Antifreeze proteins (AFPs) production is a survival strategy of psychrophiles in ice. These proteins have potential in frozen food industry avoiding the damage in the structure of animal or vegetal foods. Moreover, there is not much information regarding the interaction of Antarctic bacterial AFPs with ice, and new determinations are needed to understand the behaviour of these proteins at the water/ice interface.Different Antarctic places were screened for antifreeze activity and microorganisms were selected for the presence of thermal hysteresis in their crude extracts. Isolates GU1.7.1, GU3.1.1, and AFP5.1 showed higher thermal hysteresis and were characterized using a polyphasic approach. Studies using cucumber and zucchini samples showed cellular protection when samples were treated with partially purified AFPs or a commercial AFP as was determined using toluidine blue O and neutral red staining. Additionally, genome analysis of these isolates revealed the presence of genes that encode for putative AFPs. Deduced amino acids sequences from GU3.1.1 (gu3A and gu3B) and AFP5.1 (afp5A) showed high similarity to reported AFPs which crystal structures are solved, allowing then generating homology models. Modelled proteins showed a triangular prism form similar to β-helix AFPs with a linear distribution of threonine residues at one side of the prism that could correspond to the putative ice binding side. The statistically best models were used to build a protein-water system. Molecular dynamics simulations were then performed to compare the antifreezing behaviour of these AFPs at the ice/water interface. Docking and molecular dynamics simulations revealed that gu3B could have the most efficient antifreezing behavior, but gu3A could have a higher affinity for ice.AFPs from Antarctic microorganisms GU1.7.1, GU3.1.1 and AFP5.1 protect cellular structures of frozen food showing a potential for frozen food industry. Modeled proteins possess a β-helix structure, and molecular docking analysis revealed the AFP gu3B could be the most efficient AFPs in order to avoid the formation of ice crystals, even when gu3A has a higher affinity for ice. By determining the interaction of AFPs at the ice/water interface, it will be possible to understand the process of adaptation of psychrophilic bacteria to Antarctic ice.The online version of this article (doi:10.1186/s12934-017-0737-2) contains supplementary material, which is available to authorized users. Antarctica is an extreme continent with the coldest temperatures, low precipitations, dryness, and almost completely covered by ice. In this continent, microorganisms dominate the genetic pool and biomass playing an important role maintaining the operation of the ecosystem. Although different microorganisms have been isolated from several places, biological records are available for only a miniscule fraction of them from land and surrounding waters .Psychrophilic microorganisms dominate the majority of Antarctic habitats . For theInitially, antifreeze proteins (AFPs) were described in marine fishes , 5 and dAFPs are a diverse group of ice-binding proteins that prevent ice growing through the depression of the freezing point of a solution to below the melting point . This di2 hydrate slurry production piperazine-N-[2-ethane-sulfonic acid]) buffer pH 7.8 during 2 h. Samples were washed with 0.2 M mannitol–0.01 HEPES buffer, transferred to microscope slides with a small drop of water and covered with a coverslip and observed immediately in a light microscope at 4× magnification. For CV, 15 micrographs were used in order to count living (red to pink cells) and not-viable cells (not stained cells). Approximately, 70–90 cells and 110–130 cells were counted for cucumber and zucchini, respectively, in each micrograph. CV is represented as the percentage of red-stained living cells over the total number of cells counted.NR is a lipophilic dye that is uncharged and unprotonated in alkaline solutions. It diffuses across cell membranes and accumulates in vacuoles, where the acidic pH protonates the dye, appearing a dark red color in vacuoles in living cells that can maintain pH gradients . SamplesGenomic DNA from the three isolates was extracted as described above and further sequenced at Georgia Genomics Facility  using Illumina Miseq:PE300 technology  using DNA Nextera XT libraries.http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Only reads with a quality score Q >28 were considered for the final assembly. Reads were assembled de novo using Velvet assembler [Prior to assembly, all reads were trimmed in order to remove any low quality bases from 3′-end using the trimming tool TrimGalore  distribution, representing the oxygen–oxygen atomic distances among water molecules, were used to describe the water state in the solvation shells surrounding the AFPs.Radial distribution function (RDF) g(r), implemented in VMD, specifically, the g103 isolates were obtained from different places in insular and continental Antarctica during Chilean Antarctic Scientific Expeditions ECA 50–52, including Union Glacier, King George Island, Greenwich Island, Deception Island and Livingstone Island. Repetitive freezing–thawing cycles were employed to select cryoprotectants-producing bacteria, obtaining 17 isolates able to survive to this selection procedure  allowed identifying four putative AFP-coding genes. Two different sequences denominated gu3A (413 amino acids) and gu3B (360 amino acids) found in GU3.1.1 genome did match to the same sequence in the database (GenBank accession ALG05172.1), which corresponds to a protein annotated as an ice-binding protein from an unidentified bacterium (not published), with 53 and 63% identity, respectively. Meanwhile, only one sequence denominated afp5A (479 amino acids) could be identified as an ice-binding protein in AFP5.1 genome, nevertheless, these sequence matched to a well studied AFP from the Antarctic sea ice bacterium p. SLW05  with 55%oryensis . MultiplThe sequence analysis of putative AFPs from GU3.1.1 and AFP5.1 using NCBI’s conserved Domains Database (CDD) revealed that they share a large conserved domain of unknown function DUF3494), which is common to the type 1 ice-binding proteins [4, which Flavobacterium frigoris PS1 (PDB Id: 4NU3), having GMQE scores of 0.75  and 0.76 , respectively. 3D model of afp5A was generated using the crystal of an antifreeze protein from the Antarctic sea bacterium Colwellia sp. (PDB Id: 3WP9), having a GMQE score of 0.77 . The oligomeric state inferred from the templates for the three models is monomeric. Ramachandran plots for the generated models revealed that majority of amino acids (>90%) are positioned in favorable regions for the three structures , which corresponds to the radial distribution as a function of the distance between two oxygen atoms from water molecules, was used as a measure of the ice formation. It has been well reported that radial distribution function for crystalline ice is markedly different from that of liquid water [OO(r) plot is representing is the probability of finding another oxygen atom at a given radial distance from the first, but considering only water molecules within a distance of 15 Å of the protein. The RDF patterns observed in Fig. OO(r) analysis considering the solvation shell of 15 Å shed light on a possibly more efficient antifreezing behavior of gu3B. RDF for this protein shows a lower peak near 4 and 5 nm compared to gu3A and afp5A, revealing a lower of ice-like water molecules around this protein. In contrast, radial distribution peaks from afp5A and gu3A are both higher compared to those of gu3B. Captures of the final frames obtained with molecular dynamics are shown in Fig. Besides the orientation and positioning of proteins over the ice layer, no further inferences concerning antifreeze behavior could be made from docking results. Coordinates obtained were used as starting point for molecular dynamics simulations over growing ice lattices. Calculations were performed according to protocol described in methods for the three systems: gu3A-ice; gu3B-ice; afp5A-ice. After 150 ns dynamics simulations, it was possible to observe the ice growth in each system but no significant difference could be observed with the naked eye. Thereby, as a way to describe the structural arrangement of ice-like water molecules in the solvation shells surrounding the proteins, radial distribution functions g(r) were used. In particular, gid water  and has id water . We analPsychrophilic organisms produce AFPs in order to prevent ice growth during freezing. Some studies have demonstrated that these proteins are extremely effective in the inhibition of recrystallization of ice even at very low concentrations . HoweverTenebrio molitor [Choristoneura fumiferana [T. molitor and C. fumiferana. Moreover, the majority of these residues are arranged in TXT motif, but some of them are distributed in almost perfect TXT motifs, where threonine residues are spaced for two amino acids. These motifs have been implicated in ice binding; due to their replacement for leucine strongly decrease the antifreeze activity [AFPs have been isolated and characterized from different sources, including fish, insects, plants among others, and correspond to a structurally diverse group of proteins. These proteins are different in their structures, from amino acid sequences to tertiary structure, and they all bind to ice crystals in order to reduce the freezing point of water , but som molitor  and Chormiferana . These lactivity . Ice–AFPactivity . AFPs stMolecular dynamics simulations of AFPs allowed establishing interaction of Antarctic AFPs from isolates GU3.1.1 and AFP5.1 at the water/ice interface  indicate positions with fully conserved residues; colons (:) indicate conservation of residues with strong similar properties; periods (.) indicate conservation of weakly similar residues. Alignment was performed using ClustalW.Additional file 2: Figure S2. Ramachandran plot analysis of the dihedral angles PSI (ψ) and PHI (ɸ) of the generated models for (a) afp5A, (b) gu3A and (c) gu3B obtained by RAMPAGE. The three plots show favorable positioning of amino acids for the different models generated.Additional file 3: Figure S3. Radial distribution functions of a) water at 298 K (liquid water) and b) 220 K (ice) determined experimentally from X-ray and neutron diffraction by Soper [http://rkt.chem.ox.ac.uk/lectures/liqsolns/liquids.html.by Soper . Images


