In agreement with studies of other TSEs and species, we found reduced brain levels of copper and increased levels of manganese associated with disease in our hamster model. We also found that the in situ levels of these metals in brainstem were sufficient to image by X-PEEM. Using immunolabeled prion plaques in directly adjacent tissue sections to identify regions to image by X-PEEM, we found a statistically significant relationship of copper-manganese dysregulation in prion plaques: copper was depleted whereas manganese was enriched. These data provide evidence for prion plaques altering local transition metal distribution in the TSE-infected central nervous system. Accumulation of aggregates rich in an abnormally folded form of the prion protein characterize the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs). The molecular triggers of plaque formation and neurodegeneration remain unknown, but analyses of TSE-infected brain homogenates and preparations enriched for abnormal prion protein suggest that reduced levels of copper and increased levels of manganese are associated with disease. The objectives of this study were to: (1) assess copper and manganese levels in healthy and TSE-infected Syrian hamster brain homogenates; (2) determine if the distribution of these metals can be mapped in TSE-infected brain tissue using X-ray photoelectron emission microscopy (X-PEEM) with synchrotron radiation; and (3) use X-PEEM to assess the relative amounts of copper and manganese in prion plaques  Sc, which typically accumulates in diseased nervous tissue, often as large plaques , indicating that the decline in color intensity on the manganese image was approximately 0.005 less for one region versus the other for every 0.10 unit increase in copper.Sc exists in a low copper, high manganese state in vivo and supports the hypothesis (4) that, while copper is important for the function of PrPC; the conversion of PrPC to PrPSc may require manganese. While X-PEEM does not permit quantitative analysis of metal concentrations, our data indicate significantly increased manganese and diminished copper content in prion plaques compared to surrounding tissue. These data further confirm that PrPin situ analysis of copper and manganese levels in prion plaques and illustrates the specific localization and sizes of copper-poor and manganese-rich regions. Whether these changes to brain metal content are a cause or consequence of TSE infection, and the extent that these altered metal concentrations contribute to the tissue damage and cell loss remains to be elucidated. This study has investigated plaques in only one brain region and only two of many potential metals that may influence TSE pathogenesis. A comprehensive study designed to assess the distributions of iron [Sc molecule and suggest a role for these metals in TSE neuropathology. Our data are also consistent with previous studies, both in vivo and in vitro, that suggest a role for manganese in prion infections and our data demonstrates that increased levels of manganese, with concommitent decreased levels of copper, can be localized to brain regions containing prion plaques.Our study provides the first  of iron , zinc [2 of iron ,27 and o of iron , is warr


L-3,4-dihydroxy-6-[18F]fluoro-phenylalanine ([18F]FDOPA) positron emission tomography (PET) as a method for assessing the severity of dopaminergic dysfunction in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats by comparing it with quantitative biochemical, immunohistochemical, and behavioral measurements.We evaluated the utility of 18F]FDOPA-PET, measurement of striatal dopamine (DA) and DA metabolite levels, tyrosine hydroxylase (TH) immunostaining, and methamphetamine-induced rotational testing.Different doses of 6-OHDA  were unilaterally injected into the right striatum of male Sprague-Dawley rats. Dopaminergic functional activity in the striatum was assessed by [18F]FDOPA in the bilateral striatum was observed in rats pretreated with both aromatic L-amino acid decarboxylase and catechol-O-methyltransferase (COMT) inhibitors. Unilateral intrastriatal injection of 6-OHDA produced a significant site-specific reduction in [18F]FDOPA accumulation. The topological distribution pattern of [18F]FDOPA accumulation in the ipsilateral striatum agreed well with the pattern in TH-stained corresponding sections. A significant positive relationship was found between Patlak plot Ki values and striatal levels of DA and its metabolites (r = 0.958). A significant negative correlation was found between both Ki values (r = -0.639) and levels of DA and its metabolites (r = -0.719) and the number of methamphetamine-induced rotations.Accumulation of [Ki values determined using [18F]FDOPA-PET correlated significantly with the severity of dopaminergic dysfunction. [18F]FDOPA-PET makes it possible to perform longitudinal evaluation of dopaminergic function in 6-OHDA-lesioned rats, which is useful in the development of new drugs and therapies for Parkinson's disease (PD). Parkinson's disease is a progressive, chronic neurodegenerative disorder with movement dysfunction that primarily affects the elderly . The fouL-DOPA). Although L-DOPA remains the most effective therapy, chronic treatment causes adverse effects, such as wearing-off phenomenon and dyskinesia fluoro-phenylalanine ([18F]FDOPA), is one of the most widely used PET tracers for studying the brain dopaminergic system. Accumulation of [18F]FDOPA in the brain reflects its transport, decarboxylation, and vesicular uptake in the nigrostriatal presynaptic nerve terminals. In PD patients, [18F]FDOPA uptake in the striatum is reduced [18F]FDOPA uptake, especially in the putamen [11C]dihydrotetrabenazine ([11C]DTBZ), which binds specifically to vesicular monoamine transporter 2 (VMAT2) in dopaminergic synaptic vesicles, and [18F]β-CFT (2-β-carbomethoxy-3β-(4-fluorophenyl)tropane or WIN-35,428), a dopamine transporter (DAT)-selective radioligand. A significant correlation between [11C]DTBZ-binding and clinical motor asymmetry and a clear association of the severity of rigidity and hypokinesia with reduced [18F]β-CFT uptake in the putamen have been reported PE2I, a selective DAT radioligand. Though DAT ligands are reportedly superior for detecting decreases in DA neurons [18F]FDOPA enables examination of dopaminergic neuron functions including transport, decarboxylation, and vesicular uptake, which is useful for assessing the neuroprotective effects of therapeutic agents, as well as evaluating the differentiation of grafted cells in living brains. Especially, in the cell transplantation experiments into the striatum or gene therapy experiments, it is considered that evaluation of overall dopaminergic neuron functions using [18F]FDOPA-PET is useful for interpreting these therapeutic effects on differentiation or enhancement of dopaminergic neurons, whereas the accumulation of DAT ligands in the striatum means the expression of functional DAT protein, which is nothing more than a proof of the function of DA reuptake.Recent developments in small animal PET imaging instrumentation have enabled noninvasive, quantitative, and repetitive visualization of biological functions in living animals. From a translational research perspective, reported -32. Inajreported  reported neurons -35, FDOPA-PET which present no images of the specific uptake of [18F]FDOPA were reported to date [18F]FDOPA-PET has also not been reported. In this study, we quantitatively analyzed [18F]FDOPA-PET in unilaterally 6-OHDA-lesioned rats, and report the first accurate evaluation of the severity of dopaminergic dysfunction using [18F]FDOPA-PET.The severity of lesions induced by 6-OHDA injection is generally evaluated by biodistribution analysis, behavior ,36. A fe to date ,27. Therw/v) L-ascorbic acid . Methamphetamine sulfate  was dissolved in saline. Entacapone , a peripheral catechol-O-methyltransferase (COMT) inhibitor, and carbidopa (Sigma), a peripheral aromatic L-amino acid decarboxylase (AADC) inhibitor, were suspended in 0.25% sodiumcarboxymethylcellulose. All solutions and suspensions were prepared fresh.6-OHDA  was dissolved in saline  containing 0.2%  was dissolved in acetic acid and acetyl [18F]hypofluorite was bubbled through the solution at 130°C for 10 min. The reaction mixture was then hydrolyzed in 6 M HCl at room temperature for 3 min. The product was purified using high-performance liquid chromatography (HPLC) on a COSMOSIL 5C18-MS-II column (20 mm (i.d.) × 250 mm, 5-μm, Nacalai Tesque, Kyoto, Japan) using 0.1% acetic acid as the mobile phase. The purified fraction was mixed with 25% ascorbic acid  and sterilized by filtration before administration. Each solution was prepared on the day of the experiment. The pH of the final solution for animal use was around 6.0. Specific radioactivity at the time of injection ranged from 11 to 70 MBq/μmol. The radiochemical purity was over 99.5%.FDOPA.Images were analyzed using PMOD software . MRI images were collected 7 weeks after rats were 6-OHDA unilaterally lesioned and were manually aligned in space using a rat brain atlas . MRI ima18F]FDOPA in normal or lesioned striatum tissues was calculated by Patlak plots [18F]FDOPA administration was used. The [18F]FDOPA influx constant was determined using the following equation:Uptake of [ak plots  using PMCTissue(t) represents the tissue radioactivity concentration at time t as determined by PET image analysis. CReference(t) represents the reference tissue (cerebellum) radioactivity concentration at time t as determined by PET image analysis. Ki represents the [18F]FDOPA influx constant calculated from the regression slope. V represents the initial distribution volume in the tissue of interest at time 0, calculated from the Patlak plot y-intercept.where 18F]FDOPA by peripheral COMT and DDC. Venous blood was collected from the tail vein after the emission scan and centrifuged at 12 000 rpm for 2 min at 4°C. A fivefold volume of cold 0.2 M perchloric acid containing 100 μM Na2EDTA was then added and the resulting suspension was centrifuged at 15,000 rpm for 15 min at 4°C, filtered through a 0.45 μm PTFE filter , and an aliquot was injected onto a HPLC system  with a UG-SCA30 NaI(TI) positron detector . Radio-metabolites were separated on an EICOMPAK SC-5ODS column (3.0 mm (i.d.) × 150 mm; Eicom Corporation, Kyoto, Japan) using 0.1 M sodium citrate-acetate buffer (pH 3.5) containing 17% methanol, 0.88 mM sodiumoctanesulfonic acid, and 0.013 mM Na2EDTA as the mobile phase. The analysis was carried out at a column temperature of 25°C and a flow rate of 0.5 ml/min. Major 18F peaks were identified by comparing retention times with those of authentic metabolite standards. The individual peak areas of the radiometric chromatograms were corrected for radioactive decay to the retention time of the radio-metabolite for each sample and expressed as fractions of total radioactivity.Plasma radio-metabolites were analyzed to confirm the effect of pretreatment with carbidopa and entacapone on the metabolism of FDOPA-PET studies using sham-operated, unlesioned control rats pretreated with carbidopa and entacapone indicated that [18F]FDOPA accumulates highly in the bilateral striatum rather than other regions, including the cerebellum. In rats unilaterally lesioned with 28 μg of 6-OHDA, [18F]FDOPA uptake in the striatum was significantly lower in the ipsilateral (right) side compared to the contralateral (left) side of the injection  was detected in the plasma from the rat pretreated with carbidopa and entacapone. This peak was small in the plasma from the rat pretreated with carbidopa only, and there was a later peak with a retention time identical to that of authentic 3-O-methyl-FDOPA (OMFD) (6.0 min) (data not shown).