We examine the hypothesis that certain molecules proposed to be used selectively by nociceptors fail to express on muscle spindles afferents but appear on other afferents from the same muscle.Despite the clinical significance of muscle pain, and the extensive investigation of the properties of muscle afferent fibers, there has been little study of the ion channels on sensory neurons that innervate muscle. In this study, we have fluorescently tagged sensory neurons that innervate the masseter muscle, which is unique because cell bodies for its muscle spindles are in a brainstem nucleus  in 90% of MeV neurons, although ICa were inhibited by GABAB receptor activation. All MeV afferents had brief (1 msec) action potentials driven solely by tetrodotoxin (TTX)-sensitive Na channels and no MeV afferent expressed either of three ion channels  thought to be transducers for nociceptive stimuli, although they did express other ATP and acid-sensing channels. Trigeminal masseter afferents were much more diverse. Virtually all of them expressed at least one, and often several, of the three putative nociceptive transducer channels, but the mix varied from cell to cell. Calcium currents in 80% of the neurons were measurably inhibited by μ-opioids, but the extent of inhibition varied greatly. Almost all TG masseter afferents expressed some TTX-insensitive sodium currents, but the amount compared to TTX sensitive sodium current varied, as did the duration of action potentials.MeV muscle afferents perfectly fit expectations of cells with a non-nociceptive sensory modality: Opiates failed to inhibit calcium channel currents (Most masseter muscle afferents that are not muscle spindle afferents express molecules that are considered characteristic of nociceptors, but these putative muscle nociceptors are molecularly diverse. This heterogeneity may reflect the mixture of metabosensitive afferents which can also signal noxious stimuli and purely nociceptive afferents characteristic of muscle. The proprioceptive afferents located in the MeV include those arising from the masseter muscle dec-8-yl]benzeneacetamide), deltorphin II, capsaicin, laminin, polylysine were from Sigma. Buffer salts were from Sigma. F-12, L-15 and fetal bovine serum were from GIBCO. Tetrodotoxin and human NT-3 were from Alomone Laboratories. NGF (mouse 2.5 S) was from either Upstate or Sigma. Papain and collagenase were from Worthington Biochemical Corporation , dispase was from Roche Applied Sciences.DAMGO , U-69593 ((+)--MC, LAN & EWM declare that they have no conflicts of interest relating to this work.MC performed experiments and wrote the draft of the paper, LAN performed experiments and EWM conceived the study. All the authors were involved in the experimental design, data analysis and writing the final paper. All the authors have all seen and approved the final paper.


Drosophila Gene Disruption Project in an effort to identify the different types of genes required for glutamate receptor cluster development.A clear picture of the mechanisms controlling glutamate receptor expression, localization, and stability remains elusive, possibly due to an incomplete understanding of the proteins involved. We screened transposon mutants generated by the ongoing To enrich for non-silent insertions with severe disruptions in glutamate receptor clustering, we identified and focused on homozygous lethal mutants in a collection of 2185 BG and KG transposon mutants generated by the BDGP Gene Disruption Project. 202 lethal mutant lines were individually dissected to expose glutamatergic neuromuscular junctions, stained using antibodies that recognize neuronal membrane and the glutamate receptor subunit GluRIIA, and viewed using laser-scanning confocal microscopy. We identified 57 mutants with qualitative differences in GluRIIA expression and/or localization. 84% of mutants showed loss of receptors and/or clusters; 16% of mutants showed an increase in receptors. Insertion loci encode a variety of protein types, including cytoskeleton proteins and regulators, kinases, phosphatases, ubiquitin ligases, mucins, cell adhesion proteins, transporters, proteins controlling gene expression and protein translation, and proteins of unknown/novel function. Expression pattern analyses and complementation tests, however, suggest that any single mutant – even if a mutant gene is uniquely tagged – must be interpreted with caution until the mutation is validated genetically and phenotypically.types of genes identified, rather than the identities of individual genes. This genomic approach, which circumvents many technical caveats in favor of a wider perspective, suggests that glutamate receptor cluster formation involves many cellular processes, including: 1) cell adhesion and signaling, 2) extensive and relatively specific regulation of gene expression and RNA, 3) the actin and microtubule cytoskeletons, and 4) many novel/unexplored processes, such as those involving mucin/polycystin-like proteins and proteins of unknown function.Our study identified 57 transposon mutants with qualitative differences in glutamate receptor expression and localization. Despite transposon tagging of every insertion locus, extensive validation is needed before one can have confidence in the role of any individual gene. Alternatively, one can focus on the  The vast majority of fast synaptic transmission in the mammalian central nervous system is glutamatergic. Proper expression, localization, and regulation of glutamate receptors are critical for brain development and plasticity. Not surprisingly, the molecular mechanisms controlling glutamate receptor expression, localization, and stability are of great interest. The most common recent approach to understanding these mechanisms has been biochemical: Proteins are identified based on a biochemical interaction with a glutamate receptor subunit. This approach has identified a number of important candidates, some of which have been subsequently shown to play important roles in glutamate receptor trafficking and stability at the PSD .C. elegans. Studies using C. elegans have highlighted the importance of ubiquitination, CamKII, and PDZ proteins in controlling glutamate receptor number, and identified a novel protein required for nematode glutamate receptor function [C. elegans are accessible for electrophysiology only with great difficulty. The subunit composition of C. elegans receptors in vivo has also not yet been determined, which hinders the study of subunit-specific trafficking mechanisms.An alternative approach is forward genetics: Mutant animals are screened for alterations in glutamate receptor localization. Perhaps the most well-known glutamate receptor trafficking protein identified via genetics is stargazin, which is disrupted in stargazer mutant mice . The idefunction -7. UnforDrosophila are not only amenable to powerful genetics, but also contain glutamatergic neuromuscular junctions (NMJs) that are individually identifiable and accessible throughout development to high-resolution electrophysiological and microscopic techniques. These techniques have revealed a significant amount of information concerning this popular model synapse, including the exact subunit composition of glutamate receptors in vivo. Drosophila NMJs contain two subtypes of postsynaptic glutamate receptor, which are molecularly, pharmacologically, and spatially distinct [Drosophila genome also encodes receptor subunits with high similarity to mammalian AMPA, delta, and NMDA receptor subunits, but these proteins are not found postsynaptically in the NMJ. Virtually nothing is known about the molecular mechanisms that control Drosophila glutamate receptor expression, localization, and stability.distinct -11. The Drosophila glutamate receptor expression, localization, and stability, we are screening transposon insertion mutants generated by the Berkeley Drosophila Gene Disruption Project [To determine the molecular mechanisms controlling  Project . This muDrosophila mutants with abnormal glutamate receptor cluster development. Elimination of Drosophila NMJ glutamate receptors results in paralysis and embryonic lethality [GT1 and SuPor-P insertion mutants, since these transposons were engineered for maximal gene disruption [We sought to identify ethality . Mutatioethality -10. Any sruption ,14. Of 2sruption -18. MorpFor each of the 202 balanced lines, several  homozygous mutant animals, along with control heterozygous siblings, were manually dissected at the latest viable stage to expose NMJs on ventral longitudinal muscles, then fixed and stained using anti-HRP antibodies that recognize all neuronal membrane  and anti-GluRIIA antibodies. After dissection, fixation, and staining, each of the 202 rebalanced mutant lines was examined for changes in NMJ morphology and glutamate receptor expression using laser-scanning confocal fluorescent microscopy. 57 of the 202 rebalanced lethal mutants (28%) displayed consistent  defects in glutamate receptor expression without severe presynaptic morphological abnormalities . Examples of some of these phenotypes, from both embryonic and larval NMJs, are shown in Fig. All phenotypes could be classified into one of two broad categories: 1) loss of glutamate receptors , or 2) gain of glutamate receptors (more clusters or larger clusters). We relied on cluster size and number because cluster size and number measurements (compared to fluorescence intensity measurements) avoid a requirement for fluorescence intensity calibration or problems associated with potential differences in background immunofluorescence between genotypes. Assuming constant receptor density, cluster size should be directly proportional to the number of clustered receptors. In support of this assumption, immunoreactive cluster sizes correlate well with high-resolution patch-clamp electrophysiological measurements between genotypes ,19,20 anP{SUPor-P}KG00333, which shows a complete loss of A-type receptors despite the presence of morphologically normal presynaptic terminals  of the mutants with disruptions in glutamate receptor clusters showed a qualitative loss in receptors. Severe loss of GluRIIA was always associated with embryonic/L1 lethality. One example is als Fig. , left. SP{SUPor-P} vriKG01220, is shown in Fig. P{SUPor-P}KG00212, all of the mutants that displayed an increase in GluRIIA immunoreactivity were viable as third instar larvae and also displayed an increase in the number of presynaptic boutons. This presynaptic phenotype is consistent with previous Drosophila studies showing that overexpression of postsynaptic GluRIIA causes presynaptic overgrowth [A minority (16%) of third instar viable mutants showed a qualitative increase in GluRIIA immunoreactivity. An example of one of these mutants, P-screen database  and/or BLAST searches with flanking genomic sequence from iPCR results to identify which gene was mutated by the inserted P-element. Putative functions were assigned to each of these genes based on previous publications, FlyBase annotations, and/or Genbank BLAST searches. We binned each of the insertion site genes into one of the following functional categories: 1) extracellular matrix proteins (mucins), 2) cell adhesion proteins 3) cytoskeleton proteins, cytoskeletal regulators, and adaptor proteins, 4) kinases and phosphatases, 5) ubiqitination proteins (ubiquitin ligases), 6) transporters/pumps, 7) proteins involved in gene expression and protein translation, 8) enzymes, and 9) proteins of unknown/novel function. These categories, and the relative number of proteins in each category, are illustrated by Figure For all of the mutants identified in our screen, the Gene Disruption Project has precisely determined the genomic insertion site of the transposon using inverse PCR. In the vast majority of cases, inverse PCR results were consistent with a unique insertion, and in most cases the flanking genomic sequence revealed that the insertion was in an annotated gene. For each of the mutants identified in our screen, we used the BDGDP Drosophila gene in which each transposon is inserted, and the mouse homolog of each of those genes .Table A definitive test of whether any particular gene is required for glutamate receptor cluster development requires precise excision of each P-element insertion, or transgenic rescue, followed by re-examination of the NMJ at the same developmental stage – a task which is not practical on a genomic scale. However, we performed two broad types of checks to identify potential caveats in our results: expression analysis and complementation tests for lethality.Drosophila glutamate receptors are expressed in neurons and muscles. Thus, any gene required for expression and/or localization of glutamate receptors should be expressed in neurons and/or muscles. The Drosophila gene expression database (accessed at: ) describes expression patterns of approximately 3300 different Drosophila genes (~24% of the genome), as determined by embryonic in situ cDNA hybridization [dization . Of thesP-element mobilizations that generated each of the P-element alleles for the gene disruption project may have also created second-site mutations in an unknown locus. Spontaneous mutations can also be easily stabilized and propagated in a stock carrying a balanced lethal mutant chromosome such as those screened here. To estimate the frequency of background mutations in this transposon mutant collection, we performed complementation tests to determine whether the P-element insertions failed to complement lethal alleles of the same insertion locus. Of the 57 mutants identified in our screen, 20 were tested for complementation to deficiencies that remove the insertion site. Ten (50%) of the complementation tests failed, suggesting that lethality in half of the mutants may be caused by a mutation other than the P-element insertion. These results are consistent with those from a related screen for presynaptic morphology mutants , which included complementation tests to over 80 genes. Complementation did not appear to be biased for any particular type of gene . We did not systematically test for complementation of the receptor cluster phenotypes.Although all of the mutants identified in our screens displayed a receptor phenotype, and all of the mutants contain a unique P-element insertion, an important genetic caveat is that the receptor phenotype may not actually be caused by the transposon insertion. For example, the In an effort to identify new genes required for glutamate receptor cluster development, we screened lethal transposon insertion mutants for alterations in postsynaptic glutamate receptor clusters. 