Cerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families.GAD1, which encodes a glutamate decarboxylase isoform (GAD67), involved in conversion of the amino acid and excitatory neurotransmitter glutamate to the inhibitory neurotransmitter γ-aminobutyric acid (GABA).Here we present data that refine this locus to a 0.5 cM region, flanked by the microsatellite markers D2S2345 and D2S326. The minimal region contains the candidate gene 67 was detected, which segregated with CP in affected individuals.A novel amino acid mis-sense mutation in GAD67 and the more widely studied GAD65 homologue encoded by the GAD2 gene, are described in patients with Stiff-Person Syndrome (SPS), epilepsy, cerebellar ataxia and Batten disease. Further investigation seems merited of the possibility that variation in the GAD1 sequence, potentially affecting glutamate/GABA ratios, may underlie this form of spastic CP, given the presence of anti-GAD antibodies in SPS and the recognised excitotoxicity of glutamate in various contexts.This result is interesting because auto-antibodies to GAD Cerebral palsy (CP) is a term used to define a group of disorders  characteDepending on the overall clinical picture, CP can be sub-classified into a number of phenotypic groups ,9. DyskiKuban and Leviton  suggesteGAD1, which codes for the 67 kDa isoform of L-glutamate decarboxylase (GAD EC:4.1.1.15).A non-progressive, autosomal recessive, symmetrical spastic CP locus has been mapped to a 5 cM region between D2S124 and D2S333, at 2q24-31.1 (LOD score of 5.75) in consanguineous families originating from the Mirpur region of Pakistan (OMIM:603513) . AffecteGAD1 and GAD2 (at chromosome 10p11, encoding a 65 kDa GAD isoform), have presumably arisen by duplication and been conserved during evolution, as indicated by their sequence homology [N-termini demonstrate ~23% homology, but their C-termini, which contain the catalytic site, have ~73% amino acid sequence identity between the isoforms [GAD1 in familial spastic CP probands and unaffected family members.GAD requires the cofactor pyridoxal 5'-phosphate (PLP) and catalyses the production of Gamma-aminobutyric acid (GABA) from glutamate . Two sephomology  and the homology . Their Nisoforms . GABA anisoforms . Both isisoforms . GAD actThe oldest affected male diagnosed with non-progressive, spastic CP, demonstrated global developmental delay, with no associated neurological abnormalities and moderate mental retardation. His affected younger sister was also diagnosed with spastic CP, global developmental delay and moderate mental retardation.The oldest non-progressive, spastic CP female has severe mental retardation, and is confined to a wheelchair. The next oldest spastic CP male has severe mental retardation, mild hypertonia and ataxia of the upper limbs. The next oldest spastic CP male is a dizygotic twin born by Caesarean section. This patient on presentation demonstrated developmental delay, mild hypertonia and ataxia of the upper limbs. The youngest affected female is not able to walk or stand unaided and has severe developmental delay. Details of the clinical picture in these pedigrees have been described previously ,14.. Clones were grown up in casamino acid selective broth overnight and harvested by centrifugation. The pellet was then washed twice in 0.5 ml 100 mM Tris-HCl, pH7.5, 0.5 M EDTA buffer. After a second round of centrifugation the pellet was resuspended in molten 1% LMP agarose in 5 mM Tris-HCl, pH7.5, 0.05 M EDTA, 10 mM NaCl with 100 μg of Zymolase. The agarose was cooled and the resultant plugs were incubated overnight at 37°C in 50 ml of 0.5 M EDTA, 10 mM Tris-HCl, pH7.5, 10 mM NaCl. The buffer was replaced with fresh solution to which 100 μl of 40% Sarkosyl NL30 and 50 μl Proteinase K (1 mg/ml) was added and the plugs were incubated overnight at 50°C.ICI and CEPH YAC libraries were screened by PCR amplification of STSs that were mapped between D2S2157 and D2S385. The positive clones  were obtained from the UK HGMP Resource Centre The YAC chromosomal DNA was purified and separated for sizing by CHEF electrophoresis. The switching angle was 120°, using a CHEF™ electrophoresis tank (Bio Rad), run for 16 hours at 6 V/cm, 10°C with an initial pulse time of 30 sec to a final pulse time of 90 sec. DNA was stained with (20 mg/ml) ethidium bromide solution (BDH) for 2 hours and visualized under ultra-violet illumination. Southern blotting and membrane hybridisation of CHEF gels were performed on Hybond-N™ membranes (Amersham). DNA was immobilised by heating the membrane to 80°C in vacuum for 1 hour in a gel dryer (Bio-Rad). Radio-labelled YAC vector-specific probes were generated using the Megaprime random primer kit (Amersham) as described in the manufacturer's instructions, using PCR products as a template. Sizes were estimated based on comparisons with the known sizes of the native yeast chromosomes., , . These primers were used to amplify microsatellite marker alleles from individuals by PCR. Individual alleles were identified by denaturing polyacrylamide gel electrophoresis on an ABI Prism 377 sequencer and analysed using Genescan™ and Genotyper™ (version 1.1.1) software (Applied Biosystems).The microsatellite markers: Cen-D2157, D2S124, D2S2330, CHLC.GATA71B02 (D2S1776), D2S2345, CHLC.GATA71D01, D2S294, AFMA109YC1, D2S376, D2S2284, D2S2177, D2S2194, D2S333, D2S2302, D2S2381, D2S326, AFMA304WB1, D2S138, D2S148, D2S300-Tel, were amplified using fluorescently labelled primers (Lifetech) previously designed by the Whitehead Institute or on the GDB database GAD1 exon sequences were amplified by PCR using the primers described in Table GAD1 were designed from the sequence of BAC RP11-570c16 and obtained from Lifetech (Table Primers used to amplify the exons of , in an attempt to form an ordered BAC/YAC contig across the minimum region of interest. Selected YACs from this contig were sized (Table An integrated YAC (ICI and CEPH mega YAC libraries) and RP-9 PAC (HGMP Resource Centre) contig for the interval D2S2157 to D2S385 was constructed C , onto our YAC/PAC contig. ESTs were then collated according to their expression profiles to produce a "binned" EST map, on which candidate gene selection could be based. This reduced the number of hypothetical genes in the region and allowed the combination of genetic, physical mapping and expression data, into a single comprehensive map.The Goldenpath Human Genome Working Draft Assembly 2001, is an annotation of the Washington BAC contig, combining sequence data of BACs, ESTs, known genes and hypothetical genes. We mapped ESTs and known, uncharacterised or hypothetical genes on the basis of sequence homology . The 67 kDa (594 AA residues) form [GAD1) [25) produced from an alternatively spliced GAD1 transcript of 2 kb that contains an in-frame stop codon. This GAD1 splice variant has only been found in human islets, testis and adrenal cortex, although the homologue is present in fetal mouse brain [67 and GAD65 consist of two major sequence domains  demonstrate ~23% homology. These N-terminal domains are thought to be responsible for sub-cellular targeting and the formation of GAD65–GAD67 heterodimers [C-terminal domains (AA residues 96–585 in GAD65 and 102–594 in GAD67) contain the catalytic site, with ~73% sequence identity between the isoforms [es) form  is localm [GAD1) . There is Figure . The N-trodimers . The C-tisoforms  , which responds to short-term changes in neuronal activity and is more responsive to levels of PLP [67 predominantly exists bound to the PLP cofactor (holoGAD), providing a constitutive level of GABA production [et al. [25 is expressed in a temporally controlled manner, in the developing striatum and cortex in rodents, suggesting this may provide a mechanism of regulating GABA production in differentiating neurons.In the CNS, GADe neuron . This sue neuron . This iss of PLP . On the oduction . Bond et [et al.  showed tet al. [Gad2-/- mice are slightly more susceptible to seizures, consistent with an excitatory increase in the relative ratio of glutamate/GABA. However, they showed no obvious overall change in neuronal GABA content. Therefore GAD67 alone appears to produce sufficient GABA for effective neurotransmission [Gad1-/- mice demonstrated a decrease of ~20% in total glutamate decarboxylase activity at birth. This was assayed by the conversion of 14C-labelled glutamate to 14CO2 in the presence of PLP. There was also a marked (7%) reduction in total GABA content in cerebral cortex homogenate measured by liquid chromatography [67 in non-neural tissues [Asada et al.  undertoosmission . Gad1-/-tography . Unfortu tissues . It is o tissues ,30.GAD1 mutation was previously suspected of being the cause of PDE. Linkage of pyridoxine-dependent epilepsy has however been reported to 5q31.2-31.3, with GAD1 and GAD2 excluded [Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disorder OMIM:266100), characterized by generalized seizures during the first hours of life. The associated pathology may result from an alteration in the binding of the co-factor PLP to GAD. Interestingly epilepsy is commonly associated with CP and grand mal epilepsy developed at age six months in the two linked pedigrees described here [0, charac65 and GAD67 have been identified as auto-antigens in "Stiff Person Syndrome" (SPS, OMIM:184850), and in cerebellar ataxia [65 and GAD67 [GADr ataxia -34. GABAnd GAD67 -37. Thisnd GAD67 . Hyperexnd GAD67 .65 auto-antibodies in the CSF of ataxic and SPS patients selectively suppress GABA-mediated transmission in cerebellar Purkinje cells, without affecting glutamate-mediated transmission [A receptors [Anti-GADsmission ,40. Low smission . The exaeceptors . This prCLN3 gene are thought to be responsible for the neurodegenerative disorder Batten disease (OMIM:204200). In cln3-knockout mice autoantibodies to GAD65 have been reported to be associated with brain tissue and result in inhibition of GAD activity [cln3-knockout mice and along with anti-GAD65 autoantibodies in Batten disease patients may contribute to the associated preferential loss of GABAergic neurons.Mutations in the activity . These mDrugs, which potentiate the action of GABA, such as benzodiazepines and baclofen, ameliorate muscle rigidity and spasticity. These GABA agonists are thought to counter disinhibition of the velocity-dependent increase in skeletal muscle during stretch reflexes, observed in spasticity, which is the result of inadequate presynaptic inhibition of the muscle spindles ,35,37. γGAD1 sequence change G(36)C, which segregates with autosomal recessive spastic CP in 4 affected siblings. This nucleotide substitution causes a missense mutation, changing serine (12) to a cysteine in the N-terminal domain. This serine residue is conserved between all mammals (human/mouse/rabbit/pig) for which data are available. The association of GAD67 with membranes requires formation of heteromeric links with GAD65, which are mediated via their N-terminal domains. The N-terminus of GAD65 is palmitoylated and binds to the cellular membrane. The first 27 amino acids appear to be essential in this function [65, and the inability of residues 1–29 of GAD67, to substitute for this region, highlights the potential impact on cellular localisation of a nucleotide substitution in this domain [65 also undergoes phosphorylation of the first four serine residues in the N-terminal domain. These post-translational modifications highlight the importance of the flexibility and accessibility of this domain. N-terminal epitopes of GAD65, in the region corresponding to the residue, which undergoes mutation in GAD67, are particularly prominent autoantigens [We have identified a function . The pals domain . GAD65 aantigens .Gad1 knockout where complete loss of Gad1 enzymatic function  resulted in a cleft palate phenotype and neonatal death [25 splice variant of GAD67, also contains the S(12)C amino acid substitution in affected CP patients. This truncated variant is identical to the first 213 amino acids of GAD67, with the addition of an extra 11 C-terminal residues. It lacks the binding site for the cofactor PLP and is believed to lack any GAD activity [25 is not known, but it may compete with GAD67 for incorporation into protein complexes. Therefore the presence of an N-terminal mutation would affect both GAD25 and GAD67, and may disrupt a complex regulatory mechanism for GAD67.S(12)C amino acid substitution may thus produce subtle effects on cellular localisation, protein-protein interactions and/or protein processing, with a subsequent effect on GABA production. This is not inconsistent with the mouse al death ,30. Ther67 (S12C) and GAD25 (S12C), as recombinant proteins, to assess catalytic activity and binding properties with respect to their normal counterparts. However, it may well prove difficult to detect subtle effects based on sub-cellular localisation