Efforts to unravel the mechanisms underlying taste sensation (gustation) have largely focused on rodents. Here we present the first comprehensive characterization of gene expression in primate taste buds. Our findings reveal unique new insights into the biology of taste buds. We generated a taste bud gene expression database using laser capture microdissection (LCM) procured fungiform (FG) and circumvallate (CV) taste buds from primates. We also used LCM to collect the top and bottom portions of CV taste buds. Affymetrix genome wide arrays were used to analyze gene expression in all samples. Known taste receptors are preferentially expressed in the top portion of taste buds. Genes associated with the cell cycle and stem cells are preferentially expressed in the bottom portion of taste buds, suggesting that precursor cells are located there. Several chemokines including CXCL14 and CXCL8 are among the highest expressed genes in taste buds, indicating that immune system related processes are active in taste buds. Several genes expressed specifically in endocrine glands including growth hormone releasing hormone and its receptor are also strongly expressed in taste buds, suggesting a link between metabolism and taste. Cell type-specific expression of transcription factors and signaling molecules involved in cell fate, including KIT, reveals the taste bud as an active site of cell regeneration, differentiation, and development. IKBKAP, a gene mutated in familial dysautonomia, a disease that results in loss of taste buds, is expressed in taste cells that communicate with afferent nerve fibers via synaptic transmission. This database highlights the power of LCM coupled with transcriptional profiling to dissect the molecular composition of normal tissues, represents the most comprehensive molecular analysis of primate taste buds to date, and provides a foundation for further studies in diverse aspects of taste biology. Taste is fundamental for the selection of nutritious foods and rejection of poisonous or harmful substances Macaca fascicularis). A close relative to humans that diverged 25 million years ago, the macaque represents a model system for human physiology, prefers a similar omnivorous diet, and shares an overall 93% genomic sequence identity (97.5% identity in orthologous genes). Sequencing of the macaque genome was recently completed Characterization of gene expression in mammalian taste buds has largely been limited to rodents. Here, we report the results of a systematic and comprehensive survey of gene expression in taste buds isolated from a primate, the cynomolgus macaque  To identify taste bud-associated genes, we compared gene expression between isolated taste buds and lingual epithelium lacking taste buds. Since taste cells are specialized neuroepithelial cells, we focused on genes expressed at significantly higher levels in taste buds versus adjacent non-gustatory lingual epithelium. This allowed us to eliminate genes expressed in both locations and to identify genes involved in pathways and processes specific to taste.In the first set of microarray experiments, we generated gene expression data from ten lingual epithelium (LE) samples and ten taste bud (TB) samples - six from FG papilla and four from CV papilla. All CV samples were collected from macaques also used to generate FG samples. We collected 4,700–6,400 estimated cells per sample that yielded sufficient total RNA for use in our GeneChip analyses see . Cell nuDuring histological analyses of a set of TB-associated genes identified from this first microarray set, we discovered that certain transcripts were specifically enriched in cells located at the bottom of the taste bud, suggesting divergent gene expression between the top and bottom portions of taste buds. This observation prompted us to collect a second set of LCM samples isolated from either the top (n = 4) or bottom (n = 4) fractions of CV TB . These st-test). The pairwise comparisons were: CV TB versus LE, FG TB versus LE, CV TB top versus LE, and CV TB bottom versus LE. Finally we selected probe sets representing genes significantly expressed at specific sites using the following inclusion values: expression ratio ≥5-fold, p value ≤0.05 to generate a TB-associated probe set list. The list of TB-associated probe sets was then extensively annotated by reference to the human genome and RefSeq database. Database annotation included: probe set ID, gene title, gene symbol, pairwise comparisons with corresponding p-values, known or predicted location of gene product, and functional classification of gene product. Finally, redundant probe sets were removed resulting in a database that contained 2,382 TB-associated genes. The complete TB-associated gene database is shown in TB-associated genes were identified in a multi-step process. First, we eliminated poorly performing probe sets (those with a mean intensity ≤25 in TB samples). Then using a series of pairwise comparisons between different sample sets we calculated fold change and p values (Student's Humans have ∼30 known taste receptor/candidate taste receptor genes: TAS1R1 (umami), TAS1R2 (sweet), TAS1R3 (umami and sweet co-receptor); PKD2L1 and PKD1L3 (candidate sour), and 25 TAS2Rs (bitter). The Affymetrix Rhesus macaque array contains probe sets corresponding to 26 of these genes, summarized by their TB versus LE expression ratios in We sought to identify genes expressed exclusively or predominantly in the bottom or top portions of CV TB. A total of 159 TB-associated genes exhibited intra-TB expression differences : 79 genes in the CV TB bottom fraction  and 80 gGenes with the highest differential expression in TB relative to LE were identified. Next, we identified genes expressed exclusively or predominantly in CV but not FG taste buds and vice versa. A total of 54 TB-associated genes were site-specifically expressed : 23 genes in CV TB  and 31 gTo better classify and organize the large list of genes identified, we annotated all genes in the database to assign a primary function and tabulated the results . Five fuWe identified 90 neuronal-associated genes including many not previously associated with taste buds . Expressin situ hybridization in macaque CV papillae (Identification of a large group of genes associated with the immune system (n = 43) was both interesting and surprising. Multiple chemokine and cytokine genes are expressed in taste buds. CXCL14 represents the most highly expressed TB-associated gene . CXCL14 papillae  which shGenes in this functional class (n = 27) were defined as those expressed at sensory sites distinct from the taste bud including the olfactory epithelium (n = 4), ear (n = 4), eye (n = 13), and multiple sensory sites (n = 6). SLIT and NTRK-like family, member 6 (SLITRK6), is expressed at multiple sensory sites  during mouse development in conjunction with leucine rich repeat neuronal 3 (LRRN3) in situ hybridization, IKBKAP was found selectively expressed in taste cells that express PKD1L3 in macaque CV taste buds  . Mutatioste buds .in situ hybridization, we determined that KIT is expressed in a subset of TRPM5 cells   . By labee cells) .Genes encoding both peptide hormones (n = 7) and hormone receptors (n = 5) are abundantly expressed in taste buds. In some cases, ligand/receptor pairs such as growth hormone releasing hormone and its receptor (GHRH/GHRHR) and growth hormone 1 and its receptor (GH1/GHR) are co-expressed and exhibit similar expression profiles. Taste buds also express proopiomelanocortin , parathyroid hormone-like hormone (PTHLH), and oxytocin (OXT) as well as receptors for insulin (INSR) and secretin (SCTR). Other endocrine-associated genes encode either receptors  or ligands .We used a systematic approach to generate a high quality database of primate taste bud gene expression. The database represents the first genome-wide survey of gene expression in taste buds from a primate (cynomolgus macaque) and is a significant milestone in defining the molecular components underlying the processes of taste. The limited abundance and difficulty in isolating primate taste buds devoid of contaminating lingual epithelial cells that has hindered molecular analysis of primate taste cell gene expression was overcome by using the macaque as a source of tissue and LCM as the method of tissue collection. Two additional factors enabled accurate, sensitive and comprehensive transcriptional profiling of primate taste buds: rapid tissue collection resulting in minimal post-mortem RNA degradation, and the recent development of a genome-wide microarray specific for the macaque.Our results demonstrate the power of transcriptional profiling of microdissected tissue to better understand the components and pathways active in that tissue. While these techniques have previously been applied primarily to diseased and pathogenic tissues Spatial patterns of gene expression.We observed region-preferential expression of taste receptors in macaque taste buds. TAS1R1 was expressed at higher levels in FG TB and T2Rs were expressed at higher levels in CV TB, similar to rodents Our data has revealed the dynamic nature of taste buds. During development, taste buds arise from epithelium in a complex process involving a variety of factors including SHH, PTCH1, bone morphogenic proteins and neurotrophic factors in situ hybridization data indicate that KIT is expressed exclusively in TAS1R1 (umami) cells. While the functional role of KIT in taste cells has yet to be determined, KIT may modulate umami cell differentiation and development. Indeed, patients treated with the KIT antagonist imatinib mesylate (Gleevec) reported alterations in taste perception The tyrosine kinase receptor KIT controls stem cell survival and development in bone marrow, skin, and gut Mutations in IKBKAP cause familial dysautonomia, a disease that leads to loss of taste buds and nerve fibers innervating taste buds KIT and IKBKAP are representative of many genes we found expressed in taste cells dedicated to distinct taste modalities. This finding supports the labeled line model of taste coding in the periphery In addition to factors expressed within taste cell progenitors, taste buds require nerve innervation to survive. In this context, the presence of several genes associated with axonal growth and guidance including semaphorins  Despite originating from epithelium, taste buds express genes associated with neurons and central nervous system development, maintenance, and function. The achaete-scute complex-like 1 (ASCL1 or MASH1) is highly expressed in primate taste buds and participates in commitment and differentiation of distinct neuronal cell types including sensory neurons Several genes associated with the immune system are present in the taste bud database. We did not detect significant expression of genes encoding leukocyte markers such as immunoglobulins, T cell receptor-associated, or myelomonocytic-associated proteins in taste buds. Furthermore, we did not observe leukocyte infiltrates in taste bud sections, implicating taste cells as the origin of immune gene transcripts. Also, genes were selected because they were expressed in taste buds and not significantly expressed in adjacent LE. The taste bud represents a potentially favorable site