Drosophila is a model system for tubular organ development, few studies have addressed the development of the fly's oviduct. Recent studies in Drosophila have identified mating-responsive genes and proteins whose levels in the oviduct are altered by mating. Since many of these molecules  have known roles in the differentiation of muscle and epithelia of other organs, mating may trigger similar differentiation events in the oviduct. This led us to hypothesize that mating mediates the last stages of oviduct differentiation in which organ-specific specializations arise.In both vertebrates and invertebrates, the oviduct is an epithelial tube surrounded by visceral muscles that serves as a conduit for gamete transport between the ovary and uterus. While Using electron- and confocal-microscopy we identified tissue-wide post-mating changes in the oviduct including differentiation of cellular junctions, remodeling of extracellular matrix, increased myofibril formation, and increased innervation. Analysis of once- and twice-mated females reveals that some mating-responsive proteins respond only to the first mating, while others respond to both matings.We uncovered ultrastructural changes in the mated oviduct that are consistent with the roles that mating-responsive proteins play in muscle and epithelial differentiation elsewhere. This suggests that mating triggers the late differentiation of the oviduct. Furthermore, we suggest that mating-responsive proteins that respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Taken together, our results establish the oviduct as an attractive system to address mechanisms that regulate the late stages of differentiation and maintenance of a tubular organ. Most internal organs, including the vascular and respiratory systems and the gastro-intestinal and urinary-genital tracts are comprised of a single epithelial tube or a network of tubes. Tubular organs serve as conduits for the transport of gases, liquids, or solutes, and serve as barriers between biological compartments. To create tubes with specific flow and barrier properties, the morphology of the tube must be precisely specified during development and modulated by physiology. To accommodate specific physiological roles, tissue-specific programs for differentiation are employed at the last stages of development. While much is known about the molecular and cellular basis of tube formation -6, littlDrosophila an excellent model for understanding organogenesis in higher animals. In Drosophila, the best understood tubular organs from a developmental point of view are the trachea and salivary gland. Studies of these organs reveal a general program for tubular organ development, in which combinatorial expression of global patterning genes specifies positions within the embryo for the subsequent activation of tissue-specific early genes and transcription factors. This program results in the activation of downstream genes involved in terminal differentiation of organ-specific specializations such as the cuticle that lines the tracheal lumen [The conservation of genes and similarity in tubular organ design across taxa make al lumen ,2,4,7-9.Drosophila female reproductive tract is another tubular system, consisting of the uterus and a common oviduct (the main tube) that branches into two lateral oviducts. Regional differences in function are observed along the length of the tract, with egg activation occurring largely at the proximal end, in the lateral oviducts, and fertilization occurring at the distal end, in the uterus [The e uterus ,11. UnliDrosophila, mating induces changes in female behavior and physiology via molecules transmitted in the seminal fluid. These changes are rapid and lead to a mated female state which is profoundly different from the unmated female state. While an unmated female lays few eggs and readily accepts the courtship efforts of a male, a mated female exhibits increased egg-laying and actively rejects males [In ts males -19. Micrts males ,21. Rapits males ,23.In the upper reproductive tract , mating induces an increase in immune related transcripts and down regulates transcription factors involved in cell growth and differentiation. At the protein level mating induces increased abundance of proteins associated with muscle assembly and function and cytoskeletal proteins associated with epithelial morphogenesis . Since mOur results show that the reproductive tract is an attractive system to address mechanisms that regulate the late stages of tissue differentiation in a tubular organ. Unlike other tubular organs, the last differentiation stage of the oviduct is triggered by an extrinsic cue (mating). This makes it possible to experimentally control the onset of differentiation, with an opportunity to independently examine the effects of mating and age. In addition, it allows us to examine processes essential for reproduction.Our previous molecular profiling showed that mating promotes changes in actin-based cytoskeletal molecules and suggests that mating triggers molecular changes and tissue remodeling in the female reproductive tract that mediate its progression to a mature functional stage . To gainIn nearly all mated reproductive tracts processed for microscopy (8/9), an egg was located in one of the lateral oviducts, whereas an egg was never observed in the oviduct of unmated reproductive tracts (5/5) Figure . This obBecause 3-day-old mated females lay eggs, it is unclear whether the lack of lumenal material and increase in lumen size in mated females occurred before or after the passage of eggs. We suggest that mating directly or indirectly induces morphological changes in the oviduct that facilitate egg passage through the duct. Taken together, our observations lead us to propose that the oviduct lumen is closed and/or obstructed in the unmated reproductive tract, and that mating induces changes in the epithelia and/or muscle that "open" the oviduct lumen.Drosophila, most ectodermally derived epithelia (such as the epidermis and trachea), with a few exceptions, are joined apically by a belt-like adherens junction called the zonal adherens junction (ZA) followed basally by a septate junction (SJ) [To determine whether mating induces specific morphological changes in the oviduct epithelia post-mating, we next examined the ultrastructure of the oviduct epithelia in unmated and mated reproductive tracts. Since molecular profiling demonstrates that proteins associated with cellular junctions such as α- and β-Spectrin (Spec), Cora, and Nrg  increaseion (SJ) . Our anaDrosophila [Based on their ultrastructure, two types of SJs, smooth and pleated, can be distinguished in osophila . Smooth osophila ,28, and Given the presence of extensive SJs in the oviduct and the role of SJs in regulating apical secretion of extracellular matrix molecules in other epithelia (e.g. trachea), we asked if mating modulates apical secretion in the oviduct epithelia. Our ultrastructural analysis reveals that different regions of the oviduct display different apical membrane morphology (i.e microvilli or pleats)  called the basal lamina that covers the apical and/or basal membranes and the concomitant development of hemi-adherens junctions (HAJs). HAJs connect the cell cytoskeleton with the ECM and are formed at virtually all cell surfaces that contact an ECM. HAJs can be distinguished at the ultrastructural level as a patch-like, electron dense undercoat of the plasma membrane that opposes the basal lamina ; Figure . In the ame time . Becauseame time ,29, thisOne of the most striking post-mating changes observed in the oviduct epithelia was the appearance of numerous HAJs along the basolateral membrane in the upper oviduct Figure . The impmef2 and mlp84B genes that regulate muscle differentiation, are expressed and increased post-mating in the oviduct, as well as in the sperm storage regions of the reproductive tract [While we uncovered post-mating changes in the oviduct epithelia that might facilitate its transition to a high egg-laying state, this transition may also be mediated by changes in oviduct muscle properties and/or activity. The oviduct is lined by circular muscle fibers with supercontractile characteristics . Our preve tract ,23. Thisve tract . Like epve tract . Since tve tract , we hypoDrosophila reveal that the fly's oviduct receives aminergic, peptidergic and glutamatergic input [Drosophila, different types of boutons are formed by neurons that express different neurotransmitters and modulators [bullwinkle (bwk) gene [Bwk encodes a HMG-box containing putative transcription factor [th abdominal segment. These muscles are innervated by boutons that increase in number during metamorphosis [Drosophila demonstrate that OA inhibits oviduct contraction, while glutamate activates oviduct contraction [Drosophila, electrical stimulation of the posterior abdominal nerve gives rise to a series of muscle contractions in the oviduct followed by a period of muscle fatigue or relaxation [bwk expressing neurons that innervate the oviduct, Rodriguez-Valentin et al. [Nerve-muscle interactions play an important role in regulating adult muscle development and refining the final pattern of innervation ,35. Giveic input ,36-39. Idulators -42. By sdulators  report tdulators  further dulators ,43. In an factor . To deten factor . Type I n factor . To deteorphosis . No signtraction ,43,48. I+ pump α subunit (ATPα), another protein associated with septate junctions in epithelia, is not a mating-responsive protein and was used as a control. Using western blots, we first determined the abundance of the cytoskeletal proteins in oviducts of 3-day-old unmated and mated females at 6 hrs post-mating. We confirmed the proteomic results of Kalpenikov et al. [To gain insights into the role of cytoskeletal protein enrichment ; additio in mediav et al.  and founTo determine whether the increased abundance of mating-responsive proteins persists for longer times post-mating, we examined oviducts of 10-day-old females that mated once at 3 days of age, and calculated the abundance of the mating-responsive proteins relative to their level in oviducts of 3-day-old unmated females. We found no change or a slight increase in the relative abundance of all cytoskeletal proteins except Mlp84B at 7 days post-mating Figure . StrikinTo determine if the changes in cytoskeletal protein abundance are mating-dependent we measured their abundance in the oviducts of unmated 5- and 10-day-old females. We calculated their abundance relative to their level in oviducts of unmated 3-day-old females Figure . We ratiTaken together, our results suggest that mating is essential to fine-tune the levels of the mating-responsive proteins examined in this study. Because the changes in cytoskeletal protein abundance are different in unmated and mated females, this suggests that the post-mating changes are mating-dependent. Furthermore, we suggest that these post-mating changes are linked to changes in oviduct function.Drosophila, female fecundity decreases with age [p < 0.0001). Twice-mated 10-day-old females also laid about 50% more eggs than once-mated females of the same age , but about 20% fewer than laid by once-mated 3-day-old females during the three