This study evaluated prototype multichannel nonlinear frequency compression (NFC) signal processing on listeners with high-frequency hearing loss. This signal processor applies NFC above a cut-off frequency. The participants were hearing-impaired adults (13) and children (11) with sloping, high-frequency hearing loss. Multiple outcome measures were repeated using a modified withdrawal design. These included speech sound detection, speech recognition, and self-reported preference measures. Group level results provide evidence of significant improvement of consonant and plural recognition when NFC was enabled. Vowel recognition did not change significantly. Analysis of individual results allowed for exploration of individual factors contributing to benefit received from NFC processing. Findings suggest that NFC processing can improve high frequency speech detection and speech recognition ability for adult and child listeners. Variability in individual outcomes related to factors such as degree and configuration of hearing loss, age of participant, and type of outcome measure. Hearing aid technology provides hearing-impaired (HI) individuals with level- and frequency-dependent amplification. For most hearing aid users, hearing aids provide the most gain at higher frequencies, because hearing loss tends to increase with frequency. Current hearing aids have a limited ability to provide sufficient gain for less intense high-frequency sounds . This liLarge variability in aided listening performance is often observed for individuals with severe to profound, high-frequency hearing loss. Individual performance with hearing aids can be influenced by both the audibility of high-frequency signals, as well as the listener's proficiency in extracting useful information from the audible signals. Providing audibility through amplification in the high-frequencies for severe or profound HI listeners still remains a controversial topic. Some studies on the relationship between audibility and speech recognition suggest that providing audibility at frequencies where a hearing impairment is severe provides little or no speech recognition benefit; this is thought to be due to limitations in the perceptual abilities of the listener in extracting information from high-frequency energy . Other sSeveral studies have found significant adult/child differences in the bandwidth required for accurate fricative recognition for listeners with moderate to moderately-severe hearing loss , 2004. IFrequency-lowering has been suggested as one possible alternative to overcoming bandwidth limitations . The tertransposition shifts high-frequency sounds to lower frequency regions by a fixed amount. Early attempts at frequency transposition technology included the use of a modulated carrier frequency . Early studies indicated mixed outcomes with the body-worn FT-40 on adults and children, concluding that the FT-40 system was suitable for a select group of listeners . AVR SonThe Widex Inteo device utilizes spectral analysis to identify the frequency region with peak intensity above a cut-off frequency (i.e. peak frequency). Field studies indicated that 33% of subjects (N = 16), with sloping high-frequency hearing loss preferred listening to conversational speech with the transposer enabled than without . Case stcompression is an alternative frequency-lowering technology. This technology compresses the output bandwidth of the signal by a specified ratio. If applied across the entire frequency range of the device, frequency compression can alter the positions of vowel formants in the frequency domain. Therefore, recent attempts at frequency compression have used a multichannel approach rather than a single-channel approach  and 11 children . Participants were recruited from The University of Western Ontario (UWO) H.A. Leeper Speech and Hearing Clinic, as well as from local audiology clinics and schools. Pure–tone air and bone conduction thresholds were measured bilaterally at all octave and inter-octave frequencies between 250 and 6000 Hz for each participant using a Grason-Stadler 61 audiometer. Air conduction thresholds were obtained using Etymotic Research ER-3A insert earphones coupled to each participant's personal earmolds. Bone conduction thresholds were obtained using a Radioear B-71 bone oscillator. Audiometric testing was completed in a double-walled sound booth. Participants presented with sloping high-frequency hearing losses that ranged from moderately severe to profound in the better ear, and were sensorineural in nature for all but one participant with a mixed loss.This study used a modified withdrawal design, with both single- and double-blind outcome measures, as follows . ParticiA withdrawal study includes three measurement intervals: baseline, treatment, and withdrawal of treatment. In this study, the comparison of interest is benefit with NFC versus benefit without NFC. This was scored using the withdrawal versus treatment intervals (see Results section for details). The withdrawal versus treatment comparison loads any advantage due to practice and acclimatization effects on the withdrawal condition. Therefore, this study employed a more stringent evaluation of NFC benefit, compared to a baseline versus treatment comparison.Blinding in hearing aid efficacy research is necessary to avoid spurious labelling effects that are akin to placebo effects . In the For the CP hearing aid fittings, the prescriptive targets and clinical protocols from the desired sensation level (DSL) method version 5.0 were used DSL v5: . PrototyFor fitting, age-dependent prescriptive targets were matched using simulated real ear measures incorporating individual real ear to coupler differences (RECDs). Note that the DSL v5 algorithm prescribes more gain for children than for adults . The AudCustom software was used to enable the NFC processor while holding gain and amplitude compression parameters constant. Two NFC parameters were programmable: (1) the cut-off frequency, which determined the start of the upper band; and (2) the compression ratio, which determined the amount of frequency compression applied to the upper band. The cut-off frequency and compression ratios were determined on an individual basis as follows. The goal was to provide more audibility of high-frequency speech cues, compared to the CP fitting, while limiting negative effects such as poor sound quality or confusion of /s/ with /∫/. The fitter was instructed to evaluate the audibility of the peaks of average-level conversational speech, and of live voice productions of both /s/ and /∫/, on the same display used to fit the hearing aids without NFC. The fitter then enabled NFC and re-evaluated using the same signals to determine if NFC produced an increase in audibility for high-frequency speech energy. Listening checks and aided spectra were used to judge whether the current NFC setting caused confusion and/or spectral overlap of /s/ and /∫/, as judged by the fitter. Fitter judgments were used so that fitting could proceed even if the wearer was too young to participate in the fitting process via subjective comments. Wearer feedback was elicited if the participant could provide it, sometimes resulting in fine tuning. Tuning was most often aimed at reducing the amount of NFC, in response to reports of perceived slurring or excessive audibility of high frequency sounds. In these cases, fitters aimed to provide enough NFC to provide audibility of new sounds without slurring, at an acceptable level. In this process, the better ear was used to select initial NFC settings for both ears, in order to provide symmetrical frequency lowering to the binaural system. Final fittings were symmetrical in all participants , with the exception of one participant with a significant asymmetry in audiometric thresholds . For these tests, stimuli were digitized at a sampling frequency of 48828 Hz and routed through a computer-controlled psychoacoustic workstation (TDT) to the external inputs of a clinical audiometer. The outputs of the audiometer were routed to power amplifiers . The majority of the outcome measures utilized the front speaker only, with the exception of the plural recognition task (described below). The power amplifiers were routed to loudspeakers arranged at 72 degree spacing, one metre from the participants' test location. Participants were seated in an adjustable chair, facing a loudspeaker and computer display, adjusted in height to the level of the loudspeaker.Aided detection thresholds for speech sounds were measured in the sound field using an adaptive, computer-controlled version of the Ling six-sound test . The /∫/The consonant recognition task was a modified version of The University of Western Ontario Distinctive Features Differences test (UWO-DFD) . The UWOA plural recognition task was included to assess participant ability to use the fricatives /s/ and /z/ as bound morphemes. Stimuli for this task were chosen to be similar to those used in previous research to test sensitivity to high-frequency audibility in children who use hearing aids . The tashttp://homepages.wmich.edu/~hillenbr/voweldata.html). For the purpose of this study, vowel stimuli included five items , spoken by both adult female and child female talkers. These stimuli had energy at the second formant that spanned the range from roughly 1800 to 3400 Hz, and third formant energy from 2800 to 3700 Hz  at Western Michigan University . All other aspects of hearing aid processing  were matched between these two programs. The hearing aids automatically started up in program one; therefore listeners were required to use the program switch to select each program for comparison purposes. Participants indicated which program they preferred overall. Participants were given the option of choosing ‘same’ if they felt there was no difference between the programs being compared.Two analysis strategies were used. First, an analysis of group-level results was completed. Second, results for individual participants were analysed using single subject design methods, specifically using a modified two standard deviation band technique . ContribA repeated measures ANOVA was completed with processor type (CP versus NFC) and phoneme (/s/ or /∫/) as within-subject variables, and age group (adults versus children) as a between-subjects variable. Significant simple main effects were found for the processor type as well the phoneme type  = 42.97, p < .001; F = 6.84, p = .02). A repeated measures ANOVA was completed with processor type (CP versus NFC) and test type  as within-subject variables, and age group (adults versus