for pathogen entry via the taste pore. Interestingly, the chemokine CXCL14 is the highest expressed taste-bud associated gene in our database. While the receptor for this chemokine is unknown, it is broadly expressed in adult tissues  and has been reported to be a chemoattractant for dendritic cells and monocytes Expression of complement components  may also represent chemotactic signals to the taste pore. Local production of these factors as well as cytokine receptors of the interferon-signaling cascade by taste bud cells Expression of hormone receptor and ligand transcripts, normally associated with endocrine glands, within taste buds adds to a growing body of evidence for a cephalic response to feeding. It is well known that plasma insulin levels rise rapidly following ingestion of a carbohydrate rich meal and prior to the subsequent rise in plasma glucose levels Our database of taste bud gene expression will open new lines of investigation and lead to a better understanding of taste bud physiology in normal and diseased states. Of considerable interest are the 349 TB-associated genes in the database with unknown function. Analysis of these and other functional classes will reveal additional interesting pathways and processes active in taste buds. We have confirmed the expression of most genes of interest described in this study in human taste buds isolated by LCM, thereby confirming the macaque gene array findings. In addition, we are currently mapping more genes to specific taste cell populations and have identified new markers of known taste cell types as well as markers that define new taste bud cell types. Some of these markers may potentially define additional types of cells that may mediate novel taste modalities. Because the database contains most TB-associated genes, genes for novel taste receptors should be represented. Indeed, we have used the database to identify novel, candidate taste receptors. The identification of growth factors and growth factor receptors in taste buds raises the possibility of developing therapies to increase the survival and replenishment of taste bud cells. Since taste sensation declines with age Macaca fascicularis; 2.6–4.5 years old; males and females) was collected post-mortem by Charles River  and Covance  from animals scheduled for euthanasia for other purposes. Tissue was obtained with a post-mortem interval of 10 minutes, embedded in OCT freezing medium , and frozen in liquid nitrogen. Human tongue samples were purchased from Zoion Diagnostics  as OCT embedded blocks and stored at −80°C. Human taste tissue was obtained from Caucasian post-mortem donors less than 30 years of age, who were non-smokers with no known alcohol or drug use, with a post-mortem interval less than 5 hours.All primate samples were collected in compliance with applicable federal, state, and local laws and regulations (CFR 1985 and PHS 1996) according to IACUC recommendations and oversight at both Charles River and Covance. All human samples were collected with full written consent and with the approval of the Zoion Diagnostics institutional review board (IRB), an external independent IRB and the IRB or Human Studies Committee (HSC) at the organizations directly involved with the collection for final approval. Taste tissue from cynomolgus macaques (LCM was used to isolate taste tissue from macaque and human samples. Tissue sections (10–12 um thick) were cut on a Leica CM1850 cryostat, collected on RNase-free membrane slides , and stained with cresyl violet using the Ambion LCM staining kit  as per the manufacturer's instructions. Taste bud and lingual epithelial areas were isolated using a MMI Cellcut laser microdissection system on an Olympus IX71 inverted microscope and collected on MMI reaction tube adhesive lids. For macaque, both CV and FG papillae were used; for human, only CV papillae were used. Material from multiple sections was pooled, and all LCM samples were collected within 2 hr of sectioning. One paired TB pool and LE pool were collected per donor from multiple donors. Following collection, total RNA from taste bud and lingual areas was separately purified using a Qiagen microRNeasy kit  and evaluated using an Agilent 2100 Bioanalyzer with a Series II RNA 6000 Pico Assay. Total RNA was amplified to generate cDNA using the WT Ovation Pico system . Microarray gene expression data was generated from the macaque samples by Gene Logic using Affymetrix GeneChip® Rhesus Macaque Genome Arrays . All microarray experiments in this study were done in accordance with MIAME guidelines and the gene expression data sets presented have been deposited in the GEO database (GEO series entry GSE16485). Semi-quantitative PCR expression data was generated from amplified human cDNA samples using the Mx3000P® QPCR System  and inventoried TaqMan assays .t-tests. Genes were defined as TB-associated if they met the following inclusion criteria: minimum mean TB expression ≥25, fold expression difference (TB versus LE) ≥5 with a p value ≤0.05. Genes were selected from each set of pairwise comparisons to give 4 lists  that were subsequently combined to give a single non-redundant master list.Microarray data was analyzed using GeneSpring GX software . Raw data from the Rhesus Macaque Genome Arrays were processed using GC-RMA normalization Fresh frozen sections (10–12 µm thick) were attached to RNase-free SuperFrost Plus slides  and processed for in situ hybridization as described Figure S1Principal component analysis of microarray data.(7.92 MB TIF)Click here for additional data file.Table S1Microarray sample information.(0.02 MB XLS)Click here for additional data file.Table S2Macaque taste bud gene expression database of 2,382 genes.(0.74 MB XLS)Click here for additional data file.Tabe S3Genes associated with the bottom fraction of circumvallate taste buds.(0.03 MB XLS)Click here for additional data file.Table S4Genes associated with the top fraction of circumvallate taste buds.(0.03 MB XLS)Click here for additional data file.Table S5Representation of 2,382 taste bud-associated genes by functional classification.(0.02 MB XLS)Click here for additional data file.


Permethrin is the active component of topical creams widely used to treat human scabies. Recent evidence has demonstrated that scabies mites are becoming increasingly tolerant to topical permethrin and oral ivermectin. An effective approach to manage pesticide resistance is the addition of synergists to counteract metabolic resistance. Synergists are also useful for laboratory investigation of resistance mechanisms through their ability to inhibit specific metabolic pathways.Sarcoptes scabiei variety canis was noted when any of the three synergists were used in combination with permethrin compared to median survival time of mites exposed to permethrin alone (p<0.0001). Incubation of mite homogenates with DEF showed inhibition of esterase activity (37%); inhibition of GST activity (73%) with DEM and inhibition of cytochrome P450 monooxygenase activity (81%) with PBO. A 7-fold increase in esterase activity, a 4-fold increase in GST activity and a 2-fold increase in cytochrome P450 monooxygenase activity were observed in resistant mites compared to sensitive mites.To determine the role of metabolic degradation as a mechanism for acaricide resistance in scabies mites, PBO (piperonyl butoxide), DEF  and DEM  were first tested for synergistic activity with permethrin in a bioassay of mite killing. Then, to investigate the relative role of specific metabolic pathways inhibited by these synergists, enzyme assays were developed to measure esterase, glutathione S-transferase (GST) and cytochrome P450 monooxygenase (cytochrome P450) activity in mite extracts. A statistically significant difference in median survival time of permethrin-resistant These findings indicate the potential utility of synergists in reversing resistance to pyrethroid-based acaricides and suggest a significant role of metabolic mechanisms in mediating pyrethroid resistance in scabies mites. Sarcoptes scabiei variety canis was noted when any of the three synergists were used in combination with permethrin compared to survival time of mites exposed to permethrin alone (p<0.0001). These results indicate the potential utility of synergists in reversing tolerance to pyrethroid-based acaricides . To further verify specific metabolic pathways being inhibited by these synergists, enzyme assays were developed to measure esterase, glutathione S-transferase (GST) and cytochrome P450 monooxygenase activity in scabies mites. Results of in vitro enzyme inhibition experiments showed lower levels of esterase activity with DEF; lower levels of GST activity with DEM and lower levels of cytochrome monooxygenase activity with PBO. These findings indicate a metabolic mechanism as mediating pyrethroid resistance in scabies mites.Synergists are commonly used in combination with pesticides to suppress metabolism-based resistance and increase the efficacy of the agents. They are also useful as tools for laboratory investigation of specific resistance mechanisms based on their ability to inhibit specific metabolic pathways. To determine the role of metabolic degradation as a mechanism for acaricide resistance in human scabies, PBO (piperonyl butoxide), DEF  and DEM  were used with permethrin as synergists in a bioassay of mite killing. A statistically significant difference in survival time of permethrin-resistant  Sarcoptes scabiei. The mite lives in the skin of hosts where it passes through a series of life stages . A severe manifestation of the disease i.e., crusted scabies can occur which may predispose to streptococcal pyoderma and the subsequent development of acute post streptococcal glomerulonephritis, acute rheumatic fever and rheumatic heart disease in vitro acaricide sensitivity has demonstrated increased tolerance to permethrin of scabies mites collected from indigenous communities across northern Australia (unpublished data).Scabies is an infectious skin disease caused by the ectoparasite, Pyrethroids constitute one of the most important classes of insecticide, accounting for 17% of the world insecticide market Blattella germanica, pyrethroid resistance has been demonstrated to be mediated by multiple mechanisms including target site insensitivity, decreased cuticular penetration, and enhanced metabolism by monooxygenases and esterases kdr gene and metabolic degradation by esterases have both been identified as mediators of pyrethroid resistance Myzus persicae has been shown to be conferred by both mutations in the kdr gene and hyperproduction of esterases Various resistance mechanisms such as behavioural (avoidance of treated surfaces), physiological (reduced penetration and/or increased excretion of insecticides), reduced sensitivity of target site  and metabolic degradation (by hyperproduction of enzymes) mediate pesticide resistance of arthropods. Target site insensitivity and metabolic degradation have been demonstrated to play major roles in conferring resistance to pyrethroids in some arthropods. In the German cockroach, Pediculus humanus capitis) to counteract resistance. Examples of over-the –counter treatment products for headlice are: Pronto (33% pyrethrins and 4% PBO) , RID (33% pyrethrins