Lawsonia intracellularis. In vitro studies have shown close bacterium-cell interaction followed by cellular uptake of the bacterium within 3 h post inoculation (PI). However, knowledge of the initial in vivo interaction between porcine intestinal epithelium and the bacterium is limited. The aims of the present study were to evaluate the usefulness of a ligated small intestinal loop model to study L. intracellularis infections and to obtain information on the very early L. intracellularis-enterocyte interactions.Porcine proliferative enteropathy in pigs is caused by the obligate, intracellular bacterium L. intracellularis inocula was applied to 10-11-week-old pigs. The inocula were 1) wild type bacteria derived from overnight incubation of L. intracellularis bacteria from spontaneous disease, 2) crude vaccine bacteria (Enterisol® Ileitis Vet), and 3) vaccine bacteria propagated in cell culture. The bacteria-enterocyte interaction was visualised using immunohistochemistry on specimens derived 1, 3 and 6 h PI respectively.A ligated small intestinal loop model using three different Although at a low level, close contact between bacteria and the enterocyte brush border including intracellular uptake of bacteria in mature enterocytes was seen at 3 and 6 h PI for the vaccine and the propagated vaccine inocula. Interaction between the wild-type bacteria and villus enterocytes was scarce and only seen at 6 h PI, where a few bacteria were found in close contact with the brush border.L. intracellularis interacts with villus enterocytes within 3 to 6 h after inoculation into intestinal loops and that the bacterium, as shown for the vaccine bacteria, propagated as well as non-propagated, was able to invade mature enterocytes. Thus, the study demonstrates the early intestinal invasion of L. intracellularis in vivo.The ligated intestinal loop model was useful with respect to maintaining an intact intestinal morphology for up to 6 h. Furthermore, the study demonstrated that  Lawsonia intracellularis is the infectious cause of proliferative enteropathy (PE) in pigs and a range of other animal species [in vitro isolation and cultivation depends on cell culture [in vitro has established the basis for vaccine development [in vivo is limited. However in vitro studies have shown close bacterium-cell interaction followed by cellular uptake of the bacterium within 3 h post inoculation (PI) [The bacterium  species ,2. The b culture . The sucelopment ,5. Knowlion (PI) . Recentlion (PI) .Brachyspira hyodysenteriae and Salmonella Typhimurium [et al. [L. intracellularis and enterocytes at 1 h PI but found no intracellular uptake of L. intracellularis or bacteria-enterocyte interactions. The aims of the present study were to evaluate the usefulness of an intestinal loop model to investigate L. intracellularis infections and to obtain information on very early L. intracellularis-enterocyte interactions. Compared to the study performed by McOrist et al. [L. intracellularis and the intestinal epithelium in the loops were extended to 1, 3 and 6 h. Moreover three different preparations of L. intracellularis inoculums were used at each point.Intestinal loop models have previously demonstrated their usefulness in studies of himurium -11. McOr [et al.  used ligt et al.  the expoL. intracellularis infection after a medicated eradication program. Twenty blood samples and 10 faecal samples from pigs with body weights (BW) of 30 to 60 kg were sampled twice from the herd and tested by ELISA and PCR methods as described elsewhere [L. intracellularis infection. All samples tested negative.Four pigs were purchased from a high health (specific pathogen free (SPF)) herd considered to be free of lsewhere ,14 to en® vet. 200 mg/ml, Novartis, Copenhagen, Denmark), given at a dosage of 20 mg/kg BW intramuscularly for 4 consecutive days. Faecal samples taken before and after medication were all found negative for L. intracellularis by PCR. To avoid adverse effect of the antibiotic treatment on the study, treatment with tiamulin was ceased at least 7 days before inoculation.The pigs were acclimatised for 2 weeks before entering the study. Clinical signs of disease were not observed during this period. As a precaution, all pigs were medicated with tiamulin at arrival  with free access to water and straw. The animals were fasted from the day before experimentation with free access to water with glucose added. The pigs were 10-11-week-old (BW 26 to 31 kg) at the time of surgery. The experimental study was approved by the Danish Animal Experiments Inspectorate under the Ministry of Justice.The four pigs were housed together and fed a standard diet L. intracellularis associated with PE in the material was confirmed by immunofluorescense (IF) using an anti-L. intracellularis monoclonal antibody (Law1-DK) [2 and MgCl2 diluted 1:10 in Milli Q water, with 5 mM EDTA  and incubated at 37°C for 80 min with occasional stirring. Detached epithelial cells and L. intracellularis bacteria were harvested by centrifugation at 5000 g for 30 min. The cells were resuspended in 100 ml Dulbecco's Modified Eagle medium (DMEM)  with 5% fetal bovine serum (FBS) , 1% L-glutamine , 2% amphotericin B , gentamycine 50 μg/ml  and vancomycine 100 μg/ml  and incubated overnight at 37°C, in an atmosphere of 8.8% CO2 and 8.0% O2. Next day the inocula were centrifuged at 5000 g for 30 min and resuspended in 50 ml of DMEM with 5% FBS and the epithelial cells were lysed by forcing the suspension through a 3.5 inch 22 Gauge spinal syringe . In vitro cell culture inoculations have shown an initial intracellular replication of similar level using this method compared to crude mucosal scraping (data not shown). Compared to crude mucosal scraping, the described method provides rather homogenous inoculum.Prior to the trial, porcine small intestines having PE were collected from a herd that had previously delivered infectious materials for successful experimental infections ,15. The Law1-DK) ,17. The L. intracellularis live vaccine  was purchased and held at 5°C until use. Immediately before inoculation into intestinal loops 0.8 g of freeze dried vaccine were dissolved in 5 ml of DMEM with 5% FBS. This corresponds to four doses according to manufacturer.A commercial L. intracellularis vaccine were produced by suspending 0.4 g of freeze dried vaccine in DMEM with 5% FBS and 1% L-glutamine and inoculating the suspension into a McCoy cell culture (ATCC number: CRL-1696), T-80 bottles with 15 ml medium seeded with 2 × 105 cells per ml from the day before. The infected cell cultures were incubated at 37°C, in 8.8% CO2 and 8.0% O2. Passage of infection was done by scraping of McCoy cells, which were lysed by forcing the suspension through a 3.5 inch 22 Gauge spinal syringe. Cell debris were removed by centrifugation at 150 g for 5 min, bacteria were harvested by centrifugation at 5000 g for 20 min. The bacterial pellet was re-suspended in 3 ml of medium and re-inoculated onto new cell cultures as described above. At the day of inoculation, two cell culture bottles with massive growth of L. intracellularis were used. The cells were scraped from the bottom and lysed as described above. Cells and bacteria were centrifuged at 5000 g for 20 min, where after the pellet was re-suspended in 10 ml of medium.Infected cell cultures based on the L. intracellularis in the different inocula was determined by serial 1:10 dilutions in sucrose potassium glutamate (SPG) with 5% FBS. Ten μl of each dilution were added to each well in a six-well glass slide and examined by indirect IF [L. intracellularis bacteria was counted at 40× objective magnification in 10 view fields corresponding to 1/25 of a well. The concentrations in the different types of inocula are shown in Table The concentration of irect IF . The numet al. [et al. [Isoflurane inhalation anaesthesia and surgical procedures were done as described by Grøndahl et al.  and modi [et al. . IsotoniLoops were inoculated for 1, 3 and 6 h for each inoculum. Initially four lower jejunal and four upper jejunal loops were made   for 20 min followed by washing in TBS 3 × 5 min. Slides were incubated with 0.05% protease  in TBS for 10 min followed by washing in TBS 3 × 5 min. Slides were incubated 1 h with polyclonal rabbit anti L. intracellularis antibody [+goat anti-rabbit conjungate . After washing for 3 × 5 min in TBS, reaction was developed for 15 min with a solution of 3-amino-9-ethylcarbozole (AEC)  followed by washing in TBS 3 × 5 min, counterstained by Mayer's haematoxylin and mounted with glycergel . All procedures were undertaken at a room temperature around 20°C.The loop specimens, each consisting of two full cross sections, were cut in 5 μm thick sections and mounted on Super Frost*/plus slides . Mounted slides were heated to 60°C, deparaffinised and rehydrated in xylene, graded series of alcohol and finally in water. Endogenous peroxidase activity was inhibited by incubation with 0.6% HL. intracellularis antigens were evaluated for both intestinal lumen and mucosa.Slides were evaluated by light microscopy using 40× and 63× objectives. In tissue from mock inoculated loops and inter-loop segments, absence of L. intracellularis antigen in the intestinal lumen and mucus overlying villus epithelium and in the crypts was noted but considered as a passive presence due to inoculation.In inoculated loops the presence of intracellular bacteria was evaluated, including a specific search for bacteria in the brush border with no free space in between enterocytes and the bacterium Figure  and 1D. L. intracellularis antigen was not found by IHC in the negative control loops or in the inter-loop segments.Ligation was found to induce grossly visible local mesenteric oedema and decreased intestinal wall tonus. Pallor of the intestinal wall indicating inadequate blood supply did not occur, congestion of mesentery and intestinal vessels remained at a low level and mild stromal oedema was the only histologically circulatory associated lesion Figure . TogetheL. intracellularis bacteria  were found in villus enterocytes 6 h PI  following oral inoculation with r et al. , which pr et al.  or olderr et al. , we beliL. intracellularis interacts with villus epithelium resulting in subsequent uptake in mature enterocytes. Furthermore, this study shows the usefulness of a pig ligated intestinal loop model as an alternative to in vitro models in investigating early bacteria-host cells interactions in L. intracellularis infections. However the limited number of bacteria seen in close association with or intracellular in enterocytes limits the models usefulness with regard to investigating factors enhancing or blocking cellular uptake.The study shows that as early as 3 to 6 h after inoculation into intestinal loops, The authors declare that they have no competing interests.TSB designed the study, prepared the inoculum, performed the surgical procedures, sampled materials, did the initial histopathological and immunohistochemical evaluations, participated in interpretation of results and drafted the manuscript. KS participated in designing the study and participated in the surgical procedures and drafting of the manuscript. JSA and TKJ participated in designing the study, interpretation of results and drafting of the manuscript. All authors read and approved the final manuscript.


