Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS) is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine. Burkholderia pseudomallei and B. mallei, respectively. B. pseudomallei is endemic in the soils of South East Asia and Northern Australia and its occurrence has been reported in other tropical and subtropical regions [B. pseudomallei by direct inoculation from soil or water into skin abrasions or by inhalation [Melioidosis and glanders are two debilitating and often fatal diseases in humans and animals caused by  regions . Humans halation ,3. Sympthalation . Melioidhalation . Despitehalation .B. mallei, found in equines and solipeds. B. mallei occasionally infects humans, such as laboratory workers and those in close contact with infected animals [Glanders is a zoonotic disease caused by  animals . Infecti animals . The sym animals . Glander animals .B. pseudomallei and B. mallei pose a significant threat to human and animal health, and there is legitimate concern that these bacteria could be misused as bioterrorism agents. In fact, B. mallei was weaponized and used in the US Civil War, World War I and II [B. pseudomallei [I and II , it has I and II  and thatdomallei . Based odomallei .B. pseudomallei and B. mallei possess several virulence determinants including capsular polysaccharides (CPS), type III & VI protein secretion systems, and quorum sensing that play an important role in their intracellular lifestyle, evading the host immune response, and persistence in vivo [B. pseudomallei and B. mallei [Both  in vivo . In addi. mallei –16. The Burkholderia thailandensis is an environmental saprophyte that is closely related to B. mallei and B. pseudomallei, encompassing about 99% gene similarity. When first isolated, it was incorrectly identified as B. pseudomallei due to the similar characteristics of the two species [B. thailandensis to assimilate L-arabinose, in contrast to B. pseudomallei, which lacks the entire arabinose-assimilation operon [B. thailandensis is structurally similar to these pathogenic species as well [B. thailandensis does not require strict biocontainment in comparison to other virulent Burkholderia spp. Thus, it is considered an attractive surrogate for studying various diagnostic aspects and as a model for vaccine development studies against B. pseudomallei and B. mallei [ species . A notabn operon . The LPS as well ,19. Cons. mallei ,20–23.Neisseria meningitidis (Menomune), Streptococcus pneumonia (PCV7), and Haemophilus influenzae type b (Hib) [Bacterial polysaccharides are known to be immunodominant and protective antigens to several infectious agents. A number of polysaccharide conjugate vaccines are currently licensed to combat serious infections, which include vaccines to  b (Hib) . The abi b (Hib) . Antibod b (Hib) .B. pseudomallei have been investigated as subunit vaccines [B. pseudomallei and B. mallei polysaccharides are immunologically important and potentially viable vaccine candidates.Surface polysaccharides of vaccines , and pasvaccines –30. ThesB. pseudomallei and B. mallei are facultative intracellular pathogens, an antibody response alone in melioidosis and glanders patients might not be sufficient in controlling and eliminating the infection. A common drawback of many of the current vaccine approaches is their lack of ability to stimulate cell-mediated immunity [B. pseudomallei has been evaluated in murine models of melioidosis using multiple routes of immunizations; these have provided partial protection against challenge with virulent B. pseudomallei [Burkholderia strains as vaccines in humans.Given that immunity . The prodomallei –35. HoweSalmonella-based live vector vaccines have been extensively developed and used for delivering foreign antigens to the immune system against a number of pathogens [Salmonella strains were reported to strongly trigger innate immune responses, as they express LPS and flagella and contain stimulatory CpG motifs that stimulate Toll-like receptors. In addition, these strains also induce strong Th1-type responses in mice [athogens –38. Atte in mice ,40 and h in mice ,42.B. mallei was originally described by Burtnick et al. [B. mallei O antigen synthesis locus, which resides on the plasmid p1C3, to the attenuated aroA mutant of Salmonella enterica serovar Typhimurium, SL3261. This construct combined the adjuvant properties and advantages of the live-attenuated Salmonella strain as a delivery vehicle for the B. mallei O-antigen. LPS isolated from the recombinant strain, SL3261/p1C3 was reactive with monoclonal antibodies specific to B. mallei O antigen. This attenuated strain was delivered intranasally and evaluated for its protective efficacy in mice against lethal intranasal infection with B. thailandensis as a model of melioidosis and glanders.The identification of the biosynthetic gene cluster responsible for addition of O antigen onto the lipid A-core of k et al. . Here, wUse of animals in this study was reviewed and approved by the University of Virginia Institutional Animal Care and Use Committee (IACUC) under protocol number 2844-02-11. All mice were kept under specific pathogen-free conditions, and all guidelines for humane endpoints were strictly followed. All animal experiments were conducted in accordance with the “Public Health Service Policy on Humane Care and Use of Laboratory Animals” by NIH, “Animal Welfare Act and Amendments” by USDA, “Guide for the Care and Use of Laboratory Animals” by National Research Council (NRC).B. mallei O antigen mouse monoclonal IgG antibody and 3B3-5, an anti-B. pseudomallei O antigen mouse monoclonal IgG antibody were used in Western blots and ELISA. Pp-PS-W, a mouse monoclonal IgM antibody specific to O-PS-II, a CPS of B. pseudomallei, was used in ELISA (kindly supplied by Dr. Paul Brett (University of South Alabama) [5C8-1C3, an anti-Alabama) ).B. mallei was originally described by Burtnick et al. [aroA mutant S. enterica serovar Typhimurium strain SL3261 [The identification of the biosynthetic gene cluster responsible for the addition of O antigen onto the lipid A-core of k et al. . This lon SL3261  via trann SL3261 , resultiSalmonella enterica serovar Typhimurium strain SL3261, containing the plasmid p1C3 expressing B. mallei O antigen (vaccine), or SL3261 not containing the plasmid (vector), were used for immunization. All strains were grown overnight in Luria broth (LB) or LB supplemented with 100 μg/ml ampicillin, respectively. Both strains were subcultured and grown to an OD650 of 0.5. Bacteria were then washed twice and resuspended in phosphate-buffered saline (PBS), pH 7.4, and adjusted spectrophotometrically to obtain the desired immunization dose. For infection, B. thailandensis E264 [600 of 0.5. Cells were harvested by centrifugation and washed twice in PBS. Before administration to animals, bacterial cells were adjusted spectrophotometrically to obtain the desired density.sis E264  was growB. mallei (ATCC 23344) or B. pseudomallei (ATCC 23343) was incubated overnight with vigorous shaking. Cell pellets were obtained by centrifugation and LPS was extracted using a modified hot aqueous-phenol procedure [2, 1 mM CaCl2), and 2 μg/ml of DNase I from bovine pancreas was added followed by incubation at 37°C for 2 h. RNase A was then added to the mixture (2 μg/ml) and the incubation continued for 2 h. Proteinase K was then added to a final concentration of 20 μg/ml and the mixture was incubated for at least 3 h at 50°C. The samples were clarified by centrifugation and LPS was isolated from the supernatants as precipitated gels following repeated ultracentrifugation at 100,000 X g and 4°C until the A260 and A280 were less than 0.02. After the final spin, the gelatinous pellets were resuspended in pyrogen-free water and lyophilized. To remove contaminating phospholipids, lyophilized LPS samples were repeatedly extracted with 90% ethanol.LB inoculated with rocedure . FollowiB. mallei O-antigen, SDS-PAGE and Western blot analyses were performed as previously described [Salmonella organisms were separated SDS-PAGE and analyzed by Western blotting. As controls, LPS purified from B. mallei and B. pseudomallei were used.To confirm the expression of escribed . Whole-cad libitum; mice were given one week of acclimation before the experiments were initiated. BALB/c mice were anesthetized intraperitoneally with 200 μl of ketamine (6.7 mg/ml) and xylazine (1.3 mg/ml) in a 0.9% saline solution prior to vaccination. Mice were intranasally instilled with 20 μl (10 μl per nostril) of PBS, 107 colony forming units (CFU) SL3261, or 107 CFU SL3261/p1C3. Boosters were performed using the same protocol as the initial vaccination, approximately 14 days after the initial vaccination. Blood was collected via the lateral tail vein at four weeks post-immunization. The serum was separated and used for detection of antigen-specific antibodies by enzyme-linked immunosorbent assay (ELISA). B. thailandensis E264 was used for all infection studies in immunized mice. The LD50 was determined according to the method of Reed and Muench [6 CFU. Six weeks post initial vaccination, mice were anesthetized as described above and 5 x 106 CFU of B. thailandensis (5 x LD50) were given in a 20 μl volume (10 μl per nostril). Mice were closely monitored and checked every six hours for signs of morbidity up to 150 hours during the course of the infection. Mice that became morbid with ruffled fur, shaking, unresponsive to touch, unable to move, and unable to obtain food and water were humanely euthanized by CO2.Four- to six- week old female BALB/c mice were obtained from Harlan Sprague-Dawley Farms . The University of Virginia Animal Care and Use Committee approved all procedures concerning the use of mice for this study. Mice were housed at the University of Virginia under SPF conditions, and were fed d Muench  and calcB. mallei LPS, or 10 μg/ml of B. pseudomallei LPS (as extracted above) in PBS supplemented with 20 mM MgCl2 pH 7.2 for 1 h at 37°C or with ~5 x 107 CFU/well of B. thailandensis E264 overnight at 4°C. Following a 1 h blocking step with 5% skim milk / PBS-0.05% Tween-20 (PBS-T) at 37°C. Serum samples were serially diluted in PBS- 2% bovine serum albumin (PBS-B) and 100 μl were placed into each well in either B. mallei LPS-coated, B. pseudomallei LPS-coated, or B. thailandensis-coated plates, in duplicate. Mouse monoclonal antibodies (mAb) 5C8-1C3, 3B3-5, and Pp-PS-W were used as positive controls. After overnight incubation at 4°C, the plates were washed three times with PBS-T and air-dried. Secondary antibodies ) were added to individual plates, diluted 1:5000 in PBS-B, and incubated at 37°C for 1 h. The microwells were washed again, and the enzymatic reaction was developed by the addition of 4-nitrophenol phosphate disodium salt hexahydrate (PNPP) diluted to 1 mg/ml in PNPP substrate solution . The reaction was terminated by the addition of 3 M NaOH to each well, and the results were recorded by using a Thermo microplate reader  measuring the colorimetric reaction at OD405 with the data was displayed using SOFTmax Pro version 1.1 software.Immulon 2 HB ELISA plates  were coated with 100 μl of either 10 μg/ml 2 asphyxiation, after which bacterial loads were quantified by collecting the nasal wash (NW) and harvesting organs. For the NW, an 18-G catheter was placed at the oropharyngeal opening of the mouse and 0.5 ml of PBS-B was flushed through the nasal passage and collected. Lungs, livers, and spleens were aseptically removed from mice, weighed, and homogenized in 1 ml PBS-B. The CFU of B. thailandensis E264 were determined by plating 10-fold serial dilutions of the homogenates on tryptic soy agar (TSA) and Ashdown’s agar plates, which is a selective culture medium for the isolation and characterization of Burkholderia spp. Final results were expressed as CFU/ml for NW and CFU/g for organs.Prior to sample collection, mice were euthanized by CO6 CFU B. thailandensis (5 x LD50), lungs of BALB/c mice were isolated in their entirety. Under sterile conditions, the trachea of each