Colony-stimulating Factor Activity Research Articles

Study of cerebrospinal fluid adenosine deaminase activity for differential diagnosis of tuberculous and non-tuberculous meningitis

Background: Diagnosing a case of tuberculous meningitis (TBM) has always been challenging. Numerous previous studies have demonstrated that cerebrospinal fluid-Adenosine deaminase (CSF-ADA) estimation is useful in the diagnosis of TBM and can differentiate TBM from other types of meningitis. Aims and Objectives: The current study aims to look for a simple, rapid, cost-effective, and specific test to differentiate tubercular etiology from other causes of meningitis. Materials and Methods: A total of 80 patients admitted to the hospital with signs and symptoms of meningitis were selected and divided into two groups: Group 1 (Cases): 40 patients of TBM, Group 2 (Control): 40 patients of pyogenic and viral meningitis. The CSF ADA was estimated by enzymatic method, CSF Protein by turbidimetric method, and CSF Glucose by Glucose oxidase (GOD) and Peroxidase (POD) method. Results: CSF ADA levels (mean ± SD) in the TBM and non-TBM groups were 10.874+7.72 IU/L and 2.44+1.757 IU/L, respectively (highly significant, P<0.001). With the CSF ADA cut off as >6 IU/L, the sensitivity of the test was 95%, and the specificity of 92.5%. On comparison of the values of CSF ADA in the two groups, the t value is −6.75, and the difference in these two values was found to be highly significant (P<0.01). Conclusion: ADA estimation in CSF is a simple, inexpensive, rapid, and specific method for making a diagnosis of tuberculous etiology in TBM. As a screening test, the determination of ADA activity in CSF can help the physician diagnose TBM early and reduce irreversible brain damage and neurologic sequelae by early administration of anti-tuberculous medications.

Alpha‐synuclein co‐pathology in Alzheimer’s disease identified by RT‐QUIC identification

AbstractBackgroundalpha‐synuclein co‐pathology may influence Alzheimer’s disease (AD) phenotype but its presence has never been tested in large series of patients. Real‐time quaking‐induced conversion (RT‐QuIC) recently provided evidence of alpha‐synuclein seeding activity in CSF and olfactory mucosa of patients with alpha‐synucleinopathiesMethoda preliminary set of 82 AD patients (mean age 70,3 + 7.3 years) underwent CSF analyses for Tau, P‐tau and A‐beta amyloid and an extensive cognitive, behavioral and motor assessment. RT‐QuiC for alpha‐synuclein was performed on CSF samples.Resultalpha synuclein RT‐QuIC resulted positive in thirty‐nine out of 82 AD patients (47.6%). AD patients with positive alpha‐synuclein were comparable for age and disease severity those negative to RT‐QuIC assay including Tau/P‐Tau and A‐beta CSF markers in cross‐sectional analyses but exhibited higher prevalence of parkinsonism (p=0.02).Conclusionthese preliminary findings indicate that alpha‐synuclein co‐pathology might be detected in more than one third of Alzheimer’s disease patients. These findings might potentially explain the clinical variability of AD including age at onset, progression and response to treatment observed in clinical series. Larger longitudinal studies are warranted to confirm and extend these findings.

PKCα/ZFP64/CSF1 axis resets the tumor microenvironment and fuels anti-PD1 resistance in hepatocellular carcinoma

Despite remarkable advances in treatment, most patients with hepatocellular carcinoma (HCC) respond poorly to anti-programmed cell death 1 (anti-PD1) therapy. A deeper insight into the tolerance mechanism of HCC against this therapy is urgently needed. We performed next-generation sequencing, multiplex immunofluorescence, and dual-color immunohistochemistry and constructed an orthotopic HCC xenograft tumor model to identify the key gene associated with anti-PD1 tolerance. A spontaneously tumorigenic transgenic mouse model, an invitro coculture system, mass cytometry, and multiplex immunofluorescence were used to explore the biological function of zinc finger protein 64 (ZFP64) on tumor progression and immune escape. Molecular and biochemical strategies like RNA-sequencing, chromatin immunoprecipitation-sequencing and mass spectrometry were used to gain insight into the underlying mechanisms of ZFP64. We showed that ZFP64 is frequently upregulated in tumor tissues from patients with anti-PD1-resistant HCC. Elevated ZFP64 drives anti-PD1 resistance by shifting macrophage polarization toward an alternative activation phenotype (M2) and fostering an inhibitory tumor microenvironment. Mechanistically, we primarily demonstrated that protein kinase C alpha (PKCα) directly phosphorylates ZFP64 at S226, leading to its nuclear translocation and the transcriptional activation of macrophage colony-stimulating factor (CSF1). HCC-derived CSF1 transforms macrophages to the M2 phenotype to drive immune escape and anti-PD1 tolerance. Notably, Gö6976, a protein kinase inhibitor, and lenvatinib, a multi-kinase inhibitor, reset the tumor microenvironment and restore sensitivity to anti-PD1 by blocking the PKCα/ZFP64/CSF1 axis. We propose that the PKCα/ZFP64/CSF1 axis is critical for triggering immune evasion and anti-PD1 tolerance. Inhibiting this axis with Gö6976 or lenvatinib overcomes anti-PD1 resistance in HCC. Despite remarkable treatment progress, most patients with hepatocellular carcinoma respond poorly to anti-PD1 therapy (a type of immunotherapy). A deeper insight into the tolerance mechanisms to this therapy is urgently needed. Herein, we unravel a previously unexplored mechanism linking tumor progression, macrophage polarization, and anti-PD1 resistance, and offer an attractive novel target for anti-PD1 combination therapy, which may benefit patients with hepatocellular carcinoma.

