Structural variation among genomes is now viewed to be as important as single nucleoid polymorphisms in influencing the phenotype and evolution of a species. Segmental duplication (SD) is defined as segments of DNA with homologous sequence.Plutella xylostella, Danaus plexippus, Bombyx mori, Manduca sexta and Heliconius melpomene) to understand their potential impact on the evolution of these species. We find that the SDs content differed substantially among species, ranging from 1.2% of the genome in B. mori to 15.2% in H. melpomene. Most SDs formed very high identity (similarity higher than 90%) blocks but had very few large blocks. Comparative analysis showed that most of the SDs arose after the divergence of each linage and we found that P. xylostella and H. melpomene showed more duplications than other species, suggesting they might be able to tolerate extensive levels of variation in their genomes. Conserved ancestral and species specific SD events were assessed, revealing multiple examples of the gain, loss or maintenance of SDs over time. SDs content analysis showed that most of the genes embedded in SDs regions belonged to species-specific SDs (“Unique” SDs). Functional analysis of these genes suggested their potential roles in the lineage-specific evolution. SDs and flanking regions often contained transposable elements (TEs) and this association suggested some involvement in SDs formation. Further studies on comparison of gene expression level between SDs and non-SDs showed that the expression level of genes embedded in SDs was significantly lower, suggesting that structure changes in the genomes are involved in gene expression differences in species.Here, we performed a systematic analysis of segmental duplications (SDs) among five lepidopteran reference genomes  contains supplementary material, which is available to authorized users. Segmental duplications (SDs) are DNA fragments with near-identical sequences that are greater than 1Kb . The outPlutella xylostella), Monarch butterfly (Danaus plexippus), silkworm (Bombyx mori), Carolina sphinx moth (Manduca sexta), and postman butterfly (Heliconius melpomene), to understand the roles of SDs during the evolution of Lepidoptera. Our analysis revealed that duplication activities varied in terms of number of base pairs or events among these different species. The marked difference of transposable elements (TEs) content in the flanking regions of SDs among these species of Lepidoptera suggested various formation mechanisms of SDs. Our functional analysis of the SDs indicated that gene families embedded in the SDs were different among the five genomes and these gene families may be related to species-specific adaptive evolution.Characterization and annotation of SDs are important for understanding the structure and evolution of a genome and have been explored in many organisms’ whole genomes –15. Few P. xylostella, B. mori, D. plexippus, M. sexta, and H. melpomene, were used to construct the SD map. The genome and predicted transcripts of diamondback moth was downloaded from DBM database (http://www.insect-genome.com/) [http://silkworm.genomics.org.cn/) [http://www.butterflygenome.org/) [http://www.insect-genome.com/) [ftp://ftp.bioinformatics.ksu.edu/pub/Manduca/OGS2/).The five Lepidoptera insect species, me.com/) . The othorg.cn/) , Heliconme.org/) , Monarchme.com/)  and Carohttp://www.repeatmasker.org/, version 4.0.6). We then used silkworm TE dataset [−20).We used the Whole-Genome Assembly Comparison (WGAC) method to detect the segmental duplications in the five Lepidoptera species. The insect genomes were first masked at 15% divergence level from transposable elements (TEs), high-copy repeats or simple sequence repeats (SSR) using RepeatMasker  , 18, 21,p < 0.05).This analysis showed the GO terms that were significantly enriched among genes within SDs. Pfam was also used to annotate the function of the genes in the SDs [Gene content of segmental duplications was accessed using the GFF files obtained from the dataset above (see data sources). We also assessed whether the molecular function, biological process, and pathway terms were over-represented in SDs using Blast2Go . For eac the SDs .10(RPKM). Gene expression levels within and outside SDs regions as well as the variables were compared using a T-test with a Bonferroni correction.We collected the RNA-seq data from published sources to access the gene expression level within and outside SDs regions. These data included different tissues or different developmental stages of diamondback moth , silkworBombyx mori to 15.2% in Heliconius melpomene (Table M. sexta to 93% in D. plexippus)  and silkworm genome [The analysis of the length of SDs in all five species indicated that the Lepidoptera genomes were significantly poor in large blocks (>4 Kb)  to mask the transposable elements , high-copy repeats or simple sequence repeats (SSR). Then silkworm TE dataset [P. xylostella: 3.12%, D. plexippus: 2.05%, M. sexta: 4.97% and H. melpomene: 2.25%). Osanai-Futahashi et al. [H. melpomene may result from some TEs left in the genome.We use RepeatMasker  to 1817 in H. melpomene (e.g. 1817 SDs from H. melpomene shared with other Lepidoptera genomes)  Fig. .Fig. 2DuD. plexippus and H. melpomene) shared more SDs with each other than with the other species . Since the time of Lepidoptera speciation is relatively long, we cannot test the complete phylogeny of SDs, and a greater number of sequenced Lepidoptera genomes would be necessary to elucidate this aspect.We then analyzed the SDs that were only lost in Noctuoidea and Bombycoidea , except for silkworm . Only B. mori and M. sexta showed significant differences (p = 1.218e-05 and p = 0.03909).We speculated that the “shared” SDs between species might represent the “ancestral sequences” as they remained conserved in the genomes during the evolution of Lepidoptera. To test this speculation, we analyzed the alignment identity of “shared SDs” and “unique SDs” and found that “shared SDs” had significantly higher identity comparing with the “unique SDs” , structural constitute of cuticle (GO: 0042302), and nucleic acid binding (GO: 0003676)  to annotate all the genes and showed that each species had different GO enrichments and gene families . The prothoracicotropic hormone (PTTH) is well studied in tobacco hornworm (Rountree and Bollenbacher 1986) and in M. sexta, it is related to molting and metamorphosis [D. plexippus, we identified the GO enrichment of glucuronosyltransferase activity (GO: 0015020). In silkworm, UDP-glucuronosyltransferase (UGT) plays a role in detoxification processes, such as minimizing the harmful effects of ingested plant allelochemicals [B. mori, consistent with [In orphosis , 40. In hemicals . Also, whemicals , 43. In ent with , we idenP. xylostella, B. mori, M. sexta, D. plexippus and H. melpomene, respectively). In P. xylostella, Yu et al. [To further clarify the functions of SDs in each Lepidoptera species, we annotated the gene functions in the SDs regions using Pfam and although the GO enrichments differed among species, some of the gene families embedded in the SDs were the same for the five species , which may be involved in immunity [B. mori, M. sexta, D. plexippus and H. melpomene, respectively).We also identified the trypsin in the five species . A recent study (data unpubl.) of transcription factors in diamondback moth indicates that zinc-finger proteins may be expanded, also suggesting their potential important functions in the DBM. The zinc-finger has been shown to function in a variety of biological processes, such as DNA-binding, RNA-binding, protein-protein interactions, developmental processes and differentiation [Some species-specific genes in SDs regions were also identified including 13 Lepidopteran-specific Lipoprotein_11 in silkworm. Zhang et al.  have shontiation . FurtherH. melpomene . We found that the gene expression levels embedded within our SDs regions were significantly lower than that of other genes located elsewhere in the genome. This was true for all analyzed available developmental stages or tissues   Fig. . For exaSDs Fig. .Fig. 4ExA possible reason for this may be that some regulation mechanisms control the gene expression within SDs. Based on our analysis above, we found that some TEs were enriched in the SDs as well as the SDs’ flanking regions Table . In silkP. xylostella, D. plexippus, B. mori, M. sexta and H. melpomene). We found that the SDs contents greatly varied among the five species. Comparative analyses of SDs showed that most of them arose after the divergence of each lineage. The most closely related species based on the phylogenetic tree also shared more common SDs. Conserved ancestral SDs and species specific SD events were assessed, revealing multiple examples of gain, loss or maintenance of SDs over time. The results indicated that SDs might have undergone loss or gain during the evolution of the genome. We further analyzed the genes embedded in SDs regions and the result showed that most of the genes were located in the species-specific SDs (“Unique” SDs). Functional analysis of these genes suggested their potential roles in the lineage-specific evolution. Comparison of gene expression between SDs and non-SDs showed that the expression levels of genes embedded in SDs were significantly lower, suggesting that structural changes in the genomes were involved in gene expression differences within each species. Our results suggested that SDs might have been involved in the species-specific evolution. They thus provide a valuable resource beyond the genetic mutation to explore the genome structure for future Lepidoptera research.Structural variation between genomes is important in phenotype differentiation and genome evolution. Here, we performed a comparative analysis of segmental duplications (SDs) among five lepidopteran reference genomes Additional file 2: Table S2.Genes embedded in the SDs regions. (XLSX 81 kb)Additional file 3: Table S3.Gene duplicates located in the SD intervals. (XLSX 68 kb)Additional file 4: Table S4.p < 0.01 were shown. (DOCX 20 kb)GO enrichment for proteins within the SDs regions among the five studied Lepidoptera species. Only Additional file 5: Table S5.Gene families that were found to be different among the SDs regions from the five studied genomes. (XLSX 10 kb)


Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen associated with community-acquired infections and nosocomial infections. From 2010 to 2015, K. pneumoniae testing was included into the exiting diarrhea-syndrome surveillance with objective to estimate the prevalence of K. pneumoniae in diarrhea-syndrome patients, test antibiotics susceptibility and investigate molecular characteristics.K. pneumoniae strains were tested for antibiotics susceptibility by minimal inhibitory concentration (MIC) method, and subtyped by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).Stool specimens from diarrhea-syndrome outpatients were cultured and identified the pathogens by the Vitek2 Compact instrument. The isolated K. pneumoniae strains were identified from 4340 stool specimens of outpatients who visited sentinel hospitals in Beijing during 2010–2015. All strains were sensitive to gentamicin, nalidixic acid, ciprofloxacin, ceftriaxone, cefotaxime, cefepime, imipenem. The highest resistance rate of K. pneumoniae strains was 100% to amoxicillin–clavulanate, followed by 72.7% to ampicillin. These 22 K. pneumoniae strains were characterized into 21 different PFGE types and 20 MLST types with less similarity.22 K. pneumoniae in stool specimens from outpatients with diarrhea syndromes was about 0.5% in Beijing. Less similarity of the isolated strains indicated the unlikely long-term circulating of K. pneumoniae in the community. ST23 was the most common genotype. Drug resistance of the community-acquired K. pneumoniae was not a serious problem in comparing with hospital-acquired infections. High vigilance in the community-acquired K. pneumoniae strains and investigation of pathogens’ microbiological characteristics are valuable for signals detection for drug resistance in population and strains variation.The detection rate of  Klebsiella spp. is ubiquitous in nature. Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen associated with both community-acquired and nosocomial infections, causing pneumoniae, abscess, bacteremia, and urinary tract infections [fections , and occfections , 3.Escherichia coli, Salmonella, and Shigella spp. [K. pneumoniae might impose an increasing risk of infections in communities, from 2010 to 2015, K. pneumoniae testing was included into the exiting diarrhea-syndrome surveillance with objective to estimate the prevalence of K. pneumoniae in diarrhea-syndrome outpatients, test antibiotics susceptibility and investigate microbiological characteristics.The surveillance on diarrhea-syndrome outpatients included any age people in Beijing, involving 245 sentinel hospitals from all 16 districts. Stool specimens were collected from diarrhea-syndrome outpatients in sentinel hospitals [Salmonella, Shigella spp., diarrheagenic Escherichia coli, Vibrio parahemolyticus, and K. pneumoniae) by the Vitek2 Compact instrument . The isolated K. pneumoniae strains were proceeding for antibiotics susceptibility test and molecular characterization.Stool specimens from diarrhea-syndrome outpatients were collected through Beijing diarrhea-syndrome surveillance from 2010 to 2015. Diarrheagenic bacteria strains were cultured and isolated from stool specimens firstly. Any isolated positive bacteria strains were further identified the pathogens  method. The MICs were interpreted by the standards of Clinical and Laboratory Standards Institute (CLSI) document M100-S26:2016 [Escherichia coli strain ATCC 25922 was in use as quality-control strains for the antibiotics susceptibility testing. MIC level at or above 2 μg/mL for cefotaxime and ceftriaxone indicates the strains possibly produced extended-spectrum beta-lactamases (ESBL), which needs further confirmation. MIC for ceftazidime combination with clavulanate decrease at least three twofold concentration in contrast with the MIC value for ceftazidime alone  confirms the existing ESBL production strains [Antibiotics susceptibility testing for the K. pneumoniae strains were subtyped by pulse field gel electrophoresis (PFGE) method using restriction endonucleases XbaI [XbaI (Fermentas) was used to digest the prepared genomic DNA and resultant DNA fragments were separated in a PFGE CHEF-DR III system (Bio-Rad Laboratories) in 0.5× Tris–borate–EDTA buffer at 120 V for 18.5 h, with pulse times ranging from 6 to 36 s. PFGE dendrogram was generated by BioNumerics version 7.1  with the unweighted pair-group method (UPGMA) and Dice coefficient. Isolates that exhibited a PFGE profile with more than 80% similarity were considered as closely related strains [ses XbaI . The res strains .K. pneumoniae strains using seven housekeeping genes  [K. pneumoniae strains were amplified by PCR and sequenced. The allele sequences and sequence types (STs) were determined by the Institute Pasteur Klebsiella MLST database (http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html). BioNumerics version 7.1 software was used to create the minimum spanning tree. In the minimum spanning tree, the founder ST was defined as the greatest number of single-locus variants. Types were represented by circles and the size of a circle indicated the number of strains with this particular type.Multilocus sequence typing (MLST) was performed to subtype nd tonB) . Seven gK. pneumoniae strains were identified: one strain in 2010, three strains in 2011 and 2012 respectively, five strains in 2013, nine strains in 2014, and one strain in 2015; 16 out of 22 positive K. pneumoniae were isolated from April to October, while six positives identified in low-incidence seasons 2010–2015.In the 4340 stool specimens collected in the surveillance period from 2010 to 2015, 22 K. pneumoniae strains were all sensitive to gentamicin, nalidixic acid, ciprofloxacin, ceftriaxone, cefotaxime, cefepime, imipenem, followed by 95.5% to trimethoprim–sulfamethoxazole, cefoxitin, and 63.6% to sulfisoxazole. The highest resistance rate of K. pneumoniae strains was 100% to amoxicillin–clavulanate, and followed by 72.7% to ampicillin. Moreover, all 22 K. pneumoniae strains were sensitive to ceftriaxone, cefotaxime, the MIC was less than 1 μg/mL, and no ESBL production strain was identified.Antibiotics susceptibility is shown in Table K. pneumoniae strains isolated during 2011–2014 had over 80% similarity, which categorized into one PFGE cluster (cluster A), while other strains had less similarity , including three ST23 strains and one ST65. In addition to identifying ST23 and ST65 which are prevail in Asia countries, ST2362, ST2363, ST2364, ST2365, ST2366, ST2367, ST2368, ST2369, and ST2370 were discerned for the first time. In the minimum spanning tree, there were two clone groups: one clone group contained ST23 and ST1660, and the other clone group contained ST17 and ST20 , which further demonstrated the existence of the community-acquired K. pneumoniae infection [K. pneumoniae in stool samples ranges from 5 to 38% in hospital patients [K. pneumoniae.The detection rate of nfection . The prepatients , which eK. pneumoniae in the community. This study identified ST23 as the most common genotype, which were reported in many previous hospital-acquired infection studies in Asia [rmpA gene for aerobactin, which more likely caused community-acquired infection [Less similarity of the strains typed by PFGE or MLST indicated the unlikely long-term transmission of  in Asia , 13. Notnfection , 15 and K. pneumoniae was not a serious public health problem. All the strains were sensitive to some antibiotics  universally in use for clinical treatment. However, hospital-acquired K. pneumoniae infection with resistance to multiple antibiotics agents has been increasing [Klebsiella species strains  caused several predominant nosocomial infections [K. pneumoniae strains, but the diarrhea-syndrome surveillance identified serious drug resistance for Shigella spp. [K. pneumoniae strains can provide a significant signal of drug resistance in population.No ESBL production strains existed from stool specimens of diarrhea-syndrome outpatients in recent years illustrated that the community-acquired creasing . The Klefections . This stlla spp. . Given tlla spp. , monitorK. pneumoniae in stool specimens from outpatients with diarrhea syndromes was about 0.5% in Beijing. Less similarity of the isolated strains indicated the unlikely long-term transmission of K. pneumoniae in the community. ST23 was the most common genotype. Drug resistance of the community-acquired K. pneumoniae was not a serious problem in comparing with hospital-acquired infections. High vigilance in the drug resistance of the community-acquired K. pneumoniae strains can provide a significant signal of drug resistance in population.The detection rate of


Pseudomonas aeruginosa as serious threat for human health. It is, however, unclear, whether intestinal MDR P. aeruginosa carriage is associated with inflammatory responses in intestinal or even systemic compartments. In the present study, we generated with respect to their microbiota “humanized” mice by human fecal microbiota transplantation of secondary abiotic mice. Following peroral challenge with a clinical P. aeruginosa isolate on two consecutive days, mice harboring a human or murine microbiota were only partially protected from stable intestinal P. aeruginosa colonization given that up to 78% of mice were P. aeruginosa-positive at day 28 post-infection (p.i.). Irrespective of the host-specificity of the microbiota, P. aeruginosa colonized mice were clinically uncompromised. However, P. aeruginosa colonization resulted in increased intestinal epithelial apoptosis that was accompanied by pronounced proliferative/regenerative cell responses. Furthermore, at day 7 p.i. increased innate immune cell populations such as macrophages and monocytes could be observed in the colon of mice harboring either a human or murine microbiota, whereas this held true at day 28 p.i. for adaptive immune cells such as B lymphocytes in both the small and large intestines of mice with murine microbiota. At day 7 p.i., pro-inflammatory cytokine secretion was enhanced in the colon and mesenteric lymph nodes, whereas the anti-inflammatory cytokine IL-10 was down-regulated in the former at day 28 p.i. Strikingly, cytokine responses upon intestinal P. aeruginosa colonization were not restricted to the intestinal tract, but could also be observed systemically, given that TNF and IFN-γ concentrations were elevated in spleens as early as 7 days p.i., whereas splenic IL-10 levels were dampened at day 28 p.i. of mice with human microbiota. In conclusion, mere intestinal carriage of MDR P. aeruginosa by clinically unaffected mice results in pro-inflammatory sequelae not only in intestinal, but also systemic compartments.The World Health Organization has rated multi-drug resistant (MDR)  Pseudomonas aeruginosa constitute strictly aerobic Gram-negative bacteria that normally inhabit the soil and surfaces in aqueous environments. Their adaptive capabilities and intrinsic antibiotic resistance support survival in nature as well as in a plethora of settings including healthcare facilities  or exogenously   . Intestiividuals . Disruptividuals . We have. jejuni . Conversteriosis .P. aeruginosa carriers with an intact intestinal microbiota composition. In the present study we therefore investigated, whether mice harboring a human vs. murine microbiota could be stably colonized by a clinical MDR P. aeruginosa strain and whether bacterial carriage was associated with intestinal or even systemic inflammatory host responses.To date, however, no data are available regarding host immune responses in otherwise healthy All animal experiments were conducted according to the European Guidelines for animal welfare (2010/63/EU) with approval of the commission for animal experiments headed by the “Landesamt für Gesundheit und Soziales” . Animal welfare was monitored twice daily by assessment of clinical conditions and weight loss of mice.ad libitum). Cultural and culture-independent  quality control measures revealed virtual absence of bacteria in fecal samples as described earlier . Secondary abiotic mice with a virtually depleted microbiota were generated as described previously . In brie earlier .ad libitum). Fresh fecal samples free of enteropathogenic bacteria , viruses  and parasites  were collected from five individual healthy human volunteers, dissolved in sterile PBS , aliquoted and stored at -80°C as described earlier  methods. Bacterial groups varied less than 0.5 logarithmic orders of magnitude between independent experiments. To assure proper establishment of the human or murine microbiota in the murine host, mice were kept for 3 weeks until P. aeruginosa infection. Immediately before peroral P. aeruginosa challenge individual fecal samples were collected for quantitative molecular analyses of main intestinal bacterial communities as described elsewhere . Notably, the clinical bacterial strain was tested resistant against piperacillin/tazobactam, ceftazidime, cefepim ± cefoxitin, imipenem, ciprofloxacin, gentamycin, tobramycin, amikacin, trimethoprim/sulfamethoxazole, and aztreonam (according to EUCAST interpretation guidelines) and displayed antimicrobial sensitivity to fosfomycin and colistin only (P. aeruginosa strain was grown on cetrimide agar (Oxoid) for 48 h in an aerobic atmosphere at 37°C.The MDR tin only . Prior i9 CFU of the MDR P. aeruginosa strain by gavage in a total volume of 0.3 mL PBS as reported earlier  and cetrimide agar and incubated in an aerobic atmosphere at 37°C for 48 h as described previously  as described earlier .Mice were sacrificed 28 days p.i. by isoflurane treatment . Cardiac blood (for serum) and tissue samples from spleen, MLN, ileum and colon were removed under sterile conditions. Intestinal samples were collected from each mouse in parallel for microbiological, immunological and immunohistochemical analyses.ex vivo biopsies were used for in situ immunohistochemical analysis as reported previously  were determined by an independent blinded investigator.Five μm thin paraffin sections of colonic and ileal eviously ,b. In brEx vivo biopsies (approximately 1 cm2) derived from colon and ileum , as well as MLN and spleen were placed into 24-flat-bottom well culture plates  containing 500 mL serum-free RPMI 1640 