Political instability and the civil war in Southern Sudan have resulted in numerous atrocities, mass violence, and forced migration for vast parts of the civilian population in the West Nile region. High exposure to traumatic experiences has been particularly prominent in the Ugandan and Sudanese of the West Nile Region, representing an indication of the psychological strain posed by years of armed conflict.In this study the impact of traumatic events on the prevalence and severity of posttraumatic stress disorder (PTSD) in a random sample of 3.339 Ugandan nationals, Sudanese nationals, and Sudanese refugees (1.831 households) of the West Nile region is assessed.Results show a positive correlation between the number of traumatic events and the number of endorsed PTSD symptoms. Of the 58 respondents who experienced the greatest number of traumatizing experiences, all reported symptoms which met the DSM-IV criteria for PTSD.There is a clear dose-effect relationship between traumatic exposure and PTSD in the studied populations with high levels of traumatic events. In this context, it is probable that any individual could develop PTSD regardless of other risk-factors once the trauma load reaches a certain threshold. The debate about the impact of traumatic life events on psychiatric disorders has a long tradition in psychiatry. The introduction of posttraumatic stress disorder (PTSD) into the Diagnostic and Statistical Manual of Mental Disorders (DSM-III ) manifesContrary to this assumption, the following research showed that the development of chronic PTSD is rather the exception than the rule after the experience of a traumatic event. Community studies in the US showed that whereas more than 50% of the population reported the experience of a traumatic event, the prevalence of PTSD was not higher than 7.8% . Among tA popular and intuitively plausible assumption in this context is the dose-response model of PTSD. This hypothesis predicts that the probability for the development of PTSD after the experience of a traumatic event mainly depends on the severity of trauma exposure. Some studies tried to test this hypothesis by relating the objective severity of the traumatic event to symptoms of PTSD. However, the empirical evidence for this model is scarce, with some findings supporting this hypothesis but many failing to confirm a relationship with meaningful effect sizes ,9.The probability of detecting a relationship between trauma exposure and PTSD depends on the range and variance of traumatic exposure that is present in the population studied. Studies investigating the relationship between the objective severity of single events and PTSD are restricted to a narrow variance of traumatic exposure. Community studies that assess trauma exposure across different types of traumatic events should be more adequate to examine a dose-effect hypothesis. From a worldwide perspective, even community studies in industrialized countries are restricted to a relatively narrow range of trauma exposure. In contrast, community studies in civil populations affected by war enable the examination of a much wider range of traumatic exposure. These populations present a continuum of subjects ranging from individuals without any history of traumatic events to victims with a history of high numbers of severe events that are rarely to be found in communities without a history of war. Studying a community sample of Cambodian refugees who had fled the Pol Pot regime, Mollica  actuallytraumatic event types experienced or witnessed so far. We considered this measurement to be more reliable than assessing the frequency of traumatic events as many survivors of civil wars reported countless exposures to specific traumatic events. To examine the impact of recent traumatic exposure, we also assessed the traumatic event types experienced or witnessed in the last year.Consequently, it can be hypothesized that each individual who has experienced or is experiencing traumatic events will develop PTSD after reaching a certain threshold of traumatic exposure. As this threshold is probably very high, a large number of subjects exposed to a large variance of traumatic events is necessary to test this hypothesis. We examined the dose-response relationship in the context of a large survey in the West-Nile regions of Sudan and Uganda. The study included Ugandan nationals with a quite peaceful development in the last decade, as well as Sudanese nationals living in the Southern Sudan war region and Sudanese refugees who had fled to Uganda. Among these groups we expected a sufficient variance of traumatic exposure to test for the specified dose-response hypothesis, including an adequate number of subjects who had to experience a series of extremely severe traumatic events. Cumulative trauma exposure was estimated by assessing the number of different As part of a study designed to better understand the impact of forced migration on fertility, mortality, violence and traumatic stress among Sudanese nationals living in southern Sudan and Ugandan nationals and Sudanese refugees living in northern