Pneumocystis pneumonia (PCP) is an increasing problem amongst patients on immunosuppression with autoimmune inflammatory disorders (AID). The disease presents acutely and its diagnosis requires bronchoalveolar lavage in most cases. Despite treatment with intravenous antibiotics, PCP carries a worse prognosis in AID patients than HIV positive patients. The overall incidence of PCP in patients with AID remains low, although patients with Wegener's granulomatosis are at particular risk.In adults with AID, the risk of PCP is related to treatment with systemic steroid, ill-defined individual variation in steroid sensitivity and CD4+ lymphocyte count. Rather than opting for PCP prophylaxis on the basis of disease or treatment with cyclophosphamide, we argue the case for carrying out CD4+ lymphocyte counts on selected patients as a means of identifying individuals who are most likely to benefit from PCP prophylaxis.Corticosteroids, lymphopenia and a low CD4+ count in particular, have been identified as risk factors for the development of PCP in adults with AID. Trimethoprim-sulfamethoxazole (co-trimoxazole) is an effective prophylactic agent, but indications for its use remain ill-defined. Further prospective trials are required to validate our proposed prevention strategy. Pneumocystis carinii was identified a hundred years ago by Chagas  and recoPneumocystis pneumonia is caused in humans by the recently renamed Pneumocystis jiroveci Frenkel 1999), previously known as Pneumocystis carinii and now thought to be related to fungi on the basis of DNA analysis, despite morphological similarities to protozoa . The org99, previPneumocystis pneumonia is almost always exclusive to immunocompromised hosts. Two thirds of cases occur in HIV positive patients and constitutes the initial manifestation of AIDS in 46% of these patients . A thirdP. jiroveci trophozoites proliferate and attach to type I alveolar pneumocytes causing desquamation, leading to a foamy eosinophilic exudate visible on hematoxylin-eosin staining and a "honey comb" appearance of lung tissue. Both antibody and cell mediated immunity have been postulated as being involved in host protection .HIV-negative patients with PCP are older (48–61 years) than HIV positive patients. Males are affected more often than females . The clinical course is typically more acute, with an average duration of 6–13 days. The most frequent clinical symptoms are dyspnoea (63–100%), cough (55–74%), weakness (47%), loss of appetite (38%) expectoration 22–25%), sweating (19%), weight loss (13%), haemoptysis (9%) and thoracic pain (9%). Physical findings include fever >38°C (63–85%), râles (55–66%), tachycardia (25%) and tachypnoea (22%). Chest radiography may show bilateral abnormalities (68–88%), interstitial opacities (64–80%) and alveolar opacities (31–47%), but may be normal (5–7%) -16,18,21–25%, sweP. jiroveci infection is diagnosed in most cases by bronchoalveolar lavage, which, depending on the staining method used, has been reported to have a sensitivity of 81 to 90% and a specificity of 90 to 100% . Other mTwo antibiotic regimes have been shown to work, co-trimoxazole and parenteral pentamidine isethionate. Efficacies are comparable, although side-effects are particularly common with co-trimoxazole in HIV associated PCP and affect up to 60% of such patients . These iThe prognosis of HIV negative PCP is worse than for those HIV positive, with intensive care admission in 31–60%; mechanical ventilation in 14–64%; overall mortality 19–47%, rising to 50–71% on intensive care, although some series suggest an improved mortality in recent years ,27. PoorWhile PCP in HIV positive patients has been associated with a high incidence of relapse after successful therapy, this has not been seen in patients with AID, even without secondary prophylaxis and despite ongoing immunosuppressive treatment. This is thought to be related to better clearance of organism, confirmed by repeat bronchoalveolar lavage .There is no specific surveillance system in the United Kingdom (UK) for PCP, other than in HIV infection. Although laboratories are invited to report isolates of P. jiroveci to the Communicable Disease Surveillance Centre, it is estimated that only a fifth of clinically diagnosed cases of PCP are reported . As the The overall incidence of PCP from 1990 to 1999 has declined in the UK , as a reAttempts have been made to characterise the incidence of PCP amongst patients with AID, usually by retrospective analysis of case records. Ward & Donald's review of 223 cases of PCP in AID is the largest series and covered 2.6 million hospitalisations over the period 1983 to 1994 . They foPneumocystis pneumonia in AID is unusual in the absence of steroid treatment. Corticosteroids have been recently administered in over 90% of cases in most series ,17,18,22A number of cytotoxic and other immunosuppressive agents commonly used in the treatment of AID are frequently associated with PCP, including cyclophosphamide, azathioprine, methotrexate and ciclosporin . Cycloph3) is almost a prerequisite, with 91% of patients exhibiting a low lymphocyte count. Fifty percent of such PCP patients have total lymphocyte counts of <400 cells/mm3 [3 was recorded in ten (83%) of 12 patients with PCP, but such a low lymphocyte count was recorded in 11 (34%) of 32 with Wegener's unaffected by PCP [3 which captured 4 out of 6 cases with PCP and SLE, but only 1 of 20 patients with SLE unaffected by PCP.Data on PCP associated with AID indicates lymphocytopenia  and have a particularly poor prognosis, as discussed earlier.Any method used to select patients for prophylactic treatment needs to be assessed against set standards and have a high sensitivity and specificity. The standards should address the percentage of PCP cases captured by the selection criteria  and the risk of the condition in the selected group (which should be significant). In HIV patients who meet the criteria for PCP prophylaxis as set out by the US Public Health Service , the ann3 would capture 91% of cases of PCP in all HIV negative patients, but would also include 39–46% of patients on systemic steroids, most of whom would be unaffected by PCP. Administering prophylaxis to such large numbers of patients would unnecessarily expose patients to drug side-effects and potentially encourage drug resistance. However, analysis of their data reveals that the subgroup of patients with AID who developed PCP had CD4+ counts of <250 cells/mm3 and six out of eight had counts <200 cells/mm3 [In the study by Mansharamani et al, their proposed cut off of <300 CD4+ cells/mmells/mm3 .Given the laboratory costs, we would argue in favour of performing CD4+ counts after one month's immunosuppression only on patients who satisfy the following three screening criteria:• Steroid dosage >15 mg prednisolone or equivalent/day• >three months corticosteroid treatment proposed3• total lymphocyte count <600 cells/mm3 might then warrant the use of prophylactic co-trimoxazole, if the annual risk of PCP in these patients is greater than 9%. Most cases of PCP in patients with AID would be captured by these criteria, according to published series.A CD4+ count <200 cells/mm3, information which is currently unavailable.Clearly, further prospective investigation is required to gather sufficient data to validate any selection method. To justify our proposed threshold for prophylaxis we would need to know the risk of PCP for patients on steroid-based immunosuppression for AID with CD4+ counts of <200 cells/mmP. jiroveci infection in HIV negative patients presents more acutely and has a worse prognosis. The incidence of PCP in patients with AID is low, although these patients still represent a considerable proportion of all HIV negative cases. It is important to have a high index of suspicion of PCP when treating AID with steroid based immunosuppressive regimes, as early treatment could improve prognosis. The risk of infection is related to treatment with systemic steroid, ill-defined individual variation in steroid sensitivity and CD4+ lymphocyte count. Effective and relatively safe prophylaxis is available. Rather than opting for PCP prophylaxis on the basis of disease or treatment with cyclophosphamide, we argue the case for carrying out CD4+ lymphocyte counts on selected patients as a means of identifying individuals who are most likely to benefit from PCP prophylaxis. Further prospective trials are required to validate our proposed prevention strategy.The authors declare that they have no competing interests.AJ Carmichael initiated the discussion, appraised results, lead departmental debates and helped revise the manuscript. ES carried out the literature search, presented at meetings and wrote the original manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Molecular oxygen may be necessary for life but with its beneficial properties comes formation of potentially toxic reactive oxygen species. One of the ways in which bacteria protect themselves is explained Nevertheless, with the beneficial properties of O2 comes the inadvertent formation of reactive oxygen species, including superoxide (O−2), hydrogen peroxide (H2O2), and hydroxyl radical (•OH); these differ from O2 in having one, two, and three additional electrons, respectively  groups of cysteinyl amino acid residues in proteins. Elevated levels of H2O2 also are associated with the oxidation of other amino acids, leading to the formation of methionine sulfoxide and a variety of carbonyls. Lastly, because of its extreme reactivity, •OH targets all of the major macromolecules of cells: RNA, DNA, protein, and lipids. The extent to which membrane lipids are targets appears to depend on the presence of polyunsaturated fatty acids in lipids, which are not as prevalent in bacteria as they are in mammals.The major target of Oubstrate . Since sth media . H2O2 foE. coli  in the presence of NADPH and glutathione reductase. There are two thioredoxins in E. coli that also function to reduce disulfide bonds. Reduced thioredoxin is regenerated by thioredoxin reductase and NADPH. The fact that NADPH is required to maintain the reduced state of glutathione and thioredoxin indicates that the response to oxidative stress is coupled to the physiological status of core pathways that generate NADPH.Another component to the oxidative stress response is the reduction of oxidized thiols that arises through one of the mechanisms described below. The tripeptide glutathione and the thiol reductants glutaredoxin and thioredoxin are key to the restoration of thiols to their reduced state (SH) . E. coli2O2. The reaction of cysteinyl thiolates with H2O2 can lead to the formation of different modifications, such as sulfenic acid (–SOH), sulfinic acid (–SO2H), and sulfonic acid (–SO3H), as well as disulfide bond formation (–S–S–) and glutathione conjugation (–S–GSH)     .Given the reversible nature of most forms of thiol oxidation, it has been suggested that thiol modifications can play roles in signal transduction that are similar to protein