Purpose. To preliminarily evaluate the feasibility and accuracy of using rapid prototyping drill templates (RPDTs) for C1 lateral mass screw (C1-LMS) and C2 pedicle screw (C2-PS) placement. Methods. 23 formalin-fixed craniocervical cadaver specimens were randomly divided into two groups. In the conventional method group, intraoperative fluoroscopy was used to assist the screw placement. In the RPDT navigation group, specific RPDTs were constructed for each specimen and were used intraoperatively for screw placement navigation. The screw position, the operating time, and the fluoroscopy time for each screw placement were compared between the 2 groups. Results. Compared with the conventional method, the RPDT technique significantly increased the placement accuracy of the C2-PS (p < 0.05). In the axial plane, using RPDTs also significantly increased C1-LMS placement accuracy (p < 0.05). In the sagittal plane, although using RPDTs had a very high accuracy rate (100%) in C1-LMS placement, it was not statistically significant compared with the conventional method (p > 0.05). Moreover, the RPDT technique significantly decreased the operating and fluoroscopy times. Conclusion. Using RPDTs significantly increases the accuracy of C1-LMS and C2-PS placement while decreasing the screw placement time and the radiation exposure. Due to these advantages, this approach is worth promoting for use in the Harms technique. In 2001, Harms and Melcher first reported –5. HowevOver the past 2 decades, rapid prototyping drill templates (RPDTs) have been used for assisting screw placement in AAF surgeries –20. By un = 11) and the RPDT navigation group (n = 12). Thin-layer CT scans (0.625 mm) and 3D reconstruction images of all the craniocervical cadaver specimens were obtained preoperatively to observe the atlantoaxial anatomy of each specimen. In the RPDT navigation group, the CT scans of each craniocervical cadaver specimen were further exported as digital imaging and communications in medicine (DICOM) files, which were used for constructing the RPDTs.In all, 23 formalin-fixed craniocervical cadaver specimens  were obtained with permission from the Department of Anatomy and Pathology of Xi'an Jiaotong University. The 23 cadaver specimens were randomly divided into two groups: the conventional method group . The image thresholds were set to 226–3071 to mark the bony structures. By using the “Edit Masks,” “Region Growing,” and “Calculate 3D” tools to edit the images, high-quality 3D models of the C1 and C2 were obtained Figures . In addiThe 3D models of the C1, C2, and cylinder were exported as stereolithography files and then imported into Geomagic Studio 12 . The “Advanced Object Mover” tool was used to design the optimal position of the cylinder, simulating the optimal screw entry point and angle Figures . Then, aIn the conventional method group, after the posterior atlantoaxial bony structures of the cadaver specimens were exposed, the surgeons (Li and He) used Harms technique  to placeIn the RPDT navigation group, aside from exposing the posterior atlantoaxial bony structures of the specimen as in the conventional method group, the soft tissue on the posterior part of the C1 and the lamina and spinous process of the C2 was removed to completely expose the bony surfaces. Then, the RPDT was placed on the corresponding bony surface. At this point, the surgeons ensured that the RPDTs fit properly and stably on the bony structures, that is, made lock-and-key contact. The screw holes were drilled via RPDT navigation Figures , and theAfter all the screws were inserted into the targeted vertebrae, the C1 and C2 were separated from the specimen, and the authors directly observed and assessed the positions of the screws.Grade 1: the entry point of the C1-LMS is projected onto the middle 50% of the junction of the C1 posterior arch, and the trajectory of the C1-LMS is in the middle 50% of the corresponding lateral massGrade 2: the entry point of the C1-LMS is projected onto the peripheral 50% of the junction of the C1 posterior arch, and the trajectory of the C1-LMS is in the peripheral 50% of the corresponding lateral massGrade 3: the entry point of the C1-LMS is projected onto the external portion of the junction of the C1 posterior arch, or the screw perforates the cortical bone of the C1 lateral massThe positions of the C1-LMSs were assessed in the axial and sagittal plane. In the axial plane, the positions of the C1-LMSs were classified into the following 3 grades :Grade 1:Grade 1: the entry point and trajectory of the C1-LMS are in the upper 50% of the posterior inferior portion of the C1 lateral massGrade 2: the entry point and trajectory of the C1-LMS are in the lower 50% of the posterior inferior portion of the C1 lateral massGrade 3: the entry point of the C1-LMS is under the inferior articular process of the C1 lateral mass, or the screw perforates the cortical bone of the C1 lateral massIn the sagittal plane, the positions of C1-LMSs were also classified into 3 grades, as follows :Grade 1:Grade 1: the screw is completely within the pedicle and the centrumGrade 2: the screw perforates the cortical bone of the vertebra, but more than 50% of the screw diameter remains within the vertebraGrade 3: the screw perforates the cortical bone of the vertebra and more than 50% of the screw diameter is outside the vertebraGrade 4: the screw completely perforates the cortical bone of the vertebraThe positions of the C2-PSs were classified into the following 4 grades , 24:Gradt-tests were used to analyse differences in the operating and fluoroscopy times of screw placement between the conventional method group and the RPDT navigation group. The Mann–Whitney U test was used to analyse the differences in screw placement accuracy between the 2 groups. p value of less than 0.05 was considered statistically significant.The data are presented as the mean ± SD, and SPSS 18.0 was used for the statistical analysis. Independent-sample In the conventional method group, a total of 22 C1-LMSs and 22 C2-PSs were placed into the targeted vertebras. In the axial plane, 16 C1-LMSs (72.7%) were classified as grade 1, and 4 (18.1%) were classified as grade 2. Two C1-LMS (9.2%) were classified as grade 3 because the entry point was too lateral. In the sagittal plane, 20 C1-LMSs (90.1%) were classified as grade 1, and 2 (9.9%) were classified as grade 2. No C1-LMSs were classified as grade 3. Regarding C2-PS placement, 16 C2-PSs (72.7%) were completely inside the cortical bones and were classified as grade 1. Four (18.3%) and 1 (4.5%) C2-PSs partially perforated the cortical bones and were classified as grades 2 and 3, respectively. One C2-PS (4.5%) was completely inside the transverse foramen and was classified as grade 4 .In the RPDT navigation group, a total of 24 C1-LMSs and 24 C2-PSs were placed into the targeted vertebrae. In the axial plane, all the C1-LMSs (100%) were in the middle of the lateral mass and classified as grade 1. In addition, in the sagittal plane, all the C1-LMSs (100%) were in the upper 50% of the posterior inferior portion of the C1 lateral mass and classified as grade 1. No C1-LMSs were classified as grade 2 or 3. Regarding the C2-PS placement, 23 C2-PSs (95.8) were completely inside the cortical bones and classified as grade 1. There was 1 C2-PS (95.8%) that partially perforated the cortical bones and was classified as grade 2. The analysis of the outcomes showed that the application of RPDTs improved the accuracy of C2-PS and C1-LMS placement in the axial plane compared with the conventional method. However, RPDTs did not significantly increase the accuracy of C1-LMS placement in the sagittal plane .The average operating time for screw placement in the RPDT navigation group was 2.3 ± 0.76 minutes (range: 1 to 3.5 minutes), which was significantly shorter than that in the conventional method group . The average fluoroscopy time for each screw placement in the RPDT navigation group was 0.4 ± 0.8 (range: 1 to 3 for each cadaver specimen), which was also significantly lower than that in the conventional method group  .AAF surgery was performed using the conventional method in a 61-year-old female craniocervical cadaver specimen . After tAAF surgery was performed in a 54-year-old male craniocervical cadaver specimen using RPDTs for screw placement navigation . After tIn Harms technique, malpositioned screws can occasionally cause severe complications . GunnarsOver the past 2 decades, RPDTs have been applied in AAF surgeries to help surgeons place screws accurately –20. GoffOther screw fixation techniques in AAF surgeries also benefit from the RPDT technique, including VPAS , 22, PS A well-designed RPDT structure is critical for outstanding navigation accuracy. Aside from the surface-contact structure, some other design tips should be included in the RPDT construction workflow. First, when performing the preoperative CT scan of the target vertebrae, thin-layer CT scanning must be used because it yields more detailed information about the bony surface, which is essential for constructing a highly accurate 3D model of the target vertebrae . Second,In the present study, 2 C1-LMSs were placed too laterally in the axial plane in the conventional method group, which could carry a potential risk for VAI. In contrast, using the RPDT technique for navigation, all of the C1-LMSs were placed in the middle of the C1 lateral mass, indicating that RPDTs enable highly accurate navigation and decrease the risk of neurovascular injuries in C1-LMS placement. In the sagittal plane, there were no significant differences between the RPDT navigation and conventional method groups. However, all the screws in the RPDT group were optimally positioned, that is, in the upper 50% of the posterior inferior portion of the C1 lateral mass, which also demonstrated the applicability of the RPDT technique for protecting the C2 nerve root from screw-related injuries , 9. One Regarding the position assessment of the C2-PSs, 23 of 24 screws (95.8%) in the RPDT group were completely inside the pedicle and the cortical bone of the centrum, which was a significantly greater proportion than that in the conventional method group (72.7%). In the RPDT group, 1 screw deviated from the designed trajectory and partially perforated the corresponding pedicle (grade 2). In this case, we rechecked the vertebra and found some soft tissue on the corresponding C2 lamina, which caused the RPDT to fit the C2 improperly, providing an incorrect navigation direction to the surgeons. Thus, it is imperative to completely remove the soft tissue on the corresponding bony surfaces to ensure that the RPDT properly fits the vertebra and provides an accurate navigation direction to the surgeons. In addition to absolutely remove the soft tissue to ensure optimal RPDT accuracy, some other precautions should also be noted to obtain optimal navigation results. One is that the RPDT should be constructed to be more than 2 mm in thickness; otherwise the RPDT may be too soft and deform during the screw hole drilling procedure, which may result in an incorrect screw hole direction being drilled. The other is that, during the screw hole drilling procedure, the RPDT should be fixed on the bony surface by hands or forceps such that it cannot move and change the navigational direction.Studies evaluating the accuracy of RPDTs in AAF surgeries have used CT scans as the gold standard for assessing screw position because CT scans can clearly demonstrate the spatial relationship between the screws and the vertebrae –22. HoweAs shown in the results, in addition to providing a high navigation accuracy, the RPDT technique also significantly decreased the operating time and radiation exposure compared to the conventional method. When using the conventional method for screw placement, surgeons usually need to use intraoperative X-rays to repeatedly evaluate the position of the hand drill or the screw, repeatedly adjusting the entry point and trajectory to obtain the best position; this process increases the radiation exposure of the patients and surgeons as well as the length of the operation . By usinThe RPDT technique also has some limitations. One limitation is that constructing an RPDT is time consuming , 21, 27.The use of an RPDT significantly increases the accuracy of C1-LMS and C2-PS placement. Meanwhile, this technique can also decrease both screw placement time and radiation exposure. Due to these advantages, the RPDT approach is worth promoting for use in Harms technique.