[n Figure . The difn Figure . Figure 18F]FDOPA in the contralateral (left) striatum was high and similar in all rats, while uptake was low in the ipsilateral (right) striatum and along the 6-OHDA injection site, especially in the dorsal part of the striatum, which was consistent with immunostaining results.Figure Ki, the [18F]FDOPA influx constant, were significantly lower for the ipsilateral striatum compared to the contralateral side. One-way ANOVA revealed that 6-OHDA treatment significantly reduced the Ki values for the ipsilateral striatum , but not for the contralateral striatum  were plotted against the 6-OHDA dose  striatum compared to the contralateral (left) striatum, suggesting that 6-OHDA administration induced degeneration of the dopaminergic synaptic terminals Figure . Values m Figure . The inde Figure . The meaThe DA content in the ipsilateral striatum was significantly lower than in the contralateral striatum in the 6-OHDA-injected groups Figure . Inter-gKi R/L and the DA-metabolites R/L. The Ki R/L negatively correlated with the common logarithm of the number of methamphetamine-induced rotations  by AADC in the peripheral tissues and to [18F]OMFD by COMT [18F]FDOPA-PET , [18F]FDOPA, [18F]OMFD, and [18F]FDOPAC are present in the striatum of rats pretreated with both carbidopa and entacapone, while [18F]OMFD is the major striatum metabolite in rats pretreated only with carbidopa 30 min after [18F]FDOPA injection. Therefore, the same components of 18F-radioactivity might have contributed to the striatum radioactivity we observed.Radio-metabolite analysis of plasma sampled after the emission scan (90 min) revealed that 8FFDOPA w18F]FDOPA accumulation we observed in both the dorsal and ventral striatum (nucleus accumbens) agreed well with TH immunostaining of the corresponding sections, indicating that small animal PET imaging can provide detailed information regarding tracer location in the brain. This is particularly useful for evaluating the dopaminergic function of grafted cells because the accumulation of [18F]FDOPA at graft sites would suggest the existence of TH-positive neurons that have differentiated from grafted cells.The topological distribution of [18F]FDOPA influx rate constant Ki for the left striatum of all groups and the right striatum of the control group ranged from 15.7 to 16.7 × 10-3 min-1. Using a noninvasive approach to determine Ki, Takikawa et al. reported a mean KiOCC value of 6.6 ± 1.8 × 10-3 min-1 in healthy volunteers pretreated with carbidopa FDOPA-PET is also useful for screening striatal lesions .It is noteworthy that there was a significant positive correlation between the Ki values correlated well with striatal levels of DA and its metabolites, the linear regression curve did not pass through the origin. Even in the 28 μg group, which showed the most severe lesions, the right/left ratio of Ki values ranged from 25.0% to 39.5%, whereas the striatal DA-metabolites R/L ranged from 4.9% to 29.8%. Similarly, Ito et al. [14C]L-DOPA-specific uptake did not intersect with the origin. One possible explanation for this result is that the [18F]FDOPA-PET Ki value reflects its transport and decarboxylation not only by the dopaminergic neurons, but also by serotonergic terminals, capillary endothelium, pericytes, and different types of non-monoaminergic neurons in the striatum, since these have been reported to show DDC activity and to be sites of L-DOPA conversion to DA after dopaminergic denervation [Although the right/left ratio of o et al.  reportedervation -56.Ki R/L values correlated linearly with the common logarithm of the number of rotations induced by methamphetamine. A similar correlation between percent of striatal [14C] L-DOPA-specific uptake and the number of methamphetamine-induced rotations was reported by Ito and colleagues [18F]FDOPA-PET is ideal for noninvasive, accurate quantitative evaluation of dopaminergic function. It might be quite valuable to measure the recovery of dopaminergic function in cell transplantation experiments using 6-OHDA-lesioned rats, since discrepancies between recovery of motor deficit and the number of TH+ cells counted in postmortem analysis have been reported FDOPA-PET for imaging dopaminergic function in unilaterally 6-OHDA-lesioned rats. The severity of the dopaminergic denervation determined by [18F]FDOPA-PET correlated well with that determined by widely used biochemical, immunohistological, and behavioral assays. [18F]FDOPA-PET enables noninvasive, quantitative evaluation of dopaminergic function in 6-OHDA-lesioned rats and will be a useful tool in the development of new therapies for PD.This is the first report demonstrating the utility of [The authors declare that they have no competing interests.18F]FDOPA. MG performed tyrosine hydroxylase immunohistochemical study. YKuw provided intellectual input, performed data quality assurance of all the experiments. YWad helped perform PET scan and image reconstruction. HO is the co-PI of this study and involved in the design and interpretation of the analysis as well as in writing manuscript. YWat is the PI of the study and is involved in all aspects of this work from design to writing and approving the final content of the manuscript.All authors accept full responsibility for the accuracy of the raw data and data analysis, and exercised authority in manuscript preparation and the decision to submit the manuscript for publication. KK designed and performed all animal experiments, analyzed the data, and wrote the manuscript. TT helped the study design, animal experiments, and kinetic analysis and critically reviewed the manuscript. YKat helped animal experiments, radio-metabolite analysis, and monoamine quantitation. TK assisted in conducting rotational behavior test and gave valuable input on biological aspects. RZ and KT contributed to the labeling synthesis of [


To further explore pulmonary activin A regulation, we examined AMs in bronchoalveolar lavage (BAL) from wild-type C57BL/6 compared to GM-CSF knockout mice which exhibit a PAP-like histopathology. Both human PAP and mouse GM-CSF knockout AMs are deficient in the transcription factor, peroxisome proliferator activated receptor gamma (PPARγ).Activin A is a pleiotrophic regulatory cytokine, the ablation of which is neonatal lethal. Healthy human alveolar macrophages (AMs) constitutively express activin A, but AMs of patients with pulmonary alveolar proteinosis (PAP) are deficient in activin A. PAP is an autoimmune lung disease characterized by neutralizing autoantibodies to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). Activin A can be stimulated, however, by GM-CSF treatment of AMs In vitro studies confirmed that IFNγ stimulated activin A in wild-type AMs while antibody to IFNγ reduced activin A in GM-CSF knockout AMs. Both IFNγ and Activin A were also reduced in GM-CSF knockout mice in vivo after intratracheal instillation of lentivirus-PPARγ compared to control lentivirus vector. Examination of other M1 markers in GM-CSF knockout mice indicated intrinsic elevation of the IFNγ-regulated gene, inducible Nitrogen Oxide Synthetase (iNOS), CCL5, and interleukin (IL)-6 compared to wild-type. The M2 markers, IL-10 and CCL2 were also intrinsically elevated.In sharp contrast to human PAP, activin A mRNA was elevated in mouse GM-CSF knockout AMs, and activin A protein was increased in BAL fluid. Investigation of potential causative factors for activin A upregulation revealed intrinsic overexpression of IFNγ, a potent inducer of the M1 macrophage phenotype, in GM-CSF knockout BAL cells. IFNγ mRNA was not elevated in PAP BAL cells. Data point to IFNγ as the primary upregulator of activin A in GM-CSF knockout mice which in addition, exhibit a unique mix of M1-M2 macrophage phenotypes. Activin A, a pleiotrophic cytokine belonging to the transforming growth factor-beta (TGF-β) superfamily, is synthesized by many cell types throughout the body ,2. The mMaintenance of lung homeostasis is a complex process dependent upon a network of interacting cells and cytokines. GM-CSF is required for alveolar macrophage (AM) function and pulmonary homeostasis . In genePreviously we reported that healthy human AMs synthesize activin A in response to GM-CSF but AMs of patients with PAP are deficient in activin A . In addiUnlike previous findings of activin A deficiency in human PAP , activinin vitro in either wild-type or GM-CSF knockout AMs (data not shown). Elevated IFNγ has been reported in lungs of GM-CSF knockout mice [Intrinsic factors that could potentially affect activin A levels were subsequently investigated in GM-CSF knockout mice. Macrophage colony-stimulating factor (M-CSF) has been reported to be upregulated in GM-CSF knockout mice . Examinaout mice  thereforIn contrast to results from GM-CSF knockout mice, examination of IFNγ expression in human BAL cells from PAP patients revealed no significant increase compared to healthy controls Figure .in vitro cultures of wild-type AMs indicated that IFNγ (100 U/ml) significantly (p < 0.05) increased activin A expression  or anti-IFNγ. ELISA analysis of conditioned media indicated that anti-IFNγ reduced activin A protein synthesis compared to irrelevant Ig  Policy on humane care and use of laboratory animals and were approved by the institutional animal care committee. The GM-CSF knockout mice were generated by Dr. Glenn Dranoff and have been previously described . Controlescribed . Recombiin vitro culture, BAL cells were plated into 24-well plates  or chamber slides  as previously described [The protocol was approved by the East Carolina University Institutional Review Board and written informed consent was obtained from all patients and control subjects. Healthy control subjects had no history of lung disease and were not on medication. PAP subjects were recruited from patients undergoing routine clinical evaluation. The diagnosis of idiopathic PAP was confirmed by histopathological examination of material from open lung or transbronchial biopsies as previously described . Alveolaescribed .Total RNA was extracted from BAL cells or cultured alveolar macrophages and analyzed by Q-PCR as previously described . RNA speThe self-inactivating lentivirus expression vector used here has been described previously . ConstruActivin A or follistatin proteins (pg/ml) in BAL fluids or conditioned media from cultured alveolar macrophages were quantified by ELISA according to the manufacturer’s instructions .Immunocytochemistry for IFNγ was carried out on cytospin samples from freshly isolated BAL cells using rat anti-mouse IFNγ  followed by goat anti-rat IgG (Invitrogen) as described . Slides Data were analyzed by student’s t-test using Prism software (GraphPad). Values from treated cells were compared to untreated. Significance was defined as p ≤ 0.05.AM: Alveolar macrophage; GM-CSF: Granulocyte macrophage colony stimulating factor; PAP: Pulmonary alveolar proteinosis; BAL: Bronchoalveolar lavage; PPARγ: Peroxisome proliferator activated receptor; IFNγ: Interferonγ; iNOS: Inducible nitric oxide synthetase.The authors declare that they have no competing interests.HD contributed to acquisition of the data, analysis and interpretation of data, drafting of the manuscript and final approval of the version to be published; BPB contributed to the design, analysis and interpretation of data, drafting of the manuscript and final approval of the version to be published; AM contributed to the conception and design, acquisition of the data, analysis and interpretation of data and final approval of the version to be published; AGM contributed to acquisition of the data and final approval of the version to be published; MSK contributed to the acquisition of data and final approval of the version to be published; MJT contributed to the conception and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript and final approval of the version to be published. All authors read and approved the final manuscript.