phosphorylation/dephosphorylation . In suppE. coli and a variety of other bacteria. OxyR contains two critical cysteines that are oxidized to form an intramolecular disulfide bond when cells encounter peroxide stress , and zinc is released upon oxidation of the thiols. For both proteins, oxidation and zinc release are associated with an opening of the protein structure. For Hsp33, this structural change allows for dimerization and activates its chaperone activity  formed by the oxidation of a variety of organic molecules in the cell or in the environment. The B. subtilis OhrR transcription regulator contains only a single cysteine that forms a relatively stable sulfenic acid upon its reaction with H2O2 or organic peroxides (The peroxide-sensing repressor OhrR from phaseoli  and Bacisubtilus  can be ieroxides . OxidatiIn this issue, 2O2. We do not currently understand all of the features that determine the reactivity of a particular thiol to H2O2 (As illustrated by the examples above, an array of chemical modifications obtained by oxidizing cysteinyl residues has been exploited in combating oxidative stress. Yet it is important to note that not all cysteinyl residues of proteins are readily oxidized by oxidants such as H to H2O2 . The pKaE. coli has also been shown to contain a H2O2-labile zinc site, although the loss of zinc does not change its reductase activity (The added complexity of the cysteine targets that compose part of a Zn binding site found for Hsp33 and RsrA raises questions about the function of the zinc. Perhaps Zn binding provides some additional control over the reactivity of the cysteine thiols, or perhaps the loss of the zinc facilitates conformational changes. Recently, the oxidative, stress-induced thioredoxin-2 from activity . Thus, t2O2 and the experimental challenges associated with their detection has made it difficult to catalog all the proteins that are modified and all the types of modifications that exist. In this issue, The extent of thiol oxidation within the cell remains another open question. The variety of modifications that arise from treatment with HSaccharomyces cerevisiae requires a peroxidase denoted Gpx3 or Orp1 (2O2 reacts with a cysteine in Orp1, forming an unstable sulfenic acid intermediate that then reacts with a cysteinyl residue of Yap1 to form an intermolecular disulfide. The disulfide undergoes an exchange with a second cysteine within Yap1 to form an intramolecular disulfide that locks Yap1 in a confirmation that masks the nuclear export signal (The importance of monitoring transient changes in cysteines is highlighted by the recent finding that oxidation of the Yap1 activator of antioxidant genes in the yeast  or Orp1 . In thist signal . Thus, mThe examples mentioned here illustrate the versatile potential of thiol modifications. Given the reversibility of thiol oxidations and the wide range of structural constraints that can be imposed by the formation of a sulfenic or sulfinic acid or a disulfide bond, we predict there will be many more examples of regulation by thiol modification.


Helicobacter pylori causes chronic gastritis, peptic ulcer disease, gastric carcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. It infects over 50% of the worlds' population, however, only a small subset of infected people experience H. pylori-associated illnesses. Associations with disease-specific factors remain enigmatic years after the genome sequences were deciphered. Infection with strains of Helicobacter pylori that carry the cytotoxin-associated antigen A (cagA) gene is associated with gastric carcinoma. Recent studies revealed mechanisms through which the cagA protein triggers oncopathogenic activities. Other candidate genes such as some members of the so-called plasticity region cluster are also implicated to be associated with carcinoma of stomach. Study of the evolution of polymorphisms and sequence variation in H. pylori populations on a global basis has provided a window into the history of human population migration and co-evolution of this pathogen with its host. Possible symbiotic relationships were debated since the discovery of this pathogen. The debate has been further intensified as some studies have posed the possibility that H. pylori infection may be beneficial in some humans. This assumption is based on increased incidence of gastro-oesophageal reflux disease (GERD), Barrett's oesophagus and adenocarcinoma of the oesophagus following H. pylori eradication in some countries. The contribution of comparative genomics to our understanding of the genome organisation and diversity of H. pylori and its pathophysiological importance to human healthcare is exemplified in this review.The human gastric pathogen  Helicobacter pylori is a bacterium that colonizes the harshly acidic milieu of the human stomach. More than half of the world's population carries this infection. Infection rates vary among the developed and developing countries of the world. H. pylori infection is on a steep decline in most of the western countries mainly due to the success of combination therapies and improved personal hygiene and community sanitation to prevent re-infection. However, the situation is not improving in many of the developing countries due to failure of treatment regimes and emergence of drug resistance. The infection in some cases leads to chronic superficial gastritis, chronic active gastritis, peptic ulcer disease and gastric adenocarcinoma  were characterized and compared [H. pylori is a freely recombining or panmictic organism [H. pylori genomes [H. pylori chromosome. Recombinational events including the presence of insertion elements, pathogenicity islands, horizontally acquired genes (restriction recombination genes), mosaics and chromosomal rearrangements were frequently annotated in subsequent bioinformatics based attempts. It has been argued that such diversity is a result of a lack of direct competition between strains, even when resident within different individuals within the same community [H. pylori genome [H. pylori has extensive, non-randomly distributed repetitive chromosomal sequences, and that recombination between identical repeats contributes to the variation within individual hosts [H. pylori is representative of prokaryotes, especially those with smaller (<2 megabases) genomes, that have similarly extensive direct repeats, suggests that recombination between such direct DNA repeats is a widely conserved mechanism to promote genome diversification [H. pylori has been termed as a panmictic organism [H. pylori populations exist [H. pylori in some populations shows a clonal descent and suggest that a large array of H. pylori clonal lineages co-exist, which evolve in isolation from on another [H. pylori isolates have been shown to exhibit little genetic heterogeneity, based on fingerprint profiles [H. pylori isolates undergo 'microevolution' and give rise to sub-species during prolonged colonisation of a single host [H. pylori population to specific host niches, facilitated by unknown advantages conferred to them by select plasticity region genes. Bjorkholm et al. [cag pathogenicity island (cag PAI) in one isolate and not the other. As the cagA gene and cag PAI are principle virulence factors within the strains, the excision or abrogation of the cag PAI within a strain may indicate that attenuating the virulence of a strain could be a favourable adaptation.So how do a few criptome ,24. Whercriptome ,26. Bothcompared ,28. As Horganism  the quesorganism ,28. Straorganism ,30, in w genomes  and reclommunity . Howeveri genome . It has al hosts . That H.fication . In addiorganism ,35, it ins exist -39. Rece another . Moreoveprofiles ,40. Funcprofiles , the traprofiles , the ideprofiles ,30 and tprofiles . One of gle host -46. The m et al.  demonstrH. pylori is a model for studying genome plasticity in the colonization of individual hosts. For H. pylori, neither point mutation, nor intergenic recombination requiring the presence of multiple colonizing strains, is sufficient to fully explain the observed diversity.Within the bacterial populations, genome content may not be fixed, as changing selective forces favour particular phenotypes; however, organisms well adapted to particular niches may have evolved mechanisms to facilitate such plasticity. The highly diverse H. pylori genomes sequenced to date are each from ethnic Europeans, and genomic comparisons modelled on these data are sufficient to identify novel loci from new strains, especially from understudied Asian populations. However, these genome sequences may not be fully representative of the entire diversity of the gene pool. Identification and characterization of such loci which are more abundant in the Asian gene pool may lead to newer insights into the mechanisms of H. pylori colonization, carriage and virulence in the countries of Asia which are more seriously under threat from H. pylori. Therefore, future high throughput efforts involving a large number of strains are clearly needed. Taking the Indian example for instance, according to the Ethnologue database , there are about 1683 languages and dialects ('mother tongues') in this country and H. pylori diversity therefore can be assumed to coincide with this figure. So one has to roughly look at the inter-strain genomic diversity contributed by approximately 1683 different strains representing each dialect and or a community.The two Nonetheless, genotypic data from each geographic area or a community is extremely vital and might constitute a missing piece of a large, biologic jigsaw puzzle.H. pylori harbours a dedicated type IV apparatus, the comB gene cluster [comB gene cluster is essential for the bacterium to take up plasmid and chromosomal DNA during natural transformation. To identify the genes essential for natural transformation competence in H. pylori, genetic approach of transposon shuttle mutagenesis has been used and the comB locus was located, consisting of orf2 and comB1-comB3 [Agrobacterium tumifaciens type IV secretory apparatus) as comB7, comB8, comB9 and comB10 respectively . One of the principal virulence factors of H. pylori, the cagA antigen is contained in the 40 kb cag-PAI. The tyrosine-phosphorylated cagA protein is translocated to the epithelial cells by the type IV secretion system (forming a sort of syringe like structure) [cag-PAI interfere with tyrosine phosphorylation and induction of interleukin-8 secretion [cag-PAI are essential for cagA translocation and/or interleukin 8 induction, a complete mutagenesis of the cag-PAI was performed [H. pylori [Molecular analysis of bacterial transport has been attempted in several bacterial pathogens. Among such transport systems, Type IV secretion systems have been described in greater detail in diverse bacteria. In ructure) -53. Uponecretion . In receerformed . In gene. pylori . All the. pylori . However. pylori .H. pylori 26695 has revealed a sheathed, surface organelle, coded by the cag-PAI genes, HP0527 (forms sheath around the pilus