and 4% PBO) , A-200 (33% pyrethrins and 4% PBO)  available in the US and BanLice Mousse (pyrethrin 1.65 mg and 16.5 mg PBO) (Pfizer) available in Australia An effective way to manage pesticide resistance is the coformulation of synergist with the pesticide to counteract metabolic resistance. Synergists act by blocking metabolic pathways that would otherwise break down pesticides, thus restoring susceptibility to the agent. Piperonyl Butoxide (PBO) is co-formulated with insecticides such as carbaryl, methomyl, fenvalerate, permethrin, parathion, malathion and dimethoate; S,S,S-tributyl phosphorotrithioate (DEF) is co-formulated with malathion and permethrin; and Diethyl maleate (DEM) is co-formulated with parathion, malathion and dimethoate Apis mellifera, to pyrethroids is largely reversed by PBO and at low levels by DEF, suggesting a significant role of cytochrome P450s and a lesser role of esterases in detoxification of the chemical Helicoverpa armigera) were shown to be inhibited by PBO Blatella germanica, PBO and DEF were tested as synergists to propoxur (a carbamate insecticide). Both were shown to inhibit cytochrome P450 monooxygenases at different levels In addition, insecticide synergists have been used to investigate resistance mechanisms and to identify the specific metabolic pathways targeted. Tolerance of honey bees, kdr gene associated with permethrin resistance in a population of scabies mites Sarcoptes scabiei var canis was used to investigate the role of metabolic degradation as a contributory mechanism to observed permethrin resistance. PBO, DEF and DEM were tested as synergists to permethrin to determine if susceptibility to the acaricide can be restored, and to explore their activity in inhibiting specific metabolic pathways.Previously, we identified a single nucleotide polymorphism (SNP) in the Sarcoptes scabiei) were collected from two sources: permethrin naïve mites (Sarcoptes scabiei var. suis) were collected from a colony maintained on pigs  while permethrin resistant mites (Sarcoptes scabiei var. canis) that had been maintained under permethrin treatment for many years were collected from rabbits Live scabies mites ; Diethyl Maleate ; and S,S,S tributylphosphorotrithioate . All other chemicals and reagents used in enzyme assays were purchased from Sigma.Reagents used in the bioassays were: 5% Permethrin ; Benzyl Benzoate ; and mineral oil (Sigma).In preliminary experiments, a small number of mites (30/synergist) were exposed to each synergist alone  to determine maximum concentration that the mites could tolerate and remain alive for 24 hours (data not shown). The result of this preliminary experiment led us to select a concentration (30 mM) of each synergist for use in subsequent bioassays.Acaricide bioassays were performed on live scabies mites as previously described Scabies mites were homogenized on ice in 100 ul 0.02M phosphate buffer pH 7.0 (esterase assay), 0.05M Tris-HCl buffer pH 7.5 (GST assay) or 0.1M sodium phosphate buffer pH 7.6 (cytochrome P450 assay), using a hand-held battery operated homogenizer . After homogenization, the tubes were spun at 13,000×g at 4°C for 5 minutes. Mites used for cytochrome P450 assay were spun at 1000×g at 4°C for 15 mins. The protein concentration of the lysate was determined using the Nanodrop ND-100 Spectrophotometer . Mite homogenates used in all enzyme assays were normalized to 2 mg/ml protein concentration.Twenty microliters of mite homogenate supernatant was incubated with varying dilutions  of enzyme inhibitors  for 30 minutes at room temperature. Enzyme assays were performed as outlined below:Esterase activity was determined using alpha-napthyl acetate as the substrate which forms a diazo-dye complex with Fast Blue RR Salt. Colorimetric formation of the complex is directly proportional to esterase activity and is measured continuously in a kinetic assay as described by Gunning et al, 1996 To mite homogenate (with and without inhibitor) set up in triplicate, 200 ul of substrate  was added. Change in absorbance was measured at 450 nm at 14 second intervals for 10 minutes in a kinetic microplate reader . As an assay control, esterase from rabbit liver, diluted 1∶10 ((0.10 mg/ml) and 1∶100 (0.01 mg/ml) was included in each experiment.GST activity was measured by using monochlorobimane (MCB) as the substrate which forms a stable fluorescent conjugate with reduced glutathione. The enzymatic conversion of MCB to bimane-glutathione is measured in an endpoint fluorescence assay as described by Nauen and Stumpf, 2002 To mite homogenate (with and without inhibitor) set up in triplicate, 200 ul of substrate (3 mg reduced glutathione dissolved in 10 ml of 0.05M Tris-HCl buffer pH 7.5+500 ul of 1.5 mg/ml of monochlorobimane (MCB) in methanol) was added. Mite homogenate and substrate were incubated for 20 minutes at room temperature. Fluorescence intensity was read on a microplate reader (Polarstar) with 465 Emission and 390 Excitation filters. As an assay control, GST from equine liver, diluted 1∶10 (0.10 mg/ml) and 1∶100 (0.01 mg/ml) was included in each experiment.Cytochrome P450 activity was measured by using 7-ethoxy coumarin as the substrate which results in a fluorescent 7-hydroxycoumarin. The O-deethylation of 7-EC is measured in an endpoint fluorescent assay as described by Stumpf and Nauen, 2001 To mite homogenate (with and without inhibitor) set up in triplicate, 40 ul of 0.1M sodium phosphate buffer pH 7.6, 2 ul of 20 mM 7-EC in acetone and 10 ul of 10 mM Aqueous NADPH was added. The enzyme and substrate reaction mixture was incubated for 30 mins at 30°C with shaking. Excess NADPH was removed by adding 10 ul of 100 mM oxidized glutathione and 13 ul of 10 U/ul of glutathione reductase and the reaction incubated for 10 mins at room temperature. Fluorescence intensity was read immediately after incubation using 465 Emission and 390 Excitation filters in a microplate reader (Polarstar). As an assay control, human Cytochrome P450 isoform diluted 1∶10 (0.10 mg/ml) and 1∶100 (0.01 mg/ml) was included in each experiment.Survival time of both permethrin susceptible and permethrin resistant mites over the 24-hour exposure to acaricide and acaricide+/−synergist are shown in t test.To investigate possible metabolic pathways inhibited by synergists, enzyme inhibition assays were performed. Homogenate supernatant from resistant mites were incubated with the synergists  acting as inhibitor and enzyme levels were compared with and without inhibitors. Homogenate supernatants from susceptible mites were also incubated with the synergists for comparison. Enzyme inhibition experiments were repeated several times and showed consistent results. Significant differences in enzyme activities with and without inhibitors were determined by Esterase activity in resistant scabies mites was significantly inhibited by DEF , and to lesser degree by PBO . Inhibition of esterase activity in resistant mites by DEM was unremarkable . A decrease in esterase activity was likewise observed in susceptible mites with PBO, DEF and DEM. However, the decrease was not statistically significant  . VaryingGST levels of resistant scabies mites were significantly inhibited by DEM  and to lesser degree by PBO  and DEF . PBO, DEF and DEM decreased GST activity in sensitive mites but the decrease was not statistically significant  . A dose-Cytochrome P450 monooxygenase activity in resistant mites was significantly inhibited by PBO , DEF  and DEM  at 3mM concentration. In sensitive mites, CYP450 monooxygenase activity is significantly inhibited by DEM ; by DEF  and PBO  at the same concentration of inhibitor . ResultsBoophilus microplus, high levels of esterase activity have been observed in another two strains in the absence of this mutation Varroa destructor has been associated with sodium channel insensitivity as well as elevated cytochrome P450s and high levels of esterases Detoxifying enzymes and target alteration are equally important mechanisms of insecticide degradation in ticks and mites. While a point mutation in the Vssc gene has been associated with pyrethroid resistance in two strains of cattle ticks, kdr gene associated with permethrin resistance, in the population of Sarcoptes scabiei var canis mites studied here and developed a highly sensitive high resolution melt (HRM) assay for its detection We have previously addressed target alteration as a mechanism of pyrethroid resistance in scabies mites. A non-synonymous SNP was identified in the Sarcoptes scabiei mites, commonly used synergists PBO, DEF and DEM were used in acaricide bioassays. Enzyme assays were performed using three biochemical markers such as esterases, GSTs and cytochrome P450s linked to insecticide resistance to further define specific metabolic pathways inhibited in a population of resistant scabies mites.To better understand metabolic mechanisms of pyrethroid resistance in In this study, significantly higher levels of esterase, GST and cytochrome P450 monooxygenase activity in resistant mites were observed when compared to sensitive mites suggesting metabolic degradation as a mechanism of pyrethroid resistance in this population of scabies mites. It was also observed that PBO, DEF and DEM when used as synergists to permethrin in bioassays, significantly decreased the survival times of resistant mites. These findings are in agreement with results of enzyme inhibition experiments that showed inhibition of cytochrome P450 monooxygenase activity (81%) by PBO, inhibition of GST activity (73%) by DEM and inhibition of . esterase activity (36%) by DEF when homogenate supernatant of resistant mites were incubated with the synergists. These results suggest possible roles of GSTs, esterases and cytochrome P450 monooxygenases in detoxifying permethrin in this population of resistant mites.In initial assays of cytochrome P450 we failed to detect enzyme activity in both sensitive and resistant mites using homogenates spun at high speed. However, in subsequent assays using mite homogenate spun at a much lower speed (1000×g), positive results were observed Overall, these findings have demonstrated potential utility of synergists in reversing resistance to acaricide and validated metabolic mechanisms of pyrethroid resistance in scabies mites. The results of this study also support our hypothesis that permethrin resistance in this population of scabies mites may be mediated by multiple mechanisms including target alteration, as demonstrated in our previous study The addition of a synergist to topical creams containing permethrin as the active ingredient may result in better clinical management of acaricide resistant scabies. Improved management of permethrin resistant head lice infestations has already been achieved by adding PBO as a synergist to pyrethrin based head lice shampoos and are now available as over the counter products This work now leads us to consider undertaking in-vitro susceptibility and pesticide synergist studies on clinical isolates from scabies-endemic communities in remote Aboriginal communities in Northern Australia where mass drug administration is underway. These studies will determine the potential role of metabolic degradation as a mechanism of permethrin resistance in human scabies mites.