Uganda, we interviewed 3371 individuals from 1842 households in the Ugandan and Sudanese populations in the West Nile. Interviews were structured and were administered in the native languages of Lugbara or Juba Arabic. The study's design involved a multi-stage sampling design.The full training of the interviewers took two months. The project objectives and the rationale behind the structure of the survey instrument as well as that of each question in the questionnaire were discussed in detail. Great attention was also paid to issues such as initial contacts, maintaining a professional attitude while in the field, avoiding influencing the respondent, and reducing interviewer and courtesy biases. The importance of collecting information by means of standardized questions so that the same question was asked to all respondents is stressed and questioning and probing skills were developed. Supervisors were instructed separately on data collection guidelines, their roles and their responsibility to ensure data quality. Keeping in mind the sensitive nature of some of the questions regarding violence and trauma and the fact that the team members were from the study population and probably had experiences similar to the respondents, a workshop on sexual and gender-based-violence was conducted by a consultant to the UNICEF office in Kampala, before the survey. The aim of this workshop was to increase awareness and sensitivity of the team towards respondents and their experiences. Another consultant to the project reviewed the team's interviewing skills and the project's data quality control measures just before the start of the survey. Problem areas were identified and remedied.Data were complete and analyzed for N = 3179 respondents: 2,540 (75 %) of the respondents were women (15–50 years of age) and 831 (25%) were men (20–55 years of age). Details of the sampling, translation and assessment procedures, as well as the socio-demographic characteristics of the populations, have been described elsewhere [ever (i.e., lifetime experience) and if it happened in the past year. PTSD in respondents was assessed using the Posttraumatic Stress Diagnostic Scale (PDS), modified for assessment by trained lay interviewers [Traumatic events were assessed using a checklist consisting of possible war and non-war related traumatic event types . The checklist was compiled after interviews with key informants (security personnel, doctors, community leaders, women's representatives) and 30 respondents from all three populations about their personal history of stressful events. Following these interviews, the single events obtained in these studies were rated as being potentially traumatic by experts. The following pilot checklist was pre-tested among further 44 Ugandans and Sudanese in areas not selected for the survey and modified according to the suggestions of the respondents. A primary item analysis based on inter-item correlations led to the exclusion of some events that were obviously not directly related to traumatic histories, e.g. the experiencing of witchcraft. Events included 19 experienced events and 12 witnessed events. Respondents were asked for each event type if they had experienced or witnessed such an event rviewers . The PDSConfidentiality was assured and it was explained that researchers were not working for any UN or Ugandan government organization. Informed consent was obtained using a standardized form explaining the potential risks of participation and explaining that no compensation would be provided. Informed consent forms were signed by the respondent and a witness; fingerprints were taken from illiterate respondents. No financial incentives were provided and respondents were informed that no improvements in living conditions were to be expected as a result of participating in the survey. Respondents were provided with referrals to counseling services provided by NGOs where available.As no major clustering effects were expected in this large sample, statistical analyses were carried out on unweighted data. To examine the relationship between continuous PTSD symptoms and the number of event types reported, we correlated the PDS score and its subscales, intrusion, avoidance and arousal with the number of event types. The number of event types in life correlated with the frequency of intrusions (r = .49), hyperarousal r = .41) and avoidance (r = .47), all P < 0.001. The PDS sumscore correlated significantly (P < 0.001) with the number of traumatic events in the past year (r = .45) and for the whole life , these groups were merged to two groups of 12–19 and 20–27 event types. Figure Overall, 31.6% of the male and 40.1% of the female respondents (N = 3179) fulfilled the DSM criteria for a PTSD-diagnosis. We divided the whole population studied in the survey into different groups based on the number of traumatic event types reported, separately for the events reported for High prevalence rates of PTSD have been reported for three different population groups in the West Nile: Sudanese nationals (44.6%), Sudanese refugees (50.5%) and Ugandan residents (23.2%) . Here