E. coli-based cell-free system for the first time to express recombinant human TNF-α. Our findings revealed that cell-free expression system can be an alternative platform for producing soluble and functionally active recombinant TNF-α with a yield of 390 µg/mL in only 2 h at a temperature of 40 °C for further research and clinical trials.As a result of increasing demand for the pleiotropic cytokine TNF-α, recombinant human TNF-α protein with appropriate bioactivities was produced in several heterologous in vivo expression systems. While in vivo expression of this cytokine is laborious and lengthy, cell-free or in vitro expression system has the benefits of speed, simplicity, flexibility, focus of all the system energy on target protein synthesis alone, besides high soluble and functional protein yield. Therefore, we employed and optimized an E. coli-based cell-free protein synthesis (CFPS) system and its biological activity evaluation. The codon-optimized synthetic human TNF-α gene was constructed by a two-step PCR, cloned into pET101/D-TOPO vector and then expressed by the E. coli CFPS system. Cell-free expression of the soluble protein was optimized using a response surface methodology (RSM). The anticancer activity of purified human TNF-α was assessed against three human cancer cell lines: Caco-2, HepG-2 and MCF-7. Data from RSM revealed that the lowest value (7.2 µg/mL) of cell-free production of recombinant human TNF-α (rhTNF-α) was obtained at a certain incubation time (6 h) and incubation temperature (20 °C), while the highest value (350 µg/mL) was recorded at 4 h and 35 °C. This rhTNF-α showed a significant anticancer potency. Our findings suggest a cell-free expression system as an alternative platform for producing soluble and functionally active recombinant TNF-α for further research and clinical trials.Cell-free (in vitro) expression is a robust alternative platform to the cell-based (in vivo) system for recombinant protein production. Tumor necrosis factor-alpha (TNF-α) is an effective pro-inflammatory cytokine with pleiotropic effects. The aim of the current study was de novo optimized expression of soluble and active human TNF-α by an in vitro method in an  Tumor necrosis factor-alpha is a pleiotropic cytokine with a pro-inflammatory and immunoregulatory function ,2. It isMammalian TNF-α exists as a non-glycosylated type II transmembrane precursor protein (membrane-bound TNF-α or mTNF-α) with a molecular mass of about 26 kDa (157 amino acids in addition to a leader sequence of 76 amino acids) ,8,9. TheEscherichia coli [Lactococcus lactis [Streptomyces lividans [E. coli expression system, including gene codon optimization, choice of the strongest expression promoters and optimization of culture and induction conditions to obtain more soluble recombinant protein over inclusion bodies [E. coli systems, but have the drawbacks of high cost of cell culture and maintenance of the laboratory [As a result of increasing demand for sTNF-α protein, recombinant human TNF-α protein with appropriate bioactivities was produced in several heterologous protein expression systems such as hia coli ,14, Lacts lactis , Streptolividans , a bacullividans ,18, or mlividans . Continun bodies ,21,22. Eboratory ,24,25.E. coli, Vibrio natriegens, or Bacillus subtilis). The choice of extract source depends predominantly on the origin of the target protein, its complexity and further downstream applications [Cell-free or in vitro protein synthesis employs the cellular protein machinery to synthesize proteins directly outside the living host cells, without any constraints of cell membrane ,27,28. Iications ,28. The ications ,30,31.CFPS systems are becoming increasingly used as an alternative to in vivo systems because of the relatively high reaction speed, simplicity and flexibility; focus of all the system energy on target protein synthesis alone; easy and direct manipulation of CFPS open reaction; high soluble and functional protein yield and the possibility of using PCR fragments or mRNA directly as a template without cloning ,27,28,32The current work is the first report where the cell-free approach was employed to produce rhTNF-α protein. After optimizing cell-free expression of soluble protein using RSM, the anticancer activity of purified protein was investigated against Caco-2, HepG-2 and MCF-7. The cell-free synthesized human TNF-α demonstrated a cytotoxic effect in a concentration-dependent manner on test cancer cells. This study recommends the use of CFPS as an alternative platform for the synthesis of soluble and functionally active TNF-α products for further academic research and clinical trials.E. coli XL2-Blue MRF’ ultracompetent cells kit used for gene cloning was purchased from Stratagene .TOPO TA cloning kit  was used for gene cloning into pCR 2.1-TOPO cloning plasmid.A champion pET Directional TOPO expression kit was obtained from Invitrogen and was used for cell-free protein expression of human TNF-α under control of T7 promoter in pET101/D-TOPO expression plasmid.https://www.uniprot.org/ (accessed on 1 February 2017); subsequently, it was reverse translated using the following website: http://www.molbiol.ru/eng/scripts/01_19.html (accessed on 1 February 2017). The resulted DNA coding sequence was tested for existence of rare codons using GenScript Rare Codon Analysis Tool ) to confirm its successful expression in the E. coli extract-based CFPS system.First, the amino acid sequence of human sTNF-α was obtained from the Universal Protein Resource (UniProt) website, E. coli genes found in GenBank release 63, to obtain a DNA sequence with high-level expression by protein synthesis machinery of the E. coli CFPS system. The BioEdit program version 7 was utilized to reverse translate the amino acid sequence of human sTNF-α based on a codon usage table generated from 681 https://www.bioinformatics.org/seqext/ (accessed on 1 March 2017). This map identifies the restriction enzymes sites in the human TNF-α gene.To design cloning experiments and analyze the positive clones, a gene restriction map was generated using the Sequence Extractor website: To synthesize the codon-optimized human TNF-α gene, 20 oligonucleotides (10 forward and 10 reverse), each with average length of 40 nucleotides were designed to span most of both strands of the gene sequence as previously described ,36. OligBamHI restriction site (GGATCC) followed by an initiation ATG codon. Meanwhile, for cloning into pET101/D-TOPO, the forward PCR primer length was 22 nucleotides and contains 5′ CACC overhang followed by the BamHI restriction site and ATG codon. For amplification and subsequent cloning into pCR 2.1-TOPO plasmid, the forward PCR primer length was 22 nucleotides. It was designed to contain the XhoI restriction site (CTCGAG) and has a length of 21 nucleotides. The reverse primer designed for cloning into pCR 2.1-TOPO and pET101/D-TOPO vectors contains an Pfu DNA polymerase with proofreading properties . The human TNF-α gene was assembled by PCR using the designed overlapped oligonucleotide primers and BioReady Pfu reaction buffer with 1.5 mM MgCl2 (1 X), dNTP Mix (0.5 mM) and BioReady Pfu DNA polymerase (2.5 U). The reaction was completed to 25 µL with sterile water for injection and mixed gently by tapping PCR tube.Working on ice, the following reagents were added to a sterile DNase- and RNase-free PCR tube as follows: twenty oligonucleotide primers (5 pM/each), The cycling conditions were as follows: 35 cycles of 94 °C for 30 s, 58 °C for 2 min, 72 °C for 2 min, followed by a final extension of 72 °C for 10 min . Pfu reaction buffer with 1.5 mM MgCl2 (1 X), dNTP Mix (0.5 mM) and BioReady Pfu DNA polymerase (2.5 U). The reaction was completed to 25 µL with sterile water for injection and mixed gently. Working on ice, in a sterile DNase- and RNase-free PCR tube, the following components were mixed as follows: outermost oligonucleotide primers (20 pM/each), template , The cycling conditions were as follows: 95 °C for 3 min followed by 35 cycles of 94 °C for 30 s, different annealing temperatures  for 2 min, 72 °C for 2 min and final extension of 72 °C for 10 min .Both the assembly reaction product and the final amplified full-length gene were analyzed by 2% agarose gel electrophoresis.Pfu DNA polymerase only produces a blunt-ended PCR product and removes the 3′ adenine overhang required for TA cloning into pCR 2.1-TOPO. Accordingly, the blunt-ended PCR product of the human TNF-α gene amplified at an annealing temperature of 58 °C was reamplified using gene-specific forward and reverse primers designed for subsequent cloning into pCR 2.1-TOPO and Dream Taq DNA polymerase obtained from Thermo Fisher Scientific  (2.5 U/25 µL reaction), which is capable of adding a single 3′ adenine overhang. Subsequently, the PCR was followed by 2% agarose gel electrophoresis and the human TNF-α gene was extracted and purified from 2% agarose gel using the QIAquick gel extraction kit  according to the manufacturer’s instructions.E. coli XL2-Blue MRF’ ultracompetent cells. The positive clones were confirmed by restriction digestion and nucleotide sequencing.The purified human TNF-α amplicon was inserted into pCR 2.1-TOPO cloning vector using a TOPO TA cloning kit following the company’s instruction manual and transformed into Pfu DNA polymerase (2.5 U/25 µL reaction) to produce the blunt-ended PCR product. The PCR product was inserted into pET-TOPO expression vector as recommended by the manufacturer to yield a pET-hTNF-α expression construct. The recombinant plasmid was transformed into E. coli XL2-Blue MRF’ ultracompetent cells as a cloning host for amplification and storage of the pET-hTNF-α expression construct. The positive clones were analyzed by PCR using gene-specific forward and reverse primers designed for cloning into the pET101/D-TOPO vector and by restriction digestion in addition to nucleotide sequencing to confirm the presence and correct orientation and sequence of the insert. The gene fragment resulted from restriction cutting of the pCR 2.1-TOPO cloning vector carrying the human TNF-α correct sequence was extracted from 1.2% agarose gel. Then, it was reamplified by PCR at annealing temperature of 58 °C using gene-specific forward and reverse primers designed for cloning into the pET101/D-TOPO vector and BioReady E. coli HY kit (CFPS system derived from E. coli crude cell extract) obtained from 5 PRIME  as previously reported [E. coli S30 lysate, 10 µL reaction mix, 12 µL amino acids, 1 µL methionine, 5 µL reconstitution buffer and 10 µL of pET-hTNF-α construct at concentration of 0.5 µg dissolved in sterile water for injection. Additionally, a negative control reaction was performed using the same components except for plasmid construct. The reactions were mixed gently and incubated at 30 °C for 6 h. Afterwards, the reaction tubes were stored at −20 °C for SDS-PAGE analysis and ELISA of recombinant human TNF-α.Cell-free (In Vitro) expression of the rhTNF-α protein from the pET-hTNF-α construct was carried out using the rapid translation system RTS 100 reported . AccordiE. coli HY kit manual as follows: the soluble fraction of the cell-free synthesized human TNF-α protein was harvested by centrifugation for 5 min at 12.000 rpm and the protein was precipitated with the addition of 500 µL of −20 °C cold acetone in an Eppendorf tube, mixed and incubated on ice for 10 min. Then, it was centrifuged at 12.000 rpm for 15 min at 4 °C. The supernatant was discarded, the pellet was air dried for 15 min and resuspended in 50 µL of 1 X sample buffer. Afterwards, samples were boiled for 10 min, centrifuged and loaded onto the SDS gel.The cell-free synthesized human TNF-α protein samples were prepared for SDS-PAGE according to the RTS 100 The soluble fraction of the cell-free synthesized human TNF-α protein was harvested by centrifugation for 5 min at 12.000 rpm and the protein was precipitated with the addition of 500 µL of −20 °C cold acetone in an Eppendorf tube, mixed and incubated on ice for 10 min. Then, it was centrifuged at 12.000 rpm for 15 min at 4 °C. The supernatant was discarded, the pellet was air