changes in the mutant proteins, or changes in post-translational modification patterns.This study illustrates the difficulty of gene cloning in rare autosomal recessive diseases mapped in small, consanguineous pedigrees. Identification of an ancestral haplotype allows refinement of the locus, but this has not been possible in this example. Within the minimal linkage region, any sequence change will segregate with the disease phenotype. Detection of a nonsense mutation leading to a protein truncation would provide compelling support for a mutation as causative. However, in the present example we have not seen such a mutation in the candidate gene so far examined. We are now expressing the variant forms of GADGAD1 (G36C), into the Gad1-/- mouse to see if this can rescue the cleft palate phenotype and reveal a CP-like picture. These experiments will be reported elsewhere. However, the possibility that reduced GAD67 activity may cause CP in the patients studied herein, in a manner reminiscent of that seen in SPS, leads us to report our findings at this stage. The success reported in treating SPS with intravenous immunoglobulin [Stability of the mRNA transcripts from these variants will be assessed by transfection studies in neuronal cells. Eventually, it would be of interest to attempt knock-in experiments with globulin ,46, suggBAC Bacterial artificial chromosomeBLAST Basic local alignment search toolCEPH Centre d'Etude du Polymorphisme HumainCHEF Contour-clamped homogenous electric fieldCNS Central nervous systemCP Cerebral PalsyCSF Cerebrospinal fluidDNA Deoxyribonucleic acidEST Expressed sequence tagGABA Gamma-aminobutyric acidGAD L-Glutamate decarboxylaseGVG Gamma-vinyl-gamma aminobutyric acidHGMP Human genome mapping projectIDDM Type 1 Insulin-Dependent Diabetes MellitusMRI Magnetic resonance imagingNCBI National Centre for Biotechnology InformationPAC Plasmid artificial chromosomePCR Polymerase chain reactionPDE Pyridoxine-dependent epilepsyPFGE Pulse field gel electrophoresisPLP Pyridoxal 5'-phosphatePVL Periventricular leukomalaciaSPG Spastic paraplegiaSPS Stiff Person SyndromeSSCP Single-strand conformational polymorphismSTS Sequence tagged siteYAC Yeast artificial chromosomeThe author(s) declare that they have no competing interests.CNL carried out the molecular genetic studies, sequencing and drafted the manuscript. The YAC mapping work was undertaken by JPL and CNL. AFM, DTB and IMC conceived the study and participated in the design and coordination, they also secured financial sponsorship from the Wellcome Trust and MRC. RA, SM, ERM and CGW recruited and gained consent from the families detailed in this study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Coevolution between pairs of antagonistic species is generally considered an endless "arms race" between attack and defense traits to counteract the adaptive responses of the other species.When more than two species are involved, diffuse coevolution of hosts and parasitoids could be asymmetric because consumers can choose their prey whereas preys do not choose their predator. This asymmetry may lead to differences in the rate of evolution of the antagonistic species in response to selection. The more long-standing the coevolution of a given pair of antagonistic populations, the higher should be the fitness advantage for the consumer. Therefore, the main prediction of the hypothesis is that the consumer trophic level is more likely to win the coevolution race.We propose testing the asymmetry hypothesis by focusing on the tritrophic system plant/aphid/aphid parasitoid. The analysis of the genetic variability in the virulence of several parasitoid populations and in the defenses of several aphid species or several clones of the same aphid species could be compared. Moreover, the analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation may complement the detection of differences between host and parasitoid trophic specialization.Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots. Thus this hypothesis profoundly changes our understanding of coevolution and may have important implications in terms of pest management. Coevolution is the result of reciprocal selective pressures exerted by interacting species. Many studies have been devoted to the hypothesis of an endless "arms race" between antagonistic species, in which each species develops escalating attack and defense traits to counteract the adaptive responses of the other species. In reference to Lewis Carroll's book "Through the Looking Glass", Van Valen  named thThe RQH was initially developed in the context of multiple species interactions, to account for the constant probability of species extinction. However, as modeling the coevolution of many species involves a number of difficulties, later studies based on the RQH have mostly been limited to interactions between pairs of species. From this restricted situation, two main predictions can be made: first, arms races induce an advantage of rare genotypes of which resistance or virulence is more efficient and thus, frequency-dependent fluctuations of resistance and virulence may be predicted. Second, a geographic view of the coevolutionary process suggests a dynamic mosaic structure, with local and temporary "hot spots" of antagonistic species coevolution .However, for plant-pathogen interactions  and for Nevertheless, the RQH continues to lie at the heart of the debate concerning the coevolution of antagonistic species, probably because of the lack of plausible alternative hypotheses. Although metapopulation structures or time lags between the responses of antagonistic species to selective pressures have been evoked as reasons for the lack of clear experimental evidence in favor of the RQH , the addAdaptive responses to selective pressures exerted by antagonistic organisms are not necessarily the same for the target species and the consumer species. There is a first level of potential evolutionary asymmetry between them because the failure of virulence for a consumer is a delay in fitness acquisition whereas the failure of defense for a prey is the loss of its entire fitness. Such a difference was underlined by Dawkins  noting AExtending the field of interest to interactions and coevolution between more than two species (i.e. diffuse coevolution) may offer new perspectives. Considering the reciprocal selective pressures exerted by many species leads us to take into account the specificity of virulence and resistance. Until now, few works have been devoted to this subject (but see ). HoweveA recent model described the evolution of resistance of one host subjected to the attacks of two types of parasitoids differing in their virulence and specificity (different genotypes of a species or different species) and the evolution of virulence of one parasitoid attacking two types of hosts differing in their resistance and specificity . The resThe asymmetry hypothesis (AH) may lead to differences in the rate of evolution of the antagonistic species in response to selection. For a specialist consumer capable of target choice, chosen targets constitute a more or less "constant environment". On the other hand, for a generalist defender, the diversified, facultative and fluctuating attacks by a set of enemies constitute a "variable environment". The constancy of the environment may lead to the faster adaptation of specialists (mostly consumers under AH) than of generalists (mostly targets under AH) . Thus, uDrosophila species to encapsulate parasitoid eggs. In this case, it can be also noted that the resistance trait is not specific  when virulence traits (reviewed in [The asymmetry hypothesis seems to fit well with numerous data concerning geographic structures published in the literature (reviewed in  but see iewed in ) are divLysiphlebus testaceipes and the aphid Aphis gossypii. To measure this variability, several populations of the parasitoid collected on different clones of the aphid will be confronted to different clones of this aphid. The different clones of the aphid will be confronted to the different parasitoid populations. This system that considers intra specific genetic variability for each trophic level will be used to evaluate the fitness of the consumers and of the targets in the different parasitoid /aphid combinations. An important point is that virulence will be estimated from the success rate of parasitism (i.e. the production of offspring) whereas defense will be estimated through the measure of the aphid fitness, i.e. the number of offspring produced by the aphid whatever the outcome of the parasitism (success or failure). Two key factors of the host fitness will be considered: 1) the host survival rate in case of parasitism failure (an aphid may die or not, even if the parasitism does not result in parasitoid offspring production); and 2) the number of offspring produced by the host after parasitism: after a parasitoid's sting, the host may produce offspring until mummification in case of parasitoid embryo development or until a variable date in the case of parasitism failure. B- The analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation. The specialization of L. testaceipes on different aphid clones can lead to a neutral genetic differentiation of the parasitoid as a function of the host and the plant because the reproduction of the parasitoid occurs soon after the emergence of the adults from their host. The genetic differentiation between populations of the parasitoid sampled from different aphid clones and from different plant species could be evaluated and compared to a putative effect of isolation by distance.We propose a dedicated test of the hypothesis of the specialization of the parasitoid virulence and the absence of specialization in the defense of the aphids, against the RQH. The study will focus on the tritrophic system plant/aphid/aphid parasitoid. Two complementary experimental approaches could be considered: A- The analysis of the genetic variability of virulence and defense of the parasitoid This test may allow local verification or rejection of the predictions of the AH . When performed several times on animals from diverse geographic origins, this should eventually allow rejection of the classical interpretation of RQH and the host spots theory of coevolution .The consequences of the asymmetry hypothesis are important. Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots . HoweverThe AH cannot be applied to every situation. In the case of host-parasitoid associations, the high level of genetic variability in resistance observed within some local host populations ,21 is beAsymmetry may be suspected in cases involving successive trophic levels other than host-parasitoid associations, such as plant-insect or parasitoid-hyperparasitoid combinations, as soon as individuals of the upper trophic level can choose their target. However, random attacks, such as those due to plant pathogens, may favor more generalist traits of virulence, but the specificity of consumers, and therefore asymmetry, may be restored through indirect choices: pathogens transmitted by vectors may use or manipulate the specificity traits of the vector. Large sets of species interactions may thus lead to asymmetric coevolution.Acyrthosiphon pisum is parasitized by the hymenopteran Aphidius ervi and is specialized on different plant species. Hufbauer & Via [A. ervi than is the clover host population. If the association they studied dealt with two aphid and one parasitoid populations, under the AH, virulence specialization rather than resistance level variations can explain the observations: the parasitoid population is specialized on the clover host population, but keeps a partial virulence on the alfalfa aphid biotype. This suggests that short-term parasitoid specialization may be a key factor in biological control efficiency. For instance, consumers introduced to control a pest could rapidly specialize against non-target hosts.The AH may have consequences in terms of pest management. For instance, it is generally thought that variations in parasitism outcome result from a variability of host resistance due to the selection for higher resistance ,22. Howeer & Via  observeder & Via  also obsThe asymmetry hypothesis thus provides food for thought concerning diffuse coevolution and could be applied to domains beyond host-parasitoid coevolution. Similar thoughts may be applicable to the durability and efficiency of plant resistance or immunological responses to diseases transmitted by vectors. Its theoretical implications and its consequences in terms of population management are potentially important and remain unexplored.RQH: Red Queen HypothesisAH: Asymmetry HypothesisBoth authors have been involved in the elaboration of the hypothesis and the evaluation of its consequences, and in the drafting of the paper.


