There were no observations of serious adverse events. In the functional outcomes, the grip power of the dominant hand was increased in the EPO+G-CSF treatment group. In conclusion, this exploratory study suggests a novel strategy of EPO+G-CSF combination therapy for stroke patients.Erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF) are known to have neuroprotective actions. Based on previous reports showing the synergistic effects of EPO+G-CSF combination therapy in experimental models, we investigated the safety of EPO+G-CSF combination therapy in patients with chronic stroke. In a pilot study, 3 patients were treated with EPO and G-CSF for 5 consecutive days, with follow-up on day 30. In an exploratory double-blind study, 6 patients were allocated to treatment with either EPO+G-CSF or placebo. Treatment was applied once a day for 5 days per month over 3 months. Participants were followed up for 6 months. To substantiate safety, vital signs, adverse events, and hematological values were measured on days 0, 5, and 30 in each cycle and on day 180. Functional outcomes were determined on day 0 and 180. In the laboratory measurements, EPO+G-CSF combination therapy significantly elevated erythropoietin, CD34 Brain injury from ischemic or hemorrhagic stroke results in tissue destruction in the involved brain regions and eventually loss of motor function. Although there have been a variety of efforts to recover motor deficits through training-based interventions, conventional rehabilitation has yielded limited improvements in stroke patients. Therefore, pharmacological treatment using multiple drugs such as erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF) has been introduced in clinical trials for stroke patients ,4,5,6,7.+ hematopoietic stem cells to stimulate neutrophil progenitor proliferation and differentiation in patients with neutropenia, as well as mobilizing transplanted bone marrow stem cells in patients with hematological malignancies such as multiple myeloma [Hematopoietic growth factors such as EPO and G-CSF have particularly been utilized to increase circulating blood cells, including red blood cells, white blood cells, and platelets, by stimulating bone marrow production. EPO is commonly known for regulating red blood cell production in patients with anemia ,9, while myeloma ,11.+ hematopoietic stem cell migration from the bone marrow into the brain region through peripheral blood circulation [et al. [In addition to the peripheral effects, these hematopoietic factors also stimulate CD34culation ,13,14. Eculation ,15,16. Gculation ,17,18. Hculation . De La P [et al.  also suget al. [et al. [Ehrenreich et al.  demonstr [et al.  found thet al. [et al. [+ hematopoietic stem cells up to 15-fold at the highest dose compared with placebo, and increased total leukocyte counts up to approximately 4 times compared with pre-treatment, suggesting mobilization of hematopoietic stem cells from the bone marrow into peripheral circulation. Floel et al. [et al. [+ hematopoietic stem cells fates in the brain of subacute stroke patients, accompanied by the direct effect that given hematopoietic growth factors can cross the blood-brain barrier and act on neurons in the infarct area [Shyu et al.  suggeste [et al.  found thl et al.  showed t [et al.  revealedrct area .Likewise, hematopoietic growth factors are characterized as well-tolerated therapies in patients with stroke ,26,27,28The present investigation has been inspired by the distinctive and synergistic beneficial neuroprotective effects of EPO and G-CSF in patients with brain injury. Based on the promising results, namely that EPO in combination with G-CSF exerted synergistic effects on tissue plasticity and functional recovery in an experimental stroke model , we firsThree participants who received EPO+G-CSF treatment were included in an open-label pilot study. A total of six participants were included in an exploratory double-blinded study. Two arms (3 EPO+G-CSF and 3 placebo control) were matched at baseline . All parn = 3) showed increases of erythropoietin concentrations  versus 16.80 (10.74–16.95) mIU/mL), CD34+ hematopoietic stem cells (40.00 (27.00–63.00) versus 2.00 (1.50–4.00) cells/μL), white blood cells (44.36 (40.27–46.16) versus 6.91 (5.48–8.36) cells/μL), and neutrophils (84.20 (79.60–86.90) versus 65.40 (63.75–65.85) %) on day 5. The peaks of these four parameters had returned to baseline on day 30 , EPO+G-CSF combination therapy (n = 3) significantly elevated erythropoietin concentrations (547.00 (419.50–601.00) versus 8.89 (7.46–13.35) mIU/mL, p = 0.049) by Mann-Whitney U test, CD34+ hematopoietic stem cells (50.00 (33.00–61.00) versus 1.00 (1.00–3.00) cells/μL, p = 0.046), white blood cells (32.56 (29.59–45.68) versus 6.41 (5.93–7.44) cells/μL, p = 0.049), and neutrophils (85.80 (85.40–86.75) versus 59.30 (55.85–60.50)%, p = 0.049) on day 5 (versus 48.80 (44.45–52.75) cells/μL, p = 0.049) on day 5  kg, p = 0.049 by Mann-Whitney U test)  and modified Barthel index (MBI). This result suggested that initial conditions of groups were well matched. Six months after treatment, no significant differences had emerged with respect to MMSE and MBI between groups. Grip and pinch power  and hand dexterity evaluated by the box-and-block test in the affected hand did not show significant differences between groups . However U test) .This study is the first exploratory double-blind study based on the pilot study to evaluate long-term EPO+G-CSF combination therapy with repetitive administration in patients with chronic stroke. Although EPO and G-CSF have been commonly used individually as hematopoietic growth factors in the treatment of anemia and neutropenia, these pharmacological agents have been recently reported to have neuroprotective and regenerative potential as neurotrophic factors in stroke ,31,32. TWe found that repetitive and long-term combined use of EPO and G-CSF was well tolerated and not associated with serious adverse events, supporting the safety of dual administration in stroke patients. Although serious concerns remain for cardiovascular and hematopoietic events based on the use of hematopoietic growth factors due to increased production of peripheral blood cells, no complications leading to thromboembolic accidents or aggravation leading to mortality occurred in this study ,23,24,38+ hematopoietic stem cells, white blood cells, and neutrophils in the peripheral blood in patients with chronic stroke. All participants who received EPO+G-CSF achieved the peak values on day 5 in each cycle, followed by a hematological trend to return toward baseline within 30 days after drug administration. Despite significant increases of white blood cells and neutrophils, the EPO+G-CSF group did not show significant complications of vascular events such as thromboembolic accidents that might worsen primary stroke symptoms, consistent with previous reports [In the laboratory measurements, we found that EPO+G-CSF treatment significantly elevated erythropoietin, CD34 reports ,23. Whil reports ,4. An in reports . HoweverIn the functional outcomes of MMSE and MBI, no statistically significant difference was found between the EPO+G-CSF and placebo groups. It might be possible that chronic stroke participants, who showed no cognitive impairment and ADL with minimal assist, represent a ceiling effect to the combination therapy since they already have relatively higher functional level in the initial assessment. Although no significant differences between groups were seen with respect to functional improvement on power and dexterity in the affected hand, there was a significant improvement in the grip power of the dominant hand 6 months after EPO+G-CSF combination therapy.d-mannitol as a vehicle of G-CSF in the placebo control. Although we should have considered two vehicles , d-mannitol did not affect the double-blind test because its color and agent form were the same as normal saline, as colorless liquid.Previous studies indicate that hematopoietic growth factors encourage functional improvement in disability, dependence, and cognitive functions in patients with acute stroke or coronary artery disease ,38. BecaWe performed a pilot study followed by an exploratory double-blind study of EPO+G-CSF combination therapy in patients with chronic stroke. We conducted this trial in accordance with the Declaration of Helsinki and Good Clinical Practices. The present study was approved by Institutional Review Board of Severance Hospital, Korea Food and Drug Administration (4-2010-0468), and registered on an international registry system, ClinicalTrials.gov (NCT02018406).After obtaining signed informed consent, patients were screened using medical history, a physical assessment, routine blood laboratory tests, and brain magnetic resonance imaging (MRI) findings to confirm study eligibility.Inclusion criteria were as follows: (1) over 20 years old; (2) voluntary participants; and (3) at least 3 months after onset of ischemic or hemorrhagic stroke. Exclusion criteria were as follows: (1) under 20 years old; (2) participants who could not voluntarily consent; (3) encephalopathy from brain tumor and infection; (4) current thrombolytic therapy including warfarin or coumadin medications; (5) leukopenia, thrombocytopenia, or polycythemia; (6) malignant diseases, malignant hypertension, myeloproliferative disorder, septic embolism, or hyperkalemia; (7) hepatic or renal dysfunction with serum creatinine >3 mg/dL; (8) allergic reactions to exogenous EPO or G-CSF; (9) possessing exclusion criteria for MRI test; (10) pregnant or breast feeding in women; (11) body temperature over 38 °C; (12) blood pressure over 140/90 mmHg at pre-treatment; (13) blood pressure over 160/100 mm Hg during intervention; (14) hemoglobin > 15 g/dL at pre-treatment; (15) hemoglobin > 17 g/dL during intervention; (16) pneumonia detected by chest X-ray; and (17) recurrent history of aspiration pneumonia.In an open-label pilot study, participants consented to be treated with EPO+G-CSF for 5 consecutive days, and be followed up on day 30. In an exploratory double-blind study, following enrollment of eligible participants, randomization was carried out in an institutional clinical pharmacy. A clinical pharmacist unfamiliar with participants and investigators used a table of random sampling numbers to randomly allocate participants to either the placebo or EPO+G-CSF arm.d-mannitol solution (250 μg/mL) separately at the same time, once a day for 5 consecutive days, with follow-up on day 30.Three patients with chronic stroke participated in a pilot study. They were treated with subcutaneous recombinant human EPO  in the sodium chloride solution  and recombinant human G-CSF  in the Six patients with chronic stroke (3 EPO+G-CSF and 3 placebo) were randomly allocated into two groups in an exploratory double-blind study. The EPO+G-CSF arm were administered as the same treatment as the pilot study per cycle (one month) over a total of 3 cycles. The placebo arm had the same schedule and volume of subcutaneous saline injections as the treatment arm. These doses were in line with the published safety data ,23,24.After completing three treatment cycles, participants were followed up for six months from the start of combination therapy in the same clinic. To substantiate safety, vital signs, adverse events, and hematological tests were recorded at each follow-up visit in all patients. To measure functional outcomes, MMSE, MBI, grip and pinch power tests and box-and-block tests were performed on days 0 and 180. All patients completed a six-month follow-up .