Helicobacter pylori can be detected in normal colon or in association with colorectal neoplasia.This pilot study was carried out to determine whether Helicobacter pylori antibodies .Paraffin processed colonic tissue blocks of normal colonic mucosa (n = 60), and patients diagnosed as adenoma (n = 60), and adenocarcinoma (n = 60) were retrieved from our archive; the adenoma group included tubular (n = 20), tubulovillous (n = 20) and villous adenomas (n = 20). 4 μm sections were stained by immunohistochemical methods using anti-Helicobacter pylori were identified in tubular adenomas , tubulovillous adenomas  and adenocarcinomas  compared to controls: there was no association in numbers of Helicobacter pylori and villous adenomas .Significant numbers of Helicobacter pylori with some, but not all, colorectal neoplasms, we can not infer causality from these results. These findings need to be further substantiated with a prospective study and the use of molecular biological techniques to determine a causal association.We conclude that although, in this pilot study, there appears to be an association in the prevalence of  Helicobacter pylori (HP) as an initiator of colorectal neoplasia [Colorectal cancers develop sequentially from hyperproliferative epithelium and aberrant crypt foci through to adenocarcinomas, passing through an adenomatous stage ,2. Theseeoplasia ,9, simileoplasia ,10 is sueoplasia , as are eoplasia ,13; indeeoplasia ,14-16, ieoplasia ,17 as weeoplasia . Most aseoplasia -23 or, ieoplasia ,25 or Caeoplasia . Other seoplasia -28; indeeoplasia .In this study we have used immunohistochemical methods to interrogate normal, adenomatous colorectal tissue and colorectal adenocarcinomas for the presence of HP, using anti-HP antibodies.This pilot study was independently examined and ethically approved by the Local Research Ethics Committee, Royal Cornwall Hospital, Cornwall, UK.Samples of paraffin-embedded colorectal tissue n = 180) including normal (n = 60), adenomas (n = 60) and adenocarcinomas (n = 60) were retrieved from departmental archives. Sixty samples of each diagnosis gave an acceptable precision for the prevalence estimates. This powered the study at 0.80 (α = 0.05) to detect an absolute difference of 26% in the prevalence of the different diagnoses. Specimens were not matched for age, sex or socioeconomic status as it was thought that this would confound any comparisons; a higher prevalence in adenocarcinomas might simply reflect the greater age of the people with this type of neoplasia. However, the age and sex were adjusted for using binary logistic regression once the results had been established. The number of males and females and their mean ages in each group is shown in Table 80 includEach sample group of patients was separate and mutually exclusive. Patients with normal biopsies did not have a biopsy history of colorectal adenomas or carcinomas, or other carcinoma. The patient group with adenomas did not have a biopsy history of colorectal or other carcinoma. Patients with colorectal adenocarcinoma formed the third group: these patients did not have a biopsy history of cancer at any other site. The limitations of this study include unavailability of information relating to serology or breath test or, preceding gastric biopsy.Since there is a sequential progression of colorectal polyps to colorectal adenocarcinoma, 20 cases each of tubular, tubulovillous and villous adenomas were selected. Immunohistochemical techniques were chosen because they are more specific and sensitive than tinctorial techniques -33.Cases were identified from the histopathology database between 1996 and 2001, using appropriate T and M codes and the relevant paraffin blocks were retrieved. Topographical code T67*/68* identified biopsies from the colorectum. Morphology code M00100 identified biopsies considered normal; M80400, M82630, M82611 and M81403 detected adenomas and carcinomas.® rotary microtome. Diagnosis were confirmed with an H/E stain; the other sections were used for immunohistochemistry. H/E and immunohistochemistry were used to detect HP organisms; Giemsa was not used. The immunohistochemically stained slides were randomized, given individual research numbers and then examined by light microscopyFour 4 μm sections were cut from each of the 180 formalin fixed paraffin blocks using a Leica® antibodies were used: 1) polyclonal NCL-HPp [campylobacter jejuni; dilution 1:800), which cross-reacts with HP. The use of two antibodies allowed for identification and differentiation of HP from Campylobacter in our study material. The strategy was to see whether we could identify one or the other, using immunohistochemistry on protocol samples.Two Novocastraon 1:150),35 and, Antigen retrieval was carried out in a citrate buffer pH6.0 for 30 minutes at 430W in a domestic microwave. This method was chosen over the Novocastra recommended trypsin digestion following experiments comparing both methods, as recommended,35. PosiThe detection method followed was that provided by Dako with the ChemMate™ Envision detection kit . The CheSections were counterstained using Mayer's Haematoxylin for 1 minute and blued in Scotts Tap water. Odds ratios (OR) were estimated using the Logit procedure in SPSS11.Four cases were excluded from this analysis; two from the normal tissue group, one from the adenocarcinoma group and one from the tubular adenoma group. These were due to inadequate tissue samples.HP organisms were golden-brown "dot-like" and granular against a light blue Haematoxylin counterstain. We were unable to unequivocally detect the spiral form of HP on H/E or immunohistochemistry in any of our normal or neoplastic colonic biopsy samples; we assume from this that the "granular" HP-positive organisms represented the coccoid form -39 of thThe patient demographics and results are listed in table We have used immunohistochemical methods to demonstrate that HP organisms do reside in the colorectum associated with colorectal neoplasms. Although we can not attribute a cause-and-effect relationship in this instance, there is certainly indirect evidence demonstrating this association-25,41,42We used immunohistochemical detection methods rather than routine histochemistry (Giemsa) as the former are said to be more accurate and are Our results show that the prevalence of HP is significant in tubular and tubulovillous adenomas but not in villous adenomas; the reason for this is unclear. It may be that the microenvironment in villous adenomas does not support HP in a similar manner to absence of HP in foci of intestinal metaplasia in the stomach or, low Helicobacter DNA; however, this study could not determine a statistical association between its presence and the different Dukes stages of colorectal adenocarcinomas or, between colonic and rectal adenocarcinomas[Although some studies have not been able to substantiate an association between HP and colorectal neoplasia and HP -28 or, tarcinomas.HP-induced carcinogenesis is thought to be initiated by gastrin-induced genomic instability , its vacuolating toxin VacA -50, and Helicobacter. hepaticus[Helicobacter infection suggesting a causal association[Experimentally, 129/SvEv Rag-2-deficient mice have been shown to develop colitis and colonic cancer in the presence of hepaticus, with imsociation.Whereas we acknowledge that the presence of HP in our study does not necessarily mean that local HP effects are responsible for colorectal neoplasia, but neither does it exclude it. It is entirely possible also that these HP organisms are transiting the bowel and their localisation in colorectal neoplasia, fortuitous. We have also considered the possibility these results could reflect identification of HP or HP-like antigens in luminal debris: whist we can not exclude this, our anti-HP antibody is HP-specific. ClearlyWe have been able to identify HP organisms in colorectal neoplasms using immunohistochemical methods in this pilot study. We have also been able to demonstrate an association in their presence with tubulovillous and tubulovillous adenomas and adenocarcinomas, but not with villous adenomas. These findings need to be further substantiated with a prospective study and the use of molecular biological techniques to determine a causal association.The author(s) declare that they have no competing interests.MJ. The idea for this research was developed by JT, JM, PH and MJ with CP providing the statistical validities before, during and after the study. All slides were reviewed by MJ, PH and JM before and after immunohistochemistry. The immunohistochemistry was optimised by MJ. Randomisation of cases was done by a colleague. The authors have been variously but actively involved in its conception and design, analysis and interpretation of data, in the preparation and critical evaluation of its content, have read and approved the final manuscript.This manuscript is the essence of the MSc Thesis of We acknowledge contributions from the following sources1. Cellular Pathology Research Funds (JM is a co-signatory)2. Sunrise Appeal3. Silverfern Research.