202 lethal insertion lines, representing insertions in ~1.4% of the genome, were manually dissected and glutamate receptor clusters were examined using immunocytochemistry and confocal microscopy. This screen identified 57 mutations in 56 different loci. Transposon mutageneses are becoming increasingly popular, because a transposon insertion simultaneously mutates and 'tags' a gene. An assumption of all transposon mutagenesis (and subsequent screens) is that the insertion locus is responsible for any observed phenotype. Expression analysis showed that the expression pattern of insertion loci identified in our screen is broadly consistent with that expected from genes involved in glutamate receptor cluster development, but also not compellingly different (86% vs. 71%) from that expected from a random sampling of the genome. Furthermore, complementation tests suggest that approximately one-half of the lethality observed in our screen was not due to disruption of the insertion locus. Thus, it is impossible to definitively say which particular genes are important without extensive validation of each candidate. And even with extensive validation sufficient to confirm that a particular locus is involved, it is difficult to know whether a gene's role in receptor cluster formation is indirect with regard to glutamate receptor cluster formation (i.e. whether a gene product regulates another protein which in turn clusters receptors). Nevertheless, one cannot discount our results without also casting aside the fact that decades of forward genetic screens have successfully identified many genes critical for many different processes . But howDrosophila NMJ glutamate receptors contained the subunit GluRIIA, and that the viability of GluRIIA null mutants was due to substitution by the alternate subunit GluRIIB [Drosophila NMJ contains two independently assembled and localized subtypes of postsynaptic glutamate receptors: A-type receptors, that contain the subunit GluRIIA , and B-type receptors, which contain the subunit GluRIIB  [Should we exclude from consideration all mutants in which lethality was complemented ? This depends on the degree of coupling between two different phenotypes: lethality and abnormal glutamate receptor cluster development. To enrich for mutants with receptor cluster defects, we examined only homozygous lethal mutants, on the assumption that GluRIIA cluster defects and lethality are strongly linked. Indeed, all of the mutants showing severe loss of GluRIIA were embryonic/first instar lethal. Nevertheless, this assumption probably does not hold for all mutants. At the time this screen was initiated, it was assumed that all  GluRIIB . Thus, tGluRIIA) -10. GluRGluRIIA) ,25. Convcategories are likely to be correctly identified. Functional category analysis recognizes that not all hits will be valid, that not all genes will play roles specific to a particular process, and that many proteins are only indirectly required. Functional category analysis reveals unexplored areas of relevant biology and provides a broad roadmap for further study. For example: instead of studying the individual genes identified in Table Functional category analysis involves screening a non-redundant collection of mutants for the phenotype of interest, assigning putative functions bioinformatically, and categorizing the 'hits' by function. The goal of this approach is not to generate a definitive list of individual proteins involved in a process, but to gain insight into the types and relative numbers of proteins required. All of the categories shown in Figure Drosophila NMJ). If glutamate receptor cluster development and lethality are completely uncoupled, then our results imply that approximately 28% (57/202) of the entire Drosophila genome is required for glutamate receptor cluster development. Depending on the percentage of insertions that were too hypomorphic to show qualitatively detectable phenotypes via immunocytochemistry, that percentage could be higher. Glutamate receptor cluster formation and lethality, however, are not unrelated phenotypes. Therefore, 28% is likely to be a gross overestimate. Complete loss of Drosophila NMJ glutamate receptors results in paralysis and embryonic lethality [What percentage of the genome is involved in glutamate receptor cluster development? Our results allow this question to be addressed  is required for NMJ glutamate receptor cluster formation. This is a wide range, which in any case represents a surprisingly large number of genes. Is this reasonable? Do all of these genes represent specific machinery for glutamate receptor cluster expression and clustering? To answer these questions, it is helpful to consider the types of genes identified in the screen. As noted earlier, the role of each individual gene needs to be validated before placing too much emphasis on any particular protein's role. But a general discussion of the genes implicated is helpful for evaluation of the general results.In support of the idea that our screen correctly identified genes required for glutamate receptor cluster formation, some of the types of genes identified in our screen have been previously identified as important for postsynaptic development. Polo, for example, was identified in our screen, and mammalian polo-like kinases are receiving increasing attention as important players in synapse development . We alsoOne of the largest groups of genes identified in our screen encodes proteins that comprise or regulate the actin and/or microtubule cytoskeletons. Glutamate receptors, like many other proteins, are thought to be transported along microtubules, and anchored to the synaptic actin cytoskeleton -35. In sDrosophila GluRIIA mRNA is localized to the NMJ and locally translated [Drosophila NMJ glutamate receptor cluster formation depends on a burst of receptor subunit transcription that follows contact between pre and postsynaptice cells .Other types of genes identified by the screen are consistent with what one might expect. For example, many of the genes identified in our screen are involved in gene expression or protein translation. Mutation of a transcription factor or component of the translation machinery would be expected to disrupt many downstream things, including production of the cellular machinery required for postsynaptic development. Since our screen specifically excluded mutants which did not develop to the later stages of embryogenesis, and which did not form neuromuscular junctions, our screen may have highlighted components of pathways specific for a subset of cell differentiation steps that includes glutamate receptor cluster formation. Some of this machinery is possibly localized to the synapse. anslated . Mammalianslated -39. PrelC. elegans polycystin gene lov-1, which is localized to the ciliated sensory endings of dendrites required for male mating behavior, where it may be critical for regulating localization of other transmembrane proteins [DrosophilaAltered Synapse Development in  Sec 8 Mutants. Manuscript in preparation, and Liebl & Featherstone, unpublished) reveals that Drosophila sec 8 regulates the synaptic microtubule cytoskeleton to facilitate transmembrane protein localization  is the sole fly representative of the mammalian DLG/SAP 97/SAP102/PSD-95 protein family; DLG is important for formation of fly NMJ glutamate receptor clusters that contain the subunit GluRIIB, but not those containing GluRIIA [Drosophila NMJs.Approximately 12% of the genes identified in our screen encode proteins with unknown or novel function. A better understanding of these genes is important for understanding both those protein families and synaptogenesis – assuming these proteins are really regulators of receptor cluster formation. Even with a 50% success rate in identifying genes involved in receptor cluster formation – and the arguments and data above suggest the success rate was much higher – it is clear that novel genes encode a large fraction of proteins required for glutamate receptor cluster development in the fly NMJ. Therefore, major unexplored areas apparently may exist with regard to postsynaptic development. Interestingly, our screen did not isolate any PDZ-domain or MAGUK proteins, which are widely regarded as essential trafficking and scaffold proteins at mammalian glutamatergic synapses . However GluRIIA . Becausetypes of genes identified rather than the identities of individual genes, in an effort to gain larger insights into the entire process. Subsequent work can then be directed at investigating the processes represented by these gene types, rather than validation of individual genes that may or may not work directly or be applicable to other synapses and organisms. For example, many microtubule and actin regulators were identified in our screen. The target for these proteins, obviously, is likely to be cytoskeletal proteins instead of receptors. But their identification tells which types of cytoskeleton might be important for glutamate receptor cluster formation. Using these clues, we subsequently determined that microtubules are important for fly glutamate receptor trafficking , and that A-type receptors are anchored via the 4.1 protein coracle to postsynaptic actin [Many of the genes implicated by our screen probably do not work directly or specifically. Kinases only have to regulate something that in turn regulates receptor cluster formation. Transcription and translation factors probably control expression of multiple proteins required for PSD formation. Given the fact that many proteins definitively required for glutamate receptor cluster formation may not work directly, it is reasonable that a large percentage of the genome appears to be required. In other words, it is not difficult to identify proteins required for receptor cluster development, and we feel that the demonstration of such by itself does not give real insight into receptor cluster formation. An alternative approach, which we call 'functional category analysis' and introduce here, is to focus on the ic actin .We identified 57 transposon mutants with qualitative differences in glutamate receptor expression and localization. Mutant gene identities need to be validated despite the fact that mutant genes are tagged. Focus on the types of genes identified  may provide more useful insight into the process of glutamate receptor cluster formation, compared to focus on individual genes. Our results suggest that glutamate receptor cluster formation involves cell adhesion and signaling, extensive and relatively specific regulation of gene expression and RNA regulation, the actin and microtubule cytoskeletons, and many novel/unexplored processes such as those involving mucin/polycystin-like proteins and proteins of unknown function.NMJ staining and microscopy was performed as previously described ,19. BrieP-element mutant stocks to a balanced stock containing a deficiency that removes the insertion site. The F1 generation of each cross was examined for the presence or absence of adult flies carrying neither balancer chromosome. Thus, P-element insertion chromosomes were tested for their capacity to complement the viability of the lethal mutations in the gene carrying the P-element insertion.Complementation analysis was performed by crossing GFP-balanced P-element stocks were obtained from Bloomington Stock Center flystocks.bio.indiana.edu. 'Control' genotypes in all experiments are w1118.All FLWL performed most of the immunocytochemistry and microscopy. Analysis was jointly performed by FLWL and DEF.


