Successful treatment of chronic inflammatory diseases integrates both the cessation of inflammation and the induction of adequate tissue repair processes. Strikingly, targeting a single proinflammatory cytokine, tumor necrosis factor (TNF), induces both processes in a relevant cohort of inflammatory bowel disease (IBD) patients. However, the molecular mechanisms underlying intestinal repair following TNF blockade during IBD remain elusive. Using a novel humanized model of experimental colitis, we demonstrate that TNF interfered with the tissue repair program via induction of a soluble natural antagonist of IL-22  in the colon and abrogated IL-22/STAT3-mediated mucosal repair during colitis. Furthermore, membrane-bound TNF expressed by T cells perpetuated colonic inflammation, while soluble TNF produced by epithelial cells (IECs) induced IL-22BP expression in colonic dendritic cells (DCs) and dampened IL-22-driven restitution of colonic epithelial functions. Finally, TNF induced IL-22BP expression in human monocyte-derived DCs and levels of IL22-BP correlated with TNF in sera of IBD patients. Thus, our data can explain how anti-TNF therapy induces mucosal healing by increasing IL-22 availability and implicates new therapeutic opportunities for IBD. IBD incidence has increased worldwide profoundly over the past decades with no curative treatment currently being available. Notably, even in clinical remission stage, mucosal healing is achieved in less than half of the IBD patients, leaving the possibility of subsequent bacterial translocation, further colonic epithelial damage, and clinical relapse. Various therapies are being applied in clinical practice, but in most of the cases, they are directed towards cessation of inflammation, without actively influencing tissue repair processes.Inflammatory bowel disease (IBD) is a chronic autoimmune disease of the gastrointestinal tract driven by an aberrant immune response towards microbial constituents in genetically susceptible hosts6. TNF is a pleiotropic cytokine exhibiting both protective and pathogenic functions in vivo7. Its chronic overproduction is observed in many autoimmune diseases, such as rheumatoid arthritis, ankylosing spondylitis and IBD9. Therapeutic strategies based on TNF neutralization are highly efficient against these autoimmune diseases and can significantly decrease inflammation8.Interestingly, TNF blockade, one of the biologic therapies approved for IBD treatment, may also result in mucosal tissue repair in a significant fraction of patients10. Also, TNF controls tissue barrier functions by regulating apoptosis of intestinal epithelial cells (IECs), expression of tight junction proteins and mucus secretion13. Thus, anti-TNF therapy based on various antibodies neutralizing TNF was successfully implemented as treatment option for IBD. Apart from direct TNF neutralization, such antibodies may bind to the transmembrane form of TNF (tmTNF) expressed by monocytes and T cells and promote their depletion via antibody-induced cytotoxicity14. Consistent with this, a positive therapeutic response to anti-TNF therapy correlates with increased frequency of tmTNF-producing cells at sites of inflammation in the colon15. Despite the plethora of data on the action of TNF on various target cells, the precise in vivo action of TNF during active colitis remains scarce.TNF exhibits pleiotropic functions during intestinal inflammation. For instance, it induces production of chemokines that recruit proinflammatory myeloid cells to the colon16. The IL-22 receptor is exclusively expressed on cells of non-hematopoietic origin, such as IECs, and this receptor engagement results in cell proliferation and tissue repair upon insult. If not properly controlled, IL-22 signaling may also induce malignancy17. A soluble antagonist of IL-22 (IL-22BP) was described that may neutralize biological effects of IL-22 in vivo and, thereby, counteract the tissue repair processes18. Interestingly, IL-22BP expression is increased during IBD20, and correlation between mRNA levels of TNF and IL-22BP in colonic tissues of IBD patients has been observed20. However, the relationship between IL-22 and TNF in tissue repair remains elusive.IL-22 is a cytokine produced by various immunocytes, such as type III innate lymphoid cells (ILC3), T cells and neutrophilsHere, we show that enhanced levels of TNF during intestinal inflammation induce the expression of soluble IL-22BP in the colon and, thereby, antagonize IL-22/STAT3-mediated mucosal repair during colitis Table 21. To generate a mouse colitis model dependent solely on human TNF expression, we transferred naive T cells isolated from hTNF-KI donor animals into offspring of hTNF-KI mice crossed with Rag1−/− mice  agents with distinct pharmacological and physico-chemical characteristics that cannot be evaluated in available mouse models. Therefore, we developed a novel humanized mouse, TNF-driven model of autoimmune colitis that allows studying TNF inhibition by all clinically used anti-hTNF drugs at the stage of fully developed colitis Fig. . To thisice Fig. 21,22. Toce Figs. . The for−/− recipients, chronic autoimmune colitis developed  and molecules involved in tissue repair  were significantly upregulated in the colon of infliximab-treated mice . Notably, in this set of experiments, Infliximab-mediated recovery occurred faster, which was reflected in rapid, extensive weight gain  reduced inflammation at 3 weeks after initiation of therapy  inapy Fig.  and , while its expression by T cells remained unaffected and was much lower than by DCs  can be induced by various TLR agonists, as well as by retinoic acid, in human monocyte-derived DCs (hmoDCs)lls Fig. . Since Tlls Fig. . Of notells Fig. .Overall, our data indicate that TNF controls IL-22BP expression by DCs both in mice and humans.To further dissect the mechanism of how TNF controls IL-22BP expression during colitis, we quantified human and murine TNF protein levels in ex vivo colonic explants upon TNF ablation. Blockade of T-TNF by Infliximab not only abrogated the production of hTNF protein, but also significantly decreased the production of mTNF by other cells Fig. . On the −/− recipient mice of hTNF-KI T cells revealed significantly higher soluble murine TNF production as compared with soluble hTNF  mice into Rag1−/− recipients  and in epithelial cells  in Rag1−/− mice and induced colitis by transfer of naive T cells from WT mice. Strikingly, these experiments revealed that TNF produced by epithelial cells can control epithelial cell repair, colonic TNF production and IL-22BP colonic levels 41 and treated mice with infliximab or respective control  requires not only blockade of ongoing inflammation, but also complete restoration of tissue functions. Thus, restoration of epithelial cell functions during IBD is the major target for IBD treatment. IECs homeostasis is maintained by immune cells in the gut and by microbial composition45. Our new data further detail the mechanism of how anti-TNF therapy perpetuates epithelial tissue repair via modulation of IL-22 bioactivity. A recent study suggested a promotion of intestinal epithelial tissue repair by TNF via induction of WNT signaling32. In that study increased proliferation of epithelial cells in anti-TNF refractory patients was interpreted as indication for TNF-promoted IECs proliferation. However, the patient cohort responding to anti-TNF therapy also showed mucosal tissue repair3 and no cases of epithelial barrier leakage upon anti-TNF therapy have been reported so far, further advocating that anti-TNF treatment does induce IECs repair when it is efficacious. Intriguingly, our study employing a small cohort of IBD patients revealed a correlation between human TNF in sera and IL-22BP. At the same time, these parameters did not correlate with disease severity. Importantly, we were not able to measure bioactive TNF by existing assays because sera from IBD patients induced TNF-independent cell death of both reporter cell lines tested. Altogether, our data revealed a strong correlation between serum protein levels of TNF and IL-22BP during IBD and further studies showing whether patients with high TNF and IL-22BP levels exhibit IECs repair upon anti-TNF therapy are required.TNF is a pleiotropic cytokine that exerts both beneficial and deleterious functions during autoimmunity46. One possible scenario is that the inflammatory processes may be directly coupled to the repair program in the tissue. In line with this, our data demonstrate that TNF interferes with IL-22 bioactivity through induction of IL-22BP implying that inflammatory processes may limit tissue repair. Exploiting TNF blockade in established chronic inflammatory disorders using a novel mouse model recapitulating human pathology, we demonstrate that TNF orchestrates two distinct pathways in perpetuating chronic intestinal inflammation: first, by chemokine-mediated recruitment of inflammatory myeloid cells and second, by interference with epithelial cell proliferation via control of IL-22 activity in the colon. Thus, our data indicate that tissue repair and inflammation in some chronic inflammatory models are tightly interconnected.In order to combat invading pathogens, inflammation must be induced by immune protective mechanisms involving numerous cytokines, which drive host defense against pathogens. In such situations, all host resources should be directed towards the aim to eradicate the pathogen and induce the repair before resolution of inflammation may become deleterious36. Moreover, enhanced IL-22BP protein expression was recently reported in CD4 T cells and in DCs isolated from IBD patients20. Only T cell-derived IL-22BP expression appeared to be affected by anti-TNF therapy in one of these studies, although TNF did not induce IL-22BP in T cells directly20. Here we showed that TNF induces IL-22BP expression by DCs in vitro. Additionally, our data demonstrate that TNF blockade during colitis results in decreased IL-22BP production by intestinal DCs, but not by T cells, suggesting different modes of regulation of IL-22BP expression by these two cell types. Discrepancies between previous reports and this study could be explained by different sorting strategies: we sorted CD103+CD11c+MHCII+ cells, whereas previous works focused on CD11c+ cells only. Dissection of the mechanisms of transcriptional regulation of IL-22BP in distinct cell types is required to fully understand the contribution of each IL-22BP producing cell subset in modulating IL-22 bioactivity in vivo.IL-22BP is produced by various cell types such as DCs, T cells, eosinophils, and epithelial cellsFurther analysis of IL-22BP expression in naive animals showed that both T-cell and B-cell derived TNF contribute to control of IL-22BP expression in the spleen. T-TNF blockade during established colitis not only eliminated T-TNF, but also decreased levels of non-T cell-derived TNF possibly by limiting inflammation. Finally, genetic ablation of TNF in distinct cellular sources in this colitis model revealed that soluble TNF produced by IECs controls IL-22BP expression in the colon, while transmembrane T cell-derived TNF regulated inflammation and TNF production by IECs and, thereby, Il-22 bioactivity. Thus, TNF produced by distinct cellular sources controls IL-22BP expression in various tissues and this warrants the further dissection of contribution of its source in various disease conditions.