The pulmonary surfactant protein (SP)-A has potent immunomodulatory activities but its role and regulation during allergic airway inflammation is unknown.Aspergillus fumigatus (Af) challenge of sensitized animals.We studied changes in SP-A expression in the bronchoalveolar lavage (BAL) using a murine model of single Af provocation of sensitized mice. Decrease of SP-A was associated with influx of inflammatory cells and increase of IL-4 and IL-5 mRNA and protein levels. Since levels of SP-A showed a significant negative correlation with these BAL cytokines (but not with IFN-γ), we hypothesized that SP-A exerts an inhibitory effect on Th2-type immune responses. To study this hypothesis, we used an in vitro Af-rechallenge model. Af-induced lymphocyte proliferation of cells isolated from sensitized mice was inhibited in a dose-dependent manner by addition of purified human SP-A (0.1–10 μg/ml). Flow cytometric studies on Af-stimulated lymphocytes indicated that the numbers of CD4+ (but not CD8+) T cells were significantly increased in the parental population and decreased in the third and fourth generation in the presence of SP-A. Further, addition of SP-A to the tissue culture inhibited Af-induced IL-4 and IL-5 production suggesting that SP-A directly suppressed allergen-stimulated CD4+ T cell function.SP-A protein levels in the BAL fluid showed a rapid, transient decline that reached the lowest values (25% of controls) 12 h after intranasal We speculate that a transient lack of this lung collectin following allergen exposure of the airways may significantly contribute to the development of a T-cell dependent allergic immune response. Cells were then harvested on a PHD Harvester . [3H] thymidine incorporation was determined via liquid scintillation counting . Samples were plated in triplicates and the experiments were repeated n = 4 times unless otherwise stated.Human peripheral blood mononuclear cells were isolated from healthy volunteers using a standard protocol approved by the IRB of the University of Pennsylvania.2b isotype control  and Allophycocyanin conjugated anti-human CD8 . Data were acquired on a four-color, dual laser FACSCalibur . For FACS analysis the gated cell populations were divided into four different generations  according to their CFSE content. Isotype controls were used to distinguish positive and negative populations. The SP-A-treated samples were compared with, and data were expressed as the percentage of the non-treated samples.The effects of SP-A on lymphocyte proliferation was further assessed using CFSE labeling as previously described . SurfaceA relative quantitation of lung IL-4, IL-5, and IFN-γ mRNA expression was performed by real time PCR using TaqMan(R) Gene Expression Assay and TaqMan(R) Universal PCR Master Mix: Mm00445259, Mm00439646, Mm00434204, and Mm00801778, respectively, (Applied Biosystems) according to the manufactures instructions. In brief three RNA samples isolated from the lungs per time points, were reverse transcribed using Superscript(TM) III First Strand Synthesis System (Invitrogen). Approximately 100 ng of cDNA was used per singleplex PCR reaction in a total volume of 25 μl. Cycling was performed on an ABI SDS-7000 and ABI SDS-7500 respectively with an initial denaturation step at 95°C for 10 min and 40 cycles of 15 sec. at 95°C and 1 min. at 60°C. Every sample was run in triplicate and β-2-microglobin (Mm00437762) was used as endogenous control. The Comparative Ct Method was used to analyze the results using ABI PRISM(R) 7900 SDS software. Results are expressed as fold difference  relative to naïve animals.Splenic lymphocytes were cultured for 48 h as described. The supernatant was then removed and frozen at -80°C pending analysis. OptEIA mouse IL-4, IL-5 and IFN-γ ELISA kits (Pharmingen) were used.Data were expressed as mean ± SEM. Time courses and dose responses were compared using ANOVA. To test differences between individual groups Student's t test assuming equal variances were performed. Correlations were investigated by regression analysis. A p value of < 0.05 was considered as significant. Data were analyzed with the Sigma Stat standard statistical package (Jandel Scientific).Af challenge. Recovery was observed 72 h later (135 ± 40 %)  were compared with mice that received vehicle (glycerol) treatment instead of antigen  Fig.  and 1B. vs. 106 ± 16 p < 0.05, n = 5, Fig Af challenge.SP-A protein decreases were not associated with commensurate mRNA changes. Northern blot analysis on RNA extracted from the lung tissue of each group of sensitized mice showed that SP-A mRNA actually increased 12 h after allergen challenge in comparison with the 0 h controls  in comparison with glycerol challenge . The glycerol challenge controls were not different from mice that received no sensitization or challenge  and eosinophils  in comparison with glycerol challenged controls. These cell numbers were also reduced  by the 72 h time point.In order to examine the proinflammatory changes that were linked with the greatest fall in SP-A concentrations, we chose to study the 12 h time point. Following allergen provocation there were significant Penh increases 12 h after enge Fig . Levels Af-induced models of allergic airway inflammation [Af challenge induced a local production of predominantly Th2-type cytokines. In comparison with glycerol challenged controls there was a significant release of IL-4 (559 pg/ml vs. 3 pg/ml p < 0.01), IL-5 . There was no correlation between SP-A and IFN-γ (not shown). Further, by the 72 h time point after allergen challenge IL-4 (8.7 ± 2.7 pg/ml), IL-5 (10.3 ± 7.3 pg/ml) and Eotaxin (9.72 ± 3.5 pg/ml) returned to close to baseline. To verify that the amount of airway SP-A is negatively associated with Th2 cytokine production, we also performed quantitative real-time PCR analysis of IL-4, IL-5 and IFN-γ mRNA. These results showed that mRNA levels for the Th2 cytokines increased approximately 35 fold in comparison with naïve controls 12 h after allergen challenge and returned to control levels by 72 h . The background cpm throughout our experiments ranged between 0 and 16. The SP-A preparation had < 0.07 endotoxin units/ml. This level is consistent with other cell culture SP-A preparations [Af were markedly inhibited by SP-A in a dose-dependent manner  and ionomycin (500 ng/ml) were inhibited by SP-A (10 μg/ml) to a similar extent as the Af-stimulated lymphocytes by 66 ± 4% , G2, G3 and G4 population according to decreasing levels of CFSE expression. Cells incubated in the presence of 10 μg/ml of SP-A were compared with equivalent populations of cells stimulated in the absence of SP-A and the results are expressed as a percentage of the SP-A-free (100%) control. In comparison with medium treatment, (represented by the middle line) addition of SP-A increased the proportion of cells that remained in the G1 population in the samples stimulated by Af .To further confirm the effects of SP-A on the proliferating cells, we added an intracellular fluorescent dye 5,6-carboxy- fluorescein diacetate succinimidyl ester (CFSE) to the cell cultures. The amount of this dye is halved as the f Figure . The proAf-induced  to sensitized lymphocytes significantly inhibited IL-4 and IL-5 production both in the d Figure  and PMA/in vitro.Emerging evidence suggests that collectins provide a link between innate and adaptive immunity. The relationship of SP-A production to inflammatory changes in the lung however is poorly defined. This study reports that a transient decrease in airway SP-A levels is associated with mRNA transcription and release of Th2-type cytokines following allergen provocation. The results also demonstrate that this lung collectin has a direct negative regulatory effect on allergen-induced lymphocyte activation Af challenge but completely recovered 72 h later. The exact mechanisms that regulate the changes of SP-A concentrations in the airways are uncertain. Since reduction in the absolute amounts of SP-A was only 42%, the effect of dilution by the extravasated plasma proteins 12 h after allergen challenge could have significantly added to the fall in the fractional SP-A concentrations. Nevertheless, while further studies are needed to unravel its intricate regulation [Studies investigating allergen-induced airway inflammation previously described either significant increases ,23 or degulation , it appein vitro model where SP-A mRNA but not protein expression was induced by dexamethasone, cyclic AMP and IL-4 [SP-A changes were comparable to that of SP-B and SP-C, described previously in a similar model . Interesand IL-4 . In addiand IL-4 . Taken tAf [The inverse association we observed between levels of SP-A and the inflammatory changes 12 h after allergen provocation was used to generate our hypothesis that SP-A negatively regulates Th2 cytokine production. This hypothesis was then tested by directly adding SP-A to allergen-stimulated T cells. These results are in support of previous reports showing that administration of SP-A alleviated allergic inflammatory changes in mice (reviewed in ), howeveAf ,29 and hAf  may be oAf ,31. FurtAf  and T ceAf -12. SP-Ain vitro. Here we present for the first time the inhibitory effects of SP-A on generational progression of sensitized lymphocytes to Af re-challenge in vitro. We show that this inhibition was dose-dependent. Further, inhibition of lymphocyte activation by SP-A was not due to toxic effects since baseline proliferation of the lymphocytes was not affected. In addition, Figure Af concentrations, confirming that the antigen-responsive, stimulated T helper cell population is the target of SP-A.The mechanism of T cell susceptibility to inhibition by SP-A needs further clarifications. While the inhibitory effects of SP-A have been demonstrated in antigen-stimulated human PBMC  and moreex vivo [in vivo and that there was a significant, negative correlation between SP-A and the amount of IL-4, IL-5 and eotaxin supporting the hypothesis that this collectin exerts an inhibitory effect on Th2 processes. Indeed addition of SP-A abolished production of the Th2 cytokines IL-4 and IL-5 by Af-stimulated lymphocytes. Thus, SP-A may act as a natural immunosuppressant in the lung with direct inhibitory actions on allergen-stimulated CD4+ Th2 lymphocytes.The effects of SP-A treatment on inhibiting Th2 cytokines were previously studied ex vivo . Here weA transient decrease in airway levels of SP-A was associated with transcription of mRNA and release of the Th2-type cytokines IL-4 and IL-5 but not IFN-γ. On the other hand, addition of SP-A to lymphocyte cultures from sensitized mice suppressed allergen-induced Th2-type immune response. We speculate that this lung collectin may play a protective role to prevent allergen-inhalation provoked immune reactions and propose the novel concept that a transient SP-A deficiency contributes to the pathogenesis of the allergic airway response.Af: Aspergillus fumigatusAHR: Airway hyperresponsivenessBAL: Bronchoalveolar lavageCFSE: 5,6-carboxyfluorescein diacetate succinimidyl estercpm: Count per minuteELISA: Enzyme linked immunosorbent assayFACS: Fluorescence activated cell sorterFITC: Fluorescein isothiocyanateIg: ImmunoglobulinIL: Interleukini.n.: Intranasali.p.: IntraperitonealLPS: LipopolysaccharideMBL: Mannose binding lectinSP: Surfactant proteinPAP: Pulmonary Alveolar ProteinosisPI: PMA and ionomycinPMA: Phorbol 12-myristate 13-acetatePBMC: Peripheral Blood Mononuclear CellsTh: T helperSeth Thomas Scanlon carried out the surfactant protein analysis and the protein and phospholipids measurements. Tatyana Milovanova carried out the CFSE studies and FACS analysis. Sonja Kierstein performed the cytokine mRNA analysis. Yang Cao participated in the surfactant protein analysis and in the animal experiments. Elena N. Atochina analyzed the SP-A Western and Northern blots. Yaniv Tomer carried out the animal experiments. Scott J. Russo performed the mRNA extraction and Northern blots. Michael F. Beers participated in the design of the study and contributed to the manuscript writing with comments. Angela Haczku conceived of, designed and coordinated the study, and participated in its writing. All authors approved the manuscript.


