We use ancient DNA to compare two independent estimates of population size  and corroborate our results with gene diversity and serial coalescent simulations. Our data and analyses indicate that, with population size decreasing at times of climatic change, some species will exhibit declining gene diversity as expected from simple population genetic models, whereas others will not. While our results could be consistent with selection, independent lines of evidence implicate differences in gene flow, which depends on the life history strategy of species.Understanding how climatic change impacts biological diversity is critical to conservation. Yet despite demonstrated effects of climatic perturbation on geographic ranges and population persistence, surprisingly little is known of the genetic response of species. Even less is known over ecologically long time scales pertinent to understanding the interplay between microevolution and environmental change. Here, we present a study of population variation by directly tracking genetic change and population size in two geographically widespread mammal species  A novel analysis of paleoclimatic data, fossil abundance and contemporary and ancient DNA from different time periods introduces "phylochronology" and reveals how species respond genetically to climate change Phylogeography has advanced our understanding of the spatial distribution of genetic diversity within and between species . HoweverOur study takes advantage of a continuous, well-sampled mammalian fossil sequence spanning the last 3,000 years . Lamar Cave has an extraordinarily complete representation of the local species in the vicinity, with over 10,000 identified mammalian specimens representing over 80% of the mammal species in the local habitat . Late-HoMicrotus montanus (montane vole) and Thomomys talpoides (northern pocket gopher), species that presently are widespread in mountain habitats of western North America. Due to their preferences for wetter habitats, both responded demographically by increasing in relative abundance during wetter climates and declining during warmer climates. M. montanus showed an increase in abundance relative to other common rodents during periods of wet, cool climate in Yellowstone  techniques allows us to investigate directly the impacts of these environmental perturbations on neutral genetic diversity concurrent with these species population responses. We obtained ancient and modern mitochondrial DNA sequences from M. montanus and used previously published data for T. talpoides. Although these two species are broadly similar in body size  and are principally herbivorous, they differ in their natural history. T. talpoides is characterized by low population densities (1–62 gophers/hectare [ha]), a fossorial mode of life, maximum dispersal distances of a few hundred meters, and fiercely territorial behavior  despite the absence of obvious migration barriers.Although the population responses of behavior . Populatcalities . This spM. montanus achieves higher average population densities (60–186 voles/ha)  have found little evidence for population subdivision over the scale of hundreds of kilometers   than T. lometers , as expelometers . Such deplotypes .The primary advantage of a phylochronologic approach as opposed to a single time slice for understanding mammalian response is the ability to reveal changes in genetic variation through time. This is in contrast to modern genetic studies that seek to reconstruct demographic history based on inferences from past climate or geologic records e.g., . Our stuM. montanus and T. talpoides fossil specimens and modern population densities. (2) We calculated gene diversity over this 3,000-year period to determine the impact of environmental perturbations on genetic effective population size and gene diversity in M. montanus and T. talpoides. Unlike previous aDNA work  might influence the record of gene diversity over time. Thus, we combined four methods of estimating population size, determining statistical significance, and assessing gene diversity through time. (1) We derived independent ecological estimates of population size through time from abundances of DNA work , we usedDNA work . Our appT. talpoides, we predicted that changes in genetic variation through time would be dominated by drift, as suggested by the modern life history characteristics of small effective population size, low dispersal, and high amounts of population substructure. Therefore, as the ecological effective population size of T. talpoides declined with warmer climates, we expected genetic variation to decline. For M. montanus, we predicted that changes in genetic variation through time may be influenced more by migration, as suggested by large effective population sizes, high rates of dispersal, and low amounts of population substructure. As a result, past declines in ecological estimates of population size of M. montanus would not necessarily have resulted in a decrease in genetic variation.Despite similar population-level responses to climatic change of the late Holocene, we expected differences in gene diversity change for the two species. For e_ecol(N >> e_gen)N  . Howeveralpoides A and M. montanus B. For M.M. montanus suggests that the sequences we obtained for this study are target mtDNA. Of the 312 bp we sequenced for M. montanus, 96.5% of all the mutations were third-position codon changes, with first- and second-position mutations accounting for 3.5% and 0%, respectively. These ratios of variation are concordant with expectations for within-species variation and small overall sequence divergences . Most of the mutations are synonymous (97.7%); the transition-to-transversion ratio of the entire data set was 4.1 to 1, which is consistent with expectations for mammalian cytochrome b and evolution in other Microtus species  and translated successfully. Together with the frequency distribution of our pairwise differences, the prevalence of silent and third-position codon changes, and the standard of obtaining both forward and reverse fragments of overlapping sequence regions, these data permit us to conclude that the genetic diversity we have sampled represents authentic mitochondrial population variation and is unlikely to be from nuclear copies or pseudogenes.The genetic evidence we have assembled from ergences . Nucleot species , with an species . Fossil R2 = 0.004, not significant). All but one (out of 121) of the extraction controls were negative. When sequenced, this extraction blank BLASTed similar to Montanus townsendii, a taxon we had never worked on in the facility; this sequence has not since been amplified in the lab, and that extraction was not used further. Out of 87 successfully amplified samples and one sequence obtained from GenBank (AF119280), we identified 17 haplotypes within four haplogroups (A–D) of M. montanus  fall within haplogroups A and D, with 84% of the samples within one substitution of the locally ancestral haplotype A  and a deficit of guanine (11.4%). All mutations were synonymous; the transition-to-transversion ratio of the entire data set was 4.0 to 1.Protocols for M. montanus and T. talpoides T. talpoides (N exhibit the same trend as the genetic estimates e-tt_gen),(N are lower than those derived from genetic data e-mm_gen) for all time points except for 2,525 ybp, where observed gene diversity was significantly lower than could have been calculated given our sample size (n = 4), suggesting that we do not have sufficient power to detect processes at this time period.For ple size . Since tT. talpoides is not different given expectations from a closed population, we attempted to determine the statistical limitations of these data. Investigation of the effects of sampling for T. talpoides revealed that given the smaller number of base pairs (64 bp), we do not have enough statistical power to reject the null hypothesis. For every sampling time point, the Ewens distribution predicted that only one haplotype would be present in the genetic samples (unpublished data). As a result, the predicted gene diversity was zero for all time points. Because the observed gene diversity for T. talpoides was higher than predicted we also simulated five combinations of mutation rate and abundance, which could result in a higher predicted diversity  for 166 ybp (n = 34). Results for all five combinations predicted presence of a single haplotype. Since we do not have adequate statistical power given the genetic data for T. talpoides, we did not conduct significance tests for this species. However, the observed values of gene diversity are not unexpected from dynamics within a closed population.Although the gene diversity for M. montanus gene diversity throughout the entire time series   based on the observed change in 166 ybp) . The expM. montanus prior to 2,525 ybp. We modeled population reduction to 104,577 (0.75 × e-mm_ecol2525ybpN), 69,718 (0.5 × e-mm_ecol2525ybpN), and 34,859 (0.25 × e-mm_ecol2525ybpN) prior to 2,525 ybp. For a 75% bottleneck prior to 2,525 ybp, we were no longer able to reject the null hypothesis of a closed population. These results indicate that an extreme bottleneck where population size was reduced to 75% or more might result in the observed change in gene diversity. Our simulations reveal that unless an extreme bottleneck happened prior to 2,525 ybp, we can be confident that the observed change in gene diversity is not likely to be from events that occurred immediately prior to our historic data, and thus is due to migration or selection.Our results may depend on our assumptions of equilibrium population size prior to 2,525 ybp. We investigated sensitivity to this assumption by modeling a population bottleneck in M. montanus. Historic genetic data allow us to discriminate between population processes over millennia much better than do modern data alone.Coalescent simulations used to investigate the significance of the observed gene diversity value for the modern samples demonstrated that the null hypothesis of past population size change could be rejected at only two of the nine mutation rate and density combinations. These