We aimed to evaluate the diagnostic value of soluble interleukin-2 receptor (sIL-2R), tumor necrosis factor-α (TNF-α), procalcitonin (PCT), and combined detection for sepsis infection in patients with closed abdominal injury complicated with severe multiple abdominal injuries.n = 70) and an infection group (n = 70).One hundred forty patients with closed abdominal injury complicated with severe multiple abdominal injuries who were diagnosed and treated from 2015 to 2020 were divided into a sepsis group (p < 0.05). The receiver operating characteristic (ROC) curve showed that the areas under the ROC curve (AUCs) of sIL-2R, TNF-α, PCT and sIL-2R+TNF-a+PCT were 0.827, 0.781, 0.821, and 0.846, respectively, which were higher than those of white blood cells , C-reactive protein , serum amyloid A , and IL-6 (0.735). The AUC of the three combined tests was higher than that of TNF-α, and the difference was statistically significant (p < 0.05). There was no significant difference in the AUCs of sIL-2R and TNF-α, sIL-2R and PCT, TNF-α and PCT, the three combined tests and sIL-2R, and the three combined tests and PCT (p > 0.05). When the median was used as the cut point, the corrected sIL-2R, TNF-α, and PCT of the high-level group were not better than those of the low-level group (p > 0.05). When the four groups were classified by using quantile as the cut point, the OR risk values of high levels of TNF-α and PCT (Q4) and the low level of PCT (Q1) after correction were 7.991 and 21.76, respectively, with statistical significance (p < 0.05).The levels of sIL-2R, TNF-α, and PCT in the sepsis group were higher than those in the infection group (The detection of sIL-2R, TNF-α, and PCT has good value in the diagnosis of sepsis infection in patients with closed abdominal injury complicated with severe multiple abdominal injuries. The high concentrations of PCT and TNF-α can be used as predictors of the risk of septic infection. Closed abdominal injury with severe multiple traumas is characterized by complex and hidden conditions, and most patients are accompanied by injuries to the brain, chest, and limbs . The proSepsis is a systemic inflammatory response syndrome caused by severe infection. The main pathogens of sepsis are bacteria, followed by fungi, viruses, and parasites . Sepsis Soluble interleukin 2 receptor (sIL-2R) is a nonspecific indicator produced by lymphocytes under conditions such as malignant tumors and infection, which can reflect diseases related to lymphocyte activation and is also a marker of the activation of the immune system . Tumor nWe retrospectively analyzed 140 patients with closed abdominal injury complicated with severe multiple abdominal injuries treated in the Fifth People’s Hospital of Jiaozuo City, The First Affiliated Hospital of Henan University of Technology, the First People’s Hospital of Yancheng City, and the Tinghu People’s Hospital of Yancheng City from 2015 to 2020, including 70 patients with sepsis (sepsis group from intensive care units) and 70 patients without sepsis but with local inflammatory infection . All the medical records were confirmed by clinical symptoms, B-ultrasound, X-ray films, and laboratory, among others. The locations of abdominal injuries were the pancreas in 13 cases, the duodenum in 15 cases, colon in 32 cases, liver in 13 cases, small intestine in 19 cases, and the spleen in 48 cases; 98 cases had multiple organ injuries and 42 cases had single organ injuries. The diagnosis of sepsis was based on the Third International Consensus Diagnostic Criteria for Sepsis and Septic Shock 3.0, published in 2016.9/L or <4 × 109/L. The criteria for the sepsis group were in accordance with the signs in the infection group and a SOFA score ≥2.The infection group showed two or more of the following signs: body temperature, >38°C or <36°C; heart rate, >90 bpm; respiratory rate of >20 times/min or arterial blood carbon dioxide partial pressure of <32 mmHg; and peripheral blood leukocytes, >12 × 10The exclusion criteria were as follows: patients with solid cancer, hematologic disease, organ transplantation, with missing clinical and laboratory data, treated with antibiotics, and patients with immune deficiency.The baseline clinical data of 140 enrolled patients were collected from medical records, which included age, gender, sIL-2R, TNF-α, PCT, white blood cells (WBC), C-reactive protein (CRP), and serum amyloid A (SAA), IL-6, as well as Acute Physiology and Chronic Health Assessment (APACHE II). The included detection indexes were based on the serum samples collected for the first time within 24 h of a patient’s visit. SAA was detected by kinetics nephelometry. sIL-2R, TNF-α, and IL-6 were detected with ELISA. PCT was detected using immunofluorescence chromatography. All specimens were enrolled after obtaining informed consent from the patients or their family. Written informed consent from the participants was obtained. The study was approved by the ethics committees of the Fifth People’s Hospital of Jiaozuo City .t-test. Numeration data were analyzed using the χ2 test. Variables with a non-normal distribution were expressed as median (P25 and P75) and were compared using the non-parametric Mann–Whitney U test. GraphPad Prism software was used to generate the receiver operating characteristic (ROC) curves of each index and its combined index in order to determine the sensitivity, specificity, optimal cutoff value, Youden index, negative predictive value (NPV), and the positive predictive value (PPV) of each index in patients infected with sepsis. The area under the ROC curve (AUC) was used to judge the accuracy of the test. The combined predictors of sIL-2R, TNF-α, and PCT were calculated with binary logistic regression analysis. The AUCs were compared between each index using Z-test. Spearman’s rank correlation coefficient was used to analyze the correlation between the levels of IL-2R, TNF-α, PCT and other laboratory parameters and APACHE II. Binary logistic regression analysis was applied to evaluate the risk predictive value of the levels of sIL-2R, TNF-α, and PCT for sepsis using median (P50) and quartiles  as cut points, respectively. It was also used to calculate the values of the single-factor and multifactor adjusted odds ratios (AORs) and 95% confidence intervals (CI) based on maximum likelihood estimates. A p < 0.05 was considered statistically significant.Statistical analyses were performed with SPSS 25.0 software . Continuous data were presented as means ± standard deviations. Measurement data between the two groups were conducted using independent samples p > 0.05). The scores for WBC, CRP, SAA, IL-6, APACHE II, and SOFA in the sepsis group were all higher than those in the infection group, with statistical significance (p < 0.05).Z = −6.668, p < 0.001), as shown in Z = −5.728, p < 0.001), as shown in Z = −6.560, p < 0.001), as shown in The levels of sIL-2R in the sepsis group were higher than those in the infection group, and the difference was statistically significant . There was no correlation between the level of sIL-2R and WBC . The levels of sIL-2R in the sepsis group were positively correlated with WBC, CRP, SAA, IL-6, and APACHE II . No significant correlation between the level of sIL-2R and the SOFA score was observed for patients with sepsis , while it was not correlated with WBC, CRP, SAA, APACHE II, and the SOFA score . The TNF-α level was positively correlated with WBC, SAA, and IL-6 in the sepsis group , but there was no correlation between the level of TNF-α and CRP, APACHE II, and the SOFA score , as shown in The TNF-α level in the infected group was positively correlated with IL-6 , while it was not correlated with WBC, SAA, IL-6, APACHE II, and the SOFA score . The PCT level in the sepsis group was positively correlated with IL-6 , while it was not correlated with WBC, CRP, SAA, APACHE II, and the SOFA score , which is shown in The PCT level in the infected group was positively correlated with CRP  was used as the dependent variable; sIL-2R(X1), TNF-α(X2), and PCT(X3) were used as the independent variables. The joint predictors of sIL-2R, TNF-α, and PCT were calculated using binary logistic regression analysis, and the regression equation was 9/L, the sensitivity and specificity were 65.71% and 74.29% and the NPV and PPV were 68.4% and 71.9%, respectively. For CRP detection, when the AUC is 0.766 and the cutoff value is 85.47 mg/L, the sensitivity and specificity were 75.71% and 72.86% and NPV and PPV are 75.0% and 73.6%, respectively. When the AUC is 0.666 and the cutoff value is 123.21 mg/L, the sensitivity and specificity were 51.43% and 84.29% and the NPV and PPV are 63.4% and 76.6%, respectively. IL-6 detection with an AUC of 0.735 and a cutoff value of 9.9 pg/ml showed sensitivity and specificity values of 88.57% and 50.00% and NPV and PPV of 81.4% and 63.9%, respectively. When the AUC of the SOFA score is 1.000 and the cutoff value is 1, the sensitivity and specificity were 100% and 100% and the NPV and PPV were 100% and 100%, respectively. For sIL-2R detection, an AUC of 0.827 and a cutoff value of 1384 U/ml produced sensitivity and specificity values of 70.00% and 88.57% and NPV and PPV of 74.7% and 86.0%, respectively. When the AUC of TNF-α detection is 0.781 and the cutoff value is 14.00 pg/ml, the sensitivity and specificity were 80.00% and 68.57% and the NPV and PPV were 77.4% and 71.8%, respectively. For PCT, when the AUC is 0.821 and the cutoff value is 4.35 ng/ml, the sensitivity and specificity were 68.57% and 91.43% and the NPV and PPV were 74.4% and 88.9%, respectively. The sensitivity and specificity were 70.00% and 95.71% and the NPV and PPV were 76.1% and 94.2%, respectively, for sIL-2R+TNF-α+PCT when the AUC is 0.846 and the cutoff value is 0.70, which are shown in GraphPad Prism was used to plot the ROC curves of each index and the combined test, as shown in p > 0.05). The AUC of the combined test was greater than that of TNF-α, and the difference was statistically significant (p < 0.05), as shown in According to the data in p < 0.05), and the adjusted OR was 0.489 . Compared with low TNF-α, the risk of sepsis in patients with high TNF-α levels was 7.205 , and the adjusted OR was 1.624 . Compared with low PCT, the risk of sepsis in patients with high PCT was 8.346 , and the adjusted OR was 2.300 , which can be seen in Binary logistic regression analysis was used to evaluate the risk predictive value of the levels of sIL-2R, TNF-α, and PCT in the sepsis group. The median cutoff point (two classifications) and the quartiles  were evaluated as cutoff points (four classifications). Firstly, patients were divided into a low-level group and a high-level group according to the median values of sIL-2R , TNF-α (18.95 pg/ml), and PCT (1.815 ng/ml). Compared with low sIL-2R, the risk of sepsis in patients with high sIL-2R was 11.391 , TNF-α , PCT , the patients were divided into