EHP 113:1222–1225][.Animal studies have linked di(2-ethylhexyl) phthalate (DEHP) with reproductive and developmental toxicity, and have demonstrated an especially pronounced effect on testicular development when administered postnatally. Previous research has shown that newborns treated at neonatal intensive care units (NICUs) may receive doses of DEHP at 2–3 times the average daily adult exposure, and that these infants have relatively high urinary levels of the DEHP metabolite mono(2-ethylhexyl) phthalate (MEHP). Now researchers, using urinary MEHP as a biomarker of DEHP exposure, demonstrate for the first time that the more DEHP-containing devices are used in treating an infant, the more DEHP makes its way into the infant’s body Human DEHP exposure is widespread but generally much lower than the levels causing harm in animal studies. However, certain circumstances such as intensive medical treatment can result in higher-than-average exposure, which may be a particular risk for newborn males. DEHP is added to polyvinyl chloride (PVC) plastics for use in medical equipment including IV bags, blood bags, and various types of tubing, as well as many industrial and consumer PVC products. DEHP does not chemically link to PVC and leaches into fluids  that contact the plastic. The amount of leaching depends upon factors such as type of fluid, length of storage, and temperature.The study involved 54 newborn girls and boys receiving treatment at two Boston-area NICUs between 1 March and 30 April 2003. The infants had been admitted for various reasons, and treatment included procedures such as mechanical ventilation, enteral feedings, and cardiac catheterization.Prior to visiting the NICUs, the researchers defined low, medium, and high DEHP exposure categories based upon typical NICU procedures and equipment. Infants whose treatment consisted primarily of bottle and/or gavage feedings composed the low-exposure group. Infants in the medium-exposure group received more invasive therapies involving equipment such as an indwelling gavage tube or umbilical vein catheter. High-exposure infants experienced multiple and simultaneous invasive treatments, including endotracheal intubation and continuous umbilical vein catheterization.One researcher visited the NICUs and observed each infant for 3–12 hours over the course of 1–3 days (more than one infant was observed at a time). During the observational visits, the researcher noted the equipment being used for each infant, then assigned the infant to an exposure group accordingly. At the end of each visit, urine samples were collected for MEHP measurement.The researchers detected 10 phthalate metabolites in the samples, including 3 associated with DEHP, but focused on MEHP for data analysis since this metabolite is well studied and a proven biomarker of DEHP exposure. MEHP levels ranged from less than the level of detection to 758 nanograms per milliliter and did not vary substantially between multiple individual samples.Between the two NICUs there were 13, 24, and 17 infants in the low-, medium-, and high-exposure groups, respectively. The researchers found that infants in the high-exposure group had MEHP levels five times higher than those in the low-exposure group. MEHP levels for medium-exposure infants were twice those of the low-exposure group.The researchers indicate that the MEHP levels seen in this study are similar to those previously reported for NICU infants and higher than those reported for older children; no data are available for infants who did not need NICU care. The relevance of these exposures to health effects is unknown, and the researchers urge larger, more comprehensive studies with follow-up to determine consequences of DEHP exposure related to NICU treatment.


Following adjuvant therapy for breast cancer, some patients will die of this tumour while the remainder will die of other causes. Deaths from breast cancer tend to follow a lognormal distribution, while deaths from other causes can be approximated by national demographic data. By combining these two survival models, we have generated an age-specific method for estimating the impact of treatment on overall long-term survival. Treatment was designed to operate by one of two mechanisms: an increase in cured fraction, or an increase in median tumour-related survival time among uncured patients. This analysis revealed that, for young and middle-aged patients, an increase in cured fraction has substantially greater long-term clinical impact than an increase in median survival time. Unfortunately, the non-parametric tests traditionally used in prospective clinical trials cannot distinguish between these two mechanisms of action.


With the burgeoning immunological data in the scientific literature, scientists must increasingly rely on Internet resources to inform and enhance their work. Here we provide a brief overview of the adaptive immune response and summaries of immunoinformatics resources, emphasizing those with Web interfaces. These resources include searchable databases of epitopes and immune-related molecules, and analysis tools for T cell and B cell epitope prediction, vaccine design, and protein structure comparisons. There is an agreeable synergy between the growing collections in immune-related databases and the growing sophistication of analysis software; the databases provide the foundation for developing predictive computational tools, which in turn enable more rapid identification of immune responses to populate the databases. Collectively, these resources contribute to improved understanding of immune responses and escape, and evolution of pathogens under immune pressure. The public health implications are vast, including designing vaccines, understanding autoimmune diseases, and defining the correlates of immune protection. The immune system is the body's defense against infectious organisms and other foreign agents. The first line of defense is innate immunity, rapid nonspecific responses that allow recognition of conserved signature structures present in many microorganisms, such as lipopolysaccharides in bacterial cell walls or proteins in flagella . The secThe adaptive immune response has two major arms: the cellular immune response of T lymphocytes, and the humoral immune response of antibody-secreting B lymphocytes. In both cases the immune response is stimulated by receptor recognition of a specific small part of an antigen known as an epitope. Antibodies generally recognize intact proteins. B cell epitopes can be linear, contiguous amino acids, or they can be discontinuous amino acids that are brought together spatially in folded proteins. Discontinuous epitopes are defined through mutagenesis, competition experiments, modeling, or through cocrystallization or modeling of protein structure and docking . Even liIn contrast, T cell epitopes are short linear peptides that are cleaved from antigenic proteins, although T cell epitope generation by protein splicing is also observed . T cell + T cells is cytolytic activity resulting in apoptosis of virally infected cells [+ T cell's previous exposure to antigen and activation state [+ T cells is to produce cytokines that regulate the rest of the immune response. These functions are not exclusive, however—CD4+ T cells can induce cytolysis [+ T cells can secrete immunoregulatory factors.Two fundamental types of T cells are distinguished by expression of CD8 and CD4 proteins, which dictate whether a T cell will recognize epitopes presented by class I or class II molecules, respectively. Underlying this high-level bifurcation is a complex array of other functional markers. A key effector function of CD8on state . The priytolysis , and CD8+ T cell epitopes are processed after encapsulation by antigen-presenting cells in membrane-bound vesicles, where they are degraded by proteases into the peptide fragments that bind to MHC class II proteins. Then they are delivered to the cell surface, where class II-peptide complexes can be recognized by the CD4+ TCRs [+ T cells generally recognize viral or self antigens expressed from within a cell [+ T cells [CD4D4+ TCRs . In contn a cell , proteinn a cell  at the Cn a cell . The N tn a cell . After cn a cell ,15, althn a cell . The MHC T cells .Both B cell and T cell epitopes are constrained by sequence specificity, and mutations within and external to epitopes can result in immune escape. Obviously, mutations within an epitope can directly impact antibody-antigen interactions or epitope-MHC and TCR interactions. Mutations outside of the epitope can inhibit antibody binding through conformational changes, or inhibit proper cleavage and processing of T cell epitopes ,18. TAP Pathogen- and cancer-related immune responses are being characterized at a remarkable pace, with precise mapping of well-characterized epitopes and increasing use of full genetic typing of HLA-epitope presenting molecules, characterization of accompanying crystal structures, and definitions of escape mutations. As these elements are defined piece-by-piece in the literature, it becomes increasingly valuable to assemble the data into searchable databases and to provide computational tools to assist in interpretation of this complex information. Defining epitope sequence specificity  presents a tantalizing problem for computational biologists. The predictive amino acid patterns associated with these events are subtle, requiring sophisticated pattern recognition methods to infer directly from protein sequences which peptides have the potential to become epitopes. The complexity is compounded by the fact that recognition patterns might not be encoded by the contiguous primary sequence, but rather in local three-dimensional structure. The response to this challenging problem has resulted in an abundance of Web-based methods enabling the exploration of immunologically relevant data from a variety of perspectives. This review summarizes a sampling of particularly useful and user-friendly Web-based computational tools and searchable databases. The computational methods and databases are described and referenced in the text, and Web links are provided in summary tables. As a cautionary note, the authors have not directly tested that the functions contained in these resources will produce meaningful results, nor have we done systematic comparisons of the output of the different analysis tools; users would benefit by reading the primary literature regarding the different analyses methods if they decide to use one or more in their own work.The most thoroughly studied step of T cell epitope generation is peptide binding to MHC molecules, and the Web-based databases that include peptide-MHC data enable binding predictions. The MHCPEP database , for exaPeptide-MHC binding is the most predictable aspect of T cell epitope generation. MHC class I and class II genes are highly polymorphic, and the majority of their variable positions are located in binding pockets that restrict peptide interactions to those with particular amino acids at characteristic positions ; the setFor many MHC alleles, both simple and extended motifs are characterized and used to predict potential epitopes. For example, the SYFPEITHI database  contains+ T cell epitope database contains 3,150 entries describing 1,600 distinct MHC class I-epitope combinations (a single epitope can have multiple entries); the HCV database contains 510 entries describing 250 distinct MHC class I–epitope combinations. These databases include detailed biological information regarding the response to the epitope, including its impact on long term survival, common escape mutations, and whether an epitope is recognized in early infection; links to the primary literature; and curated alignments summarizing the epitope's global variability.The Los Alamos HIV/HCV databases offer a simple tool (MotifScan) for identifying HLA anchor-binding motifs in query proteins, highlighting them on a protein or protein alignment ,35. ThisA central assumption of the traditional prediction methods based on motif frequencies is that each position contributes independently to binding. Interactions at one site, however, can affect interactions in another site ,39. StatThe Edward Jenner Institute for Vaccine Research maintains the AntiJen database, which contains quantitative experimental binding data for peptides that bind to MHC, TAP, TCR-MHC complexes, T cell epitopes, and B cell epitopes; it also offers data on immunological protein-protein interactions. It includes more than 24,000 