Monomers of the collectin surfactant associated protein-A (SP-A) are arranged in trimers and higher oligomers. The state of oligomerization differs between individuals and likely affects SP-A's functional properties. SP-A can form aggregates together with other SP-A molecules. Here we report and assess a test system for the aggregate forming properties of SP-A in serum and broncho-alveolar lavage samples.Anti-SP-A antibodies fixed to latex beads bound SP-A at its N-terminal end and allowed the interaction with other SP-A molecules in a given sample by their C-terminal carbohydrate recognition domain (CRD) to agglutinate the beads to aggregates, which were quantified by light microscopy.SP-A aggregation was dependent on its concentration, the presence of calcium, and was dose-dependently inhibited by mannose. Unaffected by the presence of SP-D no aggregation was observed in absence of SP-A. The more complex the oligomeric structure of SP-A present in a particular sample, the better was its capability to induce aggregation at a given total concentration of SP-A. SP-A in serum agglutinated independently of the pulmonary disease; in contrast SP-A in lung lavage fluid was clearly inferior in patients with chronic bronchitis and particularly with cystic fibrosis compared to controls.The functional status of SP-A with respect to its aggregating properties in serum and lavage samples can be easily assessed. SP-A in lung lavage fluid in patients with severe neutrophilic bronchitis was inferior. Pulmonary surfactant covers the alveoli and terminal airspaces as a complex film made of lipids (90% by mass) and proteins. In the lungs, SP-A is the most abundant surfactant protein by weight. SP-A belongs like SP-D to a family of innate host defence proteins termed collectins because of the presence of a collagenous and a lectin-like domain .in vivo predominantly as octadecamers composed of six trimeric subunits forming a flower bouquet-like structure [The SP-A monomer consists of 228 amino acids and has a molecular weight of 26-32 kDa. Four domains can be differentiated in the primary structure [tructure . The C-tIn these formations SP-A participates in many physiological functions, including the interaction of the CRD with microorganisms, lectins or other molecules with attached sugar sequences. The CRD is also a ligand for phospholipids , involve2+ required was 0.5 mM for different species [2+ was enough to induce SP-A self-agglutination in physiological-ionic strength buffers at 37°C [In solution SP-A can self-agglutinate in presence of calcium via its CRD . The thr species ,19. Howe at 37°C ,21. As s at 37°C  SP-A sel at 37°C , possibl at 37°C . In addi at 37°C ,25. TherPseudomonas aeruginosa), 10 subjects with chronic bronchitis and 7 additional healthy controls. The clinical details of the subjects are depicted in Table Serum samples from four healthy individuals  were used for the establishment of the agglutination assay (see below), as described in the legends to the figures in detail. For the investigation of agglutination in patients with different diseases we used samples from 10 patients with cystic fibrosis  for 3 h. After washing 3 times, the membrane was incubated with a polyclonal recombinant rabbit-anti-human SP-A antibody  and 1% bovine serum albumin  overnight. Following 3 washes for 10 min with TBS-T (TBS with 0.05% Tween20) and incubation with the second goat-anti-rabbit antibody  and 1% BSA for 2 h, the membrane was washed finally. For signal detection the membrane was incubated for 1 min in a mixture of 500 μl of each reagent from Super Signal West Dura  and 1 ml of each reagent from ECL Western Blot Detection Reagents . Signals were measured with a DIANA III camera  by 15 min duration of exposure and evaluated by AIDA Software . The reproducibility of the slot-blot assay was good, as determined by the coefficient of variation at the highest concentration  of the standard curve.A slot blot was used to quantify SP-A concentration. 200 μl aliquots of the chromatography fractions and 100 μl standard recombinant human SP-A  were applied onto blot membranes in a Slot-Blot gadget . The membrane was blocked with blocking buffer  of a carboxylate-modified, streptavidin-labeled latex bead mixture  was incubated with 200 μl biotinylated rabbit anti-goat antibody  for 18 h at 23°C in 0.2 ml Eppendorf tubes . The mixture was washed 2 times with Hank solution  and resuspended in 600 μl Hank solution. Then 5 μl of the samples  were incubated with 2 μl bead suspension at 6.5 mM Ca2+, 3 μl pure water and 0.01 μg of the anti-SP-A antibody  for 3 h at 4°C on a microscope slide  under a cover slip . To avoid non-specific agglutination of the SP-A, all assays were done in the presence of 0.1% (v/v) Triton X-100. Under the light-optical microscope  the biggest, pure bead-agglutinate was searched and photographed. The picture was viewed by the software Adobe Photoshop  at a size of 764×573 pixels. Then, the size of the determined agglutinate was measured in pixel by drawing a square around it  were tested by using serum volumes from 0 to 0.6 μl in 0.1 μl steps Figure . The tesFirst the specificity for SP-A, and not for SP-D, and the dependency of the assay on the presence of a complete binding chain consisting of the beads coupled with the anti-goat antibody, and the anti-human-SP-A antibody was shown. Only the addition of SP-A (10 μl of a 100 ng/ml recombinant human SP-A) and the presence of all components induced agglutination Table . RecombiNext the calcium and carbohydrate dependency of the assay was assessed Table . ResultsIn 10 patients with cystic fibrosis the serum SP-A agglutination ability was measured. The size of the agglutinates was 1364 pixel ± 616 (range 667 to 2862) and did not significantly differ from that obtained with the corresponding lavage fluid .Because the size of the SP-A agglutinates varied between individuals and it is known that SP-A is present in different oligomeric forms, we assumed that differences in the structural complexity may be responsible for the variation observed. Therefore, we further analyzed the SP-A self-agglutination ability of the fractions derived from gel chromatography, containing different oligomeric structural forms of SP-A. Gel chromatography usually yields three peaks containing the main different oligomeric structures: an initial peak represented by fraction 10 (F10) containing the largest forms, i.e. octadecamers (18 mers) or more complex structural forms of SP-A, followed by the second peak represented by fraction 15 (F15), containing 6 to 12 mers, and finally the third peak represented by fraction 20 (F20) containing the smallest forms, i.e. di- and trimers or monomers. Using the SP-A self-agglutination assay, each fraction was analyzed adjusted with respect to the SP-A concentration.In sera of all three study populations, CF, bronchitis and controls, there was a significant difference regarding the SP-A self-agglutination ability between the fractions Figure . There wIn contrast to serum, the SP-A derived from patient BAL samples indicated a major impact on the ability of the fractions to induce SP-A dependent agglutination. For all BAL fractions the SP-A oligomers derived from controls agglutinated significantly better than the SP-A oligomers derived from patients. Also samples from the bronchitis population agglutinated better than those from CF patients Figure .Comparing the SP-A self-agglutination ability of the fractions between BAL and serum within the groups of CF and bronchitis patients there was also a significant difference Figure  and 4b, To test if qualitative changes within the SP-A from a specific patient might be responsible for the observed effects, certain gel-chromatography fractions of SP-A present in different patients at different levels, were isolated, the concentration adjusted and tested for their capacity to agglutinate; no differences were found (data not shown). This result suggests that the relative composition of the whole sample with respect to the different oligomeric forms is responsible for its agglutination activity and not potential differences among the oligomeric forms within a particular fraction.In conclusion, these results showed that SP-A in serum had a better functional ability than SP-A in BAL from patients with CF or chronic bronchitis, but not from controls. Also, in BAL of patients with a lung disease the self-agglutination ability of SP-A was diminished compared to controls. In all three study groups, the more complex SP-A oligomers were, the better was their self-agglutination ability.Systematic evaluation of the conditions to induce microscopically visible aggregation of SP-A showed the dependency of the test on the concentration of SP-A added into the reaction, the presence of calcium, and dose-dependent inhibition by mannose. It was established that SP-D alone, serum without SP-A or without SP-A and SP-D did not induce aggregation. Further we showed that the more complex the oligomeric structure of SP-A present in a particular sample was, the better was its capability to induce aggregation at a given total concentration of SP-A. Comparing the SP-A agglutination ability of the fractions no differences were observed for SP-A derived from serum independent of the pulmonary disease state. In contrast, in BAL fluid, it was clearly shown that patients with chronic bronchitis had some, and those with cystic fibrosis had the most severe impairments of SP-A capability to induce aggregation. Thus, the agglutination assay was measuring the ability of SP-A molecules to aggregate as an active process induced in the assay. This ability was mainly a reflection of the oligomeric structure of SP-A in the samples, further of the origin of the samples and the disease state.ex vivo analysis in very small volume samples of serum and BAL. There is also no step necessary to isolate the SP-A before the measurement. As the goal of the study was to establish the assay, the patient group sizes were relatively small. However, the results were highly reproducible. Further studies in larger populations are desirable and are underway.The strength of this approach is the specific measurement of the CRD binding of SP-A by a very simple and fast method and the possibility for Our data confirm the calcium-dependency of SP-A self-agglutination by its CRD, previously reported ,21,28. Ain vitro data, to a lesser extent than octadecamers. This data is important as for the first time ex vivo SP-A from clinically well defined patients with other lung diseases than pulmonary alveolar proteinosis (PAP) was used extensively. SP-A from PAP patients differs from normal SP-A, as the large molecular structures are mostly non-reducible [We found that also smaller SP-A structures can agglutinate, but as expected from educible ,31. Furteducible , as welleducible .SP-D is another lung collectin, also present in the alveolar space and in serum, and shows structural similarities to SP-A. SP-D monomers form trimers and four trimers assemble to a cruciform structure with CRDs at their end . SimilarThe data clearly show a local pulmonary deficiency of SP-A dependent agglutination in the pulmonary diseases investigated, whereas no abnormalities were seen in systemic circulation.In future studies it will be important to investigate the correlation of this assay with other functional properties of SP-A, and more importantly the correlation with lung function and long term disease outcome variables, in order to determine its role as a surrogate marker and potential modulator of the clinical course.In summary, we describe the characteristics of the test system to assess functional properties of SP-A in serum and lavage samples. Samples from healthy controls, bronchitis patients and patients with cystic fibrosis induced SP-A self-association; the more complex the SP-A structures were, the better were the self-agglutination abilities. Comparing the functional capabilities of corresponding SP-A structures separated from serum and BAL, in the two lung disease groups, BAL samples had inferior SP-A self-agglutination abilities. This clearly showed a strong structure - function relation of SP-A for normal subjects and some disease states with altered SP-A.SP-A; SP-D; CRDThe authors declare that they have no competing interests.SMH carried out the microscopic and biochemical studies. MG conceived the study. MG and SMH designed the experiments, performed the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/10/59/prepub


