The process involves gene fusion between a soluble receptor TNFRII with a ligand binding domain and a trimerization tag from the C-propeptide of human collagen (Trimer-Tag), which is capable of self-assembly into a covalently linked trimer. We show that the homotrimeric soluble TNF receptor (TNFRII-Trimer) produced with such method is more potent in ligand binding kinetics and cell based bioassays, as well as more efficacious in attenuating collagen-induced arthritis (CIA) in a mouse model than its dimeric TNFRII-Fc counterpart. Thus, this work demonstrates the proof of concept of Trimer-Tag and provides a new platform for rational designs of next generation biologic drugs.Tumor necrosis factor is a major pro-inflammatory cytokine which triggers various physiological consequences by binding to and trimerizing its receptors, and has been the single most sought-after drug target for intervening autoimmune diseases such as rheumatoid arthritis and psoriasis. However, current TNF-α blockers, including soluble receptor-Fc fusion and therapeutic antibodies, are all dimeric in structure, whereas their target TNF-α itself is homotrimeric in nature. Here we describe the development of a trivalent soluble TNF receptor and show that it is a more potent than the dimeric TNF receptor decoys in inhibiting TNF-α signaling both  However, an abnormal persistent release of TNF-α could result in autoimmune diseases, such as rheumatoid arthritis (RA), psoriasis, and others3. Hitherto, accumulative evidence has shown that TNF-α, the initial inflammatory cytokine, plays decisive roles in the occurrence and development of autoimmune diseases4. In vivo, biologically active TNF-α molecule exits as a homotrimer5, and activates the downstream signal pathway by binding to trimerized TNF-α receptors (TNFR) on the cell surface, leading to receptor trimerization and activation, which in turn triggers a cascade of events leading to inflammation6.Tumor necrosis factor (TNF-α) is a major pro-inflammatory cytokine produced by human body to combat infections, and it provides necessary protections for the host5. Accordingly, fusing a soluble TNF receptor with the Fc region of immunoglobulin G1, which is capable of spontaneous dimerization via disulfide bonds, allowed the secretion of dimeric soluble TNF receptor7. In comparison with the monomeric soluble receptor, the dimeric TNF Receptor II-Fc fusion has a greatly increased affinity to the homo-trimeric ligand. This provides a molecular basis for the block buster anti-TNF drug Etanercept in treating rheumatoid arthritis (RA)8. Although Enbrel was shown to have a Ki below 1 nM to TNF-α7, 25 mg twice a week subcutaneous injections, which translates to μg/mL level of the soluble receptor, are required for the RA patients to achieve clinical benefits (see www.enbrel.com). The high level of recurrent Enbrel consumption per RA patients has created a great pressure as well as high cost for the drug supply, which limits the accessibility of the drug to millions of potential patients in developing countries.Structurally, all members of TNF family of cytokines are homotrimersClearly, there is a great need to be able to create secreted homotrimeric soluble receptors or biologically active proteins, which may have perfect docking sites, hence higher affinity, to their naturally occurring homotrimeric ligands, such as TNF family of cytokines. Such trimeric receptor decoys theoretically should have a much higher affinity to their trimeric ligands than their dimeric counterparts such as etanercept Fig. . Such raApparently, one of the feasible plans to improve the current design of the TNF-α antagonist molecule may be creating a secreted form of homo-trimeric soluble receptors, or proteins with similar biological activities, containing canonical docking sites, hence with greater affinities for the natural homotrimeric ligands than that of dimeric TNFRII-Fc. Accordingly, we have developed a soluble trimeric TNFR antagonist which acquires greater affinity to its natural homo-trimeric ligands in the hope for a significant increase of its clinical benefits.9. The fibrillar types of collagen are synthesized in soluble forms of trimeric precursors procollagen, whose central triple-helical domains consist of hundreds of uninterrupted “G-X-Y” repeats (orglycine repeats), and the N- and C-terminal are flanked by non-collagenous propeptide domains10. The assembly of a trimeric pro-collagen molecule is initiated by the association of the C-propeptide domains via a single type of inter-chain disulfide bond formed between Cys47 and Cys64 among neighboring chains11, and the triple helix folds and moves toward the N-terminus in a zig-zag-pattern12. Following by the completion of its assembly, the collagen maturation requires an additional step, the proteolytic removal of the C- and N-terminal extensions. It is revealed that the C-propeptide domains are cleaved off by BMP-1 proteinases14, which recognizes a specific peptide sequence embedded in the border region between the glycine repeats and C-prodomain.To be therapeutically feasible, a desired trimerizing protein moiety for biologic drug designs should fulfill the following criteria. Ideally it should be part of a naturally secreted protein, like immunoglobulin Fc, and abundant (non-toxic) in circulation, human origin (lack of immunogenicity), relatively stable, capable of efficient self-trimerisation and pertain an optimal geometry in projecting soluble receptor to be trimerized to ensure maximum ligand binding. Collagen, being the most abundant protein in human body, is a major component of the extracellular matrix15.Recently we have demonstrated the use of the C-prodomain of human α1(I) collagen to form covalently linked TRAIL which is a member of TNF family of cytokines and showed it was fully activate in biological functions as the native non-covalently linked trimeric ligand, whereas the dimierc TRAIL-Fc fusion retained little activitiesin vitro, as well as more efficacious in attenuating collagen-induced arthritis (CIA) in an mouse model in vivo than the dimeric TNFRII-Fc fusion protein.Here we use the C-propeptide domain of human type III procollagen as a trimierzation tag for TNFRII. We provide evidence that the homotrimeric soluble TNF receptor (TNFRII-Trimer) produced with such method is more potent both in ligand binding kinetics and cell based bioassays To obtain sufficient amounts of recombinant TNFRII-Trimer fusion protein with high purity for functional analysis, we screened for high-titer production clones of TNFRII-Trimer vector-transfected CHO cells via MTX-mediated gene amplification. The resulting leading clone was adapted to serum-free medium and fed-batch cell culture from bioreactor led to high-level expression of TNFRII-Trimer Fig. . During To verify the structural feature and integrity of TNFRII-Trimer fusion protein vs TNFRII-Fc, antibodies specific to human TNFRII-domain, Fc-domain and the Trimer-Tag -domain were used to conduct the western blot analysis, respectively. As expected, the presence of TNFRII and Trimer-Tag domains of the trimeric TNFRII-Trimer fusion protein were each confirmed by the respective antibodies. As a negative control, the Trimer-Tag-specific antibody failed to detect TNFRII-Fc, while the Fc-specific antibody failed to detect TNFRII-Trimer fusion protein under non-reducing conditions Fig. . The res16 under in situ ligand-receptor binding assays after SDS-PAGE separation and transfer of the purified TNFRII fusion proteins to a PVDF membrane. The result indicated that under non-reducing condition, TNFRII-Trimer, like the TNFRII-Fc fusion protein could bind to its AP-tagged ligand, TNF-α (AP-TNF-α), whereas AP alone failed to do so  was performed to identify charge variants of TNFRII-Trimer fusion proteins. The result showed that the isoelectric point of TNFRII-Trimer was within 4.3–5.0 and that of TNFRII-Fc was within 4.5–4.8 Fig. . CompareTo determine whether TNFRII-Trimer was properly glycosylated, the resorcinol method was applied to detect sialic acid (SA) content of TNFRII-Trimer with TNFRII-Fc as a control. The SA content values for TNFRII-Trimer and TNFRII-Fc were determined to be 35.13 and 31.05 mol of sialic acid/mol of protein, respectively , the latter dissociated (Koff = 2.42 × 10−5 s−1) from TNF-α upon binding at a rate about six times slower than that of the dimeric TNFRII-Fc (Koff = 14.2 × 10−5 s−1).To determine the molecular binding kinetics of TNFRII-Trimer to TNF-α vs TNFRII-Fc, Fortebio biolayer interferometry measurement was employed. Biotin-labeled TNFRII-fusion proteins were first captured on Streptavidin (SA) sensors, and real-time binding curves were obtained by exposing the biosensor to gradient concentrations of TNF-α. As expected, TNFRII-Trimer exhibited higher ligand binding affinities than that of TNFRII-Fc with KD being 7.39 × 10in vitro by a TNF-α bioassay, we then set out to test whether TNFRII-Trimer could be therapeutically efficacious in the collagen-induced arthritis.Having demonstrated that TNFRII-Trimer had higher affinity in ligand binding as well as exhibiting higher inhibitory effects than dimeric TNFRII-Fc i.p., with dosages being both at 12.5 mg/kg showed similar pharmacokinetic profiles as shown in the drug concentration-time profile in semi-logarithmic scale  of 123 ± 10.09 μg/mL was observed, following a single i.p injection. In contrast, TNFRII-Fc had a Tmax of 4 h and Cmax of 125 ± 7.14 μg/mL. The half-lives of TNFRII-Trimer and TNFRII-Fc in DBA/1 mice were determined to be 10 hours and 30 hours, respectively by subjecting the individual slope of line into the formula of first order kinetics.To determine the dosing strategy of pharmacodynamic study, we first conducted a pharmacokinetic study of TNFRII-Trimer in DAB/1 mice, and TNFRII-Fc was also tested in parallel for comparison. The results indicated that TNFRII-Trimer and TNFRII-Fc when administrated via ale Fig. . A mono-In order to accurately compare the therapeutic efficiencies of the dimeric and trimeric TNFRII fusion protein, same dosing strategy of two drugs with equal dosage (12.5 mg/kg) and dosing frequency (once every two days) was employed in pharmacodynamic study. The result showed that treatment of CIA mice with TNFRII-Trimer led to significant reduction in the severity scores of arthritis by more than half compared to non-treatment control group (p < 0.05) Fig. . In a siin situ ligand-receptor functional staining using AP-tagged TNF-α (AP-TNF-α)  for human TNF-α ligand was over three times lower than that of the dimeric TNFRII-Fc (23.1 × 10−11 M). The increased affinity was mostly attributed to nearly 6 times slower in off rate for the bound ligand. These results confirmed our hypothesis that trimeric TNF receptor fusion protein had a better symmetry, thus tighter binding to its homotrimeric ligand, TNF-α.Molecular binding kinetics studies with Fortebio biolayer interferometry measurement confirmed the finding of cell-based bioassay. The KD of the trimeric TNFRII-Trimer  3 times shorter than that of TNFRII-Fc in DBA/1 mice. While this attribute is disadvantageous, if one considers the in vitro potency for TNFRII-Trimer (in the range of ng/mL) in blocking TNF-α, and that each mouse contains approximately 2 mL of blood, dosing at 12.5 mg/kg per animal would result in initial drug concentration of around 125 μg/mL, which would attenuate to about 4.5 μg/mL 48 hr TNFRII-Trimer. This concentration is nearly three orders of magnitude higher than its IC50in vitro. Therefore, identical dosing strategy was employed to compare TNFRII-Trimer and TNFRII-Fc pharmocodynamics studies. Consistent with our prediction, the trimeric soluble TNFRII exhibited better efficacy in suppressing disease severity compared with TNFRII-Fc. The efficacy of TNFRII-Trimer in arthritis treatment were further verified at the levels of tissue histology. Both morphological and pathological examination of immune infiltrates in the paraffin-embedded disease tissues revealed that the severity of arthritis was significantly ameliorated in mice treated with TNFRII-Trimer. Besides, our study also demonstrated that TNFRII-Trimer was safe to animals and without any hepatocyte toxicity.The therapeutic efficacy for TNFRII-Trimer Taken together, we have demonstrated that with the TRIMER tag platform from pro-collagen, any soluble receptors or secreted proteins can be efficiently trimerized into disulfide bond-lined trimmers with either type I and type III collagen C-prodomain, and expressed as secreted proteins. Such trimeric fusion proteins are trivalent in structure with a 3-fold symmetry and thus may have superior biological properties than that of either naturally occurring or existing biologic proteins. Such soluble trimeric human TNF receptors may prove to be more effective than the current dimeric soluble TNF receptor (e.g. Enbrel) or therapeutic antibodies on the market in treating autoimmune diseases such as RA. Future clinical trials should provide such pivotal information.23. Thus, in future experimentation, it will be also interesting to see if the trimeric and dimeric TNF blockers can have a synergistic effect in blocking TNF signaling in both animal models and in the clinic for treating autoimmune diseases.Although it is generally believed that TNF family of ligands are mostly homotrimeric in solution or based on x-ray crystallography, biological functional assays and structural analysis of recombinant TNF expressed in bacteria suggested that both trimeric and dimmeric forms of the ligand could co-exist25, blockade of other inflammatory cytokines such as GM-CSF signaling pathways27 and immune co-stimulators such as OX40L28, are being explored. Combinatory therapies from the above that eventually restore the normal cytokine crosstalk that went haywire in autoimmune diseases such as RA may lead to synergistic therapeutic effect or eventual cure of the disease.Although TNF antagonists have been the mainstay for the treatment rheumatoid arthritis, new venues of therapeutic strategies, such as the induction of immune tolerance29 . Purified TNFRII-Trimer was ultra-filtrated into PBS before being used for further characterization.The cDNA sequence encoding the entire C-propeptides of human α1 (III) collagen containing no glycine-repeat with a mutated BMP-1 recognition site was amplified by PCR using EST clones purchased from the American Type Culture Collection . The amplified cDNA was cloned into the pAPtag-2 mammalian expression vector , replacing the AP coding region, and creating the resulting pTRIMER (T3 version) vector. The cDNA sequence coding the entire soluble human TNFRII (aa1–256) without the trans-membrane and cytoplasmic domain was amplified by PCR and cloned into either Hind III or BglII sites of pTRIMER expression vectors to allow in-frame fusion with the TRIMER tag (C-propeptide), so TNFRII-Trimer is expressed as a secreted protein. pTNFRII-Trimer expression vector was stably transfected into GH Chinese hamster ovary CHO (dhfr−/−) cell line  using FUGENE 6  grown in IMDM with 10% FBS and 1% penicillin-streptomycin supplemented with HT . After stepwise gene amplification with increasing concentrations (0–0.5 μM) of MTX , the clone with highest TNFRII-Trimer titer determined by bioassay with a TNF-α sensitive cell line L929 was obtained. The cells were then adapted to SFM-4-CHO  serum-free medium, and TNFRII-Trimer was produced in a lL Celligen bioreactor  under Fed-batch process with Cell Boost 2  added every other day from day 3 until harvest on day14. The TNFRII-Trimer titer was monitored daily with SDS-PAGE. After 14 days of culturing in bioreactor, TNFRII-Trimer was purified to homogeneity from the conditioned medium using HiTrap BlueHP (Blue Sepharose)  under a salt gradient elution, according to the manufacturer’s instructions. The purity of TNFRII-Trimer was determined by both SDS-PAGE and SEC-HPLC30. Two μg of purified TNFRII-Trimer and TNFRII-Fc were separated on a 10% SDS-PAGE under non-reducing condition and either visualized by Coomassie Blue staining or transferred to PVDF membranes (GenHunter). The blots were then probed with 1 U/mL of either AP alone or AP-TNF-α  fusion protein, followed by visualization with AP Assay Reagent S (GenHunter).Antibodies recognize the Trimer-Tag domain and the TNFRII-domain of TNFRII-Trimer and human Fc-domain of TNFRII-Fc were used for their characterizations in this study. They were Rabbit anti-Trimer , mouse anti-TNFRII  and horseradish peroxidase (HRP) coupled goat anti-human Fc , respectively. Purified TNFRII-Trimer (0.2 μg) and TNFRII-Fc  (0.2 μg) were analyzed by western blot on a 10% SDS-PAGE under non-reducing (-mercaptoethanol) condition using the above three antibodies to verify the Trimer-tagged TNFRII. The ligand-receptor affinity blot analysis was carried out using AP assay reagent S (GenHunter) following the manufacturer’s instructions as previously describedIsoelectric focusing was performed using BioRad horizontal IEF apparatus following the manufacturer’s manual instructions. The pH gradient of precast IEF gel from Lonza was 3.0–10.0 and TNFRII-Trimer was visualized by Coomassie Brilliant Blue R-250. The sialic acid content of TNFRII-Trimer and commercial TNFRII-Fc were detected by resorcinol method. Purified TNFRII-Trimer and commercial TNFRII-Fc were mixed with resorcinol-hydrochloric acid solutions by boiling water bath for 30 minutes. After cooled, butanol-butyl acetate mixture (12:48) were used to extracting the test substance and absorbance of the upper phase was measured at a test wavelength of 580 nm by a wavelength scanner (Beckman). The sialic acid content of the fusion proteins was calculated using the formula of standard curve generated from the standards of sialic acid.17. Briefly, L929 cells (ATCC)were seeded in 96-well plates at a density of 2 × 104 cells/well. After overnight culture, L929 cells were treated with 1.5-fold serial dilutions of either TNFRII-Trimer or commercial TNFRII-Fc with 2 μg/mL actinomycin D (Sigma) and 2.5 ng/mL human TNF-α (R&D Systems). After 16 hours of incubation at 37 °C, 20 μL of 5 mg/mL MTT was added and further incubated for 4 hours. After aspirating the supernatant media from the wells, 100 μL of DMSO (Sigma) was added to dissolve Formazan crystal. The absorbance at a test wavelength of 570 nm and a reference wavelength of 630 nm was recorded.The biological activity of TNFRII-Trimer and commercial TNFRII-Fc were assessed by a MTT tetrazolium (Sigma) cytotoxicity assay as described previously31. Prior to kinetics measurements, both TNFRII-Trimer and commercial TNFRII-Fc were biotinylated with NHS-PEG4-biotin kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Unbound NHS-PEG4-biotin was removed via ultra-filtration in PBS. For kinetic analysis of biotinylated TNFRII-Trimer and commercial TNFRII-Fc with TNF-α, soluble TNFRII-Trimer and commercial TNFRII-Fc (10 μg/mL) were immobilized on streptavidin (SA) biosensors  for 300 s to ensure saturation. The 96-wellmicroplates used in the Octet were filled with 200 μL of sample or buffer per well. Following a washing step, association and dissociation measurements were carried out using serial dilutions of TNF-α (R&D Systems). The accurate kinetic parameters (Koff and Kon) and affinity constant (KD) were calculated using the Octetsoftware9.0 provided by the manufacturer.Binding kinetics measurements were performed using Bio-Layer Interferometry on FortéBio Octet QKe instrument Six-week old male DBA/1 mice were purchased from Beijing HFK Bioscience and kept under standard pathogen-free conditions in the animal care center at Sichuan University and received humane care. All animal experiments were approved by The Institutional Animal Care and Use Committee (IACUC) in Sichuan University and were conducted according to international guidelines for animal experimentation.For pharmacokinetics study, six animals were randomly divided into two groups, three in each group. Each of the three mice was dosed at 12.5 mg/kg with either TNFRII-Trimer or commercial TNFRII-Fc via i.p injections. Sera samples were collected from mouse tails at different time points after drug administration.1/2 = ln2/Ke). The areas under TNFRII-Trimer and commercial TNFRII-Fc concentrations versus time curves (AUC) were calculated by Graph Pad prism6.0 software.Serum drug levels of TNFRII-Trimer and commercial TNFRII-Fc were determined by ELISA assays. Briefly, diluted plasma samples from mice treated with TNFRII-Trimer or commercial TNFRII-Fc were added into each well of 96-well ELISA plates  which were previously coated with 1 μg/mL of human TNF-α (R&D). For TNFRII-Trimer group, following 1 hour of incubation with Rabbit anti-Trimer antibody , signals were detected with Goat anti-rabbit-IGg conjugated HRP . For TNFRII-Fc group, Goat anti-human IgG conjugated HRP  was used to detect the signals. The absorption value in 450 nm wavelength was measured with TMB-ELISA Substrate Solution (Thermo Scientific). Serum drug concentrations were calculated by using the formula of standard curve, which obtained from purified TNFRII standards. Ke was evaluated by least square regression of the points describing the terminal log-linear decaying phase . Mice in each treatment group were received 200 μL of either PBS or TNFRII-Trimer (12.5 mg/kg) or commercial TNFRII-Fc (12.5 mg/kg) every 48 h, respectively, all via i.p injections. Mice were monitored daily over a 6 weeks period from the initial immunization for signs of arthritis. Standard scoring system for the severity of arthritis and statistical analysis of arthritis scores followed the previous method18. The severity of arthritis in each mouse was determined blindly and independently by four observers, and average of scores was calculated.For pharmacodynamics study, the establishment of mouse model for CIA followed the protocol with two immunizations and treatment regimens33, which was followed with 0, no swelling or bone damage; 1, slight ankle swelling or joint space narrowing; 2, moderate bone erosion and ankle swelling; and 3, severe bone erosion. The severity of arthritis in each mouse was determined blindly and independently by four observers, and average of scores was calculated.Infrared thermography and images of radiographs of the hind paws of the CIA mice on day 41 were captured with a FLIR T600 Thermal Imaging Camera and a Kodak Point-of-Care CR140 medical X-ray system, respectively. Analysis of regional temperature was carried out by XJ-Infrared report analysis software. The severity of bone destruction in the ankle joint was obtained according to the standard scoring systemSupplementary Information


Although anti-TNF-α monoclonal antibodies are considered safe during pregnancy, there are no studies on the development of the exposed-infant immune system. The objective was to study for the first time the impact of throughout pregnancy exposure to anti-TNF-α has an impact in the development of the infant’s immune system, especially B cells and the IL-12/IFN-γ pathway.Prospective study of infants born to mothers with inflammatory bowel disease treated throughout pregnancy with anti-TNF-α . Infants were monitored both clinically and immunologically at birth and at 3, 6, 12, and 18 months.We included seven patients and eight healthy controls. Exposed infants had detectable levels of anti-TNF-α until 6 months of age; they presented a more immature B- and helper T-phenotype that normalized within 12 months, with normal immunoglobulin production and vaccine responses. A decreased Treg cell frequency at birth that inversely correlated with mother’s peripartum anti-TNF-α levels was observed. Also, a decreased response after mycobacterial challenge was noted. Clinically, no serious infections occurred during follow-up. Four of seven had atopia.This study reveals changes in the immune system of infants exposed during pregnancy to anti-TNF-α. We hypothesize that a Treg decrease might facilitate hypersensitivity and that defects in IL-12/IFN-γ pathway might place the infant at risk of intracellular infections. Pediatricians should be aware of these changes. Although new studies are needed to confirm these results, our findings are especially relevant in view of a likely increase in the use of these drugs during pregnancy in the coming years. Anti-TNF-α monoclonal antibodies (mAb) have revolutionized inflammatory bowel disease (IBD) treatment –4. IBD on = 426), OTIS registry (n = 74), and CRIB study (n = 31)] of infants exposed to anti-TNF-α drugs during, at least, the second and third trimester of pregnancy showed that both anti-TNF-α adalimumab (ADA) and infliximab (IFX) are detectable in infants from treated mothers for 12 months after birth while certolizumab was barely detectable. Infants showed no apparent major clinical consequences  related to the inhibited or modulated pathway . AlthougThe in-depth study of the effect of prenatal anti-TNF-α exposure on a developing immune system remains a relevant clinical question. Therefore, this was the aim of our study: our hypothesis was that throughout exposure during pregnancy to anti-TNF-α affects the development of the infant’s immune system, especially B-cells , 21 and We conducted a prospective study (January 2014–January 2016) of infants born to mothers with IBD who received anti-TNF-α mAb (ADA or IFX) throughout during pregnancy, including the third trimester. Infants undergoing immunosuppressive treatment or with an immunodeficiency were excluded. All IBD patients included in the study were recruited from the IBD Unit of Hospital Clinic de Barcelona (HCB), and underwent close monitoring of pregnancy and childbirth. At the time of the initiation of our study, the IBD Unit of HCB was the only institution in our region maintaining this treatment during throughout pregnancy and all patients but one fulfilling inclusion criteria were enrolled in the study.This study was carried out in accordance with the recommendations of Ley General de Sanidad (25/4/1986) Art. 10, with written informed consent from all subjects. The protocol was approved by the ethics committee of the Hospital Sant Joan de Déu (Comité Ético de Investigaciones Clínicas number PIC-50-12). All patients included in the study signed the informed consent, complying with current legislation.We included seven exposed-infants and eight healthy controls. Exposed-infants were monitored both clinically and immunologically at birth and at 3, 6, 12, and 18 months. On the delivery day, 20 ml of heparinized blood was extracted from umbilical cord blood (seven exposed-infants and eight controls). From 3 months, 6 ml heparinized blood and 2.5 ml sera without anticoagulant factor were obtained from all exposed-infants but 2, due to the mother’s refusing subsequent blood draws after an unsuccessful first attempt.Immune parameter study included: quantification of anti-TNF-α blood levels, basic T/B/NK immunophenotype, T-cell and B-cell subphenotypes with evaluation of regulatory cells (Treg and Breg), lymphocyte proliferation to mitogens, and IFN-γ/IL-12 pathway . Common procedures as immunophenotyping and cell proliferation are detailed in the supplementary methods. To facilitate reading and interpretation, populations appear in the text with a given name, while markers definitions are presented in Table 2 humidified incubator for 48 h. To assess the effect of anti-TNF-α mAb on the function of IL-12/IFN-γ pathway, the same whole blood culture was performed in two ways: using blood from exposed infants without washing and using the blood washed with PBS three times to eliminate anti-TNF-α drug. After the washing, complete medium (RPMI with 10% FBS and penicillin/streptomycin) was added to restore blood to the same volume as before the washing with PBS. In this way, we maintained cellular amounts of cells and cell concentration in the two conditions assayed. Activation conditions: medium alone, live BCG  at a multiplicity of infection of 20 BCG per leukocyte, BCG plus human recombinant IL-12p70 , BCG plus hrIFN-γ  as described elsewhere  at 48-h culture point following the manufacturer’s instructions. Briefly, supernatants were incubated for 2 h with corresponding anti-cytokine magnetic beads, and then washed with 1× washing buffer and stained with detection antibodies (provided) for 1 h. Strepatividin-PE was then added for 30 more minutes. During all incubation steps, the plate was agitated at 650 rpm. After washing, plate was agitated for 15 min at 650 rpm and read in the xMAP Luminex reader . IL-17 detection was assessed by ELISA  at 48 h culture point following manufacturer’s instructions.t-test was performed to compare different cell populations between exposed and non-exposed infants. Significance of correlation between populations/drug levels was studied with Spearman test for non-parametric populations.As data did not follow a Gaussian distribution, unpaired Statistical significance of functional studies was performed with two-way ANOVA test and Bonferroni post-test. For the comparison between the results obtained with autologous sera and washed condition, two-way ANOVA for repeated measures was used. In heat-map representation, blue corresponds with the minimum, red with the maximum, and yellow with one for each parameter.p) for immune phenotype are detailed in Tables Statistical analysis and graphical representation of the data were performed with Prism5 software  and Microsoft Excel 2010). Detailed results  with long-lasting IBD  were treated with anti-TNF-α for a period longer than 6-months prior to pregnancy. Six patients suffered from Crohn’s disease (CD) and one from extended ulcerative colitis. Five patients were treated with ADA, and two with IFX. All mothers presented supra-therapeutic levels of anti-TNF-α  before or during pregnancy. Three patients were treated with other immunosuppressive drugs—2 with azathioprine (AZA) and one with prednisolone—and two of them had active IBD during pregnancy .For all patients, the interval between the last dose of drug and the delivery was ≤7 days (range: 3–7 days). The delivery was programmed only in two patients; the five remaining mothers gave birth by caesarian section (1) or vaginal (4) delivery, with one preterm infant (35 weeks in one mother with active disease); while the remaining 4-cases delivered at 39 weeks on average (range 37–41). All newborns were normal weight for gestational age, Apgar 9/10, and did not present any malformations. Five mothers breastfed.r = 0.9 p = 0.04); this correlation was not determinable in IFX patients due to the low number of samples , slightly longer than the described half-life of immunoglobulin (Ig) G (3 cells/μl blood) and that ADA-exposed infants presented increased eosinophil counts in cord blood  , total leukocytes, lymphocytes, neutrophils, and basophils between exposed-infants and healthy controls . However, although due to sample-size limitations comparisons between ADA- and IFX-exposed infants should be approached with caution. We have observed that, compared with healthy controls, IFX-exposed infants had lower monocyte count in cord blood (0.44 vs 1.16 × 10p = 0.004), which fell within normal ranges at 12 months. No differences were observed in other lymphocyte subpopulations . One ADA-treated patient, also treated with AZA, presented very low numbers of B-cells (0.54% of lymphocytes and 32 cells/μl of blood) (this effect was not observed in the other AZA-treated infant).Absolute number of T-cells was increased in cord blood of ADA-exposed-infants  due to T helper (Th)-subset. Furthermore, CD45RO+ (memory) population frequency was decreased among Th-cells  due to a decrease in Th-early-memory-cells  while there were no significant differences in late-memory T-cells. By contrast, there was an increase in the T cytotoxic Tc-memory-cells . At 12 months of age, CD45RO+ T-cells, both Th and Tc, were in range  and switched B-cell population was smaller . Circulating marginal zone B-cells were decreased , and activated B-cells  and IgM-only B-cell-frequency  were increased. Although at some time-points, some B-cell subsets showed values outside the range, at 12–18 months all values were within age-matched ranges . An inverse correlation between mother’s anti-TNF-α trough level and Treg frequency was observed ; at follow-up, almost all Treg values were below the lower reference limit .In cord blood of anti-TNF-α-exposed-infants, Treg Figure  were dimce limit . Treg frWe evaluated anti-mycobacterial response by studying surface activation markers and cytokine secretion after whole blood cultures in the presence (non-washed condition) and absence (washed condition) of autologous sera.p = 0.004) and HLA-DR (p = 0.003) MFI (expression per cell). CD69 expression was partially recovered after drug removal (p = 0.02) although still reduced (p = 0.004); drug removal had a mild effect on HLA-DR SR reduction (p = 0.05). There was a tendency toward a reduction of CD71+ frequency SR, with (p = 0.06) or without (0.06) drug presence.Exposure to anti-TNF-α during pregnancy reduced the response after mycobacterial challenge Figure . Exposedp = 0.0002), being partially recovered after drug removal (p = 0.005) although still reduced (p = 0.02). Although without statistical significance, IL-1ß secretion was reduced (p = 0.06), and IL-6 (p = 0.06) and IL-1RA (p = 0.07) SR were increased. IL-1ß was not recovered after washing, IL-1RA SR was rescued and IL-6 secretion increased even more (p = 0.03). Without stimulation, washed samples from exposed infants produced higher amounts of IL-17 (measured by ELISA) compared with the non-washed condition (p = 0.03) and with non-exposed infants (p = 0.02). On the other hand, after BCG stimulation, IL-17 production in exposed infants was reduced, as none of the exposed infants produced any detectable IL-17 but one; however, differences did not reach statistical significance. Differences between non-washed and washed samples from exposed infants were maintained (p = 0.03) but disappeared when compared with non-exposed infants (p = 0.95) . Altogether, these data suggest that immune system activation upon mycobacterial challenge may be compromised.At birth, TNF-α induction was reduced in the exposed-infant (p = 0.006). This effect was observed in CD69 (p = 0.009) and HLA-DR (p = 0.0009) MFI, CD71+ frequency (p = 0.003), and TNF-α secretion (p = 0.02) , IFN-γ, IL-12p70, IL-1β, and TNF-α production increased while IL-6 production was stable  Figure B.All children showed normal growth and neurological development. Although one child suffered from recurrent infections from 6 to 12 months of age, no other exposed-infants manifested any significant infections, despite normal microbial exposure (attendance to daycare or siblings below 5 years old). Although our recommendation was to avoid rotavirus vaccination, it was used in four exposed-infants with no adverse effects. Atopic dermatitis was observed in four children (two of them without family history), and food allergy was diagnosed in one of them Table .In accordance with published data , 13, ant9 ANC/L in 3 and 1.1 × 109 ANC/L, and increased 3 months after birth; infectious skin lesions occurred during neutropenia. In our study, three exposed infants had normal levels of neutrophils at birth in cord blood, two had values below the reference range for the age (4.3–11.4 × 109 ANC/L): 2.78 (exposed to ADA + steroids) and 3.06 × 109 ANC/L (exposed to IFX + AZA) and one had values in the limit of the reference range: 4.73 × 109 ANC/L (exposed to ADA + AZA). We observed a decrease of neutrophil levels below the reference ranges at 3 months of age (2.2–6.3 × 109 ANC/L): one infant with severe neutropenia  and two with moderate neutropenia . None of them presented infectious skin lesions. Differences in the results may be due to differences in the drug infusion pattern. In the four cases described, the infusion of the last IFX dose was, at least, 8 weeks’ prepartum. Instead, patients included in our study received the last dose of either IFX or ADA from 3 to 7 days’ pre-partum. Also, other factors (such as prematurity and presence of positive neutrophil-specific CD16 autoantibodies) differentiate our cohort from the cases published by GuiddirT et al. However, we agree that neutrophil count should be routinely performed in infants exposed to anti-TNF-α drugs during pregnancy, especially in the event of an infection.In some patients, it has been seen that anti-TNF-α exposure during pregnancy can lead to neutropenia . NeutropWe have observed normal numbers of B cells at birth, although with a more immature phenotype. It is known that TNF-α knock out mice presented abnormal B cell structures. They lack splenic B cell follicles, organized follicular dendritic cell networks and germinal centers. These alterations lead to a decreased humoral response and increased infection risk , 21. NonBased on empirical experience [adverse event to BCG vaccine  and theoImmune system dysregulation needs to be considered: four of seven of our children presented atopy in the first year (two of them without family history), and all ADA-exposed infants had increased eosinophil counts in cord blood. Exposed infants showed an altered T- and B-regulatory compartment. There was an increased Breg frequency, a population having an anti-inflammatory role in cord blood . By cont+CD25hiCD127lowFoxP3+ T cells in all exposed infants compared to healthy controls. Interestingly, there are some publications showing an increase in Treg cells in responder patients after anti-TNF-α treatment (+) but induced (CD62L−) Treg cells  In vitro functional studies would be relevant for this purpose.We have shown that drug exposure decreases the response after a mycobacterial challenge at birth, which did not totally recover after drug cleaning. In adults, anti-cytokine biological treatments are thought to cause an immune-deficiency-like phenotype . This shAlthough this study has several strengths, including the thorough immune system analysis, it also has some limitations: our cohort of exposed infants is small, and a broader group would probably provide more robust information. Nevertheless, all observations were consistent from sample to sample. Our study control group included infants born to healthy mothers, since no IBD pregnant women with moderate-to-severe disease were without anti-TNF-α treatment; thus, we have not been able to evaluate the effect of IBD itself. Finally, immunological follow-up of healthy controls was not performed for ethical reasons.This study is the first thorough evaluation of the impact of prenatal anti-TNF-α on the immune system development of exposed-infants. Although we observed specific changes, infants were not clinically compromised. Our results aim at generating consciousness of the need to further study and follow-up on exposed-infants. The pediatrician should be informed of the mother’s mAb treatment during pregnancy, because of the impact on vaccine recommendations, especially with regards to BCG due to the observed mycobacterial-deficient response.This study was carried out in accordance with the recommendations of Ley General de Sanidad (25/4/1986) Art. 10, with written informed consent from all subjects. The protocol was approved by the ethics committee of the Hospital Sant Joan de Déu (Comité Ético de Investigaciones Clínicas number PIC-50-12). All patients included in the study signed the informed consent, complying with current legislation.AE-S performed immunological studies, carried out the analyses, drafted the initial manuscript, and approved the final manuscript as submitted. IT performed the clinical management of pregnant women, critically reviewed the manuscript, and approved the final manuscript as submitted. AD-M performed clinical follow-up of exposed infants, critically reviewed the manuscript, and approved the final manuscript as submitted. ER and MG performed the clinical management of IBD patients, critically reviewed the manuscript, and approved the final manuscript as submitted. MT, NM, and EG performed anti-TNF-α monitoring, critically reviewed the manuscript, and approved the final manuscript as submitted. JY coordinated anti-TNF-α monitoring, analyzed anti-TNF-α monitoring results, critically reviewed the manuscript, and approved the final manuscript as submitted. AP and MJ conceptualized and designed the study, critically reviewed the manuscript, and approved the final manuscript as submitted. LA performed clinical follow-up of exposed infants, conceptualized and designed the study, critically reviewed the manuscript, and approved the final manuscript as submitted.The 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.


Enterovirus 71 (EV71) is the most frequently detected causative agent in hand, foot, and mouth disease (HFMD) and is a serious threat to public health in the Asia-Pacific region. Many EV71 vaccines are under development worldwide, and although both inactivated virus vaccines and virus-like particles (VLPs) are considered to be effective, the main focus has been on inactivated EV71vaccines. There have been very few studies on EV71 VLPs. In this study, using a strategy based on HIV gag VLPs, we constructed a gag-VP1 fusion gene to generate a recombinant baculovirus expressing the EV71 structural protein VP1 together with gag, which was then used to infect TN5 cells to form VLPs. The VLPs were characterized using transmission electron microscopy, electrophoresis and staining with Coomassie blue, and Western blotting. Mice immunized with gag-VP1 VLPs showed strong humoral and cellular immune responses. Finally, immunization of female mice with gag-VP1 VLPs provided effective protection of their newborn offspring against challenge with a lethal dose EV71. These results demonstrate a successful approach for producing EV71 VP1 VLPs based on the ability of HIV gag to self-assemble, thus providing a good foundation for producing high-titered anti-EV71 antibody by immunization with VLP-based gag EV71 VP1 protein. Picornaviridae that was first reported and then isolated in the year 1969 [Human enterovirus 71 (EV71) is a non-enveloped RNA virus belonging to the family ear 1969 . It has ear 1969 , 3. Althear 1969 , 5. SeveDuring the last 10 years, large-scale outbreaks of HFMD have been reported with increasing prevalence in China , 9. The The genome of EV71 encodes a single large polyprotein, which, during infection, is first cleaved into three smaller polyproteins (P1 to P3). The P1 polyprotein is then cleaved by protease 3CD to generate four structural proteins  and seven nonstructural proteins. The structural proteins constitute the viral capsid, and the nonstructural proteins participate in virus replication –13. Of tVirus-like particles (VLPs) are structurally similar to native virions but are non-infectious due to the lack of a virus genome –19. ReceThe structural polyprotein Pr55gag of the human immunodeficiency virus type 1 (HIV-1) directs the assembly of the virion. The Pr55gag protein is the precursor of four major structural proteins, MA (p17 matrix), CA (p24 capsid), NC (p7 nucleocapsid), and LI (p6 linker), as well as the small spacer peptides p1 and p2 . The unpIn the present study, we constructed and characterized EV71 VLPs that were made based on a type I gag VLP strategy and produced in insect cells. The VLPs were made using full-length gag molecules fused at their C-terminus with EV71 VP1. We first tested whether fusion of the EV71 VP1 gene to the C-terminus of gag results in incorporation as an in-frame fusion. We then found that immunization with these VLPs generate a specific cellular and humoral immune response in mice. Furthermore, we found that injection of female mice with this type of VLP via the intramuscular (i.m.) route provided effective protection of their newborn offspring against challenge with a lethal dose of EV71. Antisera against these VLPs were also able to passively protect newborn mice against challenge with a lethal dose of EV71.RD (rhabdomyosarcoma) cells were purchased from ATCC and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. TN5 cells were cultured in SF900 II insect cell culture medium.The VP1 gene and gag gene fragments were obtained by PCR amplification from the plasmid PCAGGs-VP1 and PCAGGs-gag, respectively (kindly provided by Dr. Ching from Emory University). The gag-VP1 fusion gene was amplified using an overlapping-PCR method. The primers used were as follows: P1, CCGGATTCATGGGAGATAGGGTGGCAGATGTAAT; P2, GATCACCACTCTTGGATCCGGTGGCGGCGGCGTGAGAAAC; P3, GTTTCTCACGCCGCCGCCACCGGATCCAAGAGTGGTGATC; P4, CCCAAGCTTTTACTAACTGGTCTCCTCCAAAGAGAGAAT. The gag and gag-VP1 genes were each cloned into the pFastBacI vector (Invitrogen). The resultant pBac-gag and pBac-gag-VP1 plasmids were used to generate gag and gag-VP1 baculoviruses. The recombinant baculoviruses were obtained and amplified by infecting TN5 cells.g for 4 h through a 15–50% discontinuous sucrose gradient. The layer containing the concentrated virus was pelleted at 100,000 × g for 1 h and then resuspended in phosphate-buffered saline (PBS).EV71 was propagated in RD cells. Briefly, virus harvested from infected RD cells was concentrated by precipitation with 8% polyethylene glycol 8000 (Sigma) and then centrifuged at 100,000 × TN5 cells were infected with gag-VP1 baculovirus or gag baculovirus at a multiplicity of infection of 5, and the culture medium containing VLPs was collected at 72 h postinfection. Cell debris was removed by high-speed centrifugation, and the supernatant containing the VLPs was concentrated using a GE QSM-03SP/50quixstandTM Benchtop System and further purified by ultracentrifugation at 28,000 rpm for 2.5 h in an SW-41 rotor (Beckman) through a 15-50% discontinuous sucrose gradient. The layer containing the purified VLPs was suspended and washed with PBS, centrifuged at 28,000 rpm for 30 min, and resuspended in PBS. A BCA assay kit was used to determine the protein concentration of the VLP preparation.TEM was performed as described previously . BrieflyBriefly, VLPs were subjected to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and followed by Coomassie blue staining and Western blotting. The primary antibody for the Western blot assay was mouse anti-VP1 monoclonal antibody or anti-gag polyclonal antibody. The secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG. Protein bands were detected using ECL Detection Reagent (Sigma).Specific-pathogen-free female BALB/c mice were purchased from the animal center of the Fourth Military Medical University . Animal experiments were performed according to the current experimental protocols involving animal study approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. Six-to-eight-week-old female BALB/c mice were randomly divided into four groups (10 mice in each group), and each mouse was immunized twice at 4-week intervals by intramuscular injection with 10 μg of gag VLPs, 10 μg of gag-VP1 VLPs, 10 μg of inactivated EV71 (in-EV71) or 100 μL of PBS. Blood samples were taken through retro-orbital bleeding two weeks after each immunization (week 2 and week 6) and also collected at the end of weeks 8, 10 and 12. The sera were inactivated at 56 °C for 30 min and stored at -80 °C.Three or four mice in each group were sacrificed, and their splenocytes were isolated at week 12 to detect cellular immune responses. The remaining mice were used to mate and breed neonatal mice for lethal virus challenge four weeks after the booster immunization, and the neonatal mice were monitored for 15 days.450) was recorded.EV71 or gag-specific IgG titers in the mouse sera were determined by ELISA. In brief, a 96-well plate was coated overnight at 4°C with purified EV71 or gag protein at a concentration of 0.8 μg/well. After three times washing with 200 μl of PBST, the plate was blocked with 100 μl of 2% BSA for 1 h at 37°C. After another three washes with PBST, serum samples were serially diluted and added to the wells. The plates were incubated at 37 °C for 1 h, after which HRP-conjugated goat anti-mouse IgG was added as a secondary antibody. After three washes, the color was developed using TMB/HCL and the optical density at 450 nm (OD50) was then added to the diluted serum and mixed. The serum-virus mixture was incubated at 37 °C for 1 h to neutralize the infectious virus and then added to the cells, which were cultured for another 3 days. The highest dilution that resulted in a 50% reduction of CPE was considered the neutralization titer.For measurement the neutralization titer of serum from immunized mice, RD cells were plated in a 96-well cell culture plate and allowed to attach overnight until the cell density reached 80%. Serum was serially diluted twofold (from 8 to 256) with DMEM containing 2% FBS, and the same volume of purified EV71 virus  for stimulation. After incubation at 37°C for 48 h, the plate was centrifuged at 1000 rpm for 5 min, and the cell culture supernatant was collected carefully and assayed for interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-γ, using cytokine ELISA kits (eBioscience).To detect the cytokines produced after immunization, splenocytes were isolated from each immunized mouse, mixed, and cultured in 96-well microplates  at a density of 2 × 105 splenocytes were then added to each well. Subsequently, inactivated EV71 virus at a concentration of 200 μg/ml was added to stimulate the effector cells for 48 h. After three washes, biotin-labeled anti-IL-4 or anti-IFN-γ antibody was added. After the spots were developed and dried, spot-forming cells were counted using an automated ELISPOT reader .The production of IL-4 and IFN-γ was also measured using the ELISPOT method. Briefly, 96-well microplates were coated with anti-IL-4 or anti-IFN-γ monoclonal antibody and incubated in a humidified box overnight at 4°C. Plates were blocked with 2% BSA for 1 h at 37°C, and 5 × 106 PFU /ml, and each of the neonatal mice was challenged orally with 10 μl of the virus within 3 days after birth. Neonatal mice were monitored daily for 15 days, and the mortality rate was recorded.Four weeks after the second immunization, immunized female mice were allowed to mate. The EV71 challenge strain was diluted to 1050) of EV71. All mice were monitored daily for clinical symptoms and death for 15 days after challenge.A passive protection assay was performed to further evaluate the protective efficacy of serum antibodies raised by vaccination. One-day-old newborn mice (6 mice per group) were inoculated intraperitoneally (i.p.) with sera from mice immunized with gag-VP1 VLPs, inactivated EV71 virus or PBS. Four hours post-inoculation, the suckling mice were challenged i.p. with ten times the 50% lethal dose  (10 μg/mouse) and received a booster immunization 4 weeks later. Mice immunized with PBS were used as negative controls. For serological analysis, blood was collected at the end of weeks 0, 2, 6, 10 and 12. The results of ELISA analysis Fig. B and C sA neutralization assay was also performed to detect neutralizing antibodies (NAbs) induced by the gag-VP1 VLPs. Two weeks after the primary immunization, serum from mice inoculated with gag-VP1 VLPs showed an NAb titer of 1:32, and 2 weeks after the booster immunization, the NAb titer reached almost 1:128, which is slightly lower than the in-EV71-induced NAb titer (Fig. To examine the T cell response induced by gag-VP1 VLPs or gag VLPs, we measured the cytokines produced by VLP-stimulated spleen cells using a cytokine ELISA. Figure To further evaluate EV71-specific T cell responses induced by different vaccination regimens, spleen cells from immunized mice were harvested, and IFN-γ and IL-4-secreting cells were detected by ELISPOT assay after stimulation with EV71 VLPs. As illustrated in Fig. To investigate the passive protection efficacy against homologous virus challenge, adult female mice were injected intramuscularly with gag-VP1 VLPs, in-EV71, gag VLPs or PBS, and allowed to mate after the second immunization. The neonatal mice were then challenged within 72 hours of birth with a lethal dose of EV71 and subsequently monitored daily for survival and clinical symptoms for 15 days. Newborn mice born to female mice that had been pre-immunized with PBS or gag VLPs gradually developed severe manifestations including reduced mobility, a ruffled coat, and limb weakness, and eventually died, with a final mortality rate of nearly 70% at the end of day 15. In contrast, all of the neonatal mice born to mice injected with gag-VP1 VLPs survived, with only transient or minor symptoms. The immune-protective efficacy reached nearly 80%, which is slightly lower than the survival rate of the group immunized with inactivated EV71 (Fig. 50 of EV71 within 4 h. As shown in Fig. In another experiment, immune sera from animals that had been given gag-VP1 VLPs or inactivated EV71 were injected i.p. into one-day-old neonatal mice, which were then challenged with 10 LDAll of the above results show that maternal neutralizing antibodies were able to protect newborn mice against lethal challenge by EV71.HFMD has been ranked as one of the infectious diseases with the highest incidence in China, and was included in class C of infectious diseases in 2008. During the years 2008-2012, 7,200,092 probable cases of HFMD were reported according to the China CDC surveillance system . EV71 anHIV-1 Pr55gag-based VLPs are prime candidates for delivering foreign polypeptides, and there are two ways to produce VLPs using HIV gag. In the present study, we constructed an in-frame gag-VP1 fusion to generate rBv gag-VP1 using the Bac-to-Bac system, and this recombinant virus was used to infect insect cells to form type I VLPs based on the ability of the gag protein to self-assemble.The gag-VP1 VLPs were then used to immunize BALB/c mice, and serum samples from immunized mice were collected and assayed. The titer of EV71-specific IgG clearly increased 2 weeks after booster immunization, as measured by ELISA, and antibodies remained detectable for more than 12 weeks. More importantly, EV71-specific neutralizing antibodies induced by gag-VP1 VLPs were able to protect mice against a lethal challenge with EV71. These results demonstrated that immunization with gag-VP1 VLPs induces a high-level and persistent antibody response. Humoral immune responses are essential for protecting against EV71 infection; however, several studies have also indicated that the cellular immune response affects the severity of HFMD , 38. Difin vivo protection by gag-VP1 VLPs against viral challenge in animal models. Newborn mice born to mothers that had been pre-immunized with gag-VP1 VLPs or pre-inoculated with gag-VP1 antisera were able to survive until the end of day 15 post-challenge.We also observed In summary, gag-VP1 VLPs was successfully produced using an insect cell expression system. The induction of both humoral and cellular responses by this vaccine was evaluated, and its ability to confer passive protection to newborn mice against a lethal dose of EV71 was determined. The VLPs elicited a strong antibody response as well as a cellular immune response resulting, in antisera with high neutralization titers. The antisera were able to protect newborn mice in a passive protection experiment. As VLPs are considered safer than inactivated virus vaccines, the use of gag-based VLPs containing EV71 VP1 should be considered for HFMD vaccine research.


Meta‐analyses conventionally weight study estimates on the inverse of their error variance, in order to maximize precision. Unbiased variability in the estimates of these study‐level error variances increases with the inverse of study‐level replication. Here, we demonstrate how this variability accumulates asymmetrically across studies in precision‐weighted meta‐analysis, to cause undervaluation of the meta‐level effect size or its error variance (the meta‐effect and meta‐variance).c. 20%. Meta‐analysis of two‐sample designs from 20 studies, with sample sizes of 3–10 observations, undervalued the meta‐variance by 15%–20% for the log response ratio (lnR); it undervalued the meta‐effect by c. 10% for the standardized mean difference (SMD).Small samples, typical of the ecological literature, induce big sampling errors in variance estimation, which substantially bias precision‐weighted meta‐analysis. Simulations revealed that biases differed little between random‐ and fixed‐effects tests. Meta‐estimation of a one‐sample mean from 20 studies, with sample sizes of 3–20 observations, undervalued the meta‐variance by For all estimators, biases were eliminated or reduced by a simple adjustment to the weighting on study precision. The study‐specific component of error variance prone to sampling error and not parametrically attributable to study‐specific replication was replaced by its cross‐study mean, on the assumptions of random sampling from the same population variance for all studies, and sufficient studies for averaging. Weighting each study by the inverse of this mean‐adjusted error variance universally improved accuracy in estimation of both the meta‐effect and its significance, regardless of number of studies. For comparison, weighting only on sample size gave the same improvement in accuracy, but could not sensibly estimate significance.R, adjusted weighting also improved estimation of between‐study variance by DerSimonian‐Laird and REML methods. For random‐effects meta‐analysis of SMD from little‐replicated studies, the most accurate meta‐estimates obtained from adjusted weights following conventionally weighted estimation of between‐study variance.For the one‐sample mean and two‐sample lnWe recommend adoption of weighting by inverse adjusted‐variance for meta‐analyses of well‐ and little‐replicated studies, because it improves accuracy and significance of meta‐estimates, and it can extend the scope of the meta‐analysis to include some studies without variance estimates. This weighting aims to minimize the variance in the meta‐estimate of effect size, thereby maximizing its precision . This bias in turn has a systematic influence on the significance of the meta‐effect. Hedges  arctan (vB/vA)  its replication has a 61% (or 70%) probability of greater inaccuracy due to vB/vA = 2 (or 4). In comparison, equal replication has a 50% probability, meaning an equal likelihood of more or less inaccuracy. The relative precision of the study B estimate set by vB/vA thus determines its probability of losing accuracy.The precision and the accuracy of an effect‐size estimate both depend on the error variance v Gauch, . Conside) Gauch, . For exa2 were estimated precisely by the sample variance s2 among observations. This is because the sample variance s2 (and not the unknown σ2) determines the estimate of the error variance: v^i=s2/ni, and hence the study weight of 1/v^i for meta‐analysis. The sample variance s2, however, is subject to variability due to sampling error in estimating σ2, which rises as an inverse function of n. This variability is described in Figure v in Y¯ estimating μ. A deviation of s2 below or above σ2 will cause a weighting by 1/v^i to over‐ or undervalue the precision, and hence accuracy of the effect‐size estimate for the study. If s2 is estimated with unbiased variability around σ2, as we may expect, even symmetrically distributed deviations will accumulate asymmetrically in the estimation of the meta‐variance to cause its undervaluation. In order to understand how this meta‐level bias arises from unbiased study‐level si2, we need to understand the relationship of the estimated meta‐variance to the true meta‐variance.This 1:1 correspondence of precision with accuracy at the study level would apply to a meta‐estimate based on inverse‐variance weighting only if the population variance σk studies included in the meta‐analysis . The true meta‐variance is likewise given by the inverse of 1/v1 + 1/v2 + … + 1/vk. Consider the simplest scenario of all studies having the same true v = σ2/n, which yields a true meta‐variance of v/k. We now assume that each si2 is an unbiased estimator of σ2, such that the arithmetic mean of v^1+v^2+…+v^k will coincide with the true v on average. It then follows directly that the estimated meta‐variance must undervalue the true meta‐variance: 1/∑ik1/v^i<v/k. This inequality applies to any set of positive numbers v^i that are not all equal, because such sets have a harmonic mean, k/∑ik1/v^i, that is always less than the arithmetic mean, v  for a meta‐analysis have been collated by systematic review and filtering to remove biases due to differences in treatment factors and levels, scales of sampling or response types .The inverse‐variance weighting has a well‐established methodology  for each study i  is a cross‐study constant, mean‐adjusted error variances remain study‐specific if studies differ in their within‐study replication. We use the inverse of this mean‐adjusted‐variance as the ‘adjusted’ weighting, and compare its performance to that of the ‘conventional’ inverse‐variance weighting. For further comparison, ‘n‐weighting’ replaces the mean‐adjustment term with unity to obtain what is sometimes referred to as a ‘nonparametric variance’  or mean μi . Study‐level parameters δ and vi, and their estimates δ^i and v^i were calculated according to the Table‐2 formulae. For lnR, population means were set with the same sign, and far enough above zero to avoid a known small‐sample bias with near‐zero means . The meta‐effect variance, meta‐v^, was estimated by Equation (3). The accuracy of the meta‐effect was measured as |meta‐δ^−δ|. The significance of the meta‐effect was estimated from Student's t = meta ‐δ^/meta‐v^, and evaluated against the parameter‐based Student's t = δ/meta‐v (both having k − 1 d.f.). Each output value was reported as the median of 10,000 trial runs. Data r script for the simulations.Meta‐analysis of each simulated dataset was the same for all estimators and types of weighting. The meta‐effect, 3.3{Y¯1,Y¯2}i and s1,s2i, to compare results by the two types of weighting. Data r script for meta‐analysis.We ran simulations on input parameter values derived from three empirical studies. As above, each simulation was repeated 10,000 times to evaluate meta‐estimates from conventional and adjusted weightings. In addition, meta‐analyses were run on the three sets of published study‐specific n1, n2}i from {56, 128} downwards, including 57 × {4, 4}, 45 × {3, 3}, 13 × {2, 2}, and 5 × {1, 1}. Cross‐study means of within‐study Y¯1,Y¯2 and s1, s2 were used to define the otherwise unknown true parameter values for input to the simulation: μ1 = 368.4, μ2 = 300.8, σ1 = 129.8, σ2 = 134.6.A meta‐analysis of 165 studies was simulated with the same replication as a fixed‐effects meta‐analysis by Ma and Chen , which en1, n2}i = 4 × {12, 12}, 3 × {6, 3}, 16 × {2, 2}. The empirical data had meta‐δ^ = −2.27 and −15.09 for Hedges’ g and Y¯1−Y¯2, respectively, and T2 = 2.39 given by the method of moments. These estimates were used to define input parameters δ = −2.27, and σ = 6.66 and τ = 1.55 in simulation runs as described above. A set of 1,000 runs was first used to obtain REML estimates of τ for each type of weighting; the simulation was then run 10,000 times to obtain meta‐estimates from each type of weighting using their associated REML estimates of τ.A meta‐analysis of 23 studies was simulated with the same sample sizes as a random‐effects meta‐analysis by Gibson et al.  estimatin1, n2}i = 20 × {50, 25}, 10 × {50, 4}, 7 × {12, 12}, 15 × {6, 6}, 13 × {4, 4}. The empirical data had meta‐δ^ = −1.42 and −4.78 for Hedges’ g and Y¯1−Y¯2, respectively, and T2 = 6.53. These estimates provided simulation inputs δ = −1.42, σ = 3.36 and τ = 2.56. Meta‐estimates for each type of weighting were again obtained using their associated REML estimates of τ.The simulation was repeated for a meta‐analysis of 65 studies with the same sample sizes as those of Gibson et al.  for meta44.1ni = 10 observations strongly overvalued the precision of the meta‐effect. Figure n‐dependent sampling error causing v^i to vary around v. The upper‐right plot shows the inverse of the harmonic mean (in blue) of the estimated variances exceeding the inverse of the parametric variance. The conventional weighting consequently undervalued the meta‐variance by 21%. Table n‐weighting, which, however, could not estimate the meta‐variance or the significance of the meta‐effect. These differences between the weightings changed negligibly with the magnitudes of μ or σ2, or their study‐level specificities.For meta‐analysis of a one‐sample mean, conventional weighting of 20 studies all with the same R, conventional weighting undervalued the meta‐variance due to n‐dependent sampling error in variance estimation. Adjusted weighting largely corrected the bias, as illustrated in Figure  = 0.2δ; at a larger τ = δ, however, the much larger between‐ than within‐study variance rendered all weightings similar and suppressed differences between the weighting types.Likewise for the two‐sample lnn for the one‐sample mean and two‐sample lnR, as illustrated in Figure g, conventional weighting principally undervalued the meta‐effect by an inverse function of n . For the random‐effects meta‐analysis of lnR, the small number of studies slightly reduced the magnitude of undervaluation of the meta‐variance caused by conventional weighting, and improved the correction by adjusted weighting. For other estimators, it slightly degraded the correction by adjusted weighting at very low replication, which nevertheless still improved on conventional weighting.The Figure g, larger magnitudes of δ reduced the undervaluation of the meta‐variance for both types of weighting, and switched to causing overvaluation when |δ| exceeded c. 1.25, regardless of replication and always with less bias for the adjusted than conventional weighting. Larger magnitudes of δ had no impact on the undervaluing of the meta‐effect by conventional weighting or its correction by weighting with inverse adjusted‐variance. For the one‐sample mean and lnR, larger magnitudes of δ made no discernible impacts on undervaluation of meta‐variances or meta‐effects.In further trials with Hedges’ 4.2g again showed bias principally in the meta‐effect , due to undervaluation of the meta‐variance by 87% combined with slight undervaluation of the meta‐effect (by 3%). The adjusted weighting completely eliminated this large bias in the meta‐variance (estimate:parameter = 1.00), reducing the significance of the meta‐effect to a slight undervaluation (t19 = 19.60).For the lnR estimators, both methods over‐ and undervalued magnitudes of τ by conventional and adjusted weightings, respectively; the adjusted weighting consistently achieved a value closer to the true τ, and substantially closer at low study‐level replication. These directions of bias were switched for two‐sample SMD at low study‐level replication, with the conventional weighting achieving a value closer to the true τ.DerSimonian‐Laird and REML estimates of between‐sample variance produced similar biases in τ according to the type of weighting Table . For one4.3R meta‐effect by 5% with conventional weighting. This weighting also undervalued the meta‐variance by 23%, causing an overvaluation of significance. The adjusted weighting reduced the magnitude of bias in the meta‐effect to a 2% overvaluation. It improved the accuracy, from |meta‐δ^−δ| = 0.019 to 0.011. It eliminated bias in the meta‐variance (estimate:parameter = 1.00), although a mean overvaluation  slightly undervalued significance, resulting nevertheless in an overall improvement in the estimate . When the adjusted weighting was applied to fixed‐effects meta‐analysis of the study‐specific Y¯1,Y¯2i and s1,s2i published in Ma and Chen . These improvements obtained from adjusted and conventional weightings that substantially over‐ and underestimating τ, at 44.7 and 0.30 respectively. Both weightings overvalued the meta‐variance , causing both to undervalue the significance of the effect . The best accuracy and significance was achieved by meta‐estimation with adjusted weighting using conventionally weighted estimation of τ , which aligned closely with adjusted weighting using the true τ = 1.55 . When applied to random‐effects meta‐analysis of the study‐specific data published in Gibson et al. . These improvements obtained from adjusted and conventional weightings that again over‐ and underestimating τ, at 10.4 and 0.30. Both weightings slightly overvalued the meta‐variance (by 1% for both weighting types), causing both to undervalue the significance of the effect . The best accuracy and significance was again achieved by meta‐estimation with adjusted weighting using conventionally weighted estimation of τ , which aligned closely with adjusted weighting using the true τ = 2.56 . When applied to random‐effects meta‐analysis of the study‐specific data published in Gibson et al.  or deflation (Hedges’ g) can result from weighting on any unbiased estimator of study‐level error variances. It arises wherever the estimate of meta‐variance derives from a harmonic mean of study‐specific estimates of error variance that are prone to sampling error. Moreover, it becomes substantial in meta‐analyses of little‐replicated studies if the variance in study‐level observations depends on study‐level replication, which will almost certainly be the case.Modern statistics of model comparison attach greater importance to magnitudes of effects than to significance Hector, . Of the g  for the variance of Fisher's z‐transformation of r, and therefore has no variance component prone to sampling error.We have demonstrated that weighting on study precision by inverse adjusted‐variance will eliminate or substantially reduce biases due to low study‐level replication. The adjustment, by cross‐study averaging of the component of error variance that is itself prone to sampling error, universally improved accuracy in estimation of both the meta‐effect and its significance. It was effective in dealing with known issues of small‐sample bias in Hedges’  Hedges,  and in lT2 estimating τ2 nevertheless increases with the inverse of k, just as si2 estimating σi2 increases with the inverse of ni , effect size and the variance(s) in observations.In conclusion, we see no reason not to adopt adjusted weighting for all meta‐analyses that are concerned with accurately and precisely estimating a meta‐effect. It addresses a bias that applies even when studies are all well‐replicated, albeit with much less influence than at small sample sizes. Moreover, it can expand the scope of meta‐analyses to include some studies that lack variance estimates, a common occurrence in primary studies (Gerstner et al., We thank Julia Koricheva for allowing us to use data from Koricheva and Gurevitch  for FiguC.P.D. conceived the idea, designed the methodology, analysed and interpreted the data, and led the writing of the manuscript. R.S. contributed to interpretation and writing of main text.http://datadryad.org/resource/doi:10.5061/dryad.5f4g6 (Doncaster & Spake, R scripts for simulations in Data Figure S1Click here for additional data file.Figure S2Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Data S1Click here for additional data file.Data S2Click here for additional data file.


Hydrophis cyanocinctus to screen bioactive compounds that antagonize TNF-α and identified a novel nine-amino-acid peptide, termed hydrostatin-TL1 (H-TL1). In enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analyses, H-TL1 inhibited the interaction between TNF-α and TNF receptor 1 (TNFR1). Further, H-TL1 attenuated the cytotoxicity of TNF-α in L929 cells as determined by the 3--2,5-diphenyl tetrazolium bromide (MTT) assay. H-TL1 also decreased the mRNA expression of TNF-α/TNFR1 downstream targets and suppressed the phosphorylation of well-characterized proteins of downstream signal transduction pathways in HEK-293 cells. In vivo data demonstrated that H-TL1 protects animals against dextran sodium sulfate (DSS)-induced acute colitis and lipopolysaccharide (LPS)-induced acute shock. Given its significant anti-inflammatory activity in vitro and in vivo, H-TL1 is a potential peptide for the development of new agents to treat TNF-α-associated inflammatory diseases.Tumor necrosis factor (TNF)-α is a pleiotropic cytokine with intense pro-inflammatory and immunomodulatory properties, and anti-TNF-α biologics are effective therapies for various inflammatory diseases such as inflammatory bowel disease (IBD) and sepsis. Snake venom, as a traditional Chinese medicine, has been used in the treatment of inflammatory diseases in China for centuries. In this research, we constructed a venom gland T7 phage display library of the sea snake  Tumor necrosis factor  is a major mediator of apoptosis as well as inflammation and immunity, and it has been implicated in the pathogenesis of a wide spectrum of human diseases such as diabetes, cancer, osteoporosis, multiple sclerosis, rheumatoid arthritis, acute shock, and inflammatory bowel disease (IBD) . As a plIn recent years, antibodies  against TNF-α have been approved for patient use ; howeverEscherichia coli (E. coli) cells, resulting in the peptide sequence fused to the C-terminus of the 10B capsid protein and expressed on the surface of the phage particles , an, an39], To assess lung damage, lung tissues were excised, inflated with 4% neutral formalin, and H&E stained 12 h following LPS injection. The lung parenchyma was examined by light microscopy and graded based on the degree of congestion, edema, inflammation, and hemorrhage. Each characteristic was scored on a scale of 0 to 3 .v/v) at room temperature for 1 h. Then, the sections were washed again with Tris-HCl 0.05 M, pH 7.6, and subsequently incubated with 3,3′-diaminobenzidine (DAB) solution in the dark at room temperature for 10 min. The sections were finally washed with Tris-HCl and counterstained with hematoxylin.An additional set of paraffin sections was used for immunohistochemical analysis of TNF-α expression. Sections (5-μm thick) were mounted on slides, cleaned, hydrated, blocked with endogenous peroxidase , washed with PBS, and then treated with citrate buffer  for target antigen retrieval. The sections were treated with a buffered blocking solution (3% BSA in PBS for 15 min), incubated for 1 h at room temperature with a primary polyclonal antibody specific to mouse TNF-α, washed with PBS, and incubated with a secondary HRP-labeled antibody (Abcam) . In survival curve comparisons, data were transformed using a Mantel-Cox log-rank test. All the statistical analyses were conducted using GraphPad Prism 5 software for Windows , and H. cyanocinctus venom gland T7 phage display library and discovered H-TL1, a natural peptide with antagonizing effects against TNF-α. We subsequently evaluated the anti-inflammatory effects of H-TL1. The in vitro results demonstrate that H-TL1 specifically disrupts formation of the TNF-α/TNFR1 complex, blocks the phosphorylation of well-characterized TNF-α/TNFR1 downstream signaling transduction pathways, and suppresses TNFR1-mediated mRNA expression of pro-inflammatory cytokines, including TNF-α. Additionally, H-TL1 treatment resulted in little cytotoxicity toward mammalian cells. In vivo data from two models of inflammation, DSS-induced colitis and LPS-induced acute shock, revealed that treatment with H-TL1 at the onset of disease markedly ameliorates the clinical manifestations and inflammation-related pathologic damage to the mouse colonic mucosa and lung parenchyma. Furthermore, in accordance with the cell experiments, treatment with H-TL1 inhibited TNF-α-mediated activation of NF-κB and MAPKs pathways and reduced the production of inflammatory mediators in injured tissues.Snake venom is primarily composed of proteins and peptides that possess a variety of biological activities. In addition, venom has been shown to have pro- and anti-inflammatory properties and has been shown to regulate the expression of inflammatory mediators such as IL-1β, IL-6, IL-10, IL-22, TNF-α, PGD2, PGE2, and TXA2 ,42,43,44H. cyanocinctus venom gland T7 phage display library, effectively antagonized the TNF-α/TNFR1 interaction and alleviated the cytotoxicity and inflammation associated with TNF-α in vitro and in vivo. These new findings emphasize the potential usefulness of H-TL1 as a promising peptide candidate for the development of new agents to treat IBD, sepsis acute shock, and other inflammatory diseases associated with TNF-α.In summary, H-TL1, screened from an


Forty-four adult Wistar rats anesthetized with sevoflurane were randomized into four groups: placebo/no hemorrhage (Plc/NH); parecoxib/no hemorrhage (Pcx/NH); placebo/hemorrhage (Plc/H); and parecoxib/hemorrhage (Pcx/H). Pcx groups received a single dose of intravenous parecoxib while Plc groups received a single dose of placebo . Animals in hemorrhage groups underwent bleeding of 30% of blood volume. Renal function and renal histology were then evaluated. Plc/H showed the highest serum levels of cytokines, suggesting that pretreatment with parecoxib reduced the inflammatory response in rats subjected to hemorrhage. No difference in tissue cytokine levels between groups was observed. Plc/H showed higher percentage of tubular dilation and degeneration, indicating that parecoxib inhibited tubular injury resulting from renal hypoperfusion. Our findings indicate that pretreatment with a single dose of parecoxib reduced the inflammatory response and tubular renal injury without altering renal function in rats undergoing acute hemorrhage.Parecoxib, a selective COX-2 inhibitor, is used to improve analgesia in postoperative procedures. Here we evaluated whether pretreatment with a single dose of parecoxib affects the function, cell injury, and inflammatory response of the kidney of rats subjected to acute hemorrhage. Inflammatory response was determined according to serum and renal tissue cytokine levels (IL-1 Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most widely prescribed therapeutic agents for reducing acute pain and are often used to improve analgesia and reduce opioid consumption, accelerating recovery after surgery –3.Conventional NSAIDs block both cyclooxygenase (COX) enzyme isoforms, COX-1 and COX-2. The potential adverse effects of cyclooxygenase-1 inhibition, such as gastric ulceration and bleeding, changes in hemostasis, and adverse renal effects, have limited the use of NSAIDs in many surgical patients , 4. Thusα, IL-1β, IL-6, and TNF-α are higher in ischemic renal injury and these cytokines are involved in different mechanisms of renal tissue injury. By contrast, IL-10 is an anti-inflammatory cytokine that inhibits cytotoxicity and inflammation pathways and protects from ischemic renal injury [Inflammation plays a major role in the pathophysiology of acute ischemic renal injury , 9. The l injury –11.Some studies suggest that selective COX-2 inhibitors worsen renal function in hypoperfusion and ischemia events –14. ThusThe aim of this study was to evaluate whether pretreatment with a single dose of parecoxib affects renal function, renal tissue injury, and inflammatory response in rats submitted to acute hemorrhage.−1 IV) after anesthesia (n = 11);placebo/no hemorrhage (Plc/NH): rats kept normovolemic, receiving 0.9% saline (2 ml·kg−1 at 2 ml·kg−1 IV) after anesthesia (n = 11);parecoxib/no hemorrhage (Pcx/NH): rats kept normovolemic, treated with parecoxib (20 mg·kg−1 IV) after anesthesia and 30 minutes before bleeding (n = 11);placebo/hemorrhage (Plc/H): rats submitted to hemorrhage of 30% of the blood volume, receiving 0.9% saline (2 ml·kg−1 at 2 ml·kg−1 IV) after anesthesia and 30 minutes before bleeding (n = 11).parecoxib/hemorrhage (Pcx/H): rats submitted to hemorrhage of 30% of the blood volume, treated with parecoxib  were randomly allocated into four groups as described below:The separation of animals in groups was performed at the beginning of the experiment using a computer program that randomly ordered a sequence of steps to be followed. None of the animals died or had to be excluded from the study.T) was monitored by a rectal thermometer and maintained between 37°C and 38°C with a thermal blanket. The internal right jugular vein was dissected and cannulated with 24GA venocath to maintain infusion of Ringer lactate solution (RL) (5 mL·kg−1·h−1) [−1 PAH and 0.25 mg·h−1 IOT in 0.9% NaCI solution until the end of the experiment [−1) of saline or parecoxib according to their distribution by groups. The solution was prepared by a technician unaware of the study and the procedure was performed blindly by the researcher. The selected dose of parecoxib was based on previous studies using this drug in models of ischemia-reperfusion in rats [Rats were submitted to inhalation anesthesia with 2,5% sevoflurane  administered by a mask nonrebreathing system under spontaneous respiration. Rectal temperature (g−1·h−1) , 16 and periment  using an in rats .T30′) in Plc/H and Pcx/H groups via carotid artery in three steps, 10 minutes apart, which corresponded to a total blood drawing of 30% of the blood volume, ending at T50′ time point. Blood volume was estimated as 6% of total animal body weight [Bleeding was initiated 30 minutes after placebo/parecoxib infusion . At this moment, rats underwent laparotomy for bilateral nephrectomy and then were sacrificed by IV injection of sodium pentobarbital. A drop of blood was also collected after venous dissection in all groups to determine the initial hematocrit . PAM was measured at T0, T30′ (onset of bleeding in hemorrhage groups), T50′ (end of bleeding in hemorrhage groups), and T80′ moments.For all groups, the hematocrit and concentrations of PAH, IOT, and serum cytokines were determined in the arterial blood collected 30 minutes after the last bleeding time point in hemorrhage groups, and 80 minutes after IOT/PAH primes and placebo/parecoxib bolus in nonhemorrhage groups  were obtained by high performance liquid chromatography (HPLC), and clearances (C) of IOT and PAH (CIOT and CPAH) was calculated according to the Fick principle:Blood samples for subsequent analysis of serum cytokines were centrifuged for 10 minutes at 1200morrhage . IOT andCPAH estimated the effective renal plasma flow (ERFP), and CIOT estimated the glomerular filtration rate (GFR). Filtration fraction (FF) was defined as GFR/ERFP (CIOT/CPAH), renal blood flow (RBF) as ERFP/(1 − Ht), and renal vascular resistance (RVR) as MAP/RBF.The analysis of hematocrit was performed by microhematocrit method  and the result was expressed as a percentage.α, IL-1β, IL-6, IL-10, and TNF-α were determined using kits for ELISA  and the result was expressed as picogram per milliliter of serum.Serum concentrations of IL-1g at 4°C, obtaining the supernatant renal proteins, which were frozen and stored at −70°C for later determination of tissue cytokine levels. The dosages of IL-1α, IL-1β, IL-6, IL-10, and TNF-α were determined in the supernatant kidney by ELISA and the result was expressed as picogram per milligram of protein.Both removed kidneys were longitudinally sectioned into two halves. One half of each kidney was taken for histological preparation. The other remaining halves were macerated and homogenized with 4 mL of PBS . The samples were centrifuged for 15 minutes at 3000The remaining kidney halves separated for histological analysis were placed in Duboscq-Brazil solution  for 24 hours and then stored in 70% ethanol. Slices with fragments from both kidneys were prepared and colored with hematoxylin/eosin. The histological slides were evaluated for the presence of vascular dilation, vascular congestion, tubular dilation, tubular degeneration, and tubular necrosis. Scores were attributed for each of these histological changes, corresponding to severity of injury: zero (0), absence of lesion; one (1), discreet lesions; two (2), moderate lesions; and three (3), intense lesions . The his−1 and a standard deviation of 0.012, statistical power of 90%, and 5% level of significance, which resulted in 11 animals per group [The sample size was determined based on mean values and standard deviations observed in the literature for GFR, considering an average difference between the mean of 0.017 mL·miner group .t-test. Attributes in the animals evaluated over time were analyzed by Profile Analysis, followed by Tukey test for the results of clearances of PAH and IOT, FF, RBF, and RVR. Histological variable values in each kidney and comparison between groups were analyzed by Chi-Square test. In all analyses, statistics were considered significant when p < 0.05.Weight was analyzed by Student's T50′ and T80′ time points . Rectal temperature remained stable in all groups . Mean arterial pressure (MAP) of hemorrhage groups (Plc/H and Pcx/H) decreased significantly after bleeding  and was e points . There wand T30′ .T80′ showing lower values than non-hemorrhage groups (Plc/NH and Pcx/NH) at this time point (Hemorrhage groups (Plc/H and Pcx/H) had a significant decrease in hematocrit at me point .α, IL-1β, IL-6, IL-10, and TNF-α were higher in Plc/H group  was significantly higher in Plc/NH as compared to groups Plc/H and Pcx/H (CIOT) was higher in Pcx/NH compared to Plc/H , due to hypovolemia. Likewise, hematocrit values of Plc/H and Pcx/H were significantly lower at end time point, if compared to control time point in the same groups or to either moment in nonhemorrhage groups. There was a slight but not significant reduction in hematocrit in nonhemorrhage groups at end time point. This may be due to hemodilution resulting from intravenous infusions during the experiment.α [α, IL-1β, IL-6, IL-10, and TNF-α higher than the other groups while Pcx/H had serum levels of cytokines similar to nonhemorrhage groups , and enhances the effects of TXA2. Inhibition of COX-2 by parecoxib suppresses tubuloglomerular feedback, increasing the NO/TXA2 ratio and inhibiting renal vasoconstriction [ feedback, parecoxib could allow greater glomerular blood flow and greater GFR in the group undergoing renal hypoperfusion.The COX-2 enzyme in the macula densa regulates tubuloglomerulartriction . Then paVascular congestion and ectasia occurred similarly in the kidneys in all studied groups. There was no leukocyte infiltration in any of the groups. The hemorrhage group pretreated with placebo presented a higher percentage of tubular dilation and degeneration if compared to the group that received parecoxib, indicating that previous use of parecoxib prevented tubular injuries in this situation. Likewise, Guedes et al.  observedβ, leading to collagen and fibrin deposition in the affected kidney [Treatment with selective COX-2 inhibitor also prevented tubular lesions and reduced interstitial fibrosis in kidney undergoing ureteral obstruction, as COX-2 triggers secretion of TGF-d kidney . Cheng ad kidney  suggestsPretreatment with a single dose of parecoxib reduced inflammatory response and histological renal injury, without altering renal function in rats subjected to acute hemorrhage. However, these short-term effects may not represent benefit of this drug in the long term. The use of this selective COX-2 inhibitor, a drug widely used to prevent and treat acute pain in large surgeries, remains controversial in situations of hemorrhagic shock.


Background. Recurrent aphthous stomatitis (RAS) is a disorder characterized by recurring ulcers involving the oral mucosa in patients with no other signs of disease. The current concept of etiopathogenesis is that RAS is a clinical syndrome with several possible etiologies. The process seen in RAS is probably initiated through an as yet unidentified antigenic stimulation of the mucosal keratinocytes, which stimulates secretion of T-cell activation cytokines ‒ interleukins and tumor necrosis factor alpha (TNF-α). TNF-α causes inflammation by its effect on endothelial cell adhesion and neutrophil chemotaxis. The rele-vance of TNF-α to the pathogenesis of RAS has stemmed from the observations that anti- TNF-α drugs such as thalidomide and pentoxifylline have been found to be effective in the treatment of RAS. Therefore, the present study was an attempt to measure the levels of salivary TNF-α in patients with RAS, which will reflect the local production of cytokines at the site of the disease. The aim was to evaluate the salivary levels of TNF-α in patients with recurrent aphthous stomatitis.Methods. The study comprised of 60 subjects, of whom 30 clinically proven RAS patients of either sex were selected as cases and 30 healthy, age- and gender- matched subjects were selected as controls. After taking informed consent, 5 mL of unstimulated saliva were collected from both the study and control group subjects. Determination of salivary TNF-α levels was carried out by Enzyme-Linked Immunosorbent Assay (ELISA) and expressed in pg/mL. Statistical analysis of the RAS and control groups was carried out using unpaired t-test. Gender-wise comparison of salivary TNF-α levels in the study and control groups was carried out using one-way ANOVA.Results. Mean salivary TNF-α levels were significantly higher in the RAS group compared to the control group (P<0.001). It was also revealed that the mean salivary TNF-α levels in females were significantly higher than in males in the study group (PP<0.05).Conclusion. It is fair to suggest that TNF-α plays a very important mediatory role in the pathogenesis of RAS and may play an important role in the search for a definitive treatment for the disease. These cells are recognized by T lymphocytes which trigger a cytotoxic response and cause the ulceration.2 The aim of this study was to estimate the salivary TNF-α levels in RAS patients prior to definitive therapy.The ulcerative process in RAS is initiated by an unknown antigenic stimulus of the mucosal keratinocytes, leading to T lymphocytic stimulation, the liberation of cytokines such as TNF-α and other interleukins, and the migration of lymphocytes, neutrophils and Langerhans cells.A cross-sectional study was conducted in the Department of Oral Medicine and Radiology, Bapuji Dental College & Hospital, Davangere, after obtaining approval from the institutional ethics committee. The time period of the study was 1 year. Based on the inclusion and exclusion criteria a total of 60 subjects were included in the study.Sample size (n) was determined using the following formula:n=26 in cases and controlsn≅ 30 cases and controls representing normal distribution.2= theoretical value of distribution, s = pooled standard deviation, d = mean expected difference (i.e. mean expected difference in TNF-α levels between in subjects and controls)][t3 Only those patients who gave a signed informed consent form voluntarily were allowed to participate in the present study.Thirty clinically proven RAS patients of either sex were selected as the study group and 30 healthy age- and gender-matched subjects were selected as the control group. Patients clinically diagnosed with recurrent aphthous stomatitis were classified under the types given by Stanley HR.Patients with aggressive and chronic periodontitis, habit of tobacco and alcohol use, significant local and systemic diseases and with a history of steroid therapy 1 month prior to the investigation were excluded.4 between 9 am and 12 pm, after one hour or more since the last food intake. After cooling for 10 minutes, the samples were centrifuged at 4000 rpm for 10 minutes; the upper parts were drawn and stored in small aliquots at –80°C. TNF-α determination was carried out employing immunoassay and quimioluminiscence techniques5 using Boster’s human TNF-α ELISA kit . The salivary samples were cultivated in the presence of specific anti-TNF-α antigen after a controlled defrost and centrifugation. The levels of TNF-α were determined with an automatic analysis unit LISA PLUS  after the application of a quimioluminiscent substrate.Under aseptic conditions, 5 mL of whole unstimulated saliva were collected into sterilized polystyrene tubes for 10 minutes by passive drool method,Data was analyzed by using SPSS 18, with unpaired t-test and one-way ANOVA.3 It was observed that 29 (97%) patients had minor form of RAS and 1 (3%) patient had major form of RAS. No patient had herpetiform type of RAS. The most common sites of involvement of the ulcers were the buccal mucosa, labial mucosa, tongue, vestibule, palate, alveolar mucosa and a combination of one or more sites  females and 11 (37.7%) males. The age of the subjects ranged from 18 to 40 years with a mean age of 25.77 years . Based ore sites .The mean value of salivary TNF-α was 86.30 pg/mL for RAS cases, whereas in controls the mean value of salivary TNF-α was 47.85 pg/mL. Unpaired t-test was used to check the difference in mean between the two groups. There was a highly significant difference (P<0.001) in the mean salivary TNF-α levels between the two groups .It was observed that the mean salivary TNF-α levels in males in the RAS group was 76.74 pg/mL, whereas in females it was 91.83 pg/mL. One-way ANOVA was used to check the difference in the mean between the two groups. There was a statistically significant difference in the mean salivary TNF-α levels between the two groups (P<0.05). However, no such difference was seen in the control group .6Recurrent aphthous stomatitis, also known as recurrent aphthous ulcers or canker sores, is among the most common oral mucosal lesions the oral physicians and dentists observe. RAS is a disorder of unknown etiology that may cause significant morbidity. One or several discrete, shallow, painful ulcers are present on the unattached oral mucous membranes. Minor aphthous ulcers typically last 7‒10 days and heal without scarring. Major aphthae may last several weeks to months and can scar when healing.7 The TNF-α cytokine, a major inflammatory mediator, induces initiation of the inflammatory process by its effect on endothelial cell adhesion and a chemotactic effect on neutrophils.7 The primary role of TNF-α is in the regulation of immune cells. Studies have shown that RAS can be prevented by treatments that prevent the synthesis of endogenous TNF-α such as pentoxifylline and thalidomide.8The immune-pathogenesis of RAS probably involves a cell-mediated immune response mechanism which causes generation of T cells and TNF-α by macrophages and mast cells.11Elevated levels of interleukin-2 (IL-2) as well as those of TNF-α (both proinflammatory cytokines) and lower levels of IL-10 (an antiinflammatory cytokine) have been reported in the lesions of RAS patients. IL-10 usually stimulates epithelial proliferation in a healing process; therefore, its low levels in RAS patients may delay epithelialization and prolong the duration of the ulcers.12 As almost no MHC antigens were detected after healing, they probably play a role in the local tissue damage by targeting these cells for attack by cytotoxic T-cells (CD8+ cells) in the ulcerative process.12TNF-α also has some important immune regulatory activities, including stimulation of class I major histocompatibility (MHC) expression. An increase in class I and class II MHC antigen expression has been detected in the basal epithelial cells in the pre-ulcerative and ulcerative stages of the RAS lesion.14 No studies contradicting the above findings have been reported in the literature.In the present study, comparison between the study and control group showed a statistically significant (P<0.001) increase in the mean TNF-α levels in the RAS group compared to the control group. This indicates an increase in the cytokine level during the active stage of the disease. Similar findings have been reported by Eguia-del Valle et al (2011), Batool Al-Ghurabei et al (2011), Boras et al (2006) and Natah et al (2000).This study also showed a statistically significant (P<0.05) increase in the salivary TNF-α levels in females compared to males in the RAS group. Interestingly there is no literature available which explains such difference. It may be because of the local or systemic and genetic factors that determine this gender discrepancy in TNF-α levels for RAS. However, there was no significant difference in the TNF-α levels between males and females in the control group.17 The development of a new class of drugs specifically designed to inhibit TNF-α production could be a new approach in the treatment of RAS, with a higher efficacy and safety compared with those drugs available commercially today. A range for salivary TNF-α in normal individuals is yet to be determined but considering that the collection of saliva is a cheap, easy and non-invasive method, further research is sure to provide a positive outcome regarding the role of salivary TNF-α in the pathogenesis and management of RAS.It is fair to suggest that TNF-α plays a very important mediatory role in the pathogenesis of RAS, as the patients with active form of the disease showed a greater elevation of TNF-α in both serum and saliva and these findings have been supported by the observations that Thalidomide, which reduces the activity of TNF-α by accelerating the degradation of messenger RNA, pentoxifylline, which inhibits TNF-α production, and levimasole, which modulates serum TNF-α production, have been found to be effective in the treatment of RAS.The findings of this study need to be carefully interpreted because of the small sample size and to the best of our knowledge, lack of sufficient studies involving the estimation of salivary levels of TNF-α in patients with RAS. Further research involving larger samples is suggested along with extensive work involving the specificity of estimation techniques of salivary TNF-α.Our study demonstrated a highly significant increase in the salivary levels of TNF-α in RAS patients compared to controls. We conclude that the estimation of salivary TNF-α levels can be used as a reliable biomarker for the progression and retrogression of RAS. All these data support the importance of TNF-α in the pathogenesis of RAS. Further research needs to be undertaken regarding the role of anti-TNF-α drugs in the treatment of RAS, which may lead to the prevention and cure of the disease in the future.None.The authors declare that the there are no conflicts of interest.