entries. The MHCPred ,51 tool The BIMAS tool ,55 ranksThe following three suites of tools allow MHC/class I epitope prediction through a combination of cleavage prediction, TAP binding, and MHC binding. The Center for Biological Sequence Analysis offers the NetChop tool ,59 for pMany of the sites listed are convenient for large-scale calculations. Some, for example SYFPEITHI and MHCPred, allow one to incorporate multiple HLA alleles for epitope prediction, while others, such as NetChop, NetMHC, NetCTL, FRAGPREDICT, and IEDP tools allow one to upload protein alignments. MotifScan, MAPPP, and the ProPred series allow both. These methods are currently being applied to peptide vaccine design and can be used to identify epitopes that have the desirable properties of promiscuous presentation by many HLAs and relative conservation ,73,74. WThe number of genetically defined MHC and HLA alleles continues to expand, with a corresponding evolving and expanding nomenclature. The European Bioinformatics Institute maintains the IMGT/HLA sequence database , which iThe National Center for Biotechnology Information (NCBI) maintains dbMHC , which ihttp://www.hiv.lanl.gov/content/immunology/hlatem/index.html). These efforts have led the HIV/HCV database team to develop computational tools to facilitate study design and analyses of experimental data of this nature. These tools could, for the most part, be applied to any pathogen or protein. PeptGen [+ T cell epitopes onto a reactive peptide.Experimental T cell response mapping efforts recently have been scaling up, including additions of variant peptides to better probe responses to variable pathogens and extensions of T cell response mapping studies to span the full proteome of pathogens for large study populations . Comple PeptGen  enables  PeptGen  can be cThere is growing interest in defining and comparing HLA allele frequencies in study populations where vaccine trials are planned; thus we have made a suite of tools to compare HLA frequencies in two populations, to identify alleles in linkage disequilibrium, and to fill in estimates of missing HLA information if full genetic typing is not feasible . Because of the high cost of genetic HLA typing, although it is desirable, the reality is that often only partial HLA genetic typing of key alleles is available. A partially described data set could provide the basis for informed guesses of the HLA genotypes superimposed onto two-digit typing—for example, by utilizing available four-digit data genetic typing data at dbMHC for different populations or a cohort subset that is fully genetically typed. Thus we have created a computational tool in which four-digit HLA allele designations are estimated from a combination of two-digit and four-digit HLA typing data. A maximum likelihood probability is assigned to each four-digit estimate, based on a combination of allele frequencies in the population and linkage disequilibrium patterns.The conformational aspects of antibody binding complicates the problem of B cell epitope prediction, making it less tractable than T cell epitope prediction. Indeed, Blythe and Flower  recentlyAlthough antibody epitope prediction is difficult, many other antibody-specific resources are available on the Web . If the We maintain comprehensive Web-searchable databases of pathogen-specific HIV  and HCV This review is intended as a portal to some of the most useful online immunological software and searchable databases. This is a rapidly expanding area—experimental advances have moved immunology into population-based studies and simultaneously have brought us to the brink of comprehensively characterizing an individual's immune response to infection. Extensive listings of T cell epitopes and HLA-binding peptides, as well as peptides that do not bind, have been an invaluable resource for motif resolution and epitope prediction. Epitope prediction in turn facilitates detection of new epitopes, vaccine design, site-directed mutagenesis (to make proteins less immunogenic), potential autoantigen identification, and the design of immune-based cancer therapies. Given the compelling nature of the problem and its suitability for computational methods, many scientists have developed interesting alternative approaches to epitope prediction in silico, and have made their methods freely available through the Web . We applPromiscuous HLA presentation and epitope prediction offers one sensible strategy for the creation of T cell vaccines ,73,74. AUnderstanding the impact of host immune-pathogen interactions on pathogen evolution, pathogenesis, and immunogen design depends on coordinated global efforts to gather and share data and requires the combined expertise of experimental and computational scientists. Only through this type of cooperation will we fully harvest the knowledge implicit in the data. The computational tools presented here are not yet ready to supplant experiment, rather they should assist in experimental design and interpretation of data. We clearly do not know all of the rules yet, for instance in peptide-MHC binding, and key questions such as what determines immunodominance in T and B cell responses are still unanswered. Yet the range and power of the tools already available through the Internet, many representing global networks and collaboration, is a testimony to the substantial progress we have made in facing emerging infectious diseases and potential biothreats with broader and deeper collective knowledge. http://www.rcsb.org/pdb) accession number of HIV-1 p24 capsid protein is 2BVO.The Protein Data Bank (


Individuals with Attention-Deficit/Hyperactive Disorder (ADHD) have unexplained difficulties on tasks requiring speeded processing of colored stimuli. Color vision mechanisms, particularly short-wavelength (blue-yellow) pathways, are highly sensitive to various diseases, toxins and drugs that alter dopaminergic neurotransmission. Thus, slow color processing might reflect subtle impairments in the perceptual encoding stage of stimulus color, which arise from hypodopaminergic functioning.1) Color perception of blue-yellow (but not red-green) stimuli is impaired in ADHD as a result of deficient retinal dopamine; 2) Impairments in the blue-yellow color mechanism in ADHD contribute to poor performance on speeded color naming tasks that include a substantial proportion of blue-yellow stimuli; and 3) Methylphenidate increases central dopamine and is also believed to increase retinal dopamine, thereby normalizing blue-yellow color perception, which in turn improves performance on the speeded color naming tasks.Requires three approaches, including:1) direct assessment of color perception in individuals with ADHD to determine whether blue-yellow color perception is selectively impaired; 2) determination of relationship between performance on neuropsychological tasks requiring speeded color processing and color perception; and 3) randomized, controlled pharmacological intervention with stimulant medication to examine the effects of enhancing central dopamine on color perception and task performanceIf substantiated, the findings of color perception problems would necessitate a re-consideration of current neuropsychological models of attention-deficit/hyperactivity disorder, guide psycho-education, academic instruction, and require consideration of stimulus color in many of the widely used neuropsychological tests. Attention-Deficit/Hyperactivity Disorder (ADHD) is a common and impairing childhood-onset disorder characterized by inattention, hyperactivity, and impulsiveness that affects 3–7% of school-aged children and tends to persist into adulthood . ConvergTo date, there is no adequate explanation to account for either the observed color processing problems in ADHD or their selective amelioration by psychostimulant medication. We propose that the color processing problems may reflect subtle detrimental effects of dopaminergic deficiency in the central nervous system on retinal dopamine, which in turn impedes the efficiency of the short-wavelength (blue-cone) color vision mechanism as well as of other visual functions. Prior to elaborating on this hypothesis, we first review the evidence of problems in color processing associated with ADHD and the existing explanatory accounts. We then outline possible empirical approaches to test this hypothesis, and discuss the theoretical and clinical significance of the hypothesis should it be substantiated.perceptual deficit in selection of visual stimuli on the basis of color (red versus blue) as well as in later semantic stages of visual selective attention [Several lines of evidence indicate impaired performance on tasks requiring rapid and/or continuous processing of colored stimuli in ADHD. For example, in a seminal study on rapid automatized naming speed, Denckla  describettention .Currently, two classes of explanation  are discernible in the literature for the observed problems in rapid processing of colored stimuli in individuals with ADHD. These theoretical accounts are not mutually exclusive, but rather they overlap and reflect perspectives taken from different research approaches and different levels of measurement.Psychological accounts attribute slow color naming to developmental immaturity. For example, the development of color naming appears to be much more difficult for young children than naming shapes or animals -27. AlsoAn existing neurobiological account of color processing problems in ADHD associates poor color naming or poor color processing performance to alterations in the underlying neural substrate. For example, slower RAN color naming speed  was found to be related to smaller anterior superior white matter volumes (both right and left hemispheres) in ADHD compared to healthy controls . DecreasCurrent explanations referring to poor sustained attention, problems in set shifting or semantic processing, or differences in white matter volumes associated with ADHD can not easily account for the dissociation between rapid naming of colored stimuli and rapid naming of letters, digits, or words. Nor can they adequately account for the indications that color processing problems appear to be greater for yellow and blue stimuli. Moreover, from a developmental perspective, the neurodevelopmental immaturity hypothesis would predict greater problems in rapid color naming in children with ADHD compared to adolescents, with possible attenuation of the deficits in adults with ADHD. To date this hypothesis has not been tested directly, but meta-analyses of cognitive deficits in children, adolescents, and young adults with ADHD indicate color naming deficits of moderate to large effect size (d = .58–.62) across the life span with no evidence of age-related changes ,40.Color perception is controlled, at least in part, by retinal dopaminergic neurons . We propTo present the detailed hypothesis, we first provide the necessary background on color perception and the role of retinal dopamine, then argue how hypo-dopaminergic functioning in ADHD will give rise to impairments in the tritan color mechanism, which in turn will influence speeded color naming on tasks involving a substantial proportion of blue and yellow stimuli.Color perception is based on the three cone photoreceptor types maximally sensitive to long, middle, and short wavelengths in the perceived light spectrum that constitute two functionally and anatomically distinct systems at the retina and lateral geniculate nucleus; a 'red-green' system with a foveal specialization in which long and middle wavelength cone signals are antagonistic, and a 'blue-yellow' pathway in which short wavelength cones are opposed by a combined signals from long and middle wavelength cones without such a foveal overrepresentation . Normal Most color perception defects  are congenital and arise from altered sensitivity defects of the L and M cones. By contrast to these red-green color vision deficits, which respectively affect 2% and 6% of the male population, congenital defects in S-cone sensitivity are rare (about 0.01%) but affect both sexes equally . AcquireColor perception problems, particularly involving the blue-yellow (tritan) mechanism, have also been linked with alterations in retinal dopamine: dopamine is a major neurotransmitter in the mammalian retina ,53. DopaAlterations in the level of retinal dopamine are reflected particularly in deficits in the short-wavelength chromatic pathway that is responsible for blue-yellow color discrimination, a system that appears to be especially