Surfactant protein D (SP-D) is a collectin with immuno-regulatory functions, which may depend on oligomerization. Anti-microbial and anti-inflammatory properties have been attributed to multimeric SP-D variants, while trimeric subunits per se have been suggested to enhance inflammation. Previously, we reported low circulating SP-D in early rheumatoid arthritis (RA), and the present investigation aims to extend these data by serial SP-D serum measurements, studies on synovial fluid, SP-D size distribution and genotyping in patients with early RA.One-hundred-and-sixty disease-modifying antirheumatic drug (DMARD) naïve RA patients with disease duration less than six months were studied prospectively for four years  trial) including disease activity measures  score), autoantibodies, x-ray findings and SP-D. SP-D was quantified by enzyme-linked immunosorbent assay (ELISA) and molecular size distribution was assessed by gel filtration chromatography. Further, SP-D Met11Thr single nucleotide polymorphism (SNP) analysis was performed.P < 0.001). SP-D increased slightly during follow-up (P < 0.001), but was still subnormal at four years after adjustment for confounders (P < 0.001). SP-D in synovial fluid was up to 2.5-fold lower than in serum. While multimeric variants were detected in serum, SP-D in synovial fluid comprised trimeric subunits only. There were no significant associations between genotype distribution and SP-D. Baseline SP-D was inversely associated to CRP and HAQ score. A similar relationship was observed regarding temporal changes in SP-D and CRP (zero to four years). SP-D was not associated to x-ray findings.Serum SP-D was significantly lower in RA patients at baseline compared with healthy controls . Within recent years, search for innate immune system abnormalities in rheumatoid arthritis (RA) has attracted considerable attention . Thus, lCollectins are pattern recognition molecules, which preferentially bind to carbohydrate moieties expressed on a variety of pathogens (pathogen associated molecular patterns (PAMPs)), thereby enhancing aggregation, opsonisation or MBL-mediated complement activation . SP-D haThe present investigation extends our previous observation by readdressing the possible association between SP-D and the Met11Thr polymorphism in early, untreated RA, and by studying the correlation between SP-D and disease activity measures and radiographic progression during a four-year interventional study on DMARD naïve patients with RA of recent onset. In addition, we compared the SP-D molecular size distribution in synovial fluid and corresponding sera.One-hundred-and-sixty RA patients were included in the multicenter, randomized, double-blinded, parallel-group, placebo-controlled CIMESTRA trial ,24. BrieThe treatment protocol compared methotrexate (MTX) plus cyclosporine vs. MTX plus placebo. During the first eight weeks patients were assessed fortnightly and every four weeks thereafter. Subsequently, whenever synovitis was present MTX dose was escalated by 2.5 mg from 7.5 mg/week to maximum 20 mg/week followed by a stepwise cyclosporine/placebo-cyclosporine increment (0.5 mg/kg) every four weeks from 2.5 mg/kg to maximum 4.0 mg/kg. In addition, intra-articular betamethasone (7 mg/l) was injected into swollen joints at any visit (maximum four joints or 4 ml per visit). During the second year, hydroxychloroquine (200 mg/day) was added and cyclosporine/placebo was tapered to zero, while MTX was continued ,24. DuriSerum was obtained from routinely drawn non-fasting blood samples collected between 08.00 a.m. to 2.00 p.m. Samples were allowed to clot at room temperature followed by centrifugation at 3,000 × g for 10 minutes. Sera were stored at -80°C.SP-D was measured at baseline, after two weeks, one and six months, and after one, two, three and four years using a five-layered sandwich ELISA as previously described . In contRadiographs of hands, wrists, and forefeet were obtained at baseline (n = 155), and annually thereafter. After four years 137 radiographs were available, but only 133 patients had radiographs available at baseline and at four years. Radiographs were scored according to Sharp-van der Heijde by an independent senior radiologist who was aware of the sequence of x-ray recordings . The ann2, and pH was adjusted to 7.9 by adding 28.5 μl 1 M TRIS pH 8.6 prior to analysis.Corresponding serum and synovial fluid samples were available from 20 RA patients with joint effusions before treatment. Synovial fluid was collected by aseptic technique before injection of glucocorticoid and stored at -80°C. Before analysis, the samples were centrifuged 30 minutes at 400 × g and subsequently the supernatant was incubated four hour at 37°C with bovine testicular hyaluronidase  to reduce viscosity  to 300 μl synovial fluid). Subsequently, they were centrifuged at 20.000 × g for 10 minutes at 4°C. The supernatant was assayed for SP-D by ELISA. The possible trapping of SP-D in the synovial fluid pellet was studied by incubating the pellet with ethylenediaminetetraacetic acid (EDTA) 0.52 M in a TRIS-buffered saline (TBS) buffer (pH 7.4) at 37°C in 30 minutes followed by centrifugation in four minutes at 20.000 × g and 4°C. A total of 50 μl of the resulting supernatant was re-calcified with 60 μl of 1 M CaClGel filtration chromatography was done on available synovial fluid samples (n = 11) and corresponding sera. Hyaluronidase-treated samples (200 μl) were applied to an analytical Superose 6 column connected to a fast-performance liquid chromatography system  using TBS (pH 7.4) containing 10 mM EDTA and 0.05% emulphogen as eluent at a flow rate of 24 ml/hr. Fractions of 0.2 ml were collected and quantified by the SP-D ELISA. SP-D was eluted as two structurally different forms with high and low molecular weight (SP-D multimers (fraction 10 to 18) and SP-D trimers (fraction 24 to 38)). Size chromatography on healthy serum followed by SDS-PAGE and Western blotting has yielded protein bands at > 250 kDa for multimeric SP-D, and 90 kDa, 43 kDa and 40 kDa for trimeric SP-D ,19.Genomic DNA was isolated from EDTA stabilized whole blood. Applied Biosystems (Assay-by-design)  designed primers and probes for the non-synonymous substitutions of DNA-bases of the SP-D gene resulting in the Met/Thr variant. The genotyping procedure has been described previously . Human lAll statistical analyses were conducted using STATA version 9.2 . Comparisons between groups were done by Mann-Whitney U-test or Fischer's Exact Test, and if analysing more than two groups, Kruskal-Wallis test was used. Spearman Rank Correlation analysis was applied when appropriate. Comparison between patients and controls was performed using linear regression models, where control twins were clustered in pairs. Linear regression was also applied in the prospective analysis of SP-D in RA patients, where repeated measurements in the individual patient were clustered. We used logistic regression to assess whether baseline SP-D could predict radiographic progression after four years with adjustment for gender, age, smoking, anti-CCP and radiographic status at baseline. Robust estimation of standard error was calculated. To approximate a normal distribution, SP-D was logarithmically transformed when used as continuous, dependent variable in linear regression analyses.2-analysis. Comparisons of genotype and allele frequencies in patients and controls were performed by logistic regression with adjustment for gender and age or by Fishers Exact test.One individual from each healthy twin pair was used for genotype and allele frequency estimation. The genotype frequencies were tested for Hardy-Weinberg equilibrium by . Completers' analysis was also performed and gave similar results (data not shown). Results are presented as median  if not otherwise stated. Of 160 patients included, 61 (38%) did not complete the four-year protocol. The reasons for drop-out were adverse events (11), treatment failure (10), patients' request (13) and other (27). Fifty-six (35%) left the study during the first two years. Patients who dropped out did not differ from completers with regard to demographic and clinical variables at baseline (data not shown). At baseline one patient had serum SP-D of 8,106 ng/ml. This patient subsequently developed severe pulmonary fibrosis and was excluded from the statistical analyses.The demographic characteristics of the RA patients at baseline and the control population are shown in Table Including patients with radiographs available at both baseline and after four years (N = 133), 53%, 23% and 49% progressed radiographically according to TSS, JSN and ES score, respectively. Of note, however, radiographic progression at four-year follow-up was small in terms of Sharp/van der Heijde units (median (iqr): TSS 2 (0 to 7) to 5 (0 to 11), JSN 0 (0 to 2) to 0 (0 to 4) and ES 2 (0 to 5) to 3 (0 to 8)).P < 0.001). This difference persisted after adjustment for age, gender and current smoking status (P < 0.001) and was also present at four years after adjustment for confounders (P < 0.001). Compared to baseline, SP-D had increased in RA patients at four years  even when adjusting for gender, age and smoking status (p < 0.001). However, at four years, SP-D was still lower in RA patients as compared to controls with adjustment for confounders (P < 0.001). There was no significant correlation between age and SP-D in the RA population . Likewise, there was no significant gender difference among RA patients. In contrast, SP-D increased significantly with age in healthy subjects , and control males had significantly higher levels of SP-D compared to females . No significant difference in SP-D at baseline was observed between patients with respect to anti-CCP, IgM-RF status or any SE present . Furthermore, SP-D did not differ between smoking SE positive vs. non-smoking SE positive patients (P = 0.13).Baseline SP-D in RA patients was 693 ng/ml  vs. 913 ng/ml  in controls (P < 0.001). We found no association between SP-D and radiographic data including estimated annual progression rate (data not shown). Baseline SP-D did not predict radiographic progression  at four years (P = 0.46)The CRP change from baseline to four years (Δ) correlated inversely to the SP-D change , Figure Synovial fluid was obtained from 20 patients at baseline. Median SP-D in synovial fluid was 275 ng/ml 221; 299). SP-D in corresponding sera was 678 ng/ml . SP-D in synovial fluid and serum levels correlated significantly . The distribution of genotypes and allele frequencies is presented in Table 4. When adjusting for gender and age, there was no overrepresentation of Thr11Thr in RA patients as compared with controls Table . CirculaBased on the structural similarity between SP-D and MBL and our preliminary report on low circulating SP-D in RA , this inThe cause of low SP-D in RA is uncertain and different mechanisms may be involved. Altered SP-D expression due to genotype abnormalities should be considered. Thus, in healthy subjects the Thr11-variant is associated with low SP-D in the circulation .In the previous study by Hoegh et al , the Thrin vitro and in vivo experiments have indicated that SP-D enhances clearance of DNA and apoptotic cells by macrophages, thereby reducing anti-dsDNA antibody generation [Decreased SP-D in RA could be attributable to increased clearance from the circulation, for example, by deposition in inflamed tissues or complex formation with, for example, microbial or cellular waste ,37. Thusneration ,39,40. Sneration ,42.In order to further elucidate the possible role of SP-D in joint inflammation, we quantified SP-D in paired serum and synovial samples and studied the molecular size distribution in serum and synovial fluid. We found a SP-D serum:synovial fluid ratio at approximately 3:1, which indicates that SP-D reaches the joint cavity by diffusion (bulk flow) . The difSP-D in serum is suggested to originate primarily from pulmonary leakage . It has When interpreting the present results, the relatively large number of drop-outs should be considered. However, there was no difference with respect to baseline characteristics between completers and non-completers and the intention to treat analysis included a large majority of the cohort.SP-D did not correlate to age in RA patients, but tended to be higher in males compared to females. By contrast, SP-D was significantly higher in control males as compared to females, and SP-D correlated positively with age. This disparity may be due to the different sizes of the RA and control populations and the relative overrepresentation of females in the RA cohort. Due to the difference in age distribution in the two populations and rather few controls aged above 50 years we used logistic regression with adjustment for gender and age instead of regular frequency matching in comparisons between controls and patients.Circulating SP-D is subnormal at disease onset and after four years treatment in RA. There were no SP-D Met11Thr associations with RA disease activity or subnormal SP-D. While SP-D did not correlate with x-ray progression, we found an inverse association between SP-D and disease activity markers suggesting that low systemic SP-D is involved in the initiation or maintenance of synovitis. Whereas both multimeric and trimeric SP-D variants occurred in serum, only low molecular forms were detected in synovial fluid where it may contribute to joint inflammation. Overall, this study suggests that SP-D is implicated in RA pathogenesis at the protein level.Anti-CCP: antibodies against cyclic citrullinated peptides; CI: confidence interval; CIMESTRA: Ciclosporine, Methotrexate, Steroid in RA; CRP: c-reactive protein; DAS: disease activity score; DMARD: disease modifying anti-rheumatic drug; ES: erosion score; HAQ: health assessment questionnaire; HLA: human leukocyte antigen; IgM-RF: IgM-rheumatoid factor; JSN: Joint Space Narrowing score; MBL: mannan-binding lectin; MTX: methotrexate; RA: rheumatoid arthritis; SE: shared epitopes; SNP: single nucleotide polymorphism; SP-D: surfactant protein -D; TBS: TRIS-buffered saline; TSS: total Sharp-van der Heijde Score; VAS: visual analogue scale.The authors declare that they have no competing interests.All authors contributed to the design of the study, and the acquisition and interpretation of data. AFC performed the statistical analysis. AFC, PJ and GL drafted the manuscript. KJ carried out the immunoassays and gel filtration chromatography. AGJ and AV evaluated the x-ray data. All authors read and approved the final manuscript.