Ranong Province in southern Thailand is one of the primary entry points for migrants entering Thailand from Myanmar, and borders Kawthaung Township in Myanmar where artemisinin resistance in malaria parasites has been detected. Areas of high population movement could increase the risk of spread of artemisinin resistance in this region and beyond.A respondent-driven sampling (RDS) methodology was used to compare migrant populations coming from Myanmar in urban (Site 1) vs. rural (Site 2) settings in Ranong, Thailand. The RDS methodology collected information on knowledge, attitudes, and practices for malaria, travel and occupational histories, as well as social network size and structure. Individuals enrolled were screened for malaria by microscopy, Real Time-PCR, and serology.P. falciparum in Site 1; 10 P. vivax, 1 Pf, and 1 P. malariae in Site 2). PCR analysis demonstrated an overall weighted prevalence of 0.5%  in the urban site and 1.0%  in the rural site for all parasite species. PCR positivity did not correlate with serological positivity; however, as expected there was a strong association between antibody prevalence and both age and exposure. Access to long-lasting insecticidal treated nets remains low despite relatively high reported traditional net use among these populations.A total of 619 participants were recruited in Ranong City and 623 participants in Kraburi, a rural sub-district. By PCR, a total of 14 (1.1%) samples were positive (2 The low malaria prevalence, relatively smaller networks among migrants in rural settings, and limited frequency of travel to and from other areas of malaria transmission in Myanmar, suggest that the risk for the spread of artemisinin resistance from this area may be limited in these networks currently but may have implications for regional malaria elimination efforts. Historically, resistance to anti-malarial drugs emerged first in the Greater Mekong Sub-region (GMS) to chloroquine, sulfadoxine-pyrimethamine (SP), and mefloquine, and population movements were partially responsible for the spread of the resistant parasites to other countries and regions ,2. SinceRanong Province in southern Thailand is known to be one of the primary points of entry for migrants entering Thailand from Myanmar. Ranong historically has had both a high incidence of malaria and a high proportion of migrants (approximately 50% of residents are from Myanmar) compared to other Thai provinces. From the routine surveillance system, the annual parasite incidence (API) trend reported from Ranong Province was 12.0, 7.1, and 9.9 per 1,000 population in 2010, 2011, and 2012, respectively. By comparison, the national API was reported to be below 1 per 1,000 during this period.Understanding the movement of migrant and mobile populations is essential to curb the potential spread of the resistant parasites, but the characteristics of this group make them inherently difficult to study. They are thought to be highly mobile, often hidden, and difficult to track with routine surveillance and to target with health interventions. Current standard cross-sectional household survey methods are inadequate to obtain representative information on this hidden, transient population due to the absence of an appropriate sampling frame. In an attempt to address some of these methodological issues, respondent-driven sampling (RDS) approach was adapted ,7 as a pRespondent-driven sampling is a modified chain-referral or snowball sampling technique used to approximate more precise estimates from hidden populations and has been used to study HIV risk groups  despite The aim of this study was to determine the migratory patterns, occupational risk, healthcare-seeking and malaria prevention behaviors, network associations, and parasite infection/exposure among mobile and migrant populations along the Thai-Myanmar border in an area with known artemisinin-resistant malaria parasites and to provide a reasonable sampling frame for estimates in these hard-to-reach populations. The need for such a survey was based on the underlying assumption that these mobile populations may represent a high-risk group and may contribute to the spread of the artemisinin resistant parasites, yet information on their malaria risk, migratory and network patterns have been limited.In October 2009, an RDS study on migrant workers was carried out in Thailand along the Thai-Cambodia border and later a similar study was implemented in Cambodia to better understand internal migration patterns in two provinces on the border in the context of artemisinin resistance ,11. WhilTwo sites  were selected in Ranong Province, a southern province in Thailand that historically has had high malaria incidence and serves as an entry point for migrants from Myanmar. Initial recruits (seeds) were selected from migrant worker populations of Ranong City (urban) and Kraburi Sub-district . Since there was not expected to be inter-mixing between the two study sites, sample size calculations were obtained for each site and sites were analyzed separately. With a migrant population proportion of 50%, design effect of 1.5, 95% confidence level, and a non-response rate of 10%, a total sample size of 600 participants was required for each study site.Project staff recruited six seeds from each selected site for diversity in gender, age, and occupation. In total, 16 seeds were required (6 seeds for Site 1 and 10 seeds for Site 2) to reach the specified sample size. For Site 2 , initial seeds were not able to recruit sufficient participants in a timely manner due to the remoteness of some locations and mobile teams were used to reach the desired sample size.The inclusion criteria for the study included: 1) not being a Thai citizen; 2) coming to find work or economic advantage, or with someone who is; 3) age of at least 18 years; 4) no prior participation in this survey; and 5) provision of informed consent prior to enrollment. Pregnant women were not excluded as they were referred to receive nationally recommended treatment if found to be positive for malaria.All enrolled participants were asked about their demographic background, migratory pattern, work history, health care seeking behavior including access and barriers to health messages, health status, and knowledge of malaria including its prevention. Data collection began on 1 May 2012 and ended on 15 July 2012 during the rainy season.To minimize data collection errors, handheld personal digital assistants (HP iPAQ model HX 2007) with automatic data checks and skip patterns in the Myanmar language were used and the interviewers received training on the handling and use of these tools. Data were exported to Microsoft EXCEL. Following data cleaning and verification, analysis was performed using the Respondent-Driven Sampling Analysis Tool (RDSAT) Version 7.1.38 . The datIn order to assess current and previous malaria exposure, participants were asked to provide a blood sample for microscopy and filter paper collection to assess for current malaria infection using PCR and previous malaria exposure using serology. Reading of thick and thin blood smears was performed by local malaria staff and microscopy positive individuals were provided with antimalarial treatment according to the Thailand National Treatment Policy. All malaria positive slides and 10% of negatives were re-read at the National Reference Laboratory in Bangkok, Thailand. For molecular analyses, standard pooled, Real-Time PCR (RT-PCR) assays were used to differentiate plasmodia infections in dried blood spots . UniversP. falciparum and P. vivax using methodologies previously described [P. falciparum and P. vivax), 1/1000 for MSP-2, 1/200 for CSP and 1/2000 for AMA . Optical density was read at 492nm and antibodies reported as titers (as determined by standard on the plate).For serology, dried blood spot samples were eluted and assayed against antigens for both escribed . BrieflyApproval was obtained by the Ethics Committee for Research in Human Subjects of the Department of Disease Control, Ministry of Public Health, Thailand (FWA 00013622) and the US Centers for Disease Control and Prevention. Prior to enrollment in the study, participants provided written informed consent in Myanmar language. The inform consent form and procedures were also approved by the Ethics Committee.A total of 619 participants were recruited in Ranong City (Site 1) and 623 participants in Kraburi sub-district (Site 2). Age distributions between the two sites were different, with proportionally younger participants in the rural site compared to the urban site . The meaAll participants were migrants from Myanmar. Migrants from other countries were not found in this study. Nearly all respondents in both sites were schooled in Myanmar (95%) and reported to be able to read Myanmar (>99%); although only 7% of migrants in Site 1 and none in Site 2 were able to read Thai. The most commonly used spoken languages among the respondents in both sites were Myanmar and Dawai. Nearly one-third of migrants in Site 1 were able to speak Thai. The majority of the respondents in both sites were long-term migrants classified as M1 (migrants living in Thailand for 6 months or more). Migrants in Site 1 reported having lived in Thailand on average 79.8 months compared to 61.6 months among migrants in Site 2. More than one-third of migrants in both sites had lived in Thailand for more than 5 years .The primary reason for participants to come to Thailand was for work; more than half were assisted by relatives already living in Thailand. Very few migrants used middlemen to broker their trip to Thailand suggesting that most travel arrangements were done on an individual basis. One-fifth (20%) of those surveyed in Site 2 reported that they were planning to move to another location (mostly back to Myanmar) suggesting a more transient population in the rural site compared to the urban site (5%). Up to 33% and 22% of migrants from Site 1 and Site 2, respectively, had ever returned back to Myanmar. This may be due to the high cost of travel to return to Myanmar—the cost per trip reported by participants ranged from 2,000 to 300,000 Kyats .Differences were detected between Ranong city (Site 1) and Kraburi sub-district (Site 2) in terms of the occupational and residency profile of the migrants . MigrantKnowledge about malaria and how it is transmitted was quite high among the migrants in both sites . While kOnly one third (33%) and 19% of migrants in Site 1 and Site 2, respectively, reported having heard a health message within past 3 months. Among those who received health messages in Site 1, the majority reported receiving those messages through health education by health workers (66%) and brochures (68%). In Site 2, the most common channels were through radio (76%) and health education by health workers (71%). Respondents in Site 1 preferred migrant volunteers (64%) and brochures (47%); whereas those in Site 2 preferred getting their information about health through health workers at facilities (57%) and the radio (49%). Malaria-related messages were more commonly heard in the rural Site 2 compared to the urban Site 1, where most health messages were about HIV/AIDS, STDs, and TB.Treatment-seeking behaviors for fever among migrant populations were generally good. Half of the respondents in Site 1 and 87% in Site 2 reported having gone to a public government hospital for their last episode of fever . In bothOwnership of at least one mosquito net was high in both sites (94% in Site 1 and 83% in Site 2) . Among tTwo-thirds of respondents in Site 1 preferred to use conventional untreated nets; whereas an overwhelming majority (82%) of respondents in Site 2 preferred to use long lasting insecticidal treated nets (LLINs). Although not specifically probed, this difference could be due to the repellency effect of the LLIN.Migrant workers in the urban site generally were employed during the day, while those in the rural site were generally working at night, mostly in the rubber plantations. Nearly all migrant workers in Site 2 reported having slept the previous night in housing structures or sleeping areas provided by their employers and having slept under a mosquito net. However, most of these mosquito nets were not LLINs even though there was a strong preference for LLINs and most mosquito nets had been purchased from the shops or market in both sites.P. vivax positive case was detected in an individual from Site 2. This individual was a 48-year old female from the Dawai ethnic group, who had lived in Thailand for 3 years, and had traveled back to Myanmar once or twice per year. However, this individual was found to be both PCR and serologically confirmed negative, which might suggest to the possibility of a false positive slide reading despite double cross-checking.Although the prevalence of malaria detected during the period of the survey was found to be very low in both sites, there was marginal statistical association (p = 0.054) between having traveled back to Myanmar in the past 12 months and serological positivity among respondents in Site 1, but not statistically different among respondents in Site 2 (p = 0.216) using multi-variate logistic regression analysis. By double-read microscopy, only one (1) P. vivax (10/14) with two P. falciparum, one mixed P. falciparum and P. vivax, and one P. malariae infections. PCR analysis demonstrated an overall weighted prevalence of 0.5%  in the urban site (Ranong City) and 1.0%  in the rural site (Kraburi) for both parasite species—although the predominant species was P. vivax, which presents a limited risk for spread of artemisinin resistance and to date has only been identified in P. falciparum.By PCR, 14 samples were found to be positive  . The majP. falciparum (24%) and P. vivax (15%) antigens, compared to the urban site . Serological reactivity to either P. falciparum or P. vivax antigens among the population in Site 2 was 31% compared to 7% in Site 1. Particularly in Site 2, there was a clear association between increasing seroprevalence and age strongly suggesting that seroprevalence may be cumulatively representing current and past exposure  or neighbors (46%) of the respondent; whereas, in Site 2, a majority of the recruiters were identified as employers . The majRespondent-driven sampling has been used for nearly 20 years for the sampling of hard-to-reach populations such as intravenous drug users and commercial sex workers ,19. ThisAccessing migrant populations is intrinsically difficult for public health programs, particularly those in the GMS, where borders are porous and often where illicit forest-related activities occur. Furthermore, migrant populations who are unregistered and without work permits or have entered the country illegally often will avoid government facilities for fear of being caught, which makes accessing these populations that much more difficult. The majority of migrant workers in both study sites have work permits and were likely more amendable to enrolling in this study. Developing innovative and effective methods of reaching migrant populations was identified as a priority for responding to artemisinin resistance , and ultP. vivax malaria case, raising the question whether microscopy alone is sufficient in such low transmission settings.To our knowledge, this is the first use of RDS to collect biological specimens for malaria from cross-border migrant populations. More than 1,200 migrants from Myanmar who are living and working in urban and rural sites in Thailand were screened using standard microscopy, q-PCR, and serological assays for the detection of malaria infection, previous exposure, and assessed for their access to malaria prevention and treatment services. Malaria prevalence detected through microscopy yielded only one P. falciparum and P. vivax yielded an interesting distribution of seropositivity when comparing between the urban and rural sites. Sero-reactivity to P. falciparum antigens ranged from 4% (2%-6%) in Site 1 compared to 24% (21%-29%) in Site 2, suggesting not surprisingly, significantly higher risks of P. falciparum infection in those residing in the rural site compared to the urban site. Similar patterns of sero-reactivity to P. vivax antigens were also observed between the two sites. These results suggest that the rural site, although having only one parasitemic individual identified through gold-standard microscopy at the time of this cross sectional survey, may still experience substantial seasonal malaria transmission as evidenced by the prevalence of antibodies in these relatively stationary migrant populations. Additional investigations may be needed to determine whether these individuals were exposed and infected in Myanmar or in Thailand, though the data suggest that cross border movement occurs less frequently in this population than previously expected. Furthermore, in Site 1, the two malaria cases detectable by PCR were P. falciparum and serologically negative; whereas in Site 2, most of the PCR positive cases were P. vivax with a much more diverse serological profile, suggesting greater exposure to malaria antigens over time.To ascertain previous malaria infection, serology has been proposed as a useful way for estimating exposure to malaria parasites in low transmission settings –26. SeroMost migrants in Ranong Province are long-term residents and have not traveled frequently back to Myanmar. According to the definition used in Thailand, these migrants would be considered M1 migrants (those who have lived in Thailand for 6 months or more). Migrants in both sites had access to and reported use of mosquito nets; however, the vast majority of these were conventional, untreated mosquito nets that were purchased in the markets. The current net culture that exists among these migrant populations is encouraging, and strategies  should be considered to replace and/or convert these conventional nets into more effective LLINs that could have greater impact on the reduction of malaria transmission. Furthermore, three-quarters of those in Site 2 were rubber tappers and alternative outdoor personal protection methods  should also need to be considered.Examination of a group’s tendency to associate within or outside one’s own networks, average network sizes, and social network analyses offer insights into the potential use of influential individuals to promote health and treatment-seeking behaviors. This study highlights that the network sizes of migrants in urban Ranong are larger and more homophilous  compared to migrants in the rural setting, who tend to have smaller network sizes and stronger affiliations with their employers. Using RDS to better understand the social and occupational networks of migrants in different settings could help malaria programs better target delivery of malaria services such as distribution of LLINs, health education and promotion, or even routine screening and treatment of migrant populations. The benefits of identifying migrants who otherwise would not have been reached through routine public services, and providing health care services for these individuals are worth considering. Furthermore, analysis of networks and mobility patterns through the use of RDS can contribute valuable epidemiological and service utilization data from these hard-to-reach populations which is increasingly critical for malaria surveillance in the context of elimination.There were challenges and limitations that may have affected the outcome of this study. Firstly, relatively high transportation costs and long travel distances to the enrollment site may have affected recruitment of participants in the more rural site. Measures were put in place to mitigate these barriers, including moving the recruitment sites closer to the migrants wherever possible. Secondly, there may be a risk of convenience sampling occurring if coupons were not distributed through networks. Therefore, the selection of seeds is critical  and shouThe low prevalence of malaria parasitemia, relatively homogenous networks among these migrants, and limited frequency of travel to and from other areas of malaria transmission, specifically Myanmar, all suggest limited potential for spread of artemisinin resistance along the Kawthaung-Ranong corridor through these populations but the frequency and extent of population mobility in this region can be variable. Described here, RDS can be a potential programmatic tool to obtain reasonably representative estimates among migrant populations, to better understand different migrant networks and behaviors, and ultimately to improve access to and delivery of malaria services to these hard-to-reach populations and their associated networks.


