Despite improvements in the safety and efficacy of endovascular procedures, considerable morbidity may still be attributed to vasospasm. Vasospasm has proven amenable to pharmacological intervention such as nitrates, intravenous calcium channel blockers (CCBs), and intra-arterial papaverine, particularly in small vessels. However, few studies have focused on medium to large vessel spasm. Here we report the use of an intra-arterial CCB, verapamil, to treat flow-limiting mechanically-induced spasm of the common carotid artery (CCA) in a primate. We believe this to be the first such report of its kind.As part of a study assessing the placement feasibility and safety of a catheter capable of delivering intra-arterial cerebroprotective therapy, a female 16 kg baboon prophylaxed with intravenous nitroglycerin underwent transfemoral CCA catheterization with a metallic 6-Fr catheter without signs of acute spasm. The protocol dictated that the catheter remain in the CCA for 12 hours. Upon completion of the protocol, arteriography revealed a marked decrease in CCA size  localized along the catheter length. Intra-arterial verapamil (2 mg/2cc) was injected and arteriography was performed 10 minutes later. Image analysis at 6 points along the CCA revealed a 21.0 ± 1.7% mean increase in vessel diameter along the length of the catheter corresponding to a 46.7 ± 4.0% mean increase in cross-sectional area. Mean systemic blood pressure did not deviate more than 10 mm Hg during the procedure.Intraluminal CCBs like verapamil may constitute an effective endovascular treatment for mechanically-induced vasospasm in medium to large-sized vessels such as the CCA. Rapid advancements in endovascular technology and techniques allow for treatment of an ever-increasing range of neurovascular diseases. Despite improvements in the safety and efficacy of these procedures, complications such as vasospasm, stroke, and perforation still occur . VasospaVasospasm complicates many disease states, particularly those affecting small vessels. Recently, treatment of small-vessel vasospasm has proven amenable to pharmacological intervention. For example, in the treatment of cerebral artery spasm, intravenous nitrates , intravePapio anubis) underwent carotid artery catheterization under general anesthesia. Since Papio anubis is regarded as vasospasm-prone (unpublished data), the animal was pre-treated with oral nimodipine , and placed on a prophylactic infusion of intravenous nitroglycerin (200 mcg/hr) and heparin (100 units/hr). To place a 6 Fr (2 mm) treatment device in the 3–4 mm right CCA [As part of a study aiming to assess the placement feasibility and safety of a catheter capable of delivering intra-arterial cerebroprotective therapy, a 16 kg female baboon . Thus, it is important to identify pharmacological agents that will relieve this vasospasm with minimal side effects.Spasm of arteries secondary to therapeutic medications or diagnostic instrumentation has long been acknowledged as a possible complication of interventional procedures. Vasospasm, in general, has been attributed to a variety of pharmacological stimuli ranging from cocaine  and alcoThere are several agents that have been shown to be effective in preventing and treating vasospasm, each of which has its limitations. Intravenous nitrates have been the mainstay of vasospasm prevention for endovascular procedures , but theTo reduce the tone of a muscular artery, a logical point of intervention is inhibition of calcium influx into smooth muscle cells. Voltage-sensitive CCBs, or 1, 4-dihydropyridines , function in this manner. Nimodipine improves outcomes after cerebral vasospasm secondary to subarachnoid hemorrhage . IntraluIn this case report we document the use of a 2 mg intra-arterial verapamil injection into the CCA of a non-human primate to acutely reverse catheter-induced vasospasm. This use is unique for several reasons. Firstly, in contrast to the report by He et al. , in whicin situ metallic catheter, we believe strongly that the vasodilation was due to the verapamil. Second, the observed mitigation of vasospasm occurred during a continuous intravenous infusion of nitrates. It is conceivable that the synergistic effects of verapamil with these nitrates actually treated the vasospasm, and not the verapamil itself. Finally, this study, by its design, does not attempt to define the long-term durability of intra-arterial verapamil. Only through additional experimentation and use will the full utility of this agent as an intraluminal treatment for vasospasm be understood.This report serves as preliminary evidence for the utility of intra-arterial verapamil in large vessel vasospasm and not the conclusive study as its scope is limited by three issues. First, by not having a control (untreated) subject, it is impossible to say for sure that the observed mitigation of vasospasm is due to the intervention and not the natural history of the disease. Considering that effects were observed in the presence of an in situ catheter-induced CCA vasospasm in a non-human primate by an intra-arterial infusion of verapamil (2 mg) without demonstrable complications. Although only an observational study in one subject, this report suggests that intra-arterial administration of verapamil may be an effective intervention for the treatment of mechanically-induced vasospasm in medium to large-sized muscular arteries and that further experimentation in this area is warranted.We describe the acute alleviation of None declared.ALC, GPC, and WJM performed the surgical procedure, delivered the critical care to the animal, composed, and revised the manuscript. LF and PM performed the angiography studies. ESC conceived the study and oversaw its design and completion.The pre-publication history for this paper can be accessed here:


Cupiennius salei about 30 groups of neural precursors are generated per hemi-segment during early neurogenesis. Analysis of the ventral neuromeres after invagination of the primary neural precursor groups revealed that secondary neural precursors arise during late embryogenesis that partially do not differentiate until larval stages.In the spider In contrast to the primary groups, the secondary invaginating cells do not detach from each other after invagination but maintain their epithelial character and form so-called epithelial vesicles. As revealed by dye labeling, secondary neural precursors within epithelial vesicles do not show any morphological features of differentiation indicating that the formation of epithelial vesicles after invagination leads to a delay in the differentiation of the corresponding neural precursors. About half of the secondary neural precursor groups do not dissociate from each other during embryogenesis indicating that they provide neural precursors for larval and adult stages.Secondary neural precursors are arrested in an immature state by formation of epithelial vesicles. This mechanism facilitates the production of larval neural precursors during embryogenesis. I discuss the evolutionary changes that have occured during neural precursor formation in the arthropod group and present a model for the basal mode of neurogenesis. The arthropods form a diverse group with a correspondingly high variation of neural structures adapted to the specialized behaviour and lifestyles of individual species. This raises the question of how developmental processes have been modified during evolution to generate the wide diversity of nervous systems seen in adult arthropods. Evolutionary modifications that lead to variations in neural structures can occur during different processes of neurogenesis. The establishment of neural networks can be influenced by changes in the generation of neural precursors, modifications of cell fates or elimination of individual neurons as well as changes in axonal guidance. A comparative analysis of neurogenesis in chelicerates and myriapods has revealed that although the developmental program is genetically conserved, there is a major difference in the recruitment of neural precursors as compared to insects and crustaceans -5. GroupDrosophila melanogaster, the neural precursors arise at stereotyped positions that are prefigured by a proneural gene (CsASH 1), while the neurogenic genes Delta and Notch restrict the proportion of cells that adopt the neural fate at each wave of neural precursor formation [Drosophila melanogaster, the Delta/Notch signalling pathway is used for a decision between two cell fates in the ventral neuroectoderm: delaminating cells become neural precursors, while cells that remain apical give rise to epidermis. This decision does not take place in the central neurogenic regions of the spider [In the spider 30 to 32 groups of neural precursors are generated per hemi-segment during neurogenesis. As in ne CsASH , while tDrosophila. However, in the adult spider Cupiennius salei the subesophageal ganglion consists of 49,000 neurons [Drosophila melanogaster, 'embryonic' neuroblasts proliferate again and give rise to larval and adult lineages after a phase of cell cycle arrest from late embryogenesis to first larval instar [Since each invagination group consists of five to nine neural precursors, it can be estimated that an embryonic hemineuromere consists of about 220 neurons on average, similar to  neurons  indicatil instar . An anall instar . In addiCupiennius salei the germband develops from aggregations of cells that form the cephalic lobe and the caudal lobe [In the spider dal lobe . One to dal lobe ,13. At tdal lobe ) a longidal lobe . The forDrosophila melanogaster (see above). A detailed analysis of the morphology of the ventral neuromeres after invagination of the neural precursor groups revealed that the cells that remain apical form secondary invagination sites  Each secondary invagination group contains up to 40 cells as compared to 5 to 8 cells that form the primary invagination sites , the secondary invagination sites appear almost at the same time Fig. . A detaiAnalysis of the mitotic pattern during neurogenesis has revealed that the formation of the primary invagination sites is not connected with cell divisions . In addiachaete-scute homologues have been identified in the spider [CsASH1 is expressed like a proneural gene in the neurogenic regions prior to formation of the primary invagination sites and is necessary for the generation of neural precursors. CsASH2, in contrast, shows a pan-neural mode of expression: it is exclusively expressed in all invaginating neural precursors. Simlar to Drosophila melanogaster, the neurogenic genes Notch and Delta restrict the proportion of cells that adopt a neural fate at each wave of neural precursor formation [Two e spider . CsASH1 achaete-scute homologues and neurogenic genes [achaete-scute homologues CsASH1 and CsASH2 and the neurogenic genes CsDelta1 and CsDelta2 is down-regulated, while CsNotch remains expressed at low levels in the ventral neuroectoderm  precursors [Notch mutant Drosophila melanogaster embryos, cells with the identity of SNS and optic lobe precursors develop at approximately normal numbers, but they do not form epithelial vesicles. Instead, these cells appear as solid, irregular clusters of apolar cells [CsNotch during development of the secondary neural precursors could not be analysed, because injection of ds CsNotch RNA leads to a premature differentiation of neural precursors due to an ealier function of CsNotch in lateral inhibition [CsNotch in the secondary invagination sites suggests a role in formation of the epithelial vesicles  were obtained from Ernst-August Seyfarth, Frankfurt, Germany. Embryos were collected as described before [Fertilized females of the Central American wandering spider d before .Whole-mount in situ hybridisations were performed as described . PhalloiAfter chemically removing the chorion, embryos were fixed in 4 % formaldehyde in PBS and 1 vol heptane. The vitelline membrane was removed with needles and the embryos stained with phalloidin-FITC. Flat preparations of these embryos were attached to a coverslip with a double-sticky tape and covered with PBS. 1,1'-dioctadecyl 3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) was dissolved in ethanol and applied with glass needles. A small droplet of DiI was injected into single or several cells of an invagination group using a 63 × water-immersion lens and a FITC filter on a Zeiss fixed stage microscope and a micromanipulator.