The increasing interest in small non-coding RNAs (ncRNAs) such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) and recent advances in sequencing technology have yielded large numbers of short (18-32 nt) RNA sequences from different organisms, some of which are derived from small nucleolar RNAs (snoRNAs) and transfer RNAs (tRNAs). We observed that these short ncRNAs frequently cover the entire length of annotated snoRNAs or tRNAs, which suggests that other loci specifying similar ncRNAs can be identified by clusters of short RNA sequences.Drosophila melanogaster, and mapped them to the Drosophila genome. Approximately 6 million perfectly mapping sequence tags were then assembled into 521,302 tag-contigs (TCs) based on tag overlap. Most transposon-derived sequences, exons and annotated miRNAs, tRNAs and snoRNAs are detected by TCs, which show distinct patterns of length and tag-depth for different categories. The typical length and tag-depth of snoRNA-derived TCs was used to predict 7 previously unrecognized box H/ACA and 26 box C/D snoRNA candidates. We also identified one snRNA candidate and 86 loci with a high number of tags that are yet to be annotated, 7 of which have a particular 18mer motif and are located in introns of genes involved in development. A subset of new snoRNA candidates and putative ncRNA candidates was verified by Northern blot.We combined publicly available datasets of tens of millions of short RNA sequence tags from In this study, we have introduced a new approach to identify new members of known classes of ncRNAs based on the features of TCs corresponding to known ncRNAs. A large number of the identified TCs are yet to be examined experimentally suggesting that many more novel ncRNAs remain to be discovered. C. elegans [Following the discovery of microRNAs (miRNAs) and the RNA interference pathway in  elegans , and the elegans ,3, have  elegans -6. Such  elegans -10. More elegans ,9,11-20,Analysis of such data has shown that miRNA-sized short RNA fragments are commonly derived from other small RNAs, notably transfer RNAs (tRNAs) and small nucleolar RNAs (snoRNAs) ,22. IndeDrosophila cell lines and tissues. Approximately 6 million tags were perfectly mapped to the D. melanogaster genome, excluding chrM , and chrU and chrUextra (which contain un-assembled and un-mapped scaffolds). Each "tag" consists of one to many reads which reflects the number of times the tag was cloned and sequenced. For tags mapping to multiple locations on the genome, the number of reads of the given tag was arbitrarily distributed evenly to each mapping locus (see Methods). The tags were then assembled into 521,302 tag-contigs (TCs), comprised of contiguously overlapping (by 1 nt or more) tags present in the same strand orientation  datasets . Several studies have identified siRNAs and piRNAs processed from transposons such as LINEs, DNAs and LTRs, and correspondingly there is considerable TC coverage over these repeat elements  of Drosophila tRNAs. Some of these TCs are longer than the mature corresponding tRNAs, which in Drosophila fall within the range of 60-100 nt. This may well reflect the detection of uncleaved precursors [Most of the TCs that overlap with well-characterized classes of ncRNAs, such as miRNAs, tRNAs and snoRNAs, have particular length and tag-depth features that are consistent with the features of the corresponding ncRNA classes. TCs overlapping miRNAs show a high peak for the range of 20-30 nt in length Fig. , while Tecursors , as thesDrosophila). The shorter TCs from tRNAs and snoRNAs that comprise the peaks on the left-hand side of Fig. TCs covering the full length of 53% of all annotated box C/D snoRNAs and 56% of annotated box H/ACA snoRNAs Fig.  are withFig. For this analysis we excluded TCs derived from annotated exons, ncRNAs, transposons or other repeats annotated in FlyBase  see Met, Fig. S1AJ809564) . Considering that most snRNAs in D. simulans were predicted from BLAST analysis with known snRNAs of D. melanogaster against the D. simulans genome sequence [Among the 164 TCs overlapping known box H/ACA snoRNAs Table , 64 are sequence , the TC AJ784386) , in human and mouse [A total of 107 box C/D snoRNAs (out of 134 annotated in FlyBase ) are oveAfter these box H/ACA and box C/D snoRNA predictions, 100,157 TCs still remain unannotated. To explore these further, we initially selected 135 highly expressed TCs with a tag-depth ≥100 as Fig. The location of group 1 TCs is strongly biased to introns of genes involved in development Table  and withThe 29 TCs in Group2 are evenly distributed in introns 13 TCs) and intergenic spacers (16 TCs), and intronic TCs and intergenic TCs do not differ in tag-depth distributions. Two of these TCs, ncRNA_12 and ncRNA_37, were detected by genomic scanning for the 18mer motif derived from the Group1 TCs when 1 mismatch was allowed. Those two TCs along with ncRNA_15 are mainly composed of sequence reads specifically obtained from S2 cells   and an endogenous-siRNA cluster. We also found that the majority of experimentally detected snmRNAs [His clusters) are overlapped by TCs, another demonstration of the validity of the approach. In fact, one TC (chr3R_3300274_3300719) overlapping snmRNA:331 corresponds to the 7SK RNA recently identified in Drosophila [snmRNA:331.TCs derived from two different classes of snoRNAs showed distinct features in their length and tag-depth distributions, and the use of these characteristic features along with the their signature motifs predicted novel snoRNAs. Proof-of-principle of this approach is provided by the successful recall of two previously known but not FlyBase-annotated snoRNAs as well as the  snmRNAs   . These pet al.  bypassed snoRNAs , but it Unlike the prediction of box C/D snoRNAs and putative ncRNAs of uncharacterized classes, the box H/ACA snoRNA prediction incorporated another filter that excluded non-intronic TCs. This was based on the fact that 92% of the known box H/ACA snoRNAs reside in introns, and reduced the number of predictions from 18  to 7. It is uncertain how many of the 10 discarded TCs (excluding one snRNA candidate) may be genuine box H/ACA snoRNAs, but the high validation rate of the intronic subset (4 out of 4 tested) indicates that the incorporation of the location filter improved the specificity of the prediction.bona fide ncRNAs that can also be re-assembled, as is evidently the case for snoRNAs. Moreover, the large amount of unannotated TCs located in introns and intergenic regions  and 7 of 11 64%) putative unclassified transcripts of either group 1 or group 3. This is a high rate of validation, given that the likelihood of detecting a signal in Northern blots is dependent on the expression level of the candidate in the tissue concerned. In fact, the confirmed candidates have generally greater tag-depths than the unconfirmed , similar to TCs covering known transposon-derived sequences, and different to the ~ 100,000 TCs which were used for this study. We also observed that a large portion (37%) of these 27,000 TCs is found within reported siRNA/piRNA clusters ,14,20,42Drosophila melanogaster short RNA into hundreds of thousands tag-contigs and associated subsets of them with known ncRNAs such as snoRNAs and tRNAs. The characteristic features of TCs overlapping with known ncRNAs were used to predict 7 and 26 box H/ACA and box C/D snoRNA candidates, respectively, in addition to one snRNA and many novel unclassified ncRNA candidates, a substantial fraction of which were experimentally validated. We conclude that deep sequencing from short reads may be used to identify new members of known and novel classes of ncRNAs, including those that are significantly longer than the reads themselves.Several studies investigating the population of small RNAs have yielded millions of sequence reads. In this study, we combined all publicly available sequence data from D. melanogaster genome sequence assembly Release 3  from the Berkeley Drosophila Genome Project. Annotation of exons, introns, UTRs, and ncRNAs are from FlyBase 5.12 [We used the ase 5.12 . MicroRNase 5.12 . Repeatsase 5.12  in FlyBahttp://www.ncbi.nlm.nih.gov/geo in SOFT format  is defined as a genomic region that has mapped sequence tags with the same strand orientation contiguously overlapping with each other by at least 1 nt , intergenic and non-repeat TCs, respectively. Intersection of TCs with other annotation was performed through local mirror of University of California, Santa Cruz Genome Browser .Among the total of 521,302 TCs, 126,962 are outside of annotated exons, ncRNAs, transposons and other repeats annotated in FlyBase [http://www.R-project.org and in-house software along with the bioinformatics toolkit, Biopieces