Such strains are uncommon causes of severe human disease but may be increasing in incidence. Clostridium difficile is a recognized pathogen in neonatal pigs and may contribute to enteritis in calves. Toxinotype V strains have been rare causes of human C. difficile–associated disease (CDAD). We examined toxinotype V in human disease, the genetic relationship of animal and human toxinotype V strains, and in vitro toxin production of these strains. From 2001 through 2006, 8 (1.3%) of 620 patient isolates were identified as toxinotype V; before 2001, 7 (<0.02%) of ≈6,000 isolates were identified as toxinotype V. Six (46.2%) of 13 case-patients for whom information was available had community-associated CDAD. Molecular characterization showed a high degree of similarity between human and animal toxinotype V isolates; all contained a 39-bp tcdC deletion and most produced binary toxin. Further study is needed to understand the epidemiology of CDAD caused by toxinotype V C. difficile, including the potential of foodborne transmission to humans. Clostridium difficile–associated disease (CDAD) is increasing in incidence and severity , BI , toxinotype III (by PCR characterization of the pathogenicity locus), and 027 (by PCR ribotyping)  and nonepidemic isolates.To better understand whether food animals could be a source of infection for humans, we investigated recent and past human CDAD caused by toxinotype V C. difficile human isolates sent to the Centers for Disease Control and Prevention (CDC) from healthcare facilities and health departments in multiple states during hospital-associated outbreaks reported from 2001 through early 2007. Second, we reviewed a database of >6,000 isolates maintained by the Hines Veterans Affairs (VA) Hospital, representing CDAD reported from multiple healthcare facilities from 1984 up to January 1, 2001. All toxinotype V isolates were obtained from patients with a diagnosis of CDAD based on clinical history  and a positive clinical laboratory test for C. difficile toxin .Case finding was performed by reviewing recent and past human isolates of interest from 2 sources. Cases were defined as patients with clinical isolates identified as toxinotype V by analysis of restriction fragment length polymorphisms (RFLPs) of toxin-encoding genes. First, we reviewed 620 2 test results by using SPSS version 13.0 .Isolates identified from the Hines VA Hospital database were designated “past,” and isolates sent from hospitals to CDC from 2001 through early 2007 were designated “recent.” Additional clinical information was obtained for recent case-patients from a standard reporting form completed by the original submitting institutions. The proportions of past and recent isolates identified as toxinotype V were compared with the χClostridium difficile Surveillance Working Group (C. difficile toxin test) occurred within <48 h after the patient was admitted to a healthcare facility, provided that it had been >12 weeks since the patient was last discharged from a healthcare facility. Cases of CDAD were considered healthcare facility–associated CDAD (HCFA-CDAD) if the patient had symptom onset >48 h after admission to a healthcare facility or was discharged from a healthcare facility within the previous 4 weeks. Cases were classified as indeterminate if symptom onset was within <48 h of a patient’s admission to a healthcare facility and 4–12 weeks since discharge from a previous admission. Case-patients were considered exposed to antimicrobial agents if the patient received a dose of any antimicrobial agent within the 30 days before symptom onset.Case-patients were categorized with regard to likely place of acquisition according to recommendations developed by the C. difficile isolates from humans and animals with clinical disease were obtained from diagnostic laboratories as previously described  and 7 recent human isolates were selected for toxin quantification. Production of toxins A and B was measured by ELISA as previously described  and 16 animal  toxinotype V isolates were randomly selected for antimicrobial drug susceptibility testing. Susceptibility to clindamycin, levofloxacin, moxifloxacin, and gatifloxacin was determined by using E-test strips  on Brucella agar plates with 5% sheep blood . Results were interpreted according to Clinical and Laboratory Standards Institute standard criteria  and recent (1.3%) isolates that were toxinotype V was statistically significant (p<0.001). Three (38%) of 8 recent cases were CA-CDAD, 7 (88%) of such patients were exposed to antimicrobial agents, and 1 (13%) patient died from complications attributed to CDAD  of 8 were binary toxin positive. All 7 past human isolates also had a 39-bp deletion in tcdC, and 7 (100%) of 7 were binary toxin positive. Thirty-three toxinotype V animal isolates were obtained; they displayed a variety of PFGE patterns  when digestion was performed with the SmaI enzyme. The first group contained 1 human isolate (REA subtype BK1) that was indistinguishable by PFGE (NAP7) from 1 porcine isolate (REA subtype BK13). The second group consisted of 5 human and 2 porcine isolates, all of which were designated NAP7 by PFGE, although REA demonstrated 4 different subtypes. The third group contained 1 human isolate and 2 porcine isolates, which were indistinguishable by PFGE and REA (NAP8 and BK6). The 9 isolates in groups 1 and 2 were only 80% similar when digestion was performed with EagI. However, the isolates in the third group were 95% similar, and 1 porcine isolate was indistinguishable (100% similarity) from 1 human isolate, even after digestion with EagI.All 8 recent human isolates had a 39-bp deletion in patterns . All, hoMedian toxin A and B production in the 21 toxinotype V isolates analyzed  was greater than that by nonepidemic toxinotype 0 isolates but less than that by epidemic toxinotype III isolates at all time points measured . The meaAntimicrobial drug susceptibility testing was performed on 14 of 15 human and 16 of 33 animal toxinotype V isolates . ResistaC. difficile, which has been reported as a cause of epidemic disease in neonatal pigs and colonization in calves during the past decade  were binary toxin positive.In a review of recent and past isolates, we identified several human cases of CDAD caused by toxinotype V strains of C. difficile is recognized as a cause of disease in several animal species  human disease caused by transmission by means of direct or indirect  contact with infected live animals, and 3) human disease linked to consumption of products from food-producing animals. Both the genetic similarity of the human and animal isolates in our study and the apparent increasing importance of toxinotype V isolates in human CDAD after their emergence in animals may suggest foodborne or other forms of animal-to-human transmission.Although C. difficile is more likely, animal contact is a more plausible means of transmission for CA-CDAD. Our results suggest that toxinotype V C. difficile may be a relatively common cause of community-associated disease. Despite evidence that only 20% of all human CDAD cases are community-associated  with an 18-bp deletion in tcdC (rather than the 39-bp deletion observed in toxinotype V strains) and with genes that encode binary toxin (The epidemiology of human CDAD has been affected by recent increases in the incidence and severity of disease. These changes have been largely attributed to the emergence of the BI/NAP1/027 C. difficile has demonstrated a similarly truncated TcdC (61 aa compared with 65 aa in BI/NAP1/027 strains and 232 aa in wild-type TcdC) (tcdC, and were binary toxin negative (Current literature suggests that this considerable truncation of TcdC may impair its negative regulatory function and contribute to the increased toxin production observed in BI/NAP1/027 strains (C. difficile (C. difficile.This study is subject to the following limitations. First, the number of toxinotype V isolates examined was small and may not be wholly representative of this strain as it manifests in human or animal disease. Furthermore, recent isolates were collected from institutions that reported healthcare-associated outbreaks of CDAD, and clinical information describing patients from whom specimens were obtained may therefore overestimate disease severity. Moreover, since recent isolates were obtained from healthcare facilities that were experiencing CDAD outbreaks, they may represent a different population of patients than the past isolates, which were obtained from a variety of sources, some of which were ongoing clinical surveillance projects and some of which were outbreak investigations. The 2 source populations, however, can be considered reasonably similar in that both represent primarily hospitalized patients. If, however, the recent database contains a substantially greater proportion of outbreak-related isolates than the past collection, this would only strengthen the evidence for the recent emergence of toxinotype V CDAD. Since outbreaks of CDAD are largely associated with the epidemic, toxinotype III strain of C. difficile that cause disease in animals, which makes it impossible to determine whether the current toxinotype V strains are new or simply newly recognized. Finally, information about human cases is limited, particularly with respect to possible routes for community acquisition of disease; thus, evidence upon which to base conclusions regarding interspecies transmission is limited.Additionally, little is known about the types of C. difficile and strains responsible for occasional human disease raises the possibility of interspecies transmission. Further studies are needed to understand the etiology of CDAD caused by toxinotype V C. difficile and the mechanisms of transmission between animals and humans, including the role of the food supply.Although relatively common in animal CDAD, toxinotype V is currently an uncommon cause of human illness, which may occur more frequently among persons without traditional risk factors associated with CDAD, such as recent exposure to a healthcare setting. In vitro toxin production results from our limited sample suggest that toxinotype V strains have the potential to cause increased severity of human disease, although further studies are needed to corroborate this association. Although they share similar clinical features, evidence suggests that the predominant strains causing CDAD in humans and different animal species are distinct (


Drosophila embryos are well studied developmental microcosms that have been used extensively as models for early development and more recently wound repair. Here we extend this work by looking at embryos as model systems for following bacterial infection in real time. We examine the behaviour of injected pathogenic (Photorhabdus asymbiotica) and non-pathogenic (Escherichia coli) bacteria and their interaction with embryonic hemocytes using time-lapse confocal microscopy. We find that embryonic hemocytes both recognise and phagocytose injected wild type, non-pathogenic E. coli in a Dscam independent manner, proving that embryonic hemocytes are phagocytically competent. In contrast, injection of bacterial cells of the insect pathogen Photorhabdus leads to a rapid ‘freezing’ phenotype of the hemocytes associated with significant rearrangement of the actin cytoskeleton. This freezing phenotype can be phenocopied by either injection of the purified insecticidal toxin Makes Caterpillars Floppy 1 (Mcf1) or by recombinant E. coli expressing the mcf1 gene. Mcf1 mediated hemocyte freezing is shibire dependent, suggesting that endocytosis is required for Mcf1 toxicity and can be modulated by dominant negative or constitutively active Rac expression, suggesting early and unexpected effects of Mcf1 on the actin cytoskeleton. Together these data show how Drosophila embryos can be used to track bacterial infection in real time and how mutant analysis can be used to genetically dissect the effects of specific bacterial virulence factors. Drosophila embryos in a novel application to look at bacterial infection in real time. Contrary to popular belief, embryonic hemocytes both recognise and ingest injected Escherichia coli. This is a dynamic process in which the bacteria are recognised by, and adhere to, the phagocytes in a process that can be dramatically seen in real