children) as a between-subjects variable. Mean values were substituted for one participant with missing data on the consonant recognition task. Raw scores for the three speech recognition tasks were converted to rationalized arcsine units (RAU) prior to analysis . ResultsIndividual scores obtained in the treatment versus withdrawal phases were analysed using confidence limits for performance change. Significant change was deemed to occur when performance in the treatment condition exceeded statistically determined confidence limits. These limits were calculated for levels of significance equivalent to the 90th, 95th, and 99th percentiles.Measures of real world preference collected via multi-memory comparison prior to the withdrawal phase are noted per participant in For the consonant recognition task, all three predictors were entered into a backward regression analysis and age group as a predictor variable was removed on step one ; HFPTA as a predictor variable was removed on step two ; audiometric drop-off as a predictor variable was removed on step three . None of the candidacy variables predicted benefit on the consonant recognition task. For the plural recognition task, all three predictor variables were entered and all were included in the final regression equation; benefit was significantly predicted by age group, magnitude of high-frequency hearing loss, and audiometric drop-off frequency. For the /∫/ detection task, all three predictors were entered and age group was removed on step one . Benefit on the /∫/ detection was significantly predicted by magnitude of high-frequency hearing loss and audiometric drop-off frequency. For the /s/ detection task, all three predictor variables were entered and age group was removed on step one . Benefit for the /s/ detection task was significantly predicted by magnitude of high-frequency hearing loss and audiometric drop-off frequency. For the self-reported preference measure, all three predictor variables were entered in the backward regression analysis; HFPTA as a predictor variable was removed in step one ; audiometric drop-off was removed in step two . Processing preference, as reported on the self-reported preference measure, was significantly predicted by age group in the multiple regression analysis.In summary, participants with certain hearing losses derived greater NFC benefit on plural recognition and detection tasks. Specifically, participants with a greater amount of high-frequency hearing loss (based on HFPTA) that occurs at higher frequencies (based on the drop-off frequency) derived greater NFC benefit. In addition, there is evidence of a significant age effect for the plural recognition task and the self-reported preference measure. This implies that children deriving greater plural recognition benefit from NFC also had stronger preference for NFC, when compared to the adults.The main purpose of this study was to examine if prototype multichannel nonlinear frequency compression (NFC) hearing-aid processing provided benefit relative to the same hearing aid fitting without NFC. The NFC processor used in this study was a multichannel (i.e. two-band) strategy that provided frequency lowering (via frequency compression) to the high-frequencies while leaving the low band unaltered in the frequency domain. This was evaluated across a range of participants with varying audiometric characteristics and ages. Results suggest that the NFC processor was, on average, effective at improving high-frequency audibility, resulting in improvements in high-frequency speech sound detection and recognition. No significant changes were observed for low frequency speech sounds (i.e. vowels), on average. Benefit observed from NFC can be attributed to the increased audibility of additional high-frequency energy, albeit presented in a lower frequency range, compared to the conventional hearing aid fittings used in this study. These results generally agree with those reported by Analyses performed at the individual level provide evidence that NFC benefit varies across individuals. The results also indicate that the degree of high-frequency hearing loss may predict NFC benefit. This observation agrees with the group findings reported by The individual results also provide evidence that the age of the participants included in this study influenced the degree of NFC benefit. Although mean high-frequency thresholds for adults and children were not significantly different, the mean thresholds were 7 to 10 dB higher for the children in the mid/high frequency range. This difference may have been a factor in outcomes reported in this study. Furthermore, adult and child participants were fitted using different levels of audibility; adults received the DSL v5 adult prescription, which provides 5 to 10 dB less gain than the corresponding paediatric prescription . This diThe variability in individual phase durations of this study precludes any speculation as to the time course of acclimatization to NFC processing. However, we would speculate that some time may be required to acclimate to the new audibility provided from NFC, just as occurs for other forms of new audibility e.g. . It is aGatehouse et al, 2006aThese data were collected using prototype hearing instruments, with pre-clinical software that allowed independent manipulation of cut-off frequency and frequency compression ratio per ear and over a wide range of values. A manual fitting approach was used to individualize settings. Since this study was conducted, several similar although not identical clinical hearing instruments have been issued by the manufacturer of these prototype hearing devices (Phonak). These devices use a very similar processing strategy for NFC, with a range of NFC settings that (1) tie the crossover frequency together with the frequency compression ratio; and (2) use a more restricted range of settings than was available in the pre-clinical software. However, the range of settings available clinically is similar to the range actually used in this project . If the clinical hearing devices are fitted to children, the default NFC settings are based on a regression analysis of the pediatric fittings described in this paper; better-ear HFPTA hearing loss values were used as the basis for calculation of NFC settings, which are the same for both ears in the case of bilateral fittings. Clinicians using this technology may choose to employ the more detailed fitting method and/or findings in the present study to better understand one possible method for fitting NFC. Knowledge outcomes presented in this paper may further support fine tuning and troubleshooting of NFC devices.On average, the NFC processor improved speech sound detection thresholds, as well as consonant and plural recognition scores; vowel perception was not significantly changed. These findings are consistent with the fitting rationale and processor used in the study, which aimed at lowering high-frequency speech energy without affecting low and mid frequency speech energy.Individual results indicated that age group and degree and configuration of hearing loss were related to NFC benefit. The following trends were observed: (1) magnitude of high-frequency hearing loss and individual benefit on plural recognition/speech sound detection tasks were related, and (2) audiometric drop-off frequency and individual benefit on plural recognition/speech sound detection tasks were related. Age group was also related to individual benefit on the plural recognition task; children were more likely to benefit compared to adults.Individual preference for NFC processing was related to age group and to benefit; children were more likely to have preference for NFC processing than were adults. Also, individual participants were more likely to prefer NFC if they benefited from it.Variance in outcome results at the individual level was considerable. Some individuals experienced greater or lesser benefit than the candidacy predictors would lead one to expect. Further research is needed to generalize predictions of candidacy for this technology.Prototype nonlinear frequency compression (NFC) technology was evaluated for 24 hearing-impaired listeners using various objective and subjective outcome measures. Results can be summarized as follows:


The relationship between diabetes and hearing loss has been debated for many years. Hyperglycemia appears to have an effect on hearing loss and the proposed mechanisms are microangiopathy, neuropathy or a combination of both. The objective of this study was to evaluate a cross section of hyperglycemic subjects with age- and sex-matched normoglycemic controls with pure tone audiometry and compare the differences.Forty-one type 2 diabetes mellitus subjects and 41 age- and sex-matched normoglycemic controls were subjected to a pure tone audiometric assessment followed by evaluation of their glycemic status and degree of glycemic control. The resulting data was statistically analyzed.The auditory thresholds in hyperglycemic subjects were higher in all age groups in all the frequencies suggestive of sensorineural hearing loss. The hyperglycemic subjects with poor control of their blood sugar levels (HbA1C > 8%) had elevated auditory thresholds in all the test frequencies. The fasting blood sugar level in hyperglycemic subjects showed a trend towards significant difference at higher frequencies, the postprandial blood sugar levels showed significant differences at higher frequencies. There was no effect of duration of diabetes mellitus on the hearing thresholds in hyperglycemic subjects.Subjects with hyperglycemia have a sensorineural hearing loss when evaluated with a pure tone audiometer in all frequencies than a normoglycemic control group. The study showed that post prandial blood sugar levels and HbA1C levels had a direct bearing on the auditory acuity of the hyperglycemic subjects. Hearing loss is a functional disability which affects a person's day-to-day activities in subtle ways. Sensorineural hearing loss involving the inner ear and its central connections is irreversible. The prevalence of hearing loss in diabetes has been shown in many studies–11 to beThe present study is undertaken to compare auditory acuity in normoglycemic and hyperglycemic subjects to find out the effect of hyperglycemia on auditory acuity.Diagnostic audiometry comprises tests that detect conductive and sensorineural hearing losses. Pure tone audiometry involves the estimation of the threshold of hearing for certain standardized stimuli via the air and bone conduction routes. An audioTo assess the degree of auditory acuity in type 2 