http://www.biopieces.org.The scatter-plots of lengths (nt) against tag-depth (log10) of TCs were generated by R A sequence tag overlapping with phastCons elements  by at leFor each of the unannotated TCs within the ranges of length and tag-depth of box C/D snoRNA-derived TCs, box C motif (RUGAUGA) and box D motif (CUGA) were searched within +/- 10 bp from the 5' end and within +/- 10 bp from the 3' end, respectively . One mishttp://amigo.geneontology.org/cgi-bin/amigo/term_enrichment.The Gene Ontology term enrichment analyses in this study were performed using GO-TermFinder  through Drosophila's late embryos (12-18 h) and S2 cells using TRIZOL reagent (Invitrogen). Fifteen micrograms of total RNA was separated on 1% denaturing agarose gels, and 25 micrograms of total RNA was on 2% agarose/formaldehyde gels and 10% denaturing polyacrylamide gels. RNA separated using denaturing agarose was then transferred to Hybond-N+ membranes  using downward capillary transfer, then UV-crosslinked and baked at 80°C for 1 hour. RNA separated by denaturing polyacrylamide gels was transferred to Hybond-Nx membranes  by use of a semidry transfer cell apparatus, and cross-linked using the EDC method as outlined in [http://web.wi.mit.edu/bartel/pub/protocols/miRNA_Nrthrns_Protocol.pdf. In brief, the pre-hybridization/hybridization buffer contained 5× SSC, 20 mM Na2HPO4 pH 7.2, 7% SDS, and 2× Denhardt's solution. Blots were pre-hybridized for at least 2 hours at 50°C, then probes which had been end-labeled with γ-32P ATP by use of T4 polynucleotide kinase (New England Biolabs), or end-labeled with α-32P dCTP by use of terminal transferase (New England Biolabs), were added to the hybridization chamber and incubated with the blots overnight at 50°C. After three washes with non-stringent wash buffer containing 3× SSC, 25 mM NaH2PO4 pH 7.5, and 5% SDS, blots were given a final wash with 1× SSC and 1% SDS. The membrane was then exposed to a phosphoimager overnight and scanned.Total RNA was extracted from lined in . AntisenThe secondary structures of 7 box H/ACA snoRNA candidates (Additional file CJ contributed to the study design, carried out the data analysis and drafted the manuscript. MAH contributed to the study design and carried out the data collection and preparation. IVM contributed to the study design and helped draft the manuscript. DJK carried out the experimental validations. JSM supervised the study and helped write the manuscript. All authors read and approved the final manuscript.IVM current address: Department of Molecular and Cellular Biology, Institute of Chemical Biology and Fundamental Medicine, 630090 Novosibirsk, Russia.Supplementary figures and tables. This file contains supplementary figures S1-S9 and tables S1-S6 in PDF format. Fig. S1: The distributions of relative tag-depths of TCs against oppositely stranded overlapping TCs; Fig. S2: Tag-depth distributions of TCs covering full-length ncRNAs; Fig. S3: Length and tag-depth distribution of highly expressed unannotated TCs; Fig. S4: Number of sequence reads per million per different samples for Group1 TCs; Fig. S5: Number of sequence reads per million per different samples for Group2 TCs; Fig. S6: Number of sequence reads per million per sample for each of tested snoRNA and unclassified ncRNA candidates; Fig. S7: The accumulation of sequence reads per each base across the three TCs; Fig. S8: Screenshot of UCSC genome browser for ncRNA_64, an intronic TC at chr3R_24973624_24973672; Fig. S9: Length and tag-depth distributions of TCs of different categories; Table S1: Coverage of TCs over transposons and exons; Table S2: Predictions of snoRNA candidates; Table S3: 18mer motif sites derived from group1 TCs with 0, 1 and 2 mismatches; Table S4: Group3 TCs mapped to existing ncRNAs; Table S5: 29 unannotated TCs in group3; Table S6: Conservation of sequence tags comprising ncRNA-derived TCs.Click here for fileList of all snRNA, box C/D and box H/ACA snoRNA candidates. This file contains genomic loci, strand, tag-depths and predicted lengths of 7 box H/ACA snoRNA candidates, 26 box C/D snoRNA candidates and 1 snRNA candidate.Click here for fileList of all group1, group2 and group3 TCs. This file contains genomic loci, strand, tag-depths and predicted lengths of all putative ncRNAs of uncharacterized classes: 8 group1 ncRNAs, 29 group2 ncRNAs and 49 group3 ncRNAs.Click here for fileList of probes for Northern blot. This file contains probe sequences for experimental validation of 4 box H/ACA snoRNA candidates, 5 box C/D snoRNA candidates, 1 snRNA candidate and 10 putative ncRNA of uncharacterized class along with their other information such as genomic loci, predicted lengths and tag-depths.Click here for fileSequences of experimentally validated novel snoRNAs and unclassified ncRNA candidates. This file contains DNA sequences of all experimentally validated ncRNAs: 4 box H/ACA snoRNAs, 3 box C/D snoRNAs, 1 snRNA and 13 ncRNAs of uncharacterized class.Click here for fileSequence conservation and secondary structure predictions of snoRNA candidates. This file contains UCSC genome browser screenshots of 7 box H/ACA snoRNA candidates and 26 box C/D snoRNA candidates showing sequence conservation and tag distributions over each candidate. The positions of box H, ACA, C and D motifs are indicated by the red boxes. Predicted secondary structures of box H/ACA snoRNA candidates are provided below each of the screenshots.Click here for file


Although substance P (SP) is an important primary afferent modulator in nociceptive processes, it is unclear whether SP regulates its own release from primary sensory neurons.2-terminal kinase inhibitor increased the GR73632-evoked SP release.Using a highly sensitive radioimmunoassay for SP, we have demonstrated that the activation of neurokinin-1 receptor by SP or GR73632 (a potent neurokinin-1 receptor agonist) triggered an increase of SP release from cultured adult rat dorsal root ganglion (DRG) neurons depending on the dose and exposure time within 60 min, and thereafter, the SP release level gradually decreased over 360 min. Accompanying the SP release, a significant reduction in the percentage of neurons expressing neurokinin-1 receptor on their membranes during exposure to SP (200 pg/dish) occurred time dependently . The GR73632-evoked  SP release was attenuated by several inhibitors for mitogen-activated protein kinase kinase, p38 mitogen-activated protein (MAP) kinase and cyclooxygenase-2 (COX-2), protein kinase C (PKC), respectively. In contrast, a c-Jun NHThese results indicate that the neurokinin-1 receptor activation by its agonists regulates the SP release process involving the activation of MAP kinases, PKCs and COX-2 from cultured DRG neurons. Briefly, 4% paraformaldehyde-fixed cultured DRG cells on coverglasses were incubated with anti-neurokinin-1 receptor  or anti-SP serum  . After tAfter a 10 min pretreatment with the presence or absence of 1 μM CP-96,345 , the cultured DRG cells were incubated in serum-free DMEM (containing peptidase inhibitors) with or without SP (200 pg/dish) for 10, 60, 180, 360 minutes, respectively. The isolation of cytosolic and membrane fractions from these DRG cells was performed with a standard cell compartment kit fractionation procedure  according to the manufacturer's instructions. Protein concentrations were determined, and then the neurokinin-1 receptor levels in the same amounts of cytosolic and membrane proteins were analyzed separately by a Western blot analysis.At the end of the SP release experiments, the cell samples were processed for Western blot analysis as previously described . PrimaryP values less than 0.05 were considered significant.The data are presented as the mean ± S.E.M. Statistical analyses were performed using a one-way ANOVA followed by Bonferroni's test. COX: cyclooxygenase; DMEM: Dulbecco's modified Eagle's medium; DRG: dorsal root ganglion; ERK: extracellular signal-regulated kinase; MAP: mitogen-activated protein; MEK: mitogen-activated protein kinase kinase; PKA: protein kinase A; PKC: protein kinase C; PLC: phospholipase C; SP: substance P.The author(s) declare that they have no competing interests.HBT participated in the design of the study, carried out all the experiments outlined in the manuscripts, performed the data analysis and wrote the manuscript. YSL designed and performed the immunocytochemical staining, and contributed to the analysis and interpretation of the data. KA designed the immunocytochemical staining, assisted with the data analysis and interpretation. YN participated in the design of the study, assisted with the data analysis and interpretation, and wrote the manuscript. All authors have read and approved the final manuscript.