results reveal that given data from only the modern samples, it was not possible to reject the null hypothesis of past population size change in M. montanus populations. Estimates of β-diversity (used here to measure haplotypic turnover) from haplotypic data reveal that turnover was highest between 2,525 and 845 ybp . Closer examination of the haplotypic distributions demonstrates that five novel haplotypes appeared by 845 ybp, three of which are ≥3.2% different from the most common haplogroup (A)  A at 2,52T. talpoides and M. montanus between 1,438 and 470 ybp, a period spanning the Medieval Warm Period  results in an increase in gene diversity. We attempted to statistically validate our results by the use of serial coalescent simulations to demonstrate that the change in gene diversity of M. montanus between 2,525 and 845 ybp is significantly different from that expected based on the decrease in ecological estimates of population size. Taken together, these results indicate a departure from conditions of equilibrium (closed population without selection) for M. montanus. Our results have the following possible explanations: (1) the sampling area for fossils changed, (2) the local population size expanded, (3) selection occurred, and/or (4) gene flow occurred.Our results demonstrate different genetic responses by two species of small mammals to changes in population size driven by climatic change. Fossil abundance data reveal population decline for both m Period . For T. Microtus oeconomus, indicate that migration occurs most frequently in and between low-density patches  and between populations (gene flow). Discrepancy between the ecological and genetic estimates of population size and significant changes in haplotypic diversity prior to the Medieval Warm Period implicate increased gene flow into the Lamar Cave M. montanus population. Additionally, the observed haplotypic turnover in the Yellowstone population during this period suggests that as abundance of M. montanus declined through the last 845 years, relatively more individuals carried newly introduced haplotypes.Here we demonstrate, using a phylochronologic approach, that it is possible to distinguish the dynamic processes that govern gene diversity over relatively short time scales (hundreds to thousands of years). We have documented environmental change, population response, genetic diversity change, and the correlations between the three. Without serial data, we would capture just a single record of these historic processes: modern genetic diversity. Although it is possible to hypothesize about historic events using modern data, phylochronology affords a unique look into the past and the potential ability to separate cause from effect. In particular, we show that M. montanus was maintained. In contrast, gene flow has not played a significant role in the recent genetic history of T. talpoides. This species, instead, responded more as a closed population over this time. The disparate nature of population response to climatic change of these two species is likely due to differences in demographic dispersal patterns between their populations. Such differences in species demography have resulted in differential genetic response to climatic change, even when ecological response is similar. Thus, genetic response to environmental change can be viewed as “individualistic,” similar to unique adjustments of species ranges  declined while others expanded. Our data show that even with a prolonged ecological population size decline, the genetic diversity of s ranges . Life hin = 8,589]) and are concordant with expectations of taxonomic diversity in montane mammal communities of western North America .Lamar Cave contains well-stratified, thoroughly radiocarbon-dated deposits, which display high fidelity to the local mammalian community , 1999. T America . The cumMicrotus samples encompass 15 of 16 radiocarbon-dated stratigraphic levels from Lamar Cave N and e-mm_ecol (M. montanus).NIn order to compare ecological estimates of effective population size to genetic estimates, we estimated mitochondrial effective population size assuming that Microtus species are cryptic with respect to these teeth. Genetic diagnosis indicated that these 78 fossil samples are derived from multiple arvicoline species, with a total of 47 M. montanus specimens (see Microtus molariform teeth) ranged from 3.5 to 15.3 mg . We used two previously described extraction methods on the fossil teeth (b from ancient samples (n = 47) using the following primers: forward primers (5′–3′), CLETH 37 TAY AAY ATA ATY GAA ACH TGA A , CLETH 37L AYG GMT CTT AYA ACA TAA TCG AAA CAT G , MMONT 1 CAG TAA TTA CAA AYC TWC TAT CA , and MMONT 3 AGT GAA TCT GAG GGG GCT TCT CAG TAG A ; reverse primers (5′–3′), ARVIC 08 CAG ATY CAY TCY ACT AGT GTT G , ARVIC 08L CTC AGA TTC ACT CTA CTA GTG TTG TG , MMONT 4 TTR TTT GAT CCT GTT TCG TGT AGG AAT A , and MMONT 2L TTG ACT GTG TAG TAA GGG TGA AAT GGG A . Attempts were made to amplify the region in two overlapping fragments using CLETH37/ARVIC 08, CLETH37L/ARVIC 08L, and MMONT 1/MMONT 2L. However, the low rate of success for MMONT 1/MMONT 2L (40%) in the fossil samples necessitated the breaking of the second fragment into two overlapping fragments using MMONT 1/MMONT 4 and MMONT 3/MMONT 2L.From the fossil material, we used the upper first molar (from only one side of the jaw per level) to avoid sampling the same individual multiple times. Some mens see . Fossil il teeth . We obtaAncient DNA samples were run on an ABI PRISM 310 Genetic Analyzer in the post-PCR lab, and modern DNA samples were run on an ABI PRISM 377 Sequencer in a separate sequencing facility . Fragments were sequenced in both directions, primer regions were overlapped, and sequences with any ambiguous sites were rerun until completely resolved in order to provide additional corroboration and eliminate ambiguity.We adhered to strict extraction and amplification protocols . The pro and 12 teeth  from the vicinity (within 10 km) of the fossil site. In addition, DNA was successfully extracted from 51 modern specimens (liver tissue and museum skins) collected within a 400-km radius of Lamar Cave. Of these 51 samples, 22 were derived from museum skins  and 29 from liver tissue  of specimens trapped in the field. We followed the animal tissue protocol using the Qiagen Dneasy Tissue Kit  on 6.5 mg of liver samples and 7.5 mg of tooth samples (n = 3). For museum samples, DNA was extracted from the ventral skin incision . We amplified the entire cytochrome b gene  for some modern skins (n = 13) and liver tissue (n = 29) with the following primers: forward primers (5′–3′), MVZ 05 CGA AGC TTG ATA TGA AAA ACC ATC GTT  and ARVIC 07 AAA GCC ACC CTC ACA CGA TT ; reverse primers (5′–3′) MICRO 06 GGA TTA TTT GAT CCT GTT TCG T  and VOLE 14 TTT CAT TAC TGG TTT ACA AGA C .Modern  genetic sampling was obtained from a variety of sources including modern skins, modern liver tissue, museum skins, and teeth derived from modern raptor pellets. DNA was successfully extracted from 16 modern skins Microtus samples, and 79 of those yielded successful amplification. Of these 79 samples, 48 were positively identified as M. montanus. Of the M. montanus samples, 41 specimens were successfully haplotyped. Sequences have been deposited in GenBank  , which is not found elsewhere in a 400-km radius around Lamar Cave. This constancy in the genetic lineage of T. talpoides within a single locality persists in spite of climatic changes and concurrent significant population and body size changes (We have built upon the previously published data set  with add= 3) see . T. talp changes .e) was used to estimate e. θSN was preferred over other estimators of θ since Hθ is biased for single locus estimates, kθ does not incorporate sequence information, and πθ has higher variance. These estimates are determined entirely from genetic data; thus we label them e-tt_gen N and e-mm_gen (M. montanus).N We assumed a range of μ   using Cody's index (β-diversity each haplotype was treated as a “species.”Arlequin v. 2.000  was used per bp) . Additio's index . For β-dN, a gene with mutation rate μ, and an infinite alleles model given a sample size n. Here we use the Ewens distribution to ascertain the ability of our data to detect variation in values of gene diversity. In addition, this distribution allows us to (1) predict the distribution of expected gene diversity at each point in time (independently) given estimates of e_ecol,N a moderate mutation rate of 4% per million years, typed sequence length , and sample size  and (2) predict change in the expected gene diversity distribution for a large sample size of 100. A modified version of MONTE CARLO , and gene diversity was calculated for each configuration. Simulations were repeated 1,000 times for each sampling time point, resulting in the 95% confidence intervals for the distribution of predicted gene diversities given a closed, selectively neutral population through time.Given the general limitations of obtaining aDNA sequence data, sample sizes will always present challenges to ancient population genetic studies. To investigate whether our samples are adequate for addressing temporal gene diversity in both species, we used a neutral population model to evaluate expected diversity. TE CARLO , 1996 waM. montanus evolution during the past 2,525 years. Simulations were repeated 1,000 times to generate genetic data and changes in gene diversity for the null hypothesis. The significance of the observed changes was then inferred by comparing it to the generated null values (we thus used a Monte Carlo significance test). Estimates of e_ecolN were used to set up a null hypothesis specifying population size change through time in a closed population. We assumed that past changes in population size between intervals were due to exponential growth or decline. The effective population size at two time points and the time between the points was used to calculate a growth rate. The estimated growth rates were 1438–2525ybpr = −0.000178, 845–1438ybpr = 0.000732, 470–845ybpr = 0.0004385, and 166–470ybpr = 0.0003212. The null hypothesis thus corresponds to effective population sizes at particular points in the past and exponential growth or decline between those intervals, and represents an ecologically realistic description of the past 2,525 years. We assumed a constant population size prior to 2,525 ybp, as we have no data before this point in time. The coalescent program SIMCOAL , we investigated the significance of observed changes in gene diversity at all nine parameter combinations over the entire time span  and between 2,525 and 845 ybp .We assessed the significance of the observed changes in gene diversity between time points using simulations. The serial coalescent was used as a framework to model  SIMCOAL  was modialescent  has beenAdditionally, we also investigated the relevance of temporal data to our ability to reject the null hypothesis. Significance tests were repeated assuming the observed data were from only the most recent genetic samples. Again, simulations were repeated for all nine combinations of mutation rate and density estimates.M. montanus specimens described in Sequences for the successfully haplotyped


CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Δex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology.JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a ~1 kb Cln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Δex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced.To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbThese findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival. CLN3 gene, which eliminates exons 7 and 8 and surrounding intronic DNA, predicting a non-functional protein product [Juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, is a recessively inherited childhood-onset neurodegenerative disorder characterized by progressive blindness, seizures, motor and cognitive decline, and early death . The pri product .The pathological hallmark of JNCL is autofluorescent ceroid lipofuscin deposits within autolysosomes that are enriched in subunit c of the mitochondrial ATP synthase complex -5. RemarCLN3-encoded protein  is a highly conserved, ubiquitously expressed, multi-pass membrane protein [btn1, the yeast CLN3 ortholog, have implicated battenin in lysosomal pH homeostasis and amino acid transport [The  protein  that loc protein -9. Batteransport ,11.Cln3Δex7/8 knock-in mice harbor the ~1 kb common JNCL mutation and express a non-truncated mutant battenin isoform that is detectable with antibodies recognizing C-terminal epitopes. Homozygous Cln3Δex7/8 knock-in mice exhibit a progressive JNCL-like disease, with perinatal onset of subunit c deposition in many cell types and later onset of neuronal dysfunction and behavioral deficits [To explore JNCL pathogenesis and battenin function, we previously generated a genetically precise JNCL mouse model. deficits . These fCln3Δex7/8 knock-in mice. Homozygous CbCln3Δex7/8 cells exhibit pathological hallmarks of the disease, and a survey of membrane organelles revealed membrane trafficking defects and mitochondrial dysfunction in homozygous mutant CbCln3Δex7/8 cells.Currently, there is no suitable neuronal cell system to investigate the impact of the common JNCL mutation on biological processes. Therefore, we have established cerebellar neuronal precursor cell lines from Cln3Δex7/8 knock-in mice, and wild-type littermates. Primary cell cultures enriched for granule neurons were transduced with retroviral vector bearing a selection cassette and temperature-sensitive tsA58 SV40 large T antigen. Growth in G418 containing medium at the permissive temperature (33°C) allowed for selection and isolation of multiple clonal nestin-positive  cerebella of homozygous and heterozygous ive Fig. , and GFAive Fig. , cell liive Fig. , but notive Fig. .Cln3Δex7/8 cells were first examined for JNCL-like characteristics. Homozygous Cln3Δex7/8 knock-in mice express multiple Cln3 mRNA splice variants and mutant battenin protein that is detectable by batp1 antibody recognizing C-terminal epitopes [Cln3Δex7/8 cells, RT-PCR and anti-battenin (batp1) immunostaining were performed. As shown in Figure Cln3 mRNA isoforms in wild-type and homozygous cells were similar to those observed in total RNA isolated from wild-type or homozygous Cln3Δex7/8 knock-in brain, respectively  and the late endosomal marker, Rab7 .Homozygous Cb epitopes . To asseely Fig. . In addiCln3Δex7/8 cells displayed autofluorescence or subunit c inclusion formation (data not shown). However, when cells were aged at confluency (3+ days post-confluency), homozygous CbCln3Δex7/8 cellular subunit c levels were elevated beyond normal wild-type levels by immunostaining .We next investigated the basis for subunit c accumulation, testing the hypothesis that cathepsin D is abnormal since it is required for ATP synthase subunit c degradation in the lysosome . We firsome Fig. . Howeverrms Fig. . Cathepsin vitro assay, cathepsin D activity in total cellular extracts was not significantly altered in homozygous CbCln3Δex7/8 cells , versus wild-type cells , although a consistent trend towards increased enzymatic activity in mutant cells was observed. Thus, cathepsin D transport and processing are disrupted in homozygous CbCln3Δex7/8 cells in a manner such that enzymatic activity appears to be relatively unaffected.The impact of the altered cathepsin D processing on enzymatic activity was next tested to determine if altered enzymatic activity accounts for inefficient subunit c turnover. In a fluorogenic Cln3Δex7/8 cells; therefore, we surveyed the subcellular distribution and morphology of membrane organelles. Components of the secretory pathway, including the ER, cis-Golgi, and trans-Golgi, did not appear altered from wild-type appearance when labeled with the respective markers, protein disulfide isomerase (PDI), GM130, and VVL (data not shown). By contrast, the lysosomal markers, Lysotracker and Lamp 2 had significantly altered signal in homozygous CbCln3Δex7/8 cells, versus wild-type cells. Wild-type cells exhibited brightly stained lysosomes that were large and clustered in the perinuclear region whereas homozygous CbCln3Δex7/8 lysosomes were lightly stained, smaller vesicles that were more diffusely scattered in the cytoplasm of the cell . It is noteworthy that Lysotracker dye, which selectively accumulates in acidic compartments, exhibited the most marked reduction in lysosomal labeling. This observation may reflect altered lysosomal pH, an established finding in JNCL [The abnormal transport and processing of cathepsin D suggested membrane trafficking disruptions in homozygous Cbell Fig. . Lamp 1  in JNCL ,15.Cln3Δex7/8 cells, as measured by dextran-FITC uptake  signal observed by immunostaining Fig. , fluid-pCln3Δex7/8 cell mitochondrial morphology and function. Mitochondrial distribution in homozygous CbCln3Δex7/8 cells was indistinguishable from wild-type and heterozygous cells; however, homozygous CbCln3Δex7/8 mitochondria appeared more elongated by grp75 marker immunostaining and TEM analysis . Moreover, compared to wild-type or heterozygous cells, homozygous CbCln3Δex7/8 cells had significantly reduced cellular ATP levels , Lamp 1 , and Lamp 2  monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, maintained by The University of Iowa, Department of Biological Sciences. Batp1 (1 μg/ml) was previously described . Anti-suCln3Δex7/8 knock-in mice have been previously described [2, in granule neuron growth media . Infection was performed the following day with defective retrovirus transducing the temperature-sensitive tsA58/U19 large T antigen and a selection neomycin-resistance cassette [Cln3 cell lines. CbCln3 cell lines were maintained on 0.01% poly-ornithine coated dishes at 30–90% confluency, in 33°C and 5% CO2 atmosphere. Passage number was recorded (up to ~20 passages), but had no apparent impact on phenotype. Neuronal differentiation was as previously described [escribed . Littermescribed . Postnatcassette , as prevcassette . Followiescribed  with theCln3Δex7/8 knock-in allele PCR genotyping was with wild-type primers, WtF (5'-CAGCATCTCCTCAGGGCTA-3') and WtR (5'-CCAACATAGAAAGTAGGGTGTGC-3') to yield a ~250 bp band and knock-in primers, 552F (5'-GAGCTTTGTTCTGGTTGCCTTC-3') and Ex9RA (5'-GCAGTCTCTGCCTCGTTTTCT-3') to yield a ~500 bp band. PCR cycling conditions were 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 35 seconds, repeated for 34 cycles. Total RNA isolation and Cln3 RT-PCR primers and procedures have been previously described [Genomic DNA was extracted from tail biopsies and cell pellets as described . escribed .4 cells per well for microscopy studies, or into 100 mm dishes  at a density of 5 × 105 cells per dish for protein extraction. Cells were typically >95% confluent one day post-plating, and the following day was considered 1-day post-confluency. At the indicated times, cells were either fixed with 4% formaldehyde in phosphate buffered saline (PBS), pH 7.4, for 20 minutes and processed for autofluorescence/subunit c immunostaining, or cell pellets were collected for total protein extraction.Cells were seeded into 4-well chamber-slides  at a density of 5 × 10Alternatively, cells were fixed with 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4 for 1 hour and subsequently post-fixed and processed for TEM analysis as described . In conf4 cells per well in 4-well chamber-slides and grown overnight at 33°C, unless indicated otherwise. Fixation was with ice-cold 4% formaldehyde in PBS, pH 7.4, for 20 minutes, or with ice-cold methanol/acetone (1:1) for 10 minutes at -20°C followed by air-drying (antibody-dependent). Cells were washed with PBS at least 2 times, 5 minutes per wash, between each of the following steps of the staining procedure: 0.1 M glycine in PBS for 5 minutes, 0.05% or 0.1% (antibody-dependent) Triton X-100 (Fisher Scientific) in PBS for 5 minutes, 2% bovine serum albumin (BSA) in PBS for 30 minutes, primary antibody diluted in 2% BSA/PBS for 90 minutes, secondary antibody diluted in 2% BSA/PBS for 60 minutes. All incubations were carried out at room temperature. Following staining procedures, slides were coverslipped with Vectashield mounting