needle) and HP0532/ cagT (forms the base of the pilus) [H. pylori to the host niches and this might mediate biological as well as transport functions of the cag-PAI encoded proteins. Computational analyses to predict the macromolecular assemblies of such apparatuses are needed to have a more simplified understanding of the entire model of the H. pylori type IV secretion mechanismVery recently, and for the first time, ultrastructure analysis of the surface of e pilus) . This stH. pylori-infected population than in the H. pylori-uninfected population [cagA gene of H. pylori is assumed as partially responsible for eliciting signaling mechanisms that lead to the development of gastric adenocarcinoma. Based on the carriage of a functional cagA as a marker for the cag PAI, the H. pylori species is divided into cagA-positive and cagA-negative strains. The cagA-positive strains are associated with higher grades of gastric or duodenal ulceration and are more virulent than the cagA-negative strains [cagA positive H. pylori in the development of atrophic gastritis, peptic-ulcer disease and gastric carcinoma [cagA gene product, cagA, is translocated to the gastric epithelial cells to undergo tyrosine phosphorylation by SRC family kinases [cagA-positive H. pylori promotes gastric carcinogenesis. Cag A is noted for its variation at the SHP2 binding site and, based on the sequence variation, it is sub- classified into two main types – East-Asian cagA and Western cagA. East-Asian cagA shows stronger SHP2 binding and greater biological activity than Western cagA. In East-Asian countries, endemic circulation of H. pylori strains that carry biologically active forms of cagA might underlie the high incidence of gastric carcinoma. One puzzling attribute of H. pylori infection is why some populations with high incidences of H. pylori infection, such as those in Japan and Korea, have high incidences of gastric carcinoma, whereas other highly infected populations, such as populations in central Africa, do not. Possible reasons could be the differences in genetic susceptibility among populations, environmental factors such as dietary habits, and strain differences of H. pylori. Among these, diversity of cagA in H. pylori strains might be involved in determination of the type and severity of disease. As discussed above, East-Asian and Western forms of cagA possess the distinctly structured tyrosine phosphorylation/ SHP2-binding sites – EPIYA-D and EPIYA-C, respectively [cagA-positive strain is associated with the mortality rate of gastric cancer in Asia. Therefore, populations infected with East-Asian cagA positive H. pylori are at greater risk for gastric cancer than those infected with Western cagA-positive strains.A large-scale prospective study revealed that the risk for development of gastric carcinoma was much greater in the pulation . The cag strains . Some eparcinoma ,62. The  kinases . Tyrosinectively . Notablyectively . FurtherH. pylori [Among Western CagA species, the number of EPIYA-C sites directly correlates with levels of tyrosine phosphorylation, SHP2-binding activity and morphological transformation . Further. pylori .H. pylori has a single copy of the vacA gene. Screening of H. pylori chromosomal fragments permitted the identification of a 3864-base pair open reading frame (vacA) that encoded the vacuolating cytotoxin [vacA gene includes a 33 amino acid signal sequence. With the exception of a hydrophobic region at the N terminus, the mature 90-kDa protein (amino acids 34 to 842) is mainly hydrophilic [H. pylori strains. The different combinations of s and m regions determine the production of cytotoxic activity. Strains with the genotype s1 m1 produce high levels of vacuolating cytotoxin in vitro. Strains with the genotype s2 produce an inactive toxin. Whereas, strains with the genotype m2 produce toxic activity with a different target cell specificity from those of m1 genotype. Genotypic variations in the vacA gene structure specific to a geographic locale have been recognised. While the vacA m1a allele is specific for the European strains [ytotoxin . The seqrophilic . The cyt strains , the vac strains . Yet ano strains . Among ovir B operon of A. tumifaciens carried in a 16.3 kb genomic fragment called tfs3  of the mucosa-associated lymphoid tissue (MALT)-type and strains from age matched patients with gastritis only [v 49%) and JHP1462 (26% v 3%). JHP950 proved specific for gastric MZBL when tested against a group of strains from patients with duodenal ulcer and patients with adenocarcinoma, with significant prevalence , and is therefore the candidate marker for gastric MZBL. Interestingly, the candidate ORF JHP950 is located in the plasticity region of the J99 genome [The plasticity region as a whole displays certain characteristics of pathogenicity islands  with relormation . Interesormation . Many geormation . Researctis only . Two ORF9 genome ,76. In vH. pylori is still controversial. However, it is accepted that this organism has colonized humans possibly for many thousands of years, and the successful persistence of H. pylori in human stomach for such a long period may be because this organism offers some advantages to the host. Unfortunately, the H. pylori infection is on steep decline in the western world. This is mainly due to the success rate of combination therapies and subsequent prevention of re-infection due to improvement in sanitation and personal hygiene. This may seem good news to many gastro-enterologists around the world, but having a H. pylori infection may be advantageous. A study has shown that H. pylori produces a cecropin-like peptide  with high antimicrobial properties [H. pylori were less likely to have diarrhoea than children without an infection [H. pylori may have beneficial properties to human hosts. Interestingly, there has been a marked decline in the instances of peptic ulcer disease and gastric cancer in the 20th century. Concurrent with this is a dramatic increase in the incidences of gastro-oesophageal reflux disease (GERD), Barrett's oesophagus and adenocarcinoma of the oesophagus in Western countries [H. pylori may in some way be associated with these diseases and perhaps capable of preventing their onset. Studies have also shown that cagA+ H. pylori strains have a more protective effect than cagA- strains [cagA+ H. pylori strains can reduce the acidity of the stomach, and it is believed that the raising of the pH by H. pylori prevents GERD, Barrett's oesophagus and adenocarcinoma of the oesophagus . Conversely, arguments have been made that, although H. pylori may prevent these reflux-associated diseases, the risks of acquiring gastric cancer via H. pylori infection far outweigh any possible benefits it may provide [H. pylori does provide protection from GERD, the notion of restriction of anti-H. pylori treatment to only a few cases (peptic ulcer disease and MALT lymphoma) could be justified [H. pylori in erosive reflux oesophagitis [How long humans carried operties . A Germanfection , implyinountries . This ob strains . The pre provide . Howeverustified . In spitphagitis -85. Howephagitis  it seemsH. pylori in non-users of non-steroidal anti-inflammatory drug (NSAID). In addition, the incidence of H. pylori-negative, non-NSAID peptic ulcer disease (PUD) (idiopathic PUD) is reported to increase with time. Moreover, it appears that H. pylori-positive ulcers are not always H. pylori-induced ulcers because there are two paradoxes of the H. pylori myth, first the existence of H. pylori-positive non-recurring ulcer and secondly, recurring ulcer after cure of H. pylori infection. To summarise, H. pylori is not the only cause of peptic ulcer disease. Therefore, it is still necessary to seriously consider the need for eradication in all cases of PUD, which may exist even after the elimination of H. pylori.On the other hand, eradication also is not an ultimate choice. Some ulcers recur even after successful eradication of H. pylori in health and disease may be fully ascertained only if we analyze genetic diversity of the pathogen as juxtaposed to the host diversity and the environment (food and dietary habits). A possible working hypothesis (that we are currently nurturing) may be that among the ocean of molecular host-pathogen interactions that could potentially occur with micro-evolution of this bacterium during long term colonization, some could prove advantageous where the bacterium and the host negotiate nearly a 'symbiotic' and balanced relationship. Such a 'friendship' might have taken thousands of years to develop. If so, why has this bacterium survived for such a long time? Microbes that have long been persisted in humans may be less harmful than recently emerged microbes, such as the human immunodeficiency viruses (HIV). This suggests that the colonization may either be beneficial or of low biological cost to the host. In addition to characterization of bacterial virulence apparatuses that are for sure linked to disease outcome, host responses to such factors must also be examined hand in hand, to completely ascertain mechanisms that lead to gastroduodenal disease. For instance polymorphisms linked to the host immune apparatus, such as IL-1β, TNF-α, and IL-10, which are responsible for elevated proinflammatory potential of the strains. These polymorphisms increase the risk for atrophic gastritis and distal gastric adenocarcinoma among H. pylori-infected persons. Cancer of stomach is a highly lethal disease and establishment of H. pylori as a risk factor for this malignancy deserves an approach to identify persons at increased risk; however, infection with this organism is extremely common and most colonized persons never develop cancer. Thus, screens to identify high-risk subpopulations must use high-resolution biological markers. Fortunately, this task appears to be highly simplified due to the availability of biological tools, which were never thought in the past. Genome sequences , quantitative phenotypes , and practical animal models (Mongolian gerbils) can be harnessed to decipher the molecular basis of H. pylori-associated malignancies, which should have direct clinical applications. It is important to gain more insight into the pathogenesis of H. pylori-induced gastric adenocarcinoma, not only to develop more effective diagnostics and treatment for this common cancer, but also to validate the role of chronic inflammation in the genesis of other tumours of the alimentary tract.In our opinion, the intricacies of the role of