Immunomodulatory Activity of Granulocyte Colony-Stimulating Factor and its Therapeutic Effect on Liver Failure

Abstract Liver failure is characterized by the rapid deterioration of liver function, often accompanied by ascites, coagulation dysfunction, hepatic encephalopathy, and other critical complications. Owing to the complex multifaceted pathogenesis and consequential clinical manifestations of the disease, liver failure displays poor prognosis and warrants comprehensive clinical treatment and management. Liver transplantation remains the only well-established treatment for liver failure. However, several factors including transplantation cost and low organ donation rates limit the rate of liver transplantation. The development of a suitable therapy for liver failure is a significant challenge and remains a cause of concern for the medical world. Granulocyte colony-stimulating factor (G-CSF), a member of the cytokine family of hematopoietic growth factors, is involved in the migration of hematopoietic stem cells into the damaged liver, and effectuates their dedifferentiation into hepatocytes. Liver regeneration involves a complex crosstalk of multiple cell types, including hepatocytes, endothelial cells, and inflammatory cells. Neutrophils and monocytes/macrophages that present different types of innate immune cells were found to play a crucial role in the progression of inflammation and restoration of the liver tissue. G-CSF, known as the most common used cytokine, may also affect these immune cells by combining G-CSF receptors on their surface. The immunomodulatory activity of G-CSF should be studied and described in order to ascertain its therapeutic effect on liver failure.

Alpha-synuclein seeds in olfactory mucosa of patients with isolated REM sleep behaviour disorder.

Isolated REM sleep behaviour disorder (RBD) is an early-stage α-synucleinopathy in most, if not all, affected subjects. Detection of pathological α-synuclein in peripheral tissues of patients with isolated RBD may identify those progressing to Parkinson's disease, dementia with Lewy bodies or multiple system atrophy, with the ultimate goal of testing preventive therapies. Real-time quaking-induced conversion (RT-QuIC) provided evidence of α-synuclein seeding activity in CSF and olfactory mucosa of patients with α-synucleinopathies. The aim of this study was to explore RT-QuIC detection of α-synuclein aggregates in olfactory mucosa of a large cohort of subjects with isolated RBD compared to patients with Parkinson's disease and control subjects. This cross-sectional case-control study was performed at the Medical University of Innsbruck, Austria, the Hospital Clinic de Barcelona, Spain, and the University of Verona, Italy. Olfactory mucosa samples obtained by nasal swab in 63 patients with isolated RBD, 41 matched Parkinson's disease patients and 59 matched control subjects were analysed by α-synuclein RT-QuIC in a blinded fashion at the University of Verona, Italy. Median age of patients with isolated RBD was 70 years, 85.7% were male. All participants were tested for smell, autonomic, cognitive and motor functions. Olfactory mucosa was α-synuclein RT-QuIC positive in 44.4% isolated RBD patients, 46.3% Parkinson's disease patients and 10.2% control subjects. While the sensitivity for isolated RBD plus Parkinson's disease versus controls was 45.2%, specificity was high (89.8%). Among isolated RBD patients with positive α-synuclein RT-QuIC, 78.6% had olfactory dysfunction compared to 21.4% with negative α-synuclein RT-QuIC (P < 0.001). The extent of olfactory dysfunction was more severe in isolated RBD patients positive than negative for olfactory mucosa a-synuclein RT-QuIC (P < 0.001). We provide evidence that the α-synuclein RT-QuIC assay enables the molecular detection of neuronal α-synuclein aggregates in olfactory mucosa of patients with isolated RBD and Parkinson's disease. Although the overall sensitivity was moderate in this study, nasal swabbing is attractive as a simple, non-invasive test and might be useful as part of a screening battery to identify subjects in the prodromal stages of α-synucleinopathies. Further studies are needed to enhance sensitivity, and better understand the temporal dynamics of α-synuclein seeding in the olfactory mucosa and spreading to other brain areas during the progression from isolated RBD to overt α-synucleinopathy, as well the impact of timing, disease subgroups and sampling technique on the overall sensitivity.

