: the tumors located proximal to or distal from the ventricles , isocitric acid , and lactic acid     . In grade IV (GBM) patients only, however, there was no significant correlation between the CSF lactic acid or citric acid level and OS. In multivariate analyses, however, the CSF levels of lactic acid and citric acid were not independent predictors of survival in glioma patients.During survival analysis using the Kaplan–Meier method for all glioma patients, no significant correlation was discovered between the overall survival (OS) and the CSF level of lactic or citric acid. However, relationships were observed between higher CSF lactic or citric acid levels and having a shorter OS  and that pre-treatment with FDG-PET provides significant additional prognostic information for high-grade gliomas [Metabolic remodeling is a predominant phenotype of malignant tumor cells (Warburg effect) and refers to the alteration of the utilization and/or synthesis of important metabolites, including glucose, fatty acids, and amino acids, by tumor cells . Aerobic gliomas . Thus, gIDH compared with wild-type IDH in only low-grade gliomas (grades I–III). We sought to determine whether the higher lactic acid levels were associated with a poor outcome in grade I–III gliomas. However, this could not be determined because most of the patients were still alive at the end of the study. Although gliomas with a mutant IDH are known to be associated with improved survival, further studies are required to determine the association between the CSF lactic acid levels and the prognosis in low-grade gliomas. Lactic acid is produced by lactate dehydrogenase (LDH) and is usually an anaerobic metabolic product that occurs when the oxygen demand of a rapidly growing tumor exceeds what its neovasculature supplies. In addition, unlike most normal tissues, malignant tumor cells convert most glucose into lactic acid, regardless of whether oxygen is available to support mitochondrial oxidative phosphorylation [+ ratio and thereby accelerates lactate dehydrogenase activity [In the present study, we identified 61 metabolites in the CSF from glioma patients using GC/MS. Previous reports detected ~40–90 metabolites using GC/MS in normal human CSF –37, and rylation . Active activity . In the The CSF levels of citric and isocitric acid, which are TCA cycle metabolites, were significantly elevated in GBMs compared with grades I–II or III gliomas. Citric acid accumulates in tissue in which the glycolytic rate exceeds the TCA cycle activity . FurtherIDH mutations were frequently observed in astrocytic and oligodendroglial tumors of grades II and III [IDH status were analyzed in grades I–III gliomas, excluding GBMs, because all GBMs did not have a IDH mutation and because several metabolites were significantly different from other grades of gliomas. Reitman et al. [IDH mutations are reported to be different among cell types [IDH mutations compared with those with a wild-type IDH. In contrast, the CSF concentrations of lactic, citric, and isocitric acid were significantly higher in gliomas with IDH mutations compared with those of a wild-type IDH. In addition, the levels of the late TCA cycle metabolites that act downstream of isocitric acid decreased in the gliomas with IDH mutations. IDH1 converts isocitrate into α-ketoglutarate, which may explain the observed lower levels of late TCA metabolites (which are downstream from α-ketoglutarate) and increased isocitrate, pyruvate, and/or citrate levels (which are upstream from isocitrate). From these data, CSF metabolite levels may reflect the metabolic changes caused by the IDH mutations in the glioma cells. Recently, biochemical studies revealed that mutant IDH1 protein gains the function to catalyze the reduction of α-ketoglutarate to 2-hydroxyglutarate (2-HG) in a NADPH-consuming manner [IDH mutation. Further studies are required to detect 2-HG in the CSF of glioma patients. Most recently, Locasale et al. [ and III . In the n et al.  reportedn et al.  recentlyll types , 47; howg manner . Sahm etg manner  reportede et al.  analyzedThe present study has several limitations. First, there are several histological types in the same WHO grade. The low-grade gliomas in this series contained four different histological types , and the grade III gliomas contained three different histological diagnoses . There may be significant differences in the metabolic profile among different histological types of gliomas. Therefore, the differences in the metabolite profiles of different histological types could have contributed to the differences observed among the different grades. Second, the relatively small sample size limited our power to detect potentially important associations between CSF metabolites and the clinical outcomes of interest. Despite these and other limitations, this is the first comparative report of the CSF metabolite profile among the various grades of gliomas. However, further studies will be needed to determine the CSF metabolome of various histological types of gliomas.In conclusion, the CSF levels of several metabolites, such as citric, isocitric, and lactic acid, were altered between glioma grades and the IDH mutation status, which may reflect the glioma cell metabolism. Our study indicates that an analysis of the CSF metabolite levels may be useful for predicting glioma malignancy and prognosis.Supplementary material 1 (DOC 168 kb)Supplementary Figure S1MRI scans of the gliomas. The MRI shows an enhanced tumor proximal to the ventricles  and distal to the ventricles . (TIFF 430 kb)Supplementary Figure S2p values were calculated using the Mann–Whitney nonparametric U test (*p < 0.05). Q-value of citric acid, isocitric acid, and fumaric acid are 0.0039, 0.0039, and 0.0706, respectively. (TIFF 67 kb)Significant differences of the CSF metabolites related to TCA cycle and glycolisis between anaplastic astrocytomas (AAs) and GBMs. The columns are the average of each group; bars, SD. Supplementary Figure S3(a-g) LDHA expression in various histological types of gliomas. a: pilocytic astrocytoma (patient-1), b: diffuse astrocytoma (patient-5), c: oligodendroglioma (patient-7), d: anaplastic astrocytoma (patient-13), e: anaplastic oligodendroglioma (patient-16), f: anaplastic ependymoma (patient-18), g: GBM (patient-26).  LDHA expression in GBM. h: LDHA is predominantly expressed in tumor cells rather than in vascular tissues. i: tumor cells near necrotic tissues markedly expresses LDHA. . (TIFF 869 kb)Supplementary Figure S4MRI (left panels) and 1H-MRS (right panels) findings for three glioma patients. Upper panels  Ependymoma (grade II) of 25-year old man (patient 10). (a) Fluid attenuated inversion recovery (FLAIR) MR image shows the tumor located in left lateral ventricle. (b) The MR spectra show that the lactic acid signal is barely detectable. In this patient, the relative value of CSF lactic acid measured by a GCMS-QP2010 Ultra is 2.13. Middle panels  Glioblastoma (grade IV) of 39-year old woman (patient 19). (c) FLAIR MRI shows the large tumor diffusely extended into the bilateral frontal lobes and corpus callosum. (d) In the MR spectra, lactic acid is detectable, but the peak is small. In this patient, the relative value of CSF lactic acid is 2.68. Lower panels  Glioblastoma (grade IV) of 54-year old woman (patient 21). (e) Gd-enhanced MRI shows heterogeneously Gd-enhanced tumor in the white matter of the left parietal lobe. (f) In the MR spectra, the peak of lactic acid is large. The relative value of CSF lactic acid is 3.07. (TIFF 325 kb)Supplementary Figure S5p = 0.004, HR = 7.81, 95 %CI: 1.53–39.8). B: High-grade gliomas (Grade III & IV) have a tendency of a shorter OS as compared with low-grade gliomas (grade I & II) . C: Survival analysis of each grades of gliomas. GBMs have a tendency of a shorter OS compared with other grades of gliomas. (TIFF 74 kb)A: In survival analysis using the Kaplan–Meier method for all glioma patients, higher age (>50) were significantly associated with having a shorter OS compared with lower age (<50) (log-rank Below is the link to the electronic supplementary material.