Autoimmune progesterone dermatitis (APD) is a condition in which the menstrual cycle is associated with a number of skin findings such as urticaria, eczema, angioedema, and others. In affected women, it occurs 3–10 days prior to the onset of menstrual flow, and resolves 2 days into menses. Women with irregular menses may not have this clear correlation, and therefore may be missed. We present a case of APD in a woman with irregular menses and urticaria/angioedema for over 20 years, who had not been diagnosed or correctly treated due to the variable timing of skin manifestations and menses. In addition, we review the medical literature in regards to clinical features, pathogenesis, diagnosis, and treatment options. While many women complain of worsening acne and water retention during their menstrual cycle, there exist a small number in whom the menstrual cycle is associated with a variety of other skin manifestations such as urticaria, eczema, folliculitis, and angioedema. This condition is known as autoimmune progesterone dermatitis (APD) due to the fact that progesterone is most frequently identified as the etiologic agent. In women with irregular menses, the diagnosis may remain elusive for years. We present a case of APD, and review the current literature in regards to clinical features, pathogenesis, diagnosis, and treatment options.A 33y/o woman with a history of endometriosis presented with complaints of chronic urticaria. The patient noted that the urticaria began at the age of 12, and did not seem to have any obvious trigger. Each individual lesion would last from 12–24 hours, and the entire episode would last 5–10 days. Lesions would usually start on the chest and then spread over the entire body. She had seen multiple physicians, including allergists and dermatologists, and had been treated with a variety of medications including certirizine, desloratadine, hydroxyzine, montelukast, ranitidine, and diphenhydramine without relief. Prednisone at high doses would provide temporary relief, and she had required multiple courses of prednisone over the past 20 years. In addition, she complained of occasional angioedema, usually at the same time as the hives but occasionally occurring when hives were not present. The patient also had acne that had been very difficult to control since her teenage years, and she noted that the acne would also respond to prednisone.Multiple lab tests over the years had been unremarkable. These included SSA/SSB, anti-Smith, ACE level, C3/C4, hepatitis B, ANA, anti double-stranded DNA, immunoglobulins, SPEP, C1 esterase inhibitor level and function, chemistry panel, liver tests, TSH, T4, thyroid antibodies, rheumatoid factor, ESR, and CBC. Skin biopsy of a lesion had been read as "chronic urticaria".Upon further questioning, it was learned that due to the patient's endometriosis, she had very irregular menstrual cycles in terms of length and timing. It was determined that the hives and/or angioedema would begin approximately 4 days prior to the onset of menses, and would last about 2 days into menses. The symptoms would not occur with every episode of menses. The patient's acne would often occur on her face during the urticarial episodes. Of note, the patient had 2 children, and during each pregnancy her hives, acne, and angioedema had been markedly improved. Because of her endometriosis, she had been started on Depo-Provera (medroxyprogesterone acetate) in her twenties. After 1 injection, she developed severe hives that lasted over 2 months and required multiple courses of prednisone. Due to the urticaria, Depo-Provera was discontinued after one injection. As the patient complained of acne, Ortho Tri-Cyclen (norgestimate/ethinyl estradiol) was initiated by her dermatologist. This treatment modality did not have any effect on the urticaria, angioedema, or acne.The patient was evaluated in our clinic. Physical examination was essentially normal, and no hives were noted. Allergy skin testing was performed with progesterone 50 mg/mL in normal saline. Prick test was normal, but a full strength intradermal test revealed a 7 mm wheal with erythema. The histamine control showed a 9 mm wheal with erythema, and saline control was negative for wheal and erythema. Two healthy controls also underwent intradermal testing to exclude irritant reaction, and were found to be negativeBased on the above results, the patient was diagnosed with autoimmune progesterone dermatitis. The patient was started on a GnRH agonist . Within one month, she noted dramatic improvement in her urticaria and angioedema. Acne was still occasionally present, but much improved. She did complain of mild hot flashes, but felt these were tolerable.In a small group of women, the menstrual cycle has been associated with a spectrum of dermatologic diseases including eczema, erythema multiforme, stomatitis, papulopustular lesions, folliculitis, angioedema, urticaria, and others Table  1-8]. A-8. A1-8]The clinical symptoms of APD  usually begin 3–10 days prior to the onset of menstrual flow, and end 1–2 days into menses. Severity of symptoms can vary from nearly undetectable to anaphylactic in nature, and symptoms can be progressive . There aThe symptoms of APD correlate with progesterone levels during the luteal phase of the menstrual cycle. Progesterone begins to rise 14 days prior to the onset of menses, peaks 7 days prior to menses, and returns to a low baseline level 1–2 days after menses begins. In studies where an etiologic agent has been sought, progesterone has been found most frequently. However, estrogen, prostacyclin, and gonadotropin levels have correlated with symptoms in some cases -21.Symptoms may first appear, improve, or worsen during pregnancy and the peripartum period ,22-24. IH2 cells could result in subsequent IgE synthesis; however this mechanism would not explain the pathogenesis in patients such as ours who have the onset of APD prior to exogenous progesterone exposure. Some authors have suggested that hydrocortisone or 17-α-hydroxyprogesterone have cross-sensitivity with progesterone and may cause initial sensitization, but this has not been observed in all studies [The exact pathogenesis of APD is unknown. If exogenous progesterones (i.e. OCPs) are initially used, it is conceivable that uptake by antigen presenting cells and presentation to T studies ,28.H1-type cytokines in APD [To further delineate the pathogenesis, antibodies against progesterone have been investigated. Using immunofluorescent techniques and basophil degranulation tests, studies have found that such antibodies do exist in certain patients with APD ,13,29. Hs in APD .Eosinophils may also be involved in the pathogenesis of APD. Eosinophilia has been correlated with cutaneous symptoms in some cases, and studies have found a decrease in total eosinophil count after therapy ,29,34. WThe diagnosis of APD requires an appropriate clinical history accompanied by an intradermal injection test with progesterone. An aqueous suspension or aqueous alcohol solution of progesterone is the preferable vehicle of testing as progesterone in oil can cause an irritant reaction , though As mentioned above, APD may be due to an immediate or delayed hypersensitivity reaction. Therefore, intradermal testing may not become positive until 24–48 hours later ,24. In aSome authors have recommended further tests to evaluate the immunologic evidence in APD. These include circulating antibodies to progesterone, basophil granulation tests, direct and indirect immunofluorescence to luteinizing cells of the corpus luteum, in vitro interferon-γ release, and circulating antibodies to 17-α-hydroxyprogesterone ,29,33,36Autoimmune progesterone dermatitis is usually resistant to conventional therapy such as antihistamines. The use of systemic glucocorticoids, usually in high doses, has been reported to control the cutaneous lesions of APD is some studies, but not in others ,10,37. ECurrent therapeutic modalities often attempt to inhibit the secretion of endogenous progesterone by the suppression of ovulation. Table Various other therapy modalities are currently used in APD, and there is no clear treatment of choice. GnRH agonists, such as buserelin and triptorelin, have been used to induce remission of symptoms by causing ovarian suppression ,11,15. HAutoimmune progesterone dermatitis is a condition seen in a small number of women who present with eczema, erythema multiforme, stomatitis, papulopustular lesions, folliculitis, angioedema, urticaria, and other skin manifestations in relation to the menstrual cycle. It is usually seen 3–10 days prior to the onset of menstrual flow, but may be difficult to recognize in women with irregular menses. The exact pathogenesis is unknown, and is thought to involve a hypersensitivity reaction to progesterone. The diagnosis of APD is made by an appropriate clinical history accompanied by an intradermal injection test with progesterone. Current treatment modalities often attempt to inhibit the secretion of endogenous progesterone, but may be unsuccessful. More research is needed into the pathogenesis of APD to most appropriately care for these patients.


