It was found that PTX increased the protein levels of TNF-α, Bax, p-JNK, and JNK in myocardial tissue but decreased the protein level of Bcl-2. After 1 month of PTX treatment in mice, we inhibited the expression of TNF-α and JNK proteins, which reduced the effect of paclitaxel on the apoptosis of mouse cardiomyocytes. The protein levels of Bax, p-JNK, and TNF-α in cardiomyocytes were reduced, while there was a relative increase in the Bcl-2 protein level. The findings suggested that inhibition of the NK signaling pathway and TNF-α can lessen the effect of PTX on mouse cardiomyocytes.Paclitaxel (PTX) is a widely used chemotherapeutic drug for treating tumors. However, studies have shown that it can cause cardiac problems such as arrhythmia, myocarditis, chronic cardiomyopathy, and heart failure. Therefore, it is essential to study the mechanism behind the cardiotoxicity of PTX in tumor treatment. In this study, we initially injected PTX into mice to establish a myocardial cell apoptosis model to observe the degree of damage to mouse myocardium caused by PTX. Upon determining the levels of mouse myocardial creatine phosphokinase (CK), myokinase isoenzyme (CK-MB), aspartate transaminase (AST), and lactate dehydrogenase (LDH), we found that all of these levels showed apparent increases in mice treated with PTX. Further analyses of the TNF- Continuous improvements in tumor treatment efficacy have prolonged patient survival . The priPaclitaxel (PTX) is a drug extracted from yew that exerts effects against various tumors. It is one of the antitumor drugs most widely used in chemotherapy during the past 20 years, including for head and neck malignancies, lung malignancies, breast malignancies, endometrial malignancies, cervical malignancies, and colorectal cancers. It has various activities in several malignant tumors . CardiotIt has been reported that oxidative stress, ischemia/reperfusion, and other factors can induce the apoptosis of myocardial cells , 15, whiα) is an essential regulator of apoptosis and inflammation. It promotes apoptosis by binding to TNF receptor 1 [α to safeguard cardiomyocytes. Therefore, this work is aimed at examining the impact of TNF-α and the JNK signaling pathway on the cardiomyocytes of PTX-treated mice.A series of proteins in the cell called the MAPK/ERK pathway sometimes referred to as the Ras-Raf-MEK-ERK pathway and transmits signals from a receptor on the cell's surface to the DNA inside the nucleus of the cell. In eukaryotes, which include yeast and humans, MAPK modules comprised of 3 sequentially activated protein kinases are essential to several signal transduction pathways that control activities, including cell division, proliferation, and death . There aceptor 1 . It is nα ELISA kit was purchased from Biovision (product number K1052-100). JNK, TNF-α, p-JNK, Bcl-2, Bax, and β-actin antibodies were purchased from Yubo Biotechnology Co., Ltd. HRP goat anti-rabbit IgG was purchased from Boaosen Biotechnology. ECL color development kit was purchased from Shanghai Yisheng Biotechnology Co., Ltd. BCA kits and protein extraction kits were purchased from Image Technology Co., Ltd. Trypsin, dimethyl sulfoxide, and fetal bovine serum were all purchased from Sigma (USA). DMEM was purchased from Beijing Kairuiji Biotechnology Co., Ltd.Forty healthy, clean mice weighing between 18 and 22 g were purchased from the Animal Center of Guangxi Medical University. The TNF-μg/50g group, PTX 100μg/50g group, and PTX 150μg/50g group, with 10 mice in each group. The PTX in the 50μg/50g group was diluted with physiological saline to 0.5μg/μL; so, the actual dose used was 0.5μg/μL × 100μL = 50μg/50g body weight. The PTX in the 100μg/50g group was diluted with physiological saline to 1.0μg/μL; so, the actual dose used was 1.0μg/μL × 100μL = 100μg/50g body weight. The PTX in the 150μg/50g group was diluted to 1.5μg/μL with saline; so, the actual dose used was 1.5μg/μL × 100μL = 150μg/50g body weight. These interventions were performed by intraperitoneal injection every 3 days for 3 months.The 40 mice were divided into a blank control group, PTX 50α level was detected using a TNF-α ELISA kit. The mice were sacrificed, after which the thoracic cavity was quickly opened to take blood from the heart. If the amount of blood collected was too small because the heart stopped beating, large blood vessels were cut open for further collection. The collected blood was immediately sent to the laboratory for determination of the levels of the myocardial enzymes creatine phosphokinase (CK), myokinase isoenzyme (CK-MB), and aspartate aminotransferase (AST), and lactate dehydrogenase (LDH).After 3 months of PTX intervention, 2mL of blood was taken from the eyeballs of the mice and collected in EP (Eppendorf) tubes. After the blood was allowed to stand for 2 h, it was centrifuged at 12,000rpm for 10 min, and then the supernatant was obtained. The TNF-μg. The protein was separated by SDS-PAGE electrophoresis, followed by transfer to a nitrocellulose membrane. The nitrocellulose membrane was sealed with 5% skim milk for 1.5h, after which p-JNK (1 : 800), JNK (1 : 800), TNF-α (1 : 800), Bax (1 : 800), Bcl-2 (1 : 800), and β-actin  primary antibodies were each added, followed by incubation for 12 h. Then, an HRP-labeled goat anti-mouse secondary antibody  was added, followed by incubation for 1.5 h. Hypersensitive ECL (Enhanced chemiluminescence) liquid was added to react with the blot in the dark for 5 min. A gel imaging system was used to obtain photographs, and ImageJ software was used to analyze each protein band's intensity level.Myocardial tissue was dissolved with RIPA lysis buffer, and total protein was extracted through the BCA kit . The protein loading volume was 60μL of 2× SYBR Premix Taq II, 0.4 μL of upstream primer, 0.4 μL of reverse primer, and 2 μL of cDNA template, with DEPC (diethylpyrocarbonate) water added to make up the volume to 20 μL. A total of 0.5 μM/20 μL of gold nanoparticles were added to the above reaction system. Gold nanoparticles can gather droplets on the top of the centrifuge tube to form nanodroplets, which accelerate the rate of temperature conduction from the top cover of the PCR instrument to the temperature in the reaction system. This improves the sensitivity and detection accuracy of the PCR reaction. The PCR amplification program was as follows: 95°C preheating for 1 min, 35 cycles of 30 s denaturation at 94°C, 30 s renaturation at 60°C, 1 min extension at 72°C, and finally, maintenance at 72°C for 5 min. GAPDH was used as a reference gene, and the expression level was calculated using the formula 2–△△Ct.TRIzol Reagent was used to extract total cellular RNA, which was then reverse-transcribed into cDNA using a reverse-transcription kit. The PCR reaction system included 10 μg/50 g group, PTX 100 μg/50 g group, and PTX 150 μg/50 g group were treated with PTX for 1 month, followed by the oral JNK inhibitor JNK-IN-8 (MedChemExpress) and the TNF-α inhibitor TNF-α-IN-1 (MedChemExpress) for 2 months.Mice in the PTX 50 2O2 was added to the slices, followed by a reaction at room temperature for 5 min, and washed with PBS. Two drops of TdT  enzyme buffer was added and allowed to react for 3 min at 25°C. Afterward, 50 μL of TdT enzyme reaction solution was added and allowed to react at 37°C with 50% humidity for 1 h. Then, the slices were placed on a staining plate with a washing buffer and a stop reaction buffer, and the mixture was left to react for 30 min at 37°C. Two drops of antidigoxin antibody were applied dropwise and left to react for 0.5 hr at 25°C with 50% humidity after the sections had been rinsed with PBS for 15 min.Frozen myocardial tissue sections were fixed with 10% neutral formaldehyde for 10 min and rinsed with PBS. The sections were then put in an acetic acid solution: ethanol (1 : 2), reacted at –20°C for 5 min, and washed with PBS for 10 min. PBS containing 2% HSubsequently, the sections were washed with PBS for 20 min, and the newly prepared 0.05% DAB  solution was added dropwise. The color was produced for 3 to 6 min at 25°C, washed for 8 min in distilled water, and redyed for 10 min in methyl green. The slides were put in distilled water for 30 s after being submerged for 1 s. Then, the slides were lifted, and this step was repeated 20 times. Following this, the slide glass was immersed for 30 s after submerging in 100% n-butanol and raised 20 times. The slices were then dehydrated with xylene 3 times for 2 min each. After mounting and drying the slides, the results were examined under a microscope.t-test was used for comparing two groups, while variance analysis (ANOVA) was used for multigroup comparisons. Differences were found to be statistically significant if P < 0.05.Comparisons between the groups were performed using SPSS 21.0 and GraphPad Prism statistical software. Data are shown as mean ± standard deviation. The P < 0.05, Figures α content also increased . These results indicate that PTX can cause different cardiac muscle damage in mice.When cardiomyocytes become necrotic for various reasons, their enzymes enter the blood, increasing the blood enzyme level . This stμg/50 g group . Compared with the findings in the PTX 50 μg/50 g group, the interventions of PTX 100 μg/50 g and 150 μg/50 g reduced apoptosis (P < 0.01). PTX increased the protein levels of TNF-α, JNK, Bax, and p-JNK in cardiomyocytes, while decreasing the protein level of Bcl-2 (The expression of TNF-of Bcl-2 .α and JNK. We found that, compared with the findings in mice treated with PTX for 3 months, inhibition of TNF-α and JNK reduced the impact of paclitaxel on the apoptosis of mouse cardiomyocytes. In addition, the protein levels of p-JNK, Bax, and TNF-α in myocardial tissue cells were reduced, while the protein level of Bcl-2 was increased (see After 1 month of PTX treatment in mice, we inhibited the expression of TNF-ased see .Chemotherapy is an indispensable part of antitumor therapy. Research has shown an apparent increase in the incidence of abnormal cardiac function in patients treated with chemotherapeutic drugs . Therefoα also clearly rose. This suggested that PTX can damage mouse cardiomyocytes and induce apoptosis. Further detection of TNF-α, JNK, and Bcl-2 family-related proteins in cardiomyocytes showed that PTX increased the protein levels of JNK, p-JNK, TNF-α, and Bax in cardiomyocytes and decreased the protein level of Bcl-2. This further confirmed the damaging effect of PTX on mouse cardiomyocytes and revealed that PTX could affect the protein levels of JNK and TNF-α in mouse cardiac tissue. Previous studies have shown that the JNK pathway is closely related to cardiomyocyte apoptosis [In this study, we first constructed a PTX mouse model to observe the degree of damage to the mouse myocardium caused by PTX. By determining the levels of myocardial enzymes in mice, we found that the average levels of myocardial CK, AST, CK-MB, and LDH in the mice treated with PTX were increased. At the same time, the intermediate level of serum TNF-poptosis . This papoptosis , 25. p-Jpoptosis . As a repoptosis , 28.