High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis

AbstractDeficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mice results in pulmonary alveolar proteinosis (PAP) from impaired surfactant catabolism by alveolar macrophages (AMs). Recently, we have shown that neutralizing anti-GM-CSF autoantibodies develop specifically in patients with idiopathic pulmonary alveolar proteinosis (iPAP). Analogous to murine PAP models, it is plausible that the autoantibodies reduce GM-CSF activity, resulting in AM dysfunction and surfactant accumulation. To examine this hypothesis, we estimated the neutralizing activity of the autoantibodies in the lungs of patients and characterized their biologic properties. GM-CSF bioactivity was completely abrogated in the bronchoalveolar lavage fluid (BALF) of patients with iPAP but not in healthy subjects. Autoantibodies were present in the alveoli in high concentrations and colocalized with GM-CSF. They recognized human GM-CSF with high avidity (KAV = 20.0 ± 7.5 pM) and high specificity, reacting with its superstructure and neutralizing GM-CSF activity to a level 4000 to 58 000 times the levels of GM-CSF normally present in the lung. Although target epitopes varied among patients, GM-CSF amino acids 78 to 94 were consistently recognized. Thus, autoantibodies bind GM-CSF with high specificity and high affinity, exist abundantly in the lung, and effectively block GM-CSF binding to its receptor, inhibiting AM differentiation and function. Our data strengthen the evidence associating anti-GM-CSF autoantibodies with the pathogenesis of this disease. (Blood. 2004;103:1089-1098)

Abstract CT149: SNDX-6352-0502 - A phase 1, open-label, dose escalation trial to investigate the safety, tolerability, pharmacokinetics and pharmacodynamic activity of SNDX-6352 monotherapy in patients with unresectable, recurrent, locally-advanced, or metastatic solid tumors

Abstract Background: Immunosuppressive tumor associated macrophages (TAMs) have been shown to correlate with poor prognosis. SNDX-6352 is a humanized, high-affinity monoclonal IgG4 antibody that blocks dual ligand activation of colony stimulating factor- 1 receptor (CSF-1R), which regulates monocyte migration, proliferation and differentiation into TAMs. By inhibiting and reducing TAM numbers, SNDX-6352 may slow tumor growth and enhance anti-tumor immunotherapeutic approaches. Methods: SNDX-6352-0502 was a multi-center Phase 1 study consisting of Phase 1a (monotherapy) and Phase 1b (combination with durvalumab) in patients with pretreated, advanced solid tumors. The primary objective was determining the MTD and RP2D of SNDX-6352 alone and in combination with durvalumab. Dose regimens of 1, 2, 3 and 6 mg/kg bi-weekly (q2wks) and 6 mg/kg once monthly (q4wks) were administered in 1a, and 1, 2, and 3 mg/kg bi-weekly in 1b. Drug pharmacokinetics (PK) and pharmacodynamic (PD) biomarkers were measured. Results: Thirty-two patients with advanced solid tumors were enrolled with a median age of 61. The median number of prior therapies was 5 (range 2-12) and 72% of patients had ECOG performance status of 1. Median exposure in terms of cycles was 2, maximum was 10. Three patients had SD greater than 4 months. Most common (&amp;gt; 10%) treatment-related AEs were fatigue (31%), periorbital edema (31%) aspartate aminotransferase increased (22%), blood creatinine phosphokinase increased (22%) nausea (13%) and decreased appetite (13%). Grade 3/4 related AEs were observed in 12 patients (38%) with ≥ 2 events in CPK (5), amylase (3), AST (3) and lipase (2). One possibly related SAE of pneumonitis at 6 mg/kg q2wks was reported. DLTs reported in two subjects were G3 fatigue at 2 mg/kg and G3 pneumonitis at 6 mg/kg q2wks. Laboratory abnormalities included increases in blood creatinine phosphokinase, ALT, AST, amylase, and lipase. These abnormalities were all asymptomatic and are consistent with the known class effect of CSF-1Ri. There was no evidence of hepatocellular toxicity. Plasma concentrations of SNDX-6352 increased in a dose-proportional manner with drug accumulation observed at &amp;gt; 1 mg/kg. Plasma CSF1 and IL-34 concentrations increased with treatment and remained elevated at all doses &amp;gt; 1 mg/kg. CSF1 receptor occupancy was saturated at 4 hours post-dose in all treatment cohorts. Circulating non-classical monocytes (CD14+CD16hi) were ablated at all dose levels within 24 hrs and remained suppressed at all doses &amp;gt;1 mg/kg. Conclusion: SNDX-6352 demonstrates tolerability at the highest dose level evaluated (6 mg/kg) with robust PD biomarker modulation at doses as low as 1 mg/kg. A RP2D of 6mg/kg q4wks will be explored in future solid tumor studies. Citation Format: Nilo Azad, Drew Rasco, Sunil Sharma, Matthew Taylor, Christine Quaranto, David L. Tamang, Robert Nordness, Michael L. Meyers, Serap Sankoh, Peter Ordentlich, Anthony W. Tolcher. SNDX-6352-0502 - A phase 1, open-label, dose escalation trial to investigate the safety, tolerability, pharmacokinetics and pharmacodynamic activity of SNDX-6352 monotherapy in patients with unresectable, recurrent, locally-advanced, or metastatic solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT149.