medium (Vector Laboratories) and analyzed on a BioRad Radiance 2100 confocal microscope (Biorad), with identical exposure settings for wild-type and mutant like images. All comparisons of wild-type and mutant staining were performed in Adobe Photoshop with identical brightness and contrast adjustments.For immunostaining, cells were seeded at a density of 3–5 × 10c Protein Assay) was separated by SDS-PAGE, for subsequent immunoblotting, as described [Total proteins were isolated from cell pellets by extraction with ice-cold 20 mM Tris, pH 7.4, 1% Triton X-100 , plus protease inhibitors . Following homogenization through a 25-gauge needle (~10 passes), extracts were centrifuged at 1000 × g for 10 minutes, at 4°C, to remove debris. Typically, 20–40 μg of protein  according to the manufacturer's recommendations. Relative fluorescence was measured using an Analyst AD plate reader (Molecular Devices) with the following filters and settings: excitation filter, 360-35; emission filter, 400-20, Flash lamp with 100 readings/well, 100 ms between readings, and 100,000 μs integration time.100 mm tissue culture dishes, which were approximately 80–90% confluent, were washed briefly with ice-cold PBS, and total protein extracts were isolated by scraping cells into 10 mM Tris, pH 7.4, 0.1% Triton X-100 followed by incubation on ice, for 20 minutes. The insoluble material was centrifuged at 14,000 g, the supernatant was isolated, and protein concentration was determined using the Bio-rad D4 cells per well in 4-well chamber-slides and grown overnight at 33°C. Growth media was exchanged for fresh, pre-warmed growth media containing 500 nM Lysotracker or 1 mg/ml dextran-FITC, and cells were incubated at 33°C for 45 minutes or 15 minutes, respectively. Following labeling, cells were immediately placed on ice and washed for 10 minutes in ice-cold dye-free media, and fixed with 4% formaldehyde in PBS, for 20 minutes on ice. Chambers were removed and slides were coverslipped with Vectashield mounting media for confocal microscopy analysis, as described above.Cells were seeded at a density of 3–5 × 10TEM photomicrographs  were taken from random grid fields. For length measurements, the longest side of each mitochondria was measured in centimeters, and along the length of the mitochondria, width measurements were taken every 2.5–4 mm (dependent on the magnification of the micrograph image). Following measurement, all numbers were normalized to reflect one magnification and data was analyzed using Microsoft Excel software. To ascertain unmagnified mitochondrial size, final measurement data, in centimeters, was converted to nanometers according to scale bar representation.® Luminescent Cell Viability kit (Promega), according to the manufacturer's recommendations. Briefly, cells were plated in a black opaque-walled 96 well plate (Packard Bioscience) at a density of 20000/well and incubated at 33°C overnight. The following day, CellTiter-GLO® Reagent was added to each well and cell lysis was induced by mixing 2 minutes. An ATP standard curve was prepared in the same plate. Before recording luminescence with a microplate luminometer , the plate was dark adapted for 10 minutes at room temperature to stabilize the luminescence signal.ATP was measured by using the CellTiter-GLO® AQueous Non-Radioactive Cell Proliferation Assay (Promega), according to the manufacturer's specifications.Cells were plated at a density of 10,000 cells/well in 96-well plates and incubated at 33°C overnight. The following day, fresh media containing varying concentrations of hydrogen peroxide was dispersed to each well. Cells were incubated in the presence of hydrogen peroxide for 24 hours, at 33°C, and viability was measured using the CellTiter-96EF participated in establishment and characterization of cell lines and performed ATP determinations. PW participated in mitochondrial analysis and immunocytochemistry. JE, TL-N, AMT, and HG participated in genotypic and additional phenotypic analysis of cell lines. DR and EC generated virus-conditioned medium for conditional immortalization of cells. MEM co-conceived of the study and assisted on drafting of the manuscript. SLC co-conceived of the study, participated in establishment and phenotypic analysis of cell lines, and drafted the manuscript. All authors read and approved the final manuscript.


Ki-1 (CD30) antigen expression is not found on peripheral blood cells but its expression can be induced in vitro on T and B lymphocytes by viruses and lectins. Expression of CD30 in normal tissues is very limited, being restricted mainly to a subpopulation of large lymphoid cells; in particular, cells of the recently described anaplastic large cell lymphoma (ALCL), the Reed-Sternberg (RS) cells of Hodgkin's lymphoma and scattered large parafollicular cells in normal lymphoid tissues. More recent reports have described CD30 expression in non-hematopoietic and malignant cells such as cultured human macrophages, human decidual cells, histiocytic neoplastic cells, mesothelioma cells, embryonal carcinoma and seminoma cells.We investigated the immunohistochemical expression of CD30 antigen in 15 paraffin-embedded tissue samples representing small intestines from fetuses after spontaneous abortion in the 8th, 10th and 12th weeks using the monoclonal antibody Ki-1. Hormones had been administered to all our pregnant women to support gestation. In addition, a panel of monoclonal antibodies was used to identify leukocytes (CD45/LCA), B-lymphocytes (CD20/L-26) and T-lymphocytes (CD3). Our findings were correlated with those obtained simultaneously from intestinal tissue samples obtained from 15 fetuses after therapeutic or voluntary abortions.The results showed that: (1) epithelial cells in the developing intestinal crypts express the CD30 (Ki-1) antigen; (2) CD30 expression in these epithelial cells is higher in cases of hormonal administration than in normal gestation. In the former cases  a mild mononuclear intraepithelial infiltrate composed of CD3 (T-marker)-positive cells accompanies the CD30-positive cells. CD30 antigen, a member of the tumor necrosis factor (TNF) receptor superfamily -3, was oCD30 appears to have an important immunoregulatory role in normal T cell development. Within the thymus, CD30L is highly expressed on medullary thymic epithelial cells and on Hassal's corpuscles .Pallesen and Hamilton-Dutoir  were theth and 12th weeks of embryo development. Goblet cells are present in small numbers after 8 weeks, Paneth cells differentiate at the base of the crypts in weeks 11 and 12, and enteroendocrine cells appear between weeks 9 and 11.Primitive crypts , appear in the postpharyngeal foregut between the 9The fact that the CD30 molecule can mediate signals for cell proliferation or apoptosis  prompted2O2 in methanol. The sections were preincubated in 20% serum of the species from which the secondary antibody was raised, and the primary antibody was applied. After overnight incubation at room temperature, the secondary biotinylated antibody was applied for 30 min. Staining was visualized with a Vector Elite System  using diaminobenzidine as the chromogen. The sections were counterstained with dilute hematoxylin. The primary antibodies used were as follows: (CD30/Ki-1) activated lymphoid cells, mouse monoclonal antibody (Novocastra); (CD45/LCA) leukocyte common antigen, mouse monoclonal antibody (Dako); (CD20/L-26) B-lymphocytes, mouse monoclonal antibody (Dako); and (CD3) T-lymphocytes, mouse monoclonal antibody (Dako). We used the high temperature antigen unmasking technique for immunohistochemical demonstration of CD30/Ki-1 on paraffin sections (Novocastra). Control slides were incubated with nonimmunized rabbit serum. An anaplastic lymphoma case-slide (positive control) was run in parallel with the assay.Samples representing 15 small intestines from fetuses after spontaneous (involuntary) abortion occurring in pregnant women treated with progesterone , and 15 small intestines from fetuses after therapeutic or voluntary abortion, were obtained in the 8th, 10th and 12th weeks of gestation. The Regional Ethics Committees approved the study. Written informed consent was obtained from all individuals and the procedures followed accorded with institutional guidelines. Small intestines were cut in 3 mm slices and fixed in 10% neutral buffered formaldehyde at 4°C for 24 h, then processed for routine paraffin embedding. Paraffin blocks were available in all cases, and 3 μm thick tissue sections were stained routinely with hematoxylin-eosin, PAS and Giemsa, and subsequently by immunohistochemistry. Immunoperoxidase labeling was performed as follows: sections were deparaffinized in 70% alcohol and endogenous peroxidase was blocked with 3% H2 of tissue section. The counted areas were selected from random tissue sections, taking into account that the ratio of the area of the intestinal stroma to the area of surface epithelium covering the crypts was representative of the entire field. Areas with obvious necrosis or haemorrhages were excluded. Statistical analysis was performed using the ANOVA test.For each sample, the CD30/Ki-1 positive population was assessed by enumeration of labeled cells in each tissue compartment for a minimum of five random fields per section viewed at 40-fold magnification through a grid. Cell numbers were calculated per mmFive microscopic fields of the small intestines were evaluated in each case without knowledge of the clinical data TABLE . Two obsIn cases of spontaneous (involuntary) abortion, immunohistochemistry revealed small clusters or scattered, large-sized CD30/Ki-1 positive cryptae cells within the intestine in all settings examined Fig. , with peIn cases of spontaneous abortion, immunohistochemistry showed a higher number of positive CD30/Ki-1 cryptae cells than at the 8th week of gestation . The number in cases of voluntary or therapeutic abortions was more or less the same as at 8th and 10th weeks, with percentages varying from 3.2 to 3.7 (mean ± sd = 3.41 ± 0.17). No differences in immune reaction were noted in the neighbouring intestinal stroma in cases of either spontaneous or voluntary/therapeutic