animal was exposed and the lungs were inflated with 0.3 ml of 10% neutral-buffered formalin, removed and immediately immersed in the same fixative. Livers and spleens were also used for histology; these organs were collected under the same conditions as the lungs. All samples were processed by standard paraffin embedding methods. Sections were cut 2 μm thick and stained with haematoxylin-eosin (H&E). The preparation of tissue sections was performed by the University of Virginia Research Histology Core Facility. Tissue sections were examined by a veterinary pathologist who was blinded to animal group assignments.24 and 72 h post-infection with 5 x 10The concentrations of the selected cytokines from pooled sera collected from PBS-, vector-, and vaccine-immunized mice were measured in duplicate using the Invitrogen Mouse Cytokine Magnetic 20-Plex Panel (Invitrogen) according to the manufacturer’s protocol. Samples were acquired using the Luminex 200 analyzer system , and the data were analyzed using xPONENT version 3.1 software .et al. [B. thailandensis E264 was engineered to be luminescent, as previously described [B. thailandensis strain, E264lux, from log-phase cultures diluted to 2 x 106 CFU/ml; diluted baby rabbit serum (1:10); 2 x107 polymorphonuclear leukocyte (PMN); and sera collected from PBS-, vector-, or vaccine-immunized mice. The percentage of killing was determined by comparing the relative luciferase units (RLU) derived from assays lacking serum to the RLU obtained from assays with vector or vaccine sera following a 120 min incubation at 37°C shaking at 250 RPM. Microtiter plates were read using an Envision Multilabel plate reader (PerkinElmer).Luminescent opsonophagocytosis assay was performed according to the method previously described by DiGiandomenico et al. . In thisescribed , where l405 and were expressed as the reciprocal dilution. The x- intercept served as the endpoint titer. Antibody titers were compared using the Kruskal-Wallis test for comparison of three groups or the Mann-Whitney U test for two group analysis. The results of survival studies were represented using Kaplan-Meier survival curves and were analyzed by the log-rank test.All analyses were performed using GraphPad Prism version 4 software. ELISA endpoint titers were calculated using the linear regression of duplicate measurements of adjusted ODB. mallei O antigen, was transferred to the live-attenuated vaccine strain Salmonella enterica serovar Typhimurium SL3261. In order to determine whether SL3261 was capable of expressing the B. mallei O antigen, LPS was extracted from strains SL3261 and SL3261/p1C3 and analyzed by immunoblotting with a monoclonal antibody (mAb) specific for the B. mallei O antigen . We also observed distinct preferences of IgG subclasses to B. mallei LPS (IgG2a> IgG1>IgG3>IgG3b) and B. thailandensis (IgG2a> IgG3>IgG1>IgG2b). A determination of the IgG1 and IgG2a isotypes ratio in the serum of vaccine-immunized mice was also performed as a surrogate for Th1 and Th2 responses. Interestingly, immunization with SL3261/p1C3 resulted in low IgG1/IgG2a ratios of 0.1 and 0.4 for reactivity with B. mallei LPS and B. thailandensis, respectively, indicative of a predominantly Th1 response.The serum IgG subtype responses to The cytokine/chemokine responses of the PBS, vector-, and vaccine-immunized mice at 4 weeks post-initial immunization were determined by quantification of a panel of Th1/Th2 cytokines in pooled sera using a mouse cytokine magnetic 20-Plex Panel. Significantly higher levels of IFN-γ, IL-12p70, IL-5, TNF-α, and IL-10 were detected in the pooled sera of vaccine and vector immunized animals when compared to the respective PBS immunized mice. Also greater serum concentrations of IL-2, IL-4, IL-6, and GM-CSF were observed in vaccine- and vector-immunized mice .B. thailandensis (5 x 106 CFU). No morbidity was observed in vaccine-immunized mice. On the other hand, all PBS-treated mice were dead at 100 h post-infection and 60% of vector-immunized mice were dead at 125 h post-infection  of B. thailandensis strains at serum dilutions up to 1:72,900 compared to limited killing observed with pooled sera from vector-immunized mice . Furthermore, no killing of strain E264lux was observed in absence of vaccine immune sera  Ty21a, which is given orally to protect against typhoid fever [S. typhi [Given the worldwide emerging infectious disease problem, bioterrorism threat, and increasing incidence of antibiotic resistance, more effective measures for prevention and treatment of id fever . Ty21a vid fever ,54. MoreS. typhi –57.Salmonella-based live vector vaccines to express O antigen of Pseudomonas aeruginosa [Salmonella heterologously expressing P. aeruginosa O antigen via multiple routes induced protective immune responses against lethal infection with P. aeruginosa in several mouse models [B. mallei LPS-based vaccine described here to determine which route of immunization provides optimal protection and also whether we observe similar results when this antigen is expressed in S. typhi Ty21a.We have previously had success using ruginosa  and haveruginosa ,58. We hImmunization with purified bacterial polysaccharides predominantly generates short-lived and low-affinity antibody responses predominated with IgM and IgG3 . It has B. mallei and B. pseudomallei is both a virulence determinant and a protective antigen [B. pseudomallei and B. thailandensis are structurally similar [B. mallei expresses O antigens that are structurally similar to those expressed by B. pseudomallei and B. thailandensis with the exception of the L-6dTalp residues lacking acetyl modifications at the O-4 position due to the absence of oacA gene in B. mallei [B. pseudomallei LPS O antigen have been recently described [B. pseudomallei species express type A O antigen, which is considered the backbone and most abundant structure among Burkholderia species [B. pseudomallei and disease severity, clinical manifestation or underlying risk factors have not been reported [B. pseudomallei and B. mallei Figs et al. have previously highlighted the antigenic similarities between the LPS isolated from B. thailandensis, B. pseudomallei, and B. mallei using serum from animals immunized with either heat-killed B. mallei 23344 or B. pseudomallei K96243 [B. thailandensis  of antisera ,71 or moreported –30,72,73B. thailandensis is capable of avoiding humoral immunity by invading epithelial cells and growing intracellularly within phagocytes [Burkholderia infections [Studies form our group have previously reported the therapeutic potentials of antisera obtained from immunized animals during acute pneumonia . Howeveragocytes . Thus, afections .B. thailandensis (>50%) was observed with vaccine serum up to 1:72,900, compared to 1:2,700 with the vector serum (We focused our analysis on the antisera induced by intranasal vaccination to examine the efficiency of vaccine immune sera, and to further investigate whether antibodies to O antigen correlates with the protection observed with active immunization. We performed opsonophagocytosis assays using pooled antisera from mice to further confirm the role of anti-LPS antibodies in protection in our model. Efficient and significant killing of or serum . No killB. mallei O-antigen antibodies of IgG1, IgG2a, and IgG3 subclasses were produced in mice in response to vaccination (B. pseudomallei [The presence of IgG1, IgG2a, and IgM in the immune serum has been previously correlated with high opsonic activity ,76. In ocination . Togethecination , which adomallei ,77.While the intraperitoneal route is commonly used in experimental animal models, the subcutaneous or intranasal route of infection are considered more physiologically relevant model for melioidosis and glanders. In endemic regions and in a potential bio-threat situation, inhalation is an important route of exposure in human melioidosis and glanders infection ,6. A strSalmonella are principally based on the ability the bacteria to persist in antigen-presenting cells during their migration to lymphatic organs of the mucosal immune system where the desired antigen can be processed and presented to naïve T and B cells [Vaccination strategies using attenuated  B cells . It is w B cells . Intrana B cells ,88. Also B cells . Therefo B cells .B. pseudomallei protein array platform and comparing sera from healthy control subjects form South East Asia, patients either recovered from or with acute melioidosis [B. mallei [Brucella abortus, the causative of Brucellosis, another major worldwide disease [Salmonella to provide protection against both infections.This same vaccine system could be used to deliver other antigens to combat glanders and melioidosis. Immunogenic protein antigens have been recognized by probing a well-characterized ioidosis ,91, and . mallei . A numbe. mallei . The rib disease . These dSalmonella expressing B. mallei LPS O antigen elicits a protective immune response at the site of infection. The ability of this vaccine to induce potent humoral- and cell-mediated immunity represents a promising first step towards the development of a platform for potential immunotherapeutics against melioidosis and glanders.In conclusion, our results demonstrate that intranasal delivery of


The present study explored, whether gramicidin triggers eryptosis. To this end phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, red blood cell distribution width (RDW) from electronic particle counting, reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, [Ca2+]i from Fluo3- and Fluo4 fluorescence, and ceramide abundance from binding of specific antibodies. As a result, a 24 h exposure of human erythrocytes to gramicidin significantly increased the percentage of annexin-V-binding cells (≥1 µg/mL), forward scatter (≥0.5 µg/mL) and hemolysis. Gramicidin enhanced ROS activity, [Ca2+]i and ceramide abundance at the erythrocyte surface. The stimulation of annexin-V-binding by gramicidin was significantly blunted but not abolished by removal of extracellular Ca2+. In conclusion, gramicidin stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect at least partially due to induction of oxidative stress, increase of [Ca2+]i and up-regulation of ceramide abundance. Despite increase of [Ca2+]i, gramicidin increases cell volume and slightly reduces RWD.The peptide antibiotic and ionophore gramicidin has previously been shown to trigger apoptosis of nucleated cells. In analogy to apoptosis, the suicidal death of erythrocytes or eryptosis involves cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress, increase of cytosolic Ca Gramicidin, a cationic cyclic peptide antibiotic  and iono2+ activity ([Ca2+]i), ceramide i, and ceramide abundance at the erythrocyte surface quantified utilizing flow cytometry.The present study explored, whether and how gramicidin stimulates eryptosis. To this end, human erythrocytes from healthy volunteers were exposed to gramicidin and phosphatidylserine surface abundance, cell volume, reactive oxygen species, i), Fluo3 and Fluo4 fluorescence were measured to quantify [Ca2+]i. As illustrated in 2+ activity ([Ca2+]i), an effect reaching statistical significance at 0.25 µg/mL gramicidin concentration. A further series of experiments explored whether gramicidin-induced cell membrane scrambling was dependent on entry of extracellular Ca2+. To this end, erythrocytes were exposed for 24 h to 2.5 µg/mL gramicidin in the presence or nominal absence of extracellular Ca2+.In order to test, whether gramicidin affected cytosolic Ca2+. However, even in the absence of extracellular Ca2+ gramicidin significantly increased the percentage of annexin-V-binding erythrocytes. Accordingly, entry of extracellular Ca2+ contributed to but did not account for the stimulation of cell membrane scrambling by gramicidin.As illustrated in 2+ entry and subsequent increase of [Ca2+]i, the ceramide abundance at the erythrocyte surface was determined utilizing a specific anti-ceramide antibody. As illustrated in As cell membrane scrambling is triggered