Anti-B cell activating factor belonging to TNF-family (BAFF) antibody therapy is indicated for the treatment of patients with active systemic lupus erythematosus (SLE). We hypothesized that the BAFF receptor, transmembrane activator and calcium-modulator and cyclophilin interactor (TACI) may be responsible for the generation of antibody secreting plasma cells in SLE. To test this hypothesis, we generated TACI deficient MRL-Fas/Lpr (LPR-TACI−/−) mouse. TACI deficiency resulted in improved survival of MRL-Fas/Lpr mice and delayed production of anti-dsDNA and anti-SAM/RNP antibodies. There was also a delay in the onset of proteinuria and the accumulation of IgG and inflammatory macrophages (Mϕs) in the glomeruli of young LPR-TACI−/− mice compared to wild-type mice. Underscoring the role of TACI in influencing Mϕ phenotype, the transfer of Mϕs from 12-week-old LPR-TACI−/− mice to age-matched sick wild-type animals led to a decrease in proteinuria and improvement in kidney pathology. The fact that, in LPR-TACI−/− mouse a more pronounced delay was in IgM and IgG3 autoreactive antibody isotypes and the kinetics of follicular helper T (Tfh) cell-development was comparable between the littermates suggest a role for TACI in T cell-independent autoantibody production in MRL-Fas/Lpr mouse prior to the onset of T cell-dependent antibody production. Although conventional agents such as corticosteroids, antimalarial drugs, and other immunosuppressive therapies provide enhanced survival in patients with SLE, their use is associated with considerable toxicity and a large proportion of patients remain refractory to the treatment5. Shortly after the discovery of B cell activating factor belonging to TNF-family (BAFF) as a cytokine critically important in B cell survival6, an association between SLE and elevated circulating BAFF was established in mouse models7 and in SLE patients9. Underscoring the role of BAFF in SLE, depletion or ablation of BAFF in lupus prone mice resulted in reduced disease severity and mortality11. In contrast to BAFF, both positive and negative correlations were observed between serum levels of the related cytokine, a proliferation-inducing ligand (APRIL), and disease severity18. Investigation of APRIL’s contribution to SLE in mouse models also produced inconclusive results. For example, neither the transgenic mice overexpressing APRIL nor the SLE-prone NZM mice with disrupted APRIL gene manifested a change in autoimmune phenotype20. However, ablation of APRIL with selective monoclonal antibody resulted in delayed SLE development in NZM mice and APRIL disruption in Nba2.Yaa mice improved SLE disease with reduced pathogenic antibody production and glomerulonephritis development22. Recognition of the importance of BAFF in the generation and/or sustainment of autoreactive B cells led to BAFF inhibition strategies as a treatment for SLE23. These efforts resulted in the approval of Belimumab  in 2011 for the treatment of SLE patients with active, autoantibody-positive disease25.Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody overproduction because of dysregulated innate and adaptive immune function26. Although the alleviation of disease symptoms with anti-BAFF therapy underscored the importance of BAFF in SLE pathogenesis, the contribution of individual receptors to BAFF action is poorly understood. Compared to healthy subjects, B cells from patients with SLE have lower or similar BAFFR expression. The expression of BCMA and TACI in the same patients have been reported as higher, lower or the same as healthy individuals31. To address the role of these receptors in SLE, several different lupus mouse strains with deficiencies in individual or combination of receptors were generated. The deletion of BAFFR alone or a loss of function mutation of BAFFR was not beneficial in any tested mouse strain33. Deletion of BCMA in B6 LPR mice resulted in increased lethality, while the course and severity of disease in BCMA-deficient NZM mice was not different than the wild-type mice34. Attempts to study the role of TACI in SLE mouse models have been less conclusive. For example, TACI deficient NZM mice survival was not affected despite manifesting with increased renal pathology32. On the other hand, using a bone marrow chimera strategy, Figgett et al. have shown that TACI deficiency prevented the onset of autoimmune manifestations in BAFF transgenic mice35. TACI deficiency was also beneficial in NZM mice deficient in both TACI and BAFFR36. Finally, a more recent study reported decreased mortality accompanied by lower levels of autoantibodies and glomerulonephritis in TACI deficient male Nba2.Yaa mice22.BAFF binds to BAFF receptor (BAFFR), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B-cell maturation antigen (BCMA), while APRIL only exhibits high affinity to TACI and BCMAfas gene37. We observed that TACI-deficient MRL-Fas/Lpr mice (LPR-TACI−/−) survived significantly longer than the wild-type mice (LPR-TACI+/+). The delayed mortality was accompanied by a delay in autoreactive antibody development and the onset of lupus nephritis. In SLE, the inflammation initiated by the deposition of immune complexes in glomeruli leads to the accumulation of M1-polarized macrophages (Mϕs), which augment kidney pathology39. Conversely, the M2-polarized Mϕs are believed to maintain a protective anti-inflammatory environment. The kidneys of 12-week-old LPR-TACI−/− mice were populated with M2-like Mϕs, while the age matched WT mice kidneys were infiltrated with M1-like Mϕs. The M2-phenotype of Mϕs isolated from LPR-TACI−/− mice is consistent with our recent discovery of a role for TACI in mediating M1 polarizing signals40. The fact that spontaneous accumulation of germinal center (GC) B cells, plasma cells, follicular T helper (Tfh) cells as well as extrafollicular Tfh (Thef) cells in LPR-TACI−/− mice was not significantly different than the wild-type mice suggested that TACI deficiency does not impact the development T cell-dependent autoreactive antibodies. Instead, TACI deficiency is likely responsible for the delay in disease onset by preventing T cell-independent autoreactive antibody production and by maintaining an anti-inflammatory environment in kidneys owing to M2 phenotype of TACI deficient Mϕs.In light of these diverse outcomes regarding the role of TACI in SLE, we sought to investigate the contribution of TACI in MRL-Fas/Lpr mice, which develop SLE like autoimmune manifestation as a result of a spontaneous mutation in the 37. To examine the impact of TACI on the phenotype of MRL-Fas/Lpr mice, we generated TACI-deficient mice on an MRL-Fas/Lpr background (LPR-TACI−/−). TACI deficiency was confirmed by evaluating surface TACI protein expression on total B cells (CD19+), plasmablasts (CD19 + CD138+), and plasma cells (CD19 − CD138+) in spleens of LPR-TACI+/+, LPR-TACI+/−, and LPR-TACI−/− mice . As in human SLE, T and B cell-hyperproliferation causes lymphadenopathy in MRL-Fas/Lpr micethy Fig. . Howeverthy Fig. . Thus, T43. Since TACI is critically important in the development and maintenance of plasma cells47, we expected TACI deficient MRL-Fas/Lpr mice to mount a lesser antibody response against nuclear material. From 6 to 12 weeks of age, serum levels of anti-dsDNA IgG and anti-RNP/Sm total antibodies increased progressively with age in all three groups  Mϕs in the kidney coincides with the onset and progression of SLE disease. Furthermore, the persistence of activated Mϕs is highly associated with poor clinical outcomesice Fig. . Moreoveice Fig. . To furtice Fig. . These gTo further interrogate the significance of TACI deficient Mϕs in the prevention of kidney dysfunction, we performed adoptive transfer experiments. Ten-week-old MRL-Fas/Lpr mice with moderate proteinuria were transferred with Mϕs isolated from kidneys of 12-week-old LPR-TACI+/+ or LPR-TACI−/− mice. Mice injected with LPR-TACI+/+ Mϕs manifested slightly elevated proteinuria as compared to PBS injected or naïve mice until 7 days after the first injection Fig. . In cont53, we next assessed the frequencies of these cells to determine whether the delayed anti-dsDNA and RNP autoreactive antibody production is associated with altered plasma cell development. As expected, in addition to total B cell numbers, plasmablasts and plasma cells populated spleens of wild-type MRL-Fas/Lpr lupus mice with age significantly more than Balb/c mice or control MRL mice  all contribute to the GC-dependent and extrafollicular autoreactive antibody production58. By using GL-7 and PNA markers  or TACI-Ig had little effect on autoantibody production in NZB/W F1 mice60. In contrast, the same adTACI construct could significantly decrease the autoantibody production in MRL Fas/Lpr mice60. The relative independence of NZB/W F1 mice from BAFF for the lupus phenotype can be explained by the fact that NZB/W F1 mice SLE pathogenesis is linked to major histocompatibility complex class II (MHCII) locus and pathogenic autoantibody production is primarily dependent on CD4+ T cells61. On the other hand, SLE development in MRL-Fas/Lpr mice is attributed to a spontaneous mutation in Fas, which mediates death signal in cells. This mutation is responsible for the accelerated onset of autoimmune disease in the lupus-prone MRL strain62. Since TACI is essential for T cell-independent humoral response43, deletion of TACI in NZM mice may not have a profound effect on T cell-dependent autoantibody production in this strain. Our analysis of GC B cells, Tfh cells and Thef cells confirmed the limited role TACI plays in T cell-dependent autoantibody production in MRL-Fas/Lpr mice. Spontaneous GC formation and Tfh as well as Thef cell development are implicated in autoreactive antibody development in patients and in lupus mice63. Indeed, we found that MRL-Fas/Lpr mice GC B cells, Tfh, and Thef cell frequencies were markedly higher than those of wild-type MRL mice and Balb/c mice as early as 6 weeks of age. TACI deficiency appears to have contained this rapid increase in the number of GC B cells and Tfh cells until 8 weeks of age, which may have contributed to the delayed onset of autoreactive antibody development in LPR-TACI−/− mice during this early period. A more profound effect of TACI deficiency on autoreactive antibody production may have been the prevention of T cell-independent autoantibody generation, since TACI is essential for the production of antibodies against polysaccharide antigens45. The elevated anti-dsDNA serum IgM and IgG3 antibodies as early as 8 weeks of age in