Anopheles gambiae at selected times during the first two days following blood ingestion, at 5 and 30 min during a 40 minute blood meal and at 0, 1, 3, 5, 12, 16, 24 and 48 hours after completion of the blood meal and compared their expression to transcript levels in mosquitoes with access only to a sugar solution.Blood feeding, or hematophagy, is a behavior exhibited by female mosquitoes required both for reproduction and for transmission of pathogens. We determined the expression patterns of 3,068 ESTs, representing ~2,000 unique gene transcripts using cDNA microarrays in adult female k-means clustering into Early Genes, Middle Genes, and Late Genes, containing 144, 130, and 139 unique transcripts, respectively. Several genes from each group were analyzed by quantitative real-time PCR in order to validate the microarray results.In blood-fed mosquitoes, 413 unique transcripts, approximately 25% of the total, were expressed at least two-fold above or below their levels in the sugar-fed mosquitoes, at one or more time points. These differentially expressed gene products were clustered using The expression patterns and annotation of the genes in these three groups (Early, Middle, and Late genes) are discussed in the context of female mosquitoes' physiological responses to blood feeding, including blood digestion, peritrophic matrix formation, egg development, and immunity. Hematophagy, blood-feeding, is a behavior exhibited by most arthropod vectors of human pathogens. In anautogenous mosquitoes, the female generally feeds to repletion on a single blood meal and then proceeds to use this nutrition as the basis for the development of a batch of eggs. The cycle of host seeking, blood feeding, egg development, and oviposition is generally called the gonotrophic cycle, a term coined by Beklemishev in 1940 [Digestion of the proteinaceous blood meal is required for oocyte development and vitellogenesis, and consequently these are coordinated processes. Multiple hormones interact to alter tissue states and to activate genes involved in these processes. The two hormones juvenile hormone (JH) and 20-hydroxyecdysone (20-E) are most fundamental to ovarian development. Within several days after emergence of female mosquitoes from the puparium, juvenile hormone (JH) stimulates the separation of ovarian follicles from germaria and limited growth of the ovarian follicle to its pre-vitellogenic resting state . OEH stiAedes aegypti, and more recently it has been investigated in An. gambiae. Despite the digestive proteolysis peak at 24 hours post blood meal, digestive enzymes exhibit two phases of transcription [Ae. aegypti there are three trypsins, early trypsin, which is constitutively expressed prior to blood feeding and two late trypsins which are blood induced. These two types of trypsins are also found in An. gambiae. The An. gambiae trypsin family includes seven genes clustered within 11 kb on chromosome 3R, in division 30A, that encode five functional proteins [An. gambiae, two of which are located in tandem on chromosome 2L, in division 25D [Prior to the blood meal, female mosquitoes access sugars for nutritional sustenance. During the first several hours following a blood meal, the mosquito undergoes physiological changes in addition to hormonal ones. Acquisition of a blood meal stimulates midgut proteolytic activity such that approximately 80% of the protein content is digested within one day -14. Sericription ,17. In Aproteins . Trypsinproteins . Trypsinproteins . In addision 25D ,20. Bothsion 25D . In contAn. gambiae midguts following blood meal ingestion. Multiple aminopeptidases have been isolated from hematophagous insects, and it has been suggested that they may play different roles in digestion [Two types of exopeptidases, carboxypeptidases and aminopeptidases, have been characterized in Anopheline mosquitoes. Edwards et al.  cloned aigestion -25. Addiigestion ,27.An. gambiae, the PM can be visualized by electron microscopy as early as 12 hours PBM and it is fully formed by 48 hours PBM [An. gambiae [In addition to dramatic changes in physiology, blood feeding also induces changes in mosquito morphology. Following gut distension by blood ingestion, midgut epithelial cells secrete a Type I peritrophic matrix (PM) that is continuous along the length of the midgut ,28,29. Pours PBM . The PM ours PBM -33. Howe gambiae . The exa gambiae ].Once the adult mosquito acquires a blood meal, she spends approximately 48 hours converting about 20% of it into egg constituents , using aAn. gambiae contains approximately 50 functional egg-production structures, the ovarioles. Each ovariole is comprised of two parts, a distal germarium and a vitellarium proximal to a common oviduct through which eggs will pass as they are laid. In the germarium, mitosis of the primordial germ cells creates a syncitium with an oocyte and seven nurse cells interconnected by intracellular bridges, or ring canals as a result of incomplete cytokinesis. Both the germ cell and the nurse cells are surrounded by a somatically derived follicular epithelium [The meroistic ovary of ithelium -39. The Ae. aegypti and Anopheles albimanus, this period appears variable, lasting 3–10 and 8–16 hours, respectively, and ends with the initiation of vitellogenin synthesis [Ingestion of a blood meal reinitiates ovarian development and follicle growth resumes. In ynthesis . In the ynthesis . FollowiOnce oocyte growth has ceased, vitellogenin synthesis terminates. This signals the onset of the post-trophic phase. During this time, the oocytes mature and eggshell structures begin to develop. The chorion, part of the eggshell, is secreted by the follicular epithelium and contains two layers, the first secreted, inner endochorion and the later secreted, outer exochorion . It is tDrosophila, developing oocytes produce the TGFα protein Gurken that binds to the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) localized to the posterior follicle cells, to initiate RAS 1 signaling. Downstream from the activation of this RTK, the GTP-binding protein RAS 1 initiates a series of enzymatic events propagated successively by three protein kinases, RAF, MAPK, and MAPK kinase (MEK), resulting in the translocation of nuclear factors and possibly the concomitant reorganization of the cytoskeleton [reviewed in [Specialization of eggshell structures necessitates communication between cells. The RAS 1 signaling cascade is an important means of communication during the processes of oocyte and eggshell patterning, as it is during eye development and differentiation of structures late in embryogenesis ,42. Duriiewed in ]. Thus, iewed in .Ae. aegypti [Vitellogenic events in the fat body have also been divided into phases: 1) Pre-vitellogenic, 2) Vitellogenic, and 3) Termination. The pre-vitellogenic phase in the fat body coincides with the pre-vitellogenic phase of the ovarian cycle. During this phase, RNA synthesis increases in the fat body and the rough endoplasmic reticulum and the Golgi complex proliferate to prepare for the production of vitellogenin. At the start of the vitellogenic phase, the release of mosquito hormones initiated by digestion signal the onset of vitellogenesis . Synthes aegypti .e.g. blood-fed vs. sugar-fed, an expression signature for each gene can be defined in response to blood feeding. Consequently, these expression patterns may indicate how these genes are regulated and interact, and also the biological processes in which the act. In this study we performed microarray analysis of genes in female mosquito abdomens during the first 48 hours after a blood meal. We have implicated many of these genes in different processes stimulated de novo by blood feeding. The elucidation of the expression profiles of abdominal genes will provide a broadened basis for understanding vector-parasite interactions. Our study certainly provides insights into the physiology of the malaria vector Anopheles gambiae.Thus, blood feeding initiates a complex series of physiological events in at least three tissues that are integrated by the actions of JH, 20-E and peptide hormones. These events may be required for parasite development; they certainly can be modulated by the presence of parasites ,48 and mAn. gambiae mosquito abdomen-derived cDNA libraries to elucidate major patterns of gene expression through 48 hours post ingestion of a blood meal. Arrays were constructed from triplicate spotted negative controls (purified water, 3 × SSC with no DNA, and empty wells), positive controls for blood-fed samples consisting of 3 clones whose ESTs corresponded to rat (R. norvegicus) α and β hemoglobin chains, and PCR-amplified fragments obtained from 1132, 721, and 1204 clones randomly picked from the sugar-fed (harvested after 30 hours at 19°C), rat blood-fed (harvested 30 hours PBM at 19°C), and P. berghei infected rat blood-fed (harvested 30 hours PBM at 19°C) abdomen libraries, respectively  using the DNAstar Seqman II software (DNAstar, CA) Table . The higAn. gambiae adult females at ten time points during and post ingestion of a blood meal to the levels in sugar-fed females. First strand cDNA was generated from total RNA collected at 5 min and 30 min after initiation of blood feeding and at 0, 1, 3, 5, 12, 24, and 48 hr post-blood meal. All cDNA populations were labeled and hybridized to arrays. For each PCR-amplified insert, Cy3 and Cy5 fluorescent dye levels were measured from 3 replicate spots on each of 50 arrays to generate average signal intensities, and an expression ratio depicting transcript fold change between sugar-fed and blood-fed mosquitoes calculated. Following quality control filtering and normalization, 456 cDNAs and the rat β-hemoglobin gene, the positive control, were expressed more than twofold above or below control, sugar-fed levels at one or more of the 10 blood-feeding time points. Following EST analysis, the 456 cDNAs were found to represent 413 unique mosquito transcripts, 10 of which were present in more than one set. This anomaly is due to EST clustering of alternatively spliced transcripts with different expression patterns. More unique transcripts are up-regulated than down-regulated in response to blood feeding, while 10% of them are both up-regulated and down-regulated over the time course of this study: 192 are up-regulated at least twofold, 173 are down-regulated at least twofold, and 48 are down-regulated and up-regulated. Bioinformatic analyses of these 413 unique transcripts showed that all sequences shared sequence identity with the An. gambiae genome  of these 413 unique differentially expressed transcripts was also conducted to support the  Figures .An. gambiae genes whose products are involved in related processes, the EST consensus sequences of the transcripts differentially expressed in these 3 patterns were annotated using sequence similarity and categorized using the molecular functions listed by the Gene Ontology Consortium (GOC) and the biological processes defined by Holt et al. [To identify t et al. . Gene anDuring the 48 hours PBM, the majority of gene products involved in metabolism were up-regulated Early and Middle Genes. Largely different metabolic biological processes were up-regulated in Early vs. Middle and Late Genes. More than half of the Early metabolic gene products, 20/29 unique transcripts, appear to be involved in carbohydrate metabolism, oxidative phosphorylation, and protein digestion. In contrast, 80% of the metabolic genes, 24/30 unique transcripts, represented in the Middle Genes contribute to various processes in protein digestion and metabolism, and metabolism of amino acids and their derivatives. One third of the Late genes involved in metabolism, 8/25 unique transcripts, are involved in protein metabolism. Five of these annotated sequences, ASs 368, 807, 1179, 279, and 922, encode products involved in post-translational modification.Reflecting the necessity of biosynthetic machinery in cell maintenance and growth, and probably also the highly conserved nature of proteins involved in housekeeping functions, the protein synthesis category contained the second largest number of genes functioning in a known process. 