The retina is a well-defined portion of the central nervous system (CNS) that has been used as a model for CNS development and function studies. The full specification of transcripts in an individual tissue or cell type, like retina, can greatly aid the understanding of the control of cell differentiation and cell function. In this study, we have integrated computational bioinformatics and microarray experimental approaches to classify the tissue specificity and developmental distribution of mouse retina transcripts.We have classified a set of retina-specific genes using sequence-based screening integrated with computational and retina tissue-specific microarray approaches. 33,737 non-redundant sequences were identified as retina transcript clusters (RTCs) from more than 81,000 mouse retina ESTs. We estimate that about 19,000 to 20,000 genes might express in mouse retina from embryonic to adult stages. 39.1% of the RTCs are not covered by 60,770 RIKEN full-length cDNAs. Through comparison with 2 million mouse ESTs, spectra of neural, retinal, late-generated retinal, and photoreceptor -enriched RTCs have been generated. More than 70% of these RTCs have data from biological experiments confirming their tissue-specific expression pattern. The highest-grade retina-enriched pool covered almost all the known genes encoding proteins involved in photo-transduction.This study provides a comprehensive mouse retina transcript profile for further gene discovery in retina and suggests that tissue-specific transcripts contribute substantially to the whole transcriptome. The retina is a well-defined portion of the Central Nervous System (CNS) that has long been used as a model for CNS development and function -4. It is; [), but none of these have provided a global view of retinal transcripts. Recently, Blackshaw et al used serial analysis of gene expression (SAGE) identified 1,051 genes that showed developmentally dynamic expression in mouse retina [The complete genome sequences of human and mouse provide a new starting point for understanding specific expressed transcripts, especially the sequences associated with development and disease. Expressed sequence tag (ESTs) databases, are the most abundant resource of gene expression data. Recently, Okazaki et al. used available data on ESTs to establish a comprehensive full-length transcript data base . There h; ; ), but e retina . Schulz e retina . Althouge retina ,16, the In the present study, we have generated mouse retina ESTs from embryonic day 13.5 (E13.5), postnatal day 1 (PN1), and adult (8 weeks old) and then analyzed about 81,000 ESTs along with other three major mouse retina libraries through an approach that integrates computational and retina tissue-specific microarray data to identify a set of candidate genes highly related to retina-specific function and retinal diseases. 33,737 non-redundant sequences were identified as retina transcript clusters (RTCs) and step by step classified into neural, retinal, late-generated retinal, and photoreceptor-enriched RTCs. This study also provides a comprehensive table of mouse retina transcript profiles that will now allow a better understanding of retinal development and function.We used a series of computational steps Fig.  and 2 to, 4,825 (13.9%) have no reference RNA sequence but do have GenScan [Among the RTCs, 12,939 (37.4%) have a reference RNA sequence in the NCBI database  GenScan  predicteTo find criteria by which we could define tissue-enriched or tissue-specific transcript clusters we carried out a comparative analysis starting with approximately 106,000 lung and 80,000 cerebellum ESTs. These sequence pools were filtered using the same procedures as for retina to give transcript clusters (TCs) for each tissue. We then collected all the sets of mouse EST data and removed from them any set that would contain a contribution from retina, lung or cerebellum. This gave a set of data containing over 2 million ESTs. We then used this total EST dataset and the individual transcript clusters (TCs) to derive a ratio for the number of copies in each TC compared with the number in the total. Any EST in a TC that was not found in the total was set to a value of 100. An EST with 5 copies in a TC and 5 copies in the total would have a ratio of 1 and an EST with 10 copies in a TC and 5 in the total would have a ratio of 2. This analysis was carried out for each of the three TCs. We then asked at what ratio were ESTs in the RTC not found in either the LTC or the CTC using the subset of sequences for which fell into UniGene clusters. At a ratio of 0.3 and above only 1.79% of RTC were shared with lung and only 3 % with cerebellum Fig . By incrWe have also examined abundance in the EST pool as a criterion for helping define tissue specific transcripts. As a reference for the validity of these criteria we tested the inclusion of a known set of 47 known photoreceptor genes. Using an enrichment ratio of 2 from the above analysis, we found that 70–80% of these known photoreceptor specific genes were included in the RTCs. By increasing the copy number to 2 or more or to 3 or more, the total pool of RTCs decreased from 9,179 to 2,629 and 826 respectively without any substantial loss of detection of the known photoreceptor specific genes Fig . The pooP = 6.8 × 10-59) as criteria. Together our analysis suggests that about 80% of tissue-specific or -enriched transcripts can be identified using the two criteria, a copy ratio of at least two in the specific tissue and at least three EST copies.We also carried out this analysis by setting the copy number to > 3 and examining the effect of varying the enrichment ratios Fig . At thisrd-1 (photoreceptor-deficient) retina as set 2 (WTretina/rd1retina). In set 1 we are comparing retinas before and after the generation of rod photoreceptors and in set 2 we were comparing wild type retinas with retinas in which the rods had degenerated. Thus, when setting log2 > = 1 as positive ratio, a double positive gene population lay at the upper-right and was considered as rod-enriched genes  for experimental confirmation. Two groups of experiments were designed to detect genes enriched in retina or photoreceptors by microarray analysis. In the first group, total RNA from postnatal day 21 (PN21) retina was isolated and compared with RNA from whole brain or pooled RNA from other organs, including heart, lung, spleen, liver, and kidney from the same animals, (designated as "body" in this study). The data from Retina/Body, Retina/Brain were analyzed individually and log2 ratio results of the two experiments are shown as a spot-plot graph Fig. . The Retnes Fig. . The micnes Fig. .P = 6.1 × 10-5) and 80% using the criterion of 1.5 . The percentage of overlap increased by increasing the EST copy number , subtracted with whole body and neuron ESTs, were compared with the microarray data from Retina/Body and Retina/Brain. The overlap between the two approaches was 70% under the microarray criterion of log2 ratio > = 1  in the subcategory Response to External Stimulus is found in RTC-L and RTC-P. The majority of RTCs in this category are the genes related to light response.RTCs were further specified as RTC-A , RTC-N (RTC-neuron enriched transcripts), RTC-R (RTC-retina enriched transcripts), RTC-L (RTC-late-born retina cell enriched transcripts), and RTC-P (RTC-photoreceptor enriched transcripts) using the methods described in Figures Biological Process and Molecular Function categories of the GO database were analyzed before subtraction and about 30 functional clusters were analyzed after subtraction and the results shown in radar graphs. Interestingly, before subtraction, a similar distribution of functional clusters appeared among the three libraries in either Biological Process  categories. After subtraction, using the criteria of > 3 EST copies and an enrichment ratio of 2, the distribution of functional clusters shifted in specific directions in Biological Process  including E13.5, E14.5, and PN1 and an adult phase, using the criteria of > 3 copies RTCs and 83% (ratio > 5) enrichment in one phase compared to the other Fig  and 4F. Cell Organization and Biogenesis clusters and 12% to Cell Cycle clusters in the embryo phase compared with 6% and 5%, respectively, in the adult phase. Conversely, about 24% of RTCs were in the Transport clusters in the adult phase and only 8.5% in the embryo phase  and Energy pathways (3.9%) were significantly higher in adult phase and in embryo phase, respectively. As expected, overall comparison of these phases shows a change from functions characteristic of a proliferating epithelium (such as cell cycle) to those characteristic of a mature retina (such as transport).To examine the different functional clusters in these two phases, categories of the GO database were analyzed. In the biological process category, 18% of RTCs belonged to ase Fig. . Althougers Fig.  in both ase Fig. . InteresThe computational processes described above classified a total of 33,737 RTCs for their statistical probability of being retina specific or retina-enriched, as listed in Seven sets of information are provided in the sine oculis (so)/Six family of genes were present in the RTC pool  as single units with non-redundant sequences, although multiple transcript clusters can be in the same UniGene cluster. Such RTCs may represent different parts of a gene, or splice variants that are not considered in the UniGene database. We have calculated that about 19,000 to 20,000 genes may be expressed during mouse retina development from embryonic day 13.5 to adult. This is about 30% more compared with Schulz's Retinome  , althougA major concern for specification of transcripts from ESTs is how to confirm a reliable result. We have used four control steps to verify the results. First, as an external negative control we compared the specificity in our target tissue, retina, and in other tissues (lung and cerebellum). Second, we used an internal positive control. We used known photoreceptor-specific genes to check their coverage rate during different steps of retina specification. The third control was to identify functional clusters through the Gene Ontology (GO) database before and after specification of ESTs in different tissues. Biological confirmation was the final control experiment in this study and used a mouse retina tissue-specific microarray. Results from all the control experiments fully support the conclusions of this study.Identification of retina disease genes is an important and immediate use for genome-wide study tools. Several approaches have been used for genome-wide hunting of such genes. However, as shown in our results, the data sets show obvious gaps among these various experimental approaches Table . KatsaniGeneration and specification of an entire transcription profile for individual tissues or cells with specific functions and morphologic identities represents the next major task in the genome era. In conclusion, this study complements and extends previous studies in a number of ways. First, we have generated a comprehensive data set of retina transcript profiles with functional and developmental explanation, as the examples shown in Figure  (kindly provided by A. Smith and P. Green). Other analyses used custom programs.Mouse retina ESTs were collected from NCBI and were filtered and cleaned up by a series of programs . Retinas were dissected without contamination from lens, iris, cornea, and ciliary body. 10 to 20 retinas or other organs were pooled for RNA isolation. All animal experiments were conducted in accordance with NIH guidelines and were approved by the IACUC of Yale University School of Medicine.Mice were purchased from the Jackson Laboratory (C57Bl/6j) or were a gift from Dr. C. Zeiss, Yale University  and purified by RNeasy mini kit (QIAGEN). 