Most studies on the resistance of mosquitoes to their malaria parasites focus on the response of a mosquito line or colony against a single parasite genotype. In natural situations, however, it may be expected that mosquito-malaria relationships are based, as are many other host-parasite systems, on host genotype by parasite genotype interactions. In such systems, certain hosts are resistant to one subset of the parasite's genotypes, while other hosts are resistant to a different subset.Anopheles gambiae were challenged with several isolates of the human malaria parasite Plasmodium falciparum .To test for genotype by genotype interactions between malaria parasites and their anopheline vectors, different genetic backgrounds  of the major African vector Averaged across all parasites, the proportion of infected mosquitoes and the number of oocysts found in their midguts were similar in all mosquito families. Both indices of resistance, however, differed considerably among isolates of the parasite. In particular, no mosquito family was most resistant to all parasites, and no parasite isolate was most infectious to all mosquitoes.These results suggest that the level of mosquito resistance depends on the interaction between its own and the parasite's genotype. This finding thus emphasizes the need to take into account the range of genetic diversity exhibited by mosquito and malaria field populations in ideas and studies concerning the control of malaria. Plasmodium parasites in their mosquito vectors [Daphnia-bacterium [In the last few years, exciting advances in the biology and molecular genetics of the development of  vectors ,2 have l vectors , bringin vectors ,5. A cru vectors ,7, givin vectors ,9. Unfor vectors . One of  vectors . Because vectors  and the  vectors , it is c vectors , snail-s vectors , bumble- vectors , Daphniaacterium ), the ouacterium . In othePlasmodium falciparum and Anopheles gambiae. Malaria genotype by mosquito genotype interactions were tested with a standard procedure of quantitative genetics from measurements of the resistance of different genetic backgrounds of the mosquito A. gambiae, a major malaria vector in sub-Saharan Africa, to different isolates of the human malaria parasite P. falciparum. The parasite isolates were obtained from naturally infected children in western Kenya that harboured gametocytes, the infective stage of the parasite. The genetic backgrounds of mosquitoes were 'mosquito families' generated as the F1 offspring of single egg-laying females. Each mosquito family was challenged with each parasite isolate, and all mosquitoes were simultaneously fed on the blood of the gametocyte carriers via membrane-feeding. This basic design was repeated three times throughout three successive experimental blocks that involved different families and isolates, giving a total of 18 mosquito families, 11 parasite isolates, and 62 specific interactions. As in previous studies [Despite its potentially important role for malaria epidemiology and control, such a genetic specificity of host-parasite compatibility between malaria parasites and their insect vectors have never been investigated. This study examines the potential for genotype by genotype interactions in the combination that is most important for the epidemiology of malaria –  studies , the res studies ,19. Thes studies .A. gambiae s.s. caught in the area surrounding Mbita, a small village on the shore of Lake Victoria in Suba District (western Kenya). These mosquitoes had been initially adapted to feed on a Parafilm® membrane, and then maintained in standard insectary conditions using a rabbit as a blood source for routine maintenance. Females of the colony were blood-fed on a rabbit and allowed to lay eggs in individual vials. Immediately after hatching, each larva was individually placed in one well of a 12-well plate with three mL of filtered lake water. They were fed daily on a standard diet of Tetramin® fish food . Adults were kept in an insectary and supplied with a 6% glucose solution and cotton soaked with distilled water. The temperature and humidity in the insectary followed the daily environmental fluctuations. So that mosquito age at pupation did not affect the success of infection, only females that pupated seven days after hatching were used. The wing length of the mosquitoes, measured from the tip (excluding the fringe) to the distal end of the allula with a precision of 0.02 mm, was used as an indication of body size [The mosquitoes used in this study came from a colony that had been established in 2001 from ody size . Where bP. falciparum carriers were recruited from the two- to 10-year old children in the rural area around Mbita, from December 2003 to January 2004. Finger-prick blood samples were collected and thick blood smears were air-dried, stained with 8% Giemsa during 15 minutes, and examined microscopically for the presence of P. falciparum. Children with asexual parasitemia (>1,000 parasites/μL) were immediately treated with sulfadoxine-pyrimethamine according to national guidelines. Asymptomatic gametocyte-positive children were recruited for the study after their parents or guardians had signed an informed consent form. The Kenyan and the United States National Institute of Health ethical review committees approved this recruitment procedure.P. falciparum isolates were collected from gametocyte carriers that had been identified one or two days before, and used to feed the mosquitoes on the same single day . The gametocyte densities were assessed just before blood withdrawal on a blood smear (as described above) by counting against 500 leukocytes, and converted to numbers of parasites per μL by assuming a standard leukocyte count of 8,000/μL. Although gametocyte densities in the venous blood and the peripheral finger-prick blood might differ, potential differences were assumed to be proportional among the isolates. A sample of five mL of venous blood was collected from each gametocyte carrier in a heparinized tube, 400 μL of which were stored at -20°C for further parasite genotyping. So that the importance of human factors such as transmission-blocking immunity [g and the autologous serum was replaced with the same volume of a pool of AB serum from two French blood donors without any malaria exposure . The mixture was used to feed mosquitoes, which had been starved for 12–16 h before blood feeding, with a standard membrane-feeding system [For logistic reasons, the experiment was repeated three times, and within each experimental block infections were done simultaneously. For each of the three blocks, immunity  was reduP. falciparum DNA was extracted from the blood samples using the QIAamp DNA blood kit following the manufacturer's instructions (Qiagen, CA). The isolates were typed using the semi-nested PCR method slightly modified from a previous study [us study  . In this analysis, the square root of the number of oocysts was used, so that the assumptions of the statistical tests  were satisified. As the study was run in three successive experimental blocks, both analyses included the effect of block, and the effects of family, isolate and their interaction. The effect of wing length was also included as a potential confounder . As diffP. falciparum oocysts, and the number of oocysts in infected mosquitoes ranged from one to 97 (mean 11.0, median 3). The prevalence and the number of oocysts differed among blocks  of the 455 mosquitoes of the study were infected by 51 of theWhile the crude variation among families  was substantial , most of this variation was due to differences among blocks  and the effect of the interaction on the number of oocysts was significant in one of the cases and showed a tendency in the other case .Separate analyses of the data for the two numbers of clones (two or three) ensured that the number of clones contained in each isolate did not confound the interpretation. The effect of the mosquito family by parasite isolate interaction on the likelihood of infection was significant in both cases  and the number of oocysts . In conclusion, the two potential confounders – number of clones per isolate and gametocyte density – had no qualitative influence on the results of the analysis.In addition, differences in gametocyte density between isolates Table  may be ePlasmodium species and strains [Plasmodium parasites [While the specificity of mosquito-malaria interactions at the species level is well documented , the pre strains . The prearasites ,31. The Plasmodium recognition by mosquitoes are still largely unknown, a group of thioester-containing proteins (TEPs) represents a promising family of candidate recognition molecules. One of them, the complement-like protein TEP1, has recently been shown to bind to and mediate the killing of the rodent malaria parasite P. berghei by the mosquito A. gambiae [TEP1 gene are associated to susceptible and refractory strains of A. gambiae [The specificity of host-parasite interactions is often postulated to occur at the level of parasite recognition. While the molecular mechanisms of  gambiae . Moreove gambiae . It is tThe mosquito genotype by parasite genotype interactions shown in this paper may help to understand some puzzling aspects of the epidemiology of malaria. Thus, even in areas with intense transmission, the probability that a mosquito becomes infected is generally low ,32. FurtPlasmodium might be misleading for the development of malaria control strategies. Indeed, most of these laboratory-based studies focus on the response of one mosquito line or colony against a single parasite strain and thus do not represent the genetic diversity of mosquitoes and parasites in natural populations [The epidemiological consequences of mosquito genotype by malaria genotype interactions are perhaps most obvious in the context of malaria control with mosquitoes transformed to be resistant against malaria. The strong genetic specificity of compatibility between parasite isolates and individual insect vectors suggests that most studies on the mechanisms underlying the resistance of mosquitoes against ulations ,13. Howeulations .P. falciparum development, a major component of its vector competence, varies considerably between different combinations of parasite isolates and individual, genetically variable, vectors. Optimal transmission may thus require some specific compatibility between the insect's and the parasite's genotypes. This result has important consequences for the epidemiology of malaria. Overall, it suggests that conclusions from a particular subset of mosquito and malaria genotypes will not necessarily hold for other combinations of genotypes. Therefore, field studies taking into account the full diversity of mosquito and parasite populations are necessary to reach valid conclusions concerning the technologies developed in laboratories for the control of malaria.This study demonstrated that the resistance of an anopheline mosquito to LL participated in the design and the coordination of the study, carried out the fieldwork, participated in the molecular analysis, performed the statistical analysis, and wrote the manuscript. JH participated in the fieldwork. PD carried out the molecular analyses and helped to draft the manuscript. LCG supervised and coordinated the fieldwork. JCK conceived and designed the study, performed the statistical analysis, and wrote the manuscript.