abortion in comparison to the 8th and 10th gestational weeks.The differences among the numbers of CD30/Ki-1 positive cells at the 8th, 10th and 12th gestational week after spontaneous abortion were statistically significant (p < 0.0001). No significant differences were observed in the numbers of these cells after voluntary or therapeutic abortions (p = 0.95).The value of the CD30 antigen as a diagnostic marker for Hodgkin's lymphoma and anaplastic large cell lymphoma is well documented ,5,31. HoCD30 appears to have an important immunoregulatory role in normal T cell development. In normal cells, this transmembrane glycoprotein can be induced on B and T lymphocytes by mitogen stimulation or viral transformation -34. cDNARecent in vitro data indicate that the CD30 receptor-ligand complex can mediate signals for cell proliferation, apoptosis and cytotoxicity in lymphoid cells ,36,37. OSecond, these findings indicate that outside the lymphatic system, CD30 antigen expression in the epithelial cells of developing intestinal crypts can mediate signals for cell proliferation and differentiation in a region where other cell types  grow throughout life.Third, CD30 expression in the epithelial cells of the developing intestinal crypts is induced by progesterone. This is a novel mechanism of CD30 induction, distinct from neoplastic transformation and viral infection of lymphocytes. The demonstration of large R-S like cells in the developing crypts within a lymphoplasmacytic infiltrate, in the same way that similar R-S like cells are observed in reactive lymph nodes, especially within the parafollicular areas, is evidence that such cells might represent the physiological counterparts of R-S cells.The possibility that CD30 is an oncofetal antigen is supported by our positive findings in fetal intestinal cryptae cells. We have so far been able to investigate only a single tissue from a small number of fetuses of early gestational age. Pallesen and Hamilton-Dutoit  examined


Pain is highly prevalent among older adults, but little is known about how patient involvement in medical decision-making may play a role in limiting its occurrence or severity. The purpose of this study was to evaluate whether physician-driven and patient-driven participation in decision-making were associated with the odds of frequent and severe pain.A cross-sectional population-based survey of 3,135 persons age 65 and older was conducted in the 108-county region comprising West Texas. The survey included self-reports of frequent pain and, among those with frequent pain, the severity of pain.Findings from multivariate logistic regression analyses showed that higher patient-driven participation in decision-making was associated with lower odds  of frequent pain, but was not significantly associated with severe pain. Physician-driven participation was not significantly associated with frequent or severe pain.The findings suggest that patients may need to initiate involvement in medical decision-making to reduce their chances of experiencing frequent pain. Changes to other modifiable health care characteristics, including access to a personal doctor and health insurance coverage, may be more conducive to limiting the risk of severe pain. Persons age 65 years and older commonly endure a multitude of chronic and debilitating conditions which contribute to persistent pain . EstimatIncreasing patients' involvement in the medical decision-making process is one potentially fruitful means of improving pain management. Several studies suggest that patients, especially those with chronic conditions, who have opportunities to participate in care have more positive health outcomes than those who do not ,7. WhileAlthough patients' participation may improve their health outcomes, the effect can be diminished among elderly patients. Elderly patients, as compared to younger patients, have been shown to be less participatory in medical-decision making -11. UsinThe primary objective of the present study was to examine how participation in decision-making was associated with the occurrence of pain among a cohort of community-dwelling elders. In contrast to previous studies, we differentiated two types of participation in decision-making. The first type is physician-driven in which the physician takes the initiative to ask questions and offer choices to patients. The second type is patient-driven, in which the patient takes the initiative to ask questions and express preferences. We hypothesized that stronger physician and patient-driven participation in decision-making would be associated with lower odds of frequent pain and, among those with frequent pain, lower odds of severe pain. We also tested for the effects of other health care factors which might be conducive to pain management, such as tenure with a personal doctor. The findings have implications for how older patients interact with their physicians as well as how physicians and clinic managers organize health services.Data were obtained through a longitudinal, population-based study of community-dwelling elders, the Texas Tech 5000 Survey. The Texas Tech 5000 Survey was conducted in a 108-county region of West Texas, a geographically and ethnically diverse area encompassing approximately half of the state's land mass. The survey has been described in detail elsewhere -21. BrieThe occurrence and severity of pain were measured in Wave 3 using items developed for and included in two nationally representative surveys of older persons, the Health and Retirement Study (HRS) and Assets and Health Dynamics among the Oldest Old (AHEAD) . First, A number of sociodemographic, health care, and health status measures were included. Sociodemographic factors were gender, age (continuous), marital status, educational status (high school graduate vs. less than high school education), and place of residence . An urban area is a metropolitan county, or a county with a total population of at least 50,000, whereas a rural area is a county with fewer than 50,000 persons. Rural counties were further classified according to whether they were frontier areas, or counties with fewer than 7 persons per square mile .Health care variables were health insurance coverage, the number of physician visits in the last 6 months, tenure with a personal doctor, an index measuring physician-driven participation in decision-making, and an index measuring patient-driven participation in decision-making. Health insurance coverage was coded as Medicaid, Medicare, Medicare plus other private or government coverage, other private or government coverage, and no insurance. Tenure with a personal doctor was measured using a single question asking if the individual had a personal doctor and, if so, the duration of tenure with the physician . An index of physician-driven participation in decision-making was created using three items taken from the Medical Outcomes Study . The phyThree questions adopted from a study of older patients' communication during medical visits  were useOverall health status  was measured using a general health item from a brief health-related quality of life instrument (the SF-12) . Mental Chi-square tests were first conducted to determine whether there was an association between each categorical sociodemographic, health care, and health status factor and frequent and severe pain. T-tests were conducted to determine if there was a difference in each continuous sociodemographic, health care, and health status factor between individuals with and without frequent pain and individuals with and without severe pain. Next, multivariate logistic regression analyses were conducted to determine if physician or patient-driven participation in decision-making were associated with the odds of frequent pain and, among those with frequent pain, the odds of severe pain. The potential for multicollinearity between the covariates was assessed by calculating their variance inflation factors; no problems with multicollinearity were found.A total of 1,333 42.5%) of the 3,135 survey participants had frequent pain. Table .5% of thAmong the 1,333 individuals with frequent pain, a total of 287 (21.5%) had severe pain. As shown in Table Findings from multivariate logistics analyses are shown in Table Physician-driven participation was not significantly associated with the odds of frequent pain. However, elders with higher patient-driven participation had lower odds  of frequent pain, confirming our hypothesis that persons who take a more active role in their medical treatment are less likely to experience pain. Individuals who had been diagnosed with arthritis at some point in their lives had a higher odds of frequent pain  than those without arthritis. Those who rated their general health as excellent , very good , good , and fair  had lower odds of frequent pain than those who rated their health as poor.Among those with frequent pain, there were no gender difference in the odds of severe pain. Persons of other race/ethnicity (primarily Black/African Americans) had a higher odds  of severe pain than non-Hispanic whites. Income, marital status, rural residence, and frontier residence were not significantly associated with the odds of severe pain.The number of physician visits was not significantly associated with severe pain. However, having insurance had a significant impact on the odds of severe pain. Compared to those without health insurance coverage, those with Medicare , Medicaid , Medicare plus other private or government coverage , and those with other private or government coverage  had a significantly lower odds of severe pain. Although physician and patient-driven participation were not significantly related to the odds of severe pain, tenure with one's personal doctor was a significant factor. Elders who had been seeing their doctor for less than 1 year , 3–4 years , or 5 or more years  had lower odds of severe pain than elders who had no personal doctor.As expected, several health status measures were also significant. Those who had arthritis had higher odds of severe pain  than those without arthritis. Finally, individuals who rated their general