Primary graft failure (PGF) is a devastating complication that occurs in the immediate postoperative period following heart transplantation. It manifests as severe ventricular dysfunction of the donor graft and carries significant mortality and morbidity. In the last decade, advances in pharmacological treatment and mechanical circulatory support have improved the outlook for heart transplant recipients who develop this complication. Despite these advances in treatment, PGF is still the leading cause of death in the first 30 days after transplantation. In today's climate of significant organ shortages and growing waiting lists, transplant units worldwide have increasingly utilised “marginal donors” to try and bridge the gap between “supply and demand.” One of the costs of this strategy has been an increased incidence of PGF. As the threat of PGF increases, the challenges of predicting and preventing its occurrence, as well as the identification of more effective treatment modalities, are vital areas of active research and development. Heart transplantation is an effective method of treatment for end-stage heart failure, with more than 5,000 transplants being conducted each year in over 300 countries . The surThe reported incidence of PGF after heart transplantation varies widely between studies with estimates ranging between 2.3 and 26% . Most ofThe changing demographics of donors and recipients observed in cardiac transplantation over the last two decades appear to be contributing to an increase in the incidence of PGF , 11, 14.PGF is a syndrome in which the transplanted heart fails to meet the circulatory requirements of the recipient in the immediate post-transplant period as a consequence of either single or biventricular dysfunction. It is manifested as hypotension and low cardiac output in the presence of adequate filling pressures . In mostA universally accepted clinical definition for PGF has been lacking and is urgently needed. Several authors have suggested minimal diagnostic criteria , 9, whic The severity of PGF can be graded according to the level of support needed to restore haemodynamic stability. In less severe cases, intravenous inotropic support with two or more agents may be sufficient to achieve this, whereas in more severe cases mechanical circulatory assistance , or any ventricular assist device) is required. A three-level grading system based on the severity of primary graft dysfunction has been developed for lung transplantation and shown to be strongly predictive of one-month mortality , 19. It Acute ischaemia-reperfusion injury with myocardial stunning has been postulated as a predominant factor in the development of PGF. The donor heart is subjected to a series of insults during the transplant process including brain death and its sequelae, hypothermic storage, warm ischaemia, and finally reperfusion. Donor hearts vary in their ability to withstand these insults. It is clear, for example, that the hearts from older donors have an increased susceptibility to PGF , 16 whicBrain death in the donor is associated with a series of events that result in impaired myocardial contractility. These events include the rapid release of catecholamines immediately after brain death contributing to myocardial ischaemia, calcium overload, calpain activation, and changes in the calcium sensitivity of contractile proteins , 22. TheMost donor hearts are stored in a cold preservation solution and transported on ice. Hypothermic storage slows but does not completely arrest cellular metabolism. Consequently, progressive ischaemic injury is an inevitable consequence of prolonged static storage. In addition, loss of normal aerobic metabolism paralyses the transmembrane Na+/K+ ATPase pump leading to cellular swelling and the switch to anaerobic metabolism during cold storage results in a rapid decline in high-energy phosphates and the development of lactic acidosis . FinallyPrimary graft failure may also occur in circumstances where the donor heart has not been subjected to substantial ischaemia-reperfusion injury. Under these circumstances, recipient factors are the principal cause of PGF. There are two clinical scenarios where this is likely to occur. The first is the presence of a fixed high pulmonary vascular resistance in the recipient –31. In tIn most instances, it is likely that the combination of donor, procedural and recipient factors leads to the syndrome of PGF. For example, an older donor heart that has been subjected to a prolonged ischaemic time may fail in a recipient with an elevated pulmonary vascular resistance whereas a younger donor heart may not. On the other hand, the same older donor heart may function adequately in a haemodynamically stable recipient with low pulmonary vascular resistance. Hence, matching donors to recipients with regard to risk factors for PGF are critical to minimising the risk of this life-threatening complication.Given the significant contribution of PGF to early mortality after cardiac transplantation, identification of predictive factors is important. Multiple risk factors for PGF have been identified by different authors. They can be divided into those that are donor related, those that are recipient related and those related to the transplant procedure. As shown in Another potent risk factor for PGF identified in multiple studies is donor heart ischaemic time, referring to the period from the arrest of the donor heart to time of graft reperfusion in the recipient. It is apparent from the ISHLT Registry that one-year mortality risk after heart transplantation increases steadily with every minute of ischaemic time in excess of 3 hours . MarascoThese data indicate that the current techniques used to preserve the donor heart during procurement and transport have limited efficacy. Unfortunately, prolonged ischaemic times in heart transplantation are sometimes logistically unavoidable. There is a clear need to develop more effective preservation strategies—either by bolstering the cardioprotective efficacy of the storage solution or through use of oxygenated ex vivo perfusion systems. Counter-intuitively, Russo et al. reported an increased risk of PGF with ischaemic time of less than 1 hour, citing the potential limited cooling period being insufficient to achieve the benefits of cellular protection with global hypothermia . Several authors have reported that donor heart dysfunction as evidenced by a low left ventricular ejection fraction on echocardiography, unstable donor haemodynamics, or the need for high doses of catecholamines is a potent risk factor for PGF , 6, 11. Donor-recipient size mismatch has also been identified as a significant contributing factor in the development of PGF. In one study, the combination of a donor-recipient weight ratio of less than 0.8 with pulmonary hypertension in the recipient (>4 wood units) was associated with PGF . SeveralThe concurrent donation of other organs may also have a role in PGF, specifically the donation of lungs . The proThe presence of ventilator or ECMO support in the recipient prior to and at the time of transplantation has been shown to be a significant risk factor for PGF , 5. ThesRisk factors do not act in isolation, and it is likely that the interaction between donor, recipient, and procedural factors is a major determinant of the risk of PGF. A clear example of this is the interaction between donor age and ischaemic time reported by Russo et al. . In thatGiven that multiple factors in the donor, the transplantation process and the recipient contribute to the risk of PGF, the development of a predictive tool, and scoring system that combines known risk factors has been reported , 40. SugThe treatment of PGF remains extremely challenging—a substantial 30-day mortality rate is seen despite intensive pharmacological as well as mechanical circulatory support  used in this critical period , 2. In mWith more severe cases of graft failure, mechanical circulatory support with intra-aortic counterpulsation or VA extracorporeal mechanical support (ECMO) may be needed to maintain haemodynamic support and perfusion of vital organs. In our institution, the decision to institute ECMO has been made early, that is, in the operating room when there has been difficulty with separating from cardiopulmonary bypass despite a trial of inotropic/vasopressor support . We beliHeart transplant recipients with PGF remain supported on ECMO until graft function improves. In our experience, this has generally been within 72 hours; however, heart recovery has been observed as early as 1 day and as late as 7 days after transplant . AssessmIn cases with pre-existing recipient pulmonary hypertension, PGF is usually manifested as right ventricular dysfunction in the immediate post-transplant period. Treatment includes administration of specific pulmonary vasodilators such as inhaled nitric oxide to lower pulmonary vascular resistance  however,PGF is the leading cause of death in the first month after heart transplantation. Although registry studies indicate that the number of early deaths due to PGF has not changed over the last two decades , 2, thisThe impact of PGF beyond the first month after transplantation is less clear, but also likely to be significant. Severe ischaemia-reperfusion injury has been shown experimentally to upregulate multiple proinflammatory mediators which may prime the graft for acute rejection , 26 and Given the cumulative impact of the multiple risk factors that contribute to the development of PGF, careful matching between donor and recipient is critical to minimising the risk of PGF. Unfortunately, the logistics of transplantation sometimes dictate that unfavourable risk factor interactions cannot be avoided. While some risk factors  are not modifiable, other risk factors  may be amenable to therapeutic intervention. The period between brain death and heart retrieval is one in which heart function can deteriorate rapidly. Optimal management of the brain dead donor during this period remains a contentious issue. More than 90% of brain dead donors receive one or more inotropic or vasopressor infusions most commonly noradrenaline . While lThe period of heart storage and transport is the second period that offers an opportunity to intervene. Currently most hearts are stored and transported in cold cardioplegic/preservation solutions. The many commercial and in-house cardioplegic/preservation solutions in routine clinical use not only emphasise the complexity of the molecular and cellular mechanisms that underlie ischaemia-reperfusion injury but also the lack of consensus as to the optimal strategy for organ preservation . CardiopATP channel, a key target of prosurvival signalling pathways [Elucidation of the mechanisms of ischaemia-reperfusion injury over this same period of time has suggested novel strategies to enhance the cardioprotective capacities of existing preservation solutions. The search for an over-arching protective strategy against cardiac reperfusion injury has been advanced by the realisation that ischemic pre- and postconditioning as well as a number of pharmacological agents that mimic these physiological strategies can activate prosurvival signalling pathways such as PI3K/Akt, ERK 1/2 and STAT3 at reperfusion . Consispathways . These fpathways . In addiAn alternative to cold static storage is ex vivo perfusion. There is limited experience with this approach in heart transplantation ; howeverIn summary, the increasing reliance on “marginal” donors to meet the ever-increasing demand for heart transplantation means that PGF is likely to remain a frequent complication. Although there have been significant improvements in the treatment of established PGF, it still carries a high morbidity and mortality. While it is possible that some cases of PGF may be prevented by careful matching of donors and recipients, complete prevention of PGF will require the development of more effective donor management and donor heart preservation strategies. These remain high-priority areas for ongoing basic and clinical research.