dried for 15 min and resuspended in PBS. Quantitative estimation of the soluble human TNF-α protein synthesized in the CFPS system was performed using a sandwich high-sensitivity human TNF alpha PicoKine ELISA kit  according to the manufacturer’s instructions. The developed yellow color was measured at 450 nm by a microplate reader . The cell-free synthesized human TNF-α concentration was calculated from a calibration curve made by using different concentrations of the recombinant human TNF-α standard.The first step was carried out using AKTAprime plus fast performance liquid chromatography (FPLC) protein separator system with an ion-exchange chromatography column packed with Q-Sepharose fast flow resin (strong anion exchanger), from GE Healthcare Life Sciences Products . The soluble protein fraction of CFPS reaction of human TNF-α was dialyzed against buffer A  at 4 °C for 24 h with stirring, using a dialysis bag with molecular weight cut-off 14–18 kDa.During these 24 h, the dialysis buffer was replaced twice with fresh buffer. Directly after dialysis, the human TNF-α soluble fraction was loaded onto a Q-Sepharose fast flow column, which was previously equilibrated by buffer A. Then, the column was washed with the same buffer until the ultraviolet (UV) absorbance at 280 nm was stable around zero and eluted with a 0–100% linear gradient of buffer B  at a flow rate of 1 mL/min. Fractions were collected, respectively, and further analyzed by 12% SDS-PAGE. At the same time, the total protein concentration was measured by Bradford assay.In the second step, the fraction containing the intended protein was pooled, then dialyzed against 0.01 M sodium phosphate buffer, pH 6 and the pH was carefully adjusted to 6. The processed fraction was applied to a heparin- Sepharose 6 fast flow affinity column equilibrated with the same buffer and the column was placed on rock and roller shaker at 4 °C for 2 h. Afterwards, the column was washed with 0.01 M sodium phosphate buffer, pH 6 to remove proteins that unspecifically to the resin and the intended protein was eluted by 0.01 M sodium phosphate buffer, pH 6 with a gradient concentration of 0.1–1 M NaCl. All fractions were collected and analyzed by 12% SDS-PAGE and Bradford assay. Percent recovery was calculated according to the following equation: recovery % = (protein concentration in the collected fraction(s)/protein concentration in CFPS reaction) × 100. Fractions containing cell-free synthesized human TNF-α protein were dialyzed against PBS, pH 7.2 and stored at −20 °C.E. coli CFPS system. A 22 fractional factorial central composite rotary design for two independent variables each at five levels was employed, indicating that 13 experiments were required as a whole. Soluble human TNF-α protein production was taken as the response and it was estimated using quantitative ELISA.A response surface methodology was used to optimize the soluble human TNF-α protein expression in Y is the measured response variable, βo, βi, βij and βii are constant and regression coefficient of the model, xi and xj represent the independent variables in coded values. The second-order polynomial coefficients were calculated via Minitab 14 software to evaluate the dependent variable response. Response surface as well as the contour plots were designed using the same software, to assess the interactions between the different variables. Analysis of variance (ANOVA) was performed so that the second-order polynomial equation fits for all response variables. The significance of the model equation and model terms were estimated by Fisher test (F-test) and its associated probability P(F), while the quality of fit of the polynomial model equation was expressed by the coefficient of determination (R2) and adjusted R2 (Adj 𝑅2). An optimization tool of Minitab 14 software was utilized, in which a combination of different optimized parameters that achieved the maximum response (soluble protein cell-free expression) was tested experimentally to confirm the model validity.The experimental data derived from the design were analyzed by multiple regressions through the least square method to fit the following second-order polynomial equation :Y = βo +Human colon cancer cell line Caco-2 HTB-37, human hepatoma cell line HepG-2 HB-8065 and human breast cancer cell line MCF-7 HTB-22 were obtained from American Type Culture Collection (ATCC), USA.2 incubator . The cells were passaged twice to thrice per week, enzymatically with 0.25% trypsin-EDTA (1 X) and subcultured in 25 cm2 tissue culture flasks. The Caco-2 cell line was cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine, while the HepG-2 and MCF-7 cell lines were maintained in RPMI-1640 augmented with 10% FBS and 2 mM L-glutamine. All the mentioned cell lines were incubated at 37 °C, in a humidified 5% COHuman peripheral blood mononuclear cells (PBMCs) were isolated according to the method described by Boyum (1968) . Briefly5 mononuclear cells/well were seeded in a 96-well cell culture plate. The cells were treated with serial concentrations of purified cell-free synthesized human TNF-α in RPMI-1640 complete medium and were incubated at 37 °C in 5% CO2 incubator for 72 h. Afterwards, 20 μL of 5 mg/mL MTT were added to each well and the plate was incubated at 37 °C for 4 h followed by centrifugation at 2.000 rpm for 10 min at room temperature. Then, the MTT solution was removed and 100 µL dimethyl sulfoxide (DMSO) were added to dissolve the formazan crystals which were produced by the viable cells. The absorbance was measured with a microplate reader at 570 nm. All the measurements were performed in duplicate and untreated cells were used as a control. Finally, the percentages of cell viability and cytotoxicity were calculated compared to the untreated cells. The values of half-maximal inhibitory concentration (IC50) and the safe dose  of cell-free synthesized human TNF-α were estimated by Graphpad Instat software version 3.The cytotoxic effect of cell-free synthesized human TNF-α on PBMCs was evaluated using 3--2,5-diphenyltetrazolium bromide (MTT) dye assay as previously described by Mosmann, 1983  and El-B3 cells/well) were seeded in 96-well cell culture plates. After 24 h, serial dilutions of EC100 of human TNF-α that caused 100% PBMCs viability  or serial concentrations of purified cell-free synthesized human TNF-α , were incubated with the three cancer cell lines for 72 h at 37 °C in 5% CO2 incubator. MTT assay was performed and the percentages of cell viability and cytotoxicity were estimated. The IC50 and EC100 values against test cancer cell lines were calculated using the Graphpad Instat software version 3. Anticancer effect of cell-free synthesized human TNF-α was assessed against Caco-2, HepG-2 and MCF-7. All cancer cell lines (5 × 103 cells/well) were seeded in 96-well cell culture plates. After 24 h, the cells were treated with the IC50 of human TNF-α and incubated for 72 h at 37 °C in 5% CO2 incubator. Subsequently, plates were centrifuged at 1.000 rpm for 5 min at room temperature and 16 µL of EtBr and AO dye solution (50 μg/mL) were added to each well containing 150 µL media. Afterwards, the plates were left for 1–2 min to allow the dye to diffuse into the cells and visualized under fluorescent phase-contrast microscope . The EtBr and AO staining method was performed in duplicates and untreated cells were used as a control.Antitumor activity of cell-free synthesized human TNF-α against Caco-2, HepG-2 and MCF-7 cancer cell lines, was determined by staining cells with ethidium bromide (EtBr) and acridine orange (AO) dye mix . All canE. coli CFPS system, in silico tools were used. The amino acid sequence of human sTNF-α containing 157 residues (amino acids: 77–233) (Uniprot identifier: P01375)  codons based on E. coli is 12%, revealing that human TNF-α gene employs tandem rare codons that may reduce the translation efficiency or even disengage the machinery of translation. In view of that, by using BioEdit, the gene was codon-optimized to allow its heterologous production in the E. coli CFPS system without affecting the functional amino acid sequence.To optimize the coding sequence for the human TNF-α gene for expression in the  P01375)  was obtaBamHI and XhoI; thus, they were used in designing gene-specific primers for subsequent cloning experiments. To synthesize the codon-optimized human TNF-α gene, oligonucleotides encoding both strands of the DNA duplex were designed as illustrated in The restriction site analysis of the codon-optimized human TNF-α coding sequence is shown in The full-length of the codon-optimized human TNF-α gene (474 bp) was synthesized by a two-step gene synthesis procedure—assembly A and ampE. coli XL2-Blue MRF ultracompetent cells. Subsequently, the positive clones were subjected to restriction digestion using the EcoRI enzyme  and heparin–Sepharose affinity chromatography. In the first step, human TNF-α protein was eluted by approximately 0.5 M sodium chloride (50% buffer B) at about 64 min A, with aThe statistical approach of RSM (many factors at a time approach) can optimize independent variables to maximize the response (the cell-free production of soluble and active human TNF-α). The central composite design (CCD) is widely used as a fractional factorial design in RSM optimization. It employs a simultaneous analysis of the individual and interaction effects of all parameters. To optimize the process parameters for the in vitro synthesis of soluble and active human TNF-α protein (the response), CCD was used.E. coli HY kit according to the instructions in the manual. Therefore, the independent variables investigated in this CCD optimization experiment were limited to incubation temperature (A) and incubation time (B); each was examined at five different levels as shown in Cell-free expression of human TNF-α protein from the pET101/D-TOPO-human TNF-α recombinant plasmid was carried out using the RTS 100 Y is the response (human TNF-α concentration in µg/mL), A and B are the coded terms for the independent variables. The positive and negative signs represent the synergistic and antagonistic effects on the response.Based on the experimental results, the following regression equation was obtained in coded form:Different combinations of the independent variables yielded cell-free synthesized human TNF-α product ranging from 7.25 to 350.03 µg/mL as depicted in p-values calculated by the model for the linear, quadratic and interactions. The significance of each term of the model was verified using the associated p-value (p < 0.05). All the terms were statistically significant except for the quadratic term of incubation time (B × B) which was not significant. The R2, Adj R2 of the model were 86.6% and 77.1%, respectively.p-value for the response surface quadratic model revealed that the regression is statistically significant. Moreover, all the model terms  were all significant. Additionally, the p-value for the lack of fit of the model was not significant (p > 0.05). The results of ANOVA for the quadratic regression model obtained from CCD employed in the optimization of human TNF-α cell-free synthesis are summarized in ri) are well distributed as positive and negative residuals in the range of −2 < ri < +2.The normal (%) probability plot of the residuals for the model is illustrated in The interactive effect of the independent variables on the human TNF-α cell-free expression was obtained in the form of a 3D-surface plot A and a 2With the purpose of confirming the optimized conditions, the cell-free expression experiment under optimum conditions was performed to validate the accuracy of the model. The predicted optimal values of the independent variables for human TNF-α cell-free expression are depicted in 50 and EC100 values, at which 50 and 100% of cells are metabolically active, were estimated as 1728.2 ± 10.74 and 250 ± 0.62 ng/mL, respectively  lower than that against PBMCs. Additionally, the IC50 value of cell-free synthesized human TNF-α against HepG-2 was significantly (p < 0.05) lower than its EC100 value against PBMCs . However, other platforms are available but are