It has some disadvantages, such as adhesion to wound surfaces and pain from the irritation caused by this dressing. The Telfa AMD®, a non-adherent wound dressing which consists of absorbent cotton fibers impregnated with polyhexamethylene biguanide enclosed in a sleeve of thermoplastic polymers, is a new option for donor-site wound care which causes less adherence to the wound. The purpose of this study was to compare clinical efficacy of these two dressings for the management of donor-site wounds. Thirty-two patients who received split-thickness skin grafts by donor site harvesting from the thigh were enrolled in this study and randomized into two groups receiving either the Bactigras® or the Telfa AMD® wound treatment. Re-epithelialization, pain, infection and cost-effectiveness analyses were compared between both groups. The results showed that there was no significant difference in age, area of donor sites or length of hospital stays between the groups (p > 0.05). However, the day of re-epithelialization (≥90%) was significantly shorter in patients treated with the Telfa AMD® compared to the Bactigras® group . The average pain score was also significantly lower in the Telfa AMD® group . There was no difference in the cost of treatment between the groups . This study indicated that the Telfa AMD® was an effective dressing for the treatment of donor-site wounds.The Bactigras The success of the procedure depends on the complete integration of the graft with the recipient bed and on the re-epithelialization of the skin graft donor site –3. TreatIn general, the methods of treating donor wounds are categorized as open, semi-open, and closed . The opeStaphylococcus aureus (MRSA). Traditionally, mesh paraffin gauze with chlorhexidine is normally used for the treatment of donor-site wounds. Chlorhexidine can bind to bacterial cell walls at low concentrations, causing an alteration of the bacterial cell osmotic equilibrium and leakage. One of the disadvantages of this traditional gauze includes adherence to wounds, which can cause trauma to epithelial cells when removed. The Telfa AMD®, a non-adherent wound dressing, consists of a thin layer of absorbent cotton fibers impregnated with polyhexamethylene biguanide (PHMB), enclosed in a sleeve of poly , a thermoplastic polymer, that is perforated in a regular pattern and sealed along two edges [Chlorhexidine is an antibacterial agent which is effective against a wide range of Gram-positive and Gram-negative bacteria, including Methicillin-resistant wo edges ,13. Polywo edges ,15. It hwo edges . PolyhexThe purpose of this study was to compare the clinical efficacy of the PHMB-containing wound dressing in the thermoplastic polymer with the paraffin tulle dressing coated with chlorhexidine, the standard treatment for skin graft donor sites in the management of donor-site wounds.2.® dressing and the Telfa AMD®. The Bactigras® dressing is commonly used as a standard protocol of donor site wound dressing at our institute, which is similar to many burn centers using paraffin tulles coated with chlorhexidine [® is commercially available as a non-adherence dressing with antimicrobial agents, which may have advantages in terms of patient comfort.The alternative dressing materials used for split-thickness skin graft donor sites present differences in healing times, infection and patient comfort. Since all of the dressings possess some unique properties, no ideal dressing is available on the market . The chahexidine . The Tel® was lower than that of the Bactigras® dressing, and after moisturizing the Telfa AMD® was easily removed without damaging the newly formed epithelium. However, the Bactigras® dressing had a greater level of adhesion to the wound surfaces, and there was a risk of damaging the delicate epithelium. ®  polymers. Since the Telfa AMD® has lower adhesion properties, it not only prevents trauma to the new and delicate epithelium during dressing removal, but it also provides a good moist environment, which is preferred for epithelial cell proliferation and migration [Our results indicated that overall wound healing, as measured by the percentage of epithelialized dermis, was faster with the Telfa AMDigration . This coigration ,19–21.® dressing has a greater absorptive effect, which resulted in a greater amount of adhesion. During dressing removal or patient movement, it is possible to damage the delicate epithelial cells which can slow wound re-epithelialization as well as increase patient discomfort. Therefore, the physical difference between these two dressings may be the reasons for our results. Moreover, a case of Pseudomonas aeruginosa infection, a bacterium which is of especial concern in patients with burns, was found in a patient treated with the Bactigras® dressing, which indicated that PHMB might be more beneficial in infection control than chlorhexidine. However, no mortality or any side effects from either of the dressings occurred in this study. With respect to bacterial growth, one patient in the Bactigras® group was found to have a local infection on the tenth day with P. aeruginosa (102), and no local infection was observed in the Telfa AMD® group. However, the microbial numbers were below the critical values. After receiving a standard systemic antibiotic, no isolated bacteria were found on the next day.On the other hand, the Bactigras® results in statistically less pain at the donor site from the first day of treatment, and the pain score was significantly reduced after three days. Even though the pain score has completely subjective characteristics, it is a reflection of how comfortable patients feel during treatment and it has been widely used in similar studies.Pain is the main cause of patient discomfort which challenges burn treatment protocols. Our results indicated that the Telfa AMD® was slightly higher than the Bactigras® dressing, which may have been due to the cost of the dressing itself.The results from the comparative cost-effective analysis showed that the cost difference between both dressings was insignificant. The total cost of patients treated with the Telfa AMD3.This was a prospective, randomized control study comprising 32 patients treated at the Siriraj Burn Unit, Thailand during December 2008–February 2010. It was designed to be open-labeled and observer blinded. The study was approved by Institutional Review Board Committee of the hospital, and written informed consent was obtained from each patient who enrolled in the study.® paraffin tulle dressing coated with chlorhexidine , and 16 donor sites were treated with the Telfa AMD® cotton fiber impregnated with PHMB . The demographic were collected from each subject in both group including age, gender, area of donor site (cm2), operative time and length of hospital stay.Twenty-two of the patients were men and ten were women, aged 16–78 years old. All monitored patients had similar burns with regard to the burn area and the depth of donor sites. Patients who need skin graft operation were randomized by computer and placed into two groups: 16 donor sites were treated with the BactigrasThe donor site was at the proximal thigh area. The patients were excluded if they were allergic to paraffin, chlorhexidine, poly  or PHMB. They were also excluded if there were lesions on both thighs, if they had psychiatric problems or multiple injuries (more than two systems involved), if they were immunocompromised, such as with renal failure, cirrhosis or malnutrition, and if they were receiving radiation or chemotherapy for malignancy. Patients with diabetes mellitus, systemic lupus erythematosus or other connective tissue diseases, and patients who had donor sites in areas other than the thigh were also excluded. Patients who did not comply with the study protocol, or who had a skin graft which had previously been harvested from the same donor site area, could not be involved in this study either.® Air Dermatome Skin Grafting System . The area of donor site (cm2) was calculated using Image J Java-based image processing program developed by the National Institutes of Heath. Immediately after harvest, the donor site was covered with a saline-soaked gauze for hemostasis until surgery was completed. The Bactigras® or Telfa AMD® dressing were applied to the donor wound covering about 1 cm of intact skin. The dressing was secured by a sterile gauze. The donor site wounds were inspected every day after operation. None of the dressings were changed until the wounds were completely dry and the dressings fell off.All of the skin grafts (0.010 inches thickness) were taken from the thigh using a ZimmerA donor site follow-up chart was used to conduct the clinical follow-up of the healing process. The information gathered in the chart included the percentage of re-epithelialization of each donor site area, the state of healthy skin on the periphery of each donor site and local signs of infection. Moreover, local pain was also followed-up using a visual analogue pain scale from 0 (no pain) to 10  which was recorded at 30 minutes after the open wound, then at days 3, 7, 14 and 21 or when the dressing fell off. Normal saline solution was used to moisturize the dressing prior to removal and it is considered as neutral solution, no interaction has been found between normal saline and all dressing materials. The patients and the observer were blinded to the type of dressing in each donor site.Infection was also evaluated by swab cultures for a microbiological analysis which was performed routinely once a week on Tuesday. A cost-effectiveness analysis was also compared between these treatment groups. The costs of the dressings, supplies and nursing labor were used to calculate the treatment cost.t-test with SPSS version 10.0 . The results were expressed as mean (±SD). A p < 0.05 was considered as statistically significant.Comparative analyses of the patients in both groups were performed using two-tailed unpaired student’s 4.® dressing and the Telfa AMD® are easy to apply in clinical practice. They can both protect wounds against mechanical trauma and provide comfort for the patients. However, the Telfa AMD® provides a shorter re-epithelialization time, prevents infection and generates lower pain level in comparison with the Bactigras® dressing. The treatment cost difference between these two dressings is negligible.Both the Bactigras


Sauropod dinosaurs are a group of herbivorous dinosaurs which exceeded all other terrestrial vertebrates in mean and maximal body size. Sauropod dinosaurs were also the most successful and long-lived herbivorous tetrapod clade, but no abiological factors such as global environmental parameters conducive to their gigantism can be identified. These facts justify major efforts by evolutionary biologists and paleontologists to understand sauropods as living animals and to explain their evolutionary success and uniquely gigantic body size. Contributions to this research program have come from many fields and can be synthesized into a biological evolutionary cascade model of sauropod dinosaur gigantism (sauropod gigantism ECM). This review focuses on the sauropod gigantism ECM, providing an updated version based on the contributions to the PLoS ONE sauropod gigantism collection and on other very recent published evidence. The model consist of five separate evolutionary cascades . Each cascade starts with observed or inferred basal traits that either may be plesiomorphic or derived at the level of Sauropoda. Each trait confers hypothetical selective advantages which permit the evolution of the next trait. Feedback loops in the ECM consist of selective advantages originating from traits higher in the cascades but affecting lower traits. All cascades end in the trait “Very high body mass”. Each cascade is linked to at least one other cascade. Important plesiomorphic traits of sauropod dinosaurs that entered the model were ovipary as well as no mastication of food. Important evolutionary innovations (derived traits) were an avian-style respiratory system and an elevated basal metabolic rate. Comparison with other tetrapod lineages identifies factors limiting body size. Dinosaurs of the clade Sauropoda were the largest terrestrial animals that ever lived Scientists from many fields of biology and other backgrounds, sometimes far removed from traditional paleontology, have become interested in sauropods, recognizing them as models for understanding