Tt, on sulfide stress cracking (SSC) behaviors was estimated mainly via double cantilever beam (DCB) and electrochemical hydrogen permeation (EHP) tests and microstructure characterization. The results indicate that the threshold stress intensity factor for SSC, KISSC, increased with increasing Tt. The overall and local H concentration around the inclusions decreased with increasing Tt, due to reductions in the amounts of solute atoms, grain boundaries and dislocations, which effectively prevented SSC initiation. Also, increasing Tt caused an increased fraction of high-angle boundaries, which evidently lowered the SSC propagation rate by more frequently diverting the propagating direction and accordingly restricted SSC propagation. The overall SSC resistance of this Q&T–treated steel was therefore significantly enhanced.A medium-carbon Cr–Mo–V martensitic steel was thermally processed by quenching (Q) at 890 °C and tempering (T) at increasing temperatures from 650 °C to 720 °C and the effect of tempering temperature,  Quenching and tempering (Q&T)–treated martensitic steels have long been widely used for important oil country tubular goods production, such as C90–C110 or higher grades for sour oil/gas well service , T125 an+→H reaction [2O3 [KISSC, increased with decreasing size of martensitic packet with high-angle boundaries [Tt and time and attributed it to a martensitic ferrite matrix with fewer dislocations and more dispersed carbides. This indicates that the SSC resistance of martensitic steels could be effectively enhanced by improving the tempering process. Among these efforts, the correlations between various metallurgical factors and the susceptibility to SSC/HIC have recently received increasing attention ,20,21,22reaction , which dreaction , by actireaction  and contreaction ,24,25. Sreaction . In contreaction ,14,15. Hion [2O3 ,18 and lion [2O3  and at tion [2O3 , even thion [2O3  and the undaries  reportedTherefore, these various metallurgical factors have exerted complex effects on the susceptibility to SSC/HIC, which were originally and essentially correlated to SSC evolution, including three stages of the entry of H atoms, SSC/HIC initiation and propagation. The effects of some metallurgical aspects  on a single stage of SSC/HIC evolution in martensitic/bainitic steels, namely the entry of H atoms and SSC/HIC propagation, were estimated in previous work  by electTt, providing a group of samples with quite different microstructural features of GBs, dislocations and precipitates. Their effects on SSC behavior, namely H mobility, SSC initiation/propagation and overall resistance to SSC (KISSC), were estimated mainly through microstructure characterization and EHP and DCB tests. The mechanisms governing these correlations are also discussed.For this attempt, a martensitic steel was thermally processed via Q&T at differing YS), tensile strength (TS) and hardness (HRC) decreased, while the elongation (EL) at rupture increased with elevating Tt. The KISSC for each Q&T–treated sample was also determined via the DCB test and is also summarized in KISSC increased from 17.16 MPa∙mm−0.5 to 33.26 MPa∙mm−0.5 with increasing Tt from 650 °C to 720 °C, indicating an enhanced overall resistance to SSC due to the increased Tt.The mechanical properties of each Q&T–treated sample are summarized in t), as shown in The instantaneous SSC propagation length for each Q&T–treated sample was measured with the DCB test interrupted, according to the NACE  TM0177 standard  and evalTt.As also indicated in da/dt and KIapplied, as shown in da/dt decreased as a result of decreasing KIapplied. With KIapplied decreased to a certain value, da/dt approached zero, where KISSC was obtained, according to the NACE TM0177 standard [da/dt decreased and KISSC increased with increasing Tt, indicating that the high-temperature tempering retarded SSC propagation and correspondingly enhanced SSC resistance.SSC propagation behaviors were also interpreted in terms of the correlation between standard . Further− or S2− to the fresh material surface. The corrosion film was subsequently removed with a solution of dilute hydrochloric acid and hexamethylene tetramine and the real SSC fracture surface is shown in Tt, is shown in Dγ, was quantitatively estimated and is summarized in Dγ remained at almost the same level of 7.5 μm under identical quenching conditions. The typical prior austenite grain (PAG) morphology of the samples quenched at an identical temperature of 890 °C, waiting for further tempering at differing Dp and Db, were quantitatively determined and are summarized in Dp and Db increased with increasing Tt from 650 °C to 720 °C. The grain boundary misorientation angle (GBMA) might play a significant role in controlling SSC resistance by affecting hydrogen permeability [fGBMA≥15°, is summarized in fGBMA≥15° increased slightly with increasing Tt from 650 to 720 °C.The PAG of martensitic steel can normally be partitioned into three types of martensitic substructures: packet, block and lath ,33. The eability  and craceability ,26. TherTt. In contrast, a small quantity of regular laths might possess a particular crystallographic orientation with high thermodynamic stability [Tt, at the expense of the regular martensitic laths. The average lath width, Wl, was further determined quantitatively and is also summarized in Wl obviously increased with increasing Tt, indicating a widening of the martensitic lath, probably due to the interface migration or the merging of two neighboring laths [TEM micrographs of each Q&T–treated sample are presented in tability  and coulng laths .Tt increased. The average equivalent diameter, D and the volume fraction, f, of the precipitates were further estimated quantitatively and are provided as the function of Tt in D and f of precipitates obviously increased with increasing Tt.There still appeared to be a large quantity of rod-like and spherical precipitated particles distributed in the martensitic laths or at their boundaries, as also shown in ρ, of each Q&T–treated sample was determined by XRD pattern, as shown in 14 to 0.62 × 1014 m−2 with increasing Tt from 650 °C to 720 °C. The present result is in agreement with a previous work by Masakatsu et al. [Tt due to the addition of 0.1 wt % vanadium to the 0.5Cr–0.7Mo steel. The dislocation density, u et al. . They reD0 and the diffusion H concentration, C0, were calculated with Equations (10) and (11) and the results are summarized in D0 increased and C0 decreased with increasing Tt, indicating lower H permeability.C0, indicating H permeability, decreased with increasing Tt, as shown in C0 is dominantly dependent on the density of reversible H traps, NT, on the assumption that the irreversible traps could be saturated by the H atoms [Tt, due to increased precipitation, as shown in NT-SA, also decreased with increasing Tt. However, the precipitates in this Q&T–treated Cr–Mo–V–Ti–B martensitic steel were highly diversified and complicated in their structure [NT-SA therefore remains challenging. Accordingly, the NT for these Q&T–treated samples at differing Tt were quantified mainly as functions of the GBs  and the dislocations, respectively, to better understand their effects on C0. H atoms . Accordi H atoms ,40,41,42tructure  and hencNT-GB, was estimated, with the grain assumed to be an approximate sphere and counted as n per cubic meter, using the following equation [VG and SG are the volume and surface area of one grain, respectively; the factor of 1/2 is due to one grain plane shared by two grains; xa is atomic distance, 0.26 nm; xGB is the width of GB, about 10 times that of xa. Equation (1) can then be simplified as: dG is the grain size. This equation indicates that the NT-GB is in inverse proportion to dG, by which NT-GB for these Q&T–treated samples were estimated and are listed in The H trap density provided by the GBs, equation :(1)NT-GBNT-dis, was estimated by:ρdis is dislocation density and the factor of 5 is assumed to be the ratio of the dislocation width to xa. The NT-dis for these Q&T–treated samples were calculated and are also listed in The H trap density provided by the dislocations, NT-MP, NT-MB and NT-ML provided by the martensitic packet, block and lath boundaries decreased with their increasing size and accordingly their decreasing number of boundaries, whereas NT-PAG remained at almost the same level. As the martensitic lath boundaries normally occupied all the PAG and the martensitic packet/block boundaries, NT-ML contained NT-PAG, NT-MP and NT-MB. In general, the PAG and the martensitic packet/block boundaries were confirmed to be HAGBs [C0 [C0. In contrast, the remnant LAGBs encircling the regular laths comprised a minor fraction of the martensitic lath boundaries, still contributing little to the C0. Thus, the final NT-GB was calculated approximately by combining the NT-ML  decreased markedly, as shown in Consequently, the amount of reversible H traps provided by the various GBs, the dislocations and even the solute atoms all decreased with increasing 2O3·SiO·CaO and the slender Al2O3·CaO·TiO particles, as shown in 2O3 [A certain number of inclusions with a large size of more than 3 µm were still present in this experimental steel, although it was prepared by melting in an electric furnace and purifying in a vacuum degassing furnace. There appeared mainly two types of complex inclusions in each DCB sample: the massive Aln in 2O3 . Cσ, of the highly stressed regions in the DCB sample can be estimated using the following equation [C0 is the diffusion H concentration, σh is the hydrostatic stress, VH is the partial molar volume of H in iron (2.1 × 10−6 m3/mol), R is the gas constant and T is the ambient temperature. As this equation indicates, Cσ decreased as the result of decreased C0. When the H-induced stress concentration exceeded the atomic binding force, SSC eventually initiated. In addition, the local H concentration, equation : (4)Cσ=Cti, which was attained by fitting the crack propagation length (Δa) vs. the time curve, as demonstrated in Susceptibility to SSC initiation can be estimated by the incubation time for SSC origination, Δa = 0, the ti for the Q&T–treated samples, tempered at 650, 700 and 720 °C was 1.5, 2.2 and 10.4 h, respectively, indicating a lowered susceptibility to SSC initiation with increasing Tt. Supposing Tt led mainly to martensitic lath polygonization and widening, providing a decreased amount of HAGBs and accordingly reversible H traps, inducing a lowered overall and local diffusion H concentration (C0 and Cσ), leading to a depressed stress concentration around the inclusions and simultaneously a prolonged incubation time and finally decreased susceptibility to SSC initiation.Therefore, the increasing To further understand the effect of GBs with certain crystallographic orientations on crack propagation, secondary microcracks underneath the SSC fracture surface of DCB samples were observed and the corresponding misorientation angles between grains adjacent to the crack were analyzed by EBSD, as shown in Hence, HAGB could play a significant role in hindering SSC propagation, whereas LAGB failed to provide an evident barrier, showing the two different impacts on crack propagation. According to previous observations , the chaTt brought about martensitic lath polygonization and widening to a higher degree, providing an increased fraction of HAGBs and also an increased mean GBMA, restraining SSC propagation by lowering their propagation rate . The chemical compositions are presented in Tt of 650, 700 and 720 °C for 60 min.The samples with dimension 300 × 100 mm were cut from the steel tube, reheated at 890 °C for 20 min and water quenched and consequently tempered at differing The conventional tensile properties of each Q&T–treated sample were determined according to the ASTM  Standard A370-10 . Each sa3COOH dissolved in distilled water saturated with 1 atm H2S. The pH value of the solution did not exceed 3 at the start and 4 at the end of the test. The tests were performed for 2 weeks at ambient temperature, during which the instantaneous applied stress intensity factor, KIapplied and the threshold stress intensity factor, KISSC, were obtained using Equations (8) and (9): P is the final equilibrium wedge lift off load, determined from an abrupt change in the slope of load-displacement curve; ai/a is the instant/final crack length of SSC for estimating KIapplied/KISSC, obtained by subtracting 6.35 mm from the distance between the slot end and the mean position of the crack front; h is the arm height; and B and Bn are the DCB specimen and