Reference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples. However the expression level of these genes may vary among tissues or cells, and may change under certain circumstances. Thus the selection of reference gene(s) is critical for gene expression studies. For this purpose, 10 commonly used housekeeping genes were investigated in isolated human neutrophils.Initial screening of the expression pattern demonstrated that 3 of the 10 genes were expressed at very low levels in neutrophils and were excluded from further analysis. The range of expression stability of the other 7 genes was (from most stable to least stable): GNB2L1 , HPRT1 (Hypoxanthine phosphoribosyl transferase 1), RPL32 , ACTB (beta-actin), B2M (beta-2-microglobulin), GAPD  and TBP (TATA-binding protein). Relative expression levels of the genes (from high to low) were: B2M, ACTB, GAPD, RPL32, GNB2L1, TBP, and HPRT1.Our data suggest that GNB2L1, HPRT1, RPL32, ACTB, and B2M may be suitable reference genes in gene expression studies of neutrophils. Neutrophils are the most numerous granulocytes in blood and are responsible for the first line of host defence. However, neutrophils have frequently been implicated in the pathogenesis of many diseases because they can produce various cytokines, chemokines and other proinflammatory mediators ,2. NumerIn this study, we investigated 10 commonly used housekeeping genes . Based on the band intensity of the PCR products, the two lowest expressed genes, two medium expressed genes and the three highest expressed genes were chosen for real-time PCR analysis. ABL1, PBGD and TUBB were excluded from further evaluation due to their extremely low expression level.2) of all the seven genes ranged from 0.976 to 0.999. Based on the slopes of the standard curves, the amplification efficiencies of the standards were from 91%~100%, which were derived from the formula E = 10 1/-slope -1. The Ct values of all the 7 genes in all the unknown samples were within 15.9 to 33.5 cycles, covered by the range of the standard curves. Electrophoresis analysis of all the amplified products from real-time PCR showed a single band with the expected sizes, and no primer dimer was observed. The dissociation plots provided by the ABI Prism 7900HT also indicated a single peak in all the reactions.Standard curves were generated by using copy number vs. the threshold cycle (Ct). The linear correlation coefficient : GNB2L1, HPRT1, RPL32, ACTB, B2M, GAPD and TBP  was calculated based on the geometric mean of the copy numbers of these 5 selected reference genes in each sample. After normalization against the NF, the ranking of the relative expression levels was (from high to low): B2M, ACTB, GAPD, RPL32, GNB2L1, TBP, and HPRT1  before the gene expression study. The results indicated our RNA samples were of good quality. Other quantification methods which need a microgram-level of RNA were not practical for our study because the amount of RNA extracted from the neutrophils from 10 ml blood was very limited (around 3–5 μg).DNA contamination is another important factor that affects the accuracy of gene expression analysis. In this study, the following steps were taken to prevent and monitor DNA contamination: (1) RNase-free DNase I treatment on all the RNA samples; 2) The primers were designed to be able to distinguish the PCR product derived from mRNA or genomic DNA . After normalization we found B2M was the most highly expressed, followed by ACTB, RPL32, GNB2L1, and HPRT1 was the lowest expressed. As the expression level of the reference genes may be an additional factor for consideration in the process of reference gene selection, this ranking of the relative expression level of the candidate reference genes may be informative for future gene expression studies in neutrophils.To our knowledge, this is the first detailed study of the stability and level of reference gene expression in neutrophils. We found GNB2L1, HPRT1, RPL32, ACTB, and B2M are good choices for reference gene(s) selection. B2M and ACTB can be used for high-abundance mRNA, RPL32 and GNB2L1 for medium-abundance mRNA, and HPRT1 for low-abundance mRNA in expression studies of neutrophils. For more accurate normalization, as suggested by other authors , we reco. Hairpin structure and primer dimerization were analyzed by NetPrimer. Primers spanning at least one intron were chosen to minimize inaccuracies due to genomic DNA contamination. The length of the primers was from 18-mer to 22-mer, GC content was from 45% to 60%, and the expected PCR products range from 114 bp to 318 bp. If the genes have pseudogenes, primers were chosen according to the alignment results between the genes and the pseudogenes, so that the primers were unique to the genes and different from the pseudogenes . Gene symbols and their full names, gene accession numbers as well as functions are listed in Table A total of 15 volunteers were recruited Table . All par- Reverse Transcriptase (Invitrogen) and random primers (Invitrogen) in a total volume of 20 μl. Reverse transcription was performed at 37°C for 1 hour followed by 72°C for 15 min.Total RNA was isolated using RNeasy Mini Kit (Qiagen) as described by the manufacturer. Genomic DNA was eliminated by RNase-free DNase I digestion (Qiagen) during the isolation procedure. Isolated total RNA was analyzed on an Agilent 2100 Bioanalyzer using the RNA 6000 pico labchip Kit (Agilent Technologies). First strand cDNA synthesis was carried out with SuperScript RNase HTo screen the basal expression patterns of the candidate genes in neutrophils, three randomly selected samples were tested by PCR with the ten primer pairs Table .Taq DNA polymerase, followed by 45 thermal cycles of 15 seconds at 94°C, 30 seconds at 58°C and 30 seconds at 72°C. Size analysis of the PCR products  was performed immediately after the real-time PCR. The temperature range used for the melting curve generation was from 60°C to 95°C. Each sample was analyzed in triplicate wells. In addition, all the reactions were further subject to electrophoresis on 2.5% agarose gels stained with ethidium bromide to confirm the expected PCR products.The expression study was performed using a 384 well plate on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) with QuantiTect SYBR Green PCR Kit (Qiagen). The reactions were performed according to the manufacturer's instructions with minor modifications. The PCR program was initiated at 95°C for 10 min to activate The amplified fragments from each primer pair were purified with QIAquick PCR purification Kit (Qiagen), and confirmed by DNA sequencing . The concentrations of the PCR products were quantified by a spectrophotometer (Perkin-Elmer Lambda 2 UV/VIS Spectrometer), which were further transformed to copy numbers based on the length and base composition of the PCR products. A ten-fold series dilution was made and 10 to 1,000,000 copies were used for generating standard curves in the real-time PCR, plotted as Ct values (cycle numbers of threshold or crossing points) versus logarithms of the given concentrations of the DNA templates.[M), which is the mean pair-wise variation for a gene compared with all other tested control genes. Genes with higher M values have greater variation in expression. The stepwise exclusion of the gene with the highest M value allows the ranking of the tested genes according to their expression stability. The proposed threshold for eliminating a gene as unstable was M ≥ 0.5. In the final analysis, genes with M value lower than 0.5 were considered as stably expressed genes, and were used for normalization factor (NF) calculation. Using the NF we calculated and ranked the expression level of all the seven genes in our samples.Gene stability was also evaluated using the geNorm software program . BrieflyACTB, beta-actin; ALB1, Abelson murine leukemia viral oncogene homolog 1; B2M, beta-2-microglobulin; cDNA, complementary DNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GNB2L1 (Guanine nucleotide binding protein, beta polypeptide 2-like 1; HPRT1, Hypoxanthine phosphoribosyltransferase 1; NCBI, National Center for Biotechnology Information; PCR, polymerase chain reactions; PBGD, porphobilinogen deaminase; RPL32, ribosomal protein L32; RT-PCR, reverse transcription-polymerase chain reactions; TBP, TATA-binding protein; TUBB, beta-tubulinXZ performed all the experimental procedures and was the primary author of the manuscript. LD participated in the study design and data analysis. AS conceived of the study, participated in the study design and coordination. All authors read and approved the final manuscript.


Streptococcus pneumoniae. The success of such vaccines has led to the inference that natural protection against invasive pneumococcal disease is largely conferred by anticapsular antibody. If this is so, one would expect that the decline in disease from different serotypes would vary significantly, and that the appearance of substantial concentrations of anticapsular antibodies would coincide temporally with the decline in age-specific incidence.Antibody to capsular polysaccharide has been the basis of several vaccines that offer protection against invasive disease from Using incidence data from the United States, we show that, on the contrary, the decline in incidence with age is quite similar for the seven most important serogroups, despite large differences in exposure in the population. Moreover, only modest increases in antibody concentration occur over the second and third years of life, a period in which serotype-specific incidence declines to less than 25% of its peak. We also present detailed data on the distribution of antibody concentrations in Israeli toddlers, which are consistent with the United States findings. The same conclusion is supported by new data on age-specific incidence in Finland, which is compared with published data on antibody acquisition in Finnish toddlers.We suggest some additional studies of the mechanisms of protection that could distinguish among potential alternative mechanisms, including acquired immunity to noncapsular antigens, maturation of nonspecific immune responses, or changes in anatomy or exposure. Anticapsular antibodies induced by vaccination can protect against invasive streptococcal disease, but natural immunity seems to involve other mechanism as well The protective effects of antibody to pneumococcal capsular polysaccharides have been appreciated since the development of serum therapy, in which passively transferred, serotype-specific antipneumococcal serum reduced mortality from pneumococcal pneumonia by half . The devStreptococcus pneumoniae as a distinct, independent pathogen [While the ability of passive or vaccine-induced anticapsular antibodies to protect against pneumococcal disease is clear, less is known about the natural development of immunity to pneumococcal disease in unimmunized persons. In unimmunized populations, the incidence of invasive disease follows a well-known age distribution, peaking in the first 2 y of life, declining by more than an order of magnitude by the second and third decades of life, and then rising at an accelerating pace, with incidence in persons over 70 y approaching that in infants . The reapathogen .The hypothesis that protection from invasive pneumococcal disease is caused by the acquisition of anticapsular antibodies directed against each of the pneumococcal serotypes yields two simple predictions about the age-specific epidemiology of pneumococcal disease. First, it predicts that the age-specific timing of the decline in invasive disease should be different for different serotypes: those that are rare, poorly immunogenic, or both should decline later in life than those that are common and immunogenic. Second, it predicts that protection against invasive disease from a given serotype should coincide temporally with the acquisition of anticapsular antibody to that serotype, both at an individual level and at a population level. We tested these predictions using data from the United States, Finland, and Israel.Incidence of invasive