Although abnormal cerebrospinal fluid (CSF) protein can be used to predict the outcome of tuberculous meningitis (TBM) and diagnose TBM, normal CSF protein remains a concern in patients with TBM. This retrospective study aimed to assess the clinical characteristics associated with normal CSF protein, to resolve the dilemma of CSF protein in the management of childhood TBM.Between January 2006 and December 2019, consecutive child patients  were included for analysis. CSF protein was tested on a chemistry analyzer using the pyrogallol red-molybdate method. Abnormal CSF protein was defined as >450 mg/L. Patient characteristics were collected from the electronic medical records. Then, characteristics associated with normal CSF protein were estimated in the study, using univariate and multivariate logistic regression analysis.2=2.486, df = 8, p = .962) revealed that vomiting  and serum glucose  were associated with the normal CSF protein in childhood TBM.A total of 125 children who met the criteria were enrolled during the study period. Twenty-nine patients had a normal CSF protein and 96 had an abnormal CSF protein. Multivariate analysis (Hosmer–Lemeshow goodness-of-fit test: χIn suspected childhood tuberculous meningitis, patients without vomiting or having low serum glucose are easy to present with normal CSF protein. Hence, when interpreting the level of CSF protein in children with such characteristics, a careful clinical assessment is required.In suspected childhood TBM, patients without vomiting or having low serum glucose are easy to present with normal CSF protein. Hence, when interpreting the level of CSF protein in children with such characteristics, a careful clinical assessment is required. Currently, tuberculosis (TB) remains one of the most important infectious diseases for children. According to a WHO report 2018), it was estimated that 10 million people fell ill with TB and 1.5 million deaths occurred due to TB, children accounted for 11% of all TB cases, and 14% of cases that died from TB were children 8, it was, and surThe diagnosis of TBM remains a difficult, especially in children. This is because that when cerebrospinal fluid is employed, routine microbiological methods such as acid-fast bacilli (AFB) smear, polymerase chain reaction (PCR), and culture usually have a low sensitivity for the diagnosis of TBM . MoreoveAbnormal CSF protein is an important clinical presentation for TBM patients. Moreover, CSF protein can be used to predict the outcome of TBM and served as a tool for the diagnosis of TBM . UnfortuThe study protocol was approved by the Ethics Committee of Shandong Provincial Chest Hospital, Jinan, China (NO. 2020XKYYEC-29) and the retrospective study was conducted in compliance with the Helsinki declaration. Due to its retrospective nature and anonymous data collection, written informed consent was exempted by the Ethics Committee.M.tuberculosis cultured from CSF. 2) conclusive: symptoms and signs of meningitis and abnormal CSF findings , plus at least one of the following i) TB suggested by abnormal radiographic features , ii) positive TB assays  using non-CSF samples.Between January 2006 and December 2019, consecutive child patients aged ≤15 years old, had a diagnosis of TBM, and were tested for CSF protein were included for the analysis. TBM was defined if one of the following criteria  was metCSF protein was tested on ADVIA 2400 chemistry analyzer  using a commercial kit . Abnormal CSF protein was defined as >450 mg/L. Patient data, such as demographic, clinical, laboratory, and radiographic features were collected from the electronic medical records. Subsequently, the characteristics associated with normal CSF protein were then estimated in the study.p value <.05 was considered significant.Statistical analyses were performed using SPSS version 16.0 . Continuous variables were expressed as mean ± standard deviation (SD) and categorical variables as count (percentages). Univariate and multivariate logistic regression analyses adjusted by age and sex were performed to determine characteristics associated with normal CSF protein, and odds ratios (OR) and corresponding 95% confidence interval (CI) were also calculated . AdditioA total of 125 children meeting the criteria was enrolled during the study period. Twenty-nine patients (23.2%) had a normal CSF protein and 96 (76.8%) had an abnormal CSF protein. n = 59, 47.2%), milliary , pleural , and lymph node .The mean age and weight were 7.6 ± 5.2 years and 24 ± 16 Kg, respectively. Of the 125 child patients, 66 patients (52.8%) were male and 102 (81.6%) were from rural areas. Additionally, other sites involved with TB were reported as pulmonary  had inpatient therapy, over half  had outpatient therapy, and a significant proportion  were reported having self-treatment. In addition, a majority  of patients experienced antibiotics therapy and a few  were administrated with anti-TB therapyThe frequencies of hospitalization were 2.2 ± 1.8, and the mean treatment delay was reported at 33 ± 35 d. Among the enrolled patients, 29 patients (23.2%) had a TB contact history and 109 patients (87.2%) were transferred from a teaching hospital. Almost all patients , headache , vomiting , cough , convulsion , coma , drowsiness , and dizziness . CSF analysis is shown in The vital signs were as follows: temperature, 37.3 ± 0.9 °C; heart rate, 100 ± 22 beats/min; respiratory rate, 22.7 ± 3.0 breaths/min; systolic pressure, 107 ± 16 mmHg; diastolic pressure, 69 ± 11 mmHg. The symptoms were as follows: fever  of them had a positive result. Other lab examinations such as blood cell count and flow cytometry, were shown in p = .004), serum albumin , serum glucose , and serum LDH .χ2=2.486, df = 8, p = .962) revealed that vomiting  and serum glucose  were associated with the normal CSF protein in childhood TBM (Further multivariate analysis (Hosmer–Lemeshow goodness-of-fit test: hood TBM .It was well characterized that elevated CSF protein levels were associated with a poor outcome in TBM. For example, raised CSF protein was recently found to be a significant predictor of blindness . TherefoIncreased level of CSF protein is known as one of the main presentations of TBM. CSF protein is composed of albumin, immunoglobulin, and transferrin, along with other enzymes and globulins. In a previous study, the onset of TBM was characterized by a rise in the alpha-1 and gamma globulin fractions and by a decrease in the beta and pre-albumin fractions. In general, the abnormal CSF protein is widely explained by brain damage, which is related to the severity of meningeal inflammation and blood-brain barrier dysfunction ,19. SeriOur data suggested that vomiting was associated with an abnormal CSF protein. Therefore, in suspected children without vomiting, CSF protein may be prone to present at a normal state. Previously, a similar finding has been reported in consistency with our findings. Usually, vomiting as a presenting symptom is associated with cranial nerve involvement . Sharma Our study demonstrated that elevated serum level of glucose is associated with abnormal CSF protein. This means that a low level of glucose occurs more commonly in patients with normal CSF protein. Identifying this characteristic is easy and would help to interpret the normal CSF protein in childhood TBM. The association between serum glucose and CSF protein may be explained by the influence of diabetes on the CSF level of protein. First, diabetes mellitus is taken as a factor of the diagnostic scoring system for TBM diagnosis . Second,Although the study has several interesting findings, some limitations are still to be considered. First, this was a single centre experience, it thus may only reflect a local epidemiological situation. Second, the study had a retrospective nature, sample size and selection bias should be taken into account. Third, the criteria for abnormal CSF protein in TBM varied widely, this may have a significant impact on the result. Fourth, associations between CSF protein and other CSF analyses were not investigated, due to its close correlations with clinical course. Therefore, further studies are needed to determine the reference of CSF protein for the diagnosis of TBM, and a large prospective study is required in the future to validate the above findings.Our study found that, in suspected childhood TBM, patients without vomiting or having low serum glucose are easy to present with a normal CSF protein. A normal CSF protein may lower the suspicion of TBM in children and result in delayed diagnosis and anti-TB treatment. Therefore, when interpreting the level of CSF protein in children with such characteristics, a careful clinical assessment is required.