vulnerable to the effects of disorders and drugs ,50,56-58The fundamental mechanisms causing a specific retinal impairment of color discrimination along the blue/yellow axis in dopaminergic disorders and acquired dyschromatopsias remain unclear. Short wavelength sensitive cones may be more fragile than long and medium wavelength sensitive cones or their relative scarcity and anatomical distribution may be responsible for the greater vulnerability of the blue-yellow perception by alterations of the dopaminergic system ,47,53,69Hypo-dopaminergic functioning has been postulated in ADHD ,70 and aWe propose that the observed impairments in individuals with ADHD on tasks requiring speeded color processing of blue-yellow stimuli might be attributable in part to hypo-functioning of both central and retinal dopamine. Critical to this hypothesis is the premise that alterations in central and retinal dopamine occur in parallel . To our knowledge there is no direct evidence that this is the case. Rather, we draw inferences from the following evidence: 1) cerebrospinal concentrations of a metabolite of CNS dopamine, homovanillic acid, correlates positively with electroretinogram blue-cone amplitude ; 2) in PBLUE in yellow ink) decrease the strength of Stroop interference compared to non-opponent color pairs  [According to this retinal dopaminergic hypothesis, deficits in blue-yellow color perception result in poor performance on many of the standard neuropsychological tasks  that include a substantial proportion of blue-yellow stimuli. Thus poor task performance may reflect subtle impairments in color vision as well as, or instead of, impairments in higher-order cognitive function. Consistent with this prediction are the recent findings that individual differences in color perception influence performance on the classic Stroop color-word task and that incongruent opponent color pairs  . A neurared ink) . Also, ired ink) . The prered ink) .Stimulant-induced increases in the availability of dopamine would be expected to be reflected in retinal dopaminergic tone and thus have therapeutic effects on the short-wavelength mechanism. Thus, methylphenidate would be expected to improve visual functioning, including the speed of color processing and naming. Indeed, there is preliminary evidence of beneficial effects of methylphenidate on color naming in children with ADHD ,23.Alterations in short-wavelengths mechanism may be gender-related. Notably, estrogen has been found to have a modulatory influence on dopamine activity  and a feThe limited data available on developmental changes in retinal and central dopaminergic mechanisms, color perception, and color naming, suggest that the retinal dopamine hypothesis will likely hold for children, adolescents, and adults with ADHD. For example, animal research indicates that dopaminergic neurons are among the first neurochemical systems to appear in the developing retina and that the neural retina and dopaminergic system interact closely in a two-way manner throughout developmental period . It is oOn the other hand, the increasing efficiency of the mature (adult) brain permits the development and use of compensatory strategies. Thus, adults with ADHD, although impaired in color perception relative to healthy peers, may be able to call upon compensatory attentional strategies to enhance performance on tasks requiring rapid perception of blue and yellow stimuli, thereby exhibiting better performance than children or adolescents with ADHD.Three strategies are proposed to provide a rigorous test of the hypothesis: 1) direct assessment of color vision pathways in individuals with ADHD versus a comparison group of healthy peers; 2) evaluation of the relationship between color vision (particularly for blue-yellow stimuli) and performance on tasks requiring speeded color processing; and 3) investigation of stimulant effects on both color vision and performance on those tasks in individuals with ADHD. Color vision can be assessed directly using clinical measures that are sensitive to problems with stimuli along the blue-yellow axis -85 as weConvergent findings from empirical investigations and neural network simulation support the hypothesis that color perception contributes to performance on neuropsychological tasks requiring speeded color naming ,71. PrelIf substantiated in larger well-controlled studies, evidence that blue-yellow color perception problems contribute to poor performance on neuropsychological tasks of executive function requiring speeded color processing, would necessitate careful consideration to stimulus color when interpreting performance on many of the standard neuropsychological tests. Also, evidence of specific impairments in blue-yellow color perception  would necessitate a reconsideration of current neuropsychological models of ADHD, which posit the core deficits to be in higher-order executive functioning and not at the level of sensory and perceptual processing. From a clinical perspective, this retinal-dopaminergic hypothesis might indicate the need to include a visual examination in the assessment of ADHD, and raises the possibility that electroretinogram blue cone amplitudes may also be a possible neurobiologic marker related to central dopamine function in ADHD, as well as in cocaine-dependent patients .ADHD: Attention-Deficit/Hyperactivity DisorderRAN: Rapid Automatized Naming TestVEP: Visual evoked potentialThe author(s) declare that they have no competing interests.RT developed the hypothesis and prepared the initial draft of the manuscript; TB refined it and conducted a preliminary test of it; DG conducted an in-depth literature search. All authors helped revising the manuscript and have read and approved the final manuscript.


The midgut undergoes histolysis and remodeling during the larval to adult transition in holometabolous insects, but the molecular mechanisms underlying this process are not well understood.hmg176. Northern and western blot analysis suggested that high levels of hmg176 are expressed in the midgut during molting, but not during metamorphosis. HMG176 protein was detected by immunofluorescence within the membrane of fat bodies and the basement membrane of the midgut of both molting and feeding larvae, but not in metamorphically committed larvae. In situ hybridization revealed that hmg176 transcripts mainly localized to the columnar cells of the midgut. Interestingly, a non-steroidal ecdysone agonist, RH-2485, significantly upregulated expression of hmg176.Using Suppression Subtractive Hybridization (SSH), we identified a 531 bp cDNA predicted to encode a 176 amino acid protein, which we call hmg176 encodes a larval-specific protein that may participate in sustaining larval midgut during larval development, possibly in response to ecdysteroid in vivo. This study will enlighten our understanding of the molecular mechanisms of tissue histolysis during metamorphosis.These observations suggest that


But animal studies involve higher phthalate exposures than humans typically experience, and there is inconclusive evidence that low exposures affect testicular cells in men. Occupational phthalate exposure tends to be greater and more consistent than the highly variable low levels seen in the general population, however, and research at a Chinese manufacturing plant now reveals that such exposure can be significantly related to decreased blood testosterone concentration EHP 114:1643–1648; Pan et al.][.Human studies have shown widespread exposure to phthalates, compounds used in the manufacture of household, consumer, and medical products. The metabolites mono-2-ethylhexyl phthalate (MEHP) and mono-The study participants included 74 men who manufactured polyvinyl chloride (PVC) flooring at a plant in Liaoning Province and 63 men employed at a construction company. All the men completed a questionnaire about lifestyle factors and provided blood and urine samples. Blood samples were analyzed for circulating amounts of free testosterone, luteinizing hormone, follicle-stimulating hormone, and estradiol. Urine analysis provided data on concentrations of MBP and MEHP, which served as biomarkers of exposure.Due to the materials involved in flooring manufacture, the men at the flooring plant were assumed to have dermal and inhalational exposure to dibutyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP), the parent compounds of MBP and MEHP. Indeed, all the participants except one construction worker had detectable levels of urinary MBP and MEHP, demonstrating that phthalate exposure was pervasive. However, PVC plant workers had up to 100-fold higher levels of MBP and MEHP and significantly lower blood testosterone concentrations, compared with construction workers.Regression analysis revealed a modest but significant decrease in testosterone as total phthalate esters increased. Based on MEHP concentrations, the investigators estimated that 40.5% of the PVC plant workers had DEHP exposure exceeding the European Union’s tolerable daily intake standard of 37.0 μg/kg body weight.The team concluded that high levels of DEHP and DBP exposure seemed to suppress testosterone production in the PVC plant workers, but it is not clear from this study what effect, if any, that might have on their fertility.


Mother-to-child transmission (MTCT) is the predominant way that children become infected with HIV. MTCT can occur prenatally during pregnancy, perinatally during labor and delivery, and postnatally through breastfeeding. In the absence of specific interventions, approximately one-third of children born to mothers who are HIV-positive become infected themselves. Specific interventions, including prenatal HIV counseling and testing, antiretroviral prophylaxis, elective cesarean delivery, and avoidance of breastfeeding, have reduced MTCT to less than 2% in developed countries. In many less-developed countries, however, these interventions are not readily available, and even effective, shorter, and cheaper antiretroviral prophylaxis strategies have not yet been widely implemented or accepted. As a result, MTCT rates in lower-income countries remain high, accounting for an estimated 2,000 new pediatric HIV infections per day.An estimated one-half of the infant infections occur during labor and delivery, but the exact mechanisms remain poorly understood. Two possible ways of transmission have been proposed: direct contact of infant mucosa with HIV-infected maternal body fluids, and placental microtransfusions after breakdown of the maternal–fetal barrier through contractions at the beginning of labor. However, the evidence implicating either transmission route has been inconclusive.To further examine a possible connection between placental microtransfusions and HIV MTCT, Jesse Kwiek and colleagues have taken advantage of a recently developed assay that provides a surrogate measure for placental microtransfusions, based on the amount of placental alkaline phosphatase (PLAP) in umbilical cord blood. PLAP is a large maternal enzyme that cannot cross the intact placental barrier. Infants produce very low levels of PLAP, and amounts found in umbilical cord blood are thought to result from leakage of maternal protein into the fetal circulation, caused by placental microtransfusions.The researchers measured PLAP activity in umbilical cord blood as an indicator of maternal–fetal microtransfusions, and related this to risk of MTCT in a case-cohort study of mothers who were HIV-positive in Malawi. In the study, 149 women randomly selected from a larger cohort of pregnant women infected with HIV served as a reference group for 36 cases of prenatal MTCT and 43 cases of perinatal MTCT. The researchers saw no correlation between PLAP levels and prenatal MTCT. However, among the cases of perinatal transmission in women who had vaginal deliveries, elevated PLAP levels were associated with higher transmission risks. The connection was also seen after adjusting for some potential confounding factors such as HIV viral RNA load and chorioamnionitis.While these are preliminary results and many questions remain, the reviewers felt that the design of the study enabled an efficient first test of the maternal–fetal microtransfusion hypothesis of MCTC. As such, it should encourage other researchers to look at this issue in their datasets. If a connection between microtransfusions and transmission is confirmed, it might help to improve the timing of short-term prophylaxis regimens and possibly lead to the development of new strategies for preventing MTCT of HIV.