69%, 11/16 unique transcripts and 53%, 10/19 unique transcripts, respectively of the Early and Middle Genes, in this category are involved in translation. Approximately 25%, 5/19 unique transcripts, and 40%, 6/14 unique transcripts, of the protein synthesis genes represented among Middle and Late genes, are required for transcription and mRNA processing. This result seems almost paradoxical because transcription and mRNA processing necessarily precede translation.A number of biological processes were related by their involvement in nuclear events or the overall activity of the cell. There were three times as many genes involved in cellular processes among the Late genes than in the other two sets combined. Although all the genes involved in the cell cycle are Middle and Late Genes, the most strikingly up-regulated cellular process genes were those involved in chromatin assembly/disassembly. 5 of the 10 cellular process Late genes (ASs 1136, 59, 592, 1011, and 1792) are involved in maintenance of chromatin structure, a biological process represented only in the Late genes. These Late genes include both histones and high mobility group proteins.The majority of significantly up and down regulated genes appear to function in egg production, either in the development and maturation of oocytes or in the fat body synthesis of products that will be deposited in oocytes. Almost 90% of them, 17/19 unique transcripts, are Middle and Late genes. These genes are described in detail in the Discussion section. In contrast, half of the genes categorized as cellular communication genes, 4/8 unique transcripts, are Early genes. The majority of the cellular communication gene products in the combined sets of Middle and Late genes, are involved in different signal transduction pathways. Additionally, almost half of the intra-/extracellular architecture maintenance genes are Early genes. This category includes a wide variety of gene products such as peritrophin, both muscle-related and cytoskeletal actins, α-catenin and β-integrin. The Middle and Late genes in this category were mainly structural and included two peritrophins (ASs 13 and 642).The biological process categorized as transport included not only the movement of ions such as zinc, sodium and potassium, but also transport of molecules via receptor-mediated endocytosis. All three transport gene products, ASs 1336, 1605, and 432, in the Middle genes are responsible for the movement of ions. In contrast, in the Early and Late genes, several gene products (ASs 1974, 1071, and 2086) appear to be involved in receptor-mediated transport via clathrin-coated vesicles.A number of genes responding to oxidative stress  were found in all three sets of genes indicating that they are transcribed throughout the 48 hours PBM. Seven additional gene products most probably involved in immunity, a response to external stress, were found among these three gene sets.Expression profiles of eight selected genes and the RP S7 control gene were confirmed using a quantitative real-time PCR strategy Figure . TranscrAn. gambiae mosquitoes representing 1906 unique transcripts were determined in the first two days following ingestion of a blood meal by microarray analysis. 413 unique transcripts were shown to be up-or down-regulated at least twofold in blood fed mosquitoes relative to sugar-fed mosquitoes at one or more of the following times: 5 min and 30 min following initiation of blood feeding and 0, 1, 3, 5, 12, 24, and 48 hr post-blood meal. These transcripts were clustered into three sets with different temporal patterns of expression that may reflect the major hormonal changes occurring within the mosquito during a gonotrophic cycle. These differentially expressed gene products were annotated putatively using sequence similarity searches and categorized by biological process to identify the major events occurring post blood meal ingestion in the female mosquito.We have determined the gene expression patterns of 3,068 abdomen-derived cDNAs from adult female Multiple hormones interact to alter tissue states and to activate genes involved in the female mosquito's digestion of a blood meal, in oocyte development and in vitellogenesis. The three sets of differentially transcribed genes discerned in this study, the Early, Middle and Late genes may reflect differential hormonal responsiveness. After acquisition of a blood meal, the transcript levels of the Early genes which were abundant during blood feeding showed general declines until 24 hours PBM, after which a subset of transcript levels began to rise again. Expression of Early genes may be linked to the relatively high titers of JH present at the beginning of the first gonotrophic cycle and may then be repressed as a result of declining JH titers or of increasing 20-E. The expression of Middle gene transcripts followed an expression pattern that reflects the titers of 20-E: levels sharply increased by 12 hours PBM, remained stable or increased only slightly until 24 hours PBM, and then declined rapidly. OEH secreted by median neurosecretory cells stimulates the ovaries to secrete 20-E during vitellogenesis and the activity of this hormone begins to rise by 3–5 hours, peaks between 12 and 24 hours, and then declines to baseline levels by 48 hours post blood ingestion . In contDrosophila minidiscs gene product showed a primary response to JH, Dubrovsky et al. [minidiscs. Additionally, the majority of mosquito gene products showing sequence similarity to maternally active Drosophila genes are categorized as Late genes. Whether transcription of Early genes is directly influenced by levels of JH or 20-E, cannot be determined easily because neither hormone is present at high levels during the first few hours following blood meal ingestion. Regardless of expression pattern, the gene products in each set reflect a diverse array of processes occurring in the female mosquito within 48 hours following initiation of blood feeding. The major processes initiated in response to blood feeding including digestion, peritrophic matrix formation, oogenesis and vitellogenesis, are discussed below with emphasis on the likely roles of particular gene products.Based on their finding that the y et al.  have sugDigestion of the two different food sources, nectar sugars and blood, requires changes in the types of enzymes present within the digestive tract of the mosquito. The numbers of genes associated with sugar and protein metabolism within each set of genes may reflect the switch from sugar to protein metabolism. For instance, there are increases in transcript abundance of genes involved in carbohydrate metabolism and oxidative phosphorylation in the Early genes. However, both Early and Middle gene categories are enriched in genes involved in protein digestion. This result stems from the involvement of certain gene products in blood meal digestion that initiate a signaling cascade resulting in the up-regulation of other related proteolytic enzymes. The Middle genes contain the majority of gene products involved in amino acid metabolism, a process that follows protein digestion, whereas by 48 hours PBM, the time at which the majority of Late genes are induced, there is a generalized decrease in digestive enzyme transcripts.An. gambiae Trypsins 1 and 2 are both induced by a blood meal and exhibit similar expression profiles although Trypsin 1 is expressed at higher levels. Muller and coworkers, using an RT-PCR strategy, showed that Trypsin 2 mRNA is present at 8, 12, 24 and 28 hours post blood meal [Considering that blood contains large quantities of protein, the mosquito requires a variety of proteolytic enzymes to digest the recently acquired meal. In the present study, 11 genes were identified whose products are most likely required for protein digestion. These include 5 previously characterized digestive enzyme genes, two trypsins, a chymotrypsin, a serine protease and a carboxypeptidase. The majority of these digestive enzyme genes were transcribed at levels greater than twofold induction after 6 hours PBM. The ood meal ,52. In oood meal  and unliet al. [Ae. aegypti. An. gambiae Trypsins 3–7 may indirectly activate transcription and increase the expression of Trypsins 1 and 2, the major endoproteolytic enzymes required for blood meal digestion [Ae. aegypti and An. gambiae, these early expressed digestive enzymes are presumed to act as signal transducers causing transcriptional up-regulation of the late expressed ones [Drosophila pumpless protein, a larval fat body-expressed enzyme involved in glycine catabolism. In the fruit fly, larvae expressing the pumpless mutation are unable to pump food from the pharynx to the esophagus [pumpless mutation, Zinke et al. [Barillas-Mury et al.  demonstrigestion . In bothsed ones ,53. Thussophagus . These me et al.  proposedAn. gambiae [In addition to the trypsins, three chymotrypsin genes have been characterized in  gambiae ,20. The  gambiae . RT-PCR  gambiae . The cDNet al. [An. gambiae carboxypeptidase A following blood meal ingestion. Northern blot analysis indicated that levels of Carboxypeptidase A mRNA rose rapidly to a ten-fold increase within 3–4 hours following a blood meal, then dropped to the pre-induction state by 24 hours PBM. We identified a carboxypeptidase gene located on chromosome 2L (AS 1742) that is transcribed in a manner similar to that of carboxypeptidase A. However another cDNA representing a carboxypeptidase (AS 44) exhibited a radically different expression pattern. Transcripts were present at low levels 1–5 hours PBM but increased more than twofold between 12 and 24 hours PBM, a pattern that resembled the enzymatic activity in An. stephensi observed by Jahan et al. [Edwards et al.  investign et al. , namely An. gambiae carboxypeptidase A, the levels of aminopeptidase peaked around 30 hours in An. stephensi [et al. [An. gambiae. We identified two aminopeptidases with at least two-fold increased expression during the 48 hours PBM. The first aminopeptidase, AS 340, reached peak transcript levels at 24 hours PBM, showing a similar expression pattern to the enzyme activity levels reported by Billingsley and Hecker [et al. [Ae. aegypti aminopeptidase N. This aminopeptidase was classified as a Late gene, due to increased transcript levels at 24 hours PBM but maximal levels were not achieved until 48 hours PBM. Jahan et al. [An. stephensi depending on whether the enzyme was soluble or membrane-associated. The soluble aminopeptidase exhibited a kinetic profile similar to AS 340 and to that presented by Billingsley and Hecker [In contrast to the tephensi . Additio [et al.  recordedd Hecker  and Lemo [et al. . In contn et al.  documentd Hecker  with peaAe. aegypti although no conclusive evidence was demonstrated. However, Geering and Freyvogel [Geering  had suggreyvogel  demonstrAn. stephensi, either associated with the lumen or with epithelial lysosomes [An. stephensi, increased from 6 hours PBM to maximal levels by 24 hours and decreased to basal levels by 36 hours PBM in the anterior midgut. The transcript abundance of the α-glucosidase, AS1786, characterized in this study followed a different pattern than the enzymatic activity of An. stephensi α-glucosidase. It showed greatest amino acid sequence similarity to Drosophila melanogaster gene product CG8690 α-glucosidase