pneumococcal disease was measured in eight sites around the United States participating in the Centers for Disease Control and Prevention's Active Bacterial Core Surveillance between 1994 and 1999. The data used here are restricted to those periods during which serotyping was routinely performed: 1994–1999 for the Georgia site, 1995–1999 for the Minnesota site, and 1998–1999 for all other sites [Antibody concentrations were measured by enzyme-linked immunosorbent assay (ELISA)  in blood samples that were obtained from 130 toddlers at enrollment and at approximately 12 and 24 mo after enrollment in a double-blind, controlled trial of a nine-valent pneumococcal conjugate vaccine. The toddlers analyzed for this study were those in the control group, which received meningococcal C conjugate vaccine; the details of the trial  were prehttp://www3.ktl.fi/stat/) identified all blood and cerebrospinal fluid isolates of S. pneumoniae obtained in the years 1995–2001. Incidence within 6-mo age groups was calculated using population denominators obtained from Statistics Finland . Since the primary purpose of examining incidence in Finland was to compare age-specific rates against published distributions of antibody concentrations for the same age groups [Mandatory reporting from all microbiological laboratories in Finland to the National Register of Infectious Disease .Incidence peaks between the ages of 9 and 15 mo, and falls in an approximately parallel fashion thereafter, for each of the seven most important serogroups  and for the remaining serogroups put together. The same pattern is observed for both pneumonia and bacteremia. For each serogroup, incidence by age 24 mo is approximately half that in the peak age group, and by 36 mo, incidence for each serogroup has fallen to 10%–25% of its peak.The consistent timing of the pattern across multiple serogroups argues for a common mechanism, rather than for independent acquisition of immunity to each serogroup as a separate event. Since most individuals do not suffer from invasive pneumococcal disease in this age range, carriage or mucosal disease (otitis media) from pneumococci may be the immunizing event for anticapsular antibodies in the general population  , are capable of protecting individuals against pneumococcal invasive disease. While studies in animals  and humaWe have recently shown that mice that are exposed thrice at weekly intervals to intranasal colonization with encapsulated pneumococci are protected against subsequent carriage, that this protection is effective for heterologous as well as homologous capsular types, and that it is effective even in MuMT mice, which lack the ability to produce antibodies . We have also shown that intranasal immunization with unencapsulated, killed pneumococci protects against nasopharyngeal colonization, in a fashion that is independent of antibody but requires CD4+ T cells at the time of challenge. The relevance of cellular immune mechanisms in protecting humans against pneumococcal colonization or disease is not known.Another candidate for a factor that may be changing with age is susceptibility to viral infections, especially influenza, which may predispose to pneumococcal colonization  or diseaExposure to pneumococci probably changes in some fashion over the first 5 y of life. However, for changes in exposure to account for the sharp drop in disease incidence following the first birthday, it would be necessary for exposure also to drop severalfold per year over this age range. Studies of pneumococcal carriage do show gradual changes in the prevalence and serotype composition of the nasopharyngeal flora in these years, but the prevalence of carriage changes much more gradually than the incidence of invasive disease .We are not aware of data that bear strongly on the plausibility of other possible mechanisms for the age-related decline in pneumococcal disease, such as changes in anatomy, physiology, receptor expression, or resident bacterial flora. However, factors other than antibody—such as innate or acquired cellular immune responses, age-related anatomical changes, or changes in exposure to pneumococci—cannot be ruled out, and more than one factor may be involved. Indeed, the peak of pneumococcal meningitis incidence in the 3–6-mo age group see  suggestsAlthough we suggest that anticapsular antibody is not primarily responsible for the age-specific decline in invasive pneumococcal disease, there is no question that the capsule is an important virulence factor that interacts with the innate and acquired immune system in a number of ways. It is clear that the pneumococcal capsule interferes with various host clearance mechanisms . It woulThe evidence adduced here is subject to several limitations. With respect to the relative timing of acquisition of protection against different serotypes, one could postulate that because some of the most common pneumococcal serotypes, such as 6B, 19F, and 23F, are also among the least immunogenic , the effThe likelihood that mechanisms other than anticapsular antibody confer immunity to pneumococcal disease has important implications with respect to vaccine design. As experience with conjugate pneumococcal vaccines in children unfolds, it is becoming increasingly clear that such a strategy suffers from several limitations, including the possibility of serotype replacement , a modest effect on nasopharyngeal colonization, limited serotype coverage, cost, and difficulties in production that have led to shortages since licensure. A better understanding of the mechanisms that underlie natural immunity to pneumococcus could pave the way for the development of more effective, species-specific pneumococcal vaccines.Streptococcus pneumoniae is a common bacterium that lives in the upper respiratory tract of many children, and some adults. The bacterium generally causes no harm in healthy individuals, but in some circumstances it can cause mild infections, such as ear infections, or more severe ones, such as lung infection (pneumonia), bloodstream infection (bacteremia), or infection of the lining of the brain (meningitis). These more severe forms, called invasive pneumococcal disease, occur especially in children, elderly people, and others with weakened immune systems. The bacterium exists in different versions, or serotypes. The different versions of the bacterium each have a different outer shell . Scientists have developed vaccines against Streptococcus pneumoniae that protect against the most common serotypes. These vaccines consist of a cocktail made up of material from the capsules of the most common serotypes. This material causes the body's immune system to produce antibodies that can fight Streptococcus pnemoniae and protect vaccinated individuals against disease caused by the common serotypes. In many developed countries vaccination is recommended for all children and elderly people.Most people get exposed to many different versions of the bacterium over the course of their lives. These encounters cause little or no disease in most people, and the risk of disease declines sharply and remains low through middle age, before climbing again in the elderly. Based on experience with vaccines, scientists have thought that this “natural” protection that develops with age was also based on antibodies against the bacterial capsule. The authors of this study wanted to test whether this was actually true.If in the healthy population protection against invasive disease is in fact due to anticapsular antibodies, one can make certain predictions about the frequency of invasive disease among certain age groups. The researchers tested those predictions against actual disease records from the United States, Israel, and Finland.The actual records did not match the predictions very well, suggesting that natural protection against invasive pneumococcal disease is not based on anticapsular antibodies alone.These results suggest that there are elements of natural protection against invasive pneumococcal disease that we do not understand yet. Moreover, these elements seem to involve more general protection against various forms of the bacterium rather than individual protection against particular serotypes.Given the importance of the disease, we should try to understand all elements of natural protection. Such understanding might help researchers develop better vaccines to prevent invasive pneumococcal disease, and maybe even improve treatment of patients who have become ill.http://www.who.int/vaccines/en/pneumococcus.shtmlWorld Health Organization information page on pneumococcal vaccines: http://www.cdc.gov/nip/publications/VIS/vis-PneumoConjugate.pdfUnited States Centers for Disease Control and Prevention factsheet on pneumococcal vaccine: http://www.hc-sc.gc.ca/english/iyh/medical/pneumococcal.htmlHealth Canada information on pneumococcal vaccine: http://www.prodigy.nhs.uk/guidance.asp?gt=Immunizations%20-%20pneumococcalInformation for health-care providers from the United Kingdom Nation-al Health Service: http://www.pneumoadip.com/PneumoADIP Web page on childhood pneumococcal disease:


Apoptosis has recently been proposed to contribute to the pathogenesis of emphysema.In order to establish if cell fate plays a role even in end-stage disease we studied 16 lungs (9 smoking-associated and 7 α1antitrypsin (AAT)-deficiency emphysema) from patients who had undergone lung transplantations. Six unused donor lungs served as controls. Apoptosis was evaluated by TUNEL analysis, single-stranded DNA laddering, electron microscopy and cell proliferation by an immunohistochemical method (MIB1). The role of the transforming growth factor (TGF)-β1 pathway was also investigated and correlated with epithelial cell turnover and with the severity of inflammatory cell infiltrate.2 = 0.8). A negative correlation was found between the TGF-β pathway (particularly TGF-βRII) and T lymphocytes infiltrate in smoking-related cases The apoptotic index (AI) was significantly higher in emphysematous lungs compared to the control group (p ≤ 0.01), particularly if only lungs with AAT-deficiency emphysema were considered (p ≤ 0.01 vs p = 0.09). The proliferation index was similar in patients and controls (1.9 ± 2.2 vs 1.7 ± 1.1). An increased number of T lymphocytes was observed in AAT-deficiency lungs than smoking-related cases (p ≤ 0.05). TGF-β1 expression in the alveolar wall was higher in patients with smoking-associated emphysema than in cases with AAT-deficiency emphysema (p ≤ 0.05). A positive correlation between TGF-βRII and AI was observed only in the control group (p ≤ 0.005, rOur findings suggest that apoptosis of alveolar epithelial cells plays an important role even in end-stage emphysema particularly in AAT-deficiency disease. The TGFβ-1 pathway does not seem to directly influence epithelial turnover in end-stage disease. Inflammatory cytokine different from TGF-β1 may differently orchestrate cell fate in AAT and smoking-related emphysema types. Pulmonary emphysema, a significant global health problem, is a pathological condition characterized by enlargement of the airspaces distal to the terminal bronchiole, destruction of the alveolar walls, without and/or with mild fibrosis . To dateRecently more attention has been paid to alveolar epithelial injury in addition to alveolar matrix destruction. The presence of apoptosis has recently been described in animal models of emphysema ,5 and inThe majority of investigations have focused the attention on smoking-related emphysema keeping in mind that cigarette smoking was the main cause of apoptotic cell death. Cigarette smoke may induce alveolar cell apoptosis either directly by a cytotoxic effect on pneumocytes or indirectly by decreasing the production of vascular endothelial growth factor (VEGF) via altered epithelial cells . To dateMoreover apoptotic phenomenon has been previously investigated in moderate/severe smoking-related forms of emphysematous lungs obtained almost exclusively from lung volume reduction surgery ,7,9. If Among the growth factors, transforming growth factor (TGF)-β1 