wild-type littermates but not in LPR-TACI−/− strains is in support of an impairment of T cell-independent humoral immune response in TACI deficient lupus mice. The delay in the emergence of the T cell-independent isotype, IgG3 in LPR-TACI−/− mice is especially noteworthy because of the well documented role of IgG3 autoantibodies in the pathogenesis of lupus nephritis66. Thus, the delay in the onset of kidney pathology and associated proteinuria in LPR-TACI−/− mice may be related to the late appearance of IgG3 antibodies in this strain. A similar conclusion was reached by Tran et al. who recently attributed the improved survival of TACI deficient male Nba2.Yaa mice to diminished T cell-independent autoreactive antibody production22.In contrast to TACI deficient NZM mice, which exhibited a clinical course identical to the wild-type mice67. In addition to the well-recognized M1-polarizing signals, such as IFN-γ and TNF-α69, systemically and locally elevated BAFF and APRIL70 may be potentiating the M1-polarization of Mϕs in SLE. We recently discovered that TACI mediates M1-polarizing signals in Mϕs of C57BL/6 mice and freshly isolated Mϕs from TACI deficient C57BL/6 mice peritoneum and skin present with phenotypic and functional characteristics of M2 Mϕs40. Analysis of young LPR-TACI−/− mice peritoneal Mϕs confirmed the preservation of M2-skewed phenotype in TACI deficient lupus mouse. Moreover, the kidneys of 12-week-old LPR-TACI−/− mice not only harbored fewer numbers of Mϕs than the kidneys of wild-type littermates, but their Mϕs also expressed elevated levels of molecules associated with the M2 phenotype. In concert with the significantly progressed proteinuria, kidneys of 12-week-old wild-type littermates manifested more severe histopathological changes along with the infiltration of M1-skewed Mϕs. The predominance of M2 polarized Mϕs in 12-week-old LPR-TACI−/− mice kidneys suggested that LPR-TACI−/− mice renal Mϕs may be responsible for the low proteinuria score in TACI deficient lupus mice. Supporting this hypothesis, not only did the transfer of Mϕs from age-matched LPR-TACI−/− mice halt the progression of proteinuria in 12-week-old LPR-TACI+/+ mice, but also diminished the renal histopathological changes. However, although TACI deficient lupus mice Mϕs present with M2 phenotype, they appear to succumb to inflammatory signals initiated with immune complexes because the increase in systemic autoreactive antibodies correlated with the progression of proteinuria and conversion of the renal Mϕ-phenotype to M1. Nevertheless, our study highlights a role for M2-polarized Mϕ-transfer as a therapeutic intervention to alleviate renal impairment in SLE disease71.In SLE, inflammatory signals initiated by immune complexes and cytokines lead to accumulation of M1 polarized Mϕs in the glomeruli and interstitial compartmentsIn summary, we discovered a novel function for TACI deficient Mϕs in protecting from renal disease in SLE. Together with the M2-skewed Mϕs, late onset of autoreactive antibodies contributes to the improved survival of LPR-TACI−/− mice. The primary reason for the delayed development of autoreactive antibodies appears to be the impairment in T cell-independent antibody production when TACI is absent. Profound differences in the IgM and IgG3 anti-dsDNA antibodies between LPR-TACI−/− and LPR-TACI+/+ mice during the early phases of disease is in support of this hypothesis. A meaningful contribution of the reduction in T cell-dependent antibody responses to the overall delay in autoreactive antibody production is unlikely, because only 6-week-old LPR-TACI−/− mice GC and Tfh frequencies were significantly less than the wild-type littermates. Interestingly, although M2-skewed Mϕs may be protecting LPR-TACI−/− mice from kidney pathology at early stages of disease, TACI deficient Mϕs were unable to resist the M1 polarizing cues associated with the deposition of immune complexes in the kidneys. Thus, in addition to the early ablation of T cell-independent autoreactive antibody production, M2-Mϕ mediated preservation of kidneys from inflammatory assault appears to contribute to the improved survival of LPR-TACI−/− mice.MRL/MpJ-FASLPR/J (will be referred to as MRL-Fas/Lpr throughout the manuscript) and MRL/MpJ (will be referred to as MRL throughout the manuscript) mice, were purchased from The Jackson Laboratory . Balb/c mice were purchased from Charles River Laboratories . We generated LPR-TACI−/− using a backcross-intercross strategy for more than 10 generations to obtain mice with a homozygous mutation of both FAS and TACI. The progeny were screened by polymerase chain reaction (PCR) amplification of tail genomic DNA using primers for the TACI wild-type gene  and TACI deficiency . TACI deficient mice were also screened for 178 microsatellite markers to select the offsprings with the highest percentage of MRL genetic background. All mice were bred and housed under specific pathogen-free conditions. Only age-matched female mice were used for experiments. All mice were bred and maintained under specific pathogen-free conditions in the animal facility of US Food and Drug Administration/Center for Biologics Evaluation and Research Veterinary Services. The breeding and use of animals were approved by and carried out within accordance of the US Food and Drug Administration/Center for Biologics Evaluation and Research Institutional Animal Care and Use Committee (permit numbers 2002–31 and 2002–37). All methods were performed in accordance with the relevant guidelines and regulations.LPR-TACI−/−, LPR-TACI+/− and LPR-TACI+/+ littermates were scored blindly for proteinuria, lymphadenopathy, and skin lesions. Cervical, brachial, and inguinal lymphadenopathy was assessed daily beginning at 6 weeks of age. Lymph nodes were palpated and the date of swollen lymph nodes in littermates was recorded. Proteinuria of female mice was measured at the ages of 3 and 20 weeks using Fisherbrand™ Urine Reagent Strips  and scored on a scale of 0–5, 0, none; 1, trace; 2, 30 mg/dl; 3, 100 mg/dl; 4, 300 mg/dl; and 5, ≥2000 mg/dl.Kidneys were fixed in 10% buffered formalin overnight, were processed, and were embedded in paraffin. Sections were stained with Hematoxylin and Eosin (HE), Periodic Acid Schiff (PAS), Periodic acid methenamine silver (PAM) and Mason’s trichrome. All slides were digitally scanned by Hamamatsu Nanozoomer-XR digital slide scanner  for semiquantitative analysis of histopathological examination. Glomerular changes, inflammatory cell infiltration, and interstitial fibrosis were evaluated and scored based on a 0–3 intervals of 0.5. 0 = no pathology, 1 = mild pathology, 2 = moderate pathology and 3 = severe pathology.Freshly isolated mouse kidneys were flash-frozen in Tissue-Tek® OCT compound in liquid nitrogen, then stored at −80 °C until cryosectioning at −20 °C. Seven µm cryosections were cut onto SuperFrost Plus slides (Menzel-Glaser), fixed in acetone for 5 min and then air-dried at room temperature. Renal immunoglobulin (Ig) and C3 deposits were detected with anti-mouse IgG-Alexa Fluor 488  or anti-mouse C3  antibodies, respectively. To detect serum autoantibodies by indirect microscopy, sera were diluted 1:50 or 1:10 in PBS and applied to HEp-2 slides and Crithidia luciliae slides , respectively. Slides were washed with PBS and stained with anti-mouse IgG-Alexa Fluor 488  and DAPI . All fluorescence slides were viewed with a BX61 fluorescence microscope . Microscope images for each experiment were uniformly analyzed using ImageJ software .Serum anti-dsDNA IgG antibodies were measured by ELISA. Briefly, calf thymic DNA  was coated on a 96-well microtiter plate  at 0.5 μg/ml with 0.1 M of carbonate-bicarbonate buffer (pH 9.6) overnight at 4 °C. Plates were blocked for 30 minutes at room temperature in 5% BSA in PBS, then washed with 0.05% Tween-20 in PBS. Diluted serum samples were added to plates in triplicate and incubated at 37 °C for 2 hours. Plates were washed with 0.05% Tween-20 in PBS and further incubated with HRP-conjugated goat antibodies directed against mouse total IgG, IgG2a, IgG2b, IgG3 and IgM  for 1 hour at room temperature. Finally, plates were washed with 0.05% Tween-20 in PBS and measured at 405 nm absorbance after developing with ABTS Simple Solution .7) were stained with fluorescent anti-mouse antibodies after blocking CD16/CD32 with Fc Block . Flow cytometric analysis of mouse splenocytes was performed using the following antibodies: Pacific blue–anti-CD19, APC–anti-CD138, BV605–anti-CD3, Percp Cy5.5 anti-GL-7, Percp Cy5.5 anti-CD44, FITC anti-62L, PE anti-PSGL-1, PE-Cy7-anti–PD-1, APC anti-CXCR5, BV421 anti-CXCR4, APC anti-CD93, Percp Cy5.5 anti-IgM, APC anti-CD206, Percp Cy5.5–anti-CD86, PE anti-IL-4R, BV421 anti-F4/80, FITC anti-CD11b  was purchased from Vector Laboratories . Qdot605 anti-CD4 antibody was purchased form Fisher Thermo . ATTO 488 anti-BAFFR antibody was from Enzo life Science Inc. . BV605 anti-IgD, FITC anti-BCMA, and PE anti-TACI antibodies were from BD Biosciences\BD Pharmingen . Stained cells were analyzed using a flow cytometer  and data were analyzed using FLOWJO version 10.1 for PC .Single cell suspensions of spleen were obtained by mechanic dissociation of tissue through a 40 μM cell strainer. Kidneys were minced and digested with 2 mg/ml collagenase D at 37 °C for 30 min. The dissociated cells were filtered through a 100 μM cell strainer. Red blood cells were then lysed using ACK lysing buffer . Spleen or kidney cells . Two hundred nanograms of total RNA was reverse-transcribed into cDNA using random hexamers with the Taqman Reverse transcription kit (Invitrogen). The expression of M1 and M2-associated genes and GAPDH were determined using Taqman Gene Expression assays and the CFX96 Touch Real-Time System . Relative expression values were determined by the 2−ΔCt method where samples were normalized to GAPDH expression6/ml) in 10 cm plates and non-adherent cells were washed away after 1 hour of culture. Mϕs were cultured for 48 hours in complete RPMI media. Cells were then washed 3 times in PBS on the plate, with the remaining cells removed using TrypLE Express Enyzme Buffer (Invitrogen). The purity was greater than 95% Mϕs (F4/80+ MHCII+ Ly6G−) as confirmed by flow cytometry. Macrophages were suspended in PBS and 5 × 106 cells in 50 μl were injected i.v. into recipient mice.Isolated F4/80+ peritoneal Mϕs were cultured  and nonparametric testing was performed by the Mann-Whitney rank sum test for two groups and by Kruskal-Wallis one-way ANOVA on ranks for three or more groups. The log-rank test with Tukey-Kramer multiplicity adjustment was performed, using SAS 9.4, to compare survival curves and lymphadenopathy development.Supplemental information