The activity of donor periodontal membrane is the key factor of autologous tooth healing. The application of digital aided design, 3D printing model and guide plate in autotransplantation of tooth (ATT) is expected to reduce the damage of periodontal membrane and preserve the activity of periodontal membrane, so as to improve the success rate of ATT. This study tried to prove the role of digital technology in improving the success rate of ATT, although there are differences in model accuracy in practice.We included 41 tooth autotransplantation cases which assisted by 3D-printed donor models and surgical guides and divided them into two groups in accordance with whether the donor tooth could be placed successfully after the preparation of alveolar socket guided by the model tooth. Then, we compared and analyzed the preparation time of alveolar socket, extra-alveolar time, and number of positioning trials of the donor tooth between the two groups. We also included a comparison of the in vitro time of the donor tooth with that of 15 min. The incidence of complications was included in the prognostic evaluation.The mean preparation time of the alveolar socket, mean extra-alveolar time of donor tooth, and mean number of positioning trials with donor tooth of 41 cases were 12.73 ± 6.18 min, 5.56 ± 3.11 min, and 2.61 ± 1.00, respectively. The group wherein the donor tooth cannot be placed successfully  spent more preparation time of alveolar socket and extra-alveolar time than the group wherein the donor tooth can be placed successfully . The number of positioning trials with the donor tooth of the group wherein the donor tooth cannot be placed successfully (3.19 ± 0.75) was higher than that of the other group (2.00 ± 0.86). There was no significant difference in survival rates between the two groups.Compared with the traditional tooth autotransplantation, the introduction of computer-aided design combined with 3D printing of the model tooth and surgical guides evidently shortens the preparation time of the alveolar socket and the extra-alveolar time of the donor tooth and reduces the number of positioning trials with the donor tooth regardless of the shape deviation between the model and actual teeth. The aim of the autotransplantation of tooth (ATT) is to replace a lost tooth with a functional tooth within the same patient for the restoration of the masticatory and aesthetic functions of the recipient site. ATT can be used to treat dental defects caused by missing teeth, deep caries, poor endodontic prognosis, and periodontitis . The sucThe traditional ATT uses the donor tooth to prepare alveolar socket directly in the process of operation, which increases the extra-alveolar time of donor tooth and inevitably causes mechanical damage to the periodontal membrane on the root of the donor tooth, thus reducing the vitality of periodontal membrane cells. The most critical factor for the success of ATT is the presence of viable periodontal ligament (PDL) on the surface of donor tooth root .Lee et al.  used spiHowever, in the actual operation process, the surgeon who only uses the 3D model tooth to assist in preparing the alveolar socket cannot confirm the position of the donor tooth designed by the computer before. Thus, the position and occlusion of the 3D model tooth can hardly be consistent with the original design. Moreover, we found that some of the model teeth printed with the volume ratio of 1:1 to the donor teeth simulated by the computer are different from the actual donor teeth. Thus, parts of the donor teeth cannot be placed successfully. Thus, a secondary preparation is needed, which may prolong the operative time and affect the prognosis of autotransplantation.All included cases use the CAD to simulate the implant site, achieve relatively appropriate occlusal of the donor tooth, and make 1:1-sized 3D models of the donor teeth and surgical guide plates for the accurate control of direction and depth of the preparation of the alveolar socket. After 1 year follow-up, the clinical effect of this technique is observed, and the preparation time of alveolar socket, extra-alveolar time, and number of positioning trials of the donor tooth are analyzed to provide reference and assistance for the application of digital technology in ATT.A total of 41 consecutive cases of ATT completed in the Department of Oral Surgery of the Hospital of Stomatology Wuhan University from May 2019 to January 2020 were included. 3D model and surgical guide plates were preformed using a CAD on the basis of preoperative CBCT data in all cases to guide the preparation of alveolar socket during surgery.Before surgery, CBCT was taken by the same CT machine  in all included cases, and parameters were consistent Fig. b. The imSurgeries were performed by one experienced doctor, and procedures were consistent. The model tooth was assisted in preparing the alveolar socket until it was in place, and the surgical guide plate was used to confirm that the model tooth was positioned in the location designed before the surgery Fig. f, h. TheThe implanted tooth was fixed with elastic wire and fluid resin Fig. j. The ocThe following information was extracted: gender, age, position of donor tooth, diagnosis of the recipient site, root development, preparation time of alveolar socket, extra-alveolar time of donor tooth, number of positioning trials with the donor tooth, successful placement of the donor tooth, performance of RCT after surgery, and imaging data.s” form. The single-sample t-test was carried out on the extra-alveolar time data, and the test value was 15. The independent sample Mann–Whitney U test was used to compare the preparation time of alveolar socket in the recipient site, extra-alveolar time, and number of positioning trials with the donor tooth between the groups wherein the donor tooth can and cannot be placed successfully. The test level was α = 0.05 on both sides. And STROBE guidelines was adopted in our study.Data were analyzed using the IBM SPSS 26.0 and represented in the “In this study, 14 males and 27 females were included. The mean age was 28.68 ± 6.75 years. The oldest age was 48 years, and the youngest age was 18 years. The clinical features are shown in Fig. The mean preparation time of the alveolar socket of 41 cases was 12.73 ± 6.18 min, and the shortest and longest preparation times were 3 and 30 min, respectively. The mean extra-alveolar time of donor tooth was 5.56 ± 3.11 min, and the shortest and longest extra-alveolar times were 1 and 15 min, respectively. The mean number of positioning trials with the donor tooth was 2.61 ± 1.00, and the minimum and maximum numbers were 1 and 5, respectively .The data of the extra-alveolar time of the donor tooth conformed to the normal distribution. The single-sample t-test was conducted on the data, and the test value was 15 Fig. b. The diIn 21 cases , the donor tooth cannot be placed successfully after the alveolar socket was prepared with the assistance of 3D model Fig. a.Fig. 4IP < 0.05).In the group wherein the donor tooth cannot be placed successfully, the mean preparation time of the alveolar socket, mean extra-alveolar time of donor tooth, and mean number of positioning trials with the donor tooth were 15.57 ± 6.14 min, 7.29 ± 2.57 min, and 3.19 ± 0.75, respectively. The data of the donor tooth extra-alveolar time in the group wherein the donor tooth cannot be placed successfully conformed to normal distribution. The single-sample t-test was conducted on the data, and the test value was 15  ATT, the best restorative way of biocompatibility, has absolute advantages over dental implants in terms of the formation of periodontal membrane healing and even pulp healing , 11. TheAn important factor for the success of ATT is to maximize the integrity and vitality of the periodontal membrane on the root surface of the transplanted teeth . A shortAnother important factor affecting the prognosis of ATT is the proper distance between the alveolar socket and the donor tooth  which caP < 0.05). A previous study showed that the probability of root resorption is remarkably reduced when the donor tooth is isolated for less than 15 min [A digital technology assisted ATT treatment approach has been developed, including CBCT analysis, simulation, and preparation of 3D model and guide plate, to reduce the extra-alveolar time of donor tooth, prevent potential damage to the PDL, and prepare the alveolar socket accurately . We usedn 15 min .P < 0.05). The mean extra-alveolar time of the donor tooth in the group wherein the donor tooth cannot be placed successfully (7.29 ± 2.57) was still less than 15 min (P < 0.05). And even if there were differences in the models, the survival rate of this group was not affected, which meaned that the model differences only extended the time of donor tooth removal and alveolar socket preparation within a certain range, but would not reduce the survival rate of tooth.The application of 3D printing technology in ATT has achieved good results, but this technology still has some limitations. For example, it takes about 1.5–2 h in each case at the segmentation, simulation and production stage. A shorter time is expected by improving the accuracy of automatic identification of the software and increasing the printing speed. The model teeth of some cases are not completely consistent with the shape of the donor tooth . We founFrom the acquisition of CT data to the implementation of the surgery, a number of potential sources of error is present at each stage of the process . We specFirst, the accuracy of CT may affect the accuracy of 3D modeling. The CT scanning layer thickness and voxel affect the image resolution . A high In addition, the computer-aided rapid prototyping technology produces complex 3D physical models by selective modification. Print layer thickness is one of the key parameters, It will affect the accuracy of the replica , 27.In addition, the horizontal layer at a time with stepwise submergence along the vertical axis, during this process, the precision of the printer may also lead to the deviation of the model .The application of digital guide plate and donor model in ATT can significantly shorten the preparation time of the alveolar socket in the recipient site and extra-alveolar time of donor tooth and will reduce the number of positioning trials with the donor tooth. Even if the current technology has certain limitations in accuracy, it still helps clinicians to improve or ensure the retention rate of ATT.


