The aim of the present study was to evaluate the relevance of this variation both theoretically and experimentally using mathematical model simulations, comparing these ratio simulations with PaO2/FiO2 ratios measured in a range of different patients.Previous studies have shown through theoretical analyses that the ratio of the partial pressure of oxygen in arterial blood  according to their PaOP < 0.005). When using this model to simulate variation in the PaO2/FiO2 ratio, disease classification changed in 30% of the patients when changing the FiO2 level.The two-parameter model gave a statistically better fit to patient data (2/FiO2 ratio depends on both the FiO2 level and the arterial oxygen saturation level. As a minimum, the FiO2 level at which the PaO2/FiO2 ratio is measured should be defined when quantifying the effects of therapeutic interventions or when specifying diagnostic criteria for acute lung injury and acute respiratory distress syndrome. Alternatively, oxygenation problems could be described using parameters describing shunt and ventilation/perfusion mismatch.The PaO The ratio has been used in numerous experimental studies to quantify pulmonary gas exchange before and after therapeutic intervention  (27 kPa ≤ PaO2/FiO2 < 40 kPa) and of adult respiratory distress syndrome (ARDS) (PaO2/FiO2 < 27 kPa) [The ratio of the partial pressure of oxygen in arterial blood (PaO example -3). The  27 kPa) .2/FiO2 ratio as a tool for assessing pulmonary gas exchange has been questioned. Using mathematical models describing gas exchange, previous authors have simulated values of the PaO2/FiO2 ratio and have shown them to vary with the FiO2 level [2/FiO2 ratio is a poor indicator of a patient's pulmonary gas exchange status in the clinic. This hypothesis is only true, however, if the simulations performed are indeed able to describe measured variations in the PaO2/FiO2 ratio, and if these variations happen within interesting ranges of FiO2. The latter of these conditions is crucial in determining whether this ratio is a useful scientific and clinical parameter.Despite its widespread use, the validity of the PaOO2 level -8. These2/FiO2 ratio depends upon the complexity of the mathematical models used. Gowda and Klocke [2/FiO2 ratio on varying FiO2 levels. This complex model has the advantage of describing pulmonary gas exchange accurately; however, its complexity means that the model is not useful for describing an individual patient in the intensive care unit. Aboab and colleagues used a simple mathematical model where an 'effective' pulmonary shunt was used to describe all ventilation/perfusion (V/Q) abnormalities in the lung [2, as shown previously [2/FiO2 ratio accurately.The ability of a particular simulation to accurately describe variation in the PaOd Klocke  used thed Klocke  to simulthe lung . This moeviously . A singl2/FiO2 ratio.Mathematical models have been proposed recently that describe the gas exchange using two parameters: a shunt value, and a second parameter describing the V/Q ratio ,12. Thes2/FiO2 ratio with the FiO2 level. To do so, we determined whether changes in the PaO2/FiO2 ratio can be described accurately by either the 'effective shunt' model or a two-parameter model describing shunt and V/Q mismatch. Unlike previous studies that have examined changes in the PaO2/FiO2 ratio with FiO2 theoretically through model simulation [2/FiO2 ratio in a range of different patients. Simulations of the PaO2/FiO2 ratio performed with the two-parameter model are compared with those using the 'effective shunt' model to investigate whether the extra complexity of the two-parameter model is justified. The models are then used to simulate whether, and under which conditions, the PaO2/FiO2 ratio varies with FiO2, to further investigate the discrepancies between the two models and whether such variation is clinically relevant.The purpose of the present article is to assess the relevance of variation in the PaOmulation -8, the pData were collected from 93 patients, most of these data being published previously ,14,15. P2 values, achieving arterial oxygen saturation (SaO2) values in the range 85–100%. The FiO2 values were selected on a patient-specific basis to cover this range, meaning that patients with more severe pulmonary disorders received higher FiO2 levels. Steady state was achieved at each FiO2 level either by waiting 5 minutes or by the presence of a stable end-tidal oxygen fraction over a 30-second period [2, end-tidal oxygen fraction), of end-tidal carbon dioxide fraction, tidal volume, and respiratory frequency, and of arterial acid–base and oxygenation status . In some patients it was necessary to administer subatmospheric oxygen fractions to achieve SaO2 in the range 85–90%, which was achieved by mixing nitrogen with air in the inspiratory gas. In 18 experiments where all patients were breathing spontaneously, arterial blood gases were only measured at two levels of FiO2. These patient cases were excluded from the current analysis, giving a total number of 116 patient cases for data analysis . The PaO2/FiO2 ratio was calculated at each level of FiO2.On each occasion patients were studied using four to eight different FiOd period . At steaThe data were analysed using two mathematical models of gas exchange: the 'effective shunt' model, used by Aboab and colleagues ; and the2 value, which describes the drop in oxygen pressure from the ventilated alveoli to the mixed blood leaving the lung capillaries; that is, the value in blood prior to the mixing of shunt. As such, ΔPO2 describes the extra oxygen pressure required at the mouth to remove an oxygenation problem due to V/Q mismatch; that is, ΔPO2 = 20 kPa means air plus 20% inspired oxygen (FiO2 = 0.41) is required.In the 'effective shunt' model, oxygenation problems are described by a single parameter ('effective shunt') quantifying the blood flowing through the lungs without being oxygenated. In the two-parameter model, a shunt parameter is included along with the parameter fA2 describing the fraction of ventilation to a compartment receiving 90% of nonshunted perfusion. An fA2 value of 0.9 gives ideal V/Q matching, while lower fA2 values indicate V/Q mismatching. An fA2 value can be transformed into a ΔPOThe 'effective shunt' model and the two-parameter model were used in three ways.2 and the PaO2/FiO2 ratio with variation in FiO2 using the two mathematical models. To do so, simulations were performed for different values of model parameters.A theoretical comparison was performed between model simulations of changes in SaO2 and the PaO2/FiO2 ratio versus FiO2. A statistical comparison of the 'goodness' of fit of the two models to the data was performed using an F test [The models were fitted to the data from each patient in turn using the least-squares method, and the root mean square of the residuals was calculated for each of the fits. Model fits were illustrated by plotting simulated and measured values of SaOn F test .2/FiO2 ratio over a range of FiO2 levels. This analysis had two aims: first, to evaluate the significance of any difference between the two models when fitted to the data; and second, to investigate whether the simulated variation in the PaO2/FiO2 ratio was relevant. The relevant range was defined on an individual patient basis as the FiO2 range that resulted in a simulated value of SaO2 within the range 92–98%. The variation in the PaO2/FiO2 ratio was then used to quantify the number of patients changing disease classification as a result of varying FiO2 levels according to the two models across the defined FiO2 range, these results being presented in a confusion matrix [2 level in the range, according to the following criteria: ARDS (PaO2/FiO2 < 27 kPa) [2/FiO2 < 40 kPa) [2/FiO2 > 47 kPa) [2/FiO2 < 47 kPa).Both models were then used to analyse the variation in the PaOn matrix . Patient 27 kPa) ,5, ALI ( 47 kPa) . Those p2 on model simulated values of SaO2 and the PaO2/FiO2 ratio.Figures 2 = 0 kPa. Simulated increased shunt depresses the shoulder of the FiO2 versus SaO2 curve, and depresses and deforms the shape of the FiO2 versus PaO2/FiO2 ratio curve. As a result, the relevant range of FiO2 (thick solid part of lines) broadens with increases in shunt. The deformation in the PaO2/FiO2 ratio curve has a characteristic shape whereby the PaO2/FiO2 first falls and then gradually rises, explained as follows. On increasing the FiO2 level, the partial pressure of oxygen in the lung capillary blood increases. As the lung capillary blood mixes with that shunted, the increase in the partial pressure of oxygen in the lung capillary blood helps to oxygenate the shunted blood, so that the PaO2 value increases little and the PaO2/FiO2 ratio falls. On increasing the FiO2 level further, the mixture of shunted and lung capillary blood reaches an SaO2 value of about 98% where the arterial blood haemoglobin is almost saturated. Further increases in FiO2 translate into increased PaO2, and hence an increasing PaO2/FiO2 ratio. It should be noted that the range of FiO2 giving 92–98% saturation may extend below atmospheric oxygen levels (FiO2 = 0.21) in patients with only mild gas exchange abnormalities or in normal subjects. The simulations in Figure 2/FiO2 ratio changes with FiO2 as found by Aboab and colleagues [2/FiO2 ratio falls by 20.5 kPa, from 45.5 kPa to 25 kPa, over the relevant range of FiO2.Figure lleagues . For exa2 or the PaO2/FiO2 ratio are quite different from the effects of shunt. The FiO2 versus SaO2 curves are shifted horizontally along the FiO2 axis with increasing V/Q mismatch. The PaO2/FiO2 ratio is increased with increasing FiO2 levels, as the absence of significant shunt means that arterial haemoglobin is saturated on small increases in FiO2. The small dip in the PaO2/FiO2 ratio seen in these curves, particularly at the 0 kPa level, is due to the 5% shunt used in these plots. For the cases simulated in Figure 2/FiO2 ratio is quite sensitive to changes in FiO2. Within the relevant range of FiO2 (thick solid part of lines) for a ΔPO2 value of 10 kPa  is shown, such that those patients with more severe lung diseases have a higher range than those with less severe disease. For each of the fits, values of model parameters are given along with the root mean square value describing the error in model fitting.Figure F test showed that the two-parameter model gave a statistically better fit to the data than the 'effective shunt' model (P < 0.005). In all cases the two-parameter model fitted the data either as well as or better than the 'effective shunt' model, as described by the root mean square. In cases where the 'effective shunt' model fitted the data well  root mean square for fitting the two-parameter model to the data was 0.5 ± 0.4%, compared with 1.4 ± 1.0% (± SD) for the 'effective shunt' model. The results of the 2 versus the PaO2/FiO2 ratio illustrated in Figure 2/FiO2 ratio, where a V/Q mismatch is present. For some patients  and from 18 to 38 (~111% increase), respectively. According to the two-parameter model, the number of patient cases classified as ALI and ARDS changed from 23 to 31 (~35% increase) and from 18 to 24 (~33% increase), respectively. According to the 'effective shunt' model, disease severity only increased with FiO2 – whereas five patient cases changed classification to a less severe disease group according to the two-parameter model.Disease classification changed in 60 of 116 patient cases (~50%) according to the 'effective shunt' model, compared with 38 of 116 patient cases (~30%) according to the two-parameter model. With an increase in the FiO2/FiO2 ratio with FiO2, and the mathematical model complexity necessary to describe this variation. For the first time this analysis has been performed not only theoretically using mathematical model simulations, but also experimentally from measurements of the PaO2/FiO2 ratio taken at different FiO2 levels.The present study has investigated the variation in the PaO2/FiO2 ratio has been investigated. This model has been shown, using an F test, to provide a statistically better fit to oxygenation data than an 'effective shunt' model, even when taking into account the degrees of freedom lost