+ hematopoietic stem cells, white blood cells including neutrophils, lymphocytes, and monocytes, red blood cells, reticulocytes, hemoglobin, hematocrit, glucose, creatinine, and C-reactive protein were conducted on days 0, 5, 30 in each cycle, and 6 months after treatment commenced. Blood samples were obtained via venous puncture by medical laboratory scientists, and analyzed according to the institutional standard guidelines as follows. (1) Serum EPO concentration was measured by an enzyme-labeled chemiluminescent immunometric assay using an immunoassay system ; (2) CD34+ cell count was performed using a flow cytometer and software ; (3) Total white blood cells and differentiated subtypes , red blood cells, reticulocytes, hemoglobin, hematocrit were measured using automated blood cell counters ; (4) Glucose was measured by the hexokinase method using a commercial kits with an automated chemistry analyzer ; (5) Creatinine was measured by the Jaffe method using a commercial kit with an automated chemistry analyzer ; (6) C-reactive protein was measured by the turbidimetric latex agglutination method, using a commercial kit with an automated biochemical analyzer .Hematological examinations to measure erythropoietin concentration, CD343). A participant moved blocks from one box to the other. The number of blocks moved within 60 seconds was counted by occupational therapists [Vital signs  and adverse events were evaluated on the 5 consecutive treatment days, day 30 in each cycle, and 6 months after treatment commenced. Vital signs were measured by nurses using an automatic blood pressure monitor  and an digital thermometer . Chest X-rays  and electrocardiograms  were evaluated to screen and exclude pneumonia and heart failure on days 0 and 180. Functional outcome measures including MMSE, MBI, grip and pinch power, and the box-and-block test for hand dexterity were evaluated on days 0 and 180. MMSE evaluates cognitive functions such as registration, attention, calculation, recall, language, ability to follow simple commands, and orientation. MMSE scores range from 0 to 30, with a lower score indicating cognitive impairment and the highest score indicating no cognitive impairment . MBI conerapists . All medU tests were used to compare groups for treatment effects. Statistical significance was defined as p < 0.05.All statistical analyses were performed with SPSS Statistics 23 . Statistical analyses were performed after all participants completed experimental procedures. Median and interquartile ranges (25th–75th percentiles) were used for the non-normally distributed variables. Mann-Whitney This study provides a novel strategy of EPO+G-CSF combination therapy for stroke patients. Based on the safety outcomes of EPO+G-CSF combination therapy in chronic stroke patients, further clinical trials are required to expand the sample size of chronic stroke patients and inclusion criteria including acute or subacute stroke patients to test the efficacy of EPO+G-CSF combination therapy. Therefore, the design of our further study will be considered as a single cycle in large numbers of stroke patients as a neuroprotective strategy. Ultimately, it will provide a definite therapeutic rationale by showing the utility of this combination therapy in a large-sample-size study. Taken together, this exploratory double-blind study after a pilot study provides the first evidence that EPO+G-CSF may have safe and beneficial therapeutic potential for chronic stroke patients.


Tuberculous pericarditis (TBP) is associated with high morbidity and mortality, and is an important treatable cause of heart failure in developing countries. Tuberculous aetiology of pericarditis is difficult to diagnose promptly. The utility of the new quantitative PCR test (Xpert MTB/RIF) for the diagnosis of TBP is unknown. This study sought to evaluate the diagnostic accuracy of the Xpert MTB/RIF test compared to pericardial adenosine deaminase (ADA) and unstimulated interferon-gamma (uIFNγ) in suspected TBP.Mycobacterium tuberculosis culture and/or pericardial histology served as the reference standard for definite TBP. Receiver-operating-characteristic curve analysis was used for selection of ADA and uIFNγ cut-points.From October 2009 through September 2012, 151 consecutive patients with suspected TBP were enrolled at a single centre in Cape Town, South Africa. P = 0.03) at an estimated background prevalence of TB of 30%. The sensitivity and negative predictive value of both uIFNγ and ADA were higher than Xpert-MT/RIF (P < 0.001).Of the participants, 49% (74/151) were classified as definite TBP, 33% (50/151) as probable TBP and 18% (27/151) as non TBP. A total of 105 (74%) participants were human immunodeficiency virus (HIV) positive. Xpert-MTB/RIF had a sensitivity and specificity ) of 63.8% (52.4% to 75.1%) and 100% (85.6% to 100%), respectively. Concentration of pericardial fluid by centrifugation and using standard sample processing did not improve Xpert MTB/RIF accuracy. ADA (≥35 IU/L) and uIFNγ (≥44 pg/ml) both had a sensitivity of 95.7% (88.1% to 98.5%) and a negative likelihood ratio of 0.05 (0.02 to 0.10). However, the specificity and positive likelihood ratio of uIFNγ was higher than ADA (96.3% (81.7% to 99.3%) and 25.8 (3.6 to 183.4) versus 84% (65.4% to 93.6%) and 6.0 (3.7 to 9.8); uIFNγ offers superior accuracy for the diagnosis of microbiologically confirmed TBP compared to the ADA assay and the Xpert MTB/RIF test. Tuberculosis (TB) is a global health priority . In deveMycobacterium tuberculosis (M. tb) and rifampicin resistance, providing a result in less than two hours . Sensitivity did not decrease significantly when definite- and probable-TB patients were combined  of PF Xpert MTB/RIF was 63.8% (52.4 to 75.1) (Table P = 0.33). For uIFNγ, both the Youden’s index (cut-point that correctly classifies the most number of patients) and optimal rule-out cut-point was 44 pg/ml, while for ADA the cut-point currently utilised in routine clinical practice is >35 IU/l [Of the 151 patients enrolled, 6.6% (10/151) and 5.9% (9/151) were not subjected to uIFNγ and ADA assays, respectively. The optimal cut-points for uIFNγ and ADA levels to maximise diagnostic accuracy were determined using the ROC-curve shown in Figure >35 IU/l  comparedP = 0.1). Similarly, although the sensitivity of the biomarkers uIFNγ and ADA was similar for both HIV-positive and -negative patients, the specificity of ADA  was lower in HIV-positive patients  of uIFNγ was 95.7% 88.1 to 98.5), which was similar to ADA using the clinical cut-point Table . However to 98.5,We further interrogated the potential clinical utility of routine  and new same-day diagnostic tools  by comparing positive (LR+) and negative (LR-) likelihood ratios Table  and posiTable The performance of the new WHO-endorsed, Xpert MTB/RIF assay has recently been reported for some types of extra-pulmonary TB such as TB lymphadenitis , pleuralThe key findings of our study are that: (1) uIFNγ offers superior accuracy for the diagnosis of microbiologically confirmed TBP compared to the new Xpert MTB/RIF test and the established ADA assay; (2) PF Xpert MTB/RIF could bacteriologically confirm a TB diagnosis  in two thirds of patients with suspected TBP; (3) PF uIFNγ offered better rule-in diagnostic utility compared to ADA in current clinical use, while both tests could rapidly rule-out TBP; (4) PF Xpert MTB/RIF, when combined with either ADA or uIFNγ, offers >97% sensitivity and specificity for TBP diagnosis; and (5) concentration of PF samples prior to Xpert MTB/RIF testing increased the number of ‘indeterminate’ tests without significantly improving diagnostic yield.T-values and liquid culture time-to-positivity using PF [Xpert MTB/RIF testing is undergoing phased implementation in a number of high burden settings for routine diagnosis of pulmonary TB ,27. Therusing PF . HoweverProof-of-principle studies in TB pericarditis have demonstrated the potential utility of using uIFNγ PF levels for diagnosis of TB pericarditis ,14,34. AAre the findings of this study generalisable to other settings, and does either Xpert MTB/RIF or uIFNγ testing potentially offer utility beyond existing same-day diagnostic tools, such as smear microscopy, PF ADA measurements and/or basic clinical information? In this study we compare the utility of Xpert MTB/RIF, uIFNγ or ADA, alone or in combination across different TB prevalence rates, focusing on the diagnostic priorities of rapid rule-in and rule-out, as well as bacteriologically confirmed diagnosis. In a high prevalence setting , Xpert MTB/RIF and uIFNγ outperforms ADA and basic clinical predictors for rapid rule-in (highest LR + and PPV). However, both ADA and uIFNγ offer equivalent rapid rule-out utility, outperforming Xpert MTB/RIF and clinical predictors. Combining Xpert MTB/RIF testing followed by ADA or uIFNγ in Xpert-negative PF maximised both sensitivity and specificity to >97% for TBP diagnosis. This may offer the best diagnostic approach in high burden settings, especially where drug-susceptibility testing is desirable, but the cost of a two test algorithm will remain a key consideration in resource-poor conditions where TB is endemic. Xpert MTB/RIF currently costs approximately US$20/test, while ADA measurement