Svante Pääbo works on the edge of what's possible. He ignites our imagination, unlocking tightly held secrets in ancient remains. By patiently and meticulously working out techniques to extract genetic information from skin, teeth, bones, and excrement, Pääbo has become the leader of the ancient DNA pack. Sloths, cave bears, moas, wooly mammoths, extinct bees, and Neanderthals—all have succumbed to his scrutiny.Pääbo see  broke grA sterile hotel lobby wasn't the venue I had hoped for in interviewing Pääbo. I would have preferred a natural history museum, or, even better, an archaeological dig to stimulate the interview juices. But, when I realized he was attending the American Society of Human Genetics meeting in San Diego last fall, I grabbed the opportunity. Though jet-lagged, he gamely agreed to a 10 p.m. interview, following the Presidential symposium and only seven hours prior to his planned surfing excursion in La Jolla.Jane Gitschier: What happened in your youth to make you so interested in Egypt?Svante Pääbo: Sometime in my late boyhood, I got very interested in archeology. I went around after big storms in Sweden to spots in which trees had fallen over. You can look at the roots for things—stone age pottery and things like that. Even in the suburbs of Stockholm, where I grew up, there was still a forest around. And you could run around and have fun. It certainly was common for kids to play “stone age” behind the school in the forest.JG: Was there something that triggered your particular interest in archeology?SP: Not really, but I think it was the realization that you could actually go out yourself and find these things!JG: And did you find stuff?SP: Yes, they are still at my mother's place, in a glass cabinet—thousands of pot shards that I collected. You can sometimes passel them together and can get part of a pot that was used 3,000 years ago. Quite fascinating.Also, my mother had taken me to Egypt because I was interested in Egyptology. I think I was 14. That made me fascinated, as so many young kids are, with Egypt and mummies and pyramids. It was mainly the trips I took to Egypt—three times with my mom.JG: Wow, was your mother into Egypt, too?SP: It was partially through my fascination, but I think she still goes to lectures on Egyptology in Stockholm.JG: Were your parents scientists?SP: Yes. I grew up with my mother. My mom and dad were not married. My mom was a chemist and worked in industry. My dad had another family, but he was a biochemist and studied prostaglandins.JG: And then you worked in biochemistry?SP: I first started studying Egyptology and things like that at the University [Uppsala] and got somehow disappointed. It was not as romantic as I thought it would be. And after a year and a half or so, I didn't know what to do, because this wasn't really “it”. So I started studying medicine because I figured I would get a profession. And it was also a way into basic research.JG: I read your paper from 1985 about sequencing the mummy remains. What was the genesis of that?SP: I knew there were hundreds and thousands of mummies around in museums and that they found hundreds of new ones every year, and molecular cloning in bacteria was a rather new thing at the time, so I found in the literature that no one had tried to extract DNA from Egyptian mummies, or any old remains actually. So I started to do that as a hobby in late evenings and weekends, secretly from my thesis advisor.JG: As a lowly graduate student, where do you find a piece of mummy to start this investigation?SP: I had studied Egyptology, so the professor of Egyptology knew me quite well. He helped me to sample a mummy in the museum in Uppsala. He also had very good connections with a very large museum in Berlin, which was East Berlin at the time. Germany has a long, long tradition in Egyptology, going back to the 19th century. After the British Museum and the Museum in Paris, the Berlin Museum has the biggest collection outside Egypt.JG: So you went with your professor to the museum in East Berlin…SP: He had convinced them of our idea in advance. We sampled, I think, 36 different mummies. Small samples, of course.JG: Had people ever looked at mummy tissue before, at things like proteins?SP: There had been some work on histology of mummies, and there had been some work on trying immunoreactivity of proteins extracted from it, with very mixed results. I don't think there were any convincing results from Egyptian mummies.JG: In what kind of state are the mummies? Are you wearing gloves or masks? What are you doing?SP: We only worked with mummies that were already unwrapped and with things that were broken, so we were not destroying anything to get to the tissues. With a scalpel we removed a little piece. It was the first time this was done, so we had no big qualms about contamination. I had no idea this could be such a big issue.JG: What did you do with these 36 scalpeled samples?SP: We screened them with histology. We looked at both with traditional stainings—hematoxylin-and-eosin staining and staining with ethidium bromide—and under UV light to see if one could see any fluorescence from DNA. In the skin of a particular mummy, you could see that the cell nuclei lit up. So, there was DNA there and at the place you would expect it to be.JG: Was your interest in this simply the challenge of getting DNA sequence out of it or was there a bigger idea?SP: It was clearly the idea that if you could study the DNA of ancient Egyptians, you could elucidate aspects of Egyptian history that you couldn't by traditional sources of archeology and the written records.JG: Do you mean the relationships between people?SP: Population history. Say, when Alexander the Great conquered Egypt, did that mean there were lots of people from Greece who actually came there and settled there? When the Assyrians came there, did that have an influence? Or was the population continuous? Political things that influenced the population.Since then it has become clear that it is almost impossible to work with human remains because of contamination. It is very hard to exclude that the DNA you look at is not contaminated with modern humans.JG: Then, how do we know that this sequence in the 1985 paper is in fact the sequence of a real Egyptian?SP: In hindsight, we don't know that. In 1985, I had no idea how hard this is [to retrieve uncontaminated ancient DNA sequences] and thus did not do the controls we now know are necessary. We've even published at a later point on this.JG: But there have been no data to refute the sequence of this mummy.SP: But nothing to prove it either! It could well have been contamination, and if that was the last that had ever been written on ancient DNA, that would have been a sad state of affairs and the end of the field.JG: Have people gone on to look at more mummy DNA since then?SP: Egyptian mummies are actually quite badly preserved; also animal mummies. This probably has to do with climate. It seems the cooler it is, the better preserved things are. We looked at a few Neanderthal remains from Israel and Palestine and they have so far not yielded any DNA.JG: What is it like to travel all over the world to try to get specimens?SP: To sample these things takes building confidence—in museum curators and archeologists and paleontologists—that we can actually get information from them. And, of course, it is a balance for a curator between a destructive sampling for scientific progress against responsibility for future generations to preserve these things. With justification, you can sometimes say that if you can just wait 30 years, methods will be so much better.What you actually do is a several stage process, where you first take very small samples, of say 10 mg, and just see if there are amino acids preserved—the amino acid profile of collagen. If there is no collagen preservation, it turns out there is hardly ever DNA. We can already exclude a lot of remains that way.And then we take samples of 100–200 mg, extract DNA, and see if we can find Neanderthal DNA. And then for the genome project where we need larger samples, we use bones that have very little morphological information. So in the Museum in Zagreb, which houses the Vindija remains, we screen bones of which it cannot be said from the morphology if they are human or animal. By doing extraction from 100 mg, you can determine the species from the mitochondrial DNA. So the paleontologists gain something—they learn what species the different bones come from, and so it is easy to justify taking half a gram from them if they turn out to be Neanderthal bones.JG: So now, back to the mummies. That was not your thesis.SP: No, but then I had to tell my thesis advisor that I had done this! He was happy that it had been successful. I don't think he would have been so happy if I had presented it before it happened.JG: Then you went to Allan Wilson's lab. What did you work on there?SP: Really developing the technology for ancient DNA. PCR had just come around, and I had tried to do PCR back in Europe with water baths. It was really when Taq polymerase came and the thermocycler, and Allan's lab was, I think, the first academic lab to have one.JG: And what organism were you working on?SP: We started again on Egyptian mummies but rather soon switched to animals of different sorts. We worked on moas from New Zealand, which are extinct flightless birds, ground sloths, and the marsupial wolf from Australia.JG: Why did you switch? Better preserved or no contamination issues?SP: Contamination with human DNA became apparent rather soon when I started using PCR, when you could repeat experiments and do lots of negative controls.JG: Was your passion, though, in these extinct animals? Or was your intent to go back to human lineages?SP: It took on its own life. It became very fascinating to develop the technology and overcome the problems with contamination, problems with errors in the sequences, things like that.JG: How did your move to Germany come about?SP: Pretty much by chance. I had a girlfriend—I've had both boyfriends and girlfriends in my life—but at some point I had a girlfriend who was from Munich. The professor of genetics there asked me to give a seminar at some point, and then he said they had this professorship coming up in a year and I ought to apply, which I did, and by the time it had all worked out … I had no girlfriend there anymore!But, it was clearly a very, very good offer. The biggest break I got in my life. I became a full professor there after being a post-doc, directly, without being an assistant professor. The opposite to your prejudice about how European science works, in that case. There was a constellation of people there who were not risk-averse.JG: How did the Institute in Leipzig come to be?SP: After German reunification, there was the political will and the money to start institutes in East Germany at the same density, according to population, as there were in West Germany. This was the chance to start a number of new Max Planck Institutes. And there was a very conscious idea to ask—in what areas of science is Germany particularly weak? And of course, anthropology is such.JG: They were weak in anthropology?SP: Absolutely. Due to what happened during Nazi times. There had been an Institute of Anthropology where Mengele was an assistant. And so no one had really wanted to touch anthropology since that time.now start an institute in evolutionary anthropology not burdened by any traditions? And the idea sort of grew among people who discussed this. If we were to do this, we would ask the question of what makes humans unique in a comparative way across different disciplines—humanities or sciences—but it should all be empirical, not just a question of philosophy.So there was a lot discussion if one would dare to do it or if it was too politically sensitive. And then, once the decision was made to actually do something in the direction of human evolution, it was in fact a big advantage that there were no big traditions. Because you could say—how would we It ended up being an institute with five departments: Paleontology; Primatology, with research sites in Africa, studying chimps and gorillas in their natural habitats and their range of behaviors; Comparative Psychology, which has a primate facility in Leipzig, the only research facility in the world with all the great apes, and it's part of the zoo. Visitors can actually observe the experiments. They do experiments in cognitive development in human children and ape children for the first 12 months of life—the very same experiments. When do you see the things that set humans apart and what are these things? And there is Comparative Linguistics—what is common to all human languages? And then Genetics.JG: What have you got your eye on, other than Neanderthals?SP: We are very interested in comparative genomics of the apes in general. We are sequencing the bonobo—the last ape that has not been sequenced—with the 454 technology. The amazing thing is with these high-throughput technologies, a lab can now take on projects that a genome center did just a few years ago.For the Neanderthal, we have to do so much sequencing that we do it with the 454 company in a collaboration. We test the libraries we make in the clean room, and when we have a good library, it goes to Bradford [Connecticut] for the production sequencing.JG: Do people come to you with crazy ideas that intrigue you?SP: Yes. Our lab pretty much functions on the ideas that are born in the group, and our lab is a little unusual in that we spend a lot of time discussing every project every week, in a group setting. All the groups that have to do with ancient DNA—all the people sit together once a week and discuss their work—particularly things that don't work. Gene expression is another day, or genomics, and it is in these sorts of eternal discussions that ideas come. It is quite rare that anyone sitting alone thinking in their room comes up with any big ideas. It's really by throwing lots of ideas around. If you have a hundred ideas on the table, then one of them turns out to be really cool.JG: What other mysteries would you like to address?SP: What one dreams about is defining the genetic changes that we all share today but that made modern humans so special. That made us colonize the whole place, every little speck of land on the planet, which, after all, archaic humans had not done. They had been around for two million years, but they never crossed the water where they couldn't see land on the other side. Modern humans have been around for a hundred thousand years and we've colonized Easter Island, right?JG: Not to mention we went to the moon.SP: Exactly! We're crazy. Nothing really stops us. So there is something really special there in how we behave—to somehow understand that!Something we also talk a lot about in the group these days is how genetic diversity is structured in humans. I think we are still far too much in the pattern of looking at diversity of different groups and the boundaries between them because of how we have sampled and how we have looked at things. I think, in a way, it is sad that people interested in population history have gone out and sampled according to preconceived ideas of what groups are there, be those linguistic groups or racial groups, and of course if you sample like that you come up with some differences between groups, and say yes, they are there. Rather than going out and just sampling without regard for anything other than geography.JG: So you mean, just getting a map and sampling a person at every grid point.SP: Yes, and the logistics of doing that over a whole continent are almost impossible.But coming back to Egypt again, what I would really like to do is have a boat and sail along the Nile from the Mediterranean, where people are really “European-like” to the source of the Nile in Lake Victoria, where people are really “African-like”, and sample every 50 kilometers along this corridor through the Sahara and just see how this transition occurs. Are there sharp borders or is there a gradient? And that would be a powerful thing to look at, and it would be feasible.JG: Well, if Craig Venter can sail around the world collecting microbial samples, you should be able to get a boat for a trip up the Nile.SP: I'm not as independently wealthy as he is!JG: Now, in our final moments, I want to ask what has been your favorite project.SP: I tend to think the current project is the favorite project. Every project has this manic thing about enormous expectations in it, that often are not borne out to the extent that you imagine, but it's what drives it. And then you come down to the reality of things.But clearly now, I would say, being able to see the Neanderthal genome is something that just a couple of years ago I wouldn't think would be possible in my lifetime. And now, it is.JG: What do you think it is about Neanderthals that excites people?SP: Quite recently, only some 2,000 generations or so ago, there were some other humans with us who were similar, but clearly distinct from us. It gives us some perspective.Sometimes I like to make the thought experiment—that they made it another 2,000 generations and were here! What consequences would that have? Would racism against Neanderthals have been even worse than the sort of racism we experience today, because they truly were a bit different, or is it that if we had had something like that that was another human form, then perhaps we wouldn't have been able to distance ourselves so much from the great apes as we do now —not making this enormous distinction we do now between what we call humans and all other organisms which we call animals.It could have gone either way—we can never know, but these are things it is interesting to think about because it puts these issues in our society in perspective. Perhaps somewhere there is the fascination.