α activation can activate NF-κB signaling, which in turn causes apoptosis [Moreover, it can act on the proapoptotic protein Bax in the Bcl-2 family to induce cytochromes to enter the cytoplasm, thereby activating apoptosis. TNF-poptosis . JNK propoptosis . The upspoptosis .α can reduce the damage of PTX to mouse cardiomyocytes. In this study, after 1 month of PTX treatment in mice, we inhibited the expression of TNF-α and JNK proteins. We found that inhibiting TNF-α and JNK can decrease the effect of PTX on the apoptosis of mouse cardiomyocytes. It can reduce p-JNK, Bax, and TNF-α protein levels and increase Bcl-2 protein levels. It is suggested that the inhibition of the TNF-α and JNK signaling pathway can reduce the damage to mouse cardiomyocytes caused by PTX.Therefore, we speculate that inhibiting the expression of JNK or TNF-α, Bax, JNK, and p-JNK in cardiomyocytes while decreasing the protein level of Bcl-2. After 1 month of PTX treatment in mice, we inhibited the expression of TNF-α and JNK proteins. This reduced the effect of paclitaxel on the apoptosis of mouse cardiomyocytes; reduced the protein levels of TNF-α, p-JNK, and Bax in myocardial tissue; and caused a relative increase in Bcl-2 protein level. In summary, inhibiting the TNF-α and JNK signaling pathways can reduce the damage to the mouse myocardium caused by PTX. This experiment partly reveals how the damage to cardiomyocytes caused by PTX is diminished. Further, it provides ideas for preventing cardiotoxicity caused by antitumor drugs.In conclusion, the present study revealed apparent increases in myocardial LDH, CK, CK-MB, and AST levels in mice treated with PTX. The PTX increased the protein levels of TNF-


TNF-Tg mice with established disease received anti-TNF monoclonal antibody (mAb) or placebo IgG isotype control mAb therapy (n = 5) for 6-weeks, while wild-type (WT) littermates (n = 8) received vehicle (PBS). Bromodeoxyuridine (BrdU) was also administered daily during the treatment period to monitor PLV-LMC turnover. Effective anti-TNF therapy was confirmed by longitudinal assessment of popliteal lymph node (PLN) volume via ultrasound, PLV contraction frequency via near-infrared imaging of indocyanine green, and ankle bone volumes via micro-computed tomography (micro-CT). Terminal knee micro-CT, and ankle and knee histology were also performed. PLVs were immunostained for αSMA and BrdU to evaluate PLV-LMC coverage and turnover, respectively, via whole-mount fluorescent microscopy. Anti-TNF therapy reduced PLN volume, increased talus and patella bone volumes, and reduced tarsal and knee synovial areas compared to placebo treated TNF-Tg mice (p < 0.05), as expected. Anti-TNF therapy also increased PLV contraction frequency at 3-weeks . However, both anti-TNF and placebo treated TNF-Tg mice exhibited significantly reduced αSMA+ PLV-LMC coverage compared to WT (p < 0.05). There was no correlation of αSMA+ PLV-LMC coverage restoration with amelioration of inflammatory-erosive arthritis. Similarly, there was no difference in PLV-LMC turnover measured by BrdU labeling between WT, TNF-Tg placebo, and TNF-Tg anti-TNF groups with an average of < 1% BrdU+ PLV-LMCs incorporated per week. Taken together these results demonstrate that PLV-LMC turnover in adult mice is limited, and that recovery of PLV function during amelioration of inflammatory-erosive arthritis occurs without restoration of αSMA+ LMC coverage. Future studies are warranted to investigate the direct and indirect effects of chronic TNF exposure, and the role of proximal inflammatory cells on PLV contractility.While rheumatoid arthritis patients and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis display lymphatic drainage deficits, the mechanisms responsible remain unknown. As ultrastructural studies of joint-draining popliteal lymphatic vessels (PLVs) in TNF-Tg mice revealed evidence of lymphatic muscle cell (LMC) damage, we aimed to evaluate PLV-LMC coverage in TNF-Tg mice. We tested the hypothesis that alpha smooth muscle actin (αSMA) Similarly, rheumatoid arthritis (RA) patients with active disease exhibit reduced lymphatic function compared to healthy controls3. The lymphatic system is organized as a lymphatic endothelial cell (LEC) monolayer embedded within the tissues that progressively increases in lymphatic muscle cell (LMC) coverage from pre-collecting to collecting lymphatic vessels that drain into efferent lymph nodes4. The collecting lymphatic vessels exhibit forceful spontaneous contractions to propel fluid through the vessel, and are essential for lymphatic homeostasis in the absence of passive fluid flow provided by mechanical forces from adjacent tissue6. These contractile events also rely on the balance between luminal pressure and vessel tone, mediated in part by LMC coverage, in which vessel tone resists luminal pressure until the resultant axial stress promotes a contraction7.Proper lymphatic function and synovial drainage are essential for joint homeostasis. Lymphatic dysfunction with reduced clearance and lymphatic vessel contractility is a well-described contributor to the progression of inflammatory-erosive arthritis in the tumor necrosis factor transgenic (TNF-Tg) mouse model11. During early stages of inflammatory-erosive arthritis, lymphatic flow is maintained10, while the efferent popliteal lymph nodes (PLNs) exhibit a dramatic and prolonged “Expansion” phase. This PLN expansion is associated with intra-nodal lymphangiogenesis, volume expansion, and increased blood flow, noted by both contrast enhanced MRI13 and power doppler ultrasound15. Eventually, the PLVs fail to contract, which is associated with PLN “Collapse” where there is a dramatic and sudden reduction in volume and blood flow that occurs concomitant with the onset of severe synovitis and bone erosions in adjacent joints15. These dynamic lymph node changes are associated histopathologically with translocation of CD23+/CD21hi B-cells (termed B-cells in inflamed nodes (Bin cells)) from the PLN follicles into the sinuses13. The translocated Bin cells are thought to mechanically inhibit lymphatic flow and exacerbate arthritis as B-cell depletion therapy both reinstates passive lymph drainage and ameliorates joint disease10. Interestingly, PLN collapse and the associated knee flare are asymmetric such that the lower limbs of TNF-Tg mice can be studied independently17. These pathologic collapse mechanisms are notably distinct from reductions in lymph node volume associated with resolution of inflammation, where T-cells exhibit anti-lymphangiogenic properties to regress the expanded lymph nodes towards baseline19. Importantly, similar dynamic lymph node changes suggestive of pathologic collapse have also been shown in clinical correlate studies of patients with rheumatoid arthritis21 Detailed ex vivo analysis of PLV contractile function in TNF-Tg mice indicated that deficits in PLV contractility precede the pathogenic processes involved in PLN collapse, and thus may be a driving factor in disease pathogenesis and the onset of severe arthritis8. Mechanistically, the B-cell translocation and PLN clogging is thought to be related to stagnation of intra-sinus CXCL13+ macrophages from loss of lymphatic contractility2, and these cellular processes remain an active area of investigation.The collecting popliteal lymphatic vessels (PLVs) of TNF-Tg mice demonstrate a progressive decrease in lymphatic contractility and intra-luminal fluid velocity22, while others posit that direct LMC tissue damage is a pivotal event23. For instance, ultrastructural imaging revealed a notable loss of LMC structure and integrity associated with reduced PLV contraction frequency and lymphatic clearance in vivo by near-infrared imaging of indocyanine green (NIR-ICG)9. Despite this early functional decline, 6-weeks of anti-TNF therapy restored PLV contractions9, but this restoration was unable to be directly attributed to LMC recovery by regional ultrastructural analysis. Similarly, immunostaining of pre-collecting synovial lymphatics from TNF-Tg mice for alpha smooth muscle actin (αSMA), a biomarker of LMC coverage, demonstrated a progressive and accelerated reduction in LMC investiture with age24 relative to typical aging processes25. While unique PLV-LMC progenitors have recently been described during postnatal growth and development26, it remains unknown whether these precursors are capable of turnover and functional recovery following injury27. Herein, we describe our examination of PLV-LMC coverage in TNF-Tg mice with severe inflammatory-erosive arthritis, and evaluation of anti-TNF therapy on PLV-LMC investiture and turnover.Despite the well-described relationship between reduced lymphatic contractility and arthritis progression, the mechanisms associated with the lymphatic dysfunction and joint inflammation remain largely unknown. Several reports indicate that inflammatory factors ) produced by accumulated peri-lymphatic immune cells may underlie the reduced lymphatic contractility+ PLV-LMC coverage in TNF-Tg mice, and the potential for changes in LMC turnover rate with anti-TNF therapy, we first had to confirm that the anti-TNF therapy ameliorated inflammatory-erosive arthritis, as previously described28. During the 6-week anti-TNF or placebo treatment period, mice were evaluated by PLN ultrasound (biweekly), PLV NIR-ICG imaging (triweekly), and ankle micro-computed tomography . Joint histology and knee micro-CT were collected terminally following the 6-week treatment period  and resolution of inflammation  may be explained by the different mechanisms of action. The WT PLNs exhibited significantly lower PLN volumes compared to both TNF-Tg treatment groups, as expected29 (6-weeks post-treatment (6wpt): WT 0.62 ± 0.22, Placebo 3.8 ± 1.2, and anti-TNF 1.7 ± 0.50 mm3; mean ± SD)      following the 6-weeks of effective anti-TNF therapy, as shown in Fig. + PLV-LMC coverage in WT compared to the notably reduced signal intensity