Diamond-Blackfan anemia and Duchenne muscular dystrophy are two rare congenital anomalies. Both anomalies occurring in the same child is extremely rare. Allogeneic hematopoietic stem cell transplantation is a well-established therapy for Diamond-Blackfan anemia. However, in patients with Duchenne muscular dystrophy, stem cell therapy still remains experimental.We report the case of a nine-year-old boy of north Indian descent with Diamond-Blackfan anemia and Duchenne muscular dystrophy who underwent successful allogeneic hematopoietic stem cell transplantation. He is transfusion-independent, and his Duchenne muscular dystrophy has shown no clinical deterioration over the past 45 months. His creatine phosphokinase levels have significantly decreased to 300 U/L from 14,000 U/L pre-transplant. The patient is 100% donor chimera in the hematopoietic system, and his muscle tissue has shown 8% to 10.4% cells of donor origin.Our patient's Diamond-Blackfan anemia was cured by allogeneic hematopoietic stem cell transplantation. The interesting clinical observation of a possible benefit in Duchenne muscular dystrophy cannot be ruled out. However, further clinical follow-up with serial muscle biopsies and molecular studies are needed to establish this finding. RPS19  [Diamond-Blackfan anemia (DBA) is a rare congenital hypoplastic anemia that usually presents in infancy and childhood. The incidence of DBA is five to 10 per one million births, and more than 600 cases have been reported worldwide. The exact cause is unclear, but the problem is a defect in one of the early steps in erythropoiesis (red blood cell (RBC)) production. In about 15% of affected children, there is a defect within a gene called ein S19) . The onlein S19) -5 and isDuchenne muscular dystrophy (DMD), which is caused by mutation of the dystrophin gene, is the most common and severe form of muscular dystrophy. This disorder is marked by progressive loss of muscle function which begins in the lower limbs and later occurs in the arms, neck and other areas. DMD occurs in approximately one of 3500 male live births and is inherited in an X-linked recessive pattern . TerminaDBA and DMD are two rare congenital anomalies. Both anomalies occurring in the same child is extremely rare. We report such an unusual case of a nine-year-old boy of north Indian descent, the third of four siblings born to healthy, non-consanguineous parents with a family history of DMD in a maternal first cousin. The boy was presented to our hospital at the age of eight months with progressive pallor and failure to thrive. He was diagnosed with DBA on the basis of peripheral blood showing normocytic, macrocytic anemia and reticulocytopenia with normocellular bone marrow showing erythroblastopenia. Though initially steroid-responsive, the boy later became unresponsive to both steroids and cyclosporine. By the fourth year of life, he was completely transfusion-dependent, and iron chelation was started only in the sixth year of life with intravenous desferrioxamine when his serum ferritin level was found to be 5100 ng/mL.RPS19 genetic studies could not be done.The patient had normal milestones until the fourth year of life. Thereafter he developed lower-limb weakness and calf muscle pseudohypertrophy. The weakness was progressive in nature, and by the age of five years the boy required support to climb stairs. His CPK level was high (5939 U/L), and his electromyogram was consistent with myopathy. He was diagnosed with DMD. A muscle biopsy was not done as the parents were unwilling to allow their son to undergo the procedure at that time. At this stage, the child was referred to our center for allo-HSCT treatment in view of his transfusion-dependent DBA with hyperferritinemia. The diagnosis was reconfirmed at our center. He also had dysmorphic facial features. However, The child had an unaffected elder brother who was a human leukocyte antigen (HLA)-identical match (6/6 antigen match) and underwent myeloablative allo-HSCT treatment. The conditioning regimen comprised busulphan (16 mg/kg over four days), cyclophosphamide (200 mg/kg over four days) and equine anti-thymocyte globulin /kg body weight. Graft-versus-host disease (GVHD) prophylaxis consisted of standard dose methotrexate and cyclosporine. Neutrophil and platelet engraftment occurred on day+11 and day+16, respectively. RBC engraftment was confirmed with a change of blood group on day+90. The child has been transfusion-free since 2 months post-transplant, and his serum ferritin levels have been reduced to 600 ng/mL with regular phlebotomies. The boy's blood counts were normal 45 months after allo-HSCT treatment: hemoglobin 13.5 g/dL, reticulocyte count 1%, white blood cell count 6.0 × 103/μL with 70% neutrophils and platelet count 1900 × 103/μL.Kalamazoo, Michigan 49001, USA) (90 mg/kg over three days). There was a minor blood group mismatch, with the recipient being in the B+ve group and the donor being in the O+ve blood group. Granulocyte colony-stimulating factor primed bone marrow was harvested from the donor while the donor was under general anesthesia. Plasma depletion was done using a Cryofuge 6000i centrifuge(by Heraeus Instruments made in Germany and supplied by Kendro Labarotories (India)) at 3000 rpm, and plasma-depleted marrow was infused with a cell dose of 6 × 10This patient was evaluated by a neurologist pre-transplant and periodically post-transplant. The patient was wheelchair-bound pre-transplant, and, 45 months post-transplant, there has been no clinical deterioration whatsoever in the boy's motor power. He continues to be wheelchair-bound and is able to sit on his own for more than three hours at a stretch. The CPK level pre-transplant had been in the range of 9000 to 14,000 U/L and showed a declining trend within four weeks of transplantation, reaching a nadir of 300 U/L by six months post-transplantation. The muscle biopsy, which was done twice at 730 and 1250 days post-HSCT, respectively, revealed mixed donor chimerism with 8% to 10% cells of donor origin. Sequential chimerism studies using whole blood established trilineage engraftment with 100% donor chimerism. There were no post-transplant complications in the form of sepsis, hepatic veno-occlusive disease or acute or chronic GVHD.Chimerism analysis performed on recipient peripheral blood samples on day+30, day+90, day+365, day+730 and day+1250 revealed complete donor chimerism post-transplant. A muscle biopsy was performed twice post-bone marrow transplantation (BMT) at days 730 and 1250 to study histopathology, dystrophin expression and chimerism status. Immunostaining for dystrophin I was reduced, dystrophin II and III were absent and there was up-regulation of utrophin.Chimerism was performed to monitor donor cell engraftment in the recipient peripheral blood and was also used to detect donor cells in the muscle biopsy from the recipient. Chimerism was carried out using recipient's hair follicle as the pre-transplant sample , peripheThe fragment of muscle biopsy taken for chimerism analysis was washed in saline seven times to remove all traces of RBCs and peripheral blood MNCs (PBMCs). The muscle tissue was homogenized and further rinsed three times in saline, following which a frozen section of the homogenized muscle was taken to check for any contamination with PBMCs Figure . GenomicGene mapper TM software v 3.5, Foster City CA 94404, USA. To determine the fraction of donor cells in the recipient's peripheral blood and muscle biopsy samples post-transplantation, the informative markers were identified and the percentage of donor cells was estimated. While the post-transplant recipient peripheral blood sample displayed complete donor chimerism, the muscle biopsy sample showed mixed donor chimerism with 8% to 10.4% donor cells on days 730 and 1250, respectively  labeled with four types of fluorescent dyes . A multiplex polymerase chain reaction assay was performed in a final reaction volume of 25 μL containing 10.5 μL of reaction mixture, 5.5 μL of primer mix, 0.5 μL of AmpFl STR Identifier kit  and 10 μL of DNA at a concentration of 0.2 ng/μL. Cycling parameters were optimized as follows: 95°C for 11 minutes (one hold), 94°C for 60 seconds, 59°C for 60 seconds, 72°C for 60 seconds and 28 cycles, 60°C for 60 minutes (two holds) in a GeneAmp PCR System 9700 (Applied Biosystems). Denaturation was performed for five minutes at 95°C using Hi-Di Formamide and GeneScan 500 LIZ Size Standard (both from Applied Biosystems). The amplicon was resolved by performing capillary electrophoresis using the ABI Prism 3100-Avant Genetic Analyzer System (Applied Biosystems) and analyzed using the Applied Biosystems Allo-HSCT is the only available curative treatment for DBA. The first "successful" allo-HSCT treatment of DBA was reported in 1976 . The patmdx mouse models of DMD, allo-HSCT from normal mice led to incorporation of donor-derived nuclei into muscle and partial restoration of dystrophin expression in the affected muscle. The canine model of DMD (canine X-linked muscular dystrophy (CXMD)) has shown a clinical course very similar to that of human DMD. In a study of seven CXMD dogs that underwent allo-HSCT from non-affected littermates, there was no increase in the number of dystrophin-positive fibers or in the amount of wild-type dystrophin RNA post-transplantation compared with pre-transplantation levels. However, another canine study demonstrated that allo-HSCT provides an immune-tolerant platform for myoblast transplantation from freshly isolated muscle-derived cells from the same HSCT donor [DMD has no known cure. Experiments with stem cells on animals with DMD have been encouraging. In CT donor .The first successful allo-HSCT in a patient with DMD was reported in 2002. That patient with DMD was diagnosed at 12 years of age and underwent BMT at one year of age for X-linked severe combined immunodeficiency syndrome. Analysis of muscle biopsies revealed the presence of donor nuclei within a small number of muscle myofibers (0.5% to 0.9%). The discovery of the donor's mesenchymal cells in the patient's muscle tissue and bone marrow 13 years after the transplant raised the hope that BMT may play a role in the treatment of DMD . Two casTo the best of our knowledge, we report the first case of a boy with two rare genetic disorders, DBA and DMD, who successfully underwent myeloablative allo-HSCT, which cured his DBA and might have had a positive effect on his DMD. Clinically, the course of his DMD has seemed milder and nearly static over the past 45 months as compared to his first cousin, who also has DMD and by the same age was unable to sit without support. In addition, CPK levels showed drastic reduction, and chimerism studies revealed 8% and 10.4% donor cells in the patient's skeletal muscle at days 730 and 1250, respectively.Though this percentage of donor cells was small, the presence of these cells in the patient's muscle tissue indicates the possibility of transdifferentiation of hematopoietic donor stem cells to skeletal muscle myocytes in the patient. However, no dystrophin expression was noted in the muscle tissue, which could possibly be due to the low level of transdifferentiation. Though this transplant was done primarily for DBA, it raises the interesting possibility of allo-HSCT's being beneficial in the treatment of DMD, which is an otherwise incurable disease with 100% mortality. However, further clinical follow-up with serial muscle biopsies and molecular studies is needed to document the extent and duration of mixed chimerism in skeletal muscle in this patient. The purpose of this case report is to describe this interesting observation of a possible benefit in DMD and not to suggest HSCT as a modality of treatment until further studies show an unequivocal benefit, given the inherent risks associated with HSCT.Written informed consent was obtained from the patient's parent for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.BVN is the first author and is responsible for the conception and design of the case report. Contributing authors SD, AS, SS, JK and DKM made substantial contributions to the design of the manuscript and the acquisition, analysis and interpretation of the data.Bone Marrow Transplantation, Acta Haematologica, Blood, Journal of the Association of Physicians of India, Indian Pediatrics. Medical Journal of Armed Forces of India and Indian Journal of Haematology & Blood Transfusion among others. VN has done two International Cancer Technology Transfer Fellowships under the technology transfer program of the International Union Against Cancer, Geneva, at King's College & Hospital, London in 2004, and at Stanford University School of Medicine, Stanford, CA, USA, in 2008. He is also the former President of the Indian Society of Haematology & Transfusion Medicine and a fellow of the American College of Physicians and the Indian Academy of Medical Sciences.VN is a consultant in Medicine & Clinical Haematology and was Head of the Department of Medicine and Head of the Department of Haematology & BMT, Army Research & Referral Hospital, New Delhi, India, which is the premier institution of the Indian Armed Forces. He was also the Dean of the Army College of Medical Sciences, New Delhi, India. Presently, he is Head of the Department of Medicine at the Armed Forces Medical College in Pune, India. He is a well-known clinician, researcher and BMT physician with over 25 years of experience. He has written more than 150 publications in various national and international journals, including All other contributing authors are either professors or associate professors in the specialties mentioned on the title page and have significant experience and have written numerous publications.