the Q1, Q2, Q3, and Q4 groups from low to high levels. Compared to the group with the lowest sIL-2R levels (Q1), the ORs of sepsis risk in the Q2, Q3, and Q4 groups were 1.000 (0.328–3.052), 6.469 (2.256–18.548), and 26.156 (7.083–96.593), respectively. The ORs after correction were 0.854 (0.224–3.253), 0.403 (0.070–2.335), and 0.681 (0.074–6.262), respectively. Compared to the group with the lowest TNF-α levels (Q1), the ORs of sepsis risk in the Q2, Q3, and Q4 groups were 2.087 (0.707–6.165), 5.333 (1.839–15.471), and 31.000 (8.195–117.272), respectively. The ORs after correction were 1.098 (0.275–4.387), 0.836 (0.193–3.617), and 7.991 (1.274–50.108), respectively. Compared to the group with the lowest PCT levels (Q1), the ORs of sepsis risk in the Q2, Q3, and Q4 groups were 1.000 (0.342–2.921), 3.059 (1.117–8.373), and 98.222 (11.694–824.999), respectively. The corrected ORs were 1.013 (0.260–3.949), 0.916 (0.213–3.942), and 21.760 (2.095–226.008), as shown in As an important complication of closed abdominal injury and severe multiple abdominal injuries, sepsis is a complex disease caused by the body’s dysfunctional response to infection and is associated with acute organ dysfunction and a high risk of death , 20. SepIn this study, we analyzed the diagnostic value of sIL-2R, TNF-α, and PCT in sepsis patients. The results showed that sIL-2R, TNF-α, and PCT in the sepsis group were significantly higher than those in the infection group, which was consistent with previous studies , 12, 13 in vivo, which can regulate immune response and call immune cells to the site of infection, and interleukin presents an inflammatory response after activation of the complement pathway  and quartiles  as cutoff points (four classifications). The results showed that the corrected sIL-2R, TNF-α, and PCT in the high-level group was not superior to those in the low-level group when the median cut point was used for the classification of the two groups. When the four groups were classified using quantiles as cut points, the OR risk values of the high levels of TNF-α and PCT (Q4) and the low level of PCT (Q1) after correction were 7.991 and 21.76, respectively, the difference being statistically significant. There was no significant difference between the other groups and the low-level group (Q1). The results showed that when PCT ≥ 6.67 and TNF-α ≥ 39.4, they could be used as predictors of the risk of sepsis.The detection of sIL-2R, TNF-α, and PCT in patients with sepsis has good value for the diagnosis of sepsis infection in patients with closed abdominal injury complicated with severe multiple abdominal injuries. High concentrations of PCT and TNF-α can be used as predictors of the risk of septic infection.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the ethics committees of the Fifth People’s Hospital of Jiaozuo City . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JW, WZ, and ZX contributed to the study concept and design, acquisition of data, analysis and interpretation of data, and drafting of the manuscript. W-wW and G-hZ contributed to statistical analysis. L-zH contributed to sample collections. A-qS and G-hZ contributed to the study concept and design, study supervision, and critical revision of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the National Natural Science Foundation of China (no. 81802084) and Jiaozuo science and technology project (no. 2020162).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.


Tumor necrosis factor (TNF) and its closely related cytokine, lymphotoxin alpha (LTα), are part of the TNF superfamily and exert their functions via both overlapping and non-redundant signaling pathways. Reported pro- and antitumorigenic effects of TNF and lymphotoxin are often context-dependent and may be contingent on a particular experimental approach, such as transplantable and chemically induced tumor models; tissue and organ specificity; types of cells producing these cytokines or responding to them; and the genotype and genetic background of mice. Here, we review the mechanisms of TNF/LTα involvement in cancer promotion and suppression as studied in mouse models. We also discuss the impact of microbiota on tumor development and manipulations of the TNF/LT system, which may be effective as anti-cancer therapy.Tumor necrosis factor (TNF) and lymphotoxin alpha (LTα) are two related cytokines from the TNF superfamily, yet they mediate their functions in soluble and membrane-bound forms via overlapping, as well as distinct, molecular pathways. Their genes are encoded within the major histocompatibility complex class III cluster in close proximity to each other. TNF is involved in host defense, maintenance of lymphoid tissues, regulation of cell death and survival, and antiviral and antibacterial responses. LTα, known for some time as TNFβ, has pleiotropic functions including control of lymphoid tissue development and homeostasis cross talk between lymphocytes and their environment, as well as lymphoid tissue neogenesis with formation of lymphoid follicles outside the lymph nodes. Along with their homeostatic functions, deregulation of these two cytokines may be associated with initiation and progression of chronic inflammation, autoimmunity, and tumorigenesis. In this review, we summarize the current state of knowledge concerning TNF/LTα functions in tumor promotion and suppression, with the focus on the recently uncovered significance of host–microbiota interplay in cancer development that may explain some earlier controversial results. Tumor necrosis factor (TNF) and lymphotoxin alpha (LTα) exist as soluble homotrimers and interact with TNF receptors (p55 TNFR1 and p75 TNFR2). LTα homotrimer also binds to herpes virus entry mediator (HVEM) as does LIGHT TNFSF14), another member of the TNF superfamily . In addi, anotherThe history of TNF is closely related to the history of cancer immunotherapy. William Coley was the first to use endotoxin-induced antitumor activity to treat inoperable sarcomas . In the Lung cancer is the most commonly diagnosed and deadly type of cancer. The influence of endogenous TNF on lung metastasis has mostly been investigated in mouse models using transplantable tumor cell lines . In an eTNF-mediated signaling on tumor cells was also implicated in the process. Knocking down TNFR2 in Lewis lung carcinoma cells combined with a low dose of recombinant mouse TNF significantly inhibited carcinoma growth in WT mice, suggesting that TNF/TNFR2 signaling in tumor cells is pro-tumorigenic in this transplantable tumor model . In lineRegarding the role of LTα, a growing amount of evidence implicates lymphotoxin in antitumor activity. For instance, inoculation of B16F10 melanoma cells into LTα-deficient mice resulted in accelerated lung metastasis as compared with littermate control mice, presumably, due to impaired natural killer (NK) cell migration to the lungs . MoreverAs already discussed, systemic TNF neutralization is a standard treatment for such autoimmune diseases as rheumatoid arthritis, psoriasis, and inflammatory bowel disease (IBD). However, one of the profound side effects of this therapy is the increased risk of skin cancer development, especially non-melanoma skin cancer ,50,51. EIt should be noted that, in these earlier studies, co-housing and/or littermate control mice were not always used, making it difficult to exclude a possible impact of microbiota variation on the inflammatory response. On the other hand, more recent experiments in a TPA/DMBA two-step skin carcinogenesis model suggested that the difference in tumor load between TNF-deficient and co-housed littermate control mice may not be as dramatic as previously reported and is microbiota-dependent A,B. The In turn, the role of lymphotoxin in the development of skin cancer has also remained somewhat controversial, primarily, due to the complexity of LT signaling . For ins+ cells. Both LTβR- and RORγt+ LTα-deficient mice developed more tumors as compared with co-housed littermate control mice -induced skin inflammation was used. The expression of IL-23, an IL-22-inducing cytokine , CXCL1, Hepatocellular carcinoma (HCC) often follows cirrhosis-driven inflammation of the liver  and may + myeloid cells was associated with enhanced hepatic tumor progression, including such antitumor effects of myeloid-cell-derived TNF as direct tumor cell apoptosis and a reduced expression of immunosuppressive molecules like TGF-β, IL-10, iNOS, IL-33, and heme oxygenase-1 [Colorectal cancer (CRC) is the third most frequently diagnosed cancer and the fourth principal cause of cancer-related deaths worldwide . There igenase-1 . More imgenase-1 . Howevergenase-1 . These cgenase-1 .Previous studies demonstrated a pro-tumorigenic effect of TNF in a mouse model of chemically induced colorectal cancer . In partIn turn, the role of lymphotoxin in the development of CRC is insufficiently studied, although the data available suggest that LTβR signaling can perform protective functions. In particular, intraperitoneal injections of anti-human LTβR agonistic monoclonal antibodies every 14 days resulted in reduced tumor growth in athymic nude mice subcutaneously inoculated with WiDr human colon adenocarcinoma, whereas a single injection of the anti-mouse LTβR agonistic monoclonal antibody caused tumor necrosis in BALB/c mice with CT26 colon carcinoma .Hematopoietic malignancies, such as leukemia, myeloma, and lymphoma, comprise eight to ten percent of all human malignancies. In the context of normal hematopoiesis, TNF was described as a T-lymphocyte differentiation factor , inhibitLeukemia derives from the bone marrow and is characterized by an overproduction of abnormal immune cells whose differentiation was arrested at a certain stage. The fitness of the abnormal cell pool depends on TNF, which regulates programmed cell death via TNFR1. TNF-deficient mice were partially protected from irradiation-induced apoptosis of bone marrow cells, indicating that TNF-mediated signal transduction is crucial for bone marrow cell death . LIGHT–LMultiple myeloma (MM) originates in the bone marrow and affects plasma cells. Cancerous plasma cells produce faulty antibodies, which can damage kidneys and other organs and accumulate in the marrow. Progression of MM at early onset is driven by abnormally low levels of apoptosis, a high mitotic rate, and increased transendothelial migration of myeloma cells, partially mediated by TNF through NF-κB activation ,101. In Myc-driven lymphoma cells expressing vascular endothelial growth factor C (VEGFC) and LTα2β1 on one hand and the corresponding VEGF receptor-3 and LTβR on high endothelial venules on the other. These interactions caused vascular reprogramming, endothelial cell proliferation, and increased angiogenesis, which, in turn, resulted in reshaping of the lymphoma’s metabolism and acceleration of malignancy [Lymphoma usually starts in the lymphatic