by ceramide even without Ca2+]i was sensitive to replacement of extracellular Na+ by K+. As illustrated in + concentration from 5 to 20 mM at the expense of extracellular Na+ did not significantly influence the effect of gramicidin on annexin V binding, forward scatter or Fluo3 fluorescence. Exposure to 40 mM extracellular K+ concentration, however, significantly augmented the effect of gramicidin on FSC, and an increase to 80 mM extracellular K+ concentration significantly augmented the gramicidin-induced increase of annexin V binding, FSC and Fluo3 fluorescence.Additional experiments explored whether the cell membrane scrambling, cell volume and i) with subsequent activation of Ca2+ sensitive K+ channels, K+ exit, cell membrane hyperpolarization, Cl− exit and thus cellular loss of KCl with osmotically obliged water [2+]i. Possibly, the cellular loss of K+ was overridden by gramicidin mediated entry of Na+. Gramicidin is a well-known ionophore permeabilizing the cell membrane for monovalent ions including K+ and Na+ [Gramicidin treatment was not followed by erythrocyte shrinkage, the second hallmark of eryptosis. Eryptosis is usually paralleled by cell shrinkage which is at least partially due to increase of cytosolic Caed water . Accordi and Na+ . Gramici+ and Na+ gradients across the plasma membrane are dissipated in apoptosis [+/K+-ATPase [+ and Na+ gradients by ionophores may lead to excessive activity of Na+/K+-ATPase. However, the proapoptotic mechanisms sensitive to Na+/K+-ATPase activity remained elusive [Kpoptosis , which ipoptosis . Compellpoptosis . Along t+-ATPase . It is t elusive .2+ permeable cation channels [2+ entry. The Ca2+ entry in turn contributes to the stimulation of gramicidin induced cell membrane scrambling. However, gramicidin triggers cell membrane scrambling even in the absence of extracellular Ca2+. Accordingly, the effect of gramicidin on cell membrane scrambling is partially independent from Ca2+ entry.The stimulation of cell membrane scrambling by gramicidin may in part be due to oxidative stress, a well-known trigger of suicidal erythrocyte death . Since echannels , oxidati2+ independent mechanisms triggering eryptosis include ceramide [Caceramide . Accordiceramide .Plasmodium activates several ion channels of the infected erythrocyte including Ca2+-permeable erythrocyte cation channels [2+ entry through those channels triggers eryptosis with rapid clearance of the infected erythrocytes from circulating blood [Eryptosis is an important physiological mechanism disposing defective erythrocytes prior to hemolysis . Hemolytchannels ,55. Ca2+ng blood . Severalng blood ,56,57,58ng blood , as wellng blood , chlorprng blood  or NO syng blood . WhetherOn the other hand, stimulation of eryptosis and subsequent clearance of phosphatidylserine exposing erythrocytes may lead to anemia as soon as the removal of eryptotic erythrocytes surpasses the formation of new erythrocytes . Moreove2+ activity and enhanced ceramide abundance at the erythrocyte surface.In conclusion, gramicidin triggers erythrocyte cell membrane scrambling, an effect paralleled by and at least partially caused by oxidative stress, increase of cytosolic Cain vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2; pH 7.4 at 37 °C for 24 h. Where indicated, erythrocytes were exposed to gramicidin  at the indicated concentrations, whereby 100 mg gramicidin was solved in 1 mL DMSO. For comparison, the effect of 1 µL DMSO/mL Ringer was tested. In some experiments the extracellular K+ concentration was increased up to 80 mM at the expense of Na+ (in order to maintain constancy of osmolarity). To achieve changes of 18 mV equilibrium potential for each step, K+ concentration was increased from 5 to 10 to 20 to 40 and to 80 mM.Fresh Li-Heparin-anticoagulated blood samples were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003 V). The blood was centrifuged at 120 g for 20 min at 21 °C and the platelets and leukocytes-containing supernatant was disposed. Erythrocytes were incubated 2 and then stained with Annexin-V-FITC  in this solution at 37 °C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur . In some experiments erythrocytes were pre-incubated in Ca2+ free solution. For determination of annexin-V-binding addition of Ca2+ was required during the 15 min incubation with FITC-annexin V. Immediately thereafter the measurement was done so that the exposure to Ca2+ was too short to trigger significant phosphatidylserine translocation.After incubation under the respective experimental condition, 150 µL suspended cells were washed in Ringer solution containing 5 mM CaClOxidative stress was determined utilizing 2',7'-dichlorodihydrofluorescein diacetate (DCFDA). After incubation, a 100 µL suspension of erythrocytes was washed in Ringer solution and then stained with DCFDA  in Ringer solution containing DCFDA at a final concentration of 10 µM. Erythrocytes were incubated at 37 °C for 30 min in the dark and then washed three times in Ringer solution. The DCFDA-loaded erythrocytes were resuspended in 200 µL Ringer solution, and ROS-dependent fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD).2 and 5 µM Fluo-3/AM and Fluo-4/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo-3/AM and Fluo-4/AM loaded erythrocytes were re-suspended in 200 µL Ringer. Then, Ca2+-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur.After incubation, erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM  and Fluo-4/AM  in Ringer solution containing 5 mM CaClTo determine ceramide abundance, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 h at 37 °C with 1 μg/mL anti-ceramide antibody  in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) at a dilution of 1:10. After two washing steps with PBS-BSA, cells were stained for 30 min with polyclonal fluorescein-isothiocyanate (FITC)-conjugated goat anti-mouse IgG and IgM specific antibody  diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. Isotype control mouse IgM  and mouse IgG  were used as negative control according to the manufacturer instructions. The samples were then analyzed by flow cytometric analysis at an excitation wavelength of 488 nm and an emission wavelength of 530 nm.et al. [et al., the % hemolysis was 1.6 ± 0.3, 1.3 ± 0.3, 3.4 ± 0.4, 5.1 ± 0.3, and 64.0 ± 7.3 at 0, 0.25, 0.5, 1.0 and 2.5 µg/mL gramicidin concentration, respectively.For the determination of hemolysis, the samples were centrifuged  after incubation under the respective experimental conditions and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis. Hemolysis is expressed in % in order to allow comparison with % annexin V binding cells. In one series of experiments, the hemolysis determined as described was compared to the method of Kahn et al. . As a reFor the determination of the mean corpuscular volume (MCV) and the red blood cell distribution width (RDW), the samples were incubated under the respective experimental conditions. After incubation for 24 h, cells were spun down  and the supernatant was discarded. Seventy microliters of the pelleted erythrocytes were analyzed using an electronic hematology particle counter .t-test as appropriate; n denotes the number of different erythrocyte specimens studied. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.Data are expressed as arithmetic means ± SD. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and 2+ activity and enhanced ceramide abundance at the erythrocyte surface.Gramicidin triggers erythrocyte cell membrane scrambling, an effect paralleled by and at least partially caused by oxidative stress, increase of cytosolic Ca


In vitro, VDR activation in breast cancer cells reduced β-catenin activation and transcriptional activity leading to elevated expression of the extracellular Wnt inhibitor dickkopf-related protein 1, and a reduction in the interaction of β-catenin with the cyclin D1 promoter. Expression of a stabilized form or β-catenin ablated the protective effects of VDR activation. Collectively, these studies delineate a protective role for VDR signaling in Ron-induced mammary tumorigenesis through disruption of β-catenin activation.The Ron receptor is upregulated in human breast cancers and correlates with enhanced metastasis and reduced patient survival. Ron overexpression drives mammary tumorigenesis through direct β-catenin activation and augmented tumor cell proliferation, migration and invasion. Ron and β-catenin are also coordinately elevated in breast cancers. The vitamin D receptor (VDR) antagonizes β-catenin signaling. Herein, we examined mammary tumor onset and progression using a Ron-driven murine model of breast tumorigenesis crossed with VDR deficient mice. VDR ablation accelerated mammary tumor onset and led to tumors that exhibited a desmoplastic phenotype and enhanced metastases. Tumor levels of active β-catenin were markedly increased in the absence of VDR.  The Ron receptor tyrosine kinase, also known as macrophage-stimulating 1 receptor (MST1R), is overexpressed in approximately 50% of human breast cancers . Similarin vitro [Oncogenic Ron activation is associated with the activation of a variety of signal transduction pathways including activation of PI3-K/Akt , 7, MAPKin vitro , which win vitro . Thus, u3 through induction of cell-cycle arrest, promotion of apoptosis, regulation of differentiation, and reduction in angiogenesis  is a nuclear hormone receptor that protects against mammary tumor formation, progression and metastasis in the presence of 1,25Diewed in -25). Ligiewed in ). For exiewed in -29. Ligaiewed in . TCF-4 iiewed in . In coloiewed in  thus phyiewed in . Given t3 treatment reduced breast cancer cell growth, migration, and invasion in Ron expressing breast cancer cells. Moreover, Ron knockdown (KD) further sensitized breast cancer cells to the growth inhibitory effects of vitamin D3, while constitutive activation of β-catenin reverted the effects of vitamin D3. Mechanistically, 1,25D3 reduced active β-catenin levels, decreased β-catenin transcriptional activity, increased expression of DKK-1, and diminished the association of active β-catenin with the cyclin D1 promoter. Thus, combinational therapies integrating Ron or receptor tyrosine kinase (RTK) antagonists with vitamin D3 or potent vitamin D3 analogs in breast cancers exhibiting Ron overexpression may provide a beneficial alternative to current standard of care chemotherapeutic agents.Herein we demonstrate that VDR limits Ron-induced mammary tumor initiation and growth by decreasing active β-catenin levels and through a reduction in β-catenin target genes. Further, vitamin DTo examine the role of vitamin D receptor (VDR) signaling in the development of Ron-mediated breast tumorigenesis, MMTV-Ron transgenic mice were crossed to mice deficient for VDR (VDR−/−) generating offspring with VDR haploinsufficiency. Littermates were intercrossed to generate VDR+/+, VDR−/− mice or mice heterozygous for a functional VDR with Ron overexpression specific to the mammary epithelium. Analysis of protein and mRNA from transgenic mouse mammary gland lysates shows Ron and VDR expression levels in the MMTV-Ron VDR−/− model Figures  and 1B.Previous studies demonstrated that tissues with targeted overexpression of Ron in the mammary epithelium develop hyperplasia by 12 weeks of age . MammaryMammary tumor formation occurred in all study mice regardless of VDR status. However, MMTV-Ron mice homozygous or heterozygous for VDR exhibited significantly longer times to palpable tumor formation compared to VDR−/− mice  mimic the Ron overexpression observed in human breast cancers and serve as an excellent model for studying Ron signaling in breast tumorigenesis . In Figu3 Figure .3. Vitamin D3 in its active form dose-dependently delayed wound closure indicative of slower migration after 8 hours  were utilized . Ron KD M Figure . Howevers Figure . To acco3 also delayed wound closure in T47D cells indicative of a reduced migratory ability in the presence of vitamin D3. Compared to shNT cells, Ron KD significantly delayed the