health as very good , good , and fair  had lower odds of severe pain than those who rated their health as poor.We found that patient-driven participation in decision-making was associated with lower odds of frequent pain, which is supported by previous research indicating that adult patients who are more actively engaged in their treatment have greater reductions in symptoms and improvement in health status , better While neither physician nor patient-driven participation in decision making were significantly associated with pain severity, another factor related to the strength of the doctor-patient relationship was significant. In the present study, having a personal doctor, no matter how long the tenure of the relationship was, reduced the odds of severe pain. The finding of a beneficial effect of having a personal doctor, at least in terms of the severity of pain, is consistent with prior studies which have shown that having a usual source of care is positively correlated with an individual's access to the health care system -33, satiIn addition to having a personal doctor, access to any type of health insurance coverage was associated with the odds of severe pain. Approximately 12 percent of persons in the study sample reported that they had no health insurance coverage at all, including Medicare, and 21.5% of those without insurance had severe pain. The percentage of patients in this group with severe pain was nearly 2 times higher than the percentage of patients in the other insurance categories. Many older persons may not be eligible for public health insurance because they have not contributed to the social security system for a minimal amount of time. This may be particularly common in the southwestern United States where there are larger numbers of Hispanic immigrants. Expansion of health insurance coverage to this group could improve their ability to visit physicians and other health providers when they experience pain and, ultimately, lead to better pain management. Further research is warranted to more clearly elucidate how characteristics of different health insurance plans, such as gatekeeping and cost sharing, affect access to physician services for pain treatment.Several demographic indicators were also significantly associated with frequent and severe pain. The gender differences beckon the question of whether medical care providers treat older women's pain less effectively or appropriately than men's. No differences were found between Hispanics and non-Hispanic whites, but other races (the majority of whom were Black/African American) had higher odds of severe pain than non-Hispanic whites. However, because of the heterogeneity of the other racial category, it is difficult to discern which particular racial groups experience severe pain. Research which includes greater numbers of other racial categories is thus warranted.In regard to health status, the results support that individuals with three or more comorbid diseases have a relatively higher odds of frequent pain and severe pain than those with no comorbid diseases. One disease, arthritis, was of particular interest and therefore was treated as a separate variable. Almost two-thirds of the subjects had arthritis, which is not unexpected for persons age 65 and older. Persons with arthritis had a much higher odds (over 3 times the odds) odds of frequent pain than individuals without arthritis. Moreover, those with arthritis had approximately 1.5 times the odds of severe pain. The magnitudes of these associations imply that efforts to more effectively treat arthritis could lead to improvements in pain management.While the present study has contributed to our understanding of the relationship between doctor-patient interactions and persistent pain, it is not without several limitations. Because the study was cross-sectional in design, it is impossible to infer any causal relationships. Although the pain measures were adapted from a nationally representative cohort study of older persons , they maDespite these potential limitations, the present study suggests that several strategies could be implemented to limit the incidence and severity of pain among community-dwelling elders. Health policy makers and insurance companies might implement new reimbursement schemes to encourage visits to a personal physicians in order to improve pain and other health outcomes. Managers of physician clinics should consider organizing practices to ensure that older patients are able to make timely appointments with a personal provider. Finally, patients themselves could help reduce their chances of having frequent pain by becoming more involved in their care. These are just a few examples of how changes to the organization and delivery of care might affect pain-related health outcomes. Future research should evaluate how a range of physician characteristics , physician clinic characteristics (e.g. solo or group practice), insurance characteristics , and patient characteristics (e.g. trust in physician) influence pain and pain treatment.The author(s) declare that they have no competing interests.TFB conceived the overall study, performed the statistical analyses, and led the drafting of the manuscript. KTX assisted with the study design, interpretation of statistical findings, and drafting of the manuscript. JH assisted with interpretation of the findings and drafting of the clinical implications. GK contributed to data management, statistical analyses, and drafting of the methods section. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


Patients with upper limb pain often have a slumped sitting position and poor shoulder posture. Pain could be due to poor posture causing mechanical changes  that in turn affect the function of major limb nerves (e.g. median nerve). This study examines (1) whether the individual components of slumped sitting  cause median nerve stretch and (2) whether shoulder protraction restricts normal nerve movements.Longitudinal nerve movement was measured using frame-by-frame cross-correlation analysis from high frequency ultrasound images during individual components of slumped sitting. The effects of protraction on nerve movement through the shoulder region were investigated by examining nerve movement in the arm in response to contralateral neck side flexion.Neither moving the head forward or trunk flexion caused significant movement of the median nerve. In contrast, 4.3 mm of movement, adding 0.7% strain, occurred in the forearm during shoulder protraction. A delay in movement at the start of protraction and straightening of the nerve trunk provided evidence of unloading with the shoulder flexed and elbow extended and the scapulothoracic joint in neutral. There was a 60% reduction in nerve movement in the arm during contralateral neck side flexion when the shoulder was protracted compared to scapulothoracic neutral.Slumped sitting is unlikely to increase nerve strain sufficient to cause changes to nerve function. However, shoulder protraction may place the median nerve at risk of injury, since nerve movement is reduced through the shoulder region when the shoulder is protracted and other joints are moved. Both altered nerve dynamics in response to moving other joints and local changes to blood supply may adversely affect nerve function and increase the risk of developing upper quadrant pain. Non-specific arm pain (NSAP), often called repetitive strain injury, describes the common problem of upper limb pain and functional impairment without objective physical findings. The contributing factors to the development of NSAP are not fully understood but ergonomic guidelines commonly suggest that good upper body posture protects against NSAP (e.g. ). In a sTogether, shoulder protraction, forward head position and flexion of the trunk form the main components of slumped sitting. The present study examines the effects of each of these components on longitudinal sliding of the median nerve using high frequency ultrasound imaging in asymptomatic normal subjects. In addition, the effects of sustained protraction on nerve sliding through the shoulder region are examined.Longitudinal nerve movement was measured using high frequency ultrasound imaging, as previously described by Dilley et al. ,6. A DiaFourteen healthy subjects, 5 male and 9 female, aged 25–38 years (mean = 32 years) were screened to exclude upper limb or cervical spine pathologies, rheumatological or neurological conditions. In each subject the nerve bed length was estimated, from the C6 spinous process to the tip of the index finger ), and used to normalise the ultrasound transducer position between individuals. All measurements were taken from the right upper limb only.The median nerve was imaged in longitudinal section in the forearm during forward head position, trunk flexion and protraction and in the forearm and upper arm during contralateral neck side flexion (CNSF). Each movement was repeated three times, including some reverse trials.Each subject (n = 8) was imaged in the proximal forearm whilst positioned upright on a chair fitted with a back and head support, hips and knees at 90° flexion, and the trunk fixed with Velcro strapping. The right upper limb was strapped to a Perspex plate in 90° flexion and 20° abduction at the glenohumeral joint, with the elbow fully extended, 45° forearm supination, and the wrist, hand and fingers in neutral. An active forward head position movement was performed, which included lower cervical spine flexion and upper cervical spine extension.Each subject (n = 8) was imaged in the proximal forearm whilst positioned upright on a chair, with hips and knees at 90 degrees flexion. The right upper limb was positioned as for the forward head position trials. The subject was taught to actively flex their trunk whilst posteriorly tilting their pelvis.Each subject (n = 13) was imaged at two locations in the forearm and positioned as for the forward head position. The distal upper arm was imaged in three of the 13 subjects. For each trial the shoulder girdle was passively protracted from neutral  by sliding the Perspex plate supporting the arm on an