system and affects immune organs, causing lymphocyte accumulation in the spleen and lymph nodes. Lymphomas are divided into Hodgkin and non-Hodgkin lymphomas. Reed–Sternberg cells, a characteristic of Hodgkin lymphomas, are giant cells usually considered to be crippled germinal center B-cells. At the cell culture level, Reed–Sternberg-cell-derived LTα acts on endothelial cells to upregulate the expression of adhesion molecules that are important for T-cell recruitment into lesional lymph nodes in Hodgkin lymphoma , as welllignancy .The pathogenic role of TNF was recently documented for clonal hematopoiesis of indeterminate potential (CHIP), an age-related condition characterized by a pool of early blood cell progenitors with a genetically distinct subpopulation of cells bearing unique mutations in DNA. This condition is usually caused by the emergence of an inactivating mutation in Tet methylcytosine dioxygenase 2 (Tet2) and is characterized by skewed myelomonocytic differentiation. It was found that chronic TNF exposure, as an “inflammaging” marker, favors the fitness of Tet2 mutant clones via conferring TNF resistance to sensitive bone marrow mutant progenitors . TNF ove+ Tregs and for increased numbers of TNFR2+ Tregs in the peripheral blood are predictors of a poor outcome in several types of cancer [+ tumor cells and TNFR2+ Tregs with an increase in the T-effector cell fraction in experiments in vitro and ex vivo [+ Tregs. Taken together, TNFR2 targeting alone, or in combination with other treatments, presents a promising strategy for cancer therapy, as it potentially may have fewer side effects and act more selectively than checkpoint inhibitors [Accumulated data suggest that signaling through TNFR2 is important for tumor expansion since TNFR2 is indispensable to Treg cell functions  and the f cancer ,116,117.f cancer , colorecf cancer ,120, ovaf cancer , Hodgkinf cancer ,123, andf cancer ,125. Sinf cancer , overexpf cancer . For insf cancer . In the  ex vivo . Finally ex vivo . Therefohibitors ,132,133.Determining the specific contribution of lymphotoxin to carcinogenesis is complicated by the fact that LT/LTβR signaling is indispensable to lymph node formation during embryogenesis ,71. In lHelicobacter pylori on gastric cancer [H. pylori led to a disturbance in Wnt/β catenin signaling in epithelium, contributing to increased tumor transformation [−/− mice are protected from the development of colorectal cancer [−/− mice appeared to be more susceptible to tumorigenesis [−/− mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis demonstrated less severe inflammation than WT mice [−/− and WT mice before and after TNBS and sham treatment revealed that WT mice have a higher Firmicutes to Bacteroidetes ratio than TNF−/− mice [Turicibacter in WT mice was increased as compared with TNF−/− mice both prior to and after colitis induction, suggesting that TNF expression may affect the bacterial composition in the gut. Finally, a recent study showed that attenuation of colitis-associated cancer due to anti-TNF treatment is microbiota-dependent, and that co-housed mice treated with anti-TNF together with untreated control mice may cancel the protective effect of anti-TNF therapy [The interaction between the microbiota and the host may not only promote immune system development and support its homeostasis , but mayc cancer . Chronicormation . In the ormation , suggestormation . Howeverl cancer , while Tigenesis , presumaigenesis  and IBD igenesis ,151. Furigenesis , in part WT mice . Deep se−/− mice . Moreove therapy .Saccharomyces boulardii was shown to reduce the TNF production in the AOM/DSS model and decrease the number of tumors [Published data support the idea that TNF modulates microbial composition and abundance and that microbiota can also affect TNF production. In the AOM/DSS model of colorectal cancer, probiotic introduction reduced the tumor burden via modulation of the immune response, including reduced TNF levels . Saccharf tumors . In a raf tumors . Microbif tumors . Taken t+ ILCs (innate lymphoid cells) is necessary for T-cell-dependent production of IgA by B-cells in the lamina propria, regulating homing of T-lymphocytes into the intestine, while membrane-bound LTαβ on RORγt+ ILC is involved in the induction of T-cell-independent IgA production [LTα-mediated signaling plays an extremely important role in the control of microbiota; nevertheless, the functions of soluble LTα homotrimer are difficult to distinguish from those of the membrane-bound lymphotoxin LTαβ heterotrimer. Most of the reported lymphotoxin functions are related to its membrane-bound form ,159. Howoduction . HoweverTNF plays an important role in human cancer development and progression  and may A growing body of data suggests that TNF is involved in the regulation of a tumor’s metastasizing ability. For instance, TNF is expressed at the invasive front of colon cancers . MoreoveIn turn, the LTα expression was found to be significantly higher in HNSCCs as compared with adjacent normal tissues  and may Multiple trials of cancer treatment based on TNF and its derivatives ) were carried out for melanoma and other solid tumors . Other sThe results of meta-analyses revealed several polymorphisms of TNF and LTα genes as predictive markers for malignancy progression. The TNF rs1800629 and rs1799724 polymorphisms may be linked to increased HCC development in non-Asian populations . Other sTNF and LT were discovered as cytotoxic substances and are considered to be promising agents for cancer therapy. With our understanding of the complexity of TNF and LT signaling, this initial concept has undergone significant changes. We now know that the pro- and antitumorigenic effects of both TNF and LT depend on the specific conditions, on the type of producer and responder cell, and on the microenvironment. A better understanding of these mechanisms in the existing mouse models in microbiota-controlled settings may provide us with further insights into the role of these cytokines in cancer development, but also define the conditions and targets for successful anticancer therapy.


The sera from pigs infected with virulent classical swine fever virus (CSFV) contain substantial amounts of tumor necrosis factor (TNF), a prototype proinflammatory cytokine with pleiotropic activities. TNF limits the replication of CSFV in cell culture. In order to investigate the signaling involved in the antiviral activity of TNF, we employed small-molecule inhibitors to interfere specifically with JAK/STAT and NF-κB signaling pathways in near-to-primary endothelial PEDSV.15 cells. In addition, we knocked out selected factors of the interferon (IFN) induction and signaling pathways using CRISPR/Cas9. We found that the anti-CSFV effect of TNF was sensitive to JAK/STAT inhibitors, suggesting that TNF induces IFN signaling. Accordingly, we observed that the antiviral effect of TNF was dependent on intact type I IFN signaling as PEDSV.15 cells with the disrupted type I IFN receptor lost their capacity to limit the replication of CSFV after TNF treatment. Consequently, we examined whether TNF activates the type I IFN induction pathway. With genetically modified PEDSV.15 cells deficient in functional interferon regulatory factor 1 or 3 (IRF1 or IRF3), we observed that the anti-CSFV activity exhibited by TNF was dependent on IRF1, whereas IRF3 was dispensable. This was distinct from the lipopolysaccharide (LPS)-driven antiviral effect that relied on both IRF1 and IRF3. In agreement with the requirement of IRF1 to induce TNF- and LPS-mediated antiviral effects, intact IRF1 was also essential for TNF- and LPS-mediated induction of IFN-β mRNA, while the activation of NF-κB was not dependent on IRF1. Nevertheless, NF-κB activation was essential for the TNF-mediated antiviral effect. Finally, we observed that CSFV failed to counteract the TNF-mediated induction of the IFN-β mRNA in PEDSV.15 cells, suggesting that CSFV does not interfere with IRF1-dependent signaling. In summary, we report that the proinflammatory cytokine TNF limits the replication of CSFV in PEDSV.15 cells by specific induction of an IRF1-dependent antiviral type I IFN response. The first line of protection of host cells from invading viruses is mediated by the innate immune system. By sensing unique pathogen-associated molecular patterns, conserved cellular pattern recognition receptors initiate multiple intracellular signaling cascades involving interferon regulatory factors (IRF) that culminate in the transcriptional activation and secretion of type I interferons (IFN-α and IFN-β) and type III IFN (IFN-λ) . Specifipro protein which interacts with IRF3 and induces its proteasomal degradation  , contains five copies of an NF-κB response element (NF-κB-RE) that drives the transcription of the firefly luciferase reporter gene. Briefly, 3 million PEDSV.15 cells in 0.4 mL ice-cold PBS were electroporated with 5 µg pGL4.32[luc2P/NF-κB-RE/Hygro] and 200 ng pGL4.75[hRluc/CMV] vector  reporter plasmids. The cells were seeded in 96-well plates (3 × 10BbsI restriction endonuclease sites of the plasmid pSpCas9(BB)-2A-GFP (PX458)  were used to clone annealed oligonucleotides coding for the selected gRNAs. Individual gRNAs were designed using the CHOPCHOP web tool  [Based on the publicly available mRNA sequences for porcine IFNAR1 (NCBI reference NM_213772.1), IRF3 (NM_213770.1) and IRF1 (XM_021080244.1) individual guide RNAs (gRNAs) for the targeting of corresponding genomic exons were designed. CRISPR/Cas9-based genome editing was performed essentially as described . The twoer 2020) . The gRNpro proteins were detected using the rabbit anti-IRF3 and anti-Npro sera as described previously [The cells were lysed with a denaturing lysis buffer composed of 62.5 mM Tris HCl pH 6.8), 2% sodium dodecyl sulfate (SDS), 10% glycerol and 0.05% bromophenol blue. The proteins were separated using 4–12% gradient SDS–polyacrylamide gel electrophoresis under nonreducing conditions  and analyzed by means of Western blotting using PVDF transfer membranes  and an Odyssey Infrared Imaging System . Porcine IRF3 and viral N, 2% sodieviously . Using tCSFV-infected pigs show elevated serum TNF, and TNF was shown to inhibit replication in cell lines ,16. In oThe JAK/STAT pathway is the key element of the signaling cascade engaged in response to type I IFN . In orde10 RLU values from CSFV-luc-infected cells pre-stimulated with LPS or pTNF in the presence of the individual JAK/STAT inhibitors at concentrations of 0.5 µM or 5 µM, respectively. Altogether, we observed a general and strong correlation between the capacities of the compounds to interfere with LPS- and pTNF-mediated antiviral signaling without any outliers that would reduce LPS activity without affecting pTNF activity. This suggests that pTNF induces classical antiviral JAK/STAT signaling in PEDSV.15 cells. The effect of selected