Vertebral sarcoidosis is an extremely rare form of osseous sarcoidosis. Although osseous sarcoidosis is almost always an incidental finding of sarcoidosis elsewhere in the body, vertebrae may be the primary disease site. Involvement of vertebrae is usually localized and sclerotic or lytic.We describe a case of extensive asymptomatic vertebral involvement by sarcoid with osteolytic lesions. Making the diagnosis requires biopsy and ruling out other commoner causes of osteolytic vertebral lesions.We report this case in the hope of expanding the knowledge of osseous sarcoidosis. Our patient was unique in that all involvement was axial with sparing of the peripheral skeleton, near absence of any other organ involvement, diffuse involvement of the whole spine and osteolytic bone lesions. Vertebral sarcoidosis is increasingly diagnosed secondary to the recent advances in imaging studies. Diagnosis is made by a combination of clinical, radiographic and histologic findings. Histopathology is the cornerstone in ruling out differentials like tuberculosis, Langerhans' cell histiocytosis and tumour related sarcoid reactions. We report extensive, asymptomatic, axial, osteolytic vertebral lesions with absence of any significant involvement of any other organ systems.A 44 year old white female with diabetes presented with symptoms and signs suggestive of acute appendicitis. Review of systems was negative for fever, cough, dyspnea, chest pain, joint pains, change in bowel habits and weight loss. Physical examination was normal except for tenderness in the right iliac fossa without guarding or rebound. The white cell count was 9130/μL. Liver and renal function tests were normal. CT of the abdomen revealed fatty liver, changes of acute appendicitis and an incidental finding of multiple lytic lesions in the vertebral bodies. She underwent appendectomy and recovered uneventfully.A follow up CT scan of spine and chest revealed small lucent lesions throughout the spine and bilateral lower lobe non-calcified pulmonary nodules. Purified protein derivative (PPD) skin testing for tuberculosis was negative. MRI of the spine revealed diffuse bone marrow heterogeneity throughout the cervico-thoracic and thoraco-lumbarspine Figure  and 1B. The patient was diagnosed with sarcoidosis. The other differentials of generalized osteolytic vertebral lesions which included tuberculosis, metastasis, multiple myeloma, lymphoma, eosinophilic granuloma and disseminated echinococcal lesion were ruled out by biopsy and investigations. To the best of our knowledge this represents the most extensive reported case of asymptomatic vertebral involvement by sarcoidosis involving cervical, thoracic and lumbar regions.The patient did not have a history of tuberculosis or exposure to the same. Skin anergy is typical in patients with sarcoidosis but they react very strongly to PPD when they develop active tuberculosis . HistopaWell defined non-caseating granulomas effectively ruled out Langerhans'-cell histiocytosis. The unifying feature of the group of diseases designated as Langerhans'-cell histiocytosis is infiltration by Langerhans' cell. Instead of the well formed granuloma seen in sarcoidosis, there is a polymorphic appearance from an admixture of Langerhans' cell, nonspecific histiocyte, lymphocytes and eosinophils. Langerhans' cell also has a characteristic morphologic appearance on high power with the nuclei often being lobulated or indented with a longitudinal groove . If prelInvolvement of bone occurs in 5% of patients with sarcoidosis. Although bony involvement may be the earliest manifestation, it usually accompanies skin lesions . The firThere are three distinct types of bone lesions – 1) lytic cortical defects which are usually rounded and heal forming punched out cysts 2) permeative defects which show progressive cortical tunneling with remodeling of trabecular and cortical architecture and 3) destructive lesions with a secondary joint surface involvement and periosteal reaction. Bone lesions imply a chronic severe disorder. Usually proximal and middle phalanges are involved. However skull, vertebra, ribs, maxilla and nasal bones may be affected. Cystic bone lesions are the predominant variant. A very small number have spine lesions . When inRadiologically cystic, lacelike honeycomb or extensive bone erosions, when accompanied by intact articular space and accompanied by soft tissue mass or tenosynovitis is virtually diagnostic of sarcoidosis. CT scan reveals osteolytic or osteosclerotic lesions. MRI in vertebral sarcoidosis may show varied T1 weighted, T2 weighted and STIR sequence images depending on nature (osteolytic vs. osteosclerotic) and activity  of lesions . FDG posBefore making a diagnosis of sarcoidosis, all other causes of non-caseating epithelioid granuloma must be eliminated. Non-caseating granulomas are seen in 4% of regional lymph nodes with carcinoma, 14% of patients with Hodgkin's disease, 7% of patients with non-Hodgkin's lymphoma, 7% of patients with primary seminoma and dysgerminoma . Tumor rTo summarize, vertebral sarcoidosis although extremely rare is being increasingly diagnosed with advances in imaging; though osseous sarcoidosis is an incidental finding of sarcoidosis elsewhere in the body, vertebrae may be the primary disease site; vertebral involvement is usually localized but may be generalized as in our patient; vertebral sarcoidosis is mostly focal osteolytic or combined – lytic and sclerotic, and rarely generalized osteolytic as seen here; and while usually symptomatic, vertebral involvement may be totally asymptomatic. Making the diagnosis requires biopsy and analysis to rule out infections, malignancy and other causes. We describe a unique case of vertebral sarcoidosis with no symptoms from bone involvement, no obvious presence of sarcoidosis in other organs except for incidental pulmonary nodules, and perhaps the most extensive asymptomatic involvement of the axial skeleton.Vertebral sarcoidosis is so rare that there are no guidelines for its treatment. Therapy includes corticosteroids, methotrexate and other disease modifying agents for control of disease activity; bisphosphonates, calcium and vitamin D for treatment of osteoporosis; and vertebroplasty for fracture. Although initially the patient preferred not to be treated being asymptomatic, she developed backache four months later and is currently on non-steroidal anti-inflammatory drug (NSAID), bisphosphonates and steroid therapy.We report this case in the hope of expanding the knowledge of osseous sarcoidosis. Skeletal sarcoidosis is usually peripheral, involves the axial skeleton secondary to other organ involvement, is often localized to a few vertebrae and is combined – osteosclerotic and osteolytic. Our patient was unique in that all involvement was axial with sparing of the peripheral skeleton and had near absence of any other organ involvement, diffuse involvement of the whole spine and osteolytic nature of bone lesions.CT-computed tomography, MRI-magnetic resonance imaging, PPD-purified protein derivative, NSAID-non-steroidal anti-inflammatory drug, FDG PET-Fluoro-deoxy-glucose Positron Emission TomographyThe author(s) declare that they have no competing interests.SN has been involved in the conception, design, drafting and revising the manuscript. SPB has revised and critically analyzed the manuscript for important intellectual content. DS has been involved with the pathological diagnosis of the patient and revision of manuscript. SAV has been involved in the diagnosis and treatment of the patient and in revising the manuscript


HER2/neu gene, the cyclooxygenase-2 enzyme, the nuclear factor kappa-B transcription factor, the protein kinases, the Bcl-2 protein, and coagulation pathways. The herbs that are traditionally used for anticancer treatment and that are anti-angiogenic through multiple interdependent processes  include Artemisia annua (Chinese wormwood), Viscum album (European mistletoe), Curcuma longa (curcumin), Scutellaria baicalensis (Chinese skullcap), resveratrol and proanthocyanidin (grape seed extract), Magnolia officinalis (Chinese magnolia tree), Camellia sinensis (green tea), Ginkgo biloba, quercetin, Poria cocos, Zingiber officinalis (ginger), Panax ginseng, Rabdosia rubescens hora (Rabdosia), and Chinese destagnation herbs. Quality assurance of appropriate extracts is essential prior to embarking upon clinical trials. More data are required on dose–response, appropriate combinations, and potential toxicities. Given the multiple effects of these agents, their future use for cancer therapy probably lies in synergistic combinations. During active cancer therapy, they should generally be evaluated in combination with chemotherapy and radiation. In this role, they act as modifiers of biologic response or as adaptogens, potentially enhancing the efficacy of the conventional therapies.An integrative approach for managing a patient with cancer should target the multiple biochemical and physiologic pathways that support tumour development and minimize normal-tissue toxicity. Angiogenesis is a key process in the promotion of cancer. Many natural health products that inhibit angiogenesis also manifest other anticancer activities. The present article focuses on products that have a high degree of anti-angiogenic activity, but it also describes some of the many other actions of these agents that can inhibit tumour progression and reduce the risk of metastasis. Natural health products target molecular pathways other than angiogenesis, including epidermal growth factor receptor, the  To progress, cancers require a source of nutrition and oxygen. Tumours that outgrow their oxygen supply cannot form masses greater than 1–2 mm without developing central necrosis. Neoplasms are genetically plastic and often adapt by switching on genes that increase their ability to invade and metastasize. A critical part of this process is the induction of local small blood vessels, termed “angiogenesis” Tumours do not grow progressively unless they induce a blood supply from the surrounding stroma. Cancers that lack angiogenesis remain dormant. Rapid logarithmic growth follows the acquisition of a blood supply. The tumour angiogenic switch seems to be activated when the balance shifts from angiogenic inhibitors to angiogenic stimulators.vegf) and angiopoietins (apns). Hypoxia stimulates these peptides; the result is a sprouting of endothelial cords. This sprouting creates profuse but immature networks of thin endothelial-lined channels, essential for tumour oxygenation. Although these networks permit progressive tumour growth, they are less efficient than the vascular supply to normal tissues. The apns recruit pericytes and initiate modelling of the vessel wall to more mature forms. Tumours often secrete a relative excess of vegf that results in disorganized and leaky vessels that cause local bleeding and edema.The process of neovascularization is subtly controlled in normal tissues by a sequence of endogenous polypeptides that are secreted during growth, healing, and tissue renewal . NeoplasAnti-angiogenic therapy has this theoretic attraction: it may be less susceptible to development of treatment resistance because it is directed to stroma rather than to genomically unstable tumour cells. Judah Folkman and his colleagues were the first to propose using inhibition of tumour vasculature formation as anticancer therapy vegf receptor in endothelial cells, thereby shutting off the tumour blood supply. When used in conjunction with chemotherapy, bevacizumab has been shown to extend life by a