rate of cell migration suggesting the importance of Ron signaling in driving the enhanced migratory phenotype (data not shown). However, compared to the respective vehicle-treated control, 1,25D3 significantly delayed wound closure further in shRon T47D cells after 24 hours compared to shNT controls, suggesting that the effect of vitamin D3 on tumor cell migration is influenced by Ron receptor status  [3 to inhibit growth in non-transfected R7 controls (NT) was almost completely rescued in R7 cells with the ΔN construct  . Interes3 for 24 hours significantly reduced β-catenin transcriptional activity  mRNA levels were upregulated with increasing concentrations of 1,25D3 in R7 cells  using an antibody to pull-down active β-catenin, eluted the antibody/agarose complex from protein and chromatin, and then performed a second immunoprecipitation with an anti-VDR antibody. Similarly, we found that treatment with 1,25D3 effectively reduced the interaction of the VDR/active β-catenin complex with the LEF-1 sequence within the cyclin D1 promoter . Therefore, we hypothesized that the ligand-dependent VDR/active β-catenin interaction prevents β-catenin from binding to TCF/LEF consensus sequences with the promoters of its target genes. Therefore, a chromatin immunoprecipitation assay was performed utilizing an anti-active β-catenin antibody and primers designed to the LEF-1 binding sequence within the mouse cyclin D1 promoter. Treatment of R7 cells with 100 nM 1,25D Figures  and 6G. r Figure .in vivo evidence for the protective role of VDR in Ron-induced tumor initiation and progression in the breast by mitigating the transcriptional activity of β-catenin.Ron expression is abnormally high in about 50% of primary human breast carcinomas . Previouin vivo data suggest that the presence of at least one VDR allele participates to delay Ron-induced mammary tumorigenesis.While all MMTV-Ron expressing study animals eventually developed hyperplasia with ductal ectasia and regression of tertiary branches, complete loss of VDR signaling was associated with earlier mammary acinar hyperplasia, seen in mice as young as 10 weeks of age. Similarly, MMTV-Ron VDR−/− mice exhibited significantly shorter tumor latency than VDR heterozygous and homozygous littermates. Together, our 3 has previously been shown to inhibit the growth of a variety of normal and transformed epithelial cells, including T47D cells . 1,25Diewed in -37). In ll lines . Additioin vivo studies with VDR deletion, loss of active β-catenin levels were observed in R7 cells treated with 1,25D3 that correlated with reduced expression of β-catenin target genes. Numerous studies have linked aberrant β-catenin signaling with epithelial to mesenchymal transition (EMT) and invasiveness in human cancer [in vitro and in an orthotopic model of breast cancer [3 in R7 cells may suggest a mechanism by which VDR mitigates metastatic burden in Ron-overexpressing breast cancers. However, future work is needed to verify the potential for reduced metastasis with VDR-mediated downregulation of TCF-7.Active β-catenin protein levels were significantly higher in VDR deficient mammary glands, which correlated with elevated levels of cyclin D1 and c-Myc. Evidence of enhanced β-catenin transcriptional activity with loss of VDR was further substantiated by significantly elevated mRNA levels of β-catenin target genes cyclin D1, TCF-7 and MMP7. Similar to the n cancer , 39. It t cancer . Moreovet cancer . Increasin vivo [3 suggests a mechanism by which ligand-dependent VDR signaling delays tumor formation and mitigates progression.In normal and transformed epithelial cells, matrix metalloproteinases (MMPs) play important roles in cell migration, invasion, proliferation, apoptosis, angiogenesis, and host defenses through various mechanisms; mainly the regulation of ECM proteins in the microenvironment (reviewed in ). Interein vivo . Enhance3 dose-dependently increased expression of the extracellular canonical Wnt inhibitor, DKK-1, in R7 cells while loss of VDR in MMTV-Ron mice reduced tumoral DKK-1 mRNA. Functionally, 1,25D3-stimulated DKK-1 expression was associated with growth inhibition. These data suggest a working model by which VDR inhibits Wnt/β-catenin signaling and breast cancer in MMTV-Ron mice through the transcriptional regulation of DKK-1 expression  is n3/g . Two hours before sacrifice, mice were injected with 3 mg/mL BrdU. All mice were maintained under specific pathogen-free conditions and were treated and euthanized in accord with protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee.VDR knockout (VDR−/−) animals  were croInguinal mammary glands were isolated and processed as previously described . Images Formalin-fixed, paraffin-embedded right inguinal mammary glands, lungs, liver, and tumors from 2, 4, 6, 8, 10 and 12-month mice were cut into 5 micrometer tissue sections and stained with hematoxylin and eosin Y for gross morphological assessment. Tissue sections from each mouse at each time point were assessed for hyperplasia in the mammary gland and metastasis in the lungs and liver. Metastases were verified histologically and by immunohistochemical staining for Ron expression. Mammary glands from eight month-old MMTV-Ron VDR+/+ and VDR−/− mice were assessed for β-catenin expression by immunohistochemistry. Briefly, antigen retrieval was performed in citrate buffer, tissues were quenched for endogenous peroxide, blocked in goat serum, and the primary antibody against β-catenin  or Ron  was incubated at 4°C overnight. Detection of β-catenin or Ron staining was detected with 3,3′-diaminobenzidine . Staining was analyzed at 40X magnification using ImageJ Software . A minimum of eight animals per genotype was evaluated with two sections per animal.Whole thoracic mammary glands from 6 month-old mice, R7 cells, T47D shNT cells or T47D shRon cells were homogenized in RIPA lysis buffer, sonicated, and measured for protein concentration using a BCA protein assay. Immunoblotting was carried out using antibodies targeting VDR-D6 (Santa Cruz), Ron (Santa Cruz), active β-catenin , total β-catenin , cyclin D1 , c-Myc , DKK-1 (Santa Cruz) and tubulin (Santa Cruz).3 or EtOH (vehicle control) were fixed in 37% formalin, digested and sheared to a length between 200 and 2000 bp. Lysates were precleared with mouse IgG and immunoprecipitated with 5 μg of an anti-active β-catenin antibody (ABC) or anti-active β-catenin followed by secondary immunoprecipitation with 5 μg of an anti-VDR (D6) antibody. Chromatin and protein crosslinks were removed with 5M NaCl and DNA was purified using the QIAquick PCR Purification Kit  according to the manufacturer's instructions. Primer sets designed to encompass the β-catenin/LEF-1 recognition sequence in the mouse cyclin D1 promoter were utilized for qRT-PCR for examination of interactions with active β-catenin. The following primer sequences were used: (5′–TTCTCTGCCCGGCTTTGAT–3′) and (5′– AACTTCAACAAAACTCCCCTGTAGTC–3′); and to an ‘off-target’ site without a β-catenin binding element approximately 4000 bp upstream, within the cyclin D1 promoter: (5′-AGAGACAGGCATCCCATTGGT–3′) and (5′-CAGGTCAGAGTGCTTGCTGATT–3′).R7 cells treated with 100 nM 1,25DTotal RNA was isolated from whole mammary glands, R7 cells, or human T47D cells using Tri-Reagent RT  and subsequently reverse transcribed into cDNA (1.5 μg) using the High Capacity cDNA Reverse Transcription Kit . Between four and eight experimental cDNA samples were analyzed for VDR, Ron, β-catenin, cyclin D1, c-Myc, MMP7, TCF-7, VEGF, and DKK-1 mRNA expression in duplicate by real time PCR analysis using FastStart SYBR Green Master . The following forward and reverse mouse primer sets were used: VDR (5′–GAAGCGCAAGGCCCTGTT–3′) and (5′–CGCTGCACCTCCTCATCTGT–3′); Ron (5′–TCCCATTGCAGGTCTGTGTAGA–3′) and (5′–CGGAAGCTGTATCGTTGATGTC–3′); β-catenin (5′–TCCCTGAGACGCTAGATGAGG–3′) and (5′– CGTTTAGCAGTTTTGTCAGCTC–3′); cyclin D1 (5′–GCGTACCCTGACACCAATCTC–3′) and (5′–CTCCTCTTCGCACTTCTGCTC–3′); c-Myc (5′–TAACTCGAGGAGGAGCTGGA–3′) and (5′–GCCAAGGTGTGAGGTTAGG–3′); TCF-7 (5′–AGCATCCGCAGCCTCAAC–3′) and (5′–AGCGGCCTGTGAACTCCTT–3′); VEGF (5′–GCAGAAGTCCCATGAAGTGA–3′) and (5′–TCCAGGGCTTCATCGTTA–3′); MMP7 (5′–CTGCCACTGTCCCAGGAAG–3′) and (5′–GGGAGAGTTTTCCAGTCATGG–3′); DKK-1 (5′– CCTTCGGAGATGATGGTTGT–3′) and (5′–GTTCTGATCGCGTTGGAAT–3′). Gene expression was normalized to 18S rRNA and reported as relative expression compared to the wild type control.R7 murine breast cancer cells were derived from a mouse mammary tumor from MMTV-Ron mice as previously described . Human TRenilla luciferase  with Lipofectamine 2000  for 48 h. The cells were then treated with 1,25D3 (100 nM) for 24 hours followed by determination of luciferase activity according to the manufacturer's instructions . Values were normalized to Renilla luciferase expression.R7 cells were dual-transfected with the TCF reporter plasmid TOP-FLASH  expressing ﬁreﬂy luciferase and 2 sets of 3 copies of the TCF binding site, and pRLTX expressing 3. After 48 hours, the media was aspirated from T47D cells and 1 mg/ml thiazolyl blue tetrazolium bromide (Sigma-Aldrich) was added for 60 minutes prior to incubation with DMSO. Absorbance was measured at 570 nm. After 72 hours, the media was aspirated from R7 cells which were then washed with 1X PBS, fixed in 4% paraformaldehyde for 10 minutes, and stained with crystal violet for 60 minutes. Images were taken at 20X using Axiovision 4.5 Software  and viable cells were counted manually using ImageJ Software .R7 and T47D cells were seeded in a 12-well or 24-well plate, respectively, at a density of 5,000 (R7) or 25,000 (T47D and T47D shRon) cells per well and allowed to adhere overnight. Fresh media was placed onto the cells with or without the indicated concentration of 1,25D3 (100 nM) for 72 hours followed by determination of cell number using a crystal violet assay. DKK-1 siRNA  was transfected into R7 cells using the Amaxa Nucleofector 2b Device according to the manufacturer's instructions. Targeted DKK-1 knockdown was confirmed via Western blot 48 hours post transfection. Cells were utilized for cell growth/viability assays with or without 1,25D3 as described above.R7 cells were transfected with a plasmid construct (5 μg/1:10^6 cells) containing a stabilized form of β-catenin (ΔN)  using th3 or EtOH (vehicle control) was added to each well at the designated concentration. Images were taken at 5X magnification at 0, 4, 8, and 24 hours after treatment. The length of each scratch was calculated from three measurements per image and percent wound closure was measured using ImageJ Software . Cell number was also measured via MTT (as described above) at the designated time points to account for differences in growth rate.R7, T47D, and T47D shRon cells were seeded in a 6-well plate labeled with a vertical line across the diameter of each well and seeded at a density of 50,000 (R7) or 100,000 (T47D and T47D shRon) cells/well. Cells were grown to 80% confluence then three horizontal scratches perpendicular to the labeled diameter line were inscribed across each well using a sterile p-200 pipette tip. The media was changed and 1,25D3 or EtOH (vehicle control). Below the insert, culture media containing 10% FBS was added as a chemoattractant. Cell invasion was quantified after 48 hours by measuring the viability (MTT) of cells that had traversed the matrigel matrix.R7 cells were seeded at a density of 50,000 cells/well in 6.5 mm transwell inserts (8.0 μm pore size) in a 24-well plate above a 70 μL-thick growth factor reduced matrigel matrix . Cells were plated in serum-free media in the presence of 100 nM 1,25DStatistical analysis was conducted using GraphPad Prism software . For observance of mammary gland hyperplasia, Chi-square analysis was utilized for identifying differences between MMTV-Ron VDR+/+ and MMTV-Ron VDR−/− mice. Student's t tests or analysis of variance (ANOVA) tests were applied to determine significant differences. Data are expressed as the mean ± standard error (SE). A P-value <0.05 was regarded as significant.


