GTFP) is an important measure of a city’s health and green performance, this study exploits the difference-in-differences method to explore the impact of pilot FTZs on urban GTFP in 280 cities in China for the period between 2005 and 2017. The results show that the green areas positively contributed to the growth of GTFP. Moreover, the outcome holds with robustness tests. Statistically, the positive effect emerged in cities during the first three years after introducing the initiative, with the effect disappearing afterward. It also had a strong positive impact in the central and western regions and in large and medium-sized cities, while the influence remained insignificant in the remaining areas in China. Furthermore, the paper also reveals that the promotion of foreign direct investment and industrial structure upgrading are the primary channels through which the positive relationship between pilot FTZs and GTFP is established.Free trade zones (FTZ) are designated areas for promoting trade openness and investment facilitation. In China, FTZs are also regarded as “green areas” in which planning actions and institutional innovations are implemented, and there is a commitment to promoting urban green and healthy development. Given that green total factor productivity ( The Chinese economy has undergone spectacular growth in recent decades, while environmental degradation from traditional extensive economic development is becoming an extremely serious problem. Environmental pollution brings challenges to the sustainable and healthy development of cities . AccordiAccording to Teifenbrun  and AkbaCompared to previous policy zones like SEZs, the opening-up of the service and financial industries has been expanded in pilot FTZs for the first time. The Chinese pilot FTZs have also implemented a series of institutional innovations in addition to possessing functions similar to those of other countries. The institutional innovations mainly include: first, optimizing government functions and improving trade and investment efficiency through one-window service; second, tax holidays and tax regimes such as reduced income tax for some enterprises in pilot FTZs; third, introducing the “Negative List” management model to provide guidance and governs industry sectors in which foreign investment is prohibited or where possible restrictions may apply. “Green development” is a major development idea of Chinese pilot FTZs. As the concepts emphasized in the “Guiding Opinions on Strengthening Ecological Environment Protection in Pilot Free Trade Zones” issued by the Ministry of Ecology and Environment (MEE) of China, the ideas of green development should penetrate to the entire process of pilot FTZs construction, and pilot FTZs must develop a modern green service industry, green manufacturing, green supply chain, and green trade in the future. Some studies also indicated that pilot FTZs make efforts to become green areas in various ways . TherefoNumerous studies have examined the economic effects of the policy zones. The literature has indicated that Chinese SEZs, EPZs, and BZs are all conducive to attracting more investors and promoting trade development ,13,14. TAlthough few studies have attempted to estimate the impact of policy zones on urban green and healthy development, many studies have explored their impact on economic growth. Most extant studies support that SEZs, EPZs, and BZs positively contribute to innovation stimulation and economic growth ,23. HoweAnother emerging stream of literature emphasizes the impact of policy zones on environmental pollution. Some researchers argue that the policy zones promote environmental protection, whereas other researchers find that these zones generate high emissions of metals and other negative products that contaminate the environment ,35. RegaGTFP) has been widely used in the research field of environmental economics to reflect the level of green development [GTFP is a new productivity indicator that comprehensively incorporates resource and environmental constraints. Specifically, the new indicator adds resource consumption as an input factor to the traditional TFP analysis framework and adds environmental pollution emissions as an undesirable output to the input-output efficiency analysis [Based on these two strands of literature, the important question that we focus on is whether the economic growth brought by pilot FTZs is at the expense of the environment or has green characteristics. Currently, green total factor productivity  methods to investigate the impact of pilot FTZs on urban green development, which can address the endogeneity problem, and ensure the reliability of the research conclusions.GTFP is explored in the fifth section. Finally, conclusions are presented in the sixth section.The structure of the rest of paper is as follows. The institutional background and theoretical hypothesis are explained in the second section. The data and methodology are reported in the third section. The empirical results are proposed in the fourth section. Exploring the mediating effect of FTZ policy on Since 1978, China has established SEZs. The zones were designed as major platforms that provided preferential policies such as tax reduction for foreign enterprises. In addition, BZs have been successfully constructed since the 1990s. Goods are exempt from duty in these zones. Further, the Chinese government set up EPZs where goods manufactured for export are exempt from tax.Government’s peculiarities and intervention play an important role in the development of these policy zones ,51,52. IUnder the positive impact and intervention of government, most of special zones in China, though differing in characteristics, are quite successful in FDI introduction and trade prosperity. However, there is still considerable room for China to expand the opening of its service industry and attract advanced foreign investment.To further accelerate the pace of opening to the outside world, on 27 September 2013, the first Pilot FTZ (Shanghai) in mainland China was founded. Up to March 2017, the Chinese government has established a total of 11 pilot FTZs. The policies of pilot FTZs include not just the free entry and exit of goods, zero-tariff policies, and other traditional policies that promote trade liberalization. More importantly, the FTZ policy is China’s first policy to expand the service industry and financial opening after joining the WTO. For example, the “Negative List” management model and “pre-entry national treatment” were adopted in pilot FTZs to achieve expanding openness in the fields of the modern services industry, such as banking, insurance, capital markets, and other fields. On the premise of complying with relevant regulations, qualified foreign-funded institutions and organizations are allowed to establish companies to conduct related business in the form of joint ventures or independent forms, while enjoying various preferential policies. In addition, the convertibility of the RMB capital account, the marketization of interest rates in the financial market, the reform of foreign exchange management, the opening of financial institutions, and the construction of international block trading platforms are all piloted in pilot FTZs. Compared with other special policy zones, FTZs are multifunctional areas that gather multiple innovational advantages, as shown in Different from the FTZs of other countries, pilot FTZs in China have implemented a series of institutional innovations to improve trade efficiency, investment efficiency, and administrative efficiency. Institutional innovation is reflected in the simplified customs clearance procedures for commodities and higher administrative efficiency. For example, pilot FTZs apply the “first entering and then declaring” process and simplify customs clearance documents, and so on. In addition, pilot FTZs have vigorously promoted the reform of e-government, improved the administrative examination and approval efficiency of the enterprises in the zone. Moreover, high-tech enterprises, financial service enterprises, cultural and creative enterprises, and many other industries in the zones can enjoy lower income tax rates. Overseas high-tech talents in pilot FTZs can also enjoy personal income tax relief. These measures have effectively promoted the development of emerging industries and economic growth in pilot FTZs.FTZs are also set as demonstration zones for protecting the environment and achieving high-quality development . “OpinioGTFP is investment facilitation. Yao et al. [GTFP. On the other hand, the inflow of FDI squeezes the market share of domestic enterprises to a certain extent, intensifies industry competition, and creates a selection mechanism for the survival of the fittest. This phenomenon strongly motivates domestic enterprises to carry out technological innovation, thereby promoting the improvement of urban GTFP. Therefore, we put forward the following hypothesis:An important influencing mechanism of pilot FTZs on urban o et al. , Qu et ao et al. , and Tano et al.  found tho et al. . At the o et al. . The twoHypothesis 1 (H1).Pilot FTZs can increase GTFP through the promotion of FDI.GTFP. As an innovation in the trade management system, pilot FTZs play an active role in breaking down trade barriers, overcoming obstacles to factor flow, and making advanced resources flow freely on an international scale. On the one hand, the main policy goal for China to set up pilot FTZs is to accelerate the opening of the service and financial industry, which is beneficial to the upgrading of the industrial structure [GTFP. On the other hand, the trade facilitation brought by pilot FTZs also brings new opportunities for industrial upgrading. The special policies of pilot FTZs can not only simplify international trade procedures and reduce international trade costs but also improve logistics efficiency, which effectively promotes the flow of high-tech production factors [GTFP. Based on the above analysis, we propose hypothesis 2. In addition, the framework of influencing mechanisms of pilot FTZs on urban GTFP is shown in Industrial upgrading is another influencing mechanism of pilot FTZs on urban tructure . Upgraditructure . Therefo factors ,63. Adva factors ,64. In a factors . With thHypothesis 2 (H2).Pilot FTZs can increase GTFP through the promotion of industrial upgrading.GTFP can accurately describe and reflect the development efficiency of urban economies under the constraints of resources and the environment [GTFP.Urban ironment . Under tk-th decision-making unit; λ represents the weight of the decision-making unit; and The traditional data envelopment analysis (DEA) model has advantages in managing the problems of multi-input indices and multi-output indices ,66; thusIn addition, Equation (1) represents the SBM model under the assumption of constant returns to scale (CRS). If the constraint condition The SBM model also has certain shortcomings. For example, its objective function is to minimize the efficiency value, that is, to maximize the inefficiency of input and output. From the perspective of the distance function, the projection point of the evaluated unit is the point on the front surface that is farthest from the evaluated unit. Tone  tried toGTFP in 280 cities of China. Referring to the practice of Chung et al. [GTFP represents the green total factor productivity index. If GTFP > 1, then the GTFP of the decision-making unit increased in the current period, and if GTFP < 1, then the GTFP of the decision-making unit decreased in the current period.The Malmquist index is based on the distance function proposed by Malmquist in 1953 . The indg et al. , the speGTFP in each year but the rate of change of the GTFP relative to the previous year. Therefore, if we set the value of the GTFP in the base year to 1 and then multiply it by the GTFP of subsequent years, we can obtain the GTFP values of 280 sample cities in China from 2005 to 2017.However, the above indicator calculated according to the Malmquist index model is not the GTFP. The Chinese government established the China (Shanghai) Pilot FTZ in September 2013. To date, China has established pilot FTZs in six batches cover 21 provinces or municipalities. Therefore, we regard FTZ policy as a quasi-natural experiment. Tan and Yan [dt represents whether city GTFP is the urban green total factor productivity measured by the super-efficiency SBM model of undesirable outputs and the Malmquist index. X represents a set of control variables, including the level of informatization of the city (Informatization), population density (Pdensity), the development level of the service industry (Service_sec), the level of science and technology (SciTec), and the scale of fixed assets (Fixed_assets). year represents the year fixed effect, μ represents the city fixed effect, and λ represents the random error term.In this paper, we use the DID method to evaluate the influence of FTZ policy on  and Yan  considerGTFP of the experimental group and the GTFP of the control group have the same change trend. Referring to Li et al. [itkD is a dummy variable that represents whether the city approved the establishment of a pilot FTZ. The value of itkD is assigned according to the following rules: is represents the specific year of the establishment of a pilot FTZ. If t − is < −5, then define itkD = 1; otherwise itkD = 0. If t − is = k, then define itkD = 1; otherwise, itkD = 0 . The process of establishing a pilot FTZ is that the local governments submit an application to the State Council of China after completing the preparations and planning plans for pilot FTZs. The State Council then sends staff to the field to conduct research and perform repeated demonstrations and communication changes involving the specific plan. Only after more than a year of revisions and necessary administrative procedures will the State Council officially approve the establishment of a pilot FTZ. In fact, when the pilot FTZ was established, many service industry and high-tech companies had made preparations in advance. Therefore, we set the year before the pilot FTZ was approved as the base year.In addition, the premise of using a DID method is to meet the parallel trend hypothesis; that is, before the impact of the pilot FTZs, the i et al.  and Becki et al. , this paGTFP of 280 cities in China from 2005 to 2017 was estimated through the SBM model and the Malmquist index. The specific input factors, desirable outputs and undesirable outputs are presented in Dependent variable. As described in GTFP is based on the DEA framework. This framework uses the non-radial and non-angle SBM model of undesirable outputs, and measures GTFP by constructing the Malmquist productivity index.Dependent variable. As described in Independent variables. The FTZ policy (dt) is set in the form of a dummy variable. For the city with a pilot FTZ, the pilot year and subsequent years are set to 1, and the other years are set to 0. Up to now, China has established six batches of FTZs. the first batch of FTZs established in 2013 include the Shanghai FTZ, the second batch of FTZs established in 2015 include Guangzhou, Shenzhen, Zhuhai, Tianjin, Fuzhou, and Xiamen FTZs, and the third batch of FTZs established in 2017 includes Shenyang, Yingkou, Dalian, Zhoushan, Kaifeng, Luoyang, Zhengzhou, Yichang, Wuhan, Xiangyang, Chongqing, Chengdu, Luzhou and Xi’an FTZs. After that, the Chinese government successively established the fourth, fifth and sixth batches of free trade zones in different cities. Due to data limitations, this paper regards only the cities with pilot FTZs in the first, second, and third batches as the research objects.Control variables. This paper includes the following control variables: the level of informatization of the city (Informatization), as measured by the number of internet users in the city (by taking the logarithm); population density (Pdensity), measured by the number of people per square kilometer; the development level of the service industry (Service_sec), as measured by the proportion of the added value of the service industry in GDP; the level of science and technology (SciTec), as measured by the amount of urban technology investment (by taking the logarithm); and the scale of fixed assets (Fixed_assets), as measured by the amount of fixed asset investment in the city (by taking the logarithm).The data source is the “China Urban Statistical Yearbook”. The sample of this paper consists of 2005–2017 data on 280 prefecture-level cities in China. The descriptive statistics of each variable are presented in GTFP, we first conducted a parallel trend test based on Equation (4). The changing trend of GTFP before and after the implementation of FTZ policy is shown in GTFP.Before using the DID method to evaluate the impact of FTZ policy on GTFP. The results illustrate that the driving effect of pilot FTZs on GTFP is significant in the first and second years. This indicates that pilot FTZs effectively attracted the agglomeration of environmentally friendly foreign capital in the zones, and supportive policies enabled the development of some high-tech industries and green industries, which, in turn, promoted urban green development. However, we also find that in the third, fourth, and fifth years after the establishment of pilot FTZs, the promotion effect was no longer significant. This may be because imperfect policy guarantee systems, especially the failure to establish an effective intellectual property system, interfered with the positive impact of pilot FTZs on GTFP [GTFP shows an upward trend from the third year. Due to the unavailability of data, we are not able to estimate the long-term effect of pilot FTZs on GTFP. Since the ideas of green development are continuously applied in the pilot FTZs, we can infer that a long-term positive effect will exist. In the future, the governments should continue to adhere to the ideas of green development in the pilot FTZs so that they can effectively drive urban green development. on GTFP . We can GTFP. In column (1), only the dummy variable (dt) is used as the independent variable for the regression analysis, and the estimated coefficient is positive and significant at the 1% level. There are three possible reasons for this result. First, there is the time trend effect. Second, there is selectivity bias; that is, the cities where pilot FTZs are located have a higher GTFP. Third, the FTZ policy significantly improved the GTFP of cities. Column (2) adds time dummies to the control variables. The regression coefficient remains significantly positive, and the regression results show that the urban GTFP level has a trend of increasing year by year excluding the first reason above. Column (3) further controls for city-specific fixed effects, and the regression coefficient is significantly positive at the 1% level. This result shows that after controlling for the differences between the experimental and control groups, the regression coefficient of dt is still significantly positive at the 1% level, which further excludes the second reason and indicates that the pilot FTZ policy has significantly improved GTFP. The control variables are added in column (4), and the results do not change substantially.To minimize the systematic differences between the experimental and control groups as much as possible and to reduce the estimation bias of the DID model, referring to Liu and Zhao , this paGTFP. The estimated results are presented in GTFP is still significantly positive after using the PSM-DID method. The differences in the regression coefficients are not substantial, and the significance is almost the same whether using the DID method or using the PSM-DID method, which verifies that the policy is beneficial in improving GTFP.This section describes use of the PSM-DID method as a robustness test to study the effects of the FTZ policy on The present research contributes to the literature on policy zones that are devoted to promoting FDI, international trade, and the environment. Moreover, on the basis of previous research concerning the effect of pilot FTZs on economic growth ,32 and eGTFP in different regions. Following the research of Gong and Shen [GTFP. The results are shown in Columns 1–3 of GTFP in central and western China, but the impact on cities in eastern China is not significant. The levels of trade openness, industrial development, and technological innovation in the eastern region are inherently high; therefore, the marginal effect of the FTZ policy on GTFP is not significant. Although the development level is lower in the central region than in the eastern region, the central region has relied on the support of national policies to seek opportunities for development in recent years. The pilot FTZ policy can effectively promote trade facilitation and the entry of high-quality foreign capital in the central region, and thus greatly promote the upgrading of the industrial structure and the green innovation of enterprises. As a region with relatively backward economic development in China, the optimization of trade administration and the facilitation of trade and investment in the pilot FTZs in the western region have released a large institutional dividend, which has a significant marginal effect on GTFP.We analyzed the heterogeneity from the aspects of location and city size. There are numerous differences in the level of economic development, marketization, and institutional quality among the various regions in China . As a reand Shen , we diviGTFP heterogeneous. Therefore, we also carried out a subsample regression according to the different sizes of cities. According to the size of the urban population, we divided the sample into large and medium-sized cities and small cities. If the population of a city is more than 500,000, we defined it as a large and medium-sized city, and if the population of a city is less than 500,000, then we defined it as a small city. The regression results are shown in Columns 4 and 5 of GTFP, while in small cities, the policy effect has not been significant. This difference may be because large and medium-sized cities have not only good basic conditions for trade and foreign investment but also a high degree of spatial agglomeration of innovation [In general, compared with smaller cities, larger cities tend to have advantages in industrial structure, resource agglomeration, and scientific and technological development ,86, whicnovation . TherefoGTFP by promoting FDI and industrial upgrading. To test this hypothesis, according to Baron and Kenny [To explain the potential mechanisms of the green economic growth impacts, we believe that pilot FTZs can enhance nd Kenny , we consM represents the mediating variable, which is defined from the perspective of FDI and industrial upgrading. FDI is measured by the amount of FDI in a city. Referring to Gan et al. [In Equations (5)–(7), n et al. , industrGTFP is considered to be the dependent variable, and the pilot FTZs are taken as the core independent variable for the regression. Second, the mediating variables are considered to be the dependent variables, and the pilot FTZs are taken as the independent variable for the regression. Finally, the pilot FTZs variable and mediating variables are both included in the regression model to observe their impacts on urban GTFP. If the coefficients The steps in testing the mediating effect are as follows. First, urban GTFP based on Equation (5), which suggests that pilot FTZs significantly improve GTFP. This result might be because the pilot FTZs expand the scale of FDI and promote industrial structure upgrading, thereby enhancing GTFP. As shown in Column 2 in GTFP by expanding the scale of FDI. By the same logic, the regression results in Columns 1, 4 and 5 indicate that pilot FTZs can enhance GTFP by promoting industrial structure upgrading.Column 1 in Although several studies, such as Jiang et al. , have exTo promote an all-dimensional, multilayered, and wide-ranging opening up to the outside world, China set up pilot FTZs after the establishment of SEZs, EPZs, and BZs. The Chinese pilot FTZs not only facilitate trade and investment liberalization and facilitation through institutional innovation but also take green development as the basic concept in the development of construction plans. For this reason, this research investigates the impact of FTZ policy on the green and healthy development of cities by using the DID and PSM-DID methods. Based on a theoretical analysis, we find that pilot FTZs have strict environmental requirements for foreign investment. In particular, pilot FTZs mainly encourage foreign investment with high-tech, high value-added, and environmentally friendly characteristics, which is beneficial to the green growth of a city. In addition, the industry planning and design sets for pilot FTZs tend to prioritize the development of the high-tech industry, high-end service industry, and financial services industry, which promotes industrial upgrading and further boosts the green development of cities. Consequently, we propose the hypothesis that pilot FTZs promote green development through FDI and industrial upgrading and confirm the mechanism through the mediation model.GTFP of cities, and the results are robust to the estimation conclusion of the PSM-DID. Second, pilot FTZs have a positive impact on urban GTFP by expanding the scale of FDI and promoting industrial structure upgrading. Third, there are heterogeneous effects according to the region and city size. The impact of FTZ policy on GTFP is significant in the central and eastern cities of China but not in the western regions. In addition, pilot FTZs have a positive effect in large- and medium-sized cities, while their effects are not significant in small cities.The evidence indicates that, first, the pilot FTZs have significantly improved the The research conclusions of this article suggest that pilot FTZs carry out trade and investment following internationally accepted green trade rules and commercial and ecological environmental management rules. The concept of green development penetrates the entire process of the pilot FTZs construction. In addition, the pilot FTZs vigorously develop the modern green service industry, green manufacturing, and green supply chain, which greatly promotes the green and healthy development of cities. Indeed, the pilot FTZs aim to develop into green areas, which has become a feature that distinguishes them from Chinese policy zones such as SEZs, EPZs, and BZs and the FTZs in other countries .European countries are at the forefront in the fields of green energy, low-carbon technology, and environmental governance technologies . China uAcademic research in the future should give attention to how the pilot FTZs can adhere to the concept of green development and continue to transform themselves into green areas in the future. Due to data limitations, our research can only examine the short-term effect of FTZs on urban green development. With more data available, future researchers will have the opportunity to explore the long-term effect of the pilot FTZs on urban green development. Moreover, evaluating the development status of green finance, environmentally conscious manufacturing, and environmental supply chains, etc., in FTZs is also an important research topic in the future.


Background. Local anesthetics (LAs) have potent anti-inflammatory properties. Inflammatory down-regulation is crucial in diseases with overactive immune reactions, such as acute respiratory distress syndrome (ARDS) and chronic inflammation. We investigated the influence of four LAs, procaine, lidocaine, mepivacaine, and bupivacaine, on the reduction of tumor necrosis factor-alpha (TNF-α) secretion in lipopolysaccharide (LPS)-activated human leucocytes. Methods. Blood samples of 28 individuals were stimulated with LPS. The reduction of TNF-α production by each of the four LAs added (0.5 mg/mL) was measured and correlated with biometric variables. A response was defined as reduction to <85% of initial levels. Results. All four LAs down-regulated the TNF-α secretion in 44–61%: Bupivacaine (44.4%), lidocaine (61.5%), mepivacaine (44.4%), and procaine . The TNF-α secretion was reduced to 67.4, 68.0, 63.6, and 67.1% of the initial values in responders. The effects in both patients and healthy persons were the same. Interindividual responses to LAs were not correlated with the duration or type of complaints, basal TNF-α serum level, sex, BMI, or age of responders. Conclusions. Four clinically relevant LAs (amid-LA and ester-LA) attenuate the inflammatory response provoked by LPS. They are potential candidates for drug repositioning in treating overactive immune reactions and chronic inflammation. Local anesthetics (LAs) possess anti-inflammatory properties in in vitro and in vivo ,2. LAs cIn vitro LAs show strong immuno-modulatory properties. Lidocaine suppresses the secretion of the cytokine TNF-α  in humanTNF-α is an important factor in inflammation and carcinogenesis. Multiple signaling pathways contribute to the enhanced transcription, translation, and release of TNF-α. Most prominent transcription factors regulating the promotor of TNF-α gene are NF-κB and AP-1, which are downstream targets of the protein kinase family’s MAPK and inhibitor of NF-κB kinase (IKK) ,16,17. TIn oncogenesis, TNF-α increases the expression of intracellular adhesion molecules such as ICAM-1. ICAM-1 is a receptor necessary for leukocyte adhesion and tumor invasion. This receptor assists with tumor extravasation via the binding of neutrophils. TNF-α also activates the src protein tyrosine kinase, a regulator of endothelial permeability. Src is a proto-oncogene regulating cell growth involved in the extravasation of tumor cells. Lidocaine and ropivacaine can decrease src activation and ICAM-1 phosphorylation . In variThe aim of this study was to expand the research investigating the capability of LAs to regulate important pro-inflammatory pathways. Whereas most previous observations were obtained using transformed  cell lines from single donors and animal models, we wanted to provide evidence for the anti-inflammatory activity of LAs in human blood samples as well. We examined various donors, addressing interindividual differences as well as the general conservation of the response. We were able to substantiate the clinical relevance of this concept by the data presented in our investigation. We investigated the widely used LAs lidocaine, procaine, mepivacaine, and bupivacaine for their ability to inhibit the TNF-α production in LPS-stimulated human WBCs, from full blood, especially monocytes. The anti-inflammatory properties of LAs have only been studied in cell lines or animal models so far. We wanted to corroborate such findings in human whole blood samples. We used a well-established assay for analyzing the secretion of TNF-α by WBC, which were stimulated with the TLR4 agonist LPS. In clinical routines, this system is used for detecting individual effects of anti-inflammatory drugs in the respective patient. It is superior to WBC extracts.We hypothesize that different LAs all directly influence the TNF-α production cycle and are therefore candidates for the suppression of overactive inflammation and tumor recurrence as well. Furthermore, we assume that there may be differences in immunoactivity between different LAs, according to their chemical structure, lipophilicity, stereochemistry, clinical effectiveness (duration of anesthesia), and pharmacokinetics. If LAs can regulate pro-inflammatory pathways, they may be candidates for adjuvant therapy regimes in severely ill SARS patients with an overactive immune response, as well as in patients with chronic (silent) inflammation.Data from 10 healthy volunteers were available from the dose-finding pretests. The availability of their biometric data was limited due to data protection rules . They we2 . Eighteen out of 23 consecutive patients who had had therapy-resistant chronic pain or inflammation for ≥6 months were included in this study. The median age of the 18 patients was 61.3 years . The median body mass index (BMI) was 24.3 kg/mPatients had suffered from chronic pain on average for 10.9 ± 7.8 years (range 6.1–29.1 years). They were treated for chronic pain (N = 13), for chronic inflammation (N = 5), e.g., of the skin, joints, visceral organs , and sinusitis. In 14 out of 18 patients, a TNF-α plasma value, a marker for silent inflammation, was available. The average TNF-α value was 6.6 ± 2.3 pg/mL. Twelve of these 14 had normal values, two patients showed a pathologic level > 8.5 pg/mL. The data of all 28 individuals are depicted in We analyzed possible patterns for each of the LA and for each of the blood samples. To illustrate this, we listed the responses per person and per LA in a “heat map”, see Twenty-three out of 28 persons (82.1%) showed an inhibition of the TNF-α secretion by one or more LA. In the blood of the other five persons there was no inhibition at all . The blood samples of 21 persons showed an inhibition by 1–3 LAs, and two persons showed an inhibition by all four LAs.The number of individuals with an inhibition of TNF-α secretion reduced to 85% or more was 12 out of 27 (44.4%) with bupivacaine co-incubation, 16/26 (61.5%) with lidocaine, 12/27 (44.4%) with mepivacaine, and 13/26 (50.0%) with procaine co-incubation .p = 0.63).In these responders, the percentage of inhibition by the four LAs was very similar, ranging from 63.6 ± 17.5% to 68.0 ± 13.5%. There was no significant difference between the percentage of inhibition in responders , as well as type and duration of the patient’s complaints .p = 0.40), type of individual (patient or control), or TNF-α serum levels. However, non-responders were older than responders for procaine (p = 0.013), and the number of patients was higher in responders than in the non-responder group for mepivacaine (p = 0.049).Most biometric and anamnestic data did not differ between non-responders and responders for the four LA. We found no significant influence on the response rates based on an individual’s sex, BMI, duration of complaints  in WBC seems to play an important role in these anti-inflammatory effects . The effThe impact of LAs on the NF-κB and the MAPK pathway was demonstrated with lidocaine, an amide-LA, and cocaine, an ester-LA . In our Little is known about the exact molecular target of LA inhibiting the production of TNF-α in this complex inflammatory cascade. For lidocaine, Lahat and coworkers showed an inhibitory effect on nuclear translocation of NF-κB in human T-cells . In rat An effect of LAs on toll-like receptors on the cell surface (TLR) has been shown . ActivatAbout the same number of individuals had an attenuation of TNF-α excretion for all LAs. We would have expected differences, i.e., between patients and healthy individuals, between amide- and ester-LAs, differences correlated with their pKa, their molecular size, or their duration of anesthetic action. Inflammation triggered by GPCRs is reduced by LAs, and the suggested target in this signal transduction pathway is Gαq ,35. The Measuring TNF-α inhibition has been used by several researchers to evaluate patients’ individual responses to immune-regulatory substances such as mistletoe lectins  or herbaWe observed a reduction in more than 50% of the blood samples for all four LAs, which is a far more dramatic effect than we had expected. All four LAs, therefore, are paramount candidates for all kinds of immune-regulative therapies. The test system reveals itself to be an appropriate test also for LA immune function testing.Individual differences of reactions in the TNF-α assay have been shown for many immune-active substances in this test system . In routIn pre-tests with dilution series, we found no cell toxicity of the test substances (microscopy with trypan blue staining) in a 1:20 dilution and higher. We therefore can exclude cytotoxic effects of LA on the WBC as a possible reason for the TNF-α inhibition.A dilution of 1:20 results in a LA concentration of 0.5 mg/mL in the test sample. This is a concentration similar to local tissue concentrations in in vivo. In therapy with LA , the common LA administration is 100 mg LA, resulting in an estimated local tissue concentration of ~5mg in 10–30 mL tissue, which is in the same range (0.5–0.013 mg/mL) as in the test sample, lasting for approximately 3–10 min. Thus, we can assume the amounts and concentrations of LA used in the tests resemble the clinically relevant doses used in daily practice.The advantage of such an observational study is the relevance of the findings for everyday practice. However, because this is a small pilot study with only 28 individuals, we need to be careful with the conclusions on the individual differences of the four LA tested, as well as on the possible covariates of the inhibition of the TNF-α secretion. The difference between the sample size of patients and healthy test persons may be questionable. We chose a 2:1 sample size model because we expected major effects in the patient group. Surprisingly, this was not the case. The high number of responders in both groups to any of the LAs tested confirms immune-regulating properties of LA, independent of the patient’s health status.Nevertheless, we were able to observe some trends towards a putative influence of covariates. Even if there was no systematic difference between patients and healthy subjects, responders to lidocaine and procaine tended to be younger than non-responders. Conversely, bupivacaine inhibition did not correlate with age, or status, and oddly, mepivacaine showed the opposite effect: Responders were older, and more of them belonged to the patients’ group. Thus, at this time we cannot deduce a correlation of TNF-α inhibition with lifetime in this pilot study. The impact of these covariates on LA’s anti-inflammatory properties may be investigated in further studies based on these findings.To our knowledge, this inhibition of TNF-α secretion by LAs has not yet been shown in human leucocytes from patients; nor have several different LAs been compared for their immune-regulatory capacity in this important inflammatory pathway.Our data reveal several questions concerning the immune-regulative characteristics of LA. If all four LAs of different molecular characteristics show TNF-α reduction, is there a correlation between molecule shape, size, chirality, or length of anesthesia, and the impact on the TLR pathway? If not, can the effects be explained by the universal membrane theory of LAs ? Can we Further studies with specific inhibitors of distinct steps within the MAPK and NF-κB cascades will reveal LA’s site of action see also .This data also provides a rationale for future clinical studies in chronic inflammation, or in conditions with overactive immune responses, such as SARS. The therapeutic effect of LAs in SARS was not the aim of the study. We therefore did not screen for COVID-19 tests, risk factors, or both in our patients. However, LAs as a co-medication in severe cases of ARDS seem to be a reasonable option . ProtectWhy might someone be a responder and why might they not? Why do some have a response to a certain LA and why do others not? As soon as we can describe more precisely which target system, and which of the two main pathways, MAPK or NF-κB, will be influenced by LA, we may be able to better explain why some individuals react to one or the other LA and some don’t. However, using this test system, we can already predict the patient’s individual response to a certain LA. Studies correlating the clinical outcome with TNF-α inhibition will further elucidate if recommendations to use a certain LA for therapeutic means are crucial for the therapeutic success. At the moment, it seems reasonable to use the most reactive LA found in an individual’s TNF-α inhibition test for therapy.We conducted an observational prospective investigation study to assess the inhibition of TNF-α by LAs. Whole blood from 10 healthy volunteers was used for dose-finding studies to determine the outer limits of cytotoxic effects with 1:2, 1:5, 1:10, 1:20, 1:40, 1:80 and 1:160 dilutions of the four LAs. A dilution of 1:20 was found to be non-cytotoxic in all four LAs. From there, this dilution was used in all patient blood specimens. Between April 2018 and March 2019, out of 96 consecutive adult patients with chronic pain of >12 months presenting in an outpatient practice specialized in chronic diseases in Karlsruhe, Germany, 23 patients had therapy-resistant pain: N = 8, vulvodynia; N = 8, chronic pelvic or genital pain; N = 5, musculoskeletal disorders; N = 3, headache/migraine, N = 3 (sum > 100% due to multimorbidity). Five patients suffered from recurrent inflammation. Therapy resistance was defined as no further reduction of pain under multimodal therapy for >6 months. Such patients are routinely assessed in our institution to estimate the effects of immune-active substances on cytokine production. We used four LAs frequently used in daily clinical practice: Procaine, an ester-bound LA frequently used in therapy with LA (TLA) , and theExclusion criteria for usage of patient’s blood specimens in this study were: Acute infectious disorders  (N = 2), current antineoplastic chemotherapy (N = 1) and hyperreactivity to LA (N = 1). LA hyperreactivity was assessed before the beginning of therapy by intracutaneous testing. Furthermore, tests with a minimal basic response of the patient’s blood to LPS stimulation  were excluded (N = 1).Eighteen out of 23 patients (78.3%) could be enrolled in the study and evaluated. The selection of patients is shown in To test the anti-inflammatory potential of the four LAs, their capacity to inhibit the secretion of the pro-inflammatory cytokine TNF-α was measured in in vitro using a solid-phase chemiluminescence assay. We used an established test system for TNF-α stimulation and inhibition ,44,45,46Induction of TNF-α by LPS was set to 100%. Relative inhibition was considered relevant when the levels were reduced to less than 85%. This cut-off was used by other groups ,44,45,46® cell culture medium  and stimulated in a Falcon® non-pyrogenic 48-well plate  with 500 pg/ mL Lipopolysaccharide  and 50 µL saline solution at 37 °C under 5% CO2 atmosphere to obtain the value after LPS stimulation.From each patient, 50 µL heparinized fresh whole blood was diluted with 500 µL RPMIIn this test system, most of the tested substances show cytotoxicity rather than inhibitory effects in concentrations higher than 1:10, see ® 1000 immunoassay system . TNF-α values are given as pg/mL.In four separate samples, 50 µL of the respective LA in a 1:20 dilution was added instead of saline solution, yielding a final LA concentration of 0.5 mg/mL. After four hours of incubation, TNF-α secretion was measured in the five supernatants (after LPS stimulation without and with one of the four LAs). We used the TNF-α CLIA assay on the IMMULITEThe patient’s records and the data from healthy individuals (as far as available) were analyzed for the following data: Age, BMI, type of complaints (chronic pain vs. chronic inflammation), duration of complaints at the time of TNF-α test, intake of corticoids, immune suppressors  or other chemotherapeutic agents. All patients were non-smokers. All test persons agreed with anonymized data storage and evaluation. Statistical analysis was performed using the statistic software R version ≥ 3.5.0 . SuccessDemographic variables were described as frequencies and percentages for categorical variables and as means and standard deviations for continuous variables. Descriptive analysis of the response comprised absolute and relative frequencies of TNF-α inhibition. Results were compared by chi squared tests.p-values < 0.05 were defined as significant.Univariate analysis of possible influence factors for TNF-α inhibition included description by relative frequencies and chi-square tests for categorical variables and mean, standard deviation and Wilcoxon-tests for continuous variables. As this is an exploratory analysis, resulting p-values are to be interpreted in a descriptive sense. This study illustrates the novel anti-inflammatory action on the TLR pathway in human blood samples for different LAs, independent of their chemical structure as amide- or ester-LAs. This finding supports a rationale for co-medication studies in chronic inflammation and in conditions with overactive immune response and tissue damage, such as ARDS. Further studies with larger cohorts can be designed on the base of this pilot study.


Journal of Hand Therapy [We recently completed a systematic scoping review on return to work after hand injury which has been accepted for publication in the  Therapy . We sear Therapy , 3. Our Our experience of identifying relevant articles for the scoping review proved to be time-consuming and frustrating. We identified almost as many appropriate articles through handsearching, as we did from the database searches themselves. For instance, 10 relevant primary articles were identified from the reference lists of the excluded systematic reviews, that we had not already found in our primary searches. Given these challenges, we cannot say with certainty that we located all relevant articles in our area of interest.After completing the review, we attempted to understand why we had had this experience. As the construction of a comprehensive search strategy is integral to the efficient completion of a comprehensive systematic review , 3, the There was little commonality in the keywords, even when papers described the same condition. From the 38 included papers, we identified 135 unique keywords with the most frequent being “work” (used 38 times in 24 papers), “hand” (used 23 times in 17 papers), “return” (used 16 times in 15 papers), and “disability” (used 12 times in 11 papers). We described these keywords diagrammatically (Fig. Our findings highlight opportunities for researchers in hand rehabilitation to collaborate and develop an agreed list of common keywords, that will ensure inclusion of their research in future systematic reviews. Hand rehabilitation, and return to employment, is integral to health , 5 and q


Background: Left bundle branch area pacing (LBBAP) is a novel physiological pacing approach.Objective: To assess learning curve for LBBAP and compare the procedure and fluoroscopy time between LBBAP and right ventricular pacing (RVP).Methods: Consecutive bradycardia patients who underwent LBBAP or RVP were prospectively recruited from June 2018 to June 2020. The procedure and fluoroscopy time for ventricular lead placement, pacing parameters, and periprocedural complications were recorded. Restricted cubic splines were used to fit learning curves for LBBAP.Results: Left bundle branch area pacing was successful in 376 of 406 (92.6%) patients while 313 patients received RVP. Learning curve for LBBAP illustrated initial (1–50 cases), improved (51–150 cases), and stable stages (151–406 cases) with gradually increased success rates , steeply decreased median procedure  and fluoroscopy time , and shortened stimulus to left ventricular activation time . LBBAP at the stable stage showed longer but close median procedure  and fluoroscopy time  compared with RVP.Conclusion: The procedure and fluoroscopy time of LBBAP could be reduced significantly with increasing procedure volume and close to that of RVP for an experienced operator. Traditional right ventricular pacing (RVP) has been extensively used in clinical practice for more than 50 years. However, RV apex pacing (RVAP) can produce a deleterious effect on cardiac function and consequently increase the risk of heart failure and atrial fibrillation, especially in patients with a high burden of ventricular pacing . Pacing His bundle pacing (HBP) has been the most physiological pacing modality since 2000 . HoweverWe prospectively enrolled consecutive patients who attempted LBBAP or RVP procedures in our working group at Fuwai Hospital from June 2018 to June 2020. All patients had symptomatic bradycardia and were indicated for pacemaker implantation according to the current American College of Cardiology/American Heart Association/Heart Rhythm Society guidelines . PatientAll procedures were performed under local anesthesia. Preventive antibiotics were administered intravenously half an hour before the procedure. Venous access was usually obtained via the left axillary vein, sometimes via the right axillary vein due to various reasons.LBBAP: All LBBAP procedures were performed by an experienced operator in RVP and HBP. Our previous study has described the LBBAP procedure by using the single-lead technique in the initial stage  were applied to confirm the RV lead position.Baseline clinical data were collected, such as demographic characteristics, medical histories, pacing indications, ECG, and echocardiographic evaluation parameters. For LBBAP, the procedure and fluoroscopy time counting began when the C315 HIS sheath was advanced and ended when the LBBAP or LVSP was achieved. For RVP, the procedure and fluoroscopy time were defined as the duration from the beginning of delivery sheath to the end of successful placement of the ventricular lead. Pacing parameters  were recorded. ECG parameters were measured at a sweep speed of 100 mm/s on electrophysiology recording systems, such as LBB potential to ventricle interval , Sti-LVAT, pQRSd, QRS axis deviation, and QRS transition zone. Periprocedural complications were documented, such as lead dislodgement and revision, lead perforation, pacing system infection, and other device-related complications.Depending on the intrinsic atrioventricular (AV) conduction interval and conduction system disease, individualized AV delay was programmed. Automatic AV search algorithm was routinely turned on in patients with intact AV conduction to avoid unnecessary ventricular pacing.t-test or analysis of variance or the Kruskal–Wallis H test. Categorical variables are expressed as frequency or percentage and compared using chi-square or Fisher exact test. We used restricted cubic splines (RCS) with four knots at the 5th, 35th, 65th, and 95th centiles to flexibly model and visualize the correlations between procedure and fluoroscopy time and numbers of procedures. Based on the learning curve, all LBBAP procedures were divided into three groups: initial (1–50 cases), improved (51–150 cases), and stable stages (151–406 cases). A two-tailed P < 0.05 was considered statistically significant. Statistical analysis was performed using R version 3.5.1 .Continuous variables are presented as mean ± SD or median with interquartile range according to the normal distribution of data. The means or medians are compared using the Student's A total of 406 patients who received LBBAP procedures during the study period were included. The mean age was 64.9 ± 14.3 years old, and male patients accounted for 48.5%. Indications for pacemaker implantation included sinus node dysfunction (SND) in 39.7% of patients and AVB in 60.3% of patients. Baseline Baseline left or right bundle branch block (LBBB or RBBB) was present in 10.5 and 23.4% of patients. Other baseline clinical features are summarized in P for nonlinearity < 0.001). The steepest part of learning curves appeared to be over the first 50 cases. The procedure and fluoroscopy time were improved over the following 100 cases (from 51 to 150) and stabilized after 150 cases (from 151 to 406).P for nonlinearity = 0.003).The changing trends of Sti-LVAT at the high output are shown in n = 50, procedure 1–50), improved stage , and stable stage . As shown in P = 0.106). The mean Sti-LVAT at the stable stage was the shortest at the high output (71.2 ± 11.6 ms) and low output (74.3 ± 16.2 ms). The mean LBB capture threshold and the mean pQRSd did not differ among the three stages (P > 0.05). The mean numbers of attempts were significantly different among the three stages . With accumulated experience, we attempted once to achieve successful LBBAP in 80% of cases in the stable stage, twice in 10% of cases, and three times or more in the rest of cases. The median procedure  and fluoroscopy time  rapidly decreased from initial to stable stages.Based on learning curves of LBBAP, three step-by-step stages were identified: initial stage . One ventricular septal perforation and one lead dislodgement occurred soon after the LBBAP procedure at the initial stage. Both patients patients had no symptoms except for having features of pacing failure. Two lead dislodgements happened in the RVP group. All lead revisions were successful without further clinical symptoms or signs.A total of 313 patients received RVP during the same period. LBBAP was more frequently performed in patients with AVB  and fluoroscopy time . However, RVP produced significantly wider pQRSd compared with that in three stages of successful LBBAP . Similar pacing parameters were observed between LBBAP at three stages and RVP ( and RVP .This single-center study firstly demonstrated learning curves of LBBAP indicated by procedure duration and fluoroscopy time and made a comparison between LBBAP at three stages and RVP. The main findings of our study are as follows: (1) for operators who are adept at pacemaker implantation, the steepest part of learning curves for LBBAP was over the first 50 cases. The procedure and fluoroscopy time could be improved further over the following 100 cases and plateaued after 150 cases; (2) using the currently available implantation tools, the success rate of LBBAP could be 94.5% or more at the stable stage, and the fluoroscopy time for the ventricular lead placement could be as short as 4.0 min; (3) although LBBAP presented statistically significantly longer procedure and fluoroscopy time than RVP did, the absolute values were very close to that of RVP. Considering the advantages of LBBAP compared with RVP, our results indicate that LBBAP might be considered as the first choice in patients with a high burden of ventricular pacing to avoid the potential risk of cardiac dysfunction induced by RVP.Left bundle branch area pacing has been recently used as an alternative physiological pacing modality to HBP in some centers. Previous studies have reported fluctuated procedure and fluoroscopy time at different performing phases , 18–20. Different techniques applied to achieve LBBAP could also affect the procedure time. Huang et al. reported the “dual lead technique” with a mean fluoroscopy time ranging from 5.1 ± 4.6 min to 6.9 ± 2.5 min , which wLeft bundle branch area pacing results in rapid electrical propagation along the conduction system fibers and presents the shortest and constant Sti-LVAT at high and low outputs . In our The learning curve for LBBAP in our study was fitted based on performance data of one operator because this novel pacing modality has not been widely extended to many doctors in our hospital. The operator performing the LBBAP procedure in our study had implanted more than 600 active-fixation ventricular leads and nearly 60 successful HBP leads. The procedure and fluoroscopy time could be greatly influenced by the experience of the operator. We speculated that operators with high volume experience of HBP might be more skilled in performing LBBAP with shorter procedure and fluoroscopy time when compared with beginners without experience of HBP. The learning curve might be steeper for beginners, but smoother for an experienced operator in HBP. The significant decrease in LBBAP attempts in our study also supported the close association between learning curves and accumulated experience of the operator. Our results may provide novel insights into the routine application of LBBAP for bradycardia patients requiring ventricular pacing.Despite the potential risk of developing cardiac dysfunction, traditional RVP is still the most widely used pacing technique . In our Several limitations should be noted. Firstly, different techniques may present various learning curves. Our study described the learning curve of an operator by using the single lead technique for LBBAP. The implant technique was slightly changed in our study with performing His potential mapping in the first less than 20 patients early in the initial stage. However, it should be a neglectable bias because all the rest of the patients underwent the same procedure without His mapping. The improved procedure and fluoroscopy time, illustrated by the fitted learning curve, were associated with accumulated procedure experience instead of changing implant techniques over time. Besides, the learning curve for LBBAP in our study was fitted based on the data of one operator. Because the main point of our study was to explore whether the procedure and fluoroscopy time of LBBAP could be compared with that of RVP or not, the data from an experienced operator in LBBAP should be more convincing than data from several beginners. Furthermore, the number of LBBAP attempts, a sensitive indicator of the experience of operators, decreased significantly over time in our study. The procedure time decreased significantly accompanied by fewer attempts. Finally, safety is also a critical concern for a new technique. The complication events in the LBBAP group were low and only two lead-related complications occurred at the initial stage in the present study. A large sample and multicenter study might be needed to investigate the change of the complication rates in different stages of LBBAP.Procedure and fluoroscopy time of LBBAP could be reduced rapidly after 50 cases and plateaued over 150 procedures, while the Sti-LVAT could be shortened further until reaching a plateau after approximately 150 procedures. Compared with RVP, LBBAP can produce a narrower pQRSd and comparable pacing parameters with acceptable procedure and fluoroscopy time.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Fuwai Hospital, National Center for Cardiovascular Diseases. The patients/participants provided their written informed consent to participate in this study.ZW and HZ contributed to design of the study and performed the statistical analysis. XL organized the database. HZ wrote the first draft of the manuscript. XF wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This work was supported by grants (grant no. 81970284) from China's National Natural Science Foundation to XF.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The first basic purpose was to organize and enhance the vast knowledge developments generated hitherto by the first pandemic that was studied “on-the-fly” all over the world. The consequent target was to investigate to what extent researchers in geography were able to draw scientifically consistent conclusions about the pandemic evolution, as well as whether wider generalizations could be reasonably claimed. This implied an analysis and a comparison of their findings. Finally, we tested what geographic approaches can say about the pandemic and whether a reliable spatial analysis routine for mapping infectious diseases could be extrapolated. We selected papers proposed for publication during 2020 and 209 articles complied with our parameters of query. The articles were divided in seven categories to enhance existing commonalities. In some cases, converging conclusions were extracted, and generalizations were derived. In other cases, contrasting or inconsistent findings were found, and possible explanations were provided. From the results of our survey, we extrapolated a routine for the production of epidemiological geography analyses, we highlighted the different steps of investigation that were attained, and we underlined the most critical nodes of the methodology. Our findings may help to point out what are the most critical conceptual challenges of epidemiological mapping, and where it might improve to engender informed conclusions and aware outcomes.The present work aims to give an overview on the international scientific papers related to the territorial spreading of SARS-CoV-2, with a specific focus upon applied quantitative geography and territorial analysis, to define a general structure for epidemiological geography research. The target publications were based on GIS spatial analysis, both in the sense of topological analysis and descriptive statistics or  Pandemics are multifactorial events with complex origin Che, , and manDuring 2020, the researchers had to set out the systematic analysis of a partially unknown phenomenon while it was and is still (quickly) evolving day by day. In hindsight, the geographic research carried out during the first year of the pandemic developed within two different contexts. The first pulse of the pandemic, approximatively identifiable with the “2020 spring wave” (from the point of view of the temperate latitudes of northern hemisphere) forced the researchers to react to an almost unknown emergency that was spreading at a fast pace across mankind. During this early stage, the virus was substantially able to propagate freely in a business-as-usual scenario. Accordingly, geographers had the opportunity to sketch out an inventory of the epidemiological dynamics within several human networks. GIS became a “radioactive tracer” potentially able to spot the “basin outlets” through which the infection was exported from one community to another, thus giving a bird’s eye view on what was happening in each territorial subset of mankind. During the second stage, also mediatically known as “second wave” (2020 autumn pulse), the context radically changed. After the failure of containment measures, mitigation interventions were massively implemented, together with an increased preparedness of the medical staff, and the virus propagation restarted its exponential behavior from endemic sources, spreading not in the absence of countermeasures, but notwithstanding them. Hence, geographers were supposed to partially readjust their perspective and to confront local variances in the pandemic propagation, whose anisotropy was deemed to mirror—more or less faithfully—unconformities in the anthropic fabric of human collectivities. Our approach was, therefore, to identify the most developed investigation pathways that epidemiological geography began to tread in the quantitative study of the COVID-19 pandemic. Clearly, such a work could not be in any case considered exhaustive. The narrower purpose was to gather a selection of sources and offer an organic summary apt to build further research perspectives. The second scope was to discover to what extent geographers and researchers were able to find and explain recognizable patterns in the pandemic spreading, and to check if any shared commonalities could be excerpted. The third objective was to look for the existence of a routine in epidemiological geography able to produce reliable scientific information, and if so, to understand which methodology could be derived. Overall, this survey provided a hint about the urgency of an increased methodological awareness while addressing the geography of infectious diseases, because we deem that GIS could be a pivotal element in this subject )] AND [#COVID OR #Coronavirus OR #SARS-COV-2]”, to investigate six search engines  and the periodical reports issued by the Institute of Development Studies (K4D COVID-19). Finally, the sole two content reviews found hitherto  or APP (Advanced Preprint Publication) or PP (Pre-Print) in the table. Likewise, some Working Papers (WP) were included. The papers were assessed by considering their study area, their scale ratio, the prevailing type of data, the methodology—divided, wherever possible, in general statistic methods and more specifically geostatistical or GIS methods—and their findings. Through this routine we sifted, screened and classified the geographic analyses with the scope to identify common research patterns, shared methods, generalizable findings and cross-cutting biases.To explore and sift the highest possible number of spatial correlation tests and pandemic pattern studies, an iterative “browsing and berrypicking” process was carried out, as suggested by a classic online exploratory research approach Bates, . A seriehttps://doi.org/10.5281/zenodo.4685964. According to the above-mentioned criteria, 209 papers were selected to undergo the survey. The trend in absolute number of spatial analyses devoted to COVID-19 geographical studies is shown in Fig. The results are available at Among the 209 output papers that complied with the selection criteria, 154 papers (≈74%) relied upon daily and/or cumulative cases for their conclusions, 48 (≈23%) extended their analysis to deceases, 8 (≈4%) to recovered, 7 (≈3%) to hospitalized, 7 (≈3%) to performed tests. In 10 cases (≈5%) the authors had to rely upon suspected cases. Only 3 (≈1%) papers attempted to make comparisons with 2003 SARS-CoV-1 confirmed cases. 10 papers (≈5%) utilized symptom-related data, and 8 (≈4%) dealt with the disaggregation of local infections and imported cases. Albeit being one of the most precious datasets, the usage of the latter was significantly lower than expected, probably because of the meteoric fading of local health authorities’ ability to keep recording this differentiation within the frantic course of the first wave of infections, and maybe, we suppose, also due to the early decision of almost all countries to give up the containment and focus only on mitigation , 74 (≈35%) papers were presented in the form of spatiotemporal analyses, while 38 (≈18%) analyzed the socioeconomic context of the pandemic. 23 papers (≈11%) considered preexisting health factors, and 14 (≈7%) the demographic structure. Environmental factors were studied by 43 papers (≈21%), while pollution by 17 papers (≈8%). Mobility was the main subject of 38 papers (≈18%). 27 out of 209 papers were included in more than one category (≈13%). A specific cross-cutting analysis is provided for each category in Paragraph 3.2, to understand the stronger or weaker explanatory efficacy reached within each subject, without detracting from the assumption that in an idiographic discipline it is usually inadvisable to generalize single case studies.Data availability and their level of disaggregation may also explain the peculiar distribution of the scale ratio chosen for the analyses Fig. . 11 studThe most studied countries Fig. a were ChAs for the methodology Fig. a, the stFurthermore, some recurring geostatistical methods were detected Fig. b: 25 paplato sensu comparable:As for the findings of the surveyed papers, we grouped them in seven thematic categories, so that their contents could be 3.2.1 Spatiotemporal analyses3.2.2 Studies on socioeconomic cofactors3.2.3 Studies on health cofactors3.2.4 Studies on demographic cofactors3.2.5 Studies on environmental and climatic cofactors3.2.6 Studies on pollution3.2.7 Mobility analysesThis category encompasses the papers that primarily focused upon the very geographical characteristic of the pandemic, i.e., its territorial propagation with the passing of time. Spatiotemporal analyses were the most frequent and multidisciplinary contributions. They tried to answer a simple but paramount question: “what did happen within a specific area?”. Research strategies were quite diverse, but all built on the widely verified assumption that pandemic data showed strong spatial autocorrelation  found that confirmed cases were spatially autocorrelated; 5 (≈7%) found that the local propagation rate of the pandemic was positively correlated with the proximity to a known outbreak; 5 (≈7%) found that the insurgence of secondary outbreaks was positively correlated to the proximity to a primary outbreak; 9 (≈12%) found that the insurgence of secondary outbreaks was positively correlated to the level of connectedness between the area of insurgence and the area affected by a primary outbreak; 12 (16%) observed that the mitigation measures had a measurable impact upon the spreading of the virus. 10 (14%) observed that at the local level, the pandemic attacked a given society starting from the wealthier neighborhoods and then propagated across the poorer areas; 5 (≈7%) noticed that the apparent primary hotspot in a given area was not the very first outbreak of the given community; 5 (≈7%) observed that, at any scale ratio, the spreading of the pandemic was sustained by the occurrence of recognizable superspreading events.Altogether, 11 papers (≈15%) were able to deal with at least some medically ascertained epidemiological links. 13 papers (≈18%) directly engaged the time dependency of the COVID-19 dynamics. 9 papers produced prospective scenarios or simulations, and 26 papers (≈35%) carried on the primary mapping of the epidemiological situation of a given area for the first time.The very first spatiotemporal studies tried to extract new information about the extent of the pandemic from geographical patterns. Many authors noticed that, during the early stage of the pandemic, in each Chinese province the confirmed case number was positively correlated to the intensity of travelers’ flows incoming from Wuhan or Hubei  had a specific a priori approach and produced one or more vulnerability maps or risk maps taking into account many of the variables that are going be mentioned below. Among the a posteriori approaches, 12 papers (≈32%) found relevant association between the pandemic spreading and population density, and 4 papers (≈10%) found relevant association between the pandemic spreading and absolute population entity. 13 papers (≈34%) found relevant association between COVID-19 incidence and household financial status. In particular, deprivation (4 papers), income inequality (5 papers) and income/GDP per capita (6 papers) were found to be associated to the pandemic spreading. 5 papers (≈13%) found relevant association with the usage of private/public transport, and 5 papers (≈13%) found relevant association with the urban/rural classification of the given administrative units. 6 papers (≈16%) found relevant association with local job market features  and 3 papers (≈8%) with the level of education. 7 other papers (≈18%) found that social connectedness and social interaction were also significantly associated with the propagation of the pandemic, and 3 papers (≈8%) that the family structure and the typology of dwelling had significant association with the infections. More widely, 6 papers (≈16%) found that the granular features of the built environment  were statistically associated to the pandemic evolution across urban populations.As expected, population density was the main investigated covariate, and it gave the strongest signal of statistical association with the pandemic spreading. Human community uneven distribution across the countries can therefore be claimed as the most general and immediate anisotropic factor regarding the asymmetrical territorial propagation of COVID-19. Other relevant covariates may follow. First, socioeconomic indicators like income, poverty, deprivation—when related to financial status at the household scale—were often found to be correlated to the pandemic dynamics. These indicators, however, did not manifest unidirectional forms of covariance: the wealthier or more deprived condition of a given urban area resulted, from time to time, in a hindering or boosting cofactor. Admittedly, the inequality—and not the absolute degree—in economic wellbeing across the communities was a good predictor of pandemic outcomes at the local level, as it can emerge within a comparative overview, and as it is indeed a secondary proxy of the primary epidemiological determinant related to the “host’s condition”. Secondly, another relevant covariate was traced in the “built environment”, a wide definition through which the researchers tried to find a connection between the physical structure of highly urbanized communities and the COVID-19 human-to-human transmission. In this case, again, the location, travel-time, density of facilities like shops, supermarkets, public transports were used as a secondary proxy for the classic epidemiological determinant related to the “host’s behavior” but also to the “host’s environment”, as the built environment tacitly determines humans’ displacements, points of gathering and occasions of diminishing the interpersonal distance below the threshold apt for an effective airborne viral transmission. It is obviously a “statistical space” where interactions are multiple and non-linear and where many uncontrollable variables have to be parametrized, but it did not prevent some researchers to draft risk maps  addressed the study of the influence of pre-existing chronic diseases on the outcome of the pandemic propagation; 3 out of them found relevant spatial association between chronic disease clusters and COVID-19 clusters. Other 5 utilized the pre-existing chronic disease patterns to extract vulnerability maps and vulnerability indexes. The most investigated chronic diseases were diabetes (6), heart diseases (5), hypertension (3), asthma (3), obesity (3); also, incidence rates of pneumonia, cancer, suicide, overdose, disability, depression, smokers, mental illness, and HIV were taken in consideration for correlation tests with COVID-19 incidence rate and above all with case-fatality rate.It may be surprising that the spatial correlation between chronic conditions was investigated (and found) in quite few cases, since the correlative nexus between the two variables was soon robustly ascertained at the individual level  addressed the risk of sanitary resource scarcity, including medical personnel and nurses. The threat of hospital saturation was the core of 6 analyses (≈26%), which intended to offer practical indications about the most fragile sanitary districts and the geographical areas which were more prone to health system collapse.The pandemic propagation was supposed to be strongly correlated with the population composition  is the most common age-specific indicator disaggregated by cohort  environmental variables could condition the virus transmission, and whether geographical patterns could be found and explained.Within the medical community, the cyclization of the SARS-CoV-2 aggressiveness was strongly expected across all the communities in the temperate latitudes , where, in the last week of March, apparent CFR reached 12%  in Western Europe.This interpretation encountered some perplexities because other almost coeval studies  and PM10 (3), but also NO2, SO2 and O3 were considered for association.In medical literature, pollution is a notorious factor of prolonged inflammation and impairment of the respiratory tract , the atmospheric concentration visibly dropped during national lockdowns  of the 38 mobility analyses worked on international flight fluxes to evaluate quantitatively the risk of importation of confirmed cases from abroad. 5 papers (≈13%) relied upon the analysis of visits/contacts with the primary outbreak of Wuhan finding positive correlation between the number of these connections and the secondary outbreak entity. 4 papers (≈11%) mapped the possible role of local transport infrastructures  in fostering the pandemic propagation. 6 papers (≈16%) considered contact-tracing measures at the individual level. More widely, 17 papers (≈45%) adopted data mining on big data repositories to detect aggregate mobility flows. 10 out of them extracted their data from social networks or data aggregators . Travel bans were the focus of 14 papers (≈37%), while, in general, 9 papers (≈24%) adopted a “scenario” study design to carry out prospective analyses and forecasting.Mobility studies in China focused upon the massive relocations that took place in connection with the Lunar New Year holydays. The recurring methodology was based on the analysis of the mobile phone big data , and their combination with transportation companies’ data. Zhou et al.,  found thcordon sanitaire was settled, this correlation faded, and new locally expressed factors took over  to occur . This is the case of spatiotemporal analyses: simple synchronic or diachronic maps were obtained, but they proved to be quite effective in generalizing (see paragraph 3.2.1). For these results, vector analysis in GIS environment appeared to be the best and the most immediate methodology  used by the researchers to produce the primary explorative mapping of affected territories.Secondarily, in most cases a geography-based method is applied with the general purpose to produce a map or a spatially-based statistic output and the specific purpose to generate new information. A first severe hindrance occurs: data are tendentially scarce, and the very first choice is to rely solely upon “Spatial autocorrelation of epidemiological data can inform more conclusions, but researchers are required to rely on strong and updated knowledge of the territory to draw out more well-grounded observations about the pandemic behavior. In this case some well-known geostatistical algorithms were massively adopted  and hotspot analysis was successfully addressed to detect and hierarchize outbreaks , following the traditional epidemiological theory, the researchers address the Host Determinant in term of population entity, age, and distribution. Health covariates (paragraph 3.2.3) are less easily available, but they can manifest strong relations: through them, the researchers, again, address the Host Determinant (in term of organism susceptibility to the infection or in term of probability to receive adequate treatment). Socioeconomical covariates are, instead, utterly uneven. With them, the Host Determinant is addressed more ambiguously . Covariates can be spatially managed with spatial bivariate or multivariate correlation; in this case, besides the traditional Pearson, Kendall, Spearman and other tests, also Anselin’s LISA method, geographically weighted regressions, and Getis-Ord statistics were frequently applied. Environmental covariates are instead more ephemeral, and their spatial analysis gave more erratic results (paragraph 3.2.5). They are used to address both the Environment and the Pathogen Determinant. Anyway, for a new disease the second is originally unknown, and only the first one can be parametrized, but it is made of territorially continuous variables not inherently attached to the host. The contribute of temperature, humidity, precipitation, and climate to the spreading can be excessively smoothed respect to other stronger host-related cofactors. Moreover, in many cases, the climatic spatial analyses, hurried by the emergency, were forced to define “static” study designs and drafted only partial snapshots of the epidemiological situation, biased by the fact that a 100% susceptible host population acted as the main driver of the pandemic  or the “metonymic” suspected covariate  into the “vector point” epidemiological variable  to have a better counterproof about the spatial covariance.Ecological regression methods could represent another efficient escape rope in epidemiological geography, as suggested by Wu et al. , becauseThat finally leads to the open question about the best method to spatially shape the COVID-19 dynamics. This question, indeed, is feeding an ongoing vigorous debate in biogeography. As SARS-CoV-2 is a new species of virus, some ecologists attempted to define its most probable areal of expansion through the species distribution models, which implies to apply the ecological regression on environmental variables  eight points routine for a “COVID-19 epidemiological-geographical study” could be drawn out from the surveyed manuscripts Fig. .In everystricto sensu, as well as to implement tools to control type I error inflation. Through a proper study design, epidemiological geography is asked to engage with a permanent and critical confrontation with cohort  and socioeconomical covariates appeared to be better to be managed, and their analysis produced consistent results, with some possibilities of generalization. Common spatiotemporal trends were that the pandemic had a first rapid stage, in which, by aggressing a given community as an exogenous disturbance, it attacked wealthy classes and areas, verisimilarly because of their higher global interconnectedness with foreign sources . In a second stage the pandemic became epidemic and took advantage of preexisting weakness lines in human societies. Everywhere an abrupt shift of epicenters towards the most fragile and deprived social classes was noticed. The latter were, in the end, far more exposed to infections and suffered from their smaller material chances to exert self-protection . Therefore, the first signs of endemism were given by the rooting of long-period outbreaks within crowded and deprived urban areas (Bermudi et al., With this survey we hopefully offered a more painstaking grounding to the systematization of the geographical analyses of the pandemic, both by letting a possible study design routine emerge from the magmatic scientific production of 2020, and by emphasizing the major known weak points that geographers encountered in their task. A limit of this study is that the chosen thematic categories might be questionable. A future effort should be to engage the systematic review of each category. The sole fact that the pandemic is still ongoing, at the time we are writing, urges to perform a radical update of our findings as soon as possible. No language filters were applied on the paper retrieval, but a strong bias may be derived using keywords in English. The number of papers circulating in the second half of 2020 was likely to be underestimated, because paper indexing in search engines may require long time to become effective. Finally, it cannot be excluded that the number of papers not finding any relevant correlation among the covariates was underestimated because of the “publication bias” (Gutiérrez-Hernández,


C25H promoter following TNF stimulation. Furthermore, loss of C25H, FAK and α5 integrin expression and inhibition of FAK and α5β1 integrin with inhibitor and blocking antibody, respectively, led to diminished TNF-mediated pro-inflammatory response. Thus, our studies show extracellular 25HC linking TNF pathway with integrin-FAK signaling for optimal pro-inflammatory activity and MAPK/NFκB-C25H-25HC-integrin-FAK signaling network playing an essential role to amplify TNF dependent pro-inflammatory response. Thus, we have identified 25HC as the key factor involved in FAK activation during TNF mediated response and further demonstrated a role of cell surface integrins in positively regulating TNF dependent pro-inflammatory response.Exaggerated inflammatory response results in pathogenesis of various inflammatory diseases. Tumor Necrosis Factor-alpha (TNF) is a multi-functional pro-inflammatory cytokine regulating a wide spectrum of physiological, biological, and cellular processes. TNF induces Focal Adhesion Kinase (FAK) for various activities including induction of pro-inflammatory response. The mechanism of FAK activation by TNF is unknown and the involvement of cell surface integrins in modulating TNF response has not been determined. In the current study, we have identified an oxysterol 25-hydroxycholesterol (25HC) as a soluble extracellular lipid amplifying TNF mediated innate immune pro-inflammatory response. Our results demonstrated that 25HC-integrin-FAK pathway amplifies and optimizes TNF-mediated pro-inflammatory response. 25HC generating enzyme cholesterol 25-hydroxylase (C25H) was induced by TNF via NFκB and MAPK pathways. Specifically, chromatin immunoprecipitation assay identified binding of AP-1 (Activator Protein-1) transcription factor ATF2 (Activating Transcription Factor 2) to the  TNFR1) and TNFR2 on the plasma membrane, which results in recruitment of multiple signal transducing adaptors that activate transcription factors AP-1 (Activator Protein-1) and NFκB  or MAPK inhibitors . These cells were then treated with TNF in presence and absence of inhibitors. To evaluate STAT1 activation by IFN-β in the presence and absence of IFNR1 blocking antibody, IFN-β protein was incubated with either control IgG or IFNR1 blocking antibody at 4°C for 1h. Following incubation, they were added to MEFs for 30mins, and the collected cell lysate was subjected to western blot analysis with phospho-STAT1 antibody. For IFNR1 blocking antibody experiments, cells were pre-treated with control IgG or IFNR1 antibody for 2h and subsequently treated with TNF in presence of control IgG or IFNR1 antibody.Medium supernatants from TNF treated cells were analyzed for 25HC, CCL3 and IL-6 levels using specific ELISA kits as per manufacturer’s instructions. ELISA values of the experimental group  represent values obtained following subtraction of background signal measured in the control group .™ XRS (BioRad) and quantified using ChemiDoc™ XRS + software Image Lab 5.1 (BioRad).Cells were washed with DPBS and lysed  for 20-30mins. Cell lysates were subjected to western blot analysis. Western blot bands were visualized using ChemiDocTotal RNA was extracted using TriZOL Reagent (Invitrogen). Isolated RNA was treated with RNase-free DNase I (Thermoscientific) and cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-qPCR was performed using Bio-Rad CFX Manager with 1X SSO Advanced Universal SYBR Green supermix (BioRad) using gene specific primers. Target genes were normalized to the house keeping gene GAPDH. The primers used for specific genes are as follows:5’ TGCTACAACGGTTCGGAGC 3’Mouse C25H forward, 5’ AGAAGCCCACGTAAGTGATGAT 3’Mouse C25H Reverse, 5’ CGACTTCAACAGCAACTCCCACTCTTCC 3’Mouse GAPDH forward, 5’ TTGGTGGTCCAGGGTTTCTTACTCCTT 3’Mouse GAPDH Reverse, 5’ GCTGGCAACGCAGTATATGA 3’Human C25H forward, 5’ ACGGAAAGCCAGATGTTGAC 3’Human C25H Reverse, 5’ GATCATCAGCAATGCCTCCT 3’Human GAPDH forward, 5’ TGTGGTCATGAGTCCTTCCA 3’Human GAPDH reverse, C25H gene promoter region using the Motif Localization Toolbox (MoLoTool) available at https://molotool.autosome.ru/ [5’-GGAGCTCCGAACCGTTGTAG-3’), 0.5μM reverse primer (5’-GAAGGAGACGGAAGGGTGAC-3’) and 2μL of DNA template. Cycling conditions were set to 95°C for 5 minutes, 40 cycles at 95°C for 20 seconds, 57°C for 20 seconds and 72°C for 20 seconds, followed by melting curve analysis. Ct values were normalized by subtracting the Ct of input DNA (non-ChIP chromatins) from the Ct of each corresponding ChIP reaction. Fold enrichment was determined by 2(CtIgG-CtATF2) using normalized Ct values.A putative binding motif for ATF2 was predicted within the mouse some.ru/ . Primerssome.ru/ . ChIP-qPsome.ru/ . Mouse e™ XRS + software Image Lab 5.1 (BioRad) and Student’s t-test was utilized to determine significance.All data were analyzed using Graphpad Prism software (6.0). For ELISA, significance test was carried out using Student’s t-test. RT-qPCR data were analyzed using one-way ANOVA multiple comparison test. One-way ANOVA multiple comparison test was used for ChIP assay data analysis. Western blot densitometric values were quantified by using ChemiDocC25H gene is induced by both type-I (IFN-β) and type-II (IFN-γ) interferons [Previous studies have shown that C25H is an interferon (IFN) inducible gene –31. C25Herferons –31. We iC25H is required for generating 25HC. Therefore, we evaluated 25HC levels in TNF treated cells. Expression of C25H by TNF was functional, since we observed production of 25HC from TNF treated MEFs  and macrSince TNF induced C25H expression and concomitantly released 25HC , we nextOne of the hallmarks of TNF signaling is activation of the transcription factor NFκB which is involved in expression of various pro-inflammatory cytokines and chemokines . TherefoFAK is a critical adaptor protein in integrin signaling pathway , 33. TNFTyrosine residues of FAK are phosphorylated following integrin activation and therefore, phosphorylated form of FAK represents the activated form of FAK , 33. To Finally, the role of 25HC in inducing FAK signaling during TNF-mediated pro-inflammatory response was investigated by evaluating FAK activation status in WT and C25H KO macrophages treated with TNF. While TNF triggered FAK activation in WT cells, as indicated by its phosphorylation, such activation was lacking in C25H KO cells . DensitoWe recently identified α5β1 integrin complex involved in triggering 25HC-mediated pro-inflammatory response during Nod2 activation and virus infection . Since TWe first used α5β1 integrin blocking antibody to assess the role of integrin during TNF signaling. Inhibition of cell surface α5β1 integrin by integrin blocking antibody in TNF treated macrophages significantly reduced CCL3 production compared to IgG-treated control cells . Thus, iThe above results suggested that lack of α5 integrin expression may also lead to loss of NFκB activation by TNF. To examine this scenario, we utilized α5 integrin knockout (α5 KO) human haploid (HAP1) cells , 34. TheEnzymatic activity of C25H is required for generation of 25HC , 35, 36.For these studies we first utilized epithelial cells deficient in type-I interferon signaling. We used type-I interferon receptor (IFNAR2) lacking U5A cells and the parental wild-type 2fTGH cells. 2fTGH and U5A cells treated with TNF were subjected to RT-qPCR analysis to detect expression of C25H. Surprisingly, lack of type-I interferon signaling did not affect C25H expression following TNF treatment . Type-I C25H gene induction following TNF treatment since there was no significant difference in C25H expression in cells treated with control IgG versus IFNAR blocking antibody  do not produce IFN-γ. Therefore, our results demonstrated interferon independent induction of C25H expression by TNF.We validated this result by using IFNAR blocking antibody. We selected MEFs for studies related to TNF-mediated C25H expression due to their robust expression of C25H following TNF treatment . The effantibody . FurtherOne study suggested that C25H mRNA expression can be modulated by TLR4 via NFκB and MAPK-dependent JNK pathways . To dateC25H gene expression.We first evaluated the role of NFκB in TNF-mediated C25H expression by treating MEFs with TNF in the presence of the NFκB inhibitor BAY-11. Blocking the NFκB pathway in TNF treated cells resulted in significant loss of C25H expression . This reC25H promoter has not been documented. Interestingly, TNF is known to activate the MAPK pathway in various cells to modulate wide spectrum of cellular and biological activities [MAPK signaling pathway regulates various cellular activities in response to diverse stimuli . The thrtivities –51. HoweC25H as a MAPK inducible gene and showed a role of p38 and JNK in inducing C25H expression in TNF treated cells. Additionally, our study suggests that while NFκB is absolutely required for C25H expression during TNF signaling, MAPK signaling plays an important role in facilitating optimal C25H expression during this event.To study the role of MAPK pathway, we treated TNF-primed MEFs with inhibitors specific for ERK, JNK and p38 pathways. Analysis of C25H expression by RT-qPCR revealed significant reduction in C25H expression following inhibition of p38 and JNK pathways . The reqC25H gene (AGTGACGTCACC) in the C25H promoter region contains the consensus sequence identified for AP-1 (5’-TGAC/GTCA-3’) [p = 6.18x10-6) is located at 73 nt from the translational initiation codon within C25H gene and is conserved in both human and mouse C25H promoter regions.Although STAT1 and NFκB transcriptional factors have been implicated in transactivation of C25H gene –31, 35, 25H gene . This ATGTCA-3’) , 53. ThiC25H promoter following TNF treatment, we performed chromatin immunoprecipitation (ChIP) assay followed by real time quantitative PCR (ChIP-qPCR). ChIP-qPCR analysis revealed recruitment of ATF2 to the C25H promoter following activation of TNF signaling  is a multi-functional pro-inflammatory cytokine that regulates wide spectrum of physiological, biological and cellular processes including immunity, inflammation, apoptosis, coagulation, endothelial cell function, insulin resistance and lipid metabolism –23. ThusTNFR1 and TNFR2 to activate a signal transduction cascade that culminates in activation of MAPK and NFκB pathways [TNF binds to its cognate cell surface receptors, pathways –13. Thespathways –23. Althpathways . We recepathways . We showOur studies have highlighted a previously unknown mechanism associated with TNF signaling. We report that 25HC-integrin-FAK signaling is an essential component of TNF-mediated pro-inflammatory response. Thus, our studies suggest MAPK/NFκB-C25H-25HC-integrin-FAK signaling network playing an important role in amplifying TNF-mediated pro-inflammatory response and during this event 25HC acts as a “bridge” to link TNF pathway to integrin-FAK pathway for pro-inflammatory response amplification.Recent studies have demonstrated a role of 25HC in modulating innate immunity. 25HC regulates immune response mediated by type-I and type-II interferons –31. FurtC25H promoter and resulting expression of C25H gene [C25H gene expression. More importantly, we showed direct binding of AP-1 transcription factor ATF2 to the C25H promoter.C25H has been reported to be a type-I (IFN-β) and type-II (IFN-γ) interferon inducible gene –31. STAT25H gene . Additio25H gene . This st25H gene . However25H gene . This st25H gene , thus suIn summary, our studies have identified MAPK/NFκB-C25H-25HC-integrin-FAK signaling network as an important regulator of TNF-mediated innate immune response.S1 Fig(A) 25HC production from RAW264.7 macrophages treated with TNF (100ng/ml) for 8h were analyzed by ELISA . (B) MEFs treated with either 25HC (80 pmol/ml) or TNF (10ng/ml) for 4h were analyzed for C25H mRNA expression by RT-qPCR . ELISA data are shown as Mean ± SEM. *p ≤ 0.05 using a Student’s t-test. RT-qPCR data are shown as Mean ± SEM.*p ≤ 0.05 using one-way ANOVA multiple comparison test.(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.