Granulocyte colony-stimulating factor (G-CSF) has exhibited efficacy at preventing the progression of pulmonary hypertension (PH); however, the exact mechanism is not completely clear. The aim of the present study was to assess whether this protective effect was mediated by the upregulation of circulating endothelial progenitor cells (EPCs) via the nitric oxide (NO) system. PH was induced in male Sprague-Dawley (SD) rats by the administration of a single subcutaneous injection of monocrotaline (MCT). The rats were treated with recombinant human G-CSF  by subcutaneous injection from day five to day seven subsequent to the injection of MCT. Nω-nitro-L-arginine methyl ester  was intragastrically administered in addition to rhG-CSF as a negative intervention. The changes in hemodynamics and histology, the number and function of circulating EPCs and the concentration of plasma NO were evaluated. With the occurrence of PH in the rat model, the number and function of circulating EPCs were demonstrated to be markedly downregulated. Moreover, a reduced plasma concentration of NO was observed, which was positively correlated with the number of circulating EPCs. Administration of rhG-CSF elevated the plasma level of NO, upregulated the number and function of circulating EPCs and effectively improved pulmonary hemodynamics and vascular reconstruction. Furthermore, the positive correlation between the levels of plasma NO and circulating EPCs was also observed in the rhG-CSF treatment group. However, the protective effect of rhG-CSF in PH was attenuated by L-NAME, which mediated the downregulation of NO and the EPCs. Thus, the present study suggests that G-CSF may attenuate the progression of MCT-induced PH by improving vascular injury repair mechanisms via the NO-mediated upregulation of EPCs. The term pulmonary hypertension (PH) describes a group of lung disorders characterized by a progressive increase in pulmonary arterial pressure. The common pathological feature of PH is pulmonary vascular remodeling. Persistent high blood pressure in the pulmonary circulation results in right ventricular hypertrophy and dilatation, leading to the eventual occurrence of right heart failure. Along with the advancement in studies on PH in recent years, the importance of endothelial progenitor cells (EPCs) has been recognized among the possible factors involved in the mechanism of PH. EPCs have been considered to be pivotal in maintaining the normal structure and function of the vascular endothelium –3, and tEPC transplantation has been shown to be effective at preventing the progression of PH in laboratory rats ,7. HowevIn the present study, we utilized a rat model of PH, created by the subcutaneous injection of monocrotaline (MCT), and treated the rats with recombinant human G-CSF (rhG-CSF). Nω-nitro-L-arginine methyl ester (L-NAME) was used concurrently as a negative intervention. The therapeutic effect, number and function of circulating EPCs and the concentration of plasma NO were evaluated, in order to enhance the understanding of the protective mechanisms of rhG-CSF in PH.Male Sprague-Dawley (SD) rats, aged 8 weeks, were purchased from Capital Medical University  and housed in specific pathogen-free units of the Division of Laboratory Animals at Capital Medical University. Thirty-two rats were randomly divided into four groups: the model [MCT and phosphate-buffered saline (PBS)], rhG-CSF treatment (MCT and rhG-CSF), L-NAME intervention  and control (PBS) groups. Each group contained eight rats. PH was induced by a single subcutaneous injection of MCT  , while PAt day 21, the rats were anesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg). A polyethylene catheter was inserted into the right ventricle (RV) via the right external jugular vein, and another was targeted at the ascending aorta via the right carotid artery. Right ventricular systolic pressure (RVSP) and mean aortic pressure (MAoP) were recorded using a polygraph .Peripheral blood was collected from the right external jugular vein into EDTA-containing tubes and 100 μl was incubated with 2 μl fluorescein isothiocyanate (FITC)-conjugated goat monoclonal antibody against mouse immunoglobulin G, and 5 μl mouse monoclonal antibody against rat vascular endothelial growth factor receptor (VEGFR)-2 . Following red cell lysis and washing with PBS, the cells were incubated for a further 20 min with 5 μl allophycocyanin (APC)-conjugated mouse monoclonal antibody against rat CD45  and 5 μl phycoerythrin (PE)-conjugated mouse monoclonal antibody against rat CD34 (BD Biosciences). The samples were subsequently centrifuged for 5 min at 300 × g and then resuspended in 500 μl PBS and evaluated using flow cytometry . Isotype controls were run in parallel and ~100,000 events were recorded. Circulating EPCs were defined as cells positive for CD34 and VEGFR-2, but negative for CD45 .Peripheral blood was centrifuged for 15 min at 1,200 × g, prior to 50 μl plasma being collected into 96-well plates for the measurement of NO. The Nitric Oxide Assay kit  was based on Greiss reagent. The concentration of NO was determined by spectrophotometry (490 nm), following the addition of 50 μl Greiss reagents I (1% sulfanilamide in 0.1 mol/l HCl) and II [0.1% N-(1-naphthyl-ethylenediamine dihydrochloride)].2). Unattached cells were removed by extensive washing on day three, and the culture medium was replaced every two days thereafter. Following seven days of culture, cells were incubated with 10 μg/ml DiI-labeled acetylated low-density lipoprotein . Four hours later, the cells were incubated with 10 μg/ml FITC-conjugated lectin from Ulex europeus agglutinin-1  for 1 h, following fixation with 4% paraformaldehyde. The cells were subsequently examined using laser scanning confocal microscopy. Differentiating EPCs were identified by double fluorescence staining, as previously described  and suspended with endothelial cell growth medium-2  in 24-well culture plates pre-coated with fibronectin (Sigma), and incubated at 37°C in a humidified environment with 5% carbon dioxide (COescribed .5 cells/ml (200 μl per well), prior to 20 μl MTT (5 g/l) being added for 4 h. Following this, the media was discarded and 100 μl dimethylsulfoxide (DMSO) was added for 10 min. The absorbance was measured at a wavelength of 490 nm. For adhesion, EPCs were incubated with EGM-2 in 24-well plates pre-coated with fibronectin (5×104/well) for 30 min. Subsequent to washing three times with PBS, the attached cells were counted in a high power field (HPF). To assess the migratory ability of the cells, a modified Boyden chamber (8-μm pore size) was used. EPCs were suspended in 100 μl EGM-2 without cytokines, plus 0.5% fetal bovine serum (FBS), in the upper chamber (5×105/ml) and placed in a 24-well culture plate containing 600 μl EGM-2. Following 24 h of incubation, the lower membrane of the chamber was fixed with 4% paraformaldehyde. Migrated cells were counted in a HPF, subsequent to staining with 0.1% crystal violet.EPCs were detached using 0.25% trypsin following seven days of culture. EPC functions, such as proliferation, adhesion and migration, were assessed as described in a previous study . With reLung tissues were removed from the rats subsequent to sacrifice by decapitaiton and fixed in 10% paraformaldehyde for 24 h. Following this, serial paraffin sections (5-μm) were stained with hematoxylin and eosin for light microscopy . The medial wall thickness of the pulmonary arteriole is expressed as: Wall thickness  = [/external diameter] × 100 .Data are presented as the mean ± standard deviation, and were statistically analyzed using SPSS statistical software . Differences were compared using one-way analysis of variance (ANOVA) tests. Correlations were calculated according to Pearson’s correlation. P<0.05 was considered to indicate a statistically significant difference.Approximately 10 days following the co-administration of MCT and PBS, rats exhibited shortness of breath and a reduction in locomotor activity and food intake compared with controls. In addition, the body weight of the model rats was shown to have decreased . The administration of rhG-CSF resulted in a marked improvement in the status of the rats, and there were no differences in body weight between the rhG-CSF treatment group (203.7±19.7 g) and the controls (P>0.05), while only the level of locomotor activity remained slightly decreased. However, under the negative intervention of L-NAME, the body weight of the rats (170.5±18.2 g) was markedly decreased compared with that of the rhG-CSF treatment group (P<0.05).Twenty-one days following the injection of MCT, the RVSP of the rats in the model group was increased compared with that of the controls , while the MAoP was decreased from 120.33±18.25 mmHg in the control group to 97.24±17.52 mmHg in the model group. This indicated the occurrence of PH. The administration of rhG-CSF led to the RVSP of the rats being decreased significantly compared with that of the model group . No differences were detected between the rhG-CSF treatment group and the controls (P>0.05). The MAoP of the rats in the rhG-CSF treatment group (113.82±21.73 mmHg) was increased compared with that of the model group (P<0.05), and was restored to the level of the controls P>0.05; .Histological examination indicated that medial hypertrophy of the pulmonary arteriole smooth muscle was evident in the model group  and thatThe protective effects of rhG-CSF on the MCT-induced rat model of PH were markedly attenuated by the negative intervention of L-NAME. The RVSP of rats in the L-NAME intervention group (51.19±2.93 mmHg) was increased significantly compared with that of the rhG-CSF treatment group (P<0.01), while the MAoP was decreased . The administration of rhG-CSF was shown to notably increase the number of EPCs (0.042±0.013%) compared with the numbers in the model and control groups (P<0.01). The intervention of L-NAME attenuated the effect of rhG-CSF on the circulating EPCs  than in the control group . The administration of rhG-CSF was shown to significantly increase the plasma concentration of NO compared with that in the model group , and no differences in NO level were observed between the rhG-CSF treatment and control groups (P>0.05). The administration of L-NAME, which is an inhibitor NO synthase, suppressed the upregulation of plasma NO mediated by rhG-CSF . FurtherFollowing seven days of culture, abundant spindle-like cells were observed to be adhered to the bottom of the culture plate . Subsequin vitro, the absorbance  of the formazan supernatant (dissolved in DMSO) was 0.49±0.04 in the model group, which was decreased compared with that of the control group . Similar results were observed with regard to adhesion ability  and migratory ability . In the rhG-CSF treatment group, these functional indices were all upregulated compared with those of the model group (proliferation, OD 0.63±0.06; adhesion, 12.35±1.82 cells/HPF; migration, 