web thickness, respectively. Three parallel DCB tests were performed for each Q&T–treated sample and the average values are reported.The susceptibility to SSC in each sample was estimated with the DCB test, according to the NACE TM0177 standard, Method D . The staΔa, as a function of immersing time, t, was measured by the NACE TM0177 standard, Method D [da/dt, was obtained by seeking derivation of the Δa vs. t curves. Finally, da/dt as a function of the corresponding KIapplied was described.In order to further understand SSC crack initiation and propagation behaviors occurring in the DCB tests and their dependence on metallurgical aspects, the crack propagation length, Method D . Then th2SO4 and 0.25 g/L CH4N2S, while the electrolyte for H expanding was an aqueous solution of 0.2 mol/L NaOH. The H charging time might depend on the purpose. The sample for the H permeation estimation was 0.8 mm thick, with the nickel-plated surface acting as the anode side. The current was measured when the background current decreased and reached a steady value of 0.1 μm/cm2 or lower.The H permeation behavior in each Q&T–treated sample was quantitatively estimated with the EHP test at 298 K using the classical Devanathan-Stachursky technique. The electrolyte for H charging was a solution of 0.5 mol/L HD0 (cm2/s), was calculated using the following equation: L is the specimen thickness (cm) and t0.63 is the time lag for 0.63 saturation of permeation current density at the steady state (s). The reversible H concentration, C0 (ppm), was estimated using the following equation:i∞ is the steady-state atomic H permeation current, μA/cm2 and F is Faraday’s constant, 9.6485 × 1010 μA·s/mol.The diffusion coefficient of H, The as-quenched sample at 890 °C was etched in a supersaturated picric acid aqueous solution for optical microscopy (OM) observations of the prior austenite grain (PAG). Each Q&T–treated sample was etched in 4% nital for martensitic packet observation in a Hitachi S-3400N scanning electron microscope (SEM). The electron back-scattered diffraction (EBSD) map was obtained by a SUPRA550 field emission scanning electron microscope equipped with a TSL  orientation imaging microscopy system to reveal the martensitic block substructure and the misorientation angles between the adjacent martensitic substructure boundaries. SSC inducing the fractured surface and the secondary crack in a cross-section of the DCB specimen after each trial were also examined by SEM and EBSD, respectively. The crystallography orientation information of the martensitic packet/block structures next to the secondary crack and their correlations were determined; Channel 5 software from Oxford-HKL was used for the orientation data processing. Each EBSD sample was polished using 0.04 µm colloidal silica slurry in a vibratory polisher for 1 h. The characteristics of the martensitic lath and the distribution, shape and chemical compositions of precipitated particles were determined by examining thin foils or carbon extraction replicas using a JEM-2010 high-resolution transmission electron microscope.Do) and volume fraction (f) of precipitated particles of different shapes, the rod-like particles were considered with the spherical particles and Do was regarded as the particle size. Then f was calculated with the following equation: N is the number of particles per area, S0 is the specific area for estimation and S is the particle area.The martensitic structure size for each Q&T–treated sample was quantitatively estimated by averaging at least 200 PAGs, 200 packets, 150 blocks and 100 laths from the OM, SEM, EBSD and TEM micrographs, respectively. For estimating the equivalent diameter (θ) range of 40–110° and at a step size of 0.02° in a Rigaku D/max-2500/PC diffractometer . In order to correct the XRD line broadening resulted from the instrument, the XRD pattern of a standard sample of Si powder which was annealed at 300 °C for 24 h was measured. This diffraction line broadening correction was performed with the data processing via the software of Jade 5 .The dislocation density of each Q&T–treated sample was determined by quantitative X-ray diffraction (XRD) analysis. XRD spectra were obtained by scanning over a scanning angle (2Tt) on SSC behavior in a Q&T–treated medium carbon Cr–Mo–V martensitic steel was estimated in this work and the conclusions are summarized as follows:KISSC and the overall resistance to SSC of this martensitic steel were obviously enhanced by increasing Tt from 650 to 720 °C.Tt led to decreases in overall and local H concentration around the inclusions, owing to martensitic lath polygonization and widening and resulted in a lowered susceptibility to SSC initiation.Increased Tt brought about a higher fraction of HAGBs and resulted in enhanced resistance to SSC propagation by more frequently diverting the propagating direction and lowering the propagation rate.Increased The effect of increasing tempering temperature (


A comparison of this case to previously published reports suggests that granulocyte colony‐stimulating factor may be associated with improved prognosis in severe antithyroid drug‐induced neutropenia, and that weight‐based doses could be an appropriate strategy. Current guidelines include strong recommendations for use of these medications in Graves' disease, thyroid storm, hyperthyroidism during pregnancy, and drug‐induced thyrotoxicosis A 44‐year‐old woman with a history of hyperthyroidism treated with propranolol was admitted to an outside hospital complaining of ongoing fatigue, headaches, palpitations, and tremor along with sudden onset vertigo and left‐sided weakness. Following discovery of a right cortical vein thrombosis with intraparenchymal hemorrhage and a generalized tonic‐clonic seizure at the referring facility, the patient was transferred to our hospital. Upon admission, she was found to have abdominal pain, temperature of 102°F, heart rate of 121 beats per minute, thyroid‐stimulating hormone (TSH) <0.01 milliunits/L, thyroxine (T4) of 6.7 ng/dL, and a triiodothyronine (T3) of 310 ng/dL, all suggestive of thyroid storm μL and an absolute neutrophil count (ANC) of 100/μL, reduced from a baseline of 8700/μL. Differential showed 2.5% neutrophils, 64.9% lymphocytes, 18.8% monocytes, 12% eosinophils, 1.6% basophils, and 0.2% immature granulocytes. Considering the temporal nature of her neutropenia in the setting of recent ATD initiation, MMI‐induced agranulocytosis was suspected  Twenty‐one days after discharge (day 30 of MMI treatment), the patient returned to the emergency department (ED) complaining of 1 day of palpitations, fever, nonproductive cough, and sore throat. By the time she presented to the hospital, she was afebrile and her symptoms had resolved. A chest x‐ray was performed showing no acute abnormalities, and blood and urine cultures were negative. Laboratories demonstrated a white blood cell count (WBC) of 4250/μL. On day 3, she received 480 mcg (5 mcg/kg) of intravenous (IV) filgrastim once daily with a goal of achieving an ANC of 5000/μL or greater. A rapid response in the patients' ANC was observed, and filgrastim was discontinued after just two doses of 480 mcg  P = 0.03), suggesting that hematopoietic growth factors may be an effective treatment for drug‐induced neutropenia.The duration of drug‐induced agranulocytosis has been reported to range from 4 to 56 days, and longer recovery times are often associated with hypoplastic bone marrow μg subcutaneously once daily) have been suggested to have contributed to this lack of effect P = 0.046), as well as shorter durations of antibiotic treatment and hospital length of stay in patients treated with 300 μg/day compared to those without growth factor treatment μL) When analyzing the effects of hematopoietic growth factors on ATD‐induced agranulocytosis, specifically, results are less clear. Some case reports and one prospective, randomized controlled trial suggested a lack of effect in reducing the duration of ATD‐induced neutropenia using G‐CSF, though the low doses used (75–250 μL), time to recovery after discontinuation of the implicated ATD and G‐CSF administration has ranged from roughly 6 to 14 days using G‐CSF doses ranging from 75 to 375 μg/day μg/day, though the relevance of the association between increased dose and recovery is unknown μg subcutaneous G‐CSF was associated with rapid improvement of neutropenia μL to >6000/μL in three patients and from 360/μL to >6000/μL in one patient. Despite the high‐dose of hematopoietic growth factor, no adverse effects were reported and authors concluded that high doses of G‐CSF in the setting of potentially life‐threatening neutropenia may be reasonable.In some patients with severe neutropenia  <0.5  or peripheral granulocyte counts <100 cells/μL had ANC recovery times that were significantly longer compared to those with a G:E greater than or equal to 0.5  or an ANC >100 cells/μL  Severe neutropenia or pronounced suppression of myeloid precursors has been associated with a prolonged recovery time and failure to respond to G‐CSF μg/day was initiated until an ANC >1000 cells/μL was achieved. It was observed that patients with greater suppression of immature granulocyte cell types reached higher concentrations of endogenous G‐CSF, correlating with longer recovery times . Authors speculated that patients with greater bone marrow suppression may have required higher doses of G‐CSF in order to reach peak concentrations equivalent to the observed endogenous peaks and to see the benefit of shortened recovery time.In another observational study of 54 patients with Graves' disease, investigators performed serial measurements of serum G‐CSF levels among six patients with MMI‐induced agranulocytosis Relative to previously published case series, a couple points are worth noting in our case. First, we used G‐CSF at a fairly aggressive dose and via the IV route. While subcutaneous dosing of G‐CSF has been found to generally be superior, IV use remains a viable clinical option Next, our patients' recovery of ANC was rapid despite a severe neutropenia. While it is possible that G‐CSF merely augmented a recovery that was already underway, it should be noted that the patients' ANC dropped further while on our clinical service. Thus, the myelosuppressive effects of ATD therapy were still acute when G‐CSF therapy was begun.Last, in the context of that rapid recovery, it is likely that our patient suffered a reduction in granulopoiesis without a significant inhibition of granulocytic precursors, as suggested by her monocytosis. A shorter duration of neutropenia may be expected in such a case. However, severe agranulocytosis, as seen in this patient, is not typically associated with such a rapid and sustained recovery in ANC. Considering that some degree of dose response has been supported by previous literature, the higher weight‐based dose used in this case almost certainly contributed to the short duration of neutropenia and the favorable outcome which was observed.μg IV (5 μg/kg) was associated with a rapid improvement in ANC, increasing from 80/μL to 11,650/μL after only 2 days of treatment.All patients receiving ATDs should be counseled to seek immediate medical attention for signs of infection, including fever and sore throat. For such patients who present with neutropenia, the offending agent should be promptly discontinued, and all ATDs subsequently avoided. Use of hematopoietic growth factors to shorten the duration of neutropenia should also be considered, especially for patients with characteristics suggesting poor prognosis. The heterogeneity of existing studies and case reports complicates the determination of best G‐CSF dosing strategy in ATD‐induced agranulocytosis, however. In our patient, a once daily IV dose of filgrastim 480 μg/kg dose of IV G‐CSF and suggests that such dosing can be associated with a rapid recovery from neutropenia after ATD‐induced agranulocytosis.In summary, the case presented here adds additional support to the literature for a 5 ARB: contributed to the literature search and was the primary author. CM: contributed to the literature search and preliminary summary, content revision. CW: contributed to article conception, content revisions and supporting materials.The authors of this presentation have nothing to disclose concerning possible financial or personal relationships with commercial entities that may have a direct or indirect interest in the subject matter of this document. All coauthors are aware of content presented here. This case study has been deemed exempt by the Institutional Review Board.