adjustable table. In three of these subjects, additional data was also obtained during ultrasound imaging. A potentiometer attached using strong thread to the acromion process allowed measurement of the amount of protraction. Protraction data was captured on to a PC and synchronised offline to the recorded ultrasound sequence.In four subjects, good quality images of the median nerve within the upper arm could be obtained. In the majority of subjects, it was difficult to acquire good quality images because of dense tissue overlying the nerve that reduced the image quality. From these images, nerve trunk bowing was measured in the distal upper arm with the shoulder girdle in the neutral and protracted positions. The maximum deviation of the nerve from a straight line across single ultrasound frames was measured offline in both positions and the difference used as a measure of additional bowing. Repeat trials were averaged.Each subject (n = 11) was imaged in the distal forearm and distal upper arm, whilst lying supine with the right upper limb abducted to 90° at the glenohumeral joint. Ninety degree abduction at the glenohumeral joint rather than 90° glenohumeral flexion was used, so that the present data could be related to previous work  which haThe head was supported on a movable plate, with the centre of rotation positioned at the C7 spinous process. In each subject, the neck was passively moved to 35° CNSF with (a) the scapulothoracic joint in neutral (i.e. relaxed lying in supine) and (b) in full protraction. This movement was repeated several times. In four subjects, a potentiometer attached to the plate allowed continuous measurement of the angle of CNSF. Joint angle data was captured on to a PC and synchronised offline to the recorded ultrasound sequence.For each procedure the range of movement was determined from pictures obtained using a digital camera. Changes in joint angle and distance for each movement were determined from skin surface markers and measured using either CorelDraw  or "tpsDig" . Measurements for the individual components of slumped sitting are summarised in figure Strain is defined by the difference in the amount of elongation that occurs at two points along a nerve divided by the distance between these two points. In practice strain was determined by using regression lines fitted to plots of nerve movement against the distance along the arm. Note that the strain estimates represent the additional strain produced by the movement rather than the total nerve strain.Comparisons of nerve movement and strain in scapulothoracic neutral and protraction during CNSF were performed using paired t-tests.Moving the head forward while maintaining the shoulder and trunk position was tested in 8 subjects. This movement produced no detectable median nerve excursion in the forearm, the average trend being a movement of 0.1 mm  occurring in a proximal direction. The repeat measure variability within subjects was very low, with a standard deviation ranging from 0–0.2 mm (mean = 0.1 mm). The mean change in the angle of lower cervical spine flexion and upper cervical spine extension was 23.6°  and 2.9°  respectively.Trunk flexion also produced minimal median nerve excursion with a mean over 8 subjects of 0.1 mm  proximal movement. The mean change in the angle of trunk flexion was 19.7° .In 13 subjects the median nerve moved in a proximal direction during shoulder protraction with more movement at proximal locations , mean in upper arm = 5.9 mm ) figure .Median nerve excursion was measured in 3 subjects with simultaneous measurement of protraction. The results revealed an initial delay of 6.5–33.0 mm  of the total protraction) before significant nerve movement occurred . The nerve straightened during protraction figure .In 11 subjects the median nerve moved in a proximal direction during 35° CNSF when the scapulothoracic joint was in neutral, as reported previously . The movMedian nerve excursion was measured in 4 subjects with simultaneous measurement of CNSF. The results revealed no obvious delay in the onset of nerve movement in protraction compared to scapulothoracic neutral. Despite less movement in protraction, the pattern of nerve movement mimicked that observed in scapulothoracic neutral figure .Nine of 11 subjects reported paraesthesia in the distribution of the median nerve dermatone once the shoulder girdle was sustained in protraction. The onset of symptoms ranged from 1 to 4 minutes. Symptoms disappeared when the shoulder was repositioned in scapulothoracic neutral.Nerves are designed to slide and stretch to accommodate joint movement. Using the method of Dilley et al. ,6, mediaAs the shoulder girdle is protracted there is a delay in nerve movement, which is followed by a steady increase in nerve excursion. During this initial toe region the median nerve appears bowed in the upper arm. The nerve trunk appears to straighten as the range of protraction progresses. It therefore seems that with the upper limb in scapulothoracic neutral and the glenohumeral joint in 90° flexion, the median nerve is unloaded. If this is the case, the strain value of 0.7% will represent the total strain.The results for CNSF provide evidence for a possible restriction within the shoulder region during shoulder protraction. With the shoulder protracted there was a 60% reduction in the transmission of nerve movement through the upper limb. Consistent with a reduction in movement, there was also significantly less strain in the forearm. The possibility that the nerve becomes unloaded when the shoulder is protracted is unlikely since it had been found that protraction itself causes some median nerve stretch. In addition, there was no obvious delay in nerve movement in response to CNSF  might reduce the space between the clavicle and the first rib, restricting nerve sliding.The components of slumped sitting  are associated with poor posture ,9-11, anProblems are more likely to result from local effects of shoulder protraction on the chords of the brachial plexus. The present study shows that protraction restricts nerve sliding through the shoulder region. Most subjects also experienced paraesthesia when maintaining shoulder protraction plus elbow extension and shoulder abduction. Therefore, sustained shoulder protraction may place the median nerve at enhanced risk of injury and possibly cause a vascular compromise. This may in turn explain for the trend that a high number of NSAP patients have poor shoulder posture. (e.g. ).The direct effects of slumped sitting on median nerve strain are not sufficient to alter nerve function. However, shoulder protraction does appear to restrict nerve sliding, and prolonged protraction leads to pareasthesias.None declared.AJ and RL participated in the study design, data collection, analysis and manuscript preparation. AD participated in the study design, analysis and manuscript preparation. BL participated in the study design and manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Median nerve sliding in the forearm during protraction. Ultrasound sequence of median nerve sliding in the distal forearm during protraction. The subject was imaged with the limb in 90° flexion and 20° abduction at the glenohumeral joint and elbow neutral. The median nerve can be seen to slide in a proximal direction as the shoulder is protracted. In a repeat of the sequence the nerve movement is tracked using cross-correlation analysis (yellow plus sign). The total nerve movement was 4.70 mm.Click here for file


Only a small percentage of patients with Alzheimer's disease benefit from current drug therapy and for only a relatively short time. This is not surprising as the goal of these drugs is to enhance existing cerebral function in Alzheimer patients and not to block the progression of cognitive decline. In contrast, immunotherapy is directed at clearing the neurotoxic amyloid beta peptide from the brain that directly or indirectly leads to cognitive decline in patients with Alzheimer's disease. The single trial of active immunization with the amyloid beta peptide provided suggestive evidence of a reduction in cerebral amyloid plaques and of stabilization in cognitive function of half the patients who developed good antibody responses to the amyloid beta peptide. However, 6% of actively immunized Alzheimer patients developed sterile meningoencephalitis that forced the cessation of the clinical trial. Passive immunotherapy in animal models of Alzheimer's disease has provided similar benefits comparable to those seen with active immunotherapy and has the potential of being effective in the half of Alzheimer's disease patients who do not make a significant anti-amyloid beta peptide antibody response and without inducing T-cell-mediated encephalitis. Published studies of 5 patients with sporadic Alzheimer disease treated with intravenous immunoglobulin containing anti-amyloid beta peptide antibodies showed that amyloid beta peptide was mobilized from the brain and cognitive decline was interrupted. Further studies of passive immunotherapy are urgently required to confirm these observations. The headline "Minimal benefit is seen in drugs for Alzheimer's disease" summarized an article in the New York Times concerning drug therapy of Alzheimer's disease . The ineIn contrast to today's drug therapy for Alzheimer's disease, immunotherapy is directed at the neurotoxic amyloid beta peptide that directly or indirectly leads to cognitive decline in patients with Alzheimer's disease. Most investigators believe that the accumulation of amyloid beta peptide in the brain of elderly adults is not only a hallmark of Alzheimer's disease but is the primary cause of cognitive decline . There iInduction or infusion of anti-amyloid beta peptide antibodies prevents the accumulation of cerebral amyloid beta peptide from the brain of mouse models of familial Alzheimer's disease and may work in humans with sporadic Alzheimer's disease ,7. Even th century in China to protect from virulent smallpox infection [Immunity was first recognized by the fact that people who recovered from an infection were often protected from re-infection. Thus, Thucydides