few months for some metastatic colorectal cancer patients Recently, targeted therapies using monoclonal antibodies that antagonize the formation of new blood vessels have been developed. One example is bevacizumab . Bevacizumab is a genetically engineered humanized monoclonal immunoglobulin G antibody that blocks the vegf:apn ratio approximates normal. At that point, pericytes are recruited, the vascular basement membrane adopts a thinner morphology and tumour oxygenation temporarily increases. This is a favourable time to apply ionizing radiation, because such radiation is preferentially lethal to replicating and well-oxygenated cells. The combination of the anti-angiogenic agent and the radiation therapy is optimally effective if this window of opportunity is exploited Anti-angiogenic therapies may also be combined with radiotherapy to improve local tumour control and reduce the risk of metastasis. During a course of radiotherapy, some tumours increase their angiogenic activity So far, the evidence suggests that single anti-angiogenic agents have limited efficacy. Natural health products contain a range of complex organic chemicals that may have synergistic activity. They may inhibit angiogenesis by interacting with multiple pathways and by acting in other ways that can affect cell signalling, the apoptotic pathway, and the interaction of cancer cells with the immune system. Some anti-angiogenic agents also have anticoagulation activity that may also be associated with a reduction in metastasis. Heparin is a well-known example of a therapy with both anticoagulation and anti-angiogenic activities.Rather than develop multiple monoclonal antibodies to target the multiple peptides and their receptors, an alternative approach might be to evaluate phytochemicals and certain animal-derived chemicals that influence multiple pathways. The science of pharmacognosy evaluates natural drugs derived from herbal remedies or phytomedicines. Minimal clinical research has been undertaken to evaluate the use of natural drugs as adjuvant therapy with conventional treatment using cytotoxic drugs and radiotherapy. Formal research is required on the timing of administration of natural health products with anti-cancer therapies. As noted in the earlier discussion of the administration of radiotherapy with anti-angiogenic treatment, timing may be critical. Anti-angiogenic natural health products may be most effective as maintenance therapy that impedes cancer recurrence following cytotoxic treatment. Human tumours can remain dormant for years because of a balance between cell proliferation and apoptosis.Normal angiogenesis is the regulated formation of new blood vessels from existing ones. It is the basis of several physiologic processes, such as embryonic development, placenta formation, and wound healing. It is a good example of how a tumour can take control of normal processes and deregulate them to its own advantage.mmp) and heparinase that result in the dissolution of the extracellular matrix (ecm). The tight junctions between the endothelial cells are disrupted. The endothelial cells can then project through the newly created spaces and organize into fresh capillary tubes that grow toward the source of the blood supply In the normal and orderly formation of new blood vessels, endothelial cells receive a stimulatory signal from angiokinins and secrete specific enzymes such as matrix metalloproteinase  is a significant indicator of poorer prognosis. Angiogenesis plays a central role in the progression of most solid tumours, including those of bladder, brain, breast, cervix, colon, lung, and prostate. Increased vascular density has also been found in the bone marrow of patients with acute myeloid leukemia and myeloma Cancer cells begin to promote angiogenesis quite early in the development of a tumour. The angiogenesis is characterized by oncogene-driven tumour expression of pro-angiogenic proteins .ecm. The endothelial cells must also undergo morphogenesis. The process of angiogenesis consists of activation and resolution phases. Activation requires initial degradation of the basement membrane, followed by endothelial cell migration, invasion of the surrounding ecm, endothelial cell proliferation, and capillary lumen formation. During resolution, the microvasculature matures and stabilizes by enclosure of the vessel by pericytes, inhibition of endothelial proliferation, reconstitution of basement membrane, and formation of gap junctions.The formation of new vasculature occurs in sequential steps. Endothelial cells must proliferate, migrate, and penetrate host stroma and the The vasculature of many solid tumours is not identical to that of normal tissue apn-2 in the tumour-associated vasculature. Differences are seen in cellular composition and permeability, in vessel stability, and in regulation of growth. The balance between factors that stimulate new blood vessel growth and those that inhibit it determines the vascular density. The inhibitory influence predominates in normal tissues; in tumours, many neoplastic cells switch from an angiogenesis-inhibiting to an angiogenesis-stimulating phenotype. That switch coincides with the loss of the wild-type allele of the TP53 tumour suppressor gene and is associated with reduced production of thrombospondin-1 (tsp-1), a controller of angiogenesis in fibroblasts Failure of resolution may be a consequence of persistent overexpression of vegf is considered essential for most cancer cell migration and for angiogenesis. A high vegf expression level is associated with worse outcome in a wide array of malignancies. Expression of vegf messenger rna is upregulated by many oncogenes  and growth factors including epidermal growth factor, transforming growth factors alpha and beta, insulin-like growth factor–1, and platelet-derived growth factor The production of The process of cancer metastasis consists of a series of interrelated sequential steps. Each step is rate-limiting and may be a target for therapy. The outcome of the process depends on both the intrinsic properties of the tumour cells and the responses of the host. The balance of these interactions varies from one tumour and patient to another. These are the major steps Transformation of normal cells into tumour cells, followed by growth. Initially depends on nutrients supplied by simple diffusion.Extensive vascularization (angiogenesis). Vascularization must occur if the tumour mass is to exceed 1 mm in diameter. The production and secretion of pro-angiogenic factors by tumour cells and host cells plays a major role in establishing a capillary network from the surrounding host tissue.Local invasion. Tumour cells use several mechanisms to invade the host stroma. Thin-walled venules, fragmented arterioles, and lymphatic channels offer little resistance to penetration and provide the most common pathways for entry of tumour cells into the circulation.Detachment and embolization. Single cells or clumps break away. Most circulating tumour cells are rapidly destroyed. Those that survive must arrest in the capillary beds of distant organs by adhering either to capillary endothelial cells or to the exposed subendothelial basement membrane.Extravasation into new host organ or tissue.Proliferation within the new host organ or tissue. To continue growing beyond the 1-mm diameter, the micrometastasis must develop a vascular network and evade destruction by host defences. The cells can then continue to invade blood vessels, enter the circulation, and produce additional metastases.infβ). A potent inhibitor of angiogenesis, infβ works by blocking interleukin-8 (IL-8), basic fibroblast growth factor (bfgf), and collagenase type v, which are all potent angiogenic factors that aid tumour development and invasiveness.The growth of many cancers is associated with an absence of the endogenous inhibitors of angiogenesis—for example, interferon beta  correlate with the metastatic characteristics of human colon cancer, and so targeting vegf or egfr may be beneficial The efficacy of anti-angiogenic compounds varies from one tumour to another. The more specific the intervention is to one domain of the angiogenic pathway, the less likely a beneficial reduction in tumour growth is to occur, because alternative pathways can compensate. If the angiogenic activity of a tumour is initiated primarily by only one or two factors, then blocking the activity of one factor may be enough to inhibit tumour growth. For example, expression of Natural health products contain a cocktail of biologic chemicals that act on multiple pathways that initiate and maintain tumour angiogenesis. In addition, we hypothesize that angiogenesis within the tumour microenvironment may be more sensitive to a cocktail of natural health products administered continuously at relatively low doses than to single-agent pharmaceutical compounds administered intermittently at higher dose levels. In general, as compared with normal tissues, tumours contain very immature blood vessels that may be relatively more susceptible to anti-angiogenic therapies, permitting a therapeutic gain et al. reported on the anti-angiogenic properties of fumagillin, a secreted antibiotic of the fungus Aspergillus fumigatus One of the first anti-angiogenic agents to be isolated was a phytochemical. In 1990, Ingber Various assays are used to screen natural health products for anti-angiogenic activity The ability to maintain endothelial cells in culture has enabled the study of endothelial cell proliferation, migration, and function. For example, anti-angiogenic activity can be assessed by evaluating the potential of a substance to inhibit endothelial cell migration across a Boyden chamber. The bovine aortic endothelial cell assay is an established system.In vitro assays are relatively inexpensive and give more rapid results. However, an ability to inhibit endothelial cell proliferation, migration, and tubule formation in vitro may not predict in vivo response. In vitro assays are a rapid method for initial screening of large numbers of agents. Definitive conclusions cannot be based on in vitro assays alone.In vivo biologic assays are more specific for detecting anti-angiogenic activity. The chick embryo chorioallantoic membrane model is an extra-embryonic membrane that is commonly used to study agents that influence angiogenesis. An angiogenic response in the form of increased vessel density around the implant occurs 72–96 hours after stimulation with an angiogenic compound. An angiostatic compound will induce the vessels around the implant to become less dense and even to disappear. Other systems include animal cornea implantation, disc angiogenesis, Matrigel  systems, and tumour xenograft models.In vivo assays provide a more complete physiologic assessment of angiogenesis, but are more time-consuming and expensive.The degree of anti-angiogenic activity is dose-dependent. Most chemotherapy drugs have anti-angiogenic activity when administered at high doses. Clinicians are especially interested in compounds that, when administered at low doses, specifically interact and antagonize the steps involved in angiogenesis. These agents may have relatively low toxicity at low doses and may exhibit a higher therapeutic gain.Most conventional chemotherapy drugs have some degree of anti-angiogenic activity as a consequence of their cytotoxic activity. Ideal botanical derivatives would specifically antagonize new vessel formation in tumours without significant toxicity to normal tissues and without major adverse reactions. The ideal agent would also inhibit tumour cell