−/− T cells were transferred into Rag1−/− recipients. Further studies are needed to uncover the exact mechanism restoration of IECs homeostasis upon cessation of colonic inflammation.Of note, we have observed a discrepancy in genetic and pharmacological TNF inhibition on IL-22BP production: blockade of T cell-derived TNF during established colitis induced tissue repair via the IL-22/IL-22BP axis, while genetic depletion of TNF in T cells did not affect IL-22BP. Further experiments revealed that IECs restitution upon anti-TNF therapy is a tightly controlled, time-limited process, since it was evident at 2 weeks, but not at 3 weeks, after initiation of the treatment and, thus, it was not evident once TNF49. Chronic intestinal inflammation induced by TNF overproduction contributed to IECs apoptosis and development of ileitis47. On the other hand, TNF protects IECs in the acute gut ischemia-reperfusion model via NFkB activation48. Our results further highlight deleterious effects of TNF on epithelial cell homeostasis in a chronic model of intestinal inflammation. Subsequent in vitro studies revealed that a chronic, but not a short term, exposure of epithelial cells to TNF resulted in decreased cell proliferation and death49. Hence TNF may exert its pathogenic functions by interfering with the maintenance of the epithelial barrier integrity in chronic inflammation.TNF blockade possesses both regenerative and destructive functions depending on the context of the inflammatory reaction50 and IL-22 may ameliorate some of these disorders51. Thus, it appears plausible that TNF affects the development of aging-associated diseases via modulation of IL-22 bioactivity. This possible link has to be validated in future studies.Chronically elevated TNF levels are associated with aging and aging-associated pathologies52. Of note, most inflammatory skin disorders are associated with IL-22 and IL-22 blockade is beneficial in these patients16. Our results provide explanation how anti-TNF treatment increases IL-22 bioavailability and, thus, exacerbates such diseases. Further studies in patients undergoing anti-TNF therapy who develop secondary autoimmune manifestations in the skin will be needed to address this.Anti-TNF therapy, although effective in many patients, is associated with unavoidable side effects, including exacerbations of preexisting skin diseasesThe objectives of the present study were to establish humanized TNF mouse models and harness them for in-depth analysis of biological effects of anti-TNF drugs and dissection of the mechanism of anti-TNF mediated epithelial cell restitution. Both males and females were used in all experiments. Animals were allocated to experimental groups based on their genotype; mice in one cage were assigned to various treatment regimens to reduce cage-to-cage effect. We did not use exclusion approaches for assignment of the animals to the experiments. For treatment with various agents, mice were blindly assigned to the respective group by an independent scientist. All injection and dissection experiments were conducted in a non-blinded fashion. The sample sizes were determined on the basis of previous experience. Blinding was used for all the mouse analysis, including histology. For each experiment, sample sizes reflect the number of independent biological replicates and are provided in figure legends. In general, sample size was chosen to use the least number of animals to achieve statistical significance according to 3 R regulations.22. Mice with ablation of TNF in T cells , with ablation of TNF in myeloid cells , with ablation of TNF in B cells , with ablation of TNF in epithelial cells , TNF KO (RRID:MGI:3582275), tmTNF KI53, hTNFRp75 KI41 and Rag1−/− (RRID:MGI:3850561) mice used in this work were previously described55. Ablation of TNF in all cells, in myeloid cells and epithelial cells of Rag1−/− mice was achieved by backcrossing TNF KO, M-TNF KO and EC-TNF KO on Rag1−/− background. hTNF-KI and hTNFRp75 KI mice on Rag1−/− background were bred for this study.Mice expressing human TNF (hTNF-KI mice) were generated in our laboratory Fig. S21,22. Miflox/flox(TNF<tm1SNed>/TNF<tm1SNed>) mice were used as wild-type (WT) controls for all experiments performed with T-TNF KO mice and as donors of naive T cells when T-TNF KO T cell transfer was performed. C57BL/6 mice were purchased from Charles River Laboratories. All mice were kept under specific pathogen-free conditions in the animal facility of German Rheumatism Research Centre (DRFZ), Berlin, and Pushchino animal facility and were used for experiments at 6-12 weeks of age. Mice from various experimental groups were cohoused to exclude effects of microbiota variations on the observed phenotype. Mice in every cage were randomly assigned to various treatments to exclude cage effect. All animal procedures were performed in accordance with German and Russian regulations of animal protection.TNFBlood samples were obtained from IBD (both UC and CD) patients attending the outpatient clinic at Charité Campus Benjamin Franklin after informed consent. The study was approved by the local ethical committee (EA4/014/18). Clinical response was routinely assessed at week 6 after initiation of anti-TNF treatment (Infliximab) and defined as a decline in the Partial Mayo Score of at least 3 points for patients with ulcerative colitis and at least 3 points in the Harvey-Bradshaw Index for patients with Crohn´s disease, or by global physician assessment. Patients were classified as responders or non-responders based on their primary response to infliximab treatment. Clinical score had been determined at the time of blood collection.+ T cells by magnetic-activated cell sorting (MACS) negative selection using anti-biotin microbeads . Briefly, 5×108-1×109 cells were incubated with biotinylated anti-CD8, anti-CD25 and anti-B220 monoclonal antibodies for 15 min at 4 °C followed by streptavidin-conjugated magnetic beads (Miltenyi Biotec) in PBS/BSA. All washing steps were performed at 1500 rpm at 4 °C in PBS/BSA. Before MACS separation, cells were filtered through 30 µm mesh filters (Miltenyi Biotec). The Automacs program possel (positive selection) was used for separation.Spleens and peripheral lymph nodes (LN)  were collected from donor mice. Cells were isolated by homogenizing spleens and LN via cell strainers (80 µm)  in PBS/BSA under sterile conditions in a Laminar Flow Box (Thermo Fischer Scientific). Cells were pelleted by centrifugation  and the supernatant was discarded. For red blood cell depletion, cells were resuspended in ACK erythrocyte lysis buffer, incubated for 5 min at 4 °C and washed. Isolated cells were enriched for CD4+ T cells was incubated with CD4-PE-Cy7, CD45RB-PE, and CD25-APC antibodies, as well as propidium iodide (PI) to exclude dead cells. Cells were further sorted by fluorescence-activated cell sorting (FACS) on FACS Aria II (BD Biosciences). Anti-CD25 staining excluded CD25+ T cells, and the CD4+CD45RBhi CD25- population was sorted with a purity of >98%.Upon magnetic sorting, the negative fraction containing CD4−/− animals were reconstituted by injection of 3 × 105 sorted CD4+CD45RBhiCD25- cells in 200 µl PBS per mouse. At the time of transfer, the recipient mice were weighed and subsequently monitored daily. Mice were sacrificed after 40–60 days post transfer. Treatment with anti-human TNF drugs or respective isotype control was performed once weight loss was >5% for twice a week for two or three weeks, as indicated, and then sacrificed. Tissue samples were removed for histology, cell isolation, preparation of colonic explants and RNA isolation.Rag1Once mice had lost >5% of initial weight, they were treated with anti-human TNF antibody (Infliximab), anti-mouse IL-22  or human Fc-IgG1 isotype control  in PBS. Anti-human TNF drugs were kindly provided by Dr. Eugen Feist . Antibodies were injected intraperitoneally (i.p.) in 100 µL PBS at a dose of 10 mg/kg body weight twice a week for two or three consecutive weeks. Anti-mouse IL-22 antibody was injected i.p. at a dose of 10 µg per mouse in 200 µL PBS. Mice were analyzed 3 days after the last injection. Anti-IL-12p40 antibody , anti-mTNF  or anti-IL-18  or respective isotype controls were used for respective cytokine blockade.+CD45RBhiCD25- T cells). The colon was cleaned of mesenteric fat using forceps and the length from the cecum to rectum was determined with a ruler. The colon was subsequently divided into four equal pieces from proximal to distal part for cell isolation, ex vivo culture, histology and RNA isolation, respectively.Mice were sacrificed and their mesenteric LN (mLN) and colons were collected. LN cells were obtained as described earlier , CD3 , KI67 , Casp3  and F4/80 , followed by Streptavidin-AP kit  and Envision PO kit . The sections were processed, stained and analyzed in a blinded fashion. Representative pictures were taken by a pathologist and were provided further to the scientist for decoding and illustrations of disease characteristics. The degree of colonic inflammation was scored using a previously described scoring system by Erben and colleagues2. Medium was collected, centrifuged, pelleted cell debris discarded and the supernatant stored at −20 °C.Colonic tissue from mice was thoroughly cleaned with cold PBS/BSA to remove excess mucus and feces. Samples were weighed and incubated in 1 mL of RPMI containing 10% FCS and penicillin/streptomycin (100U/ml) in a 48-well plate. The colonic explants were incubated for 24 h at 37 °C in 5% COFor isolation of lamina propria lymphocytes, colon samples were longitudinally cut, flushed with cold PBS/BSA and cut into small pieces. Pieces were transferred into RPMI containing 5% FCS and 5 mM EDTA and incubated for 20 min at 37 °C to remove epithelial cells and to isolate intraepithelial lymphocytes (IELs). Samples were vigorously shaken; supernatant was removed and replaced with fresh EDTA solution and the incubation was repeated. The IEL-containing supernatants were washed before further experimental procedures. Intestinal pieces were minced with scissors and scalpel and were transferred into fresh 50 mL Falcon tubes to be digested in RPMI containing 5% FCS and 1 mg/mL collagenase D  and 1 mg/mL dispase II . After 20 min of digestion at 37 °C in a shaking incubator (200 rpm), samples were thoroughly vortexed and the cell-containing supernatant was collected and washed. Undigested tissue was incubated with fresh digestion mix for another 20 min in the shaking incubator. Samples from two digestion rounds were pooled and filtered through 80 µm filters . After washing, cells were used for further experimental procedures.Splenic DCs were isolated as follows: spleens were digested in RPMI-1640 containing 10% FCS, penicillin/streptomycin (100U/ml), 0,5 mg/ml of DNAse 1  and 0,8 mg/ml of Collagenase type IV . Splenocytes were stained with antibodies against CD11c, MHCII, CD4 and CD8 , as well as propidium iodide (PI) to exclude dead cells. Cells were further sorted by fluorescence-activated cell sorting (FACS) on FACS Aria II (BD Biosciences). Resulting DC populations were sorted with a purity of >98%. Sorted cells were further activated by rmTNF  and expression of IL-22BP was analysed.Colon samples were longitudinally cut, flushed with cold PBS/BSA and cut into small pieces. Pieces were transferred into PBS with 5 mM EDTA, 1 mM DTT and incubated for 20 min at 37 °C to remove epithelial cells and to isolate IEL. Samples were vigorously shaken; supernatant was removed and replaced with fresh EDTA solution and the incubation was repeated. The IEL-containing supernatants were centrifuged and resuspended in PBS containing , 0,005 % of DNAseI (Sigma-Aldrich) and 0,3 % of Dispase II (Roche) and were and incubated for 20 min at 37 °C. Cells were washed and used for further staining.57.To assess transcriptome profiles of highly pure (>99%) live epithelial cells (EpCAM1-positive) total RNA was isolated using RNeasy Plus Micro kit (Qiagen) according to the manufacturer’s instructions. Illumina libraries were prepared using Smart-Seq v4 mRNA Ultra Low Input RNA Kit (Clontech) and Nextera XT DNA Sample Preparation Kit (Illumina), with up to 10 ng of purified cDNA, according to the manufacturer’s instructions. Libraries were paired-end sequenced (2 × 75 bp) on an Illumina NextSeq500 device. Raw sequences were mapped to mouse GRCm38/mm10 genome with TopHat2 in very sensitive settings for Bowtie2. Per gene counts were calculated using featureCounts and gene expression analyses were performed using DESeq2 1.18. Differential expression between Fc-treated and infliximab-treated ECs was regarded as significant when the adjusted p value was <0.05 and the fold change was >1.3. The resulting gene sets were further analysed for pathway enrichment by EnrichrExperiments with human blood material were approved by the Ethics Committee of the Charité - Universitätsmedizin Berlin, Germany [EA2/064/14]. The buffy coats were obtained from healthy donors through the blood bank of the German Red Cross . Donors were kept anonymous.