less common such as mammalian cell lines, insect cells, algae, yeast and cell-free expression systems [The market demand for recombinant proteins manufacturing is increasing steadily, with a sale valued at USD 1.654 billion in 2017 and anticipated to rise to USD 2.850 billion by 2022 . About h systems ,43,44. AThe use of cell-free expression systems is increasing in importance as their productivity increases and cost decreases. This approach provides unique opportunities not only for protein synthesis, but also for development of therapeutics, metabolic engineering, as well as research and education. In contrast to in vivo protein synthesis systems, CFPS platforms are of an open nature because expression occurs by use of cellular protein machinery outside living host cells without any constraints of cell membrane or other life objectives of the cell. This open nature allows for direct manipulation of cellular machinery for production of target and novel proteins, posttranslational modifications, or fluorescence labeling of proteins. Most importantly, CFPS avoids any host–target protein interactions, and therefore can produce proteins which are difficult to express in vivo because of their burden on the living cell, toxicity, or sensitivity to degradation by cellular enzymes. Such proteins comprise proteins from templates of high GC content, large proteins, antibodies and metalloproteins. Other applications of CFPS platforms include high-throughput screening, production of virus-like particles, membrane protein expression and continuous formats employed for large-scale reactions in industrial applications ,46.E. coli S30 extract-based cell-free system for the expression of soluble and active human tumor necrosis factor-alpha. To the best of our knowledge, this is the first report to produce rhTNF-α in CFPS system and optimize its expression by RSM. We previously carried out a similar application for the CFPS system based on E. coli lysate in the expression of another valuable cytokine: human consensus interferon-alpha [The aim of the present study was to construct and optimize an on-alpha .E. coli CFPS system) via computational optimization of the codon profile of the human sTNF-α gene as previously described for human sTNF-α and other recombinant proteins [We first evaded expression problems due to codon bias  PCR using codon-optimized and overlapped oligonucleotides which represent most of the sequence of both strands of the DNA duplex. In other studies, Binepal et al., 2012  and Yin E. coli HY kit. We chose E. coli crude cell extract as the CFPS system because of many factors including the absence of any posttranslational modification that is required for production of functionally active human TNF-α protein; reasonable productivity (2–20 µg/50 µL reaction) and cost (1.2 USD–12 USD/µg protein) of the system; cell-free reaction preparation being quick, simple and easy to optimize; high translation efficiency of the system and finally obtaining 390 µg/mL of soluble and functionally active human TNF-α protein in only 2 h at a temperature of 40 °C. However, this system suffers from some drawbacks such as an undefined system composition with presence of nucleases, proteases and tmRNA, low engineering flexibility and low tolerance for linear templates [Cell-free expression of human TNF-α protein from pET101/D-TOPO-human TNF-α recombinant plasmid was carried out using the RTS 100 emplates .E. coli cells with a high yield of milligram (mg) protein per liter of bacterial culture [E. coli expression system after optimization of conditions of culture and induction [E. coli crude cell extract-based CFPS system used in the present study, on the contrary, produced about 390 µg/mL of soluble and functionally active human TNF-α protein in only 2 h at a temperature of 40 °C without need for cell lysis, protein extraction, refolding or other unnecessary processes. Higher yields at incubation time of hours and higher percent recovery can even be achieved if continuous formats of this system are used and optimized. Nevertheless, the yield obtained (70.5 µg/mL after purification) in the current work is a good starting point for scaling-up and optimizing the cell-free production of human TNF-α for future industrial or commercial purposes.Although recombinant human TNF-α was previously produced in  culture ,22, the  culture ,52. Latenduction ,14. The 50 values against test cancer cell lines were much lower than that against normal human PBMCs. Moreover, early necroptotic cells with bright-green nuclei and late necroptotic cells with fragmented, orange to red nuclei were distinguished upon treatment of test cancer cell lines with the IC50 of human TNF-α protein.Experimental model validation of RSM revealed that the yield of cell-free production of rhTNF-α was nearly doubled under the optimized condition as compared to the basal condition. Experimental investigations implemented to examine the biological activity of the cell-free synthesized human TNF-α protein confirmed its significant anticancer potency and selective cytotoxicity against test cancer cell lines. Its selective cytotoxicity was observed from the fact that its ICE. coli in vivo expression systems without any problem or need for any cofactor [E. coli and eukaryotic cells share the reducing cytoplasm that strongly disfavors the formation of stable disulfide bonds in proteins, but E. coli cells do not have structures resembling the eukaryotic endoplasmic reticulum. Instead of this, E. coli has an oxidizing periplasm. Therefore, eukaryotic protein expression in bacterial periplasm  is possible. As this cytokine has only a single intramolecular disulfide bond, it is successfully formed in bacteria, not only in E. coli but also in Streptomyces lividans [E. coli as reported by de Marco, 2009 [E. coli chemical protein synthesis machinery not cells, which is the innovation; however, the capabilities of living cells are still available, combined with avoiding the negatives of cell-confining membranes and the possibility of having reducing agent(s) to maintain reduced conditions. We used E. coli and its cell-free system before in production of another recombinant cytokine with a single disulfide bond, human consensus interferon-alpha, and obtained functional antiviral and anticancer protein [E. coli cell-free systems and their in vivo counterparts share the same problem, which is a limited ability to correctly fold proteins containing multiple disulfide bonds, which is not the case here [Human TNF-α protein has a single intramolecular disulfide bond between two cysteine residues at residues 69 and 101, which was successfully formed many times in cofactor ,14,22. Elividans . The proco, 2009  and Ma eco, 2009 . The use protein ,37,55. Aase here ,57. Morease here ; thus, tE. coli crude cell extract-based CFPS system for rapid expression of soluble and functionally active human TNF-α protein, which can provide the requirements for research and therapeutic applications of TNF-α.Overall, we suggest the use of an


Alveolar-capillary leak is a hallmark of the acute respiratory distress syndrome (ARDS), a potentially lethal complication of severe sepsis, trauma and pneumonia, including COVID-19. Apart from barrier dysfunction, ARDS is characterized by hyper-inflammation and impaired alveolar fluid clearance (AFC), which foster the development of pulmonary permeability edema and hamper gas exchange. Tumor Necrosis Factor (TNF) is an evolutionarily conserved pleiotropic cytokine, involved in host immune defense against pathogens and cancer. TNF exists in both membrane-bound and soluble form and its mainly -but not exclusively- pro-inflammatory and cytolytic actions are mediated by partially overlapping TNFR1 and TNFR2 binding sites situated at the interface between neighboring subunits in the homo-trimer. Whereas TNFR1 signaling can mediate hyper-inflammation and impaired barrier function and AFC in the lungs, ligand stimulation of TNFR2 can protect from ventilation-induced lung injury. Spatially distinct from the TNFR binding sites, TNF harbors within its structure a lectin-like domain that rather protects lung function in ARDS. The lectin-like domain of TNF -mimicked by the 17 residue TIP peptide- represents a physiological mediator of alveolar-capillary barrier protection. and increases AFC in both hydrostatic and permeability pulmonary edema animal models. The TIP peptide directly activates the epithelial sodium channel (ENaC) -a key mediator of fluid and blood pressure control- upon binding to its α subunit, which is also a part of the non-selective cation channel (NSC). Activity of the lectin-like domain of TNF is preserved in complexes between TNF and its soluble TNFRs and can be physiologically relevant in pneumonia. Antibody- and soluble TNFR-based therapeutic strategies show considerable success in diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel disease, but their chronic use can increase susceptibility to infection. Since the lectin-like domain of TNF does not interfere with TNF’s anti-bacterial actions, while exerting protective actions in the alveolar-capillary compartments, it is currently evaluated in clinical trials in ARDS and COVID-19. A more comprehensive knowledge of the precise role of the TNFR binding sites versus the lectin-like domain of TNF in lung injury, tissue hypoxia, repair and remodeling may foster the development of novel therapeutics for ARDS. About a century ago the New York surgeon William Coley observes that vaccination of cancer patients with a mixture of bacterial toxins (Coley’s toxins) induces tumor remission . Six decIn the 1980s, the TNF gene is cloned by several groups in Belgium, Japan and the United States  and is fTumor necrosis factor is a key mediator for the activation and recruitment of inflammatory cells, including polymorphonuclear neutrophils (PMNs) and macrophages, to sites of infection, and is as such involved in infection control, tissue repair and healing . HoweverTumor necrosis factor expression is induced upon contact with pathogen- or danger-associated molecular patterns (PAMPs and DAMPs) in immune cells, as well as in numerous types of non-immune cells, including endothelial and epithelial cells, neurons, fibroblasts, microglia and cardiomyocytes . As summWhereas both transmembrane TNF (mTNF) and soluble TNF (sTNF) can bind to both TNF receptors, TNFR2, which is typically expressed in the membrane of immune cells, only becomes fully activated by transmembrane TNF . Both mTN-terminal extracellular domain (ECD) comprising four cysteine-rich domains , an α-helical transmembrane domain, a transmembrane segment and a cytoplasmic intracellular death domain   is a proh domain . TNFR1 ih domain . Upon bih domain . These pN-terminal ECD made by four cysteine-rich domains (CRDs) as well as an α-helical transmembrane domain. In contrast to TNFR1, the cytoplasmic domain of TNFR2 harbors no death domain   is a trah domain . TNFR2 ih domain , some soh domain . Somewhahe brain .L in lung microvascular endothelial cells (MVEC) , it nevertheless promotes TNFR1-mediated apoptosis, upon degrading anti-apoptotic factors, such as Bcl-xs (MVEC)  or TRAF-s (MVEC) .Like TNF, also the TNF receptors can be cleaved from the membrane by TACE . In addiN,N′-diacetylchitobiose and branched tri-mannoses  and capillary endothelial cells (represent a surface area around 90 m2) provides a large barrier that ordinarily prevents serum proteins and fluid from leaving the capillaries and entering the alveoli. While preventing the movement of serum proteins and liquid into the alveolar space, the barriers must allow breathing, i.e. facilitate the movement of O2 from the alveoli into the blood and CO2 from the blood into the alveoli. Disruption of the alveolar epithelial barrier results in pneumonia, but additional compromise of the endothelial barrier can lead to the acute respiratory distress syndrome (ARDS) -an underdiagnosed potentially lethal complication of severe pneumonia. COVID-19, sepsis and trauma  and Na+ release into the interstitial space via basolateral Na+-K+-ATPase in AT1/2 cells  cells ]  