could play a crucial role in the remodeling process occurring in emphysematous parenchyma. TGF-β1, other than its known profibrogenetic  and antiThe cytokine has been shown to be over-expressed in patients with a history of smoking and chronic obstructive pulmonary disease (COPD) ,17. ParaIn the present work the degree of apoptotic cell death and epithelial proliferation in the lungs of patients with different types of end-stage emphysema was studied. The severity of inflammatory cell infiltrate (ICI) was also quantified and correlated with epithelial cell turnover. Further, the TGF-β1 pathway was detected and correlated with the apoptotic index (AI), the proliferative index (PI) and the ICI.Lung tissue used in the present study comprised material from 16 patients undergoing lung transplantation for end-stage emphysema at the Thoracic Surgery Unit of the University of Padua Medical School. Cold ischemia preservation was 60 minutes and 120 minutes, respectively, for single and double lung transplantations. Small-sized pieces from all lobes were cut and immediately fixed in Karnovsky's solution for electron microscopy. The lungs were then gently fixed in 10% phosphate-buffered formalin by airway perfusion and processed for sectioning (3 μm). Samples were selected from specimens that showed features of excellent tissue preservation and adequate lung inflation. In particular, large thin blocks approximately 30 × 25 mm were cut from the subpleural areas of the apical anterior and lingular segments of the upper lobes, as well as the apical and basal segments of the lower lobes. A more centrally placed block was taken to sample the segmented airways and blood vessels. The right lung was sampled in the same way with the middle lobe being treated in the same way as the lingula . Adult cThe terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling method (TUNEL) was used to investigate the presence of apoptosis. Sections were processed in accordance with Gavrieli et al's method . BrieflyLung specimens fixed in Karnovsky's solution  for 24 hours were post-fixed with 1% osmium tetroxide  for 1 hour, and then progressively dehydrated in alcohol and embedded in epon. Semi-thin sections were stained with 0.1% toluidine blue for light microscopic examination. Ultra-thin sections were stained with uranyl acetate and lead citrate for transmission electron microscopy performed using a Hitachi H-7000 .4 DNA ligase to 500 ng of lung sample DNA (for 16 h at 16°C). These then served as primers in LM-PCR under the following conditions: hot start (72°C for 8 min), 30 cycles  and extension (72°C for 15 min). Every reaction set included thymus DNA as a positive control and normalization of the amount of reaction products. Amplified DNA was subjected to electrophoresis on 1.2% agarose gel containing ethidium bromide. Images were scanned and the DNA fragmentation levels were based on the density of the bands ranging between 1000 base pairs (bp) and 300 bp. The percentage of DNA fragmentation was quantified by scanning densitometry.The presence of oligonucleosomal-length DNA cleavage was investigated with APO-DNA1  in 12 cases  in which frozen tissue was available. Briefly, DNA was obtained from lung tissue samples using proteinase K-phenol extraction. Dephosphorylated adaptors were ligated to 5' phosphorylated blunt ends with T1-β2 and-β3 primary antibody to active TGF-β1 , with polyclonal antibody against TGF-β receptor type II  and monoclonal MIB-1 antibody , which recognizes the Ki-67 antigen in paraffin-embedded tissue sections. Immunohistochemical investigations were done on the sections from the same paraffinembedded specimens processed for TUNEL analysis.All lung sections were subjected to antigen retrieval by heating in a microwave oven on high power for 8 minutes in 0.01 mol/l citrate buffer (ph 6.0) and then incubated with a mouse monoclonal anti-TGF-βThe detection system was the Vectastain ABC kit  with 3-amino-9-ethylcarbazole  and with a mixture of 3,3'-diamino-benzidine tetra7 hydrochloride (Dako) and hydrogen peroxide as the chromogenic substrates. Sections were counterstained with Mayer's hematoxylin.In all samples, immunohistochemistry for the characterization of ICI was carried out by using the following antibody panel: CD20 (1.100), CD45RO (1.100), CD4 (1:20), CD8 (1:50), CD3 (1:100), CD68 (1:50) . The detection system was the Vectastain ABC kit, as described above.For all immunohistochemistry experiments, negative controls were performed by incubation of the sections with the omission of primary antibody and using the antibody diluents alone or the appropriate non-immune IgG in each case.For simultaneous detection of DNA fragmentation and cell proliferation a double labeling was also performed. The TUNEL technique was first performed and the staining achieved was diaminobenzidine as chromogen. For MIB1 immunolocalization in the second staining sequence the sections were stained with 5-bromo-4-chloro-3-indoxyl phosphate/nitro blue tetrazolium .Immunoassay for TGF-β1 and TGF-βRII was detected by using digital quantitative analysis  as previously described . QuantifIn each case at least 50 microscopic randomly chosen fields were analyzed. A total of 5,000 epithelial cells were counted for AI and PI and the values were expressed as percentages.To avoid observer bias the cases were coded and measurements were made without knowledge of clinical data. Differences between groups were detected using the analysis of variance for clinical data and the Kruskall-Wallis test for histological data. The Mann-Whitney U test was performed after the Kruskall-Wallis test when appropriate. The statistical tests used were two-sided.Correlation coefficients were calculated using Spearman's rank method. Probability values of 0.05 or less were accepted as significant. Group data were expressed as means and SD or as medians and range when appropriate.Major clinical data for patients with emphysema are shown in Table Average patient age was 54.4 ± 7.5 years. FEV1 mean was 19 ± 8.9 . Bilateral single lung transplantation was performed in 12 out of 16 patients. All patients had been heavy smokers: 7 were only smoking-associated emphysema cases (51 ± 28 packs-year) and 9 were both AAT-deficiency emphysema and smoking cases (55 ± 27 packs-year). For the sake of brevity, the abbreviation AAT-deficiency emphysema for smoking patients with AAT-deficiency will be used throughout the manuscript.All patients had quit smoking at least 1 year before undergoing surgery.aw) was 20,9 ± 1.5 mmHg and inspiratory oxygen fraction  was 0.4 ± 0.1.The average control patient age was 34 ± 16.8 years and cerebral trauma was the cause of death. All the donors stayed less than two days in intensive care without evidence of lung infection or other complications. During artificial ventilation, airway pressure  as compared with controls (p ≤ 0.01). An increased number of CD3 (p ≤ 0.05), CD8 (p ≤ 0.05) and CD45RO (p ≤ 0.001) was seen in AAT-deficiency emphysema compared to smoking-related emphysema . The TUNEL-positive cells were more frequently detected within more enlarged alveolar walls. Apoptotic cells and/or bodies were frequently seen in intra-alveolar lumen that presumably represented apoptotic cells detached from the alveolar wall Fig. . The PI TUNEL-positive/MIB1-negative nuclei detected by double staining were seen in all cases, whereas MIB1 was never expressed in any of the TUNEL-positive nuclei Fig. .In each group, no statistically significant correlations were found between AI and PI as well as between AI/PI and ICI.At electron microscopy typical features of early apoptosis with margination of chromatin at the nuclear membrane and late apoptosis with completely dense nuclear chromatin, including apoptotic bodies in various stages of degradation, were seen in pneumocytes, endothelial cells and fibroblasts. Typical features of reduplication of vessel basal membranes were frequently seen in cases with more evident apoptosis  a-f. Ult2 = 0.8) was seen in control lungs  in smoking-related cases. No correlation was noted between the TGF-β1 pathway (TGF-β1 and its RII) and the AI/ PI of emphysematous lungs.In emphysema patients and controls TGF-β1 and TGF-βRII were localized in bronchiolar and alveolar epithelial cells and macrophages. Quantitative analysis of TGF-β1 measured in the alveolar wall showed no statistically significant difference between emphysema patients and controls. A higher cytokine expression was noted in patients with smoking-associated emphysema compared with AAT-deficiency disease  Fig. . A positIn the present study we have analyzed for the first time apoptosis and proliferation in different types of end-stage emphysema. The detection of a high AI in emphysematous lungs even in end-stage disease emphasizes the importance of the phenomenon in the development, and overall, in the progression of emphysema.In general there are two main forms of cell death: oncosis and apoptosis. The latter process results in characteristic biochemical features and cellular morphology such as cell shrinkage condensation and fragmentation of the nucleus into well contained fragments called apoptotic bodies.Perturbation of normal rates of apoptosis has been implicated in many pathologic conditions such as neuro-vegetative, cardiovascular and liver disorders and cancer -22. As sRecently the causal role of apoptosis has been increasingly recognized in the destruction of alveolar walls and airspace enlargement -9. AmongTo avoid a bias of under or over-estimated alveolar cell apoptosis and proliferation due to regional disease activity we analyzed large specimens taken from different lung regions (upper and lower lobes). The lower AI detected in our control lungs underlines an important concept: in non-emphysematous lungs apoptosis is an irrelevant process adequately balanced by proliferation. The increased number of apoptotic cells in patients with emphysema  suggests a new mechanism, namely "epithelial apoptosis/proliferation imbalance" in the pathogenesis of disease. In our study, different from a recent clinical study by Yokohori et al , a PI siTaq polymerase-based DNA in situ ligation, DNA gel electrophoresis or electron microscopy, in order to avoid false positive labeling and to assess the reliability of apoptosis [DNA nicks may be seen in cells with DNA synthesis/repair thus sometimes producing false TUNEL positive cells. False positive TUNEL staining can also be generated through non-apoptotic mechanisms: RNA synthesis and splicing, necrosis as well as artifacts due to preservation methods. Consequently, some authors have stressed the importance of associating other techniques, such as poptosis .Our TUNEL findings have been corroborated by employing an additional quantitative apoptosis assay. Moreover, the presence of different stages of apoptosis was confirmed and the cells involved in programed cell death were well characterized by using electron microscopy investigation, which is considered the gold-standard technique for apoptotic cell detection. In our work double-immune labeling showed that all TUNEL positive cells were consistently negative for MIB1 thus suggesting the true epithelial DNA fragmentation. Although the high AI detected in our patients could be mainly explained by the high rate of apoptotic cell death, an impaired clearance mechanism of apoptotic cells/bodies should also be considered. A frequent finding of apoptotic bodies in alveolar walls and within lumen may support the latter theory as an important contributing factor for a high percentage of AI.The principal trigger of epithelial injury leading to apoptotic cell death is up to now still unclear. The cytotoxic effects of cigarette smoke, one of the most clearly proven etiologic factors in the development of emphysema and in general of COPD, have been suggested to suppress epithelial proliferation and to induce cell death. In particular oxidants and aldehydes, major constituents in the volatile phase of cigarette smoke, have been reported to induce apoptosis of lung cells .Different DNA and RNA