TDP-43 real-time quaking induced conversion reaction optimization and detection of seeding activity in CSF of amyotrophic lateral sclerosis and frontotemporal dementia patients.

The pathological deposition of the transactive response DNA-binding protein of 43 kDa occurs in the majority (∼97%) of amyotrophic lateral sclerosis and in around 45% of frontotemporal lobar degeneration cases. Amyotrophic lateral sclerosis and frontotemporal lobar degeneration clinically overlap, presenting a continuum of phenotypes. Both amyotrophic lateral sclerosis and frontotemporal lobar degeneration lack treatments capable of interfering with the underlying pathological process and early detection of transactive response DNA-binding protein of 43 kDa pathology would facilitate the development of disease-modifying drugs. The real-time quaking-induced conversion reaction showed the ability to detect prions in several peripheral tissues of patients with different forms of prion and prion-like diseases. Despite transactive response DNA-binding protein of 43 kDa displays prion-like properties, to date the real-time quaking-induced conversion reaction technology has not yet been adapted to this protein. The aim of this study was to adapt the real-time quaking-induced conversion reaction technique for the transactive response DNA-binding protein of 43 kDa substrate and to exploit the intrinsic ability of this technology to amplify minute amount of mis-folded proteins for the detection of pathological transactive response DNA-binding protein of 43 kDa species in the cerebrospinal fluid of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients. We first optimized the technique with synthetic transactive response DNA-binding protein of 43 kDa–pre-formed aggregates and with autopsy-verified brain homogenate samples and subsequently analysed CSF samples from amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients and controls. Transactive response DNA-binding protein of 43 kDa real-time quaking-induced conversion reaction was able to detect as little as 15 pg of transactive response DNA-binding protein of 43 kDa aggregates, discriminating between a cohort of patients affected by amyotrophic lateral sclerosis and frontotemporal lobar degeneration and age-matched controls with a total sensitivity of 94% and a specificity of 85%. Our data give a proof-of-concept that transactive response DNA-binding protein of 43 kDa is a suitable substrate for the real-time quaking-induced conversion reaction. Transactive response DNA-binding protein of 43 kDa real-time quaking-induced conversion reaction could be an innovative and useful tool for diagnosis and drug development in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The cerebrospinal fluid detection of transactive response DNA-binding protein of 43 kDa pathological aggregates may be exploited as a disease biomarker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients.

Transmission of CJD from nasal brushings but not spinal fluid or RT-QuIC product.

ObjectiveThe detection of prion seeding activity in CSF and olfactory mucosal brushings using real‐time quaking‐induced conversion assays allows highly accurate clinical diagnosis of sporadic Creutzfeldt–Jakob disease. To gauge transmission risks associated with these biospecimens and their testing, we have bioassayed prion infectivity levels in patients’ brain tissue, nasal brushings, and CSF, and assessed the pathogenicity of amplified products of real‐time quaking‐induced conversion assays seeded with Creutzfeldt–Jakob disease prions.MethodsWe obtained olfactory mucosal brushings and CSF from patients with a final diagnosis of sporadic Creutzfeldt–Jakob disease subtype MM1 (n = 3). Samples were inoculated intracerebrally into Tg66 transgenic mice that overexpress the homologous human 129M prion protein. The mice were evaluated for clinical, neuropathological, and biochemical evidence of prion infection.ResultsPatients’ brain tissue at 102 to 105 fold dilutions affected 47/48 Tg66 mice. In contrast, maximum acutely tolerable doses of insoluble pellets from their olfactory mucosa brushings caused evidence of prion disease in only 4/28 inoculated mice, and no effects were seen with 10‐fold dilutions. No clinical prion disease was observed in mice inoculated with antemortem CSF samples or prion‐seeded real‐time quaking‐induced conversion assay products.InterpretationPellets from patients’ olfactory mucosa brushings had ≥10,000‐fold lower infectivity per unit volume than brain tissue, while CSF lacked detectable infectivity. Nonetheless, the results suggest that appropriate precautions may be warranted in surgical interventions involving the olfactory areas. The lack of pathogenic infectivity in the real‐time quaking‐induced conversion assay products provides evidence that the assay does not replicate biohazardous prions in vitro.