turbed)] . Dependiturbed)] . Moreoveturbed)]  and fromturbed)] . Thus, t tissues . Moreove tissues . The parAlthough TNFR1 is sufficient to induce sensitized apoptosis  in primary lung mouse MVEC, the presence of both receptors is required for direct TNF-induced cell death . While TTNF-TNFR2 interactions can be either destructive or preventive . On the 2+ influx, with TAT-Gap19 protected mice against TNF-induced vascular leakage and mortality (Receptor interacting protein kinase 1(RIPK1)–dependent endothelial necroptosis underlies the TNF-induced systemic inflammatory response syndrome in mice  and the ortality . Laminarortality .2+-influx is the initial pivotal signal preceding pathways leading to contraction, including myosin light chain (MLC)-dependent mechanisms and MLC-independent microtubule rearrangement  and VEGF signaling pathways converge at the level of the secondary messenger, SAPK-2/p38, with continuous autocrine signaling by mTNF sensitizing endothelial cells to respond to VEGF by increasing their vascular permeability .2-rich environments, they use the glycolytic pathway, rather than mitochondrial respiration as the main ATP-generating pathway in quiescent and proliferating states. They likely do so in order to maximize O2 delivery to tissues and perivascular cells. This prevents the generation of high levels of reactive oxygen species in this cell compartment -mediated NO generation was shown to promote pulmonary microvessel leakage in eNOS–/– mice , suggest–/– mice . Increas–/– mice  and to i–/– mice . TNF wasactivity . All of activity . A stop activity . By contactivity .in vitro (2+ transport protein antioxidant-1 (Atox1) stimulates transcription of the NADPH oxidase organizer p47phox, thereby increasing VCAM-1/ICAM-1 expression and promoting monocyte adhesion in endothelial cells inflamed with TNF  and basolateral Na+-K+-ATPase in type I and II alveolar epithelial cells , an enzyme located in lysosomes and in the extracellular space, which catalyzes the conversion of sphingomyelin, a major component of membranes, into ceramide and phosphocholine . Low levApart from inhibiting ENaC, TNF/TNFR1 signaling can also affect mRNA and/or protein expression of other channels involved in AFC. As such, TNF impairs expression of the major water channel aquaporin 5, expressed in alveolar, tracheal, and upper bronchial epithelium, through a mechanism involving activated NF-κB , 2001. MPseudomonas aeruginosa (P. aeruginosa)-induced pneumonia model in rats, Rezaiguia et al., measured alveolar protein and liquid clearance, 24 h after intratracheal instillation of bacteria, by instilling 5% bovine albumin solution with 125I-labeled human albumin into the airspaces  and the cell surface (N) expression of ENaC , can activate amiloride-sensitive Nasubunits . This efsubunits  thus cle of ENaC . Treatme peptide . This in peptide . The eff peptide . The TIPtide  and pneumolysin . Both LLO and PLY induce Ca2+-influx, which in turn activates Ca2+-dependent PKC isozymes, such as PKC-α, involved in capillary barrier dysfunction  ratio from about 70 mm Hg -indicating a condition similar to severe ARDS- to more than 400 mm Hg, which rather corresponds to a mild hypoxia-induced lung injury where a patient can typically walk up even stairs  are changed for alanine also fail to restore oxygenation. These data strongly suggest that the significant protective effect of the TIP peptide in a severe ischemia-reperfusion lung injury model involves the lectin-like activity of TNF and is due to the activation of Na+ uptake via ENaC  and the adherens junction protein VE-cadherin . The sheAnother study documenting the anti-inflammatory capacity of the TIP peptide comes from a study using a rat model of high-altitude pulmonary edema (HAPE). In this study, treatment with TIP peptide  reduces leukocyte infiltration and pro-inflammatory cytokine levels in lavage, while improving occludin expression in alveolar epithelial cells and reducing edema formation .2 generation in glomerular endothelial cells, which in turn promotes eNOS-dependent NO generation and barrier function , as well as proteinuria and blood urea nitrogen . TIP pepfunction . Despitefunction  before iversus placebo in the mechanical ventilator over seven days. Although no significant reduction in EVLWI occurs with TIP peptide inhalation over the entire patient population, it is interesting to note that there is a positive effect in a sub-group of patients with a sequential organ failure assessment (SOFA) score >11 , accompanied by a reduced ventilation pressure , is showing promising outcomes in recent clinical trials in ARDS and lung transplantation and may represent a prototype for alternative therapeutic strategies, apart from TNF inhibitory antibodies or soluble receptor constructs, to blunt the harmful, but retain the potentially beneficial activities of TNF required in host defense in acute lung injury and ARDS, as well as in bacterial and viral pneumonia, including COVID-19 pneumonia. It should probably further be studied in important clinical settings where thus far no specific treatment exists, such as in idiopathic lung fibrosis.Much experimental and clinical work has to be done for scientists and clinicians to better understand the role of TNF in lung diseases such as ARDS, in order to be able to therapeutically modulate the cytokine and its associated inflammatory disorders and in order to improve patient outcome and as such health care. Novel mediators and pathways -like those related to the lectin-like domain of TNF- might contribute to this endeavor.JH, YH, DE, MR, and RL wrote the manuscript. RL, GC, RC, HH, SS, AL, MH, DA, MU-F, TF, TC, AV, DE, MR, and JH contributed to studies reported in the review. PE, DA, MU-F, TF and TC assisted with redaction of the manuscript for clarity and with the figures. All authors contributed to the article and approved the submitted version.RL is inventor of several patents relating to the use of the TNF-derived TIP peptide in pulmonary edema reabsorption. The remaining 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.


Uveitis is an inflammation of the iris, ciliary body, vitreous, retina, or choroid, which has been shown to be the first manifestation of numerous systemic diseases. Studies about the immunopathogenesis and treatment of uveitis are helpful to comprehend systemic autoimmune diseases, and delay the progression of systemic autoimmune diseases, respectively. Tumor necrosis factor-alpha (TNF-α), a pleiotropic cytokine, plays a pivotal role in intraocular inflammation based on experimental and clinical data. Evidence of the feasibility of using anti-TNF-α agents for uveitis management has increased. Although there are numerous studies on TNF-α in various autoimmune diseases, the pathological mechanism and research progress of TNF-α in uveitis have not been reviewed. Therefore, the objective of this review is to provide a background on the role of TNF-α in the immunopathogenesis of uveitis, as well as from bench to clinical research progress, to better guide TNF-α-based therapeutics for uveitis. Uveitis is a heterogeneous nosological entity. Although the uvea is defined as the middle membrane  of the oTumor necrosis factor-alpha (TNF-α) is an acidic protein that is mainly produced by macrophages in response to infection and inflammatory irritation. It is related to chronic inflammation and tissue damage in uveitis and is critical for initiating immunity to pathogens . TNF recAlthough uveitis represents a group of intraocular inflammatory conditions, each with its own phenotypic heterogeneity, the commonality is the increased expression of TNF-α in both the serum and aqueous humor. Over the past decade, studies have increasingly emphasized the effectiveness of anti-TNF-α agents for patients with uveitis. Although there are numerous studies on TNF-α in various autoimmune diseases, the pathological mechanism and research progress with respect to the role of TNF-α in uveitis have not been reviewed. Thus, in this review, we aimed to provide a background of the role of TNF-α in the immunopathogenesis of uveitis and an account of the progress from bench to clinical research progress to better guide TNF-α-based therapeutics for uveitis.TNF-α is a cytokine with diverse functions, including inflammation, immunity, cellular communication, cell differentiation, cell death, and survival, and a variety of signaling pathways. Although TNF was identified as early as 1975, the true identity of TNF was unclear until 1984 when Aggarwal et al. reported the isolation of cytotoxic factors, one of which was derived from macrophages, named TNF . Using tTNF-α primarily exists as a trimeric transmembrane protein, transmembrane TNF-α (tmTNF-α), which is subsequently cleaved by TNF-α converting enzyme  into a soluble form (sTNF-α) . TNF-α hWhen TNF-α binds to TNFR1, it assembles different signaling complexes consisting of the complexes I, IIa, IIb (ripoptosome), and IIc (necrosome), resulting in different functional outcomes . TNF-α cWith the binding of TNF-α to TNFR1, TRADD , receptoin vivo, but silencing IL-24 in Th17 cells enhanced the disease. Some studies on inhibiting EAU by blocking the p38 signaling pathway, cannabidiol  . By contIn 1996, a study showed that the neutralization of systemic TNF-α ameliorates the pathology of EAU, and interference with afferent processes, especially antigen priming, is important to protect against EAU through anti-TNF-α treatment . A similIn 1990, some investigators showed that TNF-α antagonists prevent adhesion molecule upregulation on the vascular endothelial cells in rheumatoid arthritis (RA) and experimental allergic encephalomyelitis . In 2011+ cells, increasing the percentage of Treg cells, and suppressing ocular inflammation during EAU  has been used to treat specific patients with uveitis over the last decades. Corticosteroids are an important component of IMT and are also the first-line treatment for uveitis. However, patients with uveitis are at risk of long-term complications caused by long-term uncontrolled inflammation and corticosteroid therapy, which can reduce the treatment success rate for the disease itself. Therapeutic strategies have evolved over the last few years, and anti-TNF-α agents have become well accepted for the treatment of refractory uveitis. Anti-TNF-α agents have fewer adverse effects than corticosteroids. Studies have shown that when used properly, dependence on corticosteroids can be significantly reduced to prevent uveitis recurrence .The first use of anti-TNF-α agents was reported in the 1980s in experimental models of sepsis . In 19851) Compared with IFX, ADA is a fully human monoclonal antibody that causes almost no allergic reactions, and subcutaneous injection is safer and more convenient than intravenous injection .2) During steroid tapering, ADA significantly reduces the relapse rate, visual deterioration, and anterior chamber flare, and has relatively good tolerance .3) The use of ADA in the treatment of uveitis associated with Behçet’s disease (BD) is not affected by the concomitant application of antirheumatic agents .4) Numerous studies have shown that ADA is superior to immunosuppressive agents in decreasing the relapse rate and occurrence of retinal vasculitis and improving visual acuity .5) ADA is safe and efficacious for the treatment of non-infectious uveitis in elderly patients .6) ADA seems to be associated with better outcomes after follow-up, although both IFX and ADA are efficacious for refractory BD-related uveitis .7) ADA plus conventional therapy outperforms conventional therapy alone in patients with retinal vasculitis due to refractory BD-related uveitis .8) In children and adolescents with active juvenile idiopathic arthritis (JIA)-related uveitis, the treatment failure rate of ADA is lower than that of the placebo .The advantages of ADA are listed as follows:1) Adverse events were reported in patients who received ADA . The mos2) The use of ADA for undifferentiated uveitis might result in premature discontinuation on account of side effects .The disadvantages of ADA are as follows:1) Non-infectious