and was categorized as an Early Gene with minimal transcript levels occurring at and after 24 hours PBM.The erythrocyte membrane contains a number of glycoproteins. It is therefore possible that enzymes normally associated with carbohydrate metabolism of nectar meals could also be involved in blood digestion. Almost all of the Simple/Complex Carbohydrate Metabolism and Transport genes identified in our study as being at least twofold upregulated were categorized as Early genes. Within the first 6 hours PBM, their transcripts are present at higher levels than in sugar-fed females and thereafter, they decrease steadily until 12 and 24 hours PBM. This expression profile does not exclude these genes from having a role in RBC glycoprotein metabolism. Several glycosidases are present within the midgut of An. gambiae peritrophic matrix protein Peritrophin 1 (Ag-Aper1) by analysis of Northern blots, and demonstrated that transcripts were present 6 hours PBM, increased by 12 hours, and remained elevated between 24–48 hours PBM. The present study identified several genes encoding proteins with a chitin-binding domain (InterPro ID IPR002557: Chitin binding Peritrophin-A) similar to the one found in Peritrophin 1. The Early gene AS 928 contains the Peritrophin-A chitin binding domain and maps to chromosome 3L in silico, corresponding to agCP10685. Another Early gene, AS 13, shows high identity to Peritrophin 1. A Middle gene, AS 516, is expressed by 6 hours PBM but does not reach maximal levels until 24 hours PBM. This gene product maps in silico to chromosome 2L, exhibits a similar transcript profile, and also shares 98% amino acid identity with Peritrophin 1. It is not clear why two sets of ESTs, both identified as Peritrophin 1, should exhibit different transcription patterns unless they are derived from differentially regulated genes. Another Middle gene, AS 1164, contains the Peritrophin-A chitin binding domain in addition to a prenyl-group binding site (InterPro ID IPR001230: CAAX box). This Middle gene may not be involved in peritrophic matrix formation but in some other process coinciding with protein digestion. Two other cDNAs, Accessions CD746211 and CD746202, both in AS 13, could also be localized to chromosome 2L. However, their ESTs exhibited greatest nucleotide identity to the gene predicted as ENSANGG00000020776 located 4 kb 3' to Ag-Aper1. These two cDNAs clustered as Early and Late genes respectively, and may represent alternatively spliced gene products.Shen and Jacobs-Lorena  characteAs early as an hour after the adult female has taken a blood meal, secretory vesicles previously present in the apical brush border of midgut epithelial cells are no longer detectable [Staubli et al., 1966 as cited in ]. These Drosophila ovarian development and early embryogenesis opens windows into interpreting our An. gambiae microarray results and understanding mosquito egg development. The majority of An. gambiae genes upregulated at least twofold following a blood meal appear to function in egg production. The only gene possibly involved in oogenesis during the early phases of the ovarian cycle is the Early gene AS 670. It shares sequence similarity with peter pan, a Drosophila gene product required during oogenesis. Oocytes in peter pan mutants often have an incorrect number of associated nurse cells, suggesting that the peter pan protein influences the separation of cells within the germaria [The extensive literature on genes involved in germaria . The ideDrosophila gene CG13388 encoding the protein kinase anchor protein 200, Akap200. Akap200 protein localizes to ring canals during oogenesis, regulates protein kinase C activity, and controls their morphology [Drosophila pendulin gene product, encoded by CG4799. This gene product is also required for assembly of fully functional ring canals. pendulin encodes an importin-α2, a protein necessary for the localization of the kelch gene product, CG7210. kelch encodes an actin organizer without which the ring canals become occluded and nurse cell-oocyte cytoplasmic transport is inhibited [pendulin gene, we did not find kelch. A Late gene product, AS 578, is the homolog of Cdc42, which encodes a small monomeric RHO GTPase involved in signal transduction. Rohatgi et al. [Cdc42 protein most likely links signal transduction to the actin cytoskeleton in Xenopus. In Drosophila ovaries, mutations in Cdc42 caused nurse cells to deflate and coalesce, and inhibited transfer of nurse cell cytoplasm to oocytes in late stage egg chambers [Several differentially expressed gene products found in the present study may be involved in the formation of ring canals, structures necessary for the delivery of maternal factors to oocytes. In particular, bulk transfer of cytoplasmic content from nurse cells to oocytes depends on actin structures . A Middlrphology . Late genhibited ,60. Thoui et al.  suggestechambers .Drosophila nurse cells transcribe the bicoid anterior determinant gene and the resulting mRNA is transported to the anterior region of developing oocytes via polarized microtubules [bicoid does not appear to be an anterior determinant in other insects, but other genes important for its localization are conserved. The Early gene AS 2047 shares similarity with the Drosophila cornichon gene, CG5855. In Drosophila, cornichon is required for formation of a functional microtubular cytoskeletal scaffold used to transport bicoid mRNA and the posterior group oskar gene product to their proper location within the embryo [D. virilis exuperantia, a gene whose product is also required for proper bicoid mRNA localization D. melanogaster [An. gambiae homolog of Drosophila Notch. In Drosophila, Notch signaling regulates a large number of ovarian events beginning with cyst development in the germarium and extending through oogenesis [Notch signaling activates transcription of its target genes are reviewed by Barolo and Posakony [Notch as a Late gene, its activities may be more restricted in An. gambiae, and/or reflect fundamental differences in ovarian biology. AS 1391, a Late gene product, shares sequence similarity with Drosophila Rab-protein 11. This Drosophila small monomeric RAB GTPase is also involved in the polarization of the microtubules for the organization of the posterior pole and for oskar localization there [otubules . bicoid e embryo . The Latnogaster ,66. The ogenesis . The mecPosakony . Since won there .Drosophila gene product Bicaudal C, a RNA binding protein that may play a role in translational silencing of maternal mRNAs in addition to its role in eggshell patterning [Bicaudal C result in premature translation of oskar mRNA before it has reached the posterior region of the oocyte [Drosophila gene product vasa (CG3506), an ATP dependent helicase involved in pole plasm assembly that may also be involved in translational modification of maternal mRNAs [Drosophila cup protein (CG11181). cup protein interacts with nanos, the posterior determinant, and a translational regulator of the gap gene hunchback mRNA during oogenesis, although the exact function of the cup protein still remains unknown [vasa also influences oocyte differentiation and the development of the Drosophila embryo body plan via translation of oskar, nanos, and gurken during oogenesis [An. gambiae Middle gene product, AS 2449, shared sequence similarity with the Xenopus laevis poly(A)-specific ribonuclease and also has mRNA binding motifs, thus it may also repress translation of mRNAs in embryos.To regulate the progress of oogenesis and embryogenesis, stored maternal mRNAs are translationally repressed during early oocyte development. The Middle gene product AS 2031 shares sequence similarity with the tterning . Mutatioe oocyte . The Midal mRNAs . The Lat unknown . A DEAD ogenesis -77. In aogenesis ,79. One nop5, encoding a maternally derived product of the Drosophila CG10206 gene, a component of the small nucleolar ribonucleoprotein (snoRNP) complex involved in rRNA processing [Drosophila minidiscs gene product, an amino acid transporter. In Drosophila ovarian nurse cells, JH induces the expression of minidisks and its transcripts are most likely transferred to the oocyte during nurse cell cytoplasmic streaming [Maternal nurse cells not only provide the biosynthetic machinery and mRNA needed for oocyte axis determination, but also many transcripts and proteins required for zygotic development through the cellular blastoderm stage. The Middle gene product AS 1032 shares sequence similarity with ocessing . The Lattreaming .Drosophila, the localization of the oocyte depends on cadherin-associated adhesion [Drosophila [Drosophila Star protein, a RAS 1 enhancer involved in the EGF receptor signaling pathway, either upstream or in parallel to EGFR, during formation of the embryonic ventral midline. Star encodes a single pass transmembrane protein that may be involved in the processing of gurken protein. The DNA damage checkpoint 14-3-3epsilon protein also participates in RAS 1 signaling, normally functioning downstream or in parallel to RAF, but upstream of transcription factors. The Middle gene AS 106 shares sequence similarity with Drosophila 14-3-3epsilon. The 14-3-3epsilon protein is also capable of binding to a large number of other proteins in a phosphorylation-dependent manner. One of its functions may be to alter the cell cycle by binding Cyclin B and appears to have homologs in most if not all eukaryotes [An. gambiae Cyclin B homolog, AS 1357, grouped as a Late gene.Similar to the oocyte, the eggshell undergoes dorsal-ventral patterning. Crucial to this process is the correct placement of the oocyte relative to the maternal somatic follicle cells. In adhesion . The Latosophila . The RASosophila ,42. The karyotes ,83. The Drosophila have led to the identification of a number of other gene products that may be involved in RAS 1 signaling. TppII (tripeptidyl-peptidase II), and smt3 (SUMO) were discovered in a search for lethal mutations that could enhance a weak RAS 1 eggshell phenotype [Drosophila tripeptidyl-peptidase II, the CG3991 gene, encoding a serine protease that degrades neuropeptide signals [Drosophila smt3, CG4494, whose product is ubiquitin-like protein that may tag proteins for nuclear localization or retention in the cytoplasm [smt3 protein may modulate activity of transcription factors in the follicle cells downstream of EGFR activation. However, we feel that smt3 is likely to be a minor player in RAS 1 signaling in the events following blood ingestion in the mosquito, because its mRNA reaches maximal expression after 12–24 hours PBM, unlike the other gene products we identified as potentially influencing RAS 1 signaling.Mutation screens in henotype . The Mid signals . The Latytoplasm ,87. smt3smt3 protein may also play a role in Toll signaling. This signal transduction pathway is known to be necessary for dorsal/ventral patterning of the Drosophila embryo. smt3 protein binds the NFκB homolog dorsal protein and targets this Rel transcription factor for migration to the nucleus [et al. [smt3 conjugation to the dorsal protein enhanced its transcriptional activity. smt3 protein may play other roles in the cell by altering the interactions of septins, cytoskeletal proteins involved in cytokinesis [Drosophila, septins have been found in the cytoplasm of nurse cells and at the baso-lateral surfaces of follicle cells [smt3 gene product in oogenesis. We also found that the Late gene AS 2034, a homolog of Drosophila Aos1, the CG12276 gene, the smt3 (SUMO) activating enzyme, was also expressed at least twofold more abundantly during the height of smt3 expression. This result reinforces the importance of smt3 in the events occurring between 24–48 hours PBM. nucleus ,89. Bhas [et al.  demonstrokinesis ,91. In Dle cells . These rAe. aegypti ornithine decarboxylase antizyme, a protein that modulates polyamine synthesis. The homologous Drosophila ornithine decarboxylase antizyme gene, formerly known as gut feeling, has been shown to be important in developing oocytes. It is one target of Sex lethal which encodes an RNA binding protein that regulates mRNA splicing and the mitotic events in early germ cells via regulating Cyclin B [Drosophila polo CG12306 gene product, a protein kinase required for cytokinesis and another regulator of Cyclin B [Drosophila black pearl CG5268 gene product. This protein contains DnaJ domains implying that it is necessary for cellular growth [black pearl RNA from various developmental stages showed two transcripts with greatest expression in Drosophila embryos 0–6 hours old [Drosophila Imaginal disc growth factor4 (Idgf4), a mitogen with a non-functional chitinase domain. Transcripts of Idgf4 are detected in Drosophila nurse cells, oocytes, and in the yolk cytoplasm of early embryos [Drosophila β Integrin. An. gambiae β Integrin may interact with the Middle gene AS 985 product to promote somatic cell adhesion and cell migration during oogenesis and embryogenesis. This is due to the similarity of the AS 985 gene to Drosophila Receptor of activated protein kinase C, RACK1. RACK1 can bind a number of different signaling and cell adhesion molecules including the activated form of protein kinase C (PKC), Src family kinases, and β Integrins [et al. [et al. [An. gambiae β Integrin gene was expressed at highest levels 48 hours PBM, whereas we found that the β integrin homolog reached at least twofold increased expression by 24 hours PBM.In addition to genes regulating the polarity of the embryo and eggshell, genes involved in cellular growth and differentiation were differentially expressed during the 48 hours PBM ingestion. AS 337 and 495 shared sequence similarity with the Cyclin B . The LatCyclin B . The Ear embryos . The Midntegrins -99. Cox  [et al.  demonstr [et al.  found thAe. aegypti [Dopa decarboxylase (Ddc) gene, Ddc is an enzyme involved in the tyrosine metabolic pathway necessary for eventual chorion melanization in Ae. aegypti, and other melanization events. The gene is up-regulated in response to blood meal with transcripts initially detectable by 12 hours PBM, and maximal levels achieved between 24 and 48 hours PBM [Ae. aegypti Dopachrome conversion enzyme [Ae. aegypti females, but becomes upregulated when they are infected with Dirofilaria [An. gambiae differs from Ae. aegypti in the substrate metabolized to produce chorionic melanin.A number of genes have been implicated in the development of the egg shell structures. Our screen does not appear to have identified any homologs of the several endochorionic structural proteins characterized in  aegypti ,103. Thi aegypti ,103. Ourours PBM . Howevern enzyme . This enofilaria . Since iDrosophila thioredoxin and Ae. aegypti 2-Cys thioredoxin peroxidase, may have roles in regulating reactive oxygen species that can be produced from the highly reactive quinones which are normally cross-linked into melanin immediately after they are formed.Several Middle and Late genes encoding antioxidants were upregulated at least twofold 12–48 hours PBM. The Middle gene, AS 2033, a glutathione S-transferase D3, and the three Late genes, ASs 1684, 35, and 2156, encoding glutathione S-transferase 1–6 class theta, and homologs of An. gambiae there is a small, polymorphic tandem array of vitellogenin genes and a single dispersed vitellogenin gene, all located on Chromosome 2R in division 18B (P. Romans and M. Sharakhova, unpublished observations). Vitellogenin mRNA becomes detectable by Northern blot analysis by 8 hrs PBM, though it is detectable earlier by RT-PCR, increases dramatically by 12 hours, reaches maximal levels by 24 hours, and declines to undetectable levels by 48 hours PBM [Paramount to the development of the embryo is the massive accumulation of vitellogenin by the oocyte. In ours PBM . Our micours PBM .Drosophila Adaptin subunit, AP-1σ, CG5864, and the Late gene, AS 2086, homolog of another Drosophila clathrin-associated protein, AP-50, CG7057.Following synthesis in the fat body, vitellogenins are released into the hemolymph. Eventually, they diffuse through channels between the cells of the follicular epithelium and are accumulated by the oocyte by receptor-mediated endocytosis in clathrin-coated pits . The incAe. aegypti, the lysosomal cathepsin D-like aspartic protease (AeLAP) exhibited a similar transcription profile to vitellogenin [et al. [Ae. aegypti protein, exhibits the same expression profile, and may be its homolog. At approximately 30 hours PBM, 6 hours after peak production of vitellogenin, the activity of four other lysosomal enzymes, arylsulfatase A, acid phosphatase-1, β-galactosidase, and Cathepsin D, has dramatically increased to reach maximal levels by 36–42 hours PBM [When vitellogenesis has ceased, the biosynthetic machinery in the fat body is degraded in lysosomes . In Ae. llogenin . Cho et  [et al.  also ideours PBM ,109. Theours PBM . It willet al. [An. gambiae by identifying 168 ESTs that differed in statistical abundance between cDNA libraries made from adult female mosquitoes fed on sugar and 24 hours PBM. Ribeiro [in silico data can complement each other. However, our study is unique in that we have determined the temporal patterns of expression of the genes we identified. The observed similarities between the gene expression patterns and production of the two principal insect hormones suggest that gene transcription may be influenced by changes in JH titers as well as by 20-E levels, a phenomenon that has been well-studied in the context of Drosophila metamorphosis and in Ae. aegypti vitellogenesis. Future analysis may reveal genes co-regulated via the same promoters. Indeed, this now appears possible for organisms whose genomes have been sequenced [Holt et al.  performe Ribeiro  extended Ribeiro  studies.equenced -114. As An. gambiae genome, culminating in the public announcement of the genome sequence in 2002 and its subsequent updates. Yet, although we have identified 413 differentially expressed gene products, we could not assign almost half of them to a biological process. Of the 200 "Unknowns," 43 unique transcripts shared no significant identity with sequences in the Nr and dbEST databases. The genes corresponding to these transcripts may be identified following the second gene build of the An. gambiae genome. Functional studies using microarray analysis verified by qRT-PCR must confirm in silico predicted annotations and provide biological information about gene products. Many of the gene products identified in this study share sequence similarity with Drosophila proteins. Much of the information generated by studies of fruit fly cell biology and development may also apply to mosquitoes, although it will be more difficult to test in An. gambiae, since it is not easily manipulated genetically. This study underscores the importance of ongoing functional studies including tissue-specific expression profiling using microarray analysis and qRT-PCR. Understanding how the events following blood feeding are related to each other on a molecular level will provide a more comprehensive picture of this unique behavior and may also delineate new vector-control strategies.Great progress has been made in the annotation of the An. gambiae which had been sugar-fed (harvested 30 hours post-eclosion), rat blood-fed (harvested 30 hours PBM), and P. berghei-infected rat blood-fed (harvested 30 hours PBM), all at 19°C (Dana, unpublished PhD thesis). Clones from all three libraries were subjected to PCR-based insert amplification using λTriplEx2 vector specific primers . Amplification reactions contained 1.0 μL eluted phage, 0.03 pmol of each primer, 1 × Taq Polymerase Buffer (Invitrogen), 3 mM MgCl2, 1 mM of each dNTP, and 0.2 U Taq Polymerase (Invitrogen), in a total volume of 100 μL. Reactions were conducted in 96-well plates on a Perkin-Elmer 9700 Thermocycler using the following cycling conditions: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing/elongation at 70°C for 2 min, and a final elongation step at 68°C for 3 min. Samples of all PCR products were electrophoresed on 1% agarose, 1 × TBE gels and visualized by ethidium bromide staining. PCR products were purified on a Beckman Biomek FX using Montage PCR 96 Cleanup kits (Millipore), eluted in 100 μL of water, evaporated overnight and the pellets resuspended in 30 μL of 3 × SSC microarray spotting buffer. A total of 3060 resuspended cDNA inserts and 108 controls were spotted in triplicate on CMT-Gaps II slides (Corning, NY) using the Affymetrix Arrayer 417 at 19 – 20°C and relative humidity between 50 – 60%. Slides were post-processed by baking at 80°C for three hours, incubation in 1% SDS for 2 min, in 95°C purified water for a further 2 min, and then plunged 20 times into 100% ethanol kept at -20°C and air-dried via centrifugation at 500 RPM for 5 min.Three cDNA libraries were constructed from abdomens of adult female Total RNA was extracted from blood-fed and sugar-fed whole adult female mosquitoes of the malaria susceptible 4Arr strain, 5–7 days post eclosion, using Trizol (Molecular Research Center, Inc) according to the manufacturer's directions. Mosquitoes were blood-fed on anesthetized white rats and maintained under conditions similar to those for sugar-fed mosquitoes, 25°C with 80% humidity and a 12-h light/dark cycle with available 20% sucrose solution, until collection. Fully-engorged mosquitoes were identified visually and harvested at the following 10 time-points: 1) 5 min after initiation of blood feeding during the acquisition of the blood meal (DBM), 2) 30 min DBM, 3) 0 hr post-blood meal (PBM), immediately after they ceased feeding on the rat, 4) 1 hr PBM, 5) 3 hr PBM, 6) 5 hr PBM, 7) 12 hr PBM, 8) 16 hr PBM, 9) 24 hr PBM, and l0) 48 hr PBM. For each blood-fed sample, total RNA was extracted from batches of approximately 10–15 females. Total RNA was also extracted from batches of 100 sugar-fed females for reference samples. RNA samples were then treated with 1.0 μL DNase I (Life Science Technology) according to manufacturer's instructions. Following DNase I treatment, total RNA was re-extracted with Trizol. First strand cDNA synthesis and labeling with Cyanine 3 (Cy3) or Cyanine 5 (Cy5), were performed on 15 μg of total RNA from each sample using the Genisphere 3DNA Array 50 kit according to the manufacturer's protocol. Hybridizations were conducted following the two step protocol recommended by the manufacturer: 1) cDNA hybridization to the amplified cDNA probes spotted on the slides, 2) hybridization of 3-DNA fluorescent dendrimers (Genisphere) to cDNAs via the capture sequences incorporated into them during first strand synthesis. All cDNA and fluorescent dye hybridizations were performed in a volume of 50 μL using the formamide-based hybridization buffer provided by the manufacturer. The cDNA hybridizations were performed at 45°C overnight. The slides were then washed according to the 3DNA Array 50 kit protocol and air dried by centrifugation for 3 min at 800 RPM. The 3-DNA hybridizations were performed at 53°C for 2 hours as described above, except that 0.5 mM DTT was added to the first two wash solutions to protect the fluorochromes from oxidation. Five replicate slides were generated for each of the ten time points for a total of 50 hybridized and labeled slides. These included two dye-swap experiments performed to eliminate dye fluorescence bias. Pilot experiments conducted with total RNA from the same sample labeled with both Cy3 and Cy5, self-self hybridization, indicated that there was no dye labeling bias following data normalization (data not shown).Following hybridization and washing, microarray slides were scanned successively at 532 and 635 nm using the Affymetrix 428 Array Scanner. Raw signal intensities were acquired using the adaptive circle algorithm and spot intensities quantified using the Jaguar 2.0 segmentation and data analysis software (Affymetrix, CA). Average signal intensities were normalized using the Loess curve for intensity dependent normalization followed by a per gene median normalization using the Genespring 5.1 software (Silicon Genetics, CA). Signal intensities were filtered such that only gene products exhibiting a raw signal intensity value greater than 300 pixels in one channel and greater than two-fold expression difference between the sugar-fed and blood-fed samples from at least one time point hybridized to the same array were utilized in further analysis. Gene expression level measurements falling outside one standard deviation from the mean signal intensity of each gene product calculated from the five replicates were excluded from further analysis. As an additional quality control, only genes whose PCR amplified products migrated as a single band in agarose gel electrophoresis and that generated high quality sequences for use in EST assembly were analyzed.k-means clustering (data not shown). From this preliminary analysis it was determined that three major clades existed and the genes were clustered using Genespring software using a k-means clustering algorithm with a centroid number of 3 and the Pearson Correlation distance metric. Finally, an independent analysis using principal components analysis (PCA) was conducted on the genes induced or repressed at least twofold, using the Genespring software.Gene products that were induced or repressed at least twofold during blood feeding were initially clustered hierarchically using the Genespring software to determine the user-defined number of centroids (clusters) to be used in Transcript levels of several selected genes were measured using SYBR dye technology (Applied Biosystems, CA) and quantitative real-time PCR (qRT-PCR) analysis in order to validate microarray data,. The Primer Express Software v. 1.5 (Applied Biosystems, CA) was used to design the following primers to nine genes: the two Early Genes agCP4871  and CG8690 , the four Middle Genes agCP8064 , agCP12846 , CG7758 , and agCP14019 , and the two Late Genes agCP14623  and agCP2112 , and the ribosomal protein S7 (RP S7) gene control . RP S7 was used as an internal control since its expression is constitutive during blood-feeding . All ampAND carried out the cDNA library construction, microarray fabrication, data analysis, and drafted the manuscript. YSH performed the microarray experiment, data analysis, and helped draft the manuscript. MKK performed the qRT-PCR experiments and MEH annotated the ESTs. BWH constructed the microarray genechips and carried out microarray data acquisition. NFL sequenced the cDNA library and JRH maintained and provided mosquito samples throughout the project. PR assisted AND and YSH to draft the manuscript and reviewed it. FHC (P.I.) initiated and supervised the project. All authors read and agreed on the final version of this manuscript.Supplementary Table S1 in a Microsoft Excel format where gene annotations for all 413 at least twofold differentially expressed gene products are given.Click here for file


Chlamydiales are familiar causes of acute and chronic infections in humans and animals. Human pulmonary emphysema is a component of chronic obstructive pulmonary disease (COPD) and a condition in which chronic inflammation manifested as bronchiolitis and intra-alveolar accumulation of macrophages is common. It is generally presumed to be of infectious origin. Previous investigations based on serology and immunohistochemistry indicated Chlamydophila pneumoniae infection in cases of COPD. Furthermore, immunofluorescence with genus-specific antibodies and electron microscopy suggested involvement of chlamydial infection in most cases of pulmonary emphysema, but these findings could not be verified by PCR. Therefore, we examined the possibility of other chlamydial species being present in these patients.Tissue samples from patients having undergone lung volume reduction surgery for advanced alpha-1 antitrypsin deficiency (AATD, n = 6) or non-alpha-1 antitrypsin deficiency emphysema (n = 34) or wedge resection for hamartochondroma (n = 14) were examined by transmission electron microscopy and PCR.Chlamydia psittaci was demonstrated by PCR in lung tissue of 66.7% AATD vs. 29.0% non-AATD emphysema patients. Partial DNA sequencing of four positive samples confirmed the identity of the agent as Chlamydophila psittaci. In contrast, Chlamydophila pneumoniae was detected only in one AATD patient. Lung tissue of the control group of non-smokers with hamartochondroma was completely negative for chlamydial bodies by TEM or chlamydial DNA by PCR.In all cases of AATD and 79.4% of non-AATD, persistent chlamydial infection was detected by ultrastructural examination. Intra-alveolar accumulation of macrophages and acute as well as chronic bronchiolitis were seen in all positive cases. The presence of Chlamydophila psittaci in pulmonary emphysema by linking this chronic inflammatory process to a chronic infectious condition. This raises interesting questions on pathogenesis and source of infection.These data indicate a role of  Chlamydiaceae are well-known etiological agents of acute and chronic infections in humans and animals [Chlamydia (C.) psittaci, the agent responsible for this disease, has had several different names and, according to a recent proposal, should now be called Chlamydophila (Cp.) psittaci [C. pneumoniae [Chlamydophila pneumoniae [Cp. pneumoniae infection based on serology was reported in severe cases of chronic obstructive pulmonary disease (COPD), in which emphysema is dominant [Cp. pneumoniae by immunohistochemical staining was elevated in lung tissue from subjects with COPD, but controls were not completely negative [Cp. pneumoniae was thought to be virulent for humans only, but recent descriptions of isolates from horse, koala, frog and reptiles suggest a wider host spectrum and even the possibility of zoonotic transmission [Several species of the family  animals ,2. The f animals . Chlamydpsittaci . A centueumoniae ,6 curreneumoniae . Meanwhidominant ,8, as wedominant  and in pdominant ,11. The negative . Initialsmission -15.Chlamydia spp. in patients having undergone lung volume reduction surgery for advanced pulmonary emphysema [Cp. pneumoniae was detected in two out of ten cases [Chlamydia spp. in over 80% [Chlamydiaceae. Here we report the results of a more detailed study involving a larger number of cases and samples including controls.Our previous investigations by means of immunofluorescence using a genus-specific antiserum against chlamydial LPS and scanning as well as transmission electron microscopy showed infection of the alveolar parenchyma and the bronchioles by mphysema ,17. Accumphysema . In prelmphysema , DNA speen cases . But thiLung tissue of adequate quality from patients with advanced emphysema undergoing lung volume reduction surgery was used for the present study ,21. SampFormalin-fixed lung tissue was embedded in paraffin wax , slides of 3–7 μm thickness were cut using a rotatory microtome  and stained by hematoxylin and eosin. For TEM, tissue was fixed in 2.5% buffered glutaraldehyde or 3.5% formaldehyde and embedded in epon after postfixation with osmium tetroxide and block contrastation with uranyl acetate. In the cases of the hamartochrondroma control group, cores with diameters of 0.60 cm and 0.24 cm were obtained from paraffin blocks using a prototypical self-made manual tissue puncher (a device for tissue microarray construction developed by M.W.). These cores were used to select an area well defined by light microscopy for PCR (0.60 cm cores) and TEM (0.24 cm cores). For TEM, tissue was dewaxed with xylene and processed as described above. Semithin sections  were stained with basic fuchsin and methylene blue to define blocks of adequate quality. Ultrathin sections from two to five blocks were stained with lead citrate and examined using a Zeiss EM 900 transmission electron microscope (Zeiss, Oberkochen, Germany).Tissue from two different resources was used. Firstly, frozen tissue was collected immediately after resection and stored at -80°C (n = 31). Secondly, paraffin-embedded tissue (PET) containing histologically discernible inflammation sites was used, and sections or 0.60 cm cores were dewaxed using xylene (n = 12 and 14 controls). In five cases, material was available as both frozen and PET. DNA was isolated from lung tissue using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the instructions of the manufacturer. Five μl of the DNA extract were used as template in PCR.C. psittaci and Cp. pneumoniae by a modified version of the nested PCR procedure described by Kaltenböck et al. [ompA gene. The first step was genus-specific amplification using primers 191CHOMP (5'-GCI YTI TGG GAR TGY GGI TGY GCI AC-3') and CHOMP371 (5'-TTA GAA ICK GAA TTG IGC RTT IAY GTG IGC IGC-3'). For the second amplification, we used 1 μl of the genus-specific product and primer combination 218PSITT (5'-GTA ATT TCI AGC CCA GCA CAA TTY GTG-3') / CHOMP336s (5'-CCR CAA GMT TTT CTR GAY TTC AWY TTG TTR AT-3') for C. psittaci, or 201CHOMP (5'-GGI GCW GMI TTC CAA TAY GCI CAR TC-3') / PNEUM268 (5'-GTA CTC CAA TGT ATG GCA CTA AAG A-3'), for Cp. pneumoniae, respectively. The sizes of specific amplicons are: 576–597 bp (genus-specific product), and 389–404 bp for C. psittaci or 244 bp for Cp. pneumoniae after nested PCR. A detailed protocol of the procedure is contained in [Samples were tested fork et al. , which tained in .C. psittaci-group [In order to discriminate the different members of the ci-group , five μlci-group . NucleotThe median value of age was 46.5 in AATD, 58.0 in non-AATD emphysema and 64.5 in hamartochondroma. The rate of females varied between 33.3% in AATD, 37.5% in non-AATD and 42.9% in hamartochondroma. Histology revealed destruction of the alveoli, intra-alveolar accumulation of macrophages, and acute alongside chronic bronchiolitis in all cases of AATD Fig.  and non-TEM images illustrate that the cell and tissue morphology appeared severely destroyed in emphysema samples. Chlamydial elementary and reticular bodies of 0.2 to 0.8 μm diameter were found on the surface of alveolar or bronchiolar epithelium and showed adherence to microvilli as previously described ,17. RetiC. psittaci-specific DNA in four (66.7%) specimens with AATD and nine (29%) with non-AATD emphysema . In five cases, where both PET and frozen tissue were examined, one patient was positive in TEM and another one in PCR.Examination by PCR revealed the presence of  in four 6.7% specema Fig. . In one Cp. psittaci.To confirm the identity of the chlamydial species, DNA from four of the positive samples, i.e. N16, N25, N26, and N33, was sequenced in the 16S rRNA signature region  indicate an association with regional activity of infection and inflammation. Besides psittacosis, Cp. psittaci has been recently associated with chronic inflammation in patients with ocular adnexal lymphomas [Cp. psittaci was found at comparable and statistically significant rates in AATD and non AATD emphysema.Higher detection rates of ymphomas . In COPDymphomas . The relymphomas  and by cymphomas . These fCp. psittaci provides an important indication on the source of infection. It is conceivable that the patients were infected through contact with birds, although this could not be verified for lack of relevant data on history. The