diabetes mellitus patients using pure tone audiometry [evaluating the frequency and intensity of hearing acuity]To compare pure tone audiometric results between diabetic type 2 patients [hyperglycemic group] and a matched control group [normoglycemic group].To record pure tone audiometry in normoglycemic subjects.To record pure tone audiometry in hyperglycemic subjectsTo make a comparative study of the auditory acuity of type 2 diabetics and normoglycemic subjects.To analyze the effect of age, glycemic status , glycemic control (HbA1C), duration of type 2 diabetes on auditory acuity.The study was conducted in a sample of 82 subjects in Bangalore. The study was approved by the institutional ethics committee of Bangalore Medical College and Rajiv Gandhi University of Health Sciences, Bangalore, India. They have been divided into two groups of 41 subjects each.Group 1 comprises of 41 normal, healthy subjects of either sex selected from the patient attenders in Victoria hospital belonging to the ages between 35 and 55 years. Group 2 comprises of 41 type 2 diabetic patients of either sex selected from the Department of Medicine Victoria Hospital, belonging to the ages between 35 and 55 years. The sample size was determined after taking into consideration the methods used in other studies. Many published studies on the prevalence of hearing loss in diabetics used a similar sample size between 20 and 45 diabetic subjects.1371011710The study comprising of 41 type 2 diabetics and 41 nondiabetics, matched with respect to age and sex are selected based on inclusion and exclusion criteria.Forty-one normal healthy subjects of either sex between 35 and 55 years who had given written consent and who were nonhypertensive were included.Forty-one type 2 diabetic patients between the ages 35 and 55 years who had given written consent and who were nonhypertensive were included. Both groups were matched with respect to age and sex.HypertensionDiabetes mellitusHistory of consumption of ototoxic drugs in past three months.History of ear surgeries performed in the past.History of ear infections in the past.History of recent infections in the nose, throat or ear.Patients having a noise induced hearing loss (as shown by pure tone audiometry at 4000 Hz.)History of consumption of ototoxic drugs in past three months.History of ear surgeries performed in the past.History of ear infections in the past.History of recent infections in the nose, throat or ear.Patients having a noise induced hearing loss (as shown by pure tone audiometry at 4000 Hz.)ARPHI [500 MK I] audiometerEar phones are used to test hearing by air conduction and a small vibrator placed over the mastoid is used test hearing by bone conduction. All audiometers incorporate a calibration circuit, which allows the output sound level to be set at each frequency. The signals presented to the subject by an audiometer are characterized by its frequency, sound pressure level and wave form which are all controlled.All the subjects included in this study are given a prepared questionnaire to answer. This questionnaire was designed to reveal the patients' assessment of hearing ability.An assessment of the hearings status using a pure tone audiometer [ARPHI 500 MK 1] is done.An audiometer [ARPHI 500 MK 1] is an electronic device that produces pure tones, the intensity of which can be increased or decreased in 5-Db steps. Air conduction thresholds are measured for tones of 250, 500, 1000, 1500, 2000, 4000 6000 and 8000 Hertz. Bone conduction thresholds and measured for 250, 500, 1000, 1500, 2000, 4000 Hertz. The amount of intensity that has to be raised above the normal level is a measure of the degree of hearing impairment at that frequency. It is charted in form of a graph called the “audiogram.” The thresholds of bone conduction are a measure of the cochlear function. The difference in the thresholds of air and bone conduction (A-B gap) is a measure of a degree of conductive deafness. The audiometer is so calibrated that hearing of a normal person, both of air and bone conduction is at 0 db and there is no A-B gap.The method is based on American Society for Speech and Hearing Association [ASHA] 1978 guidelines for manual pure tone audiometry (PTA). Masked pure tone audiometry is done if there is a difference of more than 40 dB between air conduction threshold of the test ear and the bone conduction threshold of the opposite ear, or when the air bone gap of the poorer ear under test is more than 10 dB.Student t test (unpaired and two tailed) has been used to find the significance of auditory thresholds (dB) between various categories of parameters. Analysis of variance [ANOVA] has been used to find the significance of auditory thresholds in different age groups. The effect size (d) has been used to find the effect of DM on auditory thresholds (dB).The statistical soft ware namely SPSS 11.0 and Systat 8.0 were used for the analysis of the data and Microsoft Word and Microsoft Excel have been used to generate graphs, tables, etc.Tables Differences in the fasting blood sugar and post-prandial blood sugar level between the cases and controls were statistically significant at 1% as seen in As shown in As shown in As shown in The relationship between diabetes mellitus and hearing loss is controversial, primarily because the pathogenic mechanism remains obscure. This is a case control study investigating this relationship using pure tone audiometer.Diabetes has been shown to affect hearing loss by many studies. Many have tried to identify the cause, and based on their conclusions, the probable mechanisms are microangiopathy of the inner ear, neuropathy of the cochlear nerve, a combination of both, outer hair dysfunction and disruption of endolymphatic potential.This study demonstrates a significant hearing loss in diabetes in all the frequencies tested. This could be explained by microangiopathy of the vessels to the inner ear as proposed by Wackym.The effect of age on auditory thresholds in diabetic subjects was found to be clinically and statistically insignificant. Kakarlapudi and DaltLack of glycemic control shows a positive correlation with extent of hearing loss when compared to those diabetics with good glycemic control. This is noted in all the frequencies tested. Both Kurien and Lasi6The present study supports the hypothesis that duration of diabetes does not alter hearing thresholds.578 It is57Screening of all patients with diabetes for hearing loss in a longitudinal study may provide a clearer understanding of the relationship between diabetes and hearing loss. It is the intention of the author to continue with such a study and evaluate the function of outer hair cells in a well selected diabetic population in India.Diabetes mellitus type 2 raises auditory threshold in all frequencies between 250 Hz and 8000 Hz in all age groups in this study.Patients with poor control [HbA1c greater than 8%] of their glycemic status have raised auditory thresholds.The duration of diabetes does not affect auditory thresholds significantly in this study.There was no association between the diabetic age-wise subgroups and their corresponding auditory thresholds. But in the age-wise subgroups in controls, there was an association between the subgroups and auditory thresholds. But the auditory thresholds were still within the normal limits.In this study, the auditory acuity of hyperglycemic and normoglycemic subjects were studied. The variables influencing the auditory acuity were statistically analyzed which revealed the following:These results which show the effect of hyperglycemia on auditory acuity may be explained by diabetic microangiopathy of the inner ear.The relationship between diabetes and hearing loss is controversial. The present study was undertaken to better understand the disease and its influence on hearing acuity. The study involved determination of the auditory thresholds in two groups of subjects namely, hyperglycemic and normoglycemic subjects. The hearing thresholds were evaluated in both the groups using a pure tone audiometer (ARPHI 500 MK 1). The glycemic status  and the degree of the glycemic control (HbA1C) of the hyperglycemic subjects were also assessed. The results obtained were then subjected to statistical treatment.The results showed that the hyperglycemic subjects were significantly hearing impaired than the normoglycemic group. This hearing impairment was noted in all the frequencies tested. The glycemic status and the degree of glycemic control played a significant role in reducing the auditory acuity of the hyperglycemic subjects, while the duration of hyperglycemia and the age of the subjects did not influence the hearing acuity.


Allelic loss is a common mechanism of inactivation of tumour-suppressor genes in colorectal carcinomas. A number of known or putative tumour-suppressor genes including NF1, BRCA1, NME1, NME2 and prohibitin are present on the long arm of chromosome 17, and this region has not been extensively analysed in colorectal tumours. In this study 72 colorectal carcinomas were examined for allelic loss at eight loci on chromosome 17. Allelic loss was frequent both at the p53 locus, which is known to be important in colorectal carcinoma, and also telomeric to p53 on 17p. Allelic loss continued to be present in more than 50% of cases in the pericentromeric region and on proximal 17q to the marker LEW101 (D17S40) at 17q22-23. The most telomeric markers on 17q showed lower rates of allelic loss. Analysis of cases with partial deletions which did not include the p53 locus showed a common region of overlap of the deletions centred on D17S40. This suggests the target of allelic loss on 17q is a tumour-suppressor gene in this region.


Nucleic acid extracts from transplantable carcinomas Vx7 and Vx2, long maintained in domestic rabbits, were assayed for their ability to produce papillomas in animals of this kind. The Vx7 had been serially transferred 111 times when this was attempted and Vx2 was in its 203rd generation. The nucleic acid extracts from the Vx7 carcinomas consistently yielded papillomas whereas those from Vx2 completely failed to do so. The tumorigenicity of the Vx7 extracts was slight and regression of the induced papillomas often took place, as happens not infrequently to the growths caused by nucleic acid extracts obtained directly from papillomas. Malignant conversion, a common event in tumors induced by Shope papilloma virus, was also observed to occur among papillomas which were induced by the nucleic acid extracts from Vx7 carcinomas, and which kept persisting over a year on the skin of experimental animals.