days examined   and slightly more fertile than twice-mated 10-day-old females   Figure  and 5B. ) Figure . BecauseDrosophila as a model system for organ-level biology and the emerging parallels between mammalian and Drosophila reproductive biology [Drosophila oviduct. Our results provide several lines of evidence at the molecular, morphological and physiological levels suggesting that mating induces tissue-wide differentiation in the oviduct. Moreover, we identify ultrastructural changes in the mated oviduct that are consistent with the roles that some of the mating-responsive proteins examined in this study  are reported to play in muscle and epithelial differentiation elsewhere. For example, the increased abundance of Mlp84B, a major regulator of the late differentiation pathway of muscle [Despite the use of  biology , this isf muscle  is consif muscle  is consif muscle ,28. OtheMlp84B transcription occur during periods of embryogenesis and metamorphosis when muscle is differentiating [Analysis of protein abundance following different mating regimes  gave us further insights into the possible roles that mating-responsive proteins play in the oviduct. For example, Mlp84B is only responsive to the first mating, while the epithelial proteins examined  are responsive to the first and second mating. Furthermore, the response to the second mating is different from the response to the first mating. Taken together, these results suggest that Mlp84B is required for the final maturation of the oviduct, while the epithelial proteins examined are required for both the maturation and maintenance of the oviduct at a high functional state. Moreover, the post-mating pattern of Mlp84B supports the idea that the first mating induces the final maturation of the oviduct. The rise and fall of Mlp84B abundance after the first mating Figure  parallelntiating .Using different mating regimes we tested whether the first/early mating is essential for maintenance of high reproductive output in the second mating. Our results suggest that mating at an early age is essential to achieve maximum reproductive output (i.e. high fecundity and fertility). Since the first mating increases reproductive output (evidence from our mating regime experiments), it is likely that the ultrastructural changes detected in mated 3-day-old females lead to a highly functional oviduct.We suggest that the final maturation of the oviduct includes a mating-dependent stage. We propose that during the first few days post-eclosion, the oviduct undergoes the first phase of differentiation, after which the oviduct is developmentally poised for a rapid response to an extrinsic cue (mating). Mating then triggers the second phase of maturation (tissue remodeling and modulation) which is essential for proper oviduct function Figure . We furtWhat is the benefit of mating-induced differentiation of the oviduct tissues? Unmated females are capable of laying eggs, albeit at a reduced rate as compared to mated females of the same age. One possible interpretation is that reproduction is energetically costly, thus delaying oviduct maturation until sperm is available is advantageous to the female. This may reflect the evolution of a mechanism to optimize reproductive capacity in early adulthood in short-lived animals.Drosophila affords us the opportunity to uncover the signaling pathways that coordinate these events to produce a physiologically functional organ.In summary, we have identified events at the cellular, molecular and physiological levels that are part of an efficient and specific program for reproduction. Wild-type Canton-S flies were used for the fecundity/fertility experiments and confocal analysis. Wild-type Canton-S5  flies weFor all assays (unless described differently) unmated females were placed with 3-day-old unmated males and observed until mating initiated. At the end of mating, females were aspirated into fresh vials and held for 6 hrs. At 6 hrs after the start of mating, females were placed on ice for dissection. Previous molecular studies indicate changes in protein abundance at 3 hrs post-mating. We hypothesize that these molecular changes will translate into morphological changes in the next few hours. We chose to analyze the morphology of mated females at 6 h post-mating as opposed to later times post-mating because the changes observed at later times may be due, in part, to the high rate of eggs passing through the oviduct.Drosophila medium (Sigma) on ice and processed for electron microscopy as described in [Reproductive tracts were dissected in Schneider's ribed in . Tracts 2; 0.1 mM CaCl2) with 5% (w/v) sucrose on ice, fixed in 4% paraphormaldehyde in PBS  for 45 min and then washed in PBS. The reproductive tracts were then incubated in blocking solution  for 2 hrs at room temperature. The following primary antibodies, reagents, and dilutions were used: Cy3-conjugated goat anti-HRP, 1:200 ; mouse anti-Disc Large (DLG), 1:1000 , Alexa Fluor 488-phalloidin, 1:200 . Secondary antibodies were Alexa Flour 488-conjugated Goat anti-mouse, 1:200 and Alexa Flour 546-conjugated Goat anti-rabbit, 1:200 . Reproductive tracts were incubated with the different primary antibodies (diluted in PBS + 0.2% Triton x-100) for 2 hr at room temperature, washed with PBST, incubated with secondary antibodies for 2 hrs at room temperature and washed with PBS. Reproductive tracts of the different treatments were mounted with Antifade media [Reproductive tracts were dissected in Yamamoto's Ringer  were collected and projected as a reconstructed three-dimensional image using LSM image browser  software. Image collections were identical for each of the different reproductive tract regions analyzed.To quantify the number of boutons in the lateral and common oviducts we used ImageJ software  to analyze confocal images of anti-HRP and anti-DLG labeled boutons in the oviduct. The number of boutons per unit area was quantified with the Particle Analysis Tool. Briefly, to differentiate between the boutons, the particle analysis tool requires the image to be a "binary" image , thus we first converted the images to gray scale. We then set a "threshold" range so that pixels in the image whose value lies in this range are converted to black; pixels with values outside this range are converted to white. We next defined a region of interest (ROI) within the oviduct to count particles (i.e. count boutons). This ROI was saved and served to measure the number of boutons per unit area in each treatment. For each oviduct we counted the number of anti-HRP and anti-DLG labeled boutons in two ROIs within the lateral oviducts and two ROIs in the common oviduct. One-way ANOVA (SPSS 15.0) was used to measure the difference in bouton number per unit area in different regions of the oviducts, in both unmated and mated females.3); (ii) aged for 3 days, mated with 3-day-old unmated males and their oviducts were dissected after 7 days (Once3 day10); (iii) aged for 3 days, mated first with 3-day-old unmated males and held singly for 7 days. At 10 days of age, female were mated again with 3-day-old unmated males. Oviducts were dissected at 6 hrs post-second mating (Twice3&10). We also examined 5-day-old and 10-day-old unmated females .To evaluate the effect of mating on the abundance of the cytoskeleton proteins tested, females were: (i) aged for 3 days, mated with 3-day-old unmated males and their oviducts were dissected after 6 hrs post-mating  of each band was determined using ImageJ software . We evaluated protein abundance by measuring the mean gray value of a specific band and the background. The mean gray value of the background was then subtracted from that of the measured band. Relative protein abundance in mated oviduct vs. 3-day-old unmated oviduct was then calculated. Four independent biological replicates were prepared for each mating status. The reported abundance ; (ii) aged for 10 days and mated with 3-day-old unmated males (Once10); (iii) aged for 3 days, mated first with 3-day-old unmated males, held for 7 days and mated again with 3-day-old unmated males (Twice3&10). In all cases male and female pairs were observed to record mating initiation and termination.To evaluate the effect of mating on reproductive output females were treated as follows: (i) aged for 3 days and mated with 3-day-old unmated males  to fresh vials. The number of eggs laid and the number of eclosed adults were counted from vials created at 6 hrs, 1, 2 and 3 days post-mating. To ascertain the baseline of female egg-laying, we also included in our experiment unmated females that were kept in the same conditions as mated females. The number of eggs laid by unmated females was counted from vials created at 6 hrs, 1, 2 and 3 days after placing the females in the holding vials. In addition, we also recorded the pattern of unmated female egg-laying for 10 days.To determine the effect of different mating regimes on female reproductive output (i.e. fecundity and fertility), we used One-way ANOVA (SPSS 15.0).+ pump α subunit; Mlp84B: Muscle LIM protein at 84B; DLG: Disc Large; HRP: horseradish peroxidas; UM: unmated; M: mated.Nrg: Neuroglian; Cora: Coracle; Spec: α- and β-Spectrin; SJ: septate junction; SSJ: smooth septate junction; PSJ: pleated septate junction; ZA: zonal adherens junction; AJ: adherens junction; AECM: apical extracellular matrix; ECM: extracellular matrix; HAJ: hemi-adherens junction; SAJ: spot adherens junction; OA: Octopamine; Hts: Hu-li tai shao; ATP α: NaAK and PKR contributed equally to this manuscript. AK, PKR and YH conceived and designed the project and analyzed the data. AK performed the confocal, Western blots and fertility assays. PKR and AK performed the light microscopy. PKR conducted the electron microscopy. AK, PKR and YH wrote the manuscript. RRH contributed to design of the study and revision of the manuscript. All authors participated in the discussion and approval of the final manuscript.Cellular junctions are established in the lower oviduct prior to mating. (A) Junctional complex is comprised of an apical pleated septate junction (PSJ) and a basal spot adherens junction (SAJ). (B and C) Electron micrograph of junctional complex. Pleated septate junctions contains visible septae (arrowhead) and interdigitations (*). Spot adherens junction is distinguished by an electron dense undercoat of plasma membrane (arrow). Apical membrane covered by long microvilli (MV). Bar is 0.5 μm.Click here for fileChanges in AECM and lumenal matrix in lower oviduct. Cross section of lower oviduct was traced from electron micrographs . Color coded as follows: gray = muscle; orange = epithelial soma layer; yellow = AECM and microvilli; nuclei also indicated. Areas labeled A, B and C are shown in corresponding panel of electron micrographs. (A') thick layer of AECM in this region prior to mating. Luminal matrix also observed (arrowhead) (A") higher mag of luminal matrix (*). (B') AECM is flattened against the brush-like border of microvilli (thick arrow). (B") higher magnification of AECM and overlying cuticle (arrowheads). (C') lumenal matrix detected between epithelial folds (arrow). (C") higher magnification of lumenal matrix (*) and cuticle (arrowheads). N (nuclei); M (muscle); MV (microvilli).Click here for fileThe abundance of boutons innervating the adult ventral abdominal muscles (segment V) is not changing post-mating. (A) Abdominal muscles  visualized in a dissected preparation of 3-day-old unmated female. Innervation of these muscles is visualized with anti-HRP (red). Ventral midline is indicated by dashed line. Abdominal segments 1–5 are shown. Boutons were counted in 5th abdominal segment; (B) Graph shows number of boutons per unit area in unmated and mated females. Boutons were counted in 3-day-old unmated females (UM3) and mated females at 6 h post-mating (M3). The number of boutons decreased in mated females (M3) but the difference was not significant. Standard error shown for each treatment. Higher magnification shows strings of boutons (arrow) in UM3 (C) and in M3 (D).Click here for fileTable of mating-responsive oviduct cytoskeleton proteins.Click here for fileFemale fecundity is highest in once mated 3-day-old females. Number of eggs laid by 3-day-old and 10-day-old unmated and mated females was examined. In addition, we also examined twice mated 10-day-old females (mated first at 3 days of age and mated second at 10 days of age). Number of eggs laid by unmated and mated females was counted in parallel. Four different times post-mating were examined: 6 h, 1 day (1 d), 2 days (2 d) and 3 days (3 d). (A) 3-day-old females: At all times examined except 3 d post-mating, once mated females (Once3) laid significantly more eggs than unmated females (UM3) of the same age (p < 0.005). Standard error (SE) is shown for each treatment at each time examined. (B) 10-day-old females: At all times examined except 6 hrs post-mating, once mated females (Once10) laid significantly more eggs than unmated females (UM10) of the same age (p < 0.005). At all times post-mating, 10-day-old twice mated females (Twice3&10) laid significantly more eggs than Once10 females . SE is shown for each treatment at each time examined. (C) Egg-laying pattern of unmated females: Egg-laying was recorded from day 3 to day 10 post-eclosion. Females were kept singly in vials beginning at day 3. On each day eggs were counted and females were transferred to a new vial. Variation in egg-laying was observed from individual-to-individual. However, the number of eggs laid at day 3 was significantly different (p < 0.005) from the number of eggs laid at day 4. Thereafter the number of eggs laid by unmated female at each day examined was not significantly different from the number of eggs laid at the following day. SE is shown for each day. (D) Fertility was calculated at different times post-mating. Fertility is defined as the average number of adults that eclosed from the total number of eggs laid per female at each post-mating time examined. Overall, female fertility was not significantly affected by the different mating regimes at each time examined. In only one case did we observe a significant difference in fertility (*). At 2 d post-mating, fertility was higher in Twice3&10females as compared to Once10 females (p < 0.022). See also Figure Click here for file


Maternal diabetes is a well-known risk factor for birth defects, such as heart defects and neural tube defects. The causative molecular mechanisms in the developing embryo are currently unknown, and the pathogenesis of developmental abnormalities during diabetic pregnancy is not well understood. We hypothesized that the developmental defects are due to alterations in critical developmental pathways, possibly as a result of altered gene expression. We here report results from gene expression profiling of exposed embryos from a mouse diabetes model.In comparison to normal embryos at mid-gestation, we find significantly altered gene expression levels in diabetes-exposed embryos. Independent validation of altered expression was obtained by quantitative Real Time Polymerase Chain Reaction. Sequence motifs in the promoters of diabetes-affected genes suggest potential binding of transcription factors that are involved in responses to oxidative stress and/or to hypoxia, two conditions known to be associated with diabetic pregnancies. Functional annotation shows that a sixth of the de-regulated genes have known developmental phenotypes in mouse mutants. Over 30% of the genes we have identified encode transcription factors and chromatin modifying proteins or components of signaling pathways that impinge on transcription.Exposure to maternal diabetes during pregnancy alters transcriptional profiles in the developing embryo. The enrichment, within the set of de-regulated genes, of those encoding transcriptional regulatory molecules provides support for the hypothesis that maternal diabetes affects specific developmental programs. Maternal diabetes disturbs embryonic development and can cause diabetic embryopathy, with cardiovascular malformations, neural tube defects and caudal dysgenesis as the most characteristic congenital malformations ,2. DiabeGrowing evidence suggests that maternal diabetes alters expression of developmental genes in the embryo, resulting in abnormal morphogenesis. Decreased expression of Pax3, a gene involved in neural tube defects ,22, was Mouse embryos were isolated from diabetic or control dams at embryonic day 10.5 (E10.5) because at this stage neural tube defects are easily detectable. The frequency of NTDs in diabetes-exposed embryos was approximately 17% (16/96 diabetes-exposed embryos) compared to 0% in normal pregnancies (0/220). Except where noted, no malformed embryos were used for gene expression studies. We found no significant differences in litter size or number of resorbed embryos between diabetic (n = 11) and non-diabetic pregnancies . We also did not detect any developmental delay in apparently unaffected diabetes-exposed embryos; their morphological appearance, i.e. features of brain development, limb development, and somite numbers were commensurate with developmental age . All experiments used the FVB inbred strain. We here report the results from two independent expression profiling experiments , we identified 126 genes  with expression levels that were changed in diabetes-exposed embryos by more than 2-fold relative to controls  for selected genes with potential relevance to diabetes or embryonic development Table . Embryo We cannot formally exclude altered mRNA stability as a factor causing the observed changes in mRNA levels, but it would be difficult to explain how the stability of relatively few transcripts could be altered in a selective fashion. Rather, we find that many transcription factor genes are down-regulated in their expression in diabetes-exposed embryos, and this trend is also reflected in the group of genes with 1.5- to 2-fold differences in expression; most likely therefore, the observed lower levels of gene expression are due to diminished or deregulated transcription.. A combined 22% of the genes encode cell surface receptors (10%) and signal transduction molecules (12%) that ultimately converge on transcription. The transcription factor Pax3, which was previously identified as affected by maternal diabetes [The biological roles of many products encoded by the 126 diabetes-affected genes are known  occurrence of common transcription factor binding sites (TFBS) in regulatory regions associated with multiple genes, and (ii) overrepresentation of such TFBS relative to other genes in the genome. We therefore analyzed the promoter sequences (5kb upstream of the transcription start) of our diabetes-affected genes for the presence of TFBS. As expected for genes with mostly broad expression patterns , we founBoth conditions have been reported to be associated with diabetic pregnancy ,31, and Our identification of genes whose expression is affected by exposure to maternal diabetes suggests that those genes could be involved in the developmental defects in diabetic pregnancies. This notion is supported by qualitative expression information (MGI) for 69 of these genes, with 62 detected in the embryonic CNS, and 35 in the embryonic cardiovascular system. With both CNS and heart frequently being affected in diabetic embryopathy, genes with abnormal expression in these tissues might contribute to pathogenesis of birth defects.Functional data also support this hypothesis: most remarkably, about one fourth of the diabetes-affected genes are known genes for which a functional role in embryonic development was established experimentally: in knockout mutant mice, 35 of the genes we identified have been shown, by genetics, to be required for mouse embryonic development Table . This imboth statistical significance tests. Both genes were detected in the initial diabetes-exposed versus normal comparison; we did not discover any genes that were uniquely altered in NTD embryos, implying that differences between NTD-affected and -unaffected individuals with regard to gene expression are mostly quantitative. Our results are consistent with the hypothesis that diabetes of the mother alters expression of specific known heart-defect and neural-tube-defect genes, and that these genes may be responsible for the birth defects in diabetic pregnancies.Central nervous system malformations occur in about 5% of children born to diabetic mothers , which rOur intial survey employed embryo samples that came from two pregnant females: one STZ-treated diabetic and a control untreated dam. This presents the theoretical possibility that any differences between progeny of the two dams reflect differences between pregnancies in addition to diabetic state. Also, we used individual embryo samples, and this approach is likely to incur substantial variability in the data and thus understimation of molecular changes. To address both concerns, we conducted Experiment II, in which equal amounts of RNA was pooled from three embryos of same gestational age into one sample, with each embryo derived from a different dam; for the diabetic as well as the control condition, we prepared three such pools, respectively Figure , Panel BIndeed, of the 126 genes whose expression was altered by more than 2-fold in Experiment I, 67 were also recovered above the 2-fold change cut-off in Experiment II. Of the 378 probe sets with expression level altered between 1.5-fold and 2-fold, 187 were shared. Thus, of the 504 probe sets with altered expression in Experiment I, more than half 254) were confirmed in Experiment II, providing independent validation for the major results of the first experiment. This 50% confirmation rate for independent microarray experiments in the same biological paradigm agrees well with similar findings for independent yeast microarray results . Thus, e4 were coUsing mRNA from whole individual embryos allowed us to survey a broad range of embryonic tissues that are potentially affected by maternal diabetes. This approach might have "missed" effects on genes that are expressed only in small cell populations of the embryo. However, in Experiment II, we identify 19 of the 47 published genes that were found altered more than 1.5-fold in microarray analysis of cranial neural tube tissue from diabetes-exposed embryos with neural tube defects at E 11.5 , and fouWe cannot currently distinguish which of the changes in gene expression are in direct response to the diabetic milieu, and which are indirect changes downstream of altered transcription factor expression, potentially increased hypoxia  or alterHigh glucose levels during critical periods of morphogenesis appear to be the major teratogen in diabetic pregnancy. In experimental animals, excess glucose is sufficient to cause dysmorphogenesis of embryos in glucose-injected dams or in whole embryo culture ,43-45. TDiabetes was induced in 7–9 week old female FVB mice by two intraperitoneal injections of 100 mg/kg body weight Streptozotocin in 50 mM sodium citrate buffer at pH4.5  within a one-week interval. The dams were set up for mating no earlier than 7 days after the last injection, and the day of detection of a vaginal plug was counted as day 0.5 of gestation. We used embryos only from dams (n = 11) whose blood glucose levels exceeded 250 mg/dl; average glucose levels were 148 mg/dl(± 18) before STZ treatment, 337 mg/dl(± 79) on the day of mating, and 528 mg/dl(± 70) on the day of embryo harvest.