vertebrate evolution. Research has become increasingly quantitative and model-oriented. Starting with the simple quantification of sauropod body size in comparison with other clades of vertebrates Recently a new evolutionary perspective has been brought to understanding the uniquely gigantic body size of sauropod dinosaurs The ECM was the focus of the second International Workshop on Sauropod Gigantism at the University of Bonn, Germany, in December, 2011. The workshop brought together a broad expertise on the subject, much of which is reflected in the current collection. In addition, research on sauropod dinosaurs and their gigantism continues at an amazing rate of discovery and of new insights, continuously expanding and testing the ECM. Such research includes both conventional paleontological work but also much innovative transdisciplinary work, showcased at the workshop as well as in this collection.The ECM is subdivided into of a series of evolutionary cascades The major purpose of this review paper is to test the sauropod gigantism ECM based on pertinent research published since late 2009 and in the current collection, and to present a refined version of the ECM. The review paper is also intended as an update of the Sander et al. review Many points that were expressed as hypotheses in the 2010 review paper This review paper's final function is to serve as an introduction to the Sauropod Gigantism Collection of PLOS ONE.Evolutionary cascades are hypotheses of sequentiality and cause and effect. An evolutionary cascade consists of a sequence of biological traits in which one trait is hypothesized to have been the prerequiste for the evolution of the next one, driven by selection. As stated by Westneat The concept of evolutionary cascade is related to that of evolutionary constraint Several cascades and their interplay have affected the evolutionary history of a clade. These cascades and their interplay may be described and visualized in an evolutionary cascade model such as the one for sauropod gigantism. An evolutionary cascade model is a tool that reveals the complex interplay of evolutionary constraints and historical contingencies that have allowed a lifestyle or trait to evolve. An ECM is thus a framework that explains the success and peculiarities of an animal lineage, independent of whether it is fossil or living. The nature of evolutionary cascade models, like that of all models, is heuristic, bringing interactions and constraints in an evolving lineage into sharper focus. In addition to traits and selection pressures acting on them, evolutionary cascade models can include feedback loops, making such links self-amplifying . Note thTesting an evolutionary cascade model consists of testing its components, i.e., observed and inferred traits, evolutionary causations , and feedback loops. Inferred traits can be falsified by research specifically directed at this trait or by published evidence . The samwww.paleodb.org). There are no specific trends regarding where this new material comes from, but South America probably is the leader in diversity increase, the majority of new taxa pertaining to titanosaurs. Disparity does not seem to have increased markedly through these discoveries. Here I do not offer a comprehensive review but highlight only a few important finds, particularly those extending geographic and temporal ranges.New sauropod taxa continue to be found or recognized through taxonomic work at a fast rate, underscoring the importance of sauropods in terrestrial ecosystems of the Jurassic and Cretaceous. While Mannion et al. Tapuiasaurus macedoi from the Early Creatceous (Aptian) of Brazil Atacamatitan chilensis from the Late Cretaceous of the Atacama Desert, Chile, is the first named sauropod from the western side of the Andes Angolatitan adamastor is the first sauropod from Angola and one of the few known from the Late Cretaceous of Africa Angolatitan may have been a relic form Katedocus siberiElatitan lilloiNarambuenatitan palomoiSuuwassea emilieae is a valid taxon because is not a juvenile of another Morrison Formation taxon Already diverse sauropod faunas have become even more diverse, with a new diplodocine from the Late Jurassic Morrison Formation of northern Wyoming described as Eoraptor from the Carnian (Late Triassic) Ischigualasto Formation of Argentina This small biped turns out to be one of the most basal sauropodomorph dinosaurs instead, consistent with the sistergroup relationship of theropods and sauropodomorphs Alamosaurus from New Mexico Argentinosaurus, Futalongkosaurus, and Puertasaurus. These new finds Turiasaurus , Paralitan , and Sauroposeidon , in addition to the giant Argentinian taxa mentioned above  but only become the dominant terrestrial herbivores in the Middle Jurassic after the extinction of non-sauropod sauropodomorphs All sauropod dinosaurs went extinct at the end of the Cretaceous. An analysis of Late Cretaceous sauropod diversity in southwestern Europe indicates no decline towards the K/Pg boundary Seemingly, this hypothesis about dinosaur extinction The emerging picture of sauropod diversity and biogeography also continues to solidify with a number of recent studies directed at refining our view of the patterns. The following section, on ecosystems, will explore some of the causations of these patterns. The diversity of dinosaurs, including sauropods, is commonly expressed by the total number of genera, with a 2010 census noting 175 sauropod genera, 325 theropod genera, and 223 ornithischian genera Tapuisaurus serves as a reminder of the nature of the sauropod fossil record in that the major patterns of diversification are well understood but that the specifics of time and place are just now emerging. In the broader analysis of dinosaur diversity through time, a new study Progress has been made in reconstructing sauropod diversity through time The limitations of extrapolating from present patterns to the Mesozoic may be shown by an analysis of latitudinal distribution of diversity in dinosaur faunas Improvements in our understanding of ecosystems inhabited by sauropod dinosaurs have come from two different sources: the direct evidence provided by paleontology , geology, and geochemistry, and the comparison with modern, mammal-dominated ecosystems. Whereas the former is based on generalizing from case studies, i.e., specific sauropod-bearing rock formations, the latter takes the opposite approach, using general relationships in ecosystems that are consistent with the fossil and rock record.Arguably the most important source of information about sauropod dinosaurs and their environment has been the Upper Jurassic Morrison Formation of the western United States These observations partially support (migration) and partially contradict (aridity) the assumptions made by the most refined effort to quantitatively describe a sauropod ecosystem Recent studies using the general ecological approach would suggest that limitations in food availability would have affected sauropod populations less than mammalian megaherbivore populations because of the much lower minimum population densities of the former From all of this work, it is becoming increasingly clear that the key to understanding dinosaur ecosystems is the great size disparity between neonates and adults, epitomized by sauropods . Only when researchers fully embrace this difference between dinosaurs and mammals in their analyses, will a profound understanding of dinosaurian ecosystems emerge.As originally proposed The new evidence bearing on the sauropod gigantism ECM is organized topically within the individual cascade, going up each of the cascades from the basal trait to the final one, very high body mass , 6, 7, 8One of the basic assumptions of the ECM was that the evolution of sauropod gigantism is primarily under intrinsic control, meaning that it was driven by biological factors Among the several drivers of evolutionary body size increase in dinosaurs Tyrannosaurus indeed were actively hunting their prey or if they only were scavengers. Models of carrion encounter vs. prey encounter support active hunting because large theropods would have been the last ones to have found any carrion which would have been consumed by smaller theropods and juveniles first Tyrannosaurus in the Late Cretaceous Hell Creek Formation of Montana (USA) suggests that at least adult tyrannosaurs may also have subsisted on carrion Tyrannosaurus, the sum of the evidence argues for large theropods generally having been active predators.Predation pressure by large theropods on sauropods also hinges on the question if such giants as Predation pressure on herbivorous dinosaurs, i.e., ornithischians and sauropods, thus probably explains the strong left skew seen in body size histograms of these dinosaur groups Traits of sauropod reproductive biology, i.e., the lack of parental care and the large number of small offspring, also must have resulted in increased predation pressure which in turn would have led to strong selection for larger body size. In particular, because unlike in modern meagherbivores no trophic energy was lost due to parental care Trait. Very high body massThis trait will be discussed first because all cascades culminate in it. The discussion of this trait only covers the most recent developments and literature because an in-depth review is found in Sander et al. Finds of exceptionally large sauropod individuals continue to be made (see above), making the trait “Very high body mass” immediately obvious. Compilations from the literature also drive home the point GiraffatitanGiraffatitanMuch of the work underlying the ECM requires accurate estimates of body masses of sauropods at the level of the individual. Classically, two approaches have been taken for estimating body mass in extinct tetrapods: mass estimates based on body volume estimates and mass estimates based on scaling of long bone dimensions in extant tetrapods. The most general dataset compiled so far offers a universal scaling relationship of long bone circumference and body mass in tetrapods A novel approach to “weighing” sauropods is using soil mechanics to estimate the mass of a trackmaker from the substrate deformation it caused Trait (observed and inferred). Many small offspringSauropod dinosaurs, like all extinct and living dinosaurs and all archosaurs, reproduced via ovipary, presumably being constrained to this mode of reproduction by their calcified eggshells The small size of the offspring relative to the adult led to the hypothesis Particularly, the laying of several clutches and the size difference between hatchling and adult make any form of parental care unlikely. Lack of parental care is also suggested by the burial of the egg clutches by scratch-digging of the female sauropod The possible exception to the lack of parental care may be the unburied eggs from the Argentinian locality of Auca Mahuevo Selective advantage. High rate of reproductionBased on data for extant birds and mammals, an early, seminal study Janis & Carrano Trait (inferred). Fast population recoveryJanis & Carrano Selective advantage. Reduced extinction riskIn the context of sauropod gigantism, a low reproductive output has been shown to increase the risk of extinction In sauropods, the selective advantage of a reduced extinction risk may also have resulted directly from the trait “Many small offspring”. The great size difference between hatchling and fully grown sauropods as a consequence of ovipary probably meant extensive ontogenetic niche shifting, with different life stages being adapted to different environmental conditions Trait. No masticationGiraffatitanIt is generally accepted that sauropod dinosaurs did not chew their food Trait. No gastric millIn the absence of a chewing dentition, sauropod dinosaurs classically were believed to have processed their plant fodder in a gastric mill similar to granivorous birds Selective advantage. No time needed for processingThe selective advantage of not reducing fodder particle size is that no time is needed to do so. Time needed for chewing scales positively with body mass in extant mammals While similar data about scaling of duration of gastric mill use s are lacking for birds, we cannot be sure that particle size reduction in a gastric mill limits body size. However, all birds and non-avian dinosaurs that have a gastric mill are small  or medium-sized  Trait (inferred). Fast food intakeDiplodocus