RBP4, ESR1 and IGF2, currently used in genetic tests for different purposes. Moreover, we investigated a possible effect of the interaction between RBP4-MspI and ESR1-PvuII polymorphisms. The IGF2-intron3-G3072A polymorphism is actually used to select lean growth, but other possible effects of this polymorphism on reproductive traits need to be evaluated.The aim of this work was to study the effects on litter size of variants of the porcine genes RBP4 gene was carried out. RBP4-MspI and IGF2-intron3-G3072A were genotyped in a hyperprolific Chinese-European line (Tai-Zumu) and three new RBP4 polymorphisms were genotyped in different pig breeds. A bivariate animal model was implemented in association analyses considering the number of piglets born alive at early (NBA12) and later parities (NBA3+ ) as different traits. A joint analysis of RBP4-MspI and ESR1-PvuII was performed to test their possible interaction. In the IGF2 analysis, paternal or maternal imprinting effects were also considered.Detection of polymorphisms in the genomic and cDNA sequences of RBP4 haplotypes were detected  in different pig breeds and wild boars. A significant interaction effect between RBP4-MspI and ESR1-PvuII polymorphisms of 0.61 ± 0.29 piglets was detected on NBA3+. The IGF2 analysis revealed a significant increase on NBA3+ of 0.74 ± 0.37 piglets for the paternally inherited allele A.Four different RBP4 haplotypes. This suggests an ancestral origin of the quoted haplotype. The joint use of RBP4-MspI and ESR1-PvuII polymorphisms could be implemented to select for higher prolificacy in the Tai-Zumu line. In this population, the paternal allele IGF2-intron3-3072A increased litter size from the third parity. The non-additive effects on litter size reported here should be tested before implementation in other pig breeding schemes.All the analyzed pig and wild boar populations shared one of the four detected  ESR1, PRLR, RBP4 and FSHB have been shown to have effects on litter size ranging from 0.25 to over 1 piglet per litter [The use of molecular information in pig breeding programs may enhance genetic gains by increasing the accuracy of genetic evaluation and decreasing generation intervals . More thr litter .RBP4) gene codes for a member of the RBP protein family present in the uterus and in embryos during the early stages of gestation [et al. [RBP4 as a possible candidate gene associated with litter size. Subsequently, Rothschild et al. [RBP4-MspI, on the total number of born piglets. Many other studies have shown the existence of a relationship between this polymorphism and litter size [The retinol binding protein 4  gene promotes the expression of different transcription factors involved in the reproductive function of female tissues . The ESR1-PvuII polymorphism has been studied previously in the Tai-Zumu line by our group but no significant effect on litter size was observed [et al. [RBP4-Msp I and ESR1-PvuII polymorphisms.The protein coded by the estrogen receptor 1  gene, the IGF2-intron3-G3072A SNP [A polymorphism detected in the porcine insulin-like growth factor 2 . Data from a Chinese-European composite dam line (Tai-Zumu) were provided by GENE+. This line was developed from Meishan and Jiaxing sows inseminated by hyperprolific French Large White boars, and it was selected for lean growth during nine generations . The ped999. DataGenomic DNA was isolated from blood samples according to a standard protocol . Total R2 concentrations optimized for each amplified fragment 2SO4), MgClRBP4 gene was amplified from genomic DNA samples of three Tai-Zumu individuals using the PCR protocol published by Rothschild et al [RBP4-MspI polymorphism but the exact information about its location was not available. The final sequence was submitted to GenBank (accession number: GU932906). Moreover, two overlapping RBP4 cDNA fragments spanning from exon 2 to 6 and covering the complete coding sequence (CDS) were amplified from Tai-Zumu individuals. The primer pairs .A 565 bp fragment spanning from exon 2 to 4 of the ld et al . These aRBP4 sequences obtained. One of the intronic SNP (c.249-63G>C) was identified as the RBP4-MspI polymorphism previously reported by Rothschild et al [RBP4 haplotypes were determined using the PHASE software.Five intronic and one exonic SNP were detected in the ld et al . This SN SNP c.24-63G>C waESR1-Pvu II genotyping data were taken from Muñoz et al [IGF2-intron3-G3072A polymorphism was genotyped by pyrosequencing as described by Van Laere et al. [The oz et al  and the e et al.  in a PSQA multitrait animal model was used to estimate genetic parameters. Under this approach, the numbers of piglets born alive at each one of the six parity classes (1 to 5 and ≥ 6) were treated as different traits.y1 to y≥ 6 represent litter size records (NBA) at each parity class, β1 to β≥ 6 are the vectors of fixed effects for the six different traits considered, which include the genetic line of the litter's sire (Tai-Zumu or Landrace), parity order and farm-year-season, u1 to u≥ 6 and e1 to e≥ 6 are vectors of random additive genetics and residual effects for each trait, respectively. Matrices X1 to X≥ 6 and Z1 to Z≥ 6are incidence matrixes that associate respectively elements of β1 to β≥ 6 and u1 to u≥ 6 with the records in y1 to y≥ 6.p≥ 6 is the vector of permanent environmental effects for each sow with records in the last parity class being W the incidence matrix relating the elements of p≥ 6 with the records in y≥6. The expectation of yi (i = 1 to 5 and ≥ 6) is Xiβi and the variance-covariance structure of random effects was assumed to be:where where 12) and the number of piglets born alive at the third and subsequent parities (NBA3+ ) were considered as two different traits. The reduced model can be written as:A preliminary analysis of the whole data set was performed using this multitrait model. Then, a bivariate model was used to carry out a subsequent analysis of litter size data. In this model, the number of piglets born alive at the first and second parity  and dominant (δ) effects. The value of α for each sow depends on her genotype  and δ assumes a zero value for homozygote individuals and 1 for the heterozygotes.i. Mendelian inheritance: used in the analysis of the effect of RBP4 and ESR1 polymorphisms. Besides α and δ values, additive x additive interaction (Ψ) effects are also included. Ψ are equal to -1, 0 or 1 depending on the genotypic combination of the analyzed polymorphisms ii. Mendelian inheritance with epistasis effects: used in the joint analysis of IGF2 SNP. Two association analyses were performed fitting the paternal imprinting effects. Additive and dominant effects were included in the first analysis but not in the second one. In the first analysis, imprinting effects are included (λ) for the heterozygote sows: on the one hand, λ = -1/2 or λ = 1/2 if they have inherited respectively allele G or allele A from the father and on the other hand, λ = 0 for homozygote individuals. In the second analysis, the sows that have received the paternal allele G (GG or GA) have λ = -1/2 and those that have received the paternal allele A (AA or AG) have λ = 1/2. A similar parameterization was used for maternal imprinting.iii. Paternal or maternal imprinting: used in the analysis of 2 approach to the distribution of the log-likelihood ratios. Variance components and parameter estimates were obtained using VCE-5 program [The statistical significance of each effect was tested comparing the full and reduced models by the χ program  and asso program h2 = σ2u/σ2y) for NBA at each parity class and estimated genetic correlations between parities are shown in Table 12) and the remaining in another one (NBA3+ ). Parameter estimates for both traits obtained from the whole data set are shown in Table 12 were expressed as deviation from the first parity (-0.08 ± 0.36) and on NBA3+ as deviation from the third parity . On the other hand, the estimated effect of genetic line of the litter's sire was not statistically significant, i.e. 0.25 ± 0.18 for NBA12 and -0.07 ± 0.25 for NBA3+.Estimated values of heritability . Moreover, a silent SNP, c.156G>A was detected on exon 3. In addition, two overlapping cDNA fragments of 485 and 479 bp, respectively, were amplified and sequenced. The assembled fragments form an 861 bp sequence that covers the complete CDS. As a result, the SNP c.156G>A was confirmed but no other exonic polymorphism could be detected. From the comparison of the different sequences, SNP c.111+47T>C, c.156G>A, c.248+15G>T and c.248+16A>G seem to be cosegregating in the sequenced Tai-Zumu individuals. In order to check their segregation pattern, SNP c.248+15G>T, c.248+16A>G, c.248+27A>T and c.249-63G>C (RBP4-MspI) were genotyped on different domestic pig populations  and wild boars. The results distinguished four different