well-characterized JAK/STAT inhibitors, such as fedratinib, itacitinib, neratinib, pacritinib, tofacitinib, filgotinib, baricitinib and ruxolitinib efficiently blocked both LPS- and pTNF-mediated antiviral effects, which is shown in In order to explore the roles of the type I IFN receptor and of IRF3 in antiviral IFN-β, LPS and pTNF signaling, we generated IFNAR1- and IRF3-knockout (KO) PEDSV.15 cell lines  by introducing small genetic deletions within early exons using CRISPR/Cas9 gene editing . We deteWe identified two IFNAR1-KO clones #5 and #23 carrying deletions within the IFNAR1 loci a consistFor IRF3, we identified three knockout PEDSV.15 clones with identical out-of-frame homozygous deletions of 190 nucleotides within the IRF3 open reading frame on the deduced mRNA level, leading to a frameshift mutation after the first 38 codons. The IRF3-KO clones #4 and #16 c served https://prosite.expasy.org/, accessed on 2 July 2021)  a. The clSimilar to the parent PEDSV.15 and the IRF3-KO cells see d, the IRThe induction of type I IFN depends on NF-κB, as opposed to the downstream IFN-β signaling . Therefopro through IRF3 targeting [pro inhibits the expression and nuclear translocation of IRF1, thereby suppressing the production of type III IFN [CSFV antagonizes the induction of type I IFN by means of Nargeting . In addi III IFN . Therefo III IFN , the IRFThe induction of high levels of proinflammatory cytokines including TNF is a hallmark of severe and hemorrhagic CSF following infection with highly pathogenic CSFV . SeveralFirst, we observed that different cell types responded differently to TNF. While TNF induced an antiviral state in the endothelial cell line PEDSV.15 and in the porcine MDM, TNF did not inhibit CSFV replication in the SK-6 and PK-15 cells a. This wNext, we dissected the TNF–IRF1–IFN-β signaling axis in the context of a CSFV infection of PEDSV.15 cells. More specifically, we showed that TNF, similarly to LPS, induces the expression of IFN-β transcripts by activating IRF1 and NF-κB independently. Thereby, TNF triggers type I IFN receptor-dependent JAK/STAT signaling leading to decreased CSFV replication. Synergistic antiviral effects between TNF and IFNs are known to enhance antiviral responses (reviewed in ). AlthouBesides IRF1, NF-κB is also required for the TNF-mediated antiviral effect on CSFV . Li et apro was expressed (pro [pro of CSFV inhibits IRF1 expression and nuclear translocation in porcine intestinal epithelial IPEC-J2 cells, thereby suppressing type III IFN production [Interestingly, CSFV did not interfere with TNF- or LPS-triggered IFN-β mRNA induction  that botxpressed . These rsed (pro  while thoduction . This laSeveral reports suggest altogether that the antiviral activity of TNF involves NF-κB- and IRF1-dependent signaling and type I IFN responses. In order to test this formally for CSFV, we targeted NF-κB, IRF1, IRF3 and IFNAR1-dependent JAK/STAT signaling pharmacologically or by CRISPR/Cas9-mediated gene knockout. The anti-CSFV activity of porcine and murine TNF was inhibited by antibody-mediated TNF neutralization, NF-κB and JAK/STAT inhibitors and was abrogated completely in the IRF1 and IFNAR gene knockout cells but not in the IRF3 gene knockout cells. IRF1 gene knockout prevented TNF- and LPS-mediated IFN-β mRNA induction. Interestingly, CSFV did not counteract TNF- or LPS-mediated IFN-β mRNA induction. This is consistent with CSFV targeting IRF3 for proteasomal degradation  but is i


The institution that the second author, Ursula Neumann, is affiliated with has updated their name. The correct affiliation is: Fraunhofer IIS, Fraunhofer Institute for Integrated Circuits IIS, Division Supply Chain Services, Group Data Science.


Jellyfish, marketed and consumed as food in The Far East, are traditionally processed using salt and alum mixtures. In recent years, the interest of Western consumers in jellyfish (JF) as a food source is increasing. In Europe [European Union (EU)], JF-derived food products are regulated by a novel food law, but methods for JF treatment and processing have not been developed yet. In this study, a protocol for the stabilization and processing of JF into semi-finished food products without the use of alum is proposed for the first time. Safety and quality parameters, together with a series of technological and nutritional traits, were used to monitor the proposed process and for the characterization of the JF-derived products. Calcium lactate (E327), calcium citrate (E333), and calcium acetate (E263), which are food thickening/stabilizing agents allowed by EU regulations, were used in order to control the presence of possible microbial pathogens and spoilage species. The use of calcium lactate and citrate led to an increase in texture values  and in several nutritional traits such as antioxidant activity, and protein and fatty acid content. In particular, the combination of JF treatments with calcium salts and phenolic compounds resulted in an antioxidant activity increase of up to 8-fold, protein concentration increase of up to 2.6-fold, fatty acid composition maintenance, and a ω6/ω3 ratio lower than 1. For the first time, the application of phenolic compounds to improve JF technological and nutritional features was verified. This study proposes a new procedure for JF treatment and stabilization useful for future potential food applications in Western countries. The presence of jellyfish (JF) in various marine environments seems to be increasing, mainly in response to the ongoing climate change and reduction in the number of predators. The abnormal increase in JF populations, named “blooms,” can be problematic for fishing operations, aquaculture plants, and other anthropic activities in coastal areas, mainly for small-scale fishery .JF are considered a traditional food in Asian countries, such as China, Japan, Korea, and Thailand, which host most JF consumers and producers . Althoug42O or AlNH4 , automation has been performed to reduce process time, and different organoleptic and sensorial  traits have been developed in response to buyers and emerging markets. Before consumption, salt-preserved and semi-dried JF products are partially rehydrated by soaking in water several times. These products are generally served in salads and soups, together with sauces , since they have little or no flavor. Recently, ready-to-eat or ready-to-use JF products are marketed to be consumed without soaking, accompanied by sauces .In 2015, the United States Department of Agriculture (USDA) considered edible JF similar to vegetables, such as broccoli and carrots, and included them as a natural diet food. Rehydrated JF show content of 92–96% water, 3–7% protein, and negligible carbohydrate, fat, and cholesterol levels , 12. TheEpidemiological investigations revealed that aluminum can cause memory impairment and cognitive dysfunctions, which could lead to neurodegenerative diseases such as Alzheimer's and Parkinson's disease –19. FurtCatostylus tagi JF for the preparation of a new ready-to heat snack was also proposed  to form chelate rings  (40°24'36.8“N 16°53'04.0”E) with a 3-5 cm mesh nylon fishing net, during samplings in 2018 and 2019 summer period. Alive JF were temporarily stored at 10–15°C immersed in seawater in food-grade containers and transported to the laboratory within 3 h after collection. In the laboratory, the JF were immersed in sterile seawater (SW), and the umbrella and oral arms were separated with a plastic knife to remove the content of the digestive cavity. The umbrella and oral arms were washed three times with sterile seawater  and then submitted to treatment in newly formulated brines with firming agents or stored in sterile plastic bags at −80°C.Vibrio spp. incubated at 37°C for 18–24 h; Bacillus ChromoSelect Agar  added with polymyxin B supplement for the enumeration of Bacillus spp. incubated at 30°C for 24–48 h; iron agar (Lyngby) without cysteine  for the detection and enumeration of hydrogen sulfide-producing bacteria incubated at 25°C for 48 h; Pseudomonas agar  added with CFC supplement  for the enumeration of Pseudomonas spp. incubated at 35°C for 24–48 h. The enumeration of yeast and molds were performed by incubation at 25°C for 5 days on Dichloran Rose-Bengal Chloramphenicol Agar .Microbiological analyses on JF tissue were performed following the procedure already described by Bleve et al. , with so2, 0.54% MgSO4, 0.1% CaCl2, w/v)   was addewww.technelysium.com.au) and the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) for sequence alignment, and compared with the sequences in the GenBank database (Release 233).The bacterial total genomic deoxyribonucleic acid was extracted using the Power Soil DNA Isolation Kit  following the protocol of the manufacturer. The 16S rDNA region was amplified according to Bleve et al. . The ampBrine treatments. The brines were formulated using solutions of an alpha-hydroxy organic acid calcium salt. Three different types of brines were formulated as described below, calcium citrate, calcium lactate, and calcium acetate, all from Sigma-Aldrich .The solutions of calcium citrate, lactate, and acetate were formulated using 0.1 M calcium citrate solution, 0.1 M calcium lactate hydrate solution, and 0.1 M calcium acetate solution, and the pH was adjusted to 5 using the corresponding alpha organic acid 1 M citric acid, 1 M lactic acid 85% (v/v), and 0.1 M glacial acetic acid solutions, all from Sigma-Aldrich . The JF , immediately after sampling, were extensively washed with sterile seawater (hereafter named JFSW) and used for brine treatments. In order to obtain homogeneous and comparable samples, JF specimens were dissected as described in Leone et al. . BrieflyBrine and phenol treatments. A set of JF samples was processed for 10 days in brines with the procedure described above and further treated with phenol compounds, specifically, ferulic acid  or rutin hydrate . Solutions containing each phenolic compound were prepared at the final concentration of 2 mg/g (0.2% w/w of FW) of treated JF  in steriSalinity, pH, and texture were evaluated at days 0, 1, 5, and 10 of brine treatment, and at 10 days + 1 day of incubation in the samples treated with either a ferulic acid- or rutin-containing solution.2 by operating on samples consisting of radial triangular slices of the JF umbrella. Due to the different thickness of R. pulmo umbrella, data were the means of three measurements on three regions of umbrella, such as the inner, medium, and external parts. At least three-quarters of five different JF specimens were evaluated. Firmness values were measured for each time point  and expressed as Newton (N).Salinity was measured by using a salinity refractometer for seawater and marine aquaria 0–10% hydrometer with automatic temperature compensation RHS-MR110 ATC . The texFresh JF samples were homogenized in a Waring® laboratory blender (three pulses for 15 