® . We processed 2 controls and five diabetes-exposed embryos; the latter group included two specimen with neural tube defects (NTD) so as to capture the full phenotype spectrum of diabetes-exposure in pregnancy. Individual RNA samples (5 μg) from whole embryos were reverse transcribed (Invitrogen) and labeled . In Experiment I, samples were individually hybridized to 7 Affymetrix430A2.0 chips, which were scanned using a GeneChip3000 scanner; Affymetrix GCOS imaging software was used for quality control. In Experiment II, equal amounts of RNA prepared from 3 individual embryos were pooled into one sample; each embryo was from a different pregnancy and three such pools were constructed for a total of 9 control embryos, and independently, for 9 diabetes-exposed embryos; all embryos were morphologically normal. Expression levels and "Present", "Marginal", "Absent" flags were determined with default parameters through comparison of matched and mismatched oligonucleotides for the respective gene sequence.Total RNA was isolated from embryos at embryonic day 10.5 (E10.5) using Trizol. We grouped the data for control embryos and those for diabetes-exposed embryosm respectively, and filtered results in three steps: (i) "expression", i.e. "present" or "marginal" in at least one of seven samples; (ii) "statistical significance" between control and experimental samples of P < 0.05 in both CyberT and the t-test in GeneSpring; and (iii) "fold change", i.e. difference between control and diabetes-exposed samples of beyond either two-fold or 1.5-fold. The rationale for employing complementary data analysis packages and details of data transformation have been described elsewhere [Statistical analyses were performed using GeneSpring7  and CyberT . We groulsewhere ., 15364 probes exhibited signals in at least one of the 7 samples, and 302 probe sets differed by more than two fold between these samples. Of these, 180 probe sets passed the t-test in GeneSpring , and 174 probes yielded P-values below P < 0.05 in Cyber-T. Permutation of the order of tests  identified differential signals from 2262 probe sets (Cyber-T), with 575 probes displaying differences between 1.5 and 2-fold, and 174 probes with differences greater than 2-fold between controls and exposed embryos. Regardless of order of filtering criteria, identical sets of probes were recovered, thus validating the analysis process. Between Cyber-T and GeneSpring, 145 sets passed both statistical filters, and after removal of duplicates, 126 genes were found to be differentially expressed above the 2-fold cut-off criterion.Of 22690 probe sets present on the arrays . 29687 probe sets exhibited signals in at least one of 6 samples; differences reached statistical significance at p < 0.05 for 9835 probes in the t-test  implemented in GeneSpring (GX version 9). 5915 probe sets exhibited differences greater than 1.5-fold, with 2796 differentially expressed greater than 2-fold. Cyber-T identified 13770 probe sets with statistical significance, of which 3992 exceeded the 1.5-fold change level, and an additional 4601 exceeded the greater than 2-fold criterion. After removal of internal controls, 5688 probe sets passed the filtering criteria for statistical significance in both Cyber-T and GeneSpring and exhibited >1.5-fold change between experimental and control samples, of which 2634 probe sets were identified with greater than 2-fold differential expression. Reduction of duplicates for a given gene was done by judgement call factoring in signal intensity, P-value, distribution of calls  and fold-change; only one entry per gene was retained for a total of 2231 transcripts with differential expression greater than 2-fold.For the second experiment, Affymetrix Mouse 430 2.0 arrays were used, which contain 45101 probe sets The primary data files are available at the NCBI Gene Expression Omnibus repository (Accession number GSE9675).Quantitative Real-Time PCR (Q-RT-PCR) using an ABI Prism7000 instrument was performed as described  on cDNA . Molecular function attributes were based on GO-annotation , supplemented with information from ENSEMBL and UCSC genome browsers and PubMed. Information on mutant phenotypes was obtained from MGI  for null and conditional alleles.Information on gene expression in embryos, where available, was collected from MGI  was used to identify transcription factor binding sites that are conserved between mouse and human and are over-represented in the 5 Kb upstream regions of our maternal diabetes affected genes relative to all 5 Kb upstream regions in the human genome .Whole Genome rVISTA GP performed microarray analyses and PCR assays, collected information for annotations and drafted a first version of the manuscript, JMS oversaw the statistical analysis by Cyber-T, designed experiment II and led the annotation effort, CK conceived of the study, performed the annotation for experiment II and wrote the manuscript.Genes with altered expression in diabetes-exposed embryos. The file contains a list of genes identified by microarray analysis.Click here for fileTranscription factor binding sites overrepresented in promoters of genes affected by maternal diabetes. The file contains a list of putative transcription factor binding sites and respective references.Click here for fileKnown HIF1 target genes that exhibit altered expression in diabetes-exposed embryos. The file contains a list of HIF1 target genes and respective references.Click here for fileReferences for in vivo function of genes affected in diabetes exposed embryos. The file contains a list of references for the in vivo function of particular genes.Click here for filePrimer sequences for quantitative RT-PCR. The file contains a list of RefSeqIDs, position information and sequences for primers to amplify particular genes. The amplification rate for each primer pair is also listed.Click here for file


Pouchitis is the most common long-term complication of in patients with restorative proctocolectomy and ileal pouch-anal anastomosis. Patients often develop antibiotic-dependent form of pouchitis requiring long-term antibiotic therapy for remission maintenance. Rifaximin, an oral, non-systemic, broad-spectrum antibiotic with a favorable safety profile, may be a promising candidate agent for maintenance therapy. This historical cohort open-label study investigated the efficacy and tolerability of rifaximin in maintaining symptomatic and endoscopic remission in patients with antibiotic-dependent pouchitis.Adult patients with antibiotic-dependent pouchitis received a 2-week course of various antibiotics for induction of remission. Patients in remission then began maintenance therapy with rifaximin 200 mg/day (to 1800 mg/day) for up to 24 months. Pouchitis Disease Activity Index symptom scores were assessed every 1–3 months to evaluate efficacy.Fifty-one patients began maintenance therapy with rifaximin (median dose 200 mg/day); 33 (65%) maintained remission through 3 months (primary endpoint). Of these 33 patients, 26 (79%) successfully continued maintenance for 6 months after beginning maintenance, 19 (58%) successfully continued for 12 months, and two (6%) successfully continued for 24 months. Only one patient reported an adverse event .Patients' response to rifaximin as a maintenance therapy appears to be favorable in this open-labeled trial of antibiotic-dependent pouchitis. Randomized, placebo-controlled trials with a longer follow-up are warranted. Restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) is the preferred surgical treatment for patients with medically refractory ulcerative colitis (UC), UC with dysplasia or cancer, and familial adenomatous polyposis (FAP). Pouchitis, an idiopathic inflammation of the ileal pouch frequently characterized by increased number of loose bowel movements, urgency, and abdominal cramping, is the most common long-term complication of IPAA. ,2 Up to Although many patients with pouchitis experience acute episodes with remission and relapse, up to 17% develop chronic disease that requires long-term therapy for treatment or maintenance. ,5,8-10 ARifaximin is an oral, broad-spectrum antibiotic with low systemic absorption (<0.4%) that provides high bioavailability in the gastrointestinal tract with minimal risk of systemic adverse effects. During the nearly 20 years rifaximin has been available in Europe, no clinically significant antibiotic resistance has been observed. ,15 For pGiven these encouraging preliminary data on the efficacy of rifaximin in pouchitis and concerns about antibiotic resistance and adverse effects with systemic antibiotics, rifaximin may provide an effective option for patients with antibiotic-dependent pouchitis. The present open-labeled, historical cohort study was to investigate the efficacy and safety of rifaximin in maintaining remission in patients with antibiotic-dependent pouchitis.This was a historical cohort study. Eligible patients (≥ 18 years of age) with antibiotic-dependent pouchitis were seen in our Pouchitis Clinic between July 2004 and June 2006. As a part of standard of practice, clinical, endoscopic, and histologic data for all patients were entered into the Pouchitis Registry, which was approved by the Institutional Review Board at Cleveland Clinic. Informed consent was provided by all patients.Patients were required to meet all of the following inclusion criteria: diagnosis of antibiotic-dependent pouchitis, defined as ≥ four episodes per year, each of which responded to a 2-week course of ciprofloxacin or metronidazole but recurred soon after treatment ended; frequent episodes of pouchitis requiring long-term (at least 16 weeks), continuous, low-dose antibiotics or frequent pulse therapy with antibiotics for remission maintenance; and currently symptomatic. Patients were excluded from the study if they had antibiotic-refractory pouchitis ; concurrent cuffitis, irritable pouch syndrome, or Crohn's disease of the pouch; or a prior history of adverse reactions to rifaximin.Management of patients with pouchitis followed an algorithm established for Pouchitis Clinic. The management algorithm was previously published Figure . All patTo induce remission, patients received