has recently been shown to have the second-highest tooth replacement rate known among archosaurs DiplodocusCamarasaurusNigersaurus but it was not known whether this was representative for sauropods in general because of the extremely modified dentition of this taxon Food intake rate can only be observed in extant animals, but a high food intake rate has been inferred for sauropod dinosaurs for two reasons Selective advantage. More energy from the environmentProvided that plant resources are not limited in the environment, an animal with a greater capacity for food intake rate will be able to take up more energy from the environment that an animal with a lower capacity Trait (inferred). Energetic advantageFour evolutionary cascades end in this trait, indicating that at least four traits contributed to the energetic advantage permitting sauropod gigantism, but the trait per se has not received further comparative study in extant or extinct animals since the sauropod gigantism ECM was formulated.Feedback loop. Large gut capacityIn the original version of the ECM, a feedback loop leads from the trait “Very high body mass” to the trait “No mastication” Nonetheless, isometric scaling of gut capacity would have generated the feedback loop “Large gut capacity” because of the negative allometry of BMR, but the feedback loop is probably weaker than originally envisaged. With an isometric increase in gut volume, larger animals can digest more food at the same time and thus subsist on lower-quality forage. Sauropods would have needed excessively large guts to compensate for the lack of particle reduction. In fact, the sauropod body cavity appears to have provided sufficient space for such large guts Trait. No masticationThe observed trait of no mastication has been discussed above. In addition to the selective advantage of “No time needed for food processing”, this trait provides a crucial selective advantage associated with the sauropod neck Selective advantage. No positive head allometryProsaurolophusBecause of the scaling effects surrounding mastication, extant masticators show positive head allometry Trait. Small headSauropod dinosaurs had the relatively smallest heads in length and mass of any non-avian dinosaur Selective advantage. Low momentsThe obvious selective advantages of a small head are the low moments of force that it bestows on the neck Trait. Long neckThe defining feature of sauropod dinosaurs, their uniquely long neck, received a thorough review by Taylor & Wedel . This reConsidering the importance of the neck, this collection contains no fewer than four contributions on the subject Although neck length would have been constrained by mechanical factors Selective advantage. Energy-efficient feedingThe central hypothesis of the ECM possibly is that the long neck of sauropods facilitated highly energy-efficient feeding, both by giving access to tall vegetation and by extending the reach of the head without moving the heavy body. While it is clear that a longer neck confers advantages to an animal of any size A premise of the hypothesis of energy-efficient feeding is that the main function of the long neck indeed was feeding and not some other function in physiology, reproduction or behavior. In particular, the hypothesis that sauropod neck elongation was a result of runaway sexual selection Euhelopus and GiraffatitanBrachiosaurus bearing a nine-meter neck compared to a minimally-necked one MamenchisaurusSeveral kinds of new model calculations, on the other hand, do support the hypothesized selective advantage . Model cWhile both an erect and a horizontal neck convey major energetic advantages, the crucial question of neck flexibility is still surrounded by controversy Mamenchisaurus neck and optimization of intervertebral articular surface pressure indicate a horizontal posture and partitioning of flexibility along the vertebral column, with a relatively stiff middle neck region Virtual articulation of neck vertebrae and simplified models suggests that sauropod necks were held largely horizontally and may not have been flexible enough to cover a volume but only a surface in situ should be studied to address the issue of joint cartilage thickness. In addition, a new study on ostrich neck flexibility A full understanding of sauropod neck posture and flexibility is hampered by the need to reconstruct the thickness of the cartilage covering the intervertebral joints and the zygapophyses Feedback loop. Reduced vulnerabilityLong necks, particularly when their flexibility was limited by cervical ribs Trait . Posterior shift of neck musclesPlateosaurus have greatly elongated cervical ribs, extending backwards from the vertebra over two intervertebral joints. Such posteriorly elongated cervical ribs are present in most sauropods, reaching lengths of up to 340 cm The importance of the long neck for sauropod gigantism is emphasized by a new trait (inferred), the posterior shift of neck muscles, also observed in extant birds m. longus colli group based on the homology with birds m. scaleni group based on the homology with crocodiles MamenchisaurusThese muscles either belong to the Selective Advantage. Lightens the neckAmong several beneficial effects of having long ossified cervical ribs Trait (inferred). Avian-style lungIn recent years, an avian-style respiratory system  has become the consensus inference in the respiratory biology of saurischian dinosaurs, including sauropodomorphs The notion Selective advantage. Lightens the neckAmong the four major selective advantages of an ARS for sauropods, the least obvious but possibly the most important is the effect of the ARS on neck mass. While a light-weight neck would be advantageous at any size, the long, predominantly horizontal neck of large sauropods could only evolve because of PSP, a corollary of an ARS. This statement presumanly applies to long-necked extant birds, long-necked non-avian theropods, and long-necked pterosaurs as well, although this has not been explored in the literature before. The crucial aspect is the development of diverticula of the respiratory tract that invade the medullary region of individual vertebrae. In non-pneumatized bones, this region is filled with bone marrow, but in pneumatized bones it is filled with air.per se, only in the replacement of bone marrow by air. A pneumatized vertebra thus is lighter than a non-pneumatized one, despite both having the same amount of bone tissue. Statements found even in the most recent literature that “cervical airsacs and extensive cervical diverticula … would also have served to lighten long necks” Pneumatization does not result in a decrease in the mass of the bone tissue In non-avian theropod dinosaurs, the hypothesis that PSP evolved to lighten the skeleton was tested recently Selective advantage. No dead space problemThe respiratory dead space problem is familiar to human divers and refers to the interdependency of lung volume and tracheal length. If tracheal length is artificially increased , tracheal volume may reach a limit where it takes up such a large part of the tidal volume that an insufficient volume of fresh air reaches the lung. The dead space problem affects long-necked animals as well. To avoid the dead space problem, a long neck appears only possible if the non-tracheal ventilated parts of the respiratory system  have a volume that is an order of magnitude larger than that of the trachea. Since the amniote trachea is at least as long as the neck and requires a certain minimum diameter, the long necks of sauropods meant that the non-tracheal ventilated parts of their respiratory system must have been very voluminous Selective advantage. Continuous oxygen uptakeAn unquestionable selective advantage of an ARS is continuous oxygen uptake, as in birds but unlike in mammals, in which oxygen is only extracted during the inhalation part of the breathing cycle. Since the discovery of unidirectional airflow in crocodiles Trait (inferred). High BMRThe inferred trait of a high basal metabolic rate (BMR) in sauropods has found additional support by studies published since 2009, but some evidence to the contrary has also emerged.versus their presence in non-avian dinosaurs indicates different thermophysiologies in the two groups. Improved understanding of the primary bone formation in extant tetrapods led to a refined view of the evidence for high growth rates of sauropod dinosaurs provided by bone histology Comprehensive sampling of ungulate long bone histology, both in terms of taxonomic diversity and of habitat and climate zone A high BMR requires integumentary insulation structures , at least in small animals. A well preserved small theropod fossil from the Jurassic of Germany Finally, while research on stable isotopes has long contributed to the endothermy/ectothermy debate, the limitation of this approach remains its proxy nature Body temperatures can also be calculated from maximum growth rates While there is thus strong evidence that sauropod dinosaurs had a BMR at least in the lower range of large mammals but possibly higher, a new study on growth rates Feedback loop. Low mass-specific metabolic rateThe negative allometry of BMR with body mass  and BMR in vertebrates was first explored by Case There are several points in the approach of Clarke In conclusion, the trait “High growth rate” in the evolutionary cascade has not been falsified because all studies agree that sauropod MGR experienced a manifold evolutionary increase compared to their closest living and non-dinosaurian extinct relatives.Selective advantage. High likelihood to survive to adult stageLufengosaurusIndependent of the necessity of a high BMR to achieve fast growth, the selective advantage of a high growth rate appears clear. Especially in animals that, like no other amniote, had an extreme size difference between embryo and adult Trait. Upright stanceAll large extant terrestrial tetrapods with a high BMR have an upright stance Selective advantage. Energy-efficient locomotionEnergy-efficient locomotion as a selective advantage resulting from the upright stance was discussed above, but the question could be asked whether sauropods were more efficient locomotors than extant graviportal mammals and other graviportal dinosaurs. Locomotion in sauropods can be understood from two independent and complementary lines of evidence: their skeletons and their rich track record. More efficient locomotion has not figured in previous hypotheses about sauropod gigantism, but further considerations are in order. Specifically, can we formulate hypotheses that posit that any aspect of the locomotory apparatus and locomotion facilitated the unique gigantism of sauropod dinosaurs? In particular, are there any scaling factors in locomotion that would favor larger body size over smaller body size? Negative allometry of the cost of transportation might be one such factor but it could not be detected in the study by Preuschoft et al. Future research concerning the hypothesis of “energy-efficient locomotion” could be based on quantitative biomechanical models, but it will require an improved understanding of sauropod gaits. These have not been reliably reconstructed, neither from models nor from theoretical considerations The remarkable amount of evidence that has accumulated over the last few years, and that is the focus of this collection, considerably refines the evolutionary cascade model of sauropod gigantism proposed by Sander et al. in 2010 Compared to the 2010 ECM, the cascade “Reproduction” has been refined by splitting the basal trait “Many small offspring” into three different traits and by adding a subcascade that takes into account the ecological effects of the body size difference between hatchlings and adults . The casThe original cascade “Feeding, Head, Neck” has also been split into two cascades, “Feeding”  and “HeaThe cascade “Head and neck”  probablyThe cascade “High BMR” was amended by adding “Upright stance” as an observed trait and “Efficient locomotion” as the selective advantage . Much neThe ECM for sauropod gigantism is of heuristic value for explaining the unique body size of sauropod dinosaurs and the limits to body size in terrestrial amniotes in general. However, the ECM currently does not provide information about the relative contribution of the component cascades and their basal traits to gigantism see also and if aThis discussion of the limits to body size is