uveitis, intermediate uveitis, posterior uveitis, and panuveitis in adult patients with underreaction and contraindications to steroids, as well as steroid dependence in Europe .2) Non-infectious uveitis, intermediate uveitis, posterior uveitis, and panuveitis in adult patients in the United States .3) As a first-line immunomodulator for the treatment of ophthalmic manifestations of BD .4) As a second-line immunomodulator for the treatment of uveitis associated with JIA .5) ADA is approved for RA, ulcerative colitis, psoriatic arthritis, ankylosing spondylitis (AS), Crohn’s disease, and plaque psoriasis in adults .The indications are as follows:1) IFX showed commendable efficacy for refractory non-infectious uveitis and severe uveitis cases associated with BD whether it was used as monotherapy or with other immunosuppressive agents .2) IFX showed a significantly higher capacity to resolve macular edema in treating sight-threatening retinal vasculitis when compared with the effects of ADA .3) A report indicated that IFX is effective as a treatment for visually threatening refractory posterior uveitis .4) Multiple studies have shown that IFX is superior to immunosuppressive agents in reducing recurrence rates and ameliorating visual acuity .5) Arida et al. reported that 40% of BD cases remained in remission 3 years after the discontinuation of IFX . In the The advantages of IFX are listed as follows:1) Tolerance is low owing to the relatively frequent infusion reactions .2) Tuberculosis as an adverse effect was reported in patients treated with IFX .3) One study reported a higher rate of IFX toxicity in patients with uveitis .The disadvantages of the IFX are listed as follows:1) Numerous experts have recommended IFX as first-line therapy for visually threatening BD  .2) As a second-line immunomodulator for the treatment of uveitis related to JIA .3) For the treatment of severe ocular inflammatory conditions including posterior uveitis, panuveitis, severe uveitis associated with seronegative spondyloarthropathy, and scleritis in patients requiring immunomodulation .4) Infliximab is authorized by the FDA for the treatment of RA, AS, Crohn’s disease, psoriatic arthritis, plaque psoriasis in adults, and ulcerative colitis .The indications are as follows:1) Compared with IFX, GOL is a fully human monoclonal antibody that causes almost no allergic reactions .2) GOL is effective in improving visual acuity and controlling ocular inflammation .3) GOL has been proven to be conducive to AS-related anterior uveitis, ameliorating macular edema and inflammation, and decreasing the relapse rate .4) The control of intraocular inflammation with multi-refractory uveitis associated with BD .5) GOL represents an efficacious and secure therapy choice for uveitis with a significant reduction in the frequency of ocular flares while preserving visual function with a satisfactory long-term retention rate .6) The effective treatment of JIA and idiopathic retinal vasculitis by GOL has been reported, whereas other anti-TNF-α agents are ineffective .The advantages of the GOL are listed as follows:1) CZP can be an effective alternative to long-lasting chronic relapsing uveitis .2) Some studies have shown a significant decrease in ocular flares with a satisfactory long-term retention rate with CZP compared to that with placebo .3) A national multicenter observational study supported the efficacy of CZP for the management of uveitis during pregnancy . In termin vitro neutralizing potency is higher for CZP than for ADA (4) One study showed that the relative  for ADA .5) One study observed positive outcomes using CZP as therapy for patients with refractory, non-infectious uveitis when other anti-TNF-α agents proved inadequate or when tolerance issues were present .The advantages of the CZP are listed as follows:1) Owing to its poor intraocular permeability and limited effectiveness, it is not recommended for uveitis .2) Granulomatosis, as a side effect, has been reported in the treatment of uveitis with etanercept .3) Meta-analyses have shown that etanercept is inferior to other anti-TNF-α agents for uveitis treatment .4) Paradoxical occurrences of uveitis have also been reported after etanercept administration in patients with AS-related acute anterior uveitis .5) Etanercept might be less efficient than other anti-TNF-α agents in decreasing the risk of HLA-B27-related acute anterior uveitis in patients with spondyloarthritis .The disadvantages of etanercept are listed as follows:1) Etanercept received FDA approval for RA, polyarticular JIA, AS, psoriatic arthritis, and plaque psoriasis (in patients aged 17 years and older) .2) International guidelines concluded that the use of etanercept for the treatment of uveitis is not supported .The indications are as follows:Inefficiently controlled or untreated uveitis is one of the primary causes of blindness in developed countries. Corticosteroids remain the first-line treatment; however, their chronic use can result in side effects. These complications have led investigators to seek corticosteroid-sparing treatments. Although uveitis represents a group of intraocular inflammatory conditions with distinct phenotypic heterogeneity, its common feature is increased expression of TNF-α in both the serum and aqueous humor. Over the past decade, studies have increasingly emphasized the effectiveness of anti-TNF-α agents for patients with uveitis. However, the lack of clinical trials and the rarity and heterogeneity of uveitis make their utilization in ophthalmology more challenging, particularly for first-line therapy.Most international studies have focused on ADA and IFX, which are the most commonly recently employed biological agents for patients with uveitis. Authoritative experts recommended the use of ADA in cases of nullity or intolerance to immunosuppressive agents for non-infectious non-anterior uveitis. IFX was proposed as a first-line treatment for sight-threatening uveitis associated with BD. Nevertheless, knowing which of the two has a better effect in combating uveitis is an unmet demand. ADA is well tolerated with acceptable side effect profiles, and its costs have also decreased to acceptable levels. These properties make it an excellent option as second-line and reserved steroid therapy for uveitis. However, whether the earlier introduction of ADA would confer additional benefits in the management of uveitis and the preservation of visual function is unclear. GOL seems to have more evident advantages as a therapy for spondylitis-related uveitis. In terms of pregnancy safety, CZP has favorable characteristics over other anti-TNF-α agents owing to its limited transport across the placenta.in vivo and in vitro experiments.Furthermore, there are still many questions regarding the use of anti-TNF-α agents as a therapy for uveitis, including the following: treatment duration, when to stop using, the necessity to monitor drug levels regularly, alternative biological agents if anti-TNF-α failure occurs, how to reduce the immunogenicity against anti-TNF-α molecules, and how to ameliorate efficacy. Moreover, treatment failure when using one anti-TNF-α agent does not indicate that other agents in the same group will also be ineffective. Some studies have reported that agents in a group can be replaced with each other by changing novel routes of drug administration to less intense places such as subcutaneous injections. The development of monoclonal antibodies that simultaneously recognize multiple targets allows for more effective treatment of uveitis at a lower dose than that with any single biological drug. Alternatively, a secure and efficient sustained-release device can be developed that will enable the topical treatment of idiopathic immune-mediated uveitis with immunomodulators, including biological response modifiers. The pathogenic effect of TNF-α is caused by complex signaling pathways composed of cascades of signaling molecules. When the efficacy of anti-TNF-α agents is not good, changing therapeutic targets to different signaling molecules in the pathway is also a good alternative. However, these ideas need to be verified with additional


The rabbit hemorrhagic disease virus (RHDV) vaccine platform is a nanoparticle composed of 180 copies of the viral capsid protein, VP60, self-assembled into virus-like particles (VLPs). RHDV VLPs are able to accept the simultaneous incorporation of target epitopes at different insertion sites. The resulting chimeric RHDV VLPs displaying immunogenic foreign antigens have been shown to induce specific protective immune responses against inserted heterologous T-cytotoxic and B-cell epitopes in the mouse and pig models. In this study, we explored whether RHDV-based engineered VLPs can be developed as efficient multivalent vaccines co-delivering different foreign B-cell antigens. We generated bivalent chimeric RHDV VLPs displaying two model B-cell epitopes at different surface-exposed insertion sites, as well as the corresponding monovalent chimeric VLPs. The immunogenic potential of the bivalent chimeric VLPs versus the monovalent constructs was assessed in the mouse model. We found that the bivalent chimeric VLPs elicited a strong and balanced antibody response towards the two target epitopes tested, although slight reductions were observed in the levels of specific serum antibody titers induced by bivalent chimeric VLPs as compared with the corresponding monovalent constructs. These results suggest that RHDV VLPs could represent a promising platform for the development of efficient multivalent vaccines. Virus-like particles (VLPs) are nanoparticles built from self-assembled proteins, usually viral capsid subunits, which mimic virion well-defined geometry, while being devoid of genetic material. They are outstanding and safe immunogens combining a highly ordered and particulate nature with a lack of replication ability ,2,3,4. BRabbit hemorrhagic disease virus (RHDV) VLPs represent a promising platform for multimeric antigen display ,14,15,16The ~40 nm diameter RHDV capsid comprises 90 dimers of the single capsid protein arranged in T = 3 icosahedral symmetry, forming 12 pentamers and 20 hexamers. Each monomer has three domains  Figure a, an N-tWe have previously shown that RHDV VLPs are useful as diagnostic reagents or vaccine candidates for the control of RHDV in rabbits ,24. FurtBuilding on the RHDV VLP platform proof of concept, this study further explores a relevant issue: whether RHDV-based engineered VLPs can be developed as efficient multivalent vaccines co-delivering different foreign B-cell antigens. Our aim was to evaluate if chimeric RHDV VLPs harbouring two different B-cell epitopes would be able to induce strong humoral responses against both of them, or if immune interference between the target epitopes incorporated could exert a detrimental effect in the foreign antigen-specific immune responses elicited by such bivalent vaccines. We used two model epitopes: a newly described neutralizing B-cell epitope derived from the feline calicivirus (FCV) capsid protein (epitope FCV22) ,27 and aThe B-cell epitopes: FCV22 (GSGNDITTANQYDAADIIRN), derived from FCV capsid protein , and 2L2Autographa californica nuclear polyhedrosis virus (AcMNPV), were propagated in Trichoplusia ni High five cells (H5), grown in monolayer cultures at 28 °C in TNM-FH medium , supplemented with 5% fetal calf serum .Recombinant baculoviruses expressing RHDV VLPs, generated from derivatives of Feline calicivirus (FCV), Urbana strain, kindly provided by K.Y. Green ; canine parvovirus (CPV) CPV2a strain, kindly provided by P. Rueda (Eurofins Ingenasa); and the feline cell line CRFK  were used to perform indirect immunofluorescence assays.C57BL6 female mice , 7–8 weeks old, were used to evaluate the immunogenicity of the chimeric VLP constructs.Plasmid pHAPh306GSopt, containing the gene of VP1 protein (codon-optimized for expression in insect cells), along with the coding sequence of VP2 and the 3′ untranslated region of RHDV  , was useDNA sequences corresponding to the FCV-derived epitope  and to the CPV-derived epitope (2L21 SDGAVQPDGGQPAVRN-ERATGS), flanked by sequences corresponding to amino acids GS, were incorporated at the insertion sites used in this study: between amino acid positions 306 and 307 (surface-exposed loop L1) and/or between amino acid positions 342 and 343 (surface-exposed loop L2), within the P2 subdomain of RHDV capsid protein c. PlasmiRecombinant baculoviruses were generated using the flashBACULTRA baculoviral genome , following the manufacturer’s instructions.Baculovirus infected H5 cell monolayers were harvested after incubation for 4 days at 28 °C, washed three times with 0.2 M phosphate-buffered saline for VLPs  and pellets were resuspended in distilled water. After mild sonication and treatment with DNAse I , samples were adjusted to 2% Sarkosyl , 5 mM EDTA in PBS-V, and incubated overnight at 4 °C. Next, cell lysates were clarified by low-speed centrifugation and the supernatants were centrifuged using a Beckman SW28 rotor at 27,000 rpm for 2 h. Pellets were resuspended in PBS-V, extracted with Vertrel XF , and centrifuged using a Beckman SW28 rotor at 27,000 rpm for 2 h. The pelleted material was subjected to centrifugation through a 20% (wt/vol) sucrose cushion in PBS-V at 35,000 rpm for 2.5 h using a Beckman SW55 rotor. Finally, the pellets were resuspended in PBS-V containing protease inhibitors  and stored at 4 °C. Protein concentrations of the VLP samples were determined using BCA protein assay kit, Pierce, Thermo Scientific .Recombinant protein expression was analyzed by SDS-polyacrylamide gel electrophoresis (PAGE).®® Turbo™ Transfer System . Membranes were saturated  with PBS, 5% (wt/vol) skim milk, and 0.5% (vol/vol) Tween 20 and incubated  with either a rabbit hyperimmune serum against RHDV to detect VP60 protein, 51D10 monoclonal antibody recognizing FCV22 epitope [For Western blot analyses, proteins were transferred from gels onto polyvinylidene difluoride (PVDF) membranes using Trans-Blot epitope , or 3C9  epitope . After sSamples (approximately 5 μL) were applied to glow discharged carbon-coated grids for 2 min and negatively stained with 2% (wt/vol) aqueous uranyl acetate. Images were recorded with a JEOL JEM-1011 electron microscope  operated at 100 kV, with a 4K × 2.7K ES1000W Erlangshen CCD camera  at a nominal magnification of 24,000×.Groups of eight inbred C57BL6 mice , female, 7–8 weeks old, were immunized by subcutaneous route at days 0 and 22, with 100 µg of the corresponding chimeric VLP constructs emulsified in Montanide ISA 50V2  in a 1:1 (vol/vol) ratio and sacrificed at day 40 post-immunization. Negative control groups of five mice were inoculated with PBS or the native RHDV VLPs, emulsified with the same adjuvant. Blood samples were collected at day 0 (before priming) and at day 40.2SO4 and absorbance at 450 nm was measured on a Fluostar Omega microplate reader. Antibodies against RHDV VP60 capsid protein were determined by ELISA. Here, 300 ng/well of RHDV VLPs diluted in 0.05 M carbonate-bicarbonate buffer (pH 9.6) was incubated at 4 °C overnight in Maxisorp Nunc 96-well plates . After three washes with PBS-0.1%Tween-20, wells were saturated with PBS, 5% (wt/vol) skim milk, for 1 h at 37 °C. Subsequently, sera samples were serially diluted three-fold in PBS and 3% (wt/vol) skim milk and incubated for 1 h at 37 °C. After three washes, plates were incubated with HRP-conjugated goat anti-mouse IgG  at a 1:2000 dilution for 1 h at 37 °C. Finally, plates were extensively washed and colour reaction was developed with TMB . The reaction was stopped by addition of 1.8N H4HCO3. The procedure was completed as described above for RHDV-specific antibody ELISAs.Likewise, antibodies against target epitopes FCV22 and 2L21 were determined by ELISA using 96-well High Binding plates  coated with 5 µg/well of the corresponding synthetic peptide, diluted in 30% 100 mM NHTwo control samples  were added to each plate: a monoclonal antibody specific against the target antigen .Indirect immunofluorescence assays (IFAs) were performed to assess the reactivity of the immunized mice sera with the specific antigens (FCV capsid protein and CPV VP2 protein) in the context of viral infection. Feline CRFK cell monolayers, infected either with FCV (Urbana strain) or CPV (CPV2a strain), were fixed with formalin 10% (vol/vol) on coverslips. Once fixed, cells were treated with PBS and 0.2% (vol/vol) triton X-100 for 10 min at room temperature, and then saturated with blocking buffer  bovine fetal serum, 1% (wt/vol) bovine serum albumin, and 0.1% (vol/vol) triton-X100) for 30 min. Subsequently, dilutions of sera samples in blocking buffer were incubated with fixed cells for 1 h at room temperature. After washing, Alexa Fluor 488 goat anti-mouse  was incubated at 1:500 dilution to detect previous antigen–antibody reactions, for 45 min at room temperature. Fluorescence was preserved using Prolong Gold antifade reagent . The images were obtained with an inverted fluorescence Carl Zeiss Axio Vert A1 microscope.p-values were two-sided and p < 0.05 was considered statistically significant. In the figures, p-value criteria were assigned as follows: ns (not significant) p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.Data were analyzed using GraphPad Prism Software 6.00 . Geometric mean titers (GMTs) of IgG in serum were determined for all the groups of mice. One-way analysis of variance (ANOVA), followed by Bonferroni post-hoc comparison tests, were used for comparison of antibody titers among mice groups. All The experiments involving mice immunizations were performed at CISA CSIC-INIA animal facilities, in accordance with national and European Union animal experimentation guidelines. The study was previously approved by the Ethical Committee for Animal Experimentation (CEEA 2014/018) and Biosecurity Committee of INIA (CBS 2014/015). Experimental procedures were performed according to protocols approved by the Nacional Committee on Ethics and Welfare (PROEX 228/14).We generated a set of recombinant baculoviruses expressing different VP60 insertion mutants incorporating foreign B cell epitopes either at exposed loop L1 and/or exposed loop L2 within P2 subunit of protein VP60 c,d. The SDS-PAGE analyses of H5 insect cell cultures infected with the recombinant baculoviruses showed that all the constructs were expressed at grossly similar levels a. LysateThe infected cell extracts were also subjected to Western blot analysis to verify the presence of the inserted target epitopes b. A rabbThe ability of the novel chimeric constructs to assemble into VLPs was assessed by electron microscopy . BaculovThe immunogenicity of chimeric VLPs was evaluated in vivo in the mouse model. Groups of eight C57BL/6 mice were immunized twice subcutaneously with 100 µg of each purified VLP emulsified in Montanide ISA 50V2 adjuvant . As controls, groups of five mice were immunized with PBS (C-group) or native RHDV VLPs (VP60 group) using the same adjuvant. Sera samples collected at days 0 (preimmune sera) and 40 post-immunization were assayed for antibodies against VP60 protein (RHDV VLPs)  or the FSerum IgG antibody titers were measured by ELISA, and geometric mean titers (GMTs) were calculated for all the mice groups. Preimmune and PBS negative control group sera showed no reactivity against the three antigens tested.5 to 7.22 × 105, All mice immunized with RHDV-derived VLPs elicited high titers of VP60-specific antibodies (GMTs ranging from 1.33 × 103 to 6.20 × 104), while sera from mice immunized with PBS (C-group) or native RHDV VLPs (VP60 group) showed no reactivity with the target epitopes.Regarding the antibody responses elicited against the inserted target epitopes, all chimeric RHDV VLPs displaying the FCV-derived a or the 4 and 2.59 × 104, respectively 4 and 1.87 × 104, respectively Regarding the influence of the insertion site (incorporation of target epitope in loop L1 vs. loop L2), no significant differences were observed with both target epitopes tested, as specific antibody titers elicited were similar when comparing groups 1F and 2F  induced higher specific antibody titers than those induced by chimeric VLPs harbouring the target epitope only at one insertion site per monomer, although the titer differences observed were only statistically significant in the case of the chimeric VLPs harbouring the CPV-derived epitope . When comparing humoral responses elicited by bivalent versus monovalent VLPs, titers induced by bivalent VLPs were shown to be slightly lower, suggesting some degree of immune interference between the target epitopes. However, statistically significant differences were only observed in the case of the responses induced against FCV-derived epitope . These results suggested that the bivalent chimeric RHDV VLPs allowed good immunogenicity for both target epitopes tested.Finally, concerning the effect of co-delivery of two different target epitopes, the two bivalent chimeric VLPs (1F2C and 1C2F) were shown to induce high antibody titers against both target epitopes , in up to four different insertion sites, or up to three tandem copies at one insertion site (spanning up to 62 amino acids), without disrupting the self-assemble ability of RHDV capsid protein ,15,25,54Immunization of mice with the chimeric RHDV VLPs elicited high titers of antibodies against the VLP carrier , in agreWe tested two insertion sites, incorporating the same target epitopes at each of them . The results obtained indicated both target epitopes induced similar specific antibody titers, regardless of the localization, suggesting the two insertion sites used in this study were similarly efficient for antigen display .Chimeric VLPs displaying two copies of a target epitope per RHDV capsid monomer (1F2F and 1C2C) induced higher specific antibody titers than those induced by constructs displaying only one copy of the same epitope per monomer , althougp < 0.05) observed, indicating that immunity against FCV target epitope incorporated at loop L2 was negatively affected by the presence of CPV epitope at loop L1. However, the detrimental effect was not observed when the target epitopes were allocated at the reversed positions (1F vs. 1F2C). In the case of the CPV epitope, no significant differences were observed irrespective of the insertion site used. The results suggest that different target epitopes may exhibit distinct insertion site preferences, and this should be taken into account in order to obtain optimal specific immune responses.Finally, concerning the effect of co-delivery of the two different target epitopes, we generated two bivalent chimeric VLPs incorporating both epitopes at the reversed positions (constructs 1F2C and 1C2F) in order to assess whether the inherent immunogenicity of the two epitopes used, or their localization on the VLP capsid surface, was responsible for possible differences detected in the induction of specific antibody titers. This analysis could provide relevant information about the optimal spatial arrangement for the selected target epitopes within the VLP scaffold. As shown in In summary, the results obtained indicated that the bivalent chimeric RHDV VLPs allowed good simultaneous immunogenicity for both target B-cell epitopes tested, in agreement with previous reports with other chimeric VLPs ,45,46,47In this study, we evaluated the feasibility of developing RHDV-based engineered VLPs as multivalent vaccines co-delivering different foreign B-cell antigens. We compared the immune responses elicited in mice by bivalent chimeric RHDV VLPs simultaneously displaying two model B-cell epitopes derived from FCV and CPV, with that elicited by the corresponding monovalent chimeric VLPs. Here, we show that bivalent chimeric RHDV VLPs elicited a strong and balanced antibody response towards the two co-delivered B-cell epitopes, despite slight reductions observed in the levels of specific antibody titers induced as compared with cognate monovalent constructs.We believe these results further expand the capabilities of an already suitable choice of delivery system, representing an attractive platform for new vaccine development, both in human and the veterinary field.


HNSCC is a heterogeneous disease, which arises from distinct anatomic subsites, associates with various risk factors and possesses diverse molecular pathological features. Generally, HNSCC is considered as an immunosuppressive disease, characterized by abnormal tumor immune microenvironment. The TNF family plays a crucial role in the survival, proliferation, differentiation, and effector functions in both immune and non-immune cells. However, the expression patterns of TNF in HNSCC remains to be systematically analyzed.We downloaded transcriptional profile data of HNSCC from TCGA and GEO datasets. Unsupervised clustering methods were used to identify different TNF patterns and classify patients for further analysis. PCA was conducted to construct a TNF relevant score, which we called risk score.In this study, we systematically evaluated the patterns of TNF family and tumor immune microenvironment characteristics of HNSCC patients by clustering the expression of 46 members of TNF family. We identified two subtypes with distinct clinical and immune characteristics in HNSCC and constructed a risk scoring system based on the expression profile of TNF family genes.Risk score serves as a reliable predictor of overall survival, clinical characteristics, and immune cell infiltration, which has the potential to be applied as a valuable biomarker for HNSCC immunotherapy. Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors worldwide , 2. HNSCvia either helping tumor cell resist apoptosis or inducing an immune suppressive tumor microenvironment  and a 29 TNF receptor superfamily (TNFRSF), plays a crucial role in the survival, proliferation, differentiation, and effector functions in both immune and non-immune cells . A numbeironment –14. For ironment . Anotherarcinoma . HoweverIn this study, we systematically evaluated the patterns of TNF family and tumor immune microenvironment characteristics of HNSCC patients by clustering the expression of 46 members of TNF family. We identified two subtypes with distinct clinical and immune characteristics in HNSCC and constructed a risk scoring system based on the expression profile of TNF family genes. Risk score serves as a reliable predictor of overall survival, clinical characteristics, and immune cell infiltration, which has the potential to be applied as a valuable biomarker for HNSCC immunotherapy.via the R package TCGAbiolinks (https://xenabrowser.net). All baseline information of HNSCC datasets is summarized in All data used in this study were obtained from public databases. In total, three HNSCC cohorts were included in our study . For TCGA HNSCC cohort, RNA sequencing data [fragments per kilobase of transcript per million mapped reads (FPKM) values] were downloaded Unsupervised clustering methods were used to identify different TNF patterns and classify patients for further analysis. A total of 46 TNF family genes were used to conduct the unsupervised clustering. A consensus clustering algorithm was performed using the R package ConsensuClusterPlus  and was The R package GSVA  was usedThe protein-protein interactions among TNF family proteins were identified on the STRING according to the instructions .p value <0.05 and an adjusted p value <0.2. To identify the most related pathways of TNF family genes, the gseKEGG function of the R package clusterProfiler , where i is the expression of TNF family pattern related signature genes. Risk score of TCGA and two independent GEO cohorts are in First, the prognostic analysis was performed for each gene in the 693 DEGs using a univariate Cox regression model. A total of 177 genes . All statistical p values were two-tailed, with p <0.05 considered as statistically significant. All statistical analyses were conducted using R 4.0.5 (https://www.r-project.org/). .The normality of the variables was tested using the Shapiro-Wilk normality test . For comFirstly, we depicted the multi-omics landscape of 46 TNF family proteins using data from TCGA HNSCC cohort (TNFRSF6B was excluded because of its undetectable expression in TCGA HNSC cohort). We found that the overall mutation rate of all proteins is relatively low in the HNSCC genome  among two groups. A total of 693 TNF pattern-related genes were identified  was the most frequent mutation in HNSCC, and it was considered as an early driver genomic alterations . Our anaThe tumor microenvironment (TME) in HNSCC is a complex and heterogeneous population of tumor cells and stromal cells, which include endothelial cells, cancer-associated fibroblasts (CAFs) and immune cells . GeneralConsidering the distinct characteristics of two TNF clusters, we hypothesized that DEGs between two groups might also have unique characteristics. Accordingly, we conducted unsupervised clustering analysis based on the expression of DEGs and also divided HNSCC patients into two groups, which we called TNF gene cluster A and B. Similar to the results of TNF clusters, two TNF gene clusters also had distinct clinical and immune characteristics. These results demonstrated that there were indeed two immune subtypes based on the expression profile of TNF gene family in HNSCC.Regarding the clinical significance of our study, we constructed a risk score based on the expression of DEGs that can evaluate risk of HNSCC patients individually. Indeed, the patients with high risk score had worse overall survival, which was validated by two independent GEO cohorts. Also, the patients with TP53 mutation or EGFR amplification had a significantly higher risk score and patients with HPV infection had a lower risk score, which was consistent with our TNF clusters and TNF gene clusters.However, our study also had some limitations. First, we failed to find expression profile data of HNSCC patients receiving immune checkpoint blockers to validate the predictive value of our risk score to immune checkpoint inhibitors. Second, our analysis was only based on retrospective data from public databases, which need to be further validated by prospective studies in the future.In conclusion, we identified two subtypes with distinct clinical and immune characteristics in HNSCC and constructed a risk scoring system based on the expression profile of TNF genes. Risk score is capable of serving as a reliable predictor of overall survival, clinical characteristics, and immune cell infiltration, which have the potential to be applied as a valuable biomarker for HNSCC immunotherapy.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/QL, YL, MC, and AY conceived and designed the project. QL, CH, QM, JP, WD, XX, DD, XW, XQ, WZ, DS, and MC analyzed and interpreted the data. QL, YL, MC, and AY wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the funds from the National Natural Science Foundation of China .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.