single or combination therapy with ciprofloxacin (1000 mg/day), metronidazole (1000 or 1500 mg/day), tinidazole (1000 mg/day), or rifaximin  for 2 weeks. The use of single vs. combination therapy to induce the remission was at the discretion of the treating physician, based on the pattern of patient's prior response to the antibiotic therapy. After the induction period, a repeat pouch endoscopy was performed, and mPDAI scores were determined. Patients who exhibited symptoms or endoscopic signs of pouchitis with mPDAI scores > 5 points were excluded from the maintenance phase of the study; only patients in symptomatic and endoscopic remission began maintenance therapy.During the maintenance period, all patients received rifaximin at a starting dose of 200 mg/day. As a part of standard care of practice, the patients were followed up in Pouchitis Clinic or contacted via e-mail or telephone every 1–3 months during maintenance (for up to 24 months) to assess symptoms and adverse events and to confirm treatment compliance. Doses of rifaximin were increased (up to 1800 mg/day) for patients who exhibited partial response to maintenance therapy. Treatment was discontinued for patients who were unable to maintain remission with dose escalation or who chose to discontinue therapy. For patients who failed to maintain remission or who chose to discontinue therapy before the end of the 24-month study period, mPDAI symptom scores were documented at the time of discontinuation, and repeat endoscopy was conducted, if possible.The primary efficacy endpoint was the number of patients who maintained symptomatic remission for 3 months, as determined by mPDAI symptom scores. Patients who had not maintained remission or exhibited partial response were allowed to continue maintenance therapy beyond the 3-month maintenance assessment. Secondary measures included symptom response to induction therapy, the ability to predict remission maintenance based on clinical factors, and adverse events.Wilcoxon rank sum, chi-square, or Fisher exact tests were conducted to assess differences between patients who maintained remission for 3 months and those who did not maintain remission for 3 months. Within-group differences between baseline, post-induction, and post-maintenance symptom and endoscopy scores were analyzed using Wilcoxon signed rank tests. Associations between clinical factors and the primary endpoint (remission maintenance at 3 months) were calculated using a multivariate log-binomial model.A total of 53 patients with antibiotic-dependent pouchitis were treated with induction therapy with antibiotic monotherapy or combination therapy. Fifty-one patients achieved symptomatic and endoscopic remission during the 2-week induction period and began maintenance therapy with rifaximin. The overall median duration of maintenance therapy was 8 months , and the overall median maintenance dose was 200 mg/day . Following maintenance therapy with rifaximin, endoscopy scores were obtained for 30 (60%) of the 51 patients.Of the 51 patients who began maintenance therapy, 33 (65%) were still in remission at the 3-month maintenance assessment, and 18 (35%) had relapsed within 3 months. Demographic and clinical characteristics, including age, sex, extent and duration of UC, duration of IPAA, type of pouch, IPAA stage, indication for colectomy, family history of inflammatory bowel disease, smoking, excessive use of alcohol, weekly NSAID use before beginning the study were similar for patients who maintained remission for 3 months and those who relapsed by the 3-month time point (Table P < 0.0005).As expected, patients who maintained remission for 3 months showed no symptomatic or endoscopic evidence of relapse between the end of induction and the 3-month maintenance assessment , 600 mg/day (n = 3), 800 mg/day (n = 2), 1200 mg/day (n = 1), or 1800 mg/day (n = 1). In addition, 27 (82%) of the 33 patients who maintained remission for 3 months had received monotherapy during remission induction , 600 mg/day (n = 3), or 1200 mg/day (n = 1) during the total maintenance period. Significantly fewer patients (8 [44%]) who relapsed within 3 months had received monotherapy during the remission induction period compared with patients who maintained remission at 3 months , with no significant differences between the two patient groups (P = 0.18).To determine if patients who maintained remission for 3 months responded differently to the initial 2-week induction therapy than those patients who relapsed within 3 months, symptom improvements from baseline to the end of induction were compared. At baseline, symptom scores were the same between responders and non-responders  during maintenance therapy with rifaximin 200 mg/day; this patient discontinued therapy 2 weeks after beginning maintenance.This open-label study investigated the efficacy of rifaximin (200–1800 mg/day) in maintaining remission in patients with antibiotic-dependent pouchitis. The majority (65%) of patients maintained remission for at least 3 months with rifaximin, indicated by a lack of increase in mPDAI symptom scores. The efficacy of rifaximin in maintaining symptom remission appears encouraging.®, a lyophilized bacteria product containing four strains of Lactobacillus, three species of Bifidobacterium, and Streptococcus salivarius subspecies Thermophillus, effectively maintained remission for the majority of patients with chronic or recurrent pouchitis. [Management of antibiotic-dependent pouchitis can be challenging. Because symptoms quickly recur following discontinuation of antibiotic treatments, long-term antibiotic maintenance therapy is often required. Given the potential safety concerns associated with long-term therapy with systemic antibiotics frequently administered for remission maintenance, probiotics have been investigated as antibiotic-sparing agents for maintaining remission in patients with chronic or antibiotic-dependent pouchitis. ,21,22 Rauchitis.  These fiet al. [The present open-labeled study showed that long-term maintenance with rifaximin appeared to be effective in patients with antibiotic-dependent pouchitis, which would provide useful information for our future design of randomized trials. (RVIEWER2 Q2) In contrast, a small randomized trial of oral rifaximin 1200 mg/day vs. placebo N = 18) showed a marginal therapeutic benefit in treating active pouchitis.  The dosaet al.  included showed aBecause antibiotic-dependent pouchitis requires long-term, often continuous antibiotic therapy, maintenance treatment and clinical assessments in the present study were extended up to 24 months. Patients were treated with rifaximin as long as they were in remission or until they chose to discontinue therapy. A high percentage (58%) of responsive patients were still on maintenance therapy 12 months after beginning therapy with rifaximin, with two patients continuing maintenance therapy for at least 24 months. These data suggest that rifaximin effectively maintains remission during long-term therapy, extending previous findings. In addition to being efficacious, long-term treatment with rifaximin was well tolerated. Only one patient reported an adverse event with transient facial rash. The low incidence of adverse events reported in this study is consistent with a 2-week controlled trial for travelers' diarrhea in which rifaximin (up to 600 mg/day) exhibited a safety profile similar to that of placebo, as well as with a 4-month, open-label pouchitis trial with rifaximin (up to 800 mg/day) during which no adverse events were reported. ,24The present study had several limitations, including the open-label historical cohort design, lack of standardized doses of rifaximin throughout treatment, and incomplete endoscopy data for 21 of the 51 patients, a short duration of follow-up (3 month as primary end-point), and loss of follow-up of some patients after 3 months. (RVIEWER2 Q2) Although, in our experience, the dosage required for maintaining remission varied, it is possible that relapse of pouchitis in some patients might result from under-dosing. We chose to start the agent for maintenance therapy with a small dose for its cost and potential risk for bacterial resistance after long-term use. A dose ranging study is warranted for both induction and remission for pouchitis. In addition, the follow-up was set at 3 months after beginning maintenance therapy, although patients could continue maintenance therapy with rifaximin up to 24 months. While construction of Kaplan-Meier curves and estimation of recurrence rates would have been a great addition to this analysis, we unfortunately did not have the information necessary to do time-to-event analysis. In this study, recurrence of symptoms was assessed exactly 3 months after induction therapy for all patients and the exact time of recurrence was not available. Further investigations with prolonged follow-up are needed to more adequately determine the efficacy of rifaximin for maintaining endoscopy remission and for maintenance therapy beyond 3 months.Patients' response to rifaximin as a maintenance therapy appears to be favorable in this open-labeled trial of antibiotic-dependent pouchitis. Randomized, placebo-controlled trials with a longer follow-up are warranted.Cleveland Clinic maintains policies requiring that certain disclosures of financial interests accompany manuscripts submitted for publication. These financial interests with companies must be disclosed by co-authors from Cleveland Clinic whose research is sponsored by the companies or whose products (or direct and primary competitor's products) are discussed in the manuscript.This study was supported by the internal fund. However, n accordance with this Cleveland Clinic policy, we are disclosing that we have served within the past year or will serve in the coming year in the following roles in connection with the companies listed below.All authors declared no non-financial competing interest.BS: Concept, study design and execution, patient recruitment, data entry, and preparation of manuscript. FHR: Concept, patient recruitment, and manuscript preparation. ARL: Data analysis and manuscript preparation. EQ: Patient recruitment, follow-up, and data entry.The pre-publication history for this paper can be accessed here:


Rotavirus infection is responsible for about 500,000 deaths annually, and the disease burden is disproportionately borne by children in low-income countries. Recently the World Health Organization (WHO) has released a global recommendation that all countries include infant rotavirus vaccination in their national immunization programs. Our objective was to provide information on the expected health, economic and financial consequences of rotavirus vaccines in the 72 GAVI support-eligible countries.We synthesized population-level data from various sources  for the 72 countries eligible for the support by the GAVI Alliance (GAVI-eligible countries) in order to estimate the health and economic impact associated with rotavirus vaccination programs. The primary outcome measure was incremental cost  per disability-adjusted life year (DALY) averted. We also projected the expected reduction in rotavirus disease burden and financial resources required associated with a variety of scale-up scenarios.Under the base-case assumptions (70% coverage), vaccinating one single birth cohort would prevent about 55% of rotavirus associated deaths in the 72 GAVI-eligible countries. Assuming I$25 per vaccinated child (~$5 per dose), the number of countries with the incremental cost per DALY averted less than I$200 was 47. Using the WHO's cost-effectiveness threshold based on per capita GDP, the vaccines were considered cost-effective in 68 of the 72 countries (~94%). A 10-year routine rotavirus vaccination would prevent 0.9-2.8 million rotavirus associated deaths among children under age 5 in the poorest parts of the world, depending on vaccine scale-up scenarios. Over the same intervention period, rotavirus vaccination programs would also prevent 4.5-13.3 million estimated cases of hospitalization and 41-107 million cases of outpatient clinic visits in the same population.Our findings suggest that rotavirus vaccination would be considered a worthwhile investment for improving general development as well as childhood health level in most low-income countries, with a favorable cost-effectiveness profile even under a vaccine price ($1.5-$5.0 per dose) higher than those of traditional childhood vaccines. While childhood diarrhea can be caused by multiple pathogens, including both bacteria and viruses, rotavirus is the most common cause of severe diarrhea leading to hospitalization or disease-specific death among children under 5 years of age ,2. Respo® (GlaxoSmithKline) [a two-dose monovalent live attenuated human rotavirus vaccine] and RotaTeq®  [a three-dose human-bovine reassortant pentavalent rotavirus vaccine], have been licensed and are now available in many countries [® (completed in 2008) demonstrated a reduction of rotavirus disease burden in South Africa and Malawi [Rotavirus has diverse genotypes that vary geographically and over time -6, with ountries ,9. In 20ountries ; both vaountries ,9. Clinid Malawi . Based od Malawi .Especially in light of the Millennium Development Goal (MDG) 4 of child mortality reduction, there has been an effort at the global level to accelerate introduction of rotavirus vaccines in developing countries. However, in addition to the uncertainties about ultimate efficacy in Africa and Asia, as well as uncertainties about the likelihood of uptake, the current vaccines are costly relative to traditional childhood vaccines. Undoubtedly, the poorest countries will require financial assistance. The GAVI Alliance has promised to provide financial support for rotavirus vaccination programs to developing countries, and the WHO and its partners have established global networks for surveillance of rotavirus, providing valuable data on the burden of severe types of acute rotavirus gastroenteritis . HoweverThe objective of this analysis is to use population-based data for 72 GAVI-eligible countries and provide information on the expected health, economic, and financial consequences of rotavirus vaccines.We synthesized population-level data on demographic structure, country- or region-specific disease burden, medical utilization patterns for treating rotavirus diseases, and costs in order to estimate the health and economic consequences associated with rotavirus vaccination programs in the 72 GAVI-eligible countries. (The data used for our study is openly available upon request.) Model-projected clinical outcomes include intermediate health outcomes , deaths averted, years of life saved (YLS), and disability-adjusted life years (DALYs) averted. The primary outcome measure for the cost-effectiveness analysis was incremental cost  per DALY averted. For the base-case we adopted a modified societal perspective and discounted future costs and disability-adjusted and unadjusted life years by 3% annually -15. We hWe have previously described a model of the natural history of rotavirus infection using TreeAge 2008 software , which captures details such as the age-specific risk, probability of asymptomatic cases, rate of reinfection, correlation between strength of natural immunity and the total number of previous infections, and waning of vaccine efficacy . We appl® Excel 2003 and Visual Basic for Applications 6.3 . The model employs simplifying assumptions that rely on insights from more complex natural history models [The companion rotavirus model has been developed to reflect the main features of rotavirus infection and vaccination, and to project the health and economic consequences at the population level, in settings where data are limited. The model is constructed as a static cohort simulation model and is programmed using Microsofty models ,17. The y models .Lower Panel). DALYs are calculated using the approach recommended by the Global Burden of Disease (GBD) study [The Excel-based companion model is applied to birth cohorts born in 2010) in the 72 GAVI-eligible countries. The model tracks the cohorts up to age 5, incorporating background mortality and recording population-level health and cost outcomes with and without vaccination programs. Combining country-specific or region-specific data on the incidence of rotavirus deaths, ratios of outpatient visits or hospitalizations to rotavirus deaths, and serotype distribution, the model generates estimates for the reduction in rotavirus related events requiring medical treatments  or leading to deaths. By applying the estimates to each country's age-structured population, the model transforms them into aggregated population health outcome measures, YL and DALYs  study  but are 10 in theD) study . The mod® and 3  for Rotateq®. Because this analysis is intended to provide a broad policy overview of the expected health and economic outcomes of rotavirus vaccination, and not to contextualize a detailed situation of a single specific country, we chose not to distinguish the two vaccines in terms of dose and schedule or vaccine efficacy. This decision was based in large part on the WHO SAGE recommendations and in part based on published evidence: 1) a 3-dose schedule is recommended in any setting where the two vaccines, Rotarix® and Rotateq® are interchangeably used [® trial performed in Malawi, the overall efficacy against severe rotavirus gastroenteritis of a 2-dose schedule (49.2%) showed little difference compared to the corresponding value with a 3-dose schedule (49.7%) [® or Rotateq®) at ages aligned with each countries EPI visits (or DTP schedule) ; 2) coverage is 70%, (purposefully assumed in order to illustrate differences in country-specific outcomes related to variations in epidemiology); 3) vaccine-induced immunity is durable over 5 years without waning; and 4), for both vaccines, side effects are considered negligible as long as the first and last dose of either vaccine is given at the recommended age ranges (6-15 weeks for the first dose and 8 months or 32 weeks for the last dose) [The number of doses (and schedule) recommended by the vaccine manufacturers are 2 (at 2 and 4 months) for Rotarixbly used ; 2) effibly used ; and 3)  (49.7%) . To firsst dose) . We thenWe used two different approaches for estimating vaccine efficacy. For the base-case analysis, recognizing that differences in rotavirus serotype distributions would affect the overall efficacy against severe types of rotavirus, we calculated a weighted average of serotype-specific vaccine efficacy using the country- or region-specific serotype distribution data. Data for rotavirus serotype distributions were obtained from published epidemiological studies. When country-specific data were not available, region-specific estimates reported by the global networks for rotavirus surveillance were used  . We theFor country-specific age-structure that incorporates background mortality, we used the estimates of population size  from UN World Population Prospects 2006 Revision . Age-speWe estimated age-specific incidence rates of rotavirus-associated deaths by applying regional estimates of age distribution  of severe rotavirus gastroenteritis cases obtained from published literature ,25-28 toCosts included both direct medical costs  and indirect costs (travel costs and caregivers' time costs). Since the price of a rotavirus vaccine and program costs to deliver the vaccine in local settings are not known, we use a composite cost approach to estimate the vaccination program costs among the direct, medical costs. In doing so, we distinguish cost items that are tradable  from those that are typically non-tradable . We conduct analyses assuming a 3-dose vaccination schedule with total cost per vaccinated child of I$10 and of I$25, implying vaccine prices per dose of approximately US$1.50 and US$5.00 respectively. For a composite cost of I$25 per vaccinated child, we assume the following breakdown of the total costs: rotavirus vaccine purchase of US$15.00 (3 doses at US$5.00 each); vaccine wastage of approximately US$2.65 (~15% of vaccine price); freight and insurance costs of approximately US$0.90 (~6% of vaccine price); administrative costs of I$1.50; immunization support  of I$2.95; and other programmatic costs  of I$2.00. The lower cost of I$10 was derived by assuming 3 doses of vaccine at US$1.50 each, wastage of approximately US$0.79, freight and insurance costs of approximately US$0.27, administrative costs of I$0.50, immunization support of I$2.00, and programmatic costs of I$1.94. For both composite cost estimates, we assumed that the costs reflect any initial start-up cost and that the non-tradable components  would reflect incremental costs associated with introducing rotavirus vaccines into a national immunization programs to varying extents, depending on countries' health system infrastructure.Costs for hospitalization were based on country-specific data. When data were not available, we based estimates on WHO-CHOICE data for the cost of a bed-day at a secondary hospital, conservatively assuming one pre-hospitalization consultation in an outpatient setting, one-time hospitalization, and an average length of stay of 3 days for hospitalization . The incremental cost is the sum of vaccination program costs, averted medical treatment costs for children, and averted caregivers' time and travel costs. We have also evaluated the cost-effectiveness of rotavirus vaccination from the local government perspective, including direct medical costs only and adjusting vaccine costs using the GAVI's co-financing scheme for new vaccines   from the perspective of budget holders  ,35 by ex®, we use 2006 as their start date.); 2) it takes 2 or 3 years after implementation to reach full coverage for Category 1, 4 years for Categories 2 and 3, 5 years for Categories 4-6, 6 years for Categories 7-8, and 7 years for Categories 9-10; and 3) once full coverage is achieved, the coverage level would be maintained . Our previous work , which eUnder the base-case assumptions , vaccinating one single birth cohort would prevent approximately 52% (range: 50%-59%) of severe rotavirus disease events across the 72 GAVI-eligible countries compared to no vaccination Table . HoweverTable While there is no consensus on a threshold cost-effectiveness ratio under which an intervention is cost-effective, a useful proxy suggested by WHO to compare different