Three homologous cationic porphyrins differently affect the 20S proteasome gating mechanism. meso-tetrakis(4-N-methylpyridyl)-porphyrin (H2T4) and its two ortho- and meta-isomers. We show that in a first, fast binding event H2T4 accommodates in a pocket made of negatively charged and aromatic residues present in α1 , α3 (Tyr5), α5 , α6  and α7  subunits thereby stabilizing the closed conformation. A second, slower binding mode involves interaction with the grooves which separate the α- from the β-rings. Of note, the proteasome inhibition by ortho- and meta-H2T4 decreases significantly if compared to the parent compound, thus underscoring the role played by spatial distribution of the four peripheral positive charges in regulating proteasome–ligand interactions. We think that our results may pave the way to further studies aimed at rationalizing the molecular basis of novel, and more sophisticated, proteasome regulatory mechanisms.The 20S proteasome is a barrel-shaped enzymatic assembly playing a critical role in proteome maintenance. Access of proteasome substrates to the catalytic chamber is finely regulated through gating mechanisms which involve aromatic and negatively charged residues located at the N-terminal tails of α subunits. However, despite the importance of gates in regulating proteasome function, up to now very few molecules have been shown to interfere with the equilibrium by which the catalytic channel exchanges between the open and closed states. In this light, and inspired by previous results evidencing the antiproteasome potential of cationic porphyrins, here we combine experimental  and computational  approaches to inspect proteasome inhibition by  The ability of H2T4 and its two variants to interact with the α subunits of the CP correlates with the observed binding kinetics and inhibition potencies of the investigated molecules. Cationic porphyrins, may thus represent a new class of inhibitors targeting the gate of the CP.Proteasomes are large multi-subunit proteolytic assemblies that play a key role in regulating intracellular protein homeostasis.2T4 inhibits rabbit proteasome activity both in cell lysates and in purified CP preparations. In the present study we have used a commercially purified human 20S proteasome, in order to avoid eventual interactions of porphyrins with possible contaminants of a cell extract. Indeed, the results obtained with isolated 20S proteasome, reported in 2T4 inhibits all the three proteolytic CP activities with similar potency.In a previous work,2T4 > tris-T4 > cis-T4).30Moreover, it is worth recalling that previously reported data evidenced that the gradual decrease of the number of protonated pyridine rings from four to two  correspo50 values of H2T4 with those of the ortho-H2T4 and meta-H2T4 isomeric forms. 50 values of H2T4 and its ortho- and meta- derivatives calculated for the ChT-L, T-L, and PGPH-L CP activity. Dose–response plots were fitted with eqn (2)  than the other two isomeric forms  analysis: (1) the position of positively charged substituents is not relevant for the inhibition of the PGPH-L activity of the 20S proteasome; (2) by contrast, ChT-L and T-L activity are inhibited more effectively as the positively charged substituents are moved far away from the central ring with the highest activity when they are fixed in the para position of the pyridine ring  experiments. Initially, the structure of the ligand dissolved in the buffer  has been analyzed by 1H NMR spectroscopy. In particular, H2T4 1H chemical shift assignment has been obtained as it follows: 9.09  9.27 , 8.89 , 3.69 . Afterwards, 860 nM of 20S proteasome was added to 172 μM of H2T4 (protein : ligand ratio of 1 : 200). 1H H2T4 chemical shifts do not significantly changes upon the addition of 20S proteasome, except for the pyrrole ring resonances, which display shifts downfield (from 9.09 to 9.13 ppm); on the other hand, H2T4 resonances experience an overall soft line broadening. These two effects, line broadening and chemical shift perturbations, which occurred on the proton of the same residues, confirmed the occurrence of H2T4 binding to the proteasome. To structurally characterize the H2T4–20S proteasome interaction, we apply STD (Saturation Transfer Difference) NMR spectroscopy assayIn order to identify the functional groups involved in the H2T4 proton resonances (ISTD = I0 – Isat).ISTD/I0 value, the methyl group of the N-methyl pyridine, was normalized to 100%  ; the obtained value is KI = 3.6 (±0.5) × 10–7 M.The Lineweaver–Burk plot shown in 2T4 binding to the CP have also been determined as a function of time . t = 0 both the substrate  and H2T4 at different concentrations, as reported in the figure legend; the fluorescent signal was transformed into molar concentrations of fluorescent product through a calibration curve resulting from the complete processing of known concentration of substrate.The kinetic parameters of H2T4, employing Km = 7.2 × 10–5 M, kcat = 10 s–1 , and applying values of k+I and of k–I, which were constrained to the obtained KI = (k–I/k+I). The resulting kinetic parameters for the H2T4 binding are k+I = 1.3 × 104 M–1 s–1 and k–I = 4.7 × 10–3 s–1, indicating that H2T4 porphyrin behaves as a fast-reacting inhibitor of the 20S proteasome, since the inhibition appears evident already few seconds after the inhibitor addition.The continuous lines correspond to the simulation curve at different indicated concentrations of H2T4. Notably, the porphyrin turned out to have a high propensity to self-aggregate in the presence of the crystallization buffer. Thus, the ligand concentration in the crystallization drop was infinitesimal. After a soaking time of 24 hours, a dataset was recorded to 2.8 Å resolution, but the FO-FC-electron density map did not display any striking features that could be interpreted as a porphyrin core . Therefore, in order to investigate the molecular details of the interaction of H2T4 with the human 20S proteasome, we performed molecular modeling studies. Firstly, the available experimentally determined structures of 20S were downloaded from the Brookhaven Protein Data Bank  and subjected to a structural and bioinformatics analysis. Then, an homology model of the human 20S proteasome α subunits was built . Therefore, we were able to check the validity of our molecular model against the X-ray structure. Structural parameters were compared using structure evaluator softwarei.e., helices, turns, β-strands) and an RMSD value on all alpha carbons of 0.61 Å  . Obtained results excluded all the 20S catalytic sites and prompted us to hypothesize the substrate gate (closed state) as the most probable H2T4 binding site, for the competitive inhibition of 20S catalytic activities  . Then, wtivities .i.e., human, mouse, yeast, Thermoplasma acidophilum), being involved in gate opening and functioning.2T4 nitrogens . To fully explore all possible binding sites/modes, the binding domain area was defined as the whole α ring of the human 20S proteasome. Firstly, a Monte Carlo/minimization approach for the random generation of a maximum of 20 acceptable complexes was used. To ensure a wide variance of the input structures to be successively minimized, an energy tolerance value of 106 kcal mol–1 from the previous structure was used. After the energy minimization step, the energy test, with an energy range of 50 kcal mol–1, and a structure similarity check  was applied to select the 20 acceptable structures. The resulting complexes are then subject to simulated annealing (SA) calculations. In SA the temperature is altered in time increments from an initial (500 K) to a final (300 K) temperature. The temperature of 500 K was applied with the aim of surmounting torsional barriers, thus allowing a full rearrangement of the ligand and the protein . During all calculations, constraints were applied only to hydrogen bonds (α-helices) and φ and φ torsion angles (β-sheets) of some structured regions, by using different force constant values according to the results of secondary structure prediction calculations . In particular, a force constant of 100, 10, and 1 kcal mol–1 was applied to hydrogen bonds (α-helices) and φ and φ torsion angles (β-sheets) with high, medium, and low secondary structure prediction score, respectively. The rest of the atoms of the protein was left free of constraints on its movement. The quality of the resulting complexes was then assessed by using structure evaluator software2T4 ability to inhibit the 20S proteasome acting as a “plug” of the CP entrance gate. Firstly, all the complexes generated by our dynamic docking protocol, either by Monte Carlo and by SA calculations, always presented H2T4 bound to the entrance gate of the channel, interacting with the identified N-terminal residues of the α subunits  followed by a negatively charged residue (Asp); altogether, they form a sort of ring which widens up during gate opening  and D7 (α6), and the aromatic residues F9 (α1), Y8 (α5) and Y6 (α6) , ortho-H2T4. Accordingly, meta-H2T4 and ortho-H2T4 showed an overall lower inhibitory potency with respect to H2T4 and inhibited the three catalytic activities to different extents .meta-H2T4 and ortho-H2T4 may occur through a more sophisticated interference with proteasome allosteric regulation with respect to H2T4, being the three proteasome catalytic activities connected to the opening of the gate through different allosteric mechanisms. At this regard, it is worth of note that the PGPH-L activity is the most sensitive to the presence of activators such as PA28 or SDSInterestingly, the Hulations vs.B. In Y6 (α6) . When th Y6 (α6) . In this, and α7 . In ordefor H2T4  and in a extents . Interes2T4 binds the predominanti.e., induced fit mechanism)2T4 binding to the 20S α subunits could affect the catalytic activities not only by clogging the gate and, consequently, the entry of all substrates, but also by affecting the conformational equilibrium of the 20S human proteasome and inducing a shift in the equilibrium of the “open-to-close” structural transition. Following this hypothesis and considering the results of the subsequent stopped-flow kinetic studies (see next paragraph), we can propose that the binding of H2T4 induces a conformational change that may be related to the availability of another binding site for H2T4 on the 20S proteasome. In