detection rate of chlamydiae in cases of AATD emphysema vs. non-AATD emphysema was clearly higher, thus indicating a relevant role of Cp. psittaci infection in this disorder, or at least a higher susceptibility of AATD patients for an infection of their lungs with Cp. psittaci. Further investigations concerning smokers and non-smokers, pathogenetic relevance and zoonotic implications are required. In any circumstances, Cp. psittaci has to be considered an underestimated pathogen with considerable importance in public health, although the various facets of its specific impact have yet to be evaluated.The fact that the chlamydial agent present in the emphysema tissue was identified by DNA sequencing as an avian serovar of None declared.Dirk Theegarten has designed and organized this study, done light microscopy, written most of the manuscript, participitated in its statistical analysis and reviewed results of transmission electron microscopy. Olaf Anhenn participitated in designing the study, collected the data, performed statistical analysis and reviewed the manuscript. Helmut Hotzel carried out PCR analysis and DNA sequencing. Konrad Sachse has done the sequence alignments and written the parts of the manuscript concerning molecular biology and veterinary aspects, and also participated in PCR analysis. Mathias Wagner developed and used the tissue puncher for this study. Georgios Stamatis has done lung volume reduction surgery. Alessandro Marra and Georgios Stamatis have done the clinical parts. Alessandro Marra has reviewed the history of the patients. Grigori Mogilevski performed transmission electron microscopy. All authors have discussed and approved the final manuscript.The pre-publication history for this paper can be accessed here:


There is great interest in appropriate phenotypes that serve as indicator of genetically transmitted frontal (dys)function, such as ADHD. Here we investigate the ability to deal with response conflict, and we ask to what extent performance variation on response interference tasks is caused by genetic variation. We tested a large sample of 12-year old monozygotic and dizygotic twins on two well-known and closely related response interference tasks; the color Stroop task and the Eriksen flanker task. Using structural equation modelling we assessed the heritability of several performance indices derived from those tasks.h2 = nearly 50 %). In contrast, we found little evidence of heritability on flanker performance. For both tasks no effects of sex on performance variation were found.In the Stroop task we found high heritabilities of overall reaction time and – more important – Stroop interference (These results suggest that normal variation in Stroop performance is influenced by underlying genetic variation. Given that Stroop performance is often hampered not only in people suffering from frontal dysfunction, but also in their unaffected relatives, we conclude that this variable may constitute a suitable endophenotype for future genetic studies. We discuss several reasons for the absence of genetic effects on the flanker task. The Stroop test  is arguaThe Stroop test has been used both to tap fundamentals of human information processing (e.g. ), and asThere now exist numerous versions of the Stroop test. For example, instead of using color words, researchers have adopted more ecologically relevant items, such as emotion words, pictures of food items or of threatening objects, etc. In addition, it is now also common to use computerized versions of the Stroop task, permitting a trial-by-trial analysis of performance. But what all these different Stroop versions have in common is that the subject is always presented with a stimulus that simultaneously activates two conflicting response channels; one response is activated by the instructions, whereas the other response is activated by elements in the array that strongly invite an alternative – yet incorrect – response. In order to resolve this response conflict the subject has to direct attention to task relevant information and ignore information from the task irrelevant channel. The time needed to resolve this conflict is derived using subtractive logic, and can be used as an index of the efficiency of the attentional system under investigation.A task that is less widely used in clinical circles, but that also indexes the efficiency of the frontal network is the Eriksen flanker task. In the arrow version of this task, subjects have to respond to the direction of a left or right pointing arrow, and ignore flanking arrows that point in the opposite direction as the target arrow . SimilarThe Stroop task (and, to a lesser extent, the flanker task) has thus acquired a strong neuropsychological validation, and is nowadays widely used in clinical settings. However, studies adopting an individual differences paradigm have revealed that the time needed to resolve the response conflict in the Stroop task does not predict the time needed to resolve the response conflict in the flanker task. In an earlier study we  found thFurther insight into the nature of these interference tasks might be gained by adopting a genetic perspective on normal and abnormal frontal functioning. A wealth of studies has now shown that many frontal psychopathologies are influenced by genes. For example, the heritability of ADHD is estimated to be around 80% (e.g.). In a sdrd4 and dat1, and the efficiency of the frontal executive attention network [The usefulness of measures of flanker performance in a genetically informative design was demonstrated by a series of studies conducted by Fan and co-workers. Using a sample of healthy monozygotic (MZ) and dizygotic (DZ) twins, it was tested whether genetic variation contributed to variations in performance on basic attentional tasks . These t network . Using t network  that pernormal Stroop and flanker performance is caused by genetic variation. By using monozygotic twins, who share all their genetic material, and dizygotic twins, who share on average half of their segregating genes, the influence of genetic factors and environmental factors can be teased apart. If genetic effects are important, then members of monozygotic twin pairs will be more similar than members of dizygotic twin pairs in test performance. Conversely, if MZ twins show the same degree of resemblance as DZ twins, influences of environmental factors that are shared by both twins (e.g., the school or family environment) will be important. The contributions of additive genetic factors, shared environmental factors and unique environmental factors for explaining the variance observed for these measures can be explored using this twin design. If Stroop and/or flanker performance is found to be heritable, we will have a further genetic  validation of these tasks with respect to frontal dysfunction. Furthermore, a high heritability of performance measures may ultimately help to unravel the genetic pathways of complex psychiatric traits.In this paper we ask whether variation in This paper is a follow-up to a previous paper, where we reported behavioral data on Stroop and flanker performance . The curFor the Stroop task, the data from 5 subjects (3 first born twins and 2 second born twins) could not be analyzed because they failed to comply with the instructions. Visual inspection of the data revealed that a few subjects had an extremely high Stroop interference score. Four subjects whose interference score was larger than 120 s were excluded from the analysis.Due to technical problems the data of 24 subjects for the flanker task were not stored or collected. Furthermore, there were 2 subjects who had an extremely high error score . These subjects were excluded from the analyses.Table For the flanker task we found the following effects: For the first born twins the main effect of stimulus type was significant, F(1, 128) = 463.00, p < .001. Congruent stimuli yielded faster RTs than incongruent ones (556 vs. 662 ms). The main effect of sex was not significant (p > .1), nor its interaction with stimulus type. For the second born twin the main effect of stimulus type was significant, F(1, 132) = 556.02, p < .001. Again, congruent stimuli yielded faster RTs than incongruent ones (551 vs. 653 ms). Also, the main effect of sex was significant, F(1, 132) = 5.38, p < .05. Boys were somewhat faster than girls (587 vs. 618 ms).The same analyses done on the error rates yielded a comparable pattern of results. For the first born twins the main effect of stimulus type was significant, F(1, 128) = 84.86, p < .001. Congruent stimuli yielded more correct responses than incongruent ones (98.1% vs. 94.9%). In addition, the main effect of sex was significant, F (1, 128) = 4.57, p < .05, as was its interaction with stimulus type, F (1, 128) = 5.03, p < .05. These effects indicate that boys tend to respond somewhat less accurate than girls on incongruent trials. For the second born twins also the main effect of stimulus type was significant, F(1, 132) = 115.18, p < .001. Again, congruent stimuli yielded more correct responses than incongruent ones (98.8% vs. 93.9%). The main effect of sex was not significant, but the interaction was significant, F(1, 132) = 4.05, p < .05. Again, boys tended to respond somewhat less accurate than girls on incongruent trials.In order to test whether for the flanker task there was a trade-off between response speed and accuracy, we simply correlated RTs with accuracy, separately for the first born and second born twins. A possible speed-accuracy trade-off would manifest itself as a significant negative correlation between mean reaction time and percentage of errors. For the first born twins we found a significant positive correlation (r = .37, p < .001). But this correlation appeared to be due to a handful of subjects who were both quite slow and error prone. For the second born twins the correlation was small (r < .1), and not significant. Thus, we conclude that in our sample there was no evidence of a speed-accuracy trade-off.A similar analysis was done for the Stroop task. We correlated the average completion time of Card 3 with the number of errors commited with Card 3. We did not correlate speed with the number of corrections, because these measures are not independent. For both the first born twins and the second born twins we found a significant positive correlation (r = .31, and r = .21, respectively. p's < .05). Thus, subjects who were slow also tended to be inaccurate. However, the distribution of the number of errors was rather skewed (most subjects made 0 or 1 errors), which makes it difficult to interpret these correlations. So, similar to the flanker task, we conclude that there was no evidence of a speed-accuracy trade-off.Table minus RT [congruent]). The pattern of twin correlations is hardly indicative of genetic effects on performance. Even though the MZ twin correlations on response speed were higher than the DZ correlations, the highest twin correlation was obtained with the DOS zygosity group. Furthermore, the highest twin correlation for the flanker effect was obtained with the DZF group. In addition, the twin correlations within the monozygotic groups were low. We therefore conclude that there were no genetic effects of flanker performance.Table The twin correlations obtained with the Stroop task thus appeared strongly indicative of genetic effects. Using structural equation modelling, these effects were formally tested. But prior to testing we had to establish whether there were significant differences in variances across sex and zygosity, since one of the assumptions underlying structural equation modelling is the assumption of homogeneity of variances. We conducted the Levene test on all