s under refrigerated conditions). Approximately 2 g of each homogenized sample was suspended in 2, 4, or 8 ml of MilliQ water in order to obtain a homogeneous suspension.Badfrod assay  was usedThe antioxidant activity was evaluated in each JF sample by the Trolox Equivalent Antioxidant Capacity (TEAC) method, adapted for the Infinite 200 PRO microplate reader  using the radical cation ABTS•+ and Trolox  as standard , 50. The3) according to Leone et al. . The samples were evaporated under a stream of nitrogen and dissolved in 50 μl of hexane, and 1 μl was analyzed by gas chromatography-mass spectrometry (GC-MS). GC–MS analyses were performed using an AGILENT 5977E gas chromatograph . Separation of compounds was performed on a VF-WAXms . GC parameters were as follows: the column temperature was maintained at 160°C for 1 min, programmed at 4°C/min to 240°C for 30 min. Helium was used as a carrier gas at a constant flow rate of 1 ml/min. A mass spectrometer was operated in electron impact mode with a scan range of 50–700 m/z. The temperature of MS source and quadrupole was set at 230 and 150°C. Analyses were performed in full-scan mode. Compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic standards  analyzed under the same conditions. MS fragmentation patterns were compared with those of pure compounds, and a mass spectrum database search was performed using the National Institute of Standards and Technology (NIST) MS 98 spectral database. Fatty acid composition was expressed as a percentage of the total fatty acids in each sample, while the total lipid content was expressed both as yield of lipid extraction in 100 g of fresh JF sample (mg/100 g FW) and as a percentage of dry weight (% DW).Total lipids were extracted using the modified method of Bligh and Dyer , with soe et al. , with son = 3). Statistical analysis was based on one-way analysis of variance. Tukey's post hoc method was applied to establish significant differences among the means . All statistical comparisons were performed using Sigma-Stat, version 3.11 .All data represent the mean of three independent replicates .The JF samples washed with sterile seawater, representing a mixed combination of umbrellas and oral arms, revealed the presence of bacterial pathogens belonging to staphylococci and d number . This evBacillus spp. were relatively low , whereas negligible counts of hydrogen sulfide-producing bacteria (such as Pseudomonas spp. and possibly Shewanella putrefacens/Aereomonas hydrophila), of coli-aerogenes bacteria and of yeasts were reported. The possible presence of halophilic/halotolerant microorganisms associated with the JFSW samples was tested on saline media . Both bacteria and yeasts/molds were detected with some differences derived from the medium used. Marine agar and R2A seemed more permissive than the classic media  added with sea salts for yeasts and molds  and thawing the samples previously treated with calcium salts, allowed for the achievement of further reduction in their microbial load, and kept the quality and sensory characteristics comparable to those of the samples before being frozen and thawed (data not shown).Vibrio spp., Bacillus spp., coagulase-positive staphylococci, and yeasts. After the treatment with both ferulic acid and rutin, the texture was maintained or increased slightly compared with the values of the same samples treated with the three brines on day 10  contained about 302 mg of proteins per 100 g of fresh weight .The antioxidant activity (AA) was evaluated in the JF samples and expressed as nmol of Trolox equivalent for g of fresh weight  and the ones treated with rutin after the addition of calcium acetate brine (Ca-Ac 10 d + 1d R). However, a high AA was also found in the samples incubated with calcium acetate and then with ferulic acid (Ca-Ac 10 d + 1d Fa).trans-10) was registered, as the only MUFA present, which is the product of the reaction of oleic acid skeletal isomerization reaction. Total MUFA content generally decreased in all the brine-treated JF samples, except for the samples treated with Ca-Ac and Ca-L for 5 days. In this last sample, MUFA species such as palmitoleic acid (C16:1) and vaccenic acid (C18:1 cis-11) were preserved. Furthermore, total PUFA content was reduced with almost all the treatments, except for the JF samples treated with Ca-L for 10 days, probably due to a high value of AA as described above (The fatty acid (FA) composition was expressed as a percentage and was estimated in raw JF samples washed with sterile seawater (day 0) and in all the JF samples treated with the three calcium salts on days 1, 5, and 10 of treatment . In the ed above . ConsequAs shown in This study aimed to set up a new procedure to stabilize and process JF biomass, thus developing a new treatment of JF for food uses, substantially different from the traditional methods used in Asian countries. The main phases proposed for JF preparation are shown in Salmonella and Listeria were not considered, since they were absent in all previously analyzed JF samples  were isolated from Nemopilema nomurai  derived from plants and/or by-products of the food industry as additional stabilizing agents is a very promising strategy for producing healthier JF products, and promoting a circular economy model.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.FR, SD, and GB conduction of the experiments, acquisition, and interpretation of the results. GB and AL work design, data discussion, and manuscript preparation. All authors contributed to the article and approved the submitted version.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.


Virus-like particles (VLPs) are self-assembled viral protein complexes that mimic the native virus structure without being infectious. VLPs, similarly to wild type viruses, are able to efficiently target and activate dendritic cells (DCs) triggering the B and T cell immunities. Therefore, VLPs hold great promise for the development of effective and affordable vaccines in infectious diseases and cancers. Vaccine formulations based on VLPs, compared to other nanoparticles, have the advantage of incorporating multiple antigens derived from different proteins. Moreover, such antigens can be functionalized by chemical modifications without affecting the structural conformation or the antigenicity. This review summarizes the current status of preventive and therapeutic VLP-based vaccines developed against human oncoviruses as well as cancers. The expression of viral proteins in non-human cells, their purification, and auto-assembling into hollow virus-like particles (VLPs) lacking viral genomes, represent an effective and safe strategy to mimic the native virus structure and morphology .+ T helper as well as CD8+ cytotoxic T cells . In. In+  the lymph node microenvironment . The epier cells ,39. Two Therapeutic VLP-based vaccines are designed to stimulate the adaptive immune system against specific tumor antigens in order to induce tumor regression . AntibodIn the last 40 years, VLPs have been generated as preventive vaccine strategies for several viruses involved in the pathogenesis of human cancers, ultimately resulting in preventing cancer development. Of these, only two have received approval for human use by regulatory agencies, namely HBV and HPV VLPs. All other VLP models for the remaining oncogenic viruses are still in preclinical evaluation stage .Saccharomyces cerevisiae and auto-assembling of the protein in 20 nm particles characterized by octahedral symmetric structures. The latter are safer and more immunogenic than blood-derived particles and are the most widely used today. The most recent vaccine formulation against HBV is the Sci-B-Vac, containing the S, pre-S1, and pre-S2 antigens, which are produced in mammalian Chinese hamster ovary (CHO) cells [Pichia pastoris cell-free synthesis of HBV proteins [HBV is an hepatotropic virus associated with chronic inflammation, progressive fibrosis, cirrhosis, and hepatocellular carcinoma . The firO) cells . The proO) cells . The HepO) cells . New strO) cells . The hypO) cells . Lastly,proteins , showingproteins ,56.Human papillomaviruses are non-enveloped DNA viruses representing the most common genital viral contagions. The majority of HPV infections are usually cleared in a variable period of time, ranging from a few months up to 2 years, without any intervention. However, a small percentage of infections with high-risk HPV genotypes become persistent and can cause cancer at different body sites, such as the cervix, anus, vulva, vagina, penis, or oropharynx .in vitro and the development of new models to study the assembly and structures of VLPs [VLPs for HPV were initially developed in the early 1990s, expressing viral proteins from the outer layer of the HPV virus . Since t of VLPs ,59,60. Two vaccines against HPV, derived from the viral capsid protein L1 and are named Cervarix and Gardasil, have been approved by the FDA for their ability to prevent HPV-infections and related cervical cancers . CervariE. coli, which assembled in virus-like particles assuming a pentameric structure [More recently, the structure of other oncoviruses have been characterized, including the polyomavirus major capsid protein VP1, expressed in tructure ,68. Moretructure ,70,71. Structure ,73. Lasttructure . ProgresHuman herpesvirus type 8 (HHV-8) or Kaposi sarcoma-associated herpes virus (KSHV), is a gamma herpesvirus that infects endothelial cells, epithelial cells, B-cells, and macrophages. The virus is able to promote B-cell proliferation, production of angiogenic cytokines, and VEGF expression, as well as impair the host immune system by escaping the cytotoxic immune response, the antigen presentation, and T cell activation. In particular, infected endothelial cells undergo extensive proliferation and neoplastic transformation. HHV-8 is the primary cause of classical and epidemic Kaposi’s sarcoma (KS), multicentric Castleman′s disease, primary effusion lymphoma, and a rare form of B-cell lymphoma ,85. The Only a few studies to date have been devoted toward developing a prophylactic vaccine against HHV-8, given that the incidence of HHV-8-associated tumors have been dramatically reduced by the efficacy of anti-retroviral treatment in HIV infection. Moreover, the lack of animal models for KS development represents a strong limitation in evaluating the VLP efficacy ,87. The HHV-8 glycoproteins gpK8.1, gB, and gH/gL are responsible for the virus attachment, to target cells and to elicit neutralizing antibodies. These glycoproteins have been individually fused to the Newcastle disease virus (NDV) capsid proteins and the transfection of chimeric HHV-8-NDV recombinant constructs in CHO cells yielded successful production of VLPs. The gpK8.1, gB, and gH/gL VLPs resembled the morphology of the native HHV-8 virus and were shown to bind the HMC-1 cell line, susceptible to HHV-8 infection .Preclinical studies have shown that the VLPs were able to induce neutralizing antibodies in mice and that immunization with a combined formulation of gB and gH/gL VLPs induced the best neutralizing antibody response. Moreover, the administration of the gpK8.1 VLP vaccine with gpK8.1-gB, gpK8.1-gH/gL, or gpK8.1-gB-gH/gL VLPs induced an immune response that was similar to that obtained with UV-inactivated HHV-8 . More recently, a multivalent VLP-based vaccine, incorporating four HHV-8 envelope glycoproteins, was produced by cloning a polycistronic sequence containing the gpK8.1, gB, gL, and gH glycoproteins interspersed with a picornavirus 2A self-cleaving peptide under the CMV promoter in a single plasmid . Co-tranThe human T-lymphotropic virus type 1 (HTLV-1) was the first oncogenic retrovirus to be discovered in humans . The HTLin vitro without the use of any adjuvant [Epstein–Barr virus (EBV), the first oncogenic virus identified in humans, is a gammaherpesvirus infecting epithelial and B cells. EBV principally causes mononucleosis and has been associated with diverse cancer types, such as lymphomas, gastric carcinoma, and nasopharyngeal carcinoma, diagnosed either in immune-competent or immune-compromised patients . The devadjuvant . In addiadjuvant . The gH/adjuvant . More recently, a pentavalent vaccine based on EBV glycoproteins gp350, gB, gp42, gH, and gL, exposed on the surface of VLPs, has been produced in CHO cells. This vaccine has been administered with the adjuvants aluminum hydroxide and monophosphoryl lipid A, eliciting a strong neutralizing antibody response in New Zealand white rabbits. This was shown to protect both B cells and epithelial cells from the EBV infection, better than soluble gp350 .Finally, a promising strategy is represented by a multicomponent vaccine model, in which three gp350 peptides (P1 (aa 16–29), P2 (aa 142–161), and P3 (aa 282–301) were displayed on the surface of the HBV core antigen (HBc149) in different orders. All recombinant proteins were able to self-assemble in VLPs. Among these, the HBc-149-3A (P1P2P3) and HBc-149-3B (P1P3P2) VLPs induced high titers of neutralizing antibodies against gp350 in mice and blocked EBV infection in cell culture .Hepatitis C virus (HCV) is an enveloped, positive single strand RNA virus of the Flaviviridae family, characterized by a viral life cycle fully completed in the cytoplasm. The HCV RNA genome is translated in a single polyprotein of 3011 amino acids, cleaved to produce four structural , and six non-structural  proteins ,95,96. VMerkel cell polyomavirus (MCPyV) is a non-enveloped double-stranded circular DNA virus belonging to the α-polyomavirus genus, found in approximately 80% of Merkel cell tumors . MCPyV-lVaccines capable of boosting the immune response against cancer specific antigens represent a very promising therapeutic strategy for the targeted treatment of tumors. In this respect, VLPs represent an efficient platform for the presentation of tumor-associated antigens (TAAs). This can be achieved by two experimental approaches: (1) the outer surface of VLPs can be functionalized to bind the TAAs for the optimal presentation to immune cells ; (2) the coding sequence expressing the structural viral proteins forming the VLPs is modified to generate a chimeric protein presenting the desired TAA. In the first approach, the VLPs, the peptides/proteins, and linkers are synthesized separately and then combined by cross-linking reactions to produce complex vaccine formulations. In the second one, a single recombinant protein is expressed in the cell system, retaining the ability to auto-assemble in particles, and presenting the TAA, is expressed in prokaryotic or eukaryotic cells. Breast cancer is a heterogeneous tumor characterized by a high degree of histopathological and molecular diversity between and within tumors . Due to Melanoma is the deadliest skin tumor characterized by the highest mutational burden of any cancer and numerous tumor-associated antigens, which can be used for the generation of effective therapeutic cancer vaccines . A VLP sA further model of the anti-melanoma vaccine is based on the use of cowpea mosaic virus-like particles (CPMV-VLPs), which showed promising results as an immune stimulating agent for in situ cancer vaccination . Two VLPE. Coli were able to assemble into VLPs and to slightly enhance the maturation of bone marrow-derived dendritic cells ex vivo. Notably, such VLPs induced a strong antigen-specific antitumor immunity in mice, and an innate immunity against tumors and metastasis originated from the subcutaneous implantation of B16-OVA melanoma cells [A dual-antigen vaccine strategy was developed, based on the hepatitis B virus core antigen presenting the OVA257−264 (SIINFEKL) and the gp100 (KVPRNQDWL) antigen peptides . The chima cells .Pancreatic cancer is an aggressive tumor with the worst survival rate of all cancers. The outcomes and survival benefits for pancreatic cancer patients—despite significant therapeutic developments—remain unsatisfactory, highlighting the urgent need for novel therapeutic strategies to fight this deadly disease. An immunotherapeutic approach for pancreatic cancer was developed, based on a murine Trop2 VLP approach. Indeed, the Trop2 glycoprotein is highly overexpressed on the cell surface of pancreatic cancer. Immunization of C57BL/6 mice with mTrop2 VLPs resulted in a significant reduction in tumor growth and broad activation of CD4(+) and CD8(+) T cells. In addition, the administration of Trop2 VLPs in combination with gemcitabine showed an improved survival of tumor-bearing mice . A furthHPV E6 and E7 oncoproteins are attractive targets for the development of therapeutic vaccines for HPV-associated cancers. Indeed, such proteins are tumor-specific antigens (TSAs) given that they are constitutively and specifically expressed in HPV-associated cervical cancer cells . Early cChimeric VLPs have been generated, consisting of the L1 capsid protein plus the entire E7 (11 kDa) or E2 (43 kDa) HPV protein fused to the L2 capsid protein. Efficacy was assessed in a pre-clinical setting using a preventive experimental model. C57BL/6 mice were protected from tumor growth when challenged with the HPV-E7-expressing TC-1 tumor cell line .Similarly, HPV 16 L1-based chimeric virus-like particle (cVLP) were produced in plants containing a string of T cell epitopes from HPV 16 E6 and E7 fused to its C-terminus, and their therapeutic potential and the persistence of IgG neutralizing antibodies were evaluated in a tumor model of C57BL/6 mice . The IgGAlthough approved L1 HPV-VLPs are prophylactic strategies for preventing HPV infections, as well as subsequent tumor growth, and do not express E7 epitopes, a therapeutic efficacy has been recently demonstrated . Indeed,in vivo and neutralization of HBV in vitro [The clearance of HBsAg in the blood of HBV-positive subjects could be a successful strategy for the treatment of chronic infections that are often not resolved by approved anti-HBV drugs. Indeed, high levels of HBsAg are known to inhibit adaptive, as well as innate, immune response, by leading to tolerance against HBV infection. A vaccine model has been developed to express a 13-mer peptide (SEQ13) from the HBsAg by a novel immuno-enhanced VLP carrier (CR-T3) derived from the round leaf bat HBV core antigen (RBHBcAg). The CR-T3-SEQ13 formulation, by displaying multiple copies of SEQ13, induced a potent antibody response in mice, rabbits, and cynomolgus monkeys, mediating the HBsAg clearance in vitro .in vitro, even at very low doses. For instance, the DOX-loaded VLPs killed HCC cells at IC50 values of 10–15 nM, 20 times better than free DOX [in vitro [VLPs may function as carriers (for delivery to cancer cells) of a high number of molecules, such as proteins, peptides, DNA, RNA, drugs, and imaging substances. An early study showed the expression of a targeting peptide (SP94) in the MS2 VLP to selectively deliver chemotherapeutic drugs  to human hepatocellular carcinoma cells (HCC). Such modified VLPs exhibited high avidity and specificity towards HCC with minimal uptake in healthy cells and induced selective cytotoxicity free DOX . Moreovein vitro . To study the effect of nanoparticle morphology on drug delivery, DOX was loaded on three diverse VLPs: bacteriophage MS2, tobacco mosaic virus (TMV) disks, and filamentous rods of nanophage. The MS2 is spherical in shape with a diameter around 27 nm; the TMV disks are flat and round, measuring 18 × 5 nm, and the nanophages are short filaments, measuring 50 × 6 nm. Intracranial administration of TMV disks and MS2 VLPs in U87Luc glioblastoma-bearing mice determined increased survival rates, with the TMV disk-treated mice showing the greatest efficacy . The nanOther studies have shown that particles with extended surfaces improve drug delivery due to their ability to better interact with vessel walls and to accumulate in tumor tissues. Plant viruses forming filamentous and tubular structures are good candidates for drug delivery platforms. The TMV nanotubes (300 × 18 nm in size) and filamentous PVX (515 × 13 nm) have been successful used to deliver DOX . MoreoveVLPs can be considered as alternatives to traditional vaccines based on their safety, flexibility, and distinctive immunogenic properties, but considerable limitations still persist in their production. For instance, no VLP vaccine currently used in clinical and preclinical trials has induced sterile immunity and total clearance of viral infections, perhaps because most strategies dominantly target one arm of the immune system. Production efficiency and scalability are important limits in the production of VLPs. The mammalian cells, which are the only systems to provide an intracellular environment suitable for proper protein folding and post-translational modifications of VLP-forming proteins, have a low production efficacy compared to other VLP systems. In addition, achieving efficient transfection while maintaining cell viability is necessary for successful VLP production. This requires a large number of cells to produce relatively limited amounts of VLPs . AnotherSeveral VLP-based preventive vaccines have been/are currently under development. However, only those targeting HBV and HPV have been approved for human use. All others are still at the pre-clinical stage . Regarding therapeutic approaches on VLP technology—many are in the preclinical stage and only some are in early stages of human clinical trials. The efficacy of the VLP-based vaccine is highly dependent on the selection of tumor antigens and the design of VLPs. Although there are various limitations in the development of the VLP platform, recent advances in the field, the possibility of engineering the VLPs, and the use of appropriate adjuvants are keys that could improve the design and manufacturing of VLPs for prevention and treatment of human cancers.


Furthermore, nobiletin treatment enhanced nuclear factor, erythroid 2 like 2 (NFE2L2) and heme oxygenase 1 (HMOX1) expressions in TNF-stimulated HPDLCs. In conclusion, these findings suggest that nobiletin can inhibit inflammatory responses in TNF-stimulated HPDLCs by inhibiting NF-κB and AKT1 activations and upregulating the NFE2L2 and HMOX1 expression.Nobiletin, a biologically active substance in the skin of citrus fruits, has been reported to be an effective anti-inflammatory, anticancer, and antimicrobial agent. In this study, we aimed to examine the anti-inflammatory effects of nobiletin on tumor necrosis factor- (TNF-) stimulated human periodontal ligament cells (HPDLCs). Our results demonstrated that nobiletin treatment could decrease the expressions of inflammatory cytokines (C-X-C motif chemokine ligand (CXCL)10, C-C motif chemokine ligand (CCL)2, and interleukin- (IL-) 8), matrix metalloproteinases (MMPs) (MMP1 and MMP3), and prostaglandin-endoperoxide synthase 2 (PTGS2) in TNF-stimulated HPDLCs. Moreover, we revealed that nobiletin could inhibit the activation of nuclear factor- (NF-)  The excessive immune reaction is related to periodontal tissue destruction. Tumor necrosis factor (TNF)  –3. We pr(HPDLCs) –6. Ransj(HPDLCs) . So, it C. sinensis (sweet orange), C. aurantium L. (sour orange), and C. paradisi (grapefruit). Also, citrus fruit juice contains detectable amounts (1–10 mg/g) of nobiletin [Nobiletin is a biologically active substance in the skin of citrus fruits, such as obiletin . The strobiletin , anticanmounts 1– mg/g of obiletin . For exaobiletin . Nobiletmounts 1– mg/g of We decided to perform this research as a basic study to use nobiletin for the prevention or treatment of periodontitis. In other words, we examined the effects of nobiletin on the expressions of inflammatory cytokines, MMPs, and prostaglandin-endoperoxide synthase 2 (PTGS2) in TNF-stimulated HPDLCs which are the main constituents of the periodontium. In addition, we investigated to confirm the influence of nobiletin to give the activation of the signal transduction pathways that TNF stimulation derived.α/β (#2697), phosphonuclear factor- (NF-) κB p65 (#3033), phosphoprotein kinase B (AKT1) (#4060), MAPK14 (#9212), MAPK1/MAPK3 (#4695), MAPK8 (#9252), IKK-α (#2682), NF-κB p65 (#8242), AKT1 (#2920), heme oxygenase 1 (HMOX1) (#5853), nuclear factor, erythroid 2 like 2 (NFE2L2) (#12721), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#5174) were purchased from Cell Signaling Technology . Enzyme-linked immunosorbent assay (ELISA) kits of CXCL10 , CCL2 , IL-8 , MMP1 , MMP3 , and TIMP1  were purchased from R&D Systems .Nobiletin (item number: 15421) and the antibody against PTGS2 (item number: 10112) were purchased from Cayman Chemical . Recombinant human TNF was obtained from PeproTech . Antibodies against phosphomitogen activated protein kinase (MAPK)14 (#4551), phospho-MAPK1/MAPK3 (#4370), phospho-MAPK8 (#4668), phospho-IkappaB kinase- (IKK-) μg/ml streptomycin (Invitrogen) in a 5% CO2 incubator at 37°C. HPDLCs were seeded in 24-well plates or 12-well plates at 5000 cells per cm2.HPDLCs were purchased from Lonza Japan  and cultured in Dulbecco's minimal essential medium  containing 10% fetal bovine serum , 100 U/ml penicillin, and 100 μM) and then stimulated with TNF (10 ng/ml) for 24 hours. Then, cell supernatants were collected and used for ELISA analysis. All assays were performed according to the manufacturer's protocols, and CXCL10, CCL2, IL-8, MMP1, MMP3, and TIMP1 levels were determined using the standard curve prepared for each assay. The experiments were performed in triplicate and repeated three times.The release of CXCL10, CCL2, IL-8, MMP1, MMP3, and TIMP1 in the culture medium was tested by ELISA kits. HPDLCs were seeded in a 24-well culture plate. HPDLCs were incubated with different concentrations of nobiletin  for 1 hour before the stimulation with TNF (10 ng/ml) for 15, 30, or 60 min  or 24 hours . Then, the cell supernatant was discarded and washed twice with cold phosphate-buffered saline (PBS). HPDLCs were lysed with cell lysis buffer  including protease inhibitor cocktail . Debris was removed by centrifugation at 15000 rpm for 10 min at 4°C, and the supernatants were collected. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes . The membranes were blocked with 1% skim milk for 1 hour at room temperature. Then, the membranes were incubated with the following primary antibodies at 4°C overnight: phospho-MAPK14 (1 : 1000), phospho-MAPK1/MAPK3 (1 : 2000), phospho-MAPK8 (1 : 1000), phospho-IKK-α/β(1 : 1000), phospho-NF-κB p65 (1 : 1000), phosph-AKT1 (1 : 2000), MAPK14 (1 : 1000), MAPK1/MAPK3 (1 : 2000), MAPK8 (1 : 1000), IKK-α(1 : 1000), NF-κB p65 (1 : 1000), AKT1 (1 : 2000), PTGS2 (1 : 1000), HMOX1 (1 : 1000), NFE2L2 (1 : 1000), or GAPDH (1 : 8000). The membranes were washed with Tris-buffered saline including 0.1% Tween 20 (TBS-T) 3 times and exposed to horseradish peroxidase-conjugated secondary antibodies (Sigma) for 1 hour at room temperature. The membranes were washed with TBS-T 3 times, and the protein bands were visualized by the Enhanced Chemiluminescence (ECL) Plus Western blotting detection system  according to the manufacturer's instructions. The experiments were repeated three times. The densities of bands of Western blot analysis were determined using ImageJ software .HPDLCs were pretreated with nobiletin  followed by Tukey's test. Any statistically significant difference between the groups was determined at the μM) significantly decreased CCL2 production in TNF-stimulated HPDLCs. We tested the cytotoxic effects of nobiletin (100 μM) on HPDLCs using Cell Counting Regents .To investigate the anti-inflammatory effects of nobiletin on TNF-stimulated HPDLCs, production of CXCL10, CCL2, and IL-8 was detected by ELISA assay . Levels The release of MMP1 and MMP3 in culture supernatants induced by TNF in the presence of nobiletin was determined by ELISA. As shown in μM nobiletin treatment.To assess the influence of nobiletin on the expression of PTGS2, Western blot analysis was performed . The treκB, and AKT1 in HPDLCs [μM nobiletin treatment inhibited the phosphorylation levels of MAPK1/MAPK3, IKK-α/β, and p65 NF-κB in TNF-stimulated HPDLCs. The level of AKT1 phosphorylation was slightly decreased by 50 μM nobiletin treatment, and 100 μM nobiletin inhibited AKT1 phosphorylation in TNF-treated HPDLCs (Next, we examined if nobiletin modified activations of signal transduction pathways in TNF-stimulated HPDLCs. We previously reported that TNF treatment could activate MAPK14, MAPK1/MAPK3, MAPK8, NF-n HPDLCs , 12. Thed HPDLCs .It is known that HMOX1 has anti-inflammatory roles in inflammatory lesions , and NFEPeriodontal disease is the most popular infectious disease in the world that cuts down on the quality of life by losing teeth. The strong immune reaction in periodontal lesions is related to periodontium destruction. Therefore, the discovery of a safe bioactive substance which we can give in the periodontal lesion is hoped for. It is already reported that nobiletin has anti-inflammatory effects. For example, Liu et al. reported that nobiletin could inhibit IL-6, TNF, MMP1, and MMP3 in interleukin-21-stimulated human synoviocytes . Lin et κB, and AKT1 pathways are involved in inflammatory mediator production. Therefore, previous researchers examined the effects of nobiletin on activations of signal transduction pathways. Shi et al. reported that nobiletin could inhibit MAPK1/MAPK3 and AKT1 phosphorylation in hepatocyte growth factor-treated HepG2 cells [κB in human chondrocytes [κB activations might be involved in the inhibitory effects of inflammatory mediator expressions in various types of cells.MAPK, NF-G2 cells . Xie et drocytes . ResultsκB activation, and NFE2L2 is related to HMOX1 production [κB activation in this study (κB in TNF-stimulated HPDLCs.To better understand the underlying molecular mechanisms, the NFE2L2-HMOX1 pathway was investigated. HMOX1 could inhibit NF-oduction . Therefois study . In thisκB, and AKT1 signaling pathways and enhancing HMOX1 and NFE2L2 expression. We should use nobiletin for animal models of periodontal disease at the next stage. If nobiletin could prevent the progression of periodontal disease in animal diseased models, we may use nobiletin for human periodontal disease treatment. We think nobiletin might be used for local drug delivery systems such as periodontal pocket irrigation or gel application in periodontal lesions. Further studies should be necessary to prove the hypothesis.In conclusion, this study demonstrates the anti-inflammatory activity of nobiletin in HPDLCs. Nobiletin significantly inhibits the TNF-induced inflammatory response by suppressing the MAPK1/MAPK3, NF-


Prolonged enteropathogen shedding after diarrhea complicates the identification of etiology in subsequent episodes and is an important driver of pathogen transmission. A standardized approach has not been applied to estimate the duration of shedding for a wide range of pathogens.We used a multisite birth cohort of children 0–24 months of age from whom diarrheal and monthly nondiarrheal stools were previously tested by quantitative polymerase chain reaction for 29 enteropathogens. We modeled the probability of detection of the etiologic pathogen before and after diarrhea using a log-normal accelerated failure time survival model and estimated the median duration of pathogen carriage as well as differences in subclinical pathogen carriage 60 days after diarrhea onset in comparison to a prediarrhea baseline.Cryptosporidium . The largest increases in subclinical pathogen carriage before and after diarrhea were seen for Cryptosporidium  and Shigella .We analyzed 3247 etiologic episodes of diarrhea for the 9 pathogens with the highest attributable burdens of diarrhea. The median duration of postdiarrheal carriage varied widely by pathogen, from about 1 week for rotavirus  to >1 month for Cryptosporidium and Shigella. Targeted antimicrobial therapy and vaccination for these pathogens may have a relatively large impact on transmission.Postdiarrheal shedding was widely variable between pathogens, with strikingly prolonged shedding seen for  Cryptosporidium and Shigella.In a multisite birth cohort study of children 0–24 months of age and with quantitative polymerase chain reaction detection of diarrheal pathogens, we estimate the duration of postdiarrheal carriage in low-resource settings, with strikingly prolonged shedding seen for  The application of quantitative molecular diagnostics to studies of enteric infections in children in low-resource settings has identified a strikingly high prevalence of pathogen carriage even in the absence of clinical symptoms . This suPrevious estimates of the duration of shedding have come from single-pathogen studies from heterogeneous populations and study types  and have used a wide range of diagnostics . The durThe rationale, methodology, and principal findings of the enteric pathogen analyses from MAL-ED have been previously reported , 14, 15.We tested all available diarrheal and nondiarrheal stool samples from children who completed 24 months of follow-up using custom-designed TaqMan Array Cards  that compartmentalized qPCR