+ cells from the PBMC fraction by positive magnetic bead isolation according to manufacturer’s instructions . To generate hmoDCs, 1.5 mln of monocytes were cultured in 6-well plates in RPMI-1640  supplemented with heat inactivated 10% (v/v) fetal calf serum (FCS) , 1% L-glutamine, 1% sodium pyruvate, 1% MEM non-essential amino acids, streptomycin/penicillin (100U/ml), 0.1% β- mercaptoethanol (β-ME) , recombinant IL-4 (500 U/ml) and GM-CSF , at 37 °C and 5% CO2. Medium was changed after 2 days and mo-DCs were harvested after 5 days. Cells were then stimulated with rhTNF (100 ng/ml), LPS  and Pam3Cys  for various times. Cells were then lysed using TRI reagent, mRNA was isolated and gene expression was measured by real-time PCR.Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation . After thorough washing with phosphate buffer saline containing 2% EDTA and 1% human serum, monocytes were isolated as CD145-1×106 cells were incubated in IMDM medium containing 10% FCS and penicillin/streptomycin (100U/ml) in a 96-well plate in the presence of PMA (5 ng/mL)  and Ionomycin (500 ng/mL)  for 4 h at 37 °C. For intracellular accumulation of cytokines, 10 µg/mL Brefeldin A  was added for the time of stimulation.For analysis of cytokine production, 1×10Colo205 cell line  was used to estimate IL-22 bioactivity. Briefly, 40,000 cells/well were seeded in a 96-well plate. The supernatants from colonic explants with or without addition of rmIL-22BP  or anti-IL-22  were added to the culture for 48 h. Afterwards, supernatants were collected and the amount of IL-10 produced was measured using a human IL-10 Ready-Set-Go! ELISA Kit .+CD45RBhiCD25− T cells).Splenocytes were isolated as described in (Isolation of CD45 cells/well) were plated in RPMI‐1640 complete media . Cells were stimulated with LPS , with or without infliximab or respective Fc control . Cells were incubated at 37 °C for 24 h and were stained with Propidium iodide and Annexin V-Cy5, according to the manufacturer’s instructions (Thermofisher Scientific).Cells . To distinguish viable and dead cells, live/dead fixable dead cell stain (Thermo Fischer Scientific), DAPI  or propidium iodide  was used.2O), centrifugation and incubation with respective antibodies . For this, cells were incubated with freshly prepared Fix/Perm buffer (Diluent/Concentrate 3:1) for 20 mins at 4 °C, followed by washing with Permeabilisation buffer (diluted 1:10 in HData were acquired with FACSCanto or LSRII (BD Biosciences) and analysis was performed with FlowJo software (TreeStar).In order to detect proteins in colonic explant supernatant, enzyme-linked immunosorbent assay (ELISA) for human TNF (Cat #: 88-7346-22), mouse TNF (Cat #: 88-7324-22) and mouse IL-22 (Cat#: 88-7422-22) was performed. All ELISA assays (Ready-Set-Go!) were purchased from ThermoFischer Scientific and performed according to manufacturer´s protocol. Detection of human IL-22BP was done using anti-hIL-22BP (RnDSystems: MAB1087) as capture antibody, biotinylated anti-hIL-22BP (RnDSystems: BAF1087) as detection antibody and rhIL-22BP (RnDSystems: 1087-BP) as a standard. Shortly, Nunc MaxiSorb flat bottom 96-well plates (Nunc) were coated with capture antibody diluted in coating buffer overnight. After washing and blocking with 1x assay diluent, colonic explant samples were diluted 1:2 in assay diluent and applied in duplicates. Bound protein was then detected by a protein-specific biotinylated detection antibody and avidin-horseradish peroxidase and color was developed with TMB substrate solution. Color reaction was stopped with stop solution and plates were scanned with a VMax® Kinetic Microplate reader (Molecular Devices) at respective wavelength (450 nm).To assess multiple protein biomarkers simultaneously in colonic explant supernatants, ProcartaPlex Mouse Cytokines and Chemokines (26 plex)  was performed according to manufacturer’s instructions Table . The pla2 and homogenized with mortar and pestle prior to resuspension in 1 mL of Trizol (Sigma-Aldrich: Cat #: 93289). RNA from cells was isolated by direct addition of Trizol reagent to the pelleted cells. Further, RNA was isolated by phenol-chloroform extraction. Shortly, 200 µL of chloroform was added, samples were shaken thoroughly and centrifuged (13000 rpm for 15 min at 4 °C) and subsequently RNA from the upper aqueous phase was precipitated with 700 µL isopropanol and 200 µg of glycogen to enhance precipitation. After pelleting, RNA was washed with 75 % ethanol and air-dried at 56 °C in a thermoshaker. Depending on the size, the pellet was reconstituted in 10-50 µL of DEPC-treated water (Life Technologies) and dissolved at 56 °C for 10 min. RNA concentration was determined with a NanoDrop photospectrometer (Thermo Scientific).For RNA isolation, colon pieces were frozen in liquid NFor reverse transcription, 1 µg of RNA was treated with DNase I  according to the manufacturer´s instructions. To transcribe RNA to cDNA, random hexamer primers (TIB Molbiol) and RevertAid H Minus reverse transcriptase  were used according to the manufacturer´s protocol. The generated single-stranded cDNA was directly used for quantitative Real-time PCR.cDNA transcript expression of indicated genes Table  was analp < 0.05, **p < 0.01, ***p < 0.001. Statistically evaluated values are expressed as mean with error bars representing standard deviation from mean (SEM). The Pearson correlation was used for correlative analyses. The significance level was set to p ≤ 0.05.Statistics were calculated in Graph Pad Prism (GraphPad Software). Statistical significance was determined using Student´s t-test for unpaired data and statistical significance was defined as follows: *Supplementary informationSupplementary Table


G is the genomic relationship matrix, and A for genotyped animals. Incompatibility between G and A may cause inflated genetic variance. Blending and tuning G with H is built. Alternative ways of weighting the H.Compared to BLUP, in single-step genomic BLUP, A) with an augmented pedigree–genomic relationship matrix (H). Theoretically, H does not introduce any artificially inflated variance. However, inflated genetic variances have been observed due to the incomparability between the genomic relationship matrix (G) and A used in H. Usually, G is blended and tuned with A for genotyped animals) to improve its numerical condition and compatibility. If deflation/inflation is still needed, a common approach is weighting H is built. Different ways of weighting the H. Data were simulated on ten populations and twenty generations. Responses to weighting different components of The single-step genomic BLUP (ssGBLUP) is used worldwide for the simultaneous genetic evaluation of genotyped and non-genotyped animals. It is easily extendible to all BLUP models by replacing the pedigree-based additive genetic relationship matrix ( A) and genomic (G) relationship matrix  relationship matrix in which the genetic values of non-genotyped animals were conditioned to the genetic values of genotyped animals. The resulting matrix was:This relationship matrix did not condition the distributions of breeding values for genotyped and non-genotyped animals on each other, leading to incoherencies in the joint distribution of genetic values for genotyped and non-genotyped animals. Legarra et al.  presenteH, the genomic information in G influences the relationships between non-genotyped and genotyped animals and among non-genotyped animals. Later, it was discovered that H [In matrix erting H ,4.(6)H−Note that:G is not always full-rank . To force G to be positive-definite and avoid large diagonal values of G, the first step of conditioning G often involves blending it with k < 1). Blending introduces residual polygenic effects (genetic effects not captured by genetic markers) to the evaluation model without explicitly modelling it, where the scalar k is the ratio of the polygenic to the total additive genetic variance [Matrix r clones ) and of variance .H matrix [G and G and G and G and G regress data to different means. Matrix G regresses solutions toward a founder population comprising genotyped animals [G and G to improve its consistency with G by regressing its averages to the averages of It is theoretically true that no artificially inflated variance is introduced via the H matrix . However and A22 ,7,8,9. I animals , and the animals ,10 since and A22  and rebawith A22  tuned G G into G into The 1−ωA22−1 ,12,13. A)A22−1−1 , which eG closer to G and H, because the changes made in k and k are the commonly used blending coefficients of G and Reducing k coefficient has already been chosen and does not need to be validated. The most coherent approach for finding k is by restricted maximum likelihood (REML), as proposed by Christensen and Lund [Considering the above equation, there is no legitimate reason for i et al.  studied G are not satisfactory and the weighting of the Weighting recently ,15,16,17recently  and the t in A−1 . The aimH. However, any The Equation  is unconH. Weighting 1.2.3.4.5.6.7.Weighting the components of Equation , the aim2) does not match the data or 2 may differ from the current h2 of the trait in the population. Estimating variance components is a computationally expensive process. The h2 estimate might have been from a population subset or via a matrix other than 2 estimates [This scenario is helpful when the heritability estimate  were selected for their marker breeding value and mated to the same number of randomly selected females. Genotypes in each subsequent generation were obtained by combining sampled gametes from the parents’ genotypes.g, a, and e are the vectors of effects corresponding to A and G were accommodated in the simulation. Data simulation was repeated ten times to reduce the possibility of observing the results specific to a dataset.Phenotypes were calculated as Z is the matrix relating phenotypes to animals, 1 and G was used in W is the centred and scaled genotype matrix, and p is the marker allele frequency. Markers with minor allele frequency below 0.02 were discarded before calculating G. Then, G was blended as No fixed effect was simulated, and the data were analysed using the following mixed model equations:VanRaden , where GThe simulated pedigrees had a population size of 2162.8 ± 358.3  with H that provide conditionality between the breeding values of genotyped and non-genotyped animals. Other ways of weighting the components of H.Matrices n in H−1 . Usually2 and increasing inflation due to increased dispersion. It is equivalent to adding 2 or weighting the genetic variance by 1/2 can be lower than expected. The h2 reduction is expected to be greater due to genomic selection. Change of genetic variance by genomic selection is propagated from G throughout H. The predictive ability declined with increasing Weighting easing α , which measing α . TherefoH is a genomic relationship matrix extended from G for genotyped animals to non-genotyped animals via the G is more influential in defining the variances in H than A. This was confirmed by similar trends for weighting Weighting quations ). As suc and H−1 . The slo2 was more compatible with a blended The inflation results are expected to be valid for other data as weighting H. As such,2 without blending This study does not completely rule out using Concerning pedigree and genomic errors, regardless of the emphasis given to pedigree and genomic information, genotype errors propagate through non-genotyped animals, and pedigree errors incorrectly and insufficiently propagate genotype information through non-genotyped animals. Therefore, the correctness and the completeness of pedigree and genomic information are vital for accurate and unbiased ssGBLUP evaluations.D is a diagonal matrix of positive values descending in function of the animal’s age. The researcher would need to decide the d = diag(D). With recent advances in ssGBLUP , which