proliferation through other physiologic pathways, such as intracellular signalling pathways.vegf and bfgf by the tumour cells—activate intracellular pathways that prevent apoptosis in tumour-associated endothelial cells.Multiple levels of anti-angiogenic activity may be required to overcome the development of resistance by tumour-associated endothelial cells. Survival factors—such as increased secretion of Maximal anti-angiogenic activity usually requires prolonged exposure to low concentrations of the active agent. This approach contrasts with the concept of administering maximum-tolerated doses of cytotoxic drugs to maximize tumour-cell kill. Some reports have confirmed the utility of combining low, frequent–dose chemotherapy with an agent that specifically targets the endothelial cell compartment vegf Further research is necessary to screen herbs that may be useful anti-angiogenic therapies. Our developing knowledge of cancer biology suggests that administering cytotoxic drug therapy at very high doses is not always appropriate. A new approach is to administer lower doses of synergistic organic chemicals. These complexes already exist in myriad botanicals. New laboratory techniques permit more specific assays of activity, enabling maintenance of quality assurance and consistency between batches of botanical preparations. Such quality standards will permit credible clinical trials of anti-angiogenic natural health products to be initiated. At the same time, the importance of a holistic approach to managing the patient with cancer should not be minimized. Anti-angiogenic therapies form only a small part of a complex management program. Attention to the patient’s overall health and ability to mount an immune response are subtle factors that may become more important in tipping the balance towards cancer control.Artemisia annua. Artemisinin has been used clinically as an anti-malaria drug Artemisinin is the active constituent extracted from the plant in vitro in the human umbilical vein endothelial cell (huvec) model of angiogenesis and was shown to significantly inhibit angiogenesis in a dose-dependent manner huvecs was greater than that seen with cancer cells, fibroblast cells, and human endometrial cells. Those findings indicate that the anti-angiogenic activity of artesunate is greater than its cytotoxicity.Artesunate is a semi-synthetic derivative of artemisinin. Artesunate was tested in vivo was evaluated using transplanted human ovarian cancer (HO-891) cells in nude mice. Immunohistochemical staining for microvessel CD31 antigen, vegf, and the vegf receptor  was performed. In treated mice, tumour growth was decreased and microvessel density was reduced without any toxicity to the host animals. Artemisinin also lowered vegf expression by tumour cells and KDR expression by endothelial cells. Artemisinin also has anticancer activity through other pathways. It inhibits the activation of nuclear factor kappa-B (nf-κb), an important activator protein in cancer development and progression The anti-angiogenic effect of artemisinin Viscum album is also known as Iscador . It is often used as an anticancer agent in anthroposophic and homeopathic medicine. Laboratory studies show that it is anti-angiogenic by downregulation of vegf; it also induces apoptosis of cancer cells V. album derivatives in combination with other anticancer therapies are warranted.MMP2) and upregulation of tissue inhibitor of metalloproteinase-1 (TIMP1) Curcumin is the most active curcuminoid in turmeric. It interacts with cancer cells at a number of levels and can enhance the tumoricidal efficacy of cytotoxic chemotherapy and radiotherapy vegf and bfgf vegf- and nitric oxide–mediated angiogenesis in tumours Curcumin inhibits the transcription of two major angiogenesis factors, VEGF and MMP9 genes that are associated with angiogenesis. Demethoxycurcumin is a structural analogue of curcumin isolated from Curcuma aromatica. It specifically inhibits the expression of MMP9 MMP2 and MMP9, the basis of the angiogenic switch, thereby reducing degradation of the ecm ecm. It inhibits growth factor receptors such as egfr and vegf receptor and the intracellular signalling tyrosine kinases. This cell signalling system can promote further angiogenesis through gene activation that increases levels of cyclooxygenase-2 (cox-2), vegf, il-8, and the mmpsCurcumin also downregulates the expression of the i study of curcumin found no treatment-related toxicity at doses up to 8000 mg daily. Beyond 8000 mg daily, the bulky volume of the drug was unacceptable to the patients. Serum concentration of curcumin usually peaked at 1–2 hours after oral intake of curcumin and gradually declined within 12 hours A phase vegf, bfgf, 12-lipoxygenase activity, and mmp Scutellaria baicalensis is one of the herbs found in pc-spes, a complex of Chinese herbs that is clinically active against advanced prostate cancer Baicalin and baicalein are the main derivatives of the Chinese skullcap herb. They are potent anti-angiogenic compounds that reduce huvecs and to decrease the lytic activity of mmp-2 vegf-induced angiogenesis by disruption of reactive oxygen species–dependent src kinase activation and subsequent ve-cadherin tyrosine phosphorylation Resveratrol is a phytoalexin found in grapes and wine. It has anti-angiogenic activity demonstrated by its ability to inhibit division in vegf expression induced by tumour necrosis factor alpha (tnfα). Feeding proanthocyanidins to mice with tumour xenografts reduced vegf secretion, which resulted in reduced intratumoral microvasculature vegf expression, suggesting that proanthocyanidin can induce angiogenesis as part of normal tissue healing Edible berries contain high concentrations of proanthocyanidin. The latter inhibits tgfβ) expression. It also inhibits nitric oxide synthesis and tnfα expression The seed cones of the Chinese magnolia tree contain substances that inhibit the growth of new blood vessels. Honokiol is the active ingredient. It may partly reduce angiogenesis through the regulation of platelet-derived endothelial cell growth factor and transforming growth factor beta  huvecs vegf, regulated at the level of transcription. In addition, egcg suppresses protein kinase C (pkc), another vegf transcription modulator.Tea contains polyphenols and catechins, mainly epigallocatechin-3 gallate ] has been recommended. In practice, lower total daily doses of 2–4 g standardized green tea extract (95% polyphenols and 60% catechins) are usually prescribed. Each gram of this extract provides 400–500 mg of egcg. The dose-limiting adverse effects are the gastrointestinal and neurologic effects of caffeine. However, the caffeine may potentiate the anti-angiogenic effect of egcg Inhibition of Ginkgo biloba extract has anticancer effects that are related to its gene-regulatory and anti-angiogenic properties. The Ginkgo biloba extract used in most research is EGb 761, which contains about 25% flavonoids (ginkgo-flavone glycosides) and about 5% terpenoids . The most potent flavonoid is ginkgolide B. The extract inhibits angiogenesis by downregulating vegf cox-2 and lipoxygenase-5 enzymes, egfr, the HER2 intracellular signalling pathway, and the nf-κb nuclear transcription protein Quercetin is a flavone found in apples, onions, raspberries, red grapes, citrus fruit, cherries, broccoli, and leafy greens. It inhibits angiogenesis through multiple mechanisms, including interaction with the Poria cocos is a mushroom extract that, by tradition, has anticancer activity. It inhibits platelet aggregation and appears to be anti-angiogenic by down-regulating nf κb 129The lipophilic constituents of ginseng are called saponins (or ginsenosides). These extracts possess anticancer activities in tumours that include anti-angiogenesis and induction of tumour cell apoptosis pc-spes formula that is active against prostate cancer. It contains ponicidin and oridonin, two diterpenoids that possess significant anti-angiogenic activity Rabdosia is used in certain traditions to treat cancer. It is one constituent of the Herbs that are used by tradition in China as anticancer agents have been screened for their anti-angiogenic activity fgf, vegf, tnfα, and il-1 are copper-dependent.Some cancers are associated with high serum levels of copper. The role of copper in cancer promotion through pro-inflammatory cascades and angiogenesis induction is quite well established nf-κb, which in turn controls transcription of many angiogenic factors and other cytokines.Copper chelation with tetrathiomolybdate is a promising therapy for tumour control Caryophylli flos, Cinnamomi cortex, Foeniculi fructus, and Zedoariae rhizoma—have inhibitory effects on lipid peroxidation or protein oxidative modification by copper Some angiogenic cytokines appear to have separate mechanisms of copper dependence. The inhibition of multiple angiogenic cytokines gives tetrathiomolybdate the potential to be a more global inhibitor of angiogenesis. Several aromatic herbs—such as in vitro and in vivo studies have suggested the existence of anti-angiogenic compounds in shark and bovine cartilage iii randomized controlled trial The resistance of cartilage to tumour formation has been correlated with its capacity to inhibit the formation of new blood vessels. A number of mmp-2 and to inhibition of serine elastase, of vegf binding to endothelial cells, and of tyrosine phosphorylation of the vegf receptor. Neovastat reduces the vegf-dependent increase in vascular permeability.Neovastat is kept frozen until use to maximally preserve its biologic properties. Its anti-angiogenic activity may be attributable to the presence of a metalloproteinase inhibitor with a preferential inhibition of Paradoxically, shark cartilage extracts (including AE-941) also have fibrinolytic activity iii randomized controlled trials have yet proven the utility of Neovastat for cancer treatment. Part of the funding for clinical studies of AE-941 comes from Technology Partnerships Canada (tpc), a research support program run by Canada’s federal government. The agreement is that Æterna will reimburse tpc upon commercialisation of AE-941–derived products. In January 2004, Æterna announced that development of AE-941 would be focused on non-small-cell lung cancer only No published phase Squalus acanthias vegf-induced activation of mitogen-activated protein kinase and cell proliferation in human vascular endothelial cells. Squalamine is anti-angiogenic for ovarian cancer xenografts, and it appears to enhance the cytotoxic effects of cisplatin chemotherapy, independent of HER2 status. Overexpression of HER2 is normally associated with resistance to cisplatin and promotion of tumour angiogenesis ii trial of patients with advanced small-cell lung cancer, squalamine was administered at a dose of 300 mg/m2 by continuous infusion for 5 days, with paclitaxel and carboplatin given on day 1. Patient survival data and a satisfactory safety profile indicated that the combination should be explored further Squalamine is a cationic steroid isolated from the liver of the dogfish shark, In vitro and in vivo studies are uncovering anti-angiogenic activity in many natural health products. Further preclinical research is required to define whether single compounds or complex mixtures will be optimal for clinical trials. A potential advantage of phytochemicals and other compounds derived from natural health products is that they may act through multiple cell-signalling pathways and reduce the development of resistance by cancer cells. Part 2 of this review will further discuss the latter issues.