is less than US$0.1/test. Intergam kits are not currently commercially available so the cost is unknown but likely to be only slightly more than smear microscopy. Prospective studies of the cost-effectiveness of diagnostic options are needed before it can be considered for clinical practice.Our study had a number of important limitations. This study did not optimise PF sample volumes or processing beyond comparing two volumes and a simple centrifugation step thought applicable to resource-limited settings. The use of different volumes or alternative processing methods may have improved Xpert MTB/RIF sensitivity and/or decreased the high indeterminate rate found. A low number of replicates were performed in limit of detection experiments and these findings should be confirmed in further studies. The study was conducted in a high TB and HIV burden setting, which may limit the generalisability of the findings. Performance may differ in a low TB burden setting and where HIV co-infection rates and, hence, bacterial load, are lower, such as Europe and the US. However, the use of diagnostic accuracy measures that are less affected by prevalence, such as LRs, and generating estimates across varying TB prevalence rates helps to highlight potential performance differences between low and high burden settings and, hence, improve generalisability. Whilst this is the largest study that has comprehensively evaluated several diagnostic strategies and tools in the same prospective cohort, the sample size was limited in the non-TB group. The small number of non-TB patients reflects the high burden of infectious and HIV-related disease in the South African environment . AlthougIn conclusion, uIFNγ offers superior accuracy for the diagnosis of microbiologically confirmed TBP compared to the new Xpert MTB/RIF test and the established ADA assay, performed using available Xpert MTB/RIF testing protocols without fluid-specific optimisation beyond simple centrifugation. These data suggest that the uIFNγ assay may be the optimal first line test for the diagnosis of TB pericarditis, and merits consideration for implementation in clinical practice. Furthermore, PF Xpert MTB/RIF, when combined with either ADA or uIFNγ, offers high sensitivity and specificity for TBP diagnosis. Studies are needed to test the utility and cost-effectiveness of a two-test strategy, which may be preferred in HIV-positive patients where biomarker specificity may be reduced. Collectively, these data suggest that a biomarker-oriented approach may be feasible and accurate for the diagnosis of suspected TBP in a high TB and HIV prevalence setting.M. tb: Mycobacterium tuberculosis; NPV: Negative predictive value; NYHA: New York Heart Association; PF: Pericardial fluid; PPV: Positive predictive value; SD: Standard deviation; TB: Tuberculosis; TBP: Tuberculous pericarditis; uIFNγ: Unstimulated interferon-gamma; WCC: White cell count.ADA: Adenosine deaminase; ARV: Anti-retroviral therapy; AUROC: Area under the receiver operator characteristics curve; CFU: Colony forming units; CI: Confidence interval; HIV: Human immunodeficiency virus; IQR: Interquartile range; LR-: Negative likelihood ratio; LR+: Positive likelihood ratio; KD and UG have performed consultancy work for Antrum Biotech (Pty) Ltd, a University of Cape Town co-owned spin-off company, and kits for the study were donated by the company. However, Antrum Biotech played no role in study design, data analysis or its publication. The other authors declare that they have no competing interests.SP, JGP, KD and BMM contributed to the conception and design of the study, the acquisition of data, analysis and interpretation of data, and drafting of the manuscript. ZK, RM, GT, UG, and MN contributed to the acquisition and interpretation of data and drafting of the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/12/101/prepubTable S1 Table S2-5 and Figure S1.Click here for file


In this study, we evaluated the safety and pharmacodynamic effects of the Fc-inactivated anti-β-amyloid (anti-Aβ) monoclonal antibody GSK933776 in patients with mild Alzheimer’s disease and mild cognitive impairment. Aβ and tau levels were investigated in cerebrospinal fluid (CSF), and the relationship between Aβ levels and Aβ modulation in plasma was explored. The feasibility of a continuous sampling method using a lumbar catheter was assessed.n = 6/group). The time course of plasma and CSF concentrations of GSK933776 and Aβ was assessed. Sample size was based on feasibility, and no formal statistical analyses were performed.This trial was a phase I, open-label, uncontrolled, single-dose, exploratory experimental medicine study of intravenous GSK933776 at doses of 1 mg/kg, 3 mg/kg or 6 mg/kg (1–42 (from 0 to 12 hours) by 22.8 pg/ml (6.2%), 43.5 pg/ml (9.2%) and 60.5 pg/ml (12.5%), respectively. Plasma concentrations of total Aβ18–35 and Aβ4228–42 increased immediately after infusion and CSF tau concentration increased slightly, but did not significantly change, following administration of all doses of GSK933776. Pharmacokinetics confirmed the presence of GSK933776 in the CSF, which exhibited a dose–response relationship. One patient underwent minor surgery without sequelae following a ruptured lumbar catheter.Following administration of GSK933776 at doses of 1 mg/kg, 3 mg/kg and 6 mg/kg, there were decreases from baseline in CSF AβGSK933776 demonstrated pharmacological activity and target engagement in CSF and plasma, and the continuous sampling method via a catheter successfully assessed the Aβ changes following single-dose administration of GSK933776.NCT01424436. Registered 4 August 2011ClinicalTrials.gov Identifier:  Aggregated β-amyloid peptide (Aβ) is the hallmark of Alzheimer’s disease (AD) brain pathology. As Aβ plaques are one of the underlying pathological features of AD, significant research efforts have been directed at developing antiamyloid therapies to clear or neutralise the amyloid peptide’s toxic forms ,2. Immunex vivo studies of human brain slices taken from patients who had AD, gantenerumab treatment resulted in reduction of brain Aβ levels, indicating that antibody–Aβ complexes had formed [1–42, suggesting that this antibody shifted the Aβ equilibrium sufficiently to mobilise Aβ1–42 from amyloid plaques [Immunotherapy using monoclonal antibodies directed against Aβ is considered to be the preferred method in the search for mechanistic disease-modifying treatments for AD. The key element for an immunotherapy during early clinical development is to demonstrate that the agent engages its target. In the case of anti-Aβ antibodies, the preferred target is the CNS. During the development of the human anti-Aβ antibody gantenerumab, it was initially demonstrated in mice that the antibody bound specifically to Aβ plaques . In an ad formed . Cerebro plaques .Two approaches are being used in AD clinical trials to obtain CSF samples to assess drug penetration and target engagement in the CNS. The lumbar puncture technique relies on sampling at two different time points and was used to determine CSF Aβ levels and drug concentrations in a solanezumab phase II study . AlternaGSK933776 is a humanised mouse anti-human Aβ IgG1 monoclonal antibody that binds with high affinity to the Aβ N terminus and has the Fc region engineered to substantially reduce the fixation of complement and Fc receptor binding, which is responsible for humoral adaptive immunity and cell-mediated tissue damage.1–42, the final peak/trough ratio was ≤2 at GSK933776 ≥ 3 mg/kg. For total Aβ18–35, the final peak/trough ratio was ≤2 at 6 mg/kg (manuscript in submission). The results of our present study will support the dose selection for subsequent larger-scale clinical studies assessing the effect of GSK933776 on cognitive, global and functional endpoints.Herein we present the results of an exploratory experimental medicine study conducted to assess the effects of a single-dose exposure of GSK933776 on Aβ levels in CSF, as well as its relationship with Aβ modulation, in the plasma of patients with mild AD and mild cognitive impairment (MCI). We assessed the time course and dose-dependency of Aβ and tau changes in the first 12 hours after administration of GSK933776 as well as the feasibility of using lumbar catheters to assess these changes. The choice of dose ranges tested in this study was driven by pharmacodynamics detected in a previous study conducted in patients with AD. GSK933776 administered in three repeat doses every 4 weeks at ≥0.1 mg/kg lowered free Aβ concentrations in plasma. For total AβThe study protocol was reviewed and approved by the following ethics committees: Medizinische Ethik-Kommission II, der Medizinischen Fakultät Mannheim, Mannheim, Germany; Ethik-Kommission der Medizinischen Fakultät und am Universitätsklinikum Tübingen, Germany; and Regionala Etiksprovningsnamnden Stockholm, Stockholm, Sweden. The study was conducted in accordance with Good Clinical Practice and the 2008 Declaration of Helsinki. All patients provided written, informed consent to participate in the study and, separately, to undergo genetic analysis.1–42 to <550 pg/ml in CSF and increases in total T-tau to >400 pg/ml or phosphorylated tau 181 to >70 pg/ml in CSF, which are indicative of prodromal or mild AD [prodromal Alzheimer’s disease is used to describe patients who have a hippocampal type of amnestic disorder and a biomarker indicative of the presence of AD, but who are not functionally impaired and do not meet the criteria for Alzheimer’s dementia [Men and nonfertile women ages 50 to 85 years with a clinical diagnosis of probable mild AD, Mini Mental State Examination (MMSE) score of 20 to 26 [ mild AD ,10. The dementia ,12. The dementia .The inclusion criteria for entry into this trial were that patients had to be in good health, determined on the basis of routine clinical laboratory test screening results, physical examinations, vital signs and 12-lead electrocardiograms (ECGs). Exclusion criteria included a history or evidence of any other CNS disorder that could be interpreted as a cause of dementia; Hachinski Ischemic Scale score >4; a magnetic resonance imaging (MRI) scan not consistent with AD or evidence of any other CNS condition; minimal vascular changes or more than three microhaemorrhagic lesions; and a history of psychiatric illness, cerebral haemorrhage, seizures or strokes during the 3 years preceding the study; and cardiovascular