Intrathecal B-cell activation in LGI1 antibody encephalitis.

ObjectiveTo study intrathecal B-cell activity in leucine-rich, glioma-inactivated 1 (LGI1) antibody encephalitis. In patients with LGI1 antibodies, the lack of CSF lymphocytosis or oligoclonal bands and serum-predominant LGI1 antibodies suggests a peripherally initiated immune response. However, it is unknown whether B cells within the CNS contribute to the ongoing pathogenesis of LGI1 antibody encephalitis.MethodsPaired CSF and peripheral blood (PB) mononuclear cells were collected from 6 patients with LGI1 antibody encephalitis and 2 patients with other neurologic diseases. Deep B-cell immune repertoire sequencing was performed on immunoglobulin heavy chain transcripts from CSF B cells and sorted PB B-cell subsets. In addition, LGI1 antibody levels were determined in CSF and PB.ResultsSerum LGI1 antibody titers were on average 127-fold higher than CSF LGI1 antibody titers. Yet, deep B-cell repertoire analysis demonstrated a restricted CSF repertoire with frequent extensive clusters of clonally related B cells connected to mature PB B cells. These clusters showed intensive mutational activity of CSF B cells, providing strong evidence for an independent CNS-based antigen-driven response in patients with LGI1 antibody encephalitis but not in controls.ConclusionsOur results demonstrate that intrathecal immunoglobulin repertoire expansion is a feature of LGI1 antibody encephalitis and suggests a need for CNS-penetrant therapies.

EPSTEIN BARR VIRUS INFECTION CAUSING SECONDARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS AND MULTIPLE ORGAN FAILURE

SESSION TITLE: Wednesday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/23/2019 09:45 AM - 10:45 AM INTRODUCTION: Hemophagocytic Lymphohistiocytosis (HLH) is a hyperinflammatory immune disorder explained by an overactivation of monocytes, lymphocytes and macrophages resulting in overproduction of cytokines. Secondary HLH is caused by infection, malignant tumors and autoimmune disease. Amongst these, 70% of cases are triggered by Epstein-Barr virus (EBV). We present a case of EBV infection leading to HLH and multiple organ failure. CASE PRESENTATION: 64-year-old gentleman with a past medical history significant for ischemic cardiomyopathy status post biventricular implantable cardioverter defibrillator, chronic kidney disease, peripheral arterial disease status post right above knee amputation initially presented as a transfer from an outside facility with concerns for septic shock. He was afebrile, blood pressure of 94/35 mm mercury, heart rate of 73/ minute. He required mechanical ventilation and vasopressors soon thereafter. Patient was not improving despite receiving broad-spectrum antibiotics and antifungals. Upon further investigation, his serum was positive for EBV viremia with low viral load . His liver enzymes were markedly elevated, Ferritin was >4500, triglycerides 316, Natural killer cell activity was 5 and CD25 (soluble IL-2) 14,131. Bone marrow biopsy showed increased histiocytes containing fully formed cellular debris, fully formed peripheral blood and bone marrow precursor elements representing hemophagocytosis. All other infectious, viral, fungal and tick-borne work up was negative. He was given 10 mg/meter square of dexamethasone and one dose of etoposide. He was transferred to a tertiary center for further management. Unfortunately - his condition markedly declined, and treatment was stopped prematurely with home hospice measures. DISCUSSION: HLH is the extreme end of the spectrum with uncontrolled hyperinflammation leading to multiorgan failure. In EBV-HLH the virus infects CD8+T cells. After infection, the cells express EBV latent membrane protein (LMP-1) causing massive cytokine release, promote T lymphocyte proliferation and uncontrolled activation of macrophages. Diagnosis is based on HLH-2004 which includes 5 of 8 criteria consisting of fever, splenomegaly, bicytopenias, hypertriglyceridemia, elevated ferritin, increased sCD25 (soluble IL-2), decreased or absent NK cell activity and/or Hemophagocytosis in bone marrow, CSF or lymph nodes. EBV-HLH is a rapidly progressing disease with one-year mortality as high as 75%. Treatment consists of dexamethasone and etoposide based on HLH-94 and HLH-2004 protocols. Early initiation of treatment is crucial regarding a favorable prognosis. CONCLUSIONS: HLH is a hyperinflammatory reaction most often caused by infection. High clinical suspicion is necessary when patients present with multiorgan failure with otherwise unexplained symptoms. Initiation of treatment may improve survival rates although often rapid deterioration is seen. Reference #1: Wang Y, Wang Z, Zhang J, et al. Genetic features of late onset primary hemophagocytic lymphohistiocytosis in adolescence or adulthood. PLoS One. 2014; 9(9);e107386 Reference #2: Maakaroun NR, Moanna A, Jacob JT, et al. Viral Infections associated with haemophagocytic syndrome. Rev Med Virol. 2010;20(2):93-105 Reference #3: Janka GE. Familial and acquired haemophagocytic lymphohistiocytosis. Eur J Pediatr. 2007:166(2);95-109 DISCLOSURES: No relevant relationships by Cristian Dumitrescu, source=Web Response No relevant relationships by Hafiza Wajeeha Javaid, source=Web Response No relevant relationships by Kunal Mehta, source=Web Response No relevant relationships by Farrukh Munir, source=Web Response No relevant relationships by Vipul Singh, source=Web Response