restricted to terrestrial amniotes here because so many parameters are different in the marine realm  that meaningful comparisons are not obvious. Terrestrial amniotes show the following maximum body size distribution: the largest non-avian reptiles (three clades) and birds are smaller than the largest mammals; these are smaller than the largest theropod and ornithischian dinosaurs, which in turn are smaller than the largest sauropod dinosaurs. Except for non-avian reptiles, the largest  species in these clades are herbivores and are an order of magnitude larger than the largest carnivorous members of their respective clades. In addition, studies of how body size is distributed across the size range of the clade A number of factors can be identified limiting body size based on recent research and the ECM , but a fMastication-induced positive head allometry, as predicted by scaling principles, is documented for ornithischian dinosaurs by a recent study of ontogenetic changes in the skull of a hadrosaur species The question of why no multi-tonne ground birds evolved in the early Tertiary after the demise of the non-avian dinosaurs remains prominent As shown by this example of birds and extant non-avian reptiles, but also by the many other taxa and traits in Plateosaurus, as evidenced by greatly elongated cervical ribs and their histology ProtorosaurusBeyond the notion that some in the ECM are plesiomorphic and some are derived, the question can now be addressed of when the basal traits of each cascade arose in the phylogeny and how this conincides with body size increase. As noted earlier, it will be difficult to bring these two datasets into perfect congruency because of the difficultiy of plotting the largest sauropodomorph remains from any time bin onto the phylogeny. While the traits “Ovipary”, No gastric mill”, and “No mastication” are plesiomorphic for amniotes , the aviOptimizing traits from the ECM onto a phylogeny that includes all the terminal taxa which exhibit the trait will be a fruitful avenue to explore. The ultimate test of the importance of the presumed factors in the evolution of amniote body size would be to test their contribution to body size across amniotes, using phylogenetic comparative methods.This review of the biology of the sauropod dinosaurs and the evolution of their gigantism, condensed into the sauropod gigantism ECM, serves to compile and synthesize the rapidly expanding literature on the subject, including this collection in PLoS ONE. It also serves as an update to an earlier review The rich new evidence accumulated in these last four years was then used to test the ECM by asking how this evidence impacted the component cascades and the entire ECM. Most of the inferred traits, selective advantages, and feedback loops in the ECM found support, sometimes strongly so, while in a few others (e.g. “High growth rate”) support weakened or relationships had to be rejected . The ECM was also refined by splitting up traits and adding new ones. The general conclusion of Sander & Clauss


Haemophilus influenzae to transform competent cells of a laboratory strain. To identify which of the ∼40,000 polymorphic differences had recombined into the genomes of four transformed clones, their genomes and their donor and recipient parents were deep sequenced to high coverage. Each clone was found to contain ∼1000 donor polymorphisms in 3–6 contiguous runs (8.1±4.5 kb in length) that collectively comprised ∼1–3% of each transformed chromosome. Seven donor-specific insertions and deletions were also acquired as parts of larger donor segments, but the presence of other structural variation flanking 12 of 32 recombination breakpoints suggested that these often disrupt the progress of recombination events. This is the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, connecting experimental studies of transformation with the high levels of natural genetic variation found in isolates of the same species.Many bacteria are able to efficiently bind and take up double-stranded DNA fragments, and the resulting natural transformation shapes bacterial genomes, transmits antibiotic resistance, and allows escape from immune surveillance. The genomes of many competent pathogens show evidence of extensive historical recombination between lineages, but the actual recombination events have not been well characterized. We used DNA from a clinical isolate of  Haemophilus influenzae with genomic DNA from a divergent clinical isolate and used deep sequencing to identify the recombination events in four transformed chromosomes. The results show that transformation of single competent cells is more extensive than expected, and suggests that transformation can be used as a tool to map traits that vary between clinical isolates.The ability of bacteria to acquire genetic information from their relatives—called natural competence—poses a major health risk, since recombination between pathogenic bacterial lineages can help bacteria develop resistance to antibiotics and adapt to host defenses. In this study we transformed competent cells of the human pathogen  For many bacteria, natural transformation is the dominant mode of genetic transfer between close relatives. These naturally competent bacterial species can actively take up DNA fragments from their surroundings and incorporate it into their chromosomes by homologous recombination Naturally competent bacterial cells bind double-stranded DNA fragments at the cell surface but transport only single strands into the cytoplasm  [1]–[2].During natural transformation, the extent to which incoming donor DNAs replace segments of recipient chromosomes is limited by the extent and type of sequence differences between the two, as is the case with other pathways that depend on homologous recombination The genomes of independent isolates of many bacterial species differ in two ways Haemophilus influenzae, combining inexpensive sequencing technology with the availability of complete genome sequences of divergent strains. Here we report a high coverage sequence analysis of four H. influenzae genomes derived by natural transformation of a recipient strain with donor DNA of another strain differing at ∼40,000 genetic markers .Natural genetic variation between bacterial strains has previously been used to characterize transformation at specific selected loci H. influenzae are reported to contain many non-competent cells R) did not vary when any of 4 distant markers is used for selection of transformed clones.Competent cultures of H. influenzae isolates R (novobiocin resistance) and NalR alleles from MAP7 by transformation with short PCR fragments.Accurate mapping of recombination events depends on the density and distribution of sequence differences between the donor and recipient genomes. We chose the clinical isolate 86-028NP as the source of donor DNA because, like Rd, it has been completely sequenced and annotated, and because its genome differs from Rd at about 2.4% of their alignable bases, typical for a pair of R and NalR alleles reduced transformation efficiency into recipient chromosomes by ∼3-fold, compared to Rd-derived MAP7 donor DNA.We used this doubly marked strain (NP-NN) to investigate how strongly the sequence divergence between NP-NN and Rd-RR limits transformation. R (transformants Nov1 and Nov2) or a NalR  phenotype. Acquisition and processing of sequence data for these clones is described in detail in the To identify recombination events in clones transformed with chromosomal DNA from a divergent strain, we selected four Rd-RR clones that had been transformed with NP-NN chromosomal DNA to either a NovFor each set of reads, the base corresponding to each position in each of the two reference genomes was classified as: (a) the same as the reference, (b) different from the reference, (c) ambiguous, or (d) unmapped  identify differences between the published reference genome sequences and the genomes of the donor and recipient strains we used; (2) confirm the reliability of single-nucleotide variants (SNVs) for distinguishing between donor and recipient sequences; and (3) identify positions that were systematically error-prone, ambiguous, or unmapped in the alignment of reads to references. The three steps  of these control analyses are illustrated in Aligning the sequence reads from the recipient and donor strains to their corresponding reference genomes provided two controls. First, it showed that our sequencing approach was comprehensive and accurate: at >98% of the positions the aligned reads agreed with the reference with <1% error  and S5. We next evaluated the alignment of donor and recipient reads to the alternative reference genomes , suggesting that these positions suffer from systematic and persistent sequencing or mapping artifacts , and transformant Nal1 had a donor segment spanning the NalR allele at gyrA (Segment L). Nov1 contained five additional donor segments, two separated by only 4.5 kb and the other three adjacent to the selected Segment F. Nal1 contained three other widely spaced segments in addition to the selected Segment L; one of these overlapped one of those in the Nov1 transformant by 10.9 kb . The reads from this pool were aligned to the donor and recipient references, and the allele frequencies at each position for each reference were calculated  and 3. IR and NalR alleles, with the NovR allele in the Nov1 and Nov2 transformants and the NalR allele in the Nal1 and Nal2 transformants . A more detailed analysis of sequence divergence in these regions is shown in The adjacent locations of many donor segments  likely rRecombination not only brought thousands of donor-specific SNVs into the transformant genomes but introduced several donor-specific insertions and deletions  resultinOn the other hand, indels and other structural variants between the donor and recipient chromosomes appear to have blocked progression of strand exchange in several instances . Of the The plummeting cost of deep sequencing allowed us to characterize the genome-wide consequences of natural transformation, but the ability of this analysis to account for artefacts depended on our high-coverage control sequencing of the donor and recipient genomes. Aligning these control reads to the two reference genome sequences revealed many positions prone to ambiguity or false-positive SNV calls. In the absence of these controls, such artifacts would have mistakenly been interpreted as recombination-induced mutations, since mapping reads to divergent references generated these erroneous variants, while mapping reads to highly similar references did not. The frequency of these artifacts depends not only on nucleotide divergence, but also on the spectrum of structural variation and the complexity of the genome. Analysis of such high-coverage control datasets will be essential for reference-guided assembly approaches that use data with lower coverage, such as that obtained using inexpensive multiplexing methods.H. influenzae's normal environment is the mucosal layer of the human respiratory tract, which contains abundant DNA, much of it high molecular weight like that we used H. influenzae strains and present in the human host H. influenzae cells take up more fragments when fragments are short (40 fragments of 120 bp) H. influenzae's preference for DNA containing uptake sequences (see below) will also affect both the sources and sizes of the fragments cells take up.H. influenzae are similar to those reported from analysis of naturally occurring recombination tracts observed in Neisseria meningitidis at specific lociHelicobacter pylori (∼0.5–3.5 kb) in experiments using DNAs of similar divergence to ours Analysis of recombination tracts showed that the four transformants had replaced ∼1–3% of their genomes with 3–6 segments of donor DNA ranging in length from 1.2 to 16.6 kb. The number of donor segments per transformant agrees well with the ∼3.3 fragments found to be taken up per cell in laboratory experiments using long 14.4 kb fragments Helicobacter pyloriH. influenzae donor segments is unlikely to be due to chance, because of the small number of donor segments in each transformant. More probable explanations are that (1) cytosolic or translocation endonucleases degrade incoming DNAs prior to strand exchange, or (2) sequence heterology blocks progression of strand exchange, with the heterologous sequences trimmed away by nucleases.The lengths of donor