assays for 29 enteropathogens , 18. RawCryptosporidium based on the observed long duration of postdiarrheal carriage for this pathogen.We used previously developed models to assign diarrhea etiology, classifying etiologic detections of pathogens in diarrhea as those with an episode-specific attributable fraction of at least 0.5, as previously described . For allThe primary analysis included all diarrheal episodes except those that either (1) occurred during the shedding window of an episode attributed to the same pathogen or (2) occurred at any time after an episode attributed to the same pathogen and all stool samples collected between the episodes had the pathogen detected. These repeat episodes were excluded to remove cases that could have been due to a long, lingering infection rather than a new infection; we also assessed the impact of this choice with a sensitivity analysis that included all diarrheal episodes.To estimate the probability of a positive stool as a function of time, we used the binary outcome modelFor each pathogen, this approach was used to model both the probability of a positive stool symptote . Models We used model (1) to estimate the probability of pathogen detection at any time before, during, or after the episode of diarrhea, rather than to estimate time to the end of pathogen carriage, as is done with survival analysis. We chose the specific model form, rather than for example logistic regression, because it allows for a 100% positive detection rate at time 0. We also considered a modified Weibull model; however, this model, near time 0, is sensitive to the estimated shape parameter, which does not seem biologically plausible.The median durations of pre- and postdiarrheal carriage were estimated as the time in days before and after symptom onset when there was a 50% chance of pathogen detection. Median durations of postdiarrheal carriage were also estimated for defined subgroups by including additional dichotomous covariates in the model and defining the model-predicted probabilities for each level of the covariate. The prevalence difference between 60 days after diarrhea onset and 30 days prior to diarrhea onset was calculated as the difference in the model-predicted probability of detection at those time points. Confidence intervals (CIs) were estimated by bootstrapping at the subject level. All analyses were performed using R version 3.6.0 software .Shigella (736 etiologic episodes [11.0% of episodes]), rotavirus (554 [8.3%]), sapovirus (535 [8.0%]), heat-stabile toxin-producing enterotoxigenic Escherichia coli (ST-ETEC) (452 [6.8%]), and adenovirus 40/41 (404 [6.0%]) (Cryptosporidium) and 68.4% (adenovirus 40/41) of included episodes were the first identified episode for that pathogen for that child. The mean child age at the time of diarrhea ranged from 11.2 months for Campylobacter to 16.5 months for Shigella. The majority of episodes had a duration of ≤7 days for all pathogens, ranging from 92.9% of rotavirus episodes to 84.8% of Shigella episodes  had valid qPCR results for all of the pathogens included in this analysis, including 6687 diarrheal and 34 289 monthly nondiarrheal stools. Nine pathogens were identified as etiologic in at least 1% of episodes, led by  [6.0%]) . In totaepisodes .Campylobacter jejuni/Campylobacter coli, and ST-ETEC. For all etiologies, the etiologic pathogen was re-detected from the majority of stool samples obtained within 5 days after the onset of diarrhea.First, we examined the prevalence and detection quantity of etiologic pathogens before and after each etiologic episode . There wCryptosporidium (Shigella (14.1 [9.9–18.2] days). The results of the sensitivity analysis, which included all diarrheal episodes, were very similar with the exception of adenovirus 40/41, where the median duration of carriage increased from 9.2  days when excluding repeated episodes within 60 days or without an intervening negative test to 12.4  days when all episodes were included; no other estimates changed by >1.4 days.Among etiologic episodes for each pathogen, we then modeled the probability of the detection of all pathogens during the incubation and shedding windows . The remporidium . Other pCryptosporidium , Shigella , and C. jejuni/C. coli .Because it was possible that despite a shorter median duration of carriage, a subset of children could have prolonged shedding, we also estimated the difference in pathogen carriage from baseline (30 days before the onset of diarrhea) to 60 days after the onset of diarrhea . The preShigella . There was a small but non–statistically significant reduction in the duration of carriage of Shigella and C. jejuni/C. coli for episodes treated with appropriate empiric antibiotics. As expected, the quantity of pathogen detected during diarrhea was positively associated with the duration of postdiarrheal carriage.To interrogate factors associated with the duration of carriage, we estimated the median durations of carriage stratified by age in years, first or repeat etiologic episode for each child, treatment with appropriate empiric antibiotics, and the quantity of the etiologic pathogen in the diarrheal stool . There wCryptosporidium to have the longest duration of infection prior to the onset of diarrhea, with a median duration of almost 5 days, followed by adenovirus 40/41 and ST-ETEC.Because of the temporal symmetry of both the available data , we also estimated the median duration of infection prior to the onset of diarrhea, that is, the incubation period . As expeCryptosporidium and the caliciviruses and the longer incubation periods for Cryptosporidium and adenovirus 40/41 [Cryptosporidium, Campylobacter, and Shigella, were most clearly associated with persistent shedding in this analysis [Using a large, multisite birth cohort study, with intensive surveillance for incident diarrhea and regular collection of diarrheal and nondiarrheal stools tested with quantitative molecular diagnostics, we were able to systematically estimate the duration of postdiarrheal shedding of common enteropathogens among children in low-resource settings. These data provide a reference for the duration of shedding in these high-morbidity populations. There was a wide variation in the median duration of postdiarrheal enteropathogen shedding, ranging from approximately 1 week to >1 month. Many of the estimates are consistent with expectations—for example, the long duration of postdiarrheal carriage of us 40/41 , 20, 21.analysis .Cryptosporidium is consistent with historical studies using microscopy to identify oocyst excretion, in which the incubation period was 1 week, the duration of illness was close to 2 weeks, oocyst excretion continued for approximately 1 week after illness cessation, and a subset of patients had months of ongoing oocyst excretion [Cryptosporidium have been identified, which, in addition to reducing the morbidity of an episode of cryptosporidiosis, may also help reduce the substantial reservoir of postdiarrheal shedding [The long median durations of both pre-and postdiarrheal shedding of xcretion . The durxcretion . A numbeshedding  and in tshedding .C. jejuni/C. coli was relatively short, considering the strikingly high rates of subclinical carriage with this pathogen [The duration of postdiarrheal shedding for pathogen . This supathogen , and/or pathogen , 29. AltShigella shedding 2 months after the onset of diarrhea. Persistent clinical shigellosis has been described, as well as long-term asymptomatic carrier states [Shigella occurs in a sizeable minority of children in these settings. This highlights the need for control strategies, whether appropriate antibiotic therapy or vaccines, that could help reduce pathogen transmission. Shigella vaccine development is now primarily focused on subunit vaccines that would be introduced later in the first year of life, such that indirect protection from reduced transmission may be critical for reducing the disease burden in infants [Similarly, we found that, despite a relatively short median duration of shedding, approximately 1 in 5 children had persistent r states , 30. A nr states . Our ana infants . The finCryptosporidium, norovirus, and sapovirus, the 3 pathogens with the longest median durations of postdiarrheal carriage in the present analysis [Shigella diarrhea burden, suggesting that persistent Shigella carriage occurs at a sufficiently low quantity to not obscure the inference to etiology when detected in higher quantities in diarrheal stools. This supports the value of the incorporation of pathogen quantification for etiologic studies of diarrhea in settings with high carriage [The long duration of postdiarrheal shedding for these pathogens also has implications for the design and analysis of etiologic studies of diarrhea, in particular the impact of the definition of a nondiarrheal control and the incorporation of pathogen quantity . We haveanalysis . The morcarriage , 2.Cryptosporidium in this study is consistent with a volunteer study of adults in which the median incubation period was 1 week [E. coli of 4 days is longer than is typically described in natural infection [Similar to estimates of postdiarrheal shedding, prior estimates of pathogen incubation periods come from heterogeneous sources. The long duration of prediarrheal carriage identified for s 1 week . It is as 1 week . Among vs 1 week . Meanwhinfection  or challnfection . The shoThe estimates in this study come from the modeled probability of pathogen detection, taking advantage of the random intervals between diarrhea onset and prior and subsequent stool collections. Because we had relatively few postdiarrheal stools available for each episode, we could not estimate the duration of shedding for each episode and thus explicitly describe postdiarrheal carriage for each diarrhea episode. This would require more frequent postdiarrheal stool collection, which is unlikely to be available from a single study across a wide range of etiologies. Second, detection of enteric pathogen nucleic acid by qPCR cannot distinguish viable pathogen shedding. Thus, if used to infer the duration of shedding that can be directly implicated in pathogen transmission, these results are likely overestimates. However, we would presume that the relative duration of shedding of viable pathogens would be consistent with our findings here. Finally, we could not clearly distinguish postdiarrheal shedding from repeat exposure and infection during the shedding window. However, the clear asymmetry between pre- and postdiarrheal pathogen detection, the specificity of re-detection to the etiologic pathogen, and the consistency of the findings with prior single-pathogen studies all support that many of the postdiarrheal detections represent ongoing shedding after the diarrhea episode.In summary, we were able to estimate the duration of postdiarrheal carriage of a broad range of enteropathogens. In the setting of strikingly high rates of subclinical carriage for some pathogens, clinical disease still represents a sentinel acquisition event that is associated with a substantial pathogen reservoir. Interventions targeted to identify and treat these episodes may help reduce transmission and other sequelae of subclinical infection.