Additional studies were done with rat ATII cells.Surfactant proteins are produced predominantly by alveolar type II (ATII) cells, and the expression of these proteins can be altered by cytokines and growth factors. Th1/Th2 cytokine imbalance is suggested to be important in the pathogenesis of several adult lung diseases. Recently, we developed a culture system for maintaining differentiated adult human ATII cells. Therefore, we sought to determine the effects of IL-13 and IFN-γ on the expression of surfactant proteins in adult human ATII cells Adult human ATII cells were isolated from deidentified organ donors whose lungs were not suitable for transplantation and donated for medical research. The cells were cultured on a mixture of Matrigel and rat-tail collagen for 8 d with differentiation factors and human recombinant IL-13 or IFN-γ.IL-13 reduced the mRNA and protein levels of surfactant protein (SP)-C, whereas IFN-γ increased the mRNA level of SP-C and proSP-C protein but not mature SP-C. Neither cytokine changed the mRNA level of SP-B but IFN-γ slightly decreased mature SP-B. IFN-γ reduced the level of the active form of cathepsin H. IL-13 also reduced the mRNA and protein levels of SP-D, whereas IFN-γ increased both mRNA and protein levels of SP-D. IL-13 did not alter SP-A, but IFN-γ slightly increased the mRNA levels of SP-A.in vitro. IL-13 decreased SP-C and SP-D in human ATII cells, whereas IFN-γ had the opposite effect. The protein levels of mature SP-B were decreased by IFN-γ treatment, likely due to the reduction in active form cathpesin H. Similarly, the active form of cathepsin H was relatively insufficient to fully process proSP-C as IFN-γ increased the mRNA levels for SP-C and proSP-C protein, but there was no increase in mature SP-C. These observations suggest that in disease states with an overexpression of IL-13, there would be some deficiency in mature SP-C and SP-D. In disease states with an excess of IFN-γ or therapy with IFN-γ, these data suggest that there might be incomplete processing of SP-B and SP-C.We demonstrated that IL-13 and IFN-γ altered the expression of surfactant proteins in human adult ATII cells  The alveolar type II (ATII) cell produces pulmonary surfactant and most of the surfactant proteins in the lung. The four surfactant proteins, SP-A, SP-B, SP-C and SP-D, have been shown to play pivotal roles in the regulation of surfactant lipid metabolism, lipid membrane organization and host defense in the lung . DysreguSP-A and SP-D are water-soluble and belong to the collectin subgroup of C-type lectins . SP-A geSP-B and SP-C are extremely hydrophobic and play critical roles in the biophysical functions of surfactant . PolymorIL-13 is a pleiotropic cytokine and a major effector molecule at sites of Th2 inflammation and tissue remodeling. IL-13 is a potent stimulator of eosinophilic, lymphocytic, and macrophage-dominant inflammation, mucus metaplasia, and fibrosis. -27. IL-1in vitro, IFN-γ is reported to increase SP-A protein levels by 3-fold and SP-A mRNA levels by 2.7-fold but does not alter SP-B and SP-C mRNA levels [IFN-γ is the prototypic Th1 cytokine and is known to play a key role in the regulation of diverse immune responses . DysreguA levels .in vitro [in vitro. The experiments with human ATII cells were also repeated with rat ATII cells.Although dysregulation of the Th1/Th2 cytokine is related to the pathogenesis of several adult lung diseases and alterations of surfactant proteins have been reported in variety of lung diseases, the effect of IL-13 or IFN-γ on the expression of surfactant proteins in primary adult human ATII cells has not been reported. Methods for isolating and culturing adult rat and mouse ATII cells and fetal human ATII cells have been available for years, but there has been less success in maintaining the differentiated functions of adult human alveolar epithelial cells in primary culture. A variety of methods for isolating human type II cells have been published and some of their properties have been described -47, but in vitro . TherefoWe obtained human lungs from deidentified organ donors whose lungs were not suitable for transplantation and donated for medical research through the National Disease Research Interchange  and the International Institute for the Advancement of Medicine . The Committee for the Protection of Human Subjects at National Jewish Health approved this research. We selected donors with reasonable lung function with a PaO2/FIO2 ratio of >250, no history of clinical lung disease and a chest radiograph that did not indicate infection, and a limited time on the ventilator. The gender, age, and smoking history were variable and not selection criteria. The human donors used in this study included 6 males and 6 females with age ranges from 10 to 72, and there were 7 current smokers and 5 nonsmokers. Hence, there was a significant amount of variability among the donors, as expected.We modified the human type II cell isolation method published by Fang and coworkers . Briefly2 millicell inserts  that had been previously coated with a mixture of 50% Matrigel  and 50% rat-tail collagen in DMEM with 10% FBS [The isolated cells were resuspended in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and 2 mM glutamine, 2.5 μg/ml amphotericin B, 100 μg/ml streptomycin, 100 units/ml penicillin G , and 10 μg/ml gentamicin . 4.0 million cells were plated on 4.2 cm 10% FBS . For mos 10% FBS .2 millicell inserts (Millipore Corp). 2.5 million freshly isolated viable type II cells were plated in DMEM containing 5% rat serum (RS) , 2 mM glutamine, 2.5 μg/ml amphotericin B, 100 μg/ml streptomycin, 100 units/ml penicillin G , and 10 μg/ml gentamicin (Sigma-Aldrich). After attachment for 24 h, the cells were rinsed twice with DMEM and then cultured in DMEM containing RS, glutamine, antibiotics described above and 10 ng/ml KGF for 6 d with or without recombinant rat IL-13 (20 ng/ml) or rat IFN-γ (100 ng/ml) (R&D Systems) for the last 4 d.ATII cells were isolated from pathogen-free adult male Sprague-Dawley rats  by dissociation with porcine pancreatic elastase (Roche Diagnostics) and partial purification on discontinuous density gradients by methods previously described . This reProtein and mRNA expression of corresponding genes were measured by western blotting and real-time RT-PCR according to protocols as described previously . PolyacrThe cells were fixed in 4% paraformaldehyde, and then the filters were embedded in paraffin as described . The priAll data were presented as means ± standard error of the mean. One-way ANOVA was used to compare the difference between two or more groups. Appropriate post hoc tests were selected for multiple comparison. Statistical significance was set at p < 0.05.in vitro for 8 d  with 2 or 20 ng/ml human recombinant IL-13. The protein level of mature SP-C showed significant dose-dependent down-regulation by human recombinant IL-13  Figure . Consistnd band: 0.31 ± 0.12 p < 0.001, 3rd band: 0.21 ± 0.04 p < 0.001, bottom band: 0.22 ± 0.07 p < 0.001)  Figure . In propin vitro for 8 d  with 100 ng/ml human recombinant IFN-γ for the last 4 d (n = 6). We used 4 d of 100 ng/ml IFN-γ for this experiment based on our previous experiments with the KIAD system   Figure . The immSP-B and SP-C are synthesized by ATII cell as proSP-B and proSP-C, which are proteolytically processed to mature SP-B and SP-C on route from its site of synthesis to the lamellar bodies ,54. CathABCA3 is predominantly expressed in ATII cells and has been localized to the limiting membrane of lamellar bodies, which are the main intracellular storage organelle for pulmonary surfactant . The proThe results above were all done with adult human ATII cells cultured with 2 d of TGFα and 4 d of KGF. Similar findings were observed in ATII cells cultured in KGF (K) alone, in 2 d of K and 2 d of KD (data not shown) or in 2 d of KIA and 4 d of KIAD  or a combination of several abnormalities. In this study, IL-13 reduced both mRNA and protein levels of SP-C and SP-D in human adult ATII cells. Our results are different from previous reports in rodents, which show that IL-13-overexpression in mice increases SP-A, SP-B, SP-C and SP-D  and thatin vitro . We alsoIL-13 has been known to play a pivotal role in the pathogenesis of lung disease such as asthma, IPF, viral pneumonia and COPD ,28-33. S2- and COOH- terminal peptides [SP-B and SP-C are synthesized by ATII cells as proproteins (proSP-B and proSP-C) that are processed to mature SP-B and SP-C by serial cleavage of NHpeptides ,54,61-63peptides ,61 and ipeptides ,63. We fpeptides ,65. In tpeptides , alveolapeptides . In the peptides . HoweverIFN-γ production is stimulated by the innate immune response during infection, which alters macrophage function including enhanced pathogen recognition, antigen processing and presentation, stimulation of leukocyte recruitment, and activation of microbicidal effector functions and an anti-viral state . In thisABCA3 is necessary for lamellar body biogenesis, SP-B processing, and lung development late in gestation . ABCA3 dThe results with rat cells were differed from the observations with human cells in several ways (Additional File in vitro. Our current study suggests that these cytokines imbalance, that is Th1/Th2 cytokine imbalance, might contribute to the pathogenesis of lung diseases by disordering homeostasis in the alveolar space.We demonstrate that IL-13 and IFN-γ alter the expression of surfactant proteins in human adult ATII cells The authors declare that they have no competing interests.YI carried out all human and rat ATII cells studies. YI and RJM participated in the design of the study and data analysis. All authors have read and approved of the final manuscript.Effect of IL-13 and IFN-γ on expression of surfactant proteins in adult human ATII cells cultured with a differentiation factors.Click here for fileSummary of the result from immunoblot data.Click here for fileIL-13 and IFN-γ alter surfactant proteins expression in adult rat ATII cells.Click here for file