Macrophages, the key component of the tumor microenvironment, are differentiated mononuclear phagocyte lineage cells that are characterized by specific phenotypic characteristics that have been implicated in tumor growth, angiogenesis, and invasion. CX3CR1, the chemoattractant cytokine CX3CL1 receptor, plays an important role in modulating inflammatory responses, including monocyte homeostasis and macrophage phenotype and function. However, the role of CX3CR1 in the regulation of the tumor inflammatory microenvironment is not fully understood.in vivo hepatic metastasis model, human colon carcinoma specimens, immunohistochemical staining, TUNEL staining, flow cytometry analysis, Western blotting assay and co-culture in three-dimensional peptide gel, we determined the effects of CX3CR1 on angiogenic macrophage survival and tumor metastasis.Using In this study, we found that CX3CR1 was expressed in human colon carcinomas in a histologic grade- and stage-dependent manner, and CX3CR1 upregulation in TAMs was correlated with poor prognosis. Furthermore, we showed that in a microenvironment lacking CX3CR1, the liver metastasis of colon cancer cells was significantly inhibited. The underlying mechanism is associated with decrease accumulation of angiogenic macrophages that can be partly attributed to increased apoptosis in the tumor microenvironment, thus leading to impaired tumor angiogenesis in the liver and suppressed tumor metastasis.Our results suggest a role of CX3CR1 in angiogenic macrophage survival in the tumor microenvironment contributing to tumor metastasis. Tumors exhibit a complex cellular ecology that establishes their malignant potential. In addition to genetic complexity, it has become increasingly apparent that the tumor inflammatory microenvironment plays an active role in promoting all stages of tumor progression -3. TumorIn this study, we explored whether CX3CR1, a chemoattractant cytokines receptor, regulates the TAMs subtypes in the tumor microenvironment, for several reasons. First, CX3CR1 is primarily expressed on circulating monocytes, tissue macrophages, and tissue dendritic cell populations but is also expressed on T cell and natural killer cell subsets . Second,Our study showed that enhanced CX3CR1 expression was correlated with the poor prognosis in human colon carcinoma. In mice lacking CX3CR1, the liver metastasis of colon cancer cells was significantly inhibited. Mechanistically, CX3CR1 signaling was critical for survival of angiogenic macrophages and promoting tumor angiogenesis. This work highlights the function of CX3CR1 in the tumor inflammatory response and identifies CX3CR1 signaling as the important player in the complex interactions between tumor cells and the tumor microenvironment.Because the treatment and prognosis of colorectal cancer patients depends on the tumor grade (degree of primary tumor differentiation) and tumor-node-metastasis stage (how widespread the cancer is at the time of diagnosis), we first measured the CX3CR1 expression levels in normal colon tissues and human colon cancer tissue. As shown in Figure In addition, the advanced colon carcinoma tissues were markedly and extensively infiltrated by CD68-positive macrophages, particularly along the tumor cell-invasive front; however, the number of macrophages in normal colon tissues was low Figure A. CX3CR1−/− control mice  were injected into the spleen and tumors developed in the liver. Previous studies have shown that SL4 cells form tumors in the liver of 100% of syngeneic mice when injected intrasplenically. This model is not a pathological metastatic tumor model; however, SL4 cells are a useful tool to study how injected colon carcinoma cells seed and develop into a new microenvironment in the liver . Liver t−/− mice and WT controls with SL4 cells intrasplenically. The SL4 cells generated tumors within the spleen (primary) and liver (metastasis). Multiple hepatic tumor nodules, detectable by gross inspection, were evident by 2 weeks. Livers were isolated and the incidence of hepatic metastasis was evaluated. Livers from CX3CR1−/− mice showed significantly smaller tumor foci, fewer metastatic tumors, and decreased tumor-occupied area compared with those from tumor-bearing WT mice . In particular, the proportion of infiltrating CD45+F4/80+ cells in metastatic tumors was significantly lower in CX3CR1−/− mice than that in WT mice  cells was significantly increased in metastatic tumors in CX3CR1−/− or WT mice with murine SL4 colon cancer cells in a 3D peptide gel. A peptide gel was chosen because it more accurately resembles the extracellular matrix milieu of carcinoma compared to Matrigel, which models the laminin-rich basement membrane-type extracellular matrix [−/− macrophages cocultured with SL4 cells, but not WT macrophages. Therefore, CX3CR1 is responsible for promoting macrophage survival within the tumor microenvironment, which is typically associated with TAMs that contribute to tumor development.To understand the role of CX3CR1 in macrophage survival during hepatic metastasis of colon cancer, we cocultured macrophages derived from CX3CR1r matrix . As show−/− mice could be caused by inhibition of angiogenesis. Thus, we stained slices of tumors of comparable size, grown either in WT or CX3CR1−/− mice, with an anti-CD31 antibody . As shown in Figure −/− mice than those in WT mice. Immunohistochemistry and western blot analysis showed that the levels of the proangiogenic cytokines VEGF and TGF-β were significantly decreased in metastatic liver tumors in CX3CR1−/− mice compared with those in WT mice  compared to WT mice ; the antibodies for phospho-FOXO3a, FOXO3a, Bax and Bcl-2 were from Cell Signaling Technology ; the antibodies for F4/80 and A-Caspase3 were from Abcam ; and ChemMate TM EnVision System/DAB Detection Kits were from Dako . Antibodies for PerCP/Cy5.5-conjugated CD45.2, phycoerythrin (PE)-conjugated F4/80, APC-conjugated CD11b and isotype control were from Biolegend . Antibodies for PE-conjugated CCR2, PE-conjugated VEGFR2, PE-conjugated CXCR4 and isotype control were from R&D Systems . DeadEnd™ fluorometric TUNEL system was from Promega .−/−) mice and wild-type (WT) littermates were used for all experiments. CX3CR1 knockout (CX3CR1−/−) mice were harbored a target replacement of the CX3CR1 gene by a GFP gene [−/− mice were obtained from the Jackson Laboratory and maintained in a Specific Pathogen-Free atmosphere at the Beijing Anzhen Hospital affiliated to the Capital Medical University, China. The investigations conformed to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals  and were approved by the Animal Care and Use Committee of Capital Medical University.CX3CR1 knockout  staining involved standard procedures.For immunohistochemistry, sections were incubated with the primary antibody for CX3CR1(1:200), CD68(1:200), CD31(1:200), VEGF(1:200), TGF-β(1:200), or Mac-2(1:200), then incubated with the Dako ChemMate™ EnVision System  for 30 mins. Staining was visualized with use of diaminobenzidine and counterstaining with hematoxylin. Negative controls were omission of the primary antibody, non-immune IgG or secondary antibody only; in all cases, negative controls showed insignificant staining. Images were captured with use of a Nikon Labophot 2 microscope equipped with a Sony CCD-Iris/RGB color video camera attached to a computerized imaging system and analyzed by use of ImagePro Plus 3.0  with blinding to treatment. The expression of CX3CR1, CD68, CD31, VEGF, TGF-β, or Mac-2 was calculated as proportion of positive area to total tissue area for all measurements of the section.For double immunofluorescence, tumors were excised and fixed in 4% paraformaldehyde for 2 hrs, then dehydrated with 30% sucrose/PBS and frozen in compound. Frozen tissue sections, 7 μm, were permeabilized and blocked with 0.1% Triton X-100, 0.2% bovine serum albumin, and 5% normal donkey serum in PBS, then incubated with the primary antibodies F4/80 (1:100) and CX3CR1 (1:100)overnight at 4°C, then FITC or TRITC-conjugated secondary antibody  at room temperature for 1 hr in the dark, and coverslipped with DAPI-containing mounting medium.+ F4/80+ cells to the total number of F4/80+ cells.Identification of apoptotic cells was performed using TUNEL system Kit (Promega). Briefly, frozen slides were permeabilized using 0.1% solution of Triton X-100 in sodium citrate. Consequently, slides were incubated with TUNEL reaction mixture for 1 hr at 37°C. To allow detection of apoptotic macrophages, frozen sections were incubated with the primary antibodies for F4/80 (1:100) at 4°C overnight and then with TRITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) at room temperature for 1 hr. Nuclei were stained with DAPI (Invitrogen). The apoptotic index of macrophages was assessed as the percentage of apoptotic TUNELFor extracellular staining of immune markers, single-cell suspensions were prepared by mechanical dispersion and enzymatic digestion of tumor tissues as described . BrieflyProtein extracts were were diluted with loading buffer and separated by electrophoresis on 10% SDS-polyacrylamide gels before transfer to nitrocellulose membranes. The membranes were blocked in Odyssey blocking buffer  at room temperature for 1 hr, then incubated at 4°C overnight with primary antibody: Bax (1:500), Bcl2 (1:500), p-Foxo3a (1:800), Foxo3a (1:800), A-caspase3 (1:800) or GAPDH (1:1000). The membranes were washed and incubated with fluorescent secondary antibodies  for 1 hr at room temperature at 1:5000, blots were analyzed with the Odyssey infrared imaging system and Odyssey software.−/− mice as previously described in our lab [2. At 48 hrs after co-culture, peptide gel cultures were fixed in 4% paraformaldehyde in PBS for 15 mins at room temperature and washed for 20 mins in PBS. Frozen sections, 7 μm, of peptide gel cultures were treated with 0.15 M glycine in PBS at 4°C overnight to reduce autofluorescence.Macrophages were isolated from tibias and femurs of 8-week-old WT and CX3CR1 our lab . Three-d our lab . Macroph−/− mice. Matrigel plugs contained 5 × 105 WT or CX3CR1−/− macrophages. Plugs containing PBS were used as a negative control, whereas plug containing 100 ng/ml bFGF (R&D Systems), a known angiogenic factor, was used as a positive control. Each experimental condition was analyzed in triplicate. 7 days after plug implantation, plugs were collected, immediately fixed in 10% buffered formalin. Identification of angiogenesis was performed by immunohistochemistry with the primary antibody against CD31.700 μl of Matrigel (Becton Dickinson) was injected subcutaneously in the ventral area in WT or CX3CR1t test or ANOVA, and results were considered significant at a P<0.05.Data analysis involved use of GraphPad software . Results are expressed as mean ± SEM. Differences were analyzed by TAMs: Tumor-associated macrophages; TGF: Transforming growth factor; VEGF: Vascular endothelial growth factor; CXCR: Chemokine (C-X-C motif) receptor; CCR: C-C chemokine receptor; VEGFR: Vascular endothelial growth factor receptor.The authors declare that they have no competing interest.in vitro experiments. JH and JD were involved in the conception and design of the study as well as in drafting and revising the manuscript. All authors read and approved the final manuscript.JZ and MY equally participated in the design and execution of the overall study. JS carried out the pathologic analysis. YM performed