segment found in recombinant chromosomes may underestimate the original lengths of DNA fragments participating in uptake and recombination, because clustering of donor segments suggests that longer incoming DNA fragments are often disrupted before transformation is complete. Similar clustering of donor segments was seen when recombination at a single locus was examined in Helicobacter pyloriH. pylori and H. influenzae, only single DNA strands are thought to enter the cytoplasm E. coli transduction experiments H. pylori may instead be due to the accumulation of extracellular restriction enzymes during the long transformation protocol Intracellular cleavage of incoming DNA by restriction enzymes has been proposed for competent We found no evidence that recombination preferentially occurred in regions of lower nucleotide divergence than the genome-wide average. Instead, sequence divergence varied on a scale much shorter than the donor segments, with most segments spanning local regions of both high and low divergence . Althouge.g. between the clustered segments C and D, D and E, and K and L  biases transformation to USS-containing fragments Other factors could have influenced the transformation events we observed: (1) R locus. The selection for NovR and NalR also showed that unselected events are common, as 58% of donor alleles were found in segments distant from the selected loci. On the other hand, the 11 kb overlap between the unselected donor segments A and G was unexpected given the transformation frequencies of single markers . Each recombinant clone also acquired donor-specific versions of 20–50 shared genes, and these may have altered phenotype both directly and because of new interactions with recipient alleles at unrecombined loci. Recombination breakpoints that were not at structural variation usually fell within genes  was obtained from H. O. Smith in 1988, and is separated by ∼10 passages (∼500 generations) from the KW20 Rd strain sequenced in 1995 Taxonomy ID: 71421). The donor strain NP-NN ) was derived from the clinical isolate 86-028NP Taxonomy ID: 281310); it is separated from the sequenced 86-028NP strain by ∼5 passages (∼250 generations). NP-NN was constructed by PCR-mediated transformation of 86-028NP with NovR and NalR amplicons of gyrB and gyrA, respectively, (both caused by point mutations). For the NovR allele of gyrB, a 2.6 kb fragment  was amplified from MAP7 (RR666) chromosomal DNA. For the NalR allele of gyrA, a 2.8 kb fragment  was amplified.The 9 cells) for a final DNA concentration of ∼1 genome equivalent per cell. Cells were incubated with DNA at 37°C for 20 min, diluted 1∶5 into sBHI, and incubated at 37°C for 80 min to allow expression of donor resistance alleles before dilution and plating to sBHI agar ± antibiotics −9 resistant colonies/CFU). Cells from defrosted aliquots were pelleted and resuspended in fresh MIV before transformation. Two NovR and two NalR transformant colonies . The raw sequence reads for each DNA sample  and 86-028NP complete genome sequences (NCBI genome accessions NC_000907 and NC_007416) were each used as references for read alignment, with the BWA algorithm (version 0.5.5) A combination of two criteria was used to identify differences between sequence reads and their references and to flag positions with ambiguous base identity. The first method used the SamTools (version 1.12a)Control self-alignments. Sequence differences between the Rd-RR and NP-NN strains and their respective reference sequences were identified using the following two criteria: (1) a difference was found by the SamTools consensus caller, and (2) the frequency of the same non-reference variant at that position was greater than 95%. Positions prone to sequencing and read mapping artifacts were flagged as ambiguous when the SamTools tagged a base as ambiguous, the variant frequency was between 5% and 95%, or both.Control reciprocal alignments. Differences between our donor and recipient strains were identified from the reciprocal alignments of Rd-RR reads to the 86-028NP reference genome and of NP-NN reads to the Rd reference genome. SNV positions were considered cross-validated, if both reciprocal alignments and whole-genome alignment identified the same SNV. Ambiguous positions prone to read mapping artifacts in reciprocal-alignments were also flagged using the same criteria as above.Transformant sequence reads were analyzed as above. Recombination events were identified in the transformed clones by classifying the positions of cross-validated SNVs as donor, recipient, or ambiguous. Donor segments were defined as contiguous runs of donor-specific SNVs, uninterrupted by recipient-specific SNVs . Individual donor segments breakpoints were defined by the positions of their outermost donor-specific alleles. Donor segments were then manually inspected using the Integrated Genomics Viewer For the pooled sample of four transformed clones (RR3135-RR3138), donor-specific allele frequencies were determined at each cross-validated SNV position. Non-overlapping donor segments were unambiguously identified as contiguous runs of SNV positions with ∼25% donor-specific alleles. Overlapping donor segments  were disambiguated by comparison with the segments identified in RR3135 and RR3137. Segments unique to either RR3137 or RR3138 were disambiguated using allele-specific PCR; two primer pairs were designed that contained several SNVs that distinguished Rd and NP alleles .Positions that were unmapped by reads in the reciprocal alignments  were used as markers of indel differences and other structural variation between donor and recipient, and the donor segment intervals were examined for read coverage at positions unmapped by either reciprocal alignment. Indel differences flanking the observed donor segments were also tabulated. Manual inspection of read alignments to both references used the Integrative Genome Viewer, and the “.rdiff” and “.qdiff” output from the dnadiff utility of Mummer Figure S1Read depth varies consistently across the genome. (A) shows a histogram of read depth per mapped Rd genome position. Red indicates Rd-RR, and blue indicates NP-NN. (B) shows a sliding window analysis (1 kb and 100 bp steps) of mean read depth of Rd-RR reads mapped to Rd (red) and %GC (grey) along an interval of the Rd genome. The genome-wide adjusted R2 of log(read depth) and %GC on these windows was 0.26 (using the lm function in R). (C) plots mean read depths on the same sliding windows as in B, but showing Rd-RR read depths on the x-axis and NP-NN read depths on the y-axis. The red line shows y = x. Unmapped positions were included as read depth  =  0, and mean read depths were adjusted by adding a single pseudo-count, so that read depths of 0 were plotted at 1 on a log-scale. (D) shows variation in read depth for Rd-RR reads mapped to the Rd reference genome along a representative interval. (E) shows the ratio of Nov1 to Rd-RR read depth along the same interval as (D). Read depths were first normalized to the median read depth to account for differences in sequence yields. The genome-wide correlation between read depths for these two samples was 0.98.(TIF)Click here for additional data file.Figure S2Ambiguous positions. Plot of the non-reference variant frequency at positions classified as ambiguous for the indicated set of sequence reads aligned to the two references: (A) Rd, and (B) 86-028NP. Data are tabulated in (TIF)Click here for additional data file.Figure S3Examples of two kinds of artifacts. The 250 bps shown are indicated by the arrow in A) shows a systematic sequencing error-prone site, when either NP-NN or Rd-RR reads are mapped to the 86-028NP reference. (B) shows an additional problematic site, prone to systematic misalignment when Rd-RR reads are mapped to the 86-028NP genome, but not when NP-NN reads are mapped. The red curve shows the limit of detection (1/read depth). Grey bars show positions with no detected variants (i.e. variant frequency < limit of detection). Blue lollipops show the non-reference variant frequency at positions classified as matching the reference; when the lollipop falls on the limit-of-detection line, a single non-reference variant was observed. Turquoise bars show positions classified as ambiguous (variant frequency ranged from 12% to 65%). The salmon bars for Rd-RR reads indicate positions classified as SNVs. All were in the cross-validated set of SNVs, and the donor-specific allele frequency at each exceeded 98%.(TIF)Click here for additional data file.Figure S4Density histograms of read depth per position (A and B) and non-reference variant frequency per position (C and D) when mapping control sequence reads to the 86-028NP reference.A and C show the result using NP-NN reads, while B and D show reciprocal alignment of Rd-RR reads. Blue shows positions classified as a standard ACGT base by the SamTools consensus caller, while pink shows the histogram for positions classified as non-ACGT . The percentages associated with each curve indicate the fraction of total positions in that group of positions (either ACGT or non-ACGT). Also shown in B and D is the percent of mapped positions where no non-reference variants were detected and the percent of unmapped positions.(TIF)Click here for additional data file.Figure S5Unmapped positions in reciprocal alignments mark structural variation. In (A), pink hatches mark positions along the Rd reference that were unmapped by NP-NN donor reads (but mapped by Rd-RR). In (B), light blue hatches mark positions along the 86-028NP reference that were unmapped by Rd-RR recipient reads (but mapped by NP-NN). Note the scale compresses the individual positions horizontally, so exaggerates the total fraction of unmapped positions (ositions .(TIF)Click here for additional data file.Figure S6False-positive positions. Plots of non-reference variant frequency for each individually sequenced DNA sample at positions that were classified as “false positives”, SNVs found by alignment of donor reads to the Rd reference, but not identified as SNVs by whole-genome alignment Click here for additional data file.Figure S7Zooms of the five intervals (I to V) containing donor-specific alleles in the transformants, as in , plotted against the Rd reference genome. The lower schematic shows each interval as in (TIF)Click here for additional data file.Figure S8Transformation of and near structural variation. Shows Interval II for reads from the Nov1 clone mapped to the Rd reference (top panel) and also to the 86-028NP reference (bottom panel). In each plot, the top two rows show donor- and recipient-specific SNVs in blue and red, respectively. Light blue and pink bars that span the plot show donor- and recipient-specific structural variation, respectively. The purple diamond show the position of the NovR allele. The green line plots the log2 of Nov1 read depth normalized to either Rd-RR or NP-NN read depths . Positions where the green line touches the x-axis were unmapped by Nov1 reads.(TIF)Click here for additional data file.Table S1Summary of sequencing results.(DOC)Click here for additional data file.Table S2Read depth in pileups on Rd (KW20).(DOC)Click here for additional data file.Table S3Read depth in pileups on 86-028NP.(DOC)Click here for additional data file.Table S4Non-reference variants in reads mapped to Rd (KW20).(DOC)Click here for additional data file.Table S5Non-reference variants in reads mapped to 86-028NP.(DOC)Click here for additional data file.Table S6Summary of whole-genome alignment of Rd and 86-028NP reference sequences.(DOC)Click here for additional data file.Table S7Singe-nucleotide variants between Rd and 86-028NP reference sequences.(DOC)Click here for additional data file.Table S8Structural variation between Rd and 86-028NP reference sequences.(DOC)Click here for additional data file.Table S9Donor segments in four transformants using Rd (KW20) reference coordinates.(DOC)Click here for additional data file.Table S10Donor segments in four transformants using 86-028NP reference coordinates.(DOC)Click here for additional data file.Table S11Transformation of insertions and deletions within donor segments.(DOC)Click here for additional data file.Table S12Indel and other rearrangements flanking donor segments.(DOC)Click here for additional data file.Table S13Allele-specific primers used to assign Segments M, N, and O to either clone Nov2 or Nal2.(DOC)Click here for additional data file.