Elizabethkingia miricola were confirmed in black-spottedfrog farms in China in 2016. Whole-genome sequencing revealed that thisamphibian E. miricola strain is closely related to humanclinical isolates. Our findings indicate that E. miricola canbe epizootic and may pose a threat to humans.Multiregional outbreaks of meningitis-like disease caused by Elizabethkingia is a genus of gram-negative, nonmotile,non–spore-forming bacilli that are ccasionally associated with human clinicalinfections  to further characterize this Asian epizootic isolate andphylogenetically compare it with the available typical Elizabethkingiagenomes.The black-spotted frog, Since May 2016, many black-spotted frogs in farms in Hunan Province in south-centralChina have experienced an emerging, contagious disease characterized mainly bysevere neurologic dysfunction. The first clinical sign is intermittent swimming incircles. Thereafter, the frogs develop signs of torticollis , panel ABatrachochytrium dendrobatidis andranaviruses were negative  of the 213 frogs; 90% wereDSM14571. We seleDSM14571. After 2DSM14571, we founE. miricola FL160902, we conducted WGS with theIllumina HiSeq 2500 platform , producing 2× 150-bp paired-end reads. We assembled the trimmed reads using SOAPdenovo(http://soap.genomics.org.cn/soapdenovo.html). We constructed aphylogenetic tree (E.miricola FL160902 (GenBank accession no. NHPR00000000) and otherElizabethkingia genomes. The results showed that FL160902 wasmost closely related to CSID_3000517120, a clinical isolate of E.miricola from the United States sequenced by the Centers for DiseaseControl and Prevention (CDC) (E.miricola FL160902 for pathogenicity in humans.To characterize tic tree  of orthoE. meningoseptica (previouslyFlavobacterium meningosepticum) was found to be separated into2 main hybridization groups, UBI and UBII, that were ≈40%–55%interrelated; UBII could be further divided into 4 subgroups (E. meningoseptica (E.meningoseptica isolates did not group with the otherElizabethkingia spp. and were distantly related to UBII.Considering the low DNA–DNA relatedness (<70%) between the 2 groups andphylogenomic analysis based on WGS (E.meningoseptica. The UBII subgroups branching separately supports theview that they are different Elizabethkingia species (E. anophelis and E. endophytica formed a cladewith strong support of 100%, favoring the suggestion that E.endophytica is a later subjective synonym of E.anophelis (E. miricola, which is thought tobe closely related to UBII:2 (E.miricola ATCC 33958 and BM10 should be reconsidered because theyclustered with UBII:3 and not with UBII:2 E. miricola species. Ourresults agree with Eriksen’s conclusion about the genetic diversity inElizabethkingia; a more comprehensive taxonomic system isneeded to clarify the Elizabethkingia genus (Before WGS was commonly used, E. miricola was the most predominantpathogen. The neurologic signs and pathologic brain lesions suggested thatE. miricola could break through the blood–brain barrierand damage the nervous system. The etiologic analyses combined with the results ofexperimental challenge support the conclusion that the E. miricolastrain represented by isolate FL160902 is highly contagious for frogs, especially byimmersion infection. We suspect that contaminated water is the primary vehicle oftransmission, considering the infection assay and the epidemiology in 1 farm withdifferent ponds. However, diverse transmission routes might be involved becausethere is no obvious interconnectivity among independent farms, which needs to beinvestigated further. Close attention should be paid to whether this disease affectsthe wild population of amphibians. Our results indicated the gradual expansion ofits host and suggest that amphibians may serve as a reservoir for infection inhumans. Black-spotted frog farming is a major aquaculture industry in south-centralChina; thus, animals and humans that have close contact with infected frogs shouldbe continually monitored for emerging E. miricola infections, eventhough no human E. miricola infection cases were reported relatedto frog consumption or farming in Hunan in 2016. Our results demonstrate acontagious disease in frogs caused by E. miricola that poses apotential zoonotic threat to humans, generating a need for consideration of the roleof Elizabethkingia bacteria in public health.In this natural outbreak of meningitis-like disease in cultured frogs in HunanProvince, China, in 2016, Elizabethkingiamiricola in black-spotted frogs, Hunan, China.Description of the methods used in study of


Although G-CSF is widely used to prevent or ameliorate leukopenia during cytotoxic chemotherapies, its optimal use is still under debate and depends on many therapy parameters such as dosing and timing of cytotoxic drugs and G-CSF, G-CSF pharmaceuticals used and individual risk factors of patients.We integrate available biological knowledge and clinical data regarding cell kinetics of bone marrow granulopoiesis, the cytotoxic effects of chemotherapy and pharmacokinetics and pharmacodynamics of G-CSF applications (filgrastim or pegfilgrastim) into a comprehensive model. The model explains leukocyte time courses of more than 70 therapy scenarios comprising 10 different cytotoxic drugs. It is applied to develop optimized G-CSF schedules for a variety of clinical scenarios.Clinical trial results showed validity of model predictions regarding alternative G-CSF schedules. We propose modifications of G-CSF treatment for the chemotherapies ‘BEACOPP escalated’ (Hodgkin’s disease), ‘ETC’ (breast cancer), and risk-adapted schedules for ‘CHOP-14’ (aggressive non-Hodgkin’s lymphoma in elderly patients).We conclude that we established a model of human granulopoiesis under chemotherapy which allows predictions of yet untested G-CSF schedules, comparisons between them, and optimization of filgrastim and pegfilgrastim treatment. As a general rule of thumb, G-CSF treatment should not be started too early and patients could profit from filgrastim treatment continued until the end of the chemotherapy cycle.The online version of this article (10.1007/s00432-017-2540-1) contains supplementary material, which is available to authorized users. The haematopoietic growth factor G-CSF is routinely used in cancer therapy to prevent or ameliorate leukopenic conditions. Its effectiveness has been shown in several studies  and chemotherapy  are modelled  and chemotherapy action. We briefly describe these major parts of the model and corresponding biological assumptions in the following.Granulopoietic cells originate from a pluripotent stem cell compartment. Cell division can create new stem cells or cells committed to granulopoietic lineage. All other haematopoietic lineages are neglected.Subsequent proliferation and maturation of granulopoietic cells are described by transitions of cells from the stem cell compartment to the proliferating compartments of progenitor cells, proliferating precursors (PGB), and finally, maturing precursor cells which are unable to proliferate (MGB). For the latter, a postmitotic amplification is assumed which describes a loss of mature granulocytes in bone marrow due to apoptosis  is phenomenologically modelled by an additional toxicity equation with two parameters.The chemotherapy effect is assumed to be reversible. All cell-kinetic parameters remain unchanged.Often, prednisone is applied to avoid tumour lysis syndrome. Prednisone is assumed to cause a prolonged half-life of granulocytes, and therefore, it temporarily increases granulocyte counts.The impact of cytotoxic chemotherapy on haematopoiesis is modelled by drug, drug-dose and cell-stage specific toxicity functions in the following way , HD (Hodgkin’s lymphoma), BRCA (breast cancer), NSCLC , and DLBCL (diffuse large B cell lymphoma). An overview is presented in Table S1 in Additional file 1. Data were retrieved from published figures using the software tool “YCASD” . We used the tertiles of the risk score to divide our study population into low, medium and high-risk group and propose different G-CSF schedules for them in the following.Several risk factors associated with the degree of leukopenia under chemotherapy have been identified and validated in different settings. This comprises for example pre-therapeutic risk factors such as age, sex, WHO performance status as well as intra-therapeutic risk factors such as toxic response in the first therapy cycle. A risk model accounting for these factors was proposed by Ziepert et al.  for CHOPIn order to apply our model to different risk groups, we assume that parameters regarding pharmacokinetics and pharmacodynamics of G-CSF as well as cell-kinetic parameters are constant among risk groups, but the parameters regarding chemotherapy toxicity might differ. This is motivated by observed heterogeneities regarding metabolism of cytotoxic drugs .A key feature of the model is that new, yet untested schedules of both, chemotherapy and G-CSF treatment can be simulated. This requires that toxicity parameters of the chemotherapy are available for the population of interest, i.e. data of patients under at least one G-CSF schedule (or no G-CSF) of the chemotherapy of interest are required allowing to estimate the toxicity parameters thereon. Then, alternative timing of chemotherapy or G-CSF, alternative derivatives and dosing of G-CSF can be simulated by the model. An overview of already available toxicity parameters is listed in an earlier publication of our group  measures the area between simulated cell counts and the value of 4000 leukocytes/µL. There is some evidence that the risk of infectious complications in patients depends on the depth and the duration of leukopenia . Model simulations were performed by numerical integration of the ODE system using the variable step solver from Adams and Bashford (ode113).In the past, we showed that our model can successfully predict the granulotoxic outcome of CHOP chemotherapy with G-CSF support. Some of these predictions were already tested in clinical trials resulting in improved filgrastim and pegfilgrastim schedules for CHOP chemotherapy. We present these successful predictions in brief. For CHOP-14 therapy in elderly patients we predicted that a reduced filgrastim schedule with applications at days (d) 6–12 of each cycle is also feasible compared to the standard d4–13 protocol. This was confirmed by retrospective analyses of the RICOVER trial in which both schedules were applied. We present the comparison of model and data for these scenarios in Fig. 3/mm3, grade 3 and 4 leukocytopenias, grade 4-only leukocytopenias, grade 3 and 4 infections, deaths during leukocytopenia and interventional antibiotics . According to study protocols, it is recommended to apply filgrastim at d8–15 at each cycle. Figure The ETC regimen is used as adjuvant chemotherapy of breast cancer patients in the German Breast Group , it appears to be worthwhile to modify G-CSF schedules according to the drug currently applied. We implemented a stepwise optimization for this sequential chemotherapy taking the different cytotoxicities of the drugs into account: in cycles 1–3, G-CSF should be applied from d6–10. In cycles 4–9, optimal G-CSF therapy starts on d7 with 8 injections . The optimal WBCAOC is 0.57, while that of the current standard therapy is 14.9. The optimization assuming the same G-CSF schedule in each cycle (d7–14) yields 0.70. Thus, the improvement by cycle-specific G-CSF schedules is only moderate compared to the optimal unique G-CSF schedule, which probably does not outweigh the higher organizational effort.Densification of CHOP chemotherapy from cycle duration 21 to cycle duration 14 resulted in improved outcomes of elderly NHL patients . Regarding pegfilgrastim dosing, we predict that the low-risk group can safely be treated with considerably less pegfilgrastim . To a lesser extent, this also applies for the medium- and high-risk group if pegfilgrastim is administered within the above-mentioned optimal time interval .Although the haematopoietic growth factor G-CSF is routinely applied in clinical practice since many years, its optimal use in a given clinical situation is often unknown or not well investigated. The reason is that the performance of alternative G-CSF schedules is difficult to predict in view of the strong interaction of chemotherapy-induced leukopenia, pharmacokinetic properties of G-CSF and the resulting effects on bone marrow leukopoiesis.In view of the large number of variable therapy options , it is practically impossible to study this problem solely on the basis of clinical trials. Thus, there is a strong need for predictive modelling of G-CSF applications. Pastor et al.  proposedThe model was developed on the basis of large clinical and literature data sets , 4–6 (paclitaxel) and 7–9 (cyclophosphamide), respectively. However, only small improvements were predicted compared to the d7–14 schedule.Since time-intensified CHOP is advantageous for the treatment of high-grade non-Hodgkin’s lymphoma in elderly patients Below is the link to the electronic supplementary material.