Saccharomyces cerevisiae . In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae.The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W)-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides.Our data clearly shows that integrity of N-terminal α-helix I (17–33) and α-helix III (53–83) is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in Vpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes α-helix I (17–33) and α-helix III (53–83). Furthermore, the mechanism(s) underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells. Increas protein -5. Secon protein .Schizosaccharomyces pombe (Sc. Pombe) [Saccharomyces (S.) cerevisiae [S. Pombe cells [S. cerevisiae has been proposed to result from gross mitochondrial dysfunction [Even though the molecular mechanism of Vpr-mediated cell-cycle G2 arrest is still obscure, it has been known that Vpr expression leads to inactivation of the mitotic p34cdc2/cyclinB complex in human cells ,9 as wel. Pombe) -14. Invo. Pombe) -12,14 burevisiae -17. Delerevisiae ,18. Althbe cells -25, the be cells . Accordibe cells ,27. Indefunction , rather et al. have provided evidence indicating that extracellular Vpr or peptides derived from Vpr C-terminus induce mitochondrial dysfunction in human cells by a mechanism involving a specific binding to the adenine nucleotide translocator (ANT), a component of the permeability transition pore complex (PTPC) in the mitochondrial membrane. The resulting mitochondrial membrane permeabilization (MMP) leads to a decreased membrane potential and the release of cytochrome c and apoptosis inducing factor (AIF) [In addition to nuclear import and cytostatic activities, HIV-1 Vpr exhibits cytotoxic properties. Elevated intracellular expression or addition of extracellular Vpr or derived peptides results in proapoptotic effects in human cells including neurons ,28,29 asor (AIF) ,33. Thisor (AIF) -37. In aS. cerevisiae presumably by interacting with Vpr [S cerevisiae. Results reveal that the inhibitory di-W-containing peptides target specifically a functional domain of Vpr directly involved in growth arrest in budding yeast. Furthermore, several previously well-characterized Vpr mutants unable to induce cell-cycle dysregulation and/or apoptosis in mammalian cells still exhibit strong growth arrest activity in budding yeast, indeed suggesting that Vpr carries out distinct functions in S. cerevisiae.In a previous report, we have shown that expression of genetically-selected glutathione-S-transferase (GST)-fused di-tryptophan (di-W)-containing peptides inhibited Vpr-mediated growth arrest in with Vpr . Intereswith Vpr . Even thS. cerevisiae budding yeast and selected a panel of di-W-containing GST-peptides that specifically inhibit Vpr-mediated yeast growth arrest function presumably through their ability to bind HIV-1 Vpr [We have previously used a genetic selection system in IV-1 Vpr . In thisStructural studies performed with synthetic forms of Vpr indicate that Vpr is characterized by a well-defined gamma turn 14–16)-alpha helix (α-helix I: 17–33)-turn (34–36), followed by an alpha helix(α-helix II: 40–48)-loop (49–54)-alpha helix (α-helix III: 55–83) domain and ends with a very flexible C-terminal arginine-rich sequence . The α-h-alpha heTo determine the impact of the Vpr mutations on GST-B4 peptide binding, HP-16 yeast co-transformed with mutated-Vpr expressors and either GST or GST-B4 vectors were radio-labeled in Vpr-inducible medium and subjected to GST pull-down assays Fig. , as desc-, 2% raf) or a Vpr-inducible plate . Cell growth was evaluated following an incubation of 3–5 days at 30°C , wild-type (R+) or mutated Vpr expressors were serially diluted and spotted onto either a Vpr non-inducible plate  underlying Vpr-induced cytostatic effect in budding yeast are distinct from those implicated in cell-cycle alteration and apoptosis in mammalian cells.We have previously shown that GST-fused di-W-containing peptides were able to interact with HIV-1 Vpr and as a result inhibit its multiple functions in budding yeast as well as in HIV-1 infected T cells . In the S. cerevisiae budding yeast was attributed to its last 63–96 amino acid (aa) and the critical domain was located in a conserved C-terminal HFRIGCRHSRIG sequence from aa 71 to 82 [et al., revealed that substitution mutations of aa His71 or Gly75 in this HFRIG sequence abolished Vpr cytostatic effect in budding yeast [et al showing that the Vpr F34I mutant was unable to induce a growth arrest phenotype in budding yeast [et al [Previous reports have indicated that the Vpr cytostatic activity in 71 to 82 . In contng yeast . Interesng yeast . On the t [et al , mutatiot [et al , and mayin vivo. Such a difference may specifically rely on the hydrophobic di-W motif, which is not present in UDG [One striking observation of this study is that the four mutants  located in the α-helical I and III regions of Vpr, which were defective for the cytostatic activity in budding yeast  [The rc1-407) . Plasmidrc1-407) .- have been previously described [BamHI site in front of the Vpr initiation codon, a 3'-primer (5'-GCATCGCTCGAGGATCTACTGGC-3') containing a XhoI site after the stop codon of Vpr and the complementary oligonucleotide primers containing the desired mutations. Amplified Vpr cDNA harboring specific mutations were then cloned into the p424Gal1 vector at BamHI/XhoI sites. The Vpr mutants L23F, E25K, A30F, R62P, I63K, R77Q, R77fs and R80A were previously described [S. cerevisiae HP-16 yeast colony that was resistant to HIV-1 Vpr-mediated growth arrest as previously described [The HIV-1 Vpr yeast expression plasmid  and the negative control plasmid p424Gal1-Rescribed . To geneescribed  by usingescribed ,48. The escribed  while thescribed .The rabbit anti-Vpr polyclonal serum was raised against bacterially expressed recombinant Vpr as described previously . Galacto- or Trp-/Ura-, 2% raffinose (raf+)) for 2 days. Then, suspensions of transformed HP-16 yeast cells (adjusted at similar cell densities) were serially diluted and spotted onto either a Vpr non-inducible plate  or a Vpr-inducible plate  to evaluate the growth of each co-transformed HP16 population.The experimental procedures to evaluate protein expression, Vpr growth arrest activity and the anti-Vpr activity of GST-fused di-W peptide were described previously . Briefly35S-Translabel (ICN Inc.) in Vpr-inducible medium and lysed in CHAPS buffer as previously described [HP16 co-transformants were radiolabeled with 150 μCi of escribed . Cell exescribed ,38. Brieescribed .X-J Y designed the experiments, constructed most Vpr mutants and wrote the manuscript. NR carried out the binding assays and tested the effect of Vpr mutants on yeast cells growth. JL selected and characterized the B4 GST-di-W-containing peptide. GD participated in the design of the study and critically evaluated the manuscript. EAC participated in the design of the study and coordinated it. All authors read and approved the final manuscript.


The guanine nucleotide exchange factor C3G (RapGEF1) along with its effector proteins participates in signaling pathways that regulate eukaryotic cell proliferation, adhesion, apoptosis and embryonic development. It activates Rap1, Rap2 and R-Ras members of the Ras family of GTPases. C3G is activated upon phosphorylation at tyrosine 504 and therefore, determining the localization of phosphorylated C3G would provide an insight into its site of action in the cellular context.C3G is phosphorylated in vivo on Y504 upon coexpression with Src or Hck, two members of the Src family tyrosine kinases. Here we have determined the subcellular localization of this protein using antibodies specific to C3G and Tyr 504 phosphorylated C3G (pY504 C3G). While exogenously expressed C3G was present mostly in the cytosol, pY504 C3G formed upon Hck or Src coexpression localized predominantly at the cell membrane and the Golgi complex. Tyrosine 504-phosphorylated C3G showed colocalization with Hck and Src. Treatment of Hck and C3G transfected cells with pervanadate showed an increase in the cytosolic staining of pY504 C3G suggesting that tyrosine phosphatases may be involved in dephosphorylating cytosolic phospho-C3G. Expression of Src family kinases or treatment of cells with pervanadate resulted in an increase in endogenous pY504 C3G, which was localized predominantly at the Golgi and the cell periphery. Endogenous pY504 C3G at the cell periphery colocalized with F-actin suggesting its presence at the subcortical actin cytoskeleton. Disruption of actin cytoskeleton by cytochalasin D abolished phospho-C3G staining at the periphery of the cell without affecting its Golgi localization.These findings show that tyrosine kinases involved in phosphorylation of C3G are responsible for regulation of its localization in a cellular context. We have demonstrated the localization of endogenous C3G modified by tyrosine phosphorylation to defined subcellular domains where it may be responsible for restricted activation of signaling pathways. Guanine nucleotide exchange factors (GNEFs) are components of signaling pathways that link transmembrane receptors to intracellular GTPase family members regulating a wide variety of cellular functions such as proliferation, differentiation, adhesion and apoptosis. C3G RapGEF1) is an ubiquitously expressed GNEF for Ras family proteins that particularly targets Rap1, Rap2 and R-Ras  is an ub. It has The C-terminus of C3G, which shows homology to CDC25, harbors the catalytic domain. The central region of C3G, which spans about 300 residues, has polyproline tracts with the ability to bind to SH3 domains of various proteins like Crk, p130 Cas, Grb2 and Hck ,18,27. NThe effectiveness and precision of intracellular signal transduction depends on protein-protein interactions that regulate enzyme activity as well as subcellular localization. Cell surface receptor activation leads to assembly of adaptor protein complexes at the plasma membrane, which serve to localize guanine nucleotide exchange proteins. Earlier, both endogenous as well as exogenously expressed C3G has been shown to localize to the cytoplasm and not to associate with plasma membrane ,30. SincWe have recently shown that Hck interacts with and phosphorylates C3G in vivo and we wished to determine if their interaction leads to changes in the subcellular distribution of C3G and whether pY504 C3G locates to specific subcellular domains. Cos-1 cells were transfected with C3G in the presence or absence of Hck and immunostained using anti C3G antibodies. As shown in Fig. To determine the subcellular distribution of phospho-C3G, which is known to be the activated form, specificity of a rabbit polyclonal phosphorylation site-specific antibody (pY504-C3G) was verified by examining its reactivity using cell lysates expressing C3G or Y504F-C3G alone or with Hck. As shown in Fig. We wished to determine whether C3G that colocalizes with Hck was the phosphorylated component and therefore used pY504-C3G antibodies to determine the localization of pY504 C3G in cells expressing Hck and C3G. As shown in Fig. In the above experiments phosphorylation and localization of C3G was studied using exogenously expressed protein and we wished to determine whether endogenous C3G could be phosphorylated and similarly targeted. Towards this end we checked the phosphorylation of endogenous C3G under conditions of Src and Hck overexpression or upon activation of cellular tyrosine kinases by pervanadate treatment. C3G protein is expressed as a doublet of about 140–150 kDa, which are products of