time using time-lapse confocal microscopy. In contrast, when cells of the insect pathogen Photorhabdus are introduced, the hemocytes become frozen and are unable to engulf the invading bacteria. Using both recombinant E. coli expressing the bacterial toxin Makes Caterpillars Floppy, and also purified toxin itself, we show how genetic mutants of Drosophila can be used to dissect the role of bacterial toxins in infection. Such approaches should provide a useful model in which to study infection by other pathogens and their associated toxins.The humble fruit fly has formed an important model for the study of bacterial infection both by insect specific and mammalian pathogens. However, many studies of bacterial infection rely upon death of the insect host, or actin cytoskeleton staining of specific host cells, at fixed end-points to look at infection or the mode of action of different bacterial toxins. Here, we use  Drosophila is now widely established as a useful genetic model for looking at microbial infection with recent studies now encompassing both bacterial Drosophila infection models have also begun to mimic different types of infections. For example, several groups are now developing systems in which to examine bacterial intestinal infections Drosophila, the outcomes of infection are often measured by end-points such as insect death  or changes in cell morphology at fixed periods throughout infection (often monitored by staining different elements of the cytoskeleton). Although this problem can, to some extent, be addressed by the use of reporter constructs (e.g. Diptericin-LacZ) that provide quantitative or visual read-outs from specific immune response genes, we still lack the ability to follow bacterial infections in real-time in the critical early stages of infection. We are therefore unable to visualise the outcome of the first interactions between insect phagocytes and invading microbes, interactions that will determine the future success of the infection itself. To address this need, here we use Drosophila embryos, specifically embryonic hemocytes, as models for studying the early stages of infection in real-time using time lapse confocal microscopy.Within each of these infection models, three different aspects of infection can be examined Drosophila embryos and their development are extremely well documented and recent attention has focused on the role of the embryonic hemocytes in early embryonic development. Embryonic hemocytes are highly motile macrophage-like cells that migrate throughout the developing embryo following stereotypical routes to disperse from their point of origin to eventually distribute themselves equally throughout the animal by late embryonic stages Drosophila embryos therefore represent an easily injectable, containable and closed experimental system into which bacteria can be injected and their subsequent interactions with resident hemocytes observed in real time.Although these developmental roles are well documented it is less clear how competent these cells are to respond to infection and whether they play a significant role in the immune response of the embryo. Embryonic hemocytes lend themselves beautifully to live imaging studies since fluorescent probes can be expressed specifically in these cells using hemocyte specific promoters and their movements subsequently imaged within living embryos using confocal timelapse microscopy. Drosophila as models for bacterial infections have used bacteria pathogenic to man Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus or Vibrio cholera to look at insects as models for mammalian infection Photorhabdus asymbiotica as a model system in which to study cross-talk between virulence factors developed against insects that can also be deployed against mammalian immune responses. P. asymbiotica strains have been recovered from human wounds Photorhabdus bacteria is associated with the dominant toxin Makes Caterpillars Floppy 1 or Mcf1 E. coli expressing the mcf1 gene makes this an ideal virulence factor in which to study early interactions between an insect pathogen and insect phagocytes.Many of the studies developing Drosophila embryos as models to study bacterial infection in real time. We show that embryonic hemocytes both recognise and phagocytose non-pathogenic E. coli in a Dscam independent manner. In contrast, we show that the cells of the insect pathogen Photorhabdus instantly freeze the highly mobile phagocytes. This freezing phenotype can be phenocopied either by injection of recombinant E. coli expressing the mcf1 gene, or by injection of the purified toxin itself. The ability to image these first early stages of infection therefore facilitates a direct examination of the Mcf1 virulence factor neutralizing the phagocytic role of the embryonic hemocytes. Moreover, examination of the role of Mcf1 can be dissected genetically using mutants that either interfere with its endocytosis into target cells, or Rac signalling mutants that hint at early and unexpected Mcf1 mediated effects on the phagocyte cytoskeleton.Here, we pioneer the use of in vivo detection of E. coli, strain BL21 (DE3) was transformed with a high-copy vector pRSET expressing the monomeric red fluorescence protein. Protein expression was under the control of the T7 promoter; the leaky nature of this promoter allowed basal expression of the fluorescent protein without induction and successful detection of the bacteria within the embryo. The specificity of the bacterial-hemocyte interaction was initially tested by injecting stage 15 wild-type embryos containing unlabelled hemocytes with nl quantities of highly concentrated fluorescently-labelled bacterial suspension (1010 colony forming units/ml). At this embryonic stage, hemocytes are arranged into three characteristic lines that run anterior to posterior along the ventral aspect of the embryo (7 cfu/ml) and subjected to timelapse confocal imaging. Confocal images show that hemocytes clearly locate and capture invading E. coli -superfamily receptor Down syndrome cell adhesion molecule (Dscam) for bacterial recognition in emocytes .Photorhabdus is a Gram-negative, nematode-vectored bacterium that kills a wide range of insect species Drosophila embryos containing RFP-labelled hemocytes with a GFP-labelled P. asymbiotica suspension (107 cfu/ml) had a profound effect on embryonic hemocyte motility whereby hemocytes rapidly loose their ability to migrate and apparently freeze  ts1shi. When these embryos were microinjected with Mcf1 the hemocytes were immune to the paralytic effect of the Mcf1 and continued to produce large dynamic cytoplasmic extensions appearing indistinguishable from uninjected control embryos  or constitutively active (RacV12) versions of the small GTPase, Rac, in hemocytes. It has been previously shown that hemocytes expressing dominant-negative RacN17 fail to undergo their normal developmental migrations and exhibit stunted lamellipodia formation N17 expressing hemocytes were completely resistant to the effects of Mcf1 and injection of Mcf1 into these embryos failed to cause the freezing effect observed when administered to wildtype cells , YopE (Yersinia spp.) and SptP  Mcf1 is a single toxin which facilitates persistence of Drosophila larvae have implicated the immunoglobulin (Ig)-superfamily receptor Down syndrome cell adhesion molecule (Dscam) as being an important player in the recognition of bacteria E. coli with equal efficiency to that seen in wild-type embryos. This result demonstrates an intriguing difference between the immune system operating in the embryo when compared with larvae. Embryonic hemocytes are very long lived cells that persist into larval stages and constitute the circulating population of hemocytes in a larva. Within the larva a second population of hemocytes develops in a specialised hematopoetic organ called the lymph gland. Lymph gland hemocytes are normally released from this organ during metamporphosis but can be released prematurely following parasitisation Previous studies using third instar The maturation of embryonic hemocytes as they progress through embryonic into larval stages of development is an interesting process that has received very little research attention. Recent studies have shown that when circulating within larvae, hemocytes appear substantially less motile than when they migrate throughout the embryo Drosophila embryo. This system complements the use of tissue culture systems for several reasons. First, the real-time behaviour of phagocytes in their natural environment can be monitored. This overcomes the limitations of looking at immortal cell lines of uncertain origin (eg Schneider cells) or of looking at abnormal behaviour in primary cultures of phagocytes (eg hemocytes recently bled from an insect). Second, we can use both genetic mutants and RNAi to look at effects in vivo. This contrasts to transfection experiments on cell cultures that are often transient and variable in their effects. Finally, although we cant precisely define the concentration of effector proteins delivered into the hemocyte via injection, we can say, in the case of Mcf1, that both the purified protein, the recombinant protein expressed by E. coli and the native Mcf1 expressed by P. asymbiotica all had the same phenotypic effects on the in vivo system. Moreover, these effects were all very different to those previously described for Mcf1 protein applied to primary cultures of Manduca hemocytes recorded under time-lapse photography Here we have described an in vivo system for looking at a known population of phagocytes in a closed system, the Drosophila S2 cells have been used extensively as a model system to study infection and immune responses. These cells allow for large scale screening using RNAi and have been successfully used to identify proteins involved in host interactions with important human bacterial pathogens such as Escherichia coli, Staphylococcus aureusMycobacterium spp.Legionella pneumophilaChlamydia spp. Listeria monocytogenesin vitro the situation in vivo where hemocytes can interact with other immune cells to optimize immune responses is likely to be more complex. Ultimately, to have an impact on human and animal health, the results obtained by in vitro studies need to be verified in a whole organism. The assay we present here provides a perfect model to begin to fill these gaps and should lead to a better understanding of host-pathogen interactions in the complex setting of a multicellular organism.In recent years cultured P. asymbiotica ATCC 43949 was isolated from a human leg wound in San Antonio, Texas Escherichia coli S17-1λpir pir-dependent suicide replicon pBamH7 (a kind gift from Dr Leo Erbel) which constitutively expresses green fluorescence protein (GFP). E. coli BL21 was used for cloning and constitutive expression of Mcf1 from pUC18  and adjusted to the appropriate density.P. asymbiotica via conjugation with S17-1λpir by using a membrane filter mating technique. S17-1λpir pBamH7 was inoculated into 5 ml of LB broth containing kanamycin and grown at 37°C for 16–18 h with shaking (200 rpm). P. asymbiotica was grown at 28°C for 16–18 h with shaking (200 rpm) but without antibiotic selection. 100 µl of each saturated bacterial culture was added to 3 ml of sterile 10 mM MgSO4, mixed, and filtered through a 0.45-µm-pore-size nitrocellulose filter, using a 25-mm Swinnex filter apparatus (Millipore). Control assays, using donor and recipient alone, were also performed. Filters were placed on LB plates supplemented with 10 mM MgSO4 and incubated for at least 8 h in a 37°C incubator. The filters were washed with 4 ml of sterile 0.85% NaCl, and 100 µl aliquots were spread onto LB plates containing 25 µg of rifampicin and 25 µg of kanamycin per ml. Rifampicin-resistant and kanamycin-resistant transconjugants were identified after 48 h incubation at 37°C.pBamH7 was delivered to Purification and labelling of Mcf1 was carried out as described previously serpentHemoGAL4 , equipped with a krypton-argon laser and helium-neon lasers, under 63× objective. For time-lapse movies images were obtained by taking four optical slices (each slice averaged 2×) through hemocytes collected at 120 s intervals. Compilation and processing of movies was carried out using ImageJ software.Video S1E. coli. Confocal timelapse movie of an embryo containing GFP expressing hemocytes (green) that has been subjected to injection of RFP expressing E. coli (red). In the first few frames of the movie, the bacteria are rapidly engulfed by a hemocyte. Movie duration is 50 minutes.Embryonic hemocytes engulf (1.59 MB MOV)Click here for additional data file.Video S2Photorhabdus rapidly freezes embryonic hemocytes. The movie on the left shows an embryo containing RFP expressing hemocytes (red) that has been injected with E. coli (yellow). The hemocytes are highly motile cells migrating within the embryo as they clear the invading bacteria. The movie on the right shows the effect of injection of Photorhadus (yellow) on the hemocytes (red). Hemocytes are able to bind the bacteria but appear frozen and lose their ability to move. Movie duration is 40 minutes. Both movies are running at the same speed (7 fps).(2.42 MB MOV)Click here for additional data file.Video S3E. coli producing Mcf1. Top panel shows the red channel (RFP), the middle shows the green channel (GFP) and the lower is the merge. Whilst the hemocytes (red) are able to detect the bacteria (green) they are unable to move and appear completely frozen as seen following injection with Photorhabdus (compare with Video S2). Movie duration is 50 minutes.Mcf1 causes freezing of hemocytes. Confocal timelapse movie showing embryonic hemocytes expressing RFP in an embryo injected with GFP expressing (1.07 MB MOV)Click here for additional data file.Video S4Injection of purified Mcf1 causes hemocyte freezing. Movie of GFPmoesin expressing hemocytes within an embryo after injection with 0.2 mg/ml purified Mcf1. Hemocytes are able to form actin rich protrusions but these protrusions are completely static and bear no resemblance to the highly dynamic structures normally seen in these cells (compare to Video S1). Movie duration is 60 minutes.(1.97 MB MOV)Click here for additional data file.Video S5Injection of BSA has no effect on hemocyte dynamics. Movie of GFPmoesin expressing hemocytes within an embryo following injection with 0.2 mg/ml heat inactivated BSA. Hemocytes appear completely wildtype in their morphology and actin dynamics extending large lamellipodia as they patrol their environment.(0.23 MB MOV)Click here for additional data file.Video S6Hemocyte paralysis effect of Mcf1 occurs in a dose-dependent manner. Movie of GFPmoesin expressing hemocytes within an embryo following injection with 0.1 mg/ml Mcf1. The freezing effect on hemocytes is less pronounced at the lower concentration (compare with Video S4) with many cells displaying active lamellipodial ruffling although these cells are not as dynamic as untreated cells and were still unable to migrate.(0.93 MB MOV)Click here for additional data file.Video S7Mcf1 has no effect on dorsal closure. Movie showing dorsal closure in wildtype embryos (left) and embryos injected with Mcf1 (right). Both embryos express sGMCA (constitutively expressed GFPmoesin) to enable epithelial dynamics to be visualised. Epithelial fronts migrate and fuse at the same rate in both embryos demonstrating that Mcf1 has no effect on dorsal closure. Arrow in the wildtype image highlights a dynamic hemocyte as it migrates under the epithelium. No migrating hemocytes can be seen in the Mcf1 treated embryo due to the specific freezing effect on these cells.(2.45 MB MOV)Click here for additional data file.Video S8ts1shibire injected with Mcf1. These shibire mutant hemocytes can be seen migrating normally extending large, dynamic actin rich protrusions and show no sign of the freezing phenotype observed in wildtype cells upon exposure to Mcf1 toxin. Movie duration is 40 minutes.Cellular internalisation of Mcf1 is required for hemocyte freezing. Timelapse confocal movie showing embryos containing hemocytes expressing (1.95 MB MOV)Click here for additional data file.Video S9N17 expressing cells. Movie showing RacN17 expressing hemocytes in an embryo injected with Mcf1. Due to a requirement of Rac for the formation of lamellipodia, these mutant cells are far less mobile than wildtype hemocytes and produce much smaller and less dynamic protrusions. However, despite these defects the cells are still motile after exposure to Mcf1 and there is no evidence of the cell freezing effect observed in wildtype cells . Movie duration is 60 minutes.Mcf1 induced freezing is blocked in Rac(2.59 MB MOV)Click here for additional data file.Video S10V12 expression in hemocytes also blocks freezing effect of Mcf1. Timelapse movie showing RacV12 expressing hemocytes in an embryo injected with Mcf1. Like RacN17, expression of RacV12 causes migration defects in hemocytes. Despite this when exposed to Mcf1, the mutant cells appear immune to the paralytic effect of the toxin. Movie duration is 60 minutes.Rac(2.52 MB MOV)Click here for additional data file.


The Cocaine-cues video increased craving to the same extent with placebo (68%) and with methylphenidate (64%). In contrast, SPM analysis of metabolic images revealed that differences between Neutral versus Cocaine-cues conditions were greater with placebo than methylphenidate; whereas with placebo the Cocaine-cues decreased metabolism (p<0.005) in left limbic regions  and right parahippocampus, with methylphenidate it only decreased in auditory and visual regions, which also occurred with placebo. Decreases in metabolism in these regions were not associated with craving; in contrast the voxel-wise SPM analysis identified significant correlations with craving in anterior orbitofrontal cortex (p<0.005), amygdala, striatum and middle insula (p<0.05). This suggests that methylphenidate's attenuation of brain reactivity to Cocaine-cues is distinct from that involved in craving. Cocaine-cues decreased metabolism in limbic regions (reflects activity over 30 minutes), which contrasts with activations reported by fMRI studies (reflects activity over 2–5 minutes) that may reflect long-lasting limbic inhibition following activation. Studies to evaluate the clinical significance of methylphenidate's blunting of cue-induced limbic inhibition may help identify potential benefits of this medication in cocaine addiction.Dopamine (phasic release) is implicated in conditioned responses. Imaging studies in cocaine abusers show decreases in striatal dopamine levels, which we hypothesize may enhance conditioned responses since tonic dopamine levels modulate phasic dopamine release. To test this we assessed the effects of increasing tonic dopamine levels  on brain activation induced by cocaine-cues in cocaine abusers. Brain metabolism (marker of brain function) was measured with PET and  Dopamine (DA), a neurotransmitter that processes reward and prediction of reward 18F]fluoro-D-glucose (18FDG) to measure brain glucose metabolism (marker of brain function) To test this hypothesis we assessed the effects of raising tonic DA levels by giving a low dose of oral MP (20 mg) We hypothesized that MP by increasing tonic DA levels would dampen phasic DA firing triggered by conditioned cues and thus the activation pattern elicited by cocaine-cues would be attenuated when MP was given as compared to when placebo was given. We also hypothesized that this would be associated with reduced craving.Plasma MP levels did not differ between conditions and corresponded for the Neutral and the Cocaine-cues conditions respectively, at 30 minutes post administration to: 3.0±3 and 3.3±3 ng/ml; at 60 minutes to: 5.7±3 and 5.6±4 ng/ml; at 90 minutes to: 6.4±2 and 6.1±3 ng/ml; and at 120 minutes to: 6.1±2 and 5.7±2 ng/ml. Plasma MP levels were not significantly correlated with cardiovascular, behavioral or brain metabolic measures.Heart rate and systolic blood pressure (BP) increased significantly for all conditions (pre vs post) . CompariThe craving measures (CCQ and self-reports) increased significantly with the Cocaine-cues exposure for both conditions (placebo or MP). For CCQ repeated ANOVA  differed significantly for the Cocaine-cues when given with PL (p<0.002) and when given with MP (p<0.003) but did not differ for the Neutral video conditions (PL or MP) . The selWhole brain absolute metabolic measures corresponded for Neutral with PL to 34.9±5 µmol/100 g/min, for Neutral with MP to 36.1±5 µmol/100 g/min, for Cocaine-cues with PL to 35.0±5 µmol/100 g/min and for Cocaine-cues with MP to 35.9±5 µmol/100 g/min and the measures did not differ between conditions. The SPM analysis also showed no differences between conditions for the absolute metabolic images.SPM comparisons for the normalized metabolic measures showed significant decreases in relative metabolism for the Cocaine-cues video when compared with the Neutral-video that were more extensive with Placebo than with MP. When Placebo was given decreases were significant in left limbic regions /anterior insula (BA 47 and BA 11), accumbens, posterior insula) and right parahippocampal gyrus (BA 36 and 37), and in auditory (left BA 22 and left and right BA 42) and visual areas   and with the Neutral video additional decreases in posterior cingulate (BA 30), superior temporal and postcentral gyrus , Table 4The SPM analysis of the interaction effect between video and drug condition showed that the decreases with the Cocaine-cues were attenuated when MP was given when compared to placebo. Specifically the decreases when the cocaine-cues were given with placebo versus when given with MP were significantly greater in the right and left lingual gyrus, left hippocampal gyrus, left accumbens/BA 25 region and left lateral OFC (BA 47)  were not significant either for the placebo or the MP conditions.In contrast the SPM voxel-wise correlation between changes in metabolism and self-reports of craving identified several regions where these correlations were significant but the pattern differed for placebo and for MP . For theNeither order of the conditions  nor age had a significant effect. However, covarying for gender showed a significant effect . FemalesHere we show in cocaine abusers exposed to Cocaine-cues an unexpected decrease in metabolic activity in limbic brain regions  and a blunting of these regional brain responses to Cocaine-cues when oral MP was administered but showed no effect on the subjective experience of craving. Indeed, the cocaine-cues increased craving (self-reports and CCQ) to the same extent with placebo as with MP. This dissociation would suggest that the regions that showed a Cue by Drug interaction effect (regions where responses to the Cocaine-cues were attenuated by MP), are not related to the subjective perception of craving. Indeed the lack of a correlation between the metabolic changes in these regions and the craving responses support this.When compared with the neutral video, the cocaine-cues video did not change whole brain absolute glucose metabolism, which indicates that when already watching a video the inclusion of cocaine-cues did not further affect the overall glucose utilization by the brain. This contrasts with a prior study in which we reported a 10% increase in whole brain metabolism upon exposure to the same cocaine-cues video but when compared with a baseline condition  In contrast to the lack of an effect on absolute metabolic measures, normalization of metabolism by whole brain, revealed relative decreases in several brain regions with exposure to the cocaine-cues video as compared to the neutral video. These differences were significantly larger when placebo than when MP was given. Specifically during the cocaine-cues video when given with placebo there were relative decreases in limbic regions that included left lateral OFC, left anterior and posterior insula and left nucleus accumbens (NAc), and in the auditory (left BA 22 and left and right BA 42) and visual areas . In contrast when MP was given the only significant differences were decreases in right visual (inferior BA 18) and right auditory (BA 42) regions but no differences in limbic areas.Thus, the cocaine-cues video when compared with a neutral video led to a relative decrease in activity of limbic brain regions when given with placebo. This is consistent with preclinical studies showing that exposure to cocaine-cues in chronically treated rats decreased glucose metabolism in limbic regions In interpreting the results from this study it is crucial to recognize that glucose metabolic measures reflect