weAs demonstrated, the number of traumatic events correlated equally strong with avoidance and with re-experiencing symptoms but coefficients were weaker, although still significant, for the hyperarousal cluster. These results are in agreement with , who alsTypically, even severe single traumatic event produce PTSD in not more than half of those affected. Therefore, PTSD is not an inevitable consequence of potentially traumatizing events. Results from this study, however, suggest that there may be no ultimate resilience to ward off PTSD or that a psychobiological breaking point exists for even the most resistant individual. In the three population groups that were surveyed, each respondent experiencing 28 or more different traumatic event types developed the full set of symptoms of PTSD. This cumulative trauma threshold identified in this study is very high and affected only a small minority of persons even in a war-torn population. Nevertheless, if the cumulative exposure to traumatic events is high enough, these results indicate that anybody will develop chronic PTSD. We conclude that there is no ultimate resilience to traumatic stress and that the repeated occurrence of traumatic stress has a cumulative damaging effect on the mental health of the victim. In these conditions, the effect of pre-trauma factors is reduced to the modulation of the probability of exposure to traumatic events itself. The factors that determine who is exposed to many traumatic events and who manages to flee to secure places may depend on pre-trauma psychological factors. Further studies with war-populations should examine whether the exposure to traumatic events only depends on uncontrollable external factors or whether individual factors contribute to a person's ability to seek safe places.High levels of trauma exposure is found in populations affected by civil war. We show that PTSD, the major psychological consequence of war events, is linearly correlated with traumatic exposure, thus explaining the high prevalence rates of PTSD generally found in war-torn societies. These findings highlight the need for reducing the frequent exposure to traumatic events by preventing wars, controlling the violence in wars, and providing safe and stable living environments for refugees. At the same time, the presence of high numbers of PTSD cases requires the implementation of individual and community based treatment programs. Given very limited resources in refugee communities, these centers must be created to provide short-term care and must be manageable by local personnel ,19. The The author(s) declare that they have no competing interests.FN, MS, UK & TE designed the study. FN, MS and UK composed the set of instruments. UK was responsible for original instrument translation and data collection, FN, MS, CK and TE for the validation part. FN and CK performed the data analysis. CR, TE and FN drafted the original manuscript and all authors revised and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A genome-wide study of nucleosome occupancy at yeast promoters shows that promoters that regulate active genes, contain multiple conserved motifs, or contain Rap1 binding sites tend to be depleted of nucleosomes.  PHO5 promoter in yeast is largely devoid of nucleosomes. We systematically evaluated nucleosome occupancy in yeast promoters by immunoprecipitating nucleosomal DNA and quantifying enrichment by microarrays.Although eukaryotic genomes are generally thought to be entirely chromatin-associated, the activated RPS11B promoter, by removing the Rap1 consensus sites.Nucleosome depletion is observed in promoters that regulate active genes and/or contain multiple evolutionarily conserved motifs that recruit transcription factors. The Rap1 consensus was the only binding motif identified in a completely unbiased search of nucleosome-depleted promoters. Nucleosome depletion in the vicinity of Rap1 consensus sites in ribosomal protein gene promoters was also observed by real-time PCR and micrococcal nuclease digestion. Nucleosome occupancy in these regions was increased by the small molecule rapamycin or, in the case of the The presence of transcription factor-binding motifs is an important determinant of nucleosome depletion. Most motifs are associated with marked depletion only when they appear in combination, consistent with a model in which transcription factors act collaboratively to exclude nucleosomes and gain access to target sites in the DNA. In contrast, Rap1-binding sites cause marked depletion under steady-state conditions. We speculate that nucleosome depletion enables Rap1 to define chromatin domains and alter them in response to environmental cues. Global gene-expression patterns are established and maintained by the concerted actions of transcription factors and the proteins that constitute chromatin. The global network of interactions between transcription factors and promoters in yeast is increasingly being characterized . The rolPHO5 promoter in yeast, this remodeling proceeds until essentially no nucleosomes are detected across a region of several hundred base-pairs [The nucleosome consists of approximately 146 base-pairs (bp) of DNA wrapped around an octamer of histone proteins - two each of histones H2A, H2B, H3 and H4. Eukaryotic genomes are packaged into repeating units of nucleosomes separated by around 10-80 bp of linker DNA. High occupancy by nucleosomes is thought to be generally repressive [Transcription factors and chromatin proteins each form complex regulatory networks that interact in a variety of ways ,7. TransWe used chromatin immunoprecipitation (ChIP) and DNA microarrays to evaluate nucleosome occupancy levels for essentially all promoters in yeast. Promoters that regulate active genes, contain multiple conserved motifs or recruit Rap1 tend to be relatively nucleosome-depleted. We also used real-time PCR and micrococcal nuclease digestion to show that nucleosomes are depleted in the vicinity of Rap1 consensus sites. This depletion can be partially reversed by the actions of the small molecule rapamycin or by removing Rap1-binding sites. We suggest that other transcription factors have less robust nucleosome-depleting activities than Rap1 and must therefore act collaboratively to gain access to their cognate sites in the DNA.PHO5 promoter [in vivo with histone H3 was isolated by ChIP using antibody against the carboxy terminus of histone H3 . ChIP DNA and unenriched control DNA were amplified by in vitro transcription and evaluated using microarrays. DNA associated with histone H2B was evaluated in a similar fashion using anti-FLAG antibody and a FLAG-H2B strain. H3 and H2B datasets were compiled by averaging four and three independent biological experiments, respectively. These datasets are remarkably similar as shown by a genome-wide correlation of 0.83 . dA.dT stretches destabilize nucleosome formation in vitro and in vivo [in vitro studies show that a 16-bp insertion leads to a 1.7-fold increase in accessibility of nucleosomal target sites [To identify additional determinants of occupancy, we sought sequence elements associated with nucleosome depletion. Specifically, we carried out an unbiased search for elements up to 10 bp in length that occur with higher frequency in nucleosome-depleted promoters. Two distinct categories of sequences emerged Figure . The fir in vivo ,18. The in vitro and in vivo [p < 10-5). Furthermore, multiple Rap1 sites are found in 19% of nucleosome-depleted promoters with Rap1 sites, compared to 8% of promoters with Rap1 sites overall (hypergeometric p < 10-3). These data suggest that Rap1 recruitment may lead to nucleosome loss.The second sequence element enriched in nucleosome-depleted promoters corresponds to the consensus motif for the Rap1 transcription factor. This motif commonly occurs in the promoters of ribosomal proteins genes and is required for Rap1 binding  in vivo ,20. Somep < 0.001. However, many of the implicated motifs appear in the same promoters. For example, nine of the over-represented motifs are associated with filamentation gene promoters [p <10-5; Figure p < 0.001; Figure Because only the Rap1 consensus site was identified in an unbiased search, we sought to identify additional sequence motifs by incorporating species conservation data. Specifically, we evaluated a set of 71 conserved motifs identified by Kellis and colleagues, a majority of which function in transcription factor recruitment . Nearly romoters . We therp < 0.001. Rap1 has the most significant association . Furthermore, an evaluation of nucleosome occupancy at promoters bound by both members of a cooperative pair revealed a significant association with nucleosome-depletion for 18 of the 31 pairs (hypergeometric p < 0.01). Together, these findings suggest that binding motifs and transcription factors act in combination to deplete nucleosomes and suggest a role for nucleosomes in transcription factor cooperativity [We sought an underlying binding mechanism or function common to the transcription factors we had identified. However, these factors utilize a variety of binding domains, regulate different pathways, and only a minority have significant associations with promoters of highly active genes. Nonetheless, a commonality does emerge when transcription factor cooperativity is considered. A recent informatics study by Banerjee and Zhang identified 31 functionally cooperative transcription factor pairs  on the basis of comprehensive binding and expression data . Only a rativity -25.in vivo [TUB2) with average occupancy by global analysis  growing in yeast extract/peptone/dextrose (YPD) were cross-linked in 1% formaldehyde for 15 min, washed twice in PBS, resuspended in 400 μl lysis buffer (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate) and lysed with glass beads. The resulting extract was sonicated to fragment chromatin (4 × 20 sec burst/30 sec rest with a Branson Sonifier 250 at 70% duty, power 3) and centrifuged for 15 min. Solubilized chromatin was then immunoprecipitated with polyclonal antibodies against the carboxy terminus of histone H3 . A unenriched whole-cell extract sample (WCE) was also retained as a control. After enrichment, cross-links were reversed by incubating samples in 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1.0% SDS, 150 mM NaCl at 65°C overnight. DNA was purified from ChIP and WCE samples by proteinase K treatment, phenol/chloroform extraction, ethanol precipitation, and incubation with RNAse. DNA associated with histone H2B in vivo was isolated in a similar manner from yeast containing epitope-tagged H2B [DNA associated with histone H3 eviously ,48,49. B8 cells) and whole-cell extract DNA (unenriched control) were amplified in a linear fashion as described [in vitro transcription reaction carried out with the T7 Megascript Kit (Ambion) and RNA samples were purified using an RNeasy Mini Kit (Qiagen). Amplified RNA was reverse-transcribed, incorporating amino-allyl dUTP, and the resulting DNA was fluorescently labeled by incubation with monofunctional reactive Cy5 (enriched sample) or Cy3 (unenriched control) dye as described [To obtain sufficient quantities for hybridization, immunoprecipitated DNA . The FLAG-H2B ChIP dataset was determined from three independent immunoprecipitations and hybridizations. The mRNA dataset was determined from three independent extractions and hybridizations of mRNA against genomic DNA. Relative transcription rates were determined by dividing transcript levels by half-life data collected by Wang and colleagues [Cy5 and Cy3 fluorescence were integrated for each feature using GenePix Pro Software (Axon). Data were processed and composite Cy5:Cy3 ratios determined according to protocols at the Stanford Microarray Database . Correlalleagues . A set olleagues .p < 0.001), consistent with the premise that these promoters are relatively free of nucleosomes.Z-scores were assigned to each intergenic that reflect depletion across the four H3 and three H2B ChIP experiments, using the formula Z = (x - μ)/σ where x is the average of the replicate measurements, μ is the average of all intergenics and σ is the standard error of the replicate measurements. We defined as nucleosome-depleted the 410 features with the highest Z-scores. This set, which includes 308 promoters, contains nucleosome-depleted outliers and is not inclusive of all promoters that immunoprecipitate with average or lower efficiency. The average aqueous enrichment ratio  for thesSaccharomyces Genome Database [P-values calculated by a hypergeometric probability model. The P-values reflect the extent to which observed overlaps exceed that expected under the null hypothesis that there is no relationship between the sets [p < 0.001 [Sequence elements common to nucleosome-depleted promoters were identified by searching between 10 and 500 bp upstream of gene start sites for over-represented sequences up to 10 bp in length using the GeneSpring program suite (Silicon Genetics). Enrichment was confirmed by evaluating the significance of overlap between the set of nucleosome-depleted promoters and the set of promoters containing Rap1 consensus motifs (ACACCCATACAT with up to two mismatches) or poly dA.dT stretches at least 10 bp in length (identified using PatMatch, Database ). Statisthe sets . Where sthe sets  were colthe sets . Lists o < 0.001 .-ΔΔCT method described in the Applied Biosystems User Bulletin. For each region examined, the TUB2 promoter was used as the normalizer , and the unenriched control sample was used as the calibrator. Each reported ratio represents the average of three independent ChIP experiments analyzed in duplicate by real-time PCR. The following primer pairs were used:Regions approximately 200 bp in size that span one or more Rap1 consensus sites in ribosomal protein gene promoters were amplified from ChIP and unenriched control samples using SYBR green PCR mix (Qiagen) in an MJ Research real-time PCR machine according to the manufacturers' instructions. Fold-ratios that reflect relative enrichment or depletion of a given region in the H3 or FLAG-H2B ChIP assays were determined using the 2RPS22A promoter: 5'-GCCTAAAACGCCCATAAGTT-3' and 5'-ACTGCAAACCCATATTCAAGA-3'RPS15 promoter: 5'-TACACCGCGCGTATAAATCA-3' and 5'-CCCAGCAAGGAGTTTCTCAG-3'RPS11B promoter: 5'-GAAGAAATATTTCCTTGCTGCACC-3' and 5'-AAGGGAAACGTAAAGCTATTGGAC-3'RPL23A promoter: 5'-ATTAACATCTGTACACCCCCAACT-3' and 5'-TACAGTTCGTTTCCTGCC ATATTA-3'TUB2 promoter: 5'-GGCCTAACAGTAAAGATATCCTCC-3' and 5'-GTTGTAGTAGCTGCTATGT CACTC-3'RPS11B promoter lacking the two Rap1 consensus motifs [RPS11B promoter: 5'-CTGGAAGAAATATTTCCTT GCTCTAG-3' and 5'-AAGGGAAACGTAAAGCTATTGGAC-3'.Centromeric vectors containing either a mutant s motifs  or an es600 of 1.0 were spheroplasted with zymolase (10 mg in 40 ml volume of 1 M sorbitol, 50 mM Tris pH 7.4, 10 mM β-mercaptoethanol (β-ME), at 30°C for 38 min shaking at 300 rpm), divided into five aliquots, and digested with increasing concentrations (20 U to 320 U) of micrococcal nuclease (Worthington Biochem) in 600 μl 0.5 mM spermidine, 1 mM β-ME, 0.075% NP-40. DNA from digested samples was extracted with phenol twice and chloroform once and precipitated in ethanol. Samples were washed, resuspended in 10 mM Tris pH 7.5, subjected to RNAse treatment, cleaned up with the MinElute kit (Qiagen) and run out in a 1% agarose gel. Following depurination, denaturation and neutralization of the gel, DNA was transferred onto nylon membranes by capillary action and covalently linked to the membranes by UV irradiation. Southern blotting was carried out using a DIG Luminescent Detection Kit (Roche) and DIG-labeled probe generated by PCR using the TUB2, RPS11B and RPS15 primers described above.Log-phase cultures of W303a yeast grown in 450 ml YPD to OD