medium  supplemented with penicillin  and streptomycin . After 18 h at 37°C, culture supernatants and serum samples were tested for TNF, IFN-γ and IL-10 by the Mouse Inflammation Cytometric Bead Assay  on a BD FACSCanto II flow cytometer (BD Bioscience) as described earlier  as indicated. Two-sided probability (p) values ≤ 0.05 were considered significant. Experiments were reproduced at least twice.Medians and levels of significance were determined using appropriate tests  analyses of the microbiota suspensions derived from human or murine donors revealed substantial host-specific differences in bacterial compositions. TLs and those of specific families/genera such as EB, Bif, Clocc, and Clept were higher in human as compared to murine bacterial suspensions , whereas LB or MIB were lower or even virtually absent in the former as compared to the latter . Three weeks following FMT and immediately before P. aeruginosa challenge , we further assessed respective microbiota compositions in fecal samples derived from individual mice. Molecular analyses revealed higher fecal gene numbers of B/P and Clept, but lower LB and MIB burdens as well as slightly lower total eubacterial gene numbers (less than one order of magnitude) in human microbiota transplanted mice as compared to murine counterparts . These results thus indicate stable intestinal establishment of distinct microbiota following respective FMT.In the present study we addressed whether a complex human or murine gut microbiota could sufficiently confer colonization resistance against MDR 9 viable P. aeruginosa by gavage and quantitatively assessed fecal P. aeruginosa loads thereafter. Cultural analyses revealed that as early as 48 h following the latest bacterial challenge , virtually all human microbiota transplanted mice harbored P. aeruginosa in their intestines with median loads of approximately 104 CFU per g, whereas bacterial counts were approximately two orders of magnitude lower in murine controls . Of note, by day 3 p.i. P. aeruginosa could not be detected in 40% of mice with a murine gut microbiota. Until the end of the observation period, however, fecal P. aeruginosa loads were comparable in mice with either microbiota, whereas 64.7% of mice following human FMT and 77.8% of murine controls harbored the bacterial strain in their intestinal tract at day 28 p.i. . Hence, the complex intestinal microbiota, irrespective whether of human or murine origin, prevented only a subgroup of mice from long-term intestinal MDR P. aeruginosa colonization.On two consecutive days , we perorally challenged mice with 10P. aeruginosa colonization was accompanied by distinct changes of the intestinal microbiota composition over time . Quantitative culture-independent analysis revealed that four weeks following P. aeruginosa association, intestinal gene numbers of MIB had increased in mice that had been subjected to human FMT, but not conventionally colonized mice . As early as one week following P. aeruginosa challenge, however, the respective gut microbiota did not differ from uninfected control groups, neither in mice harboring a human, nor murine microbiota . Hence, no overt changes in intestinal microbiota composition could be observed relatively early  upon P. aeruginosa colonization and were rather subtle thereafter.We next surveyed whether P. aeruginosa challenge and did not display symptoms such as weight loss, diarrhea or fecal blood (data not shown), we next assessed potential microscopic sequelae of P. aeruginosa colonization. To address this, we quantitatively determined apoptotic epithelial cells in the large intestines applying in situ immunohistochemistry. Mice following human FMT displayed more than two-fold increased caspase-3 positive apoptotic cell numbers in their colonic epithelia at both day 7 and day 28 p.i. , whereas this was only the case later in the course of colonization of murine controls . Of note, at day 7 p.i. apoptotic epithelial cells were higher in the large intestines of mice that had been challenged with human gut microbiota as compared to murine controls . In the small intestines of mice with either microbiota, however, apoptotic cells were slightly higher at day 7 p.i. as compared to non-challenged controls  and further increased until the end of the observation period .Given that mice were not clinically compromised following P. aeruginosa-induced intestinal inflammatory sequelae. Remarkably, only in mice following human FMT, Ki67 positive cells were elevated in large intestines as early as 7 day p.i. , but declined thereafter . In the small intestines, proliferative cell responses were highest early in the course of P. aeruginosa colonization, whereas Ki67 positive ileal epithelial cell numbers declined thereafter . Hence, P. aeruginosa colonization of mice resulted in increased intestinal epithelial apoptosis that was accompanied by proliferative/regenerative epithelial cell responses.We additionally assessed Ki67 positive cell numbers in intestinal epithelia indicative for proliferative/regenerative cell responses counteracting potential P. aeruginosa was accompanied by an increased influx of distinct immune cell populations into the intestinal tract, again applying quantitative in situ immunohistochemistry. Irrespective of the host-specificity of the intestinal microbiota, numbers of innate immune cells such as F4/80 positive macrophages and monocytes were elevated in the large intestinal mucosa and lamina propria at day 7 p.i., but declined thereafter . Only in mice with a murine microbiota, however, P. aeruginosa-induced adaptive immune cell responses were more pronounced later in the course of colonization as indicated by increased numbers of colonic B lymphocytes at day 28 p.i. . In the small intestinal tract, increases in both numbers of macrophages/monocytes as well as of B lymphocytes could only be observed later in the course of P. aruginosa colonization in conventionally colonized, but not human fecal microbiota transplanted mice . Hence, rather early in the course of P. aeruginosa colonization increased innate immune cell subsets could be observed in the large intestines of mice harboring either a human or murine gut microbiota, whereas this held true for adaptive immune cells such as B lymphocytes in the small and large intestinal tract of mice with a murine microbiota rather later in the course of P. aeruginosa colonization.We next addressed whether colonization of human fecal microbiota transplanted mice with MDR P. aeruginosa colonization of human vs. murine fecal microbiota transplanted mice was accompanied by pro- and/or anti-inflammatory cytokine responses within the intestinal tract. At day 7 p.i., increased concentrations of the pro-inflammatory cytokine TNF could be detected in colonic ex vivo biopsies derived from mice harboring a murine, but not human microbiota . Large intestinal secretion of the anti-inflammatory cytokine IL-10, however, was dampened at day 7 and day 28 p.i. of mice that had been subjected to human FMT  and at the later time point in mice with a murine microbiota . We further analyzed cytokine secretion in MLN. Alike the colon, TNF concentrations were elevated in MLN as early as 7 day p.i. of mice harboring a murine microbiota only (p < 0.001), but declined thereafter to basal levels . In addition, at day 7 following P. aeruginosa colonization increased IFN-γ secretion could be detected in MLN of both human and murine fecal microbiota transplanted mice , whereas IL-10 levels remained unchanged . Hence, rather early in the course of murine P. aeruginosa colonization intestinal pro-inflammatory, but not anti-inflammatory cytokine responses were induced.We next addressed whether changes in intestinal innate and adaptive immune cell numbers upon P. aeruginosa colonization of human and murine fecal microbiota transplanted mice not only induced local , but also systemic cytokine responses and therefore measured pro- and anti-inflammatory cytokine secretion in splenic ex vivo biopsies. Seven days following P. aeruginosa challenge, increased TNF concentrations could be detected in spleens derived from mice harboring a murine gut microbiota, but decreased to naive levels until day 28 p.i. . In mice that had been subjected to human FMT, a trend towards elevated splenic TNF concentrations could be observed at day 7 p.i. . Whereas INF-γ concentrations were higher in spleens of mice with a murine microbiota at both day 7 and day 28 p.i. , splenic INF-γ levels increased in human fecal microbiota transplanted mice at day 7 p.i. only and reached basal concentrations thereafter . At day 28 p.i., IL-10 was down-regulated in the spleen of mice with human FMT . Hence, cytokine responses upon intestinal colonization with MDR P. aeruginosa were not restricted to the intestinal tract, but could also be observed systemically.We finally addressed whether MDR P. aeruginosa strains has gained increasing attention not only in the medical field, but also in the general public and global health politics physiological conditions. For instance, some members of the human microbiota, particularly obligate anaerobic species might be reduced or even lost upon freezing, thawing and further processing of the fecal donor samples  and/or mifficile .P. aeruginosa challenge. On microscopic level, however, P. aeruginosa induced inflammatory sequelae could be assessed as indicated by increased apoptotic epithelial cell numbers in both small and large intestines as early as 7 days p.i., irrespective of the intestinal microbiota composition of mice. Of note, apoptosis was paralleled by increased proliferative/regenerative epithelial cell responses thereby counteracting potential cell damage. Furthermore, P. aeruginosa colonization was accompanied by a rather early innate immune response as indicated by increased numbers of macrophages and monocytes in the colonic mucosa and lamina propria of human fecal microbiota transplanted mice, whereas small and large intestinal numbers of adaptive immune cells such as B lymphocytes were elevated at the end of the observation period. Pro-inflammatory sequelae of P. aeruginosa colonization are further characterized by increased TNF secretion in both colon and MLN of mice at day 7 p.i., whereas anti-inflammatory IL-10 levels, however, remained unaffected in the intestinal tract. The P. aeruginosa induced immune responses are not surprising given the plethora of virulence factors of the opportunistic pathogen. For instance, Pseudomonas lipid A, a core component of bacterial lipopeptide, is capable of activating NFκB signaling via TLR-4 leading to pro-inflammatory cytokine secretion  upon P. aeruginosa colonization and were only minor in human fecal microbiota transplanted mice thereafter. Previous studies revealed that both acute and chronic small as well as large intestinal inflammation in mice were associated with increased luminal loads of commensal EB further perpetuating the inflammatory scenario via TLR-4 dependent signaling of lipopolysaccharide (LPS) as Gram-negative bacterial cell wall constituent  pathogens and commensal intestinal bacteria, we addressed whether changes in respective gut microbiota compositions might occur during stituent . HoweverP. aeruginosa strain. Furthermore, mere intestinal carriage of MDR P. aeruginosa pose otherwise healthy hosts at risk of developing not only intestinal but even systemic pro-inflammatory sequelae. Future studies need to further unravel the molecular mechanisms underlying the interactions between P. aeruginosa, the commensal microbiota and host immunity in health and disease in order to develop strategies preventing from colonization with MDR Gram-negative strains including P. aeruginosa.Our study demonstrates that in with a complex microbiota reconstituted secondary abiotic mice the established intestinal microbiota (irrespective whether of human or murine origin) does not sufficiently prevent the host from intestinal colonization with a MDR EvK: designed and performed experiments, analyzed data, co-edited paper. IE: performed experiments, analyzed data, co-edited paper. SB: provided advice in design and performance of experiments, co-edited paper. MH: designed and performed experiments, analyzed data, wrote paper.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.