Noise is the insidious of all industrial pollutants, involving every industry and causing severe hearing loss in every country in the world. Exposure to excessive noise is the major avoidable cause of permanent hearing impairment. Worldwide, 16% of the disabling hearing loss in adults is attributed to occupational noise, ranging from 7 to 21% in the various subregions. The estimated cost of noise to developed countries ranges from 0.2 to 2% of the gross domestic product (GDP). Noise-induced hearing loss (NIHL) is bilateral and symmetrical, usually affecting the higher frequencies  and then spreading to the lower frequencies . Other major health effects are lack of concentration, irritation, fatigue, headache, sleep disturbances, etc. The major industries responsible for excessive noise and exposing workers to hazardous levels of noise are textile, printing, saw mills, mining, etc. Hearing protectors should be used when engineering controls and work practices are not feasible for reducing noise exposure to safe levels. Earmuffs, ear plugs and ear canal caps are the main types of hearing protectors. In India, NIHL has been a compensable disease since 1948. It is only in 1996 that the first case got compensation. Awareness should be created among workers about the harmful effects of noise on hearing and other body systems by implementing compulsory education and training programs. There are very few published studies of NIHL in India. More extensive studies are needed to know the exact prevalence of NIHL among the various industries in India. Noise is the insidious of all industrial pollutants, involving every industry and causing severe hearing loss in every country in the world. Occupational hearing loss includes acoustic traumatic injury and noise-induced hearing loss (NIHL), and can be defined as a partial or complete hearing loss in one or both ears as the result of ones employment. Exposure to excessive noise is the major avoidable cause of permanent hearing impairment worldwide. NIHL is an important public health priority because as populations live longer and industrialization spreads, NIHL will add substantially to the global burden of disability. In many countries, excessive noise is the biggest compensatable occupational hazard. Worldwide, 16% of the disabling hearing loss in adults is attributed to occupational noise, ranging from 7 to 21% in the various subregions. The  The 6] TIn India, occupational permissible exposure limit for 8 h time weighted average is 90 dBA.[The noise levels in different industries is given in Audiometry is a standard test to detect and evaluate hearing loss. Audiometry is used to determine the auditory threshold of an individual to pure tones of 250-8000 Hz and sound levels between 10 (the hearing threshold of intact ears) and 110 dB . The patient should not have been exposed to noise during the previous 16 h to eliminate the effects of a temporary threshold shift. Air conduction is measured by ear phones placed on the ears, while bone conduction is measured by placing a vibrator in contact with the skull behind the ears. Each ear is evaluated separately and test results are reported on a graph known as an audiogram. Comparison of air and bone conduction allows classification of hearing loss as conductive or sensorineural. The audiogram in case of NIHL is characterized by an onset of hearing loss at 4000 Hz, visible as a dip in the audiogram. As exposure to excessive noise level continues, neighboring frequencies are progressively affected and the dip broadens, intruding into neighboring frequencies. NIHL is usually bilateral and shows a similar pattern in both the ears. The difference between the two ears should not exceed 15 dB at 500, 1000 and 2000 Hz and 30 dB at 3000, 4000 and 6000 Hz, respectively.The WHO recommended the following classification on the basis of the pure tone audiogram taking the average of the thresholds of hearing for frequencies of 500, 1000, 2000 and 4000 Hz.[To express the hearing impairment in terms of percentage, different countries and professional bodies have adopted their own system to calculate the percentage. One of the methods to find hearing impairment is as follows:From the audiogram, calculate the average of the thresholds of hearing for frequencies of 500, 1000, 2000, 4000 and 6000 Hz.Deduct from it 25 dB (as there is no impairment up to 25 dB).Multiply it by 1.5.This is the percentage of hearing impairment for that ear. Similarly, calculate the percentage of hearing impairment for the other ear. The percentage handicap of an individual is calculated using the formula given below.The published studies of NIHL in India are limited, which are cited here. In a cross-sectional study, the hearing status of tractor-driving farmers (TDFs) was compared with that of non-TDFs. All participants were interviewed for details of work routine and noise exposures. Audiogram analysis showed higher prevalence of abnormalities in TDFs. TDFs more often had a higher frequency of hearing loss when compared with non-TDFs.A study was conducted in heavy engineering industry, which included machines shop and press divisions. The sound levels ranged from 83 to 116 dBA. Hearing impairment was progressive in all the study groups.In a textile mill weavers study, the sound levels were around 102-104 dBA and the hearing acuity of the textile weavers was found to be poor. NIHL at 4000 Hz was as high as 30 dB in the age range 25-29 years, 40 dB in the age range 30-34 years and 45 dB in the age range 35-39 years.A study was conducted in a drug and pharmaceutical company where the noise levels were 100-105 dBA. Significant NIHL was found in the workers.A survey on the effects of noise pollution on traffic policemen in the city of Hyderabad, India, carried out by the Society to Aid the Hearing Impaired, revealed that 76% had NIHL. Among these, all those who had completed 5 years in the traffic wing had hearing loss in various degrees.The National institute of miners’ health (NIMH) has carried out NIHL studies in various mines. NIHL was prevalent among 12.8% of the employees. Moderate NIHL was detected in 10.2% and severe NIHL was observed in 2.6% of the employees.There are other industries like construction, printing, saw mills and crushers where the workers are exposed to high levels of noise throughout their lifetime of work. Studies regarding NIHL among the workers of these industries are not available.In India, NIHL has been a compensable disease since 1948 under the Employees State Insurance Act (1948) and the Workmen's Compensation Act (1923). But still there is very little awareness regarding this fact. Nearly 3 billion dollars has been paid as compensation for NIHL in the USA in the last two decades. In India, it was only in 1996 that the first case got compensation, and about 250 workers are receiving compensation for NIHL.The most effective way to prevent NIHL is to protect the worker from hazardous noise at the workplace. Hearing protectors should be used when engineering controls and work practices are not feasible for reducing noise exposure to safe levels. A personal hearing protection device is a device designed to reduce the level of sound reaching the eardrum. Ear muffs, ear plugs and ear canal caps are the main types of hearing protectors. To selecThe workers who will be wearing them.The need for compatibility with other safety equipment.Workplace conditions such as temperature, humidity and atmospheric pressure.A variety of style should be provided so that workers may select a hearing protector on the basis of comfort, ease of use and handling and impact on communication. Each worker should receive individual training in the selection, fitting, use, repair and replacement of hearing protectors. The most common excuses reported by workers for not wearing hearing protectors include discomfort, interference with hearing speech and warning signals and the belief of workers that there is no control over an inevitable process that causes hearing loss. Given adNoise is the hazardous industrial pollutant causing severe hearing loss in workers of every country in the world. The workers in industries like mining, construction, printing, saw mills, crushers, etc are at risk. Workers are exposed to high levels of noise throughout their lifetime of work, but there are very few NIHL studies in India to show its prevalence.Awareness should be created among workers about the harmful effects of noise on hearing and other body systems by implementing education and training programs.Research studies are needed to know the exact prevalence of NIHL among various industries in India.A national program should be established considering the amount of damage the NIHL causes to the quality of life of workers.


The pig eye is similar to the human eye in terms of anatomy, vasculature, and photoreceptor distribution, and therefore provides an attractive animal model for research into retinal disease. The purpose of this study was to characterize retinal histology in the developing and mature pig retina using antibodies to well established retinal cell markers commonly used in rodents.Eyes were enucleated from fetuses in the 9th week of gestation, 1 week old piglets and 6 months old adult animals. Eyeglobes were fixed and cryosectioned. A panel of antibodies to well established retinal markers was employed for immunohistochemistry. Fluorescently labeled secondary antibodies were used for signal detection, and images were acquired by confocal microscopy. Mouse retina at postnatal day (P) 5 was used as a reference for this study to compare progression of histogenesis. Most of the primary antibodies have previously been used on mouse tissue.Most of the studied markers were detected in midgestation pig retina, and the majority had a similar distribution in pig as in P5 mouse retina. However, rhodopsin immunolabeling was detected in pig retina at midgestation but not in P5 mouse retina. Contrary to findings in all rodents, horizontal cells were Islet1-positive and cones were calbindin-immunoreactive in pig retina, as has also been shown for the primate retina. Recoverin and rhodopsin immunolabeling revealed an increase in the length of photoreceptor segments in 6 months, compared to 1 week old animals.Comparison with the published data on human retina revealed similar marker distribution and histogenesis progression in the pig and human retina, supporting the pig as a valuable animal model for studies on retinal disease and repair. Furthermore, this study provides information about the dynamics of retinal histogenesis in the pig and validates a panel of antibodies that reliably detects developing and mature retinal cell phenotypes in the pig retina. Retinal cellular inventory and development are generally conserved across many studied vertebrate species. However, a range of histological and functional differences exist between individual species. Among nonprimate mammals, the pig eye most closely resembles the human eye, with similar size and comparable histological and physiologic features. Although the gestation period for pig 112–115 days) is significantly shorter compared to human, both human and pig retina are well developed at birth –115 daysPig models for retinitis pigmentosa, glaucoma, and retinal detachment have been developed -6. TransSeveral reported in vitro studies on pig retinal neuronal survival and physiology have used immunohistochemical tools to identify cell phenotypes in the primary retinal cell culture -19. At pThe aim of the present study was twofold: 1) to characterize antibodies to retina-specific markers commonly used in rodents as tools for further investigations in the pig; and 2) to conduct an immunohistochemical study of retinal histogenesis in the pig.All animal procedures were performed in compliance with the UK Animals (Scientific procedures) Act 1986. Mixed sex white Landrance pigs were obtained from Agri-Food and Biosciences Institute, Northern Ireland  where animals were commercially bred on slats and fed with granulated pig food. The pigs were anaesthetised with intra-muscular azoperone (15 mg/kg) and ketamine (20 mg/kg) and euthanized with intravenous or intra-cardiac euthanasia-grade pentobarbitone (100 mg/kg).Eyes were collected from embryos in the ninth gestational