Plasmodium resistance to anti-malarial drug mainstays has driven a continual effort to discover novel drugs that target different biochemical pathway (s) during infection. Plasma membrane Calcium + 2 ATPase (PMCA4), a novel plasma membrane protein that regulates Calcium levels in various cells, namely red blood cell (RBC), endothelial cell and platelets, represents a new biochemical pathway that may interfere with susceptibility to malaria and/or severe malaria.Rapid emergence of Plasmodium falciparum 3D7 strain, the Plasmodium berghei ANKA strain, and Plasmodium yoelii 17XL strain as model.This study identified several pharmacological inhibitors of PMCA4, namely ATA and Resveratrol, and tested for their anti-malarial activities in vitro and in vivo using the P. falciparum 3D7 strain in the presence of a wide concentration range of the inhibitors revealed that the parasite growth was inhibited in a dose-dependent manner, with IC50s at 634 and 0.231 µM, respectively.In vitro propagation of P. berghei ANKA for experimental cerebral malaria and P. yoelii 17XL for the effect on parasite growth, showed that the highest dose of ATA, 30 mg/kg BW, increased survival of the mice. Likewise, resveratrol inhibited the parasite growth following 4 days intraperitoneal injection at the dose of 100 mg/kg BW.The results confirmed that both compounds exhibit moderate to potent anti-malarial activities with the strongest parasite growth inhibition shown by resveratrol at 0.231 µM. In vivo models using The findings indicate that the PMCA4 of the human host may be a potential target for novel anti-malarials, either as single drug or in combination with the currently available effective anti-malarials. Malaria is currently still a major public health problem around the globe. The World Health Organization (WHO) reported that an estimated 229 million cases of malaria occurred worldwide in 2020  comparedStrategies to control malaria include early diagnosis and prompt anti-malarial treatment of infected individuals, provision of long-lasting insecticidal net (LLIN) and indoor residual spraying (IRS) to reduce human contact with infected mosquitoes. However, the rapid development of drug resistance in the malarial parasite and insecticide-resistant anopheline mosquitoes have hampered large-scale efforts at malaria control , with caATP2B4 gene encoding a calcium pump called the plasma membrane calcium ATPase 4 (PMCA4) has a very strong association with resistance against severe malaria [2+ from the cytosol of most cell types [2+ concentration in the parasitophorous vacuole within RBCs might impair parasite reproduction and maturation [2+ due to alteration of PMCA4 function or expression may affect the development and structure of intra-erythrocytic stages of the parasite [2+ [Recent genome-wide association studies have demonstrated that a common single nucleotide polymorphism (SNP) of the ll types . PMCA isll types . PMCA1 all types . An exprll types . It is kturation . Thus, rsite [2+ , 10. Thesite [2+ , 11–15.Plasmodium falciparum 3D7 in the RBC culture. This study found that both ATA and resveratrol have an inhibitory activity against P. falciparum growth in vitro, with resveratrol displaying strong inhibition at low concentration. The in vivo experiment on the effect of PMCA4 inhibitors on cerebral malaria showed a modest effect on parasite survival, while resveratrol showed significant inhibition of parasite growth at the concentration of 100 mg/kg BW.The present study was aimed at exploring the effects of treatment with PMCA inhibitors on malaria parasite growth in vitro and analysed whether treatment with specific PMCA4 inhibitor aurintricarboxylic acid (ATA) or pan-PMCA1/4 inhibitor resveratrol affected the growth of The study was approved by the Eijkman Institute Research Ethic Committee (EIREC).22H14O9) and general PMCA inhibitor Resveratrol (C14H12O3) were used in this study. ATA, purchased from Sigma-Aldrich , has been identified as a potent specific pharmacological inhibitor of PMCA4 [50 of 150 nM for PMCA4 inhibition. ATA has only a minor effect on the second isoform of PMCA expressed in the heart, PMCA1 [2O) or dimethyl sulfoxide (DMSO) for both in vitro and in vivo experiments, and further serially diluted into a working concentration using RMPI-1640 media for in vitro. Artemisinin  and sulfadoxine were used as positive control on in vitro and in vivo experiment, respectively.The PMCA4 specific inhibitor Aurintricarboxylic acid  supplemented with 10% human serum, 0.005% hypoxanthine, 0.21% NaHCO3, 0.596% HEPES, 0.25% gentamicin and 3–5% human erythrocytes. The parasites were grown in Type AB+ human blood under controlled and placed in 25 mm3 flask and incubated in a candle jar at 37 °C as described by Trager and Jensen method [d-Sorbitol 5% [The human parasite n method  but withbitol 5%  2 days bP. falciparum parasite culture reached 1–2% parasitaemia, it was sub-cultured into 96- well plates (180 µl per well) and incubated in the presence of various concentrations of ATA and resveratrol, ranged from 0 to 10–9 nM, obtained through tenfold serial dilutions. Non-parasitized erythrocytes were used as a negative control and parasitized erythrocytes without any test PMCA4 inhibitors were used as positive control. The anti-malarial activity of the PMCA inhibitors was compared with the standard drug, artemisinin. After 48-h incubation, thin Giemsa blood smears were made for each well and the parasite levels were determined by manual counting of parasite under light microscope. All experiments were done in duplicate and the 50% inhibitory concentration (IC50) of each inhibitor was determined.When the Plasmodium berghei strain ANKA and Plasmodium yoelii strain 17XL parasites , Singapore) from cryopreserved stock was thawed and passaged into C57BL/6 strain mouse as donor and monitored until the parasitaemia level between 1 and 3% [4 infected erythrocytes from the donor [1 and 3% . Approxihe donor  were inoPlasmodium berghei-infected mice were treated intraperitoneally with Aurintricarboxylic Acid (ATA) drug in various concentration  daily, started from one day before infection until 9 days post infection. In addition, anti-malarial drug sulfadoxine was used as positive control for this experiment and administered 25 mg/kg BW as its therapeutic dosage. The sulfadoxine treatment was done at 6–9 days post infection. A group without any treatment was also used as a comparison in this experiment. The survival rates of all experimental mice were observed as well as the concentration of red blood cells (RBCs) and body weight during the course of infection. The amount of RBC was examined under microscope using Niewbauer-Chamber. All the observation was followed until day 14 post infection.Plasmodium yoelii were inoculated intraperitoneally into BALB/c mice and monitored daily until parasitaemia level of 1–2% with Giemsa-stained blood smear [50 of each drug were analyzed based on parasitaemia level following 4-day treatment. The data was statistically calculated using a probit analysis.od smear . The PMCPlasmodium falciparum parasite growth in various concentrations of ATA is shown in Table –1 mM) but marked inhibition at higher doses 100 µM  that showed a significantly higher survival rate Fig. . When coAnalysis on the body weight changes and the red blood cells concentrations on the untreated and ATA-treated groups revealed a non-significant pattern in which both groups exhibit a decrease in bodyweight and red blood cell concentrations during the course of the experiment. Similar pattern was also found on the degree of parasitaemia in treated and untreated groups before the mice succumbed Table .Table 2EP. yoelii-infected mice with intraperitoneal resveratrol at the concentrations of 1 mg-100 mg/kg BW revealed a non-significant inhibition of the growth rate at day 4, except at the concentration of 100 mg/kg BW where a 37% growth inhibition was shown  and several single nucleotide polymorphisms (SNPs), namely rs10900585; rs1541254; rs1541255; and rs4951074, have been implicated in susceptibility to severe malaria in genome wide association [The identification of PMCA4 SNPs as significant genetic determinants for malaria severity has prompted the idea that PMCA4 may be a novel target for malaria treatment. PMCA4 is coded for by the ociation , 20. Theociation . Indeed,P. falciparum growth in the in vitro RBC cultures. The anti-malarial properties of the two PMCA4 inhibitors may be linked to the role of this protein in RBCs [P. falciparum merozoites into RBCs [The findings of this study show that ATA and Resveratrol exhibited a potent anti-malarial activity on  in RBCs . Inhibit in RBCs , and witnto RBCs .At the in vivo experiment, particularly to determine the effect of ATA on cerebral malaria, the effect on the mice survival was noted at higher dose. Similar pattern was also observed with resveratrol on the parasite growth. The findings are in accordance with previous finding whereby ablation of PMCA4 confer a slight protection against cerebral malaria and did not significantly alter peripheral parasite burdens , 23.50 at 150 nM at cardiac fibroblast [50 at 634 mM, but in in vivo experiment, particularly to determine its activity against cerebral malaria, its activity was modest and was only observed at higher dose. Therefore, it is suggested that either the anti-malarial property of ATA in vivo is not mediated through the PMCA4 inhibition or their different pharmacokinetic property of the compound when given intraperitoneally. For the ATA activity in vivo, delivery through oral instead of intraperitoneal injection may be considered.Aurintricarboxylic acid (ATA) was identified as a potent PMCA4 inhibitor previously with ICbroblast . ATA alsbroblast –28. ThisLikewise, resveratrol which exhibits a potent anti-malarial property in vitro has currently been available at the market as antioxidant formula indicated for a wide range of diseases. In addition to its property as pan PMCA inhibitor, the compound also demonstrated efficacy on various diseases in different dosage . The resIn conclusion, we have explored the anti-malarial activities of two compounds that are known as PMCA4 inhibitors. The results indicated that PMCA4 represents a promising novel target for anti-malarial development. Further study to determine biochemical pathways affected by the PMCA4 inhibitors to exert its anti-malarial property and drug delivery is currently on going.