5 μg total RNA with 280/260 ratios greater than 1.9 was used for array hybridization without amplification. Three to four sets of RNA were prepared from each age of retina and processed individually for microarray analysis.About 12,000 non-redundant mouse retina ESTs were generated from about 28,000 ESTs that generated from E13.5, PN1 and adult (The NIH-University of Iowa Brain Molecular Anatomy Project). 9,216 purified PCR-amplified inserts were printed by the Yale Keck Microarray Core on poly L-Lysine (Sigma) coated glass slides utilizing a GeneMachines Omnigrid robotic arrayer (GeneMachines). 3DNA Submicro EX Expression Array Detection Kits  were used for RNA labeling. Detailed microarray experimental protocols are shown in supplemental methods. Slides were scanned on a GenePix 4000B scanner and the data were manipulated with GenePix software Version 4.0 (Axon Instruments). Three or four sets of microarray data for each experiment were used for Student t test and gene collection. Gene collection methods are described in text and also please see , where C is the number of classes above the baseline total population, wi is the weight for the ith class, and yi is the observed percentage related to the feature of interest in the ith class. The value of C = 2, 4, 3, and 3, for figures yi is the proportion of the ESTs with a given feature in the ith class, with the feature being either a photoreceptor related gene or a retina disease gene. The weights are 1, 2, ..., C for the C classes.To test whether the features illustrated in Figures T, we calculated the mean and variance of T under the null hypothesis of no trend conditional on the feature distribution in the baseline total population. It can be shown that when the feature of interest is binary, i.e. a given EST either has or does not have this feature,To assess the statistical evidence of a trend in the data based on whereNi is the number of ESTs in the ith class, N0 is the number of ESTs in the total population, and y0 is the proportion of ESTs having a given feature in the total population. The statistical significance of the observed increasing trend isand the statistical significance of the observed decreasing trend iswhere Φ is the cumulative function of the standard normal distribution. Statistic calculations were done by R 1.8.1 or R 1.7.1. RTC, Retina Transcript Cluster; CNS, Central Nervous System; EST, Expressed Sequence Tag; GO, Gene Ontology; PN, PostnatalSSMZ was primarily responsible for the design, coordination, conduct, and all experiments of the studies. XYF and CJB were responsible for coordination of the studies. XX and JL were responsible for computational data analyses and software development. SSMZ and MGL were responsible for microarray experiments and analysis. MGL and HZ were responsible for statistical analysis. SSMZ, MBS, and XYF were responsible for RNA collection and original initiation and generation of mouse retina ESTs. SSMZ and CJB drafted the manuscript and figures. All authors read and approved the final manuscript.Methods for in silico purification of ESTs.Click here for fileMethods for in silico specification of RTCs.Click here for fileRTC list under criteria of RTC-A/C20A3.Click here for fileGene list of RTC-L and RTC-P.Click here for fileWhole RTC information and SAGE, RetBase data comparison.Click here for filea, Heterotrimeric guanine nucleotide-biding proteins. b, ATP-binding cassette (ABC) transporter superfamily. c, Voltage-dependent calcium channel proteins. d, Voltage-gated potassium channel proteins.Samples of the percentage of RTCs in different gene families. Click here for filea, Gene numbers of chromosome interval and concentrated retina enriched gene pool in human known retina disease gene loci. b, Concentrated ratio between retina enriched gene pool and whole interval genes of the loci for human known retina disease gene. c, Gene numbers of chromosome interval and concentrated retina enriched gene pool in some human known retina disease loci. d, Concentrated ratio between retina enriched gene pool and whole interval genes of the loci for human retina disease.Candidate genes for human retina disease loci. Click here for fileKnown human retina disease gene list used in this study.Click here for fileRecommended gene candidates for human retina disease loci.Click here for fileAdditional methods.Click here for file


With the hypothesis that SP-A could bind α1-antitrypsin, we designed a series of At an α1-antitrypsin:SP-A molar ratio of 1:1, the interaction resulted in a calcium-dependent decrease of 84.6% in the association rate constant of α1-antitrypsin for neutrophil elastase. The findings were similar when SP-A was coupled with the Z variant of α1-antitrypsin. The carbohydrate recognition domain of SP-A appeared to be a major determinant of the interaction, by recognizing α1-antitrypsin carbohydrate chains. However, binding of SP-A carbohydrate chains to the α1-antitrypsin amino acid backbone and interaction between carbohydrates of both proteins are also possible. Gel filtration chromatography and turnover per inactivation experiments indicated that one part of SP-A binds several molar parts of α1-antitrypsin.We conclude that the binding of SP-A to α1-antitrypsin results in a decrease of the inhibition of neutrophil elastase. This interaction could have potential implications in the physiologic regulation of α1-antitrypsin activity, in the pathogenesis of pulmonary emphysema, and in the defense against infectious agents. Six tri) family ,5. A com) family  is attac) family . SP-A is) family ,9. The p) family . SP-A, i) family .Pneumocystis carinii, occurs via the CRD and is inhibited by sugars or calcium chelators [Most binding to micro-organisms, including influenza and herpes simplex viruses, Gram-positive and Gram-negative bacteria, mycobacteria, fungi, and helators .1-AT. We hypothesized that, in analogy with what happens with infectious agents, SP-A could bind to α1-AT, which carries 3 biantennary or triantennary asparagine-linked carbohydrate chains [Since some SP-A is present in the alveoli in the free form, it has a chance of coming into contact with αe chains .in vitro evidence that the inhibitory activity of α1-AT towards HNE is significantly decreased in the presence of SP-A, probably as a consequence of SP-A binding to α1-AT. Such an interaction would represent a novel mechanism of regulating alveolar α1-AT. This could have relevance both for the pathogenesis of emphysema in patients with the Z α1-AT variant and for the lungs' defenses against infectious agents.In this paper we provide 2, and 0.05% w/w Triton × 100, pH 8.0 (phosphate buffer), unless otherwise specified. Lipopolysaccharide (LPS) from E. coli serotype 026:B6 (Sigma) and methyl-α-D-mannopyranoside (MNOCH3) (Sigma) were dissolved in phosphate buffer. HNE and human α chymotrypsin (αChy)  were dissolved in 50 mM sodium acetate, 150 mM NaCl, pH 5.5 and diluted with phosphate buffer. N-glycocosidase F from Flavobacterium meningosepticum  was purchased from Roche Diagnostics . Clostridium histolyticum collagenase type III (EC 3.4.24) came from Calbiochem . The chromogenic substrates MeOSucAlaAlaProValNA (for HNE) and SucAlaAlaProPheNA (for αChy), and the irreversible inhibitors MeOSucAlaAlaProValCMK (for HNE) and TosPheCMK (for αChy)  were dissolved in (CH3)2SO. Wild-type α1-antitryspin (M α1-AT) was either from ART or purified from human serum by covalent chromatography. Capillary isoelectric focusing (CIEF) with bare fused-silica capillaries filled with polyethylene oxide and carrier ampholyte solutions in the pH 3.5–5.0 range [1-AT variant. Z α1-AT variant was purified by covalent chromatography from subjects identified within the Italian screening program for α1-AT deficiency [2, 1 mM MgCl2, 2.2 M sucrose (solution A), overlaid with 10 mM Tris-HCl pH 7.4, 145 mM NaCl, 1.25 mM CaCl2, 1 mM MgCl2, 2 M sucrose (solution B) and ultracentrifuged overnight at 85,000 × g in a Ti 60 rotor (Beckman). The floating material was dispersed in water and centrifuged for 30 minutes at 100,000 × g and the pellet recovered was stored at -70°C (purified surfactant). To obtain SP-A, surfactant was injected into a 50-fold excess by volume of 1-butanol and stirred at room temperature for 30 minutes. After centrifugation, the pellet was suspended in 1-butanol and re-centrifuged at 4,000 × g for 1 hour at room temperature. The final precipitate was dried under nitrogen and then resuspended in 5 mM Tris-HCl, 145 mM NaCl, 20 mM octyl β-D-glucopyranoside, pH 7.4 (solution C). After centrifugation at 100,000 × g for 1 hour, the pellet was resuspended in 5 mM Tris-HCl pH 7.4 (solution D) and dialyzed against solution D for 48 hours with at least six changes. The final solution was centrifuged at 100,000 × g for 1 hour and the resulting supernatant, containing purified SP-A, was stored. Endotoxin-free SP-A was obtained by treatment with polymyxin-B (Sigma). Small aliquots of SP-A in solution D were incubated in a 1:1 ratio for 6 hours at 4°C with polymyxin-agarose previously equilibrated with 5 mM Tris-HCl, 100 mM octyl β-D-glucopyranoside and 2 mM EDTA, pH 7.4. Polymyxin-agarose was removed by centrifugation at 14,000 × g for 15 minutes, and the supernatant was then dialyzed against 5 mM Tris-HCl pH 7.4 for 48 hours with at least six changes and lyophilized [2, pH 7.2 (solution E), and the column was washed extensively with solution E. SP-A was finally eluted with 5 mM HEPES, 0.4% Triton × 100 and 2.5 mM EDTA, pH 7.2 (solution F).All reagents were of analytical grade, unless otherwise specified. The buffer used in all experiments was 0.2 M Na-K phosphate, with 0.5 M NaCl, 2 mM CaCl.0 range  was applficiency  from surficiency  and fromphilized ,18. For See additional file 1for more details.Native and modified proteins used in our experiments were at high degree of purification Figure . See add1-AT (1 mg/ml) in a 1:50 molar ratio was incubated for 24 hrs at 37°C in phosphate buffer. The SP-A/α1-AT mixture and single proteins were loaded in a Jasco PU 980 HPLC system  equipped with two Biosep-SEC-S 4000 columns  connected in series. Samples were eluted with 100 mM Na2HPO4, 2 mM CaCl2, pH 6.8 at a flow rate of 0.3 ml/ min, and monitored at 220 nm. The excluded (V0 = 12.43 ml) and total (Vt = 24.82 ml) volumes were determined using dextran and creatinine, respectively; a calibration curve was obtained by running through the column a set of standard proteins: α2-macroglobulin (725 kD), aldolase (158 kD), bovine serum albumin (67 kD), chymotrypsinogen (25 kD), and cytochrome C (12.5 kD). The results were reported as mean ± SD of three separate experiments. 