Automobile mechanics and sprayers are at a higher risk of exposure to hazardous chemicals such as polycyclic aromatic hydrocarbons and heavy metals which may cause adverse health outcomes. This study aimed to use reticulocyte count as an indirect measure of the haematological output in automobile mechanics and sprayers in the Cape Coast Metropolis, Ghana. This cross-sectional study recruited 130 participants: 90 cases (57 automobile mechanics and 33 automobile sprayers) and 40 controls (nonautomobile workers). Venous blood samples were drawn from the participants and examined for full blood count and absolute reticulocyte count. Semi-structured questionnaire was used to collect demographic and occupational safety information from participants.p = 0.0001; 5.04 ± 1.7 versus 3.81 ± 1.1), or sprayers . Lymphocyte, monocyte, and platelet counts were also significantly higher in controls compared to cases. Whereas RBC counts were significantly higher in controls compared to automobile mechanics , haemoglobin levels were significantly higher in automobile sprayers compared to controls . Absolute reticulocyte count was significantly higher in controls compared to cases . Among the cases however, only RBC counts were significantly lower in automobile mechanics compared to automobile sprayers . 75.6% of cases had never received occupational safety training. Whereas 35.1% of automobile mechanics routinely siphoned fuel, 36.4% of automobile sprayers never used nose masks in the discharge of their duties. Controls had significantly higher WBC counts compared to mechanics ( It was evident that both automobile mechanics and sprayers had significantly reduced haematopoietic output. Occupational safety training is not given priority and must be addressed.  Exposure to aliphatic and aromatic hydrocarbons has been noted to be deleterious to the health of humankind . As thesCertain occupations such as automobile mechanics and sprayers are prone to immediate exposure to these hazardous chemicals through either inhalation, dermal exposure, or ingestion . Their oExposure to these aromatic compounds and heavy metals may suppress the bone marrow's activity leading to ineffective hematopoiesis . Heavy mThis case-control study recruited 130 participants: 90 cases [automobile mechanics (57) and automobile sprayers (33)] and 40 controls at Siwdo in the Cape Coast Metropolis, Ghana. The Siwdo automobile workshop is the largest in the Cape Coast Metropolis. The automobile mechanics included diesel-engine mechanics, motor trucks mechanics, engine repair mechanics, differential and brakes repairers, general automobile-service-station mechanics, workshop heads, and apprentices. A simple convenience sampling technique was employed where all consecutive individuals who gave informed consent were recruited for the study. Questionnaires were also used to collect sociodemographic data and working history of each participant.2EDTA anticoagulant. Samples were analyzed using automated haematological analyzer . New methylene blue (NMB) was used to supravitally stain participants' blood samples, after which thin blood film was prepared and used to estimate the reticulocyte counts in accordance with previously published protocols . Also, significantly higher proportions of cases had no knowledge on health implications of the frequent handling of petrochemicals and petroleum products . Most of the cases did not use any safety measure in the discharge of their daily duties (74.7% mechanics versus 78.8% sprayers).The demographic characteristics of the participants are presented in p < 0.001). Also, whereas 35.1% of mechanics regularly siphoned fuel in the discharge of their duties, 36.4% of sprayers never used nose mask in their discharge of their duties.The risky occupational-related behavior of the automobile workers is presented in p = 0.0354). Sprayers also had significantly higher haemoglobin levels compared to controls . However, whereas the MCH and MCHC were all significantly increased in cases compared to controls, the RDW was significantly reduced in cases compared to controls (p < 0.0001 in each case). Additionally, the total WBC counts , absolute lymphocyte count , and MID cell count  were significantly reduced in cases compared to controls. Moreover, platelet counts were significantly reduced in both mechanics (p = 0.0006) and sprayers (p = 0.0004) compared to controls.The haematological profiles of the study participants are summarized in p = 0.0088, Supplementary Among the cases however, only RBC count was significantly higher in sprayers compared to mechanics (p < 0.0001 (controls versus mechanics), or p < 0.0001 (controls versus sprayers)]. In addition, the absolute reticulocyte counts were significantly higher in cases compared to controls .To understand how the occupational exposure impacted the haematopoietic output, the % reticulocyte count and absolute reticulocyte counts were also compared between cases and controls . The % rChronic exposure to petrochemicals may adversely impact haematopoiesis as these contain heavy metals that may be inhibitory to enzymes crucial in blood cell formation . In thisPreviously, Aleemuddin et al. suggested that exposure to the heavy metals in aromatic compounds may lead to ineffective erythropoiesis as a consequence of its suppressive activity on the bone marrow . AnotherAlthough some of the findings in the Kamal and Malik  study inIt is interesting to note that our cases had significantly reduced red cell distribution width (RDW) compared to our controls. Previous prospective studies as well as meta-analysis have suggested that high RDW was associated with increased risk of cardiovascular disease –21. AlthRisky occupational behavior such as siphoning of fuel into the mouth by automechanics and not wearing nose masks (by automobile sprayers) in the work place was very prevalent in our study population. This is not surprising considering that majority of the cases (automobile sprayers and automechanics) had never received any training in occupational safety. This prevalent practice of aspiration of petrochemicals has previously been reported by others , 23. AltThere is reduced haematopoietic output in automobile mechanics and automobile sprayers. There is also lack of adherence to occupational safety principles which requires urgent attention if long term effects of petrochemical exposures are to be prevented.