viruses have been identified as viral pathogens associated with the disease. Double-strand DNA viruses such as adenovirus have the ability to persist in airway epithelial cells long after the acute infection has cleared. The expression of adenoviral trans-activating proteins has been demonstrated in the airway epithelium of both human and animal lungs and is associated with an amplification of cigarette smoke-induced inflammatory response .Different adenovirus early proteins, in particular E4orf4, have been reported in the shutoff of host protein synthesis and in the promotion of a p53-independent cell death program . It is lin vitro and in vivo studies [As the TGF-β1 pathway is well-known for its anti-inflammatory activity, a higher epithelial expression of TGF-β1 in patients with smoking-related emphysema compared with AAT-deficiency may partially justify the different patterns of inflammation in the two types of emphysema, as found in our study. A significantly higher increase of inflammation, particularly due to T lymphocytes, was found in AAT-deficiency emphysema (panlobular type) than in smoking-related disease (centrilobular type), with the latter displaying an increased expression of the TGF-β pathway (as demonstrated by the negative correlation with T lymphocyte infiltrate). Similar findings have been previously reported in both  studies ,33. An iIn our work a direct correlation between TGF-βRII and AI was found in the control group thus showing that this cytokine could play a role in alveolar homeostasis in physiologic conditions. Instead no correlation was found between the AI and TGF-β1 pathway in either type of emphysema, suggesting that the TGF-β1 regulated mechanism is lost in the disease. Other cytokines besides TGF-β1 could be involved in uncontrolled programed cell death inducing the progressive disappearance of the alveolar unit.Decreased expression of VEGF and VEGF R2 has been demonstrated to be significantly correlated with apoptosis of both epithelial and endothelial cells in cigarette smoking-induced emphysema . It has More recently in an experimental model some authors have shown that over-expression of placenta growth factor (PIGF) causes a phenotype and pulmonary dysfunction similar to human lung emphysema by inducing apoptotic events in the alveolar septa . AlthougIn summary, our work has demonstrated for the first time that apoptotic phenomenon is extensive also in end-stage emphysema patients. This unique case series, and overall the large variety of lung tissue samples examined,  may account for the differences in our AI findings compared to other studies . The higWhether the "apoptosis/proliferation imbalance" occurs before, after or at the same time as the "elastase/antielastase imbalance" is still unknown and should be the subject of future studies.Our study had a few limitations. Firstly, the patients with AAT-deficiency can not be considered pure AAT-deficiency emphysema cases because these patients were also smokers. Panlobular emphysema occurs at a younger age in alpha-1-antitrypsin patients, especially if the patients smoke cigarettes, as in our case series . Patients with AAT-deficiency who are smokers develop lung impairment function earlier and in a more severe form than their non-smoking counterparts. Thus, it is extremely rare to have patients who are non-smokers with AAT-deficiency as candidates for lung transplantation. Secondly, the clinical characterization of the donor was poor and according to the guidelines for the selection of donor lungs, smokers were not excluded . SmokersHowever, if this was the case, the AI difference between emphysema and control patients would have been even higher because of the lower AI in healthy patients. A third potential bias is that all the donors were mechanically ventilated before lung transplantation and it is known that mechanical ventilation may induce lung apoptosis . Again, Our study analyzed apoptosis and proliferation in end-stage emphysema. In particular the work described for the first time a high AI in patients with AAT-deficiency emphysema. Ultrastructural investigation, TUNEL analysis and oligonucleosomal-length DNA laddering, performed in different lung regions were all used for detection of apoptotic phenomenon. The increase of apoptotic cells in patients with emphysema not adequately replaced by new epithelial cells suggests a new mechanism, namely "epithelial apoptosis/proliferation imbalance" in the pathogenesis of disease. More inflammation, particularly due to T lymphocytes, was observed in AAT-deficiency emphysematous lungs. An increased pro-apoptotic milieu of inflammatory related cytokines may contribute to the higher cell death rate detected in AAT-deficiency emphysema. While a direct correlation between TGF-βRII and AI was found in the control group, no relation was found between the AI and TGF-β1 pathway in end-stage emphysema, suggesting that the influence of the TGF-β pathway on epithelial turnover is lost in the disease. Knowledge of the mechanism responsible for activation and progression of the apoptotic cascade could offer new information in the near future, on more appropriate stratification and treatment of the disease.FC: conceived of the study and participated in its design and coordinationCG: substantial contribution in study design and data interpretationBB: acquisition of clinical data and critical revision for important intellectual contentFR: thoracic surgeon providing lung specimen and critical revision for important technical aspectsML: thoracic surgeon providing lung specimen and critical revision for important technical aspectsRZ: critical revision for important intellectual contentGP: acquisition of clinical dataSB: acquisition of clinical data and performed the statistical analysisMS: participated in the design of the study and gave critical revision for important intellectual contentMV: substantial contribution in study design and data interpretationAll the authors read and approved the final manuscript.


In silico mapping provides an expedient way to associate the natural diversity of phenotypic traits with ancestrally inherited polymorphisms for the purpose of dissecting genetic traits. In mouse, the current single nucleotide polymorphism (SNP) data have lacked the density across the genome and coverage of enough strains to properly achieve this goal. To remedy this, 470,407 allele calls were produced for 10,990 evenly spaced SNP loci across 48 inbred mouse strains. Use of the SNP set with statistical models that considered unique patterns within blocks of three SNPs as an inferred haplotype could successfully map known single gene traits and a cloned quantitative trait gene. Application of this method to high-density lipoprotein and gallstone phenotypes reproduced previously characterized quantitative trait loci (QTL). The inferred haplotype data also facilitates the refinement of QTL regions such that candidate genes can be more easily identified and characterized as shown for  A large resource of genetic markers, typed in 48 strains of mice, combined with statistical techniques, allow "in silico" mapping of genetic regions involved in interesting traits in mice The combined efforts of the public and private mouse genome sequencing consortiums have yielded important advances in understanding the structure and content of the genome . IdentifThe utility of this sequence-variation data is 2-fold. First, the single nucleotide polymorphisms (SNPs) identified by these sequencing projects provide denser coverage marker sets that are well suited for high-throughput genotyping systems. Currently, these benefits have only been available for crosses between the relatively few strains for which substantial polymorphism discovery has been undertaken.Second, the distribution of SNPs between any two strains, or more precisely, the lack of SNPs between two mouse strains, indicates regions of their genomes that were inherited from a common ancestor. Phenotypic differences that are traditionally mapped in QTL studies are almost exclusively due to loci inherited from different ancestral progenitors rather than new mutations . Thus, ahttp://www.jax.org/phenome) have collated multistrain phenotype data. We demonstrate that when using these datasets, in combination with new analysis methods, statistically significant associations between discrete genomic regions and biologically important phenotypes can be identified. Confirmation of these associations was obtained by comparison to data from traditional QTL mapping methods.To overcome the barriers to in silico mapping, 10,990 SNP assays have been typed against 48 mouse strains in this study. These assays provide an extensive polymorphic marker set enabling expansion of traditional mapping efforts to other strains. Wide-ranging phenotyping projects that have been coordinated by The Jackson Laboratory ((Mus spretus), represent a different species of mouse from the other lines tested. Not surprisingly, fewer genotypes were obtained from these two because of the divergent genomes  track with the phenotypic outcome with the idea that these haplotypes contain causative mutations. For in silico mapping to be successful, a requirement is that the SNP data accurately represent this ancestral relationship of the mice at the genomic level. At a gross level, this was examined by comparing branches of the phylogenic tree generated from this SNP dataset with the known breeding histories of the strains used in this study . An inspMus musculus Chromosome 9 (MMU9)  B. Based A lack of sufficient underlying SNP data to this point have prevented the thorough development and testing of an algorithm to carry out in silico mapping . Previouhttp://jaxmice.jax.org/jaxmice-cgi/jaxmicedb.cgi). Albino mice were excluded from the mapping of nonagouti because the nature of the phenotype prevents the ascertainment of agouti or nonagouti coat colors. In each case, the appropriate locus for the gene responsible for the particular trait was identified from this SNP collection with the most significant p-value  statistical model. Briefly, at each three-SNP window, a modified F-statistic based on the true genotype–phenotype pairings is calculated  and gallstone development. The average HDL values from 10-wk-old mice fed on a normal chow diet were taken from the Mouse Phenome Database . Becausep-value, a recently described method by p-value = 0.000506; −log[p] = 3.2958) was used , and seven of these regions fell within the range of traditionally identified QTL for gallstone formation or mucin accumulation, which is considered a precursor to gallstone formation , located at 89.55 Mb, is expressed in the liver and adipose tissue (http://symatlas.gnf.org) and functions by producing cyclic adenosine monophosphate . This excludes the MMUX in silico QTL since they cannot be verified from current traditional QTL data. The false positive cutoff employed here is very restrictive and could be relaxed to find additional real associations, but the chances of including false positives would then increase. For the gallstones phenotype the concordance is not demonstrated to the same level; however, the top-ranked loci still show overlap with previously defined QTL. What about the loci that do not show overlap; are they still real? From a statistical analysis it is unlikely they are false positives. In these results, 25 strains are simultaneously combined, unlike standard QTL mapping using two-strain comparisons, and some phenotype–genotype associations may occur that have not been observed by classical methods. Contributions from diverse strains that have not normally been used in F2 crosses or available in RI lines can now be incorporated.The SNP data here provide new resources for traditional mapping projects and enable development of inbred strain haplotype methods for QTL detection. The analyses presented here indicate that the inferred haplotype structures derived from this dataset provide sufficient estimation of genetic diversity/similarity to map Mendelian traits to within 1-Mb intervals. QTL can