this view, it is worth of note that our structural and bioinformatics analysis identified a cluster of negatively charged residues showing suitable inter-atomic distances for a possible interaction with the porphyrin pharmacophore at the interface between α1-β1, α2-β2, and α5-β5 subunits . The values ≈0 for the dissociation rate constants (The reaction between the 20S proteasome and the Hgeometry . This evgeometry . Notablyonstants  simply mk3 = 3.23 (±0.81) × 10–3 s–1, whose rate is independent on porphyrin concentration, likely corresponding to a subsequent slower binding of porphyrins to the 20S proteasome at site(s) topologically distinct from that of the first two events. The observed kinetic process appears to be rate-limited by a conformational change, which follows and depends on the porphyrin binding to the CP gate. This slower process, as suggested by molecular modeling studies, might be related to the availability of an additional binding site for H2T4 on the 20S proteasome (see above). As a whole, kinetic results and computational simulations appear to support each other, strengthening the proposed mechanism of interaction between H2T4 and the 20S proteasome, reinforcing the idea that this porphyrin not only acts as a “plug” of the CP gate,The absorption change is also characterized by an additional process with Meso-tetrakis(4-N-methylpyridyl) porphyrin (para-H2T4 or H2T4), meso-tetra(2-N-metil-pyridyl) porphyrin (ortho-H2T4), and meso-tetra(3-N-metil-pyridyl) porphyrin (meta-H2T4) were purchased from Midcentury.Purified human 20S proteasome and the fluorogenic substrates Suc-LLVY-AMC, Z-Leu-Leu-Glu-AMC, and Ac-Arg-Leu-Arg-AMC used to test the ChT-L, PGPH-L, and T-L activity, respectively, were purchased from Boston Biochem . in vitro by mixing 20S proteasome (2 nM) with 100 μM of the proper fluorogenic peptide in the assay buffer  in a 384 multiwell black plate. The released AMC fluorescence was recorded at 440 nm (excitation at 360 nm) for 20 min, that is a time interval over which linearity was observed in a fluorescence plate reader . A minimum of three replicates were performed for each data point. Data are expressed as normalized percentages of residual activity considering the slope of the control (fluorogenic peptide/proteasome in the absence of inhibitors) as 100% of proteasome activity. Dose–response plots of the residual proteasome activity in the presence of increasing concentration of inhibitor provides a quantitative estimate of its potency. The IC50 is defined as the concentration of the inhibitor which causes 50% reduction of activity and it is thus calculated from the x-axis value of the dose–response plot occurring at a fractional activity of 50%. The estimation of the IC50 is based on a nonlinear fit with the equation:Proteasome activity assays were performed 50 values and their standard errors were deduced from the fitting. Data relative to CP activities at different substrate/inhibitor concentrations have been analyzed by a double reciprocal Lineweaver–Burk plot, according to the following equation:0] is the enzyme concentration, ν is the observed velocity  and [S] is the substrate concentration. The resulting catalytic parameter are Km, corresponding to the apparent affinity constant (or Michaelis–Menten constant) of substrate for the free enzyme (to form the ES complex), and kcat, corresponding to the velocity of the rate-limiting step during the enzymatic activity. Inhibition assays of 20S proteasome ChT-L activity have been carried out by incubating the 20S proteasome with increasing concentrations (0.1–3 μM) of inhibitor for 30 min at 37 °C; ChT-L activity was then tested in the same way as described above by adding increasing concentrations of the substrate. The mechanism of inhibition of the 20S proteasome ChT-L activity by the inhibitors was analyzed by modified double-reciprocal plot, as from the following equations for the different inhibitory mechanisms:The midpoint of this function occurs at a fractional velocity value of 50%, corresponding to half inhibition of the target enzyme. The ICfor a competitive inhibitionKI is the inhibitor affinity constant and α is the linkage factor, representing the reciprocal effect on the substrate and inhibitor affinity constants in the non-competitive inhibition and being related to the simultaneous presence in their respective binding sites. When α is very large, binding of inhibitor severely impairs binding of the substrate and the non-competitive inhibition becomes closely similar to competitive inhibition (since in the second factor of eqn (4b) (1 + [I]/(α × KI)) ≈ 1, as in eqn (4a)); conversely, when α is very small the non-competitive model becomes nearly identical to an un-competitive model eqn (4c), since in eqn (4b) (1 + [I]/KI) ≪ (1 + [I]/(α × KI)). All curve fitting and statistical analysis were carried out using the Non Linear Fitting Tool (NLFit) and MatLab . The parametric data fitting was based on nonlinear regression and the method of least squares. Model discrimination and choice was based on the goodness of fit. The goodness of fit was evaluated by visual examination of the fitted curves, 95% confidence bounds for the fitted coefficients and statistical analysis for determining the square of the multiple correlation coefficient (R2).for non-competitive inhibition, and2T4 with 20S proteasome has been also investigated from the pre-steady-state kinetic standpoint. Briefly, these assays were performed without incubation time, by direct addition of different concentrations of H2T4 in samples where the Ch-L activity was already being monitored. The extent and the rate of inhibition were determined by following the time-dependent fluorescent signal of the cleaved substrate for 25 additional minutes. Data have been analyzed according to a second-order Runge–Kutta simulation procedure based on a simple competitive inhibition mechanism, such as in The interaction of Ht. Thus, assuming that equilibrium between [E] and [ES] is very fast (which is the main assumption of the application of the Michaelis–Menten equation), the time evolution of the product formation is described by the following equation:The time evolution of each species reported in with all populations reequilibrated at each time step.2T4 and 47.1 μL of D2O to a 3 mm NMR tube. The final tube contained 0.860 μM of 20S and 172 μM of the ligand which corresponds to a protein/ligand ratio of 1 : 200. The use of 3 mm NMR tubes is particularly useful in this type of study since it allows to minimize the amount of protein used and to reduce salt effects induced by the buffer. NMR experiments were acquired at 298 K on a Varian Inova 600 MHz spectrometer, equipped with a cold probe optimized to detect 1H nucleus, located at the Institute for the Biostructures and the Bioimages of the CNR in Naples. The 1H NMR signal assignment was obtained from the analysis of the spectra assisted by the theoretical prediction based in the molecular structure using ChemAxon software . Saturation Transfer Difference (STD) spectra1H NMR signals in the low frequency region –1 ppm. The STD factor were calculated as ASTD = (I0 – Isat)/I0 = ISTD/I0 where I0 is the intensity of the signal in the reference experiment and Isat is the intensity of the same signal in the saturated spectrum. The signal obtained with the strongest ISTD/I0 value was normalized to 100%. The relative degree of saturation for the individual protons was used to compare the STD effect.36Samples used for NMR experiments were prepared using a solution of 1.03 μM of 20S in Tris–HCl (20 mM) pH 7.6, NaCl (150 mM) and DTT (1 mM) and a solution containing the ligand at a concentration of 18.1 mM in DMSO-d6. The NMR tubes were prepared by adding 250 μL of the 20S solution, 2.9 μL of HMolecular modeling calculations were performed on SGI Origin 200 8XR12000 and E4 Server Twin 2× Dual Xeon—5520, equipped with two nodes. Each node: 2× Intel® Xeon® QuadCore E5520—2.26 GHz, 36 GB RAM. The molecular modeling graphics were carried out on SGI Octane 2 workstations.2T4  were downloaded from the Cambridge Structural Database (CSD) using the CSDS  software Conquest 1.16. meta-H2T4 and ortho-H2T4 were built by modifying the experimentally determined structure of H2T4 . The apparent pKa values of porphyrins were calculated by using ACD/Percepta software.The experimentally determined structures of Hi.e., ligand and protein), was used . Although in the subsequent dynamic docking protocol all the systems were perturbed by means of Monte Carlo and simulated annealing procedures, nevertheless, the dynamic docking procedure formally requires a reasonable starting structure. Accordingly, the putative starting complexes  were subjected to a preliminary energy minimization . After the docking procedure  the CP gate, thus leading to the simultaneous inhibition of the ChT-L, T-L and PGPH-L activities. A second much slower event relates to the subsequent adhesion of the porphyrin to the CP external surface and, in particular, to the grooves which separate the α from the β subunits. This slower binding mode, which is not directly related to the stereochemistry of the observed CP competitive inhibition by porphyrins, is nonetheless relevant, since it brings about a porphyrin-dependent conformational change of the 20S proteasome. As a whole, all data here presented lead to envisage a mechanism where H2T4 “plugs” the CP gate, behaving as a competitive inhibitor, even though, it is not a competitive inhibitor, since it does not bind to the catalytic site. This stems from the complex nature of the substrate interaction with proteasome, which is at the same time allosteric (a positive feedback loop connects the site of amide bond hydrolysis with the site of ingress/egress of substrates and products) and classical enzymatic . Therefore, though displaying a competitive-like behaviour, porphyrin also interferes with the physiological quaternary equilibrium of the 20S proteasome between “open” and “closed” conformations, shifting it toward a “closed” structure with a consequent further enhancement of the energetic barrier for the gate opening of CP. These peculiar anti-proteasome properties, coupled with their versatile chemistry, propose cationic porphyrins as a novel class of CP conformational regulators with great potentiality as “lead” pharmacophores.It is known that the 20S proteasome can cleave a wide range of proteins and peptides without the assistance of ancillary RPsSupplementary informationClick here for additional data file.