haplotypes for the quoted positions: TGAC, GGAG, GAAG and GATG. Their respective frequencies in the different populations are shown in Table After sequencing and aligning the 565 bp genomic fragment of the RBP4-MspI SNP was performed in 534 sows with 957 litter size records for NBA12 and 1043 for NBA3+. Allele 1 (frequency = 0.51) was significantly associated with a higher number of piglets born alive in the two first parities (NBA12), but not in the third and subsequent parities (NBA3+ ). The estimated additive effect on NBA12 was 0.42 piglets per litter (P≤0.016), and no dominance effects were observed , with 733 litter size records for NBA12 and 934 for NBA3+. No significant effect on litter size was evidenced. In addition, a joint analysis between RBP4-MspI and ESR1-PvuII polymorphisms was performed using data from 375 sows with 679 litter size records for NBA12 and 874 for NBA3+. The number of sows for each one of the nine genotypic combinations ranged from 12 (ESR1-PvuII AA/RBP4-MspI 22) to 81 (ESR1-PvuII AB/RBP4-MspI 12). The additive effect of RBP4-MspI on NBA12 was confirmed and a significant interaction effect was detected on NBA3+ (Table ESR1 AA/RBP4 11) and (ESR1 BB /RBP4 22) and the least prolific to the alternative combination (ESR1 BB/RBP4 11) and (ESR1 AA/RBP4 22) .In a first step, an association analysis of the IGF2 SNP effects are shown in Table 12 and 1057 records for NBA3+ , but no significant result was obtained. Otherwise, to implement a model of imprinting inheritance requires that the paternal or maternal inheritance of the alleles can be determined in the heterozygote sows. This was possible for 56 of the 264 total heterozygotes: 31 with the paternal allele G and 25 with the paternal allele A. The analysis was performed on 342 sows with 613 records for NBA12 and 710 records for NBA3+. When additive and dominant effects were taken into account, a suggestive additive effect of the paternal allele A was detected on NBA3+ . If only paternal imprinting effects are considered, a significant increase produced by the paternal allele A of the number of piglets alive was detected on NBA3+. Maternal imprinting effects were not evidenced in a complementary analysis  was detected in all the pig breeds and wild boars analyzed except Iberian pigs that only displayed the TGAC haplotype. Some authors have reported introgression of Asian alleles in many European breeds, but not in Iberian pigs [et al. [PRLR, BMPR1B, ESR1) related to reproductive traits [Porcine ian pigs -34. Our  [et al.  who repoe traits ,36.RBP4-MspI because it presents intermediate allelic frequencies in the population. Given the distribution of haplotypes observed in the Tai-Zumu population, the analysis carried out with the RBP4-MspI SNP would be equivalent to comparing haplotype TGAC to haplotypes GAAG and GATG. Individual and joint association analyses of RBP4-MspI with NBA12 and NBA3+ revealed a favourable additive effect of allele 1 on NBA12. This result is in accordance with that detected by Rothschild et al. [RBP4-MspI allele on the total number of piglets born in six lines from different genetic origins. Also, Spöter et al. [RBP4-MspI alleles were compared in control and selected lines for increased litter size did not reveal any significant result [The GATG haplotype showed a low frequency in the Tai-Zumu population Table  and thusd et al. . They har et al.  have detr et al. ,11. Expet result ,10.RBP4-MspI SNP. Besides, a possible dependence on the genetic background should be taken into account, because epistatic effects could be affecting pig prolificacy as recently reported [et al. [RBP4-MspI polymorphism on litter size depend on the genotype of the ESR1-Pvu II allele in a comparison between sows from three genotypic classes. The litter size for second and later parities of sows carrying either ESR1 allele A/RBP4 genotype 11 or ESR1 allele B/RBP4 genotype 22 was greater than that of sows grouped in the third class (ESR1 AA/RBP4 22 and ESR1 BB/RBP4 11). The results of our joint association analysis allow us to corroborate more precisely the results obtained by Gonçalves et al. [ESR1 AA/RBP4 11 and ESR1BB/RBP4 22 were the most prolific for NBA3+. These findings may reflect a physiological interaction between estrogens and RBP4 proteins. Once, the first secretion of RBP has occurred in the embryo, embryonic estrogens are secreted in the maternal uterus where they induce an increase of expression and secretion of RBP proteins. These proteins enter the embryo cells rising the RBP receptors density and allowing the embryo development to continue [RBP4-MspI and ESR1-PvuII could be implemented to improve prolificacy in Tai-Zumu pigs, although its use in other commercial populations requires confirmation of the observed interaction.These diverse results indicate that the causal mutation could be in linkage disequilibrium with the porcine reported ,39. Gonç [et al.  have pois et al.  i.e., socontinue . TherefoIGF2-intron3-G3072A on prolificacy, with uneven results in different populations, although the methodology used and the available information varied among the studies. Using a Mendelian inheritance model, Horak et al. and Katska-Kiazkiewicz et al. [IGF2 polymorphisms on litter size in Czech and Polish pigs, respectively. In addition, Rempel et al. [IGF2-intron3-G3072A in a composite pig line. Assuming an imprinting inheritance model, Buys et al. [Implementation of molecular markers in selection requires exhaustive verification in order to ensure that no undesirable effect arises in other economically important traits. So far, some studies have been developed to check the effect of z et al. ,41 have l et al.  have nots et al.  have dets et al. ,45.3+. Hence, it is clear that the results depend on the model employed. Note that imprinting phenomena could arise from CpG island methylation events that trigger the silencing of the genes on a chromosomal region [IGF2-intron3-G3072A mutation is located in a CpG island and its causality on pig lean growth has been well confirmed [IGF2-intron3-G3072A mutation could be implemented in the Tai-Zumu population due to its beneficial effects both on lean growth and litter size in third and subsequent parities.In the current study, both types of inheritance were taken into account. A significant effect was only detected under the inheritance model of paternal imprinting, i.e. an increase of 0.74 piglet on NBAl region ,47. Indeonfirmed . AlthougA multitrait model is recommended to analyze the effects of various polymorphisms on litter size since early and later parities can be partially controlled by different genes.RBP4 gene in wild boars and six porcine populations allowed to detect four haplotypes. Only one of the four detected haplotypes was shared by all the analyzed pig and wild boar populations indicating an ancestral origin of the quoted haplotype. Otherwise, RBP4-MspI does not seem to be the causative mutation associated with an increase in litter size. However, an interaction effect between RBP4-MspI and ESR1-Pvu II on NBA3+ was detected in the Tai-Zumu population. According to this, the joint use of the most favorable genotypic combination could be implemented in order to select for higher litter size.Analysis of the IGF2-intron3-3072A allele in Tai Zumu increases litter size from the third parity. The causative mutation could be situated either in the IGF2 gene or very close to this gene.Selecting the paternally inherited The authors declare that they have no competing interests.RBP4 gene, drafted and finalized the manuscript. AIF carried out the genotyping of the IGF2-intron3-G3072A polymorphism. CO and GM carried out the genotyping task of the ESR1-Pvu II polymorphism. AF performed the statistical analysis and helped to revise the manuscript. EA participated in the design of the study of RBP4 gene, helped to draft, revise and complete the manuscript. LS and CR conceived, coordinated and led the project. Besides LS participated in revising and finishing the manuscript.MM carried out the polymorphism detection and the genotyping tasks in the All authors read and approved the final manuscript.2 concentrations and amplicon sizes used for RBP4 sequencing and pyrosequencingTable S1 - Primer sequences, annealing temperatures, MgCl. This table shows primers used for RBP4 sequencing and pyrosequencing. Annealing temperature, MgCl2 concentration and amplification size are indicated for each fragment.Click here for file