Pseudoviruses were created by exchanging each variable and constant domain of JR-CSF gp120 with that of JR-FL or with mutations in putative N-glycosylation sites. The sera contained different neutralizing activities dependent on C3 and V5, C3 and V4, or V4 regions located on the glycan-rich outer domain of gp120. All sera showed enhanced neutralizing activity toward an Env variant that lacked a glycosylation site in V4. The JR-CSF gp120 epitopes recognized by the sera are generally distinct from those of several well characterized mAbs (targeting conserved sites on Env) or other type-specific responses . The activity of one serum requires specific glycans that are also important for 2G12 neutralization and this serum blocked the binding of 2G12 to gp120. Our findings show that different fine specificities can achieve potent neutralization of HIV-1, yet this strong activity does not result in improved breadth.Development of a vaccine for HIV-1 requires a detailed understanding of the neutralizing antibody responses that can be experimentally elicited to difficult-to-neutralize primary isolates. Rabbits were immunized with the gp120 subunit of HIV-1 JR-CSF envelope (Env) using a DNA-prime protein-boost regimen. We analyzed five sera that showed potent autologous neutralizing activity (IC50s at ∼10 A major challenge in developing a protective vaccine for HIV-1 is the identification of an immunogen that can elicit potent and broad-spectrum neutralizing antibodies to primary isolates The envelope glycoprotein (Env) of HIV-1 forms functional spikes that mediate virus entry into host cells. Env engages the cellular receptor, CD4, which enhances the ability of Env to bind to the coreceptor, CCR5 or CXCR4 Eliciting neutralizing antibody to conserved epitopes on the gp120 subunit of native Env is extremely challenging. Thus, the CD4-binding site (CD4BS) is a target of broadly neutralizing mAbs such as b12 Many forms of Env have been evaluated as candidate vaccines to HIV-1. Monomeric gp120 typically elicits antibodies that neutralize only sensitive or lab-adapted strains with V3 sequences similar to that of the immunogen env genes from JR-CSF (AY669726) and JR-FL (AY669728) were cloned into the pMAmp expression vector described previously Full-length Rabbit sera were obtained from immunizations carried out at Aldevron LLC  under animal use protocols 2-04-007-09-2005, 1-06-001-08-2007, and 1-07-005-10-2008 approved by the local Animal Care and Use Committee. Female New Zealand white rabbits were injected intramuscularly using the Inovio Twin Injector electroporation device with a total of 400 µg of a plasmid DNA construct encoding a codon-optimized JR-CSF gp120 DNA in the pMAmp vector. Animals were boosted with JR-CSF gp120 protein purified from CHO cells The monoclonal antibodies used in this work were obtained as follows: VRC01 env genes were cloned into the pCXAS-PXMX expression plasmid, and pseudoviruses were prepared as previously described env, were co-transfected into 293T cell cultures. Virus-containing supernatants were harvested approximately 48 h after transfection. To determine infectivity and coreceptor tropisms of variant PSVs, CD4+/U87 cells expressing either CXCR4 or CCR5 were inoculated with PSV in a 96-well format. Successful infection leads to the production of luciferase in target cells. Coreceptor tropism was assessed by evaluating luciferase activity in lysed cells 72 h post-inoculation.JR-CSF and mutant PhenoSense™ neutralization assays TZM-bl cells 2SO4 (2N) and the optical density at 450 nm was read on a microplate reader (Molecular Devices). Data were normalized to the maximal ELISA binding signal of the biotinylated mAb in the absence of competitor. Results were plotted as relative binding affinity using a four-parameter sigmoidal dose-response equation (variable slope) with GraphPad Prism Software 5.0. Each competitor was tested in duplicate.Microtiter plate wells  were coated overnight at 4°C with 30 ng JR-CSF gp120 in PBS. Biotinylated b12, 2G12 and VRC01 were produced using EZ-Link-sulfo-NHS-biotin reagent (Pierce) according to the manufacturer's instructions. The wells were washed four times with PBS containing 0.05% Tween 20 (PBST) and blocked for 1 h with 4% nonfat dry milk in PBST at 37°C. After four washes with PBST, 25 µl of sera  was added to each well and incubated for 30 min at 37°C, followed by an equal volume of biotinylated mAb at a constant concentration. The final concentrations of biotinylated Abs were previously determined to generate non-saturating binding signals. The plate was then incubated for 1.5 h at 37°C. Unbound antibody was removed by washing six times with PBST. Bound bio-mAb was detected using streptavidin-horseradish peroxidase conjugate (Jackson ImmunoResearch) diluted 1∶1000. Following incubation at room temperature for 1 h, the wells were washed six times, and developed using 50 µl tetramethylbenzidine (TMB) solution (Pierce), according to the manufacturer's instructions. After 10 min, wells containing TMB were quenched by adding 50 µl HWe found that immunization of rabbits using a DNA vaccine encoding JR-CSF gp120 followed by a boost of sequence-matched gp120 protein can elicit neutralizing antisera against pseudoviruses carrying the JR-CSF Env  [52]. WeThese five sera showed little or no neutralization of a PSV containing the JR-FL Env , Table 1The serum antibodies that neutralize JR-CSF but not JR-FL presumably target regions in Env that differ between the two viruses. We therefore made a series of PSVs with chimeric Env proteins in which each domain of the JR-CSF gp120 sequence  was replaced with the corresponding domain of JR-FL . These cThe neutralizing activities of sera 3096 and 3844 against JR-CSF substantially decreased after substitution of C3 or V5 with the corresponding JR-FL sequences . In contIncorporation of JR-FL sequences into the JR-CSF PSV could modify the conformation of the Env trimer and accessibility of neutralizing epitopes compared to wild-type JR-CSF. We therefore tested the neutralization sensitivity of the domain-swap PSVs to mAbs targeting the CD4BS , the CD4i epitope (X5), the V3 loop (F425), the silent-face glycan cluster on gp120 (2G12), and the MPER region of gp41 (2F5). JR-FL was more sensitive to VRC01, b12, and b6 , which hIndividual glycans can obscure neutralizing epitopes on Env while their removal (by mutation of the PNGS) can increase sensitivity of HIV-1 to neutralization We next examined the neutralizing activity of the rabbit sera on the JR-CSF PSVs carrying PNGS mutations . This pawww.hiv.lanl.gov; accessed on June 15, 2011.). Loss of glycosylation at these positions has previously been shown to alter PSV sensitivity to neutralizing antibodies of different specificities The PNGS at positions 156 and 197 are conserved in 94% and 98%, respectively, of Env sequences in the Los Alamos National Laboratory database . If the neutralizing antibody specificities in the undiluted rabbit serum were present at between 10–1,000 µg/ml Although JR-CSF is a primary isolate Overall, the neutralizing specificities of the rabbit sera are distinct from those of well-studied mAbs, CD4-IgG2, or a broadly neutralizing human plasma (Z23). However, use of the variant PSVs shows that some sera target similar regions of Env as mAbs or CD4-IgG2. For example, PNGS mutations at N156, N197, N301, and N332 increased sensitivity to sera 3099, as well as to b6, CD4-IgG2, F425, and X5. One possibility is that serum 3099 contains antibodies to the CD4BS with specificities similar to those of b6 or F425, as these antibodies can only neutralize when glycans are removed that would otherwise protect the CD4BS and V3, respectively. In support of this explanation is the observation that JR-FL N156Q (but not wild-type JR-FL) is hypersensitive to the rabbit sera as well as to b6 and F425. In contrast, sera 1252, 3835, and 3844 are highly active against wild-type JR-CSF but are not more potent against JR-CSF N156Q, suggesting that neutralizing antibodies directed to V3/CD4BS/CD4i are absent in these sera and/or that the C3/V4-, C3/V5- and V4-dependent antibodies dominate or mask the neutralizing activity of antibodies like F425, b6 or X5. Mutation of two glycans that comprise the 2G12 epitope (N386 and N392) also diminished the neutralizing activity of rabbit serum 3835, suggesting an overlap between the neutralizing epitopes.We mapped the Env domains and PNGS that altered the activity of the rabbit sera or select mAbs onto the gp120 core structure  includinThe rabbit sera interfere with binding of 2G12, VRC01, b12 and CD4-IgG2 to gp120 in competition ELISA experiments. In particular, 2G12 is in a unique antibody competition class and no other immunized sera, and even very few HIV-1 patient sera, have been shown to block 2G12 binding to monomeric gp120 It is important to understand how sera from experimental immunizations of animals or humans can neutralize primary HIV-1 isolates in order to develop improved vaccines. We have obtained some insight on the regions of gp120 that are targets of a neutralizing response to the primary isolate JR-CSF. Whether such autologous neutralizing specificities can be matured by specific immunization strategies into more cross-reactive antibodies remains an important and open question.Figure S1Prime-boost immunization using JR-CSF gp120 elicits a range of JR-CSF neutralizing activities. IC50 values (1/dilution) of 24 sera from rabbits immunized with a JR-CSF gp120 DNA vaccine followed by JR-CSF gp120 protein are shown. The sera are from three immunization studies . The detailed immunization regime for each study can be found in the (TIF)Click here for additional data file.Figure S2Regions of gp120 targeted by rabbit sera are distinct but show some overlap with mAbs. The results of this study are represented visually using a model of the crystal structure of JR-FL gp120 core (TIF)Click here for additional data file.