Varroa destructor populations typically exceed damaging levels in the fall. One explanation for rapid population increases is migration of mite carrying bees between colonies. Here, the degree to which bees from high and low mite donor colonies move between apiaries, and the effect visitation has on Varroa populations was monitored. More bees from low mite colonies (n = 37) were detected in receiver apiaries than bees from high mite colonies . Receiver colony Varroa population growth was associated with visitation by non-natal bees (p = 0.03), but not high mite bees alone (p = 0.19). Finally, colonies lacking robbing screens experienced faster Varroa population growth than screened neighbors (p = 0.01). Results indicate visiting non-natal bees may vector mites to receiver colonies. These results do not support the current two leading theories regarding mite immigration – the “mite bomb” theory (bees from high mite colonies emigrating to collapsing colonies), or the “robbing” theory . Potential host-parasite effects to bee behavior, as well as important management implications both for Varroa treatment regimens and breeding Varroa resistant bees are discussed.A leading cause of managed honey bee colony mortality in the US,  Crop yields are influenced by the density and quality of honey bee colonies placed in fields, groves, and orchards6. High rates of honey bee colony mortality threaten pollinator dependent crop production8. Many colony health stressors exist10; the parasitic mite Varroa destructor, however, has garnered special attention as a driver of losses13. Varroa is particularly detrimental to colony health because of the damage it causes while feeding15, and by vectoring viruses that weaken the colony17. Over 50% of samples collected in the US over the critical months of August-November have mite levels well above the recommended management threshold of 3 mites/ 100 bees18. This is indicative of two non-mutually exclusive issues: beekeepers underestimate Varroa infestation levels in their operations, and/or beekeepers’ management of infestations are failing.Honey bee provided pollination services to US crops are valued at over $14 billionVarroa management. These beekeepers experience winter losses 12.5 percentage points higher than their Varroa-managing counterparts 19. Lower colony loss rates are correlated with use of common Varroacide products; an expected result considering the robust modeling and field trials that demonstrate the benefits of managing mite populations21. Beekeepers who identity as “management free” tend to believe honey bees perform best when left alone and so do not manage for Varroa22. Untreated colonies in a landscape crowded with beekeepers, however, can represent a real risk of horizontal transmission of mites, putting nearby treated colonies at risk.Management surveys show that on average between 2010 and 2018, 53% of backyard beekeepers (beekeepers with 1–50 colonies) did not perform Varroa, infestation loads in the fall are often difficult to control. Long term studies on Varroa population growth over time, and mathematical models of Varroa population growth suggest that colonies can survive for three years with no Varroa control in temperate climates25. In reality, most beekeepers need to use multiple Varroa treatments per year to keep levels below damaging thresholds21. Further, longitudinal monitoring of Varroa loads in multiple apiaries across the US found that even after treatments, Varroa population growth rates often far exceed predicted levels based on Varroa reproduction rates alone25. This suggests that mites are immigrating into colonies from an outside source, most likely nearby colonies.Even among beekeepers who do monitor and treat for Varroa transmission27. Crowding of colonies within apiaries and throughout the landscape results in increased Varroa infestations as bees are more likely to move between colonies29. Colonies can also acquire mites when their bees rob other mite infested colonies, and/or when non-natal bees drift from other colonies31. Bees' propensity to drift into non-natal colonies increases when the bees have been parasitized by mites32. “Robbing” bees can also enter non-natal, and often weakly guarded colonies, in order to rob honey during periods of food scarcity29. Robbing behavior is especially prevalent in the fall when colonies with unchecked Varroa infestations start to collapse. These weakened colonies with elevated Varroa loads are robbed by nearby healthy colonies whose robbing bees may pick up mites and bring them back to their own colony34. Late fall is a critical time period for beekeepers as they prepare their bees for winter, ensuring food stores are adequate, mite loads are low, and colonies are healthy35. Re-infestation of Varroa during this period can undo the effects of successful management. Understanding the mechanism by which this late fall inter-apiary mite transmission occurs is critical in developing best management practices to help mitigate colony health damage.Bees often drift between colonies, representing a potential route for Varroa transmission without tracking bees31. Some studies have tracked bees, but only on a small number of colonies or within one apiary36. Here, 32 colonies in eight receiver apiaries up to 1.6 km from a central donor apiary were used to reflect realistic landscape level situations. The degree to which bees moved between apiaries from high and low mite donor colonies was tracked, and changes in Varroa loads were monitored. Additionally, robbing screens were used to keep out non-natal bees from 50% of receiver colonies. This novel application of an existing beekeeping tool provided important clues about the directionality of inter-colonial mite transmission.Past studies have attempted to document and explain inter-apiary mite transmission, but understanding its directionality requires the difficult task of tracking individual bees across the landscape. Most prior work has tracked changes in mite loads in situations hypothesized to increase drift and The majority of visitors to receiver colonies should be high mite donor beesMite loads in visited colonies should increase more than in non-visited coloniesColonies with robbing screens should exhibit smaller increases in mite loads than their unscreened counterparts, as screens would inhibit non-natal high mite bees from enteringThe “mite bomb” hypothesis: If mites were primarily transmitted by bees from high mite donor colonies, the following was expected:There should be no difference in the frequency of visitations to receiver colonies by high and low mite donor beesIncreases in mite loads in receiver colonies should not be associated with donor bee visitationVarroa population growth compared to unscreened colonies, as natal bees bringing home mites after robbing would not be deterred by the screensColonies with robbing screens should not exhibit different The “robbing” hypothesis: if mites were primarily transmitted by bees from healthy receiver colonies robbing out high mite donor colonies, the following was expected:Two inter-apiary mite transmission hypotheses were tested.Varroa treatment  from September 18th to September 24th to ensure low initial mite loads.Two types of apiaries were established for this study: eight receiver apiaries and one donor apiary. Receiver apiaries consisted of four colonies each, housed in either a single 10 frame Langstroth deep brood box (n = 28) or one 10 frame Langstroth deep and one 10 frame Langstroth medium brood box (n = 4). All receiver colonies were established from splits with new queens in August 2019 to equalize colony strength and facilitate movement into the experimental location. Each receiver colony was made of seven frames of adult bees, three frames of brood, and two frames of honey. Receiver colonies were moved into the experimental location on August 30th, and received a Varroa load > 3 mites/100 bees) and two low mite colonies (Varroa load < 1 mite/100 bees). Donor colonies were overwintered and selected based on results of alcohol washes performed in August. Low mite colonies received a half dose formic acid treatment before the experiment began (September 18th to September 24th) to ensure a low initial mite load. The donor apiary was established at the experimental location on September 27th. Low mite colonies received a second half dose formic acid treatment on October 3rd, to combat any Varroa increases they had incurred from their close proximity to the high mite colonies, as substantial drift within the donor apiary was assumed. This additional mite treatment means that low mite donor colony mite population growth rates cannot be compared to receiver colony mite population growth rates.The donor apiary consisted of two high mite colonies  from the donor apiary. Four additional receiver apiaries were placed approximately 1.6 km (1 mile) from the donor apiary . These Varroa testers have a small hole where CO2 can be injected into the cylinder. The bees received CO2 until they became unconscious (~ 5–10 s), and were then poured out onto a flat surface for painting  of the adult bee population in the tub. The lid of the tub was then lifted just enough to scoop ~ 500 bees into a small plastic cylinder  fitted with a camera module (Pi Camera 2) was programmed with OpenCV (Python 3) to detect user-specified colors. For this experiment, the RBG values associated with blue and red paint colors were used. A generous range of RBG values was used to account for variation in colors due to time of day, shade, or clouds. The cameras were programmed to capture a photo at 3 frames/second when they detected red or blue. Photos were saved with time and date stamps to help identify unique individuals. Colony entrances were reduced to limit the bees’ path of entry to within the camera’s field of view. White cardboard was mounted under cameras to provide a neutral backdrop. All 36 colonies in the experiment (donor and receiver) were mounted with a camera sensor from September 28th through November 10th Fig. . Camerase.g. grass, fallen persimmons, etc.) appeared blue or red and triggered photo capture. To eliminate irrelevant photos, if a camera contained a set of over 1,000 photos taken in a short period of time, this was deemed an unlikely true detection and ignored. Sets of photos that contained fewer than 1,000 photos were checked for true detections of painted bees. Unique individuals could be discerned with reasonable confidence because their paint marks were typically distinctive . Because the cameras were programmed with a generous range of RBG values to not miss any detections of painted bees in varying light, occasionally other colored objects in a camera’s field of vision . Robbing screens are metal mesh that block the regular colony entrance, and have a separate hidden entrance at the top of the screen. Only bees that live in the screened colony learn the new entrance, and the reduced entrance is easier for natal bees to defend, so non-natal bees are deterred from entering. Whether the left most or right most colony was screened was chosen randomly, but screening both end colonies was avoided, as unpublished data suggests that end colonies are more susceptible to receiving visiting bees (vanEngelsdorp Pennsylvania State Inspection records). After the first screened colony was randomly chosen, a second colony not adjacent to the screened colony received a robbing screen.Varroa load. An alcohol wash was performed at the beginning (September 24th) and the end (November 10th) of the experiment. Samples were checked for painted bees when processed, and no painted bees were found in receiver colony alcohol samples. Sticky boards placed under each receiver colony were changed and counted approximately every three days, dependent on weather. Each receiver colony was also manually checked for painted bees at the middle (October 23rd) and end (November 10th) of the experiment. Manual checks consisted of removing and visually inspecting every frame in each colony for painted bees.Donor and receiver colonies were monitored throughout the experiment for changes in 38. Receiver colonies were not assessed for colony size as they were identical at the start of the experiment. The goal of this study was to continue until either the high mite donor colonies collapsed (and the movement of bees from collapsing colonies could be tracked) or until the weather became so cold that bees no longer foraged regularly. Under these guidelines, the experiment was conducted from September 18th–November 10th , a second round of painting was performed. Colony size was assessed by the standard method of a frames of bees