2A mixture of SP-A (1.62 mg/ml) and α1-AT, purified SP-A, and SP-A/α1-AT complex collected from the Size Exclusion Chromatography experiments, were immobilized in 50 mM Na2CO3, pH 9.5 overnight at 4°C in a polypropylene plate . Plates were then brought at room temperature, washed with 150 mM NaCl, 0.1% Tween 20 (ELISA buffer), blocked for 1 h with 50 mM Na2CO3, 2% BSA pH 9.5, incubated for 2 hrs in the presence of primary antibodies diluted 1:500 , washed and finally reacted for 2 hrs with the appropriate biotinylated secondary antibodies diluted 1:5000 . After washing, 100 μL of avidin diluted 1:2000 were added, and samples were incubated for 30 min. Color development was achieved by incubating the samples with 1,2-phenylenediamine dihydrochloride . The reaction was stopped by addition of 100 μl of 0.5 M H2SO4 and OD was read at 490 nm with a Bio-Rad 680 Microplate Reader .250 ng of standard α1-ATs , 7.5 nM HNE was incubated for 15 min at 37°C with 0–100 nM inhibitor, in the presence of 2 mM MeOSucAlaAlaProValNA. All following kinetic experiments were derived from α1-ATs and SP-A/α1-ATs complexes . See additional file 1for more details.Rate constants were derived by competition experiments of HNE and αChy. Kinetic parameters were determined as described ,21. The 1-AT we studied whether Kass values, derived from incubating HNE with α1-AT, were modified by SP-A. Indeed we found a progressive decrease in the Kass as the SP-A concentrations increased . As expected, [Kass of Z α1-AT for HNE was 3.5 fold lower than that of the normal, M α1-AT. When Z α1-AT was coupled with increasing SP-A concentrations, a further decrease in Kass towards HNE was observed  at 37°C. The number of Cpm of the samples with SP-A were the same of wells without proteins. We concluded that our data were compatible with binding of α1-AT to SP-A.To exclude that the results were due to non-specific binding, we incubated 7.5 nM HNE with 0–100 nM α1-AT complex. As shown in Figure 1-AT (1 mg/ml), gave two peaks, one corresponding to free 1-AT (unreacted α1-AT) and one, with a theoretical molecular weight of 1,642 kD (1-AT/SP-A complex), possibly corresponding to a complex made by one molecule of SP-A (670 kD) and 18 molecules of α1-AT (54 kD), suggesting that, under the experimental conditions applied, each monomer of SP-A bound one molecule of α1-AT. Further evidence that the first peak of profile a  or partition ratio + 1) defines the number of moles of irreversible inhibitor required to completely inhibit 1 mole of target proteinase. The turnover number resulting from the interaction between unmodified SP-A and αe Figure . The sam1-AT retains its ability to inhibit HNE (Kass 3.38 × 107 M-1sec-1). We did, however, find that the inhibitory activity of α1-AT is greatly decreased in the presence of SP-A  SP-A deglycosylated and boiled with native α1-AT, and 2) both proteins deglycosylated  [1-AT is ~5 μM [The present data provide evidence for an  enzymes ,29, and d (BALf) . In addid (BALf) , whereasd (BALf) . The twod (BALf) . Since ad (BALf) ,9, its cis ~5 μM , we reasin vitro experiments indicate that the interaction between SP-A and α1-AT results in binding between them. This binding, which is calcium-dependent, appears to be complex since it could involve binding between the CRD of SP-A and carbohydrates on α1-AT, binding between SP-A carbohydrates and the protein backbone of α1-AT, and binding between the carbohydrate chains of both proteins.Indeed our 1-AT. Nevertheless, SP-A binds 11 molar parts of deglycosylated, fully active α1-AT  determinants  involved in cell adhesion during murine embryogenesis [x-Lex interactions appear to be calcium-dependent [Our experiments also suggest a possible carbohydrate/carbohydrate interaction between SP-A and αogenesis . Interesependent , by invoependent -41.It is difficult to postulate whether the three proposed mechanisms of binding take place simultaneously between native proteins. It may be that the CRD plays the main role and that the other two mechanisms are less important or take place only as artificial mechanisms once the proteins have been manipulated.1-AT. There are several known mechanisms that could explain the inactivation of α1-AT. Beside the physiologic irreversible suicide substrate mechanism by which α1-AT inhibits HNE [1-AT may also be inactivated by oxidation of methionine residue(s) located at or near the active site [1-AT inactivation is proteolytic degradation at or near the active site by a number of host and non-host, mostly microbial, proteinases [in vivo, thereby contributing to the imbalance between proteinases and inhibitors in the pathogenesis and progression of pulmonary emphysema, is still a debated issue.The binding with SP-A results in a decrease in the inhibition of HNE by αbits HNE , α1-AT mive site ,23. Anotteinases . Whether1-AT, that of Z α1-AT is further impaired by this latter's enhanced tendency to undergo spontaneous polymerization [1-AT is less efficient at inhibiting HNE, and has been demonstrated to be present in vivo, since Z α1-AT polymers have been detected in the BALf of Z α1-AT subjects with emphysema [1-AT and that the binding further reduces the Kass, which is already impaired with respect to that of α1-AT. Were this binding to happen in vivo, it would further decrease the antiproteinase activity of Z α1-AT.With respect to the inhibitory activity of αrization . This phmphysema . We foun1-AT inhibitory mechanism is open to speculation. α1-AT inactivation taking place in vitro upon interaction between the two glycoproteins seems to occur because of the functional slowdown of α1-AT in the presence of SP-A, the turnover number shifting from 1 to 24. After an initial, non-covalent, Michaelis-like complex, the reaction between α1-AT and HNE progresses, through an acyl-enzyme intermediate resulting from peptide bond hydrolysis, to either a loop-inserted covalent complex (inhibitory pathway) or a cleaved serpin and free proteinase (non-inhibitory or substrate pathway) [1-AT is 1  . The num1 Figure , indicat1 Figure . The num4 Figure , thus iny Figure .1-AT in vitro, an obvious limitation of the present paper is the lack of specific studies investigating a possible interaction between SP-A and α1-AT in vivo. Nevertheless, some indirect evidence suggesting that such an interaction might take place is available, although it is not possible to address a plausible expectation of physiologic or pathophysiologic relevance of these findings. For example, a recent report has shown that in human sputum supramolecular complexes with heparan sulfate/Syndecan-1 and proteinase and inhibitors are present [1-AT, NE as well, whose proteolytic activity is however not decreased . The large MW of SP-A makes difficult to highlight the occurrence of such supramolecular complexes including α1-AT by standard techniques [1-AT fragments were superimposed on spots of SP-A, in its upper, acidic position. These findings, confirmed by mass spectrometric MALDI-TOF analysis, would suggest a possible SP-A/α1-AT interaction taking place in vivo.In spite of the detailed dissection of the binding mechanism of SP-A to α present . These cchniques . Neverthchniques  has intr1-AT, and that this binding results in a significant decrease in the association rate constant of α1-AT for HNE. The mechanism of the binding seems to be predominantly mediated by the SP-A CRDs, as indicated by the calcium dependence and by the turnovers for inactivation, but other mechanisms may be involved, such as an interaction between SP-A carbohydrates and the α1-AT amino acid backbone or between carbohydrate chains of both glycoproteins. The presence of these complex binding mechanisms would exclude the hypothesis that the α1-AT inhibition occurred simply due to steric inhibition of the large SP-A molecule, but it would rather suggest a programmed, coordinated mechanism.We have shown that SP-A binds αin vitro interaction described here, if present in vivo, would be a novel mechanism of impairment of α1-AT inhibitory activity. It might represent a physiologic mechanism of regulating α1-AT activity, especially in acute conditions (for example during defense against infections agents) [1-AT would interfere with the physiologic role of proteinases. α1-AT is indeed a highly specialised proteinase inhibitor [1-AT downregulation  would imply the occurrence of intrinsic risks related to the overexpression of a nearly perfect and immortal inhibitor. Therefore, the formation of supramolecular complexes SP-A/α1-AT might be a sort of reserve mechanism, taking place in case of need.The  agents) , in whicnhibitor , but the1-AT deficiency, since it would contribute significantly to the complex mechanisms of imbalance between Z α1-AT and HNE in the lungs. Obviously, all these speculations need further investigations, first of all to understand whether or not SP-A/α1-AT binding is a relevant down-regulatory mechanism of α1-AT inhibitory activity in vivo.On the other hand, the interaction with SP-A would be of particular relevance in the pathogenesis of pulmonary emphysema associated with αα1-AT, α1-antitrypsinαChy, α chymotrypsin3)2SO, dimethylsulfoxide declare that they have no competing interests.MG participated in the study design, performed most experiments, and helped to draft the manuscript. AL participated in the deglycosylation experiments and carbohydrate chains isolation. PI designed the experiments for the α1-AT/SP-A complex identification, and helped to draft the manuscript. CDS participated in the kinetic studies. PR performed the experiments for the α1-AT/SP-A complex identification. DD performed the purification of SP-A and CRF. NC took part to some kinetic experiments. EP participated in the coordination of the study. AB performed the purification of SP-A and CRF, helped to draft the manuscript and critically reviewed it. ML conceived the study, participated in its design, and coordinated the manuscript final version. All authors read and approved the final manuscript.contain portion of Methods' section and include details on modification of native proteins used in the experiments and details of kinetic procedures.Click here for file