variables, separately for the first-born and the second born twins. For none of the variables the Levene test yielded a significant effect, with the possible exception of the completion time of Card 1 for the second born twins, F(3, 136) = 2.664, p = 0.05. So, we felt it was legitimate to use structural equation modelling to test for genetic effects.2 ranged from 7.732 to 8.883, df = 9, p's ranged from .448 to .561), with the possible exception of Card 1 (χ2 = 15.339, df = 9, p = 0.082), although the most parsimonious model for Card 1 fitted slightly better (χ2 = 17.240, df = 13, p = 0.189). Sex differences in parameter estimates could be discarded from the models, although they were almost significant for Card 3 (χ2 = 7.652, df = 3, p = 0.054). Common environmental influences were not necessary to describe the data, but additive genetic influences explained a significant part of the variance in all 4 variables. Heritabilities for the 3 cards were 75%, 70% and 74% respectively, with confidence intervals indicating that these were well above half the variance. Heritability of the interference effect was 49%, with a 95% confidence interval between 29 and 64%.In this study we assessed the heritability of performance on two well-known response interference tasks: the color word Stroop task and the Eriksen flanker task, using a large sample of 12-year old twins. The aim was to test whether Stroop performance and/or flanker performance could qualify as a suitable endophenotype for genetic frontal abnormalities, such as ADHD. First, we found that the time to complete each of the three cards was highly heritable. This may represent a general factor related to processing speed and/or rapid naming speed. Of greater importance was the finding that the interference score (the difference between completion times of Card 2 and 3) was also heritable: nearly 50% of the variation in performance was due to genetic variation. Thus, the efficiency of the network that deals with response conflict is -in part- under genetic influence.For the theoretically similar flanker task, however, there was little evidence of genetic influences on performance. Even though the MZ twin correlations on response speed were higher than the DZ correlations (as in ), the hiThe question now is whether Stroop and flanker performance can qualify as a suitable endophenotype of frontal pathologies. Recently a list of 5 criteria was compiled that are ideally possessed by endophenotypes . CriteriCriteria 1 and 2 of  state thWe have found evidence for the existence of strong genetic effects on conflict resolution, although the effects are task dependent. We conclude that performance on the Stroop test yields a better endophenotype for frontal (dys)function than performance on the flanker task. So, despite the overlapping regions of brain activation in the Stroop and flanker tasks, and despite their face-value similarity, we believe that these interference tasks differ in important, yet unknown ways.The subject group consisted of a group of 290 12-year old twins. There were 33 monozygotic male pairs (MZM), 24 dizygotic male pairs (DZM), 45 monozygotic female pairs (MZF), 16 dizygotic female pairs (DZF), and 27 opposite sex pairs (DOS). The twins participate in a longitudinal study of attention and attention problems. The twins are registered in the Netherlands Twin Registry (NTR), which is hosted by the Vrije Universiteit of Amsterdam . The twiTwin pairs were first asked in writing whether they were willing to participate in the study. Permission was also asked of the parents or caretakers. If permission was granted, the families received further information on the study, and were invited to come to the campus site to do the tests. The study was approved by the local Ethics Committee, and on the day of testing the children and their parents / legal representatives signed an informed consent form.Twins were tested on the same day. The children performed a range of neuropsychological tests that lasted approximately 4 hours per child. All tests were performed in the same order. Here we focus on the Stroop Color and Word Test  and the In the Eriksen flanker task subjects were presented with a horizontal array of 5 arrows. Subjects were instructed to attend to the direction of the center arrow, and ignore the 4 flanking ones. Subjects had to press the left key to a left facing center arrow, and the right key to a right facing center arrow. The flanking arrows could either all point in the same direction as the target arrow , or they all pointed in the opposite direction . Subjects received 40 congruent and 40 incongruent trials in a random order. For each trial, the computer stored the RT and whether the correct key was pressed. The number of valid cases in the flanker task is lower than in the Stroop task. This was due either to practical problems (some data were not collected or not stored on the computer), or because some subjects made an extremely high number of errors (less than 75 % correct). Further details of the Stroop task and the flanker task can be found in .In order to test for the effects of card type and sex on Stroop performance we performed an analysis of variance on the completion times with card type (1, 2, and 3) as within-subjects factor, and sex  as between subjects factor. For the flanker task, we performed an ANOVA on the mean correct response times and on the mean percentages correct, with stimulus type (congruent vs. incongruent) as within-subjects factor, and sex  as between subjects factor. The same analysis was done for the error rates. These analyses were done separately for the first-born twins and the second born twins, because twins within a family do not yield independent data. We adopted an alpha-level of .05.minus Completion time Card 2. There exists another method to determine the size of Stroop effect, which also takes into account the completion times of Card 1 [From the completion times we also calculated the size of the Stroop effect (i.e., the interference score), which is simply defined as Completion time Card 3 f Card 1 ,29. But Data from monozygotic (MZ) and dizygotic (DZ) twins were used to decompose the variance in performance on the both tasks into a contribution of the additive effects of genes, environmental influences that are shared by twins living in the same family, and environmental influences that are not shared by twins. Resemblance between MZ twins is an effect of both their common genetic constitution and their shared environment. Because DZ twins share on average half of their segregating genes, the shared environment contributes fully, but genetic factors only partly to their resemblance. Therefore, if the degree of MZ resemblance on some measure is higher than the degree of DZ resemblance we have strong evidence for the influence of genetic effects.h2 = 2(rMZ-rDZ)] [Pearson correlations were calculated for the different measures between first born and second born twins for all zygosity groups. A first indication of the heritability can be derived by doubling the difference between correlations for MZ twins and those for DZ twins [MZ-rDZ)] .a, c and e represent the influence of genes, common environment and unique environment on the phenotypes (P) of twin 1 and twin 2. The total variance of the phenotype (P) = a2 + c2 + e2. The heritability (h2) is calculated as a2/VP.A structural equation modeling approach as implemented in Mx  was useda, c and e to be equal across sexes was compared to one in which they were allowed to vary. After this, the significance of c and a was investigated by dropping them one by one from the model and comparing the fit of a full model to that of a reduced model. The chi-squared statistic is computed as twice the difference between the likelihood for the full model (-LL0) and that for a reduced or constrained model (-LL1) (χ2 = 2 × (LL0-LL1)) and is tested against the difference in degrees of freedom between the two models.To test if parameter estimates are equal for boys and girls the fit of a model with constrained parameter estimates for JFS drafted the manuscript, and performed the descriptive analyses. GCMvB performed the genetic analyses. TJCP collected the data. FCV and DIB conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.h2), common environmentability (c2), and unique environmentability (e2), for completion times of the three cards, and of the Stroop interference score. Best fitting models are shown in bold. Parameter estimates are given in percentages .Model fitting and parameter estimates Model fitting and parameter estimates of heritability (Click here for file


Intravenous morphine use is associated with elevated histamine release leading to bronchoconstriction, edema and hemodynamic instability in some patients. This study evaluated the possibility that sulfite, which is present as a preservative in many morphine preparations, might contribute to histamine release in vitro.The human mast cell line, HMC-1, was exposed to various morphine concentrations, in the absence of sulfite, under cell culture conditions. Clinically attained concentrations of morphine (0.018μg/ml and 0.45μg/ml) did not cause increased histamine release from mast cells. There was a significant increase in histamine release when the morphine concentration was increased by 1184-fold (668μg/ml morphine). Histamine release from mast cells exposed to morphine and/or sulfite required the presence of prostaglandin H synthetase. Histamine release in experiments using sulfite-containing morphine solutions was not statistically different from that observed in morphine-only solutions.in vitro experiments of the human mast cell line, HMC-1. This does not preclude the fact that sulfite may lead to elevation of histamine levels in vivo.Sulfite in sulfite-containing morphine solutions, at concentrations seen clinically, is not responsible for histamine release in  Morphine is among the most common analgesic agents used in the intensive care setting. It is usually administered parenterally as an intravenous infusion. Despite its efficacy, its use is associated with a number of hazards including respiratory depression , toleranA number of clinical studies have demonstrated an increase in plasma histamine concentration following intravenous morphine administration -7, but tIn the body, sulfites are generally oxidized by the mitochondrial enzyme sulfite oxidase and non-toxic metabolites are excreted in the urine . A smallSulfite radicals are chemically reactive and have been implicated in lipid , proteinThe potential generation of free radicals from sulfite preservatives may confound our understanding the association of morphine with histamine release in man. A number of studies have demonstrated that, when exposed to free radicals, mast cells may release histamine -26. It iTotal histamine content of mast cells was calculated by freezing and thawing suspensions for 3 cycles. Cell suspensions containing 500,000 cells/ml released 1.27μg/ml (0.21) histamine, corresponding to 2.53 pg (0.41) histamine per mast cell.Mast cells stimulated with 0.25μg/ml calcium ionophore released 63% (7.2) of total histamine.The effect of histamine release from mast cells treated with both clinically attained concentrations of morphine and concentrations above those seen clinically was determined.To chemically defined reaction mixtures, morphine was added at varying concentrations (18 ng/ml, 450 ng/ml, 6.68μg/ml and 668μg/ml). Enzymes causing free radical activation were not present. The blank sample contained no morphine. Reaction mixtures were incubated in a water bath at 37°C for 60 minutes. Experiments were performed in triplicate. Histamine release from samples was determined following derivitization with o-phthaldialdehyde and mercaptoethanol using high-performance liquid chromatography as detailed below. Histamine release from reaction mixtures was expressed as a % of total histamine present in the cell pellet.Histamine release from reaction mixtures containing morphine at all concentrations was not statistically different p > 0.1) from histamine release from blank samples  was added to the reaction mixtures described previously. Blank samples contained neither morphine nor sulfite. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as above.There was no statistical difference in histamine release at any concentration of morphine with sulfite compared with blank samples Table .Reaction mixtures containing 668μg/ml morphine with 0.1% sodium bisulfite were incubated for 5, 10, 20, 40 and 60 minutes yielded results identical to those observed in the absence of sodium bisulfite (results not shown).Prostaglandin H synthestase may catalyze free radical formation . The firProstaglandin H synthetase was added to reaction mixtures at a concentration of 25 mU. In reaction mixtures containing no drug, the enzyme was added to mast cell samples containing neither morphine nor sulfite. Controls contained no enzyme.Reaction mixtures containing the enzyme alone (no sulfite or morphine) produced similar histamine release compared to blank samples (3.2% (0.7) of total and 2.9% (2.6) of total respectively). The presence of prostaglandin H synthetase had no effect.In vivo studies show histamine release at these concentrations of morphine [in vivo concentration following an intravenous morphine bolus) and 18 ng/ml  [The effect of clinically attained concentrations of morphine in the presence of prostaglandin H synthetase on mast cell histamine release was investigated. morphine -7. Morphnfusion) . Mast ceBlank samples contained water instead of morphine. All samples contained 25 mU of prostaglandin H synthetase. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as described below. Histamine release values are shown in Table The effect of sodium bisulfite 0.1% in varying concentrations of morphine in the presence of prostaglandin H synthetase was investigated. This preservative is present in many morphine preparations and its effect on histamine release from mast cells is determined here. Morphine was added to the mast cell samples at concentrations of 668μg/ml, 6.68μg/ml, 450 ng/ml and 18 ng/ml. Sodium bisulfite was added to samples at a concentration of 0.1% of morphine concentration. Controls contained morphine only. Each sample also contained 25 mU of prostaglandin H synthetase. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as described below. Reaction mixtures containing morphine 668μg/ml with 0.1% sodium bisulfite showed a statistically significant increase in histamine release compared with blank samples (p = 0.03). There was no statistical difference in histamine release between morphine only samples at all concentrations and samples containing morphine and 0.1% sodium bisulfite  showed a significant increase in histamine release in the presence of prostaglandin H synthetase compared with no enzyme. This was true for both sulfite-free (p = 0.003) and sulfite-containing (p = 0.009) morphine solutions. This suggests that even at clinically-attained morphine concentrations, it is not only possible to cause the metabolic activation of sulfite, but also that of morphine.The effect of sulfite alone (without morphine) on histamine release from mast cells was investigated. To 4 mast cell reaction mixtures, varying concentrations of sulfite (0.018 ng/ml, 0.45 ng/ml, 0.007μg/ml and 0.67μg/ml) were added. These concentrations of sulfite correspond to the concentration of sulfite present in morphine solutions of 18 ng/ml, 450 ng/ml, 6.6μg/ml and 668μg/ml. To each sample 25 mU prostaglandin H synthetase was added. The highest concentration of sulfite (0.67μg/ml) was incubated for 10, 20, 40 and 60 minutes to assess time dependence. The reaction mixtures were incubated, centrifuged and analyzed as described below.In the presence of prostaglandin H synthetase, sulfite resulted in histamine release from mast cells. There was a significant increase in histamine release (9.7% (0.65) of total) in the reaction mixture containing 0.67μg/ml sulfite (p = 0.01). There was no statistically significant difference in histamine release compared with blank samples at all other concentrations of sulfite (0.018 ng/ml sulfite (2.8% 0.3) of total, p = 0.91), 0.45 ng/ml sulfite (1.93% (0.35) of total, p = 0.53) and 0.007μg/ml sulfite (4.5% (0.7) of total, p = 0.36)  at 10 minutes incubation, 7.7% of total (2.2) at 20 minutes, 10.5% of total (6.1) at 40 minutes and 9.7% of total (0.65) at 60 minutes.Our experiments showed similar histamine release from the mast cell line HMC-1 stimulated with morphine with or without 0.1% sodium bisulfite. Maximum histamine release was 16.1% of total in morphine only samples and 17.6% of total in morphine with 0.1% sodium bisulfite samples. This percentage is similar to other studies of histamine release in the human mast cell line HMC-1, where maximum release was 20–30% -30. In oin vivo situation, where maximum release usually occurs within 5 minutes of morphine administration, and suggests that we were not able to simulate exactly the human environment where histamine release occurs. It is likely that although the sulfite radical is formed rapidly in vitro, it may take some time for its effects to become evident [HMC-1 cells contain histamine and release their histamine content in response to various exogenous stimuli -30. Our  evident .in vivo.Metabolic activation of sodium bisulfite may occur through a variety of mechanisms. We investigated a number of enzyme systems and our final model used prostaglandin H synthetase. Other enzyme systems (horseradish peroxidase/ hydrogen peroxide and hypoxanthine/ xanthine oxidase) did release histamine in our model (results not shown), but they produced excess background interference in the chromatograms and made identification of peaks difficult. Activation of sulfite following morphine administration may not occur through this mechanism, however, and is another possible flaw in our aim to construct a model similar to the one that exists in vivo, but it has not been investigated whether sulfite, in the form of the sulfite radical, is the cause of this release. Our study finds that sulfite in sulfite-containing morphine solutions, at clinically attained concentrations, is not responsible for histamine release in in vitro experiments of the mast cell line HMC-1, but this does not preclude the fact that sulfite may lead to marked elevation in histamine levels in vivo. The release of a significant amount of histamine with either sulfite or morphine at concentrations higher than those seen clinically suggests that both substances may be responsible for histamine release from mast cells. Further clinical research studies need to be performed to examine this possibility.Morphine has been shown to cause significant histamine release in vitro experiments of the mast cell line, HMC-1, but this does not preclude the fact that sulfite may lead to marked elevation in histamine levels in vivo. Our experiments suggest that at high concentrations, both morphine and sulfite are responsible for histamine release from mast cells. Further in vivo research needs to be performed to examine this possibility.Our study finds that the sulfite in sulfite-containing morphine solutions, at concentrations seen clinically is not responsible for histamine release in 2 vented tissue culture flasks  containing Iscoves modified Dulbecco's medium (Sigma, St.Louis, MO) with 10% iron-supplemented calf serum (Hyclone, Logan, Utah) and monothioglycerol 1.2 mM (Sigma, St.Louis, MO). Incubations were conducted in a humidified atmosphere at 5% CO2 and 37°C. Cells were passed weekly and fed only when necessary (evidence of color change in media).Human leukemic mast cells, HMC-1  were kinTo pass cells, the cell suspension was placed in a 50 ml polypropylene centrifuge tube  and centrifuged at 250 × g at 4°C for 10 minutes. The culture medium was discarded and the cells were re-suspended in fresh medium. Cell density was maintained at 500,000 cells/ml.Reagent grade O-phthaldialdehyde (OPA), mercaptoethanol, dihydrogen sodium phosphate, disodium EDTA, sodium bisulfite, morphine sulfate, hydrogen peroxide, xanthine oxidase, boric acid and phosphate buffered saline (PBS) were purchased from Sigma (St.Louis, MO, USA). Methanol (HPLC grade), acetonitrile (HPLC grade) and sodium hydroxide were from Fisher Scientific (Hampton, NH). Phosphate buffered saline (PBS) pH 7.4, contained 120 mmol/L NaCl, 2.7 mmol/L KCl and 10 mmol/L phosphate. Horseradish peroxidase was purchased from Cooper Biomedical. Prostaglandin H synthetase was purchased from Calbiochem (Darmstadt, Germany).Mast cell suspensions, containing approximately 500,000 mast cells were, aliquoted into 1 ml samples. These were rinsed 4 times with 4 ml of PBS, pH 7.4. Cells were then re-suspended in 1 ml PBS. Reaction mixtures were supplemented with morphine, sulfite and prostaglandin H synthase at this stage according to experimental design, unless otherwise specified. Reaction mixtures were incubated for 60 minutes in a water bath at 37°C. Reactions were stopped by centrifugation at 250 × g for 4 minutes at 4°C and placed on ice. Then 200μl of the supernatant was collected and dried under nitrogen at 40°C.Histamine in the supernatant was measured using derivitization with OPA and mercaptoethanol (ME) as described . Dried sThe response of HMC-1 cells to the stimulant calcium ionophore was investigated to assess the releasability of HMC-1 cells. 0.25μg/ml calcium ionophore was added to mast cell suspensions. Reaction mixtures were incubated and analyzed as above.To extract the remaining histamine from the original sample, the mast cell pellet in suspension was frozen and thawed 3 times. It was then centrifuged at 250 × g for 4 minutes and 200μl of the supernatant was dried under nitrogen as described previously. Samples were re-dissolved, derivitized and analyzed as above. Histamine release during morphine incubations was expressed as a percentage of total histamine release.Histamine was measured by high-performance liquid chromatography using a Varian model 5500 chromatograph. The mobile phase was 0.1 M dihydrogen sodium phosphate and 1 mM disodium EDTA with 16% methanol and 14% acetonitrile adjusted to pH 6.4. The flow rate was 0.8 ml/minute.Separations were performed using a 15 cm × 4.6 mm, Ace 5 C18 reversed-phase cartridge column (Advanced Chromatography technologies, UK). A Zorbax C8 guard column with a 5 micron, 4.6 × 12.5 mm analytical guard-column cartridge  was used. Fluorescence detection was used with the fluorimeter (Varian 9070) set at an excitation of 348 nm and an emission of 444 nm .Chromatographic conditions involved the use of a mobile phase gradient to optimize peak separation. A linear gradient starting at 90% mobile phase, 10% acetonitrile and ending at 80% mobile phase, 20% acetonitrile took place over 20 minutes. The column was washed by increasing the acetonitrile to 30% by 22 minutes and by 23 minutes the proportions were returned to the original values. The length of each run was 30 minutes.Values are expressed as a mean +/- SD of 3 experiments. One-way analysis of variance was used to determine the significance of the difference between groups. Results were considered significantly different when p < 0.05.EG participated in experiments and drafted the manuscript. JB conceived of the study and participated in its design. CM suggested the methodology of experiments. All authors read and approved the final manuscript.