estimate by observing the top bars of each colony0th Fig. .Figure 6Varroa loads and colony size were the same between treatment groups at the starting point of the experiment. Pearson’s Chi-squared test was used to check for differences in mite loads between colonies within the donor apiary, as well as in painted bee detections of each color in receiver colonies. Generalized mixed effects models with apiary as random effects were used to compare between groups at different time points . Sampling time (start or end) was also included as a random effect to account for the pseudo replication of repeated measures of the same colonies at each time point. Models were eliminated in a stepwise fashion using ANOVAs until the simplest best fit model was identified. Spearman’s correlations were performed to check for correlations between the number of painted bees detected and the starting Varroa load.All statistical analyses were performed in R (version 3.3.3). Summary statistics are reported as mean ± SEM unless otherwise stated. Student’s t tests were used to check if t = -1, df = 1, p = 0.5). At the end of the experiment on Nov 10th, 2019, high mite donor colonies had not rapidly collapsed as anticipated, but had steadily dwindled in population until functionally dead (< 1 frame of bees) and were smaller than the low mite donor colonies . All 32 receiver colonies survived the entire length of the experiment and remained approximately the same size throughout.The experiment began on Sep 28th, 2019 with all donor colonies at the same population size . The number of detections of high or low mite bees was not correlated to high or low mite donor colony population size . Painted bees were detected in 62.5% (n = 5) of receiver apiaries and at 56.3% (n = 9) of non-screened receiver colonies.In total, 47 unique bees from donor colonies were detected by 16 camera sensors on unscreened colonies Fig. . Conside2 = 43.1, df = 1, p < 0.001). The visited apiary at the further radius only received one visitor to one of the two unscreened colonies. In all other apiaries that received donor bee visitors, both unscreened colonies were visited.There was substantial drift of bees between colonies within the donor apiary. Donor colonies were mounted with cameras, and the number of non-natal bees detected was higher than could be quantified (hundreds in each donor colony camera). Two manual checks performed of receiver colonies for painted bees did not result in any detections, suggesting painted bees did not permanently remain in non-natal colonies. With only one detection in apiaries placed at a further radius (1.6 km from donors), donor bees were much more likely to visit closer apiaries then further apiaries (χVarroa loads in low mite donor colonies remained low throughout the study (from 0.17 ± 0.17 to 1.95 ± 0.10 mites/ 100 bees), while Varroa loads in high mite donor colonies grew substantially (from 9.57 ± 5.12 to 34.8 ± 29.41 mites/ 100 bees). The two high mite colonies started with significantly different mite infestations , but their mite loads were always higher than in low mite colonies . Varroa loads in all 32 receiver colonies increased over the duration of the study . Receiver colonies also had laying queens and capped brood present throughout the study.Varroa loads compared to colonies that were not visited by high mite donor bees . Receiver colony mite population increase over the duration of the study was not affected by high mite donor bee visitation .Receiver colonies that were visited by high mite donor bees started the study with similar t = 1.34, df = 9.33, p = 0.21). Varroa loads in colonies that were visited by any donor bee, however, increased more than the mite loads in colonies not visited by donor bees . Within apiaries that were visited by donor bees, there was a positive correlation between a colony’s starting mite load and the number of non-natal bees it received . There was not a correlation between the number of non-natal bees a colony received and its ending mite load . Varroa population growth was associated with whether a colony was visited by non-natal bees or not, and not with the total number of donor bee visitations.Receiver colonies visited by any donor bee (from high or low mite donor colonies) also started the study with similar mite loads to receiver colonies that were not visited . During the study, colonies with robbing screens had a lower Varroa population growth rate . Evaluation of the daily Varroa drop from colonies via sticky board mite counts show a similar trend, with starting counts not differing between colonies with or without screens . Sticky board counts over the whole experiment show that colonies with robbing screens had consistently lower Varroa loads than colonies without robbing screens . Sticky board mite counts (mean # of mites dropped per colony per day) in the first experimental week were significantly higher than any other week . This is likely due to residual mite drop from the formic acid treatment that ended one day before sticky boards were placed on colonies.Direct measures of Varroa population growth (Fig. Varroa population growth compared to unscreened colonies (Figs. Varroa population growth.The “mite bomb” hypothesis expects that high mite donor bees will end up in receiver colonies. While high mite colonies systematically lost bees throughout this study, particularly in the last two weeks of the experiment, they were rarely detected at receiver colonies. Instead, the majority of bee visitations to receiver colonies in this study came from low mite donor colonies Fig. , and viswth Fig. . The “roes Figs. , 15, sug39. Here, no evidence of recruitment was seen as the number of visitors to each receiver colony was low, thus the visitation behavior observed did not appear as true robbing. It is likely that bees from unmarked colonies were also visiting receiver colonies, and the number of marked visitors detected is probably a small fraction of the real number of non-natal bees visiting receiver colonies. Regardless of the true number of visitors, the motive behind these non-natal bee visitations is unknown, and understanding this behavior may lead to important insights regarding horizontal transmission of mites.These results suggest the need for an alternative hypothesis, which is here referred to as colony permissiveness. Colonies that permit more presumably short term visiting bees experienced greater mite population growth than their less permissive counterparts. The term “visiting” is used here instead of robbing because robbing is typically characterized by mass recruitment of nest mates, so that many non-natal bees are stealing a colony’s resources together33. Both this prior study and the present study, however, observed that high mite colonies are more likely to receive non-natal bees than act as a source of donor bees. The present study further tested the directionality of this phenomenon with the inclusion of robbing screens, finding that screened colonies exhibited reduced Varroa population growth. Because robbing screens do not deter natal bees, non-natal visitors are more likely to be a source of mites to the colonies they visit than to bring acquired mites home.A prior study tracked the movement of bees between high and low mite apiaries and the resulting change in mite populations, and found that colonies exhibited increased mite loads after their bees visited high mite colonies, thus concluding that mite transmission was due to natal bee robbing activityVarroa/virus complex. There are two avenues where Varroa and/or viral pathogens could alter host behavior to aid in dispersal and transmission. First, elevated Varroa and/or viral loads in donor colonies could result in elevated rates of infected bees exiting donor colonies and visiting receiver colonies. These infected non-natal donor bees could transmit both mites and viruses upon entering a receiver colony. Mites can switch phoretic hosts or enter a brood cell within seconds, so even a short visitation from a mite carrying non-natal bee can result in transmission40. It was also recently demonstrated that bees infected with a Varroa vectored virus were more likely to remove themselves from their natal colony, and were more frequently admitted to non-natal colonies41. In the present study, high mite donor colonies did not produce more visiting bees than low mite donor colonies. However, viral loads of high and low mite donor colonies are unknown. It is possible that due to their close proximity and the high degree of drift within the donor apiary, low mite colonies experienced elevated viral loads while maintaining low mite loads. It is certainly possible that donor bee behavior was affected by parasite and/or pathogen loads, and further work will attempt to assess to what degree this occurred.These results provide important clues to understanding the host-parasite relationship of honey bees and the 42. Here, robbing screens were used to circumvent a colony’s natural permissiveness, making receiver colonies less accessible. The number of visitations to screened colonies is unknown, but their reduced Varroa population growth indicates that they were visited less often. While host-parasite dynamics may affect a colony’s permissiveness, another potentially important factor is the role of long term bee breeding on commercially managed bees. Beekeepers have intentionally selected and propagated gentle bees for generations. Several studies, however, have noted more defensive strains of honey bees often show greater Varroa tolerance43. It is possible that by breeding gentler bees, beekeepers have inadvertently made colonies more permissive to visits, and thus more susceptible to mite immigration.Another potential factor influencing mite population growth is the effect of parasites on colony permissiveness – willingness to accept non-natal bees. Kakugo virus, a close relative of DWV for instance, is found disproportionally in a colony’s guard bees, suggesting some evolutionary advantage to low levels of infection within coloniesVarroa loads can make colonies more permissive to non-natal bee visitation. This finding is supported by prior work which tracked drifting bees and found that high mite colonies were not more likely to produce drifting bees when compared to low mite colonies, but rather high mite colonies were more likely to receive non-natal bees than low mite colonies34. Here, a receiver colony’s permissiveness to visitors showed no bias toward low or high mite donor bees, and was associated with an increased mite load. These results suggest that elevated mite or viral loads may alter receiver colony behavior, particularly in guard bees, to admit more non-natal visitors. The importance of receiver colony behavior in this context was unexpected and thus not included in this experiment’s design. While there is evidence that mite loads in these colonies played a role in their permissiveness, other factors that may play a role, such as colony size, were not rigorously measured. As all of receiver colonies started the experiment with an approximately equal number of bees and brood, and all colonies ended with a queen and brood it seems unlikely that colony size played an important role; however, further studies to elucidate the impact of these effects are needed.Declining colony health or strength either caused by or resulting in elevated mite loads could further affect a colony’s permissiveness. The number of visitors a colony received was positively associated to its starting mite load, indicating that elevated Varroa/virus complex and honey bee relationship, hinting at intriguing evolutionary questions regarding host parasite relationships at an individual bee and colony level. Arguably more impactful, however, was the finding that robbing screens, an inexpensive and accessible piece of bee equipment, could dramatically help beekeepers keep mite levels low. While more testing and refinement of this technology is necessary before its use is widely promoted, this offers some much needed hope for an industry struggling with damages caused by Varroa.These results provide further evidence that mites transmitted between colonies and apiaries on drifting or visiting bees play an important role in driving mite population rates in some colonies. Of interest is that the data supported neither the mite bomb or robbing hypothesis currently suggested as the mechanism by which mites move between colonies. Rather, results suggest that a colony’s permissiveness predicts the rate of mite growth a colony will experience. This finding seems supported by the nascent understanding of the Supplementary information.