Evaluation of biomarkers for Sanfilippo syndrome

Sanfilippo syndrome or mucopolysaccharidosis type III (MPS III) is a childhood metabolic disorder marked by neuropathology arising due to impaired heparan sulphate (HS) catabolism. Consequently, partially degraded HS accumulates in the lysosomes of affected cells and is excreted in the urine. The measurement of HS in urine has long been considered a biomarker of Sanfilippo syndrome although it is largely non-specific. Using blood, urine and CSF collected from a cohort of Sanfilippo patients we investigated the utility of primary and secondary biomarkers to inform on disease activity. These included enzyme activity, specific oligosaccharides with non-reducing end residues reflective of the enzyme deficiency, and gangliosides. The diagnostic oligosaccharides - a HS disaccharide and tetrasaccharide - were elevated in the urine, plasma and CSF of all MPS IIIA and IIIB patients, respectively. There was no correlation between the concentrations in any of the matrices suggesting they reflect specific tissues and not overall disease burden. Enzyme activity did not inform on disease severity, with no measurable activity in CSF and activity approaching normal in MPS IIIA plasma. The concentration of gangliosides, GM2 and GM3, were significantly higher in the CSF of all MPS III subjects when compared to controls and correlated with the age of onset of first symptoms. Given that these gangliosides reflect delayed brain development they may be useful measures of disease burden, within the limitations of the clinical surrogates. Observation of these biochemical measurements in MPS III patients enrolled in clinical trials may determine whether they represent true pharmacodynamics biomarkers.

Alteration of human macrophage phenotypes by the anti-fibrotic drug nintedanib

The tyrosine kinase inhibitor, Nintedanib (NTD), has been approved for the treatment of idiopathic pulmonary fibrosis (IPF). In cell-free systems, NTD was recently shown to inhibit kinase activity of the human recombinant colony-stimulating factor 1 (CSF1) receptor (CSF1R) which mediates major functions of pulmonary macrophages. In the present study, we have investigated the effects of NTD on the phenotype of human monocyte-derived macrophages controlled by CSF1 in order to identify its anti-inflammatory properties via CSF1R inhibition. NTD (0.01 to 1 μM) prevented the CSF1-induced phosphorylation of CSF1R and activation of the downstream signaling pathways. NTD, like the CSF1R inhibitor GW2580, significantly decreased the adhesion of macrophages and production of the chemokine ligand (CCL) 2. NTD also altered the polarization of macrophages to classical M1 and alternative M2a macrophages. It reduced the secretion of several pro-inflammatory and/or pro-fibrotic cytokines (IL-1β, IL-8, IL-10 and CXCL13) by M1 macrophages but did not prevent the expression of M1 markers. While NTD (50–200 nM) partially blocked the synthesis of M2a markers (CD11b, CD200R, CD206, and CD209), it did not reduce synthesis of the M2a pro-fibrotic cytokines CCL22 and PDGF-BB, and increased CCL18 release when used at its highest concentration (1 μM). The effects of NTD on macrophage polarization only was partially mimicked by GW2580, suggesting that the drug inhibits other molecules in addition to CSF1R. In conclusion, NTD alters the CSF1-controlled phenotype of human macrophages mainly by blocking the activation of CSF1R that thus constitutes a new molecular target of NTD, at least in vitro.