Background: There is little information describing the perceptions of gastroenterology fellows and attending gastroenterologists of what constitutes effective teaching of endoscopy. We sought to identify common themes regarding endoscopy training methods and their impact among fellows and attendings.Methods: Focus group exercises and surveys were conducted among fellows, about educational resources, teaching techniques and ways of improving the teaching of endoscopy. The fellows identified the ‘best' teachers of endoscopy, who were interviewed regarding their training in endoscopy, their teaching methods, key points of information, and opinions on endoscopy curriculum.Results: Nineteen fellows (68%) had attended the American Society for Gastrointestinal Endoscopy First Year Fellows’ Endoscopy course and found it very helpful. Thirteen fellows(46%) had exposure to an endoscopy simulator, but their median duration of use was only 1 hour. Only two out of five fellowship programs used a formal endoscopic skill assessment tool and none of the programs had an endoscopy curriculum of which the fellows were aware. Fellows reported that they learned endoscopy best by performing procedures. They also volunteered that attending gastroenterologists used variable teaching methods, and might benefit from instruction on how to teach endoscopy. Ten attending gastroenterologists (77%) had received training in advanced procedures; none received formal training on teaching endoscopy: they all felt that such training would be beneficial.Conclusions: A standardized endoscopy curriculum may be beneficial to fellows, who prefer to learn endoscopy by performing procedures—but they want explicit and specific instruction. Both those attending and the fellows thought that formal instruction for attending gastroenterologists on how to teach endoscopy would be beneficial, indicating a role for a ‘teach-the-teacher' curriculum. Throughout gastroenterology fellowship training, fellows are exposed to a variety of different attending physicians who observe and direct them as they manipulate the endoscope ; howeverThis study examined how fellows are taught endoscopy throughout fellowship and the degree of variability in the methods of teaching endoscopy that exists within and between different training programs. We also attempted to identify the methods of teaching endoscopy that are considered to be effective by both fellows and attending gastroenterologists, with the objective of providing this data to instructors and trainees in the discipline; furthermore, we hope to inform further investigation in this area, as well as potential interventions such as a “teach the teacher” curriculum, to improve the teaching of endoscopy.We conducted studies with focus groups of current gastroenterology fellows involved in fellowship programs at several centers in the Boston area, including Boston Medical Center, Tufts Medical Center, Brigham and Women's Hospital, Lahey Clinic and the University of Massachusetts Medical Center. Permission to include fellows in the studies was granted by the gastroenterology program directors at these institutions. Informed consent for the surveys and focus groups was obtained from all participating fellows. The focus group sessions (45–60 minutes in duration) included identical closed- and open-ended questions covering how the fellows learned endoscopy and their perceived needs for additional curriculum (Appendix 1). The fellows were also asked to identify two to three ‘best' teachers of endoscopy at their respective institutions.In addition, the participating fellows were asked to complete a survey assessing their year of training, number of procedures performed, exposure to alternative methods in the teaching of endoscopy (e.g. the ASGE First Year Fellows’ course or simulators) and their exposure to assessment tools for upper endoscopy and colonoscopy (Appendix 2).The attending gastroenterologists identified by the fellows as the best teachers were then interviewed separately by telephone. Informed consent was obtained. The interviews included closed- and open-ended questions identical to those posed to the fellows. Participants were asked a series of questions about their training, teaching styles and overall opinions on the teaching of endoscopy (Appendix 3). All the fellows’ focus group sessions and attending interviews were recorded, transcribed and coded by the investigators. Using a grounded theory approach, a thematic analysis was conducted to define those teaching methods that the attending gastroenterologists identified as being most helpful. We utilized member checking and peer debriefing techniques to increase the validity of our findings. NVivo software  was utilized to assist with the analysis. This study was approved by the Boston University Institutional Review Board.Overall, nine first-year, eight second-year, and eleven third-year fellows participated in this study (28 of 48 possible fellows: 58% participation rate). Each completed the surveys. The survey data from the fellows revealed that 19 fellows (68%) had attended the ASGE First Year Fellows’ course and found it very helpful . Thirteen fellows(46%) had exposure to an endoscopy simulator at their institution; however, the median time spent with the simulator was one hour (IQR 1–3). Only two of the five fellowship programs used a formal endoscopic skill assessment tool and none of the programs had an endoscopy curriculum of which the fellows were aware.The focus groups revealed that the fellows consistently reported learning endoscopy best by performing procedures . They identified procedure-related goals they had set for themselves, such as getting past the pylorus or achieving unassisted cecal intubation. Whilst some fellows felt that such goals were obvious, others wanted attending gastroenterologists to be more explicit in identifying goals for them to achieve, based on their levels of competency. Fellows appreciated specific and explicit instruction, and identified instances where a lack of instruction had led to frustration or delay in developing or mastering certain techniques and concepts. They did not think that they learned differently based on their year of training, although second- and third-year fellows reported a shift in their milestones towards more advanced techniques such as polypectomy. They noted that different attending gastroenterologists had variable teaching methods or styles , which wCertain teaching techniques were noted by the fellows as being less helpful ; for exaFellows consistently agreed on the most effective teachers of endoscopy in their programs and were able to identify three attending gastroenterologists considered “best teachers” at each institution.These attending gastroenterologists comprised one clinical instructor, eight assistant professors, two associate professors, and two full professors, with 9 of13 (69%) performing advanced endoscopic procedures and 10 of13 (77%) reporting having received training in advanced procedures. Common themes from how attending physicians described their teaching styles, included maintaining a positive learning atmosphere identifying specific goals for the fellows during procedures, teaching to the level of the student, and positioning themselves next to the fellow during endoscopy .Table 3Notably, none of the attending gastroenterologists in our study were provided with any formal instruction on how to teach endoscopy, and a majority of these felt that formal teaching would have been helpful during their training. Similarly to the fellows, many of the attending gastroenterologists stressed that an endoscopy curriculum should be flexible, to allow for different styles; furthermore, most attending gastroenterologists felt that if they were to receive instruction on how to better teach endoscopy presently, it would be helpful to them. Comparisons were drawn to the move to improve detection rates for adenomas and that it would be fair to extrapolate more generally that attending gastroenterologists should also receive instruction on other parts of their practice, including how to more effectively teach endoscopy to trainees. Others observed that, while performing endoscopy becomes second nature, this is not necessarily true for effectively explaining or teaching the subject, and that instruction in this skill along with a formal endoscopy curriculum would be helpful.Our findings suggest that gastroenterology fellows are interested in a more detailed endoscopy curriculum. A majority stated that the best way to learn endoscopy is by actually performing procedures. Fellows consistently expressed interest in their instructors being more explicit and specific during endoscopy procedures. Along these lines, we also found that both attending gastroenterologists and fellows indicated that instruction on how to effectively teach endoscopy may be beneficial to those giving training. It was noted by fellows that attending gastroenterologists did not necessarily learn endoscopy in a structured way and therefore may not teach it as such; furthermore, attending gastroenterologists noted that while the practice of endoscopy becomes second nature, the explanation of endoscopy to the learner is not necessarily as intuitive.Given these findings, we believe there is a role for a teach-the-teacher curriculum. This curriculum might be derived from breaking down endoscopic procedures into key portions and skills and then having experts offer tips and detailed explanation on approach and technique. From our interviews with attending gastroenterologists identified by their fellows as “best teachers,” we have compiled a table of key information about the teaching of endoscopy which may be useful teaching tools for other gastroenterologists . Given tWe acknowledge the limitations inherent in our study, including that qualitative research precludes testing of hypotheses. Nonetheless, we consider our findings an important first step towards better understanding how trainees perceive instruction in the practice of endoscopy. We also found that those attending gastroenterologists deemed to be “best teachers” shared similar opinions about which methods work and which should be avoided. Another limitation of our study was lack of 100% participation by every fellow at each participating institution. Because our study included only programs in the Boston area, our findings may not be generally applicable; however, our findings can easily be used to initiate a conversation within other gastroenterology programs that may provide other important insights. As the ACGME moves to a milestone-based means of assessing trainees' progress, we think our findings provide a useful framework for considering the manner in which endoscopy is taught.In conclusion, a standardized endoscopy curriculum with procedural goals may be beneficial to fellows, who prefer to learn endoscopy by performing actual procedures and benefit from receiving explicit and specific instruction from attending gastroenterologists. Attendings and fellows agreed that formal instruction for attending gastroenterologists on how to teach endoscopy would be helpful, yet none of the experienced endoscopists in our study reported receiving specific instruction on how to teach endoscopy, which may indicate a role for a “teach-the-teacher” curriculum. The teaching of 'pearls of wisdom' in endoscopy that we identified, suggest there is, in practice, variability in the teaching of endoscopy techniques which may benefit both instructors and trainees and be a valuable component of endoscopy curriculum.