Periodic monitoring of CSF and blood, clinical and neurological evaluations, and magnetic resonance and ultrasound imaging of target organs demonstrated no toxicity related to treatment. AAV9-mediated gene transfer resulted in detection of sulfamidase activity in CSF throughout the study. Analysis at tissue level showed widespread sulfamidase expression and activity in the absence of histological findings in any region of encephalon, spinal cord, or dorsal root ganglia. Altogether, these results provide proof of durability of expression and long-term safety for intra-CSF delivery of AAV-based gene transfer vectors encoding therapeutic proteins to the CNS.Delivery of adeno-associated viral vectors (AAVs) to cerebrospinal fluid (CSF) has emerged as a promising approach to achieve widespread transduction of the central nervous system (CNS) and peripheral nervous system (PNS), with direct applicability to the treatment of a wide range of neurological diseases, particularly lysosomal storage diseases. Although studies in small animal models have provided proof of concept and experiments in large animals demonstrated feasibility in bigger brains, there is not much information on long-term safety or durability of the effect. Here, we report a 7-year study in healthy beagle dogs after intra-CSF delivery of a single, clinically relevant dose (2 × 10  Seven-year follow-up of dogs after intra-CSF administration of AAV9-sulfamidase vectors results in detection of sulfamidase activity in CSF and widespread transgene expression in CNS, PNS, and liver, in the absence of any adverse events. Proof of durability and safety of this gene therapy support its clinical translation to treat CNS diseases. Hence, transduction of large proportions of cells with gene therapies is not necessary to achieve full therapeutic efficacy.in vivo have been described so far. The simplest approach is based on the intravenous (i.v.) administration of adeno-associated viral vectors (AAVs) because of the ability of certain serotypes, such as serotype 9 (AAV9), to cross the blood-brain barrier (BBB) and transduce the CNS.,,Different ways of delivering genes to the CNS ,,,Vectors encoding for lysosomal enzymes can also be delivered directly to the CNS by intraparenchymal injection or by administration into the cerebrospinal fluid (CSF). The first method, currently under clinical testing for MPSIIIA  and MPSIIIB NCT03300453),00453,,,,,,,,36,,,Alternatively, we and others have demonstrated in several animal models the advantages of delivering vectors, in particular AAV9, directly into the CSF to achieve efficient and widespread CNS transduction.Sgsh), the enzyme deficient in MPSIIIA, we were the first to report whole-body correction of a LSD after delivery of AAVs to the CSF of mice.,Sgsh-encoding AAV9 vectors at a clinically relevant dose. Our study demonstrates that the procedure is safe and results in high levels of SGSH activity in CSF detectable 7 years after a single vector delivery, in the absence of any adverse events. Transgene expression and activity were detectable within the CNS and the peripheral nervous system (PNS) at the end of the study. Vector persistence and transgene expression were also detectable in liver. To the best of our knowledge this is the longest safety and durability follow-up in a large animal species after CNS-directed gene transfer.Using an AAV9 vector encoding sulfamidase (13 vg (2.78 × 1011 vg/ mL of dog brain) of AAV9 vectors carrying an optimized version of canine sulfamidase coding sequence under the control of the CAG promoter (AAV9-Sgsh) through cisterna magna injection.Three male healthy beagle dogs (dogs 1–3) were administered 2 × 10Sgsh administration, treated dogs remained clinically well, with no signs of adverse events. CSF samples were tested regularly for elevations in white blood cell (WBC) counts or total protein (TP) levels as indicators of CNS inflammation. Red blood cells (RBCs) were counted in parallel to monitor for potential blood contamination during sample withdrawal. CSF WBC and TP levels remained within normal range throughout the study . In the encephalon, 83% and 93% of samples from dogs 2 and 3 tested positive for AAV9-Sgsh vector genomes , spinal cord , and dorsal root ganglia (DRGs)  for quantification of vector genome copy number (VGCN) and sulfamidase mRNA levels. One non-injected dog was used as negative control. Similar to what was previously observed in shorter (3 month) studies in dogs,Figure 3Sgsh expression covered the whole volume of the encephalon, from areas close to the cisterna magna (site of injection into the CSF), such as cerebellum, pons, and medulla oblongata, to areas distant from the injection site, such as frontal cortex and olfactory tract  and the spinal cord . The enzymatic assay used cannot distinguish endogenous from vector-derived sulfamidase. Contrary to CSF, where background SGSH activity is very low, allowing for sensitive monitoring of transgene expression, tissue SGSH activity in the CNS is relatively high, which lowers the sensitivity of the assay to detect increments over baseline. The brain and spinal cord of 15 control animals not injected with the SGSH vectors were used to obtain reference values for endogenous activity for each region analyzed, with a sample size of at least 10 samples per region, totaling >400 measurements. In agreement with biodistribution and transgene expression data, increases in sulfamidase activity over the levels detected in control dogs were documented in >70% of the encephalon and spinal cord samples obtained from each of the injected dogs A. The maAt sacrifice, we also collected encephalon, spinal cord, and DRG samples for detailed histological analysis. Sections obtained from different regions of the CNS in which we had documented high transgene expression, including frontal, parietal, and occipital cortices, hippocampus, cerebellum, and spinal cord, were analyzed in detail after hematoxylin and eosin (H&E) and Nissl staining and immunostaining against GFAP and Iba1. Tissues showed normal structure, and no histopathological observations were made that would be suggestive of long-term product-related toxicities .We paid special attention to DRGs, given the recent reports that have uncovered histopathological findings in the DRGs of primates after intravenous or intra-CSF delivery of high doses of AAV vectors.To complete the analysis, we studied spinal cord samples corresponding to the same regions  with the same histological tools. Tissue architecture was preserved in all injected dogs; the distribution of GFAP reactivity was homogeneous, with an abundance of astrocytes but with no sign of gliosis. Iba1 staining was weak and was also homogeneously distributed. Altogether, the samples were considered to have no significant structural changes, and the analysis discarded the presence of a chronic inflammatory process.,,,,,,,,Sgsh 48 and 54 months after vector administration. Two samples, from the left median and left lateral lobes, were obtained from each animal at each biopsy time point . One non-injected dog and one dog injected in the CSF with AAV9-GFP vectors at the same dose as in the present study were used as negative controls.After vector administration into the CSF, AAV9 vectors have been systematically detected in the liver of mice, dogs, and NHPs,The results of the analysis of transgene expression by quantitative real-time PCR in these biopsies were variable. Dog 2 had the highest levels of expression in both lobes, dog 3 had detectable expression at intermediate levels in the left lateral lobe, and dog 1 showed a transcript signal in the same range as negative controls . The corAt the end of the study, vector biodistribution was evaluated in additional liver samples obtained during necropsy. To get more representative data than those provided by biopsies, 20 evenly distributed samples per lobe were collected from each dog (approximately n = 60 samples/liver). One non-injected dog was used as negative control. In agreement with the observations made in biopsy samples, vector genomes were only detected in the livers of dogs 2 and 3; for dog 1 all samples except one were negative A. RemarkAt sacrifice, we also collected liver samples for detailed histological analysis. Liver showed normal structure, and no histopathological alterations were observed that would be suggestive of long-term product-related toxicities .In an attempt to better understand the differences in transduction observed across the dogs, we did a longitudinal evaluation of neutralizing antibodies (NAbs) against AAV9 in paired CSF and serum samples. At baseline, none of the dogs had anti-AAV9 NAbs in CSF . Two of ,3,3 for male and females, respectively, at age 4 years 9 months to 18 years3 and a lifespan of ∼13 years , offers a better model to address clinically relevant questions such as