The 1555A > G mutation is the most common cause of aminoglycoside-induced and non-syndromic deafness. However, the variable clinical phenotype and incomplete penetrance of A1555G-induced hearing loss complicate our understanding of this mutation. Environmental factors, nuclear genes, mitochondrial haplotypes/variants and a possible threshold effect have been reported to may be involved in its manifestation.Here, we performed a clinical, molecular, genetic and phylogenic analysis in a six-generation Chinese family.A clinical evaluation revealed that affected individuals without aminoglycoside exposure developed hearing loss extending gradually from 12000 Hz to 8000 Hz and then to 4000 Hz. Using pyrosequencing, we detected an identical homoplasmic 1555A > G mutation in all individuals except one. We did not find any correlation between the mutation load and the severity of hearing loss. T123N coexisted with the 1555A > G mutation in six affected subjects in our pedigree. Analysis of the complete mtDNA genome of this family revealed that this family belonged to haplotype B4C1C and exhibited high penetrance. Upon the inclusion of subjects that had been exposed to aminoglycosides, the penetrance of the hearing loss was 63.6%.; without exposure to aminoglycosides, it was 51.5%. This pedigree and another reported Chinese pedigree share the same haplotype (B4C1C) and lack functionally significant mitochondrial tRNA variants, but nevertheless they exhibit a different penetrance of hearing loss.Our results imply that the factors responsible for the higher penetrance and variable expression of the deafness associated with the 1555A > G mutation in this pedigree may not be mtDNA haplotype/variants, but rather nuclear genes and/or aminoglycosides. A number of mitochondrial mutations have been described to be associated with non-syndromic and syndromic hearing loss. The 1555A > G mutation is the most common mutation attributed to aminoglycoside-induced and non-syndromic deafness. It was first described in a large Arab-Israeli family that exhibited maternally inherited non-syndromic deafness ..14].To explore the role of the GJB2 gene and the A10 S variant in the TRMU gene in the hearing loss phenotype of this pedigree, we screened GJB2 and A10 S variant in maternal individuals. The V27I (79G > A) and I203T (608T > C) in the GJB2 gene were detected as homozygous status in maternal relatives. Seven maternal relatives, six of whom suffered from hearing loss were found to be heterozygous for the T123N (368T > C) SNP G7444A, tRNACysT5802C, tRNAThrG15927A, tRNACysG5821A, ND5T12338C and ND4G11696A seemed to account for the higher penetrance and expressivities in many Chinese pedigrees associated with the 1555A > G mutation and deafness. Conversely, they postulated that the absence of those functionally significant mutations in tRNA and rRNAs or a secondary LHON mutation in these Chinese pedigrees might account for the very low penetrance of hearing loss. However, the pedigree that we studied which did not have a secondary mtDNA mutation or the above functionally significant mutations, nevertheless exhibited high levels of penetrance and phenotype of hearing loss. Thus, the role of the mitochondrial haplotype or functional variants in the penetrance and expression of hearing loss seems to be questionable based on our pedigree. Nuclear genes and aminoglycosides may be responsible for the higher penetrance and more variable clinic phenotypes.It has been suggested that mtDNA haplotypes could modulate the penetrance and phenotype of mtDNA mutations. The phylogenetic tree can be a very helpful approach to assessing the origin of an mtDNA mutation ,22. Phylhttp://davinci.crg.es/deafness[To better understand the role of nuclear genes in phenotype variation, we analyzed GJB2 in this pedigree and identified homozygous V27I (79G > A) and I203T (608T > C) and heterozygous T123N (368C > A). Six of the maternal subjects harboring heterozygous T123N had hearing loss except subject V18 (Tab. /deafness and it w/deafness,29. OverIt was reported that the A10 S mutation in TRMU gene modulated the phenotypic expression of the 1555 A> G mutation in the Israeli/European pedigrees . To asseIn our clinical evaluation, we noticed that the degree of the deafness in fourth generation was worse than in the third and fifth generations. Almost of all the affected subjects in the fourth generation presented severe or profound hearing loss with a clear history of aminoglycoside exposure or uncertain history of aminoglycoside, while those in the fourth and fifth generations without histories of aminoglycoside exposure showed normal or mild hearing loss. As II-2 was a 102-year-old woman with severe hearing loss, her hearing loss might be a consequence of age-related hearing impairment combined with the specific genetic defect. Meanwhile, we noted that IV-25 began suffering hearing loss after only two streptomycin injections at age 4. This is consistent with previous findings that Chinese patients with the 1555A > G mutation suffer severe hearing loss with immediate onset after exposure to a very small dose of aminoglycosides. In contrast, in American patients, the hearing loss is mild and progressive over many years after aminoglycoside administration . The outIn summary, we have analyzed possible modifiers associated with hearing loss due to the 1555A > G mutation. First, we found that the load of the 1555A > G mutation is not correlated with phenotype expression or penetrance in this family. Second, our pedigree exhibits a statistically different penetrance from another reported Chinese pedigree, both of which share haplotype B4C1C and lack functionally significant mitochondrial tRNA or rRNA variants. Our data further strengthen the idea that haplotype background and functional variants may not play a substantial role in the penetrance and expression of the deafness associated with the 1555A > G mutation. Rather nuclear genes and/or aminoglycoside exposure may be responsible for the higher penetrance and variability of hearing loss with the 1555A > G mutation in our pedigree. Third, the covariance analysis showed that m.1555A > G and aminoglycoside exposure are main influence factors, but no frequency specificity. The m.1555A > G places the individual at risk for hearing loss, but the severity and penetrance of hearing lossis based on the interplay of mutation with other factors such as unknown nuclear genes and/or aminoglycoside exposure.The authors declare that they have no competing interests.ZW, HL, MG conceived of the study, and participated in its design and revised the manuscript. ZW provided information of this pedigree. ZW also offered the help of clinical evaluation and coordination, and helped to draft the manuscript. YB performed the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. NC, GC, carried out epidemiological survey. WD, QL reviewed and interpreted the results. All authors had read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/129/prepub


Surfactant protein D (SP-D) is an innate immune protein that is present in mucosal lined surfaces throughout the human body, including the male reproductive tract. In the present study, we characterized the regulation of SP-D expression in the mouse and rat prostate.Real time reverse transcriptase polymerase chain reaction (RT-PCR) and immunostaining were used to characterize SP-D mRNA and protein in the mouse male reproductive tract. In order to evaluate the effects of testosterone on SP-D gene expression, we measured SP-D mRNA levels via real time RT-PCR in prostates from sham-castrated mice and castrated mice. In addition, we used a rat prostatitis model in which Escherichia coli was injected into the prostate in vivo to determine if infection influences SP-D protein levels in the prostate.We found that SP-D mRNA and protein are present throughout the mouse male reproductive tract, including in the prostate. We determined that castration increases prostate SP-D mRNA levels (~7 fold) when compared to levels in sham-castrated animals. Finally, we demonstrated that infection in the prostate results in a significant increase in SP-D content 24 and 48 hours post-infection.Our results suggest that infection and androgens regulate SP-D in the prostate. Escherichia coli is a pathogen frequently associated with both acute and chronic bacterial prostatitis in humans [E. coli has been implicated in its pathogenesis [E. coli can cause prostate pathology via the release of endotoxin over a long period of time and this, in concert with dihydrotestosterone (DHT), can lead to hyperplasia of the prostate [Although nonbacterial prostatitis is more common, prostatitis can be caused by bacterial infection that over time may lead to inflammation of the prostate . Moreoven humans ,4. Althoogenesis . It has prostate . For decprostate ,7. Recenprostate .Candida albicans and Mycobacterium tuberculosis [Surfactant protein D (SP-D) is a member of the collectin family of proteins which play an important role in innate immune responses . SP-D carculosis ,15. Recerculosis . Finallyrculosis .Chlamydia in vitro [SP-D was originally described in the lung, but recent studies have shown that SP-D is expressed throughout the body, usually at mucosal surfaces ,1920,21,2120,21,23. For Pseudomonas aeruginosa, influenza virus, or the overexpression of cytokines such as interleukin-4 can also lead to elevated SP-D levels in the lung [SP-D is expressed constitutively in the lung and pulmonary SP-D levels can vary with infection or disease . Lung SPthe lung -28.E. coli leads to an increase in SP-D protein levels in the prostate gland 24 to 48 hours post infection. These data are suggestive that SP-D produced in the prostate is regulated by androgens and influenced by infection.In the present study, we found that SP-D mRNA and protein are expressed throughout the mouse male reproductive tract. We then showed that castration increases SP-D mRNA levels in the prostate. We also demonstrate that infection of the rat prostate with ad libitum.Swiss Black mice were obtained from Taconic Laboratories  and bred at the Animal Research Facility at the University of Iowa following an approved protocol. Sham-castrated or castrated Swiss Black mice were also obtained from Taconic Laboratories. All mice were housed under pathogen-free conditions and allowed food and water ad libitum. All rat experimental protocols were performed in accordance with NIH guidelines for animal care.Adult, twelve-week old male Wistar rats were housed at an animal research facility in the Universidad Nacional de Cordoba, Cordoba, Argentina. The rats were housed under controlled conditions of 14 hours of light followed by 10 hours of darkness and allowed food and water Castrated and sham-castrated Swiss Black mice were obtained from Taconic Laboratories. The animals were housed for 12 days after surgery to ensure that testosterone was completely cleared. All animals, i.e., sham-castrated and castrated, were then sacrificed and the prostate  and lungs removed. In some cases the epididymis and bladder were removed as well. This experiment was repeated three times with 3 animals per condition .E. coli, was used to address this question. We chose to perform these studies in the rat because of the greater size of the rat prostate, which enables easier manipulation, and because the rat prostatitis experimental model has been previously characterized. A strain of uropathogenic Escherichia coli , isolated from patients with complicated urinary tract infection, was stored at -20°C and grown overnight in tryptic soy broth at 37°C when required for inoculations. Rats were anesthetized with inspired ether and subjected to laparotomy in order to expose the ventral prostate. Prostatitis was induced via injection of E. coli diluted in sterile PBS  or with the same volume of sterile PBS as a sham-infected control, using a 30 gauge needle inserted directly beneath the capsule of both prostate ventral lobes. The muscle, peritoneum and skin were then closed using a simple continuous pattern with chromic suture material. Three rats were sacrificed at 24, 48, or 72 hours after bacterial inoculation. Control, sham-infected rats were sacrificed 24 and 72 hours post-operation. No morphological differences were observed in the prostates obtained at the different time points in the control groups. The