Ricinus . The collection includes lectotype specimens of Ricinus ivanovi Blagoveshtchensky, 1951 and of Ricinus tugarinovi Blagoveshtchensky, 1951. We registered Ricinus elongatus Olfers, 1816 ex Turdus ruficollis, R. ivanovi ex Leucosticte tephrocotis and Ricinus serratus  ex Calandrella acutirostris and Calandrella cheleensis which were not included in Price’s world checklist. New records for Russia are R. elongatus ex Turdus ruficollis; Ricinus fringillae De Geer, 1778 ex Emberiza aureola, Emberiza leucocephalos, Emberiza rustica, Passer montanus and Prunella modularis; Ricinus rubeculae De Geer, 1778 ex Erithacus rubecula and Luscinia svecica; Ricinus serratus  ex Alauda arvensis. New records for Kyrgyzstan are R. fringillae ex E. leucocephalos and ex Fringilla coelebs. A new record for Tajikistan is R. serratus ex Calandrella acutirostris. The new species Ricinus vaderi Valan n. sp. is described with Calandra lark, Melanocorypha calandra; from Azerbaijan, as a type host.We revised a collection of chewing lice deposited at the Zoological Institute of the Russian Academy of Sciences, Saint Petersburg, Russia. We studied 60 slides with 107 specimens of 10 species of the genus  Ricinus De Geer, 1778 (Phthiraptera: Amblycera) is the largest genus of chewing lice found parasitizing Passeriformes [Ricinus has an anomalous distribution and occurs on approximately one-third of the 70 families of Passeriformes. Major revisions of this genus were done separately for Old World [riformes . Hopkinsriformes  noted thld World  and New Ricinus parasitizing 271 different hosts in 32 families and created 299 host-louse associations. In the years thereafter, Ricinus facetus Mey, 2007, Ricinus gutheili Mey, 2007, Ricinus nhillensis Mey, 2007, Ricinus ornatulus Mey, 2007, Ricinus ptilotulae Mey, 2007 [Ricinus ruficapillus Oniki, Mey, Willis, 2004 [Ricinus arcuatus , Ricinus diffusus , Ricinus fringillae De Geer, 1778, Ricinus invadens , Ricinus marginatus , Ricinus meinertzhageni Rheinwald, 1968, Ricinus mugimaki  and Ricinus pessimalis Eichler, 1956, [Price et al.  listed 6ey, 2007  and Riciis, 2004  were desr, 1956, , 22–25 aRicinus infest more than one-third of the families of Passeriformes (45 of 122), still in accordance with Hopkins [R. fringillae De Geer, 1778 is known from 48 bird species from 9 families [Following host nomenclature according to Clements et al. , lice of Hopkins  statemenfamilies , 19. TheRicinus ivanovi Blagoveshtchensky, 1951 and Ricinus tugarinovi Blagoveshtchensky, 1951; he also mentioned additional records of this genus in the former USSR. Although he noted that Calandrella acutirostris acutirostris Hume, 1873 (Passeriformes: Alaudidae) was a host of Ricinus serratus , this was not cited by Price et al. [R. serratus is already known from several alaudid hosts, this prompted us to inspect material acquired by Blagoveshtchensky. These specimens are deposited at the Zoological Institute of the Russian Academy of Sciences, Saint Petersburg (ZISP) and are part of a larger collection, mainly assembled by him during the 1930s through the 1970s.Blagoveshtchensky  describeRicinus spp. deposited at the ZISP. We include new country records for Azerbaijan, Kyrgyzstan, Russia and Tajikistan. Knowing that data about the biodiversity of chewing lice within the former USSR were published mainly in Russian and that the existing literature is scarcely accessible, we will refer only to those records we have been able to verify. Accordingly, species of Ricinus recorded so far in Azerbaijan are Ricinus frenatus Burmeister, 1838 ex Regulus regulus buturlini and R. fringillae ex Emberiza schoeniclus [Ricinus elongatus  ex Turdus pilaris L., 1758 and Turdus merula L., 1758; R. fringillae ex Fringilla coelebs L., 1758 and Ricinus thoracicus Packard, 1870 ex Plectrophenax nivalis  [Ricinus frenatus  ex Regulus regulus ; R. fringillae ex Fringilla coelebs, Emberiza citrinella L., 1758, Emberiza cia L., 1758 and Prunella collaris ; R. ivanovi ex Leucosticte brandti Bonaparte, 1850; R. serratus ex C. a. acutirostris and Galerida cristata ; R. tugarinovi ex Terpsiphone paradisi  [In this article, we present data obtained from the studies of oeniclus . Records., 1758) , 14. RecRicinus deposited at the ZISP were examined and described. In this article, we present all Ricinus species found in this collection. Host systematics follow Clements et al. [Chewing lice of the genus s et al. . To eachSpecies concept, morphological characters and system of chaetotaxy follow Nelson  see Fig. All meaThe newly described chewing louse species is attributed to the first author.Ricinus mounted on 60 slides and no samples in fixatives were found. The majority of these specimens (78) are females, with only 7 males and 22 nymphs. Specimens had been collected from various locations and by several collectors. Specimens from Tajikistan had mostly been examined and data presented by Blagoveshtchensky [Ricinus sp. from Melanocorypha calandra. As expected, the sex ratio is unbalanced (1–11.14) and our results correspond with Nelson [Ricinus are approximately 10 times more abundant than males.In total, we examined 107 specimens of the genus tchensky . Studiesh Nelson  and litePhthiraptera Haeckel, 1896Amblycera Kellogg, 1896Ricinidae Neuman, 1890Ricinus De Geer, 1778Pediculus dolichocephalus Scopoli, 1763: 382.Oriolus oriolus  – Eurasian Golden Oriole (Oriolidae).Type host: Type locality: NE Poland.ex O. oriolus (Passeriformes: Oriolidae), Eurasian Golden Oriole. Russia, Crimea.Material examined: 1♀ (slide no. 158) R. dolichocephalus in Russia.Remarks: According to literature to which we had access, this is the first record of Nirmus elongatus Olfers, 1816: 88.Turdus viscivorus  – Mistle Thrush (Turdidae).Type host: Type locality: Hodonín, Czech Republic.Material examined: 6♀, 1♂, 1N.ex Turdus ruficollis Pallas, 1776 (Passeriformes: Turdidae), Red-throated Thrush. Russia. Stanoviy khrebet: 9.V.1944.1♀ (slide no. MV01) ex Turdus sp. (Passeriformes: Turdidae), Russia. Okr. Turukhanska: 3♀, 1♂, 1N (slide no. MV02), 30.V.1902, Ostrovskikh P. coll.; Tomskaya gub. Elizavetinsk. zav.: 1♀ (slide no. MV03), 5.VII.1903; no specific location: 1♀ (slide no. MV04), 1907, Haritonov coll.5♀, 1♂, 1N Physostomum mystax (junior synonym of R. elongatus) on T. ruficollis. Balát [T. ruficollis as a host of R. elongatus without examining any material. In revision of Ricinus occurring in the Old World [R. elongatus is known as a parasite of 11 species of birds from four closely related families, and eight of these are from the genus Turdus (Turdidae). Bearing this in mind, we suggest that T. ruficollis be recognized as a valid host for R. elongatus.Remarks: Grube  reporteds. Balát  in his ss. Balát  and inclld World , Rheinwald World . We havePhysostomum frenatum Burmeister, 1838: 442.Type locality: Not recorded (probably Germany).Regulus regulus  – Goldcrest (Regulidae).Type host: Material examined: 10♀.ex R. regulus (Passeriformes: Regulidae), Goldcrest. Tajikistan. Zap. Tigrovaya Balka: 1♀ (slide no. 134), 21.XII.1939; 1♀ (slide no. 135), 3.I.1940; 3♀ (slide no. 136), 10.I.1947, Ivanov A. coll., det. Blagoveshtchensky.5♀, ex Regulus r. buturlini Loudon, 1911 (Passeriformes: Regulidae), Goldcrest (Caucasian). Azerbaijan. Lenkoranskiy r-n d. Alekseevka: 3♀, 10–14.III.1934 (slides no. 130–132), Shtrom Zh. coll., det. Blagoveshtchensky; Sari Island (Caspian Sea): 2♀ (slide no. 133), 14.III.1937, det. Blagoveshtchensky.5♀ Remarks: Specimens were examined and notes were given by Blagoveshtchensky , 3.Ricinus fringillae De Geer, 1778: 71.Emberiza citrinella L., 1758 – Yellowhammer (Emberizidae).Type host: Type locality: Saxony, Germany.Material examined: 30♀, 3♂, 14N.ex Carduelis flammea  (Passeriformes: Fringillidae), Common Redpoll. 17.I.1929.1♀, 4N (slide no. 2410) ex Emberiza aureola Pallas, 1773 (Passeriformes: Emberizidae), Yellow-breasted Bunting. Russia. Okr. n-Tambovska na Amure: 4.VIII.1939, Blagoveshtchensky coll.1♀, 7N (slide no. MV05) ex E. citrinella (Passeriformes: Emberizidae), Yellowhammer. Tajikistan. Dushanbe: 15.II.1947, Ivanov A. coll., det. Blagoveshtchensky.2♀, 1♂ (slide No. 156) ex Emberiza leucocephalos Gmelin, 1771 (Passeriformes: Emberizidae), Pine Bunting. Kyrgyzstan. Naryn: 9.III.1913.1N (slide no. MV06) ex Emberiza rustica Pallas, 1776 (Passeriformes: Emberizidae), Rustic Bunting. Russia. Ural Basseg: 14.VII.1948.2♀ (slide no. MV07) ex Emberiza schoeniclus  (Passeriformes: Emberizidae), Reed Bunting. Azerbaijan. Kumbashi: 28.II.1934, Shtrom Zh. coll., det. Blagoveshtchensky.1♀ (slide no. 155) ex Fringilla coelebs L., 1758 (Passeriformes: Fringillidae), Common Chaffinch.15♀, 1♂, 2N, ussia. Petrodvorec: 3♀ (slides no. MV08–10), VI.1952, Karakasheva coll; Tver’ okr.: 1N (slide no. 139), 18.IV.1907, det. Blagoveshtchensky; and 1♀, 1♂ (slide no. 137), 13.IX.1907, Plotnikov V. coll., det. Blagoveshtchensky.Ryrgyzstan. Naryn: 1♀ (slide no. MV011), the most likely III.1913.Kajikistan. k. Tazhni: 3♀ (slide no. 138), 25.II.1947, Ivanov A. coll., det. Blagoveshtchensky.TNo locality notes: 1♀ (slide no. MV12).ex F. coelebs (Passeriformes: Fringillidae), Common Chaffinch. Tsikhis-Dzhivari: 1935.Locality: 6♀, 1N (slide no. 2575) ex Passer montanus  (Passeriformes: Passeridae), Eurasian Tree Sparrow. Russia. Vologda: 1947.3♀ (slide no. MV13) ex Prunella collaris  (Passeriformes: Prunellidae), Alpine Accentor. Tajikistan. Gissarskiy khrebet pereval Anzob: VIII.1945, Ivanov A. coll., det. Blagoveshtchensky.1♀, 1♂ (slide no. 154) ex Prunella modularis  (Passeriformes: Prunellidae), Dunnock. Russia. Kavkaz: 1917, Satunin coll.3♀ (slide no. MV14) ex Turdus sp. (Passeriformes: Turdidae). Ukraine. Kiev: 3.IV.1902.1♀ (slide no. MV15) R. fringillae in Russia are Emberiza aureola, E. leucocephalos, E. rustica, P. montanus and P. modularis.Remarks: According to literature to which we had access, new host records for Ricinus ivanovi Blagoveshtchensky, 1951: 283.Leucosticte brandti Bonaparte, 1851 – Black-headed Mountain-Finch (Fringillidae).Type host: Type locality: Vewrk, Gissar’s Range, Pamir, Tajikistan.ex L. brandti (Passeriformes: Fringillidae), Black-headed Mountain-Finch. Tajikistan. Gissarskiy khrebet pereval Anzob: 19.VIII.1945, Ivanov A. coll., det. Blagoveshtchensky.Material examined: 1♀ (slide no. 152) R. ivanovi on 1♀. Unfortunately, Blagoveshtchensky did not provide a slide number. Neither Rheinwald [R. ivanovi. Nelson [R. ivanovi ex Leucosticte tephrocotis  and none from L. brandti. Nelson’s record was included by Emerson [Remarks: Blagoveshtchensky  based hiheinwald  nor Nelsheinwald  examined. Nelson  