Salmonella (NTS) infections caused primarily by S. Enteritidis and S. Typhimurium particularly in immunocompromised hosts have accounted for a large percentage of fatalities in developed nations. Antibiotics have revolutionized the cure of enteric infections but have also led to the rapid emergence of pathogen resistance. New powerful therapeutics involving metabolic enzymes are expected to be potential targets for combating microbial infections and ensuring effective health management. Therefore, the need for new antimicrobials to fight such health emergencies is paramount. Enteric bacteria successfully evade the gut and colonize their hosts through specialized virulence strategies. An important player, alanine racemase is a key enzyme facilitating bacterial survival.Non-typhoidal alr, dadX and SEN3897 to Salmonella survival in vitro and in vivo. We have shown SEN3897 to function as a unique alanine racemase in S. Enteritidis which displayed essential alanine racemase activity. Interestingly, the sole presence of this gene in alr dadX double mutant showed a strict dependence on d-alanine supplementation both in vitro and in vivo. However, Alr complementation in d-alanine auxotrophic strain restored the alanine racemase deficiency. The Km and Vmax of SEN3897 was 89.15 ± 10.2 mM, 400 ± 25.6 µmol/(min mg) for l-alanine and 35 ± 6 mM, 132.5 ± 11.3 µmol/(min mg) for d-alanine, respectively. In vitro assays for invasion and survival as well as in vivo virulence assays involving SEN3897 mutant showed attenuated phenotypes. Further, this study also showed attenuation of d-alanine auxotrophic strain in vivo for the development of potential targets against Salmonella that can be investigated further.This study aims at understanding the contribution of alanine racemase genes S. Enteritidis which had a potential effect on survival and pathogenesis in vitro and in vivo. Our results also confirmed that SEN3897 by itself wasn’t able to rescue d-alanine auxotrophy in S. Enteritidis which further contributed to its virulence properties.This study identified a third alanine racemase gene unique in The online version of this article (10.1186/s13099-018-0257-6) contains supplementary material, which is available to authorized users. Salmonella enterica are Gram-negative facultative intracellular pathogens bearing around 2400 serovars which either have certain host specificity or infect a huge variety of hosts [Salmonella consists of two species namely S. bongori and S. enterica. The two epidemiologically significant serovars are Salmonella enterica serovar Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) [S. Enteritidis infections to be 43% as compared to 19% of S. Typhimurium. These infections have raised the bar of concern for public health. Hence this has led to the identification of multiple virulence factors and pathogenicity islands for determining their role in virulence [S. enterica infects host cells with its supramolecular needle-like complex called the Type Three Secretion System (T3SS) [Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) for invasion into epithelial cells and survival within phagocytic cells respectively. Both SPI-1 [Salmonella pathogenesis which is mediated through the secretion of effectors via their respective T3SS. The injection of effectors into host cells leads to actin cytoskeletal rearrangements and membrane ruffling, thereby internalizing the bacterium and further mediating pathogen survival and infection persistence [Salmonella pathogenesis. The deletion of such virulence determinants would aid in analyzing their host interaction or contribution to microbial adaptation [Salmonellosis is one of the leading causes of mortality among infectious diseases . Salmoneof hosts . Salmoneirulence . S. entem (T3SS) . This isth SPI-1  and SPI-th SPI-1 , 9 are esistence , 11. Thoaptation .Salmonella enterica serovar Enteritidis (S. Enteritidis) and Typhimurium (S. Typhimurium) cause non-typhoidal self-limiting gastroenteritis with symptoms of fever and diarrhoea in humans [S. Typhimurium both in vitro and in vivo models [S. Enteritidis in streptomycin pretreated C57BL/6 murine models as compared to SPI-1 deficient S. Typhimurium strain [S. Enteritidis would be of significant relevance. A study reported that there are certain additional genes present in S. Enteritidis for infection establishment as compared to S. Typhimurium [S. Enteritidis contributing to greater colonization and host–pathogen interaction. A previous study from our laboratory involving a comparative genome analysis of Enteritidis and Typhimurium serovars showed 98% identity with 2% extra genetic elements likely being involved in the greater infection phenotype of S. Enteritidis [S. Enteritidis as compared to Typhimurium most of which were clustered in unique islands gained through horizontal gene transfer termed as “regions of difference” (ROD).n humans . A microo models –17. Anotm strain , 19. Henhimurium . This leeritidis . Both seS. Enteritidis which wasn’t present in S. Typhimurium. Alanine racemases (EC 5.1.1.1) are unique pyridoxal-phosphate dependent bacterial enzymes essential for the reversible racemization of l-alanine to d-form [d-alanine for peptidoglycan synthesis. Generally either the presence of one or two alanine racemase genes have been reported [Salmonella Typhimurium; there are two isoforms (non-homologous) of alanine racemase genes . The constitutively expressing alanine racemase gene; alr is essential for cell wall synthesis by forming d-alanine required for peptidoglycan cross-linking [dadX forms a secondary source for cell-wall biosynthesis though it’s basically required for l-alanine catabolism forming a substrate for d-alanine dehydrogenase (dadA). Nonetheless in S. Enteritidis; in addition to these two conserved alanine racemase genes, the presence of an additional alanine racemase gene (SEN_3897) was quite interesting and speculative in terms of its functional relevance in the pathogen. Moreover bacterial cell wall has always been an interesting target for many antibiotics and antimicrobial agents [S. Enteritidis and investigate its role in virulence with a view that it may serve as a potential therapeutic target.The objective of the present study is to investigate the role of SEN3897, an alanine racemase gene that showed significant differential expression in ROD34 island of o d-form , 22. Thereported –25. For -linking . Anotherl agents . Hence tSalmonella Enteritidis P125109 and its isogenic mutants were monitored for their growth kinetics by spectrophotometric measurements (OD600) in Luria–Bertani (LB) medium  with/without d-alanine supplementation (100 µg/ml) at 37 °C, 150 rpm. The strains were sub-cultured (1:100 ratio) from overnight grown cultures under optimum conditions and absorbance measurements and plating were performed every 1 h till 10 h. The invasion assays required the bacterial strains to be grown overnight at 37 °C 120 rpm in SPI-1 inducing medium (LB containing 0.3 M NaCl) and subsequently subcultured under the similar conditions till its exponential phase. The growth media was supplemented with suitable antibiotics when required: ampicillin (Amp), 100 µg/ml; kanamycin (Km), 50 µg/ml; chloramphenicol (Cm), 20 µg/ml and streptomycin (Sm), 50 µg/ml.The bacterial strains and plasmids used in this study are listed in Table S. Enteritidis genome were constructed by the conventional phage transduction method. Briefly, the grown culture of the recipient mutant strain (KmR) was transduced with the phage lysate of the donor mutant strain (CmR) for 15 min at 37 °C. The transduced colonies were then selected on LB plates with kanamycin (50 µg/ml) supplementation and screened in PCR with FRT-specific primers confirming two antibiotic cassettes within the chromosome  on bacterial cell integrity. The bacterial cells were pelleted at their respective time-points (1 and 10 h). The cells were resuspended and fixed in 4% PFA  for 20 min at room temperature and subsequently centrifuged and washed in PBS. After centrifugation, the bacterial cells were stained initially with DAPI (10 µg/ml) followed by PI (10 µg/ml) with incubation at room temperature for 15 min each in the dark. The excess fluorochromes from cells were removed by pelleting and subsequent washing. 10 μl of each sample was mounted onto the slides with Vectashield mounting medium (Vector Laboratories) and the coverslip was placed for further image acquisition with AMG EVOS Imaging fluorescence microscope (Thermo Fischer) to detect the necessary signals.DAPI  and PI (propidium iodide) are nucleic acid stains for differentiation of live cells from damaged/permeabilized ones. DAPI detects all cells uniformly however the nucleic acid intercalation by PI occurs only in impaired bacterial membranes , 30. Hen∆alr∆dadX (d-Ala 50 µg/ml) were subcultured to examine the effect of d-alanine supplementation  in growth. The subcultured bacterial cells were harvested at specific time-points (1 and 10 h) by centrifugation and washed with PBS to remove residual media contents. These bacterial pellets were fixed in 4% PFA  for 20 min at room temperature and subsequently centrifuged and washed in PBS. The samples were then subjected to dehydration through a gradient of ethanol washings (10–100%) at room temperature. After drying the bacterial specimens were mounted on SEM silicon substrate chips and subsequently observed and analysed in Zeiss EVO 60 scanning electron microscope .The overnight cultures of WT and SENSalmonella enterica subsp. enterica serovar Enteritidis str. P125109 were unavailable; as a result the protein sequences of locus SEN3897 (protein_id = “WP_000976814.1”), SEN4016 (protein_id = “WP_001147297.1”) and SEN1235 (protein_id = “WP_000197907.1”) were retrieved from NCBI and subjected to I-Tasser for 3D structure modelling [The X-ray crystal structures of alanine racemase proteins from odelling . The comodelling  in whichodelling .S. Enteritidis chromosomal DNA using the designed primers  Codon plus strain for protein expression and selected on kanamycin (50 µg/ml). A pure single colony was grown overnight at 37 °C and was further cultured into fresh 1L LB medium till the exponential phase (A600 ~ 0.6). With the desired cell density, the bacterial cells were induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and incubated at 18 °C for 12 h. The cells were harvested by centrifugation and resuspended in lysis buffer  containing complete protease inhibitor cocktail tablet (Roche). The cells were sonicated and centrifuged at 13000 rpm for 30 min for cell-free lysate extract. The lysate was loaded onto a column packed with Ni–NTA resin beads (Qiagen) which were already equilibrated with the same buffer. The recombinant alanine racemase proteins carried N-terminal 6X His-tag. After binding, the column was washed with the same buffer and the proteins were eluted with an elution buffer  with increasing gradient of imidazole concentration . The eluted fractions of the protein were run on a 12% SDS-PAGE for purity analysis. These fractions were pooled and buffer exchanged for testing the alanine racemase enzymatic activity.The alanine racemase genes were were PCR amplified from l- and d-alanine as substrates. The conversion of d-alanine to l-alanine resulted in the increase of absorbance at 340 nm with the formation of NADH. This mixture comprises of 0.2 U l-alanine dehydrogenase , 10 μM PLP, 20 mM d-alanine and 2.5 mM NAD+ in Tris–HCl buffer (pH 8.0). The reaction started with the addition of alanine racemase followed by incubation at 37 °C and the increase in absorbance was monitored with NADH formation.The alanine racemase activity was assayed by a modified spectrophotometric method  at 37 °Cl-alanine to d-alanine comprised of 20 mM l-alanine, 10 μM PLP, 0.2 mM NADH, 0.5 U d-amino acid aminotransferase  and 2 U l-lactate dehydrogenase  in Tris–HCl buffer (pH 8.0). The reaction was initiated with alanine racemase addition followed by incubation at 37 °C and the decrease in absorbance at 340 nm was observed with NAD+ formation to assess the alanine racemase activity. One unit of enzyme was defined as the amount of substrate  produced (μmol) per min per mg protein. The absorption spectra was obtained with Cary 