disease or diabetes.n = 6 per group) administered by intravenous pump infusion for 1 hour. Patients were followed for up to 56 days postdosing.Recruitment of patients into this open-label, single-dose study  was carried out from May 2010 to December 2011 at three sites in Germany and two in Sweden. Patients were screened on day −30 prior to dosing and randomised to receive doses of GSK933776. The patients were assigned to receive doses of GSK933776 in accordance with the randomisation schedule produced using RandAll, a web server–based clinical trials system. The doses of GSK933776 were 1 mg/kg, 3 mg/kg or 6 mg/kg  relationship of GSK933776 in CSF and plasma and the safety and tolerability of GSK933776.The primary endpoint was measurement of the levels and temporal changes of AβFor the first four patients who received the lowest dose (1 mg/kg), blood and CSF samples were collected at hourly intervals over a 30-hour time period from approximately 11 hours predosing to 19 hours postdosing. The primary sampling time period for evaluation of baseline levels was 6 to 8 hours predosing, whereby 0 to 5 hours postdosing was considered a run-in period during which we obtained stable values. The time period from 0 to 12 hours postdosing was the focus for the postdosing assessment. This duration was initially based on plasma pharmacodynamics observed in our previous study, that is, the time of onset of lowering of free Aβ (manuscript in submission). After the first four patients completed the protocol, their Aβ levels were reviewed and the sample collection times for CSF and blood for both baseline and postdosing were adjusted as follows. CSF samples were collected hourly (3 ml per sample) from 9 hours predosing to 12 hours postdosing. Blood samples were collected hourly from 6 hours predosing to 12 hours postdosing and at follow-up: once on day 7 and once on day 56 postdosing. Total tau and phosphorylated tau 181 were measured in CSF at 5 hours predosing, at the time of dosing and at 5 hours and 12 hours postdosing. Because of the short-term exposure to GSK933776, no changes in total tau or phosphorylated tau 181 were expected.1–42, total tau and phosphorylated tau 181 were measured using INNOTEST kits . For the INNOTEST assay for Aβ1–42, we used 21F12 as a coating monoclonal antibody, which is specific for the C-terminal epitope 42, and 3D5 as a detection antibody, which is specific to N-terminal epitope 1. The INNOTEST assay for phosphorylated-tau 181 uses HT7 as a coating antibody, which is specific for amino acids 159 to 163, and AT270 as a detection antibody, which is specific to phosphorylated Thr181. All CSF analyses were performed batchwise by board-certified laboratory technicians who were blinded to clinical and treatment data. Intra-assay coefficients of variation were below 10%.Insertion of the lumbar catheter was done according to local hospital protocols. Continuous CSF collection was performed according to the standard operating procedures of the study and carried out by medical staff trained in this procedure. CSF Aβg for 15 minutes at 4°C to obtain plasma. A protease inhibitor  was added to 0.5 ml of plasma. Samples were then frozen upright on dry ice or in a freezer at or below −70°C. The specificities of the capture and detection antibodies used in the plasma total Aβ measurements  were as follows. The combinations of the capture antibody 6F6 ) and detection antibody 5G5  detected all Aβ fragments containing an intact C terminus, such as Aβ1–42 and Aβ3–42, which is considered indicative of total Aβ4228–42. 6F6 and the detection antibody 4G8 (amino acids 18 to 22)  detected Aβ fragments containing the amino acid sequence 18 to 35, including Aβ1–38, Aβ1–40 and Aβ1–42, which are considered indicative of total Aβ18–35. Total assays detected both free and GSK933776-bound Aβ fragments. There was no competition between the capture antibody 6FS and GSK933776, because 6F6 recognises Aβ amino acids 28 to 35 and GSK933776 recognises Aβ amino acids 1 to 5. Total levels of Aβ18–35 and total Aβ4228–42 were measured in plasma only.For plasma preparation, blood samples were processed within 30 minutes by centrifugation at 2,000 × Blood samples were collected once prebaseline, once during GSK933776 administration, hourly for 4 hours postdosing and then 6, 8, 10 and 12 hours postdosing as well as at follow-up 7 and 56 days postdosing. CSF samples were collected every hour over a total period of 22 hours, beginning 9 hours predosing and ending 12 hours postdosing. Approximately 0.5 ml of CSF and approximately 3 ml of whole blood were collected at every time point.Concentrations of GSK933776 were determined in plasma and CSF samples using an immunoassay that detects free GSK933776 antibody. Briefly, the humanized monoclonal antibody drug specific to GSK933776 is captured with a Aβ fragment immobilized on a microtitre plate. The plate was washed removing all nonspecific material and GSK933776 is detected using an anti-human IgG (Fc specific) monoclonal antibody conjugated to a reporter tag which is developed and detected by immunoelectrochemiluminescence, resulting in a quantifiable response.Cmax); the time at which Cmax was observed, which was determined directly from raw concentration–time data (tmax); the area under the plasma and CSF concentration–time curve to the last quantifiable concentration (AUCt0–); the area under the plasma and CSF concentration–time curve from time 0 to 12 hours postdosing (AUC0–12); the time of the last observed plasma concentration (tlast); and the concentration at tlast (Ct). The following additional parameters were included for analysis of CSF pharmacokinetics: the areas under the CSF concentration–time curve from time 0 to 4 hours postdosing AUC0–4 and from 5 to 12 hours postdosing AUC5–12 and the time prior to the first measurable  concentration in CSF (tlag).Pharmacokinetics analyses of plasma and CSF GSK933776 were calculated by standard noncompartmental analysis using WinNonlin Pro 5.2 software  and were based on actual sampling times. The following parameters were measured: the first occurrence of the maximum observed plasma and CSF concentrations determined directly from raw concentration–time data (http://www.meddra.org/) criteria.Clinical chemistry and haematology tests, vital signs, 12-lead ECG readings and brain MRI were performed at screening, at intervals throughout the study and at follow-up examinations. Adverse events were coded using the Medical Dictionary for Regulatory Activities  were summarised (absolute) and are presented in individual participant plots Additional file CSF Aβ parameters were summarised using standard descriptive statistics for a range of summary measures  and are presented in individual participant plots. The weighted means were derived by calculating the AUCs using the trapezoidal rule and then dividing by the actual relevant time interval. CSF parameters (tau and phosphorylated tau 181) and plasma parameters . Nine patients were female and six were male, and all were Caucasians. Most patients carried the APOE ϵ4 allele (14 (93%) of 15 patients), and six of these patients carried two copies of the allele.Twenty-six patients were screened. Nine patients were excluded because they failed to meet the inclusion criteria, and two withdrew before dosing began Figure . Fifteenn = 4), 71.0 ± 39.75 pg/ml for 3 mg/kg (n = 5) and 153.9 ± 37.37 pg/ml for 6 mg/kg (n = 6). Mean (±SD) CSF concentrations for tau were 828.3 ± 430.29 pg/ml for the GSK933776 1 mg/kg group (n = 4), 668.2 ± 262.35 pg/ml for 3 mg/kg (n = 5) and 1,209.3 ± 262.35 pg/ml for 6 mg/kg (n = 6). Baseline data for patient CSF Aβ1–42 levels are presented in Table Mean ± standard deviation (SD) CSF concentrations for phosphorylated tau 181 were 90.6 ± 39.36 pg/ml for the GSK933776 1 mg/kg group (1–42 (0 to 12 hours) of 22.8 pg/ml (6.2%), 43.5 pg/ml (9.2%) and 60.5 pg/ml (12.5%), respectively (Table 1–42 (0 to 12 hours) across the dose ranges. Among individual patients, decreases from baseline in CSF Aβ1–42 were observed across the sampling periods after administration of GSK933776 at all dose levels. Decreases were most marked at the 6 mg/kg dose  compared with GSK933776 in plasma, which was detected immediately. This delay was reflected in the CSF/plasma ratios, which increased with time and were in the range of 0 to 0.23%. There were no relationships between changes from baseline in CSF Aβ and any of the CSF pharmacokinetics parameters. Therefore, no PK/PD modelling was performed.A summary of the most frequently reported adverse events is given in Table 1–42 were stable, and there were apparent decreases following all doses of GSK933776 from baseline in CSF Aβ1–42. These decreases seem to have been dose-dependent, with greater reductions associated with higher doses of GSK933776, and time-dependent, with greater reductions occurring 5 hours after administration. In contrast to a previous study, no diurnal variation in CSF Aβ levels was observed [Our findings in this study support target engagement of GSK933776 in CSF. The patients’ baseline levels of CSF Aβobserved . CSF tauobserved .18–35 and total Aβ4228–42 concentrations in plasma following GSK933776 treatment. The pharmacokinetics data confirmed the presence of GSK933776 in CSF, which suggests a central mechanistic model of action rather than a peripheral sink mechanism. Although these two theories are popular in the mechanism of action of AD vaccination, they are not mutually exclusive. Further studies are required to characterise the mechanistic model of GSK933776 mode of action early and late after treatment.We observed an increase in both total Aβ0–∞ and t½ values were not calculated in this study, because more samples would have been required to characterise adequately the switch from the first to the terminal exponential phase. In a previous study of GSK9933776, however, a long terminal elimination half-life (average range: 10 to 15 days) was observed (manuscript in submission). The increase in plasma levels of total Aβ18–35 and Aβ28–42 did not exhibit an apparent dose–response relationship during the short observation time in this study.The findings regarding GSK933776 plasma pharmacokinetics in this study are consistent with those in previous solanezumab single- and multiple-dose studies ,16. In aThis study confirms the presence of GSK933776 in CSF. CSF versus plasma ratios increased with time and were ranged