When a trip for food can require a three-mile flight, it pays to get the directions right, especially if you're a bee. Bees more typically forage within a 600- to 800-yard radius, expending a significant amount of energy—a fact they seem keenly aware of: for reasons that remain unclear, bees tend to ignore directions that send them to a target on water. It's been known since Aristotle's time that returning foragers dance a little jig for their hivemates, presumably regaling them with tales of nectar-laden flora. Some 2,300 years later, zoologist Karl von Frisch correlated dance choreography to the direction and distance of a food source, eventually winning the Nobel prize for his work. Since then, researchers have been working out the details of bee communication, such as how fellow foragers interpret the “waggle dance” and how dancers perceive and convey navigational details of a trip. A key aspect of this information exchange is how bees estimate distance.Recent studies suggest that bee odometers are driven by the “optic flow” experienced during flight—or, simply put, bees appear to log distance by measuring the rate that images of passing terrain move in their eye during flight. This theory comes from observations that when bees fly a given distance, they indicate a much longer distance—by performing a longer waggle—for low-flying trips than for those at higher altitudes, presumably because flying at higher altitudes limits the bees' ability to perceive changing images. Likewise, bees flying through short, narrow tunnels filled with visual elements waggle a disproportionately long distance. These observations also raise questions about which visual aspects of the environment—contrast, texture, distribution of objects—are most important to a bee's perception of image flow.To investigate the factors driving the bee trip odometer, Mandyam Srinivasan and colleagues trained bees to feed at locations along two different routes in a natural environment, then compared their waggle dances. One route was entirely over land; the other started on land, shifted to water, and ended back on land. Both routes were the same distance, about 630 yards. Bees trained to feed at a boat in the middle of a lake had no trouble getting there, which was no guarantee based on reports that bees have trouble flying across lakes, often plunging into the drink. They had less success recruiting their colleagues to share in the bounty, even though their waggles clearly placed the feeder on the lake. The length of the bees' waggle dance increased faster with distance when they flew over land than when they flew over water. This disparity indicates that land provides a stronger “odometric signal” than water. “The honeybee's odometer,” the authors explain, “runs at a slower pace when flight is over water.” Overland flights tend to offer high contrasts and rich textures, while flights over water tend to offer low contrasts and sparse textures. Most likely, it is the high contrast of land surfaces that triggers a stronger odometric signal. But land surfaces also show variation in contrast, which was reflected in the bees' dance. One section of the land-only route was a paved bicycle path, a low contrast surface that the bees waggled as a relatively shorter distance.The Life of theBee, deciding it must correspond “to senses and properties of matter wholly unknown to ourselves.” As Srinivasan and colleagues show here, the bee's view of the world indeed corresponds to a unique way of interpreting the landscape—and of sharing news of their travels with their hivemates.Whether or not the contrast theory holds, Srinivasan and colleagues conclude, differences in the visual environment trigger differences in odometric signal. The odometer racks up yards depending on the nature of the terrain, whether it be land or water, during flight. The great Belgian playwright and avid bee-keeper Maurice Maeterlinck wondered at the language of bees in his 1901 book,


Sixty cancer cell lines have been extensively characterized and used by the National Cancer Institute's Developmental Therapeutics Program (NCI-60) since the early 90's as screening tools for anti-cancer drug development. An extensive database has been accumulated that could be used to select individual cells lines for specific experimental designs based on their global genetic and biological profile. However, information on the human leukocyte antigen (HLA) genotype of these cell lines is scant and mostly antiquated since it was derived from serological typing. We, therefore, re-typed the NCI-60 panel of cell lines by high-resolution sequence-based typing. This information may be used to: 1) identify and verify the identity of the same cell lines at various institutions; 2) check for possible contaminant cell lines in culture; 3) adopt individual cell lines for experiments in which knowledge of HLA molecule expression is relevant. Since genome-based typing does not guarantee actual surface protein expression, further characterization of relevant cell lines should be entertained to verify surface expression in experiments requiring correct antigen presentation. This st0 since tiscovery   exhibited a pseudo-homozygous pattern suggestive of complete loss of heterozygosity encompassing the HLA class I and HLA class II regions. This frequency is close to the loss of haplotype that we originally described for melanoma cell lines generated at the National Cancer Institute (Bethesda, MD) [Overall, there was no evidence of contamination among the cell lines tested with clean homozygous or heterozygous combinations observed in all loci analyzed. SBT of HLA class I and HLA class II loci are reported in Table sda, MD) ,39 and ssda, MD) ,41. We cOverall, discrepancies between ATCC typings and the present typing or the unbalanced frequency of homozygosity could be related to accumulated genetic alterations between the cell lines since the time of their original expansion from the patient and should not be surprising.A particular case was represented by the NCI/ADR-RES cell line which was previously believed to be an adriamycin derivative of the breast cancer cell line MCF-7. Subsequently, it was discovered not to be related to MCF-7, but it's derivation was unclear . KaryotyTo avoid possible misinterpretations, a large number of alleles are not presented here with their definitive nomenclature but rather at a two digits level of resolution because some of the ambiguities could not be completely resolved by SBT as previously described . HoweverFinally, several new alleles were identified . Information regarding the sequence of these alleles could be obtained by directly contacting the HLA laboratory, Department of Transfusion Medicine, Bethesda, MD.Genomic DNA from the NCI-60 cell line anticancer drug discovery panel was obtained from SH of the National Cancer Institute Developmental Therapeutics Program (Bethesda, MD). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum and 5 mM L-glutamine.Genomic DNA was isolated from peripheral blood using the Gentra PUREGENE isolation kit (Gentra Systems, Minneapolis, MN, USA). The DNA was re-suspended in Tris HCl buffer (pH 8.5) and the concentration was measured using a Pharmacia Gene Quant II Spectrophotometer. The DNA was then stored at -70°C until testing.HLA class I loci sequence-based typing (SBT) was performed as previously described (. The pri


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 (Nimotop, Bayer,1 mg/kg every 4 hours for 24 hours), 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 (such as verapamil, nifedipine, nimodipine, and amlodipine), 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 (eczema, urticaria, angioedema, etc.) 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 (Guanine nucleotide binding protein, beta polypeptide 2-like 1), 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 (Table Ten housekeeping genes were selected from commonly used reference genes (ABL1, ACTB, B2M, GAPD, GNB2L1, HRPT1, PBGD, RPL32, TBP, and TUBB). 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 (University of British Columbia, NAPS Unit). 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 (Helsinki, 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 (e groups , we reste groups  (ELISA me groups ).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 (p ≤ 0.05, r2 = 0.99)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 (Millonig, pH 6.8) 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 (Hitachi Ltd., Tokyo, Japan).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 (94°C for 1 min, and 72°C for 3 min) 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 (Maxim Biotech Inc, San Francisco, CA, USA) in 12 cases (4 AAT-emphysema patients, 4 smoking-related emphysema patients and 4 controls) 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 (200 μg/ml, dilution 1:200, Santa Cruz Biotechnology Inc., Santa Cruz) and monoclonal MIB-1 antibody (1:50 Dako, Santa Barbara, CA, U.S.A.), 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 (Vector Peterborough, UK) with 3-amino-9-ethylcarbazole (for TGF-β1, TGF-βRII) 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) (Dako, Santa Barbara, CA, U.S.A.). 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 (Image Pro Plus software version 4.1, Media Cybernetics, Silver Spring MD) 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 (predicted for sex, age, and body weight). 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 (FI, O2) 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 (mean 8.8 ± 1.7 vs 5.2 ± 3.9, p ≤ 0.05) 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 (49.8 ± 5.7 yrs AAT-deficiency vs 58.2 ± 6.3 yrs smokers, p ≤ 0.01). 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.