activity over a 30 minute period and thus the limbic deactivation observed with the cocaine-cues video could reflect a longer lasting effect from exposure to conditioned-cues that may have followed an initial short lasting activation. This is plausible since DA cell firing decreases after the expectation of a predicted reward (from a conditioned stimulus) does not occur The discrepancies between our findings of decreases in limbic metabolism and prior imaging studies showing limbic activation with cues exposures are also likely to reflect the sensitivity of brain responses to the mechanisms used to elicit craving (and as importantly the control condition used to compare this) and to the imaging methods used to measure them . For example, the much higher temporal resolution of fMRI enables the detection of areas with dynamic patterns of activation/deactivation as has been reported to occur during cocaine-induced craving The cocaine-cues video when compared with the neutral video decreased relative activity in somatosensory cortices ; specifically BA 42 (auditory integration region) and inferior occipital cortex. Though we do not have an explanation for these decreases we speculate that this may reflect differences in the objects and sounds presented by both videos.In this study we also show that MP decreased metabolic activity in precentral cortex (BA 6) and posterior cingulate gyrus (BA 30) for the Neutral condition. Since the posterior cingulate is part of the DMN that gets deactivated with stimulation The Cocaine-cues significantly increased heart rate and systolic BP and the effect was more robust when MP was given than when placebo was given. This indicates that some of the peripheral responses to conditioned-cues may be amplified by MP. Indeed studies with oral MP in cocaine abusers that were given cocaine showed an enhancement of cocaine's cardiovascular effects When covarying for gender we showed a significant gender interaction effect; females were more reactive to cocaine-cues than males. This highlights the importance of future studies to separately investigate the effects of gender in cue-reactivity and in responses to addiction treatments. In contrast neither age nor sequence of scans influenced the results.Here we observed an attenuation of the limbic responses to conditioned-cues by MP that may be beneficial in decreasing the negative effects that exposure to conditioned cues may have on cocaine abusers . However clinical trials with MP for the treatment of cocaine addiction have been inconclusive A limitation for this study is that changes in brain metabolism, which average activity over 30 minutes, may lack the temporal sensitivity to identify the dynamic changes in brain activity elicited by cue exposures as detected with fMRI. On the other hand the metabolic method may help identify the longer lasting consequences that follow exposure to conditioned cues.Another limitation is the use of conscious awareness of craving as the dependent variable. Indeed it is likely that unconscious responses to conditioned-cues responses are as important in triggering drug consumption. This is particularly relevant since there is evidence that in cocaine abusers there is a disruption between interoceptive processes and subjective awareness It would have been desirable to include a control group to assess the specificity of MP effects to conditioned-cues in addicted individuals.Finally though we hypothesized that oral MP would blunt the responses to conditioned cues by increasing DA tone and blunting phasic DA signals triggered by conditioned cues we can not determine that this was the mechanism responsible for the attenuation of the regional brain metabolic changes triggered by the Cocaine-cues. PET studies to directly measure DA changes triggered by cues with and without oral MP are necessary to corroborate this.These results document a blunting of regional brain metabolic responses to cocaine-cues by oral MP but failed to corroborate our hypothesis that this would be associated with a parallel blunting in craving. The functional significance of the limbic deactivation after exposure to conditioned-cues and its blunting by MP merits further investigation.Twenty-four active cocaine abusers  who responded to an advertisement were studied. Subjects fulfilled DSM-IV criteria for cocaine dependence and were active users for at least the prior 6 months (free-base or crack) with average cocaine use of 3.5±2 grams/day; 15±5 years of abuse and average time of last cocaine use was 2±2 days. Exclusion criteria included current or past psychiatric disease other than cocaine or nicotine dependence (22 subjects were smokers); past or present history of neurological, cardiovascular or endocrinological disease; history of head trauma with loss of consciousness greater than 30 minutes; and current medical illness. Scores for the “drug” domain of the Addiction Severity Index To assess the subjective experience of craving we used an analog scale (1–10) for self-reports of “cocaine craving” and the brief version of the Cocaine Craving Questionnaire (CCQ) Heart rate and blood pressure were monitored continuously throughout the procedure from which we computed the measures obtained 40 minutes prior to the video presentation (Pre) and those obtained 40 minutes after initiation of the video (Post). For the statistical comparisons we used the difference between the Pre and the Post measures and expressed these as percent change from the Pre measures. Repeated ANOVA was used to compare differences between neutral and cocaine-cue and to assess if there was an interaction effect between video  and drug (placebo vs MP).18FDG. Details on the methods for scanning have been published 18FDG. Arterialized blood sampling was used to measure 18F in plasma. All subjects were scanned four different times each on a separate day under the following randomly ordered conditions (1) oral placebo while watching a Neutral video; (2) oral placebo while watching the cocaine-cues video; (3) oral MP (20 mg) while watching the neutral video; and (4) oral MP (20 mg) while watching the cocaine cues-video. The order of the 4 scans was randomly varied so that only two subjects ended up by having the same sequence of conditions.PET scans were conducted with a whole-body, high-resolution positron emission tomograph  using 18FDG (60 minutes after placebo or MP administration) and were continued for 25 minutes after 8FDG injection . Videos were started 15 minutes prior to injection of The data were analyzed using a general lineal model with the Statistical Parametric Mapping (SPM) software package Pearson product moment correlations were performed to assess the relationship between the difference in activation in brain regions that differed between the Neutral vs the Cocaine-cues video (with placebo or with MP) and the changes in cocaine craving. For this purpose we obtained the metabolic values in circular regions of interest (diameter 7 voxels) placed on the center of the clusters identified by SPM as differing between the Neutral and the Cocaine-cues videos. We also performed voxel-wise correlations between the changes in metabolism  and changes in measures of craving and significance was set at p<0.005 (uncorrected >100 voxels).Figure S1SPM results for the voxel-wise correlation between changes in metabolism and changes in self-reports of cocaine craving  for the Methylphenidate condition for a threshold of significance of p<0.05 uncorrected, cluster size >200 voxels.(2.06 MB TIF)Click here for additional data file.


Influenza is a contagious disease caused by a pathogenic virus, with outbreaks all over the world and thousands of hospitalizations and deaths every year. Due to virus antigenic drift and short-lived immune responses, annual vaccination is required. However, vaccine coverage is incomplete, and improvement in immunization is needed. The objective of this study is to investigate a novel method for transdermal delivery using metal microneedle arrays (MN) coated with inactivated influenza virus to determine whether this route is a simpler and safer approach than the conventional immunization, capable to induce robust immune responses and confer protection against lethal virus challenge.50 of mouse adapted Aichi virus demonstrated complete protection. Microneedle vaccination induced a broad spectrum of immune responses including CD4+ and CD8+ responses in the spleen and draining lymph node, a high frequency of antigen-secreting cells in the lung and induction of virus-specific memory B-cells. In addition, the use of MN showed a dose-sparing effect and a strong Th2 bias when compared to an intramuscular (IM) reference immunization.Inactivated A/Aichi/2/68 (H3N2) influenza virus was coated on metal microneedle arrays and applied to mice as a vaccine in the caudal dorsal skin area. Substantial antibody titers with hemagglutination inhibition activity were detected in sera collected two and four weeks after a single vaccine dose. Challenge studies in mice with 5×LDThe present results show that delivery of inactivated influenza virus through the skin using metal microneedle arrays induced strong humoral and cellular immune responses capable of conferring protection against virus challenge as efficiently as intramuscular immunization, which is the standard vaccination route. In view of the convenience of delivery and the potential for self-administration, vaccine-coated metal microneedles may provide a novel and highly effective immunization method. Transdermal delivery of bioactive compounds on the skin has been used for thousands of years for therapeutic or prophylactic purposes, and more than one billion patches are now sold annually for delivery of small molecule drugs Vaccine delivery to the skin is attractive because of the complex immunologic network that the skin provides Although vaccine delivery to the skin is attractive, vaccines are not usually administered this way in large part because convenient and reliable methods do not currently exist. Intradermal injection requires specialized training and is often unreliable In this study, we examine the use of vaccine-coated microneedles to deliver an inactivated H3N2 influenza vaccine into the skin. These micron-scale needles can be fabricated using low-cost methods for inexpensive mass production; prepared in a patch-like format for simple administration, possibly by patients themselves; and inserted painlessly across the stratum corneum and into the skin's epidermis and dermis to access the resident dendritic cells In this approach, a microneedle array is pressed manually against the skin and left in place for a few minutes while the vaccine coating dissolves off within the skin, after which the microneedles are discarded. In this study, the effectiveness of this technology was tested in mice after a single immunization of inactivated H3N2 virus and lethal challenge with a homologous mouse adapted strain. H3N2 strains are the leading cause of mortality and hospitalization rates in influenza-associated epidemics Microneedles were fabricated from stainless steel sheets  by laser cutting as described before Microneedles were coated using a dip-coating process with a specially formulated coating solution described before The coating was performed manually by dip coating using an apparatus described elsewhere To measure the amount of vaccine coated per row of microneedles, three rows from each batch of coated microneedles were submerged into 200 µl of PBS buffer per row for 5 min. Vaccine dose was determined by measuring the concentration of protein in the solution as measured by BCA protein assay  coupled with a calibration curve generated using known concentrations of Aichi virus.Madin-Darby canine kidney (MDCK) cells  were maintained in Dulbecco's Modified Eagle's Medium (DMEM)  containing 10% fetal bovine serum . Influenza virus stocks (Aichi) were prepared and purified as described 50 was determined in the same mouse strain and the viral titer was determined as mentioned above.The mouse-adapted Aichi strain was obtained by passage 8 times in Balb/c mice. The LDTo confirm the quality and purity of the virus preparation, purified virus was applied to a formvar carbon coated grid for 1 min, and immediately stained with 1% uranyl acetate for 30 s Female