from 0 to 0.23%, reflecting the delay of detection of GSK933776 in CSF by 5.0 hours to 8.5 hours .1–42 were greater with the higher dose of GSK933776 (6 mg/kg) than with the 1 mg/kg and 3 mg/kg dose groups. However, this result might have been driven by the higher baseline CSF Aβ1–42 values in the 6 mg/kg group compared with the other groups. Differing baseline CSF Aβ1–42 values were also observed between the other dose groups. However, all dose groups showed similar trends in CSF Aβ1–42 changes over time, regardless of baseline CSF Aβ1–42 values, suggesting that this limitation did not affect the interpretability of the data. This study also has a small sample size and is not formally powered; therefore, we conducted no formal statistical analyses. Despite the feasibility-driven limited sample size in this study, our sample size is similar to those of other published studies in the field [1–42 assay. The potential for spurious results induced by any possible interaction of GSK933776 with the CSF Aβ1–42 assay was not assessed, thus limiting the interpretation of the results. However, there was no apparent relationship between changes from baseline in CSF Aβ and any CSF pharmacokinetics parameters. The differences in mean baseline CSF Aβ levels observed between the dose groups were most likely due to chance resulting from the small sample size.This study has several limitations. The decreases from baseline in CSF AβAPOE ϵ4 carriers was higher in this study (14 (93%) of 15 patients) than in a typical AD population (64.8%) [APOE ϵ4 data were obtained from patients who failed screening, because pharmacogenetic samples were taken only from patients who were randomised.The observed frequency of  (64.8%) . This diWe believe that, to the best of our knowledge, this study is one of very few intervention studies in which the feasibility of using the CSF catheter to assess short-term Aβ changes has been investigated. One catheter-related serious adverse event occurred during the catheter insertion procedure in our study.The outcomes of phase III studies conducted with the Aβ monoclonal antibodies bapineuzumab and gantenerumab, as well as another pivotal study carried out with solanezumab, have failed to demonstrate unambiguous efficacy in patients with mild to moderate AD, but they have triggered significant new activity in the study of earlier stages of AD.The results of this study support pharmacological activity and target engagement of GSK933776 in both CSF and plasma. They demonstrate that the continuous sampling method using a catheter can be used successfully to assess Aβ changes after single-dose administration of an anti-Aβ antibody in patients with mild AD or MCI.last; ECG: Electrocardiogram; MCI: Mild cognitive impairment; MMSE: Mini Mental State Examination; MRI: Magnetic resonance imaging; tlag: Time prior to the first measurable (nonzero) concentration in cerebral spinal fluid; tlast: Time of the last observed plasma concentration; tmax: Time at which Cmax was observed determined directly from raw concentration time data.AD: Alzheimer’s disease; Aβ: β-amyloid; AUC0–12: Area under the plasma and cerebral spinal fluid concentration–time curve from time 0 to 12 hours postdosing; AUC0–4: Area under the cerebral spinal fluid concentration–time curve from time 0 to 4 hours postdosing; AUC0–t: Area under the plasma and cerebral spinal fluid concentration–time curve to the last quantifiable concentration; AUC5–12: Area under the plasma and cerebral spinal fluid concentration time curve from 5 hours to 12 hours postdosing; Cmax: First occurrence of the maximum observed plasma and cerebral spinal fluid concentration determined directly from raw concentration time data; CNS: Central nervous system; CSF: Cerebrospinal fluid; Ct: Concentration at tMS, XT, AY, SK, AL, MC, CH, JH and PM are GlaxoSmithKline employees. This study was funded by GlaxoSmithKline. TL, NA, AR, CAFvA and HZ were sponsored by GlaxoSmithKline. GlaxoSmithKline sponsored and paid for this study.APOE ϵ4). XT contributed to the study concept and design, partly performed the study and acquired the data. NA participated in the study design, performed the study and obtained and interpreted the data. CAFvA participated in the recruitment of study participants, partly performed the study and reviewed the data. SK was the project manager and reviewed patient data. AR generated and reviewed patient data. MC reviewed safety data. PM participated in the design of the study, performed statistical analyses and provided interpretation of the data. CH contributed to and reviewed the pharmacokinetics bioanalytical data. MS performed mathematical analysis and reviewed pharmacokinetic data. JH contributed to the design and conduct of the study. All authors read and approved the final manuscript.TL participated in the recruitment of study participants, partly performed the study and reviewed the data. AL participated in the acquisition, analysis and interpretation of pharmacodynamic data. HZ contributed to the study concept and design, biochemical analyses and critical review of the manuscript for important intellectual content. AY participated in the delivery and interpretation of genetic data (Individual patient plots of cerebrospinal fluid tau by time and dose level. GSK: GSK933776. Coloured dashed lines refer to each individual patient plot.Click here for fileIndividual patient plots of cerebrospinal fluid phosphorylated tau 181 by time and dose level. GSK: GSK933776; P-TAU: Phosphorylated tau. Coloured dashed line refers to each individual patient plot.Click here for file


Spontaneous intracranial hypotension (SIH) caused by spontaneous spinal cerebrospinal fluid (CSF) leaks produces orthostatic headaches. Although upper arm pain or paresthesia is reportedly associated with SIH from spontaneous spinal CSF leak in the presence of orthostatic headache, low thoracic radicular pain due to spontaneous spinal CSF leak unassociated with postural headache is extremely rare. We report a 67-year-old female who presented with chronic, positional radicular right T11 pain. Computed tomography myelography showed a spontaneous lumbar spinal CSF leak at L2-3 and repeated lumbar epidural blood patches significantly alleviated chronic, positional, and lower thoracic radiculopathic pain. The authors speculate that a chronic spontaneous spinal CSF leak not severe enough to cause typical orthostatic headache or epidural CSF collection may cause local symptoms such as irritation of a remote nerve root. There might be considerable variabilities in the clinical features of SIH which can present a diagnostic challenge. Most spinal cerebrospinal fluid (CSF) leaks are iatrogenic, occurring after lumbar puncture, epidural injection, or spinal surgery. Spontaneous intracranial hypotension (SIH) is a postural headache syndrome unrelated to dural puncture and it is an uncommon but increasingly recognized clinical syndrome associated with reduced CSF pressure or CSF volume caused by spontaneous CSF leaks –5. The iWhen onset of such headache is spontaneous, the diagnostic challenge is created even though SIH has been recognized for more than 6 decades and the diagnosis has been greatly aided by magnetic resonance imaging (MRI) . TypicalUntil now, the symptoms and signs of SIH from spontaneous spinal CSF leak are reported to be associated with the head and the upper extremity , 9. A thA 67-year-old female presented with a 12-month history of chronic radicular pain along the course of right T11. The pain was described as mainly aching, with extreme severity  ranging from 0 to 10). It was aggravated with position change from lying to sitting, and daily activity and walking did not influence the pain. There was no precipitating event before the onset of right thoracic pain and its onset was insidious. The pain was not relieved with nonsteroidal anti-inflammatory drugs, tramadol (300 mg/day), and 20 mg oxycodone b.i.d. and was so agonizing that treatment with repeated injections of diclofenac sodium and on a regular schedule of epidural blocks, intercostal blocks of T11, and tender point injections was done two times a week for a year. However, repeated blocks were unsuccessful. She underwent right-sided L4-5 hemilaminectomy because the pain radiated to the lateral flank and was misdiagnosed as lumbar stenosis 5 months after pain onset. Hemilaminectomy was not effective. Finally, she was referred to our outpatient clinic under the diagnosis of failed back surgery syndrome.On examination, the pain was typically present along the course of the right T11 dermatome from the lower trunk radiating to the right lower abdomen. There was no sensory disturbance including right T11 dermatome and right leg, and no allodynia, tenderness, or limitation of motion of low back and trunk was observed. Laboratory examinations were normal including the erythrocyte sedimentation rate, C-reactive protein, and blood sugar level (95 mg/dL). A Congo red stain of the abdominal fat to rule out amyloidosis was negative. Plain X-rays of lower thoracic and lumbar spine were unremarkable and no pathologic lesion to explain right T11 pain was found in the MRI of the lower thoracic and lumbar spine. Electromyography including nerve conduction study revealed no evidence of radiculopathy or peripheral neuropathy.2O in the recumbent position. The CSF examination showed 10 cells/mm3 and a protein level of 35 mg/dL. No erythrocytes were found.As an evaluation for failed back surgery syndrome, myelography computed tomography (CT) was performed. There was no lesion found causing right T11 pain. However, a prominent CSF leak was noted bilaterally at the level of L2-3  and contOn the basis of the CT myelographic finding of a CSF leak, the provisional diagnosis was spontaneous spinal CSF leak causing low thoracic radiculopathic pain. Considering the chronicity and medical intractability of the radicular pain, combined with the radiological finding of spontaneous CSF leaks, an epidural blood patch (EBP) with 15 mL of autologous blood was performed at the L1-2 level. Immediately after the blood patch, she reported about 30% relief of T11 radicular pain (4–6 on NRS-11). A repeat lumbar EBP was performed one week later, which achieved an additional 30% pain relief (2–5 on NRS-11), and a total of 60% pain relief over baseline was achieved. At 2 weeks after the blood patch, myelographic CT and enhanced lumbar spine MRI were