Intrathecal immunoglobulin G (IgG) and oligoclonal bands (OCBs) are the most consistent and characteristic features of Multiple Sclerosis (MS). OCBs in MS are considered products of clonally expanded B cells in the cerebrospinal fluid (CSF), representing the sum of contributions from B cells in the brain. However, large amounts of IgG can be eluted from MS plaques in which lymphocytes are absent, and there is no correlation between levels of plaque-associated IgG and the presence of lymphocytes. It is calculated that it would take 3.2 billion lymphocytes to generate such large amounts of intrathecal IgG (30 mg in 500 ml CSF) in MS patients. Therefore, circulating lymphocytes in CSF could only account for <0.1% of the extra IgG in MS.We analyzed clinical laboratory parameters from sera and CSF of 115 patients including 91 patients with MS and 24 patients with other inflammatory central nervous system (CNS) disorders (IC). We investigated the relationship between oligoclonal bands, IgG antibodies, CSF cells, IgG Index, albumin, and total protein.MS patients have significantly elevated serum concentrations of IgG antibodies, albumin, and total protein, lower levels of lymphocytes, albumin, and total protein in the cerebrospinal fluid, but no difference in CSF IgG concentration compared to those with other inflammatory neurological disorders. Furthermore, in MS there was no linear relationship between the numbers of OCBs, CSF lymphocytes, CSF IgG, and IgG Index, and between serum IgG and serum albumin, but significant correlation between IgG in CSF and serum, and between CSF IgG and CSF albumin.There are unique differences between MS and patients with other inflammatory neurological disorders. Our data suggest that in MS patient (a) B cells and their products in the CSF may not be the sole source of intrathecal IgG; (b) oligoclonal bands may not be the products of single B cell clones in the CSF; and (c) there is a strong connection between serum components in the peripheral circulation and the central nervous system. Increased intrathecal synthesis of IgG antibodies and the presence of oligoclonal bands (OCBs) is the most characteristic feature of MS. OCBs are associated with increased levels of disease activity and disability , a greaA distorted self-reactive IgG antibody repertoire was found in MS sera , and thWe analyzed serum and CSF clinical lab parameters in 115 patients including 91 MS and 24 IC patients. We present data supporting a complex relationship between OCBs and other CSF parameters, suggesting a strong connection between serum IgG and intrathecal synthesis of IgG in MS.All data used for the analyses were extracted from clinical laboratory reports of patients from the University of Colorado Hospital (2001–2013). Serum and CSF samples were collected with approval from the University of Colorado Institutional Review Board (COMIRB # 00–688). Data (Serum and CSF samples) were anonymized before access/analysis. The protein, albumin and IgG concentrations were determined by an independent off-site laboratory using the Siemens BN™ II nephelometry system. The presence of oligoclonal bands was analyzed by ARUP Laboratories .Most of the data were from untreated patients. Information on gender was available for 100 patients (76 MS and 24 IC) . The mean age of MS patients was 42.8, and did not differ significantly from that of IC patients (mean age 49.6). A majority  of the MS patients were of the relapsing-remitting type (RRMS). Seven had primary progressive MS, one had the relapsing progressive type, and one was diagnosed as secondary progressive MS. Controls were defined as those with other inflammatory CNS (IC) diagnoses and are listed below with number of cases in parenthesis: acute viral meningitis (2), lymphoma (1), B cell lymphoma (1), Behcet's disease (1), paraneoplastic syndrome (1), viral meningitis (1), Cryptococcal meningitis (1), chronic meningitis of unknown etiology (1), subacute sclerosing panencephalitis (2), acute disseminated encephalomyelitis (1), paraneoplastic encephalitis (1), neurosyphilis, chronic progressive meningoencephalitis (1), sarcoid (1), VZV myelopathy (1), VZV radiculomyelitis (1), VZV shingles (1), ischemic optic neuritis (1), retrobulbar optic neuritis (1), papillitis (1), and transverse myelitis (1).Sera and CSF  of a serial 3-fold dilutions were blotted in duplicate on nitrocellulose membranes. The blots were incubated in HRP-conjugated goat anti-human IgG (H+L) (Sigma), followed by detection with SuperSignal® West Pico Maximum Sensitivity chemiluminescent substrate (Thermo Scientific). Band intensity analysis was performed using FluorChem Q™ system.Logarithmic transformations were used to improve normality of serum IgG, CSF IgG, albumin, and protein. Serum albumin and total protein were squared to produce a normally distributed variable. The number of lymphocytes in CSF, OCBs, and the IgG Index could not be transformed to normality. Student’s t-test was used to compare mean differences when variables were normally-distributed. The Wilcoxon non-parametric test were used for the number of CSF cells OCBs. A permutation test for differences in means with 100,000 iterations was used to compare mean levels of total protein in serum, CSF, and IgG Index due to the small sample size in IC patients.Polynomial regression was applied with a 95% confidence band around the estimated regression lines. We minimized the mean squared error over a number of different linear regression models to identify the maximum level of the predictor where the association changed from a positive to a negative relationship. We then divided the observations into two datasets and conducted significance testing using permutation tests to obtain the p-value for the correlation coefficient, and the slope of the regression lines before and after the cut-off point. A permutation-based p-value was generated because the OCBs were highly skewed and were not normally distributed. Similar analyses were conducted for relationship between OCBs and IgG concentration, and between OCBs and IgG Index.A sensitivity analysis was conducted to understand the association of number of OCBs and CSF IgG. OCB counts were dichotomized and correlations assessed between each categorical construct and the level of CSF IgG by category.The Pearson correlation coefficient was used due to the normality of the transformed variables and the linear relationships observed in regression models.MS patients had higher incidences of OCBs and higher values of IgG Index, but have no significant difference in CSF IgG levels compared to IC patients. Although CSF IgG in MS was about half the level of IC patients, this difference did not reach significance due to the high variability in CSF IgG concentration in IC patients. The mean differences in the number of CSF lymphocytes in MS patients compared to IC reached statistical significance , but witr = 0.22, p = 0.09, n = 40), and the slope of the regression line was 0.72 (se = 0.52). However, when CSF cells > 7, the linear association was negative , and slope of regression line was -0.25 (se = 0.20)  ; however, in the 16 observations where the IgG Index is greater than 1.14 but less than 1.9, the slope decreases, but not significantly so   . For CSF 0.0001) . There w = 0.68) .r = 0.33, p = 0.002). However, in 72% of MS patients when number of OCBs >2, there was no association between number of OCBs and CSF IgG  . The sensitivity analysis revealed that using cut-off values of OCBs > 3 (n = 52) and OCBs > 4 (n = 47), there is a trend towards becoming significantly correlated. At a cut-off value of 5, the p-value reaches 0.04, and the correlation once again becomes increasingly stronger. In 63 MS patients with fewer than 10 OCBs, there is a significant correlation of 0.34 (p = 0.006). In the 25 MS patients with OCBs ≥ 10, the linear association between OCBs and CSF IgG was strong and significant .The above analyses  suggest  = 0.35) , and thep = 0.03). Furthermore, the serum IgG concentration was significantly correlated with the CSF IgG concentration in MS . To a lesser extent, the serum IgG and CSF IgG was also correlated in IC patients  (p = 0.313)  . The hig= 0.313) .r = 0.55, p < 0.0001)   . This ro = 0.53) . High co = 0.53) .r = 0.5, p = 0.0003), and to CSF albumin . However, no such relationship was found in serum between total protein and IgG, and a lower but still significant correlation was observed between serum albumin and serum total protein   . Fig 6 sElevated intrathecal IgG and OCBs in MS are considered to be an indication of localized B-cell expansion in the brain , represIn MS brains, the presence of IgG containing lymphoid cells is well established , and ecThe contradiction between the lower number of CNS cells (about 3.5 million cells in MS CSF) and higher levels of intrathecal IgG (10 mg/day), combined with the evidence that there is no correlation between plasma cells and Gd-enhancing lesions in MS, raise the question as to whether B cells in MS CNS can be responsible for the massive amounts of elevated intrathecal IgG. Tourtellotte calculated that it would take 3.2 billion lymphocytes in MS to generate such large amounts of CNS IgG, therefore circulating MS CSF lymphocytes could only account for <0.1% of the extra IgG in the CSF. This apparent knowledge gap suggests that most of the intrathecal IgG in MS may actually be derived from the blood.It is estimated that 20–40% of the total IgG in MS CSF are contributed to OCBs . HoweveThe presence of a heterogeneous composition of OCBs and IgG immune complexes in MS brain has been suggested by several previous studies. Both IgG1 and IgG3 subclasses are found in the same band, indicating a micro-heterogeneous composition of the OCBs . In addr = 0.34, p = 0.006) and serum IgG . The similar correlation patterns further suggest a strong link between the two compartments. Our results are in agreement with earlier work that there is an association between high levels of intrathecal synthesis of IgG and serum OCBs [[The vascular origin of MS has been speculated over a century ago . Here wum OCBs . Link [um OCBs  have shThe strong correlation between MS CSF IgG and albumin argues that the two parameters are interchangeable, similar as that in healthy controls , suggesCompelling evidence of serum antibody-mediated demyelination in MS comes from the classic studies of Lumsden . They iIn summary, our results suggest that B cells and their products in the CNS may not be the sole source of intrathecal IgG, and OCBs may represent IgG complexes. The peripheral blood may contribute significantly to CSF IgG in MS. The data support a disturbed systemic immunity in MS, and may provide novel opportunities for blood biomarker identification and novel therapeutic targets in MS.