rat prostatitis model is based on a protocol published by Fulmer et. al. [et al. [We wished to determine if SP-D protein expression in the prostate is altered with infection. A prostatitis model, achieved via direct infection with  et. al.  with mod2O2 in PBS for 30 minutes. Slides were rinsed with PBS and blocked by incubating in normal serum (1.5%) for 20 minutes. The slides were then incubated with a SP-D polyclonal primary antibody  at 4°C overnight. For negative controls, slides were incubated overnight with either PBS or with normal rabbit IgG instead of the primary antibody. The slides were rinsed in PBS and incubated in secondary antibody  for 30 minutes at room temperature. Slides were rinsed, incubated in Vectastain ABC reagent  for one hour at room temperature, rinsed again in PBS and then incubated in diaminobenzidine (0.7 mg/mL), a peroxidase substrate. After a final PBS rinse followed by a H2O rinse, the slides were counterstained with hematoxylin, dehydrated and mounted using Permount. As a positive control for the SP-D staining, adult mouse lung tissue sections were also immunostained using the SP-D antibody.Paraffin-embedded mouse reproductive tissues or rat prostate tissues were sectioned (7 μm thick), deparaffinized and rehydrated serially in 100%, 95%, 75%, and 50% ethanol. For antigen retrieval, the slides were boiled in 0.1 M sodium citrate, pH 6.0, for 10 minutes and then cooled for 20 minutes. The slides were then rinsed in phosphate buffered saline (PBS) and endogenous peroxidase activity quenched by incubating in 0.3% HRat prostate tissues  were homogenized in 1 mM phenylmethyl-sufonyl fluoride, centrifuged at 600 × g and the supernatant collected. Protein content was determined using a Bio-Rad Protein Assay . Equal amounts of rat prostrate homogenate proteins (40 μg) were separated by gel electrophoresis on 15% Tris-HCl polyacrylamide gels. The proteins were transferred electrophoretically at 100 V to nitrocellulose membranes and then placed in 7% non-fat dry milk diluted in 0.1% TNT  overnight to block non-specific binding. The membranes were subsequently incubated with the rabbit anti-SP-D primary antibody (Chemicon) at a dilution of 1:1000 for 1 hour at room temperature, then washed in 0.1% TNT, three times, at 15 minutes per wash. The blots were incubated with a secondary antibody  followed by exposure to X-ray film. Densitometry was then performed on the immunoreactive bands using Scion Image Software .RNA was extracted from the male reproductive tract organs of 5–6 mice and also from the prostate and lung tissues of 9 castrated and 9 sham-castrated mice by homogenizing the tissues in Trizol . RNA was extracted using chloroform and precipitated with ice-cold isopropanol. The resulting total RNA pellet was resuspended in water and quantitated by determining the absorbance at 260 nm. Two μg of total RNA from each sample were then reversed transcribed. The resulting cDNAs were diluted (1/50) and replicates for each sample were aliquoted for real time polymerase chain reaction (PCR) analysis using a Stratogene Mx 3000 P instrument. FAM-labeled primers for mouse SP-D and 18 S ribosomal RNA  and Universal Taqman master mix were purchased from Applied Biosystems, Inc. . As a control, the threshold cycle (CT) values obtained from adding increasing amounts of RNA with either SP-D primers or 18 S primers were plotted. The two primer sets produced parallel curves with similar slopes. SP-D mRNA levels were normalized to 18 S rRNA levels and then the relative SP-D mRNA levels were determined by the comparative quantitation method, according to the manufacturer User Bulletin 2 . For experiments in which the levels of SP-D mRNA in different mouse male reproductive organs were compared, the levels were expressed relative to the average level of SP-D mRNA in the prostate, which was made equal to one. For experiments in which levels of SP-D were compared in prostate and lung tissue from intact vs. castrated mice, SP-D mRNA levels are expressed relative to levels in intact tissues from sham-castrated control mice.All data were derived from at least three experiments. The data were analyzed by one-way analysis of variance followed by Student-Newman-Keuls Multiple Comparisons Test.To determine if SP-D is produced throughout the mouse male reproductive tract, real time RT-PCR was performed on RNA isolated from several male reproductive tract tissues, i.e., testis, epididymis, vas deferens, seminal vesicle, prostate, and coagulating gland. SP-D mRNA was detected in all of the tissues except for the seminal vesicle Figure . The levIn the testes, peritubular tissues including vascular endothelium and some interstitial cells (asterisks), stained strongly for SP-D protein, however Sertoli cells and spermatids also stained positively Figure , arrows.Testosterone affects the growth of the prostate gland and other organs in the male genital tract . To detein vitro [E. coli was used to address this question. Infected rats were sacrificed 24, 48 and 72 hours post-infection and their prostates removed. As a control, some rats were injected with the vehicle alone (sham-infected). SP-D protein was present in the epithelium of the prostate in sham-infected rats, primarily in the apical portion of the cells . The ventral prostate of the mouse and rat are anatomically and functionally similar [E. coli infection stimulates SP-D protein production in the rat prostate epithelium. Prostate SP-D protein levels were significantly increased early in the prostate infection, i.e., 24 hours and 48 hours post-infection. However, by 72 hours post-infection, SP-D levels in the infected rat prostate epithelium had declined. This pattern of expression was expected since components of the innate immune system are often the first responders to infection.Several authors have previously shown that SP-D protein levels can be increased by infection. For instance, SP-D protein was increased in human gastric mucosa during infection with r pylori . Pneumocuse lung . Serum luse lung . We choo similar ,42. The  similar . The mor similar -45. In tE. coli LPS. Since SP-D agglutinates bacteria, promotes phagocytosis and in some cases has direct anti-microbial affects on certain strains of E. coli, it may be beneficial for the host to increase expression of SP-D soon after an infection has begun [Cytokines have previously been shown to regulate SP-D expression . We haveas begun ,24. Consas begun .et al., it was shown that SP-D levels in serum increase with age in humans and that SP-D levels are higher in males than in females [Androgen withdrawal leads to involution of the prostate gland with alterations that include a decrease in secretion and loss of epithelial cells by apoptosis. In the present study, we found that castrated mice have increased SP-D mRNA in their prostates. Interestingly, castration of the male mouse did not change SP-D mRNA levels in the lung or in other tissues in the male reproductive tract of the mouse. Although hormonal regulation of SP-D by growth hormone has been previously reported, this is the first study to suggest that SP-D can be regulated by androgens . In a re females . These d females . It is a females . TherefoSP-D appears to be particularly important in apoptotic cell removal from the lung . Thus, iin vivo and the first to show that prostatic SP-D may be regulated by androgens, findings that may lead to a better understanding of innate immunity in the prostate gland.In summary, we detected SP-D mRNA and protein throughout the mouse male genital tract, including in the prostate. We determined that SP-D expression in the prostate is increased in castrated male mice. We also demonstrated that prostate SP-D gene expression increases in response to infection. These data are the first to suggest a role for SP-D in prostatic innate immunity The author(s) declare that they have no competing interests.All authors of this manuscript contributed to the paper by conducting the research directly, writing portions of the manuscript, or contributing to the ideas behind the different projects involved in this manuscript. REO is the primary author of this paper and helped conduct SP-D immunoblotting and immunostaining and castration experiments. KLG helped conduct SP-D immunoblotting, immunostaining and RT-PCR. AAQ and CAM conducted the prostatitis experiments and helped write some of the manuscript. JMS is the PI of the laboratory and helped conduct the SP-D RT-PCR experiments and also helped write parts of the manuscript. All authors have read and approved the final manuscript.


In this work we used steered molecular dynamics to study the mechanical unfolding of 10FNIII under tensile force applied at this RGD site. We demonstrate that mechanically unfolding 10FNIII by pulling at the RGD site requires less work than unfolding by pulling at the N- and C- termini. Moreover, pulling at the N- and C-termini leads to 10FNIII unfolding along several pathways while pulling on the RGD site leads to a single exclusive unfolding pathway that includes a partially unfolded intermediate with exposed hydrophobic N-terminal β-strands – residues that may facilitate fibronectin self-association. Additional mechanical unfolding triggers an essential arginine residue, which is required for high affinity binding to integrins, to move to a position far from the integrin binding site. This cell traction-induced conformational change may promote cell detachment after important partially unfolded kinetic intermediates are formed. These data suggest a novel mechanism that explains how cell-mediated forces promote fibronectin fibrillogenesis and how cell surface integrins detach from newly forming fibrils. This process enables cells to bind and unfold additional fibronectin modules – a method that propagates matrix assembly.Fibronectin polymerization is essential for the development and repair of the extracellular matrix. Consequently, deciphering the mechanism of fibronectin fibril formation is of immense interest. Fibronectin fibrillogenesis is driven by cell-traction forces that mechanically unfold particular modules within fibronectin. Previously, mechanical unfolding of fibronectin has been modeled by applying tensile forces at the N- and C-termini of fibronectin domains; however, physiological loading is likely focused on the solvent-exposed RGD loop in the 10 Molecular force spectroscopy studies have revealed that this module is the most compliant, and thus it has been suggested that 10FNIII unfolds in response to cell-traction forces and that this unfolding facilitates FN fibrillogenesis in vitroFN is a multidomain protein where each domain can be classified into one of three distinct types . The 10Studying the effects of mechanical strain on the structure of 10FNIII is problematic because the direct observation of short-lived, partially unfolded states is difficult to achieve experimentally. Computer simulations provide a useful approach to address this problem. Molecular dynamics simulations, for example, have shed light on the physical basis of the mechanical stability of titin – a modular protein consisting of repeating FNIII-like domains – and of FN Our data suggest that pulling