Lung disease in patients with cystic fibrosis is thought to develop as a result of airway inflammation, infection, and obstruction. Pulmonary therapies for cystic fibrosis that reduce airway inflammation include corticosteroids, rhDNase, antibiotics, and high-dose ibuprofen. Despite evidence that high-dose ibuprofen slows the progression of lung disease in patients with cystic fibrosis, many clinicians have chosen not to use this therapy because of concerns regarding potential side effects, especially gastrointestinal bleeding. However, studies have shown a low incidence of gastrointestinal ulceration and bleeding in patients with cystic fibrosis who have been treated with high-dose ibuprofen.The described case illustrates a life-threatening upper gastrointestinal bleed that may have resulted from high-dose ibuprofen therapy in a patient with CF who had undergone a pneumonectomy. Mediastinal shift post-pneumonectomy distorted the patient's esophageal anatomy and may have caused decreased esophageal motility, which led to prolonged contact of the ibuprofen with the esophagus. The concentrated effect of the ibuprofen, as well as its systemic effects, probably contributed to the occurrence of the bleed in this patient.This report demonstrates that gastrointestinal tract anatomical abnormalities or dysmotility may be contraindications for therapy with high-dose ibuprofen in patients with cystic fibrosis. Airway inflammation is thought to cause much of the lung damage in patients with cystic fibrosis (CF) ,2. Such Recent therapies for CF lung disease have been shown to preserve lung function by decreasing airway exposure to inflammation. For example, airway clearance, antibiotics , rhDNase, and oral corticosteroids have been used to help decrease airway inflammation -6. High-Pseudomonas aeruginosa pneumonia. The consolidation was unresponsive to intensive therapy including intravenous (IV) antibiotics, chest physiotherapy, systemic steroids, and rhDNase. The patient developed recurrent severe pulmonary exacerbations necessitating IV antibiotic therapy 4 times per year between the ages of 14 and 15 years. A computerized tomography scan of the chest when the patient was 15-years-old revealed complete collapse of his right lung. A ventilation/perfusion scan revealed only 8% of his ventilation and perfusion in his right chest, while the remaining 92% was in his left chest. In addition, the left lung had begun to herniate into his right chest. It was felt that the right lung was a recurrent source of infection, which was causing serious morbidity. Therefore, the patient underwent a total right pneumonectomy that he tolerated well.The patient was a teenager with CF who suffered for several years from right lower lobe consolidation, as a result of recurrent Approximately one year later, in preparation for initiating high-dose ibuprofen therapy, the patient's stool was checked for occult blood. He was found to have occasional guaiac positive stools. An upper gastrointestinal endoscopy revealed chronic esophagitis, esophageal ulcerations, and Barrett's esophagus thought to be attributable to gastroesophageal reflux. A 24-hour pH probe revealed significant gastroesophageal reflux despite anti-reflux therapy. Therefore, a fundoplication was performed in order to prevent further esophageal damage. Subsequently, it was believed that his esophageal ulcerations resolved as multiple stools were documented to be guaiac negative.High-dose ibuprofen therapy was initiated when the patient was 17-years-old, based on published recommended dosage and pharmacokinetic protocols . The patFollowing this episode the patient's ibuprofen was discontinued, and his oral medications (e.g., antibiotics and vitamins) were switched to liquid form, whenever possible. His pancreatic enzymes were administered after removing the enzyme microspheres from their capsule and mixing them in applesauce.Reported complications of pneumonectomy include mediastinal shift with herniation of the remaining lung, cardiac herniation, cardiac arrhythmias, bronchopleural fistula, esophageal motility disorders, and development of scoliosis -11. It iIn our patient, esophageal dysmotility along with the distortion of esophageal anatomy probably combined to slow the esophageal transit time of the ingested ibuprofen, which may have led to development of ulcerations as a result of prolonged concentrated contact within the esophagus. Also, the ibuprofen could have contributed to the development of ulcerations by inhibiting cyclooxygenase systemically, which decreased prostaglandin E production . In turnDelayed esophageal transit may have occurred with the patient's other oral medications prior initiation of ibuprofen. For example, his pancreatic enzymes likely were retained in the same esophageal segment, which placed him at risk of developing esophageal damage akin to the development of fibrosing colonopathy and strictures described in patients with CF who received high-dose pancreatic enzymes . The enzAccording to the 2002 CF Foundation registry, only 3.8% of patients in North America with CF were treated with high-dose ibuprofen . In 1999The initial randomized, double-blind, placebo control study of high-dose ibuprofen in CF did not demonstrate serious side effects during 4 years of therapy with ibuprofen sufficient to achieve peak plasma concentrations of 50–100 mcg/L . Seven oThe incidence of gastrointestinal disease in patients reported to the CF Foundation registry from 1996–2000, was compared between patients who were and were not taking ibuprofen . She wasThe risk of developing gastrointestinal side-effects from high-dose ibuprofen therapy is low for patients with CF. However, ibuprofen may be contraindicated for those who are at increased risk because of gastroesophageal reflux, history of gastrointestinal ulcerations, or abnormal gastrointestinal motility or anatomy.None declared.JM wrote the case report. RA treated the reported patient, and edited the report.The pre-publication history for this paper can be accessed here:


Neisseria meningitidis is a serious infection which is most common in young children and adolescents. This study investigated the relationships between the incidence and age distribution of meningococcal disease, and socioeconomic environment.Meningitis caused by An ecological design was used, including mapping using a Geographical Information System (GIS) at census ward level.Incidence of meningococcal disease was highest in the most deprived wards, with a relative risk of 1.97 (1.55 – 2.51). Mapping revealed geographical coincidence of deprivation and meningococcal disease, particularly in urban areas. Two-thirds of the increased incidence was due to cases in the under fives.The results suggest that area deprivation is a risk factor for meningococcal disease, and that its effects are seen most in young children. Neisseria meningitidis are still an important cause of morbidity and mortality in the United Kingdom, with 1548 cases notified in England and Wales in 2001[Infections caused by The infection is most common in infants and adolescents, with peaks of incidence at 0–5 years and 15–19 years. OutbreaIt is accepted that many infectious diseases preferentially affect the most disadvantaged in society. This has been discussed at historical, global, and natThis study was prompted by an impression, gained from the practice of a consultant in communicable disease control (CCDC), that infant cases of meningococcal disease came from families of low socioeconomic status, whilst older cases tended to be drawn from families of higher socioeconomic status. Studies in northeast Thames, southweThe study was ecological in design, and used census-derived area data, map data, and individual case data as described below. The geographical unit of analysis was the 1991 census ward, and the study population was the entire Eastern region population of the UK (1999 estimate 5.3 million).The Eastern region of England is made up from the counties of Hertfordshire and Essex (both adjoining Greater London to the south), Suffolk and Norfolk along the east coast, Cambridgeshire centrally and Bedfordshire to the north. Much of the area is rural, although there are several medium-sized urban areas in Essex and Hertfordshire, with links to the capital. Away from the London area, the major urban centres include Cambridge and Norwich (both with large university populations), the ports of Ipswich and Harwich, and Peterborough and Luton.Data on cases came from the Eastern region Communicable Disease Surveillance Centre (CDSC Eastern) regional database of enhanced surveillance of meningococcal disease. To be iThe regional population data were projections for 1999 based on 1991 census data, obtained from the compendium of clinical and health indicators 2001[The deprivation index used was the Townsend score, which combines local measures of unemployment, car ownership, overcrowding, and housing tenure. This meThe shape files used in the MapInfo (© MapInfo Corporation) and EpiMap2000 (in Epi Info™, Centres for Disease Control and Prevention) maps were digitised ward boundaries from the 1991 census, obtained from the EDINA (Edinburgh data and information access) UKBORDERS service. Vector-Ethical approval was obtained from the West Hertfordshire health authority local research ethics committee. An application form was submitted, along with a copy of the project protocol, and written approval was returned.Cases were mapped to census wards using their postcode georeference (NHS postcode database). Microsoft Access was used to assign the deprivation scores, to divide and manipulate ward data, and to link the geographical and attribute files. The age distribution of cases and incidences was calculated using Microsoft Excel, which was also used to create the charts.©StatsDirect Ltd), apart from the relative risk confidence intervals, which were calculated using Microsoft Excel[Statistical testing was performed using StatsDirect  enhanced meningococcal database during 1999 and 2000. Of these, 524 had some postcode details and 499 had the full postcode. 