distribution of the therapeutic vector or durability of transgene expression.Genetic diseases that affect the brain constitute important unmet medical needs. LSDs with neurological involvement, in particular, represent a complex therapeutic challenge, as the enzyme replacement therapies (ERTs) that might alleviate peripheral disease are ineffective to treat the CNS because of the presence of the BBB. The option of providing the enzyme directly to the CNS has been tested clinically, although the few trials performed so far have revealed the shortcomings associated with the implantation of the intrathecal delivery devices that are required for periodic infusion of proteins to the CSF.Previous studies have unequivocally established the potential of AAV vectors to correct CNS disease with a variety of gene transfer methodologies.,,,,,Our approach to MPSs is based on the delivery of vectors encoding the missing enzyme into the CSF. The rationale behind this choice is that the use of this fluid as the route of delivery allows for better distribution of the vectors within the CNS, the main target compartment for this disease. Although other serotypes have been delivered to the CSF,In a previous report, we demonstrated that upon delivery of AAV9 vectors encoding human sulfamidase to the CSF of dogs at the vector dose deemed therapeutic in MPSIIIA mice, activity of the enzyme in the CSF peaked within 2–3 weeks.,,,,,Sgsh mRNA expression levels, which were fairly similar across the encephalon of all 3 dogs. Although sulfamidase expression was detectable in most deep brain areas, the striatum, epithalamus, and thalamus showed very low to no detectable mRNA levels, which could be a limitation of intra-CSF-based gene therapy in transducing deep brain structures.,,Sgsh mRNA expression in spinal cord, but it is difficult to interpret whether those differences are of significance, as CSF SGSH activity levels were very similar in dogs 2 and 3. In any case, the comparative analysis of vector distribution and expression data across the animals argued against transgene silencing as an explanation for the different behavior along the years of CSF SGSH in the different dogs.AAV vectors encoding the canine sulfamidase were injected into the CSF at the same dose as that of the human sulfamidase, and animals were followed for almost 7 years. The initial kinetics of canine sulfamidase expression was similar to that of the human transgene, with an initial peak within 2–4 weeks post-administration. In contrast to the study that used human sulfamidase as a transgene, the activity of sulfamidase in the CSF of dogs injected with the canine sulfamidase-bearing vectors did not drop to pre-treatment levels after a few weeks.Sgsh expression among the dogs, with >30 samples/dog of encephalon analyzed. Differences in expression and SGSH activity are more pronounced in the spinal cord, where dog 1 shows the lowest levels for both readouts; it could be the case that cells from the spinal cord are major contributors to the output of SGSH to the CSF. Alternatively, it may be the case that transduction was overall less efficient in dog 1. Because in general dog 1 shows lower VGCN, we consider it unlikely that vector silencing underlies the more pronounced drop in CSF SGSH in this dog. Other factors, such as changes in the volume of CSF of a magnitude that could impact the concentration of the enzyme in this fluid over the course of the life of dog 1, seem unlikely.,The levels in CSF of a transgene expressed and secreted by transduced CNS cells is likely to result from a complex interaction of factors, including rate of production and clearance, both of which could be influenced by physiological and pathological processes. Along the years, a certain percentage of transduced cells could be lost not necessarily due to toxicity or inflammation but to physiological cell turnover. In this regard, after intra-CSF administration of AAV9 vectors encoding the reporter gene GFP we observed transduction of neurons and astrocytes but also of ependymal and leptomeningeal cells.,,The administration of the therapeutic product to the most challenging-to-treat anatomical compartment—the CNS—guarantees therapeutic levels of the transgene in the organ most severely affected. Additionally, the escape of vector to the circulation and subsequent transduction of the liver could provide a peripheral source of the therapeutic protein. Indeed, we were the first to report whole-body correction of a LSD upon delivery of AAV9 vectors to the CSF of MPSIIIA mice,,,Given the absence of any signs that would suggest loss of vector genomes from the liver of dog 1, our observations might be reflecting true differences in liver transduction efficacy. Evidence supporting this hypothesis was provided by the longitudinal quantification of anti-AAV9 NAbs in paired CSF and serum samples for all dogs. Although the 3 dogs were negative for NAbs in the CSF at baseline, which explained the similar level of transduction efficacy in the CNS, dog 1 showed the presence of NAbs in circulation at 1:5–1:10 titer. Although low, this titer has been described to be sufficient to block liver transduction in large animals and humans.If efficacy in mild peripheral MPSIIIA disease was not achieved because of the lack of transduction of the liver, there would always be the possibility of providing patients with intravenous ERT, at expectedly lower doses than those required to treat severe CNS pathology, and most likely under a treatment regime more favorable to patients. Finally, it should be noted that, as the CSF ultimately drains into the circulatory and lymphatic systems, a certain amount of the therapeutic protein could also pass to the periphery. In this regard, studies conducted in a dog model of late infantile neuronal ceroid lipofuscinosis (Batten disease) found significant amounts of the soluble lysosomal enzyme tripeptidyl peptidase 1 (TPP1) in heart and spleen after delivery of TPP-encoding AAV2 vectors to the CSF, an AAV serotype with minimal peripheral dissemination.,,in vivo gene transfer with AAVs, irrespective of the transgene and target organ. Whether the difference in VGCN reported in short- versus long-term studies reflects non-functional vector genomes that were not taken up by cells and are cleared with time, stabilization of only an initial fraction of the vector genomes in the nucleus, or turnover of some of the targeted cells remains to be determined for each organ.,,Besides the documentation of steady levels of therapeutic transgene through multiple years, another important outcome of this study is the demonstration of the good tolerability and long-term safety of the approach. Animals remained in an excellent health status throughout the study. We did not document any clinically relevant increase in protein levels or WBC counts at any of the time points at which CSF was sampled. Likewise, no clinically relevant alterations in biochemical or hematological parameters could be attributed to the procedure or test article administration. As further proof of long-term safety, in-life MRI and ultrasound imaging, as well as histopathological studies of post mortem specimens, revealed no evident abnormalities in the architecture of the encephalon, spinal cord, DRGs, or liver. DRG toxicity, characterized by mononuclear cell infiltrate, gliosis, neuronal loss, and axonopathy, has recently been identified as a common, albeit low in magnitude, histological finding following i.v. and intra-CSF but not intraparenchymal administration of high doses of AAV vectors to NHPs.,SGSH gene described so far are missense mutations, and the report on the largest cohort of MPSIIIA patients studied up to now showed that >98% of subjects were carriers of at least one allele corresponding to a missense mutation.In the present study, we chose to use healthy beagle dogs as a large animal model. Although there is a spontaneous dog model of MPSIIIA for which an experimental colony has been established , its use does not provide any additional benefits for the long-term evaluation of sulfamidase expression and safety. On one hand, the clinical manifestations and evolution of the disease seem to differ substantially between humans and dogs, which mostly present signs of cerebellar disease, with minimal or no behavioral alterations or somatic disease.In conclusion, our study provides strong evidence of the feasibility and long-term durability and safety of using AAV9 vectors to deliver genes to the CNS. After the administration of clinically relevant doses of vector, the detection of sustained, significant levels of sulfamidase in the CSF and of widespread transgene expression in the CNS, PNS, and liver of treated dogs in the absence of any signs of toxicity after many years of follow-up provides strong evidence supporting the use of intra-CSF AAV9-mediated gene therapy to