public health interventions is per capita GDP . In thisResults were most sensitive to vaccination cost per child, rotavirus-associated mortality, ratios of hospitalizations and outpatient visits to deaths, vaccine efficacy against severe gastroenteritis, and the discount rate. For example, at a total cost per vaccinated child of I$5, rotavirus vaccination was shown to be cost-saving in 54 countries and for the rest of the countries  the incremental cost-effectiveness ratios ranged between I$1-I$170 per DALY averted. With a total cost per vaccinated child of I$50, rotavirus vaccines were not cost-saving in any GAVI-eligible countries. Results were moderately sensitive to hospitalization, outpatient visit costs, and level of vaccine immunity waning. When we decreased age-specific vaccine efficacy by ~14% annually over a 5-year time horizon in India, for instance, the incremental cost per DALY averted (at I$25 per child) increased from I$200 to I$220. The results were robust to duration of the disease, disability weight for diarrhea, transportation cost per visit, length of stay for hospitalization, and time per outpatient visit.When we used standard age weighting (K = 1)  instead We conducted analyses using alternative vaccine efficacy data by following the WHO SAGE recommendation for efficacy data extrapolation across countries. The average rotavirus death reduction for the GAVI-eligible countries was reduced to ~41% (compared to ~52%), since the average vaccine efficacy against severe rotavirus diseases was lower using this approach than calculating based on country- or region-specific serotype distribution and serotype-specific vaccine efficacy. Accordingly, rotavirus vaccination appeared less cost-effective; for instance, assuming this alternate efficacy and I$25 per vaccinated child, only seven countries had an incremental cost per DALY averted under I$100 , depending on vaccine scale-up scenarios. Rotavirus vaccination programs would also prevent 4.5-13.3 million estimated cases of hospitalization and 41-107 million cases of outpatient clinic visits in the same population over the same intervention period.Our results also show that rotavirus vaccine programs would be considered a cost-effective public health intervention in most GAVI countries; using the WHO's cost-effectiveness threshold based on per capita GDP, the vaccines were considered cost-effective in 68 of the 72 countries (~94%). It should be noted that a rotavirus vaccination program might not prove cost-effective in countries with a relatively low rotavirus mortality rate like Cuba, Ukraine, and Moldova. Although the likelihood that a country will introduce a new vaccine or scale-up rate is not always proportionate to the level of disease burden, these results may affect policy makers' decisions whether or not to introduce a vaccine. For example, although Cuba is classified into a high demand region in a previous study  and in tIn the present analysis, the most influential factor in the cost-effectiveness results was per child vaccination costs, which includes rotavirus vaccine costs, vaccine wastage, and any activities to support the delivery of the vaccines. Since the data on country-specific incremental costs for introducing a new childhood vaccine into a national immunization program are rarely available for low-income countries, we chose to use a composite cost approach, assuming a total program cost of I$10 or I$25 per vaccinated child. Accordingly, our base-case cost-effectiveness results use standardized vaccination program costs across the 72 GAVI countries, so the main differences in the incremental cost-effectiveness ratios may be attributable to the differences in disease burden  and vaccine efficacy adjusted for serotype distributions. Note that, although the discount rate was still relatively influential, it was not as important as it is with vaccines where benefits are realized in the distant future . The results were robust to country-specific hourly wage rates or transportation costs per medical visit, which were used in calculating direct non-medical costs such as caregivers' time costs or travel costs. The results were not sensitive to assumptions about medical utilization such as average length of hospitalization or average time spent per outpatient clinic visit.Note that our base-case estimates of the main health outcome (i.e. DALYs averted) are based on uniform age weights (K = 0), primarily for normative reasons, while a majority of previous similar studies use the standard age weights (K = 1) used by the WHO GBD study . UniformAlthough the WHO SAGE recommended global use of rotavirus vaccines based on the evidence from recent clinical trials, there is a high level of uncertainty about short-term and longer-term vaccine efficacy in local settings given the variability of rotavirus serotype distributions across regions and over time. Our analysis shows the implication of following SAGE's recommendation to transfer vaccine efficacy by using data from other countries with comparable levels of childhood mortality. In a majority of the GAVI countries, the extended vaccine efficacy following the SAGE approach was lower than the weighted average of serotype-specific vaccine efficacy used in our base-case analysis. Correspondingly, the results of a secondary analysis using the SAGE approach showed the estimated avertable burden to be about 30% (0-70%) lower.Recently an increasing number of published studies have evaluated the health and economic consequences of rotavirus vaccines in different settings, using various modeling approaches ,36,39-59The cost-effectiveness profiles of rotavirus vaccines from the present study are also very similar to those for HPV vaccines reported in a previous study , which wAlthough a rotavirus program proves cost-effective over a wide range of parameter values and alternative assumptions, countries need information on affordability of the vaccines as well. That is, in most low-income countries with severe resource constraints, multiple public health priorities compete for a limited shared budget; policy makers need to know the financial costs required to implement any program as well as its value for money. In the present study, in addition to the secondary cost-effectiveness analysis from the local government perspective, we performed a budget impact analysis based on simplifying assumptions, taking into account the 'new vaccine co-financing policy' recently announced by the GAVI Alliance, in order to project financial requirements and potential savings from averted medical costs for each year of the 10-year period. Our findings suggest that, with GAVI's support, the costs to each GAVI country would be reduced to approximately I$10-I$11 when assuming a composite program cost of I$25, but that covering this would still be a substantial burden to the lowest-income GAVI countries. This type of analysis may be useful to policy makers seeking to address the question of affordability of new vaccines by comparing the budget impact forecast with their own projected total immunization budget. Assessment of affordability is not complicated when there is a single earmarked budget for a new vaccine of interest. However, if there are multiple other immunization  programs competing for a shared budget, choosing an optimal set of immunization programs under a budget constraint can be challenging. Recently there have been a number of studies aiming to help policy makers with resource allocation, taking into account multiple criteria such as ethical or political considerations in addition to technical efficiency -65.Note that the results of our financial forecasting and overall disease burden reduction are, of course, sensitive to the forecasted level of demand for rotavirus vaccine. We base our base-case scale-up scenario on local experience with other childhood vaccines and national income levels. However, there are reasons why this forecast may be over- or under-estimated. For instance, there may be discrepancies between reported and actual uptake rates in local settings simply due to the challenges of collecting health information across entire populations. Further, while we assumed that all children covered would receive a full 3 doses of vaccine, in practice dose completion rates may vary, affecting the total cost and altering cost-effectiveness (due to both changes in cost and changes in impact if vaccine efficacy with fewer than 3 doses is lower than that under a full 3-dose course). Thus, to come up with more reliable forecasts for financial requirements associated with new vaccine introduction, more research is needed to improve the quality of coverage data and to conceptualize an approach to forecasting future uptake rate more realistically. Also, some operational research is needed to identify factors that might affect the sustainability of scale-up scenarios.Our study has several limitations. First, we synthesized available data using an Excel-based model that relies on simplifying assumptions; this was a purposeful tradeoff in that, unlike our past detailed models of a single country , here weThe limitations of our study suggest that our findings should be considered for only what they are intended-broad estimate of the potential impact and value of rotavirus vaccination. Our main findings suggest that rotavirus vaccine introduction would be considered a worthwhile investment for improving general development as well as public health level in most low-income countries, with a favorable cost-effectiveness profile even under a vaccine price ($1.5-$5.0 per dose) higher than those of traditional childhood vaccines. In addition, our study provides information on an approximate future stream of financial costs that need to be included in health budgets. Further, as more evidence on vaccine efficacy and safety, particularly associated with vaccination schedule in local settings , and morThe authors declare that they have no competing interests.SK and SG conceptualized and designed the study. SK and DS acquired data and performed analyses. SK, SS, DS, and SG interpreted the data and results. SK drafted the manuscript. SG provided administrative and technical support, and DS and SS provided technical expertise in managing data quality. All authors contributed to the revision of the manuscript and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/253/prepubSupplementary information on model inputs and results. The document provides detailed information on some of the key model inputs and results, including vaccine efficacy adjusted for serotype distributions, length of stay for rotavirus associated hospitalizations, hourly wage, transportation costs, and country-specific budget impact analysis results.Click here for file