Balb/c mice   were immunized once by microneedle array (MN) or intramuscular (IM) immunization. For MN delivery, the mice were anesthetized with a ketamine and xylazine cocktail and the dorsal caudal surface was prepared to remove hair as described 50 (180 p.f.u.) of live mouse adapted Aichi virus and monitored for 14 days. As a negative control group, we included 6 naïve mice. A weight loss exceeding 25% in all infected mice was used as the experimental end-point, and mice reaching this end-point were euthanized according to IACUC guidelines. The challenged mice were monitored daily for signs of morbidity  and mortality.To determine post-challenge survival rates and immune responses, 6 mice per group were challenged 2 months after their immunization by intranasal instillation of 50 µl of virus with 5×LD50 and were sacrificed on day 4. Naïve mice and naïve mice infected with 5×LD50 were used as control groups. Lung homogenates were prepared in DMEM serum-free medium to assess the viral titers per gram of tissue by plaque assay as described above.To determine the titers of influenza virus in the lungs of immunized mice after challenge, the remaining five animals of each group were infected with 5×LDThe animals were bled on days 14 and 28 after immunization and on day 4 after challenge. Blood was collected from the retro-orbital plexus with non-heparinized microcapillaries  after systemic anesthesia. Lung suspensions were prepared by mincing through a cell strainer  and the cells were resuspended in DMEM. Lung cells were used in enzyme-linked immunospot (ELISPOT) assays whereas lung supernatants were mixed with the protease inhibitor phenylmethylsulfonyl fluoride (1 mM)  and stored at −20°C until assayed for viral titers and antibody-secreting cells. Inguinal lymph nodes and spleens were processed similarly into single cell suspensions in complete RPMI 1640 for cytokine determination All sera and lung supernatants were individually collected, and anti-Aichi specific IgG, IgG1, IgG2a and IgA antibody levels were determined quantitatively by enzyme-linked immunosorbent assay (ELISA) as described We determined the hemagglutination inhibition (HAI) titers based on the WHO protocol 50 (180 p.f.u.) mouse adapted virus. Cell suspensions (0.5–1×106/well) were stimulated in vitro in the presence of 1 µg/ml nucleoprotein (NP) peptide stimulants or Aichi virus in complete RPMI medium (cRPMI) d-restricted NP class I peptide (TYQRTRALV) and a pool of three H-2d-restricted class II peptides  Spleen and lymph node cells were prepared from mice euthanized 4 days after challenge with 5×LD6/well) in cRPMI were plated directly on coated blocked plates and were incubated at 37°C for 36 h. Virus-specific ASC were detected as spots after incubation with goat anti-mouse IgG horseradish peroxidase (Southern Biotechnology Associates) and developed with stable diaminobenzidine (Research Genetics). Samples were enumerated in an ELISPOT reader  and the results shown as the number of ASC per 106 cells.Virus-specific antibody-secreting cells (ASC) in the spleen and the lungs were determined by a modification of the ELISPOT assay 5/well) were incubated for 1 and 6 days at 37°C in cRPMI supplemented with 10% Concavalin A and lipopolysaccharide-conditioned medium (ConA media) The ELISPOT assay to detect memory B cells was carried out similarly, except that splenocytes were stimulated to induce differentiation of memory B cells into ASC. Cell suspensions . Values were considered significant for p≤0.05. Unless otherwise stated, experiments were run at least in duplicates.To evaluate the efficacy of transdermal immunization with whole inactivated A/Aichi/2/68 (Aichi) H3N2 influenza virus, we determined the hemagglutination inhibition (HAI) titers as correlates of immunity in the sera of immunized mice. Groups of mice were immunized on the caudal site of the dorsal skin using solid microneedle arrays (MN) coated with 3 or 10 µg of inactivated virus . In paraWe also measured the circulating levels of influenza-specific IgG in sera by ELISA . We obseFinally, we examined if the route of immunization influenced the isotype profile. We found that the IM-immunized groups on day 14 after immunization exhibited IgG1/IgG2a ratios of 2 and 0.3 with 3 µg and 10 µg inactivated virus, respectively . In cont50 of homologous, highly pathogenic mouse-adapted virus 2 months after immunization. All vaccinated mice survived challenge and lost less than 10% of their original body weight  showing that even at this lower vaccination dose the mice effectively cleared almost all the virus. These data indicate that MN immunization with inactivated influenza virus can successfully prevent viral replication and confer protection against lethal viral challenge similarly to IM injection.To further evaluate the protective response, we also examined the extent to which MN-induced immune responses controlled viral replication in the lungs ve group . Notably50 of mouse adapted Aichi virus 2 months after immunization demonstrated robust humoral responses that increased with dose , but showed no significant differences between the IM or MN immunizations  isolated from challenged mice on day 4 with NP Class I and II restricted peptides, and quantified their ability to produce cytokines  by ELISA . The proIn both splenocytes and LN cultures the IL-4 levels did not show any significant differences between immunized or unimmunized infected groups after NP Class II re-stimulation (data not shown). In contrast, after NP Class I re-stimulation, both cell populations showed a dose-dependent increase in IL-4 production. LN IL-4 responses increased significantly for both the MN (p = 0.06) and the IM groups (p = 0.0005). Particularly, the 10 µg MN group produced a two fold higher level of IL-4 after re-stimulation when compared to the 10 µg IM group (p = 0.0189) . ElevateIFN-γ levels did not significantly change in splenocyte cultures of any groups after NP Class I or NP Class II re-stimulation . HoweverThe sustained humoral immune responses that confer protection against influenza infection are attributed in part to long-lived antibody-secreting cells (ASC). Eight weeks after the immunization and shortly after challenge, the mice were examined for ASC in spleen and lungs. In spleen, anti-Aichi IgG ASC were only elevated in MN and IM groups which received the 10 µg dose, with no significant differences among these groups. Antigen-secreting cell numbers were very low in all other groups including immunized, naïve or infected mice .In contrast, virus-specific IgG ASC in the lungs were elevated in the MN groups at both antigen doses and in the IM group at high antigen dose, reflective of the site of infection. Importantly, the MN 3 µg group had 4-fold higher numbers than the IM group at the same dose (p = 0.05) . These dex vivo, memory B cells need antigenic stimulation to divide and differentiate into ASC In contrast to the non-dividing, terminally differentiated ASC, which spontaneously secrete large antibody amounts without any antigenic stimulation We observed a significant increase in memory B cells over a period of 6 days , whereasThis study was motivated by the need to improve immunization against influenza specifically and other diseases in general. Our main goal was to determine if MN delivery of influenza vaccine could induce comparable immune responses to the conventional IM injection, using a MN array with numerous logistical advantages. We conclude that MN immunization can indeed provide protective immunity at least as strong as that of IM injection, with the notable difference that MN vaccination shifted the immune response significantly toward a Th2 bias.More specifically, this study showed that vaccination by the new technology of vaccine-coated MN arrays could induce strong humoral and cellular immune responses that can effectively confer full protection with a single immunization. Solid metal MN coated with inactivated influenza virus conferred 100% protection against lethal viral challenge and induced short-lived antigen-secreting cells in the lungs and spleens of immunized mice as well as anti-influenza memory B cell responses. MN immunization was found to be at least as effective as the conventional IM route in eliciting similar levels of functional antibodies at low or high antigen concentrations, in clearing the virus from the lungs of infected mice, in conferring protection and in inducing short-lived as well as memory B immune responses as seen by the frequency of antigen-secreting cells in spleens of challenged mice. Other approaches for transdermal and intradermal immunization have also been recently reported Interestingly, we observed differences in the responses between IM and MN immunization that may have implications for vaccine development. In IM immunization, the serum IgG responses were dose related and the Th1 responses were predominant as shown by the isotype profile before and post-challenge and the cytokine data. In contrast, responses to MN immunization were similar to each other at both antigen concentrations and both doses were equally potent in inducing similar influenza-specific IgG titers. In addition the transdermal delivery with MN induced robust Th2 responses as seen by the IgG1/IgG2a ratios.Delivery of antigen into the skin by MN, which are inserted into the epidermis and the upper layer of the dermis, may activate both LC and dermal dendritic cells (DCs) which in turn convey the antigen to the draining lymph nodes It has been reported that elderly people, who face higher risk from influenza infection, have decreased IL-2 production, diminished cytotoxic T-lymphocyte (CTL) activity The induction of ASC in the lungs, which are the site of virus entry and replication, is important to maintain sustained immune responses after vaccination. We found that both concentrations of antigen delivered with MN induced the same numbers of ASC in the lung, whereas the 3 µg IM induced lower levels of these cells. Although we did not observe any significant IgA levels in the mucosa (data not shown), It is likely that one immunization is not sufficient to induce high IgA titers.In addition to the immunologic similarities and differences between MN and IM delivery, the use of MN presents several logistic advantages for vaccination. First, MN inherently delivers antigen to the APC in the skin by targeting mainly the epidermis and the upper layers of dermis. This approach may provide dose sparing as suggested by data shown in this study as well as previous studies Another advantage of MN-based delivery is the simplicity of MN administration compared to hypodermic injection. MN arrays can be integrated into patches that can be applied by manual pressing against the skin. Such MN patches are envisioned to require no special training and can be suitable for administration by minimally trained personnel or by patients themselves. Microneedles have also been shown to be painless in contrast to hypodermic needles Other advantages include the small package size of MN, which reduces storage, transportation and disposal volumes and associated costs. A MN patch is small enough to fit inside an envelope for delivery by the postal service and will occupy much less space in the cold chain, of critical importance in developing countries. MN should reduce the dangers of accidental and intentional hypodermic needle re-use and thereby increase safety. Coating MN with a dry vaccine formulation that inherently avoids the need for reconstitution should prolong their shelf life and resistance to elevated temperatures. When mass-produced, MN are expected to have a cost similar to that of a conventional hypodermic needle and syringe 50 levels will also provide further insight into the potency of MN immunization to induce protection. Another very important topic for future study is to determine the potential of MN immunization to enhance the breadth of immunity against homologous and heterologous influenza strains.Future MN immunization studies are warranted in other animal models including guinea pigs, pigs or ferrets, whose skin thickness is closer to that of human skin. Additional dose-response studies are needed to define the minimum antigen dose that will confer full protection against lethal viral infection. Challenge studies with higher LD