two differentially spliced mRNAs . Whole cThe localization of endogenous pY504 C3G to the Golgi was examined in Cos-1 cells transfected with Hck and VSVG-GFP. Immunostaining for pY504 C3G was seen predominantly at the cell periphery and the Golgi Fig. , which wThe localization of pY504 C3G formed by the activation of endogenous tyrosine kinases was determined in cells treated with pervanadate, which is known to activate Src family kinases, in addition to inhibiting tyrosine phosphatases -37. PervIndirect immunoflourescence was performed to determine the localization of endogenous pY504 C3G formed by the activation of intracellular tyrosine kinases. Cos-1 and HeLa cells were stimulated by pervanadate and as seen in Fig. We have observed that pervanadate treatment increased tyrosine phosphorylation of endogenous C3G. Since overexpression of Src as well as Hck results in phosphorylation of C3G, it was of interest to determine whether pervanadate induced tyrosine phosphorylation of C3G was mediated by Src family kinases within the cell. Cells were treated with PP2, a specific SFK inhibitor prior to PV treatment . As showC3G is involved in a variety of signaling pathways and therefore its dynamic localization under normal and activated situations may be physiologically relevant. In this study we demonstrate the limited subcellular distribution of Y504 phosphorylated C3G, which is predominantly targeted to the Golgi apparatus and the subcortical actin cytoskeleton. This localization has been substantiated by colocalization with a Golgi marker protein and F-actin respectively.Rap1, the substrate of C3G has been localized to the Golgi, lysosomal vesicles and cortical actin cytoskeleton . But, ofWe observed more exogenously expressed pY504 C3G in the cytoplasmic compartment under conditions of inhibition of tyrosine phosphatases suggesting that pY504 C3G may be targeted by cytosolic tyrosine phosphatases. This regulation may help in restricting the activity of C3G to specific compartments. We observe very little endogenous pY504 C3G in the cytosol when HeLa or Cos-1 cells are treated with pervanadate, which not only inactivates tyrosine phosphatases, but also activates tyrosine kinases. It is possible that upon PV treatment endogenous C3G present in the cells is phosphorylated at the sites of location of the activated kinases. Pervanadate treatment has been shown to increase phosphotyrosine staining at the cell periphery indicating activation of kinases present in this subcellular domain .Recently c-Src and Jak2 have been implicated in the phosphorylation of C3G in response to growth hormone stimulation of NIH 3T3 cells because dominant negative mutants of these kinases inhibit C3G phosphorylation . It was Adhesion dependent Src activation leads to Rap-1 activation mediated by Crk and C3G . FibroblThe activity of guanine nucleotide exchange factor C3G is known to be regulated by tyrosine phosphorylation and membrane targeting. Using phospho-specific antibodies, we directly demonstrate that expression of Src family kinases or pervanadate treatment of cells induces phosphorylation of C3G on Y504. Unlike C3G, which is mostly cytosolic, pY504C3G locates to the Golgi and subcortical actin cytoskeleton. Demonstration of the localization of the active component of C3G to the Golgi and subcortical cytoskeleton provides evidence for a possible function for C3G at these cellular compartments.HeLa and Cos-1 cells were cultured in DMEM supplemented with 10% FCS. Transfections were performed on cells grown as a monolayer in either 35 mm dishes or glass coverslips using the cationic lipid DHDEAB as described . Briefly2O2, with 100 mM solution of sodium orthovanadate. It was added to the cells within 5 mins of preparation. Golgi disruption was performed by treating the cells with 5 μg/ml of nocadazole for 30 min prior to fixation. To disrupt actin cytoskeleton, cells were treated with 1 μg/ml cytochalasin D for 20 mins. PP2 was added to cells 2 hrs before pervanadate treatment at a concentration of 10 μM to inhibit Src family kinases.Cells were subjected to pervanadate treatment by the addition of a freshly prepared solution of pervanadate at 50 μM conc. for 10 min prior to harvesting. Pervanadate stock solution (50 mM) was prepared by mixing equal volumes of 100 mM solution of HFull length human C3G cloned in pcDNA3-FLAG was kindly provided by Dr S Tanaka. Y504F mutant of C3G in which tyrosine 504 is mutated to phenylalanine cloned in a His tagged expression vector was provided by Dr M Matsuda. The wild type rat p59 Hck cDNA was cloned in the pCI plasmid (Promega) and has been described earlier . ExpressWhole cell lysates were prepared by lysing cells directly in Laemli's sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. After transfer onto nitrocellulose membranes, they were processed for western blotting using the required primary antibodies. Detection was based on either color development using alkaline phosphatase conjugated secondary antibodies or on chemiluminescence using horse radish peroxidase conjugated secondaries.Cells were processed for immunoflourescence staining as described earlier . The priGNEF – guanine nucleotide exchange factorSFK – Src family kinasePV – PervanadatepY504 C3G – Tyrosine 504 phosphorylated C3GVSVG – Vesicular stomatitis virus glycoproteinDMEM – Dulbecco's modified Eagle's mediumFITC – Fluorescein isothiocyanateVR designed and carried out the experiments, analysed the data and drafted the manuscript. GS helped with designing the experiments, analyzing the data and writing the manuscript. AR provided technical help for western blotting and indirect immunoflourescence experiments.


We sought to evaluate the hypothesis that the high incidence of cutaneous melanoma in white persons in central Alabama is associated with a predominance of Irish and Scots descent.Frequencies of country of ancestry reports were tabulated. The reports were also converted to scores that reflect proportional countries of ancestry in individuals. Using the scores, we computed aggregate country of ancestry indices as estimates of group ancestry composition. HLA-DRB1*04 allele frequencies and relationships to countries of ancestry were compared in probands and controls. Results were compared to those of European populations with HLA-DRB1*04 frequencies.Ninety evaluable adult white cutaneous melanoma probands and 324 adult white controls reported countries of ancestry of their grandparents. The respective frequencies of Ireland, and Scotland and "British Isles" reported countries of ancestry were significantly greater in probands than in controls. The respective frequencies of Wales, France, Italy and Poland were significantly greater in controls. 16.7% of melanoma probands and 23.8% of controls reported "Native American" ancestry; the corresponding "Native American" country of ancestry index was not significantly different in probands and controls. The frequency of HLA-DRB1*04 was significantly greater in probands, but was not significantly associated with individual or aggregate countries of ancestry. The frequency of DRB1*04 observed in Alabama was compared to DRB1*04 frequencies reported from England, Wales, Ireland, Orkney Island, France, Germany, and Australia.White adults with cutaneous melanoma in central Alabama have a predominance of Irish, Scots, and "British Isles" ancestry and HLA-DRB1*04 that likely contributes to their high incidence of cutaneous melanoma. The incidence of cutaneous melanoma in Alabama is high  . LifestyIt has been hypothesized that the high incidence of melanoma in Alabama, a Sunbelt state with a mild climate, is due to the settlement of Alabama by a predominance of persons of Irish and Scots descent -10. In tThe performance of this work was approved by the Institutional Review Boards of the University of Alabama at Birmingham and Brookwood Medical Center. All subjects were adults (≥ 18 years of age) who were central Alabama residents; each identified himself/herself as white. Persons with melanoma were diagnosed in routine medical care in the interval 1981 – 2002, but were otherwise unselected. We excluded persons of sub-Saharan African or African American descent, because most of these persons have non-European ancestry and their incidence of cutaneous melanoma is much lower than in white persons in central Alabama [Cutaneous melanoma was diagnosed as previously described . HLA-DRBAdult control subjects residing in central Alabama were recruited in two groups. The first group consisted of 83 spouses of persons with cutaneous melanoma and randomly recruited subjects. The second group consisted of 260 unselected volunteers who completed the present questionnaire (described below); they were recruited from hospital workers, employees of two universities, spouses of patients who attended a hematology/medical oncology outpatient clinic, and members of the general public encountered in two retail shopping malls. In both groups, we excluded persons who were known to be relatives of other study participants. We did not evaluate medical histories or perform physical examinations in control subjects. These data were pooled to yield a group of 343 unrelated controls, of whom 24 were eliminated because they did not know the country of ancestry of any of their grandparents (as indicated below). This left a group of 319 control subjects whose data were deemed evaluable for the present study. HLA typing was performed on some control subjects as indicated below.A one-page questionnaire was designed to permit each study participant to indicate the countries of ancestry of his/her paternal and maternal grandparents. This method is identical to that previously validated in a study of the ancestry of hemochromatosis probands ; in partWe defined aggregate country categories as the composite of reports from respective countries . ReportsWe tabulated the number of participants who reported specific countries of ancestry or aggregate country categories (as defined above) for one or more grandparents. This method is identical to that previously described ; in partThe questionnaire and interview reports from each participant were evaluated to yield individual country of ancestry scores that reflect proportional national ancestry, as previously described . We alsoAn index of Celtic ancestry was computed in a manner similar to that used for country of ancestry indices. In the present analysis, persons who reported one or more grandparents whose country of origin was Ireland or Scotland were defined as "Celts." Other participants were defined as "non-Celts." We did not use grandparental surnames or maiden names to quantify the degree of Celtic ancestry as did previous investigators ,19.A manual and computerized literature search was performed to identify reports of HLA-DRB1*04 phenotype frequencies in white persons with or without melanoma in countries or areas of Europe and Australia that are known to have had Celt settlements The HLA-DRB1*04 phenotype frequency among white control