The gamma-isoform of the 14-3-3 protein (14-3-3 gamma) is expressed in neurons, and could be a specific marker for neuronal damage. This protein has been reported as a detectable biomarker, especially in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) patients by Western blotting (WB) or enzyme-linked immunosorbent assays (ELISAs). Western blotting for 14-3-3 gamma is not sensitive, and the reported data are conflicting among publications. An ELISA specific for 14-3-3 gamma is not available.CJD patients  and 99 patients with other neurodegenerative diseases were examined in this study. The CSF samples obtained were analyzed by Western blotting for 14-3-3 gamma, and by ELISA for total tau protein. We evaluated the sensitivity and specificity of the newly developed sandwich ELISA for 14-3-3 gamma.The cut-off value of the 14-3-3 gamma ELISA was > 1, 683 AU/ml; and sensitivity was 95.2%, with 72.7% specificity. This specificity was the same for the total tau protein ELISA. Seven CJD cases were negative by WB but positive using the 14-3-3 gamma ELISA, indicating that the ELISA is more sensitive. All 21 cases of early stage CJD could be diagnosed using a combination of the 14-3-3γ ELISA and diffusion weighted MR imaging (DWI-MRI).The 14-3-3 gamma ELISA was more sensitive than conventional WB, and was useful for laboratory diagnosis of CJD, similar to the ELISA for the tau protein. Using DWI-MRI and these ELISA tests on CSF, diagnosis of CJD will be possible even at early stages of the disease. Hshich et al. reportedThe 14-3-3 protein has been reported to be a detectable biomarker, especially in the CSF of CJD patients, by WB or enzyme-linked immunosorbent assay (ELISA). However, the WB to detect 14-3-3 is not very sensitive enough and the data obtained to date differ among reports.Previous studies have utilized an ELISA to detect 14-3-3 in the CSF  (AdditioWe developed a specific ELISA using the gamma isoform of 14-3-3 (14-3-3γ). Additionally, results from the WB and ELISA analyses were compared. We analyzed 124 human prion disease cases and 99 human non-prion disease cases using the 14-3-3γ ELISA and assessed if quantification of 14-3-3 might be helpful in their differentiation.prion protein gene (PRNP) mutation. All 124 cases contained MM at codon 129 and EE at codon 219 of the PRNP.Patients with suspected CJD were recruited from hospitals all over Japan for the purpose of conducting biochemical CSF assays. From more than 300 requests, a follow-up study was performed with 124 CJD patients. The cases were classified as sporadic CJD (n = 114), genetic CJD , or iatrogenic CJD . A total of 114 sporadic CJD patients and 3 iatrogenic CJD patients did not express a Subjects for the present study consisted of 114 sporadic CJD patients with confirmed CJD diagnosis. All 114 sporadic patients fulfilled the WHO diagnostic criteria for CJD. All sporadic CJD cases were typical with respect to clinical findings, clinical time course, neuroimaging  and PSWCs via EEG.A total of seven sporadic CJD cases  were definite, and 107 sporadic cases were probable among the 114 sporadic CJD patients. Two cases  were definite cases among the seven genetic CJD cases, and one case was a definite among the three iatrogenic CJD cases.For control samples, CSF was collected from 99 patients who suffered from one of the following disorders: dementia of Alzheimer's type , cerebrovascular dementia , Parkinson's disease , progressive supranuclear palsy , frontotemporal lobular degeneration , Huntington's disease , corticobasal degeneration , amyotrophic lateral sclerosis , limbic encephalitis , mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes , paraneoplastic cerebellar disorder/Lambert-Eaton myasthenic syndrome , temporal epilepsy , mild cognitive impairment , and dementia etiology unknown . Additionally, CSF was obtained from healthy volunteers .All subjects were examined and CSF samples collected, divided into aliquots, and stored at -80°C until required. All assays were performed simultaneously to avoid repeated freezing and thawing of samples. The CSF samples were used within 1 month of collection.We previously reported about 21 patients that suffered from early-stage CJD, defined as cases in the 6 weeks following the onset of the disease .We have described the MRI protocol and PSWC on EEGs previously .In the CJD group, 14-3-3 was detected in all CSF samples . A cut-off  was determined by an ROC curve  as determined by the 14-3-3γ ELISA  as determined by the 14-3-3γ ELISA Table . Five ofThe results from 23/99 of the non-CJD patients demonstrated that the concentration of 14-3-3γ exceeded the cut-off limit. When the concentration of 14-3-3γ was greater than 3, 000 AU/ml, the protein in the samples could also be detected by WB. Patients diagnosed with PCD/LEMS, MELAS, limbic encephalitis, temporal epilepsy, and a part of DAT were pseudo-positive, but negative using the RT-QUIC method.We attempted to develop a sandwich ELISA using combinations of the specific γ-isoform antibodies  as determined by the conflict of interest committee of Nagasaki University and are protected by the "Policy of Conflict of Interest in Clinical Research".All authors have no disclosures and no interest in the company, entity, or organization.YM and KM: YM and KM performed the analysis of the 14-3-3γ ELISA, WB and tau protein ELISA for human prion disease in CSF. TM: TM made the development of a 14-3-3γ ELISA kit and the charge nurse of the sale company of it SS: SS is a member of the CJD Surveillance Committee in Japan from 2007 till 2011. RA: RA performed the analysis of the RT-QUIC method for detecting human prion disease in CSF. AS and YK: investigators in this study. KS: KS analyzed the 14-3-3γ ELISA, Western blots and tau protein ELISA in CSF; KS is a member of the CJD Surveillance Committee in Japan on 2012; and chief investigator of this study. NN:NN was the chief manager of this study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/11/120/prepubTable S1. The profiles of all researches in the sensitivity and specificity of ELISA kits of 14-3-3 protein in CJD patients. ELISA: WB: Western blots method, N.E.: not exminated We compared the previous reports of ELISA kits of 14-3-3 protein in CSF of CJD patients with our data. Supplementary reference. 1. Geschwind MD, Martindale J, Miller D et al. Challenging the clinical utility of the 14-3-3 protein for the diagnosis of sporadic Creutzfeldt-Jakob disease. Archives of neurology. 2003;60:813-816. 2. Kenney K, Brechtel C, Takahashi H et al. An enzyme-linked immunosorbent assay to quantify 14-3-3 proteins in the cerebrospinal fluid of suspected Creutzfeldt-Jakob disease patients. Annals of Neurology. 2000; 48:395-398. 3. Gmitterova K, Heinemann U, Bodemer M et al. 14-3-3 CSF levels in sporadic Creutzfeldt-Jakob disease differ across molecular subtypes. Neurobiology of Aging. 2009; 30(11): 1842-50.Click here for fileTable S2. The characterization of three monoclonal antibodies and three polyclonal antibodies in six isoforms of 14-3-3 protein. 14-3-3 proteins are a highly conserved family of multifunctional proteins which are primarily found in high levels in neurons. These proteins comprise seven distinct isoforms , but σ-isoform has not been detected in the human brain. We analyzed six isoforms  in human CSF. We obtained the full-length gene encoding each isoform  of 14-3-3 protein from a cDNA library. Full-length constructs encoding either the β- or γ-isoform of human 14-3-3 protein in addition to a His-tag were cloned into pcDNA6/His vector, after which the constructs were transfected into murine 293T cell lines and over-expressed. All isoforms of protein were collected and purified three times through an affinity chromatography column. We analyzed all isoforms of recombinant protein of 14-3-3 protein reacted by three monoclonal antibodies (#1-#3) and polyclonal antibodies (#4-#6). Two monoclonal antibodies (#1 and #2) and one polyclonal antibody (#6) were specific by only γ-isoform of 14-3-3 protein. But one monoclonal antibody (#3) and two polyclonal antibodies (#4 and #5) were reacted by other isoforms including γ-isoform of 14-3-3 protein.Click here for fileFigures S1a and S1b. The standard curves obtained for the sandwich ELISA (clones #1 and #6). The standard curves obtained for the sandwich ELISA (clones #1 and #6). The standard control used a recombinant γ-isoform of 14-3-3. The combination of antibodies (#1 and #6) showed a dose-dependent reaction against the γ-isoform. Detection of 14-3-3 by the Western blot method in DAT patients. Detection of 14-3-3 by the Western blot method in DAT patients. Both cases 1 and 2 were DAT patients. The data from the 14-3-3 ELISA indicated that the protein concentration in cases 1 and 2 were 3, 168 and 6, 773 AU/ml, respectively. The positive control (#3) was also included.Click here for fileFigure S2a and S2b. The relationship between the concentration of standard samples and the absorbance. The relationship between the concentration of standard samples and the absorbance. Correlation coefficient = 0.9967. The relationship between the concentration of standard samples, and the absorbance in different standard samples. The relationship between the concentration of standard samples, and the absorbance in different standard samples. These measurements were repeated five times. We acquired the almost data similar to the fifth.Click here for file


Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce.Pichia pastoris strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter). We demonstrate that homogenous Man5GlcNAc2 N-glycosylation of the secreted proteins is achieved at all specific growth rates tested.Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 P. pastoris is a robust production strain for homogenously N-glycosylated proteins.Together, these data illustrate that the GlycoSwitch-Man5  Despite the availability of five classes of heterologous protein production platforms , more than 50% of currently marketed biopharmaceuticals are produced in mammalian cell lines . This isP. pastoris as an expression system for the production of therapeutic proteins, the first Pichia product approval by the FDA was announced only very recently . This now established regulatory approval pathway, together with the recent availability of glycoengineered strains that produce heterologous proteins predominantly as single glycoforms [Although many biopharmaceutical companies have been experimenting for many years with the methylotrophic yeast ycoforms ,4, shoulycoforms  and rat ycoforms .P. pastoris strains each capable of modifying its glycoproteins with predominantly one defined N-glycan structure [P. pastoris strain capable of modifying its glycoproteins with Man5GlcNAc2 N-glycans to produce murine granulocyte-macrophage colony-stimulating factor (GM-CSF) as a test protein.Over the past decade, we have created several glycoengineered tructure ,4,6,7. RGM-CSF belongs to a family of colony-stimulating factors that regulate proliferation and differentiation of hematopoietic cells . In respS. cerevisiae. Like native GM-CSF, it has 127 amino acids and is glycosylated but differs from native GM-CSF in molecular mass and in the substitution of leucine for proline at position 23. Therapeutic GM-CSF expressed in Escherichia coli is not glycosylated, has six fewer amino acids than the native protein, and an extra methionine at position 1 [E. coli-produced GM-CSF is also known as Molgramostim (marketed as Leucomax® by Schering-Plough), but was never approved for use in the United States by the FDA because it was associated with a higher incidence of adverse effects than Sargramostim [S. cerevisiae and is marketed as Leukine® .In the clinic, GM-CSF is used for treatment of neutropenia and aplastic anemia following chemotherapy and greatly reduces the risk of infection associated with bone marrow transplantation. Its utility in myeloid leukemia treatment and as a vaccine adjuvant is well established ,13. The sition 1 . E. coliramostim . The latin vitro biological activity of glycosylated GM-CSF is due to a reduction in receptor binding affininty [Murine GM-CSF (mGM-CSF) is a 124 amino acid glycoprotein with an apparent molecular weight of 14-33 kDa . It contffininty ,18.5GlcNAc2 glycoform of mGM-CSF by a glycoengineered P. pastoris strain. This approach allowed for maximal production at high cell densities while maintaining N-glycan homogeneity.Here, we describe the design of a basic but robust fed-batch fermentation process for the production of a ManEscherichia coli MC1061 was used as the host strain for plasmid propagation. The bacteria were cultivated in LB medium  containing 100 μg/ml ampicillin.P. pastoris GlycoSwitch-Man5 strain modifies its glycoproteins predominantly with Man5GlcNAc2 N-glycans. It only differs from a previously reported GS115-derived strain in the selection marker used to engineer the N-glycosylation pathway, blasticidin instead of zeocine [The  zeocine .P. pastoris cultivation was performed in BMGY  and BMMY medium .Small-scale 2PO4, 5.0 g (NH4)2SO4, 1.0 g CaSO4.2H2O, 14.3 g K2SO4, 11.7 g MgSO4.7H2O, 40.0 g glycerol. Prior to inoculation, the pH was adjusted to 5.0 with concentrated ammonium hydroxide followed by the addition of 8.7 ml of Pichia Trace Metals .Fermentations were performed in FM22 medium , which iP. pastoris expression vector pPIC9mGM-CSF contains the mature mouse GM-CSF coding sequence fused in-frame to the S. cerevisiae alfa mating factor signal sequence under transcriptional control of the AOX1 promoter. The mGM-CSF ORF was amplified by PCR using pORF9-mGMCSF (Invivogen) as template and the oligonucleotides 5'-CTAGCTCGAGAAAAGAGAGGCTGAAGCCGCACCCACCCGCTCACCC-3' and 5'-AGTTTAGCGGCCGCTCATTTTTGGCCTGGTTTTTTGC-3' as primers (XhoI and NotI restriction sites are underlined). Subsequently, an XhoI/NotI fragment was cloned into the XhoI/NotI opened pPIC9 expression vector (Invitrogen).The P. pastoris GS115 and GlycoSwitch-Man5 as described [Linearized vectors  were transformed into escribed , resultig for 5 minutes and resuspended in BMMY medium. Induction was maintained for another 48 hours by spiking the cultures twice daily with 100 μl of 100% methanol . Subsequently, the cultures were harvested by centrifugation (3000g for 10 minutes) and the supernatants were frozen at -20°C until further use.Cells were grown in 10 ml BMGY medium at 30°C. After 48 hours of cultivation, the cells were pelleted by centrifugation at 30002 cascade. Pure oxygen was supplemented as needed to maintain the DO setpoint. A part of the off-gas was diverted to an MC-168 methanol monitor and controller (PTI Instruments) equipped with a TGS822 methanol sensor , which were used to maintain a constant level of methanol in the broth. A methanol feed pump , balance  and the MC-168 controller were interfaced with the NBS-BioCommand supervisory control and data acquisition software to make a closed-loop feed control system. The amount of methanol delivered was measured using a balance as the difference between the initial mass in the methanol tank and the current mass.Fermentations were carried out in 5-liter BioFlo III or 3000 bioreactors (New Brunswick Scientific) containing two liters of FM22 medium  supplemeP. pastoris strain Man5mGM-CSF was grown in a 1-liter shake flask with 300 ml BMGY medium for 20-24 h at 30°C and 300 rpm to an optical density (at 600 nm) of 4-8. Following microscopic examination for contamination, 100 ml of this pre-culture was used as inoculum.The To increase cell mass and simultaneously prepare the cells for induction, a glycerol fed-batch phase was performed. A 1-hour fed-batch period was used at a feed rate of 20 g 50% w/w glycerol  per hour per liter of broth. Following the fed-batch phase, a transition phase was applied to shorten the time required for the cells to fully adapt to methanol. The transition phase was initiated by the addition of 1.5 g/l methanol. Simultaneously, the glycerol feed rate was ramped down linearly from 20 g/l/h to 0 over a 3-h period. By the end of the transition phase, the cells were fully adapted to methanol, which was confirmed by a sharp drop in DO .max) to be determined. The second strategy used methanol-limited feeding to maintain the growth rate below μmax at a constant predetermined value. This was achieved by varying the methanol feed rate (F) exponentially, using the following equation [Once the cells were fully adapted to methanol, the methanol fed-batch phase was started. Methanol supplemented with 12 ml of PTM1 per liter was used. Two feeding strategies were applied. The first was a methanol-excess feed strategy in which the methanol concentration was kept constant at ~2 g/l. This allowed the maximum specific growth rate (μequation :0 is the cell density and V0 the broth volume at the start of the feeding profile (t = 0), μ is the desired specific growth rate (h-1), and νMeOH is the specific methanol consumption rate (g/g/h). The latter can be calculated once μmax and νMeOH, max have been determined empirically under methanol excess conditions. For a desired μ, νMeOH is estimated by the following equation:XX/S = μmax/νMeOH,max and is independent of μ and that the maintenance coefficient is negligible [Equation (2) is based on the assumption that the yield of biomass to substrate Ygligible .g for 30 minutes. Ammonium sulphate was added to 80% saturation and the suspension was incubated overnight to precipitate the protein fraction. The protein pellet was isolated by centrifugation at 18,000g for 30 minutes and redissolved in 90 ml of 25 mM sodium acetate pH 4.5, 0.1% CHAPS. After centrifugation at 40,000g for 30 minutes to remove a minor non-soluble fraction, this solution was desalted on a Sephadex G25 column of 475 ml  to 25 mM sodium acetate pH 4.5, 0.1% CHAPS. The desalted protein fraction was loaded on a 20 ml Source 15 S column  to remove contaminants and potential endotoxins. After equilibration, mGM-CSF was eluted by a linear gradient over 20 column volumes of NaCl from 0 to 1 M in 25 mM sodium acetate pH 4.5, 0.1% CHAPS. The obtained fractions were analyzed by SDS-PAGE. The major mGM-CSF-containing fractions were pooled and loaded onto a HiLoad 26/60 Superdex 75 prep grade size exclusion column  with PBS as the elution buffer. Following concentration of the product peak by ultrafiltration , the mGM-CSF concentration was determined using the BCA assay (Pierce) and endotoxin levels were determined with the ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript USA). The process generated 92 mg mGM-CSF from 5 liter of supernatant (18 mg/l) with a purity >95% based on SDS-PAGE and an endotoxin level of 0.15 EU/mg.All purification steps were conducted at 4°C. A GS115 mGM-CSF-secreting clone was grown in BMGY-containing shake flasks at 30°C. After 24 hours of cultivation, the cells were harvested by centrifugation and mGM-CSF protein expression was induced by resuspending the cell pellet in BMMY medium containing 2% methanol. At 24, 36 and 48 hours of induction, 2% methanol was added to maintain induction. After 60 hours, five liter of cultivation medium was then obtained upon centrifugation at 18,000E. coli (Peprotech) or in-house in P. pastoris. Following differentiation, bone marrow-derived DCs (BM-DCs) were infected for 24 h with Mycobacterium bovis BCG 1721, a derivative of M. bovis BCG Pasteur, carrying a non-restrictive rpsL alteration. Following infection, the BM-DCs were collected by centrifuging (10 min at 150g and 4°C). After washing in PBS, the cells (both infected and non-infected) were stained for different cell surface markers, i.e. CD40-biotin (Pharmingen), CD86-biotin (Pharmingen), followed by streptavidin-PE, CD80-PE (Pharmingen), CD273-PE (Pharmingen), CD274-PE (Pharmingen), CD1d-PE (eBioscience). The expression of these surface markers was checked on the CD11c positive population (a marker for DCs) with CD11c-PE-Cy5 (eBioscience), which was almost 100% in both preparations. Measurements were performed on a FACSCalibur flow cytometer (BD Biosciences) equipped with CellQuest software.Bone marrow (BM) cells (C57Bl/6 mice) were cultured for 8 days in dendritic cell (DC) culture medium: RPMI 1640 containing GlutaMAX-I, supplemented with 5% (vol/vol) FCS, 50 μM β-mercaptoethanol and 20 ng/ml mGM-CSF, the latter either produced in g and 4°C) the protein pellet was dissolved in Laemmli loading buffer supplemented with 1 M unbuffered Tris to neutralize the solution and boiled for 10 minutes. Samples were analyzed by SDS-PAGE using 15% gels casted in-house according to [Secreted proteins were TCA precipitated by adding 10% v/v 10 mg/ml deoxycholate and 10% trichloro acetic acid. After centrifugation . This was done by sampling duplicate 10 ml aliquots of the fermentation broth into preweighed 15 ml conical tubes. The samples were centrifuged at 10.000 1 OD600 .Murine GM-CSF expression levels were quantified by ELISA (eBioscience). After correction for cell density, yields were expressed as milligram per liter fermentation broth.P. pastoris pPIC9 expression vector, in-frame with the S. cerevisiae α-mating factor prepropeptide coding sequence and under transcriptional control of the P. pastoris AOX1 promoter. The resulting vector was termed pPIC9mGM-CSF . Eleven clones effectively produced mGM-CSF and the one with the highest expression level was withheld for further analysis. This strain was named GS115mGM-CSF. SDS-PAGE analysis showed that mGM-CSF was produced as three molecular species ranging in molecular weight from ~14 to ~18 kDa  to differentiate murine bone marrow-derived monocytes into dendritic cells (BM-DC). Similar expression of 6 differentiation/activation-associated cell surface markers on BM-DCs that were either differentiated with Pichia-derived or E. coli-derived mGM-CSF demonstrated that both forms are equally potent. Additionally, both sets of BM-DCs responded similarly to an M. bovis BCG infection, confirming that differentiation with P. pastoris- or E. coli-derived mGM-CSF does not induce phenotypic differences during the differentiation process  expressed mGM-CSF (data not shown). These clones were designated Man5mGM-CSF. As did the wild type strain, the GlycoSwitch-Man5 strain produced mGM-CSF as three distinct glycoforms, i.e., modified with two, one or no N-glycans  were mostly modified with a heterogeneous array of high-mannose-type N-glycans  of ~2 g/l. Only minor fluctuations in methanol concentration were observed throughout the fermentations. μmax and νMeOH,max were 0.0624 h-1 and 0.0743 g/g WCW/h, respectively. This corresponds to a YX/S (yield of biomass to substrate) of 0.8398 g/g. For comparison, the 'empty' GlycoSwitch-Man5 strain, i.e., GlycoSwitch-Man5 transformed with pPIC9, had a μmax and νMeOH,max of 0.0816 h-1 and 0.052 g/g WCW/h, respectively, corresponding to a YX/S value of 1.569 g/g. These data illustrate the burden that mGM-CSF expression places on the cells: μmax of Man5mGM-CSF is about 76% of μmax of Man5. Moreover, under excess methanol conditions, Man5mGM-CSF is about 50% less efficient in converting methanol to biomass than GlycoSwitch-Man5.To determine μmax and νMeOH,max into equations (1) and (2), the methanol feed rate, F, needed to achieve a desired μ could be estimated by the following equation:By substitution of μ-1 (25% μmax), 0.0312 h-1 (50% μmax) and 0.0468 h-1 (75% μmax). X0 and V0 were determined just prior to the start of the methanol feed profile. The actual μ values were 0.015 h-1, 0.0332 h-1, and 0.0459 h-1, which correlated very well to the desired values. Figure -1 (~25% of μmax).The desired values for μ were set to 0.0156 hi.e., after correction for cell density) was maximum by growing the cells at μ = 0.015 h-1 (24.04% μmax) during the induction phase. In this way, a volumetric yield of 760 mg/l could be achieved after ~68 h of fermentation. Although volumetric yields decreased slightly after this time point, total mGM-CSF levels continued to increase during the entire course of the fermentation. Higher growth rates resulted in decreased yields (Table -1 (53.21% μmax) and μ = 0.0459 h-1 (73.56% μmax) decreased yields by about 15.3%. Allowing the cells to achieve their maximum growth rate under methanol conditions (μmax = 0.0624 h-1) further decreased mGM-CSF yields by as much as 42% (442 mg/l). Hence, limiting the growth rate during the induction phase to ~25% of μmax resulted in maximum mGM-CSF yield. During the entire course of fermentation, total recombinant mGM-CSF levels kept increasing regardless of the specific growth rate (data not shown), indicating that proteolytic degradation was not a significant problem. This is in contrast to many other recombinant proteins produced in P. pastoris that get increasingly degraded during the fermentation [Protein production kinetics of the four fed-batch fermentation types are shown in Figure entation ,28.5GlcNAc2. Even after >100 hours of fermentation, the level of N-glycan homogeneity was comparable to the level obtained in small scale cultures [Fermentation supernatant samples were analyzed by DSA-FACE Figure . The pre6-10GlcNAc2 structures. These oligosaccharides are believed to be the result of 1) incomplete processing by the introduced α-1,2-mannosidase-HDEL, 2) interfering endogenous mannosyltransferases, or 3) a combination of those two. Importantly though, growth under fermentation conditions did not decrease the conversion efficiency of P. pastoris hypermannosyl-type N-glycans to Man5GlcNAc2 structures as compared to small scale cultivation experiments, again illustrating the robustness of this strain.Regardless of the specific growth rate, DSA-FACE analysis revealed the presence of low-abundance peaks. Several of these derive from the cultivation medium (not shown), but there is a small fraction (<10%) of larger N-glycan species corresponding to ManP. pastoris strain that predominantly modifies its glycoproteins with Man5GlcNAc2 N-glycans. We have shown that growth rate does not significantly affect N-glycan homogeneity: regardless of the specific growth rate the predominant peak during the entire course of all fermentations was Man5GlcNAc2. There was, however, a clear relationship between product yield and specific growth rate. Cultivation of the cells at ~25% of their maximal growth rate (μ = 0.015 h-1) resulted in a volumetric yield of ~760 mg mGM-CSF per liter fermentation broth after ~68 h of fermentation. Higher specific growth rates during the induction phase resulted in decreased mGM-CSF yields.In this paper, we describe a basic but robust fed-batch fermentation protocol for the secreted production of murine GM-CSF in a glycoengineered 5GlcNAc2 N-glycans across a range of fermentation conditions [To date, only one similar study has been published. In this paper, researchers at GlycoFi Inc. (a wholly-owned subsidiary of Merck & Co Inc.) describe a strain capable of producing >1g/l of human IgG1 with >90% homogeneous Mannditions . UnfortuP. pastoris N-glycosylation pathway to the Man5GlcNAc2 structure does not affect the yeast's physiology much and that such engineered strains are fully capable of producing diverse mammalian glycoproteins at high levels, with the desired homogenous N-glycosylation. As the Man5GlcNAc2 structure is the starting point for buildup of mammalian complex-type N-glycans, future studies will address the behaviour of more extensively engineered strains in high cell density cultivation.However, it is clear from both reports that conversion of the X/S: Yield of biomass to substrate (g/g); μ: Specific growth rate under methanol conditions; μmax: maximum specific growth rate under methanol conditions; νmax: maximum specific methanol consumption rate (g/g/h)EFT: elapsed fermentation time (h); F: methanol feed rate (g/h); S: methanol concentration (g/L); T: Culture time (h); V: broth volume (L); WCW: wet cell weight; X: cell density (g WCW/L); YThe authors MI, NF, JH, AVH and MMM declare that they have no competing interests. PPJ, RC and NC are either inventors or share otherwise in proceeds of licensing of patents and patent applications covering parts of the described GlycoSwitch technology.P. pastoris strains, performed the fermentations, interpreted the glycan analysis data and drafted the manuscript. MI assisted in designing the fermentation strategy. NF performed the monocyte differentiation assay and edited the manuscript. JH purified the Pichia-produced mGM-CSF and edited the manuscript. AVH was responsible for N-glycan analysis. MMM supervised the fermentations and edited the manuscript. RC conceived and initiated the study. NC supervised experiments and reviewed the final manuscript. All authors read and approved the final manuscript.PPJ constructed the mGM-CSF expression vector, generated and analyzed the


The capability of performing interval timing is essential for survival, adaptation and its impairment leads to sever cognitive and/or motor dysfunctions. Although the localization of brain regions essential for interval timing is not yet clear, some progress has been made. For example, both temporal production and temporal perception are strongly connected to striatum and its afferent projections from the substantia nigra pars compacta . Since i


In chronic inflammatory lesions there are increased numbers of macrophages with a possible contribution of enhanced survival/proliferation due, for example, to cytokine action; such lesions are often hypoxic. Prior studies have found that culture in low oxygen can promote monocyte/macrophage survival. We show here, using pharmacologic inhibitors, that the hypoxia-induced pro-survival response of macrophages exhibits a dependence on PI3-kinase and mTOR activities but surprisingly is suppressed by Akt and p38 MAPK activities. It was also found that in hypoxia at CSF-1 concentrations, which under normoxic conditions are suboptimal for macrophage proliferation, macrophages can proliferate more strongly with no evidence for alteration in CSF-1 receptor degradation kinetics. TNF promoted macrophage survival in normoxic conditions with an additive effect in hypoxia. The enhanced hypoxia-dependent survival and/or proliferation of macrophages in the presence of CSF-1 or TNF may contribute to their elevated numbers at a site of chronic inflammation. In the absence of an appropriate stimulus, macrophages and neutrophils die by apoptosis thereby providing a mechanism for the resolution of an inflammatory response Chronic inflammatory lesions rich in macrophages are often hypoxic due to reduced blood supply with this reduced tissue oxygen tension postulated to contribute to the pathology TNF is a key pro-inflammatory cytokine and its blockade can be efficacious in chronic inflammatory/autoimmune diseases, such as rheumatoid arthritis. Its neutralization can lead to reduced macrophage numbers and increased apoptosis in the rheumatoid synovium with a correlation with efficacy being noted We show here, using a series of specific pharmacologic inhibitors, that the hypoxia-induced pro-survival response of CSF-1-starved BMM exhibits a partial dependence on PI3-K and S6 kinase (S6K) activities but unexpectedly is enhanced by Akt and p38 MAPK inhibition. We also show that in hypoxia at CSF-1 concentrations, which under normoxic conditions are suboptimal for macrophage proliferation, macrophages can proliferate more strongly. We also found that TNF promoted BMM survival with an additive effect in hypoxia.This work has been approved by the University of Melbourne Animal Ethics Committee.Reagents used were as follows: recombinant human CSF-1 (Chiron), recombinant murine TNF (R&D Systems), propidium iodide (Sigma), wortmannin , LY294002 (Merck), Akt VIII (Merck), rapamycin , PD98059 (Merck), U0126 (Merck), SB203580 (Merck) and antibodies: anti-phosphotyrosine (4G10) (Millipore), monoclonal anti-CSF-1R (AFS98) (eBioscience), anti-CSF-1R and anti-Erk2 (Santa Cruz Biotech), anti-phospho-Tyr809 CSF-1R, anti-phospho-Thr202/Tyr204 Erk1/2, anti-phospho-Ser473 Akt, and anti-phospho-Tyr705 Stat3 .2) to allow for adherence. Following washing, the viable cell number was counted as the value at time  = 0.Adherent bone marrow-derived macrophages (BMM) were generated from precursors in the presence of CSF-1 by a protocol similar to one previously described 2 and 5% CO2 flow For hypoxia experiments, BMM from above were transferred from incubation overnight under normoxic conditions into a precalibrated hypoxic chamber (Coy Laboratories) with a 1% OCell viability was measured using the trypan blue exclusion method. Cells were removed from Iwaki wells using either cell dissociation buffer (Sigma-Aldrich) as per the manufacturer's instructions or by incubating cells with PBS at 37°C for 20 min, followed by gentle pipetting to dislodge cells.Briefly, dislodged BMM cells were resuspended in 50 µl of PBS, and 1 mL of ice-cold 70% ethanol was added. Cells were left overnight at 4°C and, on the following day, were pelleted, washed with PBS, and resuspended in 0.5 mL of a staining solution containing 69 µM propidium iodide and 5 µg/mL RNase A in 38 mM sodium citrate Cells were lysed directly in tissue culture dishes with Nonidet P-40 lysis buffer (20 mM Tris-HC1 (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, 1 mM sodium orthovanadate, 10 mM NaF, 10 mM β-glycerophosphate) containing Complete™ protease inhibitor cocktail (Roche) for 30 min on ice. Lysates were clarified by centrifugation at 15,000 rpm for 10 min at 4°C, and the protein concentrations measured using a protein assay kit (Bio-Rad). SDS-PAGE and Western blotting were performed by standard techniques 0/G1 DNA as an indicator of apoptosis in CSF-1-deprived BMM We showed before that the loss of viability of BMM upon CSF-1 withdrawal was reduced under hypoxic conditions Presumably the loss of viability in CSF-1-starved BMM Macrophages in tissues are likely to be exposed to the ubiquitously expressed CSF-1 Given the findings above that hypoxia can potentiate the mitogenic effects of CSF-1 on BMM, we investigated the consequences of hypoxia on signaling by the CSF-1 receptor. The magnitude and duration of CSF-1 receptor autophosphorylation at Tyr809 (within the catalytic loop of the kinase domain op/Csf1opCsf1 mice (i.e. mice with an inactivating CSF-1 mutation op/Csf1opCsf1 BMM Another cytokine to which macrophages in an inflammatory and hypoxic environment are likely to be exposed is TNF There was an additive pro-survival affect when BMM were cultured in hypoxia and in optimal TNF concentration ; a similop/Csf1opCsf1 mice with an inactivating CSF-1 mutation.The above findings extend our prior ones showing that hypoxia, in contrast to its effects on most other cell types Since expression of constitutively active Akt partially enhanced the survival of CSF-1-depleted BMM We also showed above in hypoxia that CSF-1 concentrations, which are suboptimal for BMM proliferation under normoxic conditions, BMM can in fact proliferate more strongly at least over the time scale examined. These data for hypoxia are similar to what we found with other stimuli, such as low concentrations of oxidized LDL and particulate adjuvants It was recently reported that the rate at which the EGF receptor is internalized into early endosomes, and subsequently degraded in the lysosomal compartment, is reduced in hypoxia L and Bcl-2 did not correlate with pro- or anti-apoptotic responses although there was evidence for NF-κB-dependence. It is likely that the signaling pathways governing TNF-mediated survival in macrophages will show significant differences from those utilized by CSF-1 given their vastly different receptors.In agreement with the literature for human monocytes