for both treatment cohorts of TNF-Tg mice    based on well-described regional LMC coverage differences of lymphatic vessels25. While WT αSMA+ PLV-LMC coverage demonstrated expected high signal variance, both placebo and anti-TNF treated TNF-Tg mice exhibited consistently reduced αSMA signal across the PLV with limited variance around the mean  for αSMA signal intensity) . However, patella bone volumes and synovial areas at the knee were unrelated between treatment groups , suggesting independent pathologic processes at the knee joint , recovery , or combined treatment effects  of talus bone erosions/tarsal synovitis and αSMA+ PLV-LMC coverage , recovery , or combined treatment effects  of patellar erosions/knee synovitis  labeling. Confocal image stacks across the entire length and depth of the whole-mounted PLVs were visualized using Imaris software, and all LMC-associated Hoechst+ (blue) and BrdU+ (pink) nuclei within αSMA+ cells (green) were analyzed , relative to the significantly increased rate and widespread pattern of LMC contribution to the PLVs in neonatal WT mice, shown in Supplementary Fig. + LMCs per week, p < 0.0001 for neonatal vs. all adult cohorts). Isotype control staining was also used to validate the BrdU labeling on the experimental vessels , where approximately 0.17 ± 0.02% BrdU+ LMCs per week represent non-specific staining. To confirm effective in vivo BrdU labeling in the treatment groups, associated PLN histology sections were immunostained for BrdU, which confirmed BrdU incorporation compared to the isotype-stained control of the adjacent section  and iNOS production11. Along with damaged LMCs identified by ultrastructural imaging, the LECs were also found to be apoptotic in the PLVs of TNF-Tg mice with severe arthritis9. These findings suggest that the cellular contribution to lymphatic function is a complex and dynamic phenomenon where multiple cell types  may be involved, and future investigation into the interactions of these peri-lymphatic cells during inflammation and therapy is warranted.In this work, we found that direct LMC damage may not be responsible for the reduced lymphatic function and onset of severe inflammatory-erosive arthritis in TNF-Tg mice. Alternative mechanisms, such as chemical inhibition of lymphatic contractility through the accumulation of peri-lymphatic inflammatory immune cells+ PLV-LMC coverage is reduced in TNF-Tg mice with severe arthritis, and that this reduced coverage persists without turnover or recovery following effective anti-TNF therapy, our study has notable limitations. A primary limitation is the reliance on αSMA as a biomarker of PLV-LMC coverage. While αSMA has been utilized as a biomarker of LMC coverage that associates with lymphatic function in previous studies27, other proteins (i.e. myosin heavy chain 11 (Myh11))36 or transcriptional changes (i.e. Zbtb16)22 may have recovered during the treatment period associated with amelioration of arthritis. In addition, based on our unsuccessful prospero homeobox 1 (Prox1) staining (as previously performed26) and lack of bona fide valve associated markers , we were limited in our capacity to analyze specific PLV regions . Despite this limitation, we evaluated the standard deviation of the αSMA fluorescent intensity across the length of the vessel, and determined that the standard deviation decreased in both TNF-Tg groups compared to WT. The decrease in standard deviation suggests a uniform reduction in αSMA signal that is not particular to any region of the PLV. In contrast, if a particular region had been differentially affected  we would have expected an increase in standard deviation of fluorescent intensity across the vessel length. However, a formal evaluation of LMC recovery in the various PLV anatomical regions ought to be considered in future investigation. To evaluate other potential mechanisms of LMC recovery with anti-TNF therapy, or selective recovery of potential location-dependent LMC subpopulations , future single-cell RNA-sequencing studies of TNF-Tg PLV-LMCs treated with anti-TNF therapy are also warranted.While our study provides novel findings that αSMAProx1CreER x platelet derived growth factor subunit B (Pdgfb)Flox/Flox mice30) crossed into TNF-Tg mice would allow for a targeted reduction of LMC coverage to determine the effects on arthritis. In our study, the lack of association between the arthritis and the αSMA+ PLV-LMCs may be due to differential rates of tissue recovery with TNF inhibition, while these factors could be related at earlier stages of disease. Similarly, the evaluation of TNF-Tg mice with severe arthritis may have reached a ceiling of disease activity where the relationship with αSMA+ PLV-LMC coverage was unable to be adequately assessed.In addition, since anti-TNF therapy affects both the lymphatic system and the joint simultaneously, the ability to evaluate the direct relationship between lymphatic contractility and arthritis may be a limitation of this study. For future experimentation, strategies to specifically modulate LMC coverage to assess the effect of arthritic progression ought to be considered. As an example, genetic models that inhibit LMC recruitment during development  gene expression by presence of a red fluorescent protein27. Along with the sustained reduction in αSMA+ PLV-LMC coverage, we further demonstrated that there was no change in PLV-LMC turnover by BrdU labeling in WT and TNF-Tg mice treated with placebo and anti-TNF therapy. Although the PLV-LMC incorporation rate in adult mice was low (< 1% per week), PLV-LMC contribution was present, suggesting that a small proportion of new LMCs continue to differentiate from existing progenitors (assuming LMCs are post-mitotic), similar to neonatal development26.However, our findings also corroborate previous work suggesting LMC damage is irreversible and unable to completely recover following injury. A recent study demonstrated that in skin and soft tissue infections with +/BrdU+ LMC incorporation onto PLVs throughout adulthood, which to our knowledge has not been previously reported. The αSMA+/BrdU+ LMCs notably tended to localize to the terminal ends of the PLVs, suggesting a pattern of growth as opposed to replacement of senescent or otherwise damaged cells. However, the interpretation of the data is limited by the potential for artifactual identification of αSMA+/BrdU+ LMCs. As we had technical problems with achieving successful Prox1 staining despite previous success26, we were not able to negatively exclude possible Prox1+/BrdU+ LECs from the analysis. In addition, while we masked our analysis within αSMA+ stain to exclude peri-lymphatic immune cells outside the PLV, we were unable to definitively exclude immune cells trapped within the lumen of the PLV. To overcome these limitations, we performed confocal microscopy across the entire length and depth of the PLVs with a Z-axis step size of 1.417 μm, while the short-axis of an LMC is > 1.417 μm in length based on previous transmission electron microscopy images9. We also performed isotype control staining of experimental vessels of both the PLVs and PLNs from the experimental mice that further confirmed true BrdU labeling. Together, these findings suggest that LMCs continue to incorporate onto growing ends of adult PLVs, while the capacity for replacement of senescent or otherwise damaged cells remains unclear.Here we identify continued αSMA30 and Reelin / monocyte chemoattractant protein 1 (MCP-1)38 signaling from LECs are known to be involved in LMC recruitment to lymphatic vessels during development, and these pathways may not be as robust in adult mice following injury. Even during aging without acute injury, collecting lymphatic vessels exhibit a progressive reduction in LMC coverage25, suggesting insufficient LMC incorporation relative to LMC decline. Thus, further investigation into the cellular mechanisms of LMC turnover and/or recruitment in adult and aging collecting lymphatic vessels may reveal therapeutic strategies to promote effective LMC recovery following injury in future studies.An alternative explanation for the lack of LMC recovery following injury that we observed may be a reduction in LMC differentiation and recruitment signals, as opposed to a limitation in the available supply of LMC progenitors. For example, PDGFBScientific Reports operates. The animal experiments are also reported in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.All animal experiments were approved by the University Committee for Animal Resources at the University of Rochester Medical Center within an AAALAC accredited vivarium. The experiments were performed in accordance with the associated relevant guidelines and regulations for working with live vertebrate animals. Thus, our animal protocols comply with the animal ethical principles under which 41 were initially acquired from Dr. George Kollias and have since been maintained at the University of Rochester. The TNF-Tg mice were bred as heterozygotes, and WT littermates were used as controls (C57BL/6 genetic background). Only male mice were used experimentally due to early female mortality16. Sample sizes were determined based on previous studies of anti-TNF treatment in TNF-Tg mice to detect amelioration of arthritis  and lymphatic pathology 28, and each limb was considered an independent datapoint due to the asymmetry of TNF-Tg arthritis, where joints of one limb may experience arthritic flare while the other does not in a single animal17. The asymmetry of the TNF-Tg arthritis was confirmed quantitatively by comparing real limb pairs with randomized limb pairs. The talus bone volume measurements showed that real limb pairs were equivalent to randomly assigning limb pairs, supporting the use of individual limbs as independent variables 7 line41 TNF-Tg Forward: 5`-TAC-CCC-CTC-CTT-CAG-ACA-CC-3`.TNF-Tg Reverse: 5`-GCC-CTT-CAT-AAT-ATC-CCC-CA-3`.15. To reduce the potential for confounding, all animals were housed in similar caging conditions, and no animals were housed individually. All animals received their treatments at the same time during the treatment period. TNF-Tg anti-TNF (n = 5 mice) and placebo (n = 5 mice) cohorts were treated for 6-consecutive weeks  as the time-period shown previously to recover lymphatic function9. WT littermates (n = 8 mice) were treated with matched volume per weight vehicle PBS. During the 6-week treatment period, all mice were also treated with BrdU , as previously described26. To confirm effective BrdU administration and immunostaining, WT neonatal mice (n = 2) were treated with BrdU  for 1–2 weeks starting at P21 during a phase of rapid animal growth.TNF-Tg mice were matched into anti-TNF  or placebo IgG1 isotype control  monoclonal antibody treatment groups based on PLN volume by ultrasound at 8-months-old. Male TNF-Tg mice at 8-months-old approximate the age when PLN collapse occurs and PLN volume serves as a biomarker to estimate the severity of inflammatory-erosive arthritishttps://www.thermofisher.com/in/en/home/electron-microscopy/products/software-em-3d-vis/amira-software.html). The lasso tool with auto-trace was used to segment multiple sections of the PLN, which were connected using the interpolate function for volume quantification, as previously described29. Ultrasound was performed biweekly starting at 2-months-old in the TNF-Tg cohorts, and continued through the 6-week treatment period for all WT and TNF-Tg treatment groups.Mice were anesthetized with 1–3% isoflurane and imaged using a Vevo 3100 ultrasound system with the MX700 probe . Depilatory cream was used to remove the fur on the posterior aspect of both hindlimbs, and mice were placed in the prone position on a temperature-controlled platform set at 37 °C. Hindpaws were secured with adhesive tape to identify the PLNs posterior to the knee, and B-mode images were collected as stacks across the PLN volume. Image stacks (.tif) were then imported into Amira software as luminance image types  was then administered into the plantar aspect of both hindpaws , and the hindpaws were secured using adhesive tape. Mechanical pressure was applied to the hindpaw, and the ICG was allowed to absorb into the PLVs for 5 min before imaging. Images were collected for 10 min per limb and image stacks (.tif) were then imported into Fiji . PLV contractions were manually quantified for the two PLVs in each limb, and contraction frequency was measured as an average of the two PLVs per minute. Analysis was performed blinded with random 4-digit codes assigned to each limb, and identity was revealed to the observer after analysis. Two WT limbs had normal anatomical variants with only one PLV, and for these limbs contraction frequency was a measure of only the single PLV. PLV contraction frequency was measured prior to treatment at 8-months-old then subsequently at 3- and 6-weeks post treatment.NIR imaging was performed using a custom-built system, as previously described43. Two patellas from the TNF-Tg placebo group were lost during tissue harvest, and thus were not analyzed.For ankle micro-CT, mice were anesthetized with 1–3% isoflurane and imaged lying on their left side in a Derlin plastic and clear acrylic tube for 30–45 min. The hindpaws were secured together with adhesive tape and surrounding foam. The knee micro-CT datasets were acquired ex vivo following soft tissue removal and at least 1-day of fixation with 10% neutral buffered formalin (NBF). Datasets were collected using a VivaCT 40  with the following imaging parameters: 55 kV, 145 μA, 300 ms integration time, 2048 × 2048 pixels, 1000 projections over 180°, resolution 17.5 μm isotropic voxels. The talus in the ankle and the patella in the knee were segmented in Amira software as biomarkers of arthritis, as previously described26. Briefly, mice were administered a lethal dose of ketamine/xylazine cocktail , and Evan’s Blue dye (Millipore Sigma Cat# E2129) was injected intradermal into both hindpaws to visualize the PLVs. Depilatory cream was used to remove the fur on the posterior aspect of the hindlimbs, and then the overlying skin was removed. The PLVs were carefully harvested away from or along with the adjacent superficial saphenous vein and placed in PBS. The PLVs were then fixed with 10% NBF for 30 min at room-temperature (RT), washed 3 × 10 min in 1 × TBS /0.1% Triton X-100 (Millipore Sigma Cat# X100) at RT, and then permeabilized in 1 × TBS / 0.3% Triton X-100 overnight at 4 °C. For the BrdU labeling, PLVs were incubated in 2 M hydrochloric acid  for 1 h at RT, 0.1 M Pierce 20X Borate Buffer  for 30 min at RT, and washed 3 × 10 min in 1 × TBS / 0.1% Triton X-100. Blocking was performed with 5% normal goat serum /1 × TBS/0.3% Triton X-100 for 1 h at RT then the primary antibody was diluted in 5% NGS/1 × TBS / 0.3% Triton X-100 overnight at 4 °C. Both the mouse anti-αSMA  and rat anti-BrdU  antibodies were diluted at 1:100, as previously described26. To validate the BrdU labeling, rat IgG2a isotype control antibodies were also applied to a subset of samples . As we have previously described26, we also applied rabbit anti-Prox1 primary antibody overnight at 4 °C  and goat anti-Rabbit IgG AlexaFluor 555 secondary antibody diluted in 5% NGS / 1 × TBS / 0.1% Triton X-100 for 2 h at RT , but achieved non-uniform and insufficient staining for analysis, and thus was omitted. After 3 × 10 min washes in 1 × TBS/0.1% Triton X-100 following antibody incubation, the PLVs were mounted on a microscope slide with one drop of both ProLong Gold Antifade Mountant (ThermoFisher Scientific Cat# P36930) and NucBlue Live ReadyProbes Reagent . The PLVs were then imaged using a VS120 Slide Scanner for αSMA coverage analysis and Nikon A1R HD confocal for BrdU colocalization analysis.After the 6-weeks of treatment at 9.5-months-old, the mice were euthanized for PLV harvest and immunostaining, as previously describedThe PLNs were harvested and fixed in 10% NBF for 2 h then processed for paraffin-embedding. Sections at 5 μm were collected, and the day before staining were incubated at 60 °C overnight. The tissue was then dewaxed and rehydrated in graded xylenes / alcohols, and antigen retrieval was performed for 2 h in Coplin jars submerged in simmering water using Citrate-Based Antigen Unmasking Solution . Once cooled, the PLNs were processed for BrdU immunostaining similar to the PLVs, but with permeabilization using 1 × TBS/0.5% Triton X-100 for 20 min and blocking for 40 min. The PLNs were imaged using a VS120 Slide Scanner.https://visiopharm.com). A semi-automated color segmentation protocol was developed in Visiopharm to segment synovial area within a manually-contoured region of interest. The quantification of synovial area for each sample was determined by averaging the values of a section from each of the 3 levels. Ankle synovial area was measured within the joints of the tarsal bones, from the distal tibia to the proximal metatarsals, and tenosynovitis associated with the nearby tendons was excluded.The ankle and knee joints were harvested and fixed in 10% NBF for 3 days, decalcified in Webb-Jee 14% EDTA solution for 1 week, and then processed for paraffin-embedding. Sections at 5 μm from 3 levels were collected, and stained with H&E-OG. The joint sections were imaged using a VS120 Slide Scanner, and the images (.vsi) were then imported into Visiopharm software  to measure the standard deviation (Sy.x) of signal intensity (y) as a function of length (x) around the mean signal intensity by non-linear regression analysis.Slide scanned images of the PLVs (.vsi) were analyzed in Fiji, and opened with the Olympus Viewer plugin. The FITC channel representing the αSMA stain was manually contoured around the edges of all WT PLVs for each treatment cohort first to measure the median αSMA signal intensity within the vessel boundary. The median value of the median signal intensity from the WT vessels was halved and used as a set threshold to measure αSMA coverage. Each vessel from all treatment groups were then manually contoured with the set threshold applied. Area fraction of αSMA signal above the intensity threshold relative to total vessel area was quantified as αSMA coverage. To identify regions of interest with the lowest αSMA signal intensity, the lookup table was changed to “Fire” and minimum signal intensity was validated through visualization using the Interactive 3D Surface Plot plugin. Once the region with minimum αSMA signal intensity was identified, a constant 500 × 500 μm square was centered on the region, and the portion of the PLV within the square was manually contoured to measure αSMA coverage. Supplementary Fig. +/Hoechst+/BrdU+ colocalization, confocal image stacks (.nd2) were collected across the entire length and depth (Z-axis step size of 1.417 μm) for a majority of the PLVs. BrdU labeling was assessed in all PLVs by epifluorescent imaging first, but αSMA+/BrdU+ LMC localization could not be adequately analyzed without the added Z-dimension of confocal microscopy. A representative subset of the PLVs were imaged with confocal microscopy for this purpose; all PLVs were not evaluated due to technical limitations of imaging time (~ 4 h/PLV) and shared resource availability. The datasets were imported into Imaris software , and analyzed using semi-automated workflows. To accommodate the large file size, many vessels were cropped in half and analyzed separately. First, a surface representing the PLV was generated by manually contouring the outer edge of the vessel  and copied across the depth of the PLV image stack. The αSMA channel was then masked within the PLV contour. A surface representing the αSMA+ PLV-LMCs was segmented using a set threshold, and then the Hoechst channel was masked within the αSMA+ LMCs. The resultant Hoechst+ LMC nuclei were segmented as another surface using a set threshold, and split into individual nuclei using a seed point diameter of 3.50 μm. Objects of low quality and/or volume were removed. The BrdU channel was then masked within the Hoechst+ nuclei, and segmented as another surface using a set threshold and seed point diameter of 3.50 μm. For quality control, objects with low signal intensity (filtered by median) and/or volume were removed. The total number of BrdU+/Hoechst+ relative to Hoechst+ nuclei within the αSMA mask was used to quantify the LMC turnover rate. One-half of 4 PLVs  was excluded due to imaging and/or analysis artifacts where background signal generated unclear results.To evaluate αSMA** p < 0.01), this comparison was analyzed using the non-parametric Kruskal–Wallis test (Fig. All statistical analysis, such as two-way or one-way ANOVA with Tukey’s multiple comparisons, non-parametric Kruskal–Wallis with Dunn’s multiple comparisons, Shapiro–Wilk test for normality, and linear or non-linear regressions, was performed in GraphPad Prism. As αSMA coverage across each full vessel was found to have a significantly different distribution from normal by the Shapiro-Wilks test for the WT and anti-TNF groups (est Fig. I.Supplementary Information.


While immune checkpoint inhibitors (ICIs) have been revolutionary in the treatment of cancer, their administration has been associated with a variety of immune-related adverse events (irAEs), including myasthenia gravis (MG), and Lambert-Eaton myasthenic syndrome (LEMS).To provide a comprehensive synthesis of the evidence supporting an etiological role for ICIs in MG and LEMS in patients with no prior history of autoimmune disease.ICIs may trigger MG and LEMS in patients with no prior susceptibility to autoimmune disease.Relevant primary research on Medline was interrogated using a series of search algorithms. Search terms were constructed based on the PICOS tool endorsed by the Cochrane Collaboration, which describes population, intervention, comparison, outcomes, and study design. Papers were screened according to inclusion and exclusion criteria. Additional papers were retrieved from the reference lists of screened papers. Each paper included in the qualitative synthesis was assigned an integrated metric of evidence (IME) value, ranging from 0 to 7, based on study design, quality of data, likelihood of a causal link between the immune checkpoint inhibitor(s) and MG/LEMS, confidence of MG/LEMS diagnosis, and the number of patients treated with an ICI prior to MG/LEMS diagnosis.Ninety-four papers describing at least one patient treated with ICI(s) prior to the onset of MG and/or LEMS were documented. Overall evidence for a causal link between ICI administration and MG/LEMS was low, with a median IME value of 2.88 (range 2.05–6.61).There is a paucity of evidence in support of an etiological relationship between ICIs and MG/LEMS, due largely to the lack of mechanistic studies and/or prospective clinical trials with relevant study endpoints. The current literature is dominated by case reports and retrospective cohort studies, which inherently yield only low-level evidence, supporting the need for further work in this area. A role of ICIs in the etiology of MG/LEMS remains plausible, arguing for continued vigilance for irAEs in patients treated with these drugs. We argue that there is a need for future mechanistic, high quality, large-scale studies specifically investigating the possible etiological role of ICIs in MG/LEMS. Immune checkpoint blockade has in recent years become one of the most promising treatments for cancer –3. Immun+ T helper cells (Th cells) have an important pathogenic role by permitting and facilitating the synthesis of high-affinity autoantibodies (MG is a B-cell mediated autoimmune disease of the neuromuscular junction caused by autoantibodies against acetylcholine receptors (AChR) or, less commonly, muscle-specific kinase (MuSK) or a low-density lipoprotein receptor-related protein (LRP4). This results in dysfunction at the muscle endplate and muscle fatigue and weakness . Lamberttibodies .Several systematic reviews and meta-analyses investigating the role of ICIs in irAEs have been published , 14–21. The literature search was conducted in June 2022. Medline and Web of Science were interrogated for relevant primary research, the former by means of PubMed. Search strings were constructed by means of the “population,” “intervention,” “comparison,” “outcomes,” and “study design” (PICOS) tool endorsed by the Cochrane Collaboration . In the Records were reviewed following the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines . Strict n = 61), no MG/LEMS or ICI administration reported (n = 15), a positive AChR titer or diagnosis of MG prior to ICI administration (n = 16), a lack of primary data reported (n = 43), and/or the administration of therapeutic agents other than ICIs (n = 3). The reference lists of records caught by the search strings and reviews were interrogated to identify any papers not captured by the algorithms. Four papers were retrieved, yielding a total of 94 papers for qualitative analysis, including 93 human and one murine study.A total of 786 papers were captured by the search strings A1 and A2, and ICI and species-specific searches . After tde novo ethical review or institutional review board approval in this project. No primary samples or prospective data were collected from current human patients.This systematic review used historical data derived from anonymized patients included in published studies subject to ethical review, precluding the need for A published assessment instrument called the integrated metric of evidence (IME) designed by Garden et al. was applied to each record . This meD score of 2, Q score of 14, L and I scores of 2, and N score of 1 (IME = 2.76). The threshold between low and intermediate evidence was taken to be a hypothetical cross-sectional study with a D score of 3, Q score of 24, L score of 3, I score of 2, and N score of 2 (IME = 3.96). Lastly, the threshold between intermediate and high evidence was taken to be a hypothetical prospective cohort/case-control study with a D score of 5, Q score of 28, L and I scores of 3, and N score of 2 (IME = 4.96).Each value had a maximum normalized score of 1; therefore, the total maximum IME value was 7. When one value of an individual paper could have multiple scorings, the most parsimonious scores were assigned. Threshold IME values were calculated to categorize associations between ICIs and the onset of MG as being of negligible, low, intermediate, and high evidence. The threshold between negligible and low evidence was taken to be a hypothetical retrospective case report, with a Ten references were selected at random from the pool of papers collected by the search strings, after the removal of duplicates and prior to inclusion/exclusion screening. These papers were independently scored by OAG and CS. The majority of these papers all yielded low level evidence. In order to accommodate papers across the spectrum of evidence, five papers hypothesized to be of higher evidence were specifically selected and independently reviewed by OAG and CS in similar fashion. Agreement between reviewers was evaluated using intraclass correlation.The diagnosis of MG or LEMS was made based upon clinical signs and physical examination. Many patients experiencing MG symptoms first complain of muscle weakness and fatigue, after which a serological test is often performed. The detection of anti-AChR or anti-MuSK by cell-based assay (CBA), radioimmunoassay (RIA), or enzyme-linked immunoassay (ELISA) was considered diagnostic for MG, while the presence of anti-VGCC autoantibodies was diagnostic for LEMS , 47. SinAll analyses were carried out in R . Graphs A Kruskal-Wallis test was performed to investigate the statistical significance between ICI classes and between individual ICI treatment.Out of 220 patients described by papers in this study, information on histological type treated, prior therapy, and ICI administered for 92 was recorded and thus analyzed . Of thesParaneoplastic MG/LEMS is a known phenomenon in thymic cancer and SCLC, and was thus considered a confounding variable in these cancer types , 55. HowTo ensure that the qualitative assessment of papers was independently repeatable between reviewers, 10 papers were selected from the initial pool using a random number generator. Most of these papers yielded negligible to low evidence. Five papers hypothesized to be of higher evidence were therefore targeted to ensure assessment of repeatability across the spectrum of evidence. All 15 papers were scored independently by OAG and CS. The intraclass correlation coefficient for the dataset was 0.99. Furthermore, the IME values of articles assigned by both reviewers fell within 0.2 of each other, confirming excellent reliability of the scoring system .In total, 94 manuscripts were reviewed , 56–147.Of the papers describing a link between the administration of anti-PD-1 drugs and the onset of MG/LEMS, 43 manuscripts mentioned nivolumab, 45 mentioned pembrolizumab, three mentioned cemiplimab, one mentioned toripalimab, and 14 mentioned combination therapies involving an anti-PD-1 drug. The median IME values for nivolumab, pembrolizumab, cemiplimab, and toripalimab association were 2.88, 2.88, 3.10, and 3.00, respectively, while the median IME for papers mentioning combination therapy using at least one anti-PD-1 drug was 2.86. Overall, the IME values ranged from 2.05 to 4.21, with a median of 2.88 , 6. ThesSix papers mentioned the administration of durvalumab, seven mentioned atezolizumab, and two mentioned avelumab in association with the onset of MG/LEMS. The median IME values for durvalumab, atezolizumab, and avelumab administration were 3.14, 3.00, and 3.73, respectively. One paper mentioned the administration of at least one anti-PD-L1 drug in combination therapy, of which the IME value was 4.14. The IME values ranged from 2.55 to 4.14, with a median of 3.41 , 6. ThesTwenty-one manuscripts mentioned the administration of ipilimumab, while two papers mentioned tremelimumab. The median IME values for papers describing ipilimumab and tremelimumab administration were 2.88 and 3.78, respectively. In addition, 15 papers mentioned an association between combination therapy involving at least one anti-CTLA-4 drug and the onset of