12.97±2.84 cells/HPF; P<0.01; The functions of the EPCs were all downregulated in the model group compared with those of the controls. In the test of proliferation ability In the present study, it was demonstrated that in a rat model of PH, the number and function of circulating EPCs were markedly decreased. Moreover, there was a downregulation in the plasma concentration of NO, which was positively correlated with the number of circulating EPCs. Administration of rhG-CSF elevated the plasma level of NO, upregulated the number and function of circulating EPCs and effectively improved pulmonary hemodynamics and vascular reconstruction. Furthermore, the positive correlation between the concentration of plasma NO and circulating EPCs was also observed in the rhG-CSF treatment group. However, the protective effects of rhG-CSF on PH were impaired by the L-NAME-mediated downregulation of NO and the EPCs. These results indicate that rhG-CSF prevents or attenuates the progression of MCT-induced PH by improving vascular injury repair mechanisms via the NO-mediated upregulation of EPCs.PH is characterized by the persistent contraction and medial hypertrophy of extensive pulmonary arterioles, which may thus result in the abnormal elevation of pulmonary artery pressure. Despite the fact that drug interference may improve the symptoms and decrease the occurrence of heart attack, the effects are partial and limited and right ventricular failure or mortality is likely be the fate of numerous patients in the clinic. It is therefore crucial to prevent the progression of the pathological changes in the initial stages of this disease. Studies have shown that the early pathological change in PH is the injury of the arteriolar endothelium, resulting from excessive apoptosis of the endothelial cells. This then induces vasodilatation dysfunction, the over-proliferation of vascular smooth muscle cells and fibroblasts, ultimately resulting in the occurrence of PH –21. Thusin vitro, the functions of EPCs, such as proliferation, adhesion and migration, were downregulated. It is possible that the repair processes secondary to the ongoing lesion of the vascular endothelium may lead to the large consumption of circulating EPCs and potentially exhaustion. Bone marrow EPCs and precursors, as the reservoir of circulating EPCs, may also be impaired, in number and function (EPCs, which are co-precursors with hematopoietic stem cells, have been suggested to be pivotal to the homeostasis and repair of the vascular endothelium. Studies have shown that the number of circulating EPCs was decreased in patients with PH ,6 and thfunction ,25. A reData have shown that G-CSF is able to increase the number of circulating EPCs, upregulate the maturation and proliferation capacity of EPCs  and thenet al(et al, which suggests that the same signaling pathway may be involved. Furthermore, the administration of L-NAME, which acted as an NOS inhibitor in the present study, was accompanied by reductions in the plasma concentration of NO and in the number and function of circulating EPCs, thus attenuating the protective effect of rhG-CSF in PH. This further demonstrated the protective effects of the NO-mediated upregulation of EPCs in PH.To further investigate the possible mechanism underlying the protective effect of G-CSF in PH, we measured the plasma concentration of NO in each group. It was shown that the level of plasma NO decreased significantly in the rat model of PH compared with the controls, and that the administration of rhG-CSF effectively induced the upregulation of NO and accelerated the repair of the injured pulmonary artery endothelium by mobilizing the bone marrow EPCs , while ret al demonstrin vivo studies.In conclusion, the current study indicated that the administration of rhG-CSF may represent a novel strategy for the treatment of PH. The treatment may effectively prevent the disorder by upregulating the number and function of circulating EPCs via the NO system, and then accelerate the reparation of the pulmonary artery endothelial lesion. However, the prospective efficacy and side-effects of rhG-CSF remain important issues to be investigated in advanced experimental and


This review examines the use of transporter knockout (KO) animals to evaluate transporter function at the choroid plexus . Compared to the blood-brain barrier, there have been few such studies on choroid plexus (CP) function. These have primarily focused on Pept2 (an oligopeptide transporter), ATP-binding cassette (ABC) transporters, Oat3 (an organic anion transporter), Svct2 (an ascorbic acid transporter), transthyretin, ion transporters, and ion and water channels. This review focuses on the knowledge gained from such studies, both with respect to specific transporters and in general to the role of the CP and its impact on brain parenchyma. It also discusses the pros and cons of using KO animals in such studies and the technical approaches that can be used. Choroid plexus (CP) epithelial cells and their linking tight junctions form the blood-CSF barrier  and are the primary site of CSF secretion. As with the cerebral endothelium, the site of the blood-brain barrier (BBB), the CP epithelium possesses a wide variety of transporters -4. CP trIt is important to identify which transporters are present at the CP and their role . However, the latter is complicated by the array of transporters present, overlapping substrate specificity and often a lack of specific inhibitors. One approach that has proven useful in transporter studies is the use of animals with gene deletions (e.g. KO mice) or gene mutations . This approach has been used extensively in BBB studies to examine the role of different ATP-binding cassette (ABC) transporters in drug efflux e.g. -8). Mult. Mult8])Multiple transporter KO mice have been generated. For example, there have been BBB transport studies on KO mice for Abcb1 , Abcg2 in vitro using isolated CP, with limited studies on CP epithelial cell cultures because of the large number of mice required. In vitro studies have the advantage of enabling CP study without impact from other tissues . In vivo, most investigations have looked at CP transport and CSF entry after intravenous administration of compounds. Very few have used intracerebroventricular (icv) administration to study the effects of gene deletion on clearance from CSF.In contrast, the effect of transporter KO on CP has been the subject of far fewer studies and these are reviewed below. They provide insight into the types of CP experiments that can be performed, and their limitations, as well as information on the function of particular CP transporters. Broadly, the effects of gene deletion on transport has been mostly assessed The impact of the transporter KOs in relation to CP function is described in the sections below. Figure Pept2 (SLC15a2) is a member of the proton-coupled oligopeptide transporter (POT) family which also includes Pept1, and two proton-coupled histidine transporters  ,18. All -/-) [-/- mice [14C]GlySar uptake was reduced by ~90% compared to wild-type Pept2+/+ animals GlySar into periventricular brain in adult mice. These studies are complicated by the presence of Pept2 in some neurons but did show that there is a rapid drop-off in GlySar (MW = 146) concentration with distance from the CSF system T4 entry into the brain in TTR-null mice. They also found that cortex, cerebellum and hippocampus T4 levels were similar in the TTR-null and WT mice. However, CP T4 levels were very markedly lower in null mice (14% of WT). Thus, TTR effects on T4 are probably limited to the CP-CSF system and areas adjacent to that system. Interestingly, Richardson et al. GlySar and autoradiography [14C]GlySar levels with distance from the ependyma in both Pept2-/- and WT mice with concentrations dropping by 50% over 0.53 and 0.34 mm, respectively. Thus, the impact of changes in CSF concentrations resulting from a loss of a CP transporter was likely to be limited to the periventricular zone. Milhorat et al. [We have examined the effect of Pept2 KO on cerebral cortex penetration of GlySar, cefadroxil, carnosine and 5-ALA after intravenous administration. While the accumulation of these drugs in CSF is markedly increased  to mimic drug administration. The effects of CP transporter KO on brain parencyhma may be greater for compounds normally found in the body. Thus, as noted above, Svct2 KO mice have very low brain vitamin C levels  that is Some of the potential difficulties in using whole animal KO animals to assess the effects of CP transport on brain parenchyma are exemplified by the effects of such KOs for the ABC transporters. Mrp4 is present at the CP and the BBB and in both tissues it is involved in transporting substrates back to blood ,42 makinIn general, the extent of the impact of a CP transporter knockout on the parenchyma (and periventricular zone in particular) will depend upon multiple variables. It will depend upon the magnitude of the KO effect on substrate CSF levels, the clearance via CSF flow, the rate of penetration into parenchyma across the ependymal, trapping or exclusion of the substrate from parenchymal cells, and the rate of diffusion and/or transport across the BBB. Compensatory transporters (in the absence or presence of upregulation) will also serve to confound this issue.Transporter KO animals are a useful tool in elucidating the functional role of CP transporters. This is particularly the case as we gain more understanding of the overlapping substrate specificities of different transporters and the possible effects of inhibitors on multiple transport systems. As yet, only the effects of whole animal transporter knockouts on CP transport have been examined. Under normal conditions the transporter KOs examined had no obvious phenotype associated with the CP transporter deletion, with the exception of Svct2. However, such deletions can have marked effects on the CSF levels of substrates, drug efficacy and substrate-induced neurotoxicity. The effects of CP transporter KOs on brain parenchyma substrate delivery are variable with the greatest effects on the periventricular zone rather than the deep brain structures.4: thyroxine; WT: wild-type.ABC: ATP-binding cassette; 5-ALA: 5-aminolevulinic acid; BBB: blood-brain barrier; BCSFB: blood-CSF barrier; Bcrp: breast cancer related protein; CP: choroid plexus; CSF: cerebrospinal fluid; FRα: folate receptor alpha; Rfc1: reduced folate carrier 1; GlySar: glycylsarcosine; KO: knockout; Mdr: multidrug resistance protein; Mrp: multidrug resistance associated protein; POT: proton-coupled oligopeptide transporter; siRNA: small interfering RNA; TThe authors declare that they have no competing interests.RFK and DES co-wrote this manuscript. All authors read and approved the final manuscript.RFK is Professor of Neurosurgery and Molecular & Integrative Physiology at the University of Michigan. DES is Professor of Pharmaceutical Sciences.