also be defined as inferred haplotype loci of several megabases in size. The analysis for QTL provides a rank order of significant phenotype/genotype associations, and using the gFWER method of controlling for multiple-testing error, the loci reported as statistically significant are very likely to be biologically relevant. This point is borne out by the high concordance between the in silico QTL and the traditionally determined QTL . The traEven showing that this method does find significant associations, the question arises about its general utility and applicability. The methods of in silico mapping as described here should be viewed as a complement to, and not a substitute for, traditional methods for mapping QTL. Although we have demonstrated a robust approach to in silico mapping, it would certainly not be expected to find all QTL for a given phenotype. Major contributors to phenotypic variation will show up, but weaker contributors would be expected to be lost because of the limited power of 25 strains. It would also be expected to miss QTL resulting from recent strain-specific mutations or low-frequency haplotypes. Traditional QTL methods will still be required to identify the more subtle interactions, including those involving epistasis and modifying genes.However, these methods would provide a useful starting point for a new phenotype that is being investigated, where often the first step for any QTL analysis is a strain survey to quantify the range of the phenotype. Additionally, if these analyses are overlaid with the results from a traditional two-strain QTL mapping, one of the major advantages to be gained from this approach is that associative loci are defined in terms of a few megabases instead of tens of centiMorgans.M. m. domesticus. To be successful, phenotypes must have a low intrastrain variation but sufficient variance within the strain set selected. This however, is not a requirement restricted to in silico mapping.The number and selection of strains and appropriate phenotype are also important considerations. Here we have limited our analysis of the complex traits, HDL and gallstones, to 25 strains—those that are best interrogated by this SNP set. While it is true that more strains have the potential to add greater statistical power to resolve QTL, this potential is limited by our ability to accurately represent the ancestral relationship of those additional strains. If we add more strains, but cannot accurately infer haplotype structures in those strains, we only add more noise to the analysis. The ability to detect all possible haplotypes in the utilized strains from the SNP data suffers from the availability of sequence data, currently from only four strains of mice. Because the source SNPs come from the sequencing of only four closely related strains, this current set is biased toward interrogating ancestry of The overall power of this method will only improve as the biases and limitations of the SNP panel are addressed and additional strains are genotyped and phenotyped. Unique strains would become more useful if all possible SNPs are known and the mapping is then done directly with the causative polymorphism or at least with a large unbiased set of SNPs. As resequencing of other mouse genomes progresses, the ability to correctly infer the complete number and structure of haplotypes will improve, and the number of QTL regions reaching statistically significant levels will increase.Recently, two similar studies of haplotype structures across 5-Mb regions were published, although they produced differing conclusions on how their findings might affect in silico mapping efforts . Yalcin This is not to suggest that fragmented small haplotypes are not common in the genome of the inbred mouse. This clearly does mean that there will be regions of the genome that will not be interrogated well by an in silico method. This approach is still limited by the density of this SNP map and can only be expected to visualize inferred haplotype patterns of approximately 1 Mb in size, and therefore smaller haplotype structures are hidden and potential phenotype–genotype associations will be missed. Here future, larger SNP sets that will allow more SNPs to infer haplotype will become important. However, this is the best resolved whole genome view of the diversity of the commonly used inbred strains to date.The algorithms employed here provide a starting point for further development of in silico mapping. We have shown that they can be used to identify Mendelian traits and replicate classical QTL associations. Clearly, the next goals are to validate some of the previously unreported associations, and this work is ongoing.Tas1r3 locus were gathered from sequence from multiple strains available in GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=nucleotide&cmd=search&term=tas1r3). All physical positions presented in the paper are from the Celera Mouse Genome Assembly R13.SNPs for use in genotyping were selected on a weighted basis from the Celera Mouse SNP Database containing data from the DBA/2J, A/J, C57BL/6J, 129S1/SvImJ, and 129X1/SvJ strains. Sufficient SNPs were selected for coverage of at least one SNP per 300 kb on average. The 129S1/SvImJ and 129X1/SvJ strains were considered as the same strain when their alleles agreed; preference was given first to SNPs where each allele of the SNP was represented by two strains. This was done to favor selection of SNPs representing ancestral inheritance, not recent strain-specific mutations, and to favor real SNPs as opposed to errors in sequence annotation. Additional selective value was incorporated based on whether the SNP was in a gene, how many sequencing runs supported the presence of the SNP, and the proximity of the SNP to previously selected SNPs. Additional SNPs used to characterize the Primers for PCR and single-base extension were designed by using the SpectroDESIGNER software package . Assay designs are available as Supporting Information. All SNP assays were named for their position in the genome in the following format: the chromosomal location, the Mb position on the chromosome, and the kb position with a period separating each number.Taq , 0.625 μl of 10X HotStar PCR buffer containing 15 mM MgCl2, 0.5μl PCR primers mixed together at a concentration of 1.25 μM for multiplexed reactions, and 0.325 μl of 25 mM MgCl2. Reactions were heated at 95 °C for 15 min followed by 45 cycles at 95 °C for 20 s, 56 °C for 30 s, and 72 °C for 1 min and a final incubation at 72 °C for 3 min. After PCR amplification, remaining dNTPs were dephosphorylated by adding 1.5 μl of water, 0.17 μl of homogeneous mass extend reaction buffer (Sequenom), 0.3 units of shrimp alkaline phosphatase (Sequenom), and 0.03 μl of 10 units/μl exonuclease . The reaction was placed at 37 °C for 20 min, and the enzyme was deactivated by incubating at 85 °C for 15 min. After shrimp alkaline phosphatase treatment, the genotyping reaction was combined with 0.76 μl of water, 0.2 μl of 10X Termination mix (Sequenom), 0.04 μl of 0.063 units/μl Thermosequenase (Sequenom), and 1μl of 10 mM extension primer. The MassEXTEND reaction was carried out at 94 °C for 2 min and then 99 cycles of 94 °C for 5 s, 52 °C for 5 s, and 72 °C for 5 s The reaction mix was desalted by adding 3 mg of a cationic resin, SpectroCLEAN (Sequenom), and resuspended in 30 μl of water. Completed genotyping reactions were spotted in nanoliter volumes onto a matrix arrayed into 384 elements on a silicon chip (Sequenom SpectroCHIP), and the allele-specific mass of the extension product was determined by matrix-assisted laser desorption ionization time-of-flight MS. Analysis of data was by automated allele calling from the SpectroTYPER software. All SNP data are available at NCBI dbSNP (http://www.ncbi.nih.gov/entrez/query.fcgi?db=snp) and The Jackson Laboratory Mouse Phenome Database (http://www.jax.org/phenome). Placement of the SNP data across the genome and major and minor allele distributions can be visualized using SNPview (http://snp.gnf.org).For SNP genotyping, genomic DNA from pedigreed mice  was diluted to 10 ng/μl, and 1 μl of DNA was combined with 2.45 μl of water, 0.1μl of 25 mM dNTPs , 0.03μl of 5 units/μl HotStar The use of a single marker is restrictive in the sense that it only allows a representation of the genome as diallelic. The use of windows of multiple markers enables the visualization of more complex genomic relationships between multiple strains. This more accurately models actual haplotype patterns than does a binary approach. In determining the size of the SNP window to use as a definition of inferred haplotype for purposes of the algorithm, sizes of two, three, four, and five SNPs were examined. A window of only two SNPs was still found to be too limiting. Windows of three, four, and five SNPs produced similar results, but as window size is increased biologically meaningful patterns become fragmented, creating more single-strain inferred haplotypes, resulting in an increase in noise. Singly represented haplotypes can never be informative in this analysis because the commonality of haplotypes is required to achieve significant association with a phenotype. Three SNP windows were also analyzed across the whole genome to identify mirror loci. This would be a locus that has exactly the same strain distribution pattern across all 25 strains used in an in silico run. There were no mirror loci, or 1-off, or 2-off mirror loci  that occurred outside of a 5-Mb interval.Defining the genetic measure as a categorical unit necessitated the use of an ANOVA-based model. The type of ANOVA to use was determined by the characteristics of the phenotypic values.The phenotypes studied here fell into two categories: binary or continuous. The coat color phenotype was considered as binary, where phenotypic values were set to 1 and 0. The HDL phenotype is an example of a continuous phenotype since the phenotypic values are measured on a continuous scale. Two different statistical methods were employed based on this distinction.glm with the parameter family set to binomial was used. This was followed by an application of anova.glm with the parameter test set to Chisq.When phenotypic values are binary, the appropriate statistical approach involves first fitting a binomial generalized linear model to the data. An analysis of deviance table is then computed for the fitted model. The R language function For continuous phenotypic values, a log transformation was applied to reduce the effects of outliers in the phenotypic data. Next, an F-statistic weighted for the genotypic diversity of the strains within an inferred haplotype group was used. The weighted F-statistic had the following form:whereandgn is the number of strains in a given inferred haplotype, gμ is the mean of phenotypic values in a given inferred haplotype , Tμ is the mean of all phenotypic values, k is the number of inferred haplotypes, N is the total number of data values, and gw is the weight representing the genetic diversity of the inferred haplotype. The genetic diversity ratio (gw) between two strains is the number of SNPs genome-wide in which both strains have genetic information and they disagree, divided by the total number of SNPs in which they both have genetic information. The genetic diversity coefficient for an inferred haplotype in the weighted F-statistic is the average gw between all strain pairs contained in the inferred haplotype.where p-value was computed by dividing the number of random F-statistics larger than the true F-statistic by the total number of random F-statistics (million). In this way 200 p-values were collected. The vertical heights reported in the bar graphs (see p) transform of the median of these 200 p-values. A 95% confidence interval (CI) for the p-value at this window was also calculated from this bootstrap distribution.The weighted F-statistic calculated at each SNP window determines if at least one of the inferred haplotypes has an average phenotypic value significantly different from the other inferred haplotypes. To assess the