458 cases had a ward assigned, and, of these, 451 had a Townsend score. These data losses were due to postcodes not being recorded (the major factor) or incorrect, discrepancies within the NHS postcode file, and incomplete ward deprivation data (this only relates to the 524 – 451 cases, not the bulk 773-524 cases). Further analysis is therefore restricted to the 451 cases with Townsend score. Where incidence rates are given, they will generally be underestimates of the true incidence , due to the loss of case data.It was assumed that the data losses were random with respect to the variables of interest. The age structure was well preserved despite the loss of around 40% of the initial cases. Figure Of the 1184 wards included in the analysis, 325 (27%) had at least one case of meningococcal disease in the two-year period. In these wards, the maximum number of cases was 6, and the median 1.The overall incidence for 1999 and 2000 was 7.4 cases per 100,000 per year. Within the wards containing more than one case, the median incidence was 12.9 per 100,000 per year, and ranged from 3.7 to 60.0. Given the small numbers involved, Poisson confidence intervals for these incidences are wide. In a high incidence ward (41.7 cases per 100,0000 per year), the confidence interval was 11.4 to 106.8 cases per 100,000 per year. In a low incidence ward (4.0 cases per 100,000 per year), the confidence interval was from 0.1 to 22.5 cases per 100,000 per year. The ward incidences will be underestimates due to the loss of cases described above.Figure Table Poisson regression using population density and Townsend score as predictors revealed population density to be a non-significant contributor to the variation in incidence (p = 0.086). A second model, setting incidence against Townsend score alone, suggested that ward incidence rises by 12% (9 – 16%) for every unit increase in deprivation score.The age-specific incidence rates are shown in figure The Poisson confidence intervals for these incidences are narrower than for individual wards. Two were calculated: 21.3 to 35.1 for an incidence of 27.6 per 100,000 per year, and 0.3 to 4.9 for a lower incidence of 1.7 per 100,000 per year.Figure Figure Figure The results for the regional surveillance data support the theory that meningococcal disease is associated with socioeconomic deprivation. Compared to the Welsh study this stuThe analyses of the age distribution of cases and incidences suggest that there is variation between the deprivation groups. There was a significant difference between the age-specific incidence rates between the groups, and a median age difference of five years between the most and least deprived groups (non-significant). The relative risk of disease in the most deprived wards compared to the least deprived (1.97, CI 1.55 – 2.51), is greater than that observed in the northeast Thames study (odds raAll but two (25–29 years, 35–39 years) age groups experienced higher age-specific rates in the most deprived group, but the highest case numbers were seen in the under 5 age group. The increased incidence in the under fives in the most deprived group, compared to the least deprived, accounted for 68% of the total difference in age-specific incidence. This suggests that children under five are more vulnerable to meningococcal disease in the most deprived areas. The continuation of a similar pattern when case counts, percentage and age-specific incidence were considered improves the robustness of this conclusion.Several environmental factors might contribute to the increased risk of childhood meningococcal disease in more deprived areas. Childcare arrangements, necessitated by either personal or environmental circumstances, might expose a child in a more deprived area to more potential carriers. Smoking is also known to be more common in people from disadvantaged backgrounds and a number of studies have identified passive smoking as risk factors for both nasopharyngeal carriage and meningococcal disease ,28-30.An area with a relatively greater number of under fives might have a higher overall incidence rate, simply because there are more susceptible individuals. Analysis of the percentage age distribution of the populations in each deprivation group does not reveal any marked variations. There is a slight excess of 20–29 year olds in the most deprived wards compared to the other wards, but not an excess of under fives.Mapping of the incidence rates and deprivation indices by ward definitely added value to the routine surveillance data, emphasising the focal nature of disease, and the relationship of these foci to areas of higher deprivation. Mapping incidence at ward level, rather than pinpointing individual cases as in the Welsh study, both coThis is an ecological study, so the associations shown may not be valid at an individual level. Population density might still be a factor, as the regression is subject to some autocorrelation, the population variable occurring in the incidence (dependent) and population density (predictor) terms. A separate analysis, of urban and rural areas, might show whether this is the case. The paucity of cases in rural areas might make this difficult unless several more years' data were included. The north east Thames study did, howGeographical bias in postcoded data might have contributed toward the results, as the health authority with the lowest rate of postcode inclusion, and therefore the greatest loss of data, includes many of the more affluent parts of the region. The cases supplied by this authority only accounted for 7.4% of all cases and 4.4% of postcoded cases, so this potential bias is unlikely to have had a major effect.Routine childhood immunisation with meningitis C conjugate vaccine started in November 1999, so shouMapping of deprivation indices and meningococcal cases is a useful tool in the analysis of routine surveillance data. Mapping of incidence rates revealed an association between areas of high incidence and areas of higher deprivation by Townsend score. High incidence and deprivation often coincided in urban areas. Mapping of deprivation indices also reveals areas of rural deprivation, such as the coastal band in north Norfolk.Yearly mapping of routine surveillance data can help to target control strategies for meningococcal disease locally. Analytic studies would be helpful in elucidating the mechanisms by which socioeconomic conditions influence the risk of meningococcal disease in the region. Along with the study described here, knowledge gained from such investigations could inform the work on health inequalities, and try to reduce such inequalities through health promotion and community infection control.None declared.CJW obtained and analysed the case and geographical data, performed the statistical analyses and mapping, and wrote the text of the paper. PH suggested the idea for the study and helped develop the methods by which the question was addressed. LW provided the data from the enhanced meningococcal surveillance database and advised on data quality and sources. IL provided expertise in geographical information systems and postcode geography.LW, PH and IL all advised on the design of the study, and the analysis and interpretation of the results. All authors contributed to drafting the paper and have read and approved the final draft.The pre-publication history for this paper can be accessed here:


However, a recent report suggests that E2-BSA does not compete for E2 binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E2 and E2-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E2-BSA preparations with unconjugated E2 , the latter was carefully removed by ultrafiltration.The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA . Staining was restricted to the cell membrane when E2-BSA-FITC was incubated with stable transfectants of the murine ERα within ER-negative HeLa cells and with MC7 cells that endogenously produce ERα. This staining appeared highly specific since it was competed by pre-incubation with E2 in a dose dependent manner and with the competitor ICI-182,780.As previously reported, a 10-to 1000-fold molar excess of E2-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E2-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E2. More importantly, these findings demonstrate that in intact cells that express ER, E2-BSA binding is localized to the cell membrane, strongly suggesting a membrane bound form of the ER.These results demonstrate that E These genomic estrogen effects are the well described interactions between the estrogen receptor and adapter transcription factors that result in activation or inhibition of the basal transcription protein machinery. However, there is a growing body of evidence that several rapid estrogen effects are non-transcriptional in nature. These rapid estrogen effects include changes of calcium flux in several cell types -3, MAPK 2-BSA) ,12. Howe2-BSA) . A recenremoved. . These fmembrane -17.2-BSA and E2 with the ER. We show that pre-incubation of E2-BSA with ERα results in a highly significant decrease in the binding of 3H-E2. The binding of 3H-E2 with ERα is unaffected by the simultaneous addition of E2-BSA. We also demonstrate that fluorescein conjugated E2-BSA binds to the membrane of cells that endogenously produce ERα and to HeLa cell lines stably expressing mERα.Elucidation of novel membrane-associated ER effects is crucial to our understanding of the non-genomic signaling pathways of ER and other hormone receptors. Hormone-conjugated BSA is an important tool in this pursuit. We believe the contradictory results are explained by differences in the rates of binding of the bulky E2 is covalently attached to BSA using a relatively long six atom hydrocarbon tether, the bulky BSA moiety of E2-BSA still may be interfering with the binding between the estrogen molecule and the estrogen receptor. This would result in an increase in the energy of activation required for E2-BSA binding. If so, increasing the reaction time would allow for the establishment of an equilibrium between bound and free forms of E2-BSA, maximizing the amount of E2-BSA bound to the receptor. To test this hypothesis, E2-BSA free of contaminant E2 was prepared by ultrafiltration. Competition between the purified E2-BSA and labeled E2 for binding to purified ERα was determined after E2-BSA was pre-incubated with ERα and also when added at the same time as labeled E2. As shown in figure 2 and E2-BSA had no effect on labeled E2 binding. However, a four-hour pre-incubation of E2-BSA with ER significantly decreased E2 binding. These results suggest that the large BSA molecule retards, but does not prevent binding of E2-BSA.Although E2-BSA (E2-BSA-FITC) binds to the cell membrane, suggesting that a form of the estrogen receptor is present within the cell membrane and capable of binding to extracellular E2. Specific binding of E2-BSA-FITC to this membrane-localized form of the ER would further establish that E2 conjugated to the BSA molecule is capable of binding to the ER. To examine this possibility, E2-BSA-FITC binding studies were performed with MC7 cells that contain endogenous ERα and with ER-deficient HeLa cells stably transfected with the ERα (HeLa-ERα). Expression of ERα within the HeLa cells was established by demonstrating specific binding of labeled E2 to HeLa-ERα, but not native HeLa cells . Spectra obtained from media containing E2-BSA-FITC alone and media containing E2-BSA-FITC incubated with MC7 cells are shown in figure 2-BSA-FITC was the same (p < 0.05) for solutions incubated in the presence (44,556 +/- 432) and absence (43,436 +/- 289) of MC7 cells (p, 0.05). These results demonstrate that E2-BSA-FITC is stable under the culture conditions employed for the binding experiments.The possibility that E2-BSA does not bind to purified ER in competition assays with labeled E2 [2-BSA or cold E2 were added concurrently with labeled E2. We obtained similar results under these conditions. However, pre-incubation of E2-BSA with purified ER results in significant competition with labeled E2. These conflicting results may be explained by differences in the rate of binding between E2 and E2-BSA. E2-BSA is a large, bulky molecule similar in size to the ER and is probably spherical in general structure as is the parent BSA molecule. The BSA protein conformation immediately adjacent to the covalently bound estrogen undoubtedly provides substantial steric hindrance to the proper presentation of conjugated E2 to the binding pocket of ER. The increased size of the E2-BSA molecule would also reduce the rate of its diffusion compared with the smaller E2. Correct orientation of E2 in the ER binding pocket is also impeded by the restraint on three-dimensional movement imposed by the six atom spacer used to connect BSA and E2. Lastly, the use of E2-BSA solutions that are formulated in terms of the molarity of total bound-E2 probably overestimates the amount of E2 available for binding. The rate of binding between E2 and ER can be expressed using the second order rate equation: rate = k [E2] [ER], where [E2] is the concentration of estradiol, [ER] is the concentration of the ER, and k the rate constant.The cellular effects elicited by estrogen ,18-20,3 beled E2 . The stu2-BSA is commonly composed of approximately 10 molecules of E2 attached to every BSA molecule. An E2-BSA solution equimolar in estradiol to a solution of estradiol alone would contain one-tenth the molarity of E2-BSA with respect to the concentration of estradiol alone. However the rate of E2-BSA binding is dependent upon the