treat MPSIIIA and other neurodegenerative disorders.Male healthy beagle dogs  were purchased from Isoquimen (Spain) and housed at the Veterinary School of the Universitat Autònoma de Barcelona (UAB). Animals’ health was monitored through clinical, hematological, and biochemical examination. Animals were fed once daily at 9:00 AM with 30 g/kg body weight standard dry food . For general anesthesia, dogs were premedicated with an intramuscular administration of 0.05 mg/kg of acepromazine  and 0.2 mg/kg of butorphanol . Thirty minutes later, induction was performed by i.v. administration of 4 mg/kg of propofol  and 0.5 mg/kg of diazepam . After endotracheal intubation, anesthesia was maintained by inhalation of 2% isoflurane  in 100% oxygen. Throughout all surgical procedures, temperature, cardiac and respiratory frequency, capnography, arterial pressure, pulse, and electrocardiography were monitored with a multifunctional patient Vet Care monitor . The neurological evaluation of injected dogs was performed by a diplomate veterinary neurologist from the Veterinary Clinical Hospital of the UAB. All experimental procedures were approved by the Ethics Committee for Animal and Human Experimentation of the UAB.AAV expression cassette was generated by cloning the cDNA of optimized canine sulfamidase  into an AAV backbone plasmid containing the ubiquitous CAG promoter  and the rabbit beta-globin polyA signal (rβGpA). AAV9 vectors were produced by triple transfection of HEK293 cells and purified with an optimized purification protocol with double cesium chloride gradient ultracentrifugation that results in vector preps of high purity with negligible amounts of empty capsids.13 vg/dog) was obtained by scaling up the dose previously found to be effective in mice,CSF collection and delivery of vectors was performed as previously described.General laboratory parameters and CSF TP content were measured by spectrophotometry with a Cobas Mira Analyzer (Roche) at the Clinical Biochemistry Lab of the Veterinary Clinical Hospital, UAB. Hemograms were analyzed with a SCIL VetABC hematology analyzer and CSF cell counts, WBC and RBC, were manually counted with a Neubauer chamber by the Clinical Hematology Lab.For ultrasound imaging, anesthetized dogs were positioned in dorsal recumbency and an abdominal ultrasound was performed with a high-frequency linear transducer . For brain MRI, anesthetized dogs were positioned in ventral recumbency and scans were performed with a 0.2-T permanent open magnet system . Technical parameters were (1) T1-weighted images in transverse and sagittal planes: TR = 800 ms, TE = 26 ms, FOV = 180 × 180 mm, thickness = 4.0 mm, spacing = 0.4 mm, matrix = 224 × 224, and NEX = 3; (2) T2-weighted images in transverse, sagittal, and dorsal planes: TR = 3,000 ms, TE = 80 ms, FOV = 180 × 180 mm, thickness = 4.0 mm, spacing = 0.4 mm, matrix = 224 × 224, and NEX = 1; and (3) fluid-attenuated inversion recovery (FLAIR) images in transverse planes: TR = 6,960 ms, TE = 80 ms, TI = 1,800 ms, FOV = 200 × 200 mm, thickness = 4.0 mm, spacing = 0.4 mm, matrix = 192 × 192, and NEX = 1. In addition, contrast-enhanced T1-weighted images of transverse and sagittal planes were acquired after intravenous administration of paramagnetic contrast medium . For spinal cord MRI, anesthetized dogs were positioned in dorsal recumbency and scans were performed with a 0.4-T scanner . Technical parameters were (1) T1-weighted images in transverse and sagittal planes: TR = 653 ms, TE = 23 ms, FOV = 250 × 250 mm, thickness = 4.0 mm, spacing = 0.5 mm, matrix = 288 × 220, and NEX = 2 and (2) T2-weighted images in transverse, sagittal, and dorsal planes: TR = 3,100 ms, TE = 80 ms, FOV = 150 × 150 mm, thickness = 4.0 mm, spacing = 0.5 mm, matrix = 256 × 224, and NEX = 6. Contrast-enhanced T1-weighted transverse and sagittal images were also obtained.Forty-eight and fifty-four months after vector administration, percutaneous ultrasound-guided liver biopsies were obtained under general anesthesia. Briefly, abdominal hair was clipped, and a microconvex sector transducer operating at 6 or 7 MHz was used to guide liver sampling with a 14 gauge × 9 cm Tru-Cut biopsy needle .Eighty-two months after vector administration, dogs were pre-medicated by intramuscular injection of 0.2 mg/kg butorphanol  and 0.05 mg/kg acepromazine . Thirty minutes later, euthanasia was performed with an overdose of pentobarbital  administered i.v. The encephalon, the spinal cord, the DRGs, and multiple somatic tissues were collected and either formalin-fixed or snap-frozen and stored at −80°C until use.d-glucopyranoside sodium salt  and 10 μL of complete solution (Roche) for 17 h at 47°C. The second incubation was carried out after the addition of 6 μL of PiCi buffer  in the presence of 10 U/mL of α-glucosidase (Sigma) in 0.2% BSA  for 24 h at 37°C. After the second enzymatic reaction was stopped by increasing the pH, the released fluorescence was measured with a Synergy HTX fluorimeter (BioTek Instruments). Sulfamidase activity was normalized against CSF volume (expressed as nmol/17 h/mL) or TP content (expressed as nmol/17 h/mg protein) quantified by Bradford assay (Bio-Rad).CNS and liver samples were sonicated in 100–500 μL of Milli-Q water. Enzymatic activity was assayed in supernatants of sonicated samples or CSF with a 2-step protocol using 4-methylumbelliferone-derived fluorogenic substrate (4-MU), as previously described.CNS, PNS ganglia, or peripheral tissue samples were digested overnight (ON) at 56°C in 300 μL of Tissue Lysis Solution supplemented with Proteinase K (0.2 mg/mL). Total DNA was isolated from supernatants with the MasterPure DNA Purification Kit (Lucigen). DNA was resuspended in distilled water and quantified with a NanoDrop ND-1000 spectrophotometer (NanoDrop). VGCN in 40 ng of total DNA was determined by quantitative real-time PCR using the LightCycler 480 Probes Master (Roche). For analysis of liver biopsies, primers and probe specific for sequence within the optimized canine sulfamidase cDNA were used: forward primer: 5′-GCA TCA TCG GCA AGA AAC AC-3′; reverse primer: 5′-CAG CTT GAT CCT GGT GAT GT-3′; probe: 5′-ACA CCG AAG AGA ACA GCA GCG T-3′. The analysis of terminal samples was done with primers and probe specific for the rβGpA sequence: forward primer: 5′- CTT GAG CAT CTG ACT TCT GGC TAA T-3′; reverse primer: 5′- GAT TTG CCC TCC CAT ATG TCC-3′; and probe: 5′-CCG AGT GAG AGA CAC AAA AAA TTC CAA CAC-3′. A reference standard curve was built from serial dilutions of a linearized plasmid bearing the CAG promoter, the optimized canine sulfamidase cDNA, and the rβGpA sequences spiked into 20 ng/μL of non-transduced dog genomic DNA.RPLP0 gene: forward primer: 5′-ACC TCT TTC TTC CAG GCT TTA G-3′; reverse primer: 5′- CCA CTT TGT CTC CCG TCT TAA T-3′, probe: 5′- ACC ATT GAA ATC TTG AGT GAT GTG CAG-3′.Total RNA was purified from tissues homogenized in TriPure Isolation Reagent (Roche) with the RNeasy Mini Kit (QIAGEN). RNA was quantified in a NanoDrop ND-1000 spectrophotometer (NanoDrop). cDNA was synthesized with a Transcriptor First Strand cDNA Synthesis Kit (Roche). quantitative real-time PCR was performed with the LightCycler 480 Probes Master (Roche) with the same optimized canine sulfamidase-specific primers and probe used for VGCN quantification in liver biopsies. The values obtained were normalized to the expression of the dog Tissues were fixed for 12–24 h in 10% formalin, embedded in paraffin, and sectioned. In brain and spinal cord, H&E and Nissl staining and immunohistochemical staining against Iba1 and GFAP were performed in 5-μm sections. For DRG analysis, 3 serial 3-μm sections were obtained, at 3 different levels of the ganglia, with a separation between series of 50 μm. The first slide of each series was used for H&E staining, and the consecutive sections were used for immunohistochemical detection of Iba1 and GFAP, always following the same order of tissue sections. They were incubated overnight at 4°C with rabbit anti-GFAP  or goat anti-Iba1 . The secondary antibodies were biotinylated goat anti-rabbit immunoglobulin G (IgG)  or biotinylated rabbit anti-goat IgG . The ABC peroxidase kit (Pierce) was used for immunodetection, and sections were counterstained in Mayer’s hematoxylin. Images were obtained with an Eclipse E800 optical microscope (Nikon).in vitro neutralization assay, at ISGLOBAL, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic, Barcelona.Anti-AAV9 NAb titers were determined in dog CSF and serum samples as previously described,Results are expressed as mean ± SEM. Statistical comparisons for CSF sulfamidase activity were made with unpaired two-tailed t test. Statistical significance was considered if p < 0.05. Data on sulfamidase activity in tissues were represented with a confidence interval (CI) of 95% and 99% between control (non-injected dogs) and injected dogs.