wrote of the Athens epidemic of plaque that occurred in 430 BC "the same man was never attacked twice – at least fatally" . Twelve nfection  and brounfection . Despiteth century the link between the fine complexion  of milkmaids and their exposure to cowpox (vaccinia) was recognized [The next advance in immunotherapy followed the realization that exposure to a related, less virulent, infectious organism such as cowpox, protected individuals from the virulent pathogen, smallpox. In England during the second half of the 18cognized . But it cognized . In the cognized . Most vath century, von Behring and Kitasato discovered that immunity to diphtheria and tetanus was conveyed by serum antibodies against the exotoxins of these bacteria [th century. The first successful application of immunotherapy to humans occurred in 1891 when a child suffering from diphtheria was cured by an infusion of horse serum containing anti-diphtheria antibodies. The horse serum containing anti-toxin antibodies could be lifesaving although many patients, receiving large doses of xenogeneic proteins, developed serum sickness resulting from the patients' immune response to the foreign serum. During the first part of the 20th century passive immunotherapy of other infectious diseases including pneumococal pneumonia entered clinical practice but was replaced when antibiotics became available during the second third of the 20th century.At the end of the 19Today, passive immunotherapy is attracting renewed interest as a new means to treat neurodegenerative, infectious, autoimmune, and neoplastic diseases ,20,21. PImmunotherapy of Alzheimer's disease followed the development of a mouse model of familial Alzheimer's disease. In 1995, an APP-transgenic mouse expressing a mutant, human APP gene isolated from a Swedish family with inherited Alzheimer's disease was developed . These mThese observations offered great promise for the treatment of patients with familial Alzheimer's disease and, perhaps, elderly patients with sporadic Alzheimer's disease. A clinical trial of active immunization of elderly patients with sporadic Alzheimer's disease was organized and initiated in 2001. However, there were reasons to question whether the benefits seen in a middle-aged mice model of familial Alzheimer's disease could be directly extrapolated to elderly patients with sporadic Alzheimer's disease.First of all, aging is associated with a decreasing immune response . Thus, aThe clinical trial of active immunization of patients with Alzheimer's disease with amyloid beta peptide, started in 2001, was stopped a year later after 4 patients in the actively immunized group developed sterile meningoencephalitis . ApproxiNo comprehensive report of the clinical study of active immunization has yet been published but oral presentations and published results from a subset of actively immunized Alzheimer's disease patients have provided some information . It appeIn summary, active immunization with amyloid beta peptide in elderly patients with Alzheimer's disease appears to be less effective and more toxic than in the middle-aged APP-transgenic mouse model of familial Alzheimer's disease. These differences may reflect the greater age of the patients with sporadic Alzheimer's disease and the decreased antibody responses to vaccines and the paradoxical increase in autoimmune responses in the elderly..Administration of anti-amyloid beta peptide antibodies would bypass immune senescence and would not be expected to lead to T cell-mediated encephalitis. Furthermore, anti-amyloid beta peptide antibodies not only dissolved aggregates of amyloid beta peptide in vitro but also inhibited aggregated amyloid beta peptide-mediated cytotoxicity in vitro . In vivoThe potential benefit of anti-amyloid beta peptide antibodies in humans with sporadic Alzheimer's disease was also inferred from studies of cognitive function in a subset of actively immunized patients with Alzheimer's disease . In thesPassive immunotherapy of Alzheimer's disease would require repeated administration of anti-amyloid beta peptide antibodies. For this reason, human anti-amyloid beta peptide antibodies should be used to prevent an immune response to the currently available murine monoclonal immunoglobulins. Several methods are known to obtain human anti-amyloid beta peptide antibodies: (i) purification of specific antibodies from human IVIg; (ii) humanization of murine anti-amyloid beta peptide antibodies by replacing framework portions of the murine anti-amyloid beta peptide antibodies with human framework sequences using recombinant DNA technology ; (iii) gHuman IVIg, purified from human plasma, was initially developed as replacement therapy for immunodeficient patients but IVIg has also been shown to be effective therapy in patients with a variety of autoimmune diseases . RecentlThere is no evidence that anti-amyloid beta peptide antibodies induce sterile encephalitis, observed following passive immunization of several strains of APP-transgenic mice or in the small number of elderly patients with Alzheimer's disease. However, it may be premature to conclude that passive immunotherapy with anti-amyloid beta peptide antibodies does not induce cerebral pathology. It should be remembered that most of the APP-transgenic mice strains tested do not develop amyloid vascular deposits (congophilic angiopathy) that occurs in elderly patients with Alzheimer's disease. It was reported that infusion of murine anti-amyloid beta peptide monoclonal antibodies specific for the N-terminal epitope of the amyloid beta peptide into a strain of APP-transgenic mice, which develop congophilic angiopathy, cerebral hemorrhage was observed . This unPre-clinical data and inferences drawn from immunotherapy in patients with sporadic Alzheimer's disease suggest that passive immunotherapy with anti-amyloid beta peptide antibodies is preferable to active immunotherapy for the treatment of elderly patients with sporadic Alzheimer's disease. However, which anti-amyloid beta peptide antibodies would have greatest therapeutic efficacy and least risk of untoward effects for patients with Alzheimer's disease remains to be determined. However, there is general agreement that anti-amyloid beta peptide antibodies to be administered repeatedly to patients should not stimulate an antibody response to the infused immunoglobulin. There is evidence that humans not only can make immune response to therapeutic antibodies but that such immune responses compromise the action of the therapeutic antibodies . To dateIf the therapeutic benefit of polyclonal human anti-amyloid beta peptide antibodies is confirmed, it remains to be determined which antibody or antibodies are responsible for the therapeutic effect. Whether a single monoclonal antibody will be effective in humans as it has been in APP-transgenic mice is not certain. In some infectious diseases, a single monoclonal antibody has not been less protective than a mixture of several monoclonal antibodies . Thus, wMonoclonal anti-amyloid beta peptide antibodies are known to differ in their fine specificity: isotype, affinity, as well as epitope, aggregate, and Fc specificity . WhetherMonoclonal anti-amyloid beta peptide have different specificities. There are three major epitopes on the amyloid beta peptide: (i) antibodies to the N-terminal epitope (amino acids 1–6) of the amyloid peptide bind to aggregated amyloid beta peptide in vitro as well as cerebral and vascular deposits in vivo and APP (ii) antibodies specific for the central region (amino acids 15–25) of the amyloid peptide bind to APP but not to aggregated amyloid beta peptide in vitro, amyloid plaques or vascular amyloid deposits (iii) antibodies specific for the C-terminal region have been less well studied but reported to lack a therapeutic effect in APP-transgenic mice. This may be the reason why the N-terminal-specific anti-amyloid beta peptide antibodies but not central region-specific antibodies cause cerebral hemorrhage presumably from vessels with amyloid beta deposits .Anti-amyloid beta peptide antibodies that differ in epitope and Fc specificities, dissolve cerebral amyloid plaques and block cognitive decline in APP-transgenic mice . Anti-amSurprisingly, treatment of APP-transgenic mice with anti-amyloid beta peptide antibodies specific for the central region of the amyloid peptide that do not stain cerebral amyloid plaques ex vivo and were not detectable within the brain also cleared cerebral amyloid peptide and plaques . A novelBoth N-terminal-specific and central region-specific anti-amyloid beta peptide antibodies but not C-terminal-specific anti-amyloid beta peptide antibodies also bind to APP the ubiquitous transmembrane cellular protein. C-terminal-specific anti-amyloid beta peptide antibodies can distinguish between amyloid beta 1–40 and 1–42 peptide. As amyloid beta 1–42 peptide is more neurotoxic peptide and forms the nidus of cerebral amyloid plaques antibodies to this amyloid beta peptide might be the most effective antibody for passive immunotherapy as they target the most pathogenic form of the amyloid beta peptide without binding to APP or the less pathogenic amyloid beta 1–40 peptide. However, the C-terminal specific anti-amyloid beta peptide antibodies have been reported not to clear cerebral amyloid beta peptide . RecentlFinally, there is considerable interest in the greater neurotoxicity of soluble amyloid beta peptide oligomers than either the amyloid beta monomers or fibrils . If thisPassive immunotherapy of sporadic Alzheimer's disease offers the potential of reversing the pathologic accumulation of cerebral amyloid beta peptide. To date the only preparation of human anti-amyloid beta peptide antibodies that have been reported to reverse cognitive defects in patients with sporadic Alzheimer's disease are polyclonal anti-amyloid beta peptide antibodies contained in human IVIg. Selection of human anti-amyloid beta peptide antibodies for clinical trial can be tested for therapeutic effect in vivo by their treatment of immunodeficient APP-transgenic mice.The authors declare that they have no competing interests.