Antitumor necrosis factor therapies have revolutionized the treatment of some inflammatory diseases. However, the use of these agents is associated with the development of many paradoxical autoimmune diseases. Less well-recognized is the association with sarcoidosis. We report a 55-year-old female with long-standing ankylosing spondylitis who developed persistent dry cough and dyspnea while receiving etanercept therapy. High-resolution computed tomography scanning showed mediastinal lymphadenopathy and multiple nodules in both lung fields developed two months after the administration of etanercept. Lymph node biopsy was not practicable. Histopathological examination of minor salivary gland biopsy revealed noncaseating granulomata, and the serum angiotensin-converting enzyme was very elevated. All infectious studies were negative. Etanercept was discontinued plus a course of corticosteroids with a clinical improvement, and a follow-up high-resolution computed tomography scanning 4 months later showed evident regression of mediastinal lymph nodes and pulmonary nodules. Potential pathogenic mechanisms of this paradoxical effect of tumor necrosis factor-alpha blocking agents are discussed. Its etiology remains unknown . Howevercoidosis . Our casA 66-year-old female patient without medical history or professional exposures was diagnosed with ankylosing spondylitis 12 years ago. She had formerly responded to nonsteroidal inflammatory drugs. In 2006, she started treatment with infliximab, due to poor therapeutic response to various NSAIDs. After eight years of remission, the patient had repeated flares of ankylosing spondylitis symptoms with increased inflammatory back pain. In August 2015, due to persistent clinical activity with increased acute-phase reactant levels (C-reactive protein level of 16 mg/l), the treatment with etanercept at 25 mg twice weekly was started. Following national guidelines to exclude latent tuberculosis, mycobacterium tuberculosis research in sputum and interferon-gamma test were negative and a chest radiograph was normal. Two months after etanercept therapy onset, the patient developed persistent dry cough and dyspnea without fever or night sweating. Physical examination was unremarkable apart from tachypnea and severe spinal stiffness. High-resolution computed tomography (HRCT) scanning showed mediastinal lymphadenopathy and multiple nodules in both lung fields . Echocarα. The TNF-α is the key cytokine that participates in the formation of granulomas in sarcoidosis [Sarcoidosis is a multisystem disorder characterized by the accumulation of lymphocytes, mononuclear phagocytes, and noncaseating granulomas in involved tissues. Etiology of sarcoidosis is still not identified. The role of viral, bacterial, and parasitic infections is not clearly determined. The role of some drugs and environmental exposure are still considered . Due to coidosis , 4.α agents currently available: four monoclonal antibodies  and a soluble TNF-α receptor fusion protein (etanercept) [α agents, especially infliximab and sometimes adalimumab, are effective in refractory sarcoidosis [α therapy. In a recent review of literature published on January 2016, SimJae Kyeom et al. found 59 cases of sarcoidosis occurring during anti-TNF-α therapy in the medical literature. Thirty-seven (61.6%) occurred after etanercept, 13 (21.6%) after adalimumab, and 9 (15%) after infliximab. In this study, twenty-eight had rheumatoid arthritis. The lung and/or lymph node were the most commonly affected organs (64%), followed by the skin (37%) and the eye (15%). Prognosis seems to be favorable since 52 of 59 cases displayed a partial or complete resolution after anti-TNF-α withdrawal with or without steroids [At present, there are five anti-TNF-nercept) . Anti-TNcoidosis , 7. Howeα remains unclear. However, some potential hypotheses have been proposed. Mainly followed hypothesis is that sarcoidosis is of an infectious origin favored by an immunosuppressant effect of anti-TNF-α, especially with some microorganisms that are implicated in the development of sarcoidosis (Propionibacterium acnes and Propionibacterium granulosum) [α. Currently, there are many reports supporting this latter hypothesis [α receptor and monoclonal antibodies: the antibodies neutralize both soluble and membrane forms of TNF-α, whereas etanercept neutralizes just the soluble form. This partial inhibition permits the redistribution of TNF-α in areas of elevated concentration such as the lungs and lymph nodes [The pathogenesis of sarcoidosis induced by anti-TNF-nulosum) . Anotherpothesis –11. Indeph nodes . Taking ph nodes . In summph nodes .Pulmonary sarcoidosis is an uncommon effect of anti-TNF therapy. The pathogenic mechanism is unclear. Minor salivary gland biopsy may help in diagnosis. The management requires discontinuing anti-TNF plus a course of steroids.


GCT) often comprising of CD30‐positive embryonal carcinoma (EC) components remains poor. Thus, novel treatment strategies are warranted. The antibody‐drug conjugate (ADC) brentuximab vedotin delivers the potent antimitotic drug monomethyl auristatin E (MMAE) to CD30‐expressing tumour cells. After CD30 binding, internalization and intracellular linker cleavage cytotoxic MMAE can efflux and eradicate neighbouring CD30‐negative cells. To analyse cytotoxicity and a potential bystander effect of brentuximab vedotin in GCT, we established an in vitro coculture model mimicking GCT of heterogeneous CD30 positivity and measured cell viability, proliferation and apoptosis after exposure to brentuximab vedotin and unbound MMAE by MTS‐ and flow cytometry‐based CFSE/Hoechst assay. CD30 expression being assessed by quantitative RT‐PCR and immunohistochemistry was apparent in all EC cell lines with different intensity. Brentuximab vedotin abrogates cell viability of CD30‐positive GCT27 EC line exerting marked time‐dependent antiproliferative and pro‐apoptotic activity. CD30‐negative JAR cultured alone barely responds to brentuximab vedotin, while in coculture with GCT27 brentuximab vedotin induces clear dose‐dependent cytotoxicity. Cellular proliferation and cell death are significantly enhanced in CD30‐negative JAR cocultured with CD30‐positive GCT27 compared to JAR cultured alone in proof of substantial bystander activity of brentuximab vedotin in CD30‐negative GCT. We present first evidence that in an in vitro model mimicking GCT of heterogeneous histology, brentuximab vedotin exerts potent antiproliferative and pro‐apoptotic activity against both CD30‐positive as well as CD30‐negative GCT subsets. Our results strongly support translational efforts to evaluate clinical efficacy of brentuximab vedotin in high‐risk GCT of heterogeneous CD30 positivity.Prognosis in patients suffering from high‐risk, refractory and relapsed germ cell tumours ( GCT thought to arise from primordial germ cells often display a mixed histological phenotype comprising teratoma, seminoma, choriocarcinoma, yolk sac tumour or EC components Prognosis in patients with seminomatous GCT is excellent and still favourable in non‐seminomatous GCT. In patients with metastatic, refractory or recurrent disease, outcome remains poor Today, targeted agents are the focus of anticancer drug development. Cytotoxic agents linked to monoclonal antibodies selectively deliver drugs to target sites enhancing local drug activity while reducing systemic toxicity. Brentuximab vedotin (BV) represents one of these antibody‐drug conjugates (ADC). Brentuximab vedotin targets CD30 with promising clinical responses and good tolerability in patients with refractory Hodgkin (HL) or anaplastic large cell lymphoma in vitro model mimicking GCT of mixed histology, brentuximab vedotin exerts potent antiproliferative and pro‐apoptotic activity against both CD30‐positive as well as CD30‐negative GCT subsets. Our results provide insights that substantiate early clinical efforts to translate this promising drug into the clinical setting.While first pilot studies evaluating brentuximab vedotin in adults with refractory testicular cancer have been initiated 2102EP, NT2/D1 and NCCIT cells were kindly provided by L. Looijenga (Daniel den Hoed Cancer Center/NL), TCam‐2 by J.Shipley , 833KE and GCT27 by T. Müller  and B. Köberle , respectively. JAR (HTB‐144) and JEG‐3 (HTB‐36) were purchased from American Type Culture Collection. All cell lines are known to be cisplatin sensitive. Cell lines were cultivated as described previously 4 tumour cells in PBS/1.5% BSA were cytospun at 12000 g for 5′ onto glass slides and air‐dried for 15′. Signal detection was performed semiautomatically in the Autostainer 480 S  using the Bright Vision+ polymer detection system (Medac) and the following settings: anti‐CD30 primary antibody  for 20′, enhancer for 10′, polymer (Poly‐HRP‐Goat anti‐mouse/‐rabbit‐IgG) for 20′, 3,3′‐diaminobenzidine (DAB)  for 8′. Nuclei were stained by haematoxylin for 3′.A total of 4 × 10Quantitative real‐time RT‐PCT (qRT‐PCR) was performed as described previously 3 GCT27 and 1 × 104 L540 cells were seeded for 48 hrs, 2.5 × 103 GCT27 and 8 × 103 L540 cells for 72 hrs and 2 × 103 GCT27 and 5 × 103 L540 cells for 96 hrs in a 96‐well plate in 100 μl medium at 37°C. After 4 hrs, 250 ng/ml BV and 100 pM MMAE  or the vehicle PBS was added.Cell viability was assessed by MTS analysis in the ‘CellTiter 96 Aqueous One Solution Cell proliferation Assay’  according to the supplier's instructions. To ensure exponential cell growth over time, 5 × 105 GCT27, 1 × 105 NCCIT and 0.5 × 104 JAR cells were labelled with CFSE  according to supplier's instructions and cultivated either in coculture or separately. After 12 hrs, 100 pM MMAE or 250, 500 or 1000 ng/ml BV or PBS as control was added. For flow cytometric enumeration of viable cell number and proliferation analysis, cells were washed after 24–96 hrs and resuspended in 200 μl PBS/2%FBS containing 1 μg/ml Hoechst 33258  for assessment of dead cells. Cellular proliferation was traced by progressive carboxyfluorescein succinimidyl ester (CFSE) dilution.1 × 10P‐values were performed using Microsoft Excel and GraphPad Prism. For determination of statistical significance between groups, the two‐tailed Student′s t‐test was used. P‐values < 0.05 were considered statistically significant.Calculations of mean values, standard deviation and CD30 mRNA levels. In NCCIT, NT2/D1 and 2102EP mRNA levels are 1–2 two log lower  and JAR (choriocarcinoma).The EC cell lines GCT27 and 833Ke exhibit the highest Likewise in immunohistochemical analysis Fig. B, the ECCell viability was assessed in the CD30‐positive EC line GCT27 in comparison with CD30‐positive Hodgkin line L540 by MTS assay .The proliferative capacity is comparable in the three GCT cell lines. Responsiveness to brentuximab vedotin depends on CD30 expression as well as MMAE sensitivity. During 96 hrs, untreated CD30‐positive GCT27 cells  and 68.1 ± 6.7% at 1000 ng/ml (P < 0.001).To evaluate dose‐dependent effects to brentuximab vedotin, GCT27, JAR and NCCIT cell lines were exposed for 96 hrs to 250 ng/ml, 500 ng/ml and 1000 ng/ml ADC Fig. E. As alr50 values of brentuximab vedotin in the three different cell lines: CD30‐positive and MMAE‐sensitive GCT27 cells exhibit strong vulnerability to the ADC with an IC50 value of 219.5 ng/ml. The EC cell line NCCIT presenting low CD30 mRNA expression and only focal CD30 positivity by immunohistochemistry . Cocultured viable JAR cells decline further to 30.3 ± 19.8% at 500 ng/ml and to 16.1 ± 13.1% at 1000 ng/ml brentuximab vedotin which is significantly lower than in JAR cells cultured alone with 83.3 ± 28.6% at 500 ng/ml (P = 0.02) and 48.9 ± 19.1% at 1000 ng/ml Brentuximab Vedotin (P = 0.02). Also, brentuximab vedotin reduces cell numbers of the CD30‐positive GCT27 fraction to 6.0 ± 1.4% of the untreated control at 250 ng/ml, to 4.1 ± 1.5% at 500 ng/ml and to 2.3 ± 1.8% at 1000 ng/ml.Viable cell numbers of CD30‐negative JAR cells being cultured alone for 96 hrs and treated with 250 ng/ml brentuximab vedotin are comparable to untreated control. In contrast, in coculture with the CD30‐positive GCT27 cell line, only 49.9 ± 22.1% of the JAR cells survive (P < 0.05).To assess the relative contribution of antiproliferative effects to the overall cytoreductive activity of brentuximab vedotin, the mean fluorescence intensity (MFI) of viable CFSE‐stained cells was obtained for CD30‐positive and CD30‐negative tumour cells Fig. C. InhibiCell death Fig. D was quaIn cancer of mixed histologies such as GCT, the development of antibody‐conjugated drugs faces the challenge of cytotoxin delivery to the entirety of neoplastic subpopulations. In the setting of variable target expression, potent bystander mechanisms constitute the key to therapeutic success.in vitro model, we confirm that CD30 is intensely detectable in cell lines derived from EC whereas it is sparse in seminoma and absent in choriocarcinoma. We also demonstrate different sensitivity to the spindle‐toxin MMAE both in its CD30‐conjugated and unbound form. These patterns correspond to the range of responsiveness previously described for lymphoma In testicular cancer, persistence of CD30 expression after first‐line chemotherapy is associated with a particularly poor prognosis Of note, brentuximab vedotin‐mediated cytotoxicity is already substantial in CD30‐positive EC line GCT27 at concentrations as low as 250 ng/ml. Further dose escalation to 1000 ng/ml corresponding to patients` plasma levels under standard brentuximab vedotin administration In coculture of JAR with CD30‐positive EC line mimicking GCT of mixed histology, bystander efficacy is already discernable at brentuximab vedotin concentrations as low as 250 ng/ml, too. We observed marked cytotoxicity on cocultured CD30‐negative JAR cells that exceeded the antineoplastic effect observed in single culture at all dose levels. This cumulative cytoreduction results from significant inhibition of cellular expansion as well as induction of cell death in keeping with the disruption of the microtubule architecture.After internalization of brentuximab vedotin bound to CD30‐positive tumour cells and endosomal cleavage of MMAE The different experimental models of bystander activity substantiate the clinical observation that in lymphoma patients, brentuximab vedotin has shown clinical efficacy regardless of the level of CD30 expression including CD30‐negative tumour cells in vitro observation of potent brentuximab vedotin‐mediated bystander activity that has the potential to eradicate even bulky tumours.To date, nine patients with refractory GCT were treated with brentuximab vedotin as third‐line therapy in a phase II clinical trial in vitro and in vivoin vitro (data not shown). Still in clinical studies evaluating efficacy of brentuximab vedotin in GCT, prospective assessment of MDR1 expression may be considered.In the remaining patients, serum tumour marker levels were attenuated albeit transiently indicating that resistance mechanisms may limit ADC efficacy. Indeed, resistance to brentuximab vedotin particularly in heavily pre‐treated patients is of concern. In GCT, substantial down‐regulation of CD30 surface antigen after chemotherapy has been observed Multimodality regimens are the hallmark of effective cancer therapy. First experiences indicate that the combination of brentuximab vedotin with cisplatin or radiotherapy ‐ treatment modalities of proven efficacy in GCT ‐ might be well tolerated Here, we document pronounced antiproliferative as well as pro‐apoptotic activity of brentuximab vedotin in GCT. Brentuximab vedotin effectively targets the tubulin‐toxin MMAE to CD30‐expressing GCT with substantial cytotoxicity on CD30‐negative bystander cells. These potent bystander mechanisms are a prerequisite for therapeutic success in tumours of mixed histologies such as GCT.The authors confirm that there are no conflict of interests.


In addition, the characteristics of cross-tolerance between TNF and lipopolysaccharide (LPS) as well as pathophysiological aspects of TNF tolerance are discussed. We conclude that TNF tolerance may represent a protective mechanism involved in the termination of inflammation and preventing excessive or prolonged inflammation. Otherwise, tolerance may also be a trigger of immune paralysis thus contributing to severe inflammatory diseases such as sepsis. An improved understanding of TNF tolerance will presumably facilitate the implementation of diagnostic or therapeutic approaches to more precisely assess and treat inflammation-related diseases.Tumor necrosis factor (TNF) tolerance in monocytes and macrophages means that preexposure to TNF reduces the sensitivity in these cells to a subsequent restimulation with this cytokine. Differential effects arise following preincubation with both low and high doses of TNF resulting in absolute as well as induction tolerance affecting specific immunologically relevant gene sets. In this review article, we summarize the relevance of TNF tolerance  In addition, we will refer to general features and the known molecular mechanisms underlying different forms of tolerance including the role of transcription factors, signaling systems, and receptors. Finally, the pathophysiological impact of TNF tolerance on both the resolution of inflammation and immune paralysis as well as potential diagnostic and therapeutic aspects targeting tolerance-associated mechanisms or molecules will be discussed.TNF is a potent proinflammatory master cytokine modulating inflammatory processes, and its rapid induction is fundamental for the orchestration of the immune response . The appIn vivo, TNF tolerance is characterized by organo- and cytoprotective effects, as represented by the protection of tolerized mice against subsequent injections of normally lethal TNF doses  , 9 κB , extracellular signal-regulated kinase (ERK), and p38 , 5, thatin vivo suggesting that glucocorticoids and their receptor(s) contribute to the formation of tolerance [κB translocation, and induction of anti-inflammatory  as well as downregulation of proinflammatory cytokine/chemokine  expression [In murine models, it has been shown that a blockade of the glucocorticoid receptor (GR) using the antagonist RU-38486 can prevent the occurrence of low-dose-induced TNF tolerance olerance . This mipression . For othpression . Howeverpression .β-dependent suppression of p65 phosphorylation and potential GSK3 activity  phosphorylation is completely inhibited in high-dose pretreated and restimulated cells when compared to naïve cells [Long-term preincubation with high TNF concentrations may result in the development of both absolute and induction tolerance . Followiic cells  in compaic cells . In celler level . Followive cells , 36. In ve cells .κB signaling [Due to the observation that A20 is a key molecule in the establishment of TNF/LPS cross-tolerance , its impignaling , 38 and ignaling . A20 mRNignaling , 36, an ignaling , 36 indiκB (ABIN) 1, an A20 adjuvant protein [A20 interacts and cooperates with additional factors to form the A20 ubiquitin-editing complex, that is, the adaptor molecules Tax1-binding protein 1 (TAX1BP1) and RING-finger protein (RNF) 11 , 41 as w protein , and cyl protein . ABIN1 k protein  indicati protein  suggestiIt has also been demonstrated in high-dose-tolerized primary human monocytes that protein phosphorylation is affected by protein phosphatase (PP) 1, an enzyme  characteκB-associated signaling by A20 which is supported by CYLD and ABIN1  2 . DownregThe TNF-tolerant state may further include the refractoriness towards other stimuli. For instance, cells or animals tolerized with TNF are cross-tolerant towards gram-negative bacteria , 18, LPSκB activity in the nucleus under certain conditions [κB-dependent) proinflammatory gene expression and the manifestation of a widely inert cellular state. However, since TNF tolerance and LPS/TNF cross-tolerance are established via different receptors (TNFR1 versus TLR4) [Due to the equivalent conditions they are creating especially in monocytes and macrophages, TNF tolerance and cross-tolerance appear to be based in part on overlapping or at least similarly operating molecular mechanisms. This assumption is already well substantiated in the literature revealing that both TNF- and LPS-induced tolerance/cross-tolerance are characterized by (1) decreased NF-nditions , 50, (2)nditions , 51, (3)nditions , 5, 4)  κB activnditions , (5) uprnditions , 36, andus TLR4) , 50 and us TLR4) , specifiMoreover, it has been described that the application of further agents can positively or negatively modulate the occurrence of TNF tolerance. For instance, the combined treatment of mice with TNF and leukemia inhibitory factor results in a significantly increased protective effect against a  LPS dose when compared to TNF application alone . In contTo prevent deleterious consequences of inflammatory events such as excessive or chronic inflammation, a strictly controlled and fine-tuned termination of inflammatory processes is required -55. As iκB is activated such as rheumatoid arthritis [Due to its immunomodulatory effects, TNF tolerance may comprise several clinical implications in inflammatory and malignant diseases and represents a “Jekyll and Hyde”-like cellular process. On the one hand, tolerance may be a beneficial mechanism contributing to the resolution of inflammation and the protection from sepsis-associated hyperinflammation or prolonged chronic inflammatory diseases. In this context, loss of tolerance may favour the formation of chronic TNF-dependent inflammatory diseases in which NF-rthritis , inflammrthritis , or autorthritis . On the rthritis -63 (Figurthritis . In malirthritis . For insrthritis .As summarized in this review, TNF tolerance is generally characterized by the inability of affected cells, organs, or organisms to fully respond to a restimulation with TNF following a preceding long-term incubation with the same stimulus. Thus, TNF tolerance may play a role in severe acute or chronic inflammatory diseases in which TNF is present in large amounts. As such, TNF tolerance appears to be an interesting target within the efforts to improve the therapeutic modulation of exaggerated and/or prolonged dysregulated immune responses. However, despite a variety of experimental approaches and considerable progress in the characterization of its molecular regulation in recent years, the complex network of mechanisms determining occurrence and establishment of TNF tolerance is not completely elucidated yet. In addition, the distinct role of TNF tolerance in development and progression of inflammation-related diseases as well as its clinical relevance has to be precisely established .A better characterization of the mechanisms determining TNF tolerance will improve the understanding of its clinical relevance and presumably facilitate the development of diagnostic approaches to assess different forms and states of tolerance. In addition, increased knowledge on TNF tolerance potentially offers the implementation of therapeutic approaches to treat inflammation-related diseases either by initiating or breaking the establishment of TNF tolerance.


Background: Tumor necrosis factor-α (TNF-α) inhibitors, such as infliximab, adalimumab, and certolizumab pegol are effective agents in the treatment of inflammatory bowel disease. Some individuals undergoing anti-TNF-α therapy for Crohn’s disease or ulcerative colitis develop psoriasiform lesions. This is a paradoxical finding, as classical psoriasis is known to respond to these agents.Purpose: The clinical features of anti-TNF-α-induced psoriatic dermatitis are described.Method: A 60-year-old man with Crohn’s disease treated with infliximab, who developed anti-TNF-α-induced psoriasiform dermatitis, is described.Results: The man developed erythematous skin lesions in the bilateral axillae two years after beginning infliximab treatment for Crohn’s disease. Biopsy revealed psoriasiform dermatitis, consistent with a diagnosis of anti-TNF-α-induced psoriasiform dermatitis. He was treated with clobetasol 0.05% ointment twice daily for two weeks and had significant improvement. Subsequently, he used the corticosteroid ointment two days per week and calcipotriene 0.005% ointment twice daily for five days per week to achieve and maintain clear skin.Conclusions: Anti-TNF-α-induced psoriasiform dermatitis is an infrequent complication of infliximab therapy. However, the condition may require discontinuation of the anti-TNF-α agent. Anti-TNF-α-induced psoriasiform dermatitis should be considered in the differential diagnosis when evaluating a new erythematous skin condition in an individual with a history of inflammatory bowel disease who is being treated with a TNF-α inhibitor. Tumor necrosis factor-α (TNF-α) inhibitors are used to treat both inflammatory bowel disease and psoriasis . HoweverA 60-year-old man, with an eight-year history of Crohn’s disease, had been receiving infliximab and azathioprine for two years and remained in clinical remission. He developed scaly red plaques in the bilateral axillae that were initially treated with ciclopirox amine 0.77% cream twice daily. There was minimal improvement during the next six months. When he subsequently presented for evaluation, physical examination revealed eight scaly red plaques in the left axilla and one plaque in the right axilla, all measuring between 1-3 cm in the greatest diameter. The patient consented to have his photos used in this case report Figures .Biopsy of two lesions showed similar findings Figures -8. The sCorrelation of the clinical presentation, cutaneous morphology, and pathology changes established a diagnosis of TNF-α inhibitor-associated psoriasiform dermatitis. Initial treatment was clobetasol 0.05% ointment twice daily. Two-week follow-up revealed significant improvement of the skin lesions. Subsequently, he used the corticosteroid ointment two days per week and calcipotriene 0.005% ointment twice daily for five days per week to achieve and maintain clear skin.The occurrence of psoriasis-like lesions associated with anti-TNF-α therapy is infrequent, with an incidence rate of 1.04 to 3.0 per 1,000 person-years . The devAlthough several studies have attempted to elucidate the mechanism of anti-TNF-α-induced psoriasiform dermatitis, the pathogenesis of this condition remains unclear. Recent theories postulate that increased interferon-α levels, which are associated with reduced TNF-α, may contribute to the development of psoriatic plaques -4. SysteDecreased TNF-α levels appear to be associated with increased levels of pro-inflammatory cytokines, such as interleukin-12, interleukin-17, and interleukin-23, which may also contribute to the inflammatory reaction [Although the lesions of classical psoriasis and anti-TNF-α-induced psoriasiform dermatitis appear similar, their management may differ. Classical psoriasis can be treated with a variety of topical  and/or systemic  agents. In contrast, anti-TNF-α-induced psoriasiform dermatitis may only be resolved with the discontinuation of the anti-TNF-α agent or switching to another TNF-α inhibitor , 8.As demonstrated by our patient’s case, anti-TNF-α-induced psoriasiform dermatitis may be responsive to high-potency topical corticosteroids. Consistent with the observations, several studies also report the use of topical corticosteroids as the initial treatment for anti-TNF-α-induced psoriasiform dermatitis, with response rates ranging from 40-47% , 7. In tAlthough the occurrence of psoriatic dermatitis in association with anti-TNF-α therapy for inflammatory bowel disease is a rare occurrence, this complication involves considerable morbidity. Several studies have documented the difficulty of obtaining remission from the skin lesions . Cullen,Additionally, Fréling, et al. found in a study of 538 inflammatory bowel disease patients that 59 individuals (10.1%) developed psoriasiform lesions (median follow-up period of 38.2 months) [Various risk factors appear to be associated with the development of psoriasiform skin lesions in patients receiving TNF-α inhibitors. Women appear to be more commonly affected than men . Also, iIn addition, infliximab has been implicated in the majority of cases involving anti-TNF-α-induced psoriasiform dermatitis . HoweverSeveral characteristics of anti-TNF-α-associated psoriasiform skin lesions may distinguish them from classical psoriasis. Contrary to psoriasis, in which lesions are typically found on the extensor surfaces, such as the knees and elbows, there is a high prevalence of palmoplantar and scalp involvement in the patients with anti-TNF-α-induced psoriasiform dermatitis . In addiInterestingly, most patients with anti-TNF-α-induced psoriasiform skin lesions do not have a history of psoriasis , 6. In pThe onset of psoriasiform skin lesions after initiation of anti-TNF-α therapy is highly variable. In a 2009 review of 127 cases, psoriasiform dermatitis was found to occur after an average of 10.5 months after the initiation of the biologic agents; however, some cases have been reported to occur merely days after initiation of therapy, while others do not occur until up to four years after beginning treatment [The variable onset of TNF-α inhibitor-induced psoriasiform dermatitis suggests that, in addition to genetic factors that may predispose certain individuals to developing this condition, environmental influences may also be contributing . HoweverThe development of anti-TNF-α-induced psoriasiform dermatitis has been associated with infliximab, adalimumab, and certolizumab pegol. However, TNF-α-inhibitor-induced psoriasiform dermatitis is uncommon and the skin lesions may be easily mistaken for either classical psoriasis or other dermatoses. Special attention to the characteristics and distribution of the lesions is necessary to make an accurate diagnosis. Risk factors for the development of anti-TNF-α-induced psoriasiform dermatitis include female gender, no personal history of psoriasis, and the presence of Crohn’s disease. The onset of the skin lesions is unpredictable, ranging from a few days to several years, and the presentation may be highly variable. Nevertheless, certain characteristics may indicate that the consideration of an anti-TNF-α-induced psoriasiform dermatitis is appropriate. These features include involvement of the palms, soles, and scalp regions, the presence of palmoplantar pustulosis, occurrence in individuals without a previous history of psoriasis, and the dermatosis occurring in previously uninvolved areas in patients who do have psoriasis. Treatment for classical psoriasis and anti-TNF-α-induced psoriasiform dermatitis both include topical corticosteroids. Delay in diagnosis and appropriate treatment may lead to significant morbidity in patients with TNF-α-inhibitor-induced psoriasiform dermatitis. Therefore, a high index of suspicion for anti-TNF-α-induced psoriasiform dermatitis is needed when considering the differential diagnosis for erythematous papules and plaques in a patient with inflammatory bowel disease who is receiving anti-TNF-α therapy.


Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies  were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses. We determined the binding sites of six novel human norovirus specific Nanobodies  using X-ray crystallography. The unique Nanobody recognition epitopes were correlated with their potential neutralizing capacities. We showed that one Nanobody (Nano-26) bound numerous genogroup II genotypes and interacted with highly conserved capsid residues. Four Nanobodies  bound to occluded regions on the intact particles and impaired normal capsid morphology and particle integrity. One Nanobody (Nano-14) bound contiguous to the HBGA pocket and interacted with several residues involved in binding HBGAs. We found that the Nanobodies delivered multiple inhibition mechanisms, which included steric obstruction, allosteric interference, and disruption of the capsid stability. Our data suggested that the HBGA pocket might not be an ideal target for drug development, since the surrounding region is highly variable and inherently suffers from lack of conservation among the genetically diverse genotypes. Instead, we showed that the capsid contained other highly susceptible regions that could be targeted for virus inhibition. Caliciviridae family. The human norovirus genome contains three open reading frames (ORFs), where ORF1 encodes non-structural proteins, ORF2 encodes the capsid protein (VP1), and ORF3 encodes the minor capsid protein (VP2). The virion comprises of 90 VP1 dimers that form an icosahedral particle (T = 3) 35–40 nm in diameter . An. An31]. Nano-42 bound on the bottom of the P domain and closely overlapped with Nano-85 binding site . Five diNanobodies were previously shown to aid the crystallization process by increasing protein stability and stabilizing flexible regions . We haveNano-26 binding site comprised of residues from both P domain monomers, although the majority of the P domain interactions involved only one chain (chain B). Nano-26 formed seven direct hydrogen bonds with one P domain monomer  . Both P The Nano-27 binding epitope was located on the bottom region of the P domain . InteresWe previously showed that Nano-85 was able to disassemble norovirus VLPs . To explTo investigate a temperature dependence of the Nanobody treatment, we mixed GII.10 VLPs with Nano-85 and Nano-26 at 4°C, room temperature, and 37°C for 30 minutes . Nano-85We also performed DLS measurements to quantitatively evaluate GII.10 VLP heterogeneity after Nanobody exposure. Nano-14 treated VLPs had almost identical diameters to native-size particles  . Nano-32Two Nanobodies, Nano-26 and Nano-85, exhibited broad cross-reactivities coupled with adverse effects on capsid integrity. To understand if these effects were relevant for clinically important norovirus strains, GII.4 (Sydney 2012) and GII.17 VLPs were treated with Nano-26 and Nano-85 . Both NaTo further evaluate the binding effects of Nano-26 and Nano-85 on GII.4 (2012) VLPs, we performed time-, temperature-, and concentration-dependent DLS measurements  Figs. NaIn order to examine the Nanobody effects on norovirus virions, we implemented a modified RNA exposure assay and viral loads were quantified using real-time RT-PCR. Concentrated GII.4 positive stool samples were treated with the broadly reactive Nano-26 and Nano-85, while Nano-14 was used as a negative control and 250 mM citric buffer was used as a positive control. Treated samples were then subjected to RNAse digestion. Nano-26, Nano-85, and citrate treated stool samples showed reduced genome copy numbers compared to the Nano-14 control (approx. 30 times for Nano-26 and Nano-85 and 250 times for citrate) . These rTo further investigate if Nanobody treatment could render norovirus VLPs vulnerable to proteolytic cleavage, we subjected GII.10, GII.4, and GII.17 VLPs to a 30-minute trypsin digestion after Nanobody exposure and observed the protein bands using SDS-PAGE . Nano-1450 = 0.34–2.0 μg/ml) ; GII.17 P domain Nano-4 ; GII.10 P domain Nano-42 ; GII.10 P domain Nano-14 ; GII.10 P domain Nano-32 [0.2 M magnesium formate]; and GII.10 P domain Nano-27 . Crystals were grown in a 1:1 mixture of the protein sample and mother liquor at 18°C. Prior to data collection, crystals were transferred to a cryoprotectant containing the mother liquor in 30% ethylene glycol, followed by flash freezing in liquid nitrogen.Phaser-MR [X-ray diffraction data were collected at the European Synchrotron Radiation Facility, France at beamline BM30, ID30A, ID23-1 A and processed with XDS . Structuhaser-MR  using GIhaser-MR  and refihaser-MR . AlternaThe norovirus VLP morphology was analyzed using negative stain electron microscopy (EM) as previously described . NanobodIn order to determine the effects of the Nanobodies on native virions, we collected GII.4 positive stool samples from two individuals with acute norovirus infection . A 10%  were incubated with Nanobodies (1 mg/ml) in 1:1 ratio for 30 min at 37°C. Then, trypsin-EDTA was added to final concentration of 10 μg/ml for 30 min at 37°C. The concentration of trypsin was chosen to yield only partial cleavage with visible intermediate products. After digestion, samples were loaded on the SDS-12% polyacrylamide gel and stained with coomassie stain.S1 FigNanobody binding characteristics were analyzed using GII.10 VLPs and P domain in a direct ELISA. Plates were coated with (A) GII.10 VLPs, (B) GII.10 P domain. Nano-42, Nano-14, Nano-26 were the strongest binders and detected GII.10 VLPs at a dilution of ~50 ng/ml. Nano-4 detected VLPs at a lower dilution of 0.1 μg/ml. Nano-32 detected VLPs at concentrations above 0.4 μg/ml and Nano-27 above 1.5 μg/ml. A similar binding pattern was observed with the GII.10 P domain, where Nano-42, Nano-4, Nano-14, Nano-26 detected P domain in concentrations up to 20 ng/ml. Nano-32, and Nano-27 reacted with the P domain at concentrations above 0.2 μg/ml. and 1.6 μg/ml respectively. (C-E) Nano-14, Nano-27 and Nano-32 bound only GII.10 P domain and showed no cross-reactivity to any other GII P domains (15 μg/ml) or to GI.1 and GI.11 VLPs (4 μg/ml).(TIF)Click here for additional data file.S2 Fig50) to A type saliva at 0.4, 2.6, and 3.1 μg/ml, respectively. (B) For B type saliva IC50 values for Nano-14, Nano-26, and Nano-32 were 1.1, 4.3, and 1.8 μg/ml, respectively. Nano-85 showed only weak blocking potential. Binding was expressed as a percentage of the untreated VLP binding (100%). (C) Inhibition of GII.4 VLPs (0.5 μg/ml) binding to synthetic B-tri saccharide. Both Nano-26 and Nano-85 showed a complete inhibition at 10 μg/ml and no inhibition at 1 μg/ml. (D) Inhibition of GII.4 VLPs (0.5 μg/ml) binding to synthetic B type saliva. Nano-26 and Nano-85 blocked GII.4 VLP binding with IC50 of 0.7 and 1.2 μg/ml. All experiments were performed in triplicate (error bars are shown) and the cutoff was set at an OD490 of 0.15 (dashed line).Saliva blocking assay with GII.10 VLPs (2.5 μg/ml) was performed similarly to PGM binding assay. (A) Nano-14, Nano-26, and Nano-32 inhibited 50% of the binding (IC(TIF)Click here for additional data file.S3 FigTitrations were performed at 25°C by injecting consecutive aliquots of 100–150 μM Nanobodies into 10–20 μM GII.10 P domain P domain. Examples of the titrations (upper panels) are shown. The binding isotherm was calculated using a single binding site model after subtraction of the heat of dilution (lower panels). Nano-32 binding to the P domain exhibited endothermic type of reaction, whereas all other Nanobodies showed exothermic binding reaction.(TIF)Click here for additional data file.S4 FigFor the competitive ITC measurements, the P domain was pre-mixed with Nano-4, Nano-14, Nano-26, Nano-27, and Nano-42 in a 1:1 molar ratio. Standard titrations with Nano-85 were then performed. Titrations were done at 25°C by injecting consecutive aliquots of 100 μM Nanobody into 15 μM GII.10 P domain P domain. Examples of the titrations (upper panels) are shown. (A-C) Titration to P domain Nano-4, Nano-27 and Nano-42 showed the absence of heat release associated with injections, indicating the lack of binding.  Nano-85 showed the binding to GII.10 P domain Nano-26 and Nano-14 complexes with exothermic type of reaction, which resembled the binding of Nano-85 to P domain alone. The binding isotherm was calculated using a single binding site model after subtraction of the heat of dilution (lower panels).(TIF)Click here for additional data file.S5 FigSeveral loops of GII.10 P domain in complex with Nano-32 had altered conformation compared to unliganded P domain. Loop between residues 295–300 was positioned symmetrically in both monomers which was not observed in apo-structure, but was characteristic for GII.10 P domain in complex with 30 mM B-tri saccharide (PDB code 4Z4Z). Loop 343–352 was partially disordered and deviated 4.3Å away from its position in unliganded structure. Loops 309–314, 418–420 as well as 487–491, 517–522 had slightly shifted conformation (2-3Å).(TIF)Click here for additional data file.S6 FigGII.10 VLPs were pre-incubated with Nano-85, Nano-26, or with both Nano-85 and Nano-26 for 30 min at 4°C, room temperature, and 37°C. After treatment VLPs were subjected to negative staining and examined by EM at 50,000 magnification. VLPs exposed to Nano-85 showed a temperature dependence of morphological changes. At 4°C a large portion of native 35–37 nm VLPs were visible, whereas at RT small 20–23 nm VLPs appeared and prevailed at 37°C. In case of Nano-26 and joint Nano-26 and Nano-85 treatment VLPs were largely degraded at any tested temperature.(TIF)Click here for additional data file.S7 FigGII.4 VLPs were incubated with Nano-26 (A) or Nano-85 (B) for indicated periods of time at room temperature and DLS profiles were then measured. Peaks corresponding to large molecular weight aggregates appeared after 30 seconds in case of Nano-26 treated VLPs and 15 min for Nano-85. Arrows indicate native size VLPs (35–37 nm in diameter) and small VLPs (20–23 nm).(TIF)Click here for additional data file.S8 FigHydrodynamic diameters of GII.4 VLPs treated with Nano-26 (A) or Nano-85 (B) at 4°C, RT, 37°C for 15 min were measured using DLS. VLPs had an increased particle heterogeneity at 37°C compared to 4°C and RT. Additionally, VLPs were pre-incubated with different concentrations of Nano-26 (C) or Nano-85 (D) for one hour at RT. Size distribution was altered at concentrations over 12.5 μM for Nano-26 and 50 μM for Nano-85.(TIF)Click here for additional data file.S9 FigGII.10, GII.4, and GII.17 VLPs (1 mg/ml) were treated with 1 mg/ml of Nano-14 (lane N14), Nano-26 (lane N26), Nano-85 (lane N85), or PBS for 30 min at 37C. After the treatment samples were exposed to typsin  for additional 30 min at 37C. Samples were then run on the SDS PAGE and stained with Coomassie. VLPs without trypsin cleavage correspond to the last lane. Nano-14 does not impact the rate of protease degradation, whereas Nano-26 and Nano-85 significantly increased the digestion efficiency.(TIF)Click here for additional data file.S10 FigGII.10, GII.4, and GII.17 VLPs  were serially diluted in PBS, or 1M Tris buffer (pH 10), and incubated at room temperature for one or ten hours. Standard PGM binding assay was then performed using polyclonal serum against GII.10 for detection of GII.10 VLPs and polyclonal serum against GII.4 for detection of GII.4 and GII.17 VLPs. VLPs disassembled with pH 10 for ten hours showed no binding to PGM. Incubation with pH 10 for one hour greatly reduced the binding of GII.10 VLPs and completely abolished the binding of GII.4 and GII.17 VLPs.(TIF)Click here for additional data file.


The first Food and Drug Administration-(FDA)-approved drugs were small, chemically-manufactured and highly active molecules with possible off-target effects, followed by protein-based medicines such as antibodies. Conventional antibodies bind a specific protein and are becoming increasingly important in the therapeutic landscape. A very prominent class of biologicals are the anti-tumor necrosis factor (TNF) drugs that are applied in several inflammatory diseases that are characterized by dysregulated TNF levels. Marketing of TNF inhibitors revolutionized the treatment of diseases such as Crohn’s disease. However, these inhibitors also have undesired effects, some of them directly associated with the inherent nature of this drug class, whereas others are linked with their mechanism of action, being pan-TNF inhibition. The effects of TNF can diverge at the level of TNF format or receptor, and we discuss the consequences of this in sepsis, autoimmunity and neurodegeneration. Recently, researchers tried to design drugs with reduced side effects. These include molecules with more specificity targeting one specific TNF format or receptor, or that neutralize TNF in specific cells. Alternatively, TNF-directed biologicals without the typical antibody structure are manufactured. Here, we review the complications related to the use of conventional TNF inhibitors, together with the anti-TNF alternatives and the benefits of selective approaches in different diseases. Since the discovery and identification of tumor necrosis factor (TNF) in the mid 1980s ,3 and reToday, 19 members of the TNF superfamily have been identified, based on sequence similarity with TNF. In addition, 29 interacting receptors and several molecules interacting with the cytoplasmic domain of these receptors are recognized . All memTNFRSF1A gene and the 75 kDa TNF receptor 2 (TNFR2 or CD120b), encoded by TNFRSF1B ..287].Anti-TNF drugs are real blockbusters for the pharmaceutical companies because this is the best-selling pharmaceutical drug class with sales over US $25 billion. This puts a monumental pressure on health care systems, meaning that many countries even cannot afford a decent policy around these drugs. A retrospective study performed in 2016 estimated the annual cost of the use of biologics per patients in the USA. The most used biological was etanercept (48%), followed by adalimumab (29%) and infliximab (12%) and the annual costs per treated patient were US $24,859 for etanercept, US $26,537 for adalimumab and US $26,468 infliximab .+ macrophages [Although many patients benefit from treatment with anti-TNF drugs, a big problem in the clinic remains the high number of patients that do not respond to the therapy: 13–40% of patients fail to respond to initial anti-TNF therapy (primary non-responders) and up to 50% of patients lose responsiveness during therapy (secondary non-responders) . Primaryrophages . Consequrophages . Currentrophages ,291,293.Secondary non-responsiveness can be explained by the formation of anti-drug antibodies (ADAs)  in a subset of patients and the risk of loss-of-response is increased by at least threefold when ADAs are present  Figure . ADAs weTo reduce the risk of loss-of-response, therapeutic drug monitoring (TDM) has become standard of care in the clinical setting for many clinicians, because there is a well-established correlation between serum trough levels of the drugs and clinical response . Indeed,Pneumocystis jirovecii pneumonia, histoplasmosis and aspergillosis. Risk factors to develop these infections are age and concomitant treatment with corticosteroids, and the overall risk of these infections should be considered before the treatment is started [The beneficial effects of anti-TNF medication are undeniable but there are serious concerns considering their safety. In addition to acute problems, such as infusion reactions, other severe adverse events might occur . Because started .Regarding the risk for malignancies, studies in mice attributed an important role to TNF in the process of tumor immune surveillance . HoweverNeurological side effects have been reported and recent data suggest a role for anti-TNF drugs in the induction of neurological disease, especially demyelination of the CNS as well as implications at the level of the peripheral nervous system . The preIn addition to these neurological disorders, CD patients treated with anti-TNF drugs reported fatigue which was significantly associated with the use of these drugs. Subgroup analyses also indicated that long-term therapy duration and combination without azathioprine were risk factors for the occurrence of fatigue ,315.17 pathway is proposed as a possible mechanism [In addition to the “common” adverse effects, there are also paradoxical side effects described in patients treated with anti-TNF drugs . They reechanism . A thirdechanism . These pechanism . As thisSSS-07 against RA, but no results are provided yet (NCT02460393). The bovine polyclonal milk-derived anti-TNF Ab AVX-470 can be administered orally thanks to the stability of bovine Igs in human intestinal secretions. After oral administration, this Ab remained localized in the gut, and in a double-blind, placebo controlled study AVX-470 appeared to be safe and well tolerated, and was associated with dose-dependent increases in clinical and endoscopic remission in patients with active UC (NCT01759056) [®, that are stable in the gastro-intestinal tract. These will be explored for the treatment of necrotizing enterocolitis (NEC). Unfortunately, clinical drug development goes along with many failures too: CDP571 (Humicade®), a humanized mAb against TNF failed to demonstrate clinical efficacy for sparing steroids in CD patients and further development was discontinued [ozoralizumab [VHH#1-3 antagonizes the binding of TNF to its receptors with picomolar potencies. As this drug has a different mode of binding, i.e., it can bind a single trimeric TNF and blocks two of the three receptor binding sites of TNF, it distinguishes itself from other TNF neutralizing drugs [Lactococcus lactis after oral administration, as was done with an anti-TNF Nb [Lenercept is a soluble fusion protein consisting of TNFR1 fused to the hinge region of the IgG1 Fc region. It entered clinical trials for indications such as sepsis, RA and MS. However, the clinical trial for treatment of RRMS had to be terminated due to unforeseen exacerbations of the symptoms [Hitanercept is a variant of etanercept that carries a mutation in the TNFR2 domain of the fusion protein and exhibits higher affinity to sTNF and tmTNF than etanercept [HL036 is a small TNFR1 fragment (19 kDa) with enhanced ocular tissue penetration. This drug is formulated as an ophthalmic solution and is currently under clinical investigation (phase II) for dry eyes disease (NCT03334539) after it was shown to be safe in healthy volunteers. In addition, other formulations for inflammatory ocular diseases are currently considered. Onercept is a PEGylated form of soluble human TNFR1 that was tested in psoriasis and CD. Despite promising early clinical results, onercept proved not to have an exceptional efficacy and safety profile in both diseases and therefore further development was stopped [pegsunercept, a PEGylated soluble TNFR1. It has been tested for RA, but also here, development was discontinued and this decision was based on recommendations of two separate independent Data and Safety Monitoring Boards [Azd9773 (CytoFab®) was suspended as the drug failed to show efficacy in severe sepsis and septic shock [TNF-kinoid or with CYT007-TNFQb, inducing endogenous polyclonal anti-TNF antibodies that neutralize circulating TNF in inflammatory immune-mediated diseases. With TNF-kinoid, proof-of-concept was obtained in a mouse RA model and these findings were translated into the clinic in patients that experience secondary non-responsiveness of TNF-antagonists. A phase Ia clinical trial showed that therapeutic vaccination induced dose- and schedule-dependent anti-TNF Abs in RA patients and was well tolerated. Moreover, patients with anti-TNF Abs showed a trend towards clinical improvement [CYT007-TNFQb of which the phase I/II clinical trial was discontinued in psoriasis patients [Atsttrin is a progranulin-derived engineered protein that showed efficacy against RA and osteoarthritis in preclinical models [R-7050 is a TNFR complex inhibitor that improves the outcome upon intracerebral hemorrhage, suggesting its use as adjunct therapy in the treatment of neurological injury [C87 is an TNF-TNFR interaction modulator as it directly binds to TNF and prevents TNFR signaling and subsequent Casp-8 and NF-κB activation. It was found from an initial screen of ~90,000 compounds and has in vivo potency. The only remaining challenge is to determine toxicity and stability with longer-term use [In addition to the well-known approved anti-TNF inhibitors, there are several other anti-TNF drugs developed or under development . In Chin1759056) ,263. Theontinued . Other aontinued . A highlalizumab . This dralizumab . Anotherng drugs . The easi-TNF Nb . Preclini-TNF Nb . Similarsymptoms ,153. Hitanercept ,331. In anercept . HL036 i stopped ,332,333.g Boards . Likewisic shock . Anotherrovement . The drurovement . The sampatients . Progranase NCT03539 afterl models . Up until injury . The drul injury . In addil injury ,275,337.term use .minocycline decreases TNF synthesis in addition to its bacteriostatic and anti-inflammatory actions. Additionally, it also inhibits MMPs, reduces cyclooxygenase 2 (COX-2) activity and prostaglandin E2 production, and attenuates apoptosis [curcumin (diferuloymethane) is a natural anti-inflammatory agent that inhibits TNF transcription at several levels, but mostly via inhibition of NF-κB. Consequently, curcumin also antagonizes other pro-inflammatory cytokines including IL-1β and IL-6. It is a broad-acting anti-TNF that can be orally consumed via natural food spices. Unfortunately, it is poorly soluble in water and has a poor bio-availability [Other less specific anti-TNF agents are thalidomide and its derivatives lenalidomide and pomalidomide, curcumin and minocycline. Initially, thalidomide was indicated as an effective tranquilizer and painkiller associated with enormous teratogenic side effects in human. Thalidomide is now recently re-introduced as well-known (non-specific) TNF-inhibitor as it reduces the rate of TNF synthesis by enhancing the degradation of the transcript. Currently, thalidomide is under investigation to treat neurodegenerative disorders that implicate TNF-signaling such as AD, PD and amyotrophic lateral sclerosis (ALS) as this small-drug molecule can penetrate into the brain ,341,342.poptosis . In PD mpoptosis . By contlability . Currentlability . In addilability . By incrlability . Unexpeclability ,348.The anti-TNF drugs on the market were based on the wide perception that TNF is a pathological factor, ignoring the fact that TNF can also have beneficial and unique indispensable properties e.g., in immune regulation and tissue regeneration, as discussed above ,349,350.regs with upregulation of FoxP3-dependent genes. This effect again highlights the importance of preserving the TNF/TNFR2-mediated signaling pathway. In addition, two fully human anti-TNFR1 single domain antibodies have been developed and were investigated by GlaxoSmithKline [Given that TNFR1 and TNFR2 mediate different cellular effects, it is interesting to selectively target one of the two. This field is currently actively explored via several approaches . ATROSABithKline . The firithKline ,357. HowithKline ,359. ConithKline . To dealithKline . TargetiithKline ,362. TheithKline .TNFRSF1A gene, subjected to the mouse EAE model of MS. In these transgenic mice Prophylactic as well as therapeutic ip administration of the drug prevented or halted disease development, respectively [Importantly, our research group also generated a trivalent human TNFR1 inhibiting Nanobody consisting of two paratopic TNFR1 binding Nbs linked to an anti-albumin Nb. This Nb only binds to human TNFR1 without being cross reactive for the mouse homologue. One of these Nbs also competitively inhibited the TNF/TNFR1 signaling, but the potency of this Nb was improved after incorporation in the TROS construct. This construct effectively inhibited TNFR1 signaling in vitro and ex vivo on isolated colon biopsies from CD patients . Proof-oectively . The druectively .regs, recent studies also provide new insights into the role of tmTNF/TNFR2 signaling in the suppressive activity of myeloid-derived suppressor cells (MDSCs). Indeed, MDSCs require membrane TNFR2 expression to exert optimal suppressive activity [reg expansion [regs and ameliorates established CIA in mice [regs in type I diabetes. These could be useful in patients with TNF-mediated neurodegeneration or as a correctional therapy in diabetes patients [+ T cells and TNFR2 agonism has been shown to selectively kill insulin-autoreactive CD8+ T cells in blood of diabetic patients by an altered signaling pathway [N-methyl-d-aspartate (NMDA)-induced acute neurodegeneration. Administration of ATROSAB or of a TNFR2-selective TNF mutant EHD2-scTNFRR2 reverted neurodegeneration-associated memory impairment and protected cholinergic neurons against cell death [The opposite approach that is currently investigated implies TNFR2 activation to stimulate the TNFR2-mediated protective pathways in autoimmunity and neurodegenerative diseases . It has activity ,366. Intactivity . Efficieactivity ,368. Sevxpansion . Lamonta in mice . Another in mice . TNFR2 apatients . Additio pathway . This ap pathway . In type pathway . Interesll death . In addill death ,375, andll death .regs [regs are currently investigated as cancer treatment [reg proliferation and enabled T effector cell expansion which could help amplify effective anti-tumor immune responses [In contrast to TNFR2 agonism, TNFR2 inhibition was proposed as an effective anti-cancer strategy as TNFR2 has been identified as a human cancer oncogene. Indeed, many cancer cells are characterized by TNFR2 expression that promotes the expansion of tumor cells . Recentlregs , and thereatment . TNFR2 aesponses . Furtheresponses .Mycobacterium bovis, known to stimulate the innate immune system by inducing the host to produce TNF that subsequently kills the autoreactive T cells. BCG vaccination of longstanding type I diabetic subjects led to more death insulin-autoreactive T cells and transiently induced beneficial Tregs for 4–6 weeks after vaccination [Interestingly, also vaccines that stimulate endogenous TNF release are currently evaluated as a long-term modulating immuno-intervention in clinical trials in type 1 diabetes and have been investigated in MS  ,379. Thecination . In addicination . Vaccinacination .L. monocytogenes infection [Instead of targeting the receptors, approaches that selectively target sTNF have also been proposed. One of the most promising drugs is the dominant negative peptide Xpro1595 ,383. Xprnfection . The effnfection ,165,167 nfection . Locomotnfection  and intrnfection . Icv infnfection . Periphenfection ,198  and adaptive (CD8+ T cells) effector cells as well as IECs via interacting with TNFR1 on mesenchymal cells [Cell-type restricted targeting of TNF is a new innovative approach that has been suggested by the group of Nedospasov . TNF sigial load . The sitpensable . For thepensable . In dised in EAE . Conditid in EAE . Conversd in EAE . Interesd in EAE . Intestial cells ,391,392.al cells . To estaal cells ,395. Addal cells ,396.∆ARE mice, the RA pathology is independent of TNF-mediated adaptive immune responses (T and B cells), but it is mediated by TNFR1 on joint  fibroblasts [Interestingly, in the TNF-induced shock model IEC-restricted TNFR1 expression is sufficient to induce IEC-apoptosis, but chronic targeting of IECs by endogenous TNF is not enough to induce IBD pathology indicating that TNFR1 expressed on other cell types is required for effective pathology ,69,397. roblasts ,391,398.roblasts  or CD8 Troblasts .Instead of targeted TNF neutralization, there are also several indications that might benefit from targeted TNF supply, for example to exploit its anti-cancer properties. Unfortunately, TNF’s intrinsic and unacceptable toxicity hampers its use as immunotherapeutic. This obstacle can be circumvented by the creation of Activity-on-Target cytokines (AcTakines). These immunocytokines consist of mutated cytokines with reduced binding affinity coupled to a targeting moiety that guides the cytokine to the desired cell target. The activity of the mutated cytokine is only restored after local enrichment at the targeted cell types. This strategy will greatly reduce the off-target adverse effects and improve the desired efficacy .Simultaneous blockage of multiple pathways might directly cope possible compensation mechanisms that neutralize the initial effect of the drug. Alternatively, this approach can also directly increase the potency of drugs. In the pathogenesis of psoriasis, a prominent role for TNF has been described and this is illustrated by the success of the TNF inhibitors in this disease. In addition to TNF, recent data also suggest an important role for type I IFN in psoriasis. Interestingly, blockage of TNFR1 or the IFN receptor 1 (IFNAR1) only partially protects mice against imiquimod-induced psoriasis, whereas double KO mice lacking both receptors showed superior protection in this model. This was explained by the presence of a sustained type I IFN production in TNFR1 single KO mice . As therThe establishment of a bispecific therapeutic also allows targeting of cytokine-neutralization on cytokine-producing cells cf.  or at paIt is clear from the increasing amount of studies regarding the differential roles of the TNF receptors, the TNF format, i.e., tmTNF or sTNF, and the function of TNF derived from specific cell subsets, that the use of anti-TNF drugs can be improved or adjusted to the specific disease context. Given the relatively long-time experience with the approved anti-TNF drugs, they can be considered as relatively safe; however, improvements are warranted. In addition to the disease settings for which a market authorization is settled, many other diseases in which a specific TNF subtype/receptor is involved can be approached. The examples are numerous and exceed the field of neurology although this was mainly the focus in this review. Selective TNFR1 targeting is described for dry-skin induced chronic itching , neuropareg activation, which can be very interesting in, for instance, graft-versus-host disease [reg population are attractive.Although the expression of TNFR2 is limited in healthy conditions, in (autoimmune) diseases, the expression of TNFR2 is significantly elevated. Importantly, TNFR2 has a superior toxicity profile because of its limited tissue expression compared to the ubiquitous expression of TNFR1 . As disc disease . TherapiAdditionally, as discussed, cell-specific drugs that are directed against the intended TNFR expressed by a specific cell type are interesting for future research. This would lead to local accumulation, less undesired side effects and thus safer therapies. Collectively, during the development of new innovative therapies, one can not only consider the removal of the detrimental cells or signals that are present, e.g., blockage of the inflammatory TNFR1 pathway or removal of autoreactive T cells, but also think about therapies that stimulate the cells or cellular properties that are defective such as suppressive cell types. Therefore, therapeutic treatments should always find the ideal balance between doing enough good and preventing bad.In addition, critical notes are needed, and considerable attention should be paid to adverse effects that might pop-up with TNFR1 antagonizing treatments, as the sensitivity to several infectious diseases is clearly increased in mice lacking TNFR1 . HoweverTo conclude, therapeutic manipulation of TNF remains a very attractive field, and although we know already a lot about the biology of TNF, there is a lot to uncover. This will allow us to select the appropriate treatment for a specific patient population. In addition, improved patient empowerment will drive the development of innovative medicines that deliver more relevant and impactful patient outcomes . Indeed,


To study the relationship between Mg2+ and TNF-α, we used human- or mouse-derived glial and neuronal cell lines or APP/PS1 transgenic (Tg) mice as in vitro and in vivo experimental models, respectively. Our data demonstrates that magnesium-L-threonate (MgT) can decrease the expression of TNF-α by restoring the levels of Mg2+ in glial cells. In addition, PI3-K/AKT and NF-κB signals play critical roles in mediating the effects of Mg2+ on suppressing the expression of TNF-α. In neurons, Mg2+ elevation showed similar suppressive effects on the expression of presenilin enhancer 2 (PEN2) and nicastrin (NCT) through a PI3-K/AKT and NF-κB-dependent mechanism. As the major components of γ-secretase, overexpression of presenilin 1 (PS1), PEN2 and NCT potentially promote the synthesis of Aβ, which in turn activates TNF-α in glial cells. Reciprocally, TNF-α stimulates the expression of PEN2 and NCT in neurons. The crosstalk between TNF-α and Aβ in glial cells and neurons could ultimately aggravate the development and progression of AD.Alzheimer’s disease (AD) is a neurodegenerative disease characterized by cognitive impairment. The neuropathological features of AD are the aggregation of extracellular amyloid β-protein (Aβ) and tau phosphorylation. Recently, AD was found to be associated with magnesium ion (Mg By decreasing the crosstalk between TNF-α and PEN2/NCT, MgT achieved therapeutic effects against AD.Although Mg1–42, LY294002 and Quinazoline (QNZ) were obtained from Sigma-Aldrich Corp ; MgT was purchased from Soyoung Biotechnology Company . Antibodies specific for β-actin, AKT, p-AKT, NF-κB, p-NF-κB, NCT, PEN2, TNF-α, NeuN, GFAP, Alexa Fluor 488-labeled, Alexa Fluor 555-labeled, and HRP-labeled secondary antibody were purchased from Cell Signaling Technology, Inc., . Iba-1 antibody was obtained from Merck Millipore . DAPI was obtained from Beyotime Institute of Biotechnology . All reagents for the qRT-PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories. All other reagents were from Invitrogen  unless otherwise specified.AβAll animals were handled according to the care and use of medical laboratory animals  and all experimental protocols were approved by the Laboratory Ethics Committees of China Medical University and College of Life and Health Sciences of Northeastern University.2+ (100 mg/kg/d) in drinking water (4 mg/ml) for 2 months before the expression of TNF-α, PEN2 and NCT was determined. For determining the production of Aβ, GFAP and Iba-1, mice at the age of 4 months were treated with Mg2+ (100 mg/kg/d) in drinking water (4 mg/ml) for 5 months. The average drinking water consumed per day was monitored (~6.4 ml/d) and the body weight of the mice was ~20 g. All efforts were made to minimize animal suffering and the number of animals used. The brains of mice in different groups were collected under anesthesia and perfusion-fixed as previously described  85Dbo/J (Stock Number: 004462)] (Tg) and wild-type C57BL/6 mice (WT) were originally obtained from The Jackson Laboratory . Genotyping was performed 3–4 weeks after birth. The mice were kept in cages in a controlled environment . In select experiments, mice at the age of 4 months were treated with Mg1–42 (oAβ) has been described previously  was freeze-drying in monomer form after dilution to a final concentration of 1 μg/μl in 100% hexafluoroisopropanol (HFIP) and the solution was aliquoted into sterile Eppendorf tubes. HFIP was then evaporated under vacuum and the peptide was stored at −20°C before reconstitution. For preparing oAβ, the peptide was initially resuspended in dimethyl sulfoxide (DMSO) at 20 μg/μl with water bath ultrasonication for 10 min and the solution was then diluted to a final concentration of 0.4 μg/μl in phenol red-free F-12 media and incubated at 4°C for 24 h.The method for preparing oligomers AβMgT, TNF-α, oAβ or vehicle were injected (i.c.v.) into WT mice as previously described  on 6 cm tissue culture dishes (106 cells per dish) in the appropriate medium. In a select set of experiments, the cells were grown in serum-free medium for an additional 24 h before incubation with inhibitors in the absence or presence of MgT, as previously described  using total RNA and the GoTaq one-step real-time PCR kit with SYBR green (Promega) and the appropriate primers as previously described  containing protease inhibitor cocktail . The protein content of the cell lysates was determined using a bicinchoninic acid (BCA) protein assay reagent . The total cell lysates (4 μg) were subjected to SDS-PAGE, transferred to a membrane, and probed with a panel of specific antibodies. Each membrane was only probed with one antibody. β-actin was used as a loading control. All western hybridizations were performed at least in triplicate using a different cell preparation each time.Mouse brains were collected from WT or APP/PS1 Tg mice and immobilized with 4% paraformaldehyde. Serial sections (10 μM thick) were cut using a cryostat . Slides were first rehydrated in a graded ethanol series and submerged in 3% hydrogen peroxide to eliminate endogenous peroxidase activity. TNF-α levels were determined using an immunohistochemical staining kit, following the manufacturer’s instructions . In selected experiments, the slices from the brains of human and mouse were double-stained with TNF-α (Alexa Fluor 488-labeled secondary IgG) and NeuN (Alexa Fluor 555-labeled secondary IgG) antibody as previously described , New York, USA, and Lund University , Lund, Sweden. Experiments were performed in accordance with the guidelines approved by Department of Experimental Medical Science at Lund University, Lund, Sweden and Department of Biology and Biological Engineering at Chalmers University of Technology, Göteborg, Sweden. All subjects gave written informed consent in accordance with the Declaration of Helsinki. All samples were taken from the prefrontal cortex, more specifically from gyrus rectus. The brain samples were fixed with formaldehyde solution, sectioned into 40 μm thick slices and kept in phosphate buffered saline (PBS). The serial numbers were TT4263 (early stage of AD: the patient was a 73-year-old man who was diagnosed as a mild AD patient), T4308 (middle stage of AD: the patient was an 86-year-old man who was diagnosed as a moderate AD patient), T4339 and T4304 (late stage of AD: the patients was an 88-year-old woman and an 84-year-old woman who were diagnosed as severe and end-stage AD patients).t-test, 1-way or 2-way analysis of variance (ANOVA), as appropriate . As shown in Figure 2+ in the activity of γ-secretases, we examined the protein expression of PS1 and PS2 in SH-SY5Y and n2a cells. However, MgT (50 μM) treatment did not affect the expression of PS1 and PS2  for 2 months clearly decreased TNF-α expression in the cerebral cortex and DG region of APP/PS1 Tg mice  for 2 months in 4-month-old APP/PS1 Tg mice inhibited the mRNA and protein expression of TNF-α, PEN2 and NCT  addition to oAβ-stimulated WT C57BL/6 mice  TNF-α (2 ng/5 μl) into the ventricles of C57BL/6 mice. As expected, TNF-α obviously activated the astrocytes and microglia cells in the cerebral cortex and the DG region of hippocampus compared to that of controls Figures . In agre2+ ion influx activates the PI3K/AKT signaling pathway and inhibits the NF-κB signaling pathway in glia and neurons, which in turn downregulates the expression of TNF-α, PEN2 and NCT in vitro and in vivo. The crosstalk between TNF-α and PEN2/NCT could therefore accelerate the production and accumulation of Aβ in APP/PS1 Tg mice  to APP/PS1 Tg mice for 5 months disrupted the crosstalk of TNF-α and PEN2/NCT, which alleviated the development and progression of AD.AD is pathologically characterized by the aggregation of extracellular Aβ and intracellular tangles  and soluble α-secretase cleaved APP (sAPPα) and decrease the concentrations of CTFβ accumulation and Aβ secretion in APP-expressing n2a cells. To further decipher the mechanisms, a number of studies extended these investigations to the activity of β- or γ-secretases. For example, MgT treatment was reported to stabilize β-secretase (BACE-1) expression and to reduce soluble APP and β-C-terminal fragments, which prevented and reversed cognitive deficits and synaptic loss in APP/PS1 transgenic mice  in Mg2+-deficient medium, but not in Mg2+-enriched medium (Shogi et al., 4 exposure reduced the number of activated monocytes, which produced TNF-α in intrapartum women. Importantly, maternal MgSO4 treatment ameliorated the LPS-stimulated inflammation response during pregnancy, which impaired offspring learning ability and memory (Lamhot et al., 2+ on the expression of TNF-α. In agreement with our data, the PI3-K and AKT pathways have also been reported to exert anti-inflammatory functions (Guha and Mackman, 4 inhibits the LPS-induced inflammation by activating the PI3-K/AKT pathway and inhibiting the NF-κB pathway.Apart from the roles of Mgin vitro and in vivo data were clearly in agreement with our data suggesting that TNF-α regulates the formation of Aβ in a γ-secretase-dependent manner in neurons.In view of these observations, we were prompted to investigate the relationship between TNF-α and PEN2/NCT in glia and neurons. The results demonstrated that TNF-α secreted from glia was able to stimulate the expression of PEN2 and NCT in neurons. This finding was substantiated by prior observations showing that TNF-α upregulates APP processing and Aβ deposition in human rhabdomyosarcoma (Keller et al., 1–42 had the ability to stimulate the synthesis of TNF-α in rat hippocampus, which is mainly made up of neurons. In addition, Lv et al. (1–42-treated BV2 cells. Apart from microglia cells, the injection of Aβ25–35 has been used to induce the expression of TNF-α in astrocytes of the rat brain as an experimental model of AD (Díaz et al., In view of the above observations, TNF-α may act as a connector between glia and neurons. Although we found that TNF-α was predominantly expressed in neurons, we could still not negate its existence in glial cells Figure . Since mv et al.  suggeste2+ to treat AD is simply due to its ability to disrupt the crosstalk between glia and neurons in a TNF-α- and Aβ-dependent mechanism.More interestingly, we also observed the phenomenon that TNF-α secreted from glial cells translocated and bonded to the neurons Figure . We therXY and P-PG conceived and performed all of the experiments. DZ, Y-YL and TW carried out select experiments. PW along with Z-YW conceived the experiments of this study. PW and Z-YW interpreted the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer HB and handling Editor declared their shared affiliation.


Tumour necrosis factor (TNF) is a ubiquitously expressed cytokine with functions beyond the immune system. In several diseases, the induction of TNF expression in resistance artery smooth muscle cells enhances microvascular myogenic vasoconstriction and perturbs blood flow. This pathological role prompted our hypothesis that constitutively expressed TNF regulates myogenic signalling and systemic haemodynamics under non-pathological settings. Here we show that acutely deleting the TNF gene in smooth muscle cells or pharmacologically scavenging TNF with etanercept (ETN) reduces blood pressure and resistance artery myogenic responsiveness; the latter effect is conserved across five species, including humans. Changes in transmural pressure are transduced into intracellular signals by membrane-bound TNF (mTNF) that connect to a canonical myogenic signalling pathway. Our data positions mTNF ‘reverse signalling' as an integral element of a microvascular mechanosensor; pathologic or therapeutic perturbations of TNF signalling, therefore, necessarily affect microvascular tone and systemic haemodynamics. TNF is typically viewed as an inflammatory mediator. Here the authors identify a non-inflammatory mechanism conserved across species whereby the constitutively expressed smooth muscle cell TNF mediates myogenic signal transduction in skeletal muscle resistance arteries and regulates mean arterial blood pressure. Tumour necrosis factor (TNF) is primarily characterized as an immunomodulatory cytokine3434The biology of TNF is complex89101516In this report, we show that acutely deleting the TNF gene in smooth muscle cells or scavenging TNF with the clinical therapeutic ETN reduces resistance artery myogenic responsiveness and hence, systemic blood pressure. The inhibitory effect on myogenic responsiveness is conserved across five distinct species, including humans. Changes in transmural pressure are transduced into intracellular signals by membrane-bound TNF (mTNF), thereby positioning mTNF as a mechanosensor; this ‘reverse signal' connects to the established intracellular myogenic signalling elements extracellular signal regulated kinases 1 and 2 (ERK1/2) and sphingosine kinase 1 (Sphk1). Our data, therefore, suggest that several side effects of clinical ETN therapy result from interfering with TNF's pivotal mechanosensor function and hence, microvascular tone and systemic haemodynamic. Thus, perturbing TNF signalling can potentially elicit deleterious effects in the cardiovascular system.Tnf−/− mice) do not provide an appropriate genetic correlate of pharmacological TNF inhibition: the myogenic mechanisms adapt to the inherited loss of TNF activity through the recruitment of alternative cellular signalling pathwaysTnf−/− and wild-type mice have quantitatively similar myogenic responses  induces a rapid ∼50 mm Hg drop in MAP in the light/rest phase, without reducing heart rate , suggesting that TNF scavenging does not affect the serial elastic properties of the microvascular wall.ETN's systemic effect on MAP  stronglyïve mice . The effïve mice , (ii) spïve mice , (iii) i−/− mice  and (iv)−/− mice . Further20) phosphorylation  are less conducive for animal models, because their affinities for mouse TNF are many magnitudes lower than for human TNFin vitro . As a coTNF's role as a regulator of skeletal muscle resistance artery myogenic signalling is conserved across several species, including hamsters , dogs F, pigs F and, mosThe observation that ETN abolishes myogenic responsiveness and the associated molecular/cellular events  positionTnf−/− mice, confirming that mTNF transduces the sTNFR1-Fc-generated signal , which is effectively targeted by ETN35Sphk1−/− mice, Our ETN data  and prevTnfr1/2 double knockout model (Tnfr1/2 DKO) to deprive TNF from tethering. Like the germline Tnf−/− model, the inherited loss of TNF activity through compromised tethering should recruit alternative cellular signalling pathways for the myogenic mechanism and render ETN ineffective. As predicted, ETN fails to alter myogenic vasoconstriction in cremaster arteries isolated from Tnfr1/2 DKO mice  insensitive to sTNFR1-Fc ligand stimulation and (ii) clearly utilize a canonical soluble TNF/TNF receptor  mechanism to modulate myogenic reactivity34et al.Anti-TNF therapy has revolutionized the management of many chronic inflammatory diseases and is, therefore, utilized in an ever-growing number of patients. Certain side effects 42Anti-TNF therapy can clearly worsen HF45464745Throughout this investigation, we have pharmacologically inhibited TNF with ETN, a decoy receptor construct that exhibits strong species cross-reactivity. ETN's efficacy in our mouse models was pivotal for unmasking mTNF's haemodynamic function and the underlying molecular mechanisms. There are other pharmacological mechanisms that can inhibit TNF ; however, species cross-reactivity is problematic. For example, Food and Drug Administration-mandated pharmacological reviews indicate that adalimumab has at least 1,000-fold less affinity for murine TNF, relative to human TNFin vivo. The data presented herein uncovers TNF's role as a mechanosensor in smooth muscle cells and demonstrates its pivotal contribution to the transformation of transmural stretch into intracellular biochemical signals. This highly conserved function may very well extend beyond the cardiovascular system, as the ability to sense mechanical force is an essential operation found in virtually every cell.In summary, the seminal discoveries presented here indicate that anti-TNF therapy has an unappreciated capability to modulate peripheral resistance and hence, haemodynamic parameters. The immediate clinical ramification is that anti-TNF therapeutics must be more cautiously applied and monitored, especially in patients with compromised cardiac function45ad libitum access to water and chow. The use of human subjects in this study conforms to the principles outlined in the Declaration of Helsinki and was approved by the Research Ethics Board of St. Michael's Hospital, Toronto, Canada (REB #11–198). All patients provided informed written consent through Research Ethics Board-approved consent forms before study enrolment.All procedures conform to the Guide for the Care and Use of Laboratory Animals published by the NIH . All experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Toronto and conducted in accordance with Canadian animal protection laws. All animals were housed under a standard 14:10 h light-dark cycles with Tnf knockout mice (Tnf−/−)Sphk1−/−) were a gift from Dr. Richard L. Proia Tnfrsf1atm1lmx (p55) and Tnfrsf1btm1lmx (p75) double knockout mice (Tnfr1/2 DKO) and wild-type mixed background strain controls (B6.129F2) were purchased from the Jackson laboratory .Wild-type (WT) male C57/BL6 mice (2–3 months) were purchased from Charles River Laboratories  and germline Tnf knockout mice  were generated using a previously described breeding schemeCreERT2 and Tnfflox/flox miceTnfflox/flox offspring express SMMHC-CreERT2; therefore, treatment of male mice with tamoxifen  activates the Cre recombinase and excises Tnf at the LoxP sites from smooth muscle cells, yielding Sm-TNF-KOCreERT2Tnfflox/flox mice  and wild-type littermates  served as controls.Male tamoxifen-inducible smooth muscle-specific −1): NaCl 145, KCl 4.7, CaCl2·2H2O 1.5, MgSO4·7H2O 1.17, NaH2PO4·2H2O 1.2, pyruvate 2.0, EDTA 0.02, (3-morpholinopropanesulfonic acid (MOPS) 3.0, and glucose 5.0. Briefly, mice were killed using an overdose of isoflurane; the testicles were exteriorized and carefully separated from attached connective tissue and the cremaster muscle was removed. Cremaster resistance arteries were carefully dissected in 4 °C MOPS under a surgical microscope (Leica MZ16) with a cold lighting source (Leica KL 1500 LCD). The resistance arteries were then cannulated and pressurized on a custom-made perfusion myography apparatus53Mouse skeletal muscle resistance arteries were isolated from the cremaster muscleMouse olfactory cerebral arteries were isolated from the brain−1)  were euthanized by isoflurane inhalation and cervical dislocation. Hamster gracilis arteries were rapidly dissected from surrounding skeletal muscle tissue, cannulated and cultured for 24 h under constant flow conditions (1 ml h−1) ref. . This prTissue samples from healthy adult mongrel dogs (1–3 years old) and healthy Yorkshire pigs (25–35 kg) were generously donated by Dr Paul Dorian . Dogs and pigs were anaesthetized with inhaled isoflurane and were killed by removal of the heart (dogs) or ventricular fibrillation (pigs). Once the animals had been killed, a section of gracilis muscle was removed; resistance arteries were dissected from surrounding skeletal muscle tissue, cannulated and functionally assessed by perfusion myography.2) were collected without cautery, and gradually cooled in buffer to 4 °C before the resistance arteries were dissected from the surrounding skeletal muscle tissue.With informed consent (REB #11–198), human skeletal muscle biopsies were collected from (i) cardiac patients undergoing elective coronary artery bypass graft (CABG) surgery for coronary artery disease and occlusion and (ii) patients undergoing lumbar laminectomy for vertebral disk herniation and spinal stenosis. All patients had normal ejection fraction and were not treated with anti-TNF therapeutics . SurgeonSystemic blood pressure was assessed by invasive catheterization−1 intraperitoneal between 4–5 h after light-on on consecutive days) and haemodynamic parameters were recorded throughout injections and in the follow-up period (10 s sample every 10 min continuously).Continuous haemodynamics and mouse activity were gathered using blood pressure telemetry . Briefly, mice were anaesthetized with inhaled isoflurane and the carotid arteries were carefully exposed. A gel filled catheter, attached to the blood pressure transducer (which communicates wirelessly with the receiver), was inserted into the right common carotid artery and secured with a silk suture. The blood pressure transducer was implanted in a subcutaneous pouch on the chest. Mice recovered over 7 days and, after 2–4 days of baseline recording, mice were injected with tamoxifen  and a 3 h period during the dark phase (01:00 to 04:00 active).Mice were anaesthetized (1.5–1.7% isofluorane) and were placed in a supine position on a heating pad and temperature was monitored with a rectal probe . Chest fur was removed and acoustic gel was applied to the chest wall . A Vevo 770 ultrasound system  was used and a transthoracic M-mode ultrasound was taken using an ultrasonic linear transducer scanning head set to 30 MHz. Using the long-axis view, left ventricular-end-diastolic volume (LVEDV), left ventricular-end-systolic volume (LVESV), diastolic diameter (Dd), and systolic diameter (Sd) were obtained. Stroke volume (SV) was calculated as SV=LVEDV-LVESV. Using simultaneous heart rate (HR), CO was calculated as CO=HR × SV. Ejection fraction  was calculated as EF=SV/LVEDV.Tnf−/− mice underwent surgery to induce a myocardial infarction (MI) by ligation of the left anterior descending coronary artery (LAD)19−1), boiled ETN (20 mg kg−1), Adalimumab , or equivalent volumes of saline and blood pressure was continuously monitored. A subset of HF mice was euthanized for pressure myography assessments.WT and in vivo lengths. Vessel viability was assessed using 0.3 μmol l−1 norepinephrine (in hamsters), or 10 μmol l−1 phenylephrine , or 60 mmol l−1 KCl (humans). Only vessels that showed robust constriction (>50% tone) underwent further testing.Following cannulation, myography units were transferred to a heating plate mounted on an inverted microscope (Nikon Eclipse TE2000-U); vessels were heated to 37 °C and stretched to −1in vitro, 20 mg kg−1in vivo), etanercept  and the Fc component of the human IgG1, 10–300 μg ml−1in vitro, 20 mg kg−1in vivo), the TNF-cleaving enzyme inhibitor TAPI-1 , soluble recombinant human TNF , the MEKK1 inhibitor PD98059 , and recombinant mouse TNF R1/TNFRAF1A Fc Chimera . All substances were dissolved in MOPS buffer and incubated for 30 min at 37 °C before assessing myogenic responsiveness.Pressure myography experiments were conducted in the presence of various substances including: adalimumab : NaCl 147, KCl 4.7, MgSO4·7H2O 1.17, NaH2PO4·2H2O 1.2, pyruvate 2.0, EDTA 2.00, MOPS 3.0 and glucose 5.0). Following incubation with Ca2+-free MOPS buffer, vessels underwent a step-wise increase in transmural pressure under Ca2+-free conditions and passive diameter (diamax) was recorded at each pressure. Myogenic tone (%) was calculated as: myogenic tone (%)=(diamax–diaresponse)/diamax × 100, where diaresponse is the diameter at a given pressure. For agonist-induced vasoconstriction, responses were compared with passive diameter under Ca2+-free conditions at dia60mm Hg (diamax60mm Hg) for all vascular beds. Agonist-induced responses were calculated as: tone (%)=diamax60mm Hg–diaresponse)/diamax60mm Hg × 100, where diaresponse is the diameter at a given concentration of the drug.To assess myogenic responsiveness, arteries were exposed to step-wise increases in transmural pressure . In experiments in which drugs were serially tested , responses were compared with separate experiments in which serial responses were conducted only in MOPS buffer . At the completion of each experiment, vessels were incubated in Caresponse). Myogenic responsiveness was expressed as reversal of initial distension (% RID=(dia100mmHg–diaresponse)/(dia100mmHg–dia45mmHg) × 100).To assess the myogenic response in hamster arteries, transmural pressure was increased from 45 to 100 mm Hg in a single step542+] ratio was measured by incubating pressurized mouse cremaster arteries with the calcium-sensitive Fura 2 ratiometric dye 2+, not exact concentrations. Briefly, excitation of Ca2+-bound Fura 2 (340 nm) or Ca2+-unbound Fura 2 (380 nm) was switched 2 × per second with a high-speed random access monochromator . The resulting 510 nm emission was captured by a photomultiplier for quantification and simultaneous vessel diameter measurement. The Fura 2 ratio (340/380) indicates intracellular Ca2+ concentration and was expressed as a fraction of the baseline ratio of each vessel.Intracellular [Ca−1) for 5 min vessels were immediately submerged into 4 °C protein extraction buffer containing protease and phosphatase inhibitors (Roche Scientific).Cremaster arteries from wild-type and Sm-TNF-KO mice (untreated and treated with tamoxifen for 3 days) were isolated and cannulated; only viable vessels were utilized in western blots. For sTNFR1-Fc experiments, vessels were set at 80 mm Hg transmural pressure and were incubated in MOPS or sTNFR1-Fc  containing protease inhibitor (Roche Scientific) and okadaic acid (1:20).For ETN experiments, viable wild-type mouse cremaster arteries were incubated at 60 mm Hg transmural pressure in MOPS or treatment group . Vessels that were maintained at 60 mm Hg served as controls. For Sm-TNF-KO experiments, viable cremaster arteries from untreated or TAM-treated mice were incubated at 60 mm Hg in MOPS. Following incubation, vessels underwent a single pressure step from 60 to 100 mm Hg and responses were recorded. After either 30 s or 5 min at 100 mm Hg, vessels were immediately submerged into 4 °C protein extraction buffer .20 antibody , and incubated with either horseradish peroxidase-linked anti-rabbit IgG  or horseradish peroxidase-linked anti-mouse IgG . A standard chemiluminescence procedure was used to expose X-ray film. Following assessment of phosphorylated proteins, membranes were blocked (5% skim milk) and re-probed overnight with mouse anti-p44/42 MAPK (ERK1/2) antibody  or rabbit anti-MLC20 antibody  and incubated with respective horseradish peroxidase-linked secondary antibodies. Developed Kodak photographic films were scanned into a personal computer and densitometry was quantified using image analysis software  and expressed as % phosphorylated protein/total protein.ETN experiments used a standard western blot methodWestern blots involving sTNFR1-Fc in wild-type mouse vessels, and all Sm-TNF-KO experiments, used a modified method that improved sensitivity and thereby reduced tissue requirements. Following transfer, PVDF membranes were fixed in gluteraldehyde solution  and blocked . A 3-step Western blotting method (biotin/streptavidin) was then used−1 final concentration) digestion for 20 min at 55 °C. The resulting homogenate was loaded onto the spin column and washed once with the kit-provided buffer. DNase I (0.25 Kunitz units) was then applied to the column for 20 min to remove contaminating genomic DNA. The column was then washed twice and the RNA then eluted in distilled water.Cremaster resistance artery RNA was isolated with ‘Total RNA Purification Micro' spin columns (Norgen Biotek), as directed. Briefly, cremaster arteries were homogenized in the kit-provided lysis buffer using conical glass grinders; the ground homogenate was then subjected to proteinase K (450 μg ml−1 dNTPs (Genedirex Inc.), 100 ng random heximers (Invitrogen Life Technologies), 5 μmol l−1 dithiothreitol, 400 U Superscript III reverse transcriptase (Invitrogen Life Technologies) and 80 U RNaseOut recombinant ribonuclease inhibitor (Invitrogen Life Technologies). Each RT reaction was incubated at 25 °C for 5 min and then 50 °C for 1 h; the Superscript enzyme was then inactivated by heating to 70 °C for 15 min. Immediately following the RT reaction, RNA was removed by adding 2.5 U RNase H (New England Biolabs) and incubating the reaction at 37 °C for 20 min; the RNAse enzyme was subsequently inactivated by heating to 65 °C for 15 min.Creamster artery RNA was converted to cDNA using a ‘Superscript III' reverse transcription (RT) kit (Invitrogen Life Technologies). Briefly, before starting the RT reaction, the isolated RNA was combined with deoxynucleotide triphosphates (dNTPs) and random heximers, heated to 65 °C for 5 min and then chilled on ice for 2 min. The remaining components required for the RT reaction were then added: each RT reaction (40 μl volume) contained 8 μl kit-provided 5 × buffer solution, 0.5 mmol l−1 dNTPs, 400 nmol l−1 forward and reverse primers and 1.25 U iTaq DNA polymerase. Primer sequences for each target were (5′–3′): TNF forward CCAGTGTGGGAAGCTGTCTT; TNF reverse AAGCAAAAGAGGAGGCAACA; TNFR1 forward CAGTCTGCAGGGAGTGTGAA; TNFR1 reverse TCAGCTTGGCAAGGAGAGAT; TNFR2 forward CAGGTTGTCTTGACACCCTAC; and TNFR2 reverse GCACAGCACATCTGAGCCT. Each reaction received 1 μl cDNA; negative controls received water instead of cDNA. The PCR amplification consisted of 5 min denaturation at 95 °C, followed by 35 cycles of amplification (60 s at 95 °C+30 s at 60 °C+30 s at 72 °C). The PCR products were separated on a 10% polyacrylamide gel, stained with SyberSafe DNA gel stain (Invitrogen Life Technologies) and imaged on a BioRad ChemicDoc System. Expected amplicon sizes for the targets are: TNF 100 base pairs (bp); TNFR1 124 bp; and TNFR2 92 bp.Target genes were amplified with an i-Taq DNA polymerase kit (FroggaBio). Each reaction (25 μl volume) contained: 2.5 μl kit-provided 10 × buffer solution, 0.8 mmol l−1 and diluted into ‘genomix' as described. Each reaction received 1 μl of ‘genomix' per reaction. For each sample, flox, Cre and the presence of the tamoxifen-induced knockout allele were assessed using two master mixes: Cre Genomix: 50 μl SMWT1+25 μl SMWT2+50 μl Cre(phCREAS1)+125 μl H2O, SMWT1: 5′-TGACCCCATCTCTTCACTCC-3′; SMWT2: 5′-AACTCCACGACCACCTCATC-3′, Cre(phCREAS1): 5′-AGTCCCTCACATCCTCAGGTT-3′;Tissue was isolated from tamoxifen-treated SM-TNFα-KO and control mice. Samples were kept at −80 °C to maintain tissue integrity. On the day of genotyping, a small piece of each sample was isolated on ice. DNA was isolated using the Sigma REDExtract-N-Amp Tissue PCR kit according to the manufacturer's instructions. The PCR was prepared using the REDExtract-N-Amp PCR mix. All primer stocks were 100 pmol μl2O, TNF(Flox) Fwd: 5′-TGAGTCTGTCTTAACTAACC-3′, TNF(Flox) REV: 5′-CCCTTCATTCTCAAGGCACA-3′.TNF(Flox) Genomix: 25 μl TNF(Flox) Fwd+25 μl TNF(Flox) Rev+200 μl H2O, TNF(Flox) KO49 5′-CTCTTAAGACCCACTTGCTC-3′.TNF(TAM KO) Genomix: 25 μl TNF(Flox) Fwd+25 μl TNF(Flox) KO49+200 μl HThe PCR cycle parameters were 95 °C for 5 min and 40 cycles of 95 °C for 45 s (denaturation), 58 °C for 1 min , and 72 °C for 1 min (extension). These cycles were followed by 72 °C for 7 min . A 2.5% agarose gel was made containing 1.75 μl of RedSafe (Frogga cat #21141) to separate the amplicons. The incubated PCR mix was directly loaded onto the gel with the ladder . After ∼40 min of electrophoresis (120 V) the gel was digitally imaged with a Kodak 4000, mm Pro imaging station.2. Once cells reached 80–90% confluence, they were washed with Ca2+-containing PBS and treated with sTNFR1-Fc  or PBS alone. Cells were rapidly lysed with 4 °C protein extraction buffer containing protease and phosphatase inhibitors (Roche Scientific) and scraped from the culture dish. Samples were then sonicated, boiled (95 °C 10 min), spun in a centrifuge  and supernatant was removed and underwent the three-step western blotting protocol described above.Mouse vascular smooth muscle cells were isolated from mesenteric arteriesSample size was based on previous publications192929n values represent the number of mice or arteries, as specified. Variance is similar between comparison groups. All data were analysed with a Student's unpaired or paired t-test or a one-way ANOVA, followed by Dunnett's post hoc tests. Differences were considered significant at P<0.05.All data are expressed as means±s.e.m.; The data that support the findings of this study are available within the article and its How to cite this article: Kroetsch, J. T. et al. Constitutive smooth muscle tumour necrosis factor regulates microvascular myogenic responsiveness and systemic blood pressure. Nat. Commun.8, 14805 doi: 10.1038/ncomms14805 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Tables.