MG/LEMS, of which the median IME value was 2.88. Overall, the median IME values for anti-CTLA-4 administration ranged from 2.38 to 6.61, with a median of 2.93. Interestingly, the only manuscript that described induction of experimental autoimmune MG (EAMG) explored possible mechanisms in mice and was thus able to demonstrate a high level of evidence for anti-CTLA-4 antibodies , 6 43)..43). OveOverall, 20 of the 94 manuscripts mentioned at least one type of combination therapy with or without ICI monotherapy. The most common combination was nivolumab and ipilimumab, mentioned in 13 papers. Fourteen papers mentioned combination therapy with at least one anti-PD-1 drug , one with at least one anti-PD-L1 drug , and 15 with at least one anti-CTLA-4 drug (median IME value = 2.88; There was no significant difference between the IME values of ICI classes, of individual ICIs, or of combination therapies , 6. ThesICIs are commonly used to treat cancer. Tumors activate immune checkpoints such as CTLA-4 and PD-1 to inhibit anti-tumor T cell responses , 149. ICNumerous reviews and meta-analyses have been conducted to elucidate the safety of ICIs. Hottinger outlines the many possible, albeit rare, neurological irAEs known to be associated with ICI administration, highlighting the lack of guidelines available to identify those at risk of developing irAEs . In a 20Most the papers included in our qualitative analysis yield a low to intermediate level of evidence in support of a causal link. However, we suggest that this lack of cogent evidence stems from the design of the papers themselves. Many of the studies reviewed were case reports or case series, intrinsically unable to provide convincing evidence. There was a noticeable lack of mechanistic studies that could present higher levels of evidence by virtue of study design. The sole prospective, blinded, randomized control trial that met our inclusion criteria, a paper in which EAMG was induced in mice, yielded an IME value of 6.61, demonstrating high evidence for a causal link . This obSome of the larger studies sourced by our literature search failed to satisfy our inclusion criteria and were therefore excluded from formal analysis but were considered in our qualitative review in order to maximize information derived from the dataset . These sIn recent years, the administration of ICIs has become a mainstream treatment for cancer , 163. PhThe original contributions presented in the study are included in the article/Under the supervision of OG, CS developed the search algorithms, assessed the relevance of all abstracts and full texts, analyzed and interpreted the results, and wrote the first draft of the manuscript. OG analyzed and assessed a subset of articles to ensure concordance of scores. OG and CS developed the scoring system on the basis of a published instrument created by OG and Y-MC. Y-MC helped with statistical analysis and created some of the figures. OG and JL conceptualized the idea for the manuscript and edited the manuscript. All authors contributed to the article and approved the submitted version.


Tnf from our previously established murine model of colitis-associated cancer (CAC), the Winnie-Min/+Apc line. We characterized the new Winnie-Min/+-APCTNF-KO line through macroscopical and microscopical analyses. Surprisingly, the latter demonstrated that the deletion of Tnf in Winnie-Min/+Apc mice resulted in an initial reduction in dysplastic lesion incidence in 5-week-old mice followed by a faster disease progression at 8 weeks. Histological data were confirmed by the molecular profiling obtained from both the real-time PCR analysis of the whole tissue and the RNA sequencing of the macrodissected tumoral lesions from Winnie-Min/+-APCTNF-KO distal colon at 8 weeks. Our results highlight that TNF could exert a dual role in CAC, supporting the promotion of neoplastic lesions onset in the early stage of the disease while inducing their reduction during disease progression.Colorectal cancer (CRC) is among the best examples for depicting the relationship between inflammation and cancer. The introduction of new therapeutics targeting inflammatory mediators showed a marked decrease in the overall risk of CRC, although their chemopreventive potential is still debated. Specifically, a monoclonal antibody that blocks tumor necrosis factor (TNF), infliximab, increases CRC risk in inflammatory bowel disease patients. To address the axis between TNF and CRC development and progression, we depleted the  In recent years, the incidence and mortality of cancer have been rapidly increasing worldwide, particularly in industrialized countries . SeveralInterestingly, the introduction of new therapeutic options that target either immune responses or inflammatory mediators has been shown to markedly decrease the overall risk of CRC ,11, evenMin/+Apc model [However, conflicting reports exist concerning the relationship between IFX-based therapy and an increased risk of CRC and other forms of cancer in IBD patients receiving such therapy ,22. The /+ model ,29, and Tnf from Winnie-Min/+Apc mice and characterizing the development of neoplastic lesions from the histological and molecular points of view in the new Winnie-Min/+Apc-TNF-KO murine line. Our results demonstrate a dual role for TNF in CAC pathogenesis thanks to its ability to support the promotion of neoplastic lesions onset at the early stage of the disease and its ability to reduce their incidence during disease progression.In the present study, we addressed the impact of TNF on the development and progression of CAC by genetically depleting Min/+Apc model [Min/+Apc mice lacking TNF by crossing commercially-available Tnf null mice (line TNF-KO) [Min/+Apc mice. The intent was to create the Winnie-Min/+ApcTNF-KO model, and we used the same breeding strategy that was used to generate Winnie-Min/+Apc mice, i.e., double heterozygote mutations for males  and a single heterozygote mutation (Muc2) for females. In addition, a Tnf null mutation in a homozygous state was present on both breeders to generate Winnie-Min/+Apc-TNF-KO mice and control littermates , we dev TNF-KO)  with WinMin/+Apc mice [Min/+Apc-TNF-KO mice from the reduction in body weight, colon length, and their relative ratio resulting from the missense mutation in Winnie mice . Togeth-KO mice D. This pn/+ mice , was mor 8 weeks D. Thus, n/+ mice  , while at 8 weeks, the number of genes dramatically increased up to twenty-four (one up- and twenty-three down-modulated genes) (Cdkn1a), a target of the tumor suppressor p53. Network analyses of the differentially expressed genes revealed the role of numerous interconnected genes in the free radical scavenging pathway and metabolic disease at 5 weeks and in cancer and cell death/survival pathways along with organismal injury at 8 weeks  A,B. SurpMin/+Apc-TNF-KO mice and compared these data with those obtained from the sequencing of Winnie-Min/+Apc mice of the same age. An unsupervised hierarchical clustering of the gene expression data showed distinct segregation between RNA from Winnie-Min/+Apc-TNF-KO and Winnie-Min/+Apc ACFs . The IPA core analysis revealed that the top diseases and functions associated with the dataset were deeply related to cancer and its features, cellular assembly and organization, and gastrointestinal disease .In line with the IPA prediction of inhibition for this gene, TGFβR2 resulted to be highly down-modulated in Winnie-status and the use of routinary endoscopic screening in adult IBD patients for efficiently reducing CRC risks, epidemiological studies still reveal the association between IBD and CRC [Colorectal cancer is among the best examples for depicting the relationship between inflammation and increased cancer risk. Despite the use of therapies for better control of the inflammatory  and CRC ,33,34. O and CRC ,8,35.Min/+Apc line, which is able to recapitulate the pathology and clinical progression in humans. The Winnie-Min/+Apc model demonstrates that mild but chronic intestinal inflammation is sufficient to support tumor development in genetically susceptible subjects, giving the advantage of fully dissecting the cascade of events leading to CAC onset and progression. Of note, our previously established model could also be useful for designing alternative therapies by testing new predictive biomarkers for early CAC diagnosis in IBD patients. The lack of response to biological agents  is one of the major drawbacks of this new frontier of drugs with an enormous financial cost for the health system; this is especially true for young UC patients that escape the endoscopic screening due to the presence of flat, rather than polypoid, adenomas. To resemble this subgroup of patients, we created the new Winnie-Min/+Apc-TNF-KO murine line by depleting the expression of Tnf from the Winnie-Min/+Apc line. In the present study, we analyzed the role of TNF in CAC onset and progression by taking advantage of our previous murine model of UC-induced CAC, the Winnie-Min/+Apc-TNF-KO mice with the already published Winnie-Min/+Apc model, a different modulation for the incidence, multiplicity, and tumor grading of the tumoral lesions was reported. Surprisingly, we demonstrated that the absence of TNF protected Winnie-Min/+Apc-TNF-KO mice from the early onset of dysplastic lesions at 5 weeks , whereas it induced an exacerbated progression of dysplastic lesions during the transition from early to intermediate time points (8 weeks). These results were supported by the molecular analysis of the whole colon from Winnie-Min/+Apc-TNF-KO mice relative to the Winnie-Min/+Apc mice, which showed the modulation of the free radical scavenging and metabolic disease signaling at early time points. In line with the literature, both these diseases and functions support an early onset of CRC [Cdkn1a, which showed an up-modulation. It is possible to speculate that p21 encoded by Cdkn1a could either act as an oncogene or tumor suppressor gene depending on the cellular context, subcellular localization, and post-translational modifications [Min/+Apc-TNF-KO mice relative to the Winnie-Min/+Apc mice. Surprisingly, the absence of TNF induced a more aggressive phenotype for the tumoral lesions in Winnie-Min/+Apc-TNF-KO mice because of the altered mechanisms involved in carcinogenesis, tumor microenvironment, and cellular remodeling; thus, this observation supports the presence of the “advanced malignant tumor” disease and functions among the top regulator effect networks in the context of gastrointestinal disease, which was identified by the IPA. Specifically, the more aggressive phenotype of Winnie-Min/+Apc-TNF-KO mice could be supported, among others, by the TGFβ signaling. In fact, in line with the IPA-predicted inhibition for TGFβ as an upstream regulator, TGFβR2 greatly decreased in Winnie-Min/+Apc-TNF-KO as compared with Winnie-Min/+Apc mice. This data could be explained by considering the relationship between the TGFβ gene and the expression of TNF as reported in the literature, even if different pathological contexts had been studied [Min/+Apc-TNF-KO was also supported by a paper describing the development of lethal inflammatory disease and invasive CRC in a mutant APC model of colon carcinogenesis, which was characterized by an epithelial truncation of TGFβR2 [Min/+Apc-TNF-KO mice. Of note, this interaction implies an accurate consideration for the treatment of CAC patients; in fact, the use of TGFβ inhibitors may result in a worse outcome through the enhancement of the inflammatory responses. However, a deep insight into the TGFβ signaling paradoxical dynamics related to the disease stage (early vs. late) is needed to clarify its role in CAC pathogenesis.Our results demonstrated that the deletion of TNF did not ameliorate the phenotype induced by the Winnie mutation but rather increased the frequency of dysplastic lesions in the distal tract of the large intestine relative to the control littermates. Comparing the tumorigenic potential of Winnie-t of CRC ,37. Furtications . Moreove studied ,40,41. Of TGFβR2 . Thus, tf TGFβR2 . More gef TGFβR2  and actsf TGFβR2 ,46. ThusOur investigations were performed under the relevant animal protocol, which was approved by the Institutional Animal Care Committee of the National Institute of Gastroenterology “S. de Bellis” . All of the animal experiments were carried out according to the national guidelines of the Italian Directive n. 26/2014 and were approved by the Italian Animal Ethics Committee of the Ministry of Health—General Directorate of Animal Health and Veterinary Drugs (DGSAF- Prot. 768/2015-PR 27/07/2015). All animals were maintained in a controlled environment .Min/+Apc-TNF-KO was created by breeding Winnie, Min/+Apc mice, and Tnf knockout murine line on a C57BL/6J (WT) background. The WT, Min/+Apc mice, and Tnf knockout murine lines were purchased from Jackson Laboratories  .The Winnie mice were obtained from the University of Tasmania (Dr. R. Eri’s laboratory). The new murine transgenic line Winnie-Mice were sacrificed at 5 or 8 weeks of age, and their colons were removed to evaluate the presence of dysplastic lesions. The colon lengths, weights, and ratio between the colon length and total body weight were measured as indicators of colonic inflammation. Tissue sections from the large intestine were fixed in 10% buffered formalin, dehydrated, and paraffin-embedded. We stained 3-micrometer thick sections from the proximal, medial, and distal colon regions using a hematoxylin-eosin standard protocol. Colonic tissue sections were evaluated for neoplasia. Periodic acid–Schiff (PAS) staining on distal colon sections was performed to identify mucins. Observations and imaging were performed with a Nikon Eclipse Ti2 microscope . ® reagent  according to the manufacturer’s instructions. The RNA quality was checked by the A260/280 ratio. The total RNA (1 µg) was reverse-transcribed using an iScript cDNA Synthesis kit  using random primers for cDNA synthesis. Starting at 10 ng of cDNA for sample, the gene expression analysis was performed testing four animals for each experimental group and using Colonic neoplasms Tier 1 M96 . Each plate contained internal controls to check for DNA contamination, PCR efficiency, RNA integrity, and cDNA quality; for the latter, a control RNA template was added during the reverse transcription reaction -stained slides. Specifically, the tumoral area was circled on HE slides and used to perform a manual macrodissection on 10-micrometer thick unstained sections that were corresponding and consecutive to HE slides. Under the guidance of a microscope, the pathologist first removed the normal tissue that externally surrounded the circled area and then scraped off the slide, i.e., the tumoral area, using a sterile fine blade collecting the dissected material into RNase free tubes. The procedure was performed taking care to not carry more than 30% of normal tissue within the sample. RNA was extracted from the macrodissected tissues using the Qiagen RNeasy FFPE kit  according to the manufacturer’s instructions.Next-generation sequencing experiments, comprising sample quality control, were performed by Genomix4life SRL . Indexed libraries were prepared from 20 ng of purified RNA with the TruSeq RNA Exome sample prep kit  according to the manufacturer’s instructions. Libraries were quantified using the TapeStation 4200 system  and Invitrogen Qubit fluorometer  and were then pooled such that each index-tagged sample was present in equimolar amounts, with the final concentration of the pooled samples being 2 nM. Pooled samples were subjected to cluster generation and sequencing using an Illumina NextSeq 500 system  in a 2 × 75 paired-end format at a final concentration of 1.8 pmol.Sequencing data are available at the ArrayExpress database under the accession number E-MTAB-12356.http://www.bioinformatics.babraham.ac.uk/projects/fastqc; accessed on 19 October 2022). The mapping of paired-end reads was performed using the bioinformatics tool STAR (version 2.5.1a) [http://www.ensembl.org/Mus_musculus/Info/Index; accessed on 19 October 2022). The quantification of transcripts expressed for each replicate of the sequenced samples was performed using the featureCount algorithm [Fastq underwent quality control using the FastQC tool ( 2.5.1a) , with thlgorithm . The Biolgorithm  was usedNetworks and biological functions on differential expressed genes were assessed using IPA software . The canonical pathways and networks were the results of the IPA core analysis.t-tests. Results were considered statistically significant at p < 0.05. For all other analyses, statistical evaluations were performed using the GraphPad Prism statistical software, release 8.4. Data were expressed as the mean ± SEM, and results were obtained from three consecutive and independent experiments. Before proceeding to statistical comparisons, the data normality and homogeneity of variance were checked by the Shapiro–Wilk test and F test, respectively. Unless specifically described, all data were analyzed and compared by paired, two-tailed Student’s Min/+Apc-TNF-KO mice could be an effective model for the discovery of new biomarkers to be used for the early detection of young UC patients unresponsive to anti-TNF therapy as well as for the prediction of patient prognosis in this subgroup of UC patients that rapidly develop CAC. Thus, our results could be useful not only in reducing the economic costs but also in helping the design of appropriate precision medicine protocols to prevent CAC onset and/or progression.The data reported in this study point out a dual role for TNF in CAC, promoting the onset of neoplastic lesions at early time points and controlling their later progression; however, additional studies are needed to verify the mechanisms identified by our work. Importantly, the Winnie-In conclusion, the depletion of TNF from the colon of CAC-developing mice started a cascade of events, which induced the onset of tumor lesions in the distal colon at intermediate time points; these lesions are characterized by a more aggressive phenotype. This peculiar mechanism is supported by TGFβ as defined by our molecular classification. In summary, this work provides crucial insights towards the correct management of anti-TNF therapy in UC patients.


Angina pectoris (AP) occurs when oxygen and other nutrients are insufficient to meet the metabolic needs of the heart muscle. Stable angina is the most common, while the unstable angina is less frequent. Tumor necrosis factor alpha  is a pleiotropic cytokine plays a vital function in the immune response regulation. TNF gene cluster contains many polymorphisms; the most commonly investigated polymorphism is the rs1800629 SNP. This SNP, located at − 308 position with regard to the TNF promoter region, replaces guanine (G) with adenine (A), with the allelic types − 308 G/A, and has been linked to a variety of inflammatory condition and autoimmune diseases. The − 308 G/A SNP was investigated in AP and interconnected to the TNF level to figure out the responsibilities of TNF-alpha gene polymorphism in the pathogenesis of AP.n = 100 and unstable angina n = 100) compared with 100 apparently healthy control subjects. The serum level of TNF-alpha was assessed via enzyme-linked immunosorbent assay (ELISA)/sandwich method. The genotype and allele frequency distribution of TNF-alpha rs1800629 gene polymorphism were investigated by TaqMan probe of allelic discrimination method.The current work design as a case–control study that involves 300 participant divided to 200 patients evaluated as  − 308 G/A revealed higher significantly differences of GG homozygous and GA heterozygous proportions between stable angina patients and control. The A allele was more represented as etiological allele, and G allele was represented as protective allele. The serum levels of TNF-alpha were significantly higher in subjects with genetically mutated AA genotypes than in subjects with wild GG genotypes in the study groups. ROC curve analysis found the best cutoff value of TNF-alpha level was 77.25 pg/ml.The levels of TNF-alpha were significantly higher in patients with stable and unstable angina pectoris in comparison with controls. The deviation from Hardy–Weinberg equilibrium (HWE) of TNF-alpha genotypes was obvious in control and unstable angina pectoris groups. Moreover, the significant differences between patients with AP and controls under the five genetic models consider the association between TNF-alpha (rs1800629) − 308 G/A and AP with As the results, our data observed a linked of TNF-alpha (rs1800629) − 308 G/A genetic variant with angina pectoris patients, and the A allele has been linked to the production or expression of TNF-alpha serum level and represented an etiological factor of angina pectoris.  The major cardiovascular phenotypes of ischemic heart disease (IHD) are coronary artery disease (CAD), myocardial infarction (MI), and stable/unstable angina pectoris. The etiology of IHD is largely acknowledged to be complicated and polygenic, with environmental factors such as high lipid levels, smoking, sedentary lifestyle, and infectious agents, as well as genetic susceptibility . Angina The presence of TNF-alpha polymorphisms has been linked to a variety of ailments, such as rheumatoid arthritis, types 1 and 2 diabetes, ankylosing spondylitis, sarcoidosis, and silicosis . The docEthical permissions were received from the subjects, and the study was also authorized by the Ethics Committees of the Biotechnology Research Center (BRC)/Al-Nahrain University according to the research ethics checklist of human subjects Ref. No. (M.B. 25). It was a case–control study; 300 participants including 200 subjects who had angiography that established the severity of (IHD), with criteria of 50% stenosis of at least one major coronary vessel due to atherosclerosis and assessed by two qualified cardiologists, and classify to 100 patients with stable angina and 100 patients with unstable angina were recruited from the “Iraqi center for heart disease at a surgical specialist hospital in Baghdad, Iraq” between February 2021 and August 2021 to participate in the present study. A prior history of IHD, MI, or AP is established by combining clinical data with a thorough study of medical records demonstrating diagnostic of electrocardiography (ECG) (significant elevation of ST segment), ejection fraction (EF%) as an assessment of the quantity of blood ejected by the left ventricle, and enzyme alterations. In addition, 100 apparently healthy subjects were those health issues and examination findings used to establish if the control participants who lived in the same regions as the cases were free of IHD and peripheral atherosclerotic artery disease.t-tests) and statistical test (means: difference between two independent means (two groups) were used for the analysis. The effect size d = 0.2, critical t = 0.73, α and β err prob = 0.2072386, and power (1-β err prob) = 0.81.The G*Power software 3.1.9.7 was used to estimate the sample size of the current study. Test family /sandwich method using the kit’s manufacturer’s protocols  of one step assay with ranged from 31.25 pg/ml–2000 pg/ml and sensitivity 14 pg/ml.This study has investigated the polymorphism (rs1800629) G/A substitution at position (− 308) in the promoter region of TNF-alpha gene. To extract genomic DNA from EDTA blood, the Wizard Genomic DNA Purification Kit was utilized . The thermal profile of allelic discrimination approach employing real-time PCR was used to explore this polymorphism . The sequences of primers and probes were designed for the current study using the NCBI  database and synthesized by Alpha DNA Ltd. (Canada).The reaction was conducted in a final quantity of 20 µl including 0.5 µl of each working SNP genotyping assay consist of primers (forward and reverse), probes (FAM and VIC), 10 µl TaqMan® Master Mix, 3 µl genomic DNA, and nuclease-free water and was used to complete the final volume of the reaction. Real-time PCR program cycling conditions have include initial denaturation at 94 °C for 10 min by 1 cycle accompanied by 5 cycles of denaturation at 95 °C for 20 s, annealing at 60 °C for 30 s, and extension at 72 °C for 40 s and then 35 cycles of denaturation 15 s, 94 °C/annealing 35 s, and 60 °C/extension 30 s, 72 °C.P ≤ 0.05. Determined the differences of genotypes, allele frequencies, odd ratio (OR), and confidence interval (95% CI) between the groups via calculating the Fisher’s exact probability and to detect the attributable and preventive fraction in population by using statistical software (WINPEPI). The ROC curve was used to determine and predict the best cutoff value of TNF-alpha level.Analyses of data were performed using statistical comparison between angina pectoris and control subjects. TNF-alpha levels were provided as mean ± standard deviation (M ± SD) and were significant differences assessed using one-way analysis of variance (ANOVA). Observed and expected genotype was performed by chi-squared analyses used to test the deviation of genotype distribution from Hardy–Weinberg equilibrium (HWE) when The serum level of TNF-alpha was estimated among studied groups. The findings demonstrated a significant difference of the TNF-alpha concentration in stable and unstable angina and reached about 2-folds  when compared to control group 35.98 ± 15.8) respectively. No significance has been indicated between patient groups. Table 5.8 respeP ≤ 0.05) demonstrated by Hardy–Weinberg equilibrium (HWE) analyses; therefore, the accordance with the HWE was not established and deviated from the law. No significance was observed in patients with stable angina pectoris (P > 0.05). These findings were demonstrated in Table The genetic variation rs1800629) -308 G/A of TNF-alpha was represented by three proportions , all of which corresponded to two alleles (G and A). Based on categorization findings, there was a significant difference in patient with unstable angina and control between the observed and expected genotype frequencies (9 -308 G/OR > 1 and (p ≤ 0.05). Furthermore, there were significant differences in the A allele between patients with AP and controls (198 vs. 50) with .The results in Table p ≤ 0.05). In contrast, there was statistical insignificant in the state of homozygosity mutant AA proportion between the two groups (p > 0.05). With the prevalence of the G and A alleles, the significant results were noted across patients with stable angina and controls (P ≤ 0.05) ;  in the G and A alleles, respectively.The estimation of the risk of angina pectoris suggested by the proportion level was shown in Tables P ≤ 0.05) ; , respectively. The comparison was significant in the G and A alleles between the two groups of the ; . Taking into consideration that the OR less than 1 meant the genotype or allele has a protective function in population and more than 1, the genotype or allele has an etiological or attributable function in a population.Regarding the results of Table p ≤ 0.05) with the highest level reported in the patients of angina pectoris (stable and unstable), whereas the most significant was identified in the stable and unstable angina patients through the equivalent proportions with elevated level in the AA genotype.The evidence of the associated TNF-alpha concentration and genotypes of the corresponding SNP was illustrated in Table CI: 95% (0.594–0.996).The ROC curve Fig.  showed tThe objective of this study is to investigate the link between TNF-alpha rs1800629) − 308 G/A genetic polymorphism and the risk or susceptibility to angina pectoris in its two forms in the Iraqi population, based on the supposition the IHD that include angina pectoris is a chronic inflammation condition. In this manner, the results suggest that the (rs1800629) − 308 A allelic variant was accompanied by changes TNF-alpha production and an increase in risk of angina pectoris in the Iraqi population. Several variations were identified in the promoter region of the human TNF gene, with the potential to establish structural changes inside regulatory regions, potentially disrupting the performance and management of TNF-alpha production or expression [00629 − 3Chronic inflammatory process is marked by elevated circulation levels of pro-inflammatory cytokines, adhesion molecules, and cytokine-responsive acute-phase proteins. Many of these inflammatory plasma indicators have been identified to predict future cardiovascular risk in patients with acute coronary syndrome. TNF-alpha levels in the blood have been linked to atherosclerosis of coronary artery risk factors such as dyslipidemia, overweight, and inflammation . In a saP ≤ 0.05) with OR > 1. Thereby, the results sustained with [Further suggestion by  revealedned with  demonstrOR = 1.50; 95% CI: 1.23–1.77) [Genotypes and allele frequencies of TNF-alpha gene polymorphism showed a different genetic distribution between the studied groups. Both groups of patients with angina pectoris have significant differences of genotypes and G/A allele in comparison with control. These results can give information of the susceptibility for angina pectoris in the Iraqi patients by investigating the role of risk or protective alleles of rs1800629) -308G/A at the promoter region of TNF-alpha gene. However, the possibility of attributable or etiological allele which has OR more than 1 exhibited risk factor in patients who is bearing the AA and/ or GA proportions and A allele. The results demonstrated that in the existence of the AA genotype, the concentration of TNF-alpha remained considerably higher in the examined groups, particularly in patients with unstable angina (106 ± 15.7). These observations imply that TNF-alpha may exhibit a role in the pathogenicity of angina pectoris among participants, which is genetically determined. Moreover, the (rs1800629) − 308 G/A has been the most studied variant of TNF-alpha, because numerous studies have found that the individual with the A allele possesses elevated rates of TNF-alpha in serum, significantly affecting the risk of developing CAD , 18. Man00629 -3023–1.77) . To offe23–1.77) . TNF-alp23–1.77) . InteresTo date, there were no data on the correlation of the TNF-alpha − 308 G/A with angina pectoris in the Iraqi population. This study provides a significant link between TNF-alpha -308G/A gene polymorphism and susceptibility to AP. Meanwhile, it could be suggested that A allele of − 308 G/A SNP of TNF-alpha is tightly correlated with a considerable risk of angina pectoris development according to the OR and the circulatory levels of TNF-alpha in the individuals with the mutant genotype. However, these findings of the critical concentration of TNF-alpha were recommend to be used as biomarker for angina pectoris.