significance of the computed value, the null distribution of the weighted F-statistic was simulated at each SNP window by taking a million bootstrap samples of the phenotypic values. As in the algorithm used for binary phenotypes, inferred haplotype patterns present in only one strain were not included in the calculation because they are not informative in elucidating shared ancestral blocks. From this distribution of a million random F-statistics, 200 bootstrap samples of size 1 million were computed. For each bootstrap sample, a aphs see  are the To estimate the overall false positive rate for this type of calculation, calculating a significance threshold based on the family-wise error rate (FWER) has been proposed . Others p-values, the gFWER method requires an estimate of the “supremum” (least upper bound) of expected values reported at each locus, so the most significant value is reported from the 200 bootstrap p-values  corresponding to the (1 − α) percentile was added to the null distribution  is further complicated. Nearly 11,000 hypothesis tests (one at each three-SNP window) are conducted in a single run of the algorithm. Therefore, equations that currently exist for the estimation of power for QTL mapping by traditional methods cannot be applied here because they assume that the test statistic has some previously defined parametric form. Code for the above described algorithms is available upon request.For calculation of the significance of the number of in silico QTL that overlapped with previously identified QTL for the HDL phenotype, a binomial distribution was usedp = probability of success of 0.42 (overlap with HDL QTL in previous literature).given q = probability of failure; the 0.0025 result is the probability of at least nine successes in ten trials. Only ten loci could be assessed for this result as no information is available for traditional HDL QTL present on the X chromosome.Therefore For the mapping of the retinal degeneration traits, 37 strains were used. This represented all of the strains for which information existed in The Jackson Laboratory database minus the most divergent wild-derived strains for which inference of haplotype would be expected to be most inaccurate. These strains were A/J, AKR/J, BALB/cByJ, BUB/BnJ, C3H/HeJ, C57BL/10J, C57BL/6J, C57BLKS/J, C57BR/cdJ, C57 l/J, C58/J, CBA/J, CE/J, DBA/1J, DBA/2J, FVB/NJ, I/LnJ, KK/HlJ, LG/J, LP/J, MA/MyJ, NOD/LtJ, NON/LtJ, NZB/BlNJ, NZW/LacJ, PERA/EiJ, PL/J, RIIIS/J, SEA/GnJ, SJL/J, SM/J, ST/bJ, SWR/J, WSB/EiJ, ZALENDE/EiJ, 129S1/SvImJ, and 129X1/SvJ. Because of the added complexity of the coat color traits, mapping was restricted to the 25 most related strains for which coat color phenotype could clearly be determined. For the albino analysis, 129S1/SvImJ, A/J, AKR/J, BALB/cByJ, C3H/HeJ, C57BL/10J, C57BL/6J, C57BLKS/J, C57BR/cdJ, C57 l/J, C58/J, CBA/J, DBA/1J, DBA/2J, I/LnJ, LP/J, MA/MyJ, NZB/BlNJ, NZW/LacJ, PERA/EiJ, PL/J, SEA/GnJ, SM/J, WSB/EiJ, and ZALENDE/EiJ strains were used. For the nonagouti mapping, the same strain set as the albino mapping was used except for the mice presenting the albino phenotype. The strains were 129S1/SvImJ, C3H/HeJ, C57BL/10J, C57BL/6J, C57BLKS/J, C57BR/cdJ, C57 l/J, C58/J, CBA/J, DBA/1J, DBA/2J, I/LnJ, LP/J, NZB/BlNJ, PERA/EiJ, SEA/GnJ, SM/J, WSB/EiJ, and ZALENDE/EiJ. Any mouse showing an agouti coat color was considered to be agouti for this analysis regardless of genotype at the agouti locus. Only limited phenotype data were available for saccharin preference, so again all strains with available data except the most divergent wild-derived strains for which inference of haplotype would be expected to be most inaccurate were used. These strains were A/J, AKR/J, BALB/cByJ, BUB/BnJ, C3H/HeJ, C57BL/6J, C57 L/J, CBA/J, CE/J, DBA/2J, FVB/NJ, I/LnJ, KK/HlJ, LP/J, NOD/LtJ, NZB/BlNJ, PL/J, RIIIS/J, SEA/GnJ, SJL/J, SM/J, ST/bJ, and SWR/J. For the mapping of the other complex traits, only the 25 strains with the closest ancestral relationship were used. These strains were 129S1/SvImJ, A/J, AKR/J, BALB/cByJ, BTBR T+ tf/J, C3H/HeJ, C57BL/10J, C57BL/6J, C57BLKS/J, C57BR/cdJ, C57 l/J, C58/J, CBA/J, DBA/1J, DBA/2J, I/LnJ, LP/J, MA/MyJ, NZB/BlNJ, NZW/LacJ, PERA/EiJ, PL/J, SEA/GnJ, SM/J, and WSB/EiJ.Dataset S1(16.1 MB XLS).Click here for additional data file.Figure S1M. m. domesticus strains show a much closer relationship than the other wild-derived strains to the common laboratory strains.Ancestral relationships between strains can be seen within clusters of the tree such as the fact that BALB/cByJ is a progenitor strain to SEA/GnJ. The bias of the SNP set can also be viewed by the exaggerated distance between the C57 and 129 clusters and the DBA and A/J cluster. The wild-derived strains make up the outermost cluster, but the three (2.2 MB EPS).Click here for additional data file.Figure S2p-value at each three-SNP window was calculated and plotted along the x-axis. Because any log p-value below 3 will not reach significance, calculations are halted at any locus once obtaining a log p-value of 3 becomes impossible in order to increase computational throughput. As such all log p-values below 3 are reported at 3. The false positive cutoff established by the gFWER calculation is indicated by a horizontal red line. Every quantitative trait was run twice through the algorithm to ensure consistency of results.The log (5.8 MB EPS).Click here for additional data file.Figure S3p-value is reported from each run. The significance cutoff is indicated by the vertical red line.To estimate an appropriate false positive cutoff, 10,000 genome scans are conducted on randomized datasets and the 99.5 percentile log (3.1 MB EPS).Click here for additional data file.Table S1(44 KB XLS).Click here for additional data file.Table S2(34 KB XLS).Click here for additional data file.http://www.jax.org/phenome) accession numbers for the phenomes discussed in this paper are MPD:29 and MPD:99.The Mouse Phenome Database (


The Healthcare Commission started work on 1 April 2004, taking over all the functions of the former Commission for Health Improvement , which had released the corresponding PCT ratings for 2002/2003 in July 2003.On 21 July 2004, the Healthcare Commission , with handy functions like map search (by PCT name or part of it). The maps feature a colour-blind friendly quadri-colour scheme to represent PCT star ratings. Clicking a PCT on any of the maps will display the detailed performance report of that PCT for the corresponding year.We produced two Web-based interactive maps of PCT star ratings, one for 2003 and the other for 2004 Using our Web-based interactive maps, users can visually appreciate at a glance the distribution of PCT performance across England. They can visually compare the performance of different PCTs in the same year and also between 2003 and 2004 (by switching between the synchronised 'PCT Ratings 2003' and 'PCT Ratings 2004' themes). The performance of many PCTs has improved in 2004, whereas some PCTs achieved lower ratings in 2004 compared to 2003. Web-based interactive geographical interfaces offer an intuitive way of indexing, accessing, mining, and understanding large healthcare information sets describing geographically differentiated phenomena. By acting as an enhanced alternative or supplement to purely textual online interfaces, interactive Web maps can further empower organisations and decision makers. The Healthcare Commission started work on 1 April 2004, taking over all the functions of the former Commission for Health Improvement , which had released the corresponding PCT ratings for 2002/2003 in July 2003 [On Wednesday 21 July 2004, the Healthcare Commission A star rating scheme is adopted. PCTs with the highest levels of performance in the measured areas are awarded a rating of three stars. PCTs with mostly high levels of performance, but which are not consistent across all measured areas, are awarded a rating of two stars. PCTs where there is some cause for concern about particular areas of measured performance are awarded a rating of one star. PCTs that have shown the poorest levels of measured performance or little progress in implementing clinical governance receive a rating of zero stars . and . This "geographic search" does not allow any visual appreciation of PCT performance levels, or any visual comparisons to be made between PCTs or between 2003 and 2004 result sets.The performance ratings Web pages of the Healthcare Commission and the former Commission for Health Improvement offer a very limited "geographic search" restricted to browsing results by Strategic Health Authority (SHA) . The maps use a yellow-green-blue quadri-colour scheme to represent PCT star ratings. Users can switch between the two map sets or themes, 'PCT Ratings 2003' and 'PCT Ratings 2004', within the same pane  can be organised and navigated based on their geographical attributes [Using our Web-based interactive maps, users can quickly and intuitively locate any PCT and retrieve detailed performance information about it. They can also visually appreciate at a glance the distribution of PCT performance across England; for example, one can instantly note that there were no three star (dark blue) PCTs in the London region in 2002/2003 and that this has remained unchanged in 2003/2004. Users can visually compare the performance of different PCTs in the same year and also between 2003 and 2004 (by switching between 'PCT Ratings 2003' and 'PCT Ratings 2004' themes). The performance of many PCTs has improved in 2004, whereas some PCTs, e.g., Northumberland Care Trust – Figure Web-based interactive geographical interfaces offer an intuitive way of indexing, accessing, mining, and understanding large healthcare information sets describing geographically differentiated phenomena, and can act as an enhanced alternative or supplement to purely textual online interfaces. Geographical interfaces enable instant visual comparisons to be made between different geographical areas and over time (when information sets and maps for successive periods of time are available), thus empowering organisations and decision makers. and the Healthcare Commission  respectively. The Internet addresses (URLs) of the corresponding detailed reports of PCT performance were also harvested from the same sources.Star ratings of English PCTs for the years 2002/2003 and 2003/2004 were obtained from the Web sites of the former Commission for Health Improvement . We used the 2001 Census PCT (post April 2002 change) boundary dataset, which is the copyright of the Crown/Ordnance Survey , and is freely available to the UK academic community from EDINA UKBORDERS service with the support of the ESRC and JISC . The names/boundaries and labels (codes) of few PCTs changed between 2003 and 2004, but this was properly cared for in our exercise.The maps were created in ESRI ArcView GIS Version 3.1 We inserted four new fields in the original PCT boundary dataset table to store the 2003 and 2004 star ratings and corresponding detailed report URLs for all English PCTs. The PCTs in the output maps are coloured according to the values in their star rating fields , with light colours for low ratings to dark colours for high ratings. and [We used ColorBrewer  and ) to sele and Figure ).The online interactive maps were then produced using the Demo version of alta4 HTML ImageMapper 3.5 extension for ESRI ArcView GIS 3.x .It is noteworthy that HTML ImageMapper does not require any server side software installation, and as such is much simpler to use than some other Internet GIS solutions like the client/server version of ALOV Map/TimeMap  and JShape  Java applets, which don't require any server side setup, are limited by the fact that they need to download the whole map shapefile from the Web server before they can start on the client side, and so are not suitable for large datasets (our PCT boundary dataset in ESRI shapefile format is about 50 MB in size). Other options for generating interactive Web maps from a desktop GIS are discussed in [The standalone versions of ALOV Map/TimeMap