PSEN1 mutations, in demented Down syndrome individuals and in sporadic AD subjects compared to age-matched controls. Our data suggest that APP-CTF could be a potential diagnostic biomarker for AD.This study assesses whether C-terminal fragments (CTF) of the amyloid precursor protein (APP) are present in cerebrospinal fluid (CSF) and their potential as biomarkers for Alzheimer’s disease (AD). Immunoprecipitation and simultaneous assay by Western blotting using multiplex fluorescence imaging with specific antibodies against particular domains served to characterize CTFs of APP in human CSF. We demonstrate that APP-CTFs are detectable in human CSF, being the most abundant a 25-kDa fragment, probably resulting from proteolytic processing by η-secretase. The level of the 25-kDa APP-CTF was evaluated in three independent CSF sample sets of patients and controls. The CSF level of this 25-kDa CTF is higher in subjects with autosomal dominant AD linked to  Many reports support that the determination of Aβ42 in cerebrospinal fluid (CSF) is a core biomarker for AD2. While the amount of the pathological species of Aβ is increased in the AD brain, their levels in CSF are decreased, probably due to increased brain deposition. To enable monitoring early disturbance in amyloid precursor protein (APP) and Aβ mis-metabolism additional biomarkers are needed.Accumulation of the β-amyloid peptide (Aβ) in the brain is an early and specific phenomenon associated with the pathogenesis of Alzheimer’s disease (AD)3. The membrane remaining C-terminal fragments (CTFs) are always processed by γ-secretase generating shortest intracellular domain (AICD) peptides .Other plausible biomarkers for AD are additional fragments resulting from the processing of the amyloid precursor protein (APP). APP is a large type I transmembrane spanning protein consisting of a large N-terminal extracellular domain, a hydrophobic transmembrane domain, and a short intracellular C-terminal domain. APP is usually cleaved by α-secretase , or by β-secretase , which causes the secretion of large sAPPα and sAPPβ N-terminal fragments (NTFs). CSF levels of sAPPα and sAPPβ show no change in ADet al.5). Thereby, concerted cleavage of β-secretase and α-secretase result in the secretion of sAPPβ and shorter Aβ peptides6. The presence in CSF of short NTF derivatives of APP, which generation does not involve α-secretase or BACE, has been also demonstrated7. More interestingly in the context of this report, a recently discovered alternative physiological APP processing pathway driven by an asparagine endopeptidase (AEP) named δ-secretase8 or by a metalloproteinase named η-secretase9 will generate alternative proteolytic metabolites, including NTFs and CTFs, of different molecular mass.APP can also undergo alternative proteolytic processing pathways , Down syndrome subjects with Alzheimer’s type dementia (dDS), and sporadic AD subjects (sAD).In addition to the different species of Aβ, all the large extracellular APP-NTFs have been studied in human CSF9. 2D8 and 2E9 also detected other soluble fragments around ~15 kDa, termed Aη, generated after concerted cleavage by η-secretase and α/β-secretases9. 2D8 antibody also detected Aβ monomers. Multiplex fluorescence imaging with the A8717 and 2E9 antibodies indicated that the major 25 kDa band is compatible with the CTFη; similar co-labelling was revealed with antibodies C1/6.1 and 2D8  compared to those in age-matched NC composed by non-mutation carriers from the same families  in the intensity of the 25-kDa APP-CTF band, resolved with A8717 antibody, was determined in CSF samples from dDS patients, comparing these to age-matched NC  in sAD compared to age-matched NC subjects , correlating tightly with the estimation of the levels calculated with the A8717 antibody , an early-onset form of genetically determined ADies Fig. . Down sy NC Fig. . Finallycts Fig. . CSF sam9. Canonical APP proteolysis occurs via α- and β-secretases, resulting in CTFα and CTFβ with a molecular mass lower than 15 kDa; but novel APP-CTF that migrates ~25 kDa has also been described14. In addition to the described alternative pathways for APP9, other cleavages for APP could occur since non-canonical N-terminal APP fragments have been also identified in human CSF15. The precise characterization of the APP-CTF 25-kDa fragment as a CTFη requires further confirmation. In this regard, the development of pan-specific antibodies against the predicted N-terminus of CTFη could be valuable. Similarly to the reported by others15, attempts to immunoprecipitate the 25-kDa APP-CTF for sequencing were not fruitful. Since soluble full-length APP protein is also present in CSF, forming heteromers that co-immunoprecipitate with more abundant sAPPβ and sAPPα16, an enrichment process based in available APP C-terminal antibodies could result of lower efficient and specificity for APP-CTFs. Similar handicap will difficult development of a specific ELISA assay (discussed below).Here, we demonstrate that APP-CTFs are detectable in human CSF. Particularly, the most abundant soluble APP-CTF, a 25-kDa fragment, is attributed to a CTF recently characterize as the result of proteolytic processing by η-secretase (CTFη)17. Early studies failed to detect short C-terminal fragments of APP in the medium of cultured cells18; but others suggested the release of largest APP-CTF fragments from cultured neural cells19. Moreover, a series of APP-CTFs was found to be secreted within exosomes in cultured cells20, as well AICD21. The mechanisms by which these membrane-bound proteins reached the CSF are unknown; but neuronal death may be also a major contributing factor. Regarding the cellular source of the soluble 25-kDa APP-CTF found in CSF, the presence of hitherto undocumented APP fragments, including a 25-kDa CTF, has been documented in hippocampal neurons9, but also across other multiple cell types, including bovine brain microvascular endothelial cells15. Since the δ-secretase enzyme AEP is a widely distributed22, we could not discard a non-neural source for the 25-kDa APP-CTF found in CSF. The cellular origin of the 25-kDa APP-CTF could be also relevant in order to define their potential as an AD biomarker.As an intermediate of proteolytic process, the presence of any APP-CTF in CSF was unexpected. Anyhow, the presence in CSF of soluble forms of full-length membrane proteins containing the transmembrane and intracellular domains is not an unusual finding24. The current canonical views about APP-related cleavage events are likely a partial understanding of the overall process. How secretases compete for the APP substrate and whether subcellular compartmentalization of APP and secretases is responsible of the dynamic equilibrium between secretases is under discussion25. Thus, one may speculate that the particular abundance of the 25-kDa CTFη in CSF is related with compartmentalization of secretases, resulting in prolonged time of residence of these CTFη in comparison with canonical CTFα and CTFβ. Anyhow, the other fragments derived of the further processing of CTFη by α- and β-secretases, the Aη-α and Aη-β fragments, have been also identified in human CSF with levels similar than those for Aβ9.Different APP C-terminal antibodies confirmed the 25 kDa as the predominant band in human CSF. As stated, these APP proteolytic fragments are derived from a constitutive processing step driven by a η-secretase that is partially in dynamic equilibrium with α- and β-secretase-mediated proteolysis26. Further studies will indicate if distinct pattern of APP processing in ADAD and sAD affect the generation of different APP-CTFs. Given that this is the first report that address the levels of APP-CTF in CSF, in order to prevent influence of pre-analytical confounding factors, in our study each pathological group was compared with age-matched controls obtained from the same center, with same volume of CSF taken, stored in aliquot of same volume and for similar period of time; and avoiding freeze/thaw cycles. Further studies are required to determine whether limiting pre-analytical confounding factors , have a significant impact on the measured levels of APP-CTF by Western blotting. The possibility that APP-CTF levels varies with age should also be considered and deserve a specific study with a larger number of samples.Our determination of the 25-kDa APP-CTF levels by Western blotting displayed overlapping values between sAD and controls, whereas superior discrimination was obtained for ADAD and dDS subjects, compared to their respective controls. Interestingly, APP CTFβ levels are also elevated in frontal cortex brain homogenates obtained from ADAD patientsIn our statistical analysis high levels of the 25-kDa APP-CTF in CSF correlated with increased concentration of T-tau and P-tau, but failed to correlate with Aβ42 levels. Increased concentration of T-tau in CSF is believed to reflect neurodegeneration whereas P-tau is supposed to reflect tangle pathology. Otherwise, the overall decrease in Aβ42 concentration in the CSF patients is not illustrative of imbalances in the brain APP processing, since in parallel with the increase in the generation of Aβ42, there will be an increment in Aβ aggregation with subsequent deposition. Thus, the positive correlations of the 25-kDa APP-CTF with T-tau and P-tau suggest a correlation with AD pathological process, but it is not possible to interpret links with altered APP processing. Anyhow, the mentioned correlations resulted positive only when all samples were considered, and not in control or sAD groups considered separately. Thus, these correlations obtained by pooling controls and demented subject, could be artefactual, driven by the anchoring effect of the control values. Further analysis with a large number of samples is required.16, and transmembrane domain, as well intracellular C-terminal domain could participate in APP dimerization . Indeed, intracellular CTF aggregates have been described with the participation of Aβ29 and heparan sulfate degradation products30, suggesting that other APP fragments and other proteolytic sub-products could interact with APP-CTFs. Thus, future studies should develop custom pan-specific antibodies targeting the predicted N-terminal sequence of the APP-CTFs present in CSF, maybe including pretreatment methods designed to disaggregate peptides31, for preventing co-precipitation of other APP fragments. Anyhow, even the disadvantages of Western blotting for quantitative analysis, we considered demonstrated that accumulation of APP-CTFs in CSF constitute a potential new biomarker of AD.In term to define the true potential of the 25-kDa APP-CTF as a CSF biomarker, it will be valuable to replicate our present finding using techniques such as ELISA. However, this desirable outcome will be challenging. As advanced above, we have described that the soluble full-length APP, containing the C-terminal domain, also exists in CSF as heteromeric complexes compromising other sAPP speciesIn resume, to our knowledge, the possibility that APP-CTFs can be assessed in the CSF has thus far not been considered. In this study, we demonstrate noticeable amounts of APP-CTFs in human CSF. Particularly, a 25-kDa APP-CTF appears increased in genetically determined AD, as well as in sAD. Our present findings provide sufficient evidence to justify further studies on the determination of APP-CTFs as a potential new diagnostic biomarker of AD.PSEN1 mutations and who were part of the Genetic Counseling Program for familiar dementia (PICOGEN) at the Hospital Clínic . This group included 7 subjects carrying PSEN1 mutations, and 7 age-matched non-mutation carriers from the same families (non-disease controls: NC). We also included lumbar CSF samples from 7 Down syndrome subjects with Alzheimer’s type dementia (dDS), along with 7 age-matched NC obtained from the Hospital Sant Pau . In addition, 20 subjects with sporadic AD (sAD) defined as patients with cognitive symptoms and a CSF biomarker profile indicating AD  and controls gave informed consent to participate in the study.Lumbar CSF samples were obtained from autosomal dominant AD (ADAD) subjects that were all carriers of 9. Multiplex fluorescence with two of the independent antibodies served to assess the specificity of the bands. Blots were then probed with the appropriate conjugated secondary antibodies  and imaged on an Odyssey Clx Infrared Imaging System (LI-COR Bioscences). Band intensities were analyzed using LI-COR software (Image Studio Lite). A control CSF sample was used to normalize the immunoreactive signal between blots.Samples of CSF (30 µL) were denatured at 98 °C for 5 min and were resolved by electrophoresis on 16.5% Tris-Tricine gels. Following electrophoresis, proteins were blotted onto nitrocellulose membranes . CTFs of APP were detected using the following anti-APP C-terminal antibodies: C1/6.1 , A8717 , and Y188 , as well as with the rat antibodies 2D8 and 2E9For immunoprecipitation, samples were precleared for 2 h at 4 °C by incubation with protein A-Sepharose (Sigma Aldrich Co). Immunoprecipitations were performed overnight at 4 °C by incubating 150 µL of CSF with the indicated anti-APP antibody. The antibody was previously coupled to protein A-Sepharose using dimethyl pimelimidate dihydrochloride (Sigma Aldrich Co). A rabbit IgG and a rat IgG (Vector) were used as control antibodies for immunoprecipitations. Precipitated proteins were washed with PBS and eluted with 0.1 M glycine buffer at pH 2.5. After pH neutralization, supernatants were denatured in Laemmli sample buffer at 98 °C for 5 min and subjected to SDS-PAGE. The membranes were then probed with an alternative anti-APP antibody.Total tau (T-tau), phosphorylated tau (P-tau) and Aβ1–42 (Aβ42) concentrations in CSF were measured using INNOTEST ELISA methods .t test (two-tailed) for determination of exact p values. Correlation was assessed by linear regression. Results are presented as means ± SEM.All data were analyzed using SigmaStat  by Student’s Supplemental figures