performed to assess the degree of spinal CSF leaks. There was no identifiable CSF leak at the L2-3 level. However, contrast-filling in bilateral nerve root sheaths without CSF leak was identified at T12-L1, L2-3, and L3-4 levels. The small amount of epidural contrast leakage at the level of T10 had disappeared.In the repeated enhanced lumbar spine MRI, there was no typical finding of spontaneous CSF leak, such as epidural CSF collection. However, subtle epidural enhancement was noted from T10 to L3, and a remnant of the injected epidural blood patch was observed. After achieving about 60% pain relief, she was discharged and was maintained with medication .A repeated myelographic CT was performed 3 months after the first EBP showed the same findings obtained in the second myelographic CT, bilateral contrast-filling nerve root sheaths without CSF leak evident at the T12-L1, L2-3, and L3-4, L4-5 levels. An additional blood patch was not performed as the patient could tolerate low thoracic pain (NRS 2–4/10). Thus, the pain in this patient which was of 12 months' duration and severe was relieved after two epidural blood patches to a mild to moderate level (2–4 on NRS-11) at 12-month follow-up.Intracranial hypotension (IH) is a clinical syndrome in which absolute or relative hypovolemia of CSF results in various clinical symptoms . Causes Despite recent awareness of SIH by physicians, the diagnosis of SIH can be missed initially and the diagnostic delay is significant . In a seAs a differential diagnosis of isolated T11 radiculopathic pain, both structural lesions and nonstructural peripheral neuropathic pain should be taken into account. For structural lesions such as foraminal stenosis or disc pathology, there was no obvious pathologic lesion possibly abutting the right T11 root on both MRI and myelographic CT scans. A radionuclide bone scintigraphy was negative and a neoplasm or benign tumor involving peripheral nerve root was also excluded with an enhanced MRI study. Diverse peripheral neuropathic lesion or systemic metabolic disease could involve a thoracic nerve root. Most commonly, diabetic neuropathy, nonvesicular form of herpes zoster , or sarcoid infiltration of nerve root could be considered.A syndrome of painful unilateral or symmetrical multiple neuropathy tends to occur in older patients with relatively mild or even unrecognized diabetes . HoweverAlthough zoster sine herpete (ZSH) could manifest as chronic nonvesicular thoracic radiculopathy , 16, thiSpontaneous CSF leak as a cause of the right T11 radicular pain in the present case may be questioned because there has been no reported case of SIH causing lower thoracic radiculopathy. Furthermore, there was no orthostatic headache. No typical enhanced MRI of the brain was able to be obtained and CSF pressure was normal. The only positive finding that supports the diagnosis of SIH due to spontaneous CSF leak is CT myelographic evidence of CSF leak in the thoracolumbar junction area and subsequent improvement of radicular pain after EBPs.2O , which can be unmeasurable or even negative [However, considerable variability of the clinical features of SIH has been reported –6, 8. Alnegative . HoweverIndeed, the CT myelographic findings of spontaneous CSF leak in the current case were an unexpected finding during evaluation of right T11 radicular pain. Spontaneous CSF leak was most prominent at L2-3 and there was a scant amount of epidural contrast leak at T10. The CSF leaks at L2-3 and T10 disappeared after EBP with concomitant pain relief. In addition, multiple areas of contrast-filling nerve root sheaths were observed on the left side of T12-L1, left L1, and bilateral T1 in initial examination. However, the areas with contrast-filling nerve root sheaths were found bilaterally at the level of T12-L1, L2-3, and L3-4 in the second examination and bilaterally in T12-L1, L2-3, L3-4, and L4-5 in the follow-up examination at 3 months. The exact significance of contrast-filling along the course of the nerve root sheath is uncertain. We think there is a possibility that these changes may implicate an underlying structural weakness of spinal meninges, which causes spontaneous spinal CSF leak , 9, and Relatively less attention is typically given to spinal MRI in the diagnosis of SIH, mainly because it is not particularly effective in localizing the CSF leak . HoweverIt seems that the severity of the spontaneous CSF leak in the present case was obviously milder than those reported in the literature. This CSF leak was not severe enough to cause an orthostatic headache and typical spinal MRI findings of a CSF collection epidurally and in paraspinal muscles , 22, 23.Assuming that thoracic radicular pain unassociated with anatomical pathology presents without positional headache, clinical suspicion of spontaneous CSF leak would not be possible in daily clinical practice if spinal MRI does not show an extensive epidural fluid collection or paraspinal CSF extension. As shown in present case, spontaneous spinal CSF leak as a cause of unexplained, chronic thoracic radicular pain inevitably requires a process of diagnosis of exclusion. We suggest that if unexplained thoracic radicular pain presents without orthostatic headache, even without the brain and spinal findings of SIH, spontaneous spinal CSF leak should be included in the list of differential diagnosis.The occurrence of cervical unilateral radiculopathic pain caused by spontaneous spinal CSF leak has been reported in association with a typical orthostatic headache. The development of an isolated chronic lower unilateral thoracic radicular pain without orthostatic headache may be a rare manifestation of a remotely located spontaneous CSF leak. As shown in our case, disabling lower thoracic unilateral radicular pain of 12-month duration from an L2-3 CSF leak responded at 12-month follow-up after two epidural blood patches. We speculate that a chronic, milder pattern of spontaneous spinal CSF leak may cause a radicular pain without typical symptoms of intracranial hypotension. Spontaneous spinal CSF leak may have a different presentation of remote radicular pain and presents a variability to the clinical features of spontaneous intracranial hypotension.


Batrachochytrium dendrobatidis. Events that lead to the disruption of the bacterial community may have implications for the susceptibility of amphibians to such diseases. Amphibians are often marked both in the wild and in captivity for a variety of reasons, and although existing literature indicates that marking techniques have few negative effects, the response of cutaneous microbial communities has not yet been investigated. Here we determine the effects of passive integrated transponder (PIT) tagging on culturable cutaneous microbial communities of captive Morelet's tree frogs  and assess the isolated bacterial strains for anti-B. dendrobatidis activity in vitro. We find that PIT tagging causes a major disruption to the bacterial community associated with the skin of frogs (∼12-fold increase in abundance), as well as a concurrent proliferation in resident fungi (up to ∼200-fold increase). Handling also caused a disruption the bacterial community, although to a lesser extent than PIT tagging. However, the effects of both tagging and handling were temporary, and after 2 weeks, the bacterial communities were similar to their original compositions. We also identify two bacterial strains that inhibit B. dendrobatidis, one of which increased in abundance on PIT-tagged frogs at 1 day postmarking, while the other was unaffected. These results show that PIT tagging has previously unobserved consequences for cutaneous microbial communities of frogs and may be particularly relevant for studies that intend to use PIT tagging to identify individuals involved in trials to develop probiotic treatments.Symbiotic bacterial communities play a key role in protecting amphibians from infectious diseases including chytridiomycosis, caused by the pathogenic fungus  Batrachochytrium dendrobatidis fungus  and assess the isolated bacterial strains for anti-B. dendrobatidis activity in vitro.Given the role of bacterial communities in protecting amphibians from pathogenic infection, events that lead to the disruption of the bacterial community may have implications for the susceptibility of amphibians to disease. Both in the wild and in captivity, amphibians are often marked for a variety of reasons, for example, to identify individuals, to avoid resampling, or to conduct mark-release-recapture surveys. Historically, toe clipping was often used for identification purposes, although recent advances in other, less-invasive techniques such as passive integrated transponder (PIT) tagging or visible implant elastomer (VIE) dyes are now more commonly used. The existing literature indicates that these marking techniques have very few negative effects on amphibians e.g., –19, but –The Chester Zoo Ethical Committee and the University of Manchester Ethics Committee approved this study prior to its start. This study did not require a license from the United Kingdom's Home Office as PIT tagging is an approved method for the identification of frogs, and data were collected during routine marking of frogs for identification purposes.Agalychnis moreletii frogs were used in this study: 10  in the nonmarked (control) group and 10  in the marked group. All frogs were from an F1 generation of a conservation breeding program at Chester Zoo, United Kingdom, and were maintained in 24- by 18- by 18-in. tanks with a drilled base for drainage. Frogs were maintained in groups of four, but only two frogs from each tank were used in this study, with one frog PIT tagged and the other not marked . This controlled for variation between individuals according to tank. Tanks contained a water dish and cuttings of devil's ivy (Scindapsus sp.), and frogs were fed crickets gut-loaded on fresh fruit and vegetables and dusted with Nutrobal  two to three times weekly.A total of 20 adult Agalychnis moreletii frogs were marked using PIT tags . This method was chosen as frogs were due to be rehomed at other institutions, and PIT tagging gives individuals unique codes for identification. Gloves were worn throughout marking and were changed between frogs to minimize cross-contamination. During marking the frog was restrained on a flat and stable surface, and the loaded PIT tagging needle was inserted smoothly and quickly