Objective: Multimorbidity burden across disease cohorts and variations in clinico-radiographic presentations within normal pressure hydrocephalus (NPH) confound its diagnosis, and the assessment of its amenability to interventions. We hypothesized that novel imaging techniques such as 3-directional linear morphological indices could help in distinguishing between hydrocephalus vs. non-hydrocephalus and correlate with responsiveness to external lumbar drainage (CSF responsiveness) within NPH subtypes.Methodology: Twenty-one participants with NPH were recruited and age-matched to 21 patients with Alzheimer’s Disease (AD) and 21 healthy controls (HC) selected from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database. Patients with NPH underwent testing via the NPH programme with external lumbar drainage (ELD); pre- and post-ELD MRI scans were obtained. The modified Frailty Index (mFI-11) was used to stratify the NPH cohort, including Classic and Complex subtypes, by their comorbidity and frailty risks. The quantitative imaging network tool 3D Slicer was used to derive traditional 2-dimensional (2d) linear measures; Evans Index (EI), Bicaudate Index (BCI) and Callosal Angle (CA), along with novel 3-directional (3d) linear measures; z-Evans Index and Brain per Ventricle Ratio (BVR). 3-Dimensional (3D) ventricular volumetry was performed as an independent correlate of ventriculomegaly to CSF responsiveness.Results: Mean age for study participants was 71.14 ± 6.3 years . The majority  of participants with NPH comprised the Complex subtype (overlay from vascular risk burden and AD); 12/21 (57.1%) were Non-Responders to ELD. Frailty alone was insufficient in distinguishing between NPH subtypes. By contrast, 3d linear measures distinguished NPH from both AD and HC cohorts, but also correlated to CSF responsiveness. The z-Evans Index was the most sensitive volumetric measure of CSF responsiveness (p = 0.012). Changes in 3d morphological indices across timepoints distinguished between Responders vs. Non-Responders to lumbar testing. There was a significant reduction of indices, only in Non-Responders and across multiple measures . This was due to a significant decrease in ventricular measurement (p = 0.005) that correlated to independent 3D volumetry (p = 0.008).Conclusion. In the context of multimorbidity burden, frailty risks and overlay from neurodegenerative disease, 3d morphological indices demonstrated utility in distinguishing hydrocephalus vs. non-hydrocephalus and degree of CSF responsiveness. Further work may support the characterization of patients with Complex NPH who would best benefit from the risks of interventions. In this cohort, invasive gold-standard testing may be difficult or inconclusive; the needs of such patients have elevated the importance of developing more precise risk stratification scoring and neuroimaging tools to characterize responsiveness to CSF drainage.The diagnosis of normal pressure hydrocephalus (NPH), first termed by Given early and accurate diagnosis, symptoms of Classic NPH  can be reversed through ventricular shunting . There iBy current clinical standards, 2-dimensional (2d) morphological indices such as the Evans Index (EI), Bicaudate Index (BCI) and Callosal Angle (CA), are used as diagnostic markers in differentiating NPH cohorts to AD and healthy control (HC) cohorts . HoweverOther studies involving the use of morphological indices in NPH cohorts have since supported the potential utility of novel 3-directional (3d) linear indices  toward the differential diagnosis of NPH and AD cohorts , 2017. Tvia ELD.In this study, we examined the use of a 3d morphological methodology to distinguish between cohorts with hydrocephalus vs. non-hydrocephalus and evaluated its performance in Asian patients with NPH, across both Classic and Complex subtypes . Here wevia the NPH programme at the National Neuroscience Institute, Singapore, were recruited prospectively. All patients met criteria for probable or possible NPH according to published guidelines (Twenty-four patients with probable NPH (mean age 71 ± 6.3 years) who underwent the extended CSF drainage protocol idelines , presentidelines . Partici1. AD patients had mild AD, meeting NINCDS-ADRDA criteria for probable AD and a Clinical Dementia Rating of 0.5 or 1.0  and healthy controls  were obtained from baseline scans of patients enrolled to the ADNI 1 phase of the Alzheimer’s Disease Neuroimaging Initiative (ADNI) study5 or 1.0 .TR = 7.3ms, TE = 3.3 ms, flip angle = 8°, FOV = 256 × 256 mm, voxel size = 1.0 × 1.0 × 1.0 mm). Eight patients were downgraded to the 1.5-T scanner at equivalent specifications due to institutional MR safety protocol. AD and HC participants from ADNI were scanned in a 3-T MRI scanner . MRI scanning protocols for each scanner model are available online2.MR imaging data for NPH participants were acquired with a 3-T MR Philips scanner , including 3D T1, T2, FLAIR, and DTI sequences. Three-dimensional axial T1-weighted imaging with sensitivity encoding (SENSE) was acquired (modified Frailty Index-11 (mFI-11) and its components found in Frailty was quantified using the 3  linear and 3-directional (3d) linear measurements, as well as 3-Dimentional (3D) quantitative ventricular volumes3 . This toFirstly, we reproduced the methodology of deriving measurements of morphological indices inaccordance with the work by ACPC Transform and the Resample Scalar/Vector/DWI Volume modules, planes of the T1-weighted DICOM scans were re-aligned parallel to the ACPC line for accurate replication alignment at a radiology workstation. Ruler Module was used to extract measurements for the various morphological indices. Segment Editor was used for the semi-automated segmentation of the ventricles. Segment Statistics was used to derive ventricular volumes by semi-automatic counting of voxels in segments.A flow chart of the methodology used on 3D Slicer is illustrated in Validation of our workflow on 3D Slicer was done by comparing inter-cohort morphological trends of our NPH, AD and HC cohorts to that of U test and Wilcoxon signed rank test. All statistical tests were two-tailed and statistical significance was assumed at p < 0.05. The statistical analyses were performed using R statistical software version 3.3.3. . Categorical variables were compared and analyzed with Fisher’s Exact test. Inter- and intra-group comparisons for morphological measures were tested with independent-samples Mann-Whitney n 3.3.3. 4.via ELD within our NPH cohort. A summary of the demographics of our cohort is found in Twenty-one patients  with NPH were recruited. Of these 21, six were found to be solely consistent with criteria for NPH diagnosis as per international guidelines and classified as Classic NPH; fifteen patients met both the international criteria for their clinico-radiological presentation and our described definition for Complex NPH. Subjects were classified as responders to ELD if their levels of improvement met the minimal clinically important difference (MCID) in at least one domain of NPH symptomatology sufficient to support consideration for shunt insertion. We defined the MCID using the following thresholds: an increase of ≥10% in any measure of inpatient gait, balance, or cognitive testing, matched with a ≥20% functional improvement reported by the patient or caregiver at home following discharge. Using this criteria, there were 9 Responders and 12 Non-Responders to CSF drainage (p = 1.00), and a majority of the frailer patients (mFI-11 ≥ 3) were classified as Complex NPH . The distribution of our NPH subtypes (Responders vs. Non-Responders and Classic vs. Complex groups) stratified by frailty can be found in Following frailty stratification by the mFI-11, 9/21 (42.9%) and 12/21 (57.1%) had a mFI-11 score of 0–2, and ≥3, respectively. The cohort was stratified relatively evenly with respect to ELD response p < 0.001), as seen in Both 2d and 3d linear measurements were able to distinguish between disease cohorts (NPH vs. AD), and between NPH and healthy controls  and tighter high-convexities  . Howeverp = 0.012), and non-significant decreases in the EI and BCI values  and an increase in BVR at the level of the PC (p = 0.024), due to a decrease in the ventricular component of the BVR (p = 0.005). 3D volumetric analysis also supported the significant decrease in ventricular volumes, only in Non-Responders post-drainage (p = 0.008).When we classified patients by their testing timepoints, morphological indices alone were insufficient to distinguish between Responders vs. Non-Responders to CSF drainage, at either pre-or post-ELD . HoweverIn this study, we examined the impact of multimorbidity burden, frailty risks and the efficacy of novel 3d linear measures in the Asian context of hydrocephalus vs. non-hydrocephalus and Classic vs. Complex NPH subtypes. Comorbidities, such as hypertension (81%), diabetes mellitus (33.3%) and myocardial infarction/percutaneous coronary intervention/angina (3%), were commonly found in our subjects. A higher comorbidity profile is known to decrease the chances of favorable outcomes in NPH patients who undergo ventricular shunting . After ETo investigate the frailty risks of our population, we stratified our patients by means of a well-validated frailty score, in order to quantify biological, rather than purely chronological, age . Using mThe association between changes in ventricular morphology and volumes with respect to ELD-responsiveness has been reported. Both via ELD in the Non-responder group to confirm that their lack of responsiveness was not confounded by insufficient CSF drainage during testing. However, taken together with findings from literature, our study also suggests that, using changes in 3d linear measures, it may also be possible to describe the spectrum of brain-ventricular changes across both Classic and Complex NPH subtypes. Significant changes in BVR and ventricular volumes may be present at both extremes of reversibility and irreversibility of brain injury patterns in NPH, reflecting differing underlying pathophysiological processes, from brain compression/distortion to alterations in its poroelastic properties.Interestingly, when we stratified our data according to responsiveness to ELD, this change in ventricular morphology was only significant amongst the Non-Responders. Changes in BVR values matched with a decrease in ventricular volumes, as demonstrated by via ELD. However, the differences between our two cohorts across the spectrum of NPH would be that, for the same significant volume of changes in ventricular morphology, loss of microstructural integrity would likely be less reversible than pathophysiological processes such as brain compression, stretching or tissue distortion.There is evidence from other modalities of imaging to support the impact of changes in brain microstructure on CSF movement between intracranial fluid compartments. Brain compression and stiffness in nonlinear elastic regions, as seen on Magnetic Resonance Elastography (MRE) have been hypothesized to lead to non-compliance, which may result in increased CSF drainage from the lateral ventricles, with no change in clinical response . Bugalhovia the concurrent use of 3d, as well as 3D, structural morphological indices and DTI methodology.In further unpacking such considerations, Diffusion Tensor Imaging (DTI), a methodology of modeling changes in white matter microarchitectural patterns by the use of water diffusion properties, may be a helpful adjunct. We have previously described the utility of DTI to describe differing concurrent changes in white matter injuries in response to NPH and interventions, across Classic  and Compvia 3D Slicer with manual exclusion of falsely included CSF-intensity voxels following anatomic identification by the software. This could have led to human error and inter-operator variability. However, this approach is consistent with similar methods performed via the radiology workstation. We minimized these considerations by having a standard operating protocol, refined by pilot testing and incorporating all subsequent post-processing steps within a continuous workflow for consistency, using available modules on 3D Slicer. Lastly, our patient population comprised a large proportion of patients with Complex NPH, and whilst this is reflective of the clinical practice in the Asian population, the impact of other concomitant neurodegenerative diseases may have influenced our results, as compared to the purer cohorts of Classic NPH previously published on the use of morphological indices.Limitations of this study includes the relatively small sample size of our cohort, restricting the statistical power of the study. Nonetheless, testing of morphological indices on this small disease cohort yielded significant findings, consistent with other published work, which are encouraging for future studies to be conducted on larger NPH cohorts with multimorbidity burden and overlay from neurodegenerative disorders. Secondly, our methods of ventricular segmentation were semi-automated Our study has shown that novel 3-directional (3d) linear indices can be applied to cohorts of Classic and Complex NPH, and to distinguish them from AD and HC cohorts. We also demonstrated that, contrary to traditional 2d linear measurements, these measures can also provide significant correlations to CSF responsiveness and 3D ventricular volumetry. In the context of multimorbidity burden and overlay from neurodegenerative disease, 3d morphological indices may provide a non-invasive tool to aid in the characterization of NPH cohorts at the clinical-research interface.nk330@cantab.net.The datasets presented in this article are not readily available because de-identified data is not allowed to be made available to the public according to the study’s IRB. Requests to access the datasets should be directed to NK, The studies involving human participants were reviewed and approved by the SingHealth Centralised Institutional Review Board . The patients/participants provided their written informed consent to participate in this study.NK was primarily involved in the study design, protocol development, protocol implementation and analysis of the data at study site, as well as patient recruitment. SS, CL, SK, and JK were involved in patient recruitment and data collection. AT was involved in the frailty scoring. YL was involved in the 3D volumetric analysis. SS and AK were involved in the manuscript preparation with NK. CL and SS aided with the data analysis and statistical prowess. All authors have read and approved the final 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.