M-CSF receptor signaling supports the development and survival of mononuclear phagocytes and is thought to play a role in post burn anemia by promoting myeloid lineage bias. We found M-CSF secretion was increased in burn patients and a murine model of post burn ACI, so we neutralized M-CSF in ACI mice to determine if erythropoiesis was improved. Instead, M-CSF blockade further impaired erythropoiesis and erythroid cells access to iron. M-CSF blockade enhanced inflammatory cytokine secretion, further increased systemic neutrophil counts, and led to tissue iron sequestration that was dependent, in part, on augmented IL-6 secretion which induced hepcidin. Deleterious effects of post burn M-CSF blockade were associated with arrest of an iron recycling gene expression signature in the liver and spleen that included Spi-C transcription factor and heme oxygenase-1, which promote heme metabolism and confer a non-inflammatory tone in macrophages. Hepatic induction of these factors in ACI mice was consistent with a recovery of ferroportin gene expression and reflected an M-CSF dependent expansion and differentiation of Spi-C+ monocytes into Kupffer cells. Together, this data indicates M-CSF secretion supports a homeostatic iron recycling program that plays a key role in the maintenance of erythroid cells access to iron following burn injury. ACI accounts for the majority of transfusions a burn patient receives3 and even with the adoption of conservative hemoglobin targets, patients with 20–60% total body surface area burns typically receive an amount of blood that is roughly equivalent to vascular volume4. There are no alternatives to transfusion because erythropoietin (EPO)8 and iron9 supplements do not promote effective erythropoiesis in trauma victims. Resistance to EPO and iron supplements is a hallmark of inflammation driven anemia, thus a better understanding of the inflammatory mechanisms in post burn ACI could lead to clinical interventions that reduce transfusions.The anemia of critical illness (ACI) is a rapidly developing and persistent anemia associated with inflammation that occurs in nearly every patient in an intensive care unit for more than 1 week11. Post burn G-CSF blockade largely rescued erythroid cellularity in the marrow and markedly attenuated an impairment of EPO signaling10. However, we suspected involvement of other mediators because G-CSF blockade did not restore peripheral red blood cells counts in ACI mice. Previous reports have suggested that an ineffective response to EPO following burn injury may be due to myeloid biased lineage commitment which diminishes the pool of cells available to respond to EPO in the marrow and in turn reduces the number of mature red blood cells in circulation15. Increased expression of M-CSF receptors in hematopoietic stem cells and progenitors driven by adrenergic receptor signaling is a suspected mechanism of burn induced myeloid bias15, but no prior study has explicitly tested the role of M-CSF receptor or M-CSF as a mediator of post burn ACI.Utilizing a murine burn model of ACI, we previously identified G-CSF secretion as a mechanism that leads to impaired red blood cell production in the bone marrow17, and which are known to promote a non-inflammatory phenotype in macrophages20. Iron recycling macrophages rely on Hmox1 for metabolism of heme; when Hmox1 is absent or saturated, intracellular heme accumulates and induces apoptosis in macrophages22. The critical role of Spi-C and M-CSF in the development of iron recycling macrophages is exemplified by the absence of red pulp macrophages in Spi-C deficient mice23 and severe reduction of these cells in M-CSF deficient (Op/Op) mice24. The role of heme metabolizing macrophages following burn injury has not been extensively studied but is likely important as hypoferremia26 and enhanced erythrocyte destruction is characteristic of burn injury30. Further, at least one prior study recognized Kupffer cells were depleted within hours of burn injury in association with erythrophagocytosis and suggested infiltrating monocytes may compensate by transforming into Kupffer cells31, reminiscent of the M-CSF dependent adaptation to increased heme burden that is now understood to occur in conditions that damage red blood cells16.M-CSF/M-CSF receptor signaling also plays a critical role in an adaptive response to heme burden following erythrocyte destruction that involves induction of Spi-C transcription factor (Spic) and heme oxygenase 1 (Hmox1), which promote differentiation of monocytes into iron recycling macrophages and heme metabolism respectivelyIn this study, we quantified increased M-CSF secretion in burn patients and a murine model of post burn ACI. Since M-CSF could promote myeloid bias that leads to ACI or homeostatic adaptation that supports recovery from ACI, we tested the role of M-CSF using a neutralization strategy. Our results indicate M-CSF/M-CSF receptor signaling supports homeostatic iron recycling and a non-inflammatory tone in macrophages that plays a key role in the maintenance of erythroid cells access to iron following burn injury.To assess M-CSF secretion in burn patients we collected plasma discard from routine clinical testing. Stored samples were selected for measurement based on the availability of at least four serial weekly samples from the same patient to understand temporal secretion patterns. The samples included both genders, ages from two to thirteen years old, and burn sizes from 30 to 75% of total body surface area (TBSA). The manufacturer’s reported range for healthy humans was used as a reference and a pooled plasma sample from healthy volunteers confirmed the range. We found M-CSF levels were above the expected normal range in all burn plasma samples and generally followed a pattern in which secretion was elevated early and then waned over time Fig. A. M-CSF 32  and tended to be further reduced in burn injured mice that received Anti-M-CSF  compared to burn isotype-treated mice. The major reduction of TER119+ cells following burn injury is in late stage E3 and E4 populations  in the burn anti-M-CSF group compared to the burn isotype group  and IL-6 levels by 175%   and 37% in the spleen  , Spi-C transcription factor (Spic), and heme oxygenase 1 (Hmox1) over one week post injury. We found Fpn1 expression was reduced by nearly 50% on PBD1 and then tended to recover by PBD7  to quantify Kupffer cells  and monocytes  as Spi-C positive or Spi-C negative  expression on post burn day one  full-thickness flame burn injury as previously described11. In brief, mice were anesthetized with isoflurane and covered with a flame-resistant template exposing the shaved dorsal skin. The target area was saturated with 0.5 ml of absolute ethanol and ignited for 10 s in the burn group or allowed to evaporate without ignition in the sham group. Immediately after the procedure, all mice received 0.5 ml of saline for volume resuscitation via the intraperitoneal (i.p.) route. Mice were sacrificed by i.p. injection of Euthasol .All experiments were performed according to the protocol approved by the Institutional Care and Use Committee at the University of Cincinnati (IACUC Protocol# 06-06-16-01). All methods were performed following relevant guidelines and regulations in the approved protocol and are reported according to ARRIVE guidelines. Eight-week-old female C57BL/6J mice were purchased from The Jackson Laboratory and maintained on irradiated NIH-07 Mouse/Rat Diet containing 350 mg/kg iron  for at least one week prior to experiments. B6.129S6(C)-Mice were administered 200 µg of anti-CSF1 clone 5A1  or Rat IgG1 clone HRPN  isotype control by the i.p. route in a 100 µl volume of PBS immediately post injury and then daily for 6 days. For combined M-CSF and IL-6 neutralization, mice were administered 400 µg of isotype, 200 µg of isotype and 200 µg of anti-CSF1, or 200 µg anti-CSF1 and 200 µg of anti-IL-6 clone MP5-20F3  by the i.p. route in a 200 µl volume of PBS immediately post injury and then daily for 6 days.Blood was obtained from mice by cardiac puncture and placed in EDTA coated tubes  or serum separator tubes . Cell counts were performed on an ADVIA 2120i Hematology System  within two hours of collection. Serum was stored at – 80 °C until quantification of G-CSF and IL-6 using Milliplex MAP kits , quantification of M-CSF using the Mouse M-CSF Quantikine ELISA Kit MMC00 , or determination of serum iron levels using a QuantiChrom Iron Assay Kit . All human experimental protocols were approved by the University of Cincinnati Institutional Review Board, Study Approval #2014-8661. Informed consent for collection of human plasma was waived by the University of Cincinnati Institutional Review Board because the samples were obtained as laboratory discard without individual patient identifiers . The study was carried out in accordance with all relevant guidelines, regulations, and the Declaration of Helsinki. Excess pediatric burn plasma from routine clinical testing was stored daily at − 80 °C, then thawed one time to aliquot into appropriate assay volumes and a second time immediately prior to assay using the Human M-CSF Quantikine ELISA Kit DMC00B . A pool of healthy adult human plasma from both genders served as a control to confirm the normal range specified by the ELISA kit.Bone marrow cells were flushed from each femur with 1.5 ml of HBSS and spleen cells were isolated by applying gentle circular pressure to the tissue resting atop a 70 µm filter with a syringe plug while continuously rinsing with HBSS. Liver cells were isolated by dissociation using a gentleMACS . Liver dissociation buffer contained 5 ml HBSS, 2.5 U/ml Dispase , 500 U/ml Type IV collagenase , and 100 U/ml DNase I . When red blood lysis is specified, cells were suspended in RBC Lysis Buffer  according to manufacturer’s instructions. Isolated cells were incubated in viability dye eFluor 780 (eBioscience) and then washed in ice-cold FACS buffer . Non-specific binding was blocked with rat serum and Mouse Fc Block (BD Biosciences) for 10 min on ice prior to staining with fluorescent labeled antibodies for 30 min. The antibody panel for bone marrow and spleen erythroid cell analysis included TER-119 (clone TER-119), CD71 (clone R17217), CD44 (clone IM7) and CD45 (clone 30-F11), all from eBioscience. The antibody panel for bone marrow myeloid cell analysis included TER-119 (clone TER-119), Gr-1 (clone RB6-8C5), and CD45 (clone 30-F11), from eBioscience as well as CD115 (clone T38-320) and F4/80 (clone T45-2342) from BD Biosciences. The same myeloid panel was used for spleen cells, except CD11b (clone M1/70) from eBioscience was substituted for TER-119. The panel for analysis of livers from Spi-C reporter mice included F4/80 (clone T45-2342) from BD Biosciences as well as CD45 (clone 30-F11) and CD11b (clone M1/70) from eBioscience. Cells were washed three times with ice-cold FACS buffer prior to performing flow cytometry on an LSRII or LSRFortessa from BD Biosciences or an Attune (Thermo Fisher). Data were analyzed using FCS Express 5 (DeNovo Software).® RT  and converted to cDNA with a High-Capacity cDNA Reverse Transcription Kit . Power SYBR® Green PCR Master Mix  was used to amplify DNA fragments through 40 cycles at 95 °C for 15 s followed by 60 °C for 1 min. Primers are listed in Supplementary Table Total RNA was isolated using RNAzol55, with slight modifications. Briefly, 0.1 g tissue was finely cut and incubated for 20 h in 2 ml of acid solution  at 65 °C. After cooling to room temperature, 20 µl of the acid extract was incubated with 200 µl of chromogen solution  in a 96 well plate. The OD was measured at 535 nm and concentrations determined with a four-point standard curve ranging from 0 to 0.5 µg of iron and data are reported as total iron in each tissue. For iron staining, spleens were fixed in neutral buffered 10% formalin overnight, dehydrated in ethanol, embedded in paraffin, cut into 5 µm sections, mounted, and iron was detected using the Gomori Prussian Blue Stain Kit . Whole-slide images representing entire digitized histopathological tissue sections were scanned using the Panoramic DESK Scanner  equipped with a 40x (NA 0.95) Carl Zeiss Plan-Apochromat objective lens. Images were captured using CaseViewer .Non-heme iron in tissues was quantified according to the method of Torrance and Bothwellt-test was used for two group comparisons. ANOVA and Bonferroni posttest comparing the selected groups specified in the figure legends was used for studies involving more than two groups. Significance was determined at p < 0.05.Statistical analysis was performed using GraphPad Prism 9. The Student's Supplementary Information.