Pseudomonas aeruginosa using genetically-engineered SP-A mice and a library of signature-tagged P. aeruginosa mutants. A mutant with an insertion into flgE, the gene that encodes flagellar hook protein, was preferentially cleared by the SP-A+/+ mice, but survived in the SP-A−/− mice. Opsonization by SP-A did not play a role in flgE clearance. However, exposure to SP-A directly permeabilized and killed the flgE mutant, but not the wild-type parental strain. P. aeruginosa strains with mutation in other flagellar genes, as well as mucoid, nonmotile isolates from cystic fibrosis patients, were also permeabilized by SP-A. Provision of the wild-type fliC gene restored the resistance to SP-A-mediated membrane permeabilization in the fliC-deficient bacteria. In addition, non-mucoid, motile revertants of CF isolates reacquired resistance to SP-A-mediated membrane permeability. Resistance to SP-A was dependent on the presence of an intact flagellar structure, and independent of flagellar-dependent motility. We provide evidence that flagellar-deficient mutants harbor inadequate amounts of LPS required to resist membrane permeabilization by SP-A and cellular lysis by detergent targeting bacterial outer membranes. Thus, the flagellum of P. aeruginosa plays an indirect but important role resisting SP-A-mediated clearance and membrane permeabilization.Surfactant protein A (SP-A) is an important lung innate immune protein that kills microbial pathogens by opsonization and membrane permeabilization. We investigated the basis of SP-A-mediated pulmonary clearance of  Pseudomonas aeruginosa is a ubiquitous Gram-negative pathogen and a major cause of morbidity in patients with nosocomial pneumonia and cystic fibrosis (CF). Environmental strains and most clinical isolates of P. aeruginosa involved in acute infections are flagellated and motile P. aeruginosa isolated from chronically infected CF patients are nonmotile P. aeruginosa flagellin, the major structural protein of bacterial flagella P. aeruginosa flagellum is an important virulence factor P. aeruginosa strains lacking flagella are much less readily phagocytosed by alveolar macrophages and polymorphonuclear leukocytes P. aeruginosa to evade detection and clearance by host defense mechanisms during the chronic phase of CF lung infection Escherichia coli, Bordetella pertussis, and P. aeruginosa by permeabilizing the bacterial membrane SP-A is a large oligomeric protein composed of 18 subunits, each containing a collagen-like sequence and a calcium-dependent carbohydrate recognition domain, that is intimately associated with surfactant phospholipids lining the air-liquid interface of the lung P. aeruginosa library composed of 6912 mutants containing individual gene insertions were instilled into SP-A+/+ and SP-A−/− mice. Mutants which are cleared in an SP-A-dependent manner were tested for susceptibility to SP-A-mediated opsonization, phagocytic killing and permeabilization. In this paper, we characterize a SP-A sensitive P. aeruginosa mutant identified by comparative STM, the flagellar mutant flgE.We recently adapted signature tagged mutagenesis (STM) to comparatively identify microbial factors that confer resistance or susceptibility to SP-A P. aeruginosa PA01 STM mutant library −/− but not SP-A+/+ mice, as indicated by the presence of a PCR amplified oligo tag in bacterial output from SP-A−/− mouse lungs, and the absence of a PCR amplified oligo tag in bacterial output from SP-A+/+ mouse lungs (We utilized a se lungs .5Km2 (www.pseudomonas.com), and revealed a transposon insertion into the flgE (PA1080) gene,  within the operon that encodes four genes, flgB, flgC, flgC and flgD, no polarity effect is expected. The flgE gene encodes the hook structure on the flagellum from which the flagellar filament protrudes  gene, , betweenrotrudes  [20]. ThniTn5Km2   and fliD (PAO-fliD) fliC gene encodes flagellin, which comprises the filament of a flagellum whereas the fliD gene encodes the cap of a flagellum  of onmotile , and memonmotile . The levP. aeruginosa flagella play critical roles in resistance to SP-A-mediated membrane permeability, we predicted that nonmucoid, motile revertants of CF isolates CF51 and CF90, named CF51N and CF90N, would have increased resistance to SP-A-mediated membrane permeabilization. Both revertants were nonmucoid, and determined to be motile by swimming motility assay . We examined the flagellar-deficient mutants flgE, fliC and fliD for their increased susceptibility to 0.25% SDS. As shown in P. aeruginosa mutant bacteria in SP-A null and sufficient mice for mutations with increased susceptibility to pulmonary clearance by SP-A. One SP-A sensitive P. aeruginosa mutant with a disruption in flgE, a gene required for flagellar function, was identified. Enhanced uptake by phagocytes did not play a role in the preferential clearance of flgE bacteria, consistent with previous findings that flagellum-deficient mutants are resistant to phagocytosis by macrophages and neutrophils flgE bacteria were more readily membrane permeabilized and killed by SP-A than its wild-type parental strain, PA01. The importance of flagella in resisting the membrane destabilizing activities of SP-A was verified by multiple independent methods: Firstly, we showed that additional flagellar mutants fliC, fliD and flgK were also susceptible to SP-A-mediated membrane permeabilization. Secondly, genetic complementation of the fliC mutation with a copy of wild-type fliC gene restored resistance to SP-A-mediated membrane permeabilization. Thirdly, mucoid, nonmotile CF isolates of P. aeruginosa were also membrane permeabilized by SP-A. Finally, motile, nonmucoid revertants derived from the SP-A-susceptible CF isolates were able to reacquire resistance to SP-A. These results reveal that flagellum plays critical roles in conferring resistance to membrane permeabilization by SP-A.In this study, we screened a library of motAB, δmotCD mutant, which has a flagellum but is nonmotile P. aeruginosa PA01 than its isogenic flgE mutant bacteria. Secondly, flgE bacteria contain reduced levels of LPS during both logarithmic and stationary phases of growth. Thirdly, flagellar-deficient mutants flgE, fliC and fliD were all more susceptible to 0.25% SDS, indicative of weakened outer membranes.Our genetic analysis suggests that the presence of the flagellar structure rather than the flagellar-dependent motility is largely responsible for the resistance to SP-A-mediated membrane permeabilization. This conclusion is derived from our experimental evidence showing that the δmotAB, δmotCD mutant is resistant to SP-A, the susceptibility to SP-A appears to be independent of flagellar-mediated motility functions. However, we are unable to rule out other flagellum-dependent mechanisms that may protect P. aeruginosa from SP-A-mediated membrane permeabilization, including swimming chemotaxis and swarming motility P. aeruginosa encounters after inhalation into the alveolar space. In numerous species of Gram-negative bacteria including P. aeruginosa, swarmer cells alter their cellular morphology as well as induce the expression of PmrA-B regulated LPS modification genes pmrHFIJKLMN, and other LPS biosynthesis genes that are required for successful swarming P. aeruginosa cells may be more resistant to SP-A-mediated membrane permeabilization. It is also conceivable that the loss of swimming chemotaxis in the flagellum-deficient mutants render them unable to escape alveolar microenvironments in which SP-A is enriched, such as in surfactant-containing compartments. Another possible explanation, though less likely, is the inability of flagellar mutant to form biofilms. Flagellum-mediate attachment to substrate is the first step in the initiation of biofilm formation Because the nonmotile δP. aeruginosa strains, CF51N and CF90N, were more resistant to SP-A-mediated membrane permeabilization than their mucoid, nonmotile parental isolates CF51 and CF90. This transition correlated with the reacquisition of swimming motility. However, the resistance of CF51N and CF90N to SP-A-mediated membrane permeabilization was not as prominent when compared to the laboratory wild-type strain PA01. There are several possible explanations for this discrepancy. Firstly, it is likely that PA01 and CF51 and CF90 have different levels of intrinsic resistance to SP-A as these are not isogenic strains. Secondly, it is well known that many CF isolates of P. aeruginosa harbor mutated forms of LPS. In particular, CF isolates often undergo mutations in genes that participate in the biosynthesis of the highly antigenic B-band O-antigen of LPS P. aeruginosa's resistance to SP-A We found that motile, nonmucoid revertants derived from CF P. aeruginosaP. aeruginosa to safely repress the synthesis and assembly of the energy consuming and highly proinflammatory flagella P. aeruginosa being phagocytized by SP-A-facilitated macrophage and neutrophils.Reduction in the SP-A levels of the alveolar lining fluid, as has been reported in bacterial pneumonia, adult respiratory distress syndrome and CF in vivo than in vitro. One likely explanation for the discrepancy is that our in vitro membrane permeability conditions do not accurately model the environment in the lung. It is also possible that other antimicrobial factors within alveolar space, including SP-A, SP-D, lactoferrin, β-defensins and other antimicrobial peptides and proteins P. aeruginosa in vivo. We are currently examining whether these peptides act synergistically in clearance of P. aeruginosa.Our results suggest that the killing of flagellum-deficient mutants is more robust P. aeruginosa flagella play an important role in resistance to pulmonary clearance by SP-A and in particular to SP-A-mediated membrane permeabilization. Therapeutic strategies aimed at blocking flagellar