Alzheimer's disease and neurotransmission gene variants: focus on their effects on psychiatric comorbidities and inflammatory parameters

The deficit of cholinergic activity is one of the main findings in Alzheimer’s disease (AD), and is related to the synthesis of acetylcholine, and the hydrolysing enzymes, acetylcholinesterase and butyrylcholinesterase (BuChE). Together with the Apolipoprotein E-ε4 allele (ApoE-ε4), the BuChE-K variant has been proposed to increase AD risk in certain populations. In addition, this polymorphism has been associated with a lower capacity to attenuate β-amyloid aggregation.In the present study we explored the interaction of the BuChE-K variant with its activity in CSF, conventional AD biomarkers and ApoE genotype.217 AD patients and 200 age-matched controls were genotyped for the ApoE and the BuChE-K variant. BuChE activity in CSF, as well as the levels of the CSF-AD biomarkers amyloid-beta 42 (Aβ42), total and hyperphosphorylated tau (t-tau and p-tau) were determined in 88 of these patients.The results showed no significant differences in the BuChE-K variant distribution between patients and controls. No influence of the BuChE-K variant was seen neither in CSF BuChE activity, nor in the levels of Aβ42, t-tau and p-tau in AD patients. ApoE genotype also did not seem to influence CSF BuChE activity. Interestingly, in AD patients, an association between high CSF BuChE activity and increased levels of CSF Aβ42 was shown, particularly in ApoE-ε4 allele carriers.In our population, the BuChE-K variant does not seem to confer risk for AD or to influence the activity of the enzyme in CSF. However, we demonstrated an association between BuChE activity, ApoE-ε4 genotype and CSF Aβ42 levels, highlighting the importance of assessing BuChE activity as a possible modulator of Aβ load in the brain.

P.3.24 Structural magnetic resonance imaging markers are associated with Alzheimer's disease pathway-specific polygenic scores

The deficit of cholinergic activity is one of the main findings in Alzheimer’s disease (AD), and is related to the synthesis of acetylcholine, and the hydrolysing enzymes, acetylcholinesterase and butyrylcholinesterase (BuChE). Together with the Apolipoprotein E-ε4 allele (ApoE-ε4), the BuChE-K variant has been proposed to increase AD risk in certain populations. In addition, this polymorphism has been associated with a lower capacity to attenuate β-amyloid aggregation.In the present study we explored the interaction of the BuChE-K variant with its activity in CSF, conventional AD biomarkers and ApoE genotype.217 AD patients and 200 age-matched controls were genotyped for the ApoE and the BuChE-K variant. BuChE activity in CSF, as well as the levels of the CSF-AD biomarkers amyloid-beta 42 (Aβ42), total and hyperphosphorylated tau (t-tau and p-tau) were determined in 88 of these patients.The results showed no significant differences in the BuChE-K variant distribution between patients and controls. No influence of the BuChE-K variant was seen neither in CSF BuChE activity, nor in the levels of Aβ42, t-tau and p-tau in AD patients. ApoE genotype also did not seem to influence CSF BuChE activity. Interestingly, in AD patients, an association between high CSF BuChE activity and increased levels of CSF Aβ42 was shown, particularly in ApoE-ε4 allele carriers.In our population, the BuChE-K variant does not seem to confer risk for AD or to influence the activity of the enzyme in CSF. However, we demonstrated an association between BuChE activity, ApoE-ε4 genotype and CSF Aβ42 levels, highlighting the importance of assessing BuChE activity as a possible modulator of Aβ load in the brain.

DP14 - Overexpression and activation of colony-stimulating factor 1 receptor in the SIV/macaque model of HIV infection and neuroAIDS

DP14 - Overexpression and activation of colony-stimulating factor 1 receptor in the SIV/macaque model of HIV infection and neuroAIDS

Evaluation of Adenosine Deaminase activity in CSF for diagnosis of Tuberculous Meningitis (P5.151)

Evaluation of Adenosine Deaminase activity in CSF for diagnosis of Tuberculous Meningitis (P5.151)

Strategic analysis of CSF’s for not-for-profit organizations

PurposeThe purpose of this study is to analyze how strategic planning is used as critical success factors (CSF’s) in not-for-profit (NFP) organizations. This was because many nonprofits had to innovate their operations owing to the global fiscal crises, the continuing international economic instability, natural disasters or the increasing man-made worldwide terrorism. Additionally, the objective is to identify what successful nonprofit organizations actually do to remain effective at the national association level of analysis.Design/methodology/approachA constructivist research design ideology is applied (in contrast to the customary positivist philosophy to collect quantitative). The literature is critically reviewed to identify NFP CSF’s and terms such as capacity building. NFP institutions are theoretically sampled using US-based retrospective data to identify practitioner CSF activities. Applying a constructivist research design ideology, the theoretical CSF’s from the literature review are compared to practitioner activities. Representatives of NFP organizations are invited to participate in a strategic planning exercise to identify the most important CSF’s from the literature and practice that would be needed in the future.FindingsSeven of the nine United Nations NFP capacity building CSF’s are similar to NFP nine practitioner best practices. In comparison to the general literature, NFP practitioners applied leadership, strategic planning, innovation, documented procedures/training, human/technology resource management, financial management, accountability practices, ethical standards with professional communications policies, collaborative fundraising and marketing initiatives along with performance success evaluations.Research limitations/implicationsThe sample was drawn theoretically from 44 nonprofit state-centered institutions in the USA. Although statistically the results pertain strictly to US-based nonprofits, the principles should generalize to other countries as revealed by the similarity with United Nations innovation and strategic planning recommendations.Originality/valueThe authors applied a strategic planning exercise with the 44 participants at their recommendations to prioritize the CSF’s. The result was an innovative SWOT-TOWS diagram that summarized how the nine CSF’s were prioritized and grouped into the three categories of market performance, ethical responsibility and human resources.

Intracerebral gene therapy in children with mucopolysaccharidosis type IIIB syndrome: an uncontrolled phase 1/2 clinical trial

Mucopolysaccharidosis type IIIB syndrome (also known as Sanfilippo type B syndrome) is a lysosomal storage disease resulting in progressive deterioration of cognitive acquisition after age 2-4 years. No treatment is available for the neurological manifestations of the disease. We sought to assess the safety and efficacy of a novel intracerebral gene therapy. Local regulatory authorities in France allowed inclusion of up to four children in this phase 1/2 study. Treatment was 16 intraparenchymal deposits (four in the cerebellum) of a recombinant adenoassociated viral vector serotype 2/5 (rAAV2/5) encoding human α-N-acetylglucosaminidase (NAGLU) plus immunosuppressive therapy. We assessed tolerance, neurocognitive progression, brain growth, NAGLU enzymatic activity in CSF, and specific anti-NAGLU immune response for 30 months after surgery. This trial is registered with EudraCT, number 2012-000856-33, and the International Standard Clinical Trial Registry, number ISRCTN19853672. Of seven eligible children, the four youngest, from France (n=2), Italy (n=1), and Greece (n=1), aged 20, 26, 30, and 53 months, were included between February, 2012, and February, 2014. 125 adverse events were recorded, of which 117 were treatment emergent and included six classified as severe, but no suspected unexpected serious adverse drug reactions were seen. Vector genomes were detected in blood for 2 days after surgery. Compared with the natural history of mucopolysaccharidosis type III syndromes, neurocognitive progression was improved in all patients, with the youngest patient having function close to that in healthy children. Decrease in developmental quotient was -11·0 points in patient one, -23·0 in patient two, -29·0 in patient three, and -17·0 in patient four, compared with -37·7 in the natural history of the disease. NAGLU activity was detected in lumbar CSF and was 15-20% of that in unaffected children. Circulating T lymphocytes that proliferated and produced tumour necrosis factor α upon ex-vivo exposure to NAGLU antigens were detectable at 1-12 months and 3-12 months, respectively, but not at 30 months in three of four patients. Intracerebral rAVV2/5 was well tolerated and induced sustained enzyme production in the brain. The initial specific anti-NAGLU immune response that later subsided suggested acquired immunological tolerance. The best results being obtained in the youngest patient implies a potential window of opportunity. Longer follow-up is needed to further assess safety outcomes and persistence of improved cognitive development. Association Française Contre les Myopathies, Vaincre les Maladies Lysosomales, Institut Pasteur, and UniQure.

DPP4 truncated GM-CSF and IL-3 manifest distinct receptor-binding and regulatory functions compared with their full-length forms.

Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated factors [(T)-GM-CSF and- IL-3] on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and T-IL-3 had enhanced receptor binding, but decreased CSF activity, compared to their FL forms. Importantly, T-GM-CSF and T-IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and FL-IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster forming cells from patients with Acute Myeloid Leukemia (AML) regardless of cytogenetic or molecular alterations and in vivo utilizing animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared to their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.