Normal pressure hydrocephalus (NPH) is a rare condition characterized by pathologically enlarged ventricles and a normal cerebrospinal fluid (CSF) opening pressure measured by lumbar puncture. NPH typically presents as a triad of cognitive decline, gait disturbance, and urinary incontinence. Rarely, NPH can present with bulbar involvement, particularly with difficulty swallowing. Here, we present a case of NPH in a 75-year-old man who presented with an episode of choking and a recent onset of swallowing difficulties with a three-month history of ataxia and progressive memory loss. His CT scan revealed ventriculomegaly, which was consistent with the clinical presentation of NPH and was further confirmed by a normal opening pressure on the CSF tap. Furthermore, ventriculoperitoneal shunts showed a marked improvement in patients' dysphagia and the classical triad of NPH symptoms. Through this case report, we want to highlight that NPH can present as a difficulty in swallowing. Idiopathic normal pressure hydrocephalus (NPH) is a rare neurological disorder that primarily affects the elderly. NPH is caused by ventricular enlargement, which can be caused by a CSF drainage pathway blockage, reabsorption defect, or excessive fluid production . NPH canA 75-year-old retired businessman with a recent onset of choking was brought to the Accident and Emergency (A&E) Department of the Tertiary Care Hospital. He suddenly started to choke while eating dinner because he was having trouble swallowing. He told us that he has been experiencing difficulty swallowing for the past three months, which was exacerbated particularly with liquid food. Apart from that, he has a normal appetite and has not experienced any weight loss in the recent past. Upon reviewing his medical history, he reported a three-month progression of gait instability. His wife also noticed that for the last three months, he walked with an unsteady gait, with short steps, and had difficulty trying to turn while walking. She also stated that her husband has difficulty remembering names and frequently misplaces or forgets belongings. He recently forgot his address and was having difficulty finding his way back home while roaming around the neighborhood. Later, a neighbor supported him in returning to his home.2) at room temperature: 96%). A detailed neurological examination revealed that the patient had a shuffling, short-stepping gait. His Rhomberg test was negative. His sensory and motor examinations, including cranial nerves as well as deep tendon reflexes, were all normal. There was no dysmetria or any cerebellar involvement. The review of other systems was unremarkable. His score on the Mini-Mental State Examination (MMSE) was 23.On examination, his vital signs were stable , urine and electrolytes (U&E), a liver function test (LFT), a C-reactive protein (CRP), thyroid function tests, serum B12 levels, and random blood sugar. All of these test results were within normal parameters. Consequently, a CT scan of the brain was performed, which revealed dilatation of the third and lateral ventricles with no hemorrhage or ischemic infarct. No mass effect or mild line shift was noted. An Evans index ratio greater than 0.31 was observed. CT scan is shown in Figure 20 . Cerebrospinal fluid (CSF) analysis revealed a normal white blood cell count of 4 WBCs per mm3  and a mildly increased protein concentration of 65 mg/dL .On the basis of the patient's clinical manifestations and the findings of the CT scan of the brain, normal pressure hydrocephalus was suspected. Consequently, a CSF tap was performed, revealing a normal opening pressure of 15 cmHFollowing a comprehensive evaluation of the patient's clinical findings, radiological evidence, normal pressure on the CSF tap, and improvement of symptoms upon removal of 40 mL of CSF, a diagnosis of NPH was confirmed, and he was further referred to the neurosurgery team for ventriculoperitoneal (VP) shunt placement. After a successful ventriculoperitoneal shunt procedure, an immediate improvement in patient gait was noticed and later the patient was discharged home. The response to shunt placement was also assessed at three- and six-month follow-ups. The patient's gait and cognitive difficulties exhibited significant improvement. His gait improved completely, and he was able to walk and maintain his balance without assistance. In addition, his MMSE score increased from 23 to 26.Normal pressure hydrocephalus is characterized by enlarged ventricles and normal cerebrospinal fluid (CSF) pressure upon lumbar puncture. The incidence of NPH varies greatly between studies, ranging from two to 20 per million people per year . NPH is In patients with NPH, both primary (idiopathic) and secondary causes are identified. The prevalence of idiopathic NPH increases with age and is highest in persons over the age of 60. It affects both sexes with equal frequency. In a population-based study, the prevalence increased from 0.2% in those aged 70 to 79 to 6% in those over 80 . SecondaAlthough several potential mechanisms of idiopathic NPH have been proposed, no apparent cause has been identified. Several studies have found a strong correlation between decreased CSF absorption and a higher incidence of idiopathic NPH. In up to 50% of cases, arachnoid thickening is one of the autopsy findings in some patients with NPH . In one Very little is known about the presentation of NPH as bulbar symptoms, such as speech difficulties and swallowing problems. Adams noted in 1975 that a few of the patients who had the classic triad of NPH also had speech difficulties ranging from difficulty articulating or whispering until it eventually became nonexistent . They alThere are two proposed pathological reasons for bulbar dysfunction in NPH. The first one is the hypoperfusion surrounding the Sylvian fissure region, which is the site of the laryngeal motor cortex and swallowing. The perisylvian cortical hypoperfusion can be explained by Sylvian cistern dilatation, which is a typical observation in idiopathic NPH. The second cause is periventricular white matter damage (PVWM). This region has both corticospinal tract (CST) and CBT adjacent to the lateral ventricle. Hence, dilation of the ventricle stretches these fibers and has a direct impact on CBT. CBT can be skewed by either larger ventricles or decreased cerebral blood flow (CBF) . MomjianNPH is commonly identified and treated based on clinical symptoms, CSF dynamics, and brain imaging. Given the invasive nature of the treatment for NPH and the high failure rate, it is generally recommended that an additional test be performed to determine the likelihood that the patient will respond to surgery . A trialWhen doing a lumbar puncture or a lumbar drain trial, it makes sense to do routine diagnostic tests on the CSF, such as a cell count, a protein test, and a glucose test. In the majority of cases of idiopathic NPH, these test results are expected to be normal, although a mild elevation of protein in isolation is a common nonspecific finding . A ventrThis case report highlights the unusual occurrence of bulbar symptoms as part of the NPH's presentation. As a clinician, one should be aware that NPH can manifest itself in a variety of ways other than the classic triad of ataxia, memory impairment, and urinary incontinence. It must be distinguished from dementia, stroke, and other brain pathologies that frequently appear with bulbar involvement in this age group. As NPH is reversible and has a good prognosis, prompt identification and treatment are critical for patient survival.


In this study, we performed a bibliometric analysis on the metadata of literature acquired through the Scopus database (24 October 2022) with keyword combination “Laccase” AND “Pollutant” OR “Wastewater”. The included publications were filtered based on year of publication (1978–2022), types of articles  and language (English). The metadata was then exported in a CSV (.csv) file and visualized on VosViewer software. A total of 1865 publications were identified, 90.9% of which were original research articles and the remaining 9.1% were review articles. Most of the authors were from China  and India . In the case of subject area, ‘Environmental Science’ emerged with the highest published documents . The identified papers mostly cover laccase activity in degrading pollutants, and chitosan, which can be exploited for the immobilization. We encourage more research on laccase-assisted wastewater treatment, especially in terms of collaborations among organizations.Increasing trends of environmental pollution and emerging contaminants from anthropogenic activities have urged researchers to develop innovative strategies in wastewater management, including those using the biocatalyst laccase (EC 1.10.3.2). Laccase works effectively against a variety of substrates ranging from phenolic to non-phenolic compounds which only require molecular oxygen to be later reduced to H The challenge to achieve a sustainable planet has become more difficult with the increase of population resulting in increased hazardous contaminant in water environments ,2. Moreo2+ to Cu+ nucleus of this enzyme. Laccase was first introduced as an unspecific enzyme observed in Rhus vernicifera sap in Japan [Trametes versicolor is a popular source of enzyme with high catalytic degrading activity against dying agent contaminants [Pleurotus ostreatus [Trichoderma asperellum, Stemphylium lucomagnoense, and Aspergillus nidulans [The mechanism of laccase in degrading organic pollutants is dependent on redox reactions involving molecular oxygen and the reduction of Cuin Japan , and itsin Japan . Researcin Japan ,10,11. Saminants . Other tstreatus ,14 and mnidulans ) have benidulans ,17,18.P. ostreatus mutant [Bacillus licheniformis mutant [Rapid progress in genetic modifications have allowed researchers to employ these as strategies to obtain more efficacious laccase. The use of metagenomic data collected from tannery wastewater to obtain thermo-halotolerant laccase has been reported . UV lighs mutant . Mutatios mutant . Furthers mutant ,23,24. Our work aimed to provide the research landscape of laccase-assisted wastewater treatment based on bibliometric analysis. By utilizing the metadata of published studies obtained from a scientific database , a bibliometric analysis could be performed to map the research trends, especially those from newly emerging fields. The occurrence and incidence of environmental pollution have been analysed using bibliometric analysis against different contexts  ,26. In oThis study employed the Scopus database for literature search reporting laccase utilization in wastewater treatment. The keywords used in the Scopus database search were as follows: (“Laccase” AND (“Pollutant” OR “Wastewater”)) without constrains set in the publication year. The search resulted in 2073 papers, and was then filtered to include only finalized publications . Only papers written in English were included. Thereafter, we obtained 1865 papers included in this study. The workflow diagram of the literature search performed in this study has been presented in https://www.bibliometrix.org/, accessed on 17 December 