at the RGD motif leads to partial unfolding of 10FNIII along a unique and well-defined pathway. By contrast, pulling at the N- and C-termini leads to unfolding that can occur along many different pathways. Unfolding along a unique and robust pathway provides a mechanism to enable FN to reliably sample a limited number of intermediate structures that may preferentially promote FN fibril formation. These studies also reveal a novel uncoupling mechanism whereby traction-induced partial unfolding of the 10FNIII domain dislodges FN-integrin adhesions; this uncoupling permits new FN-integrin binding and propagation of fibrillogenesis.To mechanically unfold 10FNIII , two poiin vivo; slower simulations are more realistic, but they require significant computational resources. Therefore, the optimal choice of spring extension represents a compromise between two competing considerations – computational efficiency and accuracy. Prior studies demonstrate that meaningful insights into the unfolding mechanism of fibronectin modules can be obtained from simulations performed at pulling rates much faster than in vivo unfolding rates Constant velocity steered molecular dynamics (SMD) simulations were used to study structural changes in 10FNIII when tension is applied along both axes (N-to-C or N-to-RGD). Ten independent force-mediated unfolding simulations (30 ns each) were performed for each pulling axis. External force at the pulling point is provided by translating, at constant velocity, the free end of a spring  remained relatively constant; however, after 15 ns, this distance increased dramatically . This tiLastly, we note that these data suggest that 10FNIII, when subjected to mechanical unfolding at the RGD site, does not adopt structures that are more unfolded than the kinetic intermediate shown in A number of studies suggest that FN fibrillogenesis requires cell-generated traction forces that lead to the formation of partially unfolded FN intermediates A was exposed first, and in the other the C-terminal strand G was the initial strand to become exposed. Previous atomic force microscopy experiments have identified a partially unfolded intermediate adopted during force-induced unfolding of 10FNIII that may correspond to a structure that has an exposed β-strand; either at the N-terminus (strand A) or the C-terminus (strand G) G strand is relevant under physiological conditions based on an analysis of unfolding barrier heights Earlier studies have shown that 10FNIII can unfold via several different pathways when mechanical forces are applied at the N- and C-termini, and our results are consistent with these observations To gain insight into how cell traction affects the structure of 10FNIII, we modeled the interactions between the integrin receptor and the RGD motif such that force is transmitted to FN through the center Gly residue of the RGD sequence. In actuality, it is likely that the integrin-10FNIII/RGD interface involves multiple inter-atomic interactions resulting in a pulling force that may be distributed over several atoms. However, in the absence of an integrin-10FNIII crystal structure, the precise manner in which these forces are distributed over atoms in this module is unknown In both cases of pulling along either the N-to-C or N-to-RGD axis, the work put into the system is initially used to reorient the module until the end points of the axis (the anchor point and the pulling point) are aligned along the direction of pulling . Overall, the choice of the pulling direction for N-to-RGD pulling only changes the amount of time initially spent to reorient the molecule such that the anchor point-to-pulling point axis is aligned with the pulling direction, but the resulting unfolding pathways are thereafter similar (data not shown). Given the relative positions of the N-terminus and the RGD loop with respect to the direction of pulling, less than 3 ns are spent realigning the N-to-RGD axis at the beginning of the trajectories compare . During A and B . In addition, N-to-RGD pulling reliably leads to partial unfolding along one well-defined trajectory. A single pathway ensures that the same kinetic intermediates are sampled when cell-derived forces are applied at the RGD site. In addition, this intermediate contains solvent exposed hydrophobic residues within β-strands A and B . AdditioA and B . We alsoHere we focused on conformational changes in 10FNIII and its relationship to FN fibrillogenesis. It is worthwhile to note that other FN modules are likely involved in fibril formation. There are data to suggest that cryptic FN binding sites exist in various FNIII modules: 1FNIII, 2FNII, 7FNIII, 9FNIII, 10FNIII, and 15FNIII A  and living cells under isometric tension at rest The structure of 10FNIII  was taken from the crystal structure of 7–10FNIII (PDB ID 1fnf) To generate different starting structures for the pulling simulations, we performed a relatively short MD simulation on the isolated 10FNIII structure. A time step of 0.001 ps was used, and the equations of motion were integrated with a leap frog integrator. The temperature of the system was maintained by coupling the system to a heat bath at 300 K using the Berendsen method Constant velocity SMD simulations were performed by placing the Cα atom of the pulling point at the origin and the dummy atom on the z-axis. In this framework, pulling along an N-to-RGD axis corresponds to pulling the dummy atom along the +z direction. For N-to-C pulling, the protein is oriented such that the C-terminus is the pulling point and the N-terminus is the fixed point. In this framework, N-to-C pulling corresponds to pulling the C-terminus in the −z direction.2 was used. Dynamics were run as described above for the initial structure generation except that simulations were run for a total of 30 ns. The system temperature was maintained by coupling to a heat bath at 300 K, again using the Berendsen method In constant velocity SMD external force at the pulling point is provided by translating, at constant velocity, the free end of a spring (dummy atom in A) to zero. One N-to-RGD pulling simulation was performed under this altered energy function.Constant-velocity SMD pulling simulations were performed on each of the ten initial structures along both an N-to-RGD and N-to-C axis. Each simulation was run for 30 ns as outlined above, making the total simulation time, for the 20 structures, 600 ns. Unfolding trajectories and associated structures were visualized with the software program VMD (version 1.8.5) 2, KMAX = 0.6 kcal/mol/Å2, RMIN = 0 Å, RMAX = 0 Å, FMAX = 3000 kcal/mol/Å Dx,Dy,Dz) and  are the Cartesian coordinates of the dummy and pulling atoms, respectively. The components of the force on the pulling atom can be expressed as:Dv, along the z-axis, so that Dz(t) = Dv·t+Dz(0). With these definitions the magnitude of the total force on the pulling atom is given by The NOE facility in CHARMM was used to introduce an external pulling force on the dummy atom of interest, where KMIN = 0 kcal/mol/ÅThe terms in the potential energy function governing the unfolding simulation give rise to the internal forces that resist the pulling. More concretely, the force associated with the total internal energy, which is the sum of the individual energy contributions attributed to the bonded  and non-bonded  interactions, can be calculated for all the atoms in the protein throughout the unfolding trajectory. The magnitude of the internal force of 10FNIII that contribute to its mechanical stability is then calculated as described above.R, W(R), to be:minr is the value of the distance between the anchor point and the pulling point at t = 0. The function F̅z(r) is given by:D(t′) is the distance between the anchor point and the pulling point at time t′. Values of W(R) are computed from Equation 2 using the trapezoidal rule.We define the work needed to separate the anchor point and the pulling point by a distance f, as follows:SASA(0) and SASA(t) are the solvent accessible surface areas (SASAs) of the given β-strand at times 0 and t, respectively. SASAmax is the β-strand's maximum solvent accessible surface area and is computed by calculating the SASA of the strand in the initial structure when all other atoms in the protein are removed. With this definition, β-strands in the initial structures all have f = 0, while fully exposed strands have f = 1.Solvent accessible surfaces of the β-strands in the structures from the unfolding trajectories were computed using a Lee and Richard's algorithm with a 1.4 Å sphere radius and 0.025 Å accuracy Text S1(0.04 MB DOC)Click here for additional data file.Figure S1Evaluating the Role of Electrostatic Interactions Involving β-strand A. (A) Snapshots of the N-to-RGD unfolding trajectory with (right) and without (left) strand A electrostatic contributions ignored. Comparison of the unfolding trajectories for the same initial conditions, but calculated for either the normal energy potential (U) or the modified electrostatic energy potential (UE), show minimal differences between the unfolded structures. (B) Solvent accessibility factor of the β-strands. The pattern of solvent accessibility of the secondary structural elements (labeled at left) calculated every 3 ns throughout the trajectory show similarities between the two N-to-RGD unfolding profiles for normal and modified electrostatics.(0.92 MB DOC)Click here for additional data file.Figure S2Energy Contributions Significant for 10FNIII Mechanical Stability Along the N-to-RGD Axis. (A) Electrostatic energy contributions to the force profile. The black curve denotes the N-to-RGD force profile with the full potential energy function. For the same starting structure, the force profile (magenta) is calculated with the electrostatic interactions involving strand A turned off as outlined in the text. (B) Contributions due to bond length, bond angle, and van der Waals energy terms to the force profile. The black curve represents the magnitude of the external pulling force needed to unfold 10FNIII. The blue curve is the magnitude of the sum of the bond length, bond angle, and van der Waals energy contributions to the calculated internal force for the same initial structure.(0.66 MB DOC)Click here for additional data file.Movie S1Partial N-to-C Unfolding, Group 1. The anchor and pulling points are highlighted as black spheres with pulling towards the right. Initial unfolding of 10FNIII occurs at the N-terminus.(6.10 MB MOV)Click here for additional data file.Movie S2Partial N-to-C Unfolding, Group 2. The anchor and pulling points are highlighted as black spheres with pulling towards the right. Initial unfolding of 10FNIII occurs at both the N- and C- termini.(5.87 MB MOV)Click here for additional data file.Movie S3Partial N-to-RGD Unfolding. The anchor and pulling points are highlighted as black spheres with pulling towards the right. Initial unfolding of 10FNIII occurs exclusively at the N-terminus. The central glycine is labeled with a magenta sphere while Arg1445 is highlighted with a gold sphere. Note that the separation of these two residues increases following the occurrence of the kinetic intermediate (paused frame).(7.34 MB MOV)Click here for additional data file.