subjects in central Alabama is approximately 0.2148, based on the combined data from two previous reports ,20. Thust test, as appropriate. However, Student t-test can not be used to compare country of ancestry data groups in which all values are 0 (no variability). Accordingly, we arbitrarily assigned a country of ancestry datum of 0.25 for one person in each proband country group for which there were no actual country of ancestry reports. Student t-test was then performed using this modified data group; estimated p values from these tests are displayed in parentheses. A p value < 0.05 was defined as significant. Odds ratios (OR) were calculated as described by Woolf [The dataset consisted of observations on 90 cutaneous melanoma probands and two groups of controls . A computer spreadsheet  and a statistical program  were used to perform the present analyses. In a preliminary evaluation, we determined that the proportions of men and women, mean ages, country of ancestry reports, and frequencies of country of ancestry reports were not significantly different in the two control groups. Therefore, we pooled data from the two groups for comparison with those of melanoma probands. Frequencies of men and women, clinical abnormalities, and countries of ancestry were counted. General descriptive data are presented as percentages or mean ± 1 S.D. Comparisons between groups were tested for statistical differences using chi-square analysis, Fisher exact test or two-tailed Student by Woolf .There were reports from 90 evaluable probands . Their mean age at the time of participation in the present study was 49 ± 16 years (range 19 – 80 years).There were reports from 319 evaluable control subjects . The percentages of men and women in the control group were similar to those in the cutaneous melanoma probands . Their mean age at the time of participation in the present study was 43 ± 16 years (range 18 – 82 years). The mean age of the cutaneous melanoma probands was significantly greater than that of the control subjects .No person declined to participate in the study, and there were no incomplete, equivocal, or unintelligible questionnaire or interview reports. Some participants reported that they were unaware of their ancestry due to adoption, family estrangement, or disinterest in genealogy. Ninety of 110 cutaneous melanoma probands (81.8%) and 319 of 347 control subjects (91.3%) provided reports for at least one of four grandparents; these differences were significant (p = 0.0026). Data from these participants were included for further analysis. Thus, there were reports from 90 evaluable cutaneous melanoma probands and 319 evaluable control subjects. Among evaluable cutaneous melanoma probands, the mean number of countries reported was 2.2 ± 1.1 (range 1 – 5 countries). The mean number of countries reported by evaluable control subjects was similar (2.4 ± 1.1 countries (range 1 – 7 countries); p = 0.2246).These data are displayed in Table These data are displayed in Table HLA-DRB1 phenotypes were available for 63 of the 90 present cutaneous melanoma probands. We divided the probands for which we had both country of ancestry data and HLA-DRB1 phenotypes into "Celts" (n = 19) and "non-Celts" (n = 44), as defined above. There was no significant difference in the frequency of HLA-DRB1*04 in the two groups . Because this finding was unexpected and differed from that of previous reports  we then We identified HLA-DRB1*04 phenotype frequency estimates of > 0.2148 from England, Ireland, Scotland, Wales, Orkney Islands, Brest, Germany, and Australia, areas populated predominantly by persons of Celtic ancestry ,13,20,22The present results indicate that England, Ireland, Scotland and the aggregate "British Isles" are the countries of ancestry reported most frequently by cutaneous melanoma probands and control subjects in central Alabama. Germany is another country of ancestry often reported by the present study participants. These results are consistent with historical accounts of early migrations of persons of English, Irish and Scots descent into central Alabama -26, with"Native American" ancestry, especially Cherokee or Creek heritage, was reported by many of the present study participants, and this is consistent with accounts of early Alabama history ,30-32 anThe percentages of men and women were not significantly different in cutaneous melanoma probands and control subjects. Analyses of the two study groups indicate that age is not significantly correlated with country of ancestry indices. The mean age of cutaneous melanoma probands was significantly greater than that of control subjects. However, the mean number of countries reported by cutaneous melanoma probands and control subjects did not differ significantly. Thus it appears that diagnosis of cutaneous melanoma or greater age is not associated with greater interest or knowledge in personal ancestry, although this is unproven.30.0% of cutaneous melanoma probands and 30.1% of control subjects did not know the country of ancestry of any of their four grandparents and were thus declared inevaluable. Many others did not know the ancestry of some of their grandparents. Some participants reported that they were unaware of their ancestry due to adoption, family estrangement, or disinterest in genealogy. Other participants could have been incorrect in their reporting. The percentages of evaluable cutaneous melanoma probands and control subjects who reported grandparents in the "Don't Know" category were similar. Altogether, it is unlikely that exclusion of subjects who did not know the country of ancestry of each of their grandparents would significantly change the outcomes of the present study. The overall trends in frequency of country reporting and country of ancestry indices in "British Isles" and "Europe Not British Isles" categories in the present study were similar. This suggests that uncertainty of participants about the exact degree of country of ancestry of some of their grandparents was probably not a significant contributor to the major conclusions of the present study. It is possible that control subjects who were included also had cutaneous melanoma. Nonetheless, it is unlikely that identification of a presumably small number of undiscovered control subjects with cutaneous melanoma would significantly change the conclusions of the present study.Previous studies reported that Celtic ancestry is a risk factor for cutaneous melanoma, particularly when people of Celtic ancestry inhabit areas of high flux of ultraviolet radiation ,6. PersoThe present observations are consistent with those of another study in Alabama in which white persons with hemochromatosis, a disease thought to be of Celtic origin ,34, repoUsing an index of Celtic ancestry that included grandparental surnames, maiden names, and country of birth to evaluate persons in Wales, other investigators observed that "high-scoring Celts" were significantly more likely to have Fitzpatrick skin type I or II   than nonObservations from previous studies demonstrate that the frequency of positivity for the HLA-DRB1*04 phenotype is significantly greater in Alabama cutaneous melanoma probands than in control subjects ,9,20. ThInvestigators in Texas and England reported that the HLA genotype DQB1*0301 influences either susceptibility to and/or severity of melanoma ,36-38. TThe highest frequencies of HLA-DRB1*04 in Europe occur in England, Wales, Ireland, the Orkney Islands, and the Brest area of France . These gOur observations and those of others suggest that certain HLA genotypes may be markers for Celtic ancestry ,14. It hThe present results support our hypothesis, and are also consistent with the association of cutaneous melanoma with Ireland, Scotland and "British Isles" ancestry, HLA-DR phenotypes, and estimations of a northern European somatic phenotype  previously reported in white persons who reside in central Alabama ,4,8,9. TNone declared.RTA assessed the patients and participated in assessing the control population, conceived the study, participated in data collection, laboratory evaluation of the patients and controls, statistical evaluation, and wrote part of the manuscript. EHB participated in assessed the control population and in data collection. WWH participated in assessed the control population and in data collection. ALD participated in assessed the patient and control populations, in data collection and laboratory evaluation of the patients and controls. RCPG participated in conceiving the study. JCB participated in conceiving the study, assessing the control population, statistical evaluation and wrote part of the manuscript. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:


Simpson-Angus Scale (SAS) is an established instrument for neuroleptic-induced parkinsonism (NIP), but its statistical properties have been studied insufficiently. Some shortcomings concerning its content have been suggested as well. According to a recent report, the widely used SAS mean score cut-off value 0.3 of for NIP detection may be too low. Our aim was to evaluate SAS against DSM-IV diagnostic criteria for NIP and objective motor assessment (actometry).Ninety-nine chronic institutionalised schizophrenia patients were evaluated during the same interview by standardised actometric recording and SAS. The diagnosis of NIP was based on DSM-IV criteria. Internal consistency measured by Cronbach's α, convergence to actometry and the capacity for NIP case detection were assessed.Cronbach's α for the scale was 0.79. SAS discriminated between DSM-IV NIP and non-NIP patients. The actometric findings did not correlate with SAS. ROC-analysis yielded a good case detection power for SAS mean score. The optimal threshold value of SAS mean score was between 0.65 and 0.95, i.e. clearly higher than previously suggested threshold value.We conclude that SAS seems a reliable and valid instrument. The previously commonly used cut-off mean score of 0.3 has been too low resulting in low specificity, and we suggest a new cut-off value of 0.65, whereby specificity could be doubled without loosing sensitivity. Reported prevalences for neuroleptic-induced parkinsonism (NIP) in schizophrenia patients are usually in the range 19% to 36% . As NIP Simpson-Angus Scale (SAS) is a 10-item rating scale that has been used widely for assessment of NIP in both clinical practice and research settings . It consAccording to our recent study  there waAccelerometric methods have been developed to identify and monitor motor NIP symptoms, such as tremor ,19 and hThe discrepancy between SAS and DSM-IV based NIP prevalence estimates as well as other above mentioned shortcomings suggest that SAS needs an evaluation as a method to assess NIP severity and to find reliably NIP cases.Our aims were to check the internal consistency of SAS, improve the convergence between DSM IV and SAS based NIP case finding, and to evaluate how well the scale measures objective motor symptoms verified by actometry.We recruited 99 chronic schizophrenic