60 UV–Vis spectrophotometer (Agilent Technologies).The assay mixture for the conversion of 5cells/well density in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2 incubator. d-Alanine (100 µg/ml) was also added to the culture medium under specific conditions. The epithelial cultures were infected with S. Enteritidis WT, SEN∆alr, SEN∆dadX, SEN∆3897 and SEN∆alr∆dadX at an MOI 10 (multiplicity of infection). These bacterial strains were grown overnight and subcultured in SPI-1 inducing medium (0.3 M NaCl) at 120 rpm as mentioned before. The bacterial inoculums and seeded 24-well plate for attachment assay were placed on ice 15 min prior to half an hour infection time-point. In invasion assay after 1 h post-infection the cells were washed and replaced with gentamicin (100 µg/ml) supplemented DMEM with subsequent incubation for 2 h. The cells were then lysed with 0.1% sodium deoxycholate and plated with suitable dilutions for the determination of viable adherent/invasive bacterial counts. These experiments were independently conducted thrice in the form of biological triplicates.Adhesion and invasion assays were performed in human colon cancer epithelial cells (HCT116) as previously described , 36. The∆alr∆dadX and WT were also determined microscopically before lysing. The cells were fixed with 4% PFA (v/v) in PBS for 15–20 min and permeabilized with 0.1% Triton X-100 (v/v) with subsequent incubation (20 min) for Alexa546 Phalloidin (Invitrogen) actin staining. The bacterial strains were transformed with pCJLA GFP plasmid for visualization. The coverslips were mounted onto the slides with Vectashield Mounting medium (Vector Laboratories) and the images were acquired with Leica TCSSP5 inverted microscope using a 63× oil immersion objective at Central Instrumentation facility, Institute of Life Sciences, Odisha.The invasion profile of SEN5 cells/well before experimentation. Under certain requirements d-alanine (100 µg/ml) was also added to the culture media. The monolayers were infected with S. Enteritidis WT, SEN∆alr, SEN∆dadX, SEN∆3897 and SEN∆alr∆dadX individually at an MOI 10. One hour post-infection, the monolayers were washed with PBS and replaced with gentamicin supplemented DMEM (100 µg/ml) and incubated at 37 °C, 5% CO2. At varied time-points (2 h and 24 h) of post-infection the monolayers were lysed by 0.1% Triton X-100 and plated to determine the bacterial uptake and survival within phagocytic cells at 2 h and 24 h time-points respectively. The bacterial survival within the intact macrophage monolayers was also assessed microscopically at 24 h p.i before lysing (similar to the invasion assay). These assays were done thrice in biological triplicates.The uptake and survival kinetics of alanine racemase mutants was investigated in murine macrophage cell line (RAW264.7) in a 24-well plate set , 38. TheS. Enteritidis by Trizol Reagent  to determine the individual expression profile of the alanine racemase genes  in vitro. RNA extraction was later followed by DNase treatment  for pure RNA which was used for cDNA synthesis using Revert-aid cDNA Synthesis Kit . qPCR was performed in triplicates using Kapa Sybr Fast qPCR Master Mix (2×)  with normalized cDNA templates. The 16s rRNA gene was used as the housekeeping gene.RNA was extracted from logarithmic and stationary phase cultures of S. Enteritidis and mutant cultures  grown in SPI-1  and SPI-2  inducing medium for investigating their comparative SPI-1 and SPI-2 gene expression profile respectively.RNA extraction was also done from S. Enteritidis WT, SEN∆alr, SEN∆dadX, SEN∆3897 and SEN∆alr∆dadX were grown overnight and sub-cultured (1:20) for 4 h till log phase. The bacterial cultures were harvested and processed individually with equal loads (~ 107 CFU) for oral mice infection (5 in each group). Bacterial loads in feces was determined 24 h and 48 h p.i. Additionally the microbial counts in cecum, mesenteric lymph node (mLN), spleen and liver were assessed at 72 h p.i by plating appropriate dilutions of their respective homogenates on MacConkey agar plates with required antibiotic supplementation.In this study C57BL/6 specific pathogen free (SPF) mice were bred and maintained at the animal house facility of School of Biotechnology, KIIT University, Odisha for experimentation. The in vivo work followed the guidelines outlined by the Institutional Animal Ethics Committee (IAEC), KIIT University bearing the approval number: KSBT/IAEC/2014/MEET-1/A12. 6–8 week old streptomycin pre-treated mice were used in this study, as previously described . S. EnteHistopathological assessment was done as described previously , 35. 5 µAll the experiments were conducted independently in biological triplicates to represent the data sets with mean ± standard deviation. One-way analysis of variance (ANOVA) and t-tests were used to examine significant differences during treatments. GraphPad Prism version 6.0  was usedSalmonella Enteritidis, gene disruptions were carried out using the λ-Red recombination system [S. Enteritidis was studied through mutational analysis to investigate its sole functional relevance in the presence or absence of other known alanine racemases. This led to the construction of SEN ∆alr∆dadX double mutant which required exogenous d-alanine supplementation (50 µg/ml) along with antibiotics in LB agar plates. This double mutant bearing SEN3897 exclusively didn’t show any signs of growth with l-alanine (100 µg/ml) supplementation.To determine the role of three alanine racemase genes in the survival of n system  as menti∆alr (2.4 generations/h), SEN∆dadX (2.42 generations/h) and SEN∆3897 (2.21 generations/h) showed growth rates similar to SENWT (2.46 generations/h)  comparable to WT only when supplied with optimum amount of exogenous d-alanine (50 µg/ml)  supplementation displayed poor mutant growth (0.53 generations/h). The absorbance at 600 nm  increased slightly in the first hour then plateaued and consequently decreased till 10 h . However with additional d-alanine (50 µg/ml) supplementation the growth rates (2.46 generations/h) were similar to WT (2.49 generations/h)   to investigate the optimized d-alanine amount for pathogen survival. At 1 h there were no signs of any red signals for either WT or mutant strain . However during starvation (10 h), PI signal intensity increased in the mutant cells (d-Ala 5 µg/ml) possibly due to prolonged nutritional limitation . This is in line with the trend seen in Lactobacillus plantarum [To further investigate the effect of nutritional limitation on reduced viability of the mutant strain SENlantarum . The lat∆alr∆dadX due to prolonged d-alanine starvation was also validated with SEM analysis in a time-dependent kinetic study (10 h)  showed similar morphology. At 10 h certain morphological alternations were observed in the mutant strain SEN∆alr∆dadX  with an estimated score of 0.97 ± 0.05 and C-score of 1.82. Similarly, SEN3897 having a score of 0.85 ± 0.08 and C score of 1.00 showed 42% identity with Chain A, Alanine Racemase from Bartonella henselae (PDB-ID-3KW3) and SEN1235 with a C score of 1.79 and estimated score of 0.97 ± 0.05 showed 48% identity with chain A of alanine racemase from Pseudomonas aeruginosa (PDB-ID-1RCQ). The superimposition of SEN4016, SEN3897 and SEN1235 confirmed their significant structural similarities  showed significant structural identity of 91% with Chain A, Y274f Alanine Racemase from d-amino acid oxidase coupled assay at 37 °C for l-alanine to d-alanine conversion with PLP. With l-alanine as a substrate the specific activity of SEN3897 (297 µmol/(min mg)) was higher as compared to Alr (213 µmol/(min mg)) and DadX (207 µmol/(min mg)) ) relative to Alr (68.4 µmol/(min mg)) and DadX (70.8 µmol/(min mg))  of Alr and DadX was 62.1 ± 4 and 58.9 ± 2.7 mM respectively  differed for the enzymes with different enantiomeric forms ) as compared to SEN3897 (400 ± 25.6 µmol/(min mg)).To further explore this hypothesis, we investigated the in vitro alanine racemase activity of SEN3897 by a d-alanine deficiency by itself in alr dadX double mutant. To elucidate the d-alanine dependency of SEN3897, we investigated the expression levels of alr, dadX and SEN3897 in LB growth media. The expression of SEN3897 was two- and fourfold down-regulated relative to alr and dadX respectively  for investigating their role in adhesion and invasion in vitro. The HCT116 cells were infected with WT, SEN ∆alr, SEN ∆dadX, SEN ∆3897 and SEN ∆alr∆dadX with an MOI 10. To aid in comparison we normalized all bacterial counts with WT (100%). SEN ∆alr, SEN ∆dadX and SEN ∆3897 strains displayed adhesion percentages of 110, 105 and 115% respectively as compared to WT  relative to WT. SEN ∆alr∆dadX displayed a significant lower invasion percentage of 11.1% relative to WT .alr and SEN3897 single mutants showed approximately 93 and 96% uptake in RAW264.7 cells while SEN ∆dadX displayed approximately 102% uptake as compared to WT  showed a substantial lower uptake percentage of 21.3% as compared to WT  displayed significantly impaired survival within macrophages . Alr complementation in SEN ∆alr∆dadX restored the survival phenotype (55%) of the d-alanine auxotrophic strain in phagocytic cells  with GFP expressing plasmid confirmed their respective replication fold phenotype in vitro  would have growth defects due to its inability to synthesize d-alanine. This in turn might attenuate the replicative ability of the pathogen in the host cell cytosol. As expected, SEN ∆alr∆dadX displayed significantly reduced pathogenic loads of 11.1% in epithelial cells as compared to WT  before being infected with SEN ∆alr∆dadX to examine their phenotypic changes. It was observed that d-Ala supplementation increased the invasion profile of SEN ∆alr∆dadX in epithelial cells to 30.1%  before infection. It was observed that SEN ∆alr∆dadX showed reduced restoration of virulence phenotype in both HCT116 and macrophages (Data not shown). SEN ∆alr∆dadX required an optimum d-alanine (50 µg/ml) quantity for bacterial growth; however with greater concentrations of d-alanine supplementation in culture media, the virulence profile changes. Hence 100 µg/ml d-alanine supplementation was considered over 50 µg/ml d-alanine amounts in culture media for virulence restoration.Similar assays were also conducted by supplementing the tissue culture medium with alr and SEN3897 resulted in reduced pathogen invasion and survival in non-phagocytic and phagocytic cells respectively. To this effect the expression profile of SPI-1 effectors and regulators were determined by q-PCR to investigate the reduced invasion phenotype of SEN∆alr and SEN∆3897. hilA transcripts in SEN∆alr and SEN∆3897 showed significant 2.5-fold down-regulation and 2-fold down-regulation respectively relative to WT under SPI-1 conditions . Epithelial cell entry requires active SPI-1 effectors among which sipA plays a vital role. In SEN ∆3897, sipA displayed a significant fourfold reduction in expression as compared to WT  relative to WT  in the d-alanine auxotroph also showed a significant effect on Salmonella invasion and survival in epithelial cells and macrophages respectively . SEN ∆alr∆dadX showed attenuated survival within macrophages; hence SPI-2 genes were also examined through qPCR in SEN ∆alr∆dadX as they are responsible for pathogen survival within macrophages. SEN ∆alr∆dadX exhibited extremely low transcripts of SPI-2 encoded effector genes like sseJ and sseG (~18-fold decreased expression) as compared to WT  and WT. The colonization levels were comparable between WT and the mutants at 24 h post infection  were infected with ~10∆alr∆dadX portrayed significantly reduced colonization efficiency at 24 h p.i. (3-log10 fold less) and 48 h p.i. (4-log10 fold less) in comparison to WT  was also observed in cecum at 72 h post infection  in S. Enteritidis was studied through its sole chromosomal inactivation and presence irrespective of other known alanine racemases to investigate its distinguished role in the pathogen.Due to the development of drug-resistant enteric pathogens, novel drug design requires improved formulations and strategies. Bacterial cell walls have always been a fascinating target for the development of new antimicrobials . Alanineand dadX . The conand dadX , 46. Gen microbe , 24. Thi∆alr, SEN ∆dadX, SEN ∆3897), the double mutant SEN∆alr∆dadX (sole presence of SEN3897) displayed signs of growth defects, membrane integrity loss and structural modifications with the depletion of d-alanine pools. This indicated that the presence of SEN3897 only couldn’t entirely contribute to d-alanine prototrophy. One similar study had also investigated cellular damage and enhanced permeability due to d-alanine auxotrophy in A. hydrophila alanine racemase mutant [d-alanine amounts had similarly resulted in cell death with decreased optical density in many Gram-positive and Gram-negative bacteria like E. coli, Bacillus subtilis and Lactobacillus plantarum [d-alanine production in the absence of Alr. Nevertheless SEN_3897 seemingly fails in d-alanine production in the absence of both Alr and DadX. This shows that the alr gene when inactivated probably requires either another copy/isoform for survival or d-alanine supplementation in the growth medium. Some studies have reported a single deletion of alr gene to be responsible for d-alanine auxotrophy in Lactobacillus plantarum or a double knockout of two essential alanine racemases to show d-alanine dependency in Salmonella Typhimurium [d-alanine dependency which differs from other related findings.Among the mutant strains  in survival rather than the catabolic gene (dadX). d-Alanine catabolism for ATP generation could possibly be carried out by other alternative metabolic pathways in the DadX mutant. However Alr is majorly involved in cell wall biosynthesis; therefore deletion of Alr was expected to cause disruption in cell wall formation thereby affecting growth and viability. Alr mutants of M. smegmatis have d-alanine dependency [M. smegmatis have 80% reduced survival in macrophages [∆alr∆dadX led to diminished intracellular survival of pathogens in our study. This is mostly due to nutrient limitation in the phagosomal compartment as compared to the rich cytosol [3897 in SEN∆alr∆dadX. Furthermore d-alanine deficiency affected Salmonella’s entry into the rich host cytosolic compartment in SEN ∆alr∆dadX that was subsequently restored with d-Ala addition to cell culture media. Hence, this study showed that the sole presence of SEN3897 was responsible for d-alanine auxotrophy, morphological alterations and reduced pathogen survival. Further a double mutant of alr and SEN3897 was also investigated for the additive effects of mutation in contributing to invasion and survival within epithelial cells and macrophages respectively in vitro. Interestingly, SEN∆alr∆3897 showed comparable phenotypes with WT in terms of epithelial cell invasion  may not be crucial for Salmonella pathogenesis. Comparatively the presence of either alr or SEN3897 with dadX in SEN∆3897 and SEN∆alr respectively showed reduced colitis which indicates the essential involvement of SENalr and SEN3897 in virulence.Alanine racemases are ubiquitously present in all prokaryotes and their deletion has been reported to be lethal for pathogen survival in the absence of entation , 41. Impentation . Our in pendency . Additiorophages . The ina cytosol –62 whichSalmonella, SPIs are known to play a pivotal role in epithelial cell invasion and intracellular survival [alr and SEN3897 could possibly affect SPI-1 and SPI-2 through this regulatory pathway for its invasion-defective and attenuated survival phenotype respectively. H-NS is a global repressor of SPI-1 regulators  [ssaB, ssaG). H-NS silences hilA expression which affects the invasion genes [∆alr and SEN∆3897. Similar to SPI-1, we observed significant down-regulation of crucial SPI-2 effectors  in SEN ∆alr and SEN∆3897. Effectors like SseJ and SseG contribute to successful Salmonella replication by inducing SIF formation and stabilizing SCV membrane integrity [hns contributing to reduced levels of hilA and other downstream effectors in SEN∆alr∆dadX which explained the reduced phenotype of epithelial cell invasion in the alr dadX double mutant. Additionally, in this double mutant, the SPI-2 genes  also showed immense down-regulation compared to the transcript levels of these crucial effectors observed in SEN∆alr and SEN∆3897.In survival , 63, 64.C, RtsA)  as well on genes . HilA plon genes , 68. Thion genes . The invon genes . We obsentegrity , 71. Ssentegrity , 73. Ssentegrity . Hence o∆3897 at 72 h p.i. relative to WT. The attenuation in intestinal inflammation could be possibly due to the reduced replication and survival of SEN∆3897 in murine macrophages. However there wasn’t any sort of attenuation observed in cecal colonization possibly due to the differences in host system. It has been reported that SPI-1 is likely to be responsible for attenuation in gut inflammation and inflammatory responses via the needle complex, T3SS-1 [∆3897 relative to WT which likely resulted in reduced inflammatory responses in vivo. SPI-2 is also essential modulator of host inflammatory responses [∆3897 also showed a down-regulation in the expression of SPI-2 effectors . Hence investigation into its coordinated regulation by different pathogenicity islands and regulators will help us develop novel insights that may be exploited for development of antibacterial therapy against Salmonella. This study portrays a complex network of regulators and virulence-associated factors governing pathogen’s invasion and survival strategies within host [alr dadX double mutant displayed hyperattenuation in which the d-alanine synthesis route was possibly inactivated that led to its defective survival and colonization at 72 h p.i. Moreover if d-Ala was present in murine macrophages and mice models; their levels are much below threshold to support growth of the d-alanine auxotrophic strain which lacked the essential alanine racemase genes alr and dadX for infection establishment. Hence the major finding of this study stated that the sole presence of the third alanine racemase gene; SEN3897 couldn’t support pathogen survival in vivo and led to strict d-alanine auxotrophy. Moreover deletion of this gene led to defective SPI-1 and SPI-2 functioning; hence this work helped in understanding the link between metabolic genes and their role in Salmonella virulence. Cell wall auxotrophy was also studied for reduced pathogenesis in Shigella [Our in vivo studies portrayed attenuation in gut inflammation of SEN, T3SS-1 . This stesponses . SEN ∆38hin host . In vivoShigella .d-cycloserine) had major health implications on human nervous system which necessitated the requirement for novel inhibitors with better specificity and less host toxicity. Subsequently the inhibitor development through structure-based studies would examine the dimer interfaces and active site pockets of alanine racemases for use as potential targets against microbial infections. The inhibitor-protein interaction should be aimed for the discovery of new antibiotics with high selectively and specificity for alanine racemase. Another study examined a d-alanine auxotroph as a live vaccine candidate against Staphylococcus aureus infection [d-alanine auxotrophic strain in Salmonella pathogenesis. The growth of the d-alanine auxotroph is dependent on the availability of crucial metabolites in the host tissues. The double mutant auxotroph of S. aureus hence showed increased pathogenic loads in host cytosol due to d-alanine supplementation during the course of infection [d-alanine restored the attenuated profile of the auxotroph which was also observed in our study. Similar model could be replicated in vivo for modulating the virulence phenotype of the d-alanine auxotroph causing transient pathogenic survival for induction of strong immune responses. An auxotroph of Listeria monocytogenes was observed to lack immunogenic potential without d-alanine supplementation hence the vaccination strategy required the addition of the bacteria together with d-alanine [The first drug for alanine racemase was cycloserine , 77. Evenfection . Our stunfection . However-alanine . Hence aS. Enteritidis hence would aid in the development of potential therapeutics to combat non-typhoidal Salmonella infections. Salmonella displays an excellent example of regulated and coordinated expression of virulence factors contributing to its pathogenesis. This complex network involves genes central to metabolism, cell wall maintenance, quorum sensing, biofilm development and global regulation for establishment of infection through its virulence machinery. Thus Salmonella infections initiate in the intestinal lumen and then disseminate to systemic sites; hence insights into new regulatory networks and identification of novel virulence factors will help discover unique antibacterial drugs for infection control.Our findings demonstrated a significant contribution of SEN3897 in vitro and in vivo for invasion and intracellular survival due to possible modulation of SPI-1 and SPI-2 effectors. Identification of novel virulence factors like SEN3897 associated with Additional file 1: Table S1. Primer list used in this study.Additional file 2: Figure S1. PCR screening of alanine racemase gene inactivation. (A) Confirmation of PCR mediated gene replacement of alr, dadX and SEN ∆3897 in Salmonella Enteritidis by kanamycin cassette with internal kanamycin specific primers and confirmatory primers (B) PCR confirmation of double deletions  in S. Enteritidis by phage transduction with FRT specific primers for kanamycin and chloramphenicol cassettes of 1.5 kb and 1.1 kb size respectively. These ethidium bromide stained gels were analyzed by agarose gel electrophoresis. The 1 kb DNA ladder (Invitrogen) with the desired product sizes is indicated with arrows.Additional file 3: Figure S2. Growth curve experiment (cfu/mL) to check the cell viability in SEN ∆alr, SEN ∆dadX, SEN ∆3897, WT Salmonella Enteritidis and alr dadX double mutant in liquid culture with d-alanine supplementation  at different time intervals. Data is represented as mean ± SD.Additional file 4: Figure S3. (A) Sequence alignment of three alanine racemases from S. Enteritidis . Alr (SEN4016) showed 91% with Y274f Alanine Racemase from E. Coli (PDB-ID-4WR3), DadX (SEN1235) showed 48% identity with Alanine Racemase from Pseudomonas aeruginosa, (PDB-ID-1RCQ) and SEN3897 showed 42% identity with Chain A, Alanine Racemase from Bartonella henselae (PDB-ID-3KW3). (B) Ribbon protein structures determined by I-TASSER. Superimposed alanine racemase structures of Alr (green), DadX (red) and SEN3897 (turquoise Blue) denote their structural similarities with residue conservation.Additional file 5: Table S2. Active site residues of alanine racemase proteins  of Salmonella Enteritidis from CASTp analysis.Additional file 6: Figure S4. Expression of SEN3897 in WT and SEN ∆alr∆dadX bacterial strains through qRT-PCR analysis. All experiments were performed in triplicates with data represented as mean ± SD. Statistical significance: **P < 0.01.Additional file 7: Figure S5. Adhesion assay of SEN ∆alr, SEN∆dadX, SEN ∆3897 and WT in HCT116 cell-lines for 30 min at MOI 10. Statistical significance: *P < 0.05.Additional file 8: Figure S6. Survival Assay of SEN ∆alr∆dadX, Alr complemented strain  and WT in RAW264.7 in murine macrophages at 24 h time-point. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Additional file 9: Figure S7. Fluorescence microscopy images of murine macrophages RAW264.7 infected with SEN ∆alr, SEN ∆dadX, SEN ∆3897 and WT for 1 h at MOI 10, gentamicin treated for 24 h and fixed. Cell nuclei were stained with DAPI (blue) and Salmonella were GFP-tagged (green). Scale bar: 10 μm.Additional file 10: Figure S8. Colonization characteristics of SEN ∆alr, SEN ∆dadX, SEN ∆3897, SEN ∆alr∆dadX and WT: Feces from each group of 5 mice were collected and suitable dilutions  were plated on MacConkey agar plates with required antibiotics at 24 h and 48 h p.i for assessing the bacterial counts. Statistical significance: *P < 0.05, **P < 0.01.Additional file 11: Figure S9. (A) Invasion assay of SEN ∆alr∆3897 and WT in HCT116 cell-lines. SEN ∆invC acts as the negative control for invasion assay (B) Uptake Assay and (C) Survival Assay of SEN ∆alr∆3897 and WT in RAW264.7 in murine macrophages at 2 h and 24 h time-points respectively. Statistical significance: *P < 0.05, ****P < 0.0001.


In qualitative research, similarities and differences between the participant and researcher influence the research process and dynamics. Specifically, the age difference between older participants and relatively younger qualitative researchers is a common, but under-examined dynamic requiring nuanced, reflexive analysis. Using a life course conceptual framing, this study explored age-related participant-researcher dynamics in interviews from two qualitative studies of older women’s sexual experiences in later life. Participants included 25 women whose ages ranged from 55 to 93 and both studies were completed by the same researcher, a relatively younger woman (age 23 and 28 at times of data collection). A thematic analysis revealed three primary themes: 1) taking care - participants took care of the researcher by offering advice, asking about the researcher’s life, and expressing hopes for a positive future, 2) expertise – varied expertise was demonstrated by the researcher (e.g. substantive and scholarly) and participants (e.g. life experience), and 3) researcher growth - the researcher’s interviewing tactics shifted between the two studies , indicating a shift in perceptions of aging and later life. Findings indicate that older women participants and younger women researchers are bound together through the life course, by shared gendered experiences, the fact that one will eventually become the other, and the mutual sharing of expertise and caring. Gerontology researchers must actively reflect on the impact of their own identities and aging perceptions on the interviewing process in order to enhance rigor in qualitative research.


Haploidentical transplantation has been proposed as an effective treatment for severe aplastic anemia (SAA). The majority of patients have more than one HLA-haploidentical donor. Herein, we compared the outcomes between different donor-recipient relationships for optimal haploidentical donor selection in acquired SAA.We conducted a multicenter study based on a registered database of 392 patients with SAA treated with allogeneic hematopoietic stem cell transplantation  between 2006 and 2018. In total, 223 patients received grafts from father donors, 47 from mother donors, 91 from siblings, 29 from children, and 2 from collateral donors.P = 0.706). The 2-year failure-free survival (FFS) was 82.8 ± 2.7%, 86.7 ± 5.1%, 80.8 ± 4.2%, and 92.5 ± 5.1% for recipients of father, mother, sibling, and child grafts, respectively, (P = 0.508). There was no difference in the incidence of either acute or chronic graft-versus-host disease (GVHD) among the different donor sources in multivariate analyses. There were also no differences in the OS or FFS among the different donor sources in the Cox regression analysis. However, OS was significantly better in the patients with a shorter history of aplastic anemia (< 12 months), better performance status (ECOG scores 0–1), or moderate graft mononuclear cell (MNC) counts (6–10 × 108/kg), and in female recipients with male donors. The FFS was also higher in patients with a shorter history of aplastic anemia (< 12 months) and better performance status (ECOG scores 0–1).Of the 381 patients who survived more than 28 days, 379 (99.5%) recipients were engrafted. The 2-year overall survival (OS) was 86.6 ± 2.5%, 87.1 ± 4.9%, 84.3 ± 3.9%, and 92.2 ± 5.1% for recipients of father, mother, sibling, and child grafts, respectively, (Fathers, mothers, siblings, and children are all suitable haploidentical donors for patients with SAA. Severe aplastic anemia (SAA) is a life-threatening hematological disease characterized by an immune-mediated disorder of hematopoietic stem cells. Allogeneic hematological stem cell transplantation (HSCT) is recommended as the first-line treatment in young patients with an available matched sibling donor (MSD) and as the second-line treatment in older patients who failed immunosuppressive therapy (IST) , 2.However, a significant number of patients requiring urgent HSCT lack an MSD. Moreover, the long-term effects of IST are far from satisfactory due to late sequelae, including relapse and the evolution to clonal diseases.Under this condition, haploidentical donors (HIDs) have increasingly been proposed as an alternative in the absence of an MSD or a matched unrelated donor, and satisfactory outcomes after haploidentical HSCT have been recently reported –10. The Nearly all patients have at least one HLA-haploidentical related donor, and the selection algorithm of a haploidentical donor for hematological malignancies has been recommended –13. Younn = 274), Guangzhou First People’s Hospital (n = 92), and the First Affiliated Hospital of Jilin University (n = 26). In total, 392 patients were enrolled in the study. The study protocol was approved by the institutional review board. All of the patients gave their written informed consent for the procedure.This multicenter study was conducted based on a registered database. We included patients with SAA who received a haploidentical transplant between 2006 and 2018 and who received standard busulfan (BU)/cyclophosphamide (CY) conditioning at Peking University Institute of Hematology  were approved by the FDA and commercially imported .2 on days + 3, + 6, + 11). CsA was given orally once patients’ bowel function returned to normal. The target concentration of CsA was required within a year posttransplant and was tapered and discontinued over the following 2–3 months. In addition, all patients began MMF orally  on day − 9, tapered on day + 30, and discontinued on day + 60 [The transplantation procedure has been described in previous studies –10. The day + 60 , 19.9/L was achieved, and platelet recovery was defined as the first of 7 consecutive days that a platelet count ≥ 20 × 109/L without transfusion was achieved. Donor recipient chimerism was confirmed by fluorescence in situ hybridization (FISH) for donor/recipient sex-mismatched pairs and by multiplex short tandem repeat (STR) polymerase chain reaction (PCR) for donor/recipient sex-matched pairs. Primary graft failure was defined when the neutrophil counts did not exceed 0.5 × 109/L for 3 consecutive days with low/absent donor chimerism at day 28 post-HSCT. Secondary graft failure or graft rejection was defined as neutropenia (< 0.5 × 109/L) with low/absent donor chimerism in patients with a prior history of engraftment. Patients with neutropenia (< 0.5 × 109/L) but complete donor chimerism were considered to have poor graft function (PGF) [Neutrophil recovery was defined as the first of 3 consecutive days that an absolute neutrophil count ≥ 0.5 × 10on (PGF) , 22, witon (PGF) . The sevon (PGF) , 25.P < 0.10 in univariate analysis along with the donor source were included in multivariate regression. A P < 0.05 was considered significant. The data analyses were conducted primarily with SPSS software , and R software (version 2.6.1) (http://www.r-project.org).The last follow-up date was February 1, 2019. The endpoint for the study was OS and FFS. The Kruskal-Wallis rank sum test was used for continuous variables, and the chi-square test or Fisher’s exact test was used for categorical variables. All tests were two-sided. The Kaplan-Meier outcome curves for the OS, FFS, and GRFS were constructed for the 392 patients. The log-rank test was used to identify prognostic factors, and a Cox proportional hazards regression model was used to assess the relative impact of previously defined risk factors in multivariate analysis. The cumulative incidences of graft failure and GVHD were calculated in a competing risk model, with death as the competing event. All factors with P = 0.000; mother vs. sibling, P = 0.000), and the sibling donors were older than the child donors (P = 0.007). There was a higher male/female ratio of recipients in the sibling donor group, compared to the parental donor groups . The history of IST, the interval between diagnosis and transplant, and the infused cell number were similar among the different donor-recipient relationship groups. However, the median CD34+ cells were lower in the mother donor group than in the sibling donor group (P = 0.019) and the child donor group (P = 0.020).Table Of the 381 patients who survived more than 28 days, 379 (99.5%) achieved myeloid engraftment at a median time of 12 days , and 365 patients (95.8%) had platelet engraftment at a median time of 14 days . One patient failed engraftment from father donor transplantation, and she had full chimerism after a second transplant from the mother donor. One patient who did not achieve engraftment after a sister donor graft remained in graft failure after a second transplantation from the same donor. This patient eventually underwent transplantation from a matched unrelated donor and achieved hematopoietic recovery. The cumulative incidence of neutrophil recovery at 28 days post-transplant was 96.4 ± 0%, and the cumulative incidence of platelet recovery at 100 days post-transplant was 91.3 ± 0%. Of the nine patients who experienced secondary graft failure (six from father grafts and three from brother grafts), two received a second transplant from the second haplo-donor, and one received an unrelated donor graft. All three patients achieved engraftment, but two of them died from an infection. Two patients who received second transplants from the same donor and another two patients who received donor lymphocyte infusion (DLI) did not achieve engraftment. The remaining two patients did not receive either second transplants or DLI.n = 8), 6.7% (n = 1), 8.3% (n = 3), and 20.0% (n = 2) in the father, mother, sibling, and child donor groups, respectively, (P = 0.417). Of the 11 patients with graft failure, DSA was measured in four patients, and one patient with primary graft failure had positive DSA.Detection of donor-specific antibody (DSA) was performed in 196 of the 392 patients, and 14 patients (7.1%) exhibited positive DSA. The proportions of positive DSA were 6.0% (P = 0.011). In the competing risk model, mother donor transplants had a higher rate of cGVHD  but not aGVHD  compared with father donor transplants  and a higher degree of HLA mismatches  were risk factors for grade II–IV aGVHD. Patient age < 20 years  and ABO minor mismatch  remained significant prognostic factors for grade III–IV aGVHD in the multivariate model  (Table P = 0.186) or child donors . No significant factors in extensive cGVHD were identified in the multivariate analysis.Mother donor was the only risk factor for cGVHD in the Cox model  had virus infections, 85 (21.7%) had bacterial infections, and 33 (8.4%) had fungal infections. Cytomegalovirus (CMV) antigenemia occurred in 284 patients (72.4%), and eight patients developed CMV diseases . Epstein-Barr virus (EBV) antigenemia occurred in 56 patients (14.3%), and eight developed post-transplant lymphoproliferative disorders. No cases of adenovirus infection and toxoplasma were observed.In total, 52 (13.3%) patients died. The causes of death were GVHD in seven cases, infection in 20 cases, and other complications (in 25 cases). At a median follow-up of 743 days (range 125–4754 days), the OS, FFS, and GRFS were both similar among the recipients from different donor kinships. At last follow-up, 195 patients (87.4%) in the father donor group, 40 (85.1%) in the mother donor group, 77 (84.6%) in the sibling donor group, 27 (93.1%) in the child donor group, and one (50%) in the collateral donor group were alive. The long-term outcomes of the patients are shown in Fig. P = 0.032), and better performance status . The 2-year OS rate of the male-to-female transplants was higher than that of other sex-matched groups (92.1 ± 2.5% vs. 83.5 ± 2.4%) in the Kaplan-Meier survival curve, and male-to-female donations remained a significantly beneficial prognostic factor for the OS in the multivariate analysis . Moderate MNC counts of 6–10 × 108/kg were also a protective factor against death . The covariates that were significant for GRFS included ABO minor mismatch, MNC counts, and ECOG score .There were no statistically significant differences in OS or FFS between NIMA- and NIPA-mismatched sibling donor transplants . In contrast, female donors were not associated with worse survival when the recipient was male .In the subgroup analysis, for the female recipients, female donors were associated with worse OS than male donors in the multivariate analysis .In total, 29 patients received child grafts (19 from sons and 10 from daughters). There were no differences in OS, FFS, and GVHD between son donor and daughter donors , respectively. The 2-year FFS for M-M, F-M, M-F, and F-F was 82.5 ± 6.0%, 80.2 ± 9.0%, 84.6 ± 10.0%, and 71.4 ± 12.1% (P = 0.773), respectively. There were no differences in either aGVHD or cGVHD between different donor-recipient sex matches.There were 91 patients who received sibling donor grafts. Regarding male donor to male patient (M-M), female donor to male patient (F-M), male donor to female patient (M-F), and female donor to female patient (F-F), the 2-year OS was 82.1 ± 6.2%, 90.9 ± 6.2%, 92.9 ± 6.9%, and 71.4 ± 12.1% . Donor-to-recipient sex match, the duration from diagnosis to transplant, and MNC counts did not affect OS and FFS for pediatrics. ABO minor mismatch remained a significant risk factor for grade III–IV aGVHD . No significant clinical factors were found to be associated with grade II–IV aGVHD, cGVHD, and extensive cGVHD.For pediatric patients, the Cox models for OS and FFS revealed a significant influence of better performance status , and moderate MNC counts of 6–10 × 108/kg . These two factors were also significant in the Cox model for FFS . Meanwhile, we did not find any statistically significant factors for GVHD.For adult patients, the significant covariates for OS were better performance status  . Wang etIn haploidentical transplants for hematological malignancies, the number of HLA locus disparities was not significantly correlated with transplant outcomes . HoweverOur findings are in line with a study showing a higher rate of III–IV aGVHD in the minor ABO-mismatched group in 154 unrelated donor transplants . In 154 8/kg in haploidentical allogeneic transplantation for SAA, while in our clinical practice, the MNC count infused ranged from 6 to 10 × 108/kg [8/kg were better than < 6 and > 10 × 108/kg for survival. Thus, we recommended 6–10 × 108/kg as a moderate range of MNC counts. The lower CD34+ cell harvest from mother donors is an interesting phenomenon. Previous studies showed that age, sex, and body mass index (BMI) might be associated with CD34+ cell count yield, but this association is still controversial [6/kg vs. 3.33 × 106/kg, P = 0.020). In another study, the researchers observed a lower post-G-CSF CD34+ cell count in female donors than in men, and they speculated that this difference might be because female donors weighed less than male donors and received lower total amounts of G-CSF [In this SAA population, the OS and FFS were better in patients receiving moderate MNC counts. The target count of MNC was 6–8 × 10× 108/kg . In thisoversial –39. Our oversial . Howeverof G-CSF . HoweverFemale donation to a male recipient was regarded as an adverse prognostic factor in transplantation for hematological malignancies , 42 and A potential limitation of this retrospective study is that based on the current decision-making for donor selection in hematologic malignancies, mother donors are rarely selected. Therefore, improvement in the SAA survival of maternal graft transplantation might be partially underestimated. Even so, the mother donor group still demonstrated comparable outcomes with other donor groups, thus showing that maternal grafts were not inferior to grafts from other donors. Although our data showed that male donors for female recipients might be superior to female donors, this finding was drawn from limited data and is not supported by previous reports. Therefore, the results may not be generalizable to other transplant patterns, and prospective studies are warranted.In conclusion, fathers, mothers, siblings, and children are all suitable HIDs for patients with SAA. Despite the higher incidence of cGVHD in mother donors, survival outcomes were comparable for father, mother, sibling, and child donor transplantation. To reduce the risk of severe aGVHD, avoiding donor-recipient ABO blood minor mismatch should be considered. These findings provide a basis for the selection of optimal HIDs for SAA.


Two-year leukemia-free survival (LFS) rate after SCT2 was 23.5%, 23.7%, and 21.8%, respectively (P = 0.30). Multivariate analysis showed no effect of donor2 type on relapse: hazard ratio (HR) 0.89 (P = 0.57) and 1.11 (P = 0.68) for different donor and haplo-donor compared to same donor, respectively. However, donor2 did predict for non-relapse mortality (NRM) after SCT2: HR 1.21 (P = 0.50) and 2.08 (P = 0.03), respectively, and for LFS: HR 1.00 (P = 0.97) and 1.43 (P = 0.07), respectively. In conclusion, SCT2 with the same or different matched donor is associated with similar outcomes in patients with relapsed AML. Non-T-depleted haplo-identical transplant may be associated with higher NRM, similar relapse rate and with no better results in this setting.Second allogeneic stem-cell transplantation (SCT2) is a therapeutic option for patients with AML relapsing after a first transplant. Prior studies have shown similar results after SCT2 from the same or different donor; however, there are limited data on second non-T-depleted haplo-identical transplant in this setting. We retrospectively analyzed SCT2 outcomes in 556 patients, median age 46 years, relapsing after first transplant given in CR1. Patients were divided into three groups based on SCT2 donor (donor2): same donor ( However, relapse remains the main cause of treatment failure. There is no established standard of care therapy for patients relapsing after SCT. Patients can be treated with a spectrum of treatments including palliative care, withdrawal of immune-suppression therapy, low dose or intensive chemotherapy, hypomethylating agents or other targeted therapies, donor lymphocyte infusion (DLI), a second allogeneic transplant, or combinations of these therapies. However, the outcome of these patients remain poor, in particular in patients relapsing within the first 6 months after SCT2. In all, prolonged survival can be achieved only in patients achieving a second complete remission and supported by a form of cellular therapy such as DLI or second SCT3.Allogeneic hematopoietic stem-cell transplant (SCT) is a potentially curative treatment for acute myeloid leukemia (AML). Marked improvement in the rates of non-relapse mortality (NRM) after SCT has been achieved in the last decade13. Advanced age and second SCT from an unrelated donor have also been described as predictors of inferior outcome. Most studies have not shown an advantage of switching to a different donor, attempting at enhancing a stronger or different graft-versus-leukemia (GVL) effect. However, there are so far limited data on the use of haplo-identical SCT as a second SCT in patients relapsing after a first SCT from a matched donor15.Several studies have shown that the major predictors of outcome after a second SCT are the duration of remission after the first SCT and the status of disease at the second SCT16. Several studies have shown comparable outcomes of AML patients given first SCT from haplo-identical donors and unrelated and even matched sibling donors19. There is still controversy, whether haplo-identical SCT is associated with a stronger GVL effect than matched donor SCT21. If so, it is conceivable that haplo-identical SCT may result in better outcome in high-risk settings such as a second SCT after failure of a first SCT from an HLA-matched donor.The use of haplo-identical SCT has markedly increased in the last decade with the introduction of non-T-depleted platforms and in particular with the use of post-transplant cyclophosphamideIn the current study we show that a second SCT from a haplo-identical donor is not associated with improved outcome. It is not associated with stronger GVL, but NRM is increased in comparison with matched donor SCT, and therefore leukemia-free survival (LFS) is lower.This is a retrospective multicenter analysis. Data were provided and approved for this study by the acute leukemia working party (ALWP) of the European Society for Blood and Marrow Transplantation (EBMT). The study protocol was approved by the institutional review board at each site and complied with country-specific regulatory requirements. All patients provided written informed consent authorizing the use of their personal information for research purposes. Eligibility criteria included patients with de-novo or secondary AML who relapsed after allogeneic SCT from an HLA-matched sibling or unrelated donor, and were given a second SCT from the same or a different donor, in any disease status, between the years 2006 and 2016. Patients were required to engraft after SCT1, to relapse before SCT2 and to be given SCT2 within 300 days after relapse. Patients were divided into three groups based on the donor selected for SCT2: same donor group, different HLA-matched donor group, and second haplo-donor group. Haplo-identical donors were defined as two or more mismatches from a related donor. There was no haplo-identical unrelated donor. The EBMT database does not include enough information on kinship of haplo-identical donors. Most unrelated donors were 9–10 HLA matched. Variables collected included recipient and donor characteristics for both transplants, disease features, transplant related factors including drugs and total doses used in the conditioning regimen, and outcome variables.22. GVHD prophylaxis was selected according to the participating center policy and consisted of a calcineurin inhibitor (cyclosporine A or tacrolimus) with short-term methotrexate or mycophenolate mofetil in most HLA-matched transplants. Antithymocye globulin (ATG) was allowed according to the participating center policy. Haplo-identical transplants were all non-T cell depleted based on either post-transplant cyclophosphamide (PTCy) or ATG. No ex vivo manipulation was allowed for any SCT.The conditioning regimen was selected according to the participating center discretion. Dose intensity was defined according to standard criteria based on the reversibility and expected duration of cytopenia after SCTDisease relapse was defined according to standard hematological criteria. NRM was defined as death of any cause in the absence of prior disease recurrence. LFS was defined as survival without relapse. Overall survival (OS) was calculated from the day of SCT until death of any cause or last follow-up. The events for analysis of GVHD-free relapse-free survival (GRFS) were relapse, death of any cause, acute GVHD grade III–IV, or extensive chronic GVHD, which ever occurred first. Patients with no event were censored at last contact. Acute and chronic GVHD were graded according to standard criteria.23 while NRM, relapse, and GVHD were estimated using cumulative incidence analysis considering competing risks24. Univariate comparisons were done using the log-rank test for LFS and OS, and Gray’s test for GVHD, relapse incidence, and NRM. For all univariate analyses, continuous variables were categorized and the median used as a cut-off point. Multivariate analyses were performed using Cox proportional hazards. Variables were included in the multivariate model if they were conceptually important or if they differ in term of distribution between the three groups. Results are expressed as hazard ratio (HR) with 95% confidence interval. To test for a center effect, we introduced a random effect or frailty for each center into the model. All P values were two-sided and values <0.05 were considered statistically significant. Statistical analyses were performed with SPSS 24.0  and R 3.4.1 software packages (R Core Team (2017). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/).The primary end-point of the study was LFS after SCT2. Secondary endpoints included acute and chronic GVHD, NRM, relapse incidence, OS, and GRFS. All outcomes were measured from the time of stem cell infusion. The three donor groups were compared by the Chi-square method for qualitative variables, and Mann–Whitney test for continuous parameters. LFS, OS, and GRFS were estimated using the Kaplan–Meier methodn = 294) or a matched unrelated donor  and given SCT2. Patient characteristics are outlined in Table n = 163, sib/sib-112, UD/UD-51), (2) different HLA-matched donor , and (3) second haplo-donor . All haploSCT were non-T-depleted. There were some differences between the three groups in the timing of relapse and SCT2. The median time from SCT1 to relapse was similar: 10.6, 12.5, and 9.3 months, respectively (P = 0.14), and 36%, 28%, and 35%, respectively, relapsed within 6 months of SCT1 (P = 0.13). However, the median time from relapse to SCT2 was shorter for the same donor group: 2.8, 3.7, and 3.5 months, respectively (P < 0.001), and the median time between SCT1 and SCT2 was longer for the different donor group: 14.3, 17.5 and 13.8 months, respectively (P = 0.03). There was no difference between the groups in patient age, gender, and disease status at SCT2. The Karnofsky performance status at SCT2 was better for the haplo-identical donor group (P = 0.001). The conditioning regimen intensity for SCT1 or SCT2 was similar. However, more patients in the different matched group were given in vivo T cell depletion (mostly ATG) in SCT2. All haplo-identical SCT2 were non-T depleted; 70% were given PTCy, 25% ATG, and 5% both. The median follow-up was 52.0 months (0.9–131.8), 30.5 months (1.0–135.1), and 33.0 (1.0–73.9) following the three donor groups, respectively (P = 0.05).The study included 556 patients with AML relapsing after a first allogeneic SCT SCT1) given in CR1 from an HLA-matched sibling . Multivariate analysis identified acute GVHD after SCT1 as a risk factor for acute GVHD after SCT2  and the use of in vivo T cell depletion  were associated with a lower risk for acute GVHD after SCT2 (data not shown). The second donor type group was not predictive.The cumulative incidence of acute GVHD grade II–IV was 35.9% , 32.7% , and 20.1%  in the same donor, different matched donor, and haplo-identical donor groups, respectively (P = 0.42). Multivariate analysis identified chronic GVHD after SCT1 as a risk factor for chronic GVHD after SCT2 . The use of in vivo T cell depletion  was associated with a lower risk chronic GVHD after SCT2 (data not shown). The second donor type group was not predictive.The cumulative incidence of chronic GVHD was 36.6% , 31.0% , and 25.1%  in the same donor, different matched donor, and haplo-identical donor groups, respectively (P = 0.28). Among the various causes of NRM there was a trend for higher cumulative incidence of death from infection in the haplo-identical group, rates been 10.6% , 17.1% (95% CI 12.9–21.8), and 20.7% (95% CI 12.6–30.2), respectively (P = 0.08). The cumulative incidence of death due to GVHD was not different between the groups, 7.4% , 10.9% (95% CI 7.4–15.1), and 9.3% (95% CI 4.0–17.4), respectively (P = 0.59). Table P = 0.0003] and SCT2 from haplo-identical donors  were associated with increased risk, while MAC during SCT1 was associated with a lower risk of NRM after SCT2 .The 2-year NRM rate was 25.1% , 26.9% , and 33.9%  in the same donor, different matched donor, and haplo-identical donor groups, respectively . Multivariate analysis identified relapse within 6 months of SCT1 as associated with higher risk of relapse after SCT2 . Unrelated donor at SCT1 , chronic GVHD after SCT1 , and SCT2 in CR2  were factors associated with reduced risk of relapse after SCT2 , 49.3% , and 44.2%  in the same donor, different matched donor, and haplo-identical donor groups, respectively . One hundred and eighty-seven patients are alive and 369 have died. The major causes of death were disease recurrence , GVHD , infection , veno-occlusive disease of the liver , and others . The 2-year LFS rate was 23.5% , 23.7% , and 21.8%  in the three groups, respectively . The 2-year OS rate was 36.4% , 28.7% , and 23.3%  in the three groups, respectively .The median follow-up after SCT2 was 52 months , 30.5 months , and 33.0 months  following the three donor type groups, respectively , P = 0.07], and SCT2 from haplo-identical donors  were associated with inferior LFS, while SCT2 in CR2 was associated with better LFS after SCT2 . Similar factors predicted for 2-year OS with HR 1.50 , P = 0.008), 1.13 , P = 0.03), 1.61 , P = 0.03), and 0.62 , P = 0.0004), respectively.Table P = 0.16). Multivariate analysis identified SCT2 in CR2  as associated with better GRFS after SCT2 (data not shown). Haplo-identical donor was associated with a trend for inferior GRFS .The 2-year composite outcome of GRFS was 14.1% , 17.0% , and 13.7%  in the same donor, different matched donor, and haplo-identical donor groups, respectively . However, second haplo-identical donors were associated with higher NRM and lower LFS.4. Younger age, relapse beyond 6 months of the first SCT and SCT2 in CR were the most important prognostic factors for survival, but a change of donor did not result in better outcome. With the improvement in HLA typing and donor registries and with the introduction of RIC regimens the use of second SCT from unrelated donors and after a first SCT from unrelated donors markedly increased13. Christopeit et al.7 reported the first large study including unrelated donors for a second SCT (104 of 179 second transplants)7. The 2-year OS after SCT2 was 25%, 39% after sibling, and 19% after unrelated donor SCT2). Similarly, the favorable prognostic factors were longer prior remission, CR at SCT2 and SCT1 from a sibling. Selecting a new donor did not change outcome in the entire group. There was a better outcome when changing from a sibling to a different sibling but this was based on a very small group. There was also an advantage in changing MUD to a different MUD that was limited to patients with no prior chronic GVHD. Ruuto et al. summarized 2632 second transplants for relapse of various hematological malignancies reported to the EBMT9. The 5-year OS was 20%. There was a trend for lower risk of relapse when changing a donor; however, this was counter balanced by a trend for higher NRM, and overall survival was similar. Most other studies have also failed to show a difference in outcome with donor change12.Historically, second transplants were performed mostly from siblings, using the same donor and mostly myeloablative conditioning. Eapen et al. reported the first large series of 279 patients with acute and chronic leukemia having a second SCT from a sibling donor (in 15% a different donor). The 5-year rates of relapse, NRM, and LFS were 42%, 30%, and 28%, respectively15. HLA disparity can contribute to allo-reactivity and GVL. The frequency of donor T cell precursors directed against minor histocompatibility antigens or leukemia-specific antigens that mediate GVL in the matched donor transplant setting is several logs lower than the frequency of T cells directed against major HLA differences25. Loss of the unshared haplotype is a common mechanism for leukemia immune escape after haplo-identical donor transplant26. Therefore, switching to a different haplo-identical donor may be an effective strategy in a second transplant21. Using a haplotype mismatched donor may also offer new targets for GVL after failure of a first matched transplant and improve outcome21. However, the current study fails to show better GVL with a second haplo-donor, and since a second haplo-identical transplant was associated with more NRM, outcome was inferior. Mismatched HLA can be a target for natural killer (NK) cell allo-reactivity in T cell-depleted haplo-identical transplant with no post-transplant immune-suppression27 but this NK cell activity is controversial in the T cell-replete setting28. The use of post-transplant cyclophosphamide may even abrogate T cell precursors that are rapidly dividing after the mismatched transplant such as those against major HLA antigens, more than those that are less frequent. However, a more precise and individualized study of HLA antigens expression by leukemia blasts at relapse could guide in the future a personalized choice of donor for SCT2 searching for specific HLA-mismatches towards a disease immune-edited by previous mismatched donor.Switching to a haplo-identical donor after a matched donor may theoretically offer an advantageA haplo-identical donor is more rapidly available than when searching for a different unrelated donor. Despite that, in the current study the time from relapse to SCT2 was similar between different matched donor (mostly unrelated) and haplo-identical donors, suggesting that a haplo-identical donor was chosen only after failure in allocating a different matched donor. This may explain in part the higher NRM with haplo-identical donors. Randomized prospective studies comparing these different donors are needed to overcome these limitations of retrospective registry analysis.15). The conditioning regimen used in the first or second transplant had no impact on subsequent survival.Similarly to previous studies, the major factors predicting outcome after SCT2 were the biological aggressiveness of the underlying leukemia as reflected by the time to relapse after SCT1 and the ability to achieve a second remission prior to SCT2 . It seems this effect carries over after relapse during further treatment with SCT2 or DLI. The conditioning regimen and immune-suppressive therapy after SCT2 did not eliminate this GVL effect, or alternatively a supportive cytokine or allo-immune milieu induced by prior chronic GVHD continues to influence donor cells whether from the same or a different donor. However, not all studies reported a similar effect of chronic GVHD. Ruuto et al. reported, in a very large series of second SCT for various hematological malignancies, that prior chronic GVHD was associated with higher NRM and lower survival after SCT2 (ref. 9). More studies are needed to confirm if this observation is limited to patients with acute leukemia or what is the validity of this finding. There are no data supporting donor selection based on prior GVHD. Patients with prior GVHD had similar outcomes with same or different donors9. In the current study we were unable to perform a subset analysis based on prior GVHD due to patient numbers and there was no interaction between second donor and prior GVHD.Chronic GVHD after SCT1 was associated with a lower rate of relapse after SCT2 although not with statistically significant improvement in OS. Two recent studies reported similar observations. The pediatric diseases working party of the EBMT determined in a group of 373 children with acute leukemia following a second SCT that the prognostic factors for improved survival were longer duration (>1 years) between transplantations, chronic GVHD after the first SCT, CR at SCT2 (in ALL), and age >12 years (in AML)In conclusion, a second SCT from the same or a different matched donor is a valid therapeutic option for patients with AML relapsing after a first matched donor transplant with equivalent outcome. A second transplant from a haplo-identical donor was not associated with better outcome than other donor sources in this setting.


A 67-year-old male was referred to the outpatient clinic of our hospital with crescendo angina pectoris since one month. He had been diagnosed with atrial fibrillation in a general practice three days earlier and treatment with metoprolol and apixaban was initiated. He was an otherwise healthy and active cyclist who smoked a cigar on occasion. There were no other risk factors for coronary artery disease. An echocardiogram performed five years ago because of persistent bradycardia, was normal.Physical and laboratory examination on admission did not reveal any abnormalities. His initial troponin I level was 11 ng/l, with repeat troponin I concentration found at 14 ng/l . The resting electrocardiogram at presentation is shown in Figs. What is your diagnosis based on the electrocardiographic findings?What would be your next step or treatment?You will find the answer elsewhere in this issue.


Cryptococcus neoformans is the respiratory tract, and by hematogenous spread, it causes systemic symptoms. The presence of CSF leak is described to have predisposed our immunocompetent patient to infection by this organism possibly through direct spread. The gold standard for diagnosing CSF leak is by cisternography. In this case, we added a technique where nasal gauze is inserted during the procedure and scanned afterwards for dye, thus increasing the confidence of diagnosis of CSF leak through the nares. Prompt diagnosis and treatment is key to prevent adverse outcomes, and we propose that in patients with cryptococcal meningitis without any identifiable risk factor, evaluation for CSF leak should be considered especially with history of head trauma.Immune deficiency is usually the underlying predisposing factor for cryptococcal meningitis, though there have been case reports of immunocompetent patients presenting with same. The portal of entry for  Cryptococcus neoformans is commonly associated with immunodeficiency states, both human immunodeficiency virus (HIV) and non-HIV related. Cerebrospinal fluid (CSF) leak, although rare, is a well-recognized cause of headache with annual incidence of spontaneous CSF leak estimated to be 5/100,000 [Cryptococcus neoformans, the organism has not been reported to cause central nervous system (CNS) infections through direct spread. We report to the best of our knowledge, the first known case of CSF leak detected with our new cisternographic technique, predisposing a patient to cryptococcal meningitis in the absence of another immune compromise.Cryptococcosis, an infection caused by the encapsulated yeast 9/1 (84.7% neutrophils), serum sodium 138 mEq/l, potassium 4.1 mEq/l, and creatinine 0.7 mg/dl; all liver function tests were within normal limits.A 50-year-old Hispanic female presented to the emergency room (ER) with complaint of severe headache for 1 day. The headache was described as throbbing with associated photophobia and multiple episodes of nonbloody vomiting. She reported a remote history of closed head trauma and intermittent rhinorrhea, especially on leaning forward, with spontaneous resolution a few months prior to this presentation. Her medical history was significant for intermittent “migraine” and coronary artery disease. Physical examination was notable for photophobia, otherwise no nuchal rigidity or focal motor or sensory neurological deficits; the rest of the examination and vital signs were unremarkable. Laboratory parameters revealed hemoglobin 11.6 g/dl, white cell count (WBC) 13.1 × I02O, CSF fluid analysis with WBC 1850 cells/uL , red blood cells 5, protein 175 mg/dl , glucose 40 mg/dl , cryptococcal CSF antigen detected with a titer of 1 : 1024 , negative bacterial antigen, and no growth on bacterial CSF culture. Patient also had a negative HIV serology confirmed by nondetectable HIV ribonucleic acid (RNA) and an absolute CD4 count of 1168.She was admitted to the medical floor where a lumbar puncture was performed with an opening pressure of 35 cm HWith the patient's history suspicious for traumatic CSF leak, she had a magnetic resonance imaging (MRI) of the head and sinus that were both unrevealing. Given the low sensitivity of a regular MRI in CSF leak diagnosis, a computed tomography (CT) cisternogram was performed. It revealed evidence of leakage of intrathecally injected contrast into the right nasal cavity via the right olfalctory fossa, between the anterior ethmoidal cells and the crista galli bone . This waShe was initially started on antimicrobial treatment for presumptive bacterial meningitis and later transitioned to induction therapy for cryptococcal meningitis with liposomal amphotericin B and flucytosine (5-fluorocytosine) after preliminary results of her CSF analysis were obtained. Patient subsequently had an endoscopic-assisted transnasal repair of CSF leak with abdominal fat graft using brainlab navigation system by combined neurosurgical and ear, nose, and throat (ENT) teams. With completion of 2 weeks of induction therapy with liposomal amphotericin B and flucytosine, she demonstrated clinical improvement, and two repeat lumbar punctures during the course of admission showed normalized opening pressure and negative cryptococcal antigen. Fluconazole was then started for consolidation, and she was followed-up on discharge in the outpatient clinic until completion of an 8-week course of fluconazole. Patient has remained symptom free two years after completion of treatment.Cryptococcus neoformans, the causative organism of cryptococcosis, with the following immunodeficiency states described in literature: HIV infection, organ transplantation, chronic steroid use, and sarcoidosis [Immune compromise is a common underlying predisposition for infection by coidosis . There hcoidosis . Immunodcoidosis . Abbas ecoidosis . CSF leaCryptococcus neoformans causing meningitis through this route in an immunocompetent patient. With CNS involvement and meningitis, elevated intracranial pressure, defined as opening pressure of >20 cm H2O measured in a reclining position, is present in more than 50% of patients regardless of HIV status [Cryptococcal infections have a wide range of clinical presentations varying from asymptomatic respiratory tract colonization to disseminated infection . VariousV status  and is gV status . ClinicaV status . Other cV status .Haemophilus influenzae and Streptococcus pneumoniae being the most common pathogens [Cryptococcus neoformans as a causative organism for meningitis in a patient with CSF leak.In patients with persistent CSF leakage, meningitis poses a considerable risk and carries a 10% mortality  with Haeathogens . We are Trichosporon species infection or disinfectants, soap, or starch [Trichosporon species and no other underling immunocompromise that would predispose her to trichosporonosis; also, no contamination with disinfectants/soap and interference with hydroxyethyl starch were encountered. As part of the initial workup to assess immune status, HIV infection was excluded with negative HIV serologic and RNA testing. Her normal CD4 count also excluded idiopathic T-cell deficiency. Peculiar to our case is a previously unrecognized risk factor for cryptococcal meningitis, a CSF leak, and given her history of traumatic rhinorrhea with postural headache, this was further worked up with a cisternogram to confirm diagnosis and necessitate repair.For the routine detection of cryptococcal antigens in CSF, certain modalities are available and include historical staining with Indian ink and observation under light microscopy. With almost 100% sensitivity and 98% specificity, newer latex agglutination tests are currently recommended. Definitive diagnosis is made by growing the yeast in CSF culture . We suspr starch . In our Postural headache is an important feature of CSF leak ; howeverPrompt recognition and treatment is required as cryptococcal meningitis is fatal without treatment . The hosThe impact of aggressive management of intracranial pressure in HIV-negative patients is unclear and has not been consistently employed ; the samCryptococcal meningitis is known to be fatal without treatment; prompt recognition is key with regards to changing the clinical course outcome of this deadly disease. Though immune deficiency including HIV has been known as a predisposing factor, there have been reported cases of immunocompetent patients presenting with cryptococcal meningitis. Both spontaneous and head trauma-related CSF leaks are rare conditions, however, when unrecognized, could lead to life-threatening complications. Early recognition is key and could lead to interventions resulting in excellent outcomes. Postural headache, history of trauma/surgery (recent and remote), and the presence of unusual causes of meningitis should prompt workup for CSF leak. CT cisternography remains the gold standard for diagnosing CSF leak, and we propose adding the gauze scanning technique, which is innovative, inexpensive, and efficient.


Background: Several lines of evidence support the hypothesis of an autoimmune origin of Gilles de la Tourette-Syndrome (GTS). Accordingly, in a recent study we detected positive oligoclonal bands (OCB) in cerebrospinal fluid (CSF) in >30% of adult patients indicating an intrathecal antibody synthesis. However, until today no corresponding antibodies could be identified. The aims of this study were to replicate our findings of positive OCB in an independent sample and to detect CSF autoantibodies.Methods: In this prospective study, 20 adult patients with GTS  were included. All patients were thoroughly clinically characterized. Magnetic Resonance Imaging (MRI) and CSF standard measurements were performed. Isoelectric focusing on polyacrylamide gels with silver staining was used to detect OCB. To examine specific and unspecified autoantibodies, we used transfected Human Embryonic Kidney (HEK) cells expressing different surface antigens , indirect immunofluorescence on different brain tissue sections, and enzyme-linked visualization. Additionally, we differentiated Glioma stem cells SY5Y (human neuroblastoma) using retinoic acid and astrocytes (rat).Results: CSF analyses showed positive OCB (type 2) in 4/20 patients (20%). Using transfected HEK cells we did not find specific surface-autoantibodies. Immunohistochemistry on tissue-sections, SY5Y Glioma stem-cells, and astrocytes showed no specific binding patterns either.Conclusions: Our results corroborate previous findings and demonstrate positive OCB in a substantial number of patients with GTS . Although this is the largest study investigating CSF autoantibodies in GTS using several techniques, we failed to detect any specific or unspecified autoantibodies. It is t that fluificance , alteredificance . Finallyificance , perinatificance , and immificance . Accordiificance  and seveificance . Furtherificance , C-reactificance , increasificance  in patieificance  suggestiificance , see belIn line with these findings, the PANDAS  concept has been suggested, based on the hypothesis that diseases associated with tics and/or obsessive compulsive disorder (OCD) might be caused by group A streptococcal (GAS) infections . The facIn order to further explore immunological changes in GTS, in a recent study, we analyzed autoantibodies in sera of 51 patients, but failed to detect any abnormalities for N-methyl-D-aspartic acid- (NMDA), contactin-associated protein related 2- (CASPR2), Leucin-rich glioma inactivated protein (LGI1), or gamma-aminobutyric acid (GABAB1/B2) . In contUnfortunately, the vast majority of studies investigating antibodies in GTS is limited by the fact that blood sera have been used, but not cerebrospinal fluid (CSF). This is of paramount importance, since in neuropsychiatric disorders results from CSF are more meaningful, not only because the blood-brain barrier segregates blood from CSF limiting antibodies and other determinants to pass, but also the fact that CSF contains less proteins than blood, which may influence the results. Even though in a recent case-control study in five children with GTS  no CSF specific oligoclonal bands (OCB) could be detected , in a reThe aim of this study was to perform extensive CSF analyses in a group of adult patients with GTS. We expected to replicate recent findings of positive OCB  and to dIn this prospective study, we included 20 adult patients  with GTS according to DSM-5. In all cases, the diagnosis was confirmed by one of the authors (KMV). Patients were recruited from our Tourette outpatient clinic at the Hannover Medical School and internet calls between 10/2015 and 3/2016. All examinations were conducted in accordance with the declaration of Helsinki and after approval of the Local Ethics Committee (no. 6987) of the Hannover Medical School. All patients gave written informed consent before entering the study. The inclusion criteria were: (i) age >18 years, (ii) confirmed diagnosis of GTS, (iii) no other severe psychiatric or neurological diseases such as schizophrenia, alcoholism, epilepsy, and mental retardation as well as no (autoimmune) diseases that are known to cause CSF changes including OCB. Additionally, all patients were screened for contraindications of a lumbar puncture such as disturbances of blood coagulation , Partial Thromboplastin Time (PTT), and thrombocyte count), increased intracranial pressure, and spinal tumors.Before lumbar puncture, in all patients cerebral Magnetic Resonance Imaging (MRI) was performed to exclude (i) brain pathologies associated with increased intracranial pressure, and (ii) immunological diseases known to cause positive OCB such as multiple sclerosis (MS). All images were acquired on a 3T Siemens Skyra MRI scanner equipped with 32 channel head coils. For structural images, T1 weighted images were acquired by means of a MPRAGE sequence .All patients were carefully clinically assessed using the following standardized assessments: (i) Yale Global Tic Severity Scale—otal Tic Score (YGTSS-TTS)  to measuAfter lumbar puncture, CSF samples were immediately frozen at −80°C for up to 4 months prior to analyses.CSF was analyzed by standard methods assessing routine CSF parameters including (i) CSF cell count using a Fuchs-Rosenthal counting chamber, (ii) manual assessment of cytology and (iii) blood-CSF barrier function by CSF-serum albumin quotients QAlb , 37]. Th. Th37)].For the assessment of well-known specific CSF autoantibodies, we used transfected Human Embryonic Kidney Cells , which express either NMDA-, CASPR2-, LGI1-, AMPA- or GABAB1/B2. The HEK-cells were incubated with 30 μl undiluted CSF and washed afterwards to detect antibodies in CSF. Bound autoantibodies in CSF were labeled with secondary fluorescein-conjugated goat anti-human antibodies . As a positive control we used the NMDAR antibody provided in the kit .To detect antibodies targeting unknown antigens, we used indirect immunofluorescence and incubated (30 min) tissue sections from monkey cerebellum , hippocampus (rat), basal ganglia (rat), and Eu90-cells (as negative control) with undiluted patient CSF. In order to visualize bound antibodies, we used fluorescein isothiocyanate (FITC) marked anti-human IgG  provided by the kit as secondary antibody. To control for immunofluorescence staining artifacts, we used enzyme-linked visualization by incubating tissue sections from cerebellum (monkey) with 1:1 diluted patient CSF and anti-human IgG antibodies, adding 3 3′-diaminobenzidine (DAB) as dye . 1:100 diluted Anti-Yo and anti-Hu positive sera (of patients with immunoblot-confirmed antibodies) were used as positive controls. Antinuclear antibodies were described if a typical nuclear binding to all cell nuclei was present in tissue sections.To measure IgG binding to cell surface neuronal antigens, we differentiated a human Glioma cell line . Therefore, SY5Y cells were incubated for 3 days in nutritional medium with 10 μl retinoic acid . After plating on poly-l-lysine-coated wells (20.000 cells/well), we incubated the cells with 200 μl undiluted patient CSF. Antibody binding was visualized by fluorescing goat-anti human IgG  as secondary antibody. Cells were double labeled using rabbit-anti Microtubule-Associated Protein 2  as second primary antibody and fluorescing goat-anti rabbit  as second secondary antibody. Cells incubated with Hu- and Yo-receptor positive sera (patient sera with immunoblot-confirmed antibodies), diluted 1:10 using phosphate buffered saline and triton 0.1%, served as positive controls.Since astrocytes closely interact with neurons and regulate synaptic transmission and plasticity, we additionally assessed antibody binding on this cell type. On that account, we prepared astrocytes from newborn rat brains. Preparations were done in accordance with the international guidelines for the use of laboratory animals. Therefore, 50,000 astrocytes/well were plated and incubated with nutritional medium (DMEM + 1% Pen/Strep + 10% FCS) for 3 days. Assessed by Glial Fibrillary Acid Protein, the purity of astrocyte cultures was over 95%. For detection of antibody binding, the astrocytes were incubated with undiluted patient CSF and fluorescing goat anti-human IgG  as secondary antibody. Anti-Glial Fibrillary Acid Protein antibodies  as a second primary antibody and fluorescing goat anti-rabbit  as a second secondary antibody were used to label astrocytes. As positive control we used aquaporin 4 (AP4) positive serum . Additionally, CSF of all patients were examined for AP4- and MOG-antibodies using a commercially available AP4-antibody kit .Referring to the PANDAS hypothesis of streptococcal infections in GTS, in addition, we measured serum anti-streptolysin titers. Based on recent findings of positive dopamine-2 receptor antibodies , we incup-value < 0.05 was considered significant. All statistical analyses were performed using two-tailed testing. As all dependent variables were normally distributed (tested using Kolmogorow-Smirnow test), parametric tests were used throughout. Due to the small sample sizes, we assumed variance homogeneity for all tests.All statistics were calculated using SPSS. For statistical analysis a We included 20 patients with GTS  with a mean age at tic onset of 7.7 years . Mean tic severity according to YGTSS-TTS was 23.2 . All other clinical details are summarized in T1-weighted cerebral MRI did not reveal significant abnormalities suggesting increased intracranial pressure or an immunological or inflammatory disease in any of the patients.In none of the patients an elevated CSF cell count (≥5 cells/μl) was found. Using QAlb, CSF analyses showed a slightly dysfunctional blood-CSF-barrier in 4/20 patients . In ten patients (50%), no OCB (type 1) could be detected. In four patients , we found positive OCB in CSF only (type 2) indicating intrathecal IgG synthesis. One of these patients also showed an activated lympho-monocytic cell profile. None of the patients showed OCB type 3 . Identical OCB in serum and CSF (type 4) were observed in six patients (30%).Evaluation of the transfected HEK cells revealed no specific binding patterns to any of the expressed surface antigens as depicted in In two patients (10%), we identified positive ANA in sera and CSF (no OCB type 2), but failed to detect any other antibody binding pattern in any of the tissue sections . By usinTo evaluate immune reactivity against different cells in the central nervous system, we used a human Glioma stem cell line (SY5Y). We detected positive ANA in the same two patients (10%), where we also identified positive ANA using immunoreactivity on tissue sections (see above). No other antibody binding was found as depicted in When comparing patients' CSF-incubated and dyed astrocytes to aquaporin 4 positive controls, we were unable to detect antibodies . PresencMeasuring anti-streptolysin titers, five patients (25%) demonstrated elevated values (>200 IU/ml). Noteworthy, two of these patients (10%) also showed positive OCB type 2 in CSF and another two patients mirrored OCB in CSF and serum (pattern type 4). Only two patients (no. 9 and 13) reported a history of streptococcal infections in childhood. However, none of these showed elevated anti-streptolysin titers. None of the patient's sera demonstrated positive dopamine-2 antibodies.p = 0.93). We also did not find any other significant differences between patients with and without positive OCB referring to tic severity (according to YGTSS-TTS), pre-monitory urges (PUTS), and comorbidities including OCD (Y-BOCS), ADHD , depression (BDI-II), anxiety (BAI) and psychological distress and psychiatric disorders (BSI)  and without OCB  corroborating recent findings from our group in an independent sample of patients with GTS [positive OCB type 2 in 8/21 of patients (38%)] . HoweverWhen combining results from our recent  and currOur findings are completely in line with results from a recent study based on the Swedish National Patient Register, where the authors describe a general increased risk (36%) for autoimmune diseases in patients with chronic tic disorders  and the In two patients (10%) we detected positive ANA. Since the prevalence rate of positive ANA is about 27% in the general population  and becaThe second main result is the lack of autoantibodies detectable with the methods as described above. This is the first study investigating CSF samples for antibodies targeting both known and unknown antigens in a large number of adult patients with GTS. Although we used a wide spectrum of different methods including HEK cells expressing specific antigens, tissue sections of rat cerebellum, basal ganglia, and hippocampus as well as differentiated human Glioma stem cells and rat astrocytes, we failed to detect any CNS specific autoantibodies. In particular, we used not only tissue sections, but also living cells preventing possible loss of immunological reactivity to isolation or fixation processes. We primarily focused on IgG antibodies targeting cell surface antigens on live cells due to their functional impact on living cells.This negative result can be interpreted in different ways: (i) a true absence of an autoantibody-related mechanism, at least directed to a single antigen; (ii) a failure to detect the target antigen on the cells and tissues used in this study, and (iii) the use of insufficient methods to detect autoantibodies.Based on the autoimmune hypothesis for GTS, immunomodulatory interventions commonly used in autoimmune diseases have also been suggested for the treatment of GTS including plasmapheresis –49, the The following limitations of our study have to be addressed: (i) the sample size was quite small. However, relatively high effort and the invasive procedure related to lumbar puncture have to be taken into account as well as the fact that in clinical routine CSF analysis is not recommended in patients with tic disorders. When combining data from our two independent samples in GTS, our sample consists of 41 patients, of whom nearly one third exhibits positive OCB; (ii) although patients received a compensation fee for participating in the study, we do not believe that this has caused any bias in patient selection; and (iii) antibody binding was not tested on human brain sections. Yet this is due to the limited availability of these sections. A possible approach to margin this limitation is the use of CSF on protein arrays containing large numbers of CNS proteins. Furthermore, the prior fixation of tissue might have biased the results by possible loss of immunoreactivity. Data presented are based on immunochemistry results. Antibody detection via fluorescent cell sorting is possible  yet the In conclusion, although we failed to detect any specific autoantibodies, our finding of positive CSF OCB in a subset of patients points a possible humoral immune contribution to GTS and therefore supports the assumption of autoimmune processes being involved.All datasets for this study are included in the manuscript and the supplementary files.This study was carried out in accordance with the recommendations of the Local Ethics Committee (no. 6987) of the Hannover Medical School with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Local Ethics Committee of the Hannover Medical School.KM-V and K-WS contributed conception and design of the study. CB, KM-V, K-WS, JK, PS, TS, MS, and CS contributed acquisition of data and organized the database. CB wrote the first draft of the manuscript. All authors contributed analysis and interpretation of data and contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.