Mycobacterium abscessus and contained a novel plasmid, pMAB02, in addition to the previously described pMAB01 plasmid. One lineage, which emerged just prior to the initial outbreak, is responsible for the epidemic of post-surgical wound infections in Brazil. Phylogenetic analysis indicates that multiple transmission events led to its spread. The presence of a novel plasmid and the reduced genome size suggest that the lineage has undergone adaptation to the surgical niche.An epidemic of post-surgical wound infections, caused by a non-tuberculous mycobacterium, has been on-going in Brazil. It has been unclear whether one or multiple lineages are responsible and whether their wide geographical distribution across Brazil is due to spread from a single point source or is the result of human-mediated transmission. 188 isolates, collected from nine Brazilian states, were whole genome sequenced and analysed using phylogenetic and comparative genomic approaches. The isolates from Brazil formed a single clade, which was estimated to have emerged in 2003. We observed temporal and geographic structure within the lineage that enabled us to infer the movement of sub-lineages across Brazil. The genome size of the Brazilian lineage was reduced relative to most strains in the three subspecies of  AAI, average amino acid identity; ANI, average nucleotide identity; CDS, coding sequence; DCC, dominant circulating clone; MABSC, Mycobacterium abscessus species complex; MST, Minimum Spanning Tree; T7SS, type VII secretion system.http://www.ebi.ac.uk/ena/data/view/PRJEB7058).1. All Genomes sequenced in this study have been deposited in the European Nucleotide Archive; accession number ERP006740 (http://www.ebi.ac.uk/ena/data/view/GCA_900169205.1).2. The reference selected from the newly sequenced isolates and its annotation can accessed through European Nucleotide Archive accession number GCA_900169205; chromosome accession FWDG01000001; plasmid accession FWDG01000002 (http://www.ebi.ac.uk/ena/data/view/PRJEB2779).3. This research was supplemented by sequences from a global population study available from the European Nucleotide Archive; accession number ERP001039 (https://www.ncbi.nlm.nih.gov/nuccore/CP003699.2).Genome sequence GO 06 is available from GenBank; accession number CP003699.2 (https://www.ncbi.nlm.nih.gov/nuccore/ATFQ00000000.1).Genome sequence CRM-0020 is available from GenBank; accession number ATFQ00000000.1 (https://www.ncbi.nlm.nih.gov/nuccore/AGQU00000000.1).Genome Sequence 47J26 is available from GenBank; accession number AGQU00000000.1  consists of three subspecies, M. abscessus subspecies abscessus (M. a. abscessus), M. abscessus subspecies bolletii (M. a. bolletii) and M. abscessus subspecies massiliense (M. a. massiliense)  [We examined whether there was a temporal signal within the Brazilian lineage using root-to-tip linear regression analysis . This shia beast . The bea 2001.3) , just prMinimum spanning trees were used to identify the potential transmission route of the lineage between states. Given that the genetic data demonstrates that the outbreaks in Brazil are caused by a single lineage, we investigated whether there was evidence that it has adapted to the surgical niche within Brazil. blastn search against the PHAST database showed that neither of these deletions had nucleotide similarity to any known phage genes. Del_12078_1#71_02620_02653 had 89 % ANI identity to the variable region containing insertion sequence ISMab1 [Genome comparisons between representatives of the two sub-clusters within DCC , the Brazilian lineage and the out-group of these clades revealed that 14 insertions and deletions had occurred , but thae ISMab1 . GO-termblastn search of the GenBank database with the second contig showed a complete match  to a sequence described as Mycobacterium phage Adler (GenBank accession number KC960489.1). The presence of plasmid genes such as parA, parB, traI and traM, a lack of core phage genes (Table S4) and evidence from read pair information that it was circular suggested this contig was most likely a plasmid, which we designated pMAB02.There was also evidence that the Brazilian lineage had gained genetic material; a newly described 95 804 kb plasmid , found wblast search for pMAB01 and pMAB02 within the global population dataset, including the Brazilian isolates, showed that pMAB01 was unique to the Brazilian lineage but was only present in 117 of the Brazilian isolates. Although it is likely that this plasmid may be being lost in culture, there is some evidence of geographical loss of pMAB01, with a cluster of isolates from Rio Grande do Sul all having lost the plasmid (n=189), except the previously sequenced G0-06 [parA and parB, as well as genes involved in conjugation. We also observed the presence of a complete T7SS .Bryant JM, Grogono DM, Rodriguez-Rincon D, Everall I, Brown KP et al. GenBank. https://www.ncbi.nlm.nih.gov/nuccore/CP003699.2 (2014).Raiol T, Ribeiro GM, Oliveira JVA, Sandes EFO, Melo ACMA et al. GenBank. https://www.ncbi.nlm.nih.gov/nuccore/ATFQ00000000.1 (2013).Davidson RM, Reynolds PR, Farias-Hesson E, Duarte RS, Jackson M https://www.ncbi.nlm.nih.gov/nuccore/AGQU00000000.1 (2012).Chan J, Halachev M, Yates E, Smith G, Pallen M. GenBank.


Encapsulation of structurally and functionally intact chloroplasts within coacervate micro-droplets is used to prepare photosynthetically active membrane-free protocells. Encapsulation of structurally and functionally intact chloroplasts within coacervate micro-droplets is used to prepare membrane-free protocells capable of light-induced electron transport. Ex situ stabilization and integration of biological components and systems such as biomolecules, organelles and cells are important challenges for the development of new living technologies based on biocatalysis, biosensing, artificial organs, therapeutics, environmental and industrial process monitoring, and energy conversion.via complex coacervation.Herein we employ a novel approach to the assimilation of biological organelles into synthetic protocells by using polymer-rich liquid micro-droplets that are spontaneously assembled in aqueous solution Spinacia oleracea (spinach) and selectively isolated using discontinuous Percoll density gradients.a. As the isolated chloroplasts exhibited a net negative surface potential of ca. –25 mV   = 1.1 g mL–1)ρ = 1.06 g mL–1) was confirmed macroscopically when the mixed dispersions were vortexed and allowed to sediment for a few minutes in an Eppendorf tube to produce a green chloroplast-rich coacervate phase , Bris-SynBio, Marie-Curie Individual Fellowship  and University of Bristol (A. J. P. and S. A. D.) for financial support and James Ashbolt for his assistance with preliminary proof of principle experiments.There are no conflicts to declare.Supplementary informationClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.Supplementary movieClick here for additional data file.