concentration of E2-BSA (rate = k [E2-BSA] [ER]). Even if every collision between E2-BSA and ER produced binding as successful as collisions between E2 alone and the ER, an E2-BSA solution equimolar in E2 would have approximately one-tenth the rate.Commercially available E2-BSA to ER compared with free E2. However, once binding has occurred, the stability of the E2 molecule in the ER binding pocket may be only modestly impaired. This may explain how pre-incubation with E2-BSA results in successful binding, whereas immediate addition of E2-BSA does not have sufficient time to establish successfully bound forms. A similar rationale may explain our results and those of other investigators [2-BSA-FITC only to cells that express ERα. These studies typically employ at least a 30-minute incubation time with E2-BSA-FITC, which may be sufficient to result in significant binding. These factors strongly suggest that the rate of binding is an important consideration in experiments assessing potential interactions between E2-BSA and ER.Taken together, these factors reduce the binding efficiency of Etigators  that dem2-BSA activation of non-genomic effects has recently been reported that strongly supports the existence of a membrane ER. Immunocytochemistry using antibodies specific to several domains of the ERα stained only on the membrane of GH3 cells [2 conjugated to peroxidase also bound only to the membrane of pancreatic islet cells and this binding was competed by E2 [2-BSA-FITC was shown to stain only the membrane of ER deficient CHO cells transiently transfected with ERα and ERβ [2-BSA-FITC membrane staining is absent with ER deficient HeLa cells and present only on the membrane of cells that endogenously produce ER or HeLa cells that stably express mERα. Taken together these data strongly suggest that non-genomic effects of E2 are at least partially mediated by a membrane associated ER. However, whether the receptor is the classical nuclear ER translocated to the membrane or an ER unique to the membrane remains unanswered.Although an estrogen receptor has not been directly isolated and characterized from the cell membrane, evidence other than EH3 cells . Membraned by E2 . The mem and ERβ . Moreove and ERβ . Lastly,2-BSA can bind to the estrogen receptor but the rate of binding is impeded due to steric and other considerations. Commercially available forms of the reagent are contaminated with dissociable E2 and should be purified prior to studies designed to demonstrate effects mediated through a membrane ER. Although we demonstrate that classical nuclear ERs can be translocated to the membrane, the conclusive identity of the endogenous membrane receptor awaits purification and sequencing of the putative membrane ER protein.The results presented here suggest that EFull-length cDNA encoding the mouse ERα was cloned into a vector containing the CMV promoter driving the neomycin resistance gene (pcERα). HeLa cells maintained in MEM containing 10% fetal bovine serum under 5% CO2 were transfected with pcERα and successful transfectants (HeLa-ERα) were selected by survival in media containing the neomycin analog, G418 (400 ug/ml).2-BSA (10-5 M in estrogen dissolved in 50 mM tris, pH 8.5, Sigma) was added to a centrifugal filter unit with a MW cut-off of 3,000 (Millipore) and centrifuged at 14,000 × g until 50 ul of retentate remained. The retentate was washed 3 times with 350 ul of buffer, recovered and volume adjusted to 400 ul.400 ul of E3H-labeled E2 (NEN, specific activity 48 Ci/mmol, 10-8 M) was incubated with recombinant ERα (.035 pM, Alexis Corp) for four hours at room temperature in binding buffer (10 mM tris, 10% glycerol, 2 mM DTT, and 1 mg/ml BSA). The binding of labeled E2 to ERα was competed by various concentrations of ultrafiltered E2-BSA or E2 (10-9 to 3.5 × 10-6 M in E2) added four hours prior to or concurrently with the addition of labeled E2. ERα was precipitated by the addition of a hydroxyapatite slurry (50% v/v in TE) and centrifugation at 10,000 × g. The pellet was washed three times with wash buffer (40 mM tris, 100 mM KCl, 1 mM EDTA, and 1 mM EGTA) and 3H-E2 binding determined by liquid scintillation counting.-8 M (in estrogen) E2-BSA conjugated to FITC (E2-BSA-FITC, Sigma, 10 moles E2 and 3.5 moles FITC per mole BSA) or BSA-FITC (Sigma, equimolar to E2-BSA-FITC with respect to BSA) for 30 minutes at 4°C. Binding of E2-BSA-FITC to MC7 cells was competed by a 30 minute pre-incubation with E2, ICI-182,780, or E2-BSA (Sigma, 10-7 to 10-9 M). Cells were fixed and FITC staining visualized by confocal microscopy. Images were digitized, inverted to black on white, and pixel density for each cell determined by averaging the density across the cell membrane at four orthogonal points (Scion Image, Scion Corp).HeLa-ERα cells or mammary tumor cells (MC7, ATTC) were plated on glass cover slips and incubated with 500 ul of 102-BSA-FITC during the incubation with MC7 cells was assessed by HPLC. E2-BSA-FITC (500 ul, 10-8 M in estrogen) was added to empty wells and to wells containing MC7 cells prepared as above for 30 minutes at 4°C. 10 ul of supernatant was resolved using a C-18 reverse phase column (Xterra C-18 RP, 5 um, 4.6 mm × 250 mm, Waters). A multiple solvent deliver system (BIO CM 4000, Milton Roy) provided a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at a flow rate of one ml/minute. Peaks were detected by a scanning fluorescence detector (model 747, Waters) at an excitation wavelength of 495 nm and emission wavelength of 519 nm. Area under the curve was calculated using standard algorithms (Millenium Software). Assays were performed in triplicate.The stability of E6 cells) incubated with several concentrations of 3H 17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. Data was analyzed by Scatchard analysis.Subconfluent HeLa-ERα or native HeLa cells were trypsinized and aliquots (2 × 10None declared.DB wrote the manuscript and performed binding assays. MK generated the stable cell lines. YT performed binding assays.


Smac/Diablo is a recently identified protein that is released from mitochondria after apoptotic stimuli. It binds IAPs, allowing caspase activation and cell death. In view of its activity it might participate in carcinogenesis. In the present study, we analyzed Smac expression in a panel of cervical cancer patients.We performed semi quantitative RT-PCR on 41 cervical tumor and 6 normal tissue samples. The study included 8 stage I cases; 16 stage II; 17 stage III; and a control group of 6 samples of normal cervical squamous epithelial tissue.Smac mRNA expression was below the detection limit in the normal cervical tissue samples. In contrast, 13 (31.7%) of the 41 cervical cancer biopsies showed detectable levels of this transcript. The samples expressing Smac were distributed equally among the stages (5 in stage I, 4 in stage II and 4 in stage III) with similar expression levels. We found no correlation between the presence of Smac mRNA and histology, menopause, WHO stage or disease status.de novo in a subset of cervical cancer patients, reflecting a possible heterogeneity in the pathways leading to cervical cancer. There was no correlation with any clinical variable.Smac is expressed  Apoptosis is an evolutionarily conserved biological process that plays a fundamental role in development and tissue homeostasis in metazoans . This tyOne IBM protein is the recently identified Smac/DIABLO ,7. Smac in vitro and in vivo; however, further studies are required prior to clinical testing [IAPs are highly expressed in human tumor cells -17, cont testing -22.Recently, Sekimura and colleagues found that Smac expression was significantly lower in primary lung cancers than in normal tissue ; patient2.Cervical cancer cell lines (HeLa, SiHa, CaSki and CaLo) were obtained from ATCC and cultured as monolayers in Dulbecco Modified Eagle's Medium (DMEM) containing 10% (V/V) fetal bovine serum (GIBCO, Bethesda, MD, USA) at 37°C in a humidified atmosphere of 5% (V/V) COForty-one cervical cancer samples were obtained from the Instituto Nacional de Cancerologia of Mexico. Written consent was obtained from patients before the samples were collected. Tumors were staged according to the International Gynecology and Obstetric Federation (FIGO) system. The samples comprised 8 at stage IB, 16 at stage IIB and 17 at stage IIIB; and a control group comprising 6 samples of normal cervical squamous epithelial tissue  classification system Table .RNA extraction and RT-PCR analysis were performed as described previously . BrieflyThe PCR cycle parameters for Smac were: 10 min enzyme activation at 95°C followed by 3 cycles of 30 s at 95°C and 2 min at 72°C, then 30 cycles of 30 s at 95°C and 30 s at 68°C, and finally 5 min at 72°C. The corresponding parameters for GAPDH were: 10 min enzyme activation at 95°C followed by 25 cycles of 30 s at 95°C, 30 s at 60°C and 30 s at 72°C. The products were electrophoresed on 1% agarose gels and stained with ethidium bromide. Smac mRNA data were expressed as ratios between the densitometric values (Scion Image software) of Smac gene expression. The PCR products were normalized to the amplified GAPDH, the internal reference gene. Gene expression measurements were repeated at least twice.chi square tests (stage, histology of tumors, menopause and current status). Kaplan-Meier curves for status were generated and log rank was used to test for differences. The mean follow-up was 14.7 months. The statistical package Intercooled Stata 7.0 was used for analyses and statistical significance was accepted when the p value was less than 0.05.To detect a correlation between pathological tumor parameters and normalized Smac expression we used ANOVA (stage, current disease and menopause status) and To ascertain whether Smac is expressed in cervical cancer we performed semiquantitative RT-PCR analyses on a panel of cervical cancer lines, including HeLa, SiHa, CasKi and CaLo cells. As shown in Figure Next, we measured Smac mRNA levels using the same approach in 41 cervical tumor and 6 normal cervical samples. To ensure accurate determinations and to verify equal RNA input, GAPDH mRNA was amplified simultaneously. Figure Tumors proliferate beyond the constraints that limit growth in normal tissue. Therefore, the resistance of tumor cells to apoptosis is an essential feature of carcinogenesis. This has been confirmed by the finding that deregulated proliferation alone is not sufficient for tumor formation because there is concomitant induction of cell death . Overexpde novo.Smac is a recently identified proapoptotic protein that interacts with and inhibits several IAPs, including survivin ,11. It hUnfortunately, we found no correlation between Smac expression and any clinical variable. This could be attributed to differences in tissue expression of IAPs, which are reported to have different binding affinities for Smac. On the other hand, alternative IAPs such as the recently identified Omi/Htra2  might plCancer treatment by chemotherapy and γ-irradiation kills cells primarily by the induction of apoptosis. However, few tumors are wholly sensitive to these therapies, and the development of resistance to therapy is an important clinical problem. Failure to activate the apoptotic programme represents an important mode of drug resistance in tumor cells . Modulatde novo. This finding contrasts with a previous report for lung cancer [During cervical cancer progression, a subset of tumors express the apoptotic protein Smac g cancer , underliThe author(s) declare that they have no competing interests.JMZ Conceived and coordinated the study.VAML: Conceived and coordinated the study. Statistical AnalysisMEC: Performed RT-PCR assaysDCL: Provided the clinical samples and coordinated patient studyCMLG: Coordinated patient assessment, ethical guidelines.JGGS: Provided clinical assessmentThe pre-publication history for this paper can be accessed here:


To study the effect of retinal/ vitreoretinal surgeries on corneal elevations.Patients who underwent retinal/ vitreoretinal surgeries were divided into 3 groups. Scleral buckling was performed in 11 eyes (Group 1). In 8 (25%) eyes, vitreoretinal surgery was performed along with scleral buckling (Group 2). In 12 eyes, pars plana vitrectomy was performed for vitreous hemorrhage (Group 3). An encircling element was used in all the eyes. The parameters evaluated were best-corrected visual acuity (BCVA), change in axial length, and corneal topographic changes on Orbscan topography system II, preoperative and at 12 weeks following surgery.There was a statistically significant increase in anterior corneal elevation in all the three groups after surgery (p = 0.003, p = 0.008 & p = 0.003 respectively). The increase in posterior corneal elevation was highly significant in all the three groups after surgery (p = 0.0000, p = 0.0001 & p = 0.0001 respectively). The increase in the posterior corneal elevation was more than the increase in the anterior elevation and was significant statistically in all the three groups .Retinal/ vitreoretinal surgeries cause a significant increase in the corneal elevations and have a greater effect on the posterior corneal surface. Changes in corneal curvature and axial length have been reported following scleral buckling procedure using keratometer -7. VideoAll these studies have reported a change in the corneal curvature in its anterior surface. However, no study has been performed to evaluate the posterior corneal topographic changes with scanning slit topography system following retinal surgeries. Hence, we made an attempt to study the effect of various retinal surgeries on posterior corneal surface using Orbscan II topography system.A prospective study was performed by enrolling patients admitted in the Retina Services of Rajendra Prasad Centre for Ophthalmic Sciences, New Delhi for retinal/ vitreoretinal surgeries. Thirty one eyes of 31 patients who underwent retinal/ vitreoretinal surgery and came for regular follow-up as per schedule during the period between December 2001 and October 2002 were included in the study. Only those patients were included who had not undergone any previous ocular surgery & did not have any corneal pathology. An informed consent was obtained from all the patients.The patients were evaluated preoperatively on parameters of best-corrected visual acuity (BCVA), axial length (AL) measured by Ultrasound A-scan instrument (Sonomed, Inc., NY) and detailed corneal examination on slit-lamp biomicroscope and on Orbscan topography system II . The parameters that were evaluated by Orbscan topography system were anterior elevation, posterior elevation and simulated keratometry.All surgeries were performed by a single surgeon (LV) under local anesthesia by peribulbar injection of 6 ml of 2% xylocaine and 2 ml of 0.5% bupivacaine. Eyes with fresh retinal detachment with clear media and absence of advanced proliferative vitreoretinopathy underwent scleral buckling procedure (Group I, n = 11). In all these eyes, the break/ s were localized, cryotherapy was performed and subretinal fluid was drained. Only circumferential buckle of silicone of style 276  was used and radial buckle or sponge was not used in any eye. The size of the buckle was 90° to 360° depending upon the requirement in individual cases. Encircling element of style 240  was used in all the eyes undergoing scleral buckling.In eyes with associated vitreous hemorrhage or advanced proliferative vitreoretinopathy changes along with retinal detachment, vitreoretinal surgery was performed along with scleral buckling (Group II, n = 8). Pars plana vitrectomy was performed and vitreoretinal membranes were removed either by peeling or by segmentation or delamination. Air fluid exchange was performed followed by Air-Silicone oil exchange. In eyes with only vitreous hemorrhage without the presence of retinal detachment, only pars plana vitrectomy was performed (Group III, n = 12). An encircling element of style 240  was used in all these eyes to counter the anterior traction that could not be fully released by vitrectomy in order to avoid lens damage.The intraoperative details including the nature of surgery, size of the buckle, encircling element, drainage of subretinal fluid, vitrectomy & use of silicone oil or gas, were noted. Postoperative treatment included topical ciprofloxacin 0.3% QID, topical dexamethasone 0.1% QID and topical Homatropine 2% QID.The patients were evaluated at 12 weeks following surgery on similar (preoperative) parameters.The data of all the patients were managed on an excel spreadsheet. All the entries were checked for any possible keyboard error. Preoperative and postoperative measurements in the three retinal surgery groups were summarized by mean and standard deviation. Changes following surgery within each group were assessed using paired 't' test. Preoperative and postoperative values in the three groups were compared using one way analysis of variance (ANOVA), followed by bon ferroni correction for multiple comparison. For the three retinal surgery groups, median was computed for increase in various parameters due to surgery. Kruskal Wallis one way analysis of variance was used to compare median increase in the three groups. STATA 7.0 statistical software was used for data analysis. In this study, p-value smaller than 0.05 was considered as statistically significant.The mean age of the patients was 45.96 ± 15.17 (range: 18–78) years and majority (83.87%) of the patients (N = 31) were males. Of these, right eye was operated in 19 (61.29%) patients.The mean preoperative best-corrected visual acuity (BCVA) was hand motion close to face in 26 eyes; counting finger near to face in 4 eyes and 1/60 on snellen's acuity chart in 1 eye. The mean decimal postoperative BCVA was 0.20 ± 0.12 at 12 weeks follow-up after surgery.The mean preoperative anterior corneal elevation as recorded by Orbscan II topography system in group I was 0.006 ± 0.007 mm, which increased to 0.024 ± 0.013 mm at 12 weeks after surgery (p= 0.003). In group II, it increased from 0.009 ± 0.006 mm preoperatively to 0.021 ± 0.010 mm at 12 weeks (p= 0.008) and in group III, it increased from 0.003 ± 0.004 mm preoperatively to 0.012 ± 0.007 mm at 12 weeks follow-up (p = 0.003). On comparative evaluation between the groups, the change in anterior corneal elevation was significant between group I and III (p = 0.04).The mean posterior elevation in group I increased from a preoperative value of 0.016 ± 0.010 mm to 0.043 ± 0.007 mm at 12 weeks after surgery (p= 0.0000). In group II, it increased from 0.014 ± 0.006 mm preoperatively to 0.043 ± 0.007 mm at 12 weeks (p= 0.0001) and in group III it increased from a preoperative value of 0.012 ± 0.005 mm to 0.029 ± 0.006 mm at 12 weeks after surgery (p = 0.0001). A comparative analysis between the groups indicated that the increase in posterior corneal elevation between groups I & III and groups II & III were found to be highly significant .Again, the increase in the posterior corneal elevation was greater than the increase in the anterior elevation in all the 3 groups and on comparative evaluation, the difference in the increase in posterior and anterior elevation was significant statistically in each group .The mean corneal astigmatism in group I increased from 0.89 ± 0.54D preoperatively to 2.50 ± 1.39D at 12 weeks follow-up (p= 0.004). In group II, the average corneal astigmatism increased from 0.87 ± 0.30D to 3.38 ± 2.15D at 12 weeks (p= 0.01) and in group III, the mean preoperative and postoperative corneal astigmatism was 0.85 ± 0.55D and 1.37 ± 0.87D respectively (p= 0.02). A comparative analysis of the change in corneal astigmatism following surgery between groups II & III was significant statistically (p= 0.02).The mean preoperative axial length in group I was 23.27 ± 0.79 mm which increased to 23.98 ± 0.76 mm at 12 weeks after surgery (p= 0.009). The mean preoperative and postoperative (12 weeks follow-up) axial length in group II were 23.92 ± 1.32 mm and 25.94 ± 2.96 mm respectively (p= 0.03). The mean preoperative and postoperative axial length in group III were 22.69 ± 0.87 mm and 22.71 ± 0.83 mm respectively (p= 0.79). Comparative analysis of increase in axial length following surgery between groups I & III and groups II & III were found to be significant statistically .Retinal surgery with or without the use of encircling and buckling elements for external tamponade can alter the shape of the globe. This may cause changes in the refractive status of the eye. Scleral buckling is known to cause a change in the shape of the sclera and can cause induced refractive changes, including astigmatic and nonastigmatic changes -10.We have used Orbscan slit scanning system II to evaluate the corneal topographic changes following retinal/ vitreoretinal surgeries. The data accumulated by Orbscan may be limited by factors such as the accuracy of the system which is ± 20 μm, the measurement noise which leads to both positive and negative difference in the height of the posterior corneal surface and the necessity of aligning the posterior surface before and after surgery which may be a source of error,12. HoweIn the present study, there was a significant increase in both anterior and posterior corneal elevation as detected by scanning slit topography (Orbscan II topography system) following surgery. This increase in the anterior and posterior corneal elevation is probably due to the use of encircling element and/ or buckle in retinal surgeries resulting in corneal steepening. We noted that the change in the posterior elevation was more significant than anterior elevation. A comparative analysis between the three groups indicated that there was no significant difference in the anterior elevation; however the posterior elevation was significantly more in eyes with buckle. It is possible that the buckle and the encircling element have a greater effect on the posterior corneal surface. The increase in the anterior and posterior corneal elevation might be one of the contributing factors for the non improvement of visual acuity in an eye that has undergone retinal/ vitreoretinal surgery. This change might escape detection by routine videokeratography. The anterior protrusion of the corneal back surface induces an increase in the negative power of the corneal surface. Assessing the corneal surface by keratometry or placido disc videokeratography may provide inadequate information regarding refractive change caused by corneal surface alteration that results in retinal/ vitreoretinal surgery.There was an increase in mean corneal astigmatism following surgery in all the groups. This is due to the effect of encircling element or buckle on the corneal surface. Studies have reported that induced astigmatism has been associated with radial scleral buckles,13, circIn retinal/ vitreoretinal surgeries, the encircling band creates a circular indentation of the eye, thereby increasing its anteroposterior axial length; the myopic shift may be upto 3 diopters (D),10,13,16Retinal/ vitreoretinal surgeries result in an increase in the elevation of the corneal surfaces. These changes are more pronounced on posterior corneal surface.None declared.RS designed the study and performed the data collection. NS wrote the manuscript. LKV performed the retinal surgeries. RMP performed the statistical analysis and RBV followed up the patients.