under the skin into the tibiofibular leg muscle . The PIT tagging process was standardized and took approximately 1 min per individual. To ensure that any changes in microbial communities could be attributed to PIT tagging, nonmarked (control) frogs were handled using the same methods and for the same amount of time as marked individuals but without the insertion of a PIT tag. All frogs were monitored for 2 weeks after marking for any signs of adverse reaction, of which none were observed.2 solution, and serial dilutions were constructed up to a concentration of 10−2. Concentrations of 10−1 and 10−2 were plated out on low-nutrient agar . Bacterial and fungal colony growth of different morphotypes  was counted until negligible new growth was observed . Frogs were checked daily throughout the study and for 2 weeks after for any signs of adverse reaction to the swabbing protocol, of which none were observed.Microbial communities were collected from the skin of all frogs at 1 day before marking, 1 day after marking, and then 2 weeks postmarking to determine if communities had returned to their original compositions. Microbial communities were collected using similar methods as previously described in Antwis et al. . Brieflyhttp://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify each morphotype to genus level. Morphotypes with 99% sequence similarity or greater were considered the same species  according to the manufacturer's instructions using universal primers 27F (5′-GTGCTGCAGAGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-CACGGATCCTACGGGTACCTTGTTACGACT-3′) . rRNA fr species .B. dendrobatidis global panzootic lineage (GPL) isolate AUL 1.2  was used to conduct in vitro chytrid challenges for this study. The chytrid culture was grown in 1% TGhL  liquid medium at 18°C until maximum zoospore production was observed (at around 3 days). Three milliliters of the chytrid culture was spread across the surface of 1% tryptone–1% agar plates and left to dry under a sterile hood, ensuring that plates did not dry out completely but were not too wet as to cause the bacterial streaks to run  and morphotype richness  were then calculated for each sample, along with the total fungal abundance.Bacterial abundance data were Poisson distributed, and so the effect of sampling point , treatment group (marked or control), surface (dorsum or ventrum), gender , and all possible interactions were analyzed using JMP 10 software with a general linear model (GLM) with a Poisson distribution. All terms were highly significant (see Results), and so the percent change in bacterial abundance from premarking to 1-day postmarking was analyzed in JMP 10 using a GLM including all variables and possible interactions.The effect of sampling point, treatment group, surface, gender, and all possible interactions on bacterial morphotype richness was analyzed in JMP 10 using a GLM with stepwise selection , which produced a final model containing surface, gender, and their interaction.Data were combined for the dorsal and ventral surfaces of each frog, and differences in the overall bacterial community compositions according to treatment and gender were analyzed for each sampling point separately using the Adonis function of the Vegan package in RStudio. The variation in bacterial community structure according to sampling point and treatment group was visualized using nonmetric multidimensional scaling (NMDS) in RStudio. In addition, the relative abundance of each bacterial morphotype was calculated for each treatment group within a sampling point and plotted in GraphPad Prism, version 6.B. dendrobatidis bacterial morphotypes identified in this study (see Results). These data were Poisson distributed, and so the percent change in bacterial abundance from premarking to 1 day postmarking was analyzed in JMP 10 using a generalized linear model with a Poisson distribution including treatment and gender.Combined data for the dorsum and ventrum of each individual were also used to determine changes in the abundances of the two anti-Fungal abundance data were Poisson distributed, and so the effect of sampling point, treatment group, surface, gender, and all possible interactions were analyzed in JMP 10 using a generalized linear model with a Poisson distribution.Brevundimonas sp. (KC853150), Chryseobacterium sp. (KC853148), Citrobacter sp. (KC853149), Gordonia sp. (KF444801), Paracoccus sp. strain A (KF444802), Paracoccus sp. strain B (KC853138), Microbacterium sp. strain A (KC853147), Microbacterium sp. strain B (KF444800), and Micrococcus sp. (KF444803).Sequences of the 16S rRNA genes of the following bacteria were deposited in GenBank (accession number): A. moreletii frogs in this study: Brevundimonas sp. (GenBank accession number KC853150), Chryseobacterium sp. (KC853148), Citrobacter sp. (KC853149), Gordonia sp. (KF444801), Paracoccus sp. strain A (KF444802), Paracoccus sp. strain B (KC853138), Microbacterium sp. strain A (KC853147), Microbacterium sp. strain B (KF444800), and Micrococcus sp. (KF444803).A total of nine bacterial morphotypes were isolated from 2 = 355,845.7, df = 23, P < 0.0001), with highly significant effects of all variables  and interactions (P < 0.0001). The GLM for the percent change in bacterial abundance from 1 day premarking to 1 day postmarking was statistically significant . A Student's post hoc analysis showed a significant difference between control and marked frogs (P = 0.024) and a significant interaction between treatment and surface (P = 0.041), with a significant increase in the bacterial abundance associated with the ventral surfaces of frogs that were marked (∼12-fold) at 1 day postmarking . Student's post hoc analyses showed that the ventral surfaces of frogs supported a significantly greater richness than the dorsal surfaces (P = 0.0038) (P = 0.0097). Although there was no significant effect of sampling point or treatment on morphotype richness, Once model reduction had been conducted, the GLM for morphotype richness containing only gender and surface was statistically significant ( 0.0038)  and thatF1,19 = 1.516, P = 0.148), gender , or their interaction . At 1 day postmarking there was a highly significant difference in the overall composition of the bacterial community associated with frogs according to treatment  and a marginally significant interaction between treatment and gender  but no effect of gender alone . At 2 weeks postmarking, there were no significant differences in the overall bacterial community compositions according to treatment , gender , or their interaction . The ordination plot from the NMDS analysis (Microbacterium sp. A (KC853147) and Citrobacter sp. (KC853149), whereas the control frogs supported a greater abundance of other morphotypes such as Gordonia sp. (KF444801), Paracoccus sp. A (KF444802), and Micrococcus sp. (KF444803). Both groups of frogs (marked and control) showed changes in the relative abundances of morphotypes at 1 day postmarking, in particular, an increase in Paracoccus sp. B (KC853138). At 2 weeks postmarking, the relative abundance of morphotypes associated with marked frogs was very similar to that of control frogs , with treatment, gender, and their interaction terms all highly significant (P < 0.0001). Overall Citrobacter sp. KC853149 was significantly more abundant at 1 day postmarking on frogs that were PIT tagged and for males more so than for females.The generalized linear model for change in abundance of Brevundimonas sp. KC853150 was not statistically significant , indicating that the abundance of this anti-B. dendrobatidis morphotype did not change for either treatment group at 1 day postmarking.The generalized linear model for change in abundance of 2 = 4,360.5, df = 11, P < 0.0001). Effect tests show a significant effect of treatment (P < 0.0001) and surface (P < 0.0001) and a significant interaction between sampling point and treatment (P < 0.0001) and between sampling point and surface (P < 0.0001). The abundance of fungal colonies isolated from the ventral surfaces of marked frogs at 1 day postmarking was ∼200-fold greater than that at premarking and ∼12-fold greater than that for the dorsal surfaces  . In addiNine bacterial morphotypes were isolated in this study, which is a relatively low number compared to some studies  significantly increased in abundance at 1 day postmarking for frogs that were PIT tagged although this might be expected, given the significant increase in bacterial abundance overall for PIT-tagged frogs (B. dendrobatidis strain (Brevundimonas sp. KC853150) was not affected by treatment or sampling point during the study although this was a rare bacterial morphotype and so may have been relatively unaffected by the handling and PIT tagging  is not clear. The act of tagging  may have led to changes in hormone production or other biochemical signals  that altered the host biology  or interacted directly with the bacterial community –39. More–B. dendrobatidis bacteria). This may be due to the much greater snout-vent length of females than of males of A. moreletii, meaning that there was a greater surface area for microbes to attach. Gender differences were not seen in the closely related Agalychnis callidryas frogs . Future studies may need to take into account potential differences in microbial communities between genders , particularly in the development of probiotic treatments.A number of the results presented here indicate differences between genders  on the ventrum than the dorsum, possibly arising from the greater fluxes of water and solutes experienced across the ventral surfaces of frogs, leading to richer and more abundant bacterial communities , as well40–43–40–In conclusion, PIT tagging causes a major disruption to the bacterial community associated with the skin of frogs, as well as a concurrent proliferation in resident fungi. This may be particularly relevant for studies that intend to use PIT tagging to follow individual animals involved in field studies for probiotic treatments, as changes in bacterial communities may be confounded by the method used to mark amphibians. Investigation into the mechanism that drives this phenomenon is warranted, in addition to studies that determine whether PIT tagging increases the susceptibility of amphibians to infectious diseases. Furthermore, studies are required to determine the effects of other marking techniques on the bacterial communities of amphibians.