Here, a comparative analysis is performed with the model coleopteran insect Tribolium casteneum to elucidate the metabolic pathways in L. aquatilis. Based on a template luciferin biosynthetic pathway, combined with a range of protein and pathway databases, and various prediction tools for functional annotation, the candidate genes, enzymes, and biochemical reactions involved in luciferin metabolism are proposed for L. aquatilis. The candidate gene expression is validated in the adult L. aquatilis using reverse transcription PCR (RT-PCR). This study provides useful information on the bio-production of luciferin in the firefly and will benefit to future applications of the valuable firefly bioluminescence system.Bioluminescence, which living organisms such as fireflies emit light, has been studied extensively for over half a century. This intriguing reaction, having its origins in nature where glowing insects can signal things such as attraction or defense, is now widely used in biotechnology with applications of bioluminescence and chemiluminescence. Luciferase, a key enzyme in this reaction, has been well characterized; however, the enzymes involved in the biosynthetic pathway of its substrate, luciferin, remains unsolved at present. To elucidate the luciferin metabolism, we performed a  Photinus and Photuris from North America  utilizing high-throughput sequencing technology  according to the manufacturer’s protocol. The total RNA was treated with DNase I  as described in the manufacturer’s protocol. Pooled RNA sample was sent for sequencing at Macrogen (South Korea) using Illumina HiSeq 2000 Sequencing System . Quality and quantity of RNA were measured using an Agilent 2100 Bioanalyzer . The mRNA was converted into a library of template molecules using the reagents provided in the Illumina®TruSeq™ RNA Sample Preparation Kit (Illumina). The poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were converted into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then went through an end repair process using an End Repair (ERP) mix. A single ‘A’ nucleotide was added to the 3′ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. Adapters were then added to the ends of the ds cDNA, preparing them for hybridization onto a flow cell. DNA Fragments were enriched by PCR and subsequently sequenced using Illumina HiSeq 2000 Sequencing System  which is able to generate paired-end read with 2 × 100 base pairs (bp) read length.www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were trimmed and cleaned using Trimmomatic . The transcript abundance, Fragments Per Kilobase of transcript per Million mapped reads  was used to determine the quality of the RNA sequencing data . Protein coding genes were identified via TransDecoder under the following criteria: a minimum length of 100 amino acids, a default log-likelihood score of greater than 0 and matches within the Pfam (pfam.xfam.org/) and UniProtKB/Swiss-Prot databases (web.expasy.org/docs/swiss-prot_guideline.html).All protein-coding genes were predicted and extracted from assembled transcripts using TransDecoder  and BlastKOALA  tools in the metabolic pathway database, KEGG, were also used for functional annotation with a 50% identity cutoff. The annotated genes obtained from the KEGG database were categorized based on their functional roles. In addition, the protein-coding genes annotated via TransDecoder were further analyzed using KEGG taxonomic mapping.To assign protein functions, non-redundant protein (NR), and Uniprot databases were used for functional annotation. The cut-off values for each search were adjusted according to the size of the databases. An annotation with the bigger NR database was performed using a minimum amino acid sequence identity of 50% and E-value of 1E–10. Subsequently, the annotation with the smaller Uniprot database was performed using a minimum amino acid sequence under identity of 25% and E-value of 1E–05. In addition, GhostKOALA  with E-value of 1E–05 as a cut-off.The result from KEGG taxonomic mapping showed that the majority of the protein-coding genes in L. aquatilis was identified based on the template luciferin pathway by L. aquatilis, protein sequences of L. aquatilis obtained from this study were blasted against three different protein and pathway databases, NR, KEGG, and Uniprot using BLASTP. The protein sequences that showed the best hits under the highest identity to one of the NR, KEGG, or Uniprot databases were retained. The candidate genes that have functions associated with luciferin were then selected. Moreover, literature search and manual curation were also performed to identify all possible putative enzymes involved in each step of the luciferin metabolic pathway.The candidate luciferin metabolic pathway of L. aquatilis, a different developmental stage, i.e., adult stage, which is also bioluminescent was selected for RT-PCR analysis. Total RNA was extracted from an adult L. aquatilis using the same method as previously described. The first strand DNA was amplified using RevertAid first strand cDNA . Reverse transcription reaction was performed using 2 μg of total RNA and oligo-dT primer as described in the user manual. PCR was then performed using Phire Hot Start II DNA Polymerase . The PCR reaction included 2 μL of the reverse transcriptase reaction mix, 1X Phire Reaction Buffer, 200 μM of each dNTPs, 0.5 μM of each primer , 14,855 (75.17%), and 4,976 (25.18%) annotated protein-coding genes were obtained from NR, Uniprot, and KEGG, respectively . To furttegories . The gentegories . These rbowringi  and Tominanensis . In contnanensis . Differepunctata , hippodanvergens , and propunctata  were idepunctata . In the detected . Due to Tribolium castaneum, followed by Solenopsis invicta , Acyrthosiphon pisum , Apis mellifera , Bombyx mori , and the others   matched the genes found in other arthropods. Of 13,927 genes, 70.48% observably matched the genes found in  21.01%) . This reT. castaneum showed the highest homologues with L. aquatilis (76.45%). Although this T. castaneum is not bioluminescence, it is a model organism of Coleopteran insect, providing the most extensive list of proteins with which to compare; therefore, we selected this species for the comparative analysis. A comparative protein sequence analysis between these two species was then performed to identify conserved genes and their functions. The bidirectional best hits (BBH) analysis was performed between L. aquatilis  and T. castaneum . As a result, a total of 8,020 conserved genes were identified  and T. castaneum , similar trends were found as illustrated in T. castaneum than in L. aquatilis. In contrast, the numbers of genes in the metabolism of cofactors and vitamins, amino acid metabolism, metabolism of terpenoids and polyketides, metabolism of other amino acids, and glycan biosynthesis and metabolism were higher in L. aquatilis than in T. castaneum. It is worth noting that our transcriptome data was obtained only from the larvae of the firefly, but the data set from T. castaneum included annotated protein data from the genome that represents the proteins in both adult and larval stages (ftp://ftp.ncbi.nih.gov/genomes/Tribolium_castaneum/protein/). Not surprising, the genes involved in energy metabolism are likely to be higher in number in T. castaneum. In many insects, the processes during larval stages are mainly involved in food and energy storage for intense growth, while reproduction tends to be the main focus during the adult stage , an aggregation pheromone produced by male T. castaneum , phenoloxidase (EC: 1.14.18.1), luciferase (EC: 1.13.12.7), thioesterase (EC: 3.1.2.20), and luciferin regenerating enzyme (LRE). Most of the candidates obtained for this study used integration of NR and Uniprot, whereas none of the candidates were identified using KEGG. This result indicated that the KEGG database contains limited information about protein functions associated with bioluminescence, whether for fireflies or any other bioluminescent insects  and c11559 (FPKM: 1.3) from the transcriptome data. However, hydroquinone has other functions in the developmental pathway of arthropods as it can also be used to crosslink proteins in the cuticle . In fireflies, p-benzoquinone is a precursor for luciferin biosynthesis . In the larvae of many genera including Lampyris, Luciola, and Nyctophila, pleural defensive organs have been identified which secrete a repellent substance used as a defensive mechanism . In addition, five putative luciferases were identified in this study, i.e., c12163 (FPKM: 17), c14996 (FPKM: 5.31), c18617 (FPKM: 1.09), c13390 (FPKM: 3.73), and c14833 (FPKM: 5.58)  , with muM: 5.58) . Howevercruciata . Both Lccruciata . In addicruciata . Howevercruciata . Apart fnthetase . Moreoventhetase , suggestT-LRE from Lampyris turkestanicus (LRE from Luciola cruciata (L. aquatilis remains to be confirmed.The last step in the luciferin metabolic pathway is the recycling of oxyluciferin. Oxyluciferin is reported to be recycled into 2-cyano-6-hydrobenzothiazole (CHBT) by luciferin regenerating enzyme LRE;  Gomi & . The CHBstanicus , with 78cruciata  with 74.L. aquatilis. All candidate genes involved in the luciferin metabolic pathway are expected to be expressed in both larval and adult stages as fireflies at these two developmental stages are bioluminescence. Of seven candidate luciferase genes, only the two with the highest identity with known firefly luciferases were selected for RT-PCR analysis. An elongation factor 1-alpha (αEF1) gene  was selected as an internal control because it was presented in all three databases. It is not a common reference gene, but it was used because it is one of the most stable reference genes in another coleopteran, C. bowringi (tubulin and GAPDH, were shown to be less stable (The expression of 11 candidate genes identified in this study was verified using RT-PCR . This vabowringi . On the s stable . The resL. aquatilis larvae. Here, we proposed a list of enzymes involved in the firefly luciferin metabolic pathway. The expression of these enzyme-encoding genes is demonstrated in the adult stage of the firefly to confirm our transcriptome results. Although candidate enzymes of the luciferin biosynthetic pathway have been identified in this study, the actual function of these enzymes still need to be verified. Gene knockout will be performed in the fireflies to confirm the functions of these candidate genes by using the recent genome editing technology. Moreover, gene expression analyses will also be performed to confirm the involvement of these enzymes in the luciferin metabolic pathway. By elucidating the luciferin biosynthetic pathway, the development of firefly bioluminescence applications will be extended. Currently, applications using the firefly bioluminescence reporter system primarily rely on commercially synthesized luciferin to generate luminescence. Many applications can benefit from the elucidation of luciferin biosynthesis. Autoluminescence of modified organisms is ideal for this reporter system. Other bioluminescence systems, e.g., bacterial lux that can be seen without any light sources, have also been used in similar applications. Recently, a group of researchers generated an autoluminescent plant from a bacterial lux system (in vivo bioluminescence assay (In this study, we identify candidate genes involved in the firefly bioluminescence reaction from transcriptome data of bioluminescent x system . Howeverce assay . In contce assay . Prior ace assay , but as 10.7717/peerj.2534/supp-1File S1Click here for additional data file.10.7717/peerj.2534/supp-2File S2Click here for additional data file.10.7717/peerj.2534/supp-3File S3Click here for additional data file.10.7717/peerj.2534/supp-4File S4Click here for additional data file.


Human skin encodes a plethora of touch interactions, and affective tactile information is primarily signaled by slowly conducting C-mechanoreceptive afferents. We show that electrical stimulation of low-threshold C-tactile afferents produces markedly different patterns of activity compared with high-threshold C-mechanoreceptive nociceptors, although the populations overlap in their responses to mechanical stimulation. This fundamental distinction demonstrates a divergence in affective touch signaling from the first stage of sensory processing, having implications for the processing of interpersonal touch. C-mechanoreceptors in humans comprise a population of unmyelinated afferents exhibiting a wide range of mechanical sensitivities. C-mechanoreceptors are putatively divided into those signaling gentle touch  and nociception , giving rise to positive and negative affect, respectively. We sought to distinguish, compare, and contrast the properties of a population of human C-mechanoreceptors to see how fundamental the divisions between these putative subpopulations are. We used microneurography to record from individual afferents in humans and applied electrical and mechanical stimulation to their receptive fields. We show that C-mechanoreceptors can be distinguished unequivocally into two putative populations, comprising CTs and CMs, by electrically evoked spike latency changes (slowing). After both natural mechanical stimulation and repetitive electrical stimulation there was markedly less latency slowing in CTs compared with CMs. Electrical receptive field stimulation, which bypasses the receptor end organ, was most effective in classifying C-mechanoreceptors, as responses to mechanical receptive field stimulation overlapped somewhat, which may lead to misclassification. Furthermore, we report a subclass of low-threshold CM responding to gentle mechanical stimulation and a potential subclass of CT afferent displaying burst firing. We show that substantial differences exist in the mechanisms governing axonal conduction between CTs and CMs. We provide clear electrophysiological “signatures” (extent of latency slowing) that can be used in unequivocally identifying populations of C-mechanoreceptors in single-unit and multiunit microneurography studies and in translational animal research into affective touch. Additionally, these differential mechanisms may be pharmacologically targetable for separate modulation of positive and negative affective touch information.NEW & NOTEWORTHY Human skin encodes a plethora of touch interactions, and affective tactile information is primarily signaled by slowly conducting C-mechanoreceptive afferents. We show that electrical stimulation of low-threshold C-tactile afferents produces markedly different patterns of activity compared with high-threshold C-mechanoreceptive nociceptors, although the populations overlap in their responses to mechanical stimulation. This fundamental distinction demonstrates a divergence in affective touch signaling from the first stage of sensory processing, having implications for the processing of interpersonal touch. Studies1) Can all C-mechanoreceptive afferents be identified and distinguished by the marking technique? 2) Can they be differentiated and categorized into separate populations by profiles of latency changes during repetitive electrical stimulation? 3) How do these responses correspond with categorization by natural stimulation across afferents with different mechanical response properties?Human C-mechanoreceptors may form a single population of mechanically responsive C-afferents, with CTs and CMs being those with greater and lesser mechanical sensitivity, respectively, or form two (or more) separate populations, divisible on the basis of their mechanically and/or electrically evoked responses. Understanding fundamental divisions between C-mechanoreceptor subpopulations will enhance our understanding of the distinctions between touch and pain signaling and may facilitate selective targeting of these systems. The present study aimed at characterizing a range of C-mechanoreceptive afferents with differing mechanical sensitivities, using latency changes during electrical stimulation. Specific hypotheses were tested, based on the following questions: The experiment was approved by the University of Gothenburg ethics committee and performed in accordance with the Declaration of Helsinki, and written informed consent was obtained. Microneurographic axonal recordings were made from the left antebrachial cutaneous nerve  in 20 sessions from 19 healthy human participants . Single-unit recordings were gained from CT and CM afferents in the forearm through an insulated, high-impedance tungsten recording electrode (FHC). Nerve signals were amplified, band-pass filtered (0.2–4 kHz), and digitized (20.8 kHz) with a Power1401 and Spike2 software (CED). C-mechanoreceptive units were identified by stroking or pinching the skin innervation territory, and single-unit waveforms were identified online with a combination of threshold crossing and template matching (Spike2).The point of maximal tactile sensitivity in the receptive field was identified, and the monofilament with minimal bending force that reliably evoked a response from the unit was determined. To assess unit responsiveness to innocuous mechanical stimulation, a soft brush was stroked slowly across the receptive field. Units were initially classified as CT if they responded to the brush stimulus, typically with a burst of spikes, and CM if they did not respond . Spike s1) marking stimulation, to investigate the dependence of latency shifts on spikes evoked by natural stimulation; 2) a standard 2-Hz protocol, which is effective at identifying CMs; and 3) high-frequency stimulation, to examine stimulus entrainment and latency shifts at physiological discharge frequencies and near the refractory period.The effect of electrical receptive field stimulation on a single unit’s axonal conduction velocity was explored with three protocols. Constant-current electrical stimulation (0.5-ms pulse width) was delivered through two uninsulated tungsten electrodes (FHC) inserted obliquely into the skin and advanced to within ~1 mm of the point of maximum tactile sensitivity on the skin surface, with a reference electrode inserted in the same manner 5–10 mm medial/lateral to this . The eleMarking stimulation consisted of repetitive 0.25-Hz electrical stimulation  delivereTwo-hertz electrical stimulation, over a period of 3 min, was used , followeHigh-frequency electrical stimulation consisted of four pulses at frequencies of 10, 20, 50, 100, or 200 Hz, with 30-s recovery and three repeats of all frequencies . All trains were delivered in a randomized order.Analysis of microneurography data was performed in Spike2 with off-line template matching. The presence of spikes and their latency (to spike negative-going peak) were analyzed with a custom-written script. Latency changes were represented as a percentage relative to the first spike elicited by each train of stimulation. Analyses of marking data involved counting the number of spikes evoked by a mechanical stimulus in the period between two electrical stimuli, with the latency change expressed as a percentage of the response before tactile stimulation. Marking responses that caused accumulative latency shifts or collisions were not included in the analyses.P < 0.05 was considered significant, by two-tailed parametric tests. Linear regressions were compared by ANCOVA.One unit (CM) was excluded from the 2-Hz analysis because of stimulation failures, and one CM unit showed latency jumps (abrupt shifts from a distinct response latency) during high-frequency stimulation, presumably reflecting activations of another axonal branch and causing longer-latency responses. Occasional latency jumps were observed in other units see C during C and D), 19 were classified initially as CT and 17 as CM, with their receptive fields indicated in A and B. All units were identified as slowly conducting based on their clear, delayed responses to mechanical stimulation , although CTs had significantly smaller spike amplitudes  provided positive identification of all C-mechanoreceptive units. Spike latency changes to electrical stimulation were seen when delivering natural stimulation concomitantly. The number of spikes evoked by natural stimulation was strongly correlated with the observed change in latency to electrical stimulation for both CTs  and CMs . The slopes show that each additional spike caused a latency shift of 0.5% in CMs but only a 0.05% shift in CTs.The marking technique , A and BA and B; in this recording the CT readily responded to low-intensity (5.4 mN) monofilament stimulation, and both units responded to a stronger (78 mN) monofilament stimulation (C and D). Both CMs with low mechanical thresholds (2.5 mN) showed typical CM latency changes (>20%) during 2-Hz stimulation. A subpopulation of CTs (n = 4) generated additional spikes that were not time-locked to the 2-Hz electrical stimulus, and the most pronounced example of this is shown in During 2-Hz electrical stimulation, a dramatically different pattern of spike latency changes was seen between two populations of C-mechanoreceptors, corresponding to CT and CM units C. An examulation A. The CTmulation B. This dmulation , C and DP < 0.001), but no significant differences were observed at 100 Hz. CTs showed significantly less accumulative slowing (the difference between the second and third pulse and between the third and fourth pulse) at 10, 20, and 50 Hz  but only between the third and fourth pulse at 100 Hz (P < 0.05).Few differences were observed in the ability of CT and CM units to follow high-frequency (>50 Hz) electrical stimulation A, and thWe found that human C-mechanoreceptive afferents could be separated unequivocally into two populations on the basis of their responses to electrical stimulation. Mechanical stimulation produced some inconsistencies with the classification. Brush stimulation separated the C-mechanoreceptors into a population of CTs (brush responsive) and CMs (brush unresponsive), although a few CMs responded weakly to the brush. Mechanical monofilament thresholds separated the populations, where CTs had thresholds of <5 mN but a few CMs fell into this range. These findings demonstrate the similarities and differences between properties of C-mechanoreceptive afferents; therefore care must be taken when classifying C-mechanoreceptors.The marking technique was sensitive enough to detect the latency shift evoked by a single spike in CMs , but to CT responses to 2-Hz electrical stimulation were distinct from CMs and all other C-fiber afferent and efferents . There aLatency changes in mechanosensitive C fibers reflect changes in mechanical thresholds and excitability . We predDisparities in conduction latency changes between CTs and CMs during repetitive stimulation are likely produced by differences in the expression of ion channels or ionic transporting mechanisms. A difference in voltage-gated ion channels is suggested by the narrower somatic spike widths in CLTMs than in CHTMs in the rat , and we Identifying and characterizing fundamental divisions between C-fiber populations in the periphery provides opportunities to selectively modulate affect at the first stage of encoding. Targeting receptors at the periphery is particularly advantageous to avoid systemic side effects. Thus abhorrent pain signaling, as seen in various neuropathies , 2014, mThe marking technique and 2-Hz electrical stimulation protocols provide unequivocal separation of C-mechanoreceptors into putative gentle touch-signaling CT and nociceptive-signaling CM populations; however, we show overlap in some C-mechanoreceptive afferent physiological response properties. Our findings enable the unambiguous identification of CTs, which can be applied to future human multiunit microneurography recordings and provides a framework for comparing animal CLTMs to human CTs. The intrinsic axonal conduction differences highlight the fundamental differences between peripheral afferents signaling positive (CT) and negative (CM) affective touch. The underlying mechanisms may be pharmacologically targetable for control over the selective modulation and excitability of CT and CM firing, especially in pathological situations involving tactile dysfunction, such as allodynia.This work was supported by the Swedish Medical Research Council (Grants 62X-3548 to J. Wessberg and 2010-2607 to H. Olausson), Sahlgrenska University Hospital (ALFGBG Grant 3161 to J. Wessberg), and the Medical Research Council, UK (Centenary Award Fellowship Grant MR/J500446/ to R. H. Watkins).No conflicts of interest, financial or otherwise, are declared by the author(s).R.H.W., J.W., H.B.W., J.P.D., H.O., R.D.J., and R.A. conceived and designed research; R.H.W., J.W., H.B.W., J.P.D., and R.A. performed experiments; R.H.W., J.P.D., and R.A. analyzed data; R.H.W., J.W., H.B.W., J.P.D., H.O., R.D.J., and R.A. interpreted results of experiments; R.H.W. and R.A. prepared figures; R.H.W., J.W., H.B.W., J.P.D., H.O., R.D.J., and R.A. drafted manuscript; R.H.W., J.W., H.B.W., J.P.D., H.O., R.D.J., and R.A. edited and revised manuscript; R.H.W., J.W., H.B.W., J.P.D., H.O., R.D.J., and R.A. approved final version of manuscript.


Leishmania. Infections are initiated when a sand fly vector inoculates Leishmania parasites into the skin of a mammalian host. Leishmania causes a spectrum of inflammatory cutaneous disease manifestations. The type of cutaneous pathology is determined in part by the infecting Leishmania species, but also by a combination of inflammatory and anti-inflammatory host immune response factors resulting in different clinical outcomes. This review discusses the distinct cutaneous syndromes described in humans, and current knowledge of the inflammatory responses associated with divergent cutaneous pathologic responses to different Leishmania species. The contribution of key hematopoietic cells in experimental cutaneous leishmaniasis in mouse models are also reviewed and compared with those observed during human infection. We hypothesize that local skin events influence the ensuing adaptive immune response to Leishmania spp. infections, and that the balance between inflammatory and regulatory factors induced by infection are critical for determining cutaneous pathology and outcome of infection.The leishmaniases are diseases caused by pathogenic protozoan parasites of the genus  Leishmania spp. protozoa are inoculated into mammalian skin during a sand fly blood meal at the onset of infection. Although most Leishmania spp. are introduced through the skin, the clinical syndrome caused by these protozoa can be widespread, influenced most heavily by the identity of the infecting species. Most fatalities are due to visceral leishmaniasis, in which Leishmania spp. parasites disseminate from the site of skin infection to visceral organs where they can cause pathology [Leishmaniasis is the group of diseases initiated through the bite of a female phlebotomine sand fly vector. Leishmaniasis is endemic in over 98 countries, and constitutes a widespread disease burden affecting many regions of the world . With thathology .The most common form of leishmaniasis is localized cutaneous leishmaniasis (LCL), one type within a larger category of tegumentary leishmaniasis. During uncomplicated LCL, parasites remain localized in skin tissue and lead to chronic slowly healing skin ulcers. The lesions of LCL are painless and can self-heal without treatment. However, disease resolution can take several months and leave disfiguring scars . In 2013Leishmania infection. The species of parasite is the most important determinant of clinical outcome. Species of Leishmania that typically cause the different clinical forms of tegumentary leishmaniasis in humans are listed in A range of cutaneous manifestations occurs due to Leishmania species. Genome sequences from multiple strains within species causing different forms of the disease have shown only a few differences [Leishmania species that cause divergent disease [Leishmania species have been due to differences in gene copy number and chromosomal duplication events. Beyond that, post-transcriptional modifications have been identified between species [There is considerable variety in disease manifestations beyond merely the species identity. Even within a species it has been difficult to discern the determinants that lead to different disease manifestations. Identification of parasite genetic determinants affecting phenotype has been hampered by the high degree of homology between ferences . Further disease . Most of species ,33.Leishmania antigen, when infected with L. (L.) amazonensis most often develop LCL. In contrast, patients with impairment of the T cell response often develop an anergic syndrome termed diffuse cutaneous leishmaniasis characterized by multiple nodular lesions full of parasites. Currently, it is not well defined what drives these differences in clinical outcome. It is known that parasite strain, genetically or environmentally determined host immune response, and vector components all contribute [In addition to the parasite, host immune response plays a key role in the clinical presentation of leishmaniasis. For instance, subjects with evidence of cell-mediated immunity against ntribute ,34,35,36ntribute ,38,39,40Leishmania promastigotes, vector components, and host skin cells, all serving to influence the innate immune activation cascade. It has become evident that the sand fly salivary components inoculated with the parasite have potent immunomodulatory properties, some of which are being exploited in vaccine development [Despite their different clinical manifestations, all forms of vector-borne leishmaniasis start with a similar series of events in host skin. These include the initial interactions among elopment . Furtherelopment ,42.Leishmania parasites ultimately establish intracellular infection among macrophages, the primary Leishmania host cell housing a majority of parasites throughout chronic infection. These local events ultimately lead to presentation of Leishmania antigen to T lymphocytes to initiate a T cell-mediated immune response. Research in animal models has pointed towards a role for early tissue signals surrounding these events in polarization of the adaptive immune response, a response that guides the course of ensuing disease. A similar role of early events in human leishmaniasis remains difficult to discern [Leishmania species, and examine how host cutaneous responses contribute to or protect from disease.Following this,  discern ,45,46,47L.) denotes Leishmania species within the Leishmania subgenus and (V.) denotes Leishmania species within the Viannia subgenus.The terminologies that describe different clinical forms of leishmaniasis involving the skin can be confusing. Because “cutaneous leishmaniasis” (often abbreviated “CL”) often refers to a specific localized entity described below as Localized Cutaneous Leishmaniasis, “tegumentary leishmaniasis” will be used as an overall term for all forms of leishmaniasis skin involvement. Disseminated forms of tegumentary leishmaniasis called “disseminated cutaneous leishmaniasis” and “diffuse cutaneous leishmaniasis” are very different, but both have been abbreviated as DCL in the literature. Herein we will use the newer term “disseminated leishmaniasis” for disseminated cutaneous leishmaniasis, abbreviated DL. Diffuse cutaneous leishmaniasis is an anergic form sometimes called “anergic diffuse cutaneous leishmaniasis” or ADCL. Herein we will refer to this entity as ADCL, though diffuse cutaneous leishmaniasis is most often abbreviated DCL in the literature. Another disseminated form of leishmaniasis that manifests in mucus membranes is often called mucocutaneous leishmaniasis (abbreviated MCL) because it follows cutaneous lesions. The disease is also called “mucosal leishmaniasis” abbreviated ML, as the simultaneous occurrence of cutaneous and mucosal leishmaniasis is rare and because mucosal disease may develop in the absence of concomitant or previous LCL . ML is tLeishmania species (L. (V.) braziliensis), and can precede lesion formation [Staphylococcal species being most common [L. (V.) braziliensis who were treated before ulceration had a higher treatment fail rate compared to those treated after ulceration (75% vs. 25.8%) [LCL is the most prevalent clinical manifestation of leishmaniasis. It is generally non-life threatening but can be socially stigmatizing. LCL can result from infection with any one of a number of  species . LCL symormation . The lesormation . LCL cant common . For reat common . Indeed,. 25.8%) . Lesions. 25.8%) ,56. ThisLeishmania replication is associated with development of Leishmania-specific T lymphocytes producing tumor necrosis factor (TNF) and interferon gamma (IFNγ). IFNγ promotes parasite control by activating infected macrophages, the primary host cell, to produce microbicidal effectors that can kill intracellular parasites [H)17-type signature was not observed in LCL lesions in this study, an observation relevant to the localized form of disease [L. (V.) braziliensis infection, compared with healthy subject monocytes [Control of arasites . Factorsarasites ,58,59. Aarasites ,60,61. Tarasites . A T hel disease ,64. Acti disease , is also disease ,66. Afte disease . Despiteonocytes .Leishmania antigen, indicative of a functional cellular immune response [H1-type) response formed by asymptomatic hosts is not as robust as those who develop active disease, with smaller DTH induration, lower levels of antigen-induced IFNγ and TNF production by peripheral blood mononuclear cells (PBMCs), and higher IL-10 production [Leishmania antigens may contribute. Naïve PBMCs from cohorts of healthy volunteers exposed to L. (V.) braziliensis or L. (L.) amazonensis antigen generate responses that can be stratified into high-IFNγ producing and low-IFNγ producing groups [Subclinical infection is common in LCL and other forms of leishmaniasis, and can be detected by a lack of lesions or scars suggestive of healed CL, and lack of a history of chronic ulcer but a positive delayed type hypersensitivity (DTH) skin response to leishmanial antigen, or production of IFNγ by blood cells stimulated with response ,69,70. Hoduction . What drg groups ,73. Monog groups .Leishmania spp. result from a balance between pro- and anti-inflammatory factors  transcript forkhead box P3 (FOXP3) is increased intralesionally during chronic LCL due to L. (V.) guyanensis [REGS from these patients suppressed IFNγ production by autologous CD4+ T cells in vitro. The expression of these pro- and anti-inflammatory cytokines by LCL patient PBMCs is sensitive to the local cytokine environment [It has become clear that the outcome of tegumentary infections with the  factors . In infephocytes ,75. Duriphocytes ,77. Simiyanensis . TREGS fironment . In circironment . It may L. (V.) braziliensis transcripts had a unique transcriptional signature compared with lesional skin that did not contain L. (V.) braziliensis transcripts [L. (V.) braziliensis infected patient lymph nodes during the progression from early pre-lesional to lesional phases of diseases [Leishmania antigen to CD4+ T cells and elicit both pro-inflammatory and regulatory cytokine production [L. (V.) braziliensis specific IgG levels are high during the more severe Mucosal Leishmaniasis and decrease with time post treatment [Recently, RNA-seq of LCL patient biopsies revealed lesional skin containing detectable nscripts . Althougnscripts . This isdiseases . B cellsoduction . Furtherreatment . Thus, B+ T cells in the pathology of LCL has been well documented. While there is no association between CD8+ T cell activation and the inflammatory response in the pre-ulcerative phase of the disease, there is an association between the intensity of the inflammation and the frequency of CD8+ T cells expressing granzyme [+ T cells kill L. (V.) braziliensis infected cells but fail to kill intracellular parasites [The role of CD8granzyme . Althougarasites ,86.Leishmania species belonging to the Viannia subgenus. In regions where L. (V.) braziliensis is endemic, approximately 1–10% of LCL patients progress to ML. Known risk factors include sex , increased age, malnutrition, size and number of LCL lesions, lesions above the belt, and inadequate therapy [Viannia subgenus, with L. (V.) braziliensis causing the highest number of cases [Mucosal leishmaniasis  can occu therapy ,88,89. M therapy ,88,90. I therapy . ML is mof cases ,91,92. Mof cases ,94,95.Leishmania antigen-stimulated TNF and IFNγ responses of PBMCs are elevated, while IL-10 expression is reduced [H17 cells, which may be involved in ML pathogenesis [+ T cells expressing TNF and IFNγ in circulation compared with LCL patients and, contrary to LCL patients, ML patients do not show a positive correlation between the frequency of TNF+ monocytes and IL-10+ monocytes [When it is allowed to progress, ML can be highly disfiguring and accompanied by extensive tissue destruction. Lesions contain active parasite-responsive T cells and other inflammatory cells, but the actual burden of detectable parasites is low . Compare reduced . ML lesiogenesis ,64. ML ponocytes . As a reonocytes . PBMCs fonocytes  and ML ponocytes . The unrLeishmania Viannia subgenus, disseminated leishmaniasis  has emerged as highly prevalent in some regions [L. (V.) braziliensis isolates from DL patients induce higher production of TNF and IFNγ than L. (V.) braziliensis isolates from LCL patients [L. (V.) braziliensis isolates causing different disease forms differ at a genomic level [Although ML was previously the most prevalent metastatic form of cutaneous leishmaniasis due to species of the  regions . DL is c regions ,99. Lesi regions ,27. Para regions ,27. The  regions . Patient regions , but may regions ,27,100.  