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As a study limitation, although the sample size calculation has been observed, we had a convenience sample; all children who did not cry during evaluation and whose parents agreed to participate were included. In this context, we believe that the study has a good external validity for similar conditions; that is, children attending public and private day care centers in medium-sized cities. Thus, there is a gap for future studies related to fine motor development in children attending day care centers of different quality or in settings different from the one evaluated.

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Factors such as higher maternal education, extended stay in day care, and day care centers of better quality, especially regarding language stimulation, were associated with fine motor performance of children attending day care centers. Therefore, it is important that children attending public or private day care centers receive good stimulus to promote child development.

other

Rheumatoid arthritis (RA) is a prevalent autoimmune disease affecting up to 1% of the adult population in developed regions (1, 2). Its main characteristic is a symmetrical polyarthritis involving predominantly the hand and foot joints, although every organ system may be involved. The disease is more frequently diagnosed in women than in men and is more common with increasing age (1, 3). The potential causes of RA are diverse in nature (4, 5), but a role for T-cells, antibody-producing B-cells, but also monocytes/macrophages has been suggested (1, 6). Interestingly, in many female RA patients, an improvement in RA disease severity is reported during pregnancy, as well as a relapse thereof after delivery (7–10). The reasons for these changes in disease activity are still poorly understood, but are of importance to understand the etiology of RA, and may possibly provide leads for new modes of (personalized) treatment.

review

Glycosylation, the process of co- and posttranslational protein modification with complex carbohydrates, plays an important role in the interaction, function, and solubility of proteins (11–13). It is expected that more than half of all proteins is glycosylated with one or more N-glycans (14), and commonly observed glycoforms range from high-mannose- to complex type with two to four antennae (branching N-acetylglucosamines) (Figure 1) (12, 15). These structures may be extended by additional monosaccharides such as a bisecting N-acetylglucosamine, as well as galactoses, N-acetylneuraminic acids (sialic acids), and fucoses in a variety of different positions and linkages. This leads to a large N-glycan diversity, and may also lead to the formation of specific epitopes such as sialyl-Lewis X, which can be recognized by E-selectin (12, 16).

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Schematic overview of glycosylation traits derived from human serum proteins. N-Glycan structures are generalized into high-mannose type (M), hybrid type (Hy), and complex type (C) by the number of mannoses (green circle) and antennary N-acetylglucosamines (blue square). High-mannose-type N-glycans can have up to nine mannoses, whereas each antennary N-acetylglucosamine can be terminally substituted with a galactose (yellow circle), and sialic acid (magenta diamond). Sialic acids (S) can either be α2,3-linked (L) or α2,6-linked (E). N-Glycans can further be modified with a fucose (F), optionally at an antennary N-acetylglucosamine (Fa), and structures may as well be bisected (B). In case of derived traits, the subject of the calculation is represented by the last letter, e.g., sialylation (S), and the group on which it is calculated by the preceding letters, e.g., triantennary fucosylated species (A3F). This, for instance, translates A3FGS into the sialylation per galactose within triantennary fucosylated species.

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Various proteins have already been found to display altered glycosylation with RA and its disease activity. For instance, the N-glycosylation of the fragment-crystallizable (Fc) portion of immunoglobulin G (IgG) shows to differ in galactosylation, bisection, and fucosylation (17–23), and the acute-phase protein alpha-1-acid glycoprotein (also known as orosomucoid) shows differences in antennarity (i.e., the ratio between di-, tri-, and tetraantennary glycans) and fucosylation with RA as well as changes throughout pregnancy (24, 25). Although such an analysis of single proteins is highly informative, additional insights may be gained by a systemic glycomics approach that covers a broad range of protein-linked glycan modifications.

review

The total serum N-glycome (TSNG) comprises the N-glycans from all serum proteins, which are to a large extent liver- (acute-phase proteins) and plasma cell-derived (antibodies) (15, 26). Interestingly, the TSNG has been shown, in a small sample set, to differ between healthy individuals and those with RA, and undergoes clear alteration throughout pregnancy and the following postpartum period (27, 28). However, it is hitherto unknown which TSNG characteristics are associated with the disease activity of RA, and changes thereof throughout pregnancy. Recent developments in mass spectrometry (MS)-based high-throughput glycosylation analysis have provided the opportunity to acquire information on TSNG N-glycan complexity, antennarity, galactosylation, fucosylation, as well as on the presence and linkage of sialic acids (α2,6- vs. α2,3-linkage) (29, 30). The latter appears to be of high immunological relevance, since the α2,3-linked sialic acids are required for sialyl-Lewis X formation implicated in the interaction with selectins (29, 30).

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The objective of the work presented here is to assess the differences in serum N-glycosylation throughout pregnancy and the postpartum period in RA patients and to identify the glycosylation properties associated with the changes in disease activity [DAS28(3)-CRP] during pregnancy. To achieve this, we studied the N-glycosylation of sera from 253 RA and 32 control pregnancies at seven time points before, during, and after pregnancy by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and report the disease- and pregnancy-associated changes of 78 N-glycan species and 91 glycosylation traits derived thereof.

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The current research is embedded in the PARA study, a nationwide prospective cohort on pregnancy and RA (21, 31). The cohort consisted of serum samples of 253 pregnancies from 219 RA patients, collected between 2002 and 2009 (31). In addition, 32 pregnancies of healthy Caucasian volunteers without adverse obstetric histories were included and followed from the first trimester of pregnancy. Of each patient, at least one sample was obtained during pregnancy and one postpartum, with a minimum of three samples per patient. Only completed pregnancies were included and all patients fulfilled the 1987 ACR criteria for RA. Disease activity was assessed using the disease activity score (DAS) in 28 joints, incorporating the swollen joint count, the tender joint count and the C-reactive protein (CRP) level [DAS28(3)-CRP]. The study was in compliance with the Helsinki Declaration and was approved by the Ethics Review Board at the Erasmus University Medical Center, Rotterdam, the Netherlands. Written and informed consent was obtained from all research participants included in this study. For quality control purposes, 111 plasma standards (Visucon-F frozen normal control plasma, pooled from 20 human donors, citrated, and buffered with 0.02 M HEPES, obtained from Affinity Biologicals, Ancaster, ON, Canada) and 40 PBS blanks were distributed across the 21 96-well sample plates.

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SDS and analytical grade ethanol were obtained from Merck (Darmstadt, Germany). Disodium hydrogen phosphate dihydrate (Na2HPO4 × 2H2O), potassium dihydrogenphosphate (KH2PO4), sodium chloride, Nonidet P-40 substitute (NP-40), 1-hydroxybenzotriazole monohydrate 97% (HOBt), 50% sodium hydroxide (NaOH), and super-DHB were obtained from Sigma-Aldrich (Steinheim, Germany). HPLC-grade acetonitrile (ACN) was purchased from Biosolve (Valkenswaard, the Netherlands), and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) hydrochloride was obtained from Fluorochem (Hadfield, UK). Peptide-N-glycosidase F (PNGase F) was obtained from Roche Diagnostics (Mannheim, Germany) and ultrapure water (MQ) was generated from a Purelab Ultra system (Veolia Water Technologies, Ede, the Netherlands), which was maintained at 18.2 MΩ at 25°C.

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N-Glycans were released from serum proteins using PNGase F, as was previously described (32). In summary, 6 µL serum of each sample was denatured by addition of 12 µL of 2% SDS, followed by incubation for 10 min at 60°C. The release step was performed by the addition of 12.6 µL releasing mixture, which consisted of 2.5× PBS containing 2% NP-40 and 0.4 mU PNGase F, followed by 16 h incubation at 37°C. After release, the samples were stored at −20°C or transferred to the robot platform to be used directly.

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Further sample preparation was performed on an automated system, as previously described (30). In short, linkage-specific sialic acid stabilization (ethyl esterification) was performed by adding 60 µL 250 mM EDC 250 mM HOBt in ethanol to 3 µL released glycan sample. This mixture was incubated for 75 min at room temperature, after which 120 µL ACN was added. To purify the samples, a GHP membrane (GHP plate, Pall AcroPrep Advance 96 Filter plate, Pall Corporation, Ann Arbor, MI, USA) was prewetted with 70% ethanol, activated with MQ and equilibrated with 100% ACN. The complete sample was transferred to the GHP plate and incubated for 5 min. Subsequently the GHP plate was washed three times with 100 µL 96% ACN. Elution was performed by adding 30 µL of MQ to the GHP plate, incubating at room temperature for 5 min, followed by centrifugation into a PCR plate.

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From each purified sample, 10 µL was premixed with 10 µL matrix consisting of 5 mg/mL super-DHB in 99% ACN with 1 mM NaOH. Of this mixture, 2 µL was spotted onto a MALDI target plate (800/384 MTP AnchorChip, Bruker Daltonics, Bremen, Germany) and was left to dry. MALDI-TOF-MS spectra were recorded on an UltrafleXtreme mass spectrometer with a Smartbeam-II laser (Bruker Daltonics) in reflectron positive mode, controlled by flexControl 3.4 (Build 135). Measurements were performed within a range from m/z 1,000 to 5,000, accumulating 10,000 laser shots at a frequency of 1,000 Hz and with 100 shots per raster spot using a random walking pattern.

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Average spectra were separately created for all healthy samples and for all RA samples. Within these two averages, 98 signals were manually assigned to putatively originate from glycan compositions. Using flexAnalysis 3.4 (Bruker Daltonics), the spectra were transformed into text format (x, y). Massytools (version 0.1.7.1 beta) was used to perform calibration and integration of the text files (33). Specifically, calibration was performed with a high precision calibration list, requiring at least 5 calibrants to be present at a RMS S/N ≥ 6 (119 spectra were excluded during this step, which included all 40 blanks). The putative glycan structures were extracted covering 95% of the isotopic envelope, summing per isotope the background-corrected area.

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Spectra with quality control values below 3 SD of the mean values [i.e., fraction of spectrum in analytes, fraction of analyte area above S/N 9, and highest main peak (H5N4E2) S/N] were excluded from further analysis (13 spectra). Putative glycan signals were excluded from further analysis if failing to be present in 15% or more spectra of any analytical group (healthy, RA, standard) with an S/N ≥ 6 and a ppm error of at most 15 (which led to the exclusion of 21 signals). After curation, we retained 1,841 spectra with 78 analytes (Table S1 in Supplementary Material). Single N-glycan areas were normalized to the sum of all areas. To prevent outlier influence on the data analysis, individual glycosylation values surpassing 5 SD from their mean were removed from further analysis (118 values). Derived traits were calculated from the single glycans (Table S2 in Supplementary Material).

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Measurement repeatability was assessed by calculating the mean, SD, and CV for all glycans and derived traits within the plasma controls (Table S3 and Figure S1 in Supplementary Material). Statistical analyses were performed using R 3.1.2 in an environment of RStudio 0.98.1091 (RStudio Team, Boston, MA, USA) (34). Figures were annotated with glycan symbols created in GlycoWorkbench 2.1 following the nomenclature proposed by the Consortium for Functional Glycomics (35, 36). For all statistical analyses, glycosylation values were scaled and centered, making regression effect sizes (B) representative of a 1 SD change in the glycan value.

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Association between glycosylation and, respectively, pregnancy, RA, and DAS28(3)-CRP was explored by mixed linear regression. To establish the effect of pregnancy on glycosylation, a binary time point classification was constructed between (A) the first and third trimesters of pregnancy (coded, respectively, as 0 and 1), (B) the third trimester of pregnancy and 6 weeks postpartum (respectively, 0 and 1), and (C) the third trimester of pregnancy and 26+ weeks postpartum (respectively, 0 and 1). With an added random intercept per individual, glycan variables were used as outcome variable and time point classification as predictor variable (model: glycosylation ~ β1·time point) (Table S4 in Supplementary Material). The effect of RA (healthy = 0; RA = 1) on glycosylation was modeled by applying a random intercept for all time points, using glycosylation as outcome variable and RA as predictor variable (model: glycosylation ~ β1·RA) (Table S5 in Supplementary Material). The effect of DAS28(3)-CRP on glycosylation was modeled by either having a random intercept per time point (to analyze between individuals) or a random intercept per individual (to analyze within individuals), using glycosylation as outcome variable and DAS28(3)-CRP as predictor variable [model: glycosylation ~ β1·DAS28(3)-CRP] (Table S6 in Supplementary Material).

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To validate associations between glycosylation and disease activity during pregnancy, the difference in DAS28(3)-CRP [ΔDAS28(3)-CRP] and glycosylation (Δglycosylation) was established between the first trimester and third trimester of pregnancy (third trimester − first trimester). The association between these was established by linear regression [Δglycosylation ~ β1·ΔDAS28(3)-CRP] (Table S7 in Supplementary Material). This process was similarly performed for the determination of the relationship between ΔDAS28(3)-CRP and Δglycosylation during the postpartum period, which was established between the third trimester of pregnancy and 12 weeks postpartum time point (12 weeks postpartum − third trimester). The time point at 12 weeks postpartum was chosen for the calculation because it displayed the highest disease activity after delivery, since most patients had already restarted anti-rheumatic therapy afterward.

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To explore the association between serum protein N-glycosylation and improvement of RA disease activity during pregnancy, we investigated by MALDI-TOF-MS the TSNG of 285 pregnancies embedded in the PARA study (characteristics of the study population can be found in Table 1, medication details in Table 2) (31). This MS methodology allowed us to obtain information on 78 glycan compositions (Figure 2; Table S1 in Supplementary Material), including discrimination between sialic acid linkage isomers, and to calculate biologically relevant ratios between subsets of glycans in the form of 91 derived traits (Table S2 in Supplementary Material) (29). Data quality was confirmed by the repeated measurement of a standard sample (Figure S1 and Table S3 in Supplementary Material).

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Comparison of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectra obtained from released N-glycans from serum proteins after linkage-specific sialic acid esterification and HILIC enrichment. The shown spectra are derived from an individual with rheumatoid arthritis who has been diagnosed with ACPA and RF, and has been classified as a responder by the EULAR response criteria. Notable differences between the first trimester (top) and third trimester (bottom) spectra include galactosylation (e.g., between m/z 1,485.5, 1,647.6, and 1,809.6), antennarity (e.g., m/z 2,940.1 vs. 2,301.8), α2,3-linked sialylation (e.g., m/z 2,255.8 vs. 2,301.8), and α2,6-linked sialylation (e.g., m/z 1,982.7 vs. 2,301.8).

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The association of pregnancy and the postpartum period with serum protein N-glycosylation was established by mixed linear regression. Within individuals, comparison was made between the first and third trimester (representative of pregnancy; respectively, coded 0 and 1; adjusted for individual), third trimester and 6 weeks postpartum, and third trimester and 26+ weeks postpartum (Table S4 in Supplementary Material).

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With progression of pregnancy, a marked alteration in overall number of antennae per N-glycan was observed (Figure 3, top row). During pregnancy, within the complex type glycans (C), the relative abundance of tri- and tetraantennary N-glycans (CA3, CA4) showed to increase at the expense of diantennary species (CA2) (βCA2 = −1.05 SE ± 0.06; βCA3 = 1.08 ± 0.06; βCA4 = 0.70 ± 0.06), followed by a slow recovery after delivery (βCA2 = 0.94 ± 0.07; βCA3 = −0.98 ± 0.07; βCA4 = −0.56 ± 0.08; Table S4 in Supplementary Material).

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Overall glycosylation changes throughout pregnancy and the postpartum period shown in boxplots. Displayed are the glycan traits related to antennarity (top row), and the glycosylation traits likely to be of immunoglobulin origin (bottom row) (15, 26). Depicted are healthy controls (in white) and patients with rheumatoid arthritis (RA) (in gray) at preconception (pc), trimesters 1 through 3 (tm1, tm2, tm3), 6 weeks postpartum (pp1), 12 weeks postpartum (pp2), and 26+ weeks postpartum (pp3). CA2, diantennary species within complex type; CA3, triantennary species within complex type; CA4, tetraantennary species within complex type; A2S0F, fucosylation of non-sialylated diantennary species; A2FS0G, galactosylation of non-sialylated fucosylated diantennary species; A2FSB, bisection of sialylated fucosylated diantennary species. Red brackets indicate statistically significant findings between time points (horizontal), or between healthy individuals and RA patients (vertical).

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For the serum N-glycosylation characteristics likely originating from the Fc portion of IgG (diantennary N-glycans without sialylation; A2S0) (15, 26), we confirmed with progressing pregnancy a decrease in fucosylation (A2S0F, βA2S0F = −0.59 ± 0.05) and increase in galactosylation (A2FS0G, βA2FS0G = 0.59 ± 0.04), both rapidly reversing after delivery (βA2S0F = 0.67 ± 0.04; βA2FS0G = −0.81 ± 0.04) (Figure 3, bottom row).

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For the serum N-glycosylation characteristics likely originating for a large part from IgG-Fab, IgM, and IgA (fucosylated diantennary N-glycans with sialylation; A2FS) (15, 26), we observed a decrease in bisection with trimester progression (A2FSB, βA2FSB = −0.56 ± 0.04) and a rapid return to pre-pregnancy levels at 6 weeks postpartum (βA2FSB = 1.11 ± 0.03).

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Notably, whereas α2,6-linked sialylation remained relatively stable throughout pregnancy (Figure 4, bottom row), a drastic increase of α2,3-linked sialylation (e.g., per galactose on di- or triantennary glycans, respectively, A2GL and A3GL) was observed up to the third trimester (βA2GL = 1.11 ± 0.05; βA3GL = 1.16 ± 0.05), followed by a rapid return to baseline levels at the first time point after delivery (βA2GL = −1.19 ± 0.05; βA3GL = −1.42 ± 0.04) (Figure 4, top row).

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Sialylation changes throughout pregnancy and the postpartum period shown in boxplots. Whereas α2,3-linked sialylation (L) displays the most prominent change throughout pregnancy (top row), α2,6-linked sialylation shows the most distinction between patients and controls (bottom row). Separation is made between pregnancies of patients with rheumatoid arthritis (RA) (in gray) and healthy controls (in white). A2GL, α2,3-linked sialylation per galactose of diantennary species; A3GL, α2,3-linked sialylation per galactose of triantennary species; A4GL, α2,3-linked sialylation per galactose of tetraantennary species; A2GE, α2,6-linked sialylation per galactose of diantennary species; A3GE, α2,6-linked sialylation per galactose of triantennary species; A4GE, α2,6-linked sialylation per galactose of tetraantennary species. Red brackets indicate statistically significant findings between time points (horizontal), or between healthy individuals and RA patients (vertical).

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By mixed linear regression, glycosylation values were compared between individuals with RA and controls (healthy = 0, RA = 1; adjusted for pregnancy) (Table S5 in Supplementary Material). For immunoglobulin type glycosylation, we observed in RA a lower galactosylation (A2FS0G, βA2FS0G = −0.44 ± 0.07), higher bisection (A2FS0B, βA2FS0B = 0.55 ± 0.08), and a higher sialylation per galactose (A2FGS, βA2FGS = 0.75 ± 0.08). In addition, differences were found in several non-fucosylated diantennary traits (higher A2F0G, A2F0GS, and A2GE, lower A2F0B, A2F), all mainly driven by elevated levels of H5N4E2 (βH5N4E2 = 0.97 ± 0.07) in RA patient serum. For tri- and tetraantennary N-glycosylation, we observe with RA a higher fucosylation (e.g., A3F, βA3F = 0.34 ± 0.08; A4F, βA4F = 0.56 ± 0.08) particularly within species with α2,3-linked sialylation (A3LF, βA3LF = 0.35 ± 0.08; A4LF, βA4LF = 0.58 ± 0.08), suggesting an increase in sialyl-Lewis X/A.

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In addition, we compared by mixed linear regression the association of glycosylation with RA disease activity, as assessed by DAS28(3)-CRP, both between and within individuals with RA (respectively, adjusted for time point and individual) (Figure S2 and Table S6 in Supplementary Material). The increase in DAS28(3)-CRP associated with both the decrease in IgG-Fc-type galactosylation (e.g., A2S0G, βA2S0G = −0.34 ± 0.02) and the decrease in fucosylation of non-α2,3-sialylated triantennary species (A3L0F, βA3L0F = −0.13 ± 0.03). Increasing with DAS28(3)-CRP were the bisection of IgG-Fc-type glycans (A2F0S0B, βA2F0S0B = 0.25 ± 0.03) as well as the sialylation and fucosylation of tri- and tetraantennary species (e.g., A3FGS, βA3FGS = 0.26 ± 0.02; A4F, βA4F = 0.13 ± 0.03; A4GS, βA4GS = 0.17 ± 0.02). Glycosylation features associating with DAS28(3)-CRP only within (and not between) individuals throughout pregnancy were the fucosylation of (α2,6-)sialylated diantennaries (A2SF, βA2SF = −0.12 ± 0.02) and α2,6-sialylation of triantennary non-fucosylated species (A3F0GE, βA3F0GE = 0.14 ± 0.02).

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Finally, between trimesters 1 and 3 (response timeframe) as well as between trimester 3 and 12 weeks postpartum (flare timeframe), we employed linear regression to confirm the within-individual association of DAS28(3)-CRP [ΔDAS28(3)-CRP] with glycosylation (Δglycosylation) (Table S7 in Supplementary Material). In both timeframes, ΔDAS28(3)-CRP showed a negative association with the Δgalactosylation of IgG-Fc type species (ΔA2S0G, responder, βΔA2S0G = −0.29 ± 0.07; flare, βΔA2S0G = −0.35 ± 0.07; ΔA2FS0G, responder, βΔA2FS0G = −0.29 ± 0.07; flare, ΔA2FS0G; βΔA2FS0G = −0.33 ± 0.07), whereas the sialylation of triantennary fucosylated species showed in both cases a positive association (ΔA3FGS, responder, βΔA3FGS = 0.26 ± 0.07; flare, βΔA3FGS = 0.35 ± 0.07) (Figure 5). The flare timeframe additionally showed a positive association of ΔDAS28(3)-CRP with the (α2,6)-sialylation of (fucosylated) diantennary glycans (ΔA2GE, βΔA2GE = 0.27 ± 0.07; ΔA2FGS, βΔA2FGS = 0.24 ± 0.07).

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The relation between changing rheumatoid arthritis disease activity [ΔDAS28(3)-CRP] and changing glycosylation. Distinction is made between the timeframes to assess responder status (the change between first and third trimester; top row) and flare status (the change between third trimester and 12 weeks postpartum; bottom row). ΔA2S0G, the change in galactosylation per antenna of diantennary non-sialylated species; ΔA2FS0G, the change in galactosylation of diantennary fucosylated non-sialylated species; ΔA3FGS, the change in sialylation per galactose of triantennary fucosylated species. The significance of the models (p) arrives from linear regression, the correlation coefficients (r) from Pearson correlation.

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The decrease of RA disease activity during pregnancy and the flare following delivery are reproducible clinical observations that are mechanistically poorly understood (8–10). To expand our understanding of protein glycosylation that associates with RA disease activity, we studied the total serum N-glycosylation changes occurring throughout pregnancy and the postpartum period of 253 pregnancies of patients with RA, along with 32 control pregnancies.

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To interpret the information contained in this study, several aspects of our MALDI-TOF-MS analysis need to be kept in mind. First, MS assesses glycan chemical compositions and not structures, although sialic acid linkage information is provided by the employed derivatization technique (29). Other structural characteristics are presumed based on a wide array of literature on biosynthetic pathways, and on experiments with enzymatic digestion, nuclear magnetic resonance spectroscopy, and MS fragmentation (12, 15, 38–40). Second, the changes observed in the released N-glycan samples could have originated not only from changes in the glycosylation of proteins but also from changes in the abundance of those glycoproteins. Derived traits have been constructed to reflect differences in biosynthesis, and our current day understanding of the relative contribution of specific glycoproteins and tissues to the serum N-glycome (15, 26). Third, the mass spectrometric analysis did not provide quantitative ratios of N-glycosylation, but the direction and magnitude of observed changes is expected to be biologically representative, as suggested by method comparisons (41).

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In addition, it has to be noted that the clinical associations found in the study may, in part, originate from treatment differences between individuals during the study period. For example, methotrexate treatment typically only starts after delivery, potentially influencing the time point comparisons. However, this confounding effect is expected to be limited due to the relatively low number of individuals undergoing the treatment. Details on the medication can be found in Table 2.

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Previously, we found glycosylation changes throughout healthy pregnancies, and have in RA patients studied the specific glycosylation of IgG-Fc throughout pregnancy (20, 21, 27, 28, 42, 43). In the current study, the first application of a similar MALDI-MS approach with automated sample preparation, we achieved the analysis of the total serum N-glycosylation throughout the pregnancies of both healthy controls and RA patients, in total leading to the analysis of 1,770 clinical samples (30). Since the current cohort also contains the control individuals (without RA) from prior studies, these previous results were confirmed, but we could also for the first time show that comparable TSNG changes can be observed in RA patients.

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As such, we detected with the progression of pregnancy an increase of galactosylation (A2FS0G) as prior reported for the Fc part of IgG, as well as a decrease in bisection (A2FSB) (18, 20, 21). Additionally, we observed with pregnancy an overall increase in glycan branching (from CA2 to CA3 and CA4) and an increase of α2,3-linked sialylation (L, in part at the expense of α2,6-sialylation, E). In all cases, the postpartum period led to the return to the values before or at the beginning of pregnancy. The increased branching observed in the TSNG could have originated from the abundance and antennarity of acute-phase proteins such as alpha-1-acid glycoprotein, for which an increased serum level and N-glycan branching has been reported with pregnancy (24, 25). On the other hand, we did not observe the decrease in fucosylation reported for the same protein, potentially obscured by the increased fucosylation of other proteins, and have yet to identify the source of the substantial increase of α2,3-linked sialylation up to the third trimester. In literature, it has been reported that IgG and alpha-1-acid glycoprotein glycosylation may be affected by estrogens (44, 45), known to change significantly throughout pregnancy (46), and it is conceivable that other proteins within the TSNG are under similar high-level control.

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The total serum N-glycosylation changes with pregnancy showed remarkably comparable between RA patients and healthy controls, but baseline differences could be detected. For example, RA patients displayed a lower degree of IgG-Fc-type galactosylation and higher bisection when compared to controls (A2FS0G, A2FS0B). A decreased IgG-Fc galactosylation and increased bisection are well-known to associate with a variety of inflammatory conditions, including RA, inflammatory bowel disease, and aging (18, 47, 48), and the same glycosylation phenotypes appear detectable in our TSNG study as well (20, 21). The mechanisms by which IgG-Fc glycosylation may affect inflammatory processes remain for a large part to be elucidated, but increased galactosylation and decreased fucosylation have been implicated in increased FcγRIIa and FcγRIIIa binding and antibody-dependent cellular cytotoxicity (49–51).

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Furthermore, RA patients showed in our study a higher α2,6-sialylation and lower fucosylation of diantennary glycans (A2GE, A2F) compared to healthy controls, mainly driven by the N-glycan composition H5N4E2, as well as a substantially higher (multi-)fucosylation of tri- and tetraantennary glycans [A3F(a), A4F(a)]. The increased A3/A4 fucosylation strongly suggests the upregulation of sialyl-Lewis X, which has been reported with inflammatory arthritis for several acute-phase proteins, e.g., alpha-1-acid glycoprotein, haptoglobin, alpha-1-antichymotrypsin, and transferrin (52–56). The sialyl-Lewis X on glycans is known to bind to E-selectin, an inducible receptor expressed by endothelial cells (16). This interaction is implicated in the homing of immune cells to a site of inflammation (57), as well as cancer metastasis (58), and may play a role in RA as well. For instance, the alpha-1-acid glycoprotein observed within RA synovial fluid is thought to be of hepatic origin (59), meaning its circulatory variant might make use of a glycan-mediated mechanism for transportation toward the inflamed synovial tissue.

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Next to a negative association with the galactosylation of IgG-Fc-type N-glycans, we additionally report a positive association between (Δ)DAS28(3)-CRP and the sialylation of triantennary fucosylated glycans (A3FGS), notably of the α2,6-linked variety. This is in contrast to the TSNG changes throughout pregnancy, which shows marked increase of α2,3-linked sialylation but not an association with disease activity. A recent study, which compared total plasma N-glycosylation with the levels of various metabolic and inflammatory markers, has indicated a link between A3FGS and CRP, suggesting that the CRP component of DAS28(3)-CRP may in part be responsible for the association found within the current study (60). While the protein source of A3FGS remains unknown, future studies will have to reveal its biomarker potential and facilitate our understanding of RA disease severity.

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To summarize, by performing MS-based total serum protein N-glycosylation analysis we detected (1) changes in protein glycosylation throughout pregnancy, (2) glycosylation differences between healthy individuals and RA patients, and (3) glycosylation traits coinciding with the pregnancy-associated changes in RA disease activity. While we confirmed in serum the IgG glycosylation phenotypes that were prior reported to associate with RA disease activity, our glycomics approach has additionally allowed the detection of changes that are presumably independent from IgG, namely, the sialylation of fucosylated triantennary N-glycans (A3FGS).

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The study was in compliance with the Helsinki Declaration and was approved by the Ethics Review Board at the Erasmus University Medical Center, Rotterdam, the Netherlands. Written and informed consent was obtained from all research participants included in this study.

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All authors contributed to writing the manuscript. KR, GV, and AB designed and performed the experiments and analyzed the data. MB and YB facilitated the automated sample preparation. JH and RD collected and maintained the clinical cohort. MW and RD supervised the study.

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KR and MW are inventors on a patent application on sialic acid derivatization by ethyl esterification. All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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T lymphocytes are central players of the adaptive anti-tumor immune response. Activation of T lymphocytes requires two signals: the first one is originating from the T-cell receptor after interaction with an antigen-derived peptide presented by major histocompatibility complex, while the second is delivered upon interactions between co-receptors on T cells and their ligands on antigen-presenting cells (APCs) or target cells . Co-receptors, also called immune checkpoints, can be divided into stimulatory and inhibitory molecules. The CD28 was the first identified co-stimulatory molecule and the interaction between CD28 and B7.1/B7.2 is one of the dominant pathways required for full activation of naive lymphocytes [2, 3]. In contrast, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 protein (PD-1), when interacting with their ligands B7.1/B7.2 and PDL-1/PDL-2 respectively, restrain the activation of lymphocytes T and are thus considered as co-inhibitory molecules [4–7].

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It is known that manipulation of checkpoint cell-surface signaling molecules can exert potent anticancer effects. Immune checkpoint therapy has recently reached important clinical advances [8, 9]. Indeed, it has been reported that cancer patients treated with anti-CTLA-4 (Ipilimumab) or anti-PD-1 (Nivolumab or Pembrolizumab) blocking antibodies showed an increase of the antitumor T-cell response and a higher rate of disease free survival [10–13]. Additionally, antibodies blocking PD-L1 were shown to be superior to Ipilimumab by having a better safety profile. Therapies combining both Ipilimumab and Nivolumab in patients with metastatic melanoma, have shown significant increase of the progression-free survival rate. However, this combination was also associated with substantial immune related toxicities [12, 14–17].

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Alternative non-antibody based therapies constitute also a promising approach to target immune checkpoints [18, 19]. Indeed, several examples of small compounds such as sulphonamide derivatives , tri-aromatic structures [18, 21], linear, cyclic macrocyclic peptides [18, 22], hydrolysis-resistant D-peptide , peptidomimetics and cyclic peptidomimetics , as antagonist of PD-1/PD-L1 have already been reported. Some of these compounds are highly effective in antagonizing PD-1 signaling and inhibiting tumor growth and metastasis in preclinical models of cancer. Moreover, those compounds are well tolerated with no obvious toxicity [15, 18].

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Among the growing family of inhibitory receptors, the B and T lymphocyte attenuator (BTLA), which interacts with herpes virus entry mediator (HVEM), inhibits T-cell proliferation and cytokine production [24, 25], suggesting that BTLA might be an interesting target in immunotherapy . Naive human CD8+ T cells express high levels of BTLA, which is downregulated upon CD8 differentiation. However, BTLA is not downregulated in melanoma specific CD8+ T cells and remains susceptible to functional inhibition upon HVEM ligation. HVEM is frequently expressed on melanoma cells, suggesting that the BTLA/HVEM pathway might play a role in the inhibition of efficient immune responses against cancer. Preventing/targeting the BTLA/HVEM interaction can reverse the inhibitory functions of BTLA, thus triggering the immune response against cancer. Additionally, in vitro studies using antibodies to block binding of BTLA to HVEM led to increased melanoma specific CD8+ effector T-cells proliferation and enhanced cytokines production [27–29].

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The crystal structure of BTLA/HVEM complex shows specific details of the proteins interaction. BTLA binds to a fragment of HVEM(26–33) which is located in the Cysteine Rich Domain 1 (CRD1) . We used that information to design and characterize peptide-based inhibitors of BTLA/HVEM complex formation. We confirmed that the short fragment of HVEM(23–39) binds to BTLA and blocks the BTLA/HVEM interactions. Further evaluation of individual contribution of each residue from the HVEM(23–39) fragment by a single alanine substitution approach showed that cysteine residues have a key role in the binding to BTLA. Finally, we showed that free cysteine amino acid can disrupt the BTLA/HVEM complex formation, highlighting a cysteine-related artefact in vitro. Overall, these results might be helpful for the design of new compounds targeting BTLA protein.

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The recombinant human BTLA protein used in affinity tests was purchased form Novoprotein (Company product code: C563). For ELISA, recombinant human BTLA-Fc, HVEM-Fc as well as anti-human BTLA (#7.1) blocking monoclonal antibody (mAb) and anti-human HVEM (#11.8) non-blocking mAb were described previously . Anti-HVEM mAb was biotinylated using the Biotin Labeling Kit-NH2 (Abnova, #KA0003), according manufacturer’s instruction.

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The crystal structure of BTLA/HVEM complex (A and B chains) served as a starting point for the modeling (Protein Data Bank ID 2AW2) . The atoms of the HVEM B chain were removed, except for residues 23–39. The protonation state of the histidines was set depending on the environment, to optimize hydrogen bonds. All simulations were carried out using GROMACS 4.5 molecular modeling package using the CHARMM22 force field . The structure was located in the center of a TIP3P water box in which the distance between the solute and the edges was 1.4 nm. Na+ and Cl- ions were added to neutralize the system. Subsequently the system was subjected to 1000 steps of steepest descent minimization. The structures were then equilibrated according to standard equilibration procedures. The system heating was performed using simulated annealing method by increasing the temperature in four steps: from 0K to 50K, then from 50K to 100K, from100K to 200K and finally from 200K to 300K. Every step took 100ps and at the end of the procedure the system was additionally coupled to the target 300K temperature for 100ps. The force constant of 1000kJ/mol*nm was applied to keep the atoms in their positions during the heating procedure. The same force constant was kept also during the first constant volume equilibration simulation during 100ps, followed by another 100ps simulation in the NVT scheme with lowered force constant for position restraint to 300kJ/mol*nm. The final step of equilibration consisted in a simulation in the NPT scheme with Nose-Hoover thermostat and Parinello-Rahman barostat with 300K and 1Pa for 500ps and no position restraints imposed on atoms.

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The production molecular dynamics simulations were done using the NPT scheme with no restraints on the atoms. For the production molecular dynamics simulation, the Verlet leap frog integrator was used. A 12Å cutoff was applied on non-bonded interactions. The bond length was constraint. A time step of 2fs was used with a length of the simulation of 10ns. For the hydrogen bonds analysis, we used 250 structures extracted every 40ps from the molecular dynamics simulation trajectory.

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Peptides were synthesized by solid phase peptide synthesis (SPPS) using semiautomated peptide synthesizer Millipore 9050 Plus PepSynthesizer (Millipore Corporation, Burlington, VT, USA) and general conditions of solid-phase synthesis . Synthesis was performed on a TentaGel R RAM resin (0.19 mmol/g), using 9-fluorenylmethoxycarbonyl/tert-butyl (Fmoc/tBu) chemistry with the following side chain protected amino acid derivatives: Fmoc-Pro-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Tyr(tBu)-OH. Acetylation of the N-terminal amino group was performed using 1-acetylimidazole (1.10 g/1 g of resin at room temperature for 24 h). The peptide was cleaved from the resin for 2 h using a mixture of 88% TFA, 5% fenol, 5% deionized water and 2% triisopropylsilane (10 ml/1 g of resin at room temperature for 2 h). After filtration of the exhausted resin, solution was concentrated in vacuum, and the residue was triturated with Et2O. The precipitated peptide was centrifuged for 15 min, 4000 rpm, followed by decantation of the ether phase from the crude peptide (process was repeated three times). After evaporation of Et2O, the peptide was dissolved in H2O and lyophilized.

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Before the purification the peptides were dissolved in H2O, DTT was added (6-fold excess with respect to purified peptide) and the mixture was sonified for 30 min in 60°C. Purification of the crude peptide was carried out by using RP-HPLC on a semi-preparative Phenomenex Luna C8(2) (250 mm x 20 mm, 5 μm) column. A linear gradient from 5% B to 50% B in A in 150 min was used. The aqueous system (A) consisted of 0.1% (v/v) TFA solution in water, whereas the organic phase (B) was 80% acetonitrile in water, containing 0.08% (v/v) TFA. Purification was monitored by UV absorption at a wavelength of 222 and 254 nm. The purity of the peptide was verified by LC-ESI-IT-TOF/MS (Shimadzu, Shimpol, Warsaw, Poland), and by using RP-HPLC with a Kromasil C8 analytical column (250mm x 4.6 mm, 5 μm), where a gradient of 5% to 100% B in A in 60 min was employed, with A and B as described above.

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The formation of the disulfide bond in peptide HVEM(23–39) was performed after purification. The peptide was dissolved in a mixture of H2O and methanol (1:9, v:v), the final volume was 1.5 l and the concentration of the peptide was established at 40 mg/l. The pH was adjusted and kept between 8 and 9 by using ammonia whilst stirring the solution at room temperature for 3 days in the presence of atmospheric oxygen. Reaction progress was monitored by analytical RP-HPLC in a gradient of 5% to 100% B in A in 60 min. When the reaction of oxidation was completed the solvents were evaporated and the peptide was lyophilized.

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96-well round bottom plates (medium binding; Greiner Bio-One) were coated with 400 ng/well of recombinant BTLA-Fc protein in PBS (50 μL/well) and incubated overnight at 4°C. Wells were washed twice with PBS-T (phosphate buffer saline with 0.1% Tween-20, pH 7.4) and blocked with 1% milk in PBS-T for 1 hour at 37°C. After 2 washes with PBS-T, the peptides were titrated down in triplicates from 10 to 0.1 mg/well in 1% milk/PBS-T and incubated for 2 hours at 37°C. Maximum binding of HVEM to BTLA was controlled by adding 1% milk/PBS-T to negative control wells, whereas maximum inhibition was monitored by adding anti-BTLA 7.1 mAb (0.1 μg/well). Next, the plate was washed and incubated with recombinant HVEM-Fc protein at 400 ng/well in 1% milk/PBS-T for 1 hour at 37°C. Biotinylated anti-HVEM 11.8 antibody was then added (biotinylated preparation diluted at 1/10 000 in 1% milk/PBS-T) and incubated for 1 hour at 37°C. Streptavidin horseradish peroxidase (Dako) was finally added at a 1/8000 dilution in 1% BSA/PBS-T for 30 min at 37°C and detected with TMB (Biorad). The substrate reaction was blocked with 0.2 M H2SO4 and the absorbance was read at 450 nm and 630 nm. The percent of inhibition was calculated assuming that the anti-BTLA 7.1 antibody inhibit the complex at 100% and milk/PBS-T in 0%. Statistical analysis of the results was performed using the one-way analysis of variance (one-way ANOVA).

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The immobilization of BTLA protein in a microcolumn was performed using the NHS-activated acid-coupled Sepharose 4B (Sigma Aldrich). BTLA protein was dissolved in 100 μl of coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) and was added to 0.06 g NHS-activated Sepharose in 100 μl of coupling buffer. The coupling reaction was performed for 1 hour in 25°C and the mixture was transferred into the microcolumn (MoBiTec, Goettingen, Germany). The microcolumn with immobilized protein was washed alternately with 10 ml of solution blocking buffer (0.1 M amino ethanol, 0.5 M NaCl, pH 8.3) and washing buffer (0.1 M CH3COONa, 0.5 M NaCl, pH 4). This step was repeated 4 times. The microcolumn was stored in PBS (pH 7.4).

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Peptide affinity assay using NHS-activated Sepharose microcolumn was already described elsewhere [36, 37]. The peptides were added onto BTLA-Sepharose column equilibrated in ammonium hydrogen carbonate (NH4HCO3, pH 7.4) and incubated for 2 h at 25°C with gentle shaking. After this time the unbound peptide was removed and the column was washed with 100 ml NH4HCO3 and 20 ml of H2O. Dissociation of the protein—peptide complex was performed using 0.1% TFA in H2O (2 x 0.5 ml) by 15 minutes. All fractions were analyzed using mass spectrometry (MALDI TOF/TOF™ 5800, AB SCIEX).

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The presence of free thiols was investigated using Ellman’s assay. DTNB (Sigma Aldrich) was first dissolved in reaction buffer (0.1 M sodium phosphate, pH 7.4, containing 1 mM EDTA) at a concentration of 4 mg/ml. Then peptides and recombinant BTLA-Fc, alone or mixed together (ratio 1:1) were added in DTNB reaction buffer. After 15 minutes of incubation, the SH content of protein samples was then assessed by the measurement of absorbance at 412 nm. For each sample, the absorbance value from the blank was subtracted.

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293T cells expressing BTLA (generous gift from Dr. C. Krummenacher, University of Pennsylvania, USA) were maintained in culture with RPMI 10% FCS supplemented with 0.2 mg/ml hygromycin B. 2x105 293T cells expressing BTLA were incubated with rhHVEM-Fc at 8μg/ml in PBS 0.5% BSA for 30min at 4°C. Cells were then washed and incubated with goat anti-human Fc conjugated with AF647 for 20min at 4°C (Jackson ImmunoResearch, #109-606-170). Inhibition of HVEM-BTLA binding by different peptides was assessed by pre-incubating the cells with the different peptides at 5 mg/ml for 1h at 4°C. BTLA expression was controlled by anti-BTLA APC-conjugated antibody (Biolegend, #344510). Maximum inhibition of binding was determined by adding anti-BTLA7.1 antibody for 1h at 4°C at 2μg/ml before adding rhHVEM-Fc. Sample acquisition was performed on the Gallios Flow-Cytometer (Beckman Coulter) and data were analyzed using the FlowJo Software (TreeStar).

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The crystal structure of BTLA/HVEM complex is stored under ID 2AW2 in the Protein Data Bank . The structure indicates that only the first CRD1 part of human HVEM interacts with BTLA. The domain contains four short β-strands, that are connected by three disulfide bonds, formed between cysteine residues 4–15, 16–29 and 19–37. The fragment of the CRD1 domain of HVEM comprising residues 23–39 forms a β-hairpin structure and the 35–39 fragment builds an anti-parallel inter-molecular β-sheet with G° strand of BTLA in the center of the interaction interface . Moreover, the HVEM(23–39) fragment comprises two hot-spots of the BTLA/HVEM interaction: Tyr23 and Val36. We sought to determine whether the HVEM(23–39) fragment could bind BTLA. We therefore performed the molecular dynamics simulation of the BTLA structure and the HVEM(23–39) fragment (Ac-YRVKEACGELTGTVCEP-NH2). The simulation starting point was a native-like conformation of HVEM fragment, bound to BTLA as in the BTLA/HVEM complex structure (Fig 1). The complex was stable during the 10ns long molecular dynamics simulation. The averaged RMSD calculated for the backbone atoms of the superimposed over carbons alpha structures was 1.2Å. Besides, the hydrogen bonds interactions observed in the starting structure were almost all preserved during the simulation (S1 Table). This computational results suggests that the HVEM(23–39) may stably interact with BTLA.

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HVEM(23–39) peptide is shown in dark blue ribbon, BTLA in beige and the side chains of residues involved in the inter-molecular hydrogen bonds in stick representation with nitrogen atoms colored in blue, oxygen colored in red and hydrogen atoms in white. Hydrogen bonds are represented by light blue lines.

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The designed HVEM(23–39) peptide was synthesized and an affinity test was performed using microcolumn with the immobilized BTLA protein. Three fractions were analyzed: supernatant, last wash and elution by using mass spectrometry techniques. In the supernatant fraction the signal m/z corresponding to the excess of the HVEM(23–39) peptide was observed (Fig 2A). In the last wash fraction, no signal was observed confirming that the excess of peptide was removed (Fig 2B). The presence of m/z signal in elution fraction indicated that the complex between BTLA and HVEM(23–39) was formed (Fig 2C).

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Microcolumn affinity test results for binding of HVEM(23–39) fragment to BTLA: (A) supernatant, (B) last wash and (C) elution fractions. The MS measurements were done with the use of MALDI TOF/TOF 5800 (ABSciex, Germany). As a matrix α-cyano-4-hydroxycinnamic acid (CHCA, 10 mg/ml, Sigma-Aldrich) was used. The measurements were done in reflector positive mass mode with previous mass calibration with commercial standard peptide mixture (The Peptide Mass Standards Kit for Calibration of AB SCIEX MALDI-TOF™ Instruments).

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Next, HVEM(23–39) was analyzed in an competitive ELISA test, in order to determine whether this fragment may bind to BTLA and inhibit BTLA/HVEM interaction. Fig 3 shows that the HVEM(23–39) can partially prevent the ligation of HVEM to BTLA, in a dose dependent manner, while scrambled peptide (Ac-ELCAGPVTRKVECTYGE-NH2) has no effect. Moreover, the scrambled peptide (named Ctrl peptide in figures) did not interact with BTLA immobilized in microcolumn (Table 1).

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Inhibition of the BTLA/HVEM interaction was assessed by ELISA (at least two experiments in triplicate). The graph shows percentages of inhibition of the BTLA/HVEM ligation, relative to the negative control (PBS), in the presence of different concentrations (5, 1 and 0.1 mg/mL) of HVEM(23–39) and corresponding scrambled peptide (Ctrl peptide). The gray and dotted back lines correspond to the percentages of inhibition observed with an anti-BTLA blocking antibody (Mean +/-SEM). *: p< 0.05 following non-parametric One-way ANOVA and Dunn’s post-test.

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To assess the importance of each amino acid residues in HVEM(23–39) interaction with BTLA protein, single alanine-substituted HVEM(23–39) variants were synthesized [38, 39] and tested in the BTLA/HVEM ELISA. While no or minor diminution in the inhibition of BTLA/HVEM binding was found with the majority of alanine-substituted fragments compared to the native HVEM(23–39), we observed a significant reduction of the inhibition, in a dose depend manner, for the two peptides where cysteine in position 29 (C29A) or 37 (C37A) was replaced by an alanine (Fig 4). This suggests the possible importance of cysteine residues in inhibition BTLA/HVEM complex formation by the HVEM(23–39) peptide.

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The relative involvement of alanine-substituted HVEM(23–39) peptide analogs was assessed by ELISA (at least two experiments in triplicate) in the presence of graded peptide concentrations (5, 1 and 0.1 mg/mL). The percentage of inhibition of the BTLA/HVEM binding was calculated in relation to the negative control (PBS). The gray and dotted back lines correspond to the percentages of inhibition observed with an anti-BTLA blocking antibody (Mean +/-SEM). Statistical analysis was performed by comparing each concentration of modified peptide to the same concentration of wild-type peptide HVEM(23–39). **: p< 0.01 following non-parametric One-way ANOVA and Dunn’s post-test.

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To further determine the role of cysteine residues from HVEM(23–39) peptide in the inhibition of BTLA/VEM interaction, several HVEM(23–39) analogues were designed and synthesized (Table 2). Initially, we aimed to test, if the Cys29 residue could be replaced with aromatic residue that could interact with His127 residue of BTLA to improve the interaction of the peptide. To test this, we synthesized the HVEM(23–39) C29Y peptide. To additionally probe the importance of the cysteine interactions, we replaced the Cys29 residue with the serine residue, which is of similar size and polarity. We assumed that the C29S analog could mimic the cysteine interactions but bears no free sulfhydryl group that could interact non-specifically with BTLA. We were also wondering, if even shorter HVEM-derived peptide could inhibit the interactions between BTLA and HVEM. We synthesized therefore 9 amino acid-long peptides comprising the ®-strand interface of HVEM, HVEM (31–39) fragment.

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The analogs (Table 2) were tested in the ELISA tests and compared to the HVEM(23–39) and scrambled peptide (Fig 5). We found that both substitutions of Cys29 significantly abrogated the blocking capacity of the HVEM(23–39) peptide. This could indicate the lack of specific interactions of Cys29 residue and point to the importance of the free sulfhydryl group for the binding of peptides. Moreover, albeit not significant, the shorter synthesized peptide: HVEM(31–39) that lacks the Cys29, showed a lower percentage of inhibition than the HVEM(23–39). To further test the importance of Cys37, we synthesized and tested the its analog (HVEM(31–39) C37S). We found that this peptide had no blocking capacity. Finally, the cyclic peptide HVEM(23–39) (C29-C37), encompassing blocked sulfhydryl groups, was also tested, but no blocking of BTLA/HVEM interaction was observed (Fig 5). Of note, in CD spectrum, formation of the disulfide bridge in HVEM(23–39) peptide did not significantly change the peptide structure (S1 Fig).

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Increasing concentrations of HVEM(23–39) or HVEM(31–39) modified peptides, where one or both cysteines were substituted, were tested in ELISA and compared to corresponding wild-type and scrambled (Ctrl peptide) peptides (at least two experiments in triplicate). The percentage of inhibition of the BTLA/HVEM binding was calculated in relation to the negative control (PBS). Statistical analysis was performed by comparing each concentration of each peptide to the same concentration of wild-type peptide HVEM(23–39). **: p< 0.01 following non-parametric One-way ANOVA and Dunn’s post-test.

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Then those analogs were tested in affinity test. We observed that peptides, which could inhibit the BTLA/HVEM interactions in ELISA tests (Fig 5), were also bound by BTLA protein immobilized in microcolumn (Table 1). The exception was cyclic peptide HVEM(23–39) (C29-C37) which interacted with BTLA in affinity test but in ELISA test its inhibitory effect was not observed. The results suggest that the binding site of cyclic peptides and HVEM(23–39) on the BTLA surface is different.

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Since the cyclic peptide C29-C37, where cysteine residues were oxidized, does not block the BTLA/HVEM binding, we wondered whether the blocking ability of HVEM(23–39) fragment may be due to the capacity of HVEM-based peptide cysteines to reduce the cysteine disulfide bridges present on the BTLA surface, or possibly make disulfide bridges with these BTLA cysteines instead. Therefore, the effect of free cysteine and methionine amino acids or oxidized dimer of cysteine (cystine) on the BTLA-HVEM binding was analyzed. Results showed that cysteine with free sulfhydryl group targeted BTLA protein and prevented partially the binding of HVEM, whereas for the methionine and cystine, the inhibitory properties were not observed (Fig 6A). An Ellman’s assay was then performed on both HVEM(23–39) peptide and recombinant BTLA-Fc in order to quantify free thiol groups. In contrast to BTLA-Fc, we observed free thiol groups in the HVEM(23–39) peptide. However, after 2 hours of incubation, the concentration of free thiol groups in HVEM(23–39) peptide was lower, suggesting that a fraction of the peptide is oxidized. Besides, when the HVEM(23–39) peptide was incubated with BTLA-Fc, we also observed a decrease in the concentration of free thiol groups over time, which was more pronounced than in the condition where the peptide was alone (Fig 6B). These results suggest that BTLA protein may form disulfide bonds with the HVEM(23–39) peptide, lowering the amount of free thiol groups in the peptide.

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(A) The effect of increasing concentrations (5, 1 and 0.1 mg/mL) of free amino acids were tested in BTLA/HVEM ELISA (three experiments in triplicate). The percentage of inhibition was calculated in relation to the negative control (PBS). The gray and dotted back lines correspond to the percentages of inhibition observed with an anti-BTLA blocking antibody (Mean +/-SEM). Statistical analysis was performed by comparing each concentration of residue or peptide to the same concentration of Cys. (B) Assessment of free thiol groups over time in rhBTLA-Fc protein and HVEM(23–39) peptide alone or mixed together. **: p< 0.01 and ***: p< 0.001 following One-way ANOVA and Dunn’s (A) or Dunnett’s (B) post-test.

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In order to be in a more physiological setting, we developed a cellular assay, where 293T cells expressing BTLA were stained with rhHVEM-Fc followed by AF647-conjugated anti-human IgG antibody. Using this assay, we tested HVEM(23–39) fragment as well as HVEM(23–39) (C29-C37), scrambled peptide (Ctrl), cysteine and methionine amino acids. Surprisingly, in contrast to ELISA tests, almost no blocking of the BTLA/HVEM binding was observed with HVEM(23–39) peptides. However, the free cysteine amino acid blocked BTLA/HVEM complex formation (Fig 7).

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293T cells expressing human BTLA were incubated with peptides or free amino acids (5mg/mL) prior labeling with rhHVEM-Fc and AF647-conjugated anti-human IgG antibody (at least two experiments in triplicate). The graph shows the Geometric Fluorescence Intensity (GMFI). *: p< 0.05 and ***: p< 0.001 following non-parametric One-way ANOVA and Dunn’s post-test.

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Recently, activatory and inhibitory immune receptors and their ligands emerged as promising targets for cancer immunotherapies. Indeed, treatment based on the blockade of immune checkpoints such as PD-1 or CTLA-4, have demonstrated major clinical benefit for cancer patients [40–42]. It is known that BTLA is involved in the negative regulation of T-cell responses by interacting with HVEM [27, 43], highlighting BTLA as a potential target for cancer immunotherapy. Our study focused on the design of blocking peptides of the interaction between BTLA and HVEM, based on the amino acid sequence of HVEM binding fragment. The crystal structure of BTLA/HVEM complex indicated that in the interaction between the two proteins, two fragments of BTLA: (35–43) and (118–128) bind with HVEM: (26–33) and (33–38) respectively. Therefore, HVEM(23–39) fragment was synthesized and its interaction between BTLA and HVEM(23–39) was confirmed by using affinity chromatography. The competitive ELISA tests showed also that the peptide could inhibit the proteins binding. However, we showed using competitive ELISA and cellular assay that this blocking capacity was mainly due to cysteine residues present in the peptide sequence or in solution. Compaan and co-workers showed that the interactions between BTLA and HVEM proteins are primarily stabilized by main chain hydrogen bonds and includes relatively few side chain contacts . The conformation of the HVEM(23–39) peptide is significantly different than the conformation of the same fragment in the native protein due to the fact that it is not stabilized by disulfide bridges and the peptide poses two cysteine residues with free sulfhydryl groups. In the monomeric crystal structure of the extracellular domain of BTLA protein, six cysteine residues form three disulfide bonds between residues 72–79, 34–63, and 58–115. The Cys58–Cys115 disulfide bond is completely buried (solvent accessible surface is 0% for both cysteine) in the hydrophobic core of BTLA . The Cys58–Cys115 (solvent accessible surface is 2,9% and 17,9%) and more Cys34–Cys63 (solvent accessible surface is 45,1% and 33,3%) disulfide bonds are exposed to the solvent and could be sensitive for the other, free or high reactive sulfhydryl group. Therefore, we propose that free sulfhydryl groups of HVEM(23–39) peptide or free cysteine amino acid compete, in our in vitro assays, with disulfide bridges (Cys58–Cys115 or Cys34–Cys63) of the BTLA protein to form covalent S-S bond between HVEM(23–39) peptide or cysteine amino acid and BTLA protein. This may most likely change the structure of BTLA, thus hindering the binding of HVEM. Yet, we observed that the scrambled peptide, which also contains free thiol groups as confirmed by Ellman’s assay (S2 Fig), lacks blocking activity. In order to reduce the disulfide bonds on BTLA, peptides have to pre-dock to the surface of the protein. We hypothesize that scrambled peptide may have a lower affinity to BTLA, compared to HVEM(23–39) peptide. As a consequence of the weaker binding to BTLA the scrambled peptide may not be able to reduce the exposed disulfide bonds of BTLA and form disulfide bonds with the protein.

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Finally, the fact that this phenomenon was not observed in the cellular assay might be caused by a spatial arrangement of the BTLA protein on the cell surface making the disulfide bridges on the BTLA surface less accessible for the tested peptides. We hypothesize that the small, compared with peptides, cysteine amino acid could reach the BTLA surface despite the spatial hindrance, hence only activity of cysteine amino acid was observed in the cellular test.

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The copaiba trees belong to the genus Copaifera, family Fabaceae, and subfamily Caesalpinoideae. The genus was described the first time by Marcgraf and Piso in 1638, who employed the name “Copaiba” without designating the species . In 1760, Nicolaus Joseph Von Jacquin described the species Copaiva officinalis in the work Enumeratio Systematica Plantarum . Afterwards, in the year 1764, Carl von Linnaeus did a more detailed study of the genus in the work Species Plantarum, in which he described the type species Copaifera officinalis (Jacq.) L. . There are more than 70 Copaifera species distributed throughout the world, with widespread occurrence in Central and South America; there are also four species found in Africa and one species found on the island of Borneo, situated in the Pacific Ocean . Brazil is the country with the greatest biodiversity of Copaifera with 26 species and 8 varieties .

review

The vernacular name copaíba probably originated from the Tupi-Guarani and alludes to the names used by indigenous peoples, copaíva and copahu (kupa’iwa and kupa’u, respectively), which refers to the tree exudate, in reference to the oil stored in its interior . Sixteenth-century records produced by chroniclers during the Brazilian colonization report the widespread use of copaiba oil among the natives as anti-inflammatory and healing agents, and also for esoteric purposes, such as aphrodisiac and contraceptive . This natural product is known and valued to the present day, mainly in the Amazon region, where the rural population has little access to industrialized pharmaceutical products and public health care .

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The copaiba trees have shrub or arboreal habits, can reach up to 40 m height and 4 m diameter at breast height (dbh), have slow growth, and can live up to 400 years . Their cylindrical trunks contain intercellular secretory channels arranged in bands of marginal axial parenchyma, the lumen from secretory cells is formed schizogenously, and the oleoresin is synthesized in parenchyma cells of the canal. The species have alternate leaves, which are pinnate with 2–12 pairs of leaflets (opposite, alternate, or subopposite), usually glabrous, and may have translucent points and glands at the base of the marginal vein; they have small and interpetiolar stipules and are generally deciduous. The inflorescences are alternate panicles and the flower buds are protected by small bracts; they have small flowers, numerous and sessile, which are monoclamids with a tetramer chalice that forms short tubes and contains internally hirsute sepals. The androecium holds 10 free stamens, glabrous fillets, and oblong and rimose anther; and the gynoecium presents a sessile ovary with two elongate ovules, filiform style, and globular and papillary stigma. The fruits are bivalved, dehiscent, laterally compressed, and monospermic. The seed is a pendulum, oblong-globose, covered by abundant white or yellow aril, and lacking endosperm .

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Although the Copaifera genus has been extensively studied taxonomically, there are still difficulties in identifying some species, mainly due to their intricate floral morphology and absence of reproductive structures in the samples studied. With regard to the Amazonian species, the scarcity of field information and illustrations of specimens comprise the main limitations for botanical descriptions of the group. These taxonomic problems have restricted the advance of chemical and pharmacological research, limited the industrial and rational uses of resin oils and wood, and have also hampered the development of projects, plans for sustainable management, and conservation of commercially targeted species .

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The main economic contributions of Copaifera species have been wood and oleoresins. Among Copaifera species that are used in the production of oleoresins, C. reticulata is the most frequent, representing 70% of the production . Copaiba oleoresin is one of the most important renewable natural remedies for the indigenous people from the Amazon region and its use is widely diffused due its various pharmacological properties . The oleoresin is a transparent, colored liquid with variable viscosity, and is constituted by a nonvolatile fraction composed of diterpenes and a volatile fraction composed of sesquiterpenes . Its chemical profile may vary according to species, seasonal and climatic characteristics of the environment, soil type and composition, and rainfall index. Biotic pressures, such as insect predation and pathogen infection, also cause differences in oleoresin composition . The extraction of copaiba oil is done through the perforation of the trunk with a punch, and the resin is collected with the help of a polyvinyl chloride (PVC) pipe, through which the oil flows and is then stored. This practice is mainly done by plant extraction; therefore, the product of several trees is often mixed, resulting in an additional obstacle to the botanical identity of the copaiba trees. In addition, the lack of parameters to characterize the oil and to perform quality control of the botanical drug also constitutes an obstacle for the registration and exportation of herbal products containing copaiba .

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The genus Copaifera is native to tropical regions of Latin America, an area of great species diversity . Distributed widely in the Americas, stretching from Mexico to northern Argentina, the genus also occurs in West Africa and Asia . The greatest richness of species occurs in Brazil, where they are distributed from the north to the south of the country. The most common species are C. multijuga Hayne, which is found in the Amazonas, Pará and Rondônia states; C. reticulata Ducke that occurs in Amapá, Pará and Roraima;, and C. langsdorffii Desf., which can occur from the northern to southern regions of Brazil . Other species have more restricted distribution, such as C. guyanensis Desf. (Amazonas), C. majorina Dwyer (Bahia), C. cearensis Huber ex Ducke (Ceará, Bahia, Piauí and Rio de Janeiro), C. elliptica Mart. (Goias and Mato Grosso), C. paupera (Herzog) Dwyer (Acre), and C. lucens Dwyer (Bahia, Espírito Santo, Minas Gerais, Rio de Janeiro, São Paulo) . Although many species of Copaifera have wide occurrence within the Brazilian territory, and may occur in different phytogeographic domains (e.g., C. langsdorffii), some feature endemism, such as C. trapezifolia Hayne, which occurs in an extremely disturbed region of the Atlantic rainforest, of which only 11.6% of the natural vegetation cover remains . Thus, morphological, physiological, and ecological studies are highly relevant for the preservation of species and their natural environment . A study conducted in the Minas Gerais state on the geographical distribution and environmental characteristics of arboreal species showed that C. langsdorffii has wide occurrence throughout the whole state, where latosol type soil predominates, but additionally has a preference for ustic soils (62%) .

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In relation to the ecological group, copaíba are classified as long-living, late secondary, and climax tree species, demanding of light but tolerant to shade . They are considered generalists because they are adapted to a wide variety of environments. They can occur in floodplains, riparian forest, and streams of the Amazon basin and the forests of the Cerrado in the center of Brazil . C. langsdorffii, for example, has great ecological plasticity, occurring in several biomes, such as Cerrado, Atlantic Forest, Caatinga, and Amazon rainforest . Copaifera species have great plasticity in relation to edaphic conditions; they occur in areas with fertile soil and well-drained soil and in areas with very poor acidic soils, such as Cerrado fields. They grow well on sandy and clayey soils and generally occupy the forest canopy .

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Phenological studies on Copaifera are important for the rational use of the species and for the preparation of management plans . The reproduction of copaibas occurs from the fifth year of growth after planting in a climax forest ecosystem . C. multijuga, commonly found in the Amazon, blooms in the rainy season—between the months of December–April—and fructifies between April and July . Blooming of C. reticulata occurs from January to March, with fruiting from March to August, lasting into October . C. langsdorffii, observed in the Tijuca Forest, Rio de Janeiro, blooms between March and April and fructifies between August and September. Another survey carried out near Campinas, São Paulo state, showed that flowering of C. langsdorffii occurs in the middle of the rainy season (December–February), with development of fruit during the dry season (April–September) . The phenophases of C. officinalis were monitored in the municipality of Boa Vista (Roraima state, Brazil), and showed that the flowering of the species occurs between the months of September and November and the fruiting from November to March. Depending on the stage of fruit ripening, the dehiscence can begin in January, in which the seeds enveloped by the aril are exposed, allowing for their dispersal .

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Copaifera is a hermaphrodite plant of mixed reproduction with a predominance of allogamy. The trees are generally bee pollinated (melittophily), and Apis mellifera and Trigona spp. are its main pollinating agents . C. langsdorffii has high fecundity, producing large quantity of fruits in a short period of time. Its seeds have low nutritional value, mainly composed of carbohydrates, but can attract a wide variety of animals with a general diet . The dispersion of the copaiba seeds occurs mainly in zoocoric and barocoric forms . Some vertebrates, such as birds and mammals, have been observed visiting the fruits of Copaifera . Its seeds have morphological characteristics that fit the ornithocoria syndrome, mainly because they are black with colored, fleshy arils, which, after being swallowed, can be regurgitated intact and remain viable for germination . A study revealed that 10 species of Passeriformes, such as Ramphastos toco, Cyanocorax cristatellus, and Turdus rufiventris, visited the fruits of C. langsdorffii. Likewise, monkeys of the species Eriodes arachnoides and Cebus paella also eat the fruits of C. langsdorffii . Copaiba seeds may also present hydrocoric dispersion due to their frequent occurrence near waterways . Copaiba seeds are of conventional behavior and may be conserved in the long term ex situ, with dormancy due to the deposition of coumarin in the tegument, and its germination is of the epigene type . A tree can produce from 2 to 3 kg of seeds .

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The population density of copaiba trees in an area is usually very low. It is possible to find only one tree every 5 ha, but they may occur in densities of one to two trees per hectare. The production of oleoresin by species is fairly variable and can be influenced by genetic differences among species, habitat, soil, and intensity of exploitation . The production of oleoresin per tree ranges from 100 mL to 60 L per year. In addition, not all trees produce oil . Therefore, detailed investigations regarding extraction methods and equipment that do not harm the plant, correlation of genetic data to botanical identification of species, floristic inventory of copaiba populations, and ecological studies on its ecosystems are indispensable for the sustainable and rational use of this resource .

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In Pará state (Amazon region, Brazil), people of all ages and social classes consider copaiba one of the most important natural remedies from the Amazon region. Several parts and preparations of the plant are used in folk medicine . The oleoresin or bark decoction is used as an anti-inflammatory and contraceptive by native people from the Brazilian Amazon. The topical application of oil on the skin serves to heal wounds. It is used in massages on the head to cure paralysis, pains, and convulsions. In Amapá state, it is recommended to soak a cotton ball in oil and place on tumors, ulcers, or hives. The daily intake of two drops of oil mixed with one tablespoon of honey is indicated for inflammation, syphilis, bronchitis, and cough . In Venezuela, the oil is used to prepare a patch that is applied to heal ulcers and wounds, and the decoction of the bark in the form of a bath is used to combat rheumatism, to wash infected wounds such as dog bites, and to use as an anti-tetanus . A tea from the seeds is also used as a purgative and for treatment of asthma. In northern Brazil, the practice of “embrocation” (applying oil directly to the throat) is common to treat throat infections . In Belém, the “garrafada”—an infusion of the bark sold in bottles—is currently used as a substitute for the oleoresin due to the difficulty in obtaining the oil in the city .

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Copaiba has a wide range of ethnopharmacological indications, including for the treatment of: cystitis, urinary incontinence, gonorrhea, and syphilis; respiratory ailments, including bronchitis, strep throat, hemoptysis, pneumonia, and sinusitis; infections in the skin and mucosa, such as dermatitis, eczema, psoriasis, and wounds; ulcers and lesions of the uterus; leishmaniasis and leucorrhea; anemia; headaches; and snake bites. It is also used for its aphrodisiac, stimulant, anti-inflammatory, antiseptic, anti-tetanus, antirheumatic, antiherpetic, anthelminthic, anticancer, antitumor (prostate tumors), and antiparalytic properties . Copaifera species are used by people of Igarapé Miri (Pará state) for healing wounds .

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Studies have shown that the ingestion of high doses of copaiba oil can cause adverse side effects, such as gastrointestinal irritation, sialorrhea, and central nervous system depression. A dose of 10 g may cause symptoms of intolerance, nausea, vomiting, colic and diarrhea, and exanthema. Prolonged use may cause kidney damage and topical reactions in susceptible individuals . Thus, the advance in pharmacological and quality control studies of copaiba formulations sold at herbal markets is indispensable for the safe use of this plant drug.

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The species of Copaifera are intensively pursued for inclusion in the cosmetics market due to their therapeutic properties and fragrant value of their oils . Copaiba oil is currently used in the cosmetic industry as a fixative for perfumes and perfuming soaps . As an emollient, bactericidal, and anti-inflammatory agent, copaiba oil is used in the production of soaps, lotions, creams and moisturizers, bath foams, shampoos, and hair conditioners . In addition, it aids in the treatment of dandruff and acne . Despite its fragrant value, little information regarding its odorant potential is available in the literature .

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As a renewable source of hydrocarbons, the use of copaiba oil as an ecologically clean fuel has been evaluated. Experimental plantations were started in the early 1980s near Manaus, Brazil to test its viability as an alternative energy source to fossil fuels . For potential use as fuel, a combination with diesel oil in a ratio of 9:1 (diesel oil to copaiba) has been recommended . Various reports indicate that the liquid can be poured directly into the fuel tank of a diesel-powered car and the vehicle will run normally, with a bluish exhaust smoke being the only noticeable difference . Traditionally, the oil is used in lamps as fuel for lighting .

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The copaiba trees are considered hardwoods with high demand due to their properties of strength, as well as insect and xylophagous fungi repellency. The wood is saturated with oil and resin and has been used in both shipbuilding and civil construction, especially in the manufacture of steam caves, pool cues, and decorative and furniture coverings. It is also used in the preparation of lumbers, rafters, door and window frames, and boards in general, including for agricultural implements, general carpentry, flooring furniture, coatings, lamination, plywood sheets. The wood has a high content of lignin and is very good for the production of alcohol and charcoal. C. langsdorffii has traditionally been exploited extensively for charcoal in the Cariri Region, south of Ceará .

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In southern Pará state, farmers have used copaiba oil to prevent foot-and-mouth infection in cattle. The oil is poured on the floor next to the salt lick so that when cattle approach to eat salt, they step in oil soaking their feet . When wounded, some animals lick and rub their bodies in the oil that flows from the trees .

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Hunters often hunt under the copaiba tree during fruiting because the seeds and oil attract animals . The oleoresin is used in the photographic industry to improve image clarity in areas of low contrast and resolution. The resin has also been used in paper making, as an additive for butadiene in the production of synthetic rubber, as a source of a chiral substrate in the synthesis of biomarkers of sediment and oil residues, and as fixative in the manufacture of varnish, perfume, and paints used in the painting of porcelain, fabrics, and for dying cotton yarn .

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The major components of the essential oils from Copaifera species are summarized in Table 1. In general, copaiba oils derived from Copaifera oleoresins are rich in sesquiterpene hydrocarbons and often dominated by β-caryophyllene . Some copaiba oils, however, also show significant concentrations of diterpene acids, which are generally analyzed as their methyl esters . A perusal of internet sources of copaiba oil suggests that the most important commercial sources of copaiba oil are C. langsdorffii, C. officinalis, and C. reticulata, and the most prized copaiba oils are rich in β-caryophyllene. The oleoresin essential oils from these three Copaifera species can have as much as 33% (C. langsdorffii), 87% (C. officinalis), and 68% (C. reticulata) β-caryophyllene (see Table 1).

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The oleoresins of several Copaifera species have been shown to be rich sources of clerodane, kaurane, and labdane triterpenoids (Figure 1, Figure 2 and Figure 3, Table 2). In particular, C. langsdorffii resin is composed of biologically active copalic acid and kaurenoic acid . C. multijuga and C. paupera resins are also good sources of copalic acid.

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Copaifera oleoresins have shown remarkable biological activities, many of which have been attributed to diterpenoid acids (see Table 3). Generally, Copaifera oleoresins and their diterpenoid constituents have shown antibacterial, anti-inflammatory, antileishmanial, antiproliferative, antitrypanosomal, and wound-healing activities.

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Several Copaifera oleoresin oils have shown in vitro antiparasitic activity against Leishmania amazonensis promastigotes, including C. cearensis, C. langsdorffii, C. lucens, C. martii, C. multijuga, C. officinalis, C. paupera, and C. reticulata . The resin oil of C. martii showed in vivo antileishmanial activity in a mouse model and C. reticulata resin oil showed activity against L. amazonensis axenic amastigotes (IC50 = 15.0 μg/mL) and intracellular amastigotes (IC50 = 20 μg/mL) . Diterpenoids isolated from C. officinalis—agathic acid, alepterolic acid, kaurenoic acid, methyl copalate, pinifolic acid, and ent-polyalthic acid—showed antileishmanial activity against L. amazonensis promastigotes .

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Copaifera oleoresins and diterpene acids have also shown antitrypanosomal activities. C. duckei and C. reticulata resins showed in vitro activity against T. evansi trypomastigotes . The diterpene acids—agathic acid, copalic acid, alepterolic acid, kaurenoic acid, methyl copalate, pinifolic acid, and ent-polyalthic acid—all showed antitrypanosomal activity against T. cruzi, including in the epimastigote, trypomastigote, and amastigote forms of the protozoan .

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