Davallia mariesii Moore (Drynaria rhizome extract (DRE)) is widely known for its efficacy in treating inflammation, arteriosclerosis, and bone injuries. This study evaluated whether treatment with DRE inhibited FcɛRI-mediated allergic responses in the RBL-2H3 mast cells and investigated the early- and late-phase mechanisms by which DRE exerts its antiallergic effects. IgE anti-DNP/DNP-HSA-sensitized RBL-2H3 mast cells were tested for cytotoxicity to DRE, followed by the assessment of β-hexosaminidase release. We measured the amounts of inflammatory mediators  and examined the expression of genes involved in arachidonate and FcεRI signaling pathways. In addition, we confirmed the antiallergic effects of DRE on passive cutaneous anaphylaxis (PCA) in mice. DRE inhibited RBL-2H3 mast cell degranulation and production of allergic mediators in them. In early allergic responses, DRE reduced expression of FcεRI signaling-related genes  and extracellular signal-regulated kinase phosphorylation in mast cells. In late allergic responses, DRE reduced PGD2 release and COX-2 expression and cPLA2 phosphorylation in FcɛRI-mediated mast cells. Lastly, 250–500 mg/kg DRE significantly attenuated the IgE-induced PCA reaction in mice. These findings provide novel information on the molecular mechanisms underlying the antiallergic effects of DRE in FcɛRI-mediated allergic responses. Davallia mariesii Moore, known as Drynaria rhizome (DR) (“gol-se-bo” in Korean and “gu-sui-bu” in Chinese), is a traditional Korean and Chinese medicine known for its efficacy in treating inflammation, osteoporosis, traumatic brain injury, and arteriosclerosis [Allergies have become a worldwide clinical health problem, and their incidence is rapidly increasing due to various factors . Allergiclerosis , 8. It iclerosis . In addiclerosis . HoweverεRI complex, formed by binding of IgE to FcεRI and expressed on cellular surfaces, triggers early and late allergic responses and chronically prolongs inflammation by secreting chemical mediators and cytokines [εRI, a receptor expressed on the cell membranes of mast cells, comprises α and β subunits and two γ subunits. The α chain is involved in extracellular binding of IgE to antigens, whereas the β and γ chains mediate intracellular signaling [2) [β subunits of FcεRI receptors in mast cells stimulated by an antigen [εR, which is a high-affinity IgE receptor. The binding of Syk to phosphorylated ITAMs is regulated by two SH2 domains; it causes structural changes in Syk, resulting in increased enzymatic activity [γ1, and Akt, form Syk-receptor complexes, thereby activating the overall signaling system [Mast cells, which are rich in granules in their cytoplasm, are found in body areas, such as the skin surface, the mucosa of the gastrointestinal tract, serous membranes, and in the vicinity of lymphatic vessels and blood vessels, that come in contact with external stimuli. IgE antibodies, compound 48/80, protein kinase C activator, and calcium ionophores have been reported to induce mast cell degranulation , 12. In ytokines . FcεRI, ignaling . Histamiling [2) . Cyclooxling [2) . These m antigen . Lyn phoactivity . Subsequg system .α, IL-4, and IL-6, in IgE-mediated RBL-2H3 mast cells. Furthermore, we assessed the roles of DRE in the FcεRI signaling pathway  and the arachidonate signaling pathway  as well as its effects on IgE-mediated allergic responses in RBL-2H3 mast cells.In the present study, we hypothesized that DRE influences the phosphorylation of Src family kinases and antiallergic effects, including early- and late-phase reactions, in mast cells, ultimately exerting beneficial effects against IgE-mediated allergies by reducing mast cell degranulation. To test this hypothesis, we examined the effect of DRE supplementation on proinflammatory cytokines, including TNF-α medium), Dulbecco's phosphate-buffered saline, fetal bovine serum (FBS), and antibiotics  were purchased from GE Healthcare Life Sciences . Dinitrophenyl human serum albumin (DNP-HSA), DNP-immunoglobulin E (IgE anti-DNP), and dexamethasone were obtained from Sigma-Aldrich . 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco . D. mariesii Moore (Drynaria rhizome extract (DRE)) was obtained from Korean Medicine Application Center .Rat basophilic leukemia mast cell line RBL-2H3 was obtained from the American Type Culture Collection . Minimum essential medium alpha modification (MEM-μm) . Samples were freeze-dried and stored at −20°C before use. Sample acquisition was 5.2 g, and the yield was 17.5%. The powder of DRE was dissolved in 10% DMSO solution for all experiments.DR was obtained from Yeongcheon hyundai herbal market  and verified by Professor Ki Hwan Bae, Chungnam National University, Republic of Korea. To prepare the DRE, dried DR (30.0 g) were placed in 1000 mL distilled water and then extracted by 3 h of heating at 115°C . Following extraction, the solution was filtered using standard testing sieves (150 α medium supplemented with 10% heat-inactivated FBS containing 1% antibiotics (ABS). Prior to the experiments, 3 × 105 cells were seeded on a six-well plate and grown to confluence for 24 h. At day 2 post-confluence, the medium was replaced with the MEM-α medium (10% FBS and 1% ABS) containing IgE anti-DNP (0.1 μg/mL) for 16 h. The medium was replaced with the serum-free medium (MEM-α medium with 1% FBS and 1% ABS) containing DRE (100–500 μg/mL) and dexamethasone (100 nM) for 1 h and then treated with DNP-HSA (0.1 μg/mL) for 4 h and/or 30 min. The cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2.Rat RBL-2H3 mast cells were grown in MEM-DRE was prepared in a 10% dimethyl sulfoxide (DMSO) solution at a concentration of 100 mg/mL. RBL-2H3 mast cells were tested with increasing concentrations of DRE for 1 h. Cell viability was analyzed after adding 0.5 mg/mL MTT in each well, followed by incubation for 40 min at 37°C. After removing the medium, the cells were lysed with DMSO. Absorbance at 570 nm wavelength was measured using a microplate reader.μL of 4-methyl-umbellyferyl-N-acetyl-β-d-glucosaminidase (10 mM p-NAG) in sodium citrate buffer  and then incubated for 1 h at 37°C, according to the method described by Park et al. [The culture medium of IgE-sensitized RBL-2H3 mast cells treated with DRE was mixed with 25 k et al. . The rea2, TNF-α, IL-4, and IL-6 levels in the culture medium of IgE-sensitized RBL-2H3 mast cells treated with DRE were determined using enzyme-linked immunosorbent assay kits, as per the manufacturer's instructions.Histamine, PGD2, anti-phospho-Lyn, anti-phospho-Syk, anti-phospho-Fyn, anti-phospho-PLCγ1, anti-phospho-ERK, anti-phospho-Akt, and anti-α-tubulin antibodies  were used to detect COX-2, p-cPLA2, p-Lyn, p-Syk, p-Fyn, p-PLCγ1, p-ERK, p-Akt, and α-tubulin, respectively. α-Tubulin was used as protein loading control. Blots were observed using a western blot detection kit . Protein bands were quantified using Image Lab software .The cells were scraped from the plates using RIPA Lysis Buffer  containing a protease and phosphatase inhibitor cocktail . After incubation on ice for 30 min, the cell lysates were centrifuged at 14,000 rpm for 20 min at 4°C. Proteins were quantified using BCA protein assay . The protein lysates were resolved on 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane. Anti-COX-2, anti-phospho-cPLAn = 25; 5 weeks old) were randomly assigned to five groups  after 1 week adaptation period: control group (CTL), IgE anti-DNP/DNP-HSA group (IgE anti-DNP/DNP-HSA), IgE anti-DNP/DNP-HSA treated with 10 mg/kg dexamethasone group (Dex), IgE anti-DNP/DNP-HSA treated with 250 mg/kg DRE group (DRE 250), and IgE anti-DNP/DNP-HSA treated with 500 mg/kg DRE group (DRE 500). DRE was prepared in 0.5% low-viscosity carboxymethyl cellulose sodium salt (CMC), and CTL and IgE anti-DNP/DNP-HSA groups received equivalent volumes of vehicle (0.5% CMC). The mice were housed under standard laboratory conditions (21°C–24°C and 40%–60% humidity) and were maintained at a 12 h light/12 h dark cycle (lights on at 8:00), with ad libitum access to food and water. All experiments were approved by the Committee on Animal Experimentation and Ethics of KIOM.Male ICR mice (μg/mL) was subcutaneously injected into the ears of ICR mice. At day 1, IgE-sensitized mice were administered oral DRE (250 or 500 mg/kg) or dexamethasone (10 mg/kg). One hour later, DNP-HSA (300 μg/mL) containing 1% Evans blue was injected into the tail veins. After 1 h, the mice were anesthetized with CO2 and tissues from the treated ears were obtained. The Evans blue dye was removed by the ear tissue, which were then incubated with 1 mL formamide at 63°C for 16 h. The mixtures were centrifuged at 17,000 ×g for 10 min at 4°C. Absorbance at 620 nm wavelength was measured using a microplate reader.The PCA reaction was evaluated as previously described . IgE antp < 0.05 as the criterion for significance.Statistical analysis was performed using GraphPad Prism version 5 . The results are presented as means ± SE. Differences between the experimental groups were analyzed using one-way analysis of variance with Bonferroni's post hoc testing and μg/mL , and an 84% decrease at 500 μg/mL (p < 0.0001)  . These rα, IL-4, and IL-6 in IgE-mediated RBL-2H3 mast cells. DRE at all concentrations significantly reduced the levels of TNF-α released by mast cells . Concentrations of Evans blue were significantly lower in the DRE 250 group  and the DRE 500 group  than in the IgE anti-DNP/DNP-HSA group, but no significant difference was observed between the Dex and IgE anti-DNP/DNP-HSA groups (19.20 ± 2.20 μg/ear)  .εRI receptors on their cell surface; when antigens cross-link with IgE bound to this receptor, activation begins and degranulation proceeds [εRI interaction and the signal transduction pathways for the degranulation process [Mast cells are the main model for studies on inflammatory and allergic diseases, such as asthma, allergic rhinitis, tissue modification, and rheumatoid arthritis . Their mproceeds . It is w process .β-hexosaminidase along with histamine. Therefore, this enzyme is often used as a marker of mast cell degranulation or histamine release [εRI signaling-related genes known to influence the concentrations of inflammatory cytokines. Histamine released upon mast cell degranulation induces permeability hyperactivity, dilation of blood vessels, secretory action of secretory cells on mucosal surfaces, and a contraction effect on bronchial smooth muscles, leading to an immediate hypersensitivity reaction [In this study, we found that DRE prevented IgE-induced allergic responses and also observed remarkable reductions in degranulation. Degradation of granules in mast cells or basophils leads to the release of the enzyme  release . Hexosam release . Using treaction .α, IL-4, and IL-6 secretion was inhibited in a concentration-dependent manner. The cytokines TNF-α, IL-4, and IL-6 are secreted mainly by T cells; they promote and sustain the inflammatory response, resulting in local and systemic effects in the human body [α is a member of a growing family of peptide mediators and plays a crucial role in the pathogenesis of many acute and chronic inflammatory conditions. It is not only a potent inducer of other inflammatory cytokines, including IL-4 and IL-6, but it is also a self-secretory mediator [The results of the present study also demonstrated that histamine concentration decreased with DRE treatment. In addition, TNF-man body , 26. TNFmediator . IL-6 ismediator . IL-4 alεRI signal cascade. IgE-induced mast cell degranulation is associated with FcεRI receptor activation, which induces the release of various inflammatory mediators, including TNF-α, leukotrienes, and prostaglandins via Lyn/Syk pathway phosphorylation [εRI receptor activation [εRI, by DRE [2 expression and reduced the levels of the COX-2 product PGD2, which is enhanced in activated immune cells, including mast cells. The suppressive effects of DRE on PGD2 formation may contribute to its increased antiallergic activity, as PGD2 may mediate the inflammation associated with IL-4 [εRI signal cascade and inhibition of the arachidonate signaling pathway.One possible mechanism for DRE-induced antiallergic activity may be its effect on the Fcrylation . Thus, Ltivation . In the tivation . Further, by DRE . Furtherith IL-4 . These fWe next examined how DRE suppresses IgE-mediated PCA in mice. PCA is characterized by an immediate skin reaction at a localized IgE-mediated allergic response in vivo, typically with increased vascular leakage in the skin that can be assessed by an intravenous injection of Evans blue . In vivoα, IL-4, IL-6, and PGD2, caused by antigen-IgE interaction. DRE may be a useful candidate for antiallergic drugs; because it is a natural product, it may be less toxic than current antiallergic drugs.DRE has antiallergic effects in both IgE anti-DNP/DNP-HSA-activated RBL-2H3 mast cells and IgE anti-DNP/DNP-HSA-mediated PCA reactions. These antiallergic effects of DRE are because of the inhibition of the degranulation process and the production of allergenic mediators, such as histamine, TNF-