week (GW9), 1 week old piglets (1W), and 6 months (6M) old animals. Three pigs from each age group were analyzed.Postnatal day (P) 5 in the mouse was used as control for antibody functionality and comparison of developmental stage. Wild type C57BL6 mice were bred in 12 h dark/light regime with free access to food and water. P5 mice were euthanized by intraperitoneal injection with pentobarbitone. The eyes from three P5 mice from different litters were analyzed. Comparison with adult mouse retina was performed using the data from published literature which is accordingly cited in the results section.2HPO4, 2mM KH2PO4 pH 7.4) for 1–4 h at room temperature. After fixation the specimens were cryoprotected in 10% sucrose for 6 h followed by 30% sucrose overnight. The eyecups were embedded in optimal cutting temperature compound  and snap frozen in an isopentane bath on dry ice. Transverse 16 µm cryosections were cut, mounted onto Superfrost Plus glass slides  and stored at −80 °C until use.Eyes were enucleated from euthanized pigs, and the cornea and the lens were removed. The eyecups were fixed in 4% paraformaldehyde in PBS  in PBS. Slides were then washed 3 times for 5 min in PBS at room temperature and cell nuclei were counterstained with 10 µg/ml propidium iodide  in PBS containing 10 µg/ml RNase A (Sigma-Aldrich) for 30 min at room temperature, or with 5 μM DAPI  for 10 min. Slides were mounted in fluorescent mounting medium . Negative immunohistochemistry (IHC) controls were performed in parallel by omission of primary antibody. Immunoreactive cells were visualized and images recorded with an inverted confocal microscope  using the Nikon Confocal Microscope EZ-C1 software. Representative images were taken from the midperipheral region of the retina.Slides were thawed at room temperature and post-fixed in 4% formaldehyde  in PBS for 20 min at room temperature. After rinsing in PBS, sections were blocked for 1 h in 10% normal goat serum, 0.3% Triton X-100, 0.01% NaNAntibodies to intermediate filament protein nestin and the homeodomain transcription factor Pax6 were used to identify retinal progenitor cells in the developing pig retina . Nestin In GW9 pig retina, Pax6 strongly labeled most cells in the GCL, cells in the inner portion of the neuroblast layer (NBL) and some cells in the prospective outer plexiform layer (OPL) . The rouThe antibodies to POU domain transcription factor Brn3a and intermediate neurofilament (NF)-160 were chosen to characterize ganglion cells in the developing, postnatal and mature pig retina . The actBrn3a immunoreactivity was found exclusively in the GCL in GW9 pig retina . The obsIn GW9 pig retina, NF-160 staining was observed in the GCL, IPL, and in some differentiating amacrine and horizontal cells . A similA weak AP2α-positive signal was found in sparse cells in the GCL of GW9 pig retina . In P5 mTo characterize developing neurons in the INL, namely amacrine and horizontal cells, we used antibodies against calretinin, calbindin, and the 65 kDa isoform of glutamic acid decarboxylase GAD65; . Since tIn the developing pig retina at GW9, calretinin immunoreactivity was found in a subpopulation of cells in the GCL and rare cells in the inner NBL, suggestive of amacrine phenotypes . Sparse A subpopulation of cells in the GCL and some cells in the developing OPL were strongly calbindin-immunopositive in GW9 pig retina . Weak stIn the GW9 pig retina, GAD65 immunostaining was found in amacrine and ganglion cell bodies, with stronger signal localized to the IPL . Some GAIn the GW9 pig retina, moderate to intense Islet1 immunoreactivity was observed in the GCL and in some amacrine cells at the inner border of the NBL . StronglWeak PKCα immunolabeling was detected in the GCL, IPL, inner NBL, and in the prospective OPL of GW9 pig retina . P5 mousAn antibody to calcium binding protein recoverin, which labels both rods and cones, was used as an early marker of photoreceptor differentiation . Rod phoIn the GW9 pig retina, strong recoverin immunoreactivity was detected in putative differentiating photoreceptors, localized to the apical surface of the NBL, and in sparse cells found deeper in the inner NBL . RecoverIn GW9 pig retina, sparse elongated rhodopsin-immunoreactive cells were observed localized to the prospective ONL, suggestive of early differentiating rod photoreceptors . Some ruNo labeling with GNAT2 antibody was observed in the GW9 pig retina. Similarly, mouse cones at P5 were not positive for this marker (not shown). Cone outer segments in both 1W and 6M pig retina were strongly immunoreactive for GNAT2 . The sizNTR) and glutamine synthetase (GS) were used to characterize Müller glia, whereas an anti-GFAP antibody was used to label astrocytes. In the GW9 pig retina, strong P75NTR staining was present in the neurofiber layer  rat retina  , but conThe majority of investigated markers showed the same pattern of immunolabeling in both 1W and 6M pig retina. However, differences were found suggesting that some degree of retinal maturation occurs during the postnatal period. The same is true for the human retina, which reaches maturity by 5 years of age . For exaThe data presented herein should prove useful in future investigations that use pigs as a preclinical model system for retinal disease and repair. Similarity with human retina makes such studies a viable alternative to expensive and ethically problematic experimentation on nonhuman primates.


Tinnitus disturbs lives and negatively affects the quality of life of about 2% of the adult world population. Research has shown that the main cause of tinnitus is hearing loss. To analyze a possible association of the degree of hearing loss with the severity of tinnitus, we have performed a retrospective study using admission data on 531 patients suffering from chronic tinnitus. We have found that 83% of our tinnitus patients had a high frequency hearing loss corresponding to a noise-induced hearing loss (NIHL). There was a significant correlation between the mean hearing loss and the tinnitus loudness (p < 0.0001). Interestingly, patients suffering from decompensated chronic tinnitus had a greater degree of hearing loss than the patients with compensated form of tinnitus. In addition, we demonstrate that the degree of hearing loss positively correlates with the two subscales  of the Tinnitus Questionnaire. Our retrospective study provides indirect evidence supporting the hypothesis that the degree of noise-induced hearing loss influences the severity of tinnitus. This perception can be induced by various dysfunctions on several levels of the peripheral or central auditory pathway . RegardlThe onset of tinnitus can have various basis such as neurologic, traumatic, infectious or drug-related , howeverBetween 57% and 76% of tinnitus patients were shown to have NIHL –11. ThesTo test our hypothesis we used a retrospective study using data acquired from 531 tinnitus patients. This data were randomly collected on the admission of patients who reported to the day ward of Tinnitus Center at the Charité - Universitätsmedizin in Berlin between January 2008 and March 2010. We have analyzed general audiometric and tinnitus-oriented psychometric parameters.2.2.1.We have obtained the data from 531 tinnitus patients (sub-acute and chronic) admitted to a 7-day multimodal tinnitus therapy. The data were collected on the admission day. The data were collected between January 2008 and March 2010. Of 531 tinnitus patients, 280 were women and 251 men. The mean age of the patients was 49 years .2.2.Audiometric tests were performed using routine pure tone audiogram (0.5–8 kHz). In addition, tinnitus frequency (kHz) and tinnitus loudness (dB HL) were matched for each patient.2.3.Tinnitus-related distress was assessed using validated standard German version of the tinnitus questionnaire (TQ). The TQ is composed of 52 items allocated to six scales: emotional distress, cognitive distress, intrusiveness, auditory perceptual difficulties, sleep disturbances and somatic complaints . The sev2.4.Groups were compared using the Mann-Whitney U-test. Spearman's rank correlation was used to test the statistical relationship between hearing loss and tinnitus parameters or tinnitus severity. P < 0.05 was the level of significance; the confidence intervals were 95% .3.3.1.Of all 531 tinnitus patients tested, 441  had a hearing loss in the upper frequencies , corresp3.2.Of 441 tinnitus patients with NIHL-like audiogram, 319 patients had pure tone tinnitus whereas 122 patients had noise tinnitus. Forty-seven percent had unilateral tinnitus and 53% had bilateral tinnitus.The median value of the tinnitus frequency measured on the left or right side of patients with pure tone tinnitus was 6.0 kHz . There wThe mean tinnitus-related distress of the 441 NIHL-like audiogram /tinnitus patients was 34.2 points (SD = 16.3) as measured using the TQ. The values did not differ between female and male patients except for the subscale “somatic complaints” with woman having significantly greater scores than the male patients . Of the 3.3We found a close correlation between the mean hearing loss and the tinnitus loudness on the right and left sides . Within single frequencies, the closest correlation emerged between the tinnitus loudness and the hearing loss measured at 6 kHz .3.4Hearing loss was of significantly higher degree in patients with decompensated tinnitus than in patients with compensated tinnitus . This meDuring the analyses of separate TQ subscales, we found a correlation between the hearing loss and the TQ subscales “intrusiveness” and “auditory perceptual difficulties” . We found no correlation between tinnitus frequency or loudness and distress caused by tinnitus.3.5.Our recent study  revealed3.6.We compared the tinnitus-related distress between the patients with left-sided, right-sided and bilateral tinnitus. Despite matching values of the hearing loss, tinnitus frequency and tinnitus loudness, the TQ score was significantly higher in patients who had tinnitus on their left side as compared to the right-sided tinnitus (p < 0.05) . Within 4.et al. [et al. study, there were 38% of patients with acute (duration of less than three months) and 36% of patients with sub-acute tinnitus (between four and twelve months). Of our patients, none suffered from acute tinnitus. Based on the above evidence, one is tempted to cautiously speculate that the tinnitus associated with hearing loss has a greater chance of chronification than the tinnitus without hearing loss. Obviously, more studies are needed to validate this observation. In the other study, Attias and colleagues have indentified a subgroup of patients with tinnitus, who were actively looking for a remedy. This group was called “Tinnitus Seeking Help Group” (n = 355) [Our present work was aimed at analyzing the association between noise, the grade of the noise-induced hearing loss and the severity of tinnitus. Our results partially corroborate the findings of others by demonstrating a strong bond between the noise-induced hearing loss and tinnitus . In factet al. . The samn = 355) . IntriguWe found a significant correlation between the mean hearing loss and the tinnitus loudness (p < 0.0001).The fact that the loudness of tinnitus is increased in patients with coexisting hearing loss has been already known , but theNoise pollution and occupational noise were demonstrated to induce asymmetric hearing loss, with the left side being affected more than the right one . AlthougNoise—either occupational or environmental noise pollution—is a major preventable cause of the hearing loss and therefore of chronic tinnitus. The consequences of noise exposure last for life, as the sensory auditory epithelium never regenerates. The price to pay for the noise exposure includes not only a limited ability to hear but also increased chance to develop chronic decompensated tinnitus, which separates the affected persons from normal social activities and significantly decreases the quality of life.5.Our study demonstrates that the chain of events started by overexposure to noise not only induces the hearing loss but also tinnitus with its co-morbidities, finally resulting in a decreased quality of life. The degree of hearing impairment is reflected by the severity of tinnitus.