In an ensemble exposed to these laser beams, the donor excitation is modulated at f1, and the acceptor excitation, and therefore the degree of saturation of the excited electronic state of the acceptors, is modulated at f2. Since the ensemble contains donor acceptor pairs engaged in FRET, the released donor fluorescence is modulated not only at f1 but also at the beat frequency Δf: = |f1 − f2|. The depth of the latter modulation, detectable via a lock-in amplifier, quantitatively indicates the FRET efficiency.The process of radiationless energy transfer from a chromophore in an excited electronic state (the “donor”) to another chromophore (an “acceptor”), in which the energy released by the donor effects an electronic transition, is known as “Förster Resonance Energy Transfer” (FRET). The rate of energy transfer is dependent on the sixth power of the distance between donor and acceptor. Determining FRET efficiencies is tantamount to measuring distances between molecules. A new method is proposed for determining FRET efficiencies rapidly, quantitatively, and non-destructively on ensembles containing donor acceptor pairs: at wavelengths suitable for mutually exclusive excitations of donors and acceptors, two laser beams are intensity-modulated in rectangular patterns at duty cycle  In 1923, based on theoretical considerations by Klein and Rosseland   measurements  at confocal spatial resolution for quantitative measurements, since FRET-based [Ca2+] indicators are now available  measurements employing the “Cameleon” dye, with which CFP and YFP can be excited selectively by specific Ti-Sap laser wavelengths [Using one or two phase modulating EOMs, a beat situation could be simulated. The phase modulation would accelerate or retard one or both of the laser beams. Thus, a beat can be simulated by electronic means, provided two titanium sapphire lasers are available and the two chromophores in the respective donor acceptor pair can be excited selectively, each chromophore by one of the beams. The method then could for example, be used for semi-quantitative or even quantitative [Caelengths ,13.The system can be extended to include appropriate numbers of laser lines, EOMs, PMTs, and LIAs, in order to quantitatively determine competing FRET processes in one sample. In this case, several different donors interact with one acceptor or one donor interacts with several different acceptors. It is essential that there be available illumination and detection wavelength bands for the involved chromophores, which must not cause too much mutual cross-excitation or cross-channel detection. In a situation where for example, there would be one donor interacting with three different acceptors, one laser line and EOM would be needed for the donor and one laser line and EOM would be needed for each acceptor, in addition to one LIA for each acceptor and one LIA for each FRET process, requiring altogether four laser lines, four EOMs, three PMTs, and seven LIAs.i.e., triangular co-localization situations.While it is not a problem to understand that the method can be used in situations, where, for example, each of the donor molecules interacts with an acceptor of either the first, second, or third type, the method would even allow determination of how much FRETing would occur between the donor and several acceptors if, in an ensemble, a large fraction or even all of the donor molecules could enter FRET processes with two acceptors, at which the donors would be in FRET range distance to acceptors molecules of both types at the same time, The outline of a new, non-destructive method for the quantitative and fast measurement of FRET processes has been presented. Experiments will have to prove whether the method will be useful in praxi. The method is independent of any specific method of observation and may be applied to imaging as well as purely photometric methods. The method promises to be a useful tool, especially in high-resolution light microscopy.


Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology remains unknown in a large proportion of cases. We identified and characterized the full genome of a novel cyclovirus (tentatively named cyclovirus-Vietnam [CyCV-VN]) in cerebrospinal fluid (CSF) specimens of two Vietnamese patients with CNS infections of unknown etiology. CyCV-VN was subsequently detected in 4% of 642 CSF specimens from Vietnamese patients with suspected CNS infections and none of 122 CSFs from patients with noninfectious neurological disorders. Detection rates were similar in patients with CNS infections of unknown etiology and those in whom other pathogens were detected. A similar detection rate in feces from healthy children suggested food-borne or orofecal transmission routes, while high detection rates in feces from pigs and poultry  suggested the existence of animal reservoirs for such transmission. Further research is needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus. Acute central nervous system (CNS) infections cause substantial morbidity and mortality, but the etiology frequently remains unknown, which hampers development of therapeutic or preventive strategies. Hence, identification of novel pathogens is essential and is facilitated by current next-generation sequencing-based methods. Using such technology, we identified and characterized the full genome of a novel cyclovirus in cerebrospinal fluid (CSF) specimens from two Vietnamese patients with CNS infections of unknown etiology, which was subsequently detected in none of 122 CSF specimens from patients with noninfectious neurological disorders but 4% of 642 CSF specimens from Vietnamese patients with suspected or confirmed CNS infections. Similar detection rates in feces from healthy children suggested food-borne or orofecal transmission routes, while frequent detection in feces from Vietnamese pigs and poultry  suggested the existence of animal reservoirs for such transmission. Further studies are needed to address the epidemiology and pathogenicity of this novel, potentially zoonotic virus. Acute central nervous system (CNS) infections cause substantial morbidity and mortality in children and adults, especially in tropical regions. While the causative microorganisms include a wide variety of bacterial, parasitic, fungal, and particularly viral pathogens, the majority of cases remain undiagnosed, despite extensive diagnostic efforts . The ideCyclovirus in the family Circoviridae. In addition, we report the results of initial studies to explore the pathogenic potential of this virus in humans as well as the existence of possible animal reservoirs for zoonotic transmission.Using a combination of an amplified fragment length polymorphism (AFLP)-based virus discovery assay (VIDISCA) and next-generation sequencing , we set Escherichia coli-like sequences suspected to be derived from VIDISCA-454 (454: Roche 454 Genome Sequencer FLX System) reagents (data not shown). Of these, a 190-bp sequence was identified in a pool consisting of 5 CSF samples that showed 82% identity to a replication-associated gene (Rep) of cycloviruses (CyCV). To confirm this VIDISCA-454 read, a specific PCR  CSF specimens generated 321,000 reads, which were reduced to 161,000 sequence reads for further analysis after excluding Circovirus and Cyclovirus in the family Circoviridae with the typical feature of two open reading frames (ORFs) encoding two major proteins, the replication-associated (Rep) and capsid (Cap) proteins, arranged in opposite directions  . Ana. AnaCyclTwenty-nine cyclovirus species have been proposed thus far –5, 7. Wh–Streptococcus pneumoniae, Haemophilus influenzae, or Streptococcus suis serotype 2) and 5/121 (4.1%) patients with laboratory-confirmed or clinically suspected tuberculous meningitis (CT) values was not different between specimens of unknown etiology and those diagnosed with other pathogens (data not shown). Details on characteristics and clinical outcome of CyCV-VN patients are presented in To study the prevalence of CyCV-VN in CSF, we first determined the frequency of viral DNA detection by CyCV-VN-specific real-time PCR in 642 randomly selected CSF samples from patients with suspected acute CNS infections enrolled in three descriptive studies in southern and central Vietnam from which the acute-infection CSF samples were selected for our virus detection experiments. We detected CyCV-VN in 26 of 642 (4%) acute-infection CSF specimens, including 10 of 273 (3.7%) CSF specimens from patients with CNS infections of unknown etiology and 16 of 369 (4.3%) from patients in whom laboratory-confirmed CNS infection with other pathogens was established. The latter included 4/140 (2.9%) patients with confirmed viral encephalitis , 7/108 (6.5%) patients with bacterial meningitis  fecal specimens from healthy children.Previous reports have shown the detection of diverse cycloviruses in farm animal meats . The exiIn order to explore the genetic relationship between the cycloviruses detected in human and animal specimens, we obtained additional 6 full-genome sequences (4 from human CSF and 2 from chicken and pig feces), 1 nearly full-genome sequence from a duck fecal sample, and 19 partial Rep gene sequences . Pairwise comparison of the obtained sequences revealed that the obtained human and animal cyclovirus sequences belong to the same CyCV-VN species, as reflected by a degree of sequence similarity of >97% between the detected strains (data not shown). This low intraspecies sequence divergence of the CyCV-VN is consistent with that of other cyclovirus species –5. The r–Cyclovirus of the family Circoviridae that we tentatively named CyCV-VN.The availability of highly sensitive molecular methods such as PCR and sequence-independent techniques based on next-generation sequencing technology allows enhanced detection of (novel and unknown) pathogens in clinical specimens, which is essential to guide prevention strategies, aid clinical management, and identify the sources of outbreaks . Using aCircoviridae is a family of nonenveloped viruses containing a circular single-stranded DNA genome and is subdivided into two genera, Circovirus and Gyrovirus, both of which have been associated only with disease in animals, including birds and pigs. Avian circoviruses have been associated with beak abnormalities, lethargy, and anorexia in pigeons and production losses and mortality in geese . However, it should be noted that 88% of 122 CSF samples from patients without CNS infections were collected over a short period of time (August to October 2011), possibly introducing bias.After initial discovery and characterization, we detected CyCV-VN by PCR in a total of 12 CSFs from 273 patients with acute encephalitis of unknown etiology. We also tested the CSF from patients with other laboratory-confirmed CNS infections and found a detection rate similar to that in patients with unknown etiology . In contrast, CyCV-VN DNA was not detected in 122 CSF specimens from patients with noninfectious CNS conditions , which is asymptomatically present in blood of 7 to 78% of people, with occasional detection in CSF –21.–CyCV-VN DNA was also detected in throat and rectal swabs of CSF-positive patients as well as in fecal specimens of healthy children. This suggests a possible food-borne or fecal-oral transmission route for infection, although detection in feces of healthy children may also merely reflect consumption of CyCV-VN-infected food rather than true infection.To explore the existence of possible animal reservoirs of CyCV-VN for such transmission, we tested fecal samples from animals in Dong Thap Province, where the index patient lived, and found remarkably high detection rates in fecal samples collected from pigs, ducks, and chickens, ranging from 42 to 100%. Sequence identities between CyCV-VN strains in humans and animals were high (around 98%)  (n = 291) (unpublished data), and a study (01SS) of children and adults with suspected CNS infections admitted to 13 provincial hospitals in central and southern Vietnam between 2008 and 2010   on children with presumed viral encephalitis admitted to Children’s Hospital Number One in Ho Chi Minh City in 2004 (n = 194) ; a study= 1,241) .Acute-infection CSF specimens were collected, divided into aliquots, and stored at −80°C until analysis. In addition, acute-infection blood specimens and rectal and throat swabs were collected from children participating in the PVE study. Detailed demographic and clinical data, including routine blood and CSF hematology and chemistry laboratory investigations, were collected on case record forms at enrollment and during follow-up.Streptococcus pneumoniae, Streptococcus suis serotype 2, Haemophilus influenzae, and Neisseria meningitidis. Additional diagnostic tests for flaviviruses (generic), influenza A virus, and cytomegalovirus were performed in the PVE and BMD studies. Parechoviruses and Semliki Forest virus were also tested for in the PVE study  were selected.From these case series, CSF specimens with unknown etiology were selected for VIDISCA-454 analysis, for which the following selection criteria were used to increase the probability of detecting a novel viral pathogen causing relevant disease: (i) fever or a history of fever (≥37.5°C), (ii) illness onset of ≤5 days, (iii) CSF white cell count between 6 and 1,000 cells/µl, (iv) normal CSF glucose (CSF glucose/blood glucose ratio ≥ 0.5), and (v) absence of an etiological diagnosis by the methods described above. In total, 125 CSF specimens from patients with CNS infections with unknown etiology  or as pools of ≤5 specimens .The selected CSF specimens were analyzed by VIDISCA-454 either as single samples (The VIDISCA-454 procedure was done as previously described . In brie2 (provided with the polymerase), 1× PCR buffer (provided with the polymerase), and 3 µl of extracted NAs. PCR was performed in a Mastercycler  with an initial polymerase activation step at 95°C for 14 min 30 s, followed by 45 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Confirmation of amplified product was done by sequencing, using BigDye Terminator v1.1 cycle sequencing kit  in an ABI377 automatic sequencer (Applied Biosystems), following the manufacturer’s instructions.To confirm the presence of the cyclovirus-like sequence generated by high-throughput sequencing, specific PCR primers (CyCV31-53F and CyCV178-53R) -labeled probe (CyCV65-96)   and neighbor-joining in MEGA version 4  to remove adaptor sequences and DNA fragments of ersion 4 , respectAll clinical studies were reviewed and approved by the Institutional Review Boards of collaborating hospitals/authorities and the Oxford Tropical Research Ethics Committee (OxTREC), University of Oxford, United Kingdom.The GenBank accession numbers for the CyCV-VN sequences are KF031465–KF031492.Figure S1Nucleotide sequence alignment showing sequence identity between primers/probe of CyCV-VN PCR used and CyCV-VN and CyCV-20  sequences. Degenerate nucleotides: D = A, G, or T; M = A or C; S = G or C. 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Objectives. To determine how beta-amyloid 1-42 (Aβ 1-42), total tau (tTau), and phosphorylated tau (pTau) levels in CSF behave in a cohort of Thai patients from the Memory Clinic in Bangkok, Thailand. Methods. During 2009–2011, twenty eight subjects from the memory clinic at Siriraj Hospital had CSF analysis for AD biomarkers. Aβ 1-42, tTau, and pTau (at amino acid 181) were measured in CSF by ELISA technique. Results. Mean of Thai mental state examination (TMSE) of 28 Thai cohort was 16.48 (6.63). Fourteen had AD, ten had non-AD dementia, and four non-cases were those with subjective memory complaint (SMC) without dementia. Mean CSF Aβ 1-42, tTau, ptau (181), and pTau/Aβ 1-42 in the AD group were 241.36 (60.14) pg/mL, 222.79 (212.24) pg/mL, 40.79 (27.84) pg/mL, and 0.18 (0.12) accordingly. Mean CSF Aβ 1-42, tTau, pTau (181), and pTau/Aβ 1-42 in the non-AD dementia group were 430.40 (125.18) pg/mL, 349.30 (692.16) pg/mL, 36.80 (14.90) pg/mL, and 0.09 (0.04) accordingly. Mean CSF Aβ 1-42, tTau, pTau (181), and pTau/Aβ 1-42 in the non-cases with SMC without dementia were 499.75 (93.44) pg/mL, 137.25 (62.74) pg/mL, 31.75 (17.48) pg/mL, and 0.06 (0.02). There is significant difference (P < 0.05) among the 3 groups in CSF Aβ 1-42 and pTau/Aβ 1-42. We propose mean + 1.5 SD of CSF Aβ 1-42 in AD group (331.57 pg/mL) to be the cut-off point in Thai subjects. Conclusion. There are significant different in CSF Aβ 1-42 and CSF p-tau/Aβ 1-42 among those with AD, non-AD dementia and non cases with SMC without dementia in Thai cohort. Cut-off point of CSF Aβ 1-42 of 331.57 pg/mL is suggested in Thai study. The hyperphosphorylated tau (pTau) is a characteristic component of NFT. The pTau is a fraction of concentrated total Tau (tTau) protein in the NFT. Previous studies demonstrated that cerebrospinal fluid biomarkers of Aβ 1-42, tTau, and pTau (at amino acid 181) can differentiate individuals with AD from healthy controls with a good sensitivity and specificity, but cut-off levels differ between test centers [ The Hallmark pathology of Alzheimer's disease (AD) is senile plaques (SPs) and  neurofibrillary tangles (NFTs) . The pri centers . Culturaβ 1-42, total tau (tTau), and phosphorylated tau at threonine 181 (pTau 181) between AD, non-AD dementia, and noncases. We hope to look at possible new cut-off point of CSF biomarkers in our Thai population cohort.We aimed to compare CSF levels of Aβ 1-42, tTau, and pTau (at amino acid 181) were measured in CSF by enzyme-linked immunoassay (ELISA) technique of the INNOTEST, Ghent, Belgium. All patients underwent a standardized clinical assessment, including medical history, physical and neurological examination including Thai Mental State Examination  [r = −0.679, P < 0.001) with the Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE) in an earlier study in Thai cohort [P < 0.05. Post hoc analysis of Dunnett T 3 equal variance not assumed was used. The controls in this study were defined by those without dementia but have memory problems. This study obtained approval to study from the local ethical committee at Siriraj Hospital. During 2009–2011, the Memory Clinic in Bangkok at Siriraj Hospital set up the investigations of cerebrospinal fluid biomarkers for the service in cases with unusual manifestation to the clinic. Twenty eight subjects had CSF analysis for AD biomarkers. CSF was obtained by lumbar puncture between the L3/L4 or L4/L5 intervertebral space, using a 25-gauge needle, and collected in 10 mL polypropylene tubes. Within 2-3 hours, CSF samples were centrifuged at 2,100 g for 10 minutes at 4°C. One milliliter of CSF was used for routine analysis. Routine CSF analysis was also done to look for infection or metastasis. Aliquots of each sample were immediately frozen at minus 80°C until further analysis. Aof MMSE) , laborati cohort . When alβ 1-42 was 343 pg/mL. Mean CSF Aβ 1-42, tTau, pTau (181), and pTau/Aβ 1-42 in the AD group were 241.36 (60.14) pg/mL, 222.79 (212.24) pg/mL, 40.79 (27.84) pg/mL, and 0.18 (0.12) accordingly. Mean CSF Aβ 1-42, tTau, pTau (181), and pTau/Aβ 1-42 in the non-AD dementia group were 430.40 (125.18) pg/mL, 349.30 (692.16) pg/mL, 36.80 (14.90) pg/mL, and 0.09 (0.04) accordingly. Mean CSF Aβ 1-42, tTau, pTau (181), and pTau/Aβ 1-42 in those non cases with SMC without dementia were 499.75 (93.44) pg/mL, 137.25 (62.74) pg/mL, 31.75 (17.48) pg/mL, and 0.06 (0.02). Twenty eight patients with memory problems underwent CSF biomarker analysis. The consensus diagnoses revealed that fourteen had AD, ten had non-AD dementia, and four did not meet the criteria for dementia. Among 14 AD, eight were early onset dementia . Twelve AD presented with magnetic gait and memory problems. Two had only memory problems. Among ten non-AD dementia, two were EOD (1 CJD and 1 FTD). The diagnoses of non-AD dementia were 5 FTD, 2 DLB, 1 CJD, 1 VaD, and 1 NPH. Five non-AD dementia presented with abnormal gait, four with behavioral problems, and one with both behavioral problems and abnormal gait. Among four subjects with memory complaint without dementia, one had schizophrenia aged 24, one had chronic kidney disease and malignancy of the lungs, and two had memory complaint with hydrocephalus and cautious gait. Sixteen were women and twelve were men. Mean age of the study cohort was 68.39 (SD = 14.69) years old. The consensus diagnoses revealed that among 11 EOD, 8 had AD, 2 had FTD, and 1 had CJD. The Patient characteristics are shown in β 1-42 than those with non-AD dementia and non- cases. There was no statistical difference in the level of CSF pTau and tTau among the three groups. The results of CSF pTau/CSF Aβ 1-42 also had statistical difference among the three groups (β 1-42 and CSF pTau/CSF Aβ 1-42. We proposed a cut-off levels of CSF Aβ 1-42 of less than 331.57 pg/mL (mean+1.5 SD of that from Thai AD cohort) in our laboratory investigation. When utilizing the receiver operating characteristic (ROC) curve analysis using AD (n = 14) as diagnosis and individual with non-AD dementia (n = 10) as controls, the area under the cure (AUC) of CSF Aβ 1-42 is 0.900 . The coordinates of the ROC curve showed that CSF Aβ 1-42 cut-off level 315 pg/mL gives the sensitivity of 85.7% and specificity of 90%, and CSF Aβ 1-42 cut-off level 355 pg/mL gives the sensitivity of 92.9% and specificity of 50%.   . From ouβ 1-42, CSF tTau, and CSF pTau were useful in diagnosis of AD. Despite our small sample size, CSF Aβ 1-42 showed significant difference among AD, non-AD, and those noncases with SMC without dementia. CSF pTau/CSF Aβ 1-42 revealed significant difference between AD and non cases but showed a trend of difference between AD and non-AD groups. The cut-off point of CSF Aβ 1-42 less than 331.57 pg/mL and CSF pTau/CSF Aβ 1-42 more than 0.1085 suggest the diagnosis of AD in Thai cohort in our laboratorywith a moderate level of accuracy.We demonstrated that CSF biomarkers using CSF Aβ 1-42 less than 487 pg/mL, increased CSF phosphorylated tau (pTau 181) more than 61 pg/mL, or increased CSF total tau more than 425 pg/mL were categorized to support the diagnosis of AD, and the opposite data was against [β 1-42: amyloid fragments are plausible AD biomarkers because they represent senile plaque formation which is a hallmark pathological process in AD. However, CSF Aβ 1-42 and CSF tTau are less effective in discriminating AD from other dementias (specificities of 57% for suspected non-AD dementias) [β 1-42 and increased CSF pTau proteins are the most precise and reproducible chemical diagnostics of sporadic AD. Our study had only small cohort; we then tried to exaggerate the increased pTau in CSF by manipulation of CSF pTau/CSF Aβ 1-42. The figure of of CSF pTau/CSF Aβ 1-42 closed to 0.1 suggests AD pathology. Our data showed wide SD in CSF tTau levels in non-AD group because of 1 CJD with CSF tTau more than 2,000 pg/mL. Other review supports that extremely high CSF tTau indicates CJD [ Previous studies showed that decreased levels of CSF A against , 16. How against . CSF Aβ tau pTau 1 more thates CJD . Limitations of this study are small sample size and no autopsy confirmation of diagnosis. The strength of our study is the demonstration of real-life everyday practice of how CSF biomarkers assist the working diagnosis of dementia in Thai population. Further investigation of validation of current cut-off point in differentiating AD from non-AD and from control subjects is needed in Thai population. Additionally, we hope to engage to become a participant in the global or European quality control CSF standardization program in the near future.


Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins.Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2 Biosimilars are biologic medical products that are made by living organisms using recombinant DNA technology ,2. One oEscherichia coli (E. coli) has similar biological activity to that of the native human protein, but differs in that it contains an N-terminal methionine residue and is not glycosylated ) was purchased from Stratagene . The expression plasmid pPT, in which the g-csf (isoform B) gene was inserted into the NdeI and HindIII sites of the vector, was used for protein production as previously described , and refolded in buffer C . The refolding was carried out at 25°C for 15 h. After refolding, the solution was adjusted to pH 5.5 with phosphoric acid to separate contaminating E. coli proteins followed by centrifugation at 10,000 × g for 20 min, and then the supernatant was concentrated 20-fold by ultrafiltration system  with MWCO 10K cassettes.The cultivated cells were harvested using tubular type continuous centrifuge  at 10,000 × 4 # 214 TP 1010, 250 mm × 10 mm), and collected the fractions containing rhG-CSF. Each fraction was diluted in distilled water (pH 3.5) and concentrated by the Amicon ultrafiltration system. Elution was carried out at a flow rate of 2 mL/min at 40°C with a linear gradient of acetonitrile  for 140 min using solvent A (22.5% ACN and 0.1% TFA) and solvent B (90% ACN and 0.1% TFA). The eluted sample was detected at 280 nm absorbance.The concentrated supernatant (10 mg) was loaded on a sp-HPLC column . The amount of loaded sample was 500 mg and the column volume was 400 mL  for 145 min. The volume of each fractionation was 75 ml. Absorbance was measured at 280 nm.All experimental conditions for sample preparation and performance of prep-HPLC were similar to that of semi-prep-HPLC. The Prep-HPLC System  was performed using Kromasil C4 column  with modified method  containing 5% (w/v) dried skim milk. Subsequently, the membrane was incubated with an anti-hG-CSF polyclonal antibody  overnight at 4°C in blocking buffer. The blot was then incubated with a secondary antibody conjugated with horseradish peroxidase (1:1000 dilution) for 2 h at RT. Diaminobenzidine (DAB) was used to develop the signal. The molecular weight marker was Precision Plus Protein Dual Color Standard .l-glutamine and 1 mM Na-pyruvate at a concentration of 5.0 × 103 cells/well in a 96-well plate . The standard G-CSF (NIBSC code: 09/136) was from the World Health Organization (WHO) 2nd International Standard . Both standard G-CSF and purified rhG-CSF were serially diluted 2-fold in growth medium and added to the well at a final concentration of 1,600 pg/mL and 3.12 pg/mL, respectively, in a total volume of 100 μL/well. The culture plate was incubated in a 37°C humidified CO2 incubator with 5% CO2 for 48 h. The cell proliferation reagent WST-1  was added and the cells were incubated for an additional 4 h. Absorbance was determined at 450 nm using an microplate reader . The absorbance was directly proportional to the number of viable cells because the tetrazolium salts in the WST-1 are cleaved to formazan by mitochondrial dehydrogenases in the cells. As well, the activity of rhG-CSF was measured using a Quantikine hG-CSF enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s protocol.The bioactivity of rhG-CSF was analyzed by its ability to stimulate the proliferation of the murine myeloblastic NFS-60 cell line . G-CSF-dp<0.05.The student t-test was used to assess significant differences between the treatment groups. The criterion for statistical significance was set at E. coli JM109, the human g-csf gene was cloned into a pPT plasmid. Batch culture with E. coli JM109/pPT-G-CSF was carried out in a flask containing 50 mL of the initial medium at 250 rpm, 37°C. As shown in To express rhG-CSF molecules in E. coli JM109 were transformed properly by pPT-G-CSF plasmid, the stepwise increase strategy of temperature for the protein expression was performed with the same cells [Because me cells ,24. As sE. coli JM109. To refold, the IBs in the culture broth were harvested by centrifugation after the cells were disrupted. The harvested IBs were washed with a buffer  and refolded in a dilution step to decrease the concentration of urea. To precipitate impurities containing misfolded rhG-CSF, lipids, and other unnecessary proteins, the pH of the refolded rhG-CSF solution was stepwise lowered from 9.5 to 3 by the addition of phosphoric acid [losses, pH precipitation process was positively necessary because the purity of rhG-CSF was increased from 70% to 85% based on image analyses. Subsequently, both unrefolded and refolded rhG-CSF were analyzed by RP-HPLC. As a result, the retention time of rhG-CSF before and after refolding was shifted from 54.3 min to 52.5 min (data not shown), suggesting that the refolded form is more hydrophilic. The refolding yield was increased to 65% by RP-HPLC.The most of rhG-CSFs were detected in IBs of cultured ric acid . The optIt is not easy to separate the isoforms of rhG-CSF from homogenates because their physicochemical properties are quite similar each other. Although there was an attempt to separate isoforms from rhG-CSFs by two cationic exchange chromatography steps , it is aThe first 35 amino-terminal amino acid residues of rhG-CSF protein were in order of MTPLGPASSLPQSFLLKCLE-QVRKIQGDGAALQEK by the Edman degradation analysis, being in tune with the expected sequences. For purity analysis of rhG-CSF, SDS-PAGE analysis was performed, followed by Coomassie brilliant blue staining. As shown in The rhG-CSF was digested with endoproteinase Glu-C and analyzed by a C18 RP-HPLC column. The peaks of rhG-CSF and standard hG-CSF were overlapped in 8 IU/mg according to ELISA and the cell proliferation assay.To test the biological activity of rhG-CSF, we carried out a proliferation assay with NFS-60 cells. The cells were treated with various concentrations of standard or rhG-CSF for 48 h. Their activity was then assessed using a reagent WST-1. As shown in E. coli [rrnB P2 promoter instead of pET system, [In this study, we established the new method for the industrial-scale production and characterization of rhG-CSF. pET expression system has been extensively used for the production of rhG-CSF in E. coli ,31,32. ME. coli ,24, we c system, ,23,33. A system, ,32. Howe system, . This diE. coil leads to the unavoidable result of IBs, which should be solubilized in chaotropic agents such as high grade GuHCl or urea [High-level expression of rhG-CSF in  or urea ,26,34. R or urea ; however or urea .The function of ultrafiltration is to remove contaminants as well as to reduce volume before the refolded sample passes to an ion exchanger chromatography. Linear scale flat-sheet cassettes were chosen in this pilot-scale purification. The main advantages of this module are that they are suitable for bioprocessing industry that provide 1000-fold scaling up of ultrafiltration processes with consistent product yield and process flux . HoweverE. coil [There are several reports related to the purification of rhG-CSF ,22,36,37in vitro and slightly lower pI`s [wer pI`s . Althougwer pI`s ,22,26.nd International Standard. Above all, it is emphasized that new method for purification of rhG-CSF was cost-effective one because the overall costs of expression were reduced by omitting the addition of IPTG.Regarding the biological activity of purified rhG-CSF, it showed that rhG-CSF promotes 50% maximal growth of the cells at 13.4 pg/mL. It proves that they have extremely similar activities when compared with that of World Health Organization (WHO) 2E. coli. First, in the fed-batch culture, recombinant protein expression by a temperature shift strategy without inducer such as IPTG is a compatible method for its large-scale production. Second, the purification by a single step of prep-HPLC after the pH precipitation of the refolded samples is sufficient for the industrial-scale production. In addition, our results show that the isoforms of rhG-CSF can be separated during the prep-HPLC step. Taken together, results described here demonstrate that experimental methods and strategies described here to generate high-purity recombinant proteins may be worthful for the large-scale production.Here, we describe a detailed report about simplified large-scale refolding, purification, and characterization of rhG-CSF in