The Activity of Daily Living (ADL) is an instrument that screens elderly respondents for physical functioning and assesses whether they are dependent or independent in their daily activities. This study demonstrates a translation procedure and obtains the reliability and validity of a translated, Arabic ADL.The ADL was translated to Arabic through a forward translation method followed by a committee-consensual approach. The ADL and the Arabic Mini-Mental State Examination (AMMSE) were administered to an opportunistic sample of 354 Lebanese elderly living in nursing homes who did not have dementia.Reliability split half measures, sensitivity, and negative predictive values were high across all dimensions of the ADL with the exception of feeding. There were non-significant differences on the scored ADL between the three age groups: young age, middle age and older old. In addition, a non-significant difference was found on the scored ADL between the high and low AMMSE scores.Overall, the translated ADL was consistent and valid measure for assessing daily activities in elderly nursing home residents. As it is quick and easy to use, the ADL in Arabic could help caregivers and doctors to prescribe appropriate physical exercise for elderly Arabic speaking patients. In 2006, the number of people aged 60 years and above was 11.1% of the Lebanese population  calculatPhysical support facilities for elderly dependents are found in nursing homes (NHs) . GeneralAssessment by caregivers of the activities of daily living is important in identifying the degree of independence or dependence in NH residents . The ADLThe ADL scale was translated from English to Arabic then sampled among Arabic speaking elderly living in Lebanese NHs  bathing, 2) dressing, 3) going to toilet, 4) transferring (movement), 5) continence, and 6) feeding. It was forward translated by the author, co-author, and a third academic. All translators were native speakers of Arabic and teach in English. Each of the three translators was blinded to the results of the other translators' efforts. To validate the Arabic translations, three judges rate each of the three translations. All judges were native speakers of Arabic and were proficient in Arabic and English. One judge had a PhD in biochemistry, the second was an MD specializing in geriatric medicine, and the third was a PhD in nutrition. All judges had more than 5 years experience working in academia, the geriatric and nutritionist, on a part-time basis, worked in NHs.The judges, blinded to the translators and to each other's ratings, they rated the translated items for three factors: applicability (can these items be used by researchers in Lebanese NHs?), adaptability (are caregivers or the elderly able to answer or perform a task described in the items?), and suitability  or a 1 (complete independence). The responses on the Arabic version of the ADL were altered to 0, 0.5, or 1, with 0.5 indicating partial independence.Functional impairment scores were calculated for each item and for the overall total. A total mean percentage score  was obtained by adding the scores on the 6 items, dividing by 6 and then multiplying by 100. The lower the total ADL score, the more severe the dependence. Using an adaptation of the model developed by Johnson et al., , a scoreSensitivity and specificity are measures of accuracy: sensitivity refers to the proportion of patients with a given disease who have a positive test and specificity to the proportion of patients without the disease who have a negative test. Predictive value refers to the likelihood that a patient has or does not have the condition, given a positive or negative test result . In analWhen testing the validity of the Arabic ADL, it was expected that a high sensitivity would indicate that elderly respondents who were dependent would screen positive and that there would be a low false negative rate . To subsA research grant review committee of the World Health Organization, Eastern Mediterranean Regional Office, granted approval to carry out the study. The study was supported by the Ministry of Social Affairs, Ministry of Public Health, and National Association of Elderly Affairs. The study was partially funded by the WHO through contract # EM/07/0564956. An ethics committee did not exist at the national level or where the two authors work.In 2007 there were 44 NHs in Lebanon that assisted the elderly with feeding, shelter, physical support, and medical support. Seven field researchers moved from one region to another, starting in the North then going to the South, East, central Lebanon, and the capital, Beirut; and 36 NHs were visited.Residents who agreed to be part of the study and did not fit five exclusion criteria were included in the study. The exclusion criteria were having been institutionalized for less than 3 months, suffering from a terminal disease, being blind and or deaf, exhibiting cognitive impairment with a score less than 18 on the Arabic Mini-Mental State Examination (AMMSE), and being below 60 years of age. Of the 2094 elderly people living in the 36 NHs, 354 residents (average age 76.84) were enrolled in the study. Only three elderly who were not excluded and did not respond were excluded from the study.The authors sent a consent form to NH administrators describing the project and asking for approval to enter the NH and obtain information from the residents and the medical records. Administrators were told that all information would be anonymized. We garnered approval from 36 NH administrators which identified residents fitting the enrolment criteria. Field researchers described the project to potential participants, who were given a one-page summary of the project in Arabic and were informed that certain information would be taken from their medical records but that anonymity would be maintained. Residents who were willing to participate either signed a consent form or gave verbal consent in the presence of a nurse or caregiver. No duress was used when obtaining consent to participate or gathering responses to questions on the ADL.Participants were first given the Arabic Mini Mental State Examination (AMMSE) to determine their cognitive abilities and to identify dementia. They then completed the Arabic version of the ADL. In some cases, caregivers answered the questions in the presence of the participants. In addition, data were obtained from the medical records, including disability measures and sociodemographic information. The field researchers maintained complete respect for the rights and needs of the participants.Assessment data were coded by a data entry person. The codes were tagged to the caregiver and the NH data. The data were related through a coding scheme to maintain the anonymity of elderly, the NHs, and caregivers.The MMSE was administered to assess the cognitive level of the participants. It has concurrent and construct validity, established by Folstein et al. , and wasWhen the three judges rated the three translations for adequacy, applicability, and appropriateness, there was an 83% agreement on the adaptability of the translated items and 100% agreement on the applicability and appropriateness. The items with weak adaptability were reviewed and translated by the judges then rated again. Once the judges reached 100% agreement on adaptability, they finalized the Arabic ADL instrument. The tool was tested in 10 elderly to see if the translations could be operationally defined, and this pilot study did not identify any problems.Respondents were grouped according to age using the classification system of Vierck and Hodges . The ageThe reliability split half measure had a strong Cronbach alpha of 0.90 for the first three subscales on the ADL . The Cronbach alpha for the second three subscales was 0.65 for transferring (movement), continence, and feeding, with a correlation of r = 0.8 between the two halves.Sensitivity, specificity, and positive and negative predictive values were calculated for each of the ADL items. A total score was computed for the ADL: a total score above 3 was considered negative (i.e. no dependence) and below or equal to 3 was considered positive (i.e. indicating dependence). The positive and negative predictive values were cross-referenced against the two conditions of being crippled or having had an amputation. A high probability for sensitivity and predictor value negative was found with low probabilities for specificity and predictive value positive.The probability of the predictive value positive was very small compared to a high predictive value negative. Because of the high level of sensitivity of the ADL, there were few false negatives: the high negative predictive value therefore meant a high probability that elderly people who were not dependent screened negative on the ADL  indicated that the higher the age group, the higher the ADL and, therefore; the greater the functional dependence.To determine the validity of using an AMMSE score cut-off of 18 for cognitive impairment, we compared the total ADL score of the group with AMMSE 18–20 to a second group scoring above 20, using Student's t-test (respondents scoring below 18 were excluded from the study). The mean ADL score for the lower group (AMMSE 18–20) was 71.2 (SD = 35.1). It was higher for the higher AMMSE group at 78.69 (SD = 25.69) but this difference was not significant t(df = 250) = 1.13, p = 0.26.We looked at whether the ADL could discriminate between the older old, middle-old and young-old. No significant differences were found, F = 0.14, p = 0.86. However, the older old had lower ADL scores than the middle and younger age groups, at a non-significant level Table .Many caregivers for the elderly use general observation and clinical judgment as easy, practical and quick methods for assessing the activities of daily living. Having an objective, reliable and valid instrument for Arab speaking clients would be useful in the Mediterranean and Middle East. In our review of the literature, not one study used an Arabic, valid and reliable ADL instrument.We used the ADL, a reliable, valid instrument that has been field tested in Lebanon. We translated the ADL and assessed whether it could provide objective indications of dependence. The translation was improved by a committee of three experts who rated the items on adaptability, applicability, and appropriateness. Agreement by the judges in the first round of translation reached 94.3% on these three factors. Through a process of reiterative translation and rating, judgment reached 100% agreement.The translated ADL was administered to elderly people living in nursing homes. The high Cronbach alpha of the ADL, indicated 3 random items correlated highly with the rest of the three items.Ideally, for a test to be a maximally useful screening tool, both sensitivity and specificity should be 100%, so that every subject with the condition has a positive test , and there is a negative predictive value of 100%. Similarly, positive tests should occur only in subjects with the condition , resulting in a positive predictive value of 100%, which is generally impossible . ConsideThe negative predictive value gives confidence in the use of the chosen scoring scheme, involving scores of 0, 0.5 or 1. The 0.5 was transformed to a 0 as a positive measure of the condition and this would increase the risk of false positives. With low proportions in the sample having either of the two test conditions we chose as likely to increase dependence , an appropriate test would have a high sensitivity and a high negative predictive value, minimizing falsely negatives.Possibly because the elderly in our sample were generally independent, as shown in the mean overall ADL score Table , the feeThe literature indicates that the development of dementia is associated with disability . The ADLWe investigated whether increasing age, a known a risk factor for loss of independence, predicted a lower overall ADL score and found no association. In fact, with 8.3% of the sample being severely handicapped, dependence would be associated with the age rather than condition. However, the negative correlation found between age and the ADL percentage score is substantiated by Covinsky, et al.  that sugAssessing the activities of daily living is essential for planning geriatric interventions . For exaThe authors declare that they have no competing interests.RN, wrote and conceptualized the study presented in this paper. JD administered the questionnaires collected the data, conceptualized and edited the paper for the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Arabic ADL Scale. The Arabic ADL scale is presented with a 0, 1/2 and 1 response format.Click here for file