due to the presence of an extra parameter. This improvement in fit can be seen in the plots shown in Figure 2/FiO2 ratio with varying FiO2 level. Interpretation of the PaO2/FiO2 ratio changes in these four examples using the 'effective shunt' model would result in an overestimation of the changes in the PaO2/FiO2 ratio when varying the FiO2 level. In the remaining two cases (Figure The use of a two-parameter model of gas exchange to describe variation in the PaOs Figure  the 'eff2/FiO2 ratio results in an overestimate of the number of patient cases changing disease classification upon increasing FiO2, as illustrated in Table In general, use of the 'effective shunt' model to simulate changes in the PaO2/FiO2 ratio – the 'effective shunt' model simulating an incorrect degree of disease severity. In these patient cases the V/Q mismatch was the major cause of hypoxemia according to the two-parameter model, and this model was necessary to simulate these changes in the PaO2/FiO2 ratio. The difference in the direction of disease classification provided by these two models can be understood by looking at Figure 2/FiO2 ratio on increasing the FiO2 level in comparison with both the raw data and the two-parameter model simulation.For five patient cases, the change in disease classification simulated by the 'effective shunt' model was in the opposite direction to that shown by the measured PaO2/FiO2 ratio limits [2/FiO2 ratio. The sole difference between the criteria for ALI and ARDS, however, is the level of hypoxemia quantified by the PaO2/FiO2 ratio.The necessary criteria for diagnosing ALI and ARDS include acute onset of respiratory failure, bilateral infiltrates seen on a frontal chest radiograph and no clinical evidence of left atrial hypertension in addition to the PaOo limits . In the 2/FiO2 ratio depends on both the FiO2 level and the SaO2 level, and that, for changes in FiO2 corresponding to an SaO2 range of 92–98%, 30% of patients change disease classification due to variation in the PaO2/FiO2 ratio. The clinical and scientific utility of the PaO2/FiO2 ratio therefore seems doubtful, and at the very least the FiO2 level at which the PaO2/FiO2 ratio is measured should be specified when quantifying the effects of therapeutic interventions or when specifying diagnostic criteria for ALI and ARDS. Perhaps more appropriate would be to replace the single-parameter PaO2/FiO2 ratio description with two parameters, a parameter to describe the oxygenation problem due to V/Q mismatch and one to describe oxygenation problems due to shunt. Indeed, Riley and Cournand [The present article has shown that the PaOCournand  recognizCournand  and simpCournand , their c2/FiO2 ratio with the FiO2 level is scientifically and clinically relevant.• The variation in the PaO2/FiO2 ratio with the FiO2 level cannot be explained with an 'effective shunt' model, and requires a more complex, two-parameter, model.• The variation in the PaO2 = drop in oxygen pressure from the ventilated alveoli to the mixed blood leaving the lung capillaries oxygen; fA2 = fraction of ventilation to a compartment receiving 90% of nonshunted perfusion; FiO2 = inspired oxygen fraction; PaO2 = partial pressure of oxygen in arterial blood; SaO2 = arterial oxygen saturation; V/Q = ventilation/perfusion.ALI = acute lung injury; ARDS = acute respiratory distress syndrome; ΔPOSK, SA and SER are all shareholders of Mermaid Care APS, a company involved in the development of equipment for the measurement of pulmonary gas exchange. SA is a board member of Mermaid Care APS. All other authors declare that they have no competing interests.All authors contributed to the conception and design of the study. SK, KE and CA contributed to the data collection and clinical interpretation of the results. DSK, BWS, SA and SER contributed to the mathematical modelling, data analysis and technical interpretation of the results, including statistical analysis. DSK and SER drafted the manuscript, with all other authors being involved in its revision and approval.


Amplification and overexpression of the erbB-2 oncogene is an unfavourable prognostic marker in human breast cancer and occurs in approximately 25% of breast carcinomas. We used erbB-2 antisense oligonucleotides to inhibit the proliferation of human breast cancer cell lines. erbB-2 antisense oligonucleotides (20 microM) inhibited the growth and DNA synthesis of breast cancer cell lines with an amplified erbB-2 gene by up to 60%. Control complementary sense oligonucleotides did not inhibit cellular proliferation at the same concentration but showed inhibitory effects at higher concentrations. There was no specific effect of erbB-2 antisense oligonucleotides on breast cancer cell lines that had no amplification of erbB-2. erbB-2 antisense oligonucleotides reduced erbB-2 protein levels, measured by immunohistochemistry, in a dose-dependent manner. erbB-2 sense oligonucleotides did not decrease the levels of erbB-2 protein. These data indicate that erbB-2 antisense oligonucleotides induce a specific inhibition of erbB-2 protein expression and that erbB-2 gene overexpression is important for the proliferation of the breast cancer cells that have been selected for erbB-2 amplification.


Surfactant Protein-A (SP-A) is the most prominent among four proteins in the pulmonary surfactant-system. SP-A is expressed by alveolar epithelial cells type II as well as by a portion of non small cell lung carcinomas (NSCLC).The expression of SP-A is complexly regulated on the transcriptional and the chromosomal level. SP-A is a major player in the pulmonary cytokine-network and moreover has been described to act in the pulmonary host defense.in situ hybridization as promising methods to further elucidate the role of this molecule in adult lung diseases.By the use of cell culture or animal models the functional properties have been repeatedly shown in many aspects, often bearing surprising properties which strongly indicate the physiological importance of SP-A. To date SP-A is recognized as a molecule essential for pulmonary development, structure and function. An upcoming number of reports deals with the role of SP-A for pulmonary pathology. This article gives an overview about the state of knowledge on SP-A focused in applications for human pulmonary disorders and points out the importance for pathology-orientated research approaches using immunohistochemistry or  Since it is synthesized by humans starting in the 28me (IRDS).Pulmonary surfactant regulates dynamically the alveolar surface tension. The central role of the surfactant system for maintaining pulmonary function has been repeatedly shown by the use of cell culture or animal models .Surfactant is a complex mixture of lipids, carbohydrates and four proteins . The initial descriptions of surfactant lead back to the 1950s, but little attention was given to the surfactant proteins until the 1980s . The genin situ. The expression of SP-A is also complexly regulated on the transcriptional level. Moreover the protein-turnover and the release of SP-A into the serum represents a further point of regulation [SP-A is the major surfactant apoprotein exhibiting complex interactions and participation in processes fundamental for pulmonary structure and function with its expression restricted to alveolar epithelial cells type II. Moreover expression of SP-A was also described for a portion of NSCLC facilitating a diagnostic marker for these carcinomas ,6. Aftergulation . This soIn recent years the role of defects in the expression of SP-A in context with different pulmonary diseases has become an issue of scientific investigations. Initially numerous studies have been performed to elucidate the role of surfactant substitution in pediatrics .Pseudomonas aeruginosa [Respiratory Syncytial Virus (RSV) has been shown to be reduced and may be restored by exogenous SP-A administration [As one major function SP-A displays a protective role of the molecule in pulmonary host defense by interacting with various infectious agents such as bacteria, fungi and viruses. SP-A deficient knock-out mice – compared to wild type animals – are susceptible to infections with ruginosa  and the ruginosa . In accostration -17.Mycobacterium tuberculosis to alveolar macrophages and promoting the phagocytosis of these bacteria, SP-A is important in the pathogenesis of tuberculosis [Haemophilus influenza Type A [Pneumocystis carinii [Influenza A Virus (less efficient than SP-D) [Candida tropicalis [Aspergillus fumigatus [By mediating the attachment of rculosis -21. SP-Arculosis ,23, Haema Type A ,25, Pneu carinii ,27, Inflan SP-D) , Candidaopicalis  and Aspeumigatus .Moreover, SP-A is involved in the complex pulmonary network of cytokines as a central player, for example interacting with TNF-alpha and several interleukins ,12,14.Therefore it is likely that defects in the expression of SP-A may be important in the course of non infectious pulmonary diseases of adult patients. In the case of idiopathic pulmonary fibrosis, for example, low levels of SP-A (measured by ELISA) have been reported in broncho alveolar lavages (BAL), but elevated levels were found in the sera -34. ImmuReduced levels of SP-A have been demonstrated also in other pulmonary diseases such as the Adult Respiratory Distress Syndrome (ARDS) and in pneumonia .Keeping in mind that surfactant substitutes are widely available due to their application in pediatrics, a growing number of therapeutic possibilities may result from these findings.In sarcoidosis elevated levels of SP-A have been described  using BASince SP-A represents a central molecule in pulmonary immunoregulation as well as in host-defense it is obvious that defects in the surfactant system may have functional influence in the course of these pulmonary disorders.Another point of research concerning SP-A is the diagnostic value of this molecule, the expression of which is restricted to the lungs. It has been reported that SP-A levels in BAL or serum from patients suffering from idiopathic pulmonary fibrosis correlate with the progression of the disease and can be used to predict survival ,38. In sHowever, the choice of a suitable SP-A antibody is highly important since approaches using polyclonals display cross reactions with other tumors . This prImmunohistochemical detection of SP-A even might be utilized for forensic purposes helping to distinguish between fatal drowning and postmortem immersion .in situ, which provides evidence of the cellular activities in the context of a human native tissue. One example is shown in Fig. in situ; the reddish signals of the transcripts are visible in the cytoplasm of type II pneumocytes. When analyzing the expression of SP-A in histological sections in context with other molecules of the pulmonary cytokine network one can expect further clues for the scenarios taking place in the course of interstitial lung diseases.It is evident that SP-A is a molecule which already proves to be an interesting subject for medical research. However, the studies concerning the possible role of surfactant-defects in pulmonary diseases of adults have been performed mainly in different cell culture or animal models hardly analyzing adult human lung tissue. For these reasons SP-A is a promising target for histomorphological approaches using pathological specimens which exactly represent the scenarios of various diseases with all the different cell types involved which are difficult to simulate in models. With the modern tools of molecular pathology, the genetic activities of genes can be analyzed Taken together, SP-A is a complexly regulated molecule with surprising properties and essential importance for pulmonary development, structure and function which is getting more and more into focus concerning various diseases of the adult lung. Thus, as an outlook, it will become an issue of pulmonary pathology which might provide promising perspectives for applications in research, diagnosis and therapy.The authors declare that they have no competing interests.TG, DK and EV conceived the study. HS, MA, DSL and FS took part in the conception and writing of the manuscript. All authors read and approved the final manuscript.