P < 2.2*10−16) and CSF metastatic cells obtained before chemo/radiotherapy ; normal in inflammatory control cells (2.6 ± 0.04 ns) and cells from children without metastasis before chemo/radiotherapy  and following treatment ; and short in CSF metastatic cells obtained after chemo/radiotherapy . FLIM is a simple test that can potentially identify CSF spread of brain tumours. FLT changes in accordance with treatment, with significant prolonged median values in tumours and metastases. More accurate detection of metastatic cells may guide personalised treatment and improve the therapeutic outcome.In pediatric brain tumours, dissemination of malignant cells within the central nervous system confers poor prognosis and determines treatment intensity, but is often undetectable by imaging or cytology. This study describes the use of fluorescence lifetime (FLT) imaging microscopy (FLIM), a novel diagnostic tool, for detection of metastatic spread. The study group included 15 children with medulloblastoma and 2 with atypical teratoid/rhabdoid tumour. Cells extracted from the tumour and the cerebrospinal fluid (CSF) 2 weeks postoperatively and repeatedly during chemo/radiotherapy were subjected to nuclear staining followed by FLT measurement and cytological study. Control CSF samples were collected from patients with infectious/inflammatory disease attending the same hospital. Median FLT was prolonged in tumour cells (4.27 ± 0.28 ns;  Embryonal tumours are the second most common type after gliomas. Three-fourths of embryonal tumors are medulloblastomas3, making medulloblastoma the most common malignant tumour of childhood .Brain tumours account for at least one-fourth of all cases of cancer in the pediatric age group and are the leading cause of cancer-related death in this population4, and one-third of patients have metastatic spread already at diagnosis. Treatment consists of surgical resection, craniospinal irradiation, and aggressive chemotherapy sometimes supplemented by autologous bone-marrow transplantation. The 5-year overall survival rate ranges from 60% to 70%5. Despite recent advances in treatment, 30% to 40% of children experience tumour recurrence, and most of them die from disease especially if previously irradiated6.Medulloblastomas affect the posterior fossa7. Although ATRT appears to be chemosensitive, chemotherapy-only regimens are often insufficient9, and there are few long-term survivors. Median overall survival is around 10 months10, and with chemotherapy12, radiotherapy13, or both15, it may reach 14–18 months or more.Another highly malignant tumour of childhood is atypical teratoid/rhabdoid tumour (ATRT), usually diagnosed in the first 2 years of life16. However, current means of detection of CNS disease, mainly preoperative imaging and postoperative cytologic study of the cerebrospinal fluid (CSF), are limited and reviewer-dependent. This is crucial, because diagnostic errors may lead to under-treatment, which may be life-threatening, or over-treatment, which places patients at unnecessary risk of severe adverse effects and future disability. Therefore, novel diagnostic solutions are urgently needed.Clinically, malignant brain tumours usually metastasize within the central nervous system (CNS) and very rarely to extraneural sites. The dissemination of malignant cells within the CNS is associated with a very low survival rate17. These factors may help clinicians differentiate malignant from non-malignant cells. Fluorescence lifetime imaging microscopy (FLIM) is a novel spectroscopic technique that incorporates hitherto uncorrelated mathematical18 and physical19 methods of light-tissue interaction21. FLIM produces images based on differences in the rate of fluorescence decay, providing both temporal and spatial information on changes in the fluorescence lifetime (FLT) of fluorescently labelled components in a sample. Contrast is generated by the FLT of individual fluorophores at each pixel. FLIM has a wide range of biological and biomedical applications24, such as quantification of tissue morphology and as an add-on in high-density protein arrays25. It differs from the earlier steady-state fluorescence techniques used in the bio-sciences which are not useful for quantitative investigation of cellular function at the molecular level.Studies of light propagation in tissue models have shown that different tissues have different diffusion reflection profiles and that light propagates differently through different cell typesThe aim of the present study was to determine if FLIM technology may be used to detect and monitor metastatic cells in the CSF in children with brain tumours. The early detection of metastasis in the CSF has important implications for improving treatment regimes, monitoring of disease progression, identification of disease recurrence, and assessment of the success of a selected therapy.Seventeen children were included in the study group: 15 with medulloblastoma and 2 with ATRT. Their demographic, clinical, and treatment data are shown in Table Findings on FLIM study in the individual patients are shown in Table In most cases, the FLT value was higher at diagnosis than at the end of follow-up.Seven CSF samples were collected from patients with a noncancerous diagnosis of infection/inflammation. Median FLT, calculated per slide per patient (11–36 cells per slide) ranged from 2.49 to 2.67 ns Table .The FLIM results are summarized in Table P < 2.2*10−16) and metastatic CSF cells before treatment . Group B had a statistically significantly longer FLT than every other group. Normal/medium FLT values were seen in CSF cells obtained from children with non-metastatic disease before chemo/radiotherapy  and after chemo/radiotherapy . Short FLT values were seen in CSF cells from children with metastases after chemo/radiotherapy . Figure Prolonged FLT values were seen in both the original tumour cells  from metastatic disease at diagnosis. The ability to detect occult metastasis at diagnosis using a simple CSF examination has important therapeutic implications. FLIM also makes disease follow-up possible with repeated CSF sampling for early detection of recurrence.We found 3 different FLT measurements in CSF cells, clearly differentiating normal inflammatory non-malignant cells from malignant cells. The median FLT of metastatic CSF samples was significantly longer than that of the other CSF samples.One of the advantages of this study is that it includes 17 children with long-term and intensive follow-up, with a mean of 3 examinations per child and a total of 58 FLT measurements. Therefore, FLT results could be compared to the clinical, histological and imaging results at each time point. FLT was found to be reliable, reproducible, and highly correlated to the clinical course, treatment, and outcome.6. The presence of CNS metastases is assessed in two ways: prior to surgery by MRI of the whole craniospinal axis and approximately 2 weeks after surgery by cytologic study of CSF samples. Lumbar puncture to collect CSF cannot be performed before surgery because of the risk of brain stem herniation. In patients with metastatic disease at diagnosis, treatment is usually upgraded to include a higher dose of craniospinal irradiation and possibly autologous bone marrow transplantation following high-dose chemotherapy.According to the literature, about one-third of children with medulloblastoma have metastatic disease within the CNS at diagnosis26. Its accuracy depends largely on the quality of the sample and the experience of the cytopathologist who must, in equivocal cases, decide if the findings are pathological. Furthermore, studies have shown that M1 disease (presence of positive cells in the CSF) is also a poor prognostic marker, even in children with normal-appearing imaging studies, and is treated with high-risk treatment protocols28 similar to those used for overt metastasis4.MRI is limited in this setting because micrometastases appear as equivocal cord irregularities and may be confused with blood vessels and other structures. The cytologic study, performed when the post-surgery edema has decreased but prior to the initiation of chemo/radiotherapy, consists of microscopic examination of the CSF for appearance and staining characteristics of cells5. Over-treatment predominantly involves the dose of radiation. The higher radiation dose of the high-risk as opposed to the average-risk protocol (36.0 vs 23.4 GY CSI for patients older than 3 years) makes an enormous difference in outcome in terms of long-term side effects, including poor cognitive functioning, short stature, and hearing impairment. At the same time, under-treatment may pose a risk of reduced survival, since salvage of relapsed patients is low, particularly after prior radiation.Thus, better means to rapidly and accurately identify malignant spread of pediatric brain tumours are needed. Accuracy is essential because cells in the CSF at 2 weeks after surgery may be indicative of postoperative complications, including infection and inflammation, as well as true metastatic spread. The failure to correctly differentiate between malignant and non-malignant cells may lead to over- or under-treatmentIn the present study, we identified 3 different FLT populations that differentiated among cells of different types and at different stages. Despite the variability in each slide and the wide FLT range, the majority of the cells from each patient  were significantly indicative of the clinical stage (non-metastatic/metastatic) and response to treatment. In one of our patients  as well as longer (6 ns) FLT values of DAPI in B-cells from leukemia patients30. There are several possible causes of such restriction, namely, structural properties 31, binding properties , or simply solvent viscosity. The present results point to the viscosity of the fluorophore-hosting area as the culprit in the FLT alterations. This is in line with our previous findings of a clear correlation of FLT and processes of the peripheral blood mononuclear cell response to mitogens, as well as to antigens and phorbol esters32 and the contraction cycle of cardiac cells34.When monitoring cell functionality, cytometrists more frequently use fluorescence intensity rather than FLT. However, unlike direct fluorescent intensity measurements, which are strongly dependent on non-relevant variables , FLT is intrinsic, thereby allowing for more reliable comparisons of data obtained from different experiments and different laboratories. High FLT values in cells (6 ns vs. 2.6 ns in the present study) may point to a significant restriction on mobility of the probe imposed by its intracellular microenvironmentThe results of our FLT studies were nearly all in accordance with the cytology results. CSF samples were taken only when clinically indicated; in some patients, when the disease was clearly metastatic, no samples were obtained. Our results yielded a statistically significant difference between metastatic and local disease, and high correlations with the clinical observations and outcome were noted over the follow-up period. With a larger cohort, multidimensional scaling analysis as well as a machine-learning approach could be performed in order to quantify the power of FLIM technology and detect metastatic disease in the early stages. To overcome the variability of measurements for the same patient, we measured as many cells as possible per slide and calculated the median. Within each cell-source group (A-F), except for the non-malignant inflammatory group (group G), there was wide variability among the patients, but we found the median values of each group to be representative and useful for comparing the groups at different stages of treatment.37. There are already some reports describing the assessment of cell-free DNA in the CSF as a tumour marker39. These techniques hold great promise for the future.In the future, it may be possible to distinguish metastatic from localised disease by measuring circulating DNA in the blood or CSFAt present, in the absence of supersensitive methods to detect minute quantities of tumour in DNA or RNA in blood, the FLIM system is a promising auxiliary tool for the early and reliable detection of metastatic spread in the CSF of children with malignant brain tumours.The study was conducted at Schneider Children’s Medical Center of Israel. The study design adhered to the tenets of the Declaration of Helsinki and was approved by the Schneider Children’s Medical Center of Israel and national review boards of the Israel Ministry of Health before its initiation. Informed consent was obtained from all parents. The anonymous CSF samples were obtained under separate institutional review board approval and exempted from consent.Tumour samples were collected at primary surgery. CSF samples were collected at least 2 weeks after primary surgery, during the course of diagnostic lumbar puncture performed prior to chemo/radiotherapy. Additional CSF samples were collected after administration of chemotherapy, radiotherapy, or both as part of the routine clinical follow-up. No lumbar punctures were performed for research purposes. Demographic, imaging, and treatment data were collected from the medical files.Control samples of CSF with inflammatory cells derived from patients with non-oncological, infectious diseases attending the same hospital during the same period were collected from the hospital’s microbiology laboratory.A tissue cut of up to 10 mg from tumour samples was subjected to gentle pipetation with 1 ml pipete, in order to separate the cells. Tumour and CSF samples were centrifuged at 600 × g for 7 min. The supernatant fluid was gently removed, and 500 μl of PBS solution were added to the sediment cells. These steps were then repeated. After the second centrifuge, the supernatant fluid was gently removed, and 10 μl of a solution containing methanol and acetic acid (3:2) were added to the cells, followed by rapid transfer of 20 μl of the cells to the center of a slide. After 7 min, the slide was transferred through an ethanol gradient . Two drops of NucBlue® Fixed Cell Stain ReadyProbes® Reagent  were diluted in 1 ml phosphate-buffered saline, and 200 μl of this 4′,6-diamidino-2-phenylindole (DAPI) solution were added to the slide. After 5 min, unincorporated DAPI solution was removed, and 10 μl of ProLong® Gold Antifade Mountant Reagent  were added.2CAM MD intensified CCD camera with modulated image intensifier for detection of the fluorescence images, a modulation signal generator/power supply unit, computer, and the LI-FLIM software package for complete system control, image acquisition, and FLT calculation. The excitation source of 403 nm was set to a repetition frequency between 40 and 60 MHz for heterogeneous samples; the emission wavelengths were 420 nm and above. In slides rich with cells, 20 to 106 cells were randomly selected and measured. When there were fewer than 10 cells, all were measured. Each measurement was repeated twice. Median FLT was calculated per slide. As shown in previous studies, the FLT of free DAPI is 2 ns, and the FLT of DAPI in normal cells is 2.7 ns26. As a control experiment, cells from the same patients were measured without DAPI staining and below magnification of MCP GAIN of 750 ± 50 V. Under these conditions, no fluorescence intensity was detected. Above 750 ± 50 V, weak fluorescence intensity signals can be extracted, with average FLT of 1.57 ± 0.64 ns (data not shown). Except for this phenomenon, no autofluorescence was observed. In our experiments, we used magnification in the range of 620-730 V.The FLIM experiments were performed using an Olympus IX-81 inverted microscope with a 10X, NA = 0.4 objective . The images were acquired with a Lambert Instruments (LI) system  consisting of 3 modulated LED excitation sources at 403 nm, 468 nm, and 537 nm, a LI30. In our study, the FLT measurements of DAPI bound to DNA in tumour cells were divided into subgroups, as described in a previous study on the use of FLIM to classify B-cell chronic lymphocytic leukemia30. The FLT of the DNA binder DAPI found in healthy cells, τ = 2.66 ± 0.12 nsec (range 2.3–2.92 nsec), correlated with the FLT of DAPI in the control group. The three groups with an abnormal value were classified as short+, prolonged, and prolonged + according to their relative proximity to the normal group: τ ≪ norm (τ < 1.8 nsec), τ > norm (3 nsec < τ < 4.5 nsec), and τ ≫ norm (τ > 4.5 nsec), respectively. FLIM measurements revealed that values in the range of 1.8 nsec < τ < 2.2 nsec were related to free DAPI (not bound to DNA) and therefore were not part of the analysis (2 ± 0.12 ns). The FLT value was calculated by extracting the phase FLT (τφ) and the modulation FLT (τm), yielding a total single FLT value.It is well known from the literature that the FLT of DAPI has a heterogeneous distribution40. Patients were divided into different groups by time from diagnosis, therapy stage, and disease status. The FLT values were normalised before merging in order to prevent bias due to differences in cell number between slides. Due to the high variability of the FLT measured per slide, median and median absolute deviation were calculated per slide and per group. Median absolute deviation is insensitive to the presence of outliers, unlike the standard mean/standard deviation combination. Wilcoxon signed rank test was used to test the significance of differences in FLT distribution. We compared the control group (G) against each of the other groups, separately. We also compared each group against the CSF metastatic, pre-therapy group (B). Hierarchical clustering was performed using R framework by applying a 1-correlation distance measure and average-linkage clustering.Boxplots and density plots were generated using R statistical languageThe study was approved by the national and institutional ethics committees. Informed consent was obtained from the parents of all children. The anonymous samples of the CSF obtained for the control group were exempted from consent.


Repeated nontreponemal serologic test for syphilis titers is recommended to evaluate treatment response. However, it is unknown whether serum rapid plasma reagin (RPR) titer can serve as a surrogate for determining the efficacy of treatment in general paresis (GP) remains unknown.We retrospectively reviewed data from 105 GP patients, who were divided into two groups (62 CSF RPR+ patients and 43 CSF RPR- patients) according to reactive RPR test status in CSF. Clinical assessment included the Mini-Mental State Examination (MMSE) scores, CSF examinations , and serum tests (RPR titer and TPPA). Among the 105 GP patients, 13 CSF RPR+ patients and 6 CSF RPR- patients had a 12 months follow-up of CSF, serum measures and MMSE.P < 0.001). The number of CSF RPR+ patients with serum RPR titer≥1:32 was significantly higher when compared with CSF RPR- patients (P = 0.001). For CSF RPR+ patients, the MMSE scores improved or remained constantly after penicillin treatment. For CSF RPR+ patients, the CSF RPR titer declined four-fold in 85% (11/13) of the patients, whereas the serum RPR titer declined four-fold in only 46% (6/13) of the patients, the odds ratio is 6.4 .The median serum RPR titer was significantly higher in CSF RPR+ patients than that in CSF RPR- GP patients, 1:8 [IQR 1:4–1:32] vs. 1:4 [IQR 1:4–1:8] (A four-fold decline in CSF RPR titer is a good predictor for treatment efficacy in CSF RPR+ GP patients within 12 months after the completion of therapy.The online version of this article (10.1186/s12879-018-3062-4) contains supplementary material, which is available to authorized users. Treponema pallidum, is a major health problem worldwide. Cases of syphilitic dementia , which is characterized by rapidly progressive cognitive decline with or without psychiatric features, continue to be reported in the modern era [Syphilis, which is caused by dern era .T. pallidum Haemagglutination test (TPHA) and T. pallidum Particle Agglutination test (TPPA)), symptoms and signs of neurosyphilis and reactive venereal disease research laboratory (VDRL) in cerebrospinal fluid (CSF) or CSF white blood cells (WBCs) > 5/μl or CSF protein > 45 mg/dL are used in the diagnosis of symptomatic neurosyphilis [Serum treponemal and non-treponemal tests are the most widely used methods for syphilis diagnosis. Reactive serum treponemal test  serologic test for syphilis has been recommended to evaluate treatment response, with a four-fold decrease from baseline and/or seroreversion in 12–24 months after treatment representing an appropriate response to therapy/serologic cure .The goal of this study is to explore whether serum RPR titer could serve as a surrogate for determining the efficacy of treatment in GP.We retrospectively reviewed 105 GP patients . These GP patients were late syphilis and fulfilled all of the following criteria: (1) TPPA and RPR titer were tested in serum and CSF, and were positive in serum TPPA; 2) serologic test was based on RPR positivity in CSF, or WBC count of CSF>5 cells/μL or protein of CSF>45 mg/dL with TPPA positivity in CSF  serologi. WBC couThirteen CSF RPR+ GP patients returned for follow-up visits with lumbar puncture, MMSE scores and blood samples in 12 months after penicillin treatment   software . Associations between categorical variables were assessed using the χ2 test or Fisher’s exact test. Associations between continuous variables and categorical variables were assessed using the Mann-Whitney The study was conducted according to the principles expressed in the Declaration of Helsinki and approved by the Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University. The patients or the guardians of patients with severe cognitive impairment provided written informed consent. Lumbar puncture was also performed with informed consent.P < 0.001, see Table P = 0.001). The number of CSF RPR+ GP patients who had no abnormalities or only one abnormality on initial lumbar puncture was significantly different when compared with CSF RPR- patients .One hundred and five GP patients were divided into two groups (62 CSF RPR+ patients and 43 CSF RPR- patients) according to reactive RPR test status in CSF. The characteristics and CSF abnormalities of GP patients with CSF RPR+ and CSF RPR- are shown in Tables P < 0.001, see Table As shown in Tables Fourteen CSF RPR- GP patients  had a follow-up evaluation of serum RPR titer . The normalization of CSF protein concentration occurred in most of CSF RPR- GP patients by follow-up, which suggested that it may be a good indicator of the success of therapy in GP, although the number of this patient group by follow-up is relatively small. Further studies should be conducted to provide more evidence on this question.During the follow-up at 12 months after penicillin treatment, no significant difference was found in the four-fold decline in serum RPR titer between CSF RPR+ and CSF RPR- patients. According to our data and our analyses, for CSF RPR- patients, normalization of CSF measures, not serum RPR titer value, is a sensible way of assessing treatment efficacy. A cutoff of greater than or equal to 5 cells/μL has been the standard for determining normal CSF values. For CSF RPR- GP patients, CSF WBC count was often normal before treatment, so it is difficult to evaluate the treatment efficacy by assessing the recovery of CSF WBC. CSF protein concentration in neurosyphilis is slow to normalize and may remain elevated even after the normalization of other CSF and clinical abnormalities , 18. ThuSome limitations of our study should be considered. Specifically, a) the study is a retrospective study, in which case the association may be confounded or modified by patient characteristics; b) the large percentage of loss of follow-up leads to small number of the patients with follow-up, which has limited the statistical power; c) further studies are required to assess an association between RPR titers and the clinical features of these cases, and to identify an ideal indicator of the success of therapy for CSF-RPR- GP patients.The practical implications of our findings are that a four-fold decline in CSF RPR titer is a good predictor for treatment efficacy in CSF RPR+ GP patients within 12 months after completion of therapy. The sole use of serum RPR titer might not adequately predict treatment efficacy of GP. Considering the limitations of this study, larger samples and more follow-up data are needed for future investigation. Furthermore, the association between RPR titers and the clinical features of these cases need to systematically quantified, and more indicators of treatment efficacy need to be investigated for CSF RPR- GP patients.Additional file 1:Table S1. Follow-up of MMSE, CSF and serum measures in CSF-RPR+ patients. Thirteen CSF RPR+ GP patients returned for follow-up visits with lumbar puncture, MMSE scores and blood samples in 12 months after penicillin treatment. (DOCX 19 kb)Additional file 2:Table S2. Follow-up of MMSE, CSF and serum measures in CSF-RPR- patients. Fourteen CSF RPR- GP patients returned for follow-up visits with MMSE scores and serological tests in 12 months after penicillin treatment, while 6 of them had a follow-up evaluation of CSF. (DOCX 19 kb)