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The non-thrombotic effects of thrombin in cardiovascular tissues, as mediated via the protease activated receptors (PARs), and particularly PAR-1, have been the focus of much recent research. The aims of this study were to evaluate the effects of thrombin, a specific PAR-1 activating peptide (PAR1-AP), and a PAR-1 antagonist on human umbilical artery tone in vitro.Human umbilical artery samples were obtained from 17 women at term. Arterial rings were suspended under physiologic conditions for isometric recording. The in vitro effects of thrombin (0.5 units/mL to 3 units/mL), PAR1-AP TFLLR-NH2 [10(-9) to 10(-6) M], and PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH2) [10(-9) M to 10(-5) M] on umbilical artery tone were measured.Both thrombin and TFLLR-NH2 exerted a potent cumulative vasodilatory effect on human umbilical artery resistance (P < 0.001). The mean net maximal inhibition (MMI) for thrombin was 53.05%  at tissue bath concentration of 3 units/mL. The MMI with TFLLR-NH2 was 61.50 %  at bath concentration of 10(-6) M. In comparison to vehicle control, the PAR-1 antagonist did not show a significant relaxant or contractile effect (P > 0.05).These findings highlight a potential role for thrombin and PAR-1 receptors in vascular regulation of feto-placental blood flow in normal pregnancy, and in association with the vascular lesions associated with IUGR and pre-eclampsia. In disorders resulting in poor fetal growth, and in pre-eclampsia, thrombotic lesions are frequently observed in the maternal and fetal vascular components of the placenta, -3 and he2 (Thr-Phe-Leu-Leu-Arg-NH2), and the PAR-1 specific antagonist (N-trans cinnamoyl -p-fluoroPhe-p-quanidinoPhe-Leu-Arg-Orn-NH2) on human umbilical artery tone in vitro.There is growing evidence, from vascular tissue studies in several animal models, that non-thrombotic thrombin-mediated signalling events are central to the response to the disease process typical of vascular lesion formation in atherosclerosis . The dirThe study was carried out in the Department of Obstetrics and Gynaecology, University College Hospital Galway, Ireland between May 2002 and April 2003. Sections of human umbilical cord approximately 10 cm in length were excised from the proximal segment of the cord  immediately after elective cesarean delivery. Samples were obtained from 17 women after elective cesarean section at term and from one patient after normal vaginal delivery. All pregnancies were uncomplicated and there was no evidence of hypertensive disease or intrauterine fetal growth restriction. The mean maternal age was 32.47 years (range 25–40 years). The median period of gestation was 38 weeks (range 37–42 weeks). The reasons for cesarean section included previous cesarean section (n = 9), breech presentation (n = 6), previous myomectomy (n = 1) and unstable fetal lie (n = 1). At the time of recruitment 4 women were nulliparous and 13 women were parous.Samples were immediately placed in cold buffered Krebs Henseleit physiological salt solution (pH 7.4) of the following composition: potassium chloride 4.7 mmol/L, sodium chloride 118 mmol/L, magnesium sulphate 1.2 mmol/L, calcium chloride 1.2 mmol/L, potassium phosphate 1.2 mmol/L, sodium bicarbonate 25 mmol/L and glucose 11 mmol/L. Maternal written informed consent was obtained prior to tissue collection, and the tissue collection procedure was approved by the Research Ethics Committee at University College Hospital Galway.2 / 5% CO2) Krebs Henseleit physiological salt solution (PSS) at 37°C and pH 7.4. Rings were allowed to equilibrate for 90 minutes with regular washouts of PSS. During this interval, spontaneous tone developed. After the equilibration period, the vessel rings were challenged with 60 mM potassium chloride (KCl). Three washouts with PSS were carried out once the maximum response had reached a plateau, and a 20 minute recovery period was allowed in order that baseline be attained again. The KCl challenge was performed three times. After the last KCl challenge, 40 minutes recovery was allowed, and contraction was then stimulated by bath exposure of the vessel rings to 5-hydroxytriptamine (5-HT) (10-7 M). Once maximum contractile response to 5-HT was attained, the rings were allowed to remain at plateau for 20 minutes. Concentration- effect experiments were performed by cumulative additions of thrombin, the PAR1-AP, or the PAR-1 antagonist to the tissue bath. Thrombin was added to the tissue bath at an initial concentration of 0.5 units/mL, and this was increased at 20-minute intervals to 1 unit/mL, 2 units/mL and 3 units/mL respectively. The bath concentrations ranges investigated for PAR1-AP were 1 nanomol/L, 10 nanomol/L, 100 nanomol/L and 1 micromol/L (i.e. 10-9 - 10-6M), and for PAR-1 antagonist were 1 nanomol/L, 10 nanomol/L, 100 nanomol/L, 1 micromol/L and 10 micromol/L (i.e. 10-9-10-5M), all at 20-minute intervals.Umbilical arteries were carefully dissected free of Wharton's Jelly and cut in rings 4–5 mm in axial length. Rings were suspended individually on stainless steel hooks inserted into their lumens and mounted under 2 g (30 mN) of isometric tension, in glass-jacketed tissue baths, as previously described ,17. EachThe effects of thrombin, PAR1-AP and PAR-1 antagonist were demonstrated by expressing the mean amplitude calculated during the 20-minute period following addition of each drug concentration, as a percentage of the mean amplitude obtained in the 20 minutes prior to any drug addition. This measurement represents percentage contractility or tone, and subtracted from 100%, provides the percentage relaxation value for each bath concentration of vehicle and study compounds. The net relaxant effect of each compound was calculated by subtracting the percentage contractility value calculated for its respective vehicle (for thrombin, PAR1-AP or PAR-1 antagonist), control experiment, at each similar bath concentration. All of the umbilical artery samples used for experimentation for each compound were obtained from different women (i.e. n = 6 for PAR1-AP), for example, was achieved by using umbilical artery samples from 6 different women). The allocation of umbilical artery samples for the different experiments was entirely random.50 value for antagonist inhibition of platelet aggregation stimulated with 1 micromol/L SFLLRN agonist was determined to be 0.1 micromol/L. It was prepared as a 10 millimol/L stock solution in dimethysulphoxide (DMSO) and stored in room temperature protected from direct light. Final bath concentrations of DMSO, at the highest concentration of PAR-1 antagonist, did not exceed 1%, for both study and vehicle control strips. Serial dilutions of thrombin, PAR1-AP and the PAR-1 antagonist were made using Krebs Henseleit physiological salt solutions.Fresh Krebs Henseleit physiological salt solution was made and buffered daily. KCl solutions were prepared on the day of experimentation. A stock solution of 5-HT (Sigma-Aldrich, Dublin, Ireland) was made up in de-ionised water and diluted with Krebs solution. Thrombin was purchased from Sigma-Aldrich (Dublin, Ireland) and a stock solution of 1100 U/ml prepared in de-ionised water and stored at -20°C. PAR1-AP was purchased from Tocris Cookson Ltd (Bristol, UK) and a stock solution of 1 millimol/L was prepared using deionized water, with subsequent dilutions in PSS. The PAR-1 antagonist was prepared and assayed by methods previously described . The IC5Comparisons of measurements of amplitude for each bath concentration of thrombin, PAR1-AP and the PAR-1 antagonist, or respective control vehicle, were made using a one-way ANOVA. Post-hoc comparisons were made using the Tukey HSD test. A P value <0.05 was accepted as statistically significant. The statistical package SPSS version 10 was used for statistical calculations.A recording from control experiments (i.e. without addition of vehicle, thrombin, PAR1-AP or PAR-1 antagonist) demonstrating umbilical artery tone due to bath exposure of the ring to serotonin, for the entire duration of an experiment, is shown in Figure 2, the PAR1-AP, similarly exerted a cumulative vasodilatory effect on umbilical artery tone. A representative recording of the cumulative effects of PAR1-AP are shown in Figure -6M / 1 micromol/L) was 61.5% .TFLLR-NH2, no relaxation of umbilical artery tone in vitro was observed, in comparison to vehicle only control experiments. Figure For the PAR-1 antagonist, N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NHThis study demonstrates that thrombin exerts a potent vasodilatory effect on human umbilical artery vasculature in vitro. While it is known that thrombin has an inhibitory effect on vessel tone in other animal and human vascular tissue types -21 this 2. PAR1-APs are recently designed small synthetic peptide ligands which mimic the effects of proteases by binding directly to the activation site of the PAR-1 receptor, bypassing the need for proteolytic cleavage of the receptor. Soluble peptide ligands, as PAR-APs are, can vary greatly in potency as agonists in comparison with proteases [2 is reportedly one of the more selective PAR1-APs and is deemed most preferable for purposes of studying the physiologic role of PAR-1 [We have not elucidated the exact mechanism, or mechanisms, of the thrombin mediated alteration in vascular tone in human umbilical artery. We have however demonstrated that a similar effect is elicited by bath exposure of the arterial rings to the specific PAR1-AP, TFLLR-NHroteases . TFLLR-Nof PAR-1 . In viewof PAR-1 ,14,15 an2, did not alter umbilical artery tone. This peptide is a competitive antagonist i.e., it competes for the agonist binding site and does not appear to have any other activity [2 is inactive towards the PAR-1 receptor. The true role of PAR antagonists, in such experiments, is for the purpose of investigation of the potential effects of an agonist at a PAR subtype. Pre-incubation of the arterial rings with PAR-1 antagonist, for example, should alter the response elicited by PAR1-AP. Such experiments, with selective blockade of the PAR subtypes, in normal and diseased pregnancies, are a subject of further studies.The PAR-1 antagonist, N-trans cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NHactivity . The diractivity . The resThere are some limitations to this study. All umbilical cord artery samples were obtained at elective cesarean section. This was included in the design of the study in order to maintain uniformity in terms of mode of delivery, and, to avoid using cord samples that may have undergone excessive traction in the third stage of labor at vaginal delivery. It is unknown whether this latter point is valid, or not, in studies using in vitro umbilical preparations. We are currently performing comparative studies from samples obtained at vaginal delivery to further evaluate this matter. Secondly, the effects of thrombin and PAR-1 modulation, on smaller vessels in the feto-placenta circulation , and in association with disorders of pregnancy, would compliment our knowledge of the importance of this pathway. Finally, there are limitations in extrapolating from in vitro experiments to the in vivo situation, but the experiments conducted here represent a reliable and valid in vitro model for these vascular preparations.In conclusion, thrombin exerts a potent vasodilatory effect on umbilical artery preparations in vitro. A similar effect is also observed using a specific PAR1-AP. The potential non-thrombotic role of thrombin, and PAR subtype modulation, in regulation of the feto-placental circulation in normal pregnancy, and in pregnancies complicated by hypertensive disease or intrauterine growth restriction, is highlighted by these findings.2) and wrote the manuscript. JJM designed, supervised the study and wrote the manuscript. All authors read and approved the final manuscript.AJO'L performed the experiments and wrote the manuscript. CJO'S and NR performed the experiments. AMF analysed the data and wrote the manuscript. JTE provided the PAR-1 antagonist (N-trans cinnamoyl- p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Orn-NH