In order to characterize the cellular composition of cerebrospinal fluid (CSF) in a healthy state and in the setting of chronic pleocytosis associated with HIV-1 (HIV) infection, multi-parameter flow cytometry was used to identify and quantitate cellular phenotypes in CSF derived from HIV-uninfected healthy controls and HIV-infected subjects across a spectrum of disease and treatment. CD4+ T cells were the most frequent CSF population and the CD4:CD8 ratio was significantly increased in the CSF compared to blood (p = 0.0232), suggesting preferential trafficking of CD4+ over CD8+ T cells to this compartment. In contrast, in HIV-infection, CD8+ T cells were the major cellular component of the CSF and were markedly increased compared to HIV-uninfected subjects (p<0.001). As with peripheral blood, the CSF CD4:CD8 ratio was reversed in HIV-infected subjects compared to HIV-uninfected subjects. Monocytes, B cells and NK cells were rare in the CSF in both groups, although absolute counts of CSF NK cells and B cells were significantly increased in HIV-infected subjects (p<0.05). Our studies show that T cells are the major cellular component of the CSF in HIV-infected and uninfected subjects. The CSF pleocytosis characteristic of HIV infection involves all lymphocyte subsets we measured, except for CD4+ T cells, but is comprised primarily of CD8+ T cells. The reduced proportion of CD4+ T cells in the CSF may reflect both HIV-related peripheral loss and changes in trafficking patterns in response to HIV infection in the central nervous system. HIV-1 (HIV) infection is frequently accompanied by cerebrospinal fluid (CSF) pleocytosis that occurs early in infection and largely resolves with antiretroviral therapy (ART) We used an eight-color flow cytometry panel combined with TruCount™ beads (for cell enumeration) to create “Flow Count,” a single platform assay for the determination of both the proportion and the absolute count of the major lymphocyte populations  and that of monocytes and granulocytes. Flow Count was validated on whole blood samples of forty HIV-infected and ten HIV-uninfected subjects by comparison to standard clinical laboratory methods  at the University of California San Francisco (UCSF) CSF was obtained by lumbar puncture (LP) and blood by phlebotomy for study purposes as previously described g for 10 minutes to concentrate the cells. The supernatant was aspirated to 100 µL, and cells were re-suspended in this residual volume before transfer to a TruCOUNT™ tube for staining. CSF cells were stained in the dark for 20 minutes then re-suspended in 350 µL of 0.5% formaldehyde.Paired whole blood and CSF samples from each subject were stained in BD TruCOUNT™ tubes  for cell quantification using a “no wash” procedure to preserve cell numbers. For whole blood, antibodies were combined with 50 µL of whole blood and incubated for 20 minutes at room temperature in the dark. Erythrocytes were then lysed with 450 µL of 1X FACS Lysing Solution (BD Biosciences) and incubated for 10 minutes at room temperature. Control samples (described below) were processed in parallel without TruCOUNT™ beads. Nine milliliters of CSF was collected in a 15 mL tube and processed at 4°C. The CSF was gently vortexed to liberate cells attached to the polypropylene walls of the tube, then centrifuged at 250 Antibodies. CSF and whole blood samples were stained for Flow Count, with the following antibodies: anti-CD45 conjugated with FITC; anti-CD16 and anti-CD56, both conjugated with PE; anti-CD14 conjugated with APC; anti-CD8 conjugated with APC-Cy7; anti-CD4 conjugated with PE-Cy7; anti-CD3 conjugated with Pacific Blue  and anti-CD19 conjugated with PE-Texas Red .Controls. Unstained blood, as well as unstained and single stained BD™ CompBeads (BD Biosciences) controls were prepared for optimizing fluorescence compensation settings for flow cytometric analyses. Fluorescent minus one (FMO) controls were prepared on blood samples by omitting one antibody and used to evaluate fluorescence spill-over and assist with setting gates.– (debris and red blood cells) population. BD™ CompBeads were stained with the individual antibodies used to stain the cells and run on the same settings. Analysis and compensation of data was performed using FlowJo . The gating strategy used to define each population (Stained samples were acquired on a customized LSRII flow cytometer (BD Biosciences) within 2 hours of staining. For blood samples, 100,000 events were acquired; for CSF samples, the entire cell suspension was acquired. Threshold fluorescence was set on CD45-FITC plotted against Side Scatter (SSC) to discriminate the CD45pulation  was deveStatistical analysis was performed using Prism 5 . Non-parametric tests were used to compare group differences, either Kruskal-Wallis test to compare multiple groups with Dunn’s post hoc comparison within the analysis or Mann-Whitney U-tests to compare two groups. Linear regressions were used for graphic display to compare proportions of CD4+ and CD8+ T cells present in the blood and CSF.10, P<0.001) while plasma HIV RNA levels were not significantly different between these 2 groups. The peripheral blood CD4 concentrations were not significantly different among the three HIV-infected groups.Clinical characteristics of the 10 HIV-uninfected and 40 HIV-infected  study participants are described in Measurement of lymphocytes, granulocytes and monocytes in the CSF showed that in both HIV-infected and uninfected groups the predominant population was lymphocytes. Monocytes were a minor population  and granThe proportions and absolute counts of CSF and blood lymphocyte populations are shown in Compared with the HIV-uninfected, HIV-infected subjects had a significantly lower proportion of CSF CD4+ T cells (p<0.001) and a significantly higher proportion of CD8+ T cells (p<0.001) . In addiThe absolute counts of CSF cell populations were compared across HIV-infected subjects grouped according to ART treatment and their response to treatment . There wWithin the three HIV-infected groups, treatment was associated with lower CSF T cell and B cell, but not CSF monocyte or NK counts . In contrast, CSF NK cell, B cell, and monocyte counts did not show significant correlation with the corresponding populations present in the blood (data not shown).Since the pattern of CD4+ and CD8+ T cell changes in CSF of HIV- infected subjects were similar to those observed in the blood, linear regression was used to examine the relationship between blood and CSF cell populations. As shown in To better understand the pattern of CSF CD4+ and CD8+ T cell changes across HIV-infected groups, we examined the CSF and peripheral blood ratios of CD4+ to CD8+ T cell absolute counts (CD4:CD8) . In HIV-Since CSF WBC count has previously been shown to correlate with CSF HIV RNA level Flow cytometry has become widely used for analysis of the cellular content of CSF, and the utility of this technique for samples with low numbers of cells has increased as advances in technology have allowed more parameters per individual cell to be interrogated We found that in healthy controls the cellular composition of the CSF differs from whole blood; as previously reported in healthy controls and subjects with NIND, granulocytes are rare and CD4+T cells are the predominant population in CSF CSF pleocytosis occurs early in HIV infection and in the absence of opportunistic brain infection . Reversal of the CD4:CD8 ratio in the CSF of HIV-infected subjects is consistent with a previous study We found that the ratio of CD4+ to CD8+ T cell absolute counts (CD4:CD8) in HIV-uninfected subjects was higher in the CSF than the blood consistent with previous reports for normal controls and subjects with non-inflammatory neurological disease (NIND) B cells were extremely rare in the CSF, as previously reported in normal and NIND subjects While the absolute number of CSF WBC, lymphocytes, and T cells in our study was very similar to a previous report on the frequency of these cells in the CSF NK cell frequencies in the CSF have not been reported in HIV-infected subjects. Previous studies of normal CSF reported a very low frequency Patients in this study were all neurologically asymptomatic but most had abnormally high levels of CSF CD8+ T cells, even when virologically suppressed, and may be at risk for development of neurological impairment. While ART has drastically reduced the incidence of HIV associated dementia (HAD) In conclusion, our study provides a comprehensive analysis of all the major WBC populations in the CSF of neuroasymptomatic HIV-negative healthy controls and HIV-infected subjects using Flow Count, a polychromatic flow cytometry method. This report provides novel insight into CSF cell populations counts, and into the relationship between CSF and blood cellular composition in different settings of HIV infection and treatment. Our results indicate that even in the setting of ART, an increase in CD8+ T cells, as well as a generalized increase in most other cell types present in normal CSF, characterizes HIV infection. Whether the less frequent NK, B cell and monocyte populations are subject to the same factors that induce transmigration of T cell is not clear, but they may play an important role in the initiation and maintenance of HIV infection of the CSF and brain (monocytes), in control of that infection (NK and B cells), and in the production of potentially neurotoxic inflammatory agents. Given that a significant percentage of HIV-infected patients may have worsened cognition Figure S1Comparison of cell quantification by standard clinical laboratories and flow cytometry-based assay. Correlation of the standard clinical laboratory and the Flow Count method (Left column) and standard difference between paired results of each assay, plotted against the mean of the paired results using a Bland-Altman plot (right column). Mean bias between the 2 assays is shown as a solid line and the 95% limits of agreement is shown as a dotted line.(TIFF)Click here for additional data file.Text S1Validation of Flow Count Assay.(DOCX)Click here for additional data file.