The causes of dolphin and whale stranding can often be difficult to determine. Because toothed whales rely on echolocation for orientation and feeding, hearing deficits could lead to stranding. We report on the results of auditory evoked potential measurements from eight species of odontocete cetaceans that were found stranded or severely entangled in fishing gear during the period 2004 through 2009. Approximately 57% of the bottlenose dolphins and 36% of the rough-toothed dolphins had significant hearing deficits with a reduction in sensitivity equivalent to severe (70–90 dB) or profound (>90 dB) hearing loss in humans. The only stranded short-finned pilot whale examined had profound hearing loss. No impairments were detected in seven Risso's dolphins from three different stranding events, two pygmy killer whales, one Atlantic spotted dolphin, one spinner dolphin, or a juvenile Gervais' beaked whale. Hearing impairment could play a significant role in some cetacean stranding events, and the hearing of all cetaceans in rehabilitation should be tested. Odontocete cetaceans  use high frequency echolocation sounds for navigation and foraging and are well-known for possessing high frequency hearing abilities Hearing measurements in captive cetaceans are often performed with behavioral conditioning, where the dolphin is trained to press a response paddle or vocalize when it hears a test signal Tursiops truncatus) and 5 out of 14 (36%) rough-toothed dolphins (Steno bredanensis) examined in this study . No hearing loss was found in stranded Rissos's dolphins (Grampus griseus) from three different stranding events, two pygmy killer whales (Feresa attenuata) from a single stranding event, an Atlantic spotted dolphin , and a spinner dolphin  to profound (>90 dB loss) hearing loss was found in four out of seven (57%) of the stranded bottlenose dolphins (peak) (The audiograms of two stranded bottlenose dolphins are shown in peak) . This aupeak) . There wpeak) . HoweverThere are five main contributing factors to hearing loss in mammals: intense chronic noise, transient intense noise (e.g. explosions), age-related hearing loss (presbycusis), congenital hearing impairment, and ototoxic drug treatment It is likely that hearing loss was congenital in some of these stranding cases, especially two of the rough-toothed dolphins (Dancer and Vixen), which were estimated to each be approximately two years old at the time of stranding. Congenital hearing impairment in humans is not uncommon. In one study, 113 out of 52508  newborns screened in Texas had hearing loss Some odontocetes have social systems with strong mother-calf bonds where the mother can support the calf for years through lactation Many of the stranded dolphins in this study were being treated for various infections with antibiotics and antifungals, including the oral administration of gentamicin sulfate and itraconazole and intramuscular injections of amikacin sulfate. Aminoglycoside antibiotics, including gentamicin and amikacin, are known to cause cochlear hair cell death in mammals Steno bredanensis at MML; #932-1489-06  for Grampus griseus Riverhead Foundation; #932-1489-06  for S. bredanensis at Marine Mammal Conservancy; #932-1489-08  for S. bredanensis at Gulf World; #932-1489-08  for Grampus griseus at MML; #1053-1825-00  for all other dolphins. Hearing tests were conducted either in the water or on land, depending on the situation at the stranding facility . In all cases, a jawphone consisting of an ITC-1042 piezoceramic transducer embedded in a RTV silicone suction cup was placed on the lower jaw fat pad to deliver sound stimuli. Sound levels were calibrated underwater by measuring the sound 10 cm from the jawphone in a free field using a Reson TC4032 hydrophone. These underwater calibrations were used as the calibrations for the in-air tests. Background noise levels in test tanks were typically <45 dB re 1 µPa2/Hz at frequencies above 20 kHz. Noise levels in field test locations, which were shallow beaches , were about 85 dB re 1 µPa2/Hz at frequencies above 20 kHz.All research was approved by the IACUC of the University of South Florida and Mote Marine Laboratory with permit authorization from NOAA. Hearing tests were conducted under NOAA permits #932-1489-06  for Evoked potentials were measured in response to amplitude modulated (AM) tone pips. The AM rate was either 600 Hz or 1000 Hz, as these rates were found to yield large evoked potentials. Click evoked potentials were also measured for some dolphins. Clicks were generated by sending a 0.1 ms square wave into the transducer, which generated a click with a peak frequency of approximately 80–120 kHz. Evoked potentials were measured in response to click trains delivered at 600 Hz and 1000 Hz. All signals were generated by a Tucker-Davis Technologies (TDT) RX6 real-time processor with a 260 kHz sample rate.Silver chloride electrodes embedded in suction cups were used to measure evoked potentials after 10,000× amplification through a differential amplifier (TDT HS4-DB4). The recording electrode was placed about 5–10 cm behind the blowhole, and a reference electrode was placed approximately 40 cm behind the blowhole. The ground electrode was placed in the water for in-water tests, or on the back of the dolphin for in-air tests.Evoked potentials were averaged in response to up to 1000 presentations of the acoustic stimulus (TDT BioSig software). Fewer presentations were used when a response at one level was apparent. AEP signals were analyzed in a custom MATLAB program (BSlab) which performed a 2441-point FFT (resulting in a 10 Hz resolution) and measured the signal-to-noise ratio at the modulation frequency of the AM signals. A signal was considered to be present if the AEP signal was at least 3 dB above the background noise . This is equivalent to the F-test with an alpha of at least 0.01 Table S1Summary of odontocete audiogram results. One bottlenose dolphin, MML0701 (Filly), became entangled in fishing gear and was rescued. MML = Mote Marine Laboratory; MMC = Marine Mammal Conservancy; CMA = Clearwater Marine Aquarium; Riverhead = Riverhead Foundation; Curacao = Curacao Seaquarium. Dolphins with severe to profound hearing loss are also indicated in bold type.(0.02 MB DOCX)Click here for additional data file.


Cryptococcus neoformans is mediated in part by host factors that promote interactions between immune cells and C. neoformans yeast. Surfactant protein A (SP-A) contributes positively to pulmonary host defenses against a variety of bacteria, viruses, and fungi in part by promoting the recognition and phagocytosis of these pathogens by alveolar macrophages. In the present study we investigated the role of SP-A as a mediator of host defense against the pulmonary pathogen, C. neoformans. Previous studies have shown that SP-A binds to acapsular and minimally encapsulated strains of C. neoformans. Using in vitro binding assays we confirmed that SP-A does not directly bind to a fully encapsulated strain of C. neoformans (H99). However, we observed that when C. neoformans was incubated in bronchoalveolar fluid, SP-A binding was detected, suggesting that another alveolar host factor may enable SP-A binding. Indeed, we discovered that SP-A binds encapsulated C. neoformans via a previously unknown IgG dependent mechanism. The consequence of this interaction was the inhibition of IgG-mediated phagocytosis of C. neoformans by alveolar macrophages. Therefore, to assess the contribution of SP-A to the pulmonary host defenses we compared in vivo infections using SP-A null mice (SP-A-/-) and wild-type mice in an intranasal infection model. We found that the immune response assessed by cellular counts, TNFα cytokine production, and fungal burden in lungs and bronchoalveolar lavage fluids during early stages of infection were equivalent. Furthermore, the survival outcome of C. neoformans infection was equivalent in SP-A-/- and wild-type mice. Our results suggest that unlike a variety of bacteria, viruses, and other fungi, progression of disease with an inhalational challenge of C. neoformans does not appear to be negatively or positively affected by SP-A mediated mechanisms of pulmonary host defense.Initiation of a protective immune response to infection by the pathogenic fungus  Cryptococcus neoformans is an environmentally ubiquitous fungal pathogen that is responsible for significant morbidity and mortality in immunocompromised hosts. In humans, a pulmonary infection occurs following inhalation of C. neoformans cells. The identity of the infectious propagule remains unknown but is presumed to be either small, poorly encapsulated, desiccated yeasts or basidiospores. Hematogenous dissemination of C. neoformans from the lungs to the central nervous system can result in cryptococcal meningoencephalitis, a life-threatening complication that requires aggressive chemotherapeutic intervention C. neoformans will not only broaden our understanding of C. neoformans pathogenesis but may aide in the development of therapeutic strategies for the prevention and treatment of this important fungal disease.Pseudomonas aeruginosaStreptococcus pneumoniaeRespiratory Syncytial VirusInfluenza A virus Aspergillus fumigatusPneumocystis jiroveciin vitro studies have shown that SP-A can bind to acapsular and minimally encapsulated strains of C. neoformans but not heavily-encapsulated yeast C. neoformans remains to be fully elucidated.Surfactant protein A (SP-A) is one such innate host factor that has been shown to contribute positively to the pulmonary defenses against a diverse group of pathogenic microorganisms including, C. neoformans (H99) C. neoformans are uniformly avirulent, not clinically relevant and thus would not be suitable for investigating the role of SP-A as a mediator of host defense. Second, if a desiccated yeast form or basidiospore of C. neoformans were inhaled, rapid rehydration of capsule occurs in airway structures and the capsular structure is expected to be a major factor in the initial host-pathogen pulmonary interaction. We, therefore, initiated our study by first investigating the direct binding of SP-A to C. neoformans (H99). Using in vitro binding assays we confirmed that SP-A does not directly bind to C. neoformans but, we did discover an IgG dependent mechanism that does enable SP-A binding to C. neoformans. SP-A-/- mice and a murine cryptococcosis inhalation infection model were then used to investigate the role of SP-A in host defense against C. neoformans. These experiments revealed that SP-A does not appear to contribute to the pulmonary host defenses against C. neoformans.In the present study we investigated the effect of SP-A on host defense against an encapsulated, pathogenic strain of Cryptococcus neoformans var. grubii strains H99  was revived from 15% glycerol stocks stored at −80°C. H99 was maintained on yeast extract peptone dextrose  agar plates at 30°C. Prior to use in these studies, yeast were grown in YPD liquid medium overnight at 30°C, harvested, washed three times with sterile phosphate-buffered saline (PBS), suspended in phagocytosis buffer and counted with a hemacytometer to determine cell concentrations. For phagocytosis assays, C. neoformans were labeled with Alexa Fluor 647  per the manufacturers instructions. Stocks of Alexa Fluor 647 labeled yeast were maintained at −20°C until needed.Limulus amebocyte lysate assay  per the manufacturer's instructions. SP-A was labeled with Alexa Fluor 488  per the manufactures instructions. The ratio of moles dye per mole of protein was quantified and SP-A preparations with labeling efficiencies between 4 and 10 were used for binding assays.SP-A was isolated from the lavage fluid of alveolar proteinosis patients as previously described C. neoformans, yeast were treated with Alexa Fluor 488 labeled SP-A (5 to 20 µg/ml), anti-GXM IgG mAb 18B7  or a combination of SP-A and anti-GXM IgG for 60 minutes at 37°C with shaking. Following treatment, yeast were washed twice with 5 volumes of phagocytosis buffer (PB) (PBS +0.1% BSA +900 µM CaCl2) and binding was quantified by flow cytometric analysis . Data analysis was performed using the FlowJo software package . For fluorescence microscopy experiments, PE-Texas Red  labeled anti-GXM IgG mAb 18B7 was used to detect C. neoformans capsule. Calcofluor was used to stain the cell wall. SP-A binding assays using Escherichia coli HB101 were used to validate the binding activity of all SP-A preparations used in this study. Binding was visualized by fluorescent microscopy (Zeiss Axioskop 2 Plus microscope). Fluorescent images were captured using an AxioCam MRm  digital camera and AxioVision 4.5 software . Images were uniformly processed to adjust contrast and image clarity using Adobe Photoshop CS2.To assess SP-A binding to C. neoformans to alveolar macrophages, yeast were incubated with SP-A (5 to 20 µg/ml), anti-GXM IgG (1.0 µg/ml) or a combination of SP-A and anti-GXM IgG at 37°C for 60 minutes with shaking. Yeast were washed twice with 5 volumes of PB and added to alveolar macrophages in suspension at a multiplicity of infection of 1∶1. Yeast and alveolar macrophages were co-incubated for 60 minutes at 37°C with gentle shaking and attachment/phagocytosis was quantified by flow cytometric analysis.Alveolar macrophages were isolated from Sprague-Dawley rats . Bronchoalveolar lavage (BAL) was performed with 10 ml of PBS containing 0.2 mM EGTA (instilled 10 times). Cells were washed once with PBS and resuspended in PB. Prior to the addition of Alexa Fluor 647 labeled C. neoformans wild-type strain H99 was used to assess the immune response and susceptibility of SP-A-/- mice to infection by C. neoformans. The immune response to infection by C. neoformans was assessed by infecting groups consisting of 5 or 10 SP-A-/- or wild-type C57BL/6 mice via intranasal inhalation with 1×104 or 1×105 CFU of each strain (in a volume of 50 µl), as described previously 4 yeast for studies that assessed the host immune response was so that we could measure the immune response to a relatively small number of C. neoformans. SP-A-/- and wild-type mice were infected with an inoculum of 1×105 yeast, which is a common inoculum size used for susceptibility studies. Bronchoalveolar lavages  were performed and total lung tissues were collected for colony forming unit determination at days three and seven post-infection. These time points were chosen so as to capture early events in the immune response to C. neoformans. Mouse TNFα ELISAs (R&D Systems) were performed on bronchoalveolar lavage fluid per the manufacture's instruction. Cell differentials were performed by microscopic examination of cytospin preparations of BAL from mice seven days post infection. Slides were prepared with Wright-Giemsa stain per the manufacturers instructions. Susceptibility studies were performed with groups consisting of 5 SP-A-/- or wild-type C57BL/6 mice that were infected via intranasal inhalation with 1×105 CFU of each strain (in a volume of 50 µl), as described previously P-values<0.05 were considered statistically significant.Statistical analysis was performed using the JMP statistics package . Student's T-test or ANOVA was used to assess binding, phagocytosis and cell differential data for statistical significance. Survival statistics were performed to evaluate mouse survival data for statistical significance. in vitro, SP-A binding is restricted to acapsular and minimally encapsulated strains of C. neoformansC. neoformans yeast (1×105). SP-A binding assays using Escherichia coli HB101 were performed to confirm the binding activity of all SP-A preparations (results not shown). Our results were consistent with those of previous studies; we did not observe SP-A binding to encapsulated H99 C. neoformans yeasts (C. neoformans yeasts (1×105) were next treated with undiluted bronchoalveolar lavage fluid (BAL). In contrast to the previous conditions, we were able to detect a small amount of SP-A binding under these conditions  or left untreated as a control. Calcofluor was used to visualize the cell wall. While SP-A did not bind directly to C. neoformans cells . Attachment/phagocytosis was quantified by flow cytometry. Non-IgG opsonized C. neoformans were attached/phagocytosed by less than 1% of the alveolar macrophage population in the presence or absence of SP-A, thus percent inhibition of uptake for these conditions is presented as ∼100% (P<0.001) inhibited the uptake of anti-GXM IgG opsonized C. neoformans yeast by alveolar macrophages and that this inhibition occurred in a dose dependent manner   . These rin vitro studies suggested that the classic role of SP-A as an opsonin was unlikely to contribute to host defense against C. neoformans. In fact, its affect on IgG-mediated phagocytosis could enhance extracellular colonization and thus change the growth and dissemination of C. neoformans in vivo. Furthermore, SP-A is also known to contribute to host defense via other mechanisms, such as modulation of the immune response. Therefore, we could not predict the impact of SP-A on cryptococcosis. We explored the role of SP-A as a modulator of the early immune response against C. neoformans using a murine pulmonary infection model and an SP-A deficient mouse strain (SP-A-/-). Groups of SP-A-/- and wild-type mice were infected via intranasal inoculation with C. neoformans cells (1×104) and several defined endpoints were analyzed at days three and seven post infection.Our C. neoformans. Bronchoalveolar lavage fluid and lung tissue were collected from SP-A-/-  and wild-type  mice at days three and seven post infection. We found that SP-A deficiency did not affect the colonization of the lungs by C. neoformans (P<0.001) number of yeast were present in the lung tissue of both mouse strains, as compared to the bronchoalveolar lavage fluid  and wild-type (n = 3) mice at day seven day post infection were equivalent  differences between the numbers of C. neoformans cells phagocytosed by alveolar macrophages from SP-A-/- (n = 3), as compared to wild-type (n = 3) mice was observed  and seven  post infection  to investigate whether SP-A deficiency affected the final outcome of infection. By day 32 of infection all mice had succumbed to infection and died with no significant difference (P>0.05) in the mean time of mortality between the two groups  The inability of SP-A to bind encapsulated strains of C. neoformans capsule and then SP-A binds to bound IgG. First, we showed that SP-A binding was not inhibited by EDTA, demonstrating that the SP-A carbohydrate recognition domain was not required. This ruled out the possibility of SP-A binding to carbohydrate ligands that may have been exposed as a result of anti-GXM IgG binding. Second, SP-A binding inhibited the IgG dependent phagocytosis of C. neoformans yeast by alveolar macrophages. This observation suggested that SP-A binding functionally interfered with IgG binging to the Fcγ receptor on the cell surface of alveolar macrophages, which is required for phagocytosis in the case of C. neoformansin vivo conditions.This is the first report that has demonstrated IgG dependent binding of SP-A to a microorganism. Several lines of evidence suggest that this occurs via a mechanism in which IgG first binds to Aspergillus fumigatus conidia by alveolar macrophages and neutrophils SP-A-/- mice have shown that SP-A deficiency results in increased susceptibility to infection by P. jiroveciH. capsulatumC. neoformans. The end points analyzed in our in vivo studies clearly demonstrated an equivalent progression and outcome of disease in SP-A-/- and wild-type mice.SP-A mediated mechanisms of host defense provide protection against several pathogenic fungi. For example, SP-A enhances the phagocytosis and killing of SP-A-/- mice would be expected to produce increased levels of reactive oxygen species as compared to wild-type mice. This up regulation of reactive oxygen species however would not be expected to have a significant effect on C. neoformans colonization of the lungs since C. neoformans possesses a highly robust antioxidant defense system in vitro and in vivo results of this study suggest that unlike a variety of bacteria, viruses and other fungi, the encapsulated yeast C. neoformans does not appear to be susceptible to SP-A mediated mechanisms of innate immunity in the murine pulmonary tract.Another mechanism by which SP-A can mediate host defense is via direct modulation of alveolar macrophage function. SP-A can reduce reactive oxygen intermediate production by alveolar macrophages via the down regulation of NADPH oxidase


The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses. The APOBEC3 proteins constitute a family of seven cytidine deaminases. Cytidine deaminases are editing enzymes able to remove the amine group from cytidine in single-strand DNA (ssDNA) and RNA, converting it to uracil. APOBEC3 proteins have potent antiviral activity against retroviruses, retrotransposons, and DNA viruses. APOBEC3 generated high interest because of the ability of APOBEC3G (A3G) to inhibit HIV. APOBEC3A (A3A) is a member of the family that inhibits the human parvovirus adeno-associated virus (AAV) and the retrotransposon LINE-1. Parvoviruses are simple ssDNA viruses that do not require a retrotranscription step for their replication. In contrast to A3G, which is predominantly cytoplasmic, A3A is located in both the nucleus and cytoplasm. In addition, A3A consists of a single cytidine deaminase catalytic domain, whereas A3G has two. The dependence of the antiviral function on deaminase activity is controversial. In this study, we identify numerous A3A residues required for deaminase and antiviral activities. We show that A3A not only inhibits AAV but also the minute virus of mice (MVM). Importantly, we demonstrate that A3A does not require its deaminase activity to block the replication of both parvoviruses. Thus, exploiting the simplicity of parvoviruses together with the single-domain cytidine deaminase A3A, we are able to demonstrate that cytidine deaminase activity is not required for APOBEC3 mediated viral inhibition. Eukaryotes have evolved numerous innate immune defenses against invading pathogens. The apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) proteins comprise a family of seven cytidine deaminases 23–28-P-C-X2–4-C (where X is any amino acid). It has been proposed that the histidine and the two cysteine residues coordinate a Zn2+ ion, while the glutamic acid residue serves an essential role in catalysis as a proton shuttle The APOBEC3 proteins share structural and functional features with zinc-dependent deaminases and possess cytidine deaminase activity The mechanism of the antiviral activity of APOBEC3 proteins against retroviruses has been studied extensively Whether cytidine deamination is the principal mechanism for the antiviral activity of APOBEC3 proteins remains controversial Although it lacks activity against retroviruses, A3A is a potent inhibitor of both parvovirus and the human transposon LINE-1 A3A inhibition of parvovirus provides an attractive system in which to decipher the relative contribution of deamination and deaminase-independent mechanisms to antiviral activity. Unlike the other viruses known to be inhibited by APOBEC3 proteins, the parvoviruses replicate exclusively in the nucleus, do not pass through RNA intermediates, do not have a reverse transcription step in their replication schemes, and use DNA hairpins for priming replication in vitro and for antiviral activity in rAAV production assays. We identify mutants that lack deaminase activity but retain the antiviral effect, supporting the idea of a deamination-independent mechanism. In addition to AAV, we show that A3A inhibits DNA replication of the autonomous parvovirus MVM. Chimeric A3A/A3G proteins generated by exchanging divergent sequences demonstrate loss-of-function for A3A and gain-of-function for A3G. Our mutants also reveal residues in the linker and pseudoactive site domains that are important for deamination, target specificity and antiviral activities of A3A. Together, these studies reveal domains of A3A that are responsible for its distinct antiviral activity and suggest that A3A can inhibit parvoviruses through a mechanism separate from its function as a cytidine deaminase.To ascertain whether deaminase activity is required for inhibition of parvovirus replication, and to understand the functional significance of amino acid divergence between A3A and A3G, we analyzed the properties of a panel of A3A point mutants and A3A/A3G chimeras. The proteins were tested for deaminase activity We previously showed that A3A antiviral activity was dependent on the integrity of conserved amino acids in the active site that are responsible for proton shuttling and zinc coordination First, we determined the cellular localization of the mutant proteins in transfected U2OS and HeLa cells . Most ofin vitro deamination assay. We previously demonstrated deaminase activity for A3A packaged into HIV-1 virions in vitro transcription/translation (IVT) using wheat germ extract. An advantage of this method is that it allows assessment of proteins that are unstable or difficult to express in cells, such as FF7595LL and A3A truncations , which allowed for quantitative assessment of virus production by transduction of target cells. Immunoblotting confirmed that proteins of the expected size were expressed  production assay . The APOxpressed . Wild-tyin vitro was not caused by an altered target sequence preference. We also tested the deaminase activity of A3A mutants in cell lysates by adapting the quantitative fluorescence resonance energy transfer (FRET) assay recently developed for A3G in vitro deaminase assay and the conclusion that AAV is inhibited in the absence of deamination.To exclude differences in AAV inhibition due to disparities in protein expression levels, we compared the mutants F75L and F95L with wild-type A3A over a dose-response . ImmunobIn our previous studies of AAV production in the presence of wild-type A3A, we found no detectable evidence of AAV sequence changes but viral replication was inhibited To examine the effect of A3A and mutants on replication of wild-type parvovirus genomes, we used two different viral systems. AAV2 depends upon helper virus for replication, while MVM replicates autonomously. First we used immunofluorescence to assess the effect of APOBEC3 proteins in cells infected with AAV2 and adenovirus helper virus. As previously shown BglII), was also inhibited by A3A  was fused to the C-terminal PmlI fragment of A3G (residues 306–384) to form the chimera A3ApmlA3G. The reciprocal chimera A3GpmlA3A was also generated. We also assessed the activity of the C-terminus of A3G  and a fusion with A3A at the PmlI site (A3G-CTpmlA3A). Cellular localization of the mutants was tested by immunofluorescence in transfected cells  differ from A3G . Using ted cells . We founubstrate . Neitherubstrate . Togethein vitroin vitro assays, while we have analyzed protein from cell lysates. Furthermore, A3G-CT was shown to be significantly less active than the full-length protein Careful inspection of the deaminase assay autoradiograms . While Ain vitro assay  are important for deamination, and may play a role in antiviral function. Therefore, we determined whether the reciprocal switch (where residues in A3G were substituted with the sequences from A3A) would generate a gain-of-function . Sequencactivity . Incorpoactivity . Thus A3in vitro deaminase assay. The lack of deaminase activity on a panel of target oligonucleotides demonstrated that the F75L A3A mutant is truly deaminase-deficient, and has not simply changed its target site preference and eluded detection in the deaminase assay. Although the in vitro deaminase assay has limitations, we demonstrated that lysates containing F75L also lacked deaminase activity in the FRET assay In this report we provide multiple pieces of evidence to show that A3A inhibition of AAV can occur through a deaminase-independent mechanism. Mutation of A3A active site residues that are essential for catalytic activity  led to the loss of activity against AAV. However, other mutants (F75L and mutants in VS1 and VS2) separated the deaminase activity from the ability to inhibit AAV. Together these data indicate that the integrity of the active site is important but that deaminase activity is not required for AAV inhibition. In APOBEC1, aromatic residues analogous to F75L and F95L of A3A are required for both deaminase activity and binding to nucleic acids Deaminase-independent inhibition of ΔVif-HIV and retroelements by A3G has been controversial CCA CA. Thus, A3A residues in the C-terminus contribute to its target specificity, in agreement with data from chimeric and mutant proteins of other APOBEC3 family members In addition to providing evidence for deaminase-independent antiviral activity, our study also offers insights into the structural basis of APOBEC3 protein function. The linker and pseudoactive site domains in the N-terminus of A3G are required for HIV-1 virion incorporation in vitro deaminase activity than other APOBEC3 proteins. Deaminase activity of A3G resides in the C-terminal CDD 2–4C motif of A3A. Deletion of these amino acids in the A3A/ΔWG mutant slightly reduced in vitro deaminase activity  Although 293 cells are a standard system used to study parvovirus replication, it remains unclear whether endogenous A3A restricts parvovirus infection 2.293T, HeLa, A9 and human osteosarcoma U2OS cell lines were purchased from the American Tissue Culture Collection. Cells were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics at 37°C in a humid atmosphere containing 5% COExpression plasmids encoding cDNAs for A3A (NM_145699) and A3G (NM_021822) and the A3A mutants H70R, E72Q, and C106S in the pcDNA3.1 (+) vector with a hemagglutinin (HA) tag at the C-terminus have been previously described 6 cells/well in 6-well plates and the next day were co-transfected with pXX6 (2.25 µg), pXX2 (0.75 µg), pACLALuc (0.75 µg) and APOBEC3 expression vector (1 µg unless otherwise stated) or pcDNA3.1(+) control vector (1 µg). Dose-response titrations maintained the total amount of effector DNA by addition of pcDNA3.1(+). Transfections were performed in duplicate or triplicate using polyethyleneimine (PEI) Recombinant AAV production assays were performed as previously described 2HPO4, 1.76 mM KH2PO4, 1 mM NaO3V, 20 mM β-glycerol phosphate, 20 mM NaF, 0.1% NP40, and 0.025% Triton-X 100) supplemented with Complete protease inhibitor cocktail (Roche) for 30 min on ice. The lysates were clarified by centrifugation at 10,000×g for 20 min. Protein concentrations from whole cell lysates were quantified by BCA assay (Bio-Rad), and 20 µg of protein was loaded per well onto polyacrylamide gels. Proteins were separated in 4–12% or 12% Acrylamide Bis-Tris NuPage gels in MOPS buffer (Invitrogen) and transferred onto Hybond nitrocellulose membranes (Amersham Biosciences). Membranes were probed with anti-HA 16b12 monoclonal antibody (mAb) (Covance) and anti-Ku86 mAb (Santa Cruz). Bound antibody was detected by incubation with goat anti-mouse antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch), and the bands were visualized with enhanced chemiluminiscence reagent  followed by autoradiography.Immunoblotting was performed essentially as described APOBEC3 protein localization was determined by indirect immunoflorescence 32P-labeled luciferase probe as previously described DpnI (New England Biolabs) to remove input plasmid. DNA was processed by gel electrophoresis on a 1% agarose gel in TAE buffer. The pACLALuc plasmid was digested with SmaI as control. The gel was depurinated in 0.2 M HCl, denatured in 1 M NaCl, 0.5 M NaOH, and neutralized in 0.5 M Tris pH 7.5, 1.5 M NaCl. DNA was then transferred to a Hybond XL membrane (Amersham Biosciences) and UV-cross linked. The membrane was hybridized with a 32P labeled luciferase probe generated by PCR using the primers described in 32P dCTP using Radivue II labeling kit (Amersham), and visualized in a FLA-5100 phosphorimager (Fuji).Low molecular weight AAV episomal DNA was analyzed by Southern hybridization with a BglII were performed as previously described Southern blot replication assays of wild-type MVM and MVM-Δ5 cells/well in 6-well plates and transfected after one day with 3 µg of APOBEC3 expression vector. Two days post-transfection, the cells were rinsed twice with cold PBS and lysed for 30 min on ice in lysis buffer . Lysates were clarified by centrifugation at 13,000×g for 10 min and pre-cleared with 50 µl of High flow protein-G-Sepharose (Amersham). The lysate was incubated with anti-HA mAb 3F10 (Roche) for 2 h at 4°C. The lysate-antibody was then incubated with High flow protein-G-Sepharose for 1-2 h at 4°C. The resin was washed three times with lysis buffer. One-fifth of the resin was removed for immunoblot analysis and the remainder was washed once with deaminase reaction buffer . PAGE purified deoxyoligonucleotide  in buffer containing 20 mM Tris, pH 8.0, 1 mM DTT for 1 h at 37°C and treated with 150 mM NaOH for 1 h at 37°C. The samples were incubated at 95°C for 5 min, 4°C for 2 min and separated by 15% TBE/urea-PAGE. The gel was dried, exposed to a phosphorimager screen and analyzed using a FLA-5100 scanner (Fuji).293T cells were seeded at 5×10cleotide  was 5′-ein vitro synthesis of A3A and mutants we employed the TNT Coupled Wheat Germ Extract System (Promega) using T7 polymerase. Translation reactions were performed with non-labeled amino acids following the manufacturer's protocol in 50 µl final volume that included 1 µg of pcDNA3.1(+) plasmid encoding APOBEC3. Reactions were incubated for 90 min at 30°C. After incubation, 450 µl of TritonX-100 buffer  were added to the reactions and used to analyze deaminase activity after immunoprecipitation as described above.For 32P labeled deoxyoligonucleotide in 30 µl of deaminase reaction buffer and assayed for deaminase activity following the same procedure described above.In order to measure deaminase activity directly from IVT reactions, after 90 min incubation at 30°C translation reactions were centrifuged at 10,000×g for 1 min. An aliquot of supernatant (5 µl) was removed for immunoblot analysis. The reaction mixture (15 µl of supernatant) was incubated with 5′-end et al.6 cells/well in 6 cm plates and a day later transfected with 8 µg of APOBEC3 expression vector. After 36 to 48 h, cells were resuspended in lysis buffer (200 µl) and lysates were obtained as described above. Cell lysate (10 µl) was mixed with 70 µl of FRET deaminase buffer  containing 10 pmol of dual-labeled probe  for 7 days in order to differentiate them into macrophages. The monocyte-derived macrophages were plated in 12 well plates at 106 cells per well and cultured with or without 2000U of Universal Type I Interferon  for 20 hours. The cells were then collected in lysis buffer. Cell lysates from human peripheral blood mononuclear cells (PBMCs) were obtained from F. Chisari (The Scripps Research Institute) Human monocytes were purified from leukocyte enriched blood samples (New York Blood Center) using CD14+ magnetic beads (Miltenyi Biotec) according to manufacturer instructions. The CD14Figure S1in-vitro transcription-translation are active in UDG-dependent deaminase assays. Deaminase activity of A3A and mutant proteins generated by cell transfection and in-vitro coupled transcription-translation (IVT) was analyzed in UDG-dependent deaminase assays. (A) 293T cells were transfected with plasmids for A3A and mutants. Cells were harvested at 48 hrs post-transfection, and lysates were subject to immunoprecipitation (IP) with anti-HA antibody (3F10). 4/5 of the IP was incubated with a 5′-end 32P labeled T28TCAT28 deoxyoligonucleotide and tested in UDG-dependent deaminase assays. Arrows indicate substrate deoxyoligonucleotide and cleaved deaminated product. Bottom panel shows an immunoblot corresponding to 1/5 of the IP analyzed with an anti-HA antibody (16B12). Asterisks indicate bands corresponding to IgG light chain. A3A truncations are: TruncA (aa 1–145), TruncB (aa 1–165), and TruncC (aa 53–199). (B) Wild-type and mutant A3A proteins were synthesized by IVT as described in APOBEC3A proteins synthesized from coupled (1.92 MB PDF)Click here for additional data file.Figure S232P-labeled deoxyoligonucleotide substrates, each containing four target sites. The -1 base is shown at the top of each lane and the +1 base is shown on the left. The structure of the cleaved products is shown on the right. (B) Quantification of deaminase acitivity by FRET in cell lysates obtained from 293T cells transfected with A3A, E72Q, F75L, F95L and C106S. Cell lysates were incubated with a dual-labeled probe containing the CCCG target sequence (Lack of deaminase activity for F75L is not due to modified target sequence preference and is supported by the lack of deamination measured by FRET. (A) To rule out differences in target sequence preference, A3A and F75L were tested in UDG-dependent deaminase assays against 16 different target sites . Target sequence . The TAM(2.23 MB PDF)Click here for additional data file.Figure S3Localization of A3A/A3G chimeras. Immunofluorescence to detect localization of HA-tagged APOBEC3 and chimeric proteins (red) expressed by transfection in U2OS cells. Cell nuclei were detected by staining with DAPI (blue). (A) Localization of wild-type APOBEC3 proteins and A3A/A3G chimeras. (B) Localization of A3A mutants with sequences incorporated from A3G.(9.29 MB PDF)Click here for additional data file.Figure S4Inhibition of rAAV production by A3A/A3G chimeras in the VS1 region. Dose-response for A3A and mutant proteins in the rAAV production assay. Virus production was assessed by transduction of target cells and quantitation by luciferase assay. Panels below show immunoblots for APOBEC3 (HA) and Ku86 proteins in transfected cells.(0.39 MB PDF)Click here for additional data file.Figure S5t test.Dose-response antiviral activity of A3G-CT/KNLLCGFY. Comparison of A3A and A3G-CT/KNLLCGFY antiviral activity over a dose-response in rAAV production assays. Comparable levels of A3A and A3G-CT/KNLLCGFY resulted in ∼95% and ∼50% inhibition respectively. Bottom panels show immunoblots for A3A and A3G-CT/KNLLCGFY (HA), and Ku86 protein as a loading control. The asterisks indicate that the inhibition with A3G-CT/KNLLCGFY was statistically significant (p<0.001) when compared to mock by Student (0.53 MB PDF)Click here for additional data file.Figure S6In vitro deamination assay. Proteins were immunoprecipitated from transfected cells and incubated with the radiolabeled substrate in the standard assay. Arrows indicate the substrate and deaminated product. The panel below shows an immunoblot to detect proteins in immunoprecipitates. Arrows indicate bands corresponding to APOBEC3 proteins. Asterisk indicates bands corresponding to IgG light chain. (C) Production of rAAV in the presence of APOBEC3 proteins. Virus production was assessed by transduction of target cells and quantitation by luciferase assay. Immunoblots show similar expression levels for APOBEC3 proteins (HA) in transfected cells. Ku86 served as a loading control.Chimeric A3G proteins with sequences replaced with VS1 and VS2 from A3A. (A) Immunofluorescence to detect localization of HA-tagged APOBEC3 and chimeric proteins (red) expressed by transfection in U2OS cells. Cell nuclei were detected by staining with DAPI (blue). (B) (3.93 MB PDF)Click here for additional data file.Figure S7Antiviral activity of A3A is achieved with physiological levels of transfected A3A. (A) Antiviral activity of A3A was analyzed in a rAAVLuc production experiment over a dose response. Expression levels of A3A were analyzed by immunoblotting using an anti-HA antibody (top blot), and compared to endogenous levels of A3A expressed in human PBMCs incubated with IFNα. A3A was detected using a rabbit polyclonal antisera raised against an N-terminal A3A specific peptide (bottom blot). Arrow indicates bands corresponding to A3A. Background band below A3A is indicated with an asterisk. (B) In panel B the expression levels of A3A are compared to endogenous levels of A3A induced in human macrophages in response to IFNα. A3A was detected with polyclonal rabbit serum generated to recombinant A3A protein.(1.09 MB PDF)Click here for additional data file.Table S1Oligonucleotides used in this study. Sequences corresponding to the oligonucleotides used in this study. Name, template and type of experiments are indicated for each oligonucleotide. D.A. (deaminase assays). N/A (not applicable).(0.08 MB DOC)Click here for additional data file.


Here, we further examined rod and cone development, chromophore production by the RPE, and photoreceptor signaling to the inner retina under organ culture conditions.Retina organ cultures can be used as a valuable tool to study retina development ex vivo. Comparison between culture methods has revealed that timing the start of the culture and the presence of the retinal pigment epithelium (RPE) are critical for the development of the rods and cones, which are the two types of photoreceptors; rods can develop in the absence of the RPE, cones cannot. One of the necessary compounds produced by the RPE and essential for cone development and survival is the chromophore 11-cis retinal spectrophotometrically by measuring rhodopsin. Signal transmission in the rod pathway was studied by analyzing c-fos expression in the inner retina in response to stroboscopic illumination.Retina-RPE cultures were prepared from 7-day-old C57BL/6 pups and maintained in culture for 11 days. Rod and cone structure was analyzed by immunohistochemistry, and cell-specific mRNA expression was analyzed by quantitative real-time PCR. We quantified 11-cis retinal production ex vivo plateaued after 7 days in culture, resulting in approximately 30% of the in vivo level by day 11. The retinas responded to prolonged stroboscopic illumination with the normal nuclear expression of c-fos in cells in the inner retina.In retina-RPE cultures analyzed after 11 days in culture, rod and cone numbers exhibited a similar ratio to those observed in the intact animal. Although photoreceptor outer segments were shorter when grown ex vivo, membrane proteins, such as cone opsin and transducin, were localized appropriately to the outer segment. Relative 11-cis retinal to promote cone development and support signal transmission in the rod pathway. Organ cultures may be a powerful low-throughput screening tool to identify novel agents to promote photoreceptor cell survival and signaling.Mouse retinal structure is maintained in retina–RPE organ cultures. The RPE in organ cultures produces sufficient amounts of 11- Mouse retinas are amenable to growth in culture using either the isolated retina  or retincis retinal [cis retinal independent [cis retinal to promote cone maturation. However, in this context it is important to consider that in isolated RPE cultures, the expression of RPE65, the isomerohydrolase required for the production of the chromophore 11-cis retinal [RPE65 have significantly reduced levels of rhodopsin and opsin [For organ cultures to be a useful tool to study or manipulate photoreceptor development and function, baseline parameters first have to be established. Here, we have asked four related questions addressing different aspects of the photoreceptor cells: cell numbers, subcellular localization of cell-type specific proteins, pigment content, and postsynaptic signaling. Pertaining to the first aspect, rods outnumber cones by a ratio of 30:1 in the mouse in vivo , and a s retinal -15, wherependent . As a coependent . Here, w retinal , appears retinal ,19, age  retinal , and sou retinal . Third, nd opsin . Fourth,nd opsin -24, a mend opsin .cis retinal production sufficient for cone development and maintenance, as well as signaling from the photoreceptors to the inner retina.Taken together, we have tested and shown that commonly used organ culture conditions generate a tissue environment that supports normal rod and cone development, 11-C57BL/6 mice were generated from breeding pairs obtained from Harlan Laboratories . Animals were housed in the Medical University of South Carolina (MUSC) Animal Care Facility under a 12 h:12 h, light–dark cycle, with access to food and water ad libitum. All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the MUSC Animal Care and Use Committee.2, balanced air, 100% humidity, at 37 °C), and the medium was changed every 2 days under dim red light. No antimicotics or antibiotics were required.All chemicals and reagents used for organ cultures were tissue culture grade and were purchased from Invitrogen . Retina–RPE cultures were grown by means of the interface technique according to published protocols ,2,25 wit2HPO4(H2O), 1.8 mM KH2PO4), they were blocked with 10% normal goat serum  and 3% BSA (in PBS containing 0.4% Triton X). Tissues were incubated overnight in blocking solution containing the antibodies of interest, followed by incubation with the appropriate fluorescent-labeled secondary antibody for 2 h . Control experiments included omission of primary antibody and observation of singly-labeled slides through the appropriate filter set. Sections were mounted and analyzed by confocal microscopy  using identical settings for all slides. Images were imported into Adobe Photoshop software  for further analysis. The following primary antibodies were used in this study: rhodopsin , and cut into 14-μm cryostat sections ,25,26. ACruz, CA ,28), mouCruz, CA , mouse cCruz, CA ), and c-Cruz, CA ,32). All−1·cm−1 [Pigment measurements were performed on samples from age-matched animals and retina explant cultures. Mice were dark-adapted overnight before tissue collection, and explant cultures were manipulated under dim red light. All procedures were performed under dim red light to prevent photobleaching. Endogenous levels of pigment were determined by extraction of a homogenate of two retinas as described previously  with mod−1·cm−1 .Gnat2), forward 5′- GCA TCA GTG CTG AGG ACA AA-3′, reverse 5′- CTA GGC ACT CTT CGG GTG AG-3′; and Rpe65, forward 5′- TGC ATG CAC AGA GAC CAA CT-3′, reverse 5′- CAG TGG CAC CAT TGA CAG AA-3′. Quantitative values were obtained from the cycle number  to establish the fold difference in gene expression between the treated and untreated retina cultures [Real-time PCR was performed as previously described . In shorcultures .Stroboscopic illumination was used to study light-induced gene expression in the inner retina ,23. For t test, with p<0.05 accepted as significant.Data are expressed as means± standard deviation (SSD). Statview software  was used for statistical analysis. Comparison of two conditions was performed using the Student cis retinal. Tissues were placed in organ cultures starting at P7, a time point when inner retinal layer formation is complete, but rod precursors actively migrate through the outer plexiform layer into the outer photoreceptor layer, a process that is complete between P10 and P12 [We established a retina–RPE explant culture to analyze rod and cone development under controlled conditions. The defined media used here  contains vitamin A levels comparable to those found in mouse serum , provid and P12 . After Pcis retinal [cis retinal for cone opsin expression and cone survival [cis retinal is necessary for the localization of cone opsin and cone outer segment membrane proteins, such as cone transducin (Gnat2), to the cone outer segments of the mouse retina [Rod development occurs independent of the presence of RPE , and rod retinal . Howeversurvival  and latee retina -13. Our ucin Gnat, to the e retina , immunohe retina   and approximately 14.2±2.4 Gnat2-positive cone outer segments (p<0.05). To establish the ratio of rods:cones per window in the central retina, the number of rods was obtained by multiplying the number of horizontal rows of photoreceptor nuclei by the height in nuclei of these rows. Using this comparison we confirmed that the ratio of rods:cones is close to 30:1 in retinas collected from both in vivo and in vitro conditions . Although the rod:cone ratio was approximately 15% higher in the in vitro condition, this difference was not statistically significant, largely due to the number of mice tested under each condition (n=6). The reduced number of rods and cones as well as the shortened outer segments present in cultured photoreceptors is reflected by 2-fold to 3-fold lower levels of rhodopsin, UV and MWL opsin, and vels see .Taken together, using a snapshot at P7+11DIV as reported by others, retinal layers are retained in organ culture . While acis retinal. To determine the amount of 11-cis retinal generated in these cultures, the amount of rhodopsin was assayed in dark-adapted littermates and retinal explants at different postnatal days –PCR. At P7+11DIV, Rpe65 mRNA levels in organ cultures were approximately 2.5-fold lower than in age-matched in vivo littermates  to prolonged stroboscopic illumination. The c-fos results, which showed that signal transmission from rods to bipolar cells does occur and is strong enough to be detected, also revealed that culture conditions provide all the essential components to promote expression of the proteins required for cellular development and synaptic transmission within the rod pathway. In future experiments we wish to utilize whole-retina ERG recordings [cis retinal formation plateaus after 7DIV and both rod and cone OS are stunted suggests that while retinal development occurs in culture, maturation of the retina is arrested in culture. This may ultimately limit the useful range of the culture system to 7DIV.In summary, the RPE in vitro was found to produce between ~40 and 25% of the level of 11-cordings , which wRodent retinas can easily be grown in culture conditions. We established a retina–RPE system of explants from the early postnatal mouse eye to set a defined mimic of the in vivo condition in which mechanisms of photoreceptor development, outer segment membrane protein trafficking, and light-induced signal transmission can be studied. Since many compounds have teratogenic effects, this system will allow us to study compounds in isolation.


Aspergillus flavus I-V, Aspergillus fumigatus I-ll, Helmintosporium sp, Cladosporium sp, Mucor rouxii mutant, M. rouxii IM-80, Mucor sp-I and 2, Candida albicans and Cryptococcus neoformans was studied in this work. It was found that the biomass of C. neoformans, natural sediment, Helmintosporium sp and chitosan was more efficient to remove chromium (VI)  achieving the. following percentage of removals: 98%,98% and 63%, respectively. The highest adsorption was obtained with C. neoformans and Helmintosporiumsp at pH 2.0 and 4.0 + 0.2, respectively, at 28∘C after 24 hours of incubation, with 0.2 mg/L of cellularbiomass.The biosorption of chromium (VI) on eighteen different natural biosorbents: Natural sediment, chitosan,chitin,


Surfactant protein D (SP-D), an innate immune molecule, plays an important protective role during airway inflammation. Deficiency of this molecule induces emphysematous changes in murine lungs, but its significance in human COPD remains unclear.in vitro using an established model of isolated type II alveolar epithelial cell culture.We collected bronchoalveolar lavage fluid from 20 subjects with varying degrees of COPD (8 former smokers and 12 current smokers) and 15 asymptomatic healthy control subjects . All subjects underwent a complete medical history and pulmonary function testing. SP-D was measured by Enzyme-Linked ImmunoSorbent Assay. Statistical analysis was performed using nonparametric methods and multivariable linear regression for control of confounding. The effect of corticosteroid treatment on SP-D synthesis was studied Among former smokers, those with COPD had significantly lower SP-D levels than healthy subjects . In a multivariable linear regression model controlling for age, sex, race, and pack-years of tobacco, COPD was independently associated with lower SP-D levels  and inhaled corticosteroid use was independently associated with higher SP-D levels . To support the hypothesis that corticosteroids increase SP-D production we used type II alveolar epithelial cells isolated from adult rat lungs. These cells responded to dexamethasone treatment by a significant increase of SP-D mRNA (p = 0.041) and protein (p = 0.037) production after 4 days of culture.in vitro. Given the importance of this molecule as a modulator of innate immunity and inflammation in the lung, low levels may play a role in the pathogenesis and/or progression of COPD. Further, we speculate that inhaled steroids may induce SP-D expression and that this mechanism may contribute to their beneficial effects in COPD. Larger, prospective studies are warranted to further elucidate the role of surfactant protein D in modulating pulmonary inflammation and COPD pathogenesis.Among former smokers, COPD is associated with lower levels of SP-D and inhaled corticosteroid use is associated with higher levels of SP-D in the lung. Dexamethasone induced SP-D mRNA and protein expression in isolated epithelial cells  Chronic obstructive pulmonary disease (COPD) is characterized, in part, by an abnormal inflammatory response of the lung to noxious particles or gases , chiefly2+ dependent lectins that share col lagen-like N-terminal tails and globular lectin heads containing C-type carbohydrate recognition domains. Produced in alveolar type-II cells and Clara cells, SP-D is a 43-kD monomer that forms a higher order quaternary structure . SP-D binds to and enhances clearance of a wide variety of pathogens  for an additional 3 days. Cells were re-fed with fresh medium on day 3 and were harvested on day 0, and day 4. Three individual experiments were performed, each using duplicate cultures.Cells were isolated from adult rat lungs digested by collagenase I and collagenase IA  as previously described ,23,24). ,24. 23,2To detect intracellular surfactant proteins, the cell pellet was sonicated and the supernatant was concentrated by evaporation. Western blot analysis was performed as previously described . MembranThe 386-nucleotide rat cDNA probe for SP-D was newly prepared using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and the Retroscript Kit . Total RNA was isolated from type II cells by RNeasy kit  and Northern blot analysis was performed as previously described .In univariate analysis, SP-D levels were tested for association with age, sex, race, disease status, smoking status, total smoke exposure in pack-years, years since quit smoking, and inhaled corticosteroid use. The Wilcoxon's ranksum test or Kruskal-Wallis test were used for categorical variables with two or more than two levels, respectively, and the Spearman test for correlation was used for continuous variables. The association between disease status and SP-D level in BALF was stratified by smoking status to control for possible confounding. Among former smokers, SP-D levels approximated a normal distribution; as a result, multivariable linear regression was used to test whether the association between disease status and SP-D levels within this group was independent of the effect of other measured variables. Covariates were considered confounders if they altered the association between disease and SP-D levels by 15% or more.Further analyses included a non-parametric test for trend  for SP-DWe recruited 20 subjects with varying degrees of COPD (8 former smokers and 12 current smokers) and 15 asymptomatic healthy control subjects . Subject characteristics by disease status are listed in Table Previously published studies on BALF SP-D levels from a wide range of laboratories have shown only single sample measurements. To investigate the variability in individual BALF SP-D levels over time, we compared SP-D levels in repeated BALF samples from subjects who underwent multiple bronchoscopies as described in the Methods. One-way analysis of variance revealed marked within-subject variability over time (standard deviation 215 ng/ml) in BALF SP-D levels from repeat samples obtained within 3–9 months. This value was nevertheless lower than the between-subject variability (standard deviation 306 ng/ml). To attain the most representative measure for further analyses we used the average of SP-D levels obtained from each subject.Smoking status had a substantial impact on SP-D levels, with former smokers having levels intermediate between never smokers and current smokers . Importantly, all former smokers in this cohort had quit a minimum of 1.5 years prior to bronchoscopy, eliminating the possibility that the lower levels in former smokers were due to bias from recent cessation.Overall, subjects with COPD had lower SP-D levels in their BALF compared with healthy subjects . In order to determine if the effect of disease status was independent of the effect of smoking status, we stratified subjects by smoking status and re-examined the effect of disease status among former smokers and current smokers, separately. Among current smokers, those with COPD had only marginally lower SP-D levels in their BALF relative to healthy smoking controls, but this difference was not statistically significant (p = 0.45). However, among former smokers, COPD subjects had significantly lower SP-D levels in their BALF relative to healthy controls (p = 0.01), suggesting that COPD is associated with lower levels of SP-D in the lung independent of smoking status .To investigate whether corticosteroids would be capable of directly affecting SP-D synthesis we isolated type II alveolar epithelial cells from adult rat lungs. Cells were cultured for 4 days in the presence or absence of dexamethasone (10 ng/ml). Figure To exclude the possibility that lower levels of SP-D among former smokers with COPD were due to a non-specific loss of pulmonary proteins through epithelial destruction, we re-examined our multivariable model for former smokers using SP-D levels normalized to total protein concentration. This model yielded comparable results for the effect of disease on SP-D levels  even after controlling for age, race, pack-years of tobacco use, and ICS use . In addition, we analyzed Clara cell protein  in the BALF in comparison with SP-D levels. Although former smokers with COPD did have a small but statistically significant reduction in CCP-16 levels compared to healthy former smokers , this difference disappeared with control for any potential confounders, including age, race, sex, pack-years of tobacco use, or ICS use. This sharply contrasted with the effect of disease on levels of SP-D, which remained statistically significant despite inclusion of all of these confounders, simultaneously. Thus, the reduced SP-D levels in association with COPD appear to be a specific phenomenon rather than simply reflecting an overall decrease in pulmonary proteins due to loss of airway epithelium.To further investigate the relationship between smoking and COPD with regard to SP-D levels, we subdivided our cohort into 5 groups consisting of healthy never smokers (n = 5), healthy former smokers (n = 3), former smokers with COPD (n = 8), healthy current smokers (n = 7), and current smokers with COPD n = 12). We then analyzed pulmonary SP-D levels by this categorization and found that there was a very strong pattern of progressive decline in SP-D levels as determined by a non-parametric test for trend  had lower levels of SP-D compared with subjects with GOLD stage 2 or 3 disease, but this difference was not statistically significant. While we hypothesize that greater disease severity is associated with lower SP-D levels, our study was underpowered to detect this difference. Clearly, further studies with larger numbers of subjects will be required to ultimately prove or disprove this hypothesis.In our study, no distinction was made between COPD subjects fitting the emphysema phenotype and those fitting a more chronic bronchitic phenotype. Although SP-D may be differentially affected in these disparate phenotypes, any such difference would likely bias our study toward the null hypothesis; that is, such differences would increase the overall variability in SP-D levels among COPD subjects and thus increase the likelihood of failing to detect an association. Thus, that this variability would cause a spurious difference (i.e. a type-I error) is unlikely. Similarly, although definitive exclusion of asthma in clinical studies of COPD subjects with symptomatic airflow obstruction is quite challenging, the likelihood of asthma confounding our results is low. We carefully excluded subjects with a history of asthma, our COPD subjects were relatively old  with a substantial tobacco exposure history  and incompletely reversible airflow obstruction.It is possible that SP-D levels are lower in those with disease as a result of disease-related epithelial destruction that depletes the Clara cells and Type-II alveolar cells responsible for SP-D production. As such, SP-D could simply be acting as a marker of parenchymal destruction in COPD. Arguing against this is the fact that normalizing SP-D levels to the optical density for protein measurement did not substantially change the results of our multivariable model and that CCP-16 levels did not appear to be altered by disease independent of confounding. These results indicate that the ability to produce SP-D is unlikely to be a simple reflection of the extent of emphysematous changes in the distal air spaces.Regardless of whether lower SP-D expression in COPD subjects is due to parenchymal destruction, depletion of this protective molecule may still have a substantial effect on subsequent disease progression and susceptibility to infection. In murine models, mice deficient in SP-D display a markedly abnormal phenotype with pulmonary emphysema, accumulations of activated macrophages, and increased levels of matrix metalloproteases ,20,27-29Interestingly, in our model including all 35 subjects, African-American race was associated with higher SP-D levels independent of smoking status. While possible explanations such as differences in types of tobacco smoked or other smoking-related behaviors are plausible, this effect could potentially represent genetically regulated differences in expression. Indeed, in humans allelic variations have been shown to be strongly associated with differences in SP-D serum levels . HeidingOur multivariate analysis suggested that ICS use was independently associated with higher SP-D levels in the lung of former smokers with COPD. Corticosteroids have been shown to induce expression of SP-D in the fetal lung tissue of both rats ,33-35 anIn summary, we have demonstrated for the first time an association between decreased SP-D levels in the lung and COPD that is independent of current cigarette smoke exposure. We hypothesize that chronic exposure to cigarette smoke reduces SP-D expression and that subsequent relative deficiency of this protective molecule impairs innate immunity and contributes to the pulmonary inflammation characteristic of COPD. In this cross-sectional study, we were unable to determine the temporal association between lower SP-D levels and development of disease, making causal inference tentative at best. Larger studies with prospective follow-up are warranted to determine whether our observations are reproducible and predictive of subsequent clinical outcomes. Our study emphasizes that the relationship between COPD, corticosteroid therapy, and the structure and function of pulmonary SP-D merits further investigation.Among former smokers, COPD is associated with lower levels of SP-D and inhaled corticosteroid use is associated with higher levels of SP-D in the lung. Given the importance of this molecule as a modulator of innate immunity and inflammation in the lung, low levels may play a role in the pathogenesis and/or progression of COPD. We speculate that inhaled steroids may induce SP-D expression and that this mechanism could contribute to their anti-inflammatory effects in COPD. Larger, prospective studies are needed to further elucidate the role of surfactant protein D in modulating pulmonary inflammation and COPD pathogenesis.1: forced expiratory volume in one second; FVC: forced vital capacity; FEV1/FVC: ratio of forced expiratory volume in one second to forced vital capacity; GOLD: Global Initiative for Chronic Obstructive Lung Disease; ICS: inhaled corticosteroid; RV: residual volume; SP-D: surfactant protein D; TLC: total lung capacityBALF: bronchoalveolar lavage fluid; CCP-16: Clara cell protein 16; COPD: chronic obstructive pulmonary disease; ELISA: enzyme-linked immunosorbent assay; FEVRTS is a full-time employee of GlaxoSmithKline. MS, AH, and RAP receive funds from GlaxoSmithKline in the form of research grants.Michael Sims managed recruitment and clinical characterization of subjects, conducted all statistical analyses, and wrote the manuscript. Ruth Tal-Singer helped conceive the study and provided substantive feedback on the manuscript. Sonja Kierstein conducted all laboratory assays. Ali Musani performed all bronchoscopies and collected the BALF samples. Michael Beers assisted with data interpretation and editing of the manuscript. Rey Panettieri and Angela Haczku contributed equally to the conception of the study and editing of the manuscript. All authors read and approved the final manuscript.


The human EB1 gene product was recently found, by a yeast two-hybrid screening, to be associated with the carboxy terminus of the APC (adenomatous polyposis coli) protein, the product of a tumour-suppressor gene thought to act as a gatekeeper in colorectal carcinogenesis. Because virtually all of the APC mutations result in the synthesis of carboxy-terminal truncated proteins, mutant APC proteins are expected to lose their ability to interact with EB1 gene product. Thus, the interaction between APC and EB1 proteins may be important for the tumour-suppressor activity of APC protein, and raises the hypothesis that EB1 is also involved in sporadic colorectal tumorigenesis. To investigate this hypothesis, somatic mutations in the entire coding sequence of EB1 cDNA were searched by reverse transcriptase single-strand conformational polymorphism (SSCP) analysis in 21 sporadic colorectal cancers and seven adenomas. None of these tumours contained somatic mutation, whereas a silent cDNA variant was identified in 14% of alleles. Furthermore, to investigate whether EB1 locus was included within a region subjected to losses of heterozygosity, four polymorphism markers surrounding EB1 locus were surveyed. Only one out of 28 colorectal tumours contained a loss of heterozygosity at the D20S107 marker. In conclusion, the present findings strongly suggest that EB1 gene is not involved in somatic colorectal carcinogenesis.