regions ,75,76. Hpatients . Althougic level .L. (V.) braziliensis isolates causing distinct syndromes is unknown, at least in one region genetic variability between strains within a species contributes to the outcome of disease. L. (V.) braziliensis strains isolated from DL vs. LCL patients in the same region of Brazil showed significant enrichment for distinct polymorphisms [Some studies indicate DL is becoming more common in South America, and there is a site where it has surpassed ML in incidence in the state of Bahia, northeast Brazil . Howeverorphisms . Furtherorphisms .Genetic variability in the human population has been reported as significantly associated with DL  or with Leishmania species, which can be detected by a positive DTH response to Leishmania antigen. This contrasts with patients who contract anergic diffuse cutaneous leishmaniasis (ADCL), who have a conspicuous absence of a specific Leishmania DTH response  is a rare but severe form of LCL, characterized by the development of multiple satellite lesions that can coalesce into plaques covering large areas of skin. Lesions are predominantly nodular or papular in nature, contain an abundance of amastigotes, and do not ulcerate . Lesion  antigen ,109. Anereserved ,110,111.patients ,108,112.patients . These ipatients ,115,116.ceptor 2 . In mousceptor 2 ,119,120.L. (V.) pifanoi parasites isolated from an ADCL lesion were inoculated into human volunteers, all developed classical LCL [ADCL is rare in areas endemic for transmission of other forms of CL, suggesting there may be a contribution of parasite strain to disease. However, when ical LCL  suggestiical LCL . Howeverical LCL ,123,124.L. (L.) tropica, L. (L.) major or L. (V.) braziliensis, in decreasing order of frequency [Leishmania lesions [Leishmaniasis recidivans (LR) is a rare, chronic relapsing form of tegumentary leishmaniasis that can occur following primary infection with requency ,125,126.requency . Disease lesions . LR lesi lesions ,128,129. lesions .L. (L.) tropica long after the initial infection subsides, but a role for these cytokines in human LR has not been documented [The proximity of nascent LR lesions to primary lesion sites leads to the hypothesis that a small number of organisms survive silently within the skin at the site of initial infection, and can lead to reactivated inflammatory lesions years after initial cure, responding to an unknown stimulus. The cellular localization of parasites during periods of latency remains unknown. One hypothesis is that local trauma may play a role in reactivation of LR lesions, perhaps via inflammatory changes in the local microenvironment ,131. In cumented .L. (L.) donovani, primarily occurring in east Africa  and the Indian subcontinent  [L. (L.) donovani endemic areas, the instance of PKDL is higher in HIV-positive patients [Post Kala-Azar dermal leishmaniasis (PKDL) is a complication of visceral leishmaniasis caused by , Nepal) . There a, Nepal) . In Indi, Nepal) ,134. PKD, Nepal)  and pers, Nepal) . PKDL ca, Nepal) . A subse, Nepal) . In L.  infantum are scarce. Some cases of non-ulcerating cutaneous lesions due to L. (L.) infantum infection have been reported, but these constitute a separate disease state from PKDL [Reports of PKDL due to other visceralizing rom PKDL . PKDL syrom PKDL .Leishmania species. Different mouse strains have genetically programmed variations in the severity of infection [L. (L.) major infection has been widely used to study CL [L. (L.) major infection, developing a TH2-type CD4+ T cell response, large local ulcerating lesions, and eventually visceral dissemination of parasites. In contrast, C57BL/6, CBA, and C3H mice are relatively resistant to L. (L.) major, developing a TH1-type cellular immune response and small lesions that ultimately self-heal without dissemination [H1-type cells express IFNγ upon contact with antigen presenting cells [+ T cell deficient C57BL/6 mice experience minimal cutaneous pathology despite a high parasite burden in the ear [L. (L.) major and L. (L.) infantum murine infection, there is a prolonged period of silent parasite replication during the first weeks of infection, during which pathology is not observed. After approximately four weeks, infiltration of immune cells coincides with the onset of pathologic changes [Mouse models have been used extensively to study cutaneous and visceral leishmaniasis . As in hnfection , an obsenfection . In partstudy CL . BALB/c mination ,159,160.ng cells , which ing cells ,162. Sim the ear . Further changes ,164.L. (L.) major infection have been useful for elucidating mechanisms controlling type 1 vs. type 2 CD4+ T cell polarization, this dichotomy is not observed during murine infection with other Leishmania species. Murine infection with L. (L.) mexicana results in a chronic phenotype in most mouse strains [L. (V.) braziliensis forms small, non-ulcerative and self-healing lesions in wild type mice [L. (L.) amazonensis in different mouse strains, with higher levels of antibody being associated with susceptibility [L. (L.) donovani and L. (L.) infantum, in which murine susceptibility maps to a single locus encoding solute carrier family 11 member 1  on chromosome 1 [Although murine models of  strains . L. (V.)ype mice . There itibility . In contmosome 1 , murine mosome 1 ,169,170.+ and CD8+ T cells contribute to this protection [L. (L.) major, IL-10 blockade at chronic time points leads to sterile cure [L. (L.) major-infected IL-10 deficient mice achieve a sterile cure, but these mice do not develop immunity to reinfection, suggesting that persistent parasites may be integral to achieving long-lasting immunity [L. (L.) amazonensis or L. (L.) mexicana which are still unable to eliminate infection despite strong IFNγ production [Mouse models of CL show that treated or self-resolved mice can achieve disease resolution, measured by decreased parasite burden and lack of cutaneous lesions, but that parasites persist in the host for long periods of time ,172, as otection . The celotection ,177,178.ile cure ,173. Intimmunity . Nonetheoduction ,181,182,H response in leishmaniasis [+ T cells is one important determinant of development of TH0 cells toward either TH1 vs. TH2 poles [L. (L.) major, skin-infiltrating inflammatory monocytes differentiate locally into DCs and subsequently migrate to the draining lymph nodes, where they induce polarized CD4+ T cell responses [Early immune events within a critical 24–48 h post-infection have been implicated in instructing the polarization of the Tmaniasis . The cytH2 poles ,184,185.esponses . Thus, tL. (L.) major to mouse ears [+ T cell priming in neutrophil depleted mice [Neutrophils are the first immune cells recruited to the site of cutaneous infection after delivery of use ears ,187. Theuse ears ,188. It use ears  and signuse ears . During use ears . Dermal use ears . Along tL. (L.) major parasites and migrate from the skin to the draining lymph nodes [L. (L.) major parasites are rapidly taken up by dermal and inflammatory DC subsets, but not by LCs [L. (L.) major infection. The first showed no effect on CD4+ T cell priming or parasite burden [Leishmania spp. and participate in the immune response, although the biological relevance of these cells remains controversial.Langerhans cells (LCs) are a subset of dendritic cells resident in the epidermis whose role during cutaneous leishmaniasis is unclear. It has been demonstrated that epidermal LCs can take up ph nodes ,192. Howt by LCs ,194. Twoe burden , wherease burden . The datLeishmania infection and can internalize parasites  [Resident and recruited macrophages harbor the greatest burden of intracellular maniasis ,197. Macmaniasis . Both phmaniasis ,202,203.maniasis ,205. In  factors ,207. Pha factors . Mechani(SHIP-1) .L. (V.) braziliensis parasites when stimulated with IFNγ as well as wild-type macrophages, while iNOS-deficient macrophages were unresponsive to IFNγ activation [L. (L.) major infection lacking iNOS are susceptible, despite eliciting a robust TH1 immune response [L. (L.) amazonensis [Leishmania infection is controversial [The relative importance of NADPH oxidase vs. iNOS for macrophage microbicidal responses was explored with knockout mouse models. Macrophages lacking a functional NADPH oxidase were able to kill intracellular tivation . Additioresponse . Macrophzonensis . The extoversial ,212,213.L. (L.) major infection [L. (L.) major inoculation [L. (L.) major infection of resistant mice compared to susceptible mice, and the local parasite burden in the skin is inversely correlated with early NK cytotoxic activity in different mouse strains [L. (L.) amazonensis promastigote-infected DCs [H1 polarization of naïve CD4+ T cells [Leishmania infected macrophages (H1 cells [L. (L.) major infection [NK cells are innate immune effector cells that were shown to be protective against murine nfection ,215. NK culation ,217. NK cted DCs . These D T cells . NK cellrophages  but NK-dH1 cells ,221. Connfection . OverallLeishmania spp. infection remain to be fully explored. Keratinocytes reside in the epidermis and participate a communicative immune network in the skin, mediated in part by cytokine exchange with the local cell populations [L. (L.) major, expression of immunomodulatory mediators involved in TH1 induction were significantly highly induced in keratinocytes isolated from the infected skin of resistant mice and several were enriched in the epidermis upon RNA in situ hybridization, suggesting keratinocytes may be a rich source of skin derived cytokines in vivo [L. (L.) major [Leishmania species causing divergent clinical disease elicit distinct inflammatory responses by human keratinocytes in vitro. VL causing L. (L.) infantum-exposed human keratinocytes up-regulate pro-inflammatory cytokine gene expression with concurrent nuclear factor-κB (NF-κB) activation, while CL-causing L. (L.) major may inhibit keratinocyte activation [Leishmania, warranting future investigation in this area.Other resident cell types critical to immune responses at the site of ulations . In a mo in vivo . Interfe.) major . Experimtivation . TogetheLeishmania cause characteristic clinical syndromes. However, there is nonetheless a spectrum of outcomes of infection with each Leishmania species in both human leishmaniasis and animal models. The range of clinical outcomes, when understood, seems to depend on key contributions from infecting parasite species and a delicate balance of pro- and anti-inflammatory host immune factors in response to infection. An interesting property of the Leishmania species infections is the persistence of parasites long after clinical cure of symptomatic infection, a property that can result in downstream cutaneous manifestations, but which also may be critical for developing immune protection against re-infection. The mechanisms and cell types involved in parasite persistence, and how they lead to disease re-emergence vs. immune protection, remain poorly understood and a subject of great interest for investigators.The different species of Leishmania spp. infection. This has led to the development of improved models that better mimic natural infection conditions. Our group and others have postulated a previously unappreciated role for inflammatory tissue signals derived from resident, non-hematopoietic cells such as keratinocytes, during early stages of Leishmania infection within the skin. Additional signals generated from sand fly derived factors also cannot be overlooked. Ongoing investigations aim to determine the cellular targets of these vector elicited effects. Mounting evidence suggests the skin microenvironment has a critical influence on the course of Leishmania infection. Therefore, elucidation of early cutaneous inflammatory mechanisms is an important subject of future research in the field of leishmaniasis.Mouse models have revealed early host signals are important controllers of the clinical response to