Interleukin (IL)-6 is recognised as an important cytokine involved in inflammatory diseases of the central nervous system (CNS).To perform a large retrospective study designed to test cerebrospinal fluid (CSF) IL-6 levels in the context of neurological diseases, and evaluate its usefulness as a biomarker to help discriminate multiple sclerosis (MS) from other inflammatory neurological diseases (OIND).We analyzed 374 CSF samples for IL-6 using a quantitative enzyme-linked immunosorbent assay. Groups tested were composed of demyelinating diseases of the CNS , including relapsing-remitting MS , primary progressive MS , clinically isolated syndrome , optic neuritis ; idiopathic transverse myelitis ; other inflammatory neurological diseases ; and non-inflammatory neurological diseases . Differences between groups were analysed using Kruskal−Wallis test and Mann−Whitney U-test.CSF IL-6 levels exceeded the positivity cut-off of 10 pg/ml in 18 (51.4%) of the 35 OIND samples, but in only three (3.9%) of the 76 MS samples collected. CSF IL-6 was negative for all NIND samples tested (0/212). IL-6 cut-off of 10 pg/ml offers 96% sensitivity to exclude MS.CSF IL-6 may help to differentiate MS from its major differential diagnosis group, OIND. Multiple sclerosis (MS) is an autoimmune disease of multifactorial origin that affects the central nervous system (CNS). Cerebrospinal fluid (CSF) is a promising body fluid for the search of biomarkers associated with chronic neurological disease. The recently revised 2010 MS criteria have been simplified to allow easier diagnosis of MS without losing sensitivity or specificity. In the present study, we investigated the potential of CSF IL-6 as a biomarker of CNS inflammatory diseases by evaluating its levels in a large number of patients with a variety of CNS disorders, primarily to differentiate MS from other inflammatory conditions that may mimic MS.This study was performed on cerebrospinal fluids (CSF) collected in excess during regular spinal taps that were stored anonymously in a centralized biobank for retrospective research purposes. This research was conducted accordingly to the regulations of the Act on Medical Research Involving Human Subjects of the Geneva University Hospital (HUGO.RE.DG.0010). Neither written nor recorded verbal informed consent was compulsory for this research study involving the retrospective analysis of fluid collection, as the resulting research data set was completely anonymous. The Geneva University Hospital Medical Ethical board approved this study and consent process: Authorization # 07-261R (NAC 07-102R).This study included CSF samples from 374 patients representing 22 definite diagnoses and classified into four major diagnostic groups, which are summarised in We used the revised 2005 McDonald Criteria Owing to the asymmetric data distribution of CSF IL-6 levels, we applied non-parametric tests for statistical analysis. We used the Kruskal−Wallis test for the distribution analysis, and the Mann−Whitney U-test for the head-to-head comparisons. A p-value <0.05 was considered statistically significant. We used receiver operating characteristic (ROC) curve analysis to determine the power (sensitivity and specificity) of CSF IL-6 indices as a discriminatory diagnostic marker for MS. The area under the curve (AUC) was also evaluated. The Division of Clinical Epidemiology at the Geneva University Hospital (DC) performed all statistical analyses using SPSS (v. 18.0) statistical software.The levels of CSF IL-6 in different disease subgroups are shown in Given this cut-off value, CSF was IL-6−positive in 40% of patients with ITM  70) and 51% of patients with OIND . By contrast, CSF was IL-6−positive in only 3.9% of MS patients , and in no patients with other DD, such as ON  and CIS . IL-6−positive CSF was not observed in patients with NIND .Head-to-head comparisons among the four different groups  were observed in 60% of RRMS patients. Therefore, neither radiological nor biological patterns of inflammation observed in RRMS patients correlated with the observation of absent or low-level CSF IL-6.The clinical, biological, and radiological characteristics of DD patients are summarised in It is noteworthy that the levels of IL-6 in the three IL-6−positive MS samples were found to be low  compared with ITM and OIND IL-6−positive samples. Two of the three IL-6−positive RRMS samples were associated with an elevated WBC CSF count above the average for the group (mean: 8 WBC). Two of them were also OB-positive. The brain MRI available for these three IL-6−positive MS patients revealed no T1 gadolinium-positive lesions; this was also the case regarding the single available spinal cord MRI. This finding indicates that the elevated CSF IL-6 levels observed in the three MS patients were not correlated with biological or radiological signs of CNS/CSF inflammation.The proportion of positive CSF OB in RRMS and PPMS patients was 91%, which is compatible with observations of other MS cohorts. We then compared the usefulness of OB versus IL-6 to discriminate MS patients. We observed that the absence of CSF IL-6 (96% sensitivity) was similar to the presence of OB (91% sensitivity) to confirm MS diagnosis. In addition, ROC curve analysis demonstrated that CSF IL-6 levels were more closely linked to MS than WBC counts, when the MS and OND groups were compared (data not shown). Altogether, these results strongly suggest that the measurement of IL-6 CSF levels as a quantitative assay could be useful to support a discriminatory clinical diagnosis of MS. It is however important to underline that OB testing mainly helps distinguishing between MS and healthy subjects or non-inflammatory neurological diseases, whereas IL-6 helps in our view in distinguishing MS and OIND.The use of biomarkers is increasingly proposed as a method to refine the diagnosis and guide the treatment of numerous diseases. To date, no biomarker has had sufficient full-scale testing to qualify as a useful addition to standard diagnostic and prognostic factors, or to guide the prescription of specific treatments for MS. A recent study has shown promising results with the demonstration of the potassium channel KIR4.1 as a target of the autoantibody response in MS. ROC curves, which represent the probability that a test will yield false-positive or false-negative results for all possible cut-off values, were drawn to determine the optimal cut-off value of CSF IL-6 to discriminate MS from OIND, a group of CNS inflammatory diseases considered in the classical differential diagnosis of MS. The results presented in this study indicate that elevated IL-6 levels (>10 pg/ml) are observed almost exclusively in CSF samples from patients with OIND and ITM, which likely reflects the inflammatory characteristics of the diseases represented in these groups. Thus, the measurement of CSF IL-6 with a cut-off of 10 pg/ml yielded a sensitivity of 96% to differentiate patients with MS from those with OIND of the CNS. Notably, these results have direct clinical implications, because ITM and OIND are typical diagnoses that may mimic MS and could therefore be mistaken for MS according to clinical presentation or MRI-restricted assessment. In contrast, CSF samples from patients with ON, CIS, MS, and NIND were negative for IL-6 reactivity, with the exception of three RRMS CSF samples that exhibited low IL-6 levels. Regarding the clinical and radiological characteristics of these patients, no difference with the other samples of the group was found. A satisfactory explanation for this low CSF IL-6 positivity is thus not available. In addition, separated analysis in patients with distinct DD revealed comparable results between the subgroups.No single CSF test has proven fully reliable for distinguishing MS from OIND. Only CSF parameters such as CSF OB and IgG index are currently used to assist with MS diagnosis, although CSF analysis is no longer required for a diagnosis of MS. IL-6 is a pleiotropic cytokine that is present during inflammation. In conclusion, we propose the measurement of CSF IL-6 as a relevant screen to discriminate MS patients from patients with OIND that may classically mimic MS. Although this test lacks specificity for any particular disease, the high IL-6 in the CSF renders the likelihood of MS less probable. In addition, this test could be easily performed on a routine basis in any center that is equipped to test CSF samples. Thus, we believe that this test should be considered as an added value to the CSF OB determination to help confirm or exclude MS.