biosynthesis or formation, or aerosolizing SP-A or SP-A fragments into septic airways where SP-A levels are severely depleted, may be reasonable therapeutic approaches to explore in the future.In summary, our data suggest that All chemicals, except where noted, were obtained from Sigma Chemical Co. .P. aeruginosa PA01 strains P. aeruginosa STM library 5 into the genome of PA01 strain from Holloway et alfliCfliDfliC strain PAOC-fliC flgK, δmotAB, δmotCD and δmotAB, δmotCD mutants, which were derived from PA14, was as previously described P. aeruginosa, CF51 and CF90, were obtained from the University of Cincinnati Adult Cystic Fibrosis Center following approval by the Institutional Review Board at the University of Cincinnati. Escherichia coli K12 used as a control in some experiments was transfected with the plasmid PVL 1393  to confer ampicillin resistance. Plasmid pUCP19GFP (GFP+ Apr), which constitutively expresses gfp, was used to label the strains for microscopic studies. Bacterial strains were grown in Luria broth (LB) for 16 hr at 37°C, and then suspended in LB with 20% glycerol and frozen in aliquots at −80°C. Before each assay, aliquots of the bacteria were cultured from frozen stocks in liquid LB or on LB agar plates (1.5% agar) with or without antibiotics for 3–16 hr at 37°C with continuous shaking, and then suspended in Tris-buffered saline, pH 7.4. Bacteria were cultured to stationary growth phase (OD600≈3.0) as indicated. The optical density at 600 nm was determined by spectrophotometry and correlated with numbers of viable bacteria by colony-forming units (cfu) after plating serial dilutions on agar plates. The antibiotic concentrations used were as follows: for E. coli, ampicillin (100 µg/ml); for P. aeruginosa, carbenicillin (300 µg/ml), gentamycin (100 µg/ml), kanamycin (100 µg/ml), rifampicin (50 µg/ml) and tetracycline (100 µg/ml).All bacterial strains, plasmid vectors, and their derivatives are described in Human SP-A was purified from the lung washings of patients with alveolar proteinosis by a modification of the method of Suwabe et al, Routine protein concentrations were determined with a bicinchoninic acid protein assay kit  using BSA as a standard. Protein samples were separated on 8–16% SDS-PAGE gel and stained with Coomassie blue or silver.−/− mice (a gift of J. Whitsett/T. Korfhagen) were developed from embryonic stem cells after disruption of the mouse SP-A gene by homologous recombination and maintained by breeding with Swiss Black mice, as previously reported +/+) mice were purchased from Charles River Laboratory (Boston). All comparisons made with the SP-A−/− mice were with age and strain-matched C3H/HEN controls. All animals were housed in positively ventilated microisolator cages with automatic recirculating water located in a room with laminar, high efficiency particulate-filtered air. The animals received autoclaved food, water, and bedding. Mice were handled in accordance with approved protocols through the Institutional Animal Care and Use Committee at the University of Cincinnati School of Medicine.Swiss Black SP-AP. aeruginosa PA01 STM library and screening methods were previously described +/+ and SP-A−/−mice were anaesthetized using isofluorane and inoculated intranasally with 1×107 cells of individual STM pools of 72 mutants in 50 µl of phosphate buffered saline buffer. After 16 hr of in vivo selection, lungs were removed from sacrificed mice, homogenized, and plated on LB agar. At least 10,000 bacterial colonies were harvested for multiplex PCR as described previously in vivo passaging were subsequently retested by single PCR of bacterial cells recovered from three separate mice as previously described The 7 bacterial cells comprised of a 1:1 ratio of wild-type PA01 and flgE. Lungs were harvested 16 hr after infection for bacterial load determination. The competitive index (CI) is defined as the output ratio of mutant to wild-type bacteria divided by the input ratio of mutant to wild-type bacteria 7flgE cells were performed by the intranasal route in SP-A+/+ and SP-A−/− mice (group of five). Attenuation was defined as the log10 difference in CFU of flgE recovered from the lung tissues of SP-A+/+ and SP-A−/− mice lung tissue 16 hr after inoculation.For competition assays, mice were inoculated intranasally with 1×10in vivo phagocytosis studies, 2.5×108 GFP-expressing PA01 or flgE bacteria were intranasally inoculated into SP-A+/+ and SP-A−/− mice. Two hours later, infected lungs were lavaged with PBS to obtain alveolar macrophages. For in vitro studies, freshly recovered mouse macrophages (∼5×105 cells from 3 uninfected SPA−/− mice) were adhered to chamber plastic culture slides, coated with 5% poly-D-lysine in 200 µl RPMI , for 2 hr at 37°C in 5% CO2 and used immediately. Live GFP-expressing PA01 or flgE in RPMI were preincubated with or without 25 µg/ml human SP-A for 1 hr at 37°C with rotation. The media was removed from each well, replaced with 250 µl of opsonized PA01 or flgE, and incubated for 1 hr at 37°C in 5% CO2 at a ratio of 100 bacteria to 1 macrophage. Chamber slides were washed 3 times with PBS containing 1 mM CaCl2. Fluorescence from extracellular bacteria was quenched with crystal violet (0.8 mg/ml). Following two additional washes, cells were fixed with 1% paraformaldehyde in PBS plus 1 mM CaCl2 for 10 min, and stained with Evans blue for 2 min. The percentage of macrophages with engulfed bacteria was quantified by phase contrast fluorescence microscopy. At least 200 macrophages were counted. Human neutrophils were purified and cultured as previously described For P. aeruginosa are capable of spontaneously reverting to a nonmucoid phenotype and acquiring flagellum based motility in a static growth conditions P. aeruginosa strains, CF51 and CF90, were inoculated into 5 ml LB in 18×150 mM culture tubes. These cultures were incubated at 37°C vertically in test tube racks without shaking for 72 hr, and then plated on regular LB agar. Individual nonmucoid colonies were replicated on swimming motility agar plates (LB solidified with 0.3% agar) to confirm reversion to motility phenotype.Previous studies have demonstrated that mucoid strains of Flagellum-dependent swimming motility was measured in fresh swimming motility agar plates with or without antibiotics. Plates were inoculated with 5 µl of an overnight culture of each strain of same cell density (as determined by spectrophotometer) and then incubated at 30°C for 24–48 hr. Motility was quantitatively assessed by examining the radius of opacity from the inoculation point, indicating the extent of bacterial swimming, compared to that of the wild-type PA01.P. aeruginosa cell wall integrity was assessed by determining permeability to a phosphatase substrate, Enzyme-Labeled Fluorescence 97  as described 8 logarithmic or stationary phase bacterial cells/ml in 100 µl of 5 mM Tris and 150 mM NaCl for 15 min at 37°C, and 100 µM ELF97 phosphatase substrate was added. Fluorescence was measured at excitation and emission wavelengths of 355 and 535 nm, respectively, for a period of 120 min.The effect of the SP-A or melittin on E. coli or P. aeruginosa bacteria were washed three times with 5 mM Tris, 150 mM NaCl, and adjusted to an OD600 of 1.0. A 100 µl aliquot of the cells was added to culture tubes containing SP-A (100 µg/ml) in 100 µl of 5mM Tris, 150mM NaCl, or to buffer alone as control. The mixtures were incubated at 37°C while shaking at 300 rpm for 1 hr, and stained according to the instructions provided by the supplier. The viability of the cells was checked under a fluorescence microscope. At least 1, 000 cells were counted.The viability of SP-A exposed bacteria was determined by LIVE/DEAD baclight™ Bacterial Viability Kit . Briefly, stationary phase P. aeruginosa PA01 and flgE cells were grown to late logarithmic or stationary phase in LB at 37°C and concentrated to A560 1.5 in PBS, and verified to contain the same viable cell counts (data not shown). The resultant cell pellet from 500 µl of sample was resuspended in 100 µl of PBS and incubated with or without 100 µg/ml of SP-A at 37°C for 30 min. An equal volume of 2×sample buffer  was added. The samples were boiled for 10 min and protein was digested by the addition of proteinase K to a final concentration of 50 µg/ml at 37°C overnight. The samples were boiled again for 10 min and cooled down to room temperature. A second volume of proteinase K, equal to the first, was added and incubated at 55°C for 3 h. Gross LPS profiles were determined by silver stain after SDS-PAGE electrophoresis.Western blot analyses were performed as described t-test and one-way analyses of variance (ANOVA). A significant difference was considered to be p<0.05.Statistical analysis was performed using the Student's


The objective of this study was to assess the responsiveness of the Minimum Data Set Activities of Daily Living (MDS-ADL) Scale to change over time by examining the change in physical function in adults with moderate to severe dementia with no comorbid illness who had been resident in a nursing home for over 90 days.Longitudinal data were collected on nursing home residents with moderate (n = 7001) or severe (n = 4616) dementia in one US state from the US national Minimum Data Set (MDS). Severity of dementia was determined by the MDS Cognitive Performance Scale (CPS). Physical function was assessed by summing the seven items  on the MDS activities of daily living (ADL) Long Form scale. Mean change over time of MDS-ADL scores were estimated at three and six months for residents with moderate (CPS score of 3) and severe (CPS score of 4 or 5) dementia.Physical function in residents with moderate cognitive impairment deteriorated over six months by an average of 1.78 points on the MDS-ADL Long Form scale, while those with severe cognitive impairment declined by an average of 1.70 points. Approximately one quarter of residents in both groups showed some improvement in physical function over the six month period. Residents with moderate cognitive impairment experienced the greatest deterioration in early-loss and mid-loss ADL items  and residents with severe cognitive impairment showed the greatest deterioration in activities related to eating, a late loss ADL.The MDS-ADL Long Form scale detected clinically meaningful change in physical function in a large cohort of long-stay nursing home residents with moderate to severe dementia, supporting its use as a research tool in future studies. Dementia, characterised by a progressive decline in cognitive function, affects up to 11 percent of those 65 and over, and 25 to 47 percent of