2022). Furthermore, VosViewer 1.6.17. was used to conduct the bibliometric analysis such as the co-authorship/co-occurrence analysis and to generate data visualization.Microsoft Excel 2016 was used to analyze the selected papers and to export the graphs and tables needed to present the paper type, source journals, top 10 organizations or countries and the most cited papers. Data were double-checked using Biblioshiny  and review articles  regarding laccase and pollutant/wastewater were identified through a Scopus database search with the publication year ranging from 1978 to 2022. A majority of the identified papers  were published in the past decade (2012–2022), showing the increase of the study of lacasse with regard to its use in dealing with pollutants or wastewater , Biochemistry, Genetics and Molecular Biology , and Chemical Engineering  were the most studied subject areas among papers related to laccase and pollutants or wastewater  was the leading journal publishing the most papers, followed by the Journal of Hazardous Materials  and Chemosphere . These three journals were also the only ones to publish more than 50 papers, contributing to 15.65% of total papers published , Jiangnan University , and Tianjin University  were revealed as the three most productive organizations, as well as the only organizations with more than 90 published papers , India  and Spain  combined , the Natural Sciences and Engineering Research Council of Canada , and the European Regional Development Fund   .Biodegradation aspects of Polycyclic Aromatic Hydrocarbons (PAHs): A review, authored by Haritash, A.K. and Kaushik, C.P. in 2009, which was cited in 2072 papers, almost twice as many as Technologies for the removal of phenol from fluid streams: A short review of recent developments, written by Busca, G., et al. in 2008, cited in 1046 papers. There were only three papers cited more than 1000 times. The trend in citations continued to rise across the years, and peaked in 2022 with 11249 citations. The data of the top 10 most cited papers have been presented . The most cited paper was resented .A co-authorship analysis was conducted to evaluate the relationship among authors, organizations and countries according to the number of papers they published together. This analysis indicates the trends of collaborations and identifies the leading authors, organizations, and countries in terms of the related study, which in this case was laccase and pollutant/wastewater. Using VosViewer software, a visualization map will be generated which will also show nodes of various sizes representing the author, organization, or country. These nodes will also be connected by a line called Link Strength (LS), where thickness determines the link strength corresponding to the number of co-authored papers and how strong the collaboration is between the two authors, organizations, or countries. All links of a node will be accumulated as Total Link Strength (TLS), representing how strong the author, organization, or country is connected to others .To analyze the authors’ co-authorship, the minimum number of papers per author was set to three and the minimum times of citations of an author was set to 0. Of 5573 identified authors, 689 met the thresholds. The network visualization and overlay visualization maps are shown in Meanwhile, an organizational’ co-authorship analysis was conducted on 4165 identified organizations, with the minimum number of papers per organization set to three and the minimum times of citation of an organization was set to 0. Of 4165 organizations, 97 met the thresholds, however only 11 organizations were found to be linked to each other. The network visualization and overlay visualization maps are shown in Finally, countries’ co-authorship analysis was conducted on 89 identified countries, with the minimum number of papers per country set to three and the minimum times of citations of a country set to 0. The results of this analysis have been presented in Keyword co-occurrence analysis is a visualization approach to indicate how frequent and high the connected terms or words used in papers are related to a specific field of research. This analysis is also able to identify the development trend of the research focus, generally grouping the keywords into clusters. Using VosViewer software, each node indicates the keyword, while the link line connecting the nodes indicate the keyword relationship and their use on a paper. Both nodes and link lines are varied in size, showing the popularity of the keyword.For this study, a total of 3777 keywords were identified, with the minimum number of occurrences of a keyword set to 10. Of 3777 keywords, 100 met the threshold. The most used main keyword in the study on laccase and pollutant/wastewater was laccase (occurrences = 753), while the other included keywords were biodegradation (occurrences = 189), decolorization (occurrences = 165), bioremediation (occurrences = 141) and immobilization (occurrences = 102). These five keywords were also the only five keywords to pass the 100 occurrences-mark. The network visualization shown in The density visualization was altered to show the keyword change trends across the past 20 years, as the default range (1978–2022) did not show any significant change over the keywords use b. The anDensity visualization of the keywords that occurred in laccase-assisted wastewater treatment research has been presented in 2+/Cu+) to produce a phenolic radical. This initiates a long chain reaction which eventually induces the cleavage of the organic contaminant and the reduction of molecular O2 into H2O [Laccases (EC 1.10.3.2), a member of the multicopper oxidases family, have been reported for their efficacy in removing phenolic or non-phenolic contaminating agents which can be obtained through an isolation from plants, bacterial, or fungal species . Despiteinto H2O . In terminto H2O ,44,45,46into H2O .Bioresearch Technology published the highest number of papers about laccase and wastewater or pollutants (5.79% of 1865 papers), followed by the Journal of Hazardous Materials (5.09% of the papers). Govindwar, S.P. published the most number of papers and also recorded the highest number of citations. An author co-authorship analysis showed that Iqbal, H.M.N. and Bilal, M. had the highest number of collaborations between each other.A bibliometric analysis was performed on the 1865 identified papers reporting the utility of laccase in pollutant or wastewater treatment. This study aimed to provide a thorough assessment of the study status according to the source journals, leading organizations, countries and the collaborations between each other. The organizations publishing papers on laccase-assisted wastewater treatment were centred in China and India, with Shivaji University (India), Jiangnan University (China), and Tianjin University (China) producing the most papers compared to other institutions. China published the largest number of papers (22.3% of papers), almost twice as many as India (14.79% of papers). These two countries were also the only ones who managed to pass the 10% mark. China’s dominance may occur due to its large funding sources, one of them being the National Natural Science Foundation of China, which was also the largest funding source with regard to papers about laccase and pollutants or wastewater. The aforementioned funding source contributed to 13.67% of 1865 papers, followed by the Natural Sciences and Engineering Research Council of Canada, which contributed funding to 2.57% of papers. Of the 10 top funding sources, both Asia and Europe had three funding sources, while the rest were two funding sources from both North America and South America. The number of collaborations remained an issue among organizations, with only 0.26% of 4165 organizations having collaborations.Between 1978 and 2022, publications on laccase and pollutant or wastewater reached a total of 1865, of which 90.9% were research articles and the remaining 9.1% were review articles. The total number of identified papers is still significantly smaller compared to the 14,057 total papers that studied laccase . This shows the dominance of experimental work on laccase and pollutants or wastewater. However, the top papers found in this present study (indicated by the number of citations) were mostly review papers. Whilst most papers specifically discussed laccase activity in degrading pollutants, a paper authored by Rodríguez Couto and Toca Herrera highlighted chitin and chitosan characterizations . We alsoT. versicolor and could also perform biosorption [A visual networking analysis of the keywords on papers published from 1978 revealed that biodegradation has the highest density, followed by immobilization, decolorization, and biosorption. This suggests that the research is dominated by the utility of non-isolated laccase for the wastewater treatment ,56,57. Asorption ,62,63.To get an insight on the constrains of laccase-assisted wastewater treatment, we performed a literature search on review articles using the same previously used keyword combination, but the search was refined to only include those published between 2018 and 2023. Challenges and recommended topics for future research have been presented in Being the first study to research the bibliometric analysis on the study of laccase and pollutants or wastewater trends, this study used VosViewer to analyse the inputted data searched from the Scopus database, offering more accountability and objectivity. It is worth mentioning that we did not include book chapters, conference paper/reviews, editorial notes, erratum, letters, short surveys, or retracted documents. This study was also based on the Scopus database, and did not include other databases such as Web of Science (WoS). We also found difficulties in the visual networking analysis, including the incomplete name of affiliation in the visualization, an issue which cannot be resolved by the user manually.Bioresource Technology. Environmental Science was the most reported subject area, indicating the urgency of laccase use on wastewater treatment. Laccase immobilization is on the research spotlight, where the high occurrence of the keyword ‘chitosan’ has been found. This research, however, was still lacking in terms of collaboration between organizations, which needed to be strengthened to further ensure the betterment in terms of treating wastewater or pollutants.Laccase utilization in degrading pollutants is mostly studied by scientists in China (22.3%) and India (14.8%). The leading journal publishing research about laccase utilization in wastewater treatment was There are also several recommendations for the bibliometric analysis. Despite being user friendly, the visualization using VosViewer software restricts the user to editing the name in the presentation. In our case, we found the affiliation being presented with an incomplete name, and there was no option for manual editing. There should be a tool to analyze the quality of the paper rather than being based solely on the quantity. More importantly, the bibliometric analysis should be validated with a different approach, and in our case we used Biblioshiny.


Mycobacterium tuberculosis has been shown to be responsible for 12.5-59% of all infections; Pneumocystis jirovecii has been responsible for 20% of all non-viral infections; and Legionella pneumophila infections occur at 13-21 times the rate of the general population. This review will outline the mechanism of immune modulation caused by TNF inhibitors and how they predispose to infection with a focus on Mycobacterium tuberculosis, Legionella pneumophila, and Pneumocystis jirovecii. This review will then explore and evaluate how other immunomodulators and host-directed treatments influence these infections and the severity of the resulting infection to mitigate or treat TNF inhibitor-associated infections alongside antibiotics.Immunomodulators such as tumour necrosis factor (TNF) inhibitors are used to treat autoimmune conditions by reducing the magnitude of the innate immune response. Dampened innate responses pose an increased risk of new infections by opportunistic pathogens and reactivation of pre-existing latent infections. The alteration in immune response predisposes to increased severity of infections. TNF inhibitors are used to treat autoimmune conditions such as rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, transplant recipients, and inflammatory bowel disease. The efficacies of immunomodulators are shown to be varied, even among those that target the same pathways. Monoclonal antibody-based TNF inhibitors have been shown to induce stronger immunosuppression when compared to their receptor-based counterparts. The variability in activity also translates to differences in risk for infection, moreover, parallel, or sequential use of immunosuppressive drugs and corticosteroids makes it difficult to accurately attribute the risk of infection to a single immunomodulatory drug. Among recipients of TNF inhibitors,  Immunomodulators are compounds that can be used to either suppress or enhance the production or activity of immune factors such as antibodies, cytokines, chemokines, and immune cells. Some examples of these are tumour necrosis factor (TNF) antagonists, corticosteroids, interleukin (IL) supplements, and cytokine antagonists. Immunomodulators have found applications in the treatment of various autoimmune disorders, often targeting specific aspects of the immune system while not directly influencing others to minimize collateral damage. The altered immune response caused by immunomodulators may increase susceptibility to infectious diseases, particularly those caused by commonly encountered respiratory pathogens . The outAmong immunomodulators, TNF inhibitors are widely used as treatment for a variety of conditions involving hyperactive immune disorders such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease (IBD). Corticosteroids are often prescribed for the same diseases as TNF inhibitors; however, short courses are favored due to long-term consequences associated with corticosteroid administration. Compared to corticosteroids, TNF inhibitors have a lower risk profile, while also being more efficacious . ConsequThe purpose of this review is to summarize the differences between TNF inhibitors and therapeutic immunomodulation on predisposition to pneumonia caused by select intracellular pathogens. Then, potential host-directed treatments that could mitigate or treat TNF inhibitor-associated infections alongside antibiotics will be reviewed with a focus on metformin.Metformin will be discussed because of its status as an FDA-approved drug, its lower risk profile (including infection risk) with and without diabetes, evidence suggesting its potential as immunotherapy in the context of infections, and evidence of it ameliorating conditions treated with TNF inhibitors, making it an attractive drug for study . While tMycobacterium tuberculosis, Legionella pneumophila, and Pneumocystis jirovecii.In addition to drugs like metformin, cytokines can be supplemented or neutralized to control infections by manipulating the immune response in a way that is less permissive to pathogen growth. Some of these potential interventions will be discussed based on cytokine profiles and drug interventions from studies examining the host immune response in individuals, animal models, and cells infected with Mycobacterium spp., Legionella spp., and Pneumocystis jirovecii due to the high prevalence of latent infections and wide geographical distribution. The infectious risks of TNF inhibitors are further increased in regions of the world where these pathogens are endemic, particularly among persons from countries or regions of highly prevalent TB , with infliximab being a chimeric antibody with a murine variable region and human Fc region, and the latter two being fully-humanized IgG mAbs . EtanercSeveral studies found that the binding affinity and neutralization ability of different TNF inhibitors are comparable. However, the biophysical properties do not fully explain the differences seen in their resultant infection risks . DespiteVariabilities in efficacy and safety of TNF inhibitors may be in part due to their half-lives and the effects that are maintained upon cleavage. TNF inhibitors are susceptible to cleavage by proteases. Biancheri et al. reported that infliximab and adalimumab are degraded into an Fab and an Fc region, allowing for them to continue to neutralize TNF-α with their Fab regions. Certolizumab is unique in that it has no effector functions due to the absence of an Fc domain, making neutralization its sole function . In contThe previous discussion looked at TNF-α as a ligand, but TNF-α may act as a receptor when an agonist binds to tmTNF-α. This binding results in outside to inside signaling, inducing apoptosis of that cell . In contUltimately, differences in the structure, biochemistry, and biophysics impact the interactions made between TNF inhibitors and their physiological targets, dictating the resultant effects of each agent.In this section the mechanisms by which TNF inhibitors and other immunomodulators predispose or protect individuals from infection by select respiratory pathogens will be explored. The immunomodulators discussed in this section will include TNF-α (and TNF inhibitors), metformin, and various cytokines/cytokine mimics, and cytokine antagonists.M. tuberculosis infections can manifest across a spectrum of disease spanning from asymptomatic latency to severe pneumonia that can prove lethal if left untreated. Unlike traditional bacteria, members of the Mycobacterium genus have a unique cell wall composition that includes mycolic acid, arabinoglycan, and a modified muramic acid in addition to the traditional cell wall constituents such as N-acetyl glucosamine   . In the o 12.5%) . Despiteo 12.5%) . While gM. tuberculosis infection by interfering with granuloma formation  in mice when provided as an adjuvant by facilitating phagolysosome fusion  with those that were administered rifampicin without metformin. While it was found that METRIF did not improve sputum culture conversion, it did reduce chest cavities and inflammatory markers . Similarc effect . The dosc effect . Contrare fusion . The dife fusion . Heo et e fusion .Notably, later phases of metformin treatment may induce activation of macrophage activity, facilitating mROS production and phagocytosis . FurtherM. tuberculosis-induced sequelae, but Lee et al. believe that it is unsuitable as a general adjuvant in people affected by diabetes mellitus  over Th2-dominated responses   . By chalenhanced . This clenhanced . Taken tLegionella is the causative agent of Pontiac fever, a self-limiting flu-like illness; and Legionnaires’ Disease (LD), a severe form of pneumonia that may last several months and lead to mortality  were found to increase mortality while also doubling the likelihood of requiring intensive care unit (ICU) care in individuals coinfected with HIV and M. tuberculosis or HIV and P. jirovecii  their ability to keep infections under control and improve clinical outcomes, and 2) the pre-existing approval and existence of these drugs, bypassing the early stages of preclinical studies and phase 1 drug trials necessary to prove their safety and quickening the pace of transitioning into adjuvants. A majority of studies on Legionella focus on Legionella pneumophila, which is considered the most virulent Legionella and thus will be the topic of focus in this section.Despite most cyclines . The lacL. pneumophila serogroup 1 infections, at 13-21 times more likely than the general population  and a monocyte cell line (THP-1)  . When su (THP-1) . Multiplgionella . Moreovenhibited . Taken tL. pneumophila infection and even improve prognosis when administered prophylactically, decreasing bacterial load in a dose-dependent manner, but not when administered post-infection . The LCV protects L. pneumophila from the immune system and allows it to avoid cellular defenses, while exploiting the host cell’s functions   in individuals using TNF inhibitors in general  . In cont general . This ch general . Severalhibitors . A Spaninfection . Howeverd States . The difd States . The difpresence .P. jirovecii-infected rats improved clearance. While it was concluded that M2 polarization may be more suitable in P. jirovecii infection, the experiments involved adoptive transfer of M2 macrophages into rats 8 weeks post-infection instead of inducing differentiation with cytokine supplementation  and reactive nitrogen species (RNS) production (Currently, there are no studies looking at the effects of metformin on oduction . M1’s inoduction . It may oduction . HoweverP. jirovecii-induced sequelae (β-glucan’s ability to trigger a hyperactive immune response has been attributed to NF-κB overstimulation . In addisequelae .P. jirovecii infection must be further studied, as it is unclear what interactions may occur in the host and how the resultant interactions affect the infection. However, if metformin induces further killing, it is expected that β-glucan release upon fungal death can lead to further complications that may also be suppressed by metformin’s immunosuppressive effects.The effects of metformin on severe P. jirovecii (P. jirovecii yielded no cytokines in common with Kottom et al. (P. jirovecii caused an increase in anti-inflammatory factors IL-2, IL-4, IL-10, and IL-13 mRNA (P. jirovecii as an escape mechanism. It is also unknown whether the observed cytokine expressions impact the outcomes or if they occurred as a result of the infection. Il4 and Il10 gene transcription were found to be associated with worse symptoms and disease while Il13 transcription was associated with milder cases consisting of only fever (Il2 mRNA levels were found to be associated with more P. jirovecii-positive cases than P. jirovecii-negative cases. These positive cases also experienced milder symptoms. IL-4, IL-10, and IL-13 are known to induce a Th2-dominated response consisting of B cell activation, phagocyte deactivation, and anti-inflammation (Several genes were found by Kottom et al. to have significantly altered levels of transcription when infected with irovecii . Among tm et al. . Alshahr-13 mRNA . While tly fever . Higher ammation . This isammation . While tammation .P. jirovecii infections is, both proinflammatory and anti-inflammatory effects have been shown to be protective. IL-2 also induces B cell proliferation, allowing for antibody-mediated neutralization of glucans to reduce the magnitude of glucan-induced cytokine release syndrome (P. jirovecii infection.While traditionally considered to be a proinflammatory marker, more recent findings have shown IL-2 to promote Treg development, paradoxically suppressing the immune system . While isyndrome . We propM. tuberculosis, L. pneumophila, and P. jirovecii but more research is needed. The summarized pathways and proposed interventions can be found in The drugs and proposed treatments covered in this section are all host-targeted treatments that modulate the innate immune system with an emphasis on macrophage activation or deactivation, with the exception of metformin, which has paradoxical effects as both an immunosuppressor and immunostimulant . The stuP. jirovecii is present in a significant portion of the world’s population as latent infections, making them a concern when prescribing TNF inhibitor therapy. In the case of M. tuberculosis, the fact that trials are conducted in high-income countries with low incidence of TB infection obfuscates the risk evaluation when the risks are higher in lower-income countries.TNF inhibitors increase susceptibility to infections, especially those attributed to intracellular pulmonary pathogens. The spectrum of associated causative agents for pneumonia and relative risk of infections varies and is often confounded by parallel or sequential immunomodulatory agents administered. Each subclass has distinct characteristics and associated risks that seem to be influenced by their binding partners and the nature of the interaction. The mAb-based TNF inhibitors can be used to treat and modify the course of inflammatory conditions but cause a heightened risk of infection compared to the TNF-R-based counterpart. Evaluation of the patients’ comorbidities and risk for infection are critical to guide the choice of alternate immune modulators and determine the need to screen for latent infections as well as follow-up after initiation of therapy. P. jirovecii infection and TNF inhibitors in general. While immunosuppression is associated with increased infection risk, it can be used to ameliorate infection-induced cytokine release syndromes such as those caused by SARS-CoV-2 or Staphylococcus aureus (The three pathogens covered in this review primarily infect macrophages, but the pathogeneses are dissimilar, leading to different risks that must be considered when administering host-directed therapies. While this must be approached with caution, it appears to be beneficial to prescribe additional immunomodulators as adjuvants alongside other treatments provided to individuals who are immunosuppressed or immunocompromised. Additionally, like antibiotics, the responses induced by immunomodulators are not universally applicable. There exists potential to exacerbate diseases, as was seen by the proinflammatory M1 macrophage response in s aureus .Narrow therapeutic window and the potential to induce an overreactive immune response or severely crippled immune system dictate cautious consideration of this form of therapy. Within the context of adjuvants used alongside TNF inhibitor treatment, these proposed treatments are meant to aid in infections without triggering an onset of severe inflammation. Outside of individuals using TNF inhibitor treatment there may be room to adjust dosages to modulate the rate and magnitude of inflammation as needed. No previous studies looking at the effects of the combined use of multiple immunomodulators in an attempt to attenuate the risk of infection or damage caused by infection were found in our literature search. Therefore, it is an area that will benefit from further study.The clearance of all the pathogens that were covered in this review appear to benefit from IFN-γ supplementation. This indicates that while the viability of immunotherapy varies, select immunomodulators may find broad spectrum use, much like antibiotics. Some immunomodulators, particularly cytokine mimics, may even be able to supplement the loss of specific immune cells by replacing the key cytokines produced by the lost cell type. However, this would have limited applications in the loss of cells that directly induce cytotoxic effects such as granulocytes, macrophages, cytotoxic T cells, and natural killer cells. Nandakumar et al.’s study is of particular interest because it showed potential for activated immune cell transplant as a potential therapy for infections that may even extend to individuals who are immunocompromised, addressing the issue of insufficient immune cell counts .Of the immunomodulators covered in this review, metformin appears to be of particular interest. Several studies, as reviewed by Malik et al., have shown the protective effects offered by metformin against intracellular respiratory pathogens, both viral and bacterial, that were not mentioned within this review .Because of the difficulty in conducting studies looking at infections in individuals taking immunomodulatory agents, monitoring tools should be used to aid in both treatment and further studies. By keeping descriptive records of infections among individuals receiving long-term treatments in a surveillance or reporting program, retrospective analyses are more easily conducted, and less prominent unintended effects of drugs can be made clearer. Several of these programs have been used among the studies that have been covered in this review, but many others exist. Among these are REDCap, a web-based service that can be used to build surveys and clinical databases; SECURE-IBD, a surveillance tool for tracking COVID-19 cases in persons living with IBD; AERS, the adverse event reporting system from the US FDA; and RATIO, a French registry collecting data on infections occurring in individuals who use TNF inhibitors. Diligent usage of these tools with special attention to incidence, clinical presentation, and severity of infections, both nationally and internationally will allow better estimation of the risks associated with these immune modulators. Additionally, it also reduces the burden of having to recruit study subjects, allowing for faster data collection and larger sample sizes. This can be especially useful for stage 4 clinical trials and the early stages of pandemics to identify risks or therapeutic candidates. However, while there are many such tools that exist for these uses, the lack of a standard program results in cases being scattered across multiple databases, posing an issue if inconsistencies exist in the types of data that can be inputted.Overall, TNF inhibitors pose significant risks in infection, with TNF-R mimics posing relatively lower risks compared to α-TNF mAb’s. While etanercept appears to have to impose the lowest infection risk among TNF inhibitors, but as outlined by Gerriets et al., each drug has varying effectiveness against each condition, making it harder to reduce infections in individuals with conditions that are unable to be treated with lower infection risk agents . While eYK, ZR, and RH defined the review question and the topics to be reviewed. RH draft the manuscript. YK and ZR revised the paper critically for important intellectual content. All authors approved the final version to be published.This manuscript was also supported, in part, by the Canada Research Chairs Program for Dr. Rueda, and through the master’s scholarship for RH. Award number: 323473.The authors would like to express their gratitude to Dr. Keith Fowke for reviewing the manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Porcine circovirus type 2 (PCV2) is a highly prevalent virus in pig farms worldwide that causes significant economic losses in the swine industry. The PCV2 virus-like particles (VLPs) are potent subunit vaccines that are widely used. Currently, the adopted quality control of VLPs vaccines is mainly based in animal testing, the titration of neutralizing antibodies, or other biochemical/biophysical assays. In this study, we generated a monoclonal antibody that can distinguish assembled PCV2 VLPs from the capsid proteins. Subsequently, a convenient Sandwich ELISA was developed based on the monoclonal antibody (mAb) that recognizes the PCV2 VLPs specifically. This assay can be used for the quantity and quality control of PCV2 VLPs vaccines for both the intermediate or final products with high accuracy. Porcine circovirus type 2 (PCV2) is a highly prevalent pathogen that causes immune suppression in pigs, which results in enormous economic losses in the swine industry ,2,3,4. TThe assembled virions or virus-like particles determine the potency of vaccines . For insThe potency of vaccines is mainly evaluated by immunization and virus challenges in animals. However, these experiments require a large number of animals and a long time and have highly variable deviations ,13,14. MFreund’s complete adjuvant, Freund’s incomplete adjuvant, hypoxanthine/aminopteri n/thymidine, hypoxanthine/thymidine, and PEG3000 were purchased from Sigma-Aldrich . RPMI 1640 and fetal bovine serum were purchased from Thermo Fisher Scientific . BABL/c mice were obtained from Vital Rivea Experimental Animal Technology . The animal experiments were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute, and the methods were conducted in accordance with the approved animal ethics guidelines. The Animal Ethics Committee approval number was 211123-03.w/v) of each processed tissue sample suspension, and incubated at 37 °C for 4–5 days. Each sample was subjected to blind passages. PCR further confirmed the presence of PCV2 in the cell culture, and the isolate from the 9th and 15th passages was sequenced.In 2019, lungs and lymph nodes from 10-week-old piglets which were suspected of PCV2 infection were subjected to PCR tests. All of the PCV2-positive samples were further used for virus isolation. Briefly, PK15 cells were washed with 0.01 M PBS of pH 7.4, infected with twenty percent (PCV2 ORF2 gene was codon-optimized and synthesized by Genscript Corporation and ligated into the expression vector pET28a. The recombinant vector pET28a-PCV2-ORF2 was transformed into BL21(DE3) competent cells. Bacterias were selected on the plate containing kanamycin and culture in TB medium containing 50 PCV2 VLPs were purified as previously described . BrieflyThe purified VLPs were used as immunogen emulsified with Freund’s complete adjuvant for the first immunity and Freund’s incomplete adjuvant for the second and third immunity. Five female Balb/c mice were used via intraperitoneal injection to produce mAb. The immunized mice were euthanized, and spleen cells were fused with SP2/0 cells according to the standard procedure . The hybHRP conjugation to mAb was performed according to the following steps . The freThe purified PCV2 VLPs were used as standard control to develop sandwich ELISA. The concentration of PCV2 VLPs was determined by the Bradford method with the BCA kit .For the forced degradation by heat, the antigen reagent was incubated in a water bath at 80 °C for 15 min, 48 h, and 64 h. For the forced degradation by denaturing agent, the antigen reagent was subjected to the treatment of urea overnight at 4 °C. These treated samples were stored at 4 °C, and the concentrations were measured by the Bradford method with the BCA kit.Different concentrations of PCV2 VLPs and forced degraded VLPs were coated in ELISA plates and blocked by 5% fat-free milk in PBS. The purified mAbs were added and followed by HRP goat anti-mouse IgG antibody  for 30 min. TMB was used as an enzyme substrate, and 2 M HCl was added to stop the reaction. A spectrophotometer was used to measure the optical density (OD).The purified mAb 3H9 was used as the capture antibody, and the HRP-labeled mAb 2G8 as the detective antibody. The coating concentration of the capture antibody and the dilution of HRP-labeled 2G8 were optimized. The blocking buffer and blocking time were optimized. Two-fold serial dilutions of the antigen samples and stander reference were added to the coated plates for 1h. TMB was used as an enzyme substrate, and the reaction time for TMB was optimized.Electrophoresis was carried out with 15% polyacrylamide gels, and the gels were then stained with Coomassie stain. Western blot detection of Cap protein was performed by the same procedure without staining, and the proteins were transferred to a PVDF transfer membrane  at 120 V for 60 min. The membrane was blocked in 5% fat-free milk in PBS and then incubated with mAbs for 1h and subsequently with HRP goat anti-mouse IgG antibody  at room temperature for 1 h.PK-15 cells and PCV2 were seeded in 96 plants for 48 h before being washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Samples were permeabilized with 0.5% triton x-100 in PBS for 10 min and washed twice. The purified mAbs were added to the cells for 1 h at 37 °C, followed by FITC-conjugated goat anti-mouse antibody for 1 h at 37 °C. Cells were rinsed three times in PBS for 5 min, and imaging was performed by a fluorescent microscope in the dark.The data were analyzed by GraphPad Prism8.3.0 , and the results were presented as mean ± SD.PCV2 VLPs were used as an antigen to generate mAbs. Two positive hybrid cell lines, 3H9 and 2G8, with higher ascitic titers, were screened and purified. SDS-PAGE analysis was performed to assess the purity of the antibodies, and the results showed a 55-kDa band and a 25-kDa band, corresponding to the molecular weight of the heavy chain and the light chain, respectively A. FurtheNext, degradation experiments were performed to select mAbs capable of distinguishing the assembled PCV2 VLPs from the cap protein. PCV2 VLPs treated with heat and denaturant urea were tested in ELISA, with untreated samples as a control. The results showed that only the diluted assembled PCV2 VLPs reacted specifically with 3H9 C, suggesFor the development of a sandwich ELISA, we selected mAb 3H9 as the capture antibody and HRP-labeled 2G8 as a detection antibody. The experimental conditions were optimized as follows: the optimal coating amount of the capture antibody mAb 3H9 was 312.5 ng/well, and the optimal dilution of HRP-labeled 2G8 was 1:3200. The optimal blocking time was 2 h at 37 °C with 3% BSA in PBS. The optimal incubation time for the antigen was 1 h. The optimum reaction time for TMB was 15 min. The purified VLPs were tested by SDS-PAGE and TEM analysis A,B.The optimized sandwich ELISA assay analyzed the standard PCV2 VLPs references with two-fold dilutions. The standard curves were generated as shown in = 0.9941 D. The deTo evaluate the accuracy of the sandwich ELISA method, three quality-control samples with concentrations of 40, 10, and 2 ectively A, indicaectively , demonstTo assess whether this sandwich ELISA method was able to distinguish the assembled PCV2 VLPs from linearized or other proteins, we performed degradation experiments with PCV2 VLPs antigen treatment with heat or urea and quantified to five concentrations of dilution. Subsequently, the samples were analyzed with the sandwich ELISA method. The results showed that antigens were undetectable by the sandwich ELISA method after heat and denaturant urea treatments B, suggesWe analyzed the crude, purified, and final products of PCV2 VLPs with the sandwich ELISA and SDS-PAGE to assess whether the sandwich ELISA method works with intermediate and final productions. Crude PCV2 VLPs samples, including the total bacterial lysate, the separated supernatant, and precipitation, were assayed by grayscale analysis and ELISA. The SDS-PAGE results are shown in At present, vaccine immunization is the most effective method to prevent immunosuppressive disease caused by PCV2 and to improve the production traits of growing pigs . The potIn summary, we have demonstrated that the sandwich ELISA could be used to measure the purity and quality of the PCV2 VLPs vaccine. The bacterial proteins have no significant impact on the detection of PCV2 VLPs. Our ELISA could also serve as an alternative to the time-consuming and costly immunogenicity studies to evaluate the potency of inactivated PCV2 vaccine.In conclusion, we generated a mAb that recognizes the assembled PCV2 VLPs instead of the Cap protein. In addition, a Sandwich ELISA was developed based on the mAb. This assay can be used for the quality control of PCV2 VLPs vaccines.