institutionalized adult patients from a state nursing home in central Estonia . InclusiAn experienced clinician (SJ) assessed all the subjects to identify NIP cases in accordance with DSM-IV. The DSM-IV diagnostic criteria for other neuroleptic-induced movement disorders (NIMD) were also checked because of frequent comorbidity and common aetiology. Clinical NIP symptoms were assessed by SAS and the motor activity during rest was measured by actometry. Each item of the 10-item SAS is rated on a 5-point scale (0–4), and the mean score is obtained by adding the items and dividing by 10 . NeuroleThe actometric recording was performed during sitting in a standardized clinical interview for 30 minutes, a method described previously as measuring "controlled rest activity" ,21. ContCronbach's α was assessed to evaluate the internal consistency of the scale. The correlations between the lower limb activity (the mean of right and left ankle movement indices) and individual item scores and mean SAS scores were analysed. Differences between the NIP and non-NIP, as well as the NIMD and the non-NIMD groups in the SAS mean score and lower limb activity were analysed. The performance of SAS mean score and individual item scores in case identification was evaluated by receiver operating characteristics (ROC) analyses against DSM-IV NIP diagnosis. Validity coefficients  for different mean SAS score thresholds were calculated. To explore the discriminatory power of each single SAS item we performed ROC analyses for each item separately. We also explored the effect on the validity coefficients of merging the six rigidity items of SAS into one single item, to de-emphasise the influence of rigidity on the mean SAS score. The Spearman test was used to correlation analysis and the Mann-Whitney 2-tailed U-test for the comparison between two groups because of the non-normal distribution of the data. The software used in analyses was SPSS 11.0. .Of the 99 participants, 45 (45.5%) were male and 54 (54.5%) female. The mean age was 49.7 (SD 9.5) years. The mean continuous treatment in hospital or in nursing home was 13.6 (SD 9.0) years. Seventy-nine (79.8%) patients used conventional antipsychotics  and 20 (20.2%) used clozapine (one was receiving clozapine combined with sulpiride). Low-dose antipsychotics in this study were haloperidol, cyclopentixol, perphenazine and fluphenazine; high dose antipsychotics were chlorpromazine, thioridazine, levomepromazine, chlorprotixen and sulpiride. Sixteen (16.2%) patients were receiving combinations of typical antipsychotics (either predominantly low-dose [N = 10] or predominantly high-dose [N = 6] neuroleptic regimens), and 63 (63.6%) were receiving monotherapy . No new atypical antipsychotics were used. The mean daily chlorpromazine equivalent conditions. The prevalence of any NIMD according to DSM-IV was 61.6% in the whole sample. Cronbach's α for SAS was 0.79. dose  was 328 The SAS mean score for DSM-IV NIP patients  was significantly higher from that  of non-NIP patients . The mean scores of each single SAS item are presented in Table Actometric data was missing for one male patient due to non-co-operation. The median lower limb activity for NIP patients was not significantly higher than that of non-NIP patients . The median lower limb activity for NIMD patients was significantly higher from that of non-NIMD patients .The SAS mean score did not correlate significantly with actometric lower limb activity either in the whole population , in the NIP group , or in the pure NIP subgroup . Even after a post-hoc analysis of co-variance in the whole population, where the effect of akathisia  and tardive dyskinesia (AIMS severity score) were controlled for, no significant correlation between SAS mean score and the lower limb activity could be found .The tremor item of the SAS correlated significantly with the lower limb activity in the whole population  but not in the NIP population  or in the pure NIP subgroup . No correlation was evidenced between the hypokinesia item of the SAS and lower limb activity in the whole population  either in NIP population  or in pure NIP subgroup .No correlation was evidenced between the mean of rigidity items of the SAS and lower limb activity in the whole population  either in NIP population  or in pure NIP subgroup .ROC-curve for screening performance of SAS mean score is presented in Fig ROC-curve for screening performance of SAS mean with single averaged rigidity item was clearly inferior to the original SAS mean curve with AUC of 0.80 (CI = 0.70–0.89).The screening performances of the individual SAS items for NIP case finding are shown at Table As SAS elbow rigidity item had case finding power similar to SAS mean score, we calculated optimal cut-off for this item. Cut-off threshold of 1.5, with sensitivity of 0.826 and specificity of 0.974, was superior to cut-off threshold of 0.5 with sensitivity of 0.957 and specificity 0.553.Our study aimed to evaluate some of the characteristics of the SAS and its utility for identifying and measuring NIP in a naturalistic schizophrenia sample. The internal consistency of SAS was satisfactory, which suggests sufficient reliability for the scale. We compared the SAS with the DSM-IV to assess its discriminant validity and evaluate it in detecting NIP cases. The comparison with objective movement assessment aimed to estimate the concurrent validity of SAS in NIP severity measurement.As expected, the SAS had discriminant validity for a clinical diagnosis of NIMD. SAS mean score discriminated NIMD patients well from those without NIMD, and more specifically, also NIP patients from other patients. Actometry discriminated NIMD patients from non-NIMD patients, but did not identify DSM-IV NIP patients.According to ROC analysis the SAS had good case finding properties converging with the DSM IV NIP diagnosis. In our population the commonly used threshold 0.3 was inappropriate: according to our results the optimal cut-off point should be between 0.65 – 0.95 depending on the emphasis in the trade-off between sensitivity and specificity. We suggest that the new cut-off value for screening NIP could be 0.65, whereby specificity could be doubled without loosing any sensitivity. To be useful for diagnostic purposes a combination of high specificity and high positive predictive value (PPV) is reached at cut-off – 0.75 . To answUsing the single elbow rigidity item for case detection had the same (or slightly better) case detection capacity as the SAS mean score. This finding supports the use of elbow rigidity testing when assessing parkinsonism in clinical settings, as cut-off value 0.5 has good sensitivity and specificity for DSM-IV NIP.We found that SAS mean score did not correlate with actometric lower limb activity, and hypokinesia observed during gait item of SAS did not correlate with actometric motor activity during the 30-minute recording. There are a few explanations for that:First, actometry measures only the productive motor dimension of the parkinsonian symptoms while SAS takes into account also rigidity, gait, salivation and glabella tap, with a clear emphasis on rigidity. Lack of correlation with actometric findings in NIP subgroup indicates that tremor may not be the core feature of NIP. This is also supported by the small AUC for the tremor item of SAS.Secondly, we used lower-limb actometry while the clinical assessment by SAS and DSM-IV considered predominantly upper limbs. Parkinsonism may be more symptomatic in upper limbs, and the upper limb disturbances may have influenced our SAS and DSM IV assessments more than lower limb disturbances. Our findings indicate that lower limb actometry is not suitable for diagnosing NIP.Thirdly, diurnal naturalistic actometry may have more power in detecting hypokinesia.This study was limited to a few aspects of utility/validity of the SAS: internal consistency, convergence to DSM-IV NIP diagnosis and convergence to objectively measured motor activity. Many aspects of the scale's reliability (e.g. test-retest and inter-rater reliability) and validity (e.g. construct) were not evaluated.DSM-IV was used as a standard in this study, but there is not much data available on the validity of NIP criteria of the DSM-IV. A better golden standard in this study would probably have been an expert-consensus diagnosis. Furthermore, as there was only one rater for the scales, a cross-scale contamination issue might have occurred.It is known that with age the prevalence of spontaneous NIMD rises. Our material did not allow a thorough examination of the issue, but age correlated with SAS mean score in our sample.The measurement of motor activity here was purely quantitative; we did not assess the patterns of the disordered movements.As a conclusion, SAS seems be a reliable and a valid instrument. It performs well and similarly to DSM-IV in NIP case detection. The optimal SAS mean score cut-off value in a naturalistic population of neuroleptic-treated schizophrenia patients is higher than the commonly used 0.3. We suggest that the new cut-off value for screening NIP could be 0.65, whereby specificity could be doubled without loosing sensitivity. Combining SAS rigidity items does not seem to improve the performance of the scale.The author(s) declare that they have no competing interests.SJ contributed to the study design, collected the data, contributed to the analyses and data interpretation, made the literature search and was responsible for manuscript preparation with MMH.MMH contributed to study design, made most of the analyses and data interpretation, and was equally responsible for manuscript preparation with SJ.KT contributed to study design, statistical analysis, data interpretation and manuscript preparation.KW supervised the study design and contributed to statistical analysis, data interpretation and preparation of the manuscript.The pre-publication history for this paper can be accessed here:


There has been concern that the incidence of autism and other pervasive developmental disorders (PDDs) is increasing. Previous studies have been smaller, restricted to autism , included boys only, or have not been based on a national sample. We investigated time trends in the rates of diagnosis of pervasive developmental disorders.We analysed the rates of first diagnosis of pervasive developmental disorders among people registered with a practice contributing to the United Kingdom General Practice Research Database during the period 1988 to 2001. We included 1410 cases from over 14 million person-years of observation. The main outcome measures were rates of diagnosis of pervasive developmental disorders by year of diagnosis, year of birth, gender and geographical region.The rate increased progressively from 0.40/10,000 person-years (95% CI 0.30 to 0.54) in 1991 to 2.98/10,000 (95% CI 2.56 to 3.47) in 2001. A similar change occurred in the age standardised incidence ratios, from 35 (95% CI: 26–47) in 1991 to 365 (95% CI: 314–425) in 2001. The temporal increase was not limited to children born during specific years nor to children diagnosed in a specific time period. The rate of diagnosis of PDDs other than autism rose from zero for the period 1988–1992 to 1.06/10,000 person-years in 2001. The rate of diagnosis of autism also increased but to a lesser extent. There was marked geographical variation in rates, with standardised incidence ratios varying from 66 for Wales to 141 for the South East of England.Better ascertainment of diagnosis is likely to have contributed to the observed temporal increase in rates of diagnosis of PDD, but we cannot exclude a real increase. The term pervasive developmental disorder (PDD) refers to a range of disorders with onset in childhood characterised by abnormalities in the domains of language development, communication abilities and social interactions and by a rigid, repetitive pattern of behaviours and interests . Within There have been a number of reports that the incidence of PDD is increasing  and the As part of a case-control study to identify risk factors for autism and other PDDs , we haveThe GPRD was set up in 1987, then known as the VAMP  Research Bank . It consEach individual is assigned a unique identification number and all data that could identify individuals are removed from records before they are incorporated into the GPRD.st January 1988 to the 31st December 2001. Such children will have been eligible at some time in their life for MMR vaccination, an exposure of particular interest in the case-control study [The information analysed in the present study comprised the electronic medical records of children born in 1973 or later and registered with general practices contributing data to the GPRD during the period 1st January 1988 or the date at which the practice started contributing data to the GPRD. The end of the observation period was the date at which the practice stopped contributing data to the GPRD, or the 31st December 2001 if this was earlier. Individual patients were included in the study during the times within the observation period that they were registered with a practice contributing to the GPRD. The number of practices included in the GPRD varied during the study period, rising from 34 in 1988 to 557 in 1996 then falling to 380 by 2001.For a specific general practice, the start of the observation period for this study was taken as the later of either the 1Age- and gender-specific rates were calculated for people born in 1973 or later and registered with contributing general practices during each calendar year. Those registered for part of a particular year contributed time at risk for the period they were registered. People were classified by geographical location of their general practice by country for Wales, Northern Ireland and Scotland, and by the eight former administrative National Health Service regions for England.When a patient first registers with a practice contributing to the GPRD, past medical events and diagnoses judged to be clinically important by the general practitioner are recorded in the electronic record, with the date of each diagnosis where available. While a patient is registered with a contributing practice, all new diagnoses and all new drug therapies or changes in existing therapies are recorded contemporaneously. Cases were defined as people born in 1973 or later who had a first diagnosis of PDD entered into their general practice record while registered with a practice contributing to the GPRD during the study period. Cases were identified by searching the complete medical record of all people registered with participating practices during the study period for either OXMIS or READ clinical codes indicating a diagnosis of PDD: Appendix see . People On the basis of the recorded diagnoses patients were classified as 'autism' or 'other PDD'. Those with autistic disorders and similar presentations were grouped in the autism category and other descriptions (such as Asperger's syndrome) were grouped in the other PDD category.Crude rates and age standardised incidence ratios for first diagnosis of all PDDs, autism, and other PDDs were calculated by year of diagnosis and gender and for different geographical regions. Because of low numbers of cases in some years, indirect standardisation was used with rates during the whole study period as the standard rates. The age-standardised incidence ratios presented take into account variations in the age structure of the population in different time periods and different geographical regions. Rates of first diagnosis were calculated by year of diagnosis and year of birth, both in two year intervals.Ethical approval for the study was obtained from the Scientific and Ethical Advisory Group of the GPRD and from the ethics committee of the London School of Hygiene and Tropical Medicine.We identified 1410 persons with a first recorded diagnosis of PDD during the study period: 1097 were categorised as autism and 313 as other PDD, for 294 (94%) of whom the diagnosis was Asperger's syndrome (appendix).Table The person-years at risk varied from year to year because of changes in the number of contributing practices, which increased until the mid 1990s and then fell. During the study period incidence rates increased progressively for both males and females. The patterns of crude rates and of standardised incidence ratios were very similar. The overall rate increased seven-fold from 0.40/10,000 person-years (95% CI 0.30 to 0.54) in 1991 to 2.98/10,000 person-years (95% CI 2.56 to 3.47) in 2001. A similar change occurred in the age standardised incidence ratios, from 35 (95% CI: 26–47) in 1991 to 365 (95% CI: 314–425) in 2001. The overall male to female ratio was 4.8 and during 1991 to 2001 ranged from 2.7 to 8.4 , with no clear temporal trend.Table Table There was a striking rise in the rate of diagnoses of other PDDs: from zero for the period 1988 to 1992 rising to 1.06/10,000 person-years by 2001. Over the study period the rate of diagnosis of autism also increased substantially. The patterns were broadly similar for males and females (data not shown). Few diagnoses of other PDDs were made before 1997, but by 2001 over one third of all diagnoses were PDDs other than autism.Table There was marked geographical variation in rates of first diagnosis, with the lowest rate in Wales being less than half the rate in the South East of England . In general, the regional variations were less marked for autism than for other PDDs and the ratio of the diagnoses of autism compared with other PDDs varied greatly between areas .There was around a 10-fold increase in the rate of first recorded diagnoses of PDDs in United Kingdom general practice medical records from 1988–92 to 2000–01 table . If thesSome of the general practices contributing data to the GPRD will provide anonymised copies of hospital letters and specialist reports on individual patients. Of patients with a recorded diagnosis of PDD in the GPRD, including prevalent cases when first registered, 446 were registered with 203 general practices willing to provide this service. For 80 of these, medical records were not available as the patient was no longer registered with the general practitioner. We obtained complete case records including copies of hospital clinic letters and specialist reports for 318 (87%) of the remaining 366 people. These were reviewed by a psychologist (LH) and a random sample of 50 records were also reviewed by a child psychiatrist (EF), both of whom have long experience in the field of autism. They judged that a PDD was likely to be present in 294 children (92.5%). For theOf the 211 cases reviewed and included in this paper, 5 (2.4%) were classified as 'other-PDD' on the basis of their electronic record only, whereas in the validation, 32 (15.1%) met diagnostic criteria for a PDD other than autism. Thus it is likely that a proportion of people in the 'autism' diagnostic category in this paper have a form of PDD other than autism. The inaccuracy of diagnostic descriptions of different PDDs within the GPRD is likely to reflect changes in the definition of PDD over the past two decades, in particular a broadening of PDDs other than autism ,17. ManyIn the late 1980s and early 1990s autism was only likely to be diagnosed by specialist child psychiatrists who increased in number by 40% between 1988 and 2001 . Through the 1990s developmental and community paediatricians began to diagnose PDDs, and this, combined with increased awareness of autism and PDDs among the general public, may have contributed to increased ascertainment of the disorders [It is likely that children with PDDs other than autism will generally be first diagnosed at later ages than children with autism. Greater ascertainment of other PDDs during the latter part of the study period table  could haBecause of low numbers of cases in some years, indirect standardisation was used to calculate SIRs. When comparing SIRs, marked differences in the age distribution of the populations being compared can lead to a biased comparison . HoweverThe accuracy of the denominator data may have changed during the study period. When patients move geographical area they may delay registering with a new general practice until they have a specific reason to visit them. Thus our estimates of person-years at risk may be too low for those moving into a practice and too high for those moving out. Person-years at risk may also be inflated as patients who emigrate may omit to inform their general practitioner and, furthermore, administrative delays and errors may result in individuals being registered with more than one general practitioner. For example, in 1997 in England 51 million people were registered with a general practitioner  whereas A previous study based on the GPRD assessed time trends in the diagnosis of autism from 1988 to 1999 ,25. The This is one of the largest studies undertaken of trends in the incidence of autism and other PDDs. We describe striking increases in the rates of diagnosis of these conditions. However, much of the increase may be due to better ascertainment related to changes in diagnostic practice and improved recognition of the conditions. While review of clinical records confirmed that over 90% of diagnoses of PDDs recorded by general practitioners were likely to be correct, the nature of the study precluded us from assessing how often children with PDDs were not diagnosed. Thus the extent to which the increases in incidence we document are true increases in disease is uncertain.PDD pervasive developmental disorderGPRD General Practice Research DatabaseThe authors declare that they have no competing interests.AH, LS, EF, LR and PS designed the study. LH and EF undertook the validation of case reports. CC analysed the data. LS drafted the paper. All authors commented on drafts and approved the final version of the paper.The pre-publication history for this paper can be accessed here:Appendix: Codes used to identify cases, numbers identified, and diagnostic classification used in this paperClick here for file