APP/PS1 mice were treated for 5 months between the age of 3.5 and 8.5 months. SAR255952 reduced amyloid load in 8.5-months-old animals, but not in 5.5-months animals compared to mice treated with a control antibody (DM4). Histological evaluation confirmed the reduction of amyloid load and revealed a lower density of amyloid plaques in 8.5-months SAR255952-treated animals. The longitudinal follow-up of individual amyloid plaques by MRI revealed that plaques that were visible at 5.5 months were still visible at 8.5 months in both SAR255952 and DM4-treated mice. This suggests that the amyloid load reduction induced by SAR255952 is related to a slowing down in the formation of new plaques rather than to the clearance of already formed plaques.Extracellular deposition of β amyloid plaques is an early event associated to Alzheimer’s disease. Here, we have used  Alzheimer’s disease (AD) is the most common neurodegenerative disease of the central nervous system. It is characterized by two major microscopic lesions: β amyloid deposits in the form of extracellular amyloid β (Aβ) plaques and neurofibrillary tangles (NFTs) made of abnormal intracellular tau protein aggregates . Aβ is cIn vivo imaging of amyloid plaques is useful to evaluate anti-amyloid therapies and/or mechanisms associated with amyloid plaque production either at the clinical or preclinical levels. In humans, in vivo neuroimaging studies of amyloid plaques are performed with Positron emission tomography (PET) using different PET ligands .The ability to use MR imaging to follow-up anti-amyloid therapies is still an opened question. Two studies showed that oid load , but to 1.SAR255952 is a mouse monoclonal aglycosylated IgG1 antibody engineered to reduce the risk of vasogenic edemas and microhemorrhages that have been associated to anti-amyloid immunotherapies . These pTransgenic mouse models of amyloidosis were treated between the age of 3.5 and 8.5 months and were imaged by MRI twice at 5.5 and 8.5 months. Gd-stained longitudinal MRI allowed to follow-up individual amyloid plaques from 5.5 to 8.5 months and revealed that plaques that were visible at the first time point were still detected at the second time point. Also, MRI, as well as histology, showed a reduction of amyloid load in the SAR255952-treated animals.n = 26) and PS1 (n = 20) transgenic mice overexpressing amyloid precursor protein (APP) and/or presenilin 1 (PS1) mutations associated with familial AD [double Thy1 APP751SL Swedish (KM670/671NL) and London (V717I) mutations introduced in the human APP751 sequence] × HMG PS1M146L transgenic mouse line ; (2) APP/PS1 mice immunized with the control DM4 antibody (n = 12); (3) PS1 mice immunized with the SAR255952 antibody (n = 10) and (4) PS1 mice immunized with the control DM4 antibody (n = 10). All animals were 3.5-months-old at the beginning of the immunization and were treated for 5 months, i.e., until 8.5 months. The PS1 amyloid-free animals survived during the whole study while mortality was higher in the APP/PS1 mice  injections of a mouse monoclonal aglycosylated IgG antibody . Age-matched control mice received weekly intraperitoneal injections of a control antibody that does not recognize murine antigens . Four groups of mice were used: (1) APP/PS1 mice immunized with the SAR255952 antibody , to the animals as previously described  interfaced with a console running VnmrJ 2.3. The spectrometer was equipped with a rodent gradient insert of 700 mT/m. A birdcage coil  was used for emission and an actively decoupled mouse brain surface coil  was used for reception. MR images were recorded using a high-resolution 3D-Gradient Echo sequence with a resolution of 29∗29∗117 μm3 . MR images were recorded starting at 60 min after administration of the Gd-DOTA contrast agent used for Gd-staining. During the MRI experiment the animals were anesthetized with a mixture of isoflurane (0.75–1.5%) and carbogen (95% O2 – 5% CO2) and their breathing rate was monitored. Carbogen was used to reduce the signal coming from the circulating blood  were drawn on each of these slices . The eight slices were positioned so that the third slice was localized at the level of the anterior commissure. The coordinates of the first and last slices were thus 1.08 and -2.2 mm compared to the Bregma  using a peristaltic pump (flow rate ∼2 mL/min). The brains were then carefully extracted, post-fixed in formalin for 24-h at 4°C and finally stored in PBS at 4°C.2 in order to perform histological analysis. Formalin-fixed brains were frozen and cut along the rostro-caudal axis. Five series of 25-μm-thick coronal sections were collected. The first, second and third series were used for Nissl staining, Aβ peptide deposits staining , and immunoglobulin G (IgG) staining (antimouse secondary antibodies IHC), respectively. A flatbed scanner  was used to digitize all stained series . Thus, for each stain, 80 sections, evenly spaced by 125 μm across the entire cerebrum were digitized.The brains were then processed by NeuroScience Associates2 each) were drawn on each of these slices (two in each hemisphere). Each stained section was binarized after conversion of the images into gray scale images and thresholded. Amyloid plaques were further segmented by using a watershed algorithm. The proportion of tissue area occupied by 6E10 or anti-IgG immunoreactivity, as well as the number of amyloid plaques and their average sizes were quantified within each ROI by using the “area fraction” and “analyze particles” tools of the imageJ measurement process. The ratio (stained surfaces/ROI surfaces) was taken as a global measure of the cerebral “amyloid load” or “IgG load” of each individual mouse. Thus, for each staining, 64 observations were performed for each animal.Amyloid plaques and IgG staining were quantified from histological sections by using ImageJ and a protocol similar to that used to quantify plaques from MRI: eight coronal slices stained with 6E10 or anti-IgG, evenly spaced by 500 μm, were selected and four circular ROIs .Statistical analysis was performed using Statistica 7 software . The significance of between-group differences was tested by repeated measures ANOVA for longitudinal study and Student’s ∗29∗117 μm3. Hypointense spots were seen in the cortex , hippocampus , thalamus and septal nuclei of transgenic APP/PS1 mice after Gd-staining protocols. No hypointense spots were observed in the brain of PS1 amyloid-free mice at 5.5 and 8.5 months . Registration between MR images and histological sections confirmed that the hypointense spots on MR images were amyloid plaques .Three-dimensional MR images of APP/PS1 and PS1 mice were recorded with a resolution of 29Figures 2A,B) and SAR255952-treated animals . In both treatment groups the plaques that were present in 5.5-months-old animals were still present at 8.5 months . Some new plaques occurred between the age of 5.5 and 8.5 months .APP/PS1 mouse models of amyloidosis were passively immunized with SAR255952 or with a control (DM4) antibody from 3.5 months old until 8.5 months-old. The mice were followed-up in a longitudinal way and MR images were recorded 2 and 5 months after the beginning of the treatment, i.e., at 5.5 and 8.5 months-old. For each animal, we registered MR images recorded at 5.5 and 8.5 months in order to monitor the evolution of each hypointense spots corresponding to amyloid plaques. The visual observation of these images revealed an age-related increased in amyloid load in both DM4 . Amyloid load increased in both DM4 and SAR255952-treated animals between the ages of 5.5–8.5 months . At 8.5 months, the amyloid load detected by MRI was 24% lower in the SAR255952-treated mice compared to the DM4-treated mice . Histological evaluations confirmed the lower amyloid load in 8.5-months-old APP/PS1 animals treated with SAR255952 compared to animals treated with the control antibody . In addition, histological quantifications revealed that the number of amyloid plaques (plaque density) was reduced by 33% in the SAR255952-treated animals compared to the DM4-treated animals , whereas the size of the plaques was not significantly different in the two groups .Quantitative studies of MRI sections revealed a similar amyloid load in the 5.5-months-old animals treated with DM4 or SAR255952 antibodies  while we did not detect any plaques labeled by IgG in the DM4-treated animals . The quantification of IgG in brain sections spreading from the anterior to the posterior parts of the brain showed that IgG was equally distributed on all the brain sections . There was also a strong positive correlation between the IgG load and the amyloid load  or amyloid density  detected by histology.We stained IgG deposition in the brain to further evaluate the interaction between IgG and amyloid. In SAR255952-treated animals, IgG decorated amyloid plaques from all the brain regions examined . It can be used to monitor individual amyloid plaques in the whole brain and to assess the dynamics of their formation and clearance. Interestingly, we were able to demonstrate the very long stability (3 months) of plaques already deposited in the brain. One can argue that the registration of amyloid plaques detected from Gd-stained MRI and immunohistochemical sections was not perfect. This might be related to limitations in the precision of co-registration because of the different thickness of immunohistochemical (25 μm) and MRI sections (117 μm). This can also be related to the different mechanisms leading to plaque detection by MRI or immunohistochemistry. With Gd-stained MRI, the ability to detect the plaques is related to their hydrophobicity , could also be administered together with gadolinium to increase its uptake into the brain  as compared to doses commonly used for intra-venous administration (0.1–0.2 mmol/kg). Also, the contrast agent that we used is largely used in the clinic including for applications that are based on its penetration into the brain . Its safhe brain . Finallyhe brain .Multiple mechanisms have been involved in the efficacy of anti-amyloid immunotherapies. For example, some of them clear aggregated forms of Aβ , whereasOn the basis of our histological study, we also reported that the lower amyloid load in the SAR255952-treated animals was linked to a reduction of amyloid plaque density and not to a diminution of the size of the plaques. One explanation for this pattern of amyloid load modulation can come from recent models showing that, in the absence of therapy, amyloid load increases following three temporal phases . In the in vivo with a conventional MR contrast agent, as long as the contrast agent remains furtive for the BBB.We have shown that Gd-stained MR imaging can be used to monitor individual amyloid plaques in a longitudinal way, to test the efficacy of an anti-amyloid immunotherapy in APP/PS1 mice. This method can be used in addition to histological evaluations as a useful tool for the development of new therapies. Future improvement of the method may be based on the automatic segmentation of the amyloid load to speed-up plaque quantification after Gd-staning . Also, oMS, Thomas Debeir, TR, and MD designed the study. LP, CC, Thomas Debeir, TR provided the immunotherapy. Thomas Debeir, TR, and MD coordinated the study. MS and MD performed the MRI experiments and analysis. MS, MV, A-SH, Thierry Delzescaux, and MD designed and performed the histological analysis. MS and MD wrote the manuscript. MV, A-SH, Thomas Debeir, and TR revised the manuscript.LP, CC, TD, and TR are full employees by Sanofi. All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.