examined Emerson  in a che Emerson .Pediculus rubeculae Schrank, 1776: 115.Erithacus rubecula  – European Robin (Muscicapidae).Type host: Type locality: Moravia, Czech Republic.Material examined: 3♀.ex E. rubecula (Passeriformes: Muscicapidae), European Robin. Russia. Teberda: 13.VI.1917, Gorbunov G. coll.2♀ (slides no. MV16–17) ex Luscinia svecica  (Passeriformes: Muscicapidae), Bluethroat. Russia. Kazalinsk: 15.VI.1932, Popov coll.1♀ (slide no. MV18) R. rubeculae in Russia.Remarks: According to literature to which we had access, this is the first record of Physostomum serratum Durrant, 1906: 528.Eremophila alpestris  – Horned or Przewalski’s Lark (Alaudidae).Type host: Type locality: Ft. Collins, Colorado, USA.Material examined: 18♀, 3♂, 4N.ex Alauda arvensis  (Passeriformes: Alaudidae), Sky Lark. Russia. Verhojanskij okrug: 2.VI.1927, Tkachenko coll.1♀ (slide no. MV19) ex Calandrella acutirostris acutirostris Hume, 1873 (Passeriformes: Alaudidae), Hume’s Lark. Tajikistan. Dushanbe okr.: 18.X.1946, Ivanov A. coll., det. Blagoveshtchensky.2♀ (slide no. 151) ex Galerida cristata  (Passeriformes: Alaudidae), Crested Lark. Tajikistan. Zap. Tigrovaya Balka: 1N (slide no. 143), 29.IV.1934, and 13♀, 2♂, 4N , 9.II.1941, Sosnina E. coll., det. Blagoveshtchensky; Kirovobad: 1♀, 1♂ (slide no. 150), 29.VI.1932, Shtrom Zh. coll., det. Blagoveshtchensky; Saray-Kamar: 1♀ (slide no. 149), 29.VI.1932, Pospelova M. coll., det. Blagoveshtchensky.15♀, 3♂, 4N, C. a. acutirostris and G. cristata in Tajikistan have been recorded [R. serratus ex C. a. acutirostris was not provided in the checklist by Price et al. [Ricinus serratus is known from 14 species of birds from three families of which nine species belong to the family Alaudidae [Calandrella and besides C. a. acutirostris, the records for Calandrella cheleensis  recorded by Mey [R. serratus are easily distinguishable from other Ricinus species by the presence of unique serrated pleural nodi. We suggest C. a. acutirostris and C. cheleensis be considered as valid hosts of R. serratus.Remarks: Chewing lice on recorded . The rece et al. . Ricinuslaudidae . The worlaudidae  includedd by Mey  were misd by Mey  and thatRicinus subdiffusus Nelson, 1972: 97.Spizella passerina  – Chipping Sparrow (Emberizidae).Type host: Type locality: Hopland Field Station, Mendocino Co., California, USA.ex Emberiza sp. (Passeriformes: Emberizidae). Russia. Chukot. p-ov, Lavrentiya: 28.VII.1948, Ljobin coll.Material examined: 1♀ (slide no. MV20) Emberiza. Having just one specimen and doubtful collection techniques in the past, we suggest that R. subdiffusus not be considered as a parasite of Emberiza at this time.Remarks: Although this species infests members of the family Emberizidae, we have found no previous records of it infesting birds of the genus Nirmus thoracicus Packard, 1870: 94.Plectrophenax nivalis  – Snow Bunting (Emberizidae).Type host: Type locality: Not recorded, USA.ex P. nivalis (Passeriformes: Emberizidae), Snow Bunting. Russia. o-v Vrangelya b. Rodzhersa: 7.V.1939, Portenko coll., det. Blagoveshtchensky, slide no. 157.Material examined: 1♀ (slide no. 157) Remarks: This specimen was examined and mentioned by Blagoveshtchensky .Ricinus tugarinovi Blagoveshtchensky, 1951: 286.Terpsiphone paradisi  – Asian Paradise-Flycatcher (Monarchidae).Type host: Type locality: Kondara, Tajikistan.ex T. paradisi (Passeriformes: Monarchidae), Asian Paradise-Flycatcher. Tajikistan. Kondara: 6.VI.1944, Blagoveshtchensky coll., det. Blagoveshtchensky.Material examined: 1♀ (slide no. MV153) R. tugarinovi on 2♀, but he did not provide a slide number for the type specimen. Subsequently, Rheinwald [Ricinus, re-described R. tugarinovi without examining the type material. Since the specimen examined in this study shares notes about locality and date with two specimens examined by Blagoveshtchensky [Remarks: Blagoveshtchensky  based hiheinwald , in his tchensky , and inaurn:lsid:zoobank.org:act:F18FF85A-2A81-4CE1-8A47-4CE5E4816708Melanocorypha calandra  – Calandra Lark (Alaudidae).Type host: ex M. calandra (Passeriformes: Alaudidae), Calandra Lark. Azerbaijan. Kaspiy r. Viljash-Chay: 17–18.IV.1937, Ivanov coll. Collection of chewing lice of the Zoological Institute of the Russian Academy of Sciences, Saint Petersburg, Russia (ZISP)  and paratype ♀ with 1N (slide no. MV22) a (ZISP) .R. vaderi and Darth Vader’s helmet.Etymology: This species name is derived from Darth Vader, a fictional character in the Star Wars trilogy. The first author’s fiancée noticed a similarity between the head of the Authorship: Note that the authors of the new taxon are different from the authors of this paper; Article 50.1 and Recommendation 50A of the International Code of Zoological Nomenclature .n = 2). Head wider than longer, with characteristic shape as . Two tactile setae on coxa I. q2–q4 large and spinose, q4 sometimes absent; w series composed of four setae with anterior two spinose and twice as large as posterior two pilose setae; c1 spinose and larger than c2; c2–c4 pilose. Sternal plate as in Thorax . Setae LRicinus pattern, two setae with inner less than half the length of the outer. One pair of long setae on sternite VIII and one vulval seta. Setae in anal fringe: ADF, 41–42; AVF, 42–43. Ventral pleural chaetotaxy as in Terminal segments of abdomen as in Dimensions as follows: total length = 3.73–3.82; total width = 1.11; head length = 0.72–0.73; head width = 0.83; HI = 86–87 (head index = ratio of head length to head width × 100); labrum width = 0.36–0.37; prothorax length = 0.39–0.40; prothorax width = 0.73–0.75; distance between prosternal setae = 0.039–0.040.R. vaderi have ornamented ovoid sclerites, present only in the diffusus, serratus and subangulatus species groups. Ricinus vaderi cannot be placed into any of these groups. In the subangulatus species group, the frontal margin is continuous with that of marginal carinae, but not in R. vaderi n. sp. or the diffusus and serratus species groups. Ricinus vaderi n. sp. has a characteristic head shape with broadly concave lateral head margins; the head is wider than the length (HI = 86–87); setae along the antennal lappets are reduced with diastoma; the setal pattern on the terminal tergite iiIIi × iIIii. In the diffusus species group, the lateral margin is almost straight; HI is 100–110, with the exception of R. thoracicus (HI = 93); there is no reduction or diastoma along the antennal lappets and the pattern on terminal tergite iIIim × miIIi (where “m” means moderately long seta). Ricinus serratus, the only member of its group, has serrated pleural nodi, the head has a postfrontal constriction, the gular plate has no posterior extensions, the a1 setae are long and there is one tactile seta on coxa I.Diagnosis: Our specimens of Ricinus vaderi n. sp. is the second Ricinus species found on larks (Alaudidae).Remarks: ex Alauda arvensis  (Passeriformes: Alaudidae), Sky Lark. Russia. 1904, Haritonov coll.Material examined: 2♀ (slide no. MV23) R. serratus, the only known Ricinus from this host species, in not having a laterally positioned, serrated structure on the pleurites.Remarks: The condition of these specimens is poor and we were not able to identify them. These two specimens differ from ex Leucosticte brandti (Passeriformes: Fringillidae), Black-headed Mountain-Finch. Kyrgyzstan. Naryn: 3.III.1913.Material examined: 2N (slide no. MV24) R. ivanovi, a known Ricinus from this host species. In contrast to R. ivanovi, the heads of these nymphs are wider than the length and have broadly concave lateral margins.Remarks: These specimens are not identified to species level because of their poor condition, but they are not ex Buteo lagopus  , Rough-legged Hawk. Russia. Okhotsk: 25.V.1936Material examined: 1♀ (slide no. MV25) ex Strix aluco harmsi  , Tawny Owl (Turkestan). Russia. 17.V.1947, Ivanov coll.1♀ (slide no. MV26) Ricinus spp. 3 and 4, whose specimens differ considerably from each other probably represent two different species. They are considered as contaminants (stragglers).Remarks: Ricinidae are parasites of songbirds (Passeriformes) and hummingbirds (Apodiformes: Trochilidae); hence, Ricinus among his records. Exceptions are specimens of R. fringillae ex E. cia reported in Tajikistan [Ricinus vaderi n. sp. ex Melanocorypha calandra, and include previous records [Ricinus species in this country to three. We found only a single paper about chewing lice from Kyrgyzstan published by Kravtsova [Ricinus. Our records represent the first findings of this genus in Kyrgyzstan. In addition to records of Ricinus thoracicus Packard, 1870 on Wrangel Island [Ricinus elongatus from two hosts and R. fringillae [R. elongatus ex Turdus ruficollis, R. ivanovi ex Leucosticte tephrocotis, R. serratus on Calandrella acutirostris and C. cheleensis was not cited in Price et al.’s world checklist [R. tugarinovi and R. ivanovi.The collection of chewing lice at the Zoological Institute of the Russian Academy of Sciences, Saint Petersburg, Russia was mainly assembled by Blagoveshtchensky in the 1930s through the 1970s. He published several surveys on the biodiversity of chewing lice in the former USSR –4. We ex records , bringinravtsova , who didingillae  in CentrRicinus in a restricted sense, parasitizing only Passeriformes despite the recent study by Rheinwald [Trochiliphagus with Ricinus. In his revision of the genus Trochiliphagus, he declared that all 12 known species of the genus Trochiliphagus should be one single species Ricinus jimenezi.We consider the genus heinwald  who synoTrochiliphagus specimens from the same host, and did not examine any of the type material. This leaves some open questions: 1. Is there indeed only one species of Trochiliphagus/Ricinus parasitizing hummingbirds or more, regardless of the correct genus? 2. Is the status of the genus Trochiliphagus valid or is it indeed part of the genus Ricinus? 3. Perhaps the genus Ricinus occurring on Passeriformes could also be divided into several genera since some species groups of this genus  are more similar to those of Trochiliphagus from hummingbirds than to other species groups from passerine birds . As Rheinwald noted, application of modern enzymatic and DNA techniques may provide the answers. Therefore, until proper DNA studies are performed or at least morphological investigations of more specimens from different hummingbird species and of course of type material for the genus Trochiliphagus, we consider Rheinwald’s hypothesis is insufficiently supported and suggest resurrection of the name Trochiliphagus.Rheinwald based his study on investigation of only two