Laboratory and clinical data indicate that DCIS can progress to invasive disease. Carbohydrate-mediated cell-cell adhesion and tumor-stroma interaction play crucial roles in tumorigenesis and tumor aggressive behavior. Breast carcinogenesis may reflect quantitative as well as qualitative changes in oligosaccharide expression, which may provide a useful tool for early detection of breast cancer. Because tumor-associated carbohydrate antigens (TACA) are implicated in tumor invasion and metastasis, the purpose of this study was to assess the expression of selected TACA by lectin histochemistry on DCIS specimens from the archival breast cancer tissue array bank of the University of Arkansas for Medical Sciences.The number of breast cancer patients diagnosed with ductal carcinoma Griffonia simplicifolia lectin-I (GS-I) and Vicia vilosa agglutinin (VVA). We studied associations of lectin reactivity with established prognostic factors, such as tumor size, tumor nuclear grade, and expression of Her-2/neu, p53 mutant and estrogen and progesterone receptors.For detection of TACA expression, specimens were stained with P = 0.0002) and VVA .We observed that both lectins showed significant associations with nuclear grade of DCIS. DCIS specimens with nuclear grades II and III showed significantly more intense reactivity than DCIS cases with nuclear grade I to GS-1 (Mean-score chi-square = 17.60, DF = 2; The results suggest that the expression of VVA- and GS-I-reactive carbohydrate antigens may contribute to forming higher grade DCIS and increase the recurrence risk. An increasing number of women choose more sensitive screening with digital mammograms and magnetic resonance imaging, which has ultimately resulted in dramatic increase in the diagnosis of ductal carcinoma nt years -4. The mnt years -9. Untrent years .An increased risk of a recurrence as DCIS or invasive cancer was associated with initial DCIS lesions that were larger than 10 mm or were of high or intermediate nuclear grade . High nuTumor initiation and growth is associated with modifications to the structure of glycan residues belonging to glycoproteins, glycolipids and proteoglycans present at the cell surface . AberranVicia vilosa agglutinin (VVA) and Griffonia simplicifolia lectin-I (GS-I) is closely associated with development, progression, and metastasis of breast adenocarcinoma [A feature of adenocarcinoma cells is their high accumulation of an abnormal mucin carbohydrate-substitution pattern compared with mucin of normal epithelial cells. This phenomenon is observed in most types of human cancers including breast cancer -26. The arcinoma -30. VVA arcinoma ,31. The arcinoma -35.Because the expression of cell surface carbohydrate structures reactive with VVA and GS-I lectins has been linked to invasive breast cancer and its metastatic potential, we hypothesized that the expression of TACA reactive with these two lectins may define an aggressive phenotype in DCIS. Therefore, the aim of this study was to assess the expression of breast-cancer-related TACA markers reactive with GS-I and VVA lectins in DCIS cases in order to evaluate their presence and determine their correlations with established tumor prognostic factors.This retrospective study was conducted on archival tissue-array blocks from patients diagnosed with DCIS between 1990–2002 at the University of Arkansas for Medical Sciences. This study was approved by the University of Arkansas for Medical Sciences Institutional Review Board.Follow up information was available on all patients ranging from 5 to 16 years. Patients' age, race and tumor characteristics including tumor nuclear grade and size were obtained from the pathology data base whenever possible. For the majority of cases immunohistochemistry results of the molecular markers Her-2/neu, p53 and the estrogen and progesterone receptors were also available in pathology files. For the majority of the cases, immunohistochemistry results of the molecular markers Her-2/neu, p53 and the estrogen and progesterone receptors were also available in the pathology files. Immunohistochemical studies for estrogen, progesterone and p53 were performed on the original blocks using antibodies from Ventana Medical Systems Inc. . Anti ER clone 6F1, anti PR clone1A6 and anti-p53 clone Bp53-11 were used. Any tumor with greater than 10% positivity with estrogen and progesterone were recorded as positive. Any nuclear positivity for p53 was reported as positive. Benign breast tissue within the submitted sections was used as internal positive control according to the pathology protocols. Hercept test by Dako  was used for Her2-neu testing. The cases were scored according to the DAKO standards of reporting for this antibody. The tumor was reported as negative, if the tumor cells showed no or weak incomplete cytoplasmic membrane staining. They were reported as positive 2+, if the neoplastic cells showed complete cytoplasmic membrane staining in more than 10% tumor cells at weak or intermediate intensity and positive (3+), if the tumor cells showed complete strong cytoplasmic membrane staining. The pathologist on record (SK) reviewed all samples and verified the presence of DCIS on the core used for the analysis. The study conducted on total of 60 available DCIS cases.Staining of tumor specimen with lectins was performed based on a previously described method . BrieflyELISA was performed as described elsewhere . Brieflys). Associations between staining intensities and other ordinal categorical variables were tested for statistical significance using the Mantel-Haenszel (MH) correlation chi-square test [P values associated with the rank correlations. The significance level for correlation analyses was set at 0.5% alpha to adjust for the multiple comparisons without inflating Type II error. Staining-intensity associations with nuclear grade of DCIS were further charactserized by computing for each grade the average staining-intensity score as follows: Average score = 0 × (proportion negative) + 1 × (proportion 1+) + 2 × (proportion 2+) + 3 × (proportion 3+). Differences in average scores between grades were tested for significance using the extended Mantel-Haenszel mean-scores chi-square test [Tumor nuclear grade was determined using the Van Nuys grading system ,38. The are test . Staininare test . RecurreTable We examined reactivity of these two lectins against an array of carbohydrates by ELISA to confirm their binding specificity Figure . Both VVP values) of 0.50 (0.0002) for GS-I and 0.48 (0.0005) for VVA. At 0.5% alpha, VVA showed a significant correlation with Her2/Neu , but GS-1 did not . Neither lectin showed significant correlations with tumor size, ER, PR, or p53. Staining intensities for GS-I and VVA were positively associated with each other , suggesting that in most cases the two lectins either bind to a single structure, or to structures that somehow are co-expressed.We characterized the associations of GS-I and VVA reactivity with expression levels of ER, PR, p53 mutant, Her-2/neu, and with age, race, tumor size and nuclear grade (Table P = 0.0002), with average staining-intensity scores (percents negatively stained) of 1.82+ (14%) in grade II and 2.00+ (0%) in grade III, compared to 0.50+ (71%) in grade I , with average staining-intensity scores (percents negatively stained) of 1.81+ (10%) in grade II and 1.94+ (0%) in grade III, compared to 0.64+ (57%) in grade I . Figure P = 0.02). The effect on RFS of negativity for ER or PR was examined, but was not significant (results not shown).Of the fourteen patients with grade-I DCIS, follow-up information was available for 13. Further inspection of the data revealed that five of the 13 showed recurrence in the fourth, sixth, and seventh year of follow-up after their original diagnosis. None of the 13 showed p53 or Her-2-neu amplification, two of 13 were ER-negative and six of 13 were PR-negative. One of the13 patients died in the third year of follow-up of an unrelated cause. The effect of lectin positivity on RFS was examined using survival analysis. Figure The belief that DCIS is a precursor of invasive breast cancer necessitates the search for finding specific DCIS-associated antigens to identify patients at greater risk for development of invasive cancer and recurrence. Many studies have examined molecular markers in DCIS that are used in clinical practice for invasive breast cancer -51. An oTo search for possible DCIS-associated molecular markers that recognize high-risk DCIS patients, we performed a lectin histochemical staining focusing on breast-cancer-relevant TACAs and evaluated their correlations with tumor prognostic factors. We observed positive correlations between the expression of VVA- and GS-I-reactive structures and tumor grade in DCIS patients. Because tumor grade is the most relevant pathologic determinant of disease with invasive potential, these data suggest that glycoconjugates containing these antigens are associated with invasiveness in DCIS. Tumor grade has been shown to predict prognosis using four gene-expression-based models   and has Lack of association of reactivity of either lectin with age and race indicates that reactive structures are independent of race and age. Together, these data suggest that histological evaluation of the expression of VVA- and GS-I-reactive TACAs may serve as phenotypic markers of not only high grade DCIS as defined by nuclear grade, but they may also define DCIS cases which are at high risk for recurrence. In support of this conjecture is the observation that of a total of 13 Low-grade (grade I) DCIS cases in our study, only seven were found positive for VVA lectin and interestingly five of these seven cases were diagnosed with recurrences later in their life. The data suggest that the expression of the lectin-reactive structures in DCIS may play a role in tumor relapse. Further studies targeting low-grade DCIS specimens are needed to make conclusive remarks.The exact nature of reactive carbohydrates in our staining with VVA and GS-I lectins is not known. Our data confirms the specificity of both lectins. We observed that VVA binds to both α-GalNAc and β-GalNAc, which is consistent with data published by others . SpecifiOur results suggest that the expression of VVA- and GS-I-reactive carbohydrate antigens may contribute to forming higher grade DCIS and increase the recurrence risk. Such carbohydrates might ultimately serve as viable targets for future development of novel therapeutic strategies aiming at early stage of breast cancer. However, the elucidation of their role in biology and patho-physiology of the disease, which needs further research, precedes such efforts. The data also support the hypothesis of using the expression of these structures as prognostic markers for early stage breast cancer that necessitate conducting further studies on much larger sample size of low-grade DCIS specimens with long follow-up time to establish whether low-grade DCIS with a specific glycomic profile can be subject to relapse more often than other tumors of the same category.The authors declare that they have no competing interests.SK was involved in design of the study, pathological review of specimens, staining and scoring of samples, interpretation of results and drafting the manuscript. ES was involved in statistical analysis and drafting and preparing the manuscript. TKE was involved in design, choosing targets and overall writing and review of the manuscript. BMK was involved in design of the study, data analysis, guiding experimental work and acquiring data, interpretation of results, and drafting and preparing the final manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: