Mycobacterium tuberculosis. Tumor Necrosis Factor (TNF) is a crucial cytokine needed to control tuberculosis infection that remains a leading cause of morbidity and mortality worldwide. TNF blockade compromises host immunity and may increase the risk of reactivation of latent infection resulting in overt pulmonary, pleural and extra-pulmonary tuberculosis. While TNF signaling is mainly considered pro-inflammatory, its requirement for the anti-inflammation process involved in the resolution of infection and tissue repair is less explored. Our study analyzes the role of TNF and TNF receptors in the control of the inflammatory process associated with Bacillus Calmette-Guérin (BCG)-induced pleurisy. This study shows that the absence of TNF causes exacerbated inflammation in the pleural cavity of BCG-infected mice which is controlled by the transmembrane TNF (tmTNF) expression. The lack of TNF is associated with an impaired cellular expression and shedding of TNFR2 in the pleural cavity. The presence of tmTNF restores the normal expression of TNFR2 on myeloid cells during BCG-induced pleurisy. We also show that absence of TNFR1 affects the expression of TNFR2 on pleural cells and inflammation in the pleural cavity of BCG-infected mice. In conclusion, tmTNF but not soluble TNF prevents pleural cavity inflammation leading to attenuation and the resolution of the inflammatory process caused by mycobacterial pleurisy in association with the expression of TNFR2 on myeloid cells.Pleural tuberculosis is one of the most frequent forms of extra-pulmonary tuberculosis observed in patients infected with  Tumor Necrosis Factor (TNF) is a pleiotropic cytokine exerting a large range of activities through its receptors, TNFR1 and TNFR2. TNF is first synthetized as a transmembrane molecule (tmTNF) and then cleaved by tumor necrosis factor-alpha converting enzyme (TACE) resulting in the soluble trimeric TNF molecule (solTNF). A majority of the TNF effector functions have been attributed to the solTNF form but many are also mediated by tmTNF, especially when cell-to-cell contact interaction is involved. Both solTNF and tmTNF as well as the Lymphotoxin-alpha (LT-α) signal through TNFR1 and TNFR2 ,4.Mycobacterial tuberculosis (M. tuberculosis), which is still a major health problem [TNF is a master cytokine required for host responses against mycobacterial infections mainly caused by  problem ,5,6,7. T problem ,9,10,11. problem . The pul problem . Pleural problem . Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the live attenuated mycobacterium used as a vaccine against M. tuberculosis infection. BCG vaccination promotes Th1 type immune responses and confers non-specific protection against non-related mycobacterial infections [fections . BCG is fections . We havefections . BCG-indfections . The resfections .TNF is considered one important pro-inflammatory cytokine due to its association with pathological processes. However, as many other molecules, a paradoxical role can be attributed indicating that TNF has to be synthesized at the good moment and intensity to trigger a TNF-dependent cascade leading to anti-inflammatory process and disease resolution . The preWe have reported that BCG-induced pleurisy is lethal in TNF KO and TNFR1TNFR2 KO mice . In contBecause BCG infection activates TNF pathways, membrane TNFRs are shed and the concentration of solTNFRs can be quantified in the serum as reported in Reference . We aske+TNFR2+ pleural myeloid cells after BCG-induced pleurisy was abrogated in the complete absence of TNF expression, but fully restored in the presence of tmTNF  as compared with WT mice a. Mice eWe then asked if the reason of the absence of IFN-γ by tmTNF was due to the incapacity of tmTNF pleural cells to produce IFN-γ rather than the ability to control the inflammation process in the pleural cavity at the time point of the infection. To answer this question, we obtained pleural cells from BCG-infected mice and cultured ex-vivo with living BCG for 24 h. Our data showed that pleural cells from tmTNF KI and TNFR1 KO produced higher levels of IFN-γ compared to WT cells, even if they produced less IFN-γ than TNF KO cells, they efficiently produced this pro-inflammatory cytokine upon reactivation b. These TNF is a pleiotropic cytokine involved in many different biological systems and in the pathogenesis of several diseases. TNF is required for host defense mechanisms against mycobacterial infections and the therapeutic blockade of TNF increases the risk of infection and reactivation of a latent infection. BCG induces the release of TNF as well as TNFRs which are also shed from the cell membrane by TACE. TACE can be activated by different stimuli including mycobacterial infections and TNF ,20,21,22A majority of reported studies have explored the role of TNF and TNFRs in host defense mechanisms and the pro-inflammatory activities mediated by this potent system. However, the contribution of the TNF pathway during the resolution of a mycobacterial pleurisy is less known and merits exploration in animal models. The present study evaluates the contribution of both tmTNF and solTNF, as well as their receptors in the inflammatory process associated with BCG-induced pleurisy and its exhaustion. We explore how TNF pathway modifies the expression of both the membrane and soluble form of TNFR2 during BCG-induced pleurisy and disease resolution.Our study shows that the recruitment of inflammatory cells caused by pleural BCG infection is associated with an important shedding of solTNFR2 which is abrogated in the absence of TNF. The pleural levels of solTNFR2 are found to be regulated by both tmTNF and solTNF as well as TNFR1. The shedding of solTNFRs in both the physiological and pathological conditions has been considered a mechanism to reduce the sensitivity of cells to TNF mediated activities by blocking TNF bioactivity ,20,23. SOur results show that the majority of pleural myeloid cells express TNFR2 on the cell membrane which is sustained by tmTNF but surprisingly is independent of solTNF. We also observe that while tmTNF KI cells recover similar expression of membrane bound TNFR2 cells than WT cells, the absence of TNFR1 affects both the frequency of the cells expressing TNFR2 and the level of molecules expressed by myeloid cells. These data indicate that TNFR1 plays a role in the regulation of TNFR2 expression and shedding by pleural myeloid cells after BCG-induced pleurisy. Our results are in agreement with previous data reporting that the shedding of TNFR2 was mediated by TNF signaling through TNFR1 . HoweverM. tuberculosis infection [TNFR1 has been shown to be required for host defense against mycobacterial while TNFR2 plays a tolerogenic role during infection as it is expressed by MDSC, regulatory T cells (Tregs) and T cells ,26,27. Hnfection . In addinfection . This meWe have previously reported that non-selective inhibition of both solTNF and tmTNF increases the sensibility of mice to tuberculosis infection, in contrast, selective inhibition of solTNF sparing tmTNF activities, by using dominant-negative TNF molecules, was a good strategy to maintain immunity after mycobacterial infections . We and Δ1–9,K11E, deletion of amino acids 1 to 9 and substitution at position 11 or tmTNF KI) [Wild-type (WT) of C57BL/6 background, TNFR1TNFR2 KO and TNFR1 KO mice (The Jackson laboratory), mice deficient for TNF (TNF KO) , and tramTNF KI)  were useMycobacterium bovis BCG Pasteur strain 1173 P2 grown on Middlebrook 7H9 broth containing ADC (Difco) and at middle-log phase was frozen in aliquots and kept frozen at −80 °C until use. BCG-induced pleurisy was performed by intrapleural cavity injection of 106 CFU of M. bovis. BCG in 100 µL of saline as previously reported [The thoracic cavities were washed three times with 1 mL of 2 mmol/L EDTA-phosphate-buffered saline (PBS). After centrifugation, the first washing solution was frozen at −20 °C and used for cytokine evaluation and the pellet containing the cells were pooled to recover pleural cells .The frequency of immunological cellular subpopulations in pleural cells was analyzed by flow cytometry. Briefly, cells were stained for 30 min at 4 °C with following fluorochrome-conjugated mAb: CD11b (Clone M1/70) and TNFR2 (Clone TR75-89) . Cells were incubated with antibodies and then washed with PBA . We used a FACs CyAn flow cytometer  and analyzed the data with the FlowJo  software. We acquired 100,000 events per sample.Cytokine levels in the pleural fluid and cell supernatants were assessed by Enzyme-linked immunosorbent assay (ELISA). Conditions for ELISA were optimized using anti-TNFR2 coating  and detection antibody  from BioLegend. IFN-γ was quantified in the pleural fluid and in cellular supernatants in accordance with the manufacturer’s instructions (BioLegend). Total cells from the pleural cavity were prepared as described above. Cells were stimulated or not with BCG (multiplicity of infection MOI 1). After 24 h of culture, supernatants were recovered to quantify cytokines by ELISA.t test and multiple t test and corrections for multiple comparisons using the Holm-Sidak method. p value < 0.05 was considered as statistically significant. Quantitative and statistical analyses were performed using with Prism 5.0 . Experiments were analyzed with an unpaired Student


The regulation of cell death through apoptosis is essential to a number of physiological processes. Defective apoptosis regulation is associated with many abnormalities including anomalies in organ development, altered immune response and the development of cancer. Several signalling pathways are known to regulate apoptosis including the Tumour Necrosis Factor-α (TNF-α) and Hippo signalling pathways. In this paper we review the cross-talk between the TNF-α pathway and the Hippo signalling pathway. Several molecules that tightly regulate the Hippo pathway, such as members of the Ras-association domain family member (RASSF) family proteins, interact and modulate some key proteins within the TNF-α pathway. Meanwhile, TNF-α stimulation also affects the expression and activation of core components of the Hippo pathway. This implies the crucial role of signal integration between these two major pathways in regulating apoptosis. In a complex biological system, it is essential that the number and the type of living cells in each organ are maintained at precise levels. Therefore, it is equally important to remove unwanted cells by programmed cell death, or apoptosis, to keep the desired cell population alive. For example, during organ development apoptosis is essential in controlling organ size/cell numbers, shaping organ structure, eliminating dysfunctional cells and removing unwanted structures . DefectiThus, it is understandable that any abnormality in the regulation of apoptosis may cause many types of disease. For example, enhanced apoptosis is a feature of neurodegenerative diseases, myocardial infarction, heart failure and some types of autoimmune disease ,5. In coTherefore, precise regulation of apoptosis at the molecular and cellular level is crucial to organ homeostasis. In essence, the molecular control of apoptosis can be classified in two distinct pathways: the extrinsic and the intrinsic pathways. The fundamental difference between these two pathways is the source of the initial inducers. The extrinsic pathway is induced via stimulation of membrane receptors by extrinsic signals, for example TNF-α, Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, the intrinsic pathway is initiated by internal stimuli, such as DNA damage, hypoxia and metabolic stress, that eventually increase mitochondrial membrane permeability .However, one of the most important aspects of apoptosis regulation is the mechanism behind the transmission and translation of the initial inducer signal into the activation of the common pathways of the apoptotic machinery. The extrinsic pathway starts with the binding of apoptotic ligands to their receptors, which induces formation of death inducing signalling complex (DISC), leading to activation of caspase 8 and eventually activation of the effector caspases (caspase 3/7). The intrinsic pathway is slightly more complicated, involving members of the Bcl-2 family of proteins. Activation of this pathway leads to the release of cytochrome c due to increased mitochondrial membrane permeability. Cytochrome c interacts with apoptosis protease-activating factor 1 (Apaf-1), and forms a complex known as the apoptosome, which in turn activates the effector caspases [As with many other molecular signalling pathways, the presence of negative regulatory systems that counteract and balance the signal is very important. For example, there is balance between the pro-inflammatory and the anti-inflammatory cytokines, balance between the kinases and the phosphatases, and balance between the oncogenes and tumour suppressor genes, which are all very important in maintaining cell homeostasis. Indeed, in the case of apoptosis the presence of anti-apoptotic pathways is also essential. There are several anti-apoptotic molecules that have been very well characterised such as extracellular signal–regulated kinases 1 and 2 (ERK1/2), Akt, β-catenin and the Yes-associated protein-Tafazzin/TEA domain family member (YAP-TAZ/TEAD) complex which is closely related to the Hippo signalling pathway.The Hippo signalling pathway has attracted a lot of attention in recent years. The strong involvement of this pathway in regulating both cell survival and cell death/apoptosis is of particular importance in several different pathophysiological processes, for example in tumour formation, regeneration and inflammation . The corTNF-α is known as a pro-inflammatory cytokine, mainly produced by inflammatory cells such as monocytes, macrophages and activated T-cells, along with other cell types including endothelial cells and fibroblasts. The precursor protein of TNF-α (pro-TNF-α or membrane TNF-α) is a transmembrane protein that requires TNF-α converting enzyme (TACE) to be converted into soluble TNF-α . Both types of TNF-α exert their function by binding to TNF-receptor (TNFR). There are two types of TNF-α receptors, TNFR1 and TNFR2. TNFR 1 can be activated either by transmembrane TNF or soluble TNF, while TNFR2 is activated primarily by soluble TNF . Both TNBinding of TNF-α to the receptors leads to homotrimerization of the TNFR subunit . The deaThe TNF-α induced apoptosis pathway is well characterized. It is mainly modulated through signalling by TNFR1. Activation of this pathway involves the recruitment of molecules to form the “death inducing signaling complex” (DISC). Components of this complex include TRADD, FADD and TRAF1 . FormatiIn contrast to its role in the induction of apoptosis pathways, TNF-α may also induce cell survival via activation of the NF-κB . In addiIt is known that TNF-α has an important role in the inflammatory process. TNF-induced inflammation mainly occurs via the NF-κB and MAPK signalling pathways. However, there are several alternative pathways through which TNF-α can initiate inflammatory response. These include the induction of the sphingomyelinase pathway, which leads to the generation of diacyl glycerol and protein kinase C and eventually results in the activation of NF-κB via an alternative pathway . In addiOverall, TNF-α signalling appears to have pleiotropic roles including regulation of cell death/apoptosis, the immune response, cell survival/proliferation and protein ubiquitination. Functions of TNF-α are likely to be different depending on the cell types. It is also apparent that diverse interaction partners between components of TNF-α signalling and other major signalling molecules determine the downstream effects of the signal. One major signalling pathway to have recently come to the fore in the area of cell death, survival and proliferation is the Hippo signalling pathway. In the following section we will focus on reviewing recent findings that link the Hippo pathway and TNF-α signalling. Drosophila, with the characterization of hippo, salvador (sav) and warts via a genetic mosaic screen for potential tumor suppressor and oncogenes [Drosophila exhibited a “Hippo-like appearance” due to organ overgrowth [The Hippo pathway was first discovered in ncogenes . When thergrowth . Hippo iergrowth . The patergrowth . Drosophila ortholog [The Hippo pathway is highly conserved among metazoans. The components of the mammalian Hippo pathway are functionally very similar to their ortholog . The mamortholog . Howeverortholog . Phosphoortholog . YAP is ortholog ,21,24.There are a number of different pathways which may regulate YAP, either through the core Hippo kinase cascade or independent of the central Hippo components. These include regulation by extracellular signals, for example via the extra cellular matrix, cell to cell contact, G-protein coupled receptor (GPCR) signalling and cell polarity . The maintenance of cell polarity is important to sustain cell function. For example, epithelial cells normally attach to their neighbouring cells via complexes called adherens junctions (AJs), desmosomes, and tight junctions (TJs). The presence of TJs and AJs divide the plasma membrane into apical and basolateral domains and hence establish apical-basal polarity. Interestingly, proteins that are important in building or maintaining apical-basal polarity have been shown to modulate the Hippo pathway. These include: E-cadherin, which causes YAP inactivation ; Ajuba, Extracellular matrix components and tension have recently been regarded as important modulators of major signalling pathways. In the case of the Hippo pathway, extracellular matrix (ECM) components seem to be an important factor as well. For example Agrin, a unique component of the embryonic ECM, has recently been identified as a strong modulator of YAP activity and can thereby induce cell growth and proliferation ,34,35.An additional group of extracellular factors important for Hippo signalling are GPCR ligands such as the LPA (lysophosphatidic acid). LPA was identified as a positive modulator of YAP via binding and activation of the Gα12/13-coupled receptor, and was associated with inhibition of LATS1/2 . This fiThe physiological functions of the Hippo pathway are determined by the cellular function of its core components (for example MST1/2 and LATS1/2) and the main downstream effector YAP. The Hippo core components act as negative regulators of YAP, but also act independently of YAP to promote apoptosis ,38. MeanThe control of organ size by the Hippo pathway has been demonstrated in mice with genetic knockout of MST1 and MST2, which display profound liver overgrowth . In addiThe Ras-association domain family member (RASSF) family proteins consist of ten members (RASSF1-10), several of which are understood to regulate and interact with components of the Hippo pathway. All members of this group of proteins are known to have a Ras-association domain, which can be located either in the N-terminus, as is the case in RASSF 7–10, or in the C-terminus as seen in RASSF 1–6 ,55. A nuThe SARAH domain largely mediates interaction between RASSF proteins and members of the Hippo signalling pathway, in particular MST1 and MST2. Of the ten members of the family it is understood that RASSF1A, RASSF1C, RASSF2, RASSF3, RASSF4, RASSF5A (Nore1A), RASSF6 and RASSF7 show interactions with either MST1, MST2 or with both . In addiAs a consequence, RASSF family proteins have been implicated with a number of important cellular processes and organ function. Since the canonical Ras-Raf-Mek-MAPK pathway regulates growth and development, RASSF1A, which is closely linked to this pathway, is involved in the regulation of cell growth, proliferation and oncogenesis. RASSF1A has been shown to be a strong modulator of cardiac growth (hypertrophy), whilst the involvement of this molecule in cancer and tumorigenesis is also evident ,58.Although RASSF family protein members mainly interact with MST1/2 via a similar pattern, i.e., mediated by the SARAH domain, the implications of the interaction may be variable. RASSF1A and RASSF5 are known to positively modulate MST-kinase activity , whereasAs discussed above, both the TNF-α and the Hippo signalling pathway together with the associated RASSF family proteins, are crucial in the regulation of cell apoptosis. As cross-talk between signalling molecules is essential in achieving balance between pro- and anti- apoptotic signals, it is very important to understand if the TNF-α pathway is also linked with the Hippo-RASSF pathway. Several studies have reported indications of such interactions which will be outlined below. RASSF family members have been linked to death receptor signalling through interactions with modulator of apoptosis 1 (MOAP1) ,62. ApopThe protein kinase C conserved region 1 (C1) domain at the N-terminal region of RASSF1A is essential for its association with the TNF-R1/MOAP1 and TRAIL/MOAP1 complexes . FurtherAn interaction between RASSF1A and the components of the TNF receptor complex has also been observed in cardiomyocytes . InteresRASSF1A is also implicated in the NF-κB signalling pathway. It inhibits signal transmission from the proximal complex involving toll-like receptor/ Myeloid differentiation primary response 88 (TLR/MyD88), TRAF6 and Interleukin-1 receptor-associated kinase (IRAK) . The outOverall, the interaction between members of the RASSF family proteins and components of TNF-α signalling is evident. The physiological implications are varied depending on cell type and the organ. LATS2 is one of the central components of the Hippo pathway. It functions as a tumour suppressor and thus loss of LATS2 function is associated with many types of tumour such as soft tissue sarcomas, leukaemia and breast cancer . In addiRecently, Dong et al. have demonstrated regulation of LATS2 expression by TNF-α. By using an oral squamous cell carcinoma cell line (HN6 cells) they discovered that treatment with TNF-α not only induced LATS2 expression but also induced YAP phosphorylation and hence reduced its co-transcriptional activation . The indSimilar to its effects on LATS2 expression, TNF-α is also reported to induce YAP expression. Using a breast cancer cell line, Gao et al. observed that treatment with TNF-α or macrophage conditioned medium induces the expression level of YAP . InteresAlthough this is an interesting finding, it is contrary to the accepted view that YAP phosphorylation normally leads to its cytoplasmic retention and hence inactivation ,24. It iIn contrast to these findings in breast cancer cells, studies have also shown that TNF-α can inhibit YAP. Using the A172 glioblastoma cell line, Lu et al. found TNF-α treatment to dose-dependently promote apoptosis via increased mitochondrial fission and dysfunction. This coincided with a reduced expression of YAP, whilst YAP overexpression was shown to prevent the TNF-α induced apoptosis and mitochondrial dysfunction [Similar findings have also been observed in chondrocytes. Deng and colleagues showed robust YAP expression in young mouse cartilage, which deteriorated with age and upon surgical induction of osteoarthritis. They went on to show that treatment of primary chondrocytes with TNF-α led to degradation of YAP and TAZ via TAK1-mediated phosphorylation, independently of upstream members of the Hippo pathway. Meanwhile, YAP overexpression inhibited TNF-α induced JNK and NF-κB signalling activation, and could thus provide a target to prevent cartilage degradation in osteoarthritis . The most recent evidence describing TNF-α and YAP crosstalk was observed in endothelial cells. The role of the Hippo pathway and in particular YAP in endothelial cells has been recognised mostly during the process of blood vessel formation. Deletion of YAP in mice led to abnormal vascular development leading to alterations in retinal and brain vessels . In contIn a more recent study by Choi et al. the role of YAP in endothelial cells was characterized in the setting of vascular inflammation . During Both the TNF-α and the Hippo signalling pathways play major roles in the regulation of cell death and apoptosis. A large number of studies have characterized the upstream modulators and downstream effectors of the two pathways. It appears that several components of the Hippo pathway interact with and regulate the effectors of the TNF-α pathway. On the other hand, TNF-α itself is able to modulate the activation of LATS2 and YAP, two major components of the Hippo pathway . The close relationship between the TNF-α and the Hippo pathways may provide us with new insights into how to interfere with or modulate these pathways to produce beneficial effects. Further studies dissecting the cross-talk and intersection between these two signalling pathways are essential in order to completely understand the functional implications of the interactions, and how they can be manipulated for therapeutic purposes.


Anatomical differences between adults and adolescents and between left and right varicoceles were shown in this work. We designed a standardized and reproducible method for pressure measurement in the inguinal internal spermatic vein (ISV). We demonstrated that the mean absolute pressure in the ISV in the upright position is higher than the veno-capillary pressure in the testicle, and hence could impair spermatogenesis prompting the need for treatment in varicoceles.Histoacryl transparent and Glubran2, the current commercially available adhesives for the treatment of varicoceles, do not differ with regard to efficiency, safety and tolerance during and after embolization. Both adhesives cause a mild pain in 30% of the patients in the week after embolization. The radiation exposure is low during embolizations of varicoceles with highly viscous liquid products. Therefore, the endovascular treatment with glue is an efficient, safe and tolerable method of treatment for varicoceles when applicable. This thesis aimed at contributing to the pathophysiology and treatment of varicoceles. The research project encompasses five prospective studies and one retrospective study. The specific objectives of these clinical studies were to assess: (i) the safety, efficacy and tolerance of two types of glue and a non-gluing liquid embolic agent, in the endovascular treatment of varicoceles, (ii) to assess radiation burden for patient during varicocele embolization, (iii) to develop a standardized method to read the phlebographic anatomy of varicoceles and to test a different ontogenesis of varicoceles in adolescents and adults, and (iv) lastly to develop a standardized method to measure the intravascular pressure in the internal spermatic vein and to determine whether infertility in varicoceles can be explained by the principle of elevated hydrostatic pressure.The vast majority of adolescents with varicoceles are asymptomatic . It may An adult patient with a varicocele complains either for scrotal pain or heaviness at the scrotal sac or for infertility problems. Approximately 15% of all men in the general population have a clinical varicocele. Between 19–41% of men investigated for infertility have a varicocele . The incThe prevalence of varicoceles increased significantly at 13-years of age, somewhere at the start of puberty. Varicoceles are rarely found in boys six to nine years of age , 6, 7. TMost studies based on venography and with large number of patients report a bilateral varicocele in 25% , 9, 10. The heredity of varicoceles and the potential transmission to first-degree relatives has been little investigated. Clinical varicoceles are more prevalent among first-degree relatives of patients with known varicoceles .The etiology and pathogenesis of varicoceles cannot be explained by one single theory. Valve dysfunction, ontogenetic collateral formation and the nutcracker phenomenon seem to act synergistically. Hyperthermia, elevated hydrostatic pressure and antisperm agents are suggested as possible causes for the pathophysiology how varicoceles induce infertility. However, the combination of patient’s lifestyle, genetic factors and the consequences of reflux into the pampiniform plexus (PP) are believed to contribute to the infertility.Although venography stays the gold standard, the combination of physical examination, colour Doppler ultrasound and thermography has the highest sensitivity and specificity to diagnose a varicocele. Regarding infertility, criteria or grading, to decide which patients with a varicocele may or may not have benefit from treatment remain to be established.Treatment of varicoceles can be performed by different open, mostly surgical techniques and by percutaneous, mostly endovascular techniques. Treatment of varicoceles for infertility or to prevent infertility remains controversial, because the majority of men with varicoceles are still fertile. At the moment, inguinal or subinguinal microscopic surgery gave the highest pregnancy rates, the lowest recurrence and lowest complication rates. But retrograde superselective glue embolization or sclerosing of the ISV are the best percutaneous alternative and can be performed on an outpatient basis under local anesthesia and with faster return to normal activities than surgery.As the different surgical techniques for the treatment of varicoceles were developed in the 70–80 ties, endovascular embolization emerged as a valuable alternative.Depending on the used embolic agent the occlusion can be restricted to the main ISV or extended to the collateral venous network. The first technique is the endovascular equivalent of the surgical ISV clipping, the latter has a similar effect than an extended microsurgical dissection and clipping of all veins. The first reports of retrograde embolization of the ISV are from the late 70ties , 16. WitThere are liquid (sclerosing agents and glue) and non-liquid  agents. Many investigators use a combination of these agents to treat varicoceles. Newer products like Onyx®, vascular plugs or detachable coils are used in case reports or in unpublished series , 20. LiqSclerosing agents (sodium tetradecyl sulfate 3% (STS), sodium morrhuate, dextrose and aethoxysklerol (1–2 or 3%)) are the most commonly used liquid agents in Europe (not authorized in the USA for spermatic veins) . During Tissue-adhesives (Cyanoacrylates) were first introduced by Kunnen, et al. in 1980 . Glue isOnyx®  is a biocompatible ethylene-vinyl alcohol copolymer (EVOH) dissolved in dimethyl sulfoxide (DMSO) that in contact with blood solidifies as a kind of plastic. Onyx proved to be superior to tissue-adhesives for the occlusion and penetration of side-branches in cerebral embolization , 26. OnyGelatin sponge is a water-insoluble, hemostatic sponge prepared from purified porcine skin gelatin. Gelfoam may be cut in small pieces that are injected through a diagnostic catheter and is able to absorb and hold many times its weight of blood and other fluids within its interstices. Gelatin sponge is a non-permanent occlusive agent, and therefore more used in combination with other permanent mechanical or liquid embolic agents .sandwiched 70% dextrose (a sclerosing agent) [Detachable balloons can be navigated through an introducer catheter and detached in the ISV. Depending on the size of the varicocele and the presence of collateral vessels, one or more 1- or 2-mm detachable balloons are used. Riedl and White were the first to treat varicoceles with detachable silicone balloons , 29. Aftg agent) .Varicocele treatment with coils was introduced by Thelen, et al. who used the original 0.038-inch Gianturco coils . Other fAll percutaneous endovascular techniques have a low complication rate (<1%). Non-liquid agents  and low viscosity liquids (sclerosing agents) have a high primary failure rate that ranges from 3–28% and increases when aberrant vessels are present , 31. RecThe need for contrast agent and the radiation exposure are the main disadvantages of the endovascular percutaneous technique. Severe allergic reactions to contrast agents are possible but very uncommon. Radiation exposure during embolization, with a subsequent 0.1% life-long risk for cancer, was reported in a retrospective study . RadiatiThe advantage of percutaneous embolization is that the patient can return to normal activities faster than with surgical treatment, since there is no incision and splitting of the abdominal muscles involved .In our experience, we believe that retrograde liquid embolization under local anesthesia should be the treatment modality of choice for varicoceles. Solid embolic materials  have the risk as in surgical clipping of leaving collaterals untreated, which could be responsible for recurrences. With low viscosity liquid sclerosing agents such branches and duplications can be occluded if injected in sufficient quantity and under Valsalva control. However, their low viscosity and visibility might increase nontarget embolization. Moreover, the sclerosing agent need time to obstruct the veins, so a waiting time of about 20 minutes is required to control its effect. A higher viscosity liquid like glue has the advantage of being radiopaque and of being a quick polymerization with definitive occlusion. The polymerization time can be reduced regarding the anatomical complexity of the ISV to allow the glue to penetrate into side branches , 35.Percutaneous embolization with glue should be the first line of therapy in varicoceles. Superselective sclerotherapy is a good alternative in countries where tissue adhesives are not available.Since 1998, the FDA approved octyl-cyanoacrylates as a topical skin tissue adhesive. For butyl-cyanoacrylates this approval has been refused until 2007. As an endovascular adhesive and exclusive in cerebral interventions, only TRUFILL® n-Butyl Cyanoacrylate (n-BCA) Liquid Embolic System is FDA approved. GLUBRAN®2 is the only glue with a CE-mark for endovascular use.Polymerization of glue in vessels produces an inflammatory reaction in the wall and in the surrounding tissues of the vessel, which changes in a chronic process resulting in fibrosis. Glue has to be mixed with iophendylate (IBCA), which makes the mixture radio-opaque during embolization. Beside its radio-opacity, iophendylate could change the polymerization time.Kunnen introduced in 1980 the use of IBCA in varicocele treatment . He repoIn 1985, Kunnen described a technical success of more than 99%, 0.46% complications (two glued catheters) and a 1.6% recurrence rate in a series of 435 patients . In men Two studies reported a higher recurrence rate of 6% , 45. AltMost of the investigators choose a 1/1 glue-lipiodol mixture to obtain a rapid polymerization and avoid penetration of the glue into the PP.The largest population of varicoceles embolized with glue to date (3043 patients) is described by Kunnen in a book chapter. He reported a very low technical failure (<1%), a low complication rate (0.3%) and recurrence rate (2%) and a high pregnancy rate (50%) .There are currently two commercially available tissue-adhesives that can be used for the embolization of varicoceles. The two adhesives differ only minimally in chemical formula, but polymerize in a different way. The more recently developed Glubran2, available since 2001, would have the same occlusion features as the older glue, hystoacryl transparent. The manufacture of Glubran2 obtained a CE label for intravascular use and alleged that Glubran2 has a higher stability and a lower toxicity.We tested both glues  for differences in efficacy, safety and tolerance in a double-blind, prospective, randomized study. We found that both glues can be handled in the same way and that the embolic result is similar.Technical success was 100%, this means that in all patients we could effectively block the reflux in the insufficient ISV. The procedure was very safe with no complications caused by the glue.Concerning patients comfort we were the first to delineate a late inflammatory reaction to the cyanoacrylates. During the week after embolization, 59% of patients reported some discomfort, which was in 35% at least a bearable pain. There was no significant difference in discomfort between the two glues in the post-embolization week. Moreover, there was some mild acute inflammatory pain reaction during the embolization in 20% of the patients. There was no difference between both glue groups. Given these findings, patients should be informed that they might experience discomfort or mild pain during and after embolization of their varicocele. As this inflammatory reaction is self-limiting and mostly mild, we would not advise to prescribe NSAIDs prophylactically.Because there is no difference between Histoacryl transparent and Glubran2, both glues can be used effectively and safely. However, it is medicolegally sensible to choose Glubran2 because it has a CE approval for endovascular use.Another liquid embolic agent, which is non-adhesive, is ethylene-vinyl alcohol also called “Onyx”. Onyx is a plastic dissolved in dimethyl sulfoxide. After diffusion of the solvent, Onyx solidifies to a rigid cast, which can be pushed and extended in different directions. For these reasons, Onyx embolization has become the primary intravascular therapy for cerebral arterio-venous malformations (c-AVM) and dural fistulas (d-AVF). Because of the anatomical similarities between AVM and complex insufficient ISV, we thought that Onyx could be a potential embolic agent in complex varicoceles. Moreover, we know from extirpated AVM species that Onyx does not induce a post-embolization inflammatory reaction.In a pilot study we tested Onyx as an embolic agent in ISV embolization. We found that Onyx was efficient and indeed better tolerated in the post-embolization period. However, there was an acute pain reaction during the injection of Onyx in nine of ten patients, prompting to stop the injection temporarily in six patients. The technique to inject Onyx as we do with glue, allowing only little reflux, demonstrated to be suboptimal and ensued a higher radiation dose. Further research should concentrate on adaptation of the embolization technique as used for c-AVM and on reducing the acute pain reaction by injection of intravenous anesthetics.justification. This means that the clinical benefit of the treatment has to outweigh the possible radiation-induced risks. The treatment of varicoceles to prevent infertility or to improve fertility, is very controversial. Other studies and our study, show that the gonadal dose in varicocele embolizations is definitely far below the lower threshold corresponding to the deterministic effect of temporary sterility (150 mSv).The first basic principle of radiation protection is –6). With respect to the risk of cancer mortality a mean value of 0.06% was calculated. The latter value is much lower than other typical interventional X-ray procedures. According to the NCRP publication 168, the obtained risks in our study population are requiring a minor to moderate benefit to justify the varicocele embolization procedure. The low dose and risk values obtained are probably linked to a set of specific radiation reducing measures. The latter optimization includes the use of pulsed fluoroscopy, the application of gonadal shielding and the careful collimation of the radiation beam.Based on the in-vivo measured testes doses, we could estimate the risks of hereditary effects as being very low  and the assumed association with infertility. In adolescents, varicoceles are mostly asymptomatic. Treatment of adolescent’s varicocele is advised to anticipate testicular hypotrophy and future fertility. To justify this prophylactic treatment, it is generally assumed that varicocele in children are an early stage of the varicocele in adults. Although the adult varicocele is usually symptomatic, treatment is requested in first place because of sperm dysfunction.the easier procedure was due to the absence of competent valves and a straighter anatomy in adolescents. These observations could point at a different phlebographic anatomy between adolescents and adults, and indirectly disclose a different pathophysiology of varicoceles.At a certain moment in my practice, I had the impression that the embolization of a varicocele in a child seemed a technically less challenging procedure than in an adult. I thought that To investigate this hypothesis, we set up a retrospective study to compare phlebographic radio- anatomical landmarks between adolescents and adults. For this purpose, we used the selective phlebographies that were performed prior to the embolization of the varicocele Table  Figure .After analysis and comparison of about 500 phlebograms, we demonstrated that our suspicion was not unfounded. Adults had significantly more often a competent outflow valve than adolescents. Moreover, adult varicocele had a more complex anatomy consisting of a significantly higher number of collaterals and of a complex outflow in the renal vein. The phlebographic complexity of adult varicocele could be the remnant of an insufficient embryonic venous drainage during the ontogenesis of the left ISV.Left-sided varicocele in many adolescents seems to be more a congenital abnormality rather than a progressive chronic disease: we found a higher rate of insufficient outflow valves, congenital renospermatic bypasses and the compressive nutcracker phenomenon.On the other hand, we have arguments that right-sided varicocele might be a progressive disease. The incidence of an insufficient ISV increase with age (2.4 times more often in adults (33%) than in adolescents (14.7%). The diameter of the ISV also increases significantly with age: a fact that could point at a degenerative process with reflux and dilatation Table  Figure .Many theories have been developed about how varicoceles can cause infertility. The causal association is most probably multifactorial, combining of lifestyle factors, genetic factors and the effect of reflux of blood into the PP.counterpressure might interfere with the arterial and nutritional supply of the testicle and decrease spermatogenesis and testicle growth.The reflux of blood into the ISV is a direct result of the insufficiency of valves, a mechanism we can observe during the phlebography of varicocele patients. By reflux of warm blood, temperature in the testicle rises. Furthermore, in a varicocele patient standing upright, the hydrostatic pressure at the PP and the testicle increases according the height of the fluid column in the ISV. Venous drainage at the PP will then be impaired, and the cooling of the testicle less efficient. This hydrostatic ρ, resulting in a value of 31.5 mm Hg at the left and a value of 27 mm Hg at the right side. This hydrostatic pressure is, according to Gat, high enough to cause testicular insufficiency and infertility in varicoceles. This is the theoretical point of view.In an editorial of Andrologia, Gat titled that, “Erect posture of humans leads to infertilit.” . In two Pressure measurements in the PP or inguinal ISV of varicocele patients were previously performed by direct measurements through a needle or catheter in the PP , 48, 49.Performing pressure measurements through a microcatheter in the ISV was not an easy task. Initial results were not at consistent. We tested and calibrated our method ex-vivo and standardized the measurement method. We compared pressures obtained via a microcatheter  with those registered with two different commercial pressure wires. In-vitro  and in-vivo (endovascular in the ISV) tests demonstrated that the direct measurements (pressure wires) could be reproduced by the microcatheter measurements  Figure . MeasureFor the first time, an in-vivo experiment done in 42 patients showed that the absolute mean pressure in the inguinal left ISV is 33 mm Hg. According to the hypothesis of Gat, this pressure would only exist of a hydrostatic pressure component. However, the height of the blood column that produces this hydrostatic pressure would reach far above the renal vein, even higher than the right atrium. We disclosed that the pressure in the renal vein and at the outflow of the ISV did not change with patient’s position. It was constant at about 12 mm Hg, corresponding to the systemic venous pressure. The absolute measured pressure is therefore composed of a hydrostatic (21 mm Hg), and a systemic pressure (12 mm Hg) component. This hydrostatic pressure was compatible with the indirect calculation from the measured height of the ISV .As the normal capillary pressure of the testicle can be estimated at less than 20 mm Hg, then the hydrostatic pressure in upright standing position alone appears to be high enough to act as a counterpressure. This counterpressure could impair arterial supply and spermatogenesis in varicoceles.Only standardized pressure measurements in fertile patients without varicoceles would be able to answer whether erect posture in varicocele patients leads to infertility.The current commercially available tissue-adhesives, Histoacryl transparent and Glubran2, are equally efficient, safe and tolerable for transcatheter embolization of varicoceles. Glue can cause a mild, self-limiting pain in 1/3 of the patients in the week after embolization, independently of what product was used. Because Glubran2 has a CE label for intravascular use, it is medico-legal justified to use this glue. Worldwide, glue is not a popular embolic agent for the treatment of varicoceles, probably because it requires more skills or preconception persists about the potential complications. Controlled randomized trials, reporting the success and complications rate, as well as the pregnancy rate could lead to an FDA approval for intravascular use and contribute to the attractiveness of glue.Onyx has the potential of an efficient and safe agent for the embolization of varicoceles. Unfortunately, the acute pain reaction as well as the radiation exposure during embolization have to find a solution before Onyx can be recommended. Finally, we have to explore in which anatomical situation Onyx can constitute an advantage over the other embolics.Radiation exposure is safe during embolizations of varicoceles with highly viscous liquid products. We report a gonadal dose that is far below the lower threshold corresponding to deterministic effects. In future, by the use of the flat panel detectors, the radiation exposure may be reduced even further.In left-sided varicocele children’s spermatic vein showed more often valve insufficiency, renospermatic bypasses and nutcracker syndrome. In adults, reflux more often is caused by collateral connections. Left varicocele seems rather a congenital abnormality than an evolutionary disease as commonly believed. At the right varicocele, we have shown that the diameter of the ISV increases in adults and that a bilateral varicocele occurs 2.4 times more. As far as the right varicocele is concerned, we do have arguments for an evolutionary chronic disease. In a subsequent study, we could further investigate and discover links which might predict whether certain children may profit from a prophylactic embolization.This thesis demonstrated in-vivo that the mean absolute pressure in the inguinal ISV in the upright position is significantly high enough to build a counter pressure to the capillary pressure, and hence impair the spermatogenesis. In addition, we have invalidated Gat’s hypothesis and proven that not only the hydrostatic pressure is responsible for this elevated pressure, but the sum of the systemic and the hydrostatic pressure. We could investigate whether clinical presentation, thermography, CDUS, phlebographic anatomy or age are associated with different pressure levels. Here again, the detection of certain sub-groups could change the therapy policy.really pathological since we cannot compare with a healthy control population. We could measure the pressure in the ISV of a control group of fertile men without varicocele. It will not be an easy task to convince the ethics committee. Moreover, it will be a technical challenge to catheterize healthy men retrograde through several competent valves down to the inguinal ISV. Only then we could answer the question “does erect posture in varicoceles lead to infertility?”However, we still ignore whether the pressures we obtained, are


Crohn’s disease (CD) accounts for a variety of clinical manifestations or phenotypes that stem from chronic inflammation in the gastrointestinal tract. Its worldwide incidence is increasing including younger or childhood-onset of disease. The natural history of Crohn’s disease is characterized by a remitting and relapsing course that progresses to complications and surgery in most patients. The goals of treatment are to achieve clinical and endoscopic remission, to avoid disease progression and minimise surgical resections. Medical treatment usually features antibiotics, corticosteroids, immunomodulators . Anti-TNF (tumour necrosis factor) therapy was approved for use in Crohn’s disease in 1998, and has changed the paradigm of treatment, leading to improved rates of response and remission in patients. There are significant considerations that need to be borne in mind, when treating patients including immunogenicity, safety profile and duration of treatment. Crohn’s disease (CD) is a chronic inflammatory condition that can affect any part of the gastrointestinal (GI) tract. It is encompassed in the term “inflammatory bowel disease (IBD)”, which includes ulcerative colitis (UC) and CD and these are characterised by chronic, uncontrolled and relapsing inflammation of the GI tract. The incidence and prevalence of IBD is increasing with the highest annual incidence of CD being in North America  . It is aHuman TNF is a member of a large family of proteins and receptors that are involved in immune regulation . The secThe underlying cause of CD is not fully understood; however, the aetiopathogenesis is thought to involve an interplay between environmental triggers, dysbiosis, aberrant immune responses/immunoregulation and genetic susceptibility . CD resuAntibody neutralization studies implicated a significant role for TNF in the pathogenesis of Crohn’s disease . These wp = 0.002) and closure of all fistulas draining at baseline occurred in 55%  of the 63 patients receiving infliximab at 0, 2, and 6 weeks [Infliximab was the first biological response modifier to be used in the treatment of inflammatory bowel disease  and is a 6 weeks . Mainten 6 weeks . However 6 weeks .Adalimumab, a fully human antibody that also fixes complement and lyses cells expressing TNF-α, is administered subcutaneously (via auto-injector pen) every two weeks. Crucial studies that demonstrated evidence for Adalimumab use in CD included the CLASSIC-I trial   Table 1Table 1. p = 0.02). The PRECiSE 2 trial [p < 0.001) and remission rate  with maintenance of certolizumab following positive response to induction therapy at 26 weeks, compared to placebo. Certolizumab has also been evaluated using health related quality of life (QoL) as an outcome measure, by assessing patients’ response to treatment using the Inflammatory Bowel Disease Questionnaire (IBDQ) [p = 0.038) [Certolizumab pegol is a chimeric humanized antibody fragment against soluble (secreted) TNF and transmembrane TNF, which is attached to polyethylene glycol, PEG (“pegylated”). It differs from IFX and ADA in that it does not contain the crystallisable fragment (Fc) region of typical antibody. It also differs in function in that it does not induce apoptosis as one of its mechanisms of action, nor does it activate the complement pathway, or result in cell or antibody mediated cytotoxicity ,65. It i 2 trial  reportede (IBDQ) . Rutgeere (IBDQ)  reportede (IBDQ) . This wae (IBDQ)  of four = 0.038) . The abo= 0.038) .There are no trials comparing all three anti-TNF therapies, however there are suggestions that there are no significant differences in efficacy between infliximab and adalimumab . They arThe efficacy of anti-TNF medication has led to the introduction of biosimilars, as the patents of older anti-TNF agents have either expired or are close to expiration. Biosimilars are synthesized versions of existing biological drugs with no perceived difference in safety or efficacy. Several have been approved for treatment of CD in USA and Europe (with 19 products authorised at the end of 2015 ) and theDespite the paradigm shift seen over the last two decades with anti-TNF treatment in Crohn’s disease, the response that an individual patient will have to a specific anti-TNF and dose is difficult to predict when compared with conventional (non-biologic) therapies. Up to 30% of patients do not respond to anti-TNF therapy (primary non-responders) and almost half of the patients who experience a benefit with these drugs will lose clinical benefits within the first year, requiring dose escalation or therapy change ,81,82, tThe reasons why some patients do not respond or lose response after a successful course of therapy are not completely clear, but they are likely multifactorial and related to metabolism of the drug or to the development of antidrug antibodies . This prOther factors that may result in loss of response are potential differences in pharmacokinetic properties, including tissue penetration and mode of action of the different anti-TNF drugs. The need to develop biomarkers that can predict response to therapies will become increasingly important for personalised medicine decisions in the future. Once loss of response is confirmed, dose escalation/interval shortening and switching anti-TNF agents are recommended potential strategies .The route of administration of anti-TNF influences its pharmcokinetics. Infliximab is administered intravenously, which allows for the usage of large volumes, rapid distribution, with peak serum concentrations shortly after infusion and low variability in bioavailability . AdalimuAntibody distribution is mainly within the extracellular fluid and occurs via cellular movement from circulation to tissue, diffusion or receptor mediated endocytosis ,93. For The principles of anti-TNF dosing regimen are to achieve steady-state range of serum or tissue drug concentrations that are adequate for the drug to neutralize surplus TNF, but not so high that it affects safety because of neutralization of homeostatic concentrations of TNF required for host defence. Conversely, tissue drug concentrations should not be so low as to impair efficacy as a result of suboptimal neutralization of TNF . Within The use of TDM to optimise anti-TNF drug concentrations has a promising potential for dose optimisation in clinical practice, in view of the reported correlations between anti-TNF trough concentrations, anti-drug antibodies, and disease outcomes; however, studies in TDM are yet to demonstrate unequivocal benefit ,97. Two Clinicians and patients are often faced with the question on whether it is possible to stop anti-TNF therapy once disease remission has been achieved. However, despite all the studies that have now addressed this issue in IBD, no conclusive strategy has yet emerged . Data isIt is often empirically proposed not to routinely stop anti-TNF-α agents in IBD patients who respond, and especially in patients with disabling features of disease and/or at high-risk for relapse . The STOLong-term therapeutic use of anti-TNF carries with it safety issues which include potential for development of skin lesions, immune reactions, peri-operative complications, infections, cancers and decreased fertility/adverse effects on pregnancy ,110.Infusion reactions relate to infliximab and can be categorized based on their timing, pathogenesis, and severity . They arp = 0.008). However, multivariate analysis, demonstrated no significant increased risk of infection  due to anti-TNF therapy after controlling for factors such as disease duration, severity and concurrent corticosteroid and immunomodulator use [Susceptibility to infection is a significant concern following instigation of treatment with anti-TNF. Anti-TNF treated patients are rendered immunocompromised through their treatment and this is often in the context of combination treatment with other immunosuppressant medication . Infective complications vary from serious invasive bacterial/opportunistic infections to relatively milder cases (mild respiratory/urinary infections) . Analysiator use . In a reator use ,119. Antator use , which iator use . In viewator use .p < 0.0001) and thiopurines alone  but not with anti-TNF use alone  [p = 0.46), with no differences seen for solid tumours, non-melanoma skin cancers, and lymphoma between the two groups [Anti-TNF therapy has been theoretically linked with a propensity for malignancy, due to the suppression of TNF, and a meta-analysis in 2006 reported a 3-fold increased risk of malignancy for patients with rheumatoid arthritis undergoing anti-TNF  treatment . However = 1.00) ,123. The = 1.00) , however = 1.00) ,126. Howo groups . In a meo groups . This hao groups ,129,130.All three anti-TNF agents have been deemed safe in pregnancy, not least because of the increased risk of complications, preterm delivery and low birth weight conferred by active uncontrolled disease . HoweverIn summary, anti-TNF introduction has led to major shifts in the therapeutic paradigm that evolved from slow symptomatic clinical remission towards rapid sustained and deep remission; short- and long-term clinical, and endoscopic endpoints can now be reached that were previously unachievable ,92. Anti


Tumor necrosis factor-alpha (TNF) is a pleiotropic cytokine, which is thought to play a major role in the pathogenesis of inflammatory diseases, including allergy. TNF is produced at the early stage of allergen sensitization, and then continues to promote the inflammation cascade in the effector phase of allergic reactions. Consequently, anti-TNF treatment has been proposed as a potential therapeutic option. However, recent studies reveal anti-intuitive effects of TNF in the activation and proliferative expansion of immunosuppressive Tregs, tolerogenic DCs and MDSCs. This immunosuppressive effect of TNF is mediated by TNFR2, which is preferentially expressed by immunosuppressive cells. These findings redefine the role of TNF in allergic reaction, and suggest that targeting TNF-TNFR2 interaction itself may represent a novel strategy in the treatment of allergy. Highlights- Pleiotropic function of TNF in allergy is likely mediated by its two receptors, TNFR1 and TNFR2- Activation by TNFR1 results in the allergic inflammatory responses while TNFR2 plays a role in the immune tolerance to allergens- TNFR2 is preferentially expressed by highly suppressive and replicating Tregs and TNFR2 signaling leads to the activation and proliferation of Tregs- Targeting of TNFR2 to boost Treg activity may represent a novel strategy for treating patients with allergy.Allergy is an immune-mediated hypersensitivity to allergens. Exposure of allergens through inhalation, ingestion or skin contact leads to diseases such as asthma, allergic rhinitis, food allergy, and atopic dermatitis. Allergy is a complex disease, and both genetic and environmental factors contribute to its pathogenesis. It affects 30–35% of the population at some point in their life, with incidence continuing to rapidly grow each year. The past decades have exhibited large-scale anthropogenic changes which are currently considered the leading causes of the increasing burden of allergic diseases.The development of allergy can be divided into two phases: 1) the sensitization and memory phase, and 2) the effector phase, which can be further staged into immediate and late responses   . In one or Tregs  was dimior Tregs . In anotor Tregs . Becauseor Tregs . Furtheror Tregs .Overall a proper maturity and homeostasis of immune system of neonates during the first years of life is fundamental to minimize allergic development. Maternal allergy correlates well with impaired frequency and function of Tregs in neonates and consequently the increased susceptibility for the development of allergy in early childhood –29. A stTNF is a pleiotropic cytokine that plays important dual roles in maintaining immune homeostasis and in promoting the development of diseases. TNF is required for host defense against pathogens, immune surveillance against malignancies, as well as cell proliferation and survival . This cyDual biological functions of TNF are likely to be transduced through its two distinct receptors, TNFR1 and TNFR2. TNF and its receptors have both a membrane-bound form and a soluble form. TNFR1 shows high affinity toward both forms of TNF, while TNFR2 is only fully activated by membrane-bound TNF . Once acTNF is reported to play significant role in the pathogenesis of allergy and contributes to both early and late stages of allergy development. This is evident when allergy manifestations are inhibited in TNF-knockout (KO) mice as well as with anti-TNF treatments –37. FurtAlthough binding of TNF to its distinct receptors generally activates the same major signaling pathways (NFκB and MAPK), the distinct structure and motifs of TNFR1 and TNFR2 result in entirely different functional consequences. TNFR1 bears the death domain, which recruits several death signaling proteins such as TNFR1-associated death domain protein (TRADD), Fas associated protein with death domain (FADD) and the TNFR-associated factor (TRAF)-1 to induce inflammation and apoptosis .In the context of apoptosis, TNF-TNFR1 axis signals through caspase 3 and 8, and is mainly responsible for the host defense of pathogen  and antiUnlike TNFR1, TNFR2 lacks the death domain where the activation of TNFR2 recruits TNFR-associated factor 2 (TRAF2) that mainly promotes cell proliferation and survival . In alleDistinct functions of TNF and its receptor led to the prospective of selectively targeting TNFR1 to inhibit apoptosis, and TNFR2 to induce cell survival. This approach intends to achieve homeostasis in various autoimmune and inflammatory diseases including allergy. These disorders are primarily associated with defects in TNF signaling through TNFR2. Therefore, this particular receptor is of interest and important roles of TNF-TNFR2 interaction on various cell types are further discussed in the next section  have been established as therapeutic options in inflammatory diseases. Etanercept, a genetically engineered recombinant protein that comprises of TNFR2 and Fc portion of human IgG1, specifically binds to TNF and blocks its interaction with cell surface receptors . StudiesUnlike in allergy, TNF-TNFR2 axis in autoimmune diseases and cancer has been well established. Adalimumab has shown to expand functional Tregs through TNF-TNFR2 axis on monocytes in rheumatoid arthritis . Another+Foxp3+ Tregs after allergen challenge, partly via the activation of CD103+ DCs . It can be considered as an achievable long-term cure for allergy as basic principle in SIT, which involves inducing immune tolerance toward allergens by specifically and repeatedly administrating the causative allergen (Due to its restricted cellular expression and prominent role in immune regulation, TNFR2 is still a more attractive target for treatment in diseases, including allergy. Faustman and Davis  suggeste103+ DCs , 110. Thallergen .In allergy, TNF may have dual pro-inflammatory and anti-inflammatory activities, which are likely mediated by its two distinct receptors. TNFR2 signaling is attributable to the immunosuppressive effects of TNF and thus is protective against allergy. Consequently, targeting TNF-TNFR2 pathway may represent future direction to develop new therapies in allergy. However, as a pleiotropic cytokine, TNF and its effects on TNFR signaling is diverse and can either regulate allergic manifestations or to control disease pathogenesis. Implications of TNFR2 in health and diseases requires additional investigation to further elucidate their exact mechanisms and provide more insight for future strategies in manipulating TNFR2 for therapeutics.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Thus, ATP critically alters TNF trafficking and secretion from macrophages, inducing novel unconventional membrane TNF signaling via MVs without direct cell-to-cell contact. These data have crucial implications for this key cytokine, particularly when therapeutically targeting TNF in acute inflammatory diseases.—Soni, S., O’Dea, K. P., Tan, Y. Y., Cho, K., Abe, E., Romano, R., Cui, J., Ma, D., Sarathchandra, P., Wilson, M. R., Takata, M. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles.Cellular stress or injury induces release of endogenous danger signals such as ATP, which plays a central role in activating immune cells. ATP is essential for the release of nonclassically secreted cytokines such as IL-1β but, paradoxically, has been reported to inhibit the release of classically secreted cytokines such as TNF. Here, we reveal that ATP does switch off soluble TNF (17 kDa) release from LPS-treated macrophages, but rather than inhibiting the entire TNF secretion, ATP packages membrane TNF (26 kDa) within microvesicles (MVs). Secretion of membrane TNF within MVs bypasses the conventional endoplasmic reticulum– and Golgi transport–dependent pathway and is mediated by acid sphingomyelinase. These membrane TNF–carrying MVs are biologically more potent than soluble TNF  The majority of cytokines, including TNF, IL-6, and IL-12, are secreted via the classic pathway. This involves rapid transcription and translation of cytokines, which are conveyed from the endoplasmic reticulum (ER) to the Golgi complex. These proteins undergo sorting in the trans-Golgi network and are then transported to the cell surface via recycling endosomes  release from activated macrophages but instead preferentially packages transmembrane pro-TNF (26 kDa) within released microvesicles (MVs). We uncover a novel way of membrane TNF signaling in vivo and establish that these MVs are highly potent, inducing significant TNF-dependent inflammation and allowing long-range TNF signaling to target cells more effectively than soluble TNF. These data suggest mechanisms that potentially explain why previous anti-TNF strategies targeting soluble TNF have shown little benefit for acute inflammatory diseases such as sepsis or acute respiratory distress syndrome (ARDS)  .Animal Research: Reporting ofIn VivoExperiments guidelines . Seventy-six male C57BL/6 mice  and 6 TNF−/− mice  aged between 7 and 8 wk [for bone marrow–derived macrophage (BMDM) harvesting] or 10–14 wk (for in vivo experimentation) were used. Mice were housed in individual ventilated cages (maximum number of 5/cage) and exposed to 12-h light/dark cycles. All experiments were initiated and completed during the light cycle, and no unexpected adverse effects were observed in any of the treatment groups.All protocols were approved by the Ethical Review Board of Imperial College London, carried out under the authority of the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986, and reported in compliance with the 2 atmosphere. BMDMs were isolated and cultured as previously described by Manzanero (g 5 min at 4°C) and cultured for 5 d in 148-cm2 dishes in a humidified 5% CO2 atmosphere with medium supplemented with 50 ng/ml recombinant M-CSF  to induce differentiation of bone marrow monocytes into macrophages  (mycoplasma tested) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin-glutamine, at 37°C in a humidified 5% COanzanero . In brierophages A. Media E. coli O111:B4; InvivoGen, San Diego, CA, USA) for 1 h  to pellet MVs, which were then washed twice to remove contaminating factors . Tin vivo, randomly selected mice were anesthetized by intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg). The vocal cords were identified using a microscope and an external light source, and a fine catheter was briefly passed 1 cm below the cords. Fifty nanograms Ultrapure LPS in 25 µl was instilled intratracheally as previously described  for 7 min at 37°C. Cell pellets were isolated by centrifugation and incubated with a fluorescence-conjugated antibody cocktail containing anti-CD45 , anti-CD11b , anti-TNF , and its corresponding isotype control, IgG1κ , for 30 min at 4°C.+ and CD11b+, whereas BMDMs were identified as CD45+, CD11b+, and F4/80-positive  (Cells were incubated with BD cytofix/cytoperm (BD Biosciences) at 37°C for 10 min to permeabilize and fix them. These cells were then washed twice with permeabilization wash buffer , and antibody staining then followed as described above. RAW macrophages were identified as CD45CA, USA) A.Following doses of LPS and ATP, the lungs of mice were also removed and mechanically disrupted in BD cytofix/cytoperm using a GentleMacs dissociator . Samples were then passed through 40-µm sieves, washed, and resuspended twice in permeabilization wash buffer to yield a fixed single cell suspension. Alveolar macrophages were stained with fluorescence-conjugated antibodies of anti-CD45, anti-CD11c , anti-CD11b, anti-F4/80, and anti-TNF B.All stained samples were analyzed using flow cytometry . Cell populations were analyzed in FlowJo software for their mean fluorescence intensity (geometric mean) for surface (unpermeabilized) or total (permeabilized) TNF expression.1) size <1 µm, 2) positive for specific precursor cell markers, and 3) sensitivity to 0.1% Triton X-100 detergent  for the assessment of MV count. Forward scatter and side scatter  were used to elucidate a 1-µm gate that was delineated by specific sizing beads as previously described by Soni et al. . T. T1) sizetergent B and S2B−/− mice and wild-type (WT) C57BL/6 mice and stimulated with LPS and ATP as described above (without BB94 treatment) to generate TNF-depleted and TNF-laden BMDM-derived MVs, respectively. MVs were counted using flow cytometry, and 4 × 106 TNF-laden or TNF-depleted , th00,000 g  but is d00,000 g , and we 00,000 g . Whetherg et al. , and its (et al. . Howevervia MVs without cell-to-cell contact. These data have important, previously unappreciated biologic implications for this key proinflammatory cytokine, particularly when targeting TNF in drug applications.In summary, our results demonstrate that the danger signal ATP critically modulates cytokine secretion from macrophages, redirecting intracellular TNF trafficking and inducing a novel unconventional form of membrane TNF signaling http://www.fasebj.org to obtain this information.This article includes supplemental data. Please visit Click here for additional data file.


Salmonella Typhi or Paratyphi human challenge studies were conducted, and daily stool cultures performed. Vi-containing vaccines reduced bacterial shedding, Ty21a or an experimental vaccine did not. Higher Vi immunoglobulin G titers were associated with reduced shedding.Six  Salmonella Typhi or Paratyphi in the stool or urine leads to contamination of food or water, which is a prerequisite for transmission of enteric fever. Currently, there are limited data on the effect of vaccination or prior exposure on stool shedding.Shedding of Salmonella Typhi or Paratyphi human challenge studies were conducted between 2011 and 2017. Participants were either unvaccinated or vaccinated with 1 of 4 vaccines: Vi-polysaccharide (Vi-PS), Vi-tetanus-toxoid conjugate vaccine (Vi-TT), live oral Ty21a vaccine, or an experimental vaccine (M01ZH09). Daily stool cultures were collected for 14 days after challenge.Six P = .010 and OR, 0.41; 95% CI, 0.19–0.91, P = .029 for Vi-PS and Vi-TT, respectively). Higher anti-Vi immunoglobulin G titers were associated with less shedding of S. Typhi (P < .0001). A nonsignificant reduction in shedding was associated with Ty21a vaccine . Individuals previously exposed to S. Typhi shed less than previously unexposed individuals . Shedding of S. Typhi was more common than S. Paratyphi.There were 4934 stool samples collected from 430 volunteers. Participants who received Vi-PS or Vi-TT shed less than unvaccinated participants (odds ratio [OR], 0.34; 95% confidence interval [CI], 0.15–0.77; S. Typhi. Field trials of Vi-TT should be designed to detect indirect protection, reflecting the consequence of reduced stool shedding observed in the human challenge model.Prior vaccination with Vi vaccines, or natural infection, reduces onward transmission of  Salmonella enterica subspecies enterica serovars Typhi or Paratyphi (S. Typhi or S. Paratyphi) is estimated to be responsible for between 11.6 and 26.9 million cases of enteric fever and 75000–216510 deaths annually ..24].S. Typhi lipopolysaccharide (LPS) (Sigma L2387), S. Typhi Hd (University of Maryland 01-CVD0150622-01), S. Paratyphi A O:2  [S. Paratyphi Ha (University of Maryland CVD 1902D lot CVD141113-01) antigens were measured with an in-house ELISA in serum samples collected immediately prior to challenge [Anti-Vi immunoglobulin (Ig) G titers were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s guidelines . IgG and Health) , and S. hallenge , 19.Stool culture data were combined in mixed effects logistic regression models, which included participant-specific random intercepts to account for the multiple samples per person. Models were adjusted for the vaccine received, study, and, where applicable, challenge dose (high or low). “Day” was included in the model as a categorical factor to allow the odds of stool shedding to vary by day.An overall interaction term (day-by-vaccine) was tested to determine if vaccination altered the pattern of shedding over time. An interaction term for day-by-diagnosis status was used to compare the pattern of shedding in those who were diagnosed with enteric fever and those who remained undiagnosed at day 14.The linear predictor from the model was exported for each participant for each day and converted into a probability by taking the inverse logit. These probabilities are presented in figures with a loess smooth to illustrate the findings from the logistic regression models. Odds ratios (ORs) presented from logistic regression models represent the ratio of the odds of shedding in comparative groups on average across all 14 days.All models were fitted in SAS version 9.4, and code is displayed in the S. Typhi challenge participants and 1236 samples were from S. Paratyphi challenge participants  met the diagnostic criteria for typhoid fever [P < .0001 day-by-diagnosis interaction). The odds of shedding 1 day after challenge were 2.5 times greater in those who were later diagnosed than in those who remained undiagnosed . On day 2 odds were 9 times higher in those later diagnosed  and on day 3 were 23 times higher . In the second week after challenge, many diagnosed participants had commenced antibiotics, and shedding ceased. In the undiagnosed participants, increased shedding was observed from day 10 onward, until those participants also received treatment on day 14 . A sensitivity analysis excluding low-dose challenge gave a very similar estimate . The dose received was nonsignificant in the model . In Ty21a vaccine recipients, there was a nonsignificant reduction in shedding compared with unvaccinated controls  and nonsignificantly increased the rate of bacterial shedding compared with previously unexposed individuals . For those receiving S. Paratyphi challenge, there were no differences between groups, and a lower rate of shedding in general was observed in comparison with those exposed to S. Typhi challenge.Participants previously exposed to P = .0187). Age was not significantly related to shedding (P = .795).In 197 unvaccinated participants , the median number of samples provided was 11  for women and 12  for men. Men had proportionately more positive stool samples than women . There was no relationship between anti-LPS IgG or anti-Hd IgG and shedding after challenge with either S. Typhi or S. Paratyphi Typhi bacteremia), but shedding can also occur in the absence of bacteremia or clinical symptoms of disease. Vaccination with Vi-PS or Vi-TT significantly reduced stool shedding of S. Typhi following controlled human infection, suggesting that these vaccines are likely to reduce onward transmission of disease. The decreased shedding following vaccination with the live-attenuated Ty21a vaccine was not significant, possibly due to the small sample sizes available for these comparisons and moderate protective efficacy of Ty21a in the challenge model. In earlier challenge studies with Ty21a involving larger numbers of participants who received a freshly harvested formulation of vaccine, shedding of S. Typhi was significantly reduced in recipients of the live oral vaccine [Shedding of  vaccine .The effects of Vi-PS or Vi-TT vaccination on indirect protection and stool shedding are poorly understood. A cluster randomized control trial in Kolkata, India, demonstrated that Vi-PS vaccination can result in indirect protection against typhoid fever in unvaccinated individuals resident in population clusters randomized to Vi-PS vaccine . HoweverPseudomonas aeruginosa exotoxin A as a carrier protein was shown to have a high vaccine efficacy (VE) in Vietnam  [The reduction in stool shedding observed in individuals vaccinated with a Vi-TT conjugate vaccine is an important finding. To date, there are no completed cluster randomized trials of typhoid conjugate vaccines. However, 1 trial is ongoing in Bangladesh, and individually randomized trials are ongoing in Nepal and Malawi . A previ%–96.5%) . Vi-TT v%–96.5%) , with de%–96.5%)  and vacc%–96.5%) . In OctoS. Typhi is reduced in a manner that is proportional to the vaccine efficacy . The results presented here support such assumptions. The protection afforded by Vi-TT against typhoid infection is similar to its effect against stool shedding. When both are expressed as ORs, the effects of Vi-TT against typhoid diagnosis and shedding have largely overlapping CIs . However, more analysis is needed to examine the relationships between vaccination, stool shedding, and the development of clinical typhoid and how this may vary between the human challenge model and field settings. Additionally, the mechanism by which anti-Vi antibodies prevent stool shedding requires further investigation.Current models that predict the potential impact of Vi-conjugate vaccines account for this potential indirect protection by assuming that transmission of S. Typhi from between day 4 and day 30 post-challenge  [5 organisms; Oxford, 104), and criteria for initiating antibiotics. Of note, Ty21a appeared to provide herd immunity in field studies conducted in Chile [The nonsignificant reductions in shedding with Ty21a vaccine contrast with data from early challenge studies, where Ty21a was associated with a reduction in any stool shedding of Salmonella will act as asymptomatic short-term carriers who transiently shed in the absence of overt clinical disease; this is a factor that should be considered when determining the target population for vaccination campaigns.As was observed in early typhoid challenge studies, early shedding increased the likelihood of subsequent development of typhoid fever . InteresS. Typhi, we detected a significant reduction in the rate of stool shedding compared with naive controls. A single episode of typhoid exposure is thought to confer moderate protection against subsequent clinical disease , and mo studies . These atory IgA .S. Typhi challenge were twice as high as those following S. Paratyphi A challenge. The higher rate of shedding following typhoid challenge may reflect the higher challenge dose administered in the typhoid model (104 vs 103 colony-forming units), or differences may exist between these serovars in host–pathogen interactions and transmission mechanisms. Several studies in areas coendemic for S. Typhi and S. Paratyphi A have suggested that transmission dynamics and risk factors may differ between the 2 serovars [S. Paratyphi A cases appear to be more spatially dispersed than S. Typhi cases in an urban area [When shedding rates were compared between typhoid and paratyphoid challenge studies, rates of shedding following serovars , 33, 34.ban area , 34. Impban area .S. Typhi (Quailes) or S. Paratyphi A (NVGH308) at a single dose, which may not mirror the shedding dynamics of contemporary circulating strains in Asia and Africa, such as the multidrug-resistant associated H58 (genotype 4.3.1) strain of S. Typhi [There are several limitations to assessing stool shedding using data from human challenge studies. The population enrolled comprises adults from a nonendemic country; shedding dynamics may differ in individuals from endemic countries with prior immune priming or in children, who represent the majority of enteric fever cases (but not necessarily the majority of shedders). In this analysis, data were pooled across 6 studies conducted over 6 years. While all samples were processed in the same laboratory using consistent protocols, study-to-study variation may still exist in the sensitivity of stool testing; thus, “study” was adjusted for in all models. Furthermore, challenge was conducted using only a single strain of S. Typhi . These lS. Typhi .In summary, we have performed the first detailed model of shedding dynamics in the context of controlled typhoid and paratyphoid challenge and provide evidence for efficacy of new and existing typhoid vaccines (Vi-PS and Vi-TT) in reducing rates of shedding. These studies illustrate the value of closely monitored experimental human challenge studies in obtaining novel insights into host–pathogen interactions and microbial dynamics, which can directly inform the disease control efforts for priority pathogens.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary MaterialClick here for additional data file.


IFX, anti-drug antibodies and TNF serum concentrations were measured at day 0 (1 h after infusion), 1, 4, 7, 11, 14, 18, 21, 28 and 42. A binding model, TMDD model, and a quasi-steady state (QSS) approximation were evaluated using nonlinear mixed effects modeling (NONMEM). A two-compartment model best described the concentration–time profiles of infliximab. Typical clearance of infliximab was 0.404 L day−1 and increased with the presence of anti-drug antibodies and with lower albumin concentrations. The TMDD-QSS model best described the pharmacokinetic and pharmacodynamics data. Estimate for TNF baseline (Bmax was 19.8 pg mL−1 and the dissociation constant (Kss) was 13.6 nM. This model could eventually be used to investigate the relationship between suppression of TNF and the response to IFX therapy.Ulcerative Colitis (UC) is an inflammatory bowel disease typically affecting the colon. Patients with active UC have elevated tumor necrosis factor (TNF) concentrations in serum and colonic tissue. Infliximab is a monoclonal antibody directed against TNF and binds with high affinity. Target-mediated drug disposition (TMDD) is reported for monoclonal antibodies meaning that their pharmacokinetics are affected by high target affinity. Here, a TMDD model is proposed to describe the interaction between infliximab and TNF in UC patients. Data from 20 patients with moderate to severe UC was used. Patients received standard infliximab induction therapy (5 mg kgThe online version of this article (10.1007/s10928-019-09652-5) contains supplementary material, which is available to authorized users. Ulcerative colitis (UC) is a chronic auto-immune disease of the colonic mucosa . PatientTNF is a pro-inflammatory cytokine and has been found in increased concentrations in blood, stool, and epithelial tissue of UC patients –6. In paTNF is produced as a transmembrane cytokine (mTNF) and released in its soluble form (sTNF) after conversion by TNF converting enzyme (TACE) . InflixiThe pharmacokinetics of IFX in IBD patients have been described extensively –16. NotaMonoclonal antibodies exhibit different pharmacokinetic properties compared to small molecules. Due to their high molecular weight and hydrophilicity, distribution to peripheral tissue is limited. Also, unlike small molecules, monoclonal antibodies are not cleared via the kidneys or liver, but via alternative pathways, primarily via proteolytic catabolism after receptor-mediated endocytosis in the reticuloendothelial system. Monoclonal antibodies are designed for a specific target, which they bind to with high affinity. Target-mediated drug disposition (TMDD) is described for monoclonal antibodies that bind with high affinity to their target, to such an extent that it affects the pharmacokinetics of the drug . At low IFX binds with high affinity to its target sTNF, resulting in the formation of stable IFX-TNF complexes , 19.Based on this mechanism of action of the monoclonal antibody IFX and measured sTNF concentrations, a TMDD model could be used to describe the interaction between IFX and TNF. This would provide more insight into IFX response of IBD patients with regard to not just IFX exposure, but also their TNF concentrations.In this study, we aimed to quantify the binding of IFX to its biological target, TNF, by means of the development of a TMDD model in patients with moderate to severe UC.−1) induction therapy at week 0, 2, and 6. One patient received an additional IFX administration at day 5. Serum IFX concentrations, anti-IFX antibody status, free TNF serum concentrations, C-reactive protein (CRP) and albumin concentrations were collected at day 0 (1 h after infusion), day 1, 4, 7, 11, 14 (before and 1 h after infusion), 18, 21, 28 and 42 (before infusion). Patient characteristics are summarized in Table Data was used from a prospective cohort study with 20 anti-TNF naive patients with moderate to severe UC, as previously reported . Patient−1 with coefficient of variation (CV) of 12%. Free serum TNF concentrations were measured using an ultrasensitive immunoassay  and LoQ for TNF measurements was 10.0 fgs mL−1, with CV of 15% [Serum IFX concentrations and anti-IFX antibodies were measured with a homogenous mobility shift assay (HMSA)  , 21. WitV of 15% , 23.p value of 0.01 was used (OFV decrease > 6.63 points). Continuous covariates were modeled according to the general equation:TV is the typical value of the parameter P in a patient with median covariate value (COV) and θ is the fractional change in P with each unit of deviation from the median covariate. Categorical covariates were modeled according to the general equation:TV is the typical value of the parameter P and θcov is the fractional difference in P between categories.IFX and TNF concentrations were converted to nanomolar (nM) using their molecular weights of 149 kDa (IFX) and 52 kDa (TNF) . A populSeveral structural models were evaluated to describe the data: a binding model, a full TMDD model, and a TMDD model with quasi-steady state (QSS) approximation . The strBmax denotes the baseline serum TNF concentration, Ac represents the total IFX amount in serum, Vc the central volume of distribution of IFX and KD the equilibrium dissociation rate constant.For the binding model, serum concentrations of TNF bound to IFX were expressed in the $ERROR block as follows:ksyn (nM day−1) and degradation of TNF is represented by the first-order rate constant kdeg (day−1). TNF forms a complex with IFX with binding rate constant kon (nM−1 day−1) and the complex dissociates with dissociation rate constant koff (day−1), see Fig. In the TMDD model, synthesis is represented by the zero-order rate constant n nM day− and degrAc) and peripheral IFX (AP) amounts, expressed in nmol, are represented by Eqs. R), and complex (P) concentration over time (t), expressed in nM, are represented by Eqs. ke represents the first-order elimination rate constant of IFX and ke(P) represents the first-order elimination rate constant of the complex. Free IFX is distributed by first-order processes to the peripheral compartment (k12 and k21).In the full TMDD model, central IFX (koff/kon) and internalization rates (ke(P)/kon). In contrast to a quasi-equilibrium (QE) approximation, the QSS approximation does not assume that the rate of elimination of the complex (ke(p)) is negligible. Instead, the dissociation constant (KSS) is expressed as follows:KD denotes the equilibrium dissociation rate constant (koff/kon).A QSS approximation was evaluated to improve parameter estimation in the TMDD model . The maiCtot), free IFX concentration (C), total central IFX amount (Atot) and the peripheral IFX amount (AP), are represented by Eqs. Rtot), is represented by Eq. Total central IFX concentration  and an inability to achieve satisfactory model fit with either of these values, a sensitivity analysis was performed to assess the most likely value at which to fix kdeg to ensure reliable and accurate estimation of all remaining parameters [kdeg. The fixed kdeg values ranged from 0.02 to 40.28 day−1, with a two-fold increase between each value and the next.Due to a large difference reported rameters , 28. TheThe final model was evaluated using a visual predictive check (VPC), using 1000 simulations and the plots were stratified by compartment. Bootstrap analysis (2000 runs) was performed to test the stability and robustness of the final model parameter estimates.Pharmacokinetic/pharmacodynamic modeling was performed using nonlinear mixed effects modeling (NONMEM)  with first-order conditional estimation with interaction (FOCE + I). Pearl-speaks-NONMEM  and R  were used to visualize and evaluate the model outcomes.−1 and increased to 3.5–31 pg mL−1 at day 42 right before the next IFX administration. Antibodies-to-infliximab were detected in 7/20 patients.The dataset included 214 IFX serum concentrations, and 214 TNF serum concentrations from 20 UC patients. Unbound TNF concentrations appeared inversely related to IFX serum concentrations. Directly after IFX infusion, TNF concentrations ranged from 0.27 to 2.1 pg mLKss), the complex internalization constant (ke(P)) and of the degradation constant TNF (kdeg).Supported by the available data and the assumption that degradation of the IFX-TNF complex is not negligible, a TMDD model with QSS approximation best described the interaction between IFX and TNF serum concentrations. The TMDD-QSS approximation allowed the estimation of the steady state equilibrium constant (Kss) was 13.6 nM, ke(P) was estimated to be 0.984 day−1 and kdeg was fixed to 5.12 day−1 based on a sensitivity analysis (see Supplementary Table 1). Median baseline TNF value (Bmax) was estimated to be 0.38 pM, which is equal to 19.8 pg mL−1 and comparable to literature [CL, Vc, Vp, and Bmax. Simulated total TNF concentrations are depicted in Fig. Population steady state dissociation constant  are situated within the associated 90% confidence intervals of the prediction intervals. This indicated a good qualification of the QSS model. Bootstrap results confirmed the validity of the model  was fixed to 5.12 day−1, a biologically plausible value obtained from the sensitivity analysis (Supplementary Table 1).Evaluation of different initial estimates showed that a lower initial estimate for kdeg) and the dissociation constant of the complex of IFX and TNF (Kss).IFX is an anti-TNF agent used in patients with moderate to severe UC. In this present study we analyzed the pharmacokinetics of IFX and TNF within the first 6 weeks after start of IFX therapy in UC patients. IFX binds with high affinity to its target TNF and as a result exhibits TMDD. In this study, application of a TMDD model allowed not only the estimation of the population pharmacokinetic parameters of IFX but also the first-order elimination rate of TNF , changed by the binding of IFX to TNF in patients with CD was previously proposed . More reSeveral structural models were evaluated to describe the pharmacokinetics of IFX and TNF in this population. First, a binding model was evaluated to describe TNF concentration–time curves, in which the binding of TNF to IFX did not influence the pharmacokinetics of the latter. However, when using a binding model to describe pharmacokinetics and dynamics, total receptor (i.e. TNF) concentrations are assumed to be constant over time. As shown in Fig. A full TMDD model was evaluated, but parameter estimates were highly dependent on initial estimates, indicating poor stability of the model and, subsequently, several approximations of the TMDD model were evaluated. Both QE and QSS approximations of the TMDD model assume an equilibrium between free drug/receptor concentrations and formation of the complex. In contrast to the QE approximation, a QSS approximation does not assume the elimination constant of the complex to be negligible. Because of the assumption that IFX-TNF complex is mainly eliminated, rather than dissociated, this approximation was deemed more applicable.Kss resulted in a higher kdeg estimate, and higher initial estimates for Kss resulted in a lower kdeg estimate. To stabilize the QSS model, kdeg was fixed to a value which, in the absence of applicable values in literature, was based on a sensitivity analysis. Estimated Kss (13.6 nM) was higher than binding affinities (KD) reported for IFX and TNF, which range from 0.0273 nM to 1.92 nM [Kss was expected to be higher than KD values reported in literature because Kss represents steady-state equilibrium by taking into account not only the equilibrium constant (KD = koff/kon) but also the elimination of the complex (ke(P)/kon). Moreover, equilibrium constants are difficult to measure and are highly dependent on the type of assay performed. Furthermore, differences may exist between the in vitro and in vivo derived affinity values.Evaluation of different initial estimates showed that a lower initial estimate for  1.92 nM , 31, 32.ke(P)), i.e. 0.984 day−1. Nonetheless, little is known about the fate of the IFX-TNF complex. It is hypothesized that the complex is recycled via the neonatal Fc-receptor (FcRn), as described for free concentrations of monoclonal antibodies. Also, the IFX-TNF complex could be subject to proteolytic degradation.With the use of the QSS approximation, the elimination of the complex was assumed to be non-negligible and the proposed model was able to estimate the degradation constant (As described in Eq. ke(P)) compared to the elimination rate of TNF (kdeg), reflecting a prolonged half-life of TNF due to formation of the complex, which has been previously proposed [TNF is a pro-inflammatory cytokine and after conversion released in its soluble form, i.e. sTNF. We were able to measure free sTNF concentrations in sera of patients. Although measuring free sTNF is challenging, due to the low detection level needed, Song et al. also previously reported measurements of free sTNF in patients with Crohn’s disease and found a similar trend, i.e. decrease of free sTNF concentrations after initiation of anti-TNF therapy .Total TNproposed . Hence, proposed . In addiproposed .First, the study is limited by the relatively small samples size (n = 20), despite rich sampling. The main limitation of our study however is the absence of measured baseline TNF values. Baseline TNF serum concentrations have been found to be higher in responders compared to non-responders before treatment with IFX in patients with fistulizing CD . In addiIn conclusion, we propose a TMDD model with QSS approximation to describe the interaction between IFX and TNF. This model could eventually be used to investigate the relationship between suppression of TNF and the response to IFX therapy.Supplementary material 1 (TXT 1 kb). Supplementary file: Control streamSupplementary material 2 (TIFF 4221 kb). Supplementary Figure 1A: Goodness-of-fit plots infliximab. Goodness-of-fit plots of the final model. The dashed lines represent the line of identitySupplementary material 3 (TIFF 4422 kb). Supplementary Figure 1B: Goodness-of-fit plots TNF. Goodness-of-fit plots of the final model. The dashed lines represent the line of identitySupplementary material 4 (DOCX 19 kb)Below is the link to the electronic supplementary material.