those over the age of 85 -11.Dementia is widely prevalent in nursing homes residents ,13, howeThe Minimum Dataset Set (MDS) provides extensive data on residents of nursing homes. It was introduced in the United States in the early 1990s in response to the United States Congress in the Omnibus Budget Reconciliation Act of 1987  and inclAlthough the primary aim for the development of the MDS is to improve the quality of care in nursing homes, it also provides a unique database for researchers. A number of studies have assessed the validity of specific MDS rating scales and commented on their usefulness as research tools ,20. MDS This study aims to assess the responsiveness of the MDS-ADL scale to change over time by examining the change in physical function in adults with moderate and severe dementia with no comorbid illness who have been resident in a nursing home for over 90 days. We excluded residents with very severe dementia as they were likely to be already severely physically impaired, and those with very mild dementia as decline in ADL was less likely to be related directly to cognitive impairment.This study was a longitudinal analysis of a representative sample of nursing home residents in one US state. MDS assessments for residents with a length of stay of greater than 90 days in the year 2002 were collected from a US National Resident Assessment Instrument database  and follow-up data were extracted at three and six months . We chosIn order to minimise the effect of factors, other than cognitive impairment, in relation to change in ADL, residents were excluded if they had specific co-morbid diseases or conditions, or received specific treatments. These included history of learning disability, decreased auditory and visual acuity, problems with expressive speech, hypotension, seizure disorder, traumatic brain injury, manic depression, schizophrenia, HIV infection, septicaemia, weight fluctuation, inability to lay flat due to shortness of breath, dehydration, insufficient fluid consumption, internal bleeding, recurrent lung aspiration, end stage of disease, feeding by parenteral/IV or feeding tube, treatment including chemotherapy, radiation, tracheotomy care, transfusion, ventilator, for alcohol/drug problems, hospice care and respite care. Residents with concurrent or pre-existing limitations to mobility such as hip fracture, missing limb, bone fracture, cerebral palsy, stroke, hemiplegia/paresis, multiple sclerosis, paraplegia, Parkinson disease, or quadriplegia were also excluded. After applying these exclusions, the total number of residents available for study was 21,670. The most common reasons for exclusion were stroke (23%), presence of a learning disability (19%), impaired vision (10%) and hip fracture (5%). Nearly 40% of the sample had two or more of these exclusion criteria.Cognitive impairment was measured using the MDS Cognitive Performance Scale (CPS), which classifies all residents into seven levels of cognitive performance ranging from a score of '0' (intact) to '6' (very severe impairment). These scores are derived from four MDS variables – two cognitive items (short-term memory and decision-making), one communication item (ability to make self understood) and one ADL item (eating). The CPS measure of cognitive impairment correlates highly with well accepted and frequently used research rating scale scores, including Folstein's Mini-Mental Status Examination (MMSE) ,24-26. TChange in ADL was measured using the MDS-ADL scale, which has also been validated against a number of standardised instruments for measuring physical function -28. ThisBaseline ADL scores were documented for each long-stay resident, using data from their earliest MDS-ADL assessment. Follow-up was assembled from assessments collected three and six months after this baseline assessment. In this way, the study includes long-stay residents with a range of nursing home lengths of stay in excess of 90 days.We report here on the mean score calculated at baseline and at the three and six month follow-up, both for the overall ADL scale and for each of the seven individual ADL items. Mean change was calculated from baseline to three and six months for both cognitive impairment groups, and we report mean change, standard deviations (SD) and 95% confidence intervals (CI).The data used in this study were extracted from an aggregated anonymised database and submission to a research ethics committee was not required.The demographics and status at the three and six month follow-up are presented in Table In those with moderate cognitive impairment, mean ADL score at baseline was 14.77 (SD 7.34). The distribution of baseline scores for each ADL item is outlined in Figure Tables Residents in both cognitive groups showed a significant decline in all seven individual ADL items. The percentage of residents showing a decline ranged from 18% to 21% for the moderate cognitive impairment group and 14% to 29% for the severe impairment group at the three month assessment. This increased to ranges of 26% to 29% and 20% to 38% in the two cognitive groups, respectively, after six months. The percentage of residents who improved in each individual ADL item was smaller. Between 10% to 12% of those with moderate impairment and 9% and 13% in those with severe impairment improved at three months, and between 10% to 13% and 10% to 12% at six months  and in toileting functions (a mid loss ADL item). At six months around 26% of residents with moderate impairment experienced a decline in these functions compared to around 21% with severe impairment. Although the proportion of residents showing a decline in the function of eating (a late-loss ADL) was high for both groups, it was highest for those with severe impairment (29% moderate vs. 38% severe).This study was the first assessment of ADL change over time using the MDS-ADL Long Form scale in long-stay nursing home residents with moderate to severe cognitive impairment, with no comorbid illness. The MDS-ADL scale detected a mean decline in ADL for residents with moderate and severe impairment of 1.78 and 1.70 points respectively. A change of one point in the MDS-ADL scale denotes a clinically meaningful change. An increase of one point represents a precisely defined increased requirement for carer input confirmed as 'effective' during analysis and the extensive reviews and debriefings during the original development of the MDS-RAI . Not onlFurthermore, the study demonstrates that long-stay nursing home residents with moderate to severe cognitive impairment without comorbid illness have a high degree of physical impairment requiring assistance from staff in activities of daily living. Over time there is a general decline in ADL, indicating greater support is needed from nursing home staff. Comparison between the two dementia groups suggests a direct relationship between cognitive impairment and change in physical function over time; the more severe the cognitive impairment the more ADL items are likely to have been lost already, and the more likely they will continue to be lost in the ensuing months.A higher proportion of residents with moderate cognitive impairment experienced a decline in the early and mid-loss ADLs  compared to those more severely impaired. Residents with severe cognitive impairment showed the greatest decline in eating, a late-loss ADL. The main reason for the relatively small change in early and mid-loss ADLs  for residents with severe impairment is likely due to these residents already having experienced substantial decline in these ADLs. This pattern of ADL follows the hierarchy of early, mid and late loss ADL originally outlined by Katz  and deveTwo other studies have assessed ADL change using MDS data. One study using the MDS-ADL 20-point scale in a sample of long-stay nursing home residents with CPS scores of 0 to 5 reported a mean change of 0.95 (SD 3.0) over one year . AnotherThe main limitation of this study was that the analysis of ADL change over time does not include a number of residents who were lost to follow-up, either because they died or moved from the nursing home. However, this proportion was relatively small (19% at six months for both groups combined). Those residents who died may have shown a more marked decline in ADL compared to those who remained in the study. The study is also limited by the accuracy of the MDS dataset. However, for the cognitive function and ADL measures a high degree of reliability has been established .This study supports existing evidence that despite the decline in ADLs, some residents showed an improvement in physical function – even long-stay residents with severe cognitive impairment. Investigation of factors associated with improvement and further research into interventions that reduce ADL dependency, such as rehabilitation therapy and the prescription of pharmaceuticals that alter the rate of decline in cognitive function, will have the added benefit of reducing the ever-increasing pressures on nursing home care staff and quality of life of cognitively impaired residents. As the MDS assessment instrument is widely used in routine clinical practice and has demonstrated utility for pharmacoepidemiology -40, furtModerate to severe cognitive impairment was associated with decline in physical function, which confirms previous studies. However, further to this, we provide evidence of decline, and the rate of that decline, in a particularly vulnerable group in a relatively short period of time. This provides further supporting evidence for the use of the MDS-ADL scale as a research tool. The study also supports published research in the ordering of ADL loss into early, mid and late loss ADLs and its association with cognitive impairment. The more severe the cognitive impairment the more likely the subject is to have experienced decline in late loss ADLs. Most change in moderately impaired was in the early-loss and the mid-loss ADLs. These results could help direct the frequency and type of provision of care given to cognitively impaired residents of nursing homes and help inform future studies using MDS measures to assess the effect and cost benefits of interventions on the progression of dementia.ADL Activity of Daily LivingMDS Minimum Data SetCPS Cognitive Performance ScaleMMSE Mini-Mental Status ExaminationThis study was supported by funding from Janssen-Cilag.GIC, CLH, JNM and BEF contributed to the concept, design, data analysis and interpretation and writing of the manuscript. JA contributed to the data analysis and interpretation and writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: