Inflammation is involved in the pathogenesis of depression. A few cross-sectional population-based studies have found that depression is associated with increased levels of inflammatory markers. Soluble urokinase plasminogen activation receptor (suPAR) is known to be a stable marker for inflammation. We investigated the bidirectional association between suPAR levels and use of antidepressants.suPAR level was measured in 9305 blood donors and analysed in relation to 5-years follow-up data on purchase of antidepressants and hospital diagnoses of depression from a nationwide Danish register.st to the 4th quartile . For women, it increased by 108% from the 1st to the 4th quartile . Previous use of antidepressants was also significantly associated with higher suPAR levels (p = 0.002).For men and women without prior use of antidepressants we found a significantly higher risk for incident use of antidepressants with higher suPAR values. For men, the risk of first use of antidepressants increased by 72% from the 1High suPAR levels are associated with an increased risk for both previous and future use of antidepressants in healthy men and women. High suPAR are also associated with increased risk for a hospital diagnosis of depression. In the last decade, it has become clear that there is a biological link between inflammation and depression. In patients with depression, several studies have demonstrated increased expression of proinflammatory cytokines, chemokines, acute phase reactants, and adhesion molecules compared to non-depressed controls A few population-based studies have investigated the relationship between C-reactive protein (CRP) and de novo depression. In a study including 644 women with no prior history of depression, of whom 48 women developed depression during the 5827 person-years of follow-up, CRP was found to be an independent predictor of depressive disorder, supporting an aetiological role for inflammatory activity in the pathophysiology of depression The lack of consensus with regard to CRP and development of depression may in part be due to the high biological variation of CRP (hsCRP). In 38 healthy blood donors with 6 determinations over 22 days a biological variation of hsCRP of 50% was found SuPAR is a protein that is measurable in the circulating blood of all individuals. In contrast to most pro-inflammatory and acute response biomarkers, circadian changes in plasma suPAR is minimal, and in vitro stability is also high Elevated levels of suPAR are associated with immune activation, inflammation and a negative outcome in patients with symptoms of infection  consisting of healthy individuals, plasma suPAR levels were associated to the development of cancer, cardiovascular disease, type-2 diabetes and mortality during 12-years follow-up To test the hypothesis of inflammation and development of depression, we investigated whether the biomarker suPAR was associated with an increased risk of developing depression in a large cohort of Danish blood donors. As a proxy for depression, we used purchase of antidepressants, supplemented with a discharge diagnosis of depression.Aim of the study was to test the hypothesis, that higher levels of circulating suPAR in healthy individuals are associated with an increased risk for future use of antidepressants or a hospital diagnosis of depression. From both analyses were donors with a pre-history of antidepressant use or a previous hospital diagnosis of depression excluded. We further tested the hypothesis that prior use of antidepressants was associated with higher levels of suPAR.This study was approved by the local Medical Scientific Ethics Committee of Denmark (H-KF 01283058). All participants provided written informed consent. This study was conducted in accordance with the Declaration of Helsinki.th of January to the 24th of April 2006 were invited to participate in the study. The inclusion criterion was fulfilment of the Danish criteria for blood donation . All donors were asked about medication usage. Usage of antidepressive medicine within the last two weeks prior to visiting the blood bank was an exclusion criterion for donation and thus participation. Informed written consent was obtained from 11971 donors. Blood samples were obtained from a look-back repository where samples were available for 9312 of these donors.Blood donors, who donated blood to the Copenhagen City Blood bank (former H:S Blood bank) during a three month period from the 14All blood samples were collected in 7-ml blood EDTA anticoagulated tubes with stabilising gel . Blood samples were centrifuged to separate the plasma from the cells within 1 hour and frozen within 6 hours.Plasma suPAR was measured at Hvidovre hospital with the suPARnostic ELISA kit  according to the manufacturer instructions. The samples were measured in singlets and the interassay variation of a control plasma sample run on all plates was <10%. The kit standard curve was validated to measure suPAR levels between 0.6 and 22 ng/ml.st of December 2010. In Denmark, all antidepressive or antipsychotic medications are prescribed by doctors and can only be purchased at pharmacies. The sale of prescribed medication from the pharmacies is registered with the name of the drug, Anatomical Therapeutical Chemical code (ATC code), dose, CPR-number of the patient and the identity of the doctor prescribing the medicine.Data on purchase of antidepressive medicine and other prescription medicines from the Danish register of Medical Products From the Danish Patient Registry we obtained data on hospitalization with depression. Diagnoses were based on the International Statistical Classification of Disease (ISCD).Case persons were persons who filled prescriptions for antidepressive medication  or had a hospital discharge diagnose with the codes F.32 or F.33.When analysing incident depression, donors who purchased antidepressants before the time of suPAR measurement were excluded.Similarly were donors with a hospital discharge diagnosis of depression before the time of suPAR measurement, excluded from the analysis of incident hospitalization.In the following text, references to the purchase and use of antidepressants will be used interchangeably. Other medications were all prescription medications purchased other than antidepressants.st of December 2010 in participants who never purchased antidepressants before suPAR the measurement, according to the register.As a proxy for incident depression, we investigated the first use of antidepressants between suPAR measurement and the 31The incident use of antidepressants after suPAR measurement was illustrated according to sex-specific suPAR level quartiles by Kaplan-Meier curves and compared using a log-rank test. Cox regression analyses stratified by sex, with age as the underlying timescale, were further used to assess the effect of suPAR level on the subsequent incident use of antidepressants. The interaction between sex and suPAR in relation to time of first purchase of antidepressant medication was investigated in a model with suPAR sex and a sex-suPAR level interaction term. Analyses were conducted using the log2-transformed suPAR level as a continuous variable and with quartiles of suPAR levels. Analyses were repeated while also adjusting for the purchase of prescription medication other than antidepressants as a time-dependent variable.The same analysis was used to examine if there was an association between the suPAR level of the donors and a hospital discharge diagnosis of depression but only with suPAR as continuous variable adjusted for sex.We tested the hypothesis that a prior purchase of antidepressants was associated with increased suPAR levels. The effect of prior purchase of antidepressants on the log2-transformed suPAR level data was estimated using linear regression models adjusted for age (continuous linear variable). Separate analyses were conducted for women and men. The purchase of antidepressants was analysed as a dichotomous variable (none/any) and in categories of time from last purchase to suPAR measurement .A total of 9312 individuals with an available blood sample had their suPAR level measured. Seven blood samples were excluded due to outlying low values. Thus, the cohort consisted of 9305 individuals, including 4464 women and 4841 men. There were more women than men in the younger age group (18–30 years of age) while there were more men than women in all other age groups. The median age for women was 36 years (quartiles: 27–47) and the median age for men was 39.9 years (quartiles: 30–49).−1 to 2.58 ng/ml after which the level increased slowly to 2.71 ng/ml. For men the median suPAR value increased more evenly across the age groups from 2.08 ng/ml to 2.44 ng/ml  for women and 2.22 ng/ml (range 0.6–15.4) for men. suPAR levels increased with age in both men and women. In women there was an increase between the 18–30 and 31–40 age groups from 2.43 ng ml44 ng/ml .st of December 2010 the 9305 donors made 387.837 purchases of prescription medications, of which 6347 were for antidepressants (ATC code ‘N06A’) made by 856 individuals. There were 445 donors who purchased antidepressants before suPAR measurement and 411 donors who made their first recorded purchase after suPAR measurement.During the observation period from the first of January 1995 to the 31th suPAR level quartiles than for those in the 1st, 2nd and 3rd quartile. The association between suPAR level and first use of antidepressants persisted when stratifying by sex and adjusting for age . For women, the risk of using antidepressants for the first time increased by 108% from the 1st to the 4th quartile .The 8860 donors who had previously never purchased antidepressants at the time of suPAR measurement were included in the analysis evaluating the correlation between suPAR and the subsequent first use of antidepressants. In the 5-year period from suPAR measurement to the end of follow-up in December 2010, 411 donors (247 women and 164 men) purchased antidepressant medication. The majority made less than four purchases in the period. There was a statistically significant difference in the incidence of the first use of antidepressants among the suPAR level quartiles (p<0.0001),  for age . Both mequartile . For menThe purchase of prescriptive medication other than antidepressants was not associated with an increased incidence of first antidepressant usage in men or women and the inclusion of other medication did not change the association between the suPAR level and the first use of antidepressants . TestingAnalysis of data from the Danish Patient Register of hospitalization found an association between the suPAR levels and a hospital discharge diagnosis of depression . A higheOf 9305 donors, 445 purchased antidepressant medicine in the period from January 1995 to the day of suPAR measurement in the beginning of 2006. The majority (54.4%) of those who used antidepressants had three purchases or fewer. Thus, there was a trend towards declining effect on the suPAR level with increasing time because the last antidepressant purchase, although the associations between suPAR level and antidepressant purchase were not significantly different for the three timing categories in women.To our knowledge, this is the first larger prospective population-based study of the inflammatory marker suPAR and use of antidepressants. We investigated the association of suPAR levels and both subsequent incident use of antidepressants and prior use of antidepressants and found an association between high suPAR levels and the use of antidepressants in both directions.When analysing the incident use of antidepressants, we found a statistically significant effect of suPAR level on the risk of subsequent use of antidepressants during a five-year observation period. To our knowledge, this is the first larger prospective population-based study of the inflammatory marker suPAR and use of antidepressants. We investigated the association of suPAR levels and both subsequent incident use of antidepressants and prior use of antidepressants and found an association between high suPAR levels and the use of antidepressants in both directions. Kaplan-Meier curves depicting suPAR quartiles indicate that a high suPAR level was associated with an increased risk of antidepressant usage throughout the observation period. This is a surprising and interesting observation, suggesting that an elevated suPAR level seems to reflect a constitutively increased risk of depression.We also found that prior use of antidepressants was significantly associated with higher suPAR levels in both men and women. This could possibly be an example of reverse causality, as we cannot rule out that the suPAR level was already increased before the purchase of antidepressants. For men, the effect was greater when the purchase was temporally closer to the suPAR measurement. For women, the effect was independent of purchase timing. There was, however, a trend towards a decreased strength of the association when the time between suPAR measurement and the purchase of the antidepressant medicine increased. The timing categories were based on clinical guidelines for treatment in the acute and maintenance phases and are in accordance with prior studies Our findings are in line with studies of depression and CRP that found an association between CRP levels and prior depression It is well known that there is an increased prevalence of depression among patients with diseases such as diabetes This study is for the outcome based on register data using the purchase of antidepressant medicine as a proxy for depression. The same ATC code (N06A) has previously been used in other population-based studies of depression Thus, it seems reasonable to use the purchase of antidepressant medicine as a proxy for depression, acknowledging that milder forms of depression may not all be recorded. The sensitivity of 50% indicates that many people with depression are not prescribed medication and there can be a long delay between the onset of depression and initiation of the antidepressant use. Further, antidepressants are used for a number of indications other than major depression. Thus, we cannot completely rule out that a proportion of participants who started using antidepressants after the suPAR measurement were already depressed or had been depressed before blood collection without receiving an antidepressant at that time. Also did we in this analysis exclude all donors who had purchased antidepressants prior to the suPAR measurement. However older donors could potentially have been treated with antidepressants before the register started in 1995.A limitation of our study is the lack of information on relevant potential confounders such as smoking, alcohol consumption, BMI and physical activity. Smoking may lead to increased suPAR levels A second limitation of this study is the lack of a comparison between suPAR measurements and other components of the fibrinolytic system or other inflammatory markers previously shown to be associated with depression, such as CRP or IL-6 suPAR has not been previously investigated in depression. In neurological diseases such as Alzheimer, multiple sclerosis, HIV dementia, cerebral malaria and Creutzfeldt-Jakob disease, the presence of uPAR in macrophages/microglia within the CNS has been described We conclude that high suPAR levels are associated with an increased risk of subsequent incident purchase of antidepressants and an incident hospital diagnosis of depression. Conversely, the prior purchase of antidepressants is associated with increased suPAR levels. We are not able to identify methodological drawbacks that clearly refute these findings as artefacts, though we did not have data on a number of important potential confounders. suPAR is an inflammatory marker and high levels could thus reflect a subclinical state of inflammation that increases the vulnerability to depression. Our findings support the theory that low-grade inflammation contributes to the pathogenesis of depression, adding depression to the list of diseases associated with low-grade inflammation. Further studies are needed to characterize the exact role of suPAR in depression in clinical practice.


Basic research is a journey taken by scientists to explore our fundamental understanding of biology. The process of conducting basic research leads to new solutions and knowledge that can be applied to different problems, including many unexpected ones. One of the primary questions I have been interested in since starting my journey, upon entering graduate school, is: how do bacteria detect changes and modify themselves accordingly to survive diverse environments? This is critical during infection of the host, a situation in which the bacteria detect cues upon entering the host and additional changes during the progression of the infection. Understanding the ways that bacteria sense these changes and respond may reveal potential new strategies to combat infections and antibiotic resistance.Escherichia coli), indicating that other secondary messenger signaling molecules may exist. Since that time, a large number of other signaling molecules  have been identified by numerous labs, supporting the idea of diversity of second messenger signaling systems in prokaryotic organisms. Three basic research questions can be asked for all of these signaling molecules: 1. Which of the cellular protein(s) and RNA(s) bind these signaling molecules? 2. What are the allosteric changes on the protein and RNA upon binding to the signaling molecule? 3. How does a cell coordinate these events to form a coherent response?From the beginning of my graduate training in Olaf Schneewind’s lab during the mid-1990s, I knew that there was a growing body of knowledge about signal transduction systems used by bacteria to respond to environmental changes. One class of signal transduction systems, called the second messenger signaling system, detects changes in the environment and generates a second messenger signaling molecule in the cell. This signaling molecule then binds directly to proteins or RNA to alter their behavior, thus allowing the bacterial cell to respond to changes. This fundamental signaling process that occurs in bacteria also exists in higher organisms, so findings from prokaryotes have the potential to explain how signaling systems work in eukaryotes. The literature on secondary signaling systems in bacteria in the 1990s was predominantly based on studies of two molecules, cyclic AMP (cAMP) and “magic spot” (ppGpp), in one model bacterial organism . However, I ran into a technological roadblock, since testing even a few proteins took many months to complete. There were no feasible ways to test thousands of proteins for their ability to bind a signaling molecule in a reasonable time frame . On a dark day before the winter holidays after years of searching for a solution, I spotted a mixture of protein and the ligand on a dry nitrocellulose paper. This quick and simple process, akin to putting a paper towel on a coffee spill, easily differentiated binding proteins, since they prevented the signaling molecule from moving with the liquid wicking away on the paper. Through both serendipity and paying careful attention in the lab, I realized that this procedure, which we termed Differential Radial Capillary Action of Ligand Assay, or DRaCALA, that took only seconds to complete was a solution to my technological roadblock.Work by my student Kevin Roelofs adapted DRaCALA to screen thousands of proteins encoded on the bacterial genome. Members of my lab, including Kevin, Mona Orr, and Sarah Helman, as well as other labs have used this assay to identify a number of new protein receptors for cyclic-di-GMP and cyclic-di-AMP. In addition to these discoveries, my students Ori Lieberman and Eric Zhou have used this method to identify small molecule inhibitors that can occupy the binding site and prevent binding by the signaling molecule. Such inhibitory molecules can serve as lead compounds to treat bacterial infections and reduce antibiotic resistance. In addition, Gregory Donaldson, a former student, utilized DRaCALA to detect interactions between proteins and larger nucleic acids, indicating that the assay may allow for the rapid detection of biomarkers and pathogens in the clinical setting. The development of this simple DRaCALA technique not only enabled me to answer the question I was personally interested in but has also reduced the technological barrier for scientists interested in other signaling molecules and binding proteins. As evidenced by my adventurous journey in science, support for basic research can pave the way for discovery and applications in unforeseeable ways. I believe continued support for basic research is critical to advance our knowledge and uncover innovative solutions to existing and new problems.


The human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus associated with both proliferative and inflammatory disorders. This virus causes a persistent infection, mainly in CD4+ T lymphocyte. The ability to persist in the host is associated with the virus capacity to evade the immune response and to induce infected T-cell proliferation, once the HTLV-1 maintains the infection mainly by clonal expansion of infected cells. There are several evidences that ORF-I encoded proteins, such as p12 and p8, play an important role in this context. The present study will review the molecular mechanisms that HTLV-1 ORF-I encoded proteins have to induce dysregulation of intracellular signaling, in order to escape from immune response and to increase the infected T-cell proliferation rate. The work will also address the impact of ORF-I mutations on the humanhost and perspectives in this study field. Human T-cell lymphotropic virus type 1 (HTLV-1) was first isolated in the early 80s, in a patient with a T-cell lymphoma . HTLV-1  gag, pol, and env. In addition, the provirus presents a unique pX region, which contains the regulatory and nonstructural genes. Thus, the 3′ end of the viral genome expresses alternatively spliced mRNAs encoding proteins from open reading frames (ORFs) I–IV. ORF-I encodes the p12I protein. This protein is highly hydrophobic and presents four SH3 binding motifs, one leucine zipper domain, one leucine zipper-like domain, and two putative transmembrane domains (TM1 and TM2) [I protein undergoes complex posttranslational modifications. It undergoes proteolytic cleavage at two different sites: at position 9 (L9↓S10) and at position 29 (G29↓L30). These yields two protein forms: p8 and p12 (8-kDa and 12-kDa forms) which presents different cellular localization and function, while 12-kDa (p12) remains in the endoplasmatic reticulum (ER) and cis-Golgi compartment the 8-kDA form (p8) traffic to cell surface. Indeed, early studies have shown that the p12 cDNA expression yields two proteins [The viral genome is approximately 9 kb long and contains the typical retroviral structural and enzymatic genesand TM2) , 12. Theproteins . CurrentThe ability of HTLV-1 to achieve a persistent infection may be associated with the virus capacity to evade the immune response. The immune evasion may be also associated with a high viral replication rate that can lead to a high proviral load, which, in turn, is related with disease manifestation –9. A preThe recognition of an infected cell by CTLs is made through the specific interaction between the T-cell receptor (TCR) and the MHC-I molecule presenting an antigen.β2-microglobulin. Several pathogens developed many strategies targeting MHCI molecule in order to escape from CTLs response [The peptides (antigens) are generated by the proteasome in the cytoplasm. After, they are transported by TAP (transporter associated with antigen processing) to the ER, where they are conjugated to MHC-I molecule. These MHC-I-antigen complexes are transported to the infected cell surface for CTL recognition and activation. MHC-I is assembled in the ER lumen and presents a heavy chain (Hc) and response . It was response .The down modulation of MHC-I expression, on the other hand, makes HTLV-1 infected cells susceptible to natural killer (NK) response. However, this virus also presents mechanisms to evade NK cell activity. This ability can be also mediated by ORF-I encoded proteins and will be discussed in the next topic.In contrast, p12 expression did not alter the expression level of MHC-I and MHC-II in infected immortalized T cells. This data indicates that ORF-I encoded proteins may modulate the MHC expression only during the early stage of infection .The NK cell activation is the result of the integrated balance between the stimulation of activating and inhibitory NK receptors. Therefore, a prior recognition of the pathogen is not necessary for the NK cell activation by the infected cell. NK cells are activated by activating ligands present on the cell surface. As uninfected cells can also express these ligands, the noninfected cells have to be able to dampen NK cell cytotoxicity. This is achieved by the engagement of specific molecules, such as MHCI, to the inhibitory receptors (iNKRs) –19. Ther in vitro study, using a lentivirus vector encoding HTLV-1 p12, demonstrated that NK cells are not able to kill HTLV-1 infected primary CD4+ T cells. This inability was associated with the down modulation of ICAM-1 and ICAM-2 in the infected cell, which results in a decreased ability to form the immune synapse. This alteration was induced by HTLV-1 p12. In the same study, neither NCR nor NKG2D ligands were expressed by HTLV-1 infected T cells [Another important point to NK cell activity is the formation of the “NK-target cell immune synapse.” This strong adhesion between NK and the target cell is mediated by integrins present in NK and their natural ligands available in the target cell. This is critical to trigger NK cell response. The leukocyte function antigen 1 (LFA-1) is one of those integrins that can be found in NK cell surface and binds to intercellular adhesion molecules (ICAMs). The ICAMs are expressed by several cells, including leukocytes. Besides the formation of immune synapses, LFA-1 can also trigger intracellular signaling for cytotoxic granule polarization, which play an important role in directing the machinery of NK to target cell. An T cells . The exa2. An increase in the concentration of intracellular calcium activates the phosphatase calcineurin that dephosphorylates NFAT, which is translocated to nucleus and induces IL-2 expression [It is know that HTLV-1 infection is maintained mainly by the clonal expansion of infected T-cell . In thispression . Both NFpression , 24, 25.In fact, studies with indirect immunofluorescence and electron microscopy showed that p12 is retained in ER. The same study showed that p12 is associated with calreticulin and calnexin, which are resident ER proteins and regulate the calcium storage . This biMoreover, the p12 protein binds to the cytoplasmic domain of the IL-2 receptor (IL-2R) in the ER . The p12All these data together clearly demonstrate that ORF-I encoded proteins induce intracellular signaling modification that result in a decreased IL-2 requirement for T-cell proliferation. These HTLV-1 accessory proteins seem to confer a growth advantage to infected T cells, mainly during the early stage of infection, before the immortalized T-cell state .I clone that is not able to produce p12, although can express LFA-1, exhibited less LFA-1 mediated adhesion than T cells infected with wild type clone (ACH). In the same study the authors also demonstrated that the expression of p12 in Jurkat T cells enhanced the cell adhesion mediated by LFA-1 in a calcium dependent manner, once it was inhibited with the addition of calcium chelator, calcium channel blocker, and an inhibitor of the calcium dependent protease calpain [+ and p12− cell lines produced similar amounts of HTLV-1 particles in the supernatant of culture cells, ORF-I encoded proteins expression enhanced virus transmission more than twofold when compared to infected cells lacking p12. The same study showed that p12 expression increased the rate of syncytium formation [HTLV-1 maintains the infection almost exclusively by the clonal expansion of infected T-cell . Therefo calpain . Reinforormation . More reormation , 41. Theormation . in vitro studies and animal model. A study conducted by Collins and colleagues using a HTLV-1 molecular clone that was not able to produce p12 (ACH.p12I) demonstrated that this virus was infectious in vitro; however, it was not able to establish a persistent infection in rabbits. Indeed, after the ACH.p12I inoculation, the animals presented a reduced anti-HTLV-1 humoral response compared to those infected with the full-length clone ACH. Moreover, p19 antigen was not detected in peripheral blood mononuclear cells (PBMCs) of ACH.p12I infected animals and there was only a transient detection of HTLV-1 proviral DNA in PBMCs in those animals [ in vitro and in vivo studies may be associated with the stimulation of cell cultures with IL-2 and phytohemagglutinin. Other studies also demonstrated that different molecular clones lacking ORF-I did not alter the HTLV-1 capacity to infect, replicate, and immortalize T-cells in cell cultures [Several studies addressed the role of HTLV-1 ORF-I proteins to viral infection using animals . It is icultures , 45. In cultures . Indeed,cultures .ORF-I encoded proteins are expressed in naturally infected individuals. This was evidenced by the detection of ORF-I mRNA in HTLV-1 infected cells from HAM/TSP and ATLL patients and from asymptomatic carriers , 49. In I, observed in individuals with p12I mutant (G29 → S), would cause an increase in HTLV-1 proviral load [G29S represents another known mutation. It inhibits the production of p8 protein at the cleavage site position of p12. All these data together highlighted for the possibility that a predominance of the 12-kDa uncleaved form of p12ral load .IG29, but not the mutate p12IS29, induces a decrease in tax-mediated transactivation. A recent study showed that different ORF-I mutations are associated with distinct patterns of p12 and p8 expression. While some mutations are associated with a balanced expression of p8 and p12, some ORF-I mutations result in a predominance of p8 or p12. As expected, the mutation G29S was associated with a predominance of p12 expression. However, in the same study, G29S virus was not infectious in macaques and was poorly infectious in monocytes and HTLV-1 infected T CD4+ cells by this mutant virus were partially susceptible to CTL killing. The same study showed that the proviral load was higher in individuals with the balanced expression of those proteins. This study finally concluded that p8 and p12 are both required for HTLV-1 persistence and spread [It was also observed that the wild type p12d spread .There are also different mutations that result in premature stop codons and consequently the production of truncated p12 proteins. This truncated p12 protein has been described in strains from different countries and is not dependent on clinical presentation, once it was described in asymptomatic, HAM/TSP, and ATL individuals. Interestingly, this truncated protein was also observed in STLV. Some authors even used this fact as an argument to the hypothesis that p12 protein is not essential for virus replication. in vitro study, the culture conditions, such as the phytohemagglutinin stimulation and the media supplementation with IL-2, may interfere in the obtained results [Several studies did not evaluate isolated p12 and p8 protein functions. Consequently, the better understanding of the role and the importance of each protein on HTLV-1 infection becomes limited. Another issue is that a lot of knowledge in this field was obtained using animal model and cell culture. Animal model may not represent what really happens in natural human infections. This can be exemplified with the fact that G29S strain was not able to infect monkeys; however, it is circulating in human host . Another results . As ORF- results . ORF-I eThe role of ORF-I proteins in immune evasion and T-cell proliferation highlight the importance of these proteins, ORF-I diversity, and the proviral load in HTLV-1 infected individuals. In addition, new studies should address asymptomatic and symptomatic patients (HAM/TSP and ATL). This information is extremely important since the proviral load is the main prognostic factor for the development of complications associated with HTLV-1 infection.There are still few studies with controversial data regarding this theme. Many promising findings could help to achieve a better understanding regarding HTLV-1 pathogenesis and may help to identify a possible therapeutic target and vaccine. The disease progression in HTLV-1 infected individuals is a very complex issue. Viral, host, and environmental factors may contribute to clinical outcome. How these factors interact in each individual may determine the development of disease.


Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of basic cellular processes, including I-κB kinase (IKK) signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in leukemogenesis, but the underlying mechanisms remain unknown. IKK and its upstream kinase, transforming growth factor β activated kinase-1 (TAK1), contain ubiquitin-binding subunits, NF-κB essential modulator (NEMO) and TAK1 binding protein 2 (TAB2) respectively, which interact with K63-linked polyubiquitin. On this signaling platform, auto-phosphorylation and activation of TAK1 occurs, followed by TAK1-catalyzed IKK phosphorylation and activation. Here we demonstrate in vitro and in vivo that Tax stimulates ubiquitin E2 conjugating enzyme Ubc13:Uev1A (or Ubc13:Uev2) and ubiquitin E3 ligase ring finger protein 8 (RNF8) to assemble long and unanchored K63-linked polyubiquitin for TAK1 and IKK activation. The TAK1 so activated by Tax also promotes JNK phosphorylation. The inappropriate activation of RNF8 — an E3 ligase involved in DNA damage repair, cytokinesis, and centrosome function — by Tax can explain the pleiotropic effects of Tax on signaling pathways.


Human T-cell lymphotropic Virus Type I (HTLV-1) is a retrovirus that generates an inflammatory response that can trigger a neurological disease called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) which appears in 5-10% of infected people around the world. This inflammatory process is due the action of anti-Tax CD8+ lymphocytes that employ cytokines in order to stop the predominant infiltration of infected CD4+ T lymphocytes. IL8 is a pro-inflammatory cytokine that induces the activation and migration of neutrophils towards the sites of infection and it is continuously expressed in HTLV-1 infected cells. The SNP rs4073, located in the promoter of IL-8 gene, has been found associated to some inflammatory diseases and we hypothesized that it could be also a risk factor for the HAM/TSP development. The aim of our study was to evaluate the association of IL8 gene polymorphism SNP rs4073 and the presence of HAM/TSP. The study includes 243 infected subjects between asymptomatic (AC) and HAM/TSP patients. The genotyping of the polymorphisms was performed using the tetra-primer ARMS-PCR (Amplification Refractory Mutation System) method, 37 ancestry informative markers (AIMs) were used to correct by ethnicity and to avoid false positive results due to population stratification effect. The statistical analysis was conducted using the chi-square and Kruskal-Wallis test for possible differences among the groups. Linear and logistic regression analysis was performed using age, gender and ethnicity. The results show that none of the SNP rs4073 genotypes achieved a significant difference between AC and HAM/TSP, neither significant difference was found with proviral load (p>0.05). Our results suggest that the increase of the inflammatory response in HAM/TSP patients may not be caused by the SNP rs4073 in the promoter site of IL8, but the over expression of these gen could be possibly manage by another SNP or any other mutation in the enhancer region.


HTLV-1 is endemic in Brazil and is associated with major illnesses . Since the infection remains asymptomatic in the majority of cases and screening is only performed in blood banks, there is an ongoing vertical transmission that remains unnoticed by the public health system. Although recommended by Brazilian researchers of the field, the prenatal screening for HTLV-1 or 2 was not yet implemented throughout the country. We performed a cross-sectional analysis to verify the impact of counseling HTLV-1 seropositive women in childbearing age participating in the GIPH cohort. The counseling included recommendations of not breastfeeding, giving infant formula and preferably having the child delivered by cesarean section. Children born from HTLV-1 seropositive women were divided in two groups: (1) born before and (2) after the participation of mothers in the GIPH cohort (“GIPH babies”). We have identified 21 children born to HTLV-1 positive mothers who received no counseling and 18 “GIPH babies”, born from mothers who received counseling. 3/21 (14.3%) and 1/18 (5.6%) of the children were found positive for HTLV-1. The numbers will be expanded with active search of the remaining children not yet tested (n=15). Although the numbers are still preliminary, they point to a trend of successful counseling and avoidance of transmission of the virus. These actions should be widespread in the country to avoid the silent transmission of the virus.


The role of uric acid (UA) in the progression of chronic kidney disease (CKD) remains controversial due to the unavoidable cause and result relationship. This study was aimed to clarify the independent impact of UA on the subsequent risk of end-stage renal disease (ESRD) by a propensity score analysis.A retrospective CKD cohort was used (n = 803). Baseline 23 covariates were subjected to a multivariate binary logistic regression with the targeted time-averaged UA of 6.0, 6.5 or 7.0 mg/dL. The participants trimmed 2.5 percentile from the extreme ends of the cohort underwent propensity score analyses consisting of matching, stratification on quintile and covariate adjustment. Covariate balances after 1:1 matching without replacement were tested for by paired analysis and standardized differences. A stratified Cox regression and a Cox regression adjusted for logit of propensity scores were examined.After propensity score matching, the higher UA showed elevated hazard ratios (HRs) by Kaplan-Meier analysis . The number needed to treat was 8 to 9 over 5 years. A stratified Cox regression likewise showed significant crude HRs . Adjusted HR lost its significance at 6.0 mg/dL. The adjustment for the logit of the propensity scores showed the similar results but with worse model fittings than the stratification method. Upon further adjustment for other covariates the significance was attained at 6.5 mg/dL.Three different methods of the propensity score analysis showed consistent results that the higher UA accelerates the progression to the subsequent ESRD. A stratified Cox regression outperforms other methods in generalizability and adjusting for residual bias. Serum UA should be targeted less than 6.5 mg/dL. Impact of uric acid (UA) on the progression of chronic kidney disease (CKD) still remains controversial due to inconsistent results of the observational studies –8. The iRecently a propensity score analysis is increasingly being used to estimate causal effects in the observational studies because one can replicate the prospective randomized controlled trial by minimizing baseline confounding as much as possible . To solvWe used a retrospective CKD cohort already reported  but with3), albumin-corrected calcium (cCa), inorganic phosphorus (P), low-density lipoprotein cholesterol (LDL-C) and C-reactive protein (CRP). Urine parameters included spot urine proteinuria (expressed as gram per gram creatinine excretion) and spot urine hematuria by dipstick . Due to the distribution, C-reactive protein, proteinuria and hematuria were log-transformed for analyses.The demographic characteristics included sex, age, body mass index (BMI), original kidney disease (diabetic nephropathy or not) and systolic blood pressure (SBP). Blood parameters included hemoglobin (Hb), white blood cell (WBC), platelet (Plt), albumin (Alb), uric acid (UA), sodium (Na), potassium (K), chlorine (Cl), Na-Cl  study equation for Japanese population . The propensity score model was tested for its accuracy by c-statistics according to the area under the Receiver Operating Characteristic (ROC) curve for the threshold  and for ropriate . Standarropriate , 26. Foron (AIC) . StatistDuring the follow-up period of 4.0 ±1.6 years, 110 out of 803 patients progressed to ESRD. The demographic characteristics and baseline values were shown in Table A in p < 0.001). The relationship between baseline UA and time-averaged UA was plotted in p < 0.001). Of interest is that the regression line between time-averaged UA vs. baseline UA was less than a unity in the slope and converged at UA of 7.0 mg/dL, indicating that the patients with time-averaged UA > 7.0 mg/dL received more chance of using UA-lowering drugs (61% vs. 39%). On the other hand, the patients with baseline UA < 7.0 mg/dL showed the increase in time-averaged UA in the follow-up due to the advancement of CKD.The values of baseline UA and time-averaged UA are normally distributed as depicted in p values were all greater than 0.05. Then, two groups divided by the threshold of time-averaged UA were subjected to a Kaplan-Meier analysis Click here for additional data file.


Bone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment.® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals.A total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP2 = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24).The average TRACP level of the high-UA individuals (1.47± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) . The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an RHigh UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases. With very few exceptions, the natural history of all kinds of tumors is known to progress from localized indolent stages to aggressive metastatic stages,2. Once Tartrate-resistant acid phosphatase 5b (TRACP 5b) is generally secreted by osteoclasts during bone resorption,14. Its Uric acid (UA) is a heterocyclic compound of carbon, nitrogen, oxygen, and hydrogen with the formula C5H4N4O3. UA is a product of the metabolic breakdown of purine nucleotides. High blood concentrations of UA can lead to gout. High inRecently, we observed that sixteen cancer patients with bone metastasis diagnosed by bone scintigraphy showed no obviously increased TRACP 5b levels. Meanwhile, these patients showed higher UA level than others. Here, we investigated the correlation between the concentrations of UA and TRACP 5b in a random sample of 77 high-UA patients and determined whether TRACP 5b level was associated with UA in interference experiments.This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. All samples were collected from August 2014 to June 2015. Patients with cancer, hepatitis, renal dysfunction and inflammatory disease were excluded from serum collection. A total of 77 patients (Hyperuricemia), including 16 women and 61 men  formed the study group. Additionally, 77 healthy subjects were sampled as the control group, including 16 women and 61 men .® Assay .The alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood glucose (Glu), blood urea nitrogen (BUN), creatinine (CREA) and uric acid (UA) quantitation were analyzed using an Olympus AU5400 automatic chemical analyzer and commercial kits  according to the instruction manual. White blood cells (WBC) and neutrophils (NEU) % were counted by the Sysmex XE-2100 hematology analyzer . The levels of carcinoembryonic antigen (CEA) and alpha-fetoprotein (AFP) were measured by electrochemiluminescence immunoassay (ECLIA) on an Elecsys E-602 . Serum tartrate-resistant acid (TRACP) 5b was detected by using bone TRAPA known concentration of TRACP 5b standard sample was divided into 6 aliquots. Serial dilutions of UA  were prepared from UA standard subject. The 5 aliquots were then spiked at 3:2 with each UA solution. This generated 5 different test samples with the same TRACP 5b level, whose final UA concentrations ranged from 100 to 1600 μM. An aliquot containing DDW (double distilled water) instead of UA served as a blank. Serum TRACP 5b assay was performed according to manufacturer’s protocol. The change in TRACP 5b level caused by UA spikes was measured and marked as a function of UA. A linear formulation for the effect of UA-derived TRACP 5b was made based on the best least squares fit.The concentrations of both TRACP 5b and UA were measured in serum sample of each donor. Each high-UA individual's UA-corrected TRACP 5b concentration was calculated based on the UA-TRACP 5b correction equation. Increasing UA concentration means difference value between each high-UA individual's and mean value of healthy subjects’.Results were presented as means and ranges. Statistical analysis was performed with SPSS 16.0. Two-tailed t-tests were used for significance testing between groups of continuous data. Corrected TRACP 5b concentration was calculated according to the formula: ΔTRACP 5b (U/L) = -1.9751lgΔUA (μM) + 3.7365. For all statistical comparisons a p< 0.05 was considered statistically significant.Ethical clearance for this study was obtained from the Ethics Committee at the First Affiliated Hospital of Nanjing Medical University. Because all the samples used in this study were collected from clinical residual specimen, written informed content from each patient was waived.2 = 0.98899 . Meanwhile, these patients showed relatively higher serum UA concentrations (UA concentration: 398.4 ± 40.4 IU/ml). So we inspected that high UA concentrations were associated with decrease of TRACP 5b levels for cancer patients bone metastasis. To evaluate whether there was a correlation between high concentrations of UA and decrease of TRACP 5b levels, we randomly tested the TRACP 5b levels of 77 high-UA individuals (UA concentration: 463.5 ± 50.8 IU/ml) and 77 healthy subjects (UA concentration: 293.4 ± 56.9 IU/ml). The results showed that the average TRACP 5b levels of the high-UA individuals (1.47± 0.62 U/L) were significantly lower  than that of the healthy subjects (2.62 ± 0.63 U/L)  Fig 1A–S1 File)S1 File). 0.98899 . This im 0.98899 , which sIn this study, by analyzing the collected data and the result of an interference experiment in vitro, we illustrated that the patients with high concentrations of UA presented a false-negative decrease in TRACP 5b. Our results reminded physicians should fully consider interference of hyperuricemia, especially when TRACP 5b was used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.S1 File(PDF)Click here for additional data file.


Human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). Dysregulations of cellular signaling pathways have been considered to be important strategies for HTLV-1 induced leukemogenesis. In this study, we found that a Wnt pathway-related protein, Dishevelled (Dvl)-associating protein with a high frequency of leucine residues (DAPLE), could be associated with Tax to activate the canonical Wnt signaling. However, in the downstream of the pathway, T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1), which are two transcription factors mainly expressed in T-cells, could interact with Tax and suppress its transactivating ability. We further found virus production was greatly inhibited by TCF1/LEF1. Due to the high expression of TCF1/LEF1 in the thymus and their significant downregulation upon T-cell activation, we suspect TCF1/LEF1 may contribute to the HTLV-1 tropism on activated mature T-cells. On the other hand, HTLV-1 bZIP factor (HBZ) markedly suppressed canonical Wnt activation through targeting both TCF1 and LEF1. As a result, the canonical Wnt pathway was not activated in HTLV-1-infected cells, whereas the representative of noncanonical Wnt ligands, Wnt5a, which antagonizes canonical Wnt signaling, was overexpressed in HTLV-1 infected cells. Further experiments revealed HBZ could induce Wnt5a expression through activation of the TGF-β pathway. Importantly, knocking down of Wnt5a in HTLV-1-infected cells repressed cellular proliferation and migration. By suppressing the canonical and enhancing the noncanonical Wnt pathway, HBZ is likely able to generate a favorable environment for HTLV-1 infection and persistence.


The Notch signaling pathway plays a critical role in maintaining the balance between cell proliferation, differentiation and apoptosis, and is a highly conserved signaling pathway that regulates normal development in a context- and dose-dependent manner. Dysregulation of Notch signaling has been suggested to be key events in a variety of hematological malignancies. Notch1 signaling appears to be the central oncogenic trigger in T cell acute lymphoblastic leukemia (T-ALL), in which the majority of human malignancies have acquired mutations that lead to constitutive activation of Notch1 signaling. However, emerging evidence unexpectedly demonstrates that Notch signaling can function as a potent tumor suppressor in other forms of leukemia. This minireview will summarize recent advances related to the roles of activated Notch signaling in human lymphocytic leukemia, myeloid leukemia, stem cells and stromal microenvironment, and we will discuss the perspectives of Notch signaling as a potential therapeutic target as well. The Notch signaling pathway is highly conserved from Drosophila to human and plays an important role in the regulation of cell proliferation, differentiation and apoptosis . MoreoveFour members of Notch proteins have been identified to date in mammals, including Notch1-4 -5. The NNotch signaling has been shown to be initiated by binding of the Notch transmembrane receptors with their specific ligands between two neighboring cells . Upon acIt has been shown that Notch signaling is abnormally regulated in many human malignancies ,23. NotcAdditionally, other Notch signaling and target genes are also involved in the initiation and progression of T-ALL. It has been reported that Notch3 and Hes1 are highly expressed by T-ALL cells, as well as dramatically reduced or absent in remission . DownregInterestingly, the function of Notch signaling in leukemogenesis has been shown to be either oncogenic or tumor suppressive, and it could be context dependent ,34. NotcIn contrast with B-ALL, Notch signaling could maintain B cell chronic lymphoblastic leukemia (B-CLL) cell survival and apoptosis resistance, undoubtedly indicating an oncogenic role in B-CLL. Emerging evidence suggests that the Notch signaling network is frequently deregulated in human B-CLL with up-regulated expression of Notch1 and Notch2 as well as their ligands Jagged1 and Jagged2 . Moreove+Lin-Sca1+ cells from pre-leukemic Ctsg-PML-RARA mice [et al. have found that all four Notch homologues and Hes1 were sufficient to inhibit the growth and induced caspase-dependent apoptosis of AML, which were associated with B cell lymphoma 2 (BCL2) loss and enhanced p53/p21 expression [in vitro but lead to dramatic increases in leukemia burden in two xenograft mouse models, which was associated with p53 dysregulation [et al. also found that the Notch ligands tended to induce differentiation under the specific conditions rather than promoted the self-renewal capacity of AML cells [Knowledge about the role of Notch signaling in acute myeloid leukemia (AML) is equally poorly understood. Very recently, Jagged1 and Dll1 were shown to be expressed at significantly higher levels in acute promyelocytic leukemia (APL) samples compared with all other subtypes, as well as normal myeloid populations . InhibitARA mice . Our preARA mice . These dARA mice ; howeverARA mice -48. Kannpression . Additiogulation . The 17-gulation ,49,50. Bgulation . Tohda eML cells . OverallNotch signaling appears to play a tumor suppressive role in chronic myeloid leukemia (CML). It is reported that overexpression of the active form of Notch1 or Notch2 in K562 cells resulted in the inhibition of proliferation, accompanied by increased Hes1 mRNA level ,55. On tin vivo and downregulated as HSCs differentiated. Inhibition of Notch signaling could lead to accelerated differentiation of HSCs in vitro and depletion of HSCs in vivo[+ or CD8+) by inductive interactions from thymic stromal cells [Leukemia stem cells (LSCs) arise either from corrupted HSCs or from more differentiated and committed progenitors that acquire self-renewal potential -58. Thers in vivo,60. Furtal cells ,62, suggal cells . Notch1 al cells .+/CD38- stem/multipotential progenitor populations from AML patients compared to normal CD34+ stem cells. Recently, inactivating mutations of Notch signaling have been described in patients with chronic myelomonocytic leukemia (CMML) [In vivo studies have also revealed both oncogenic and tumor suppressive functions for Notch signaling , extramedullary hematopoieisis and the induction of CMML-like disease. Furthermore, ectopic expression of Notch1-IC or Hes1 could suppress the expression of key GM commitment genes such as Cebpα and Pu.1, and the CMML-like disease developing in the Ncstn−/− animals [et al. reported that Jagged1 was higher in Kit+Lin-Sca1+ cells from pre-leukemic Ctsg-PML-RARA mice, and both genetic and pharmacologic inhibition of Notch signaling abrogated the enhanced self-renewal seen in hematopoietic stem/progenitor cells [To date, the role of Notch signaling in LSCs has not yet been examined adequately and seems to be context dependent. Notch signaling was shown to be silenced in CD34a (CMML) . In vivog Figure . In an Mng cells . Notch1– animals . Howeveror cells . Taken tet al. also found that coculture of primary human T-ALL with a mouse stromal cell line expressing the Dll1 reproducibly allowed maintenance of T-LiC and long-term growth of blast cells through rescuing from apoptosis [in vitro studies have demonstrated that endothelial cells enhance proliferation and survival of AML cells [Leukemia cell survival relies on leukemic microenvironment, which is composed of bone marrow stromal cells (BMSCs), endothelial cells and other factors. Accumulating evidence emphasized the importance of Notch signaling in the cross-talk between leukemia cells and their stromal microenvironment. BMSCs were shown to induce upregulation of Notch signaling molecules, such as Notch1, Notch3 and4 or Jagged1/2 and Dll1 ,66. Morepoptosis . The molpoptosis ,71, lymppoptosis . In addiML cells . Our stuML cells .The specific and profound involvement of Notch signaling in various leukemic types makes it an ideal target for pharmacological intervention. Several strategies have been proposed to inhibit or modulate this signaling ,74. The Controversy will remain, as we do not understand the complexity of the Notch pathway and tools to specifically modulate the Notch pathway are still limited. Further studies assessing the levels of Notch activation and inhibition in leukemia still need to be carried out. Further advancement in understanding the molecular events of Notch signaling can potentially lead to further clinical benefit.The authors declare no competing financial interests.All authors have contributed to data preparation, drafting and revising the manuscripts. All authors have read and approved the final manuscript.


Endothelial connexin40 (Cx40) contributes to regulate the structure and function of vessels. We have examined whether the protein also modulates the altered growth of vessels in tumor models established in control mice (WT), mice lacking Cx40 (Cx40−/−), and mice expressing the protein solely in endothelial cells (Tie2-Cx40). Tumoral angiogenesis and growth were reduced, whereas vessel perfusion, smooth muscle cell (SMC) coverage and animal survival were increased in Cx40−/− but not Tie2-Cx40 mice, revealing a critical involvement of endothelial Cx40 in transformed tissues independently of the hypertensive status of Cx40−/− mice. As a result, Cx40−/− mice bearing tumors survived significantly longer than corresponding controls, including after a cytotoxic administration. Comparable observations were made in WT mice injected with a peptide targeting Cx40, supporting the Cx40 involvement. This involvement was further confirmed in the absence of Cx40 or by peptide-inhibition of this connexin in aorta-sprouting, matrigel plug and SMC migration assays, and associated with a decreased expression of the phosphorylated form of endothelial nitric oxide synthase. The data identify Cx40 as a potential novel target in cancer treatment. Angiogenesis, i.e. the growth of new capillaries sprouting from pre-existing blood vessels, is fundamental to many physiological and pathological events, including tumor survival and growth . The mecConnexins (Cx) form channels for the electrical and metabolic signaling, which coordinates the functions of individual cells , notablyin vitro and in vivo effect of a peptide which mimics extracellular portions of Cx40, and specifically targets this protein [We hypothesized that Cx40 may also participate in the angiogenic response of tumoral tissues. To test this hypothesis, we have compared WT mice, expressing control levels of Cx40, with Cx40−/− mice, which lack this Cx  and with protein , 23. TheWe compared knock-out mice which lack Cx40 and transgenic Tie2-Cx40 mice , in whicComparable observations were made after the instillation of TC-1 cells in the bladder of Cx40−/− mice Figure . In contGiven that Cx40−/− mice are hypertensive , 19 and 40Gap27, a peptide known to specifically inhibit Cx40 function [in vivo neo-angiogenesis, whichever the angiogenic stimulus tested. This was further confirmed by the absence of Vascular Endothelial Growth Factor Receptor-2 modulation in TC-1 s.c tumors of Cx40−/− mice , an intense red staining and sizable hemoglobin content of the plugs, indicating extensive angiogenesis, was observed in WT mice Figure . Both pafunction , 23, 25,−/− mice .40Gap27 peptide , were seeded onto matrigel, to form vascular-like 3D structures . The devAfter i.v. injection of FITC-labeled tomato lectin in tumor-bearing mice, we found that more vessels were labeled by the lectin in tumors grown in Cx40−/− than WT mice Figure , indicatSeven, 14 and 21 days after the s.c. implantation of TC-1 cells, we i.p. injected WT and Cx40−/− mice with cyclophosphamide (CTX). In WT mice, this treatment prolonged mice survival (14% of surviving mice at the end of the 90 days experiment), as compared to that observed for untreated WT mice, which all died within 7 weeks Figure . Under t40Gap27, an increased recruitment of A7r5 SMCs to HUVEC-made vessels was observed  underlying the connexin effects remain(s) to be fully unraveled. Knocking down of Cx40 in HUVECs using specific siRNA, was already shown to decrease the formation of capillary-like structures . We now tween EC  which motween EC , or by itween EC . Thus, o2+ [Immunofluorescence showed that the labeling of Cx40 within the ECs of these mice was similar to that seen in WT controls. Accordingly, one would expect that the angiogenic parameters we evaluated here would also be alike in Tie2-Cx40 and WT controls, which is what our data indicate. Obviously, the effects which are dependent on Cx40 do not rule out the possible concomitant involvement of the other EC connexins, whether composing gap junction channels or hemi-channels, notably because Cx40 oligomerizes with Cx37 in some connexons , and tha2+ , 21, 38,2+  and reni2+ . By anal2+  which, i2+ –45, and 2+ . Our datin vivo implantation of artificial plugs) does not support the view that the connexin effect is dependent on a specific cell type or a given host environment. Rather, our data indicate that the loss of Cx40 signaling consistently decreases the development of neo-vessels, while improving their maturation, as judged by the increased coverage of ECs by SMA-expressing contractile cells. Strikingly, this connexin-dependent effect was evident under conditions of actively stimulated angiogenesis in both tumoral and non tumoral tissues, but was not observed under the more chronic, steady state conditions of control vessels renewal, which was presumably the situation in the histologically normal regions of the murine bladders we analyzed. If validated, this differential effect could largely benefit the future therapeutic translation of the experimental approach we describe here. Indeed, since the vessels of human tumors express Cx40 , Cx40−/−  and Tie2The generation of Cx40−/− and Tie2-Cx40 mice and their genotypic characterization has been described , 35. The®, Atacand, Astra Zeneca), and blood pressure was monitored by tail cuff plethysmography method as previously described [In some experiments, WT and Cx40−/− mice received for 3 weeks drinking water containing 3 mg/kg. day Candesartan , were intraperitoneal (i.p.) injected three times, once a week, for 3 weeks  into subcutaneous TC-1 tumor-bearing mice . In otheets Cx40 , 25. The studies , 25.165  and 10 U/ml Heparin [40Gap27 or its scrambled version. The plugs were removed 1 week after injection, for evaluation of vascularization.Plugs of 0.5 mL Growth Factor Reduced matrigel  supplemented with either 500 ng/ml recombinant murine FGF-2  and 3 U/ml Heparin  or 200 ng/ml recombinant murine VEGF Heparin , were s.™-2 BulletKit™, C2517A) was added, and then changed every 3 days. In some experiments, the assay was performed on aortic rings from mice pretreated for 1 week with 100 μg of either peptide 40Gap27 [40Gap27 [Mouse aortas from 10 week-old WT and Cx40−/− mice, were cut into rings of 1 mm thickness , dropped 40Gap27  or a scr[40Gap27  or the sEthanol-fixed aortic rings and 8 μm-thick cryosections of tissues and tumors were incubated in blocking buffer  for 1 h at room temperature, and then incubated overnight at 4°C in PBS with one of the following antibodies against: CD31 , diluted 1:100; Cx40 , diluted 1:50; cleaved VEGFR2 , diluted 1:100; Von Willebrand Factor , diluted 1:500; Phospho eNOS , diluted 1:100; alpha-smooth muscle actin  diluted 1:100. Samples were then incubated for 2 h at room temperature with either Alexa-Fluor-594- or -488-coupled secondary antibodies (Life Technologies), diluted 1:500 in PBS, covered with PBS containing 50% glycerol and 0.4 μg/mL DAPI, and observed by fluorescence microscopy . Frozen sections from human bladder biopsies were also stained with hematoxylin and eosin.Hemoglobin content was quantified using the Drabkin's reagent kit . Briefly, 100 mg tumor powder or excised matrigel-plugs were homogenized in 0.5 mL 0.1% Brij-35 lysis buffer, and centrifuged for 5 min at 10,000 g. 50 μL of the supernatant was mixed with 450 μL Drabkin's reagent, and absorbance read at 540 nm. The concentration of hemoglobin was calculated from a cyan-methemoglobin standard.Mice were intravenously (i.v.) injected with 100 μg FITC-tomato lectin . Five min later, mice were perfused systemically with 4% paraformaldehyde through the left heart ventricle, and then sacrificed before tumor sampling. Tumor tissues were fixed in 4% paraformaldehyde for 2–3 additional hours, and incubated in 30% sucrose overnight at 4°C. The tissues were then embedded in OCT compound, cut at 20 μm thickness and kept frozen at −80°C. CD31 was immunostained as described above. Cell nuclei were counterstained with DAPI, and sections observed by fluorescence microscopy.in vitro angiogenesis assays, Matrigel  was coated onto 24-well culture plates and polymerized for 15 min at 37°C. HUVECs (1 × 105 cells/well) were seeded on Matrigel and incubated at 37°C for 12 h. To evaluate the effects of Cx40 inhibition on this process, similar experiments were conducted in presence of 300 μM 40Gap27 peptide [5 cells/well) were seeded into wells containing HUVECs capillary-like structures generated under control conditions or after addition of 300 μM 40Gap27 peptide or its scrambled version to the media. Motility of SMCs was assessed by time-lapse microscopy using a Cytation3 Cell Imaging Multi-Mode Reader (BioTek). Images were acquired every 5 minutes over 6–8 hours. The quantification of SMC recruitment to capillary-like structures after 6 h was performed using ImageJ software.For  peptide  or its s peptide . For SMC100 μg powder of s.c. B16-F10 tumors or confluent HUVECs collected from a 12-well plate were homogenized by sonication in SDS Lysis Buffer . Protein content was measured using a detergent-compatible protein assay kit . 25 μg proteins were loaded on a 10% polyacrylamide gel, electrophoresed and transferred onto PVDF membrane . Membranes were incubated for 1 h in TBS containing 5% milk and 0.1% Tween20 (blocking buffer). Saturated membranes were incubated overnight at 4°C with the following antibodies, against: Von Willebrand factor , diluted 1:2000; Phospho eNOS , diluted 1:1000; eNOS , diluted 1:1000; VEGFR2 , diluted 1:1000; MMP2 , diluted 1:1000; VE-cadherin , diluted 1:1000; smooth muscle actin  diluted 1:1000 or monoclonal antibody anti-alpha-tubulin . Membranes were then incubated at room temperature for 1 h with a convenient secondary antibody conjugated to horseradish peroxidase . Bands were developed using enhanced chemiluminescence , and visualized using a supercooled CCD camera . Densitometric analysis was performed using ImageLab Software (3.0.1 Bio-Rad Laboratories).t test whichever applicable. Multiple comparisons were performed using one-way ANOVA and Tukey's Multiple Comparison Test. Other statistical tests are indicated in the text or figure legends.All tissue staining measurements were made using the ImageJ software . Statistical analyses were performed using Prism 6.00 for Windows . Differences between groups were performed by Mann-Whitney test or by Student


Clustering of receptors associated with immunoreceptor tyrosine-based activation motifs (ITAMs) initiates the macrophage antimicrobial response. ITAM receptors engage Src-family tyrosine kinases (SFKs) to initiate phagocytosis and macrophage activation. Macrophages also encounter nonpathogenic molecules that cluster receptors weakly and must tune their sensitivity to avoid inappropriate responses. To investigate this response threshold, we compared signaling in the presence and absence of receptor clustering using a small-molecule inhibitor of Csk, which increased SFK activation and produced robust membrane-proximal signaling. Surprisingly, receptor-independent SFK activation led to a downstream signaling blockade associated with rapid degradation of the SFK LynA. Inflammatory priming of macrophages upregulated LynA and promoted receptor-independent signaling. In contrast, clustering the hemi-ITAM receptor Dectin-1 induced signaling that did not require LynA or inflammatory priming. Together, the basal-state signaling checkpoint regulated by LynA expression and degradation and the signaling reorganization initiated by receptor clustering allow cells to discriminate optimally between pathogens and nonpathogens.DOI:http://dx.doi.org/10.7554/eLife.09183.001 Macrophages are white blood cells that protect the body from infection by bacteria and other microbes. Proteins and sugars on the microbe bind to receptor proteins on the surface of the macrophage, which triggers the macrophage to engulf the cell and destroy it. Macrophages also release molecules that are toxic to the microbe and activate other immune responses in the body. It is vital that macrophages can tell the difference between normal body cells and microbes because if macrophages are activated at the wrong time, they can damage tissues and cause inflammatory diseases.When the receptor proteins are activated by contact with a microbe, they interact with a family of proteins called SFKs, which in turn stimulate communication systems called signaling pathways inside the macrophages that activate their immune responses. However, these responses are only triggered if many receptors are activated and clustered together. Thus, macrophages are able to identify whether a cell is a normal part of the body or is a foreign invader by sensing the number of receptors that cluster together as they bind to the cell. However, it is not clear if it is the clustering itself that signals a genuine encounter with a microbe, or whether this information comes from the strength of the response by the SFK proteins.Here, Freedman et al. addressed this question by developing a new experimental system that allows SFKs to be directly activated in macrophages in the absence of receptor clusters. The experiments used macrophages obtained from genetically engineered mice and found that the direct activation of SFK proteins alone does not fully activate signaling pathways in the macrophages. A signaling blockade occurs due to the rapid destruction of an SFK protein called LynA. When the macrophages were exposed to molecules that are signals of inflammation in the body, they produced more LynA. This allowed these macrophages to be activated even without the formation of receptor clusters by interactions with pathogens.Freedman et al.'s findings reveal that LynA acts as a checkpoint that primes macrophages to respond more aggressively when the body is under attack. Future work will be aimed at understanding how LynA is destroyed and how the detection of real pathogens overcomes the checkpoint.DOI:http://dx.doi.org/10.7554/eLife.09183.002 Macrophages are myeloid-derived hematopoietic cells that guard against infection and orchestrate a broad immune response to pathogens. Bridging the innate and adaptive immune systems, they survey tissues for signs of pathogen invasion and present antigen to T cells. When activated, they engulf and destroy foreign organisms and secrete cytokines . While mMacrophage stimulation through immunoreceptor tyrosine-based activation motif (ITAM)- and hemi-ITAM-coupled receptors initiates pathogen destruction via phagocytosis and the production of reactive oxygen species, which require activated macrophages to be physically close to the pathogen. To prevent nonproductive activation and tissue damage, macrophages must differentiate between receptor ligands displayed during a pathogen encounter  vs. environmental noise . One mechanism by which cells make this distinction is by using receptor clustering to sense the size of a potential stimulus. Large-diameter stimuli that are more likely to be associated with pathogenic particles nucleate large-scale receptor clusters, forming a signaling complex called a phagocytic synapse. In contrast, small-diameter stimuli induce only small-scale or transient receptor clusters . HereaftReceptor engagement initiates interactions with an active pool of Src-family tyrosine kinases (SFKs), which phosphorylate intracellular ITAMs within the receptor, the tyrosine kinase Syk, cytoskeleton-associated proteins, and other membrane-proximal signaling proteins . This meTo some extent, macrophages can modulate their responsiveness to activating stimuli according to their environment. For example, inflammatory cytokines secreted by other immune cells trigger a process termed inflammatory priming, a transcriptional program that increases macrophage reactivity. Priming is known to upregulate the expression of proteins involved in signal detection, pathogen killing, and stimulation of other immune cells . HoweverIt is also unclear whether strong macrophage stimuli are defined by their ability to robustly activate the SFKs or whether receptor clustering itself relays qualitative information that indicates a genuine pathogen encounter. Clustered receptors are immobilized within the cell membrane, participate in costimulatory interactions, and organize the signaling complex spatially, integrating SFK-dependent and SFK-independent steps into a productive signaling response . Until nAS’) that is sensitized to inhibition by a 3-iodobenzyl analog of the kinase inhibitor PP1 (3-IB-PP1). The bulkiness of 3-IB-PP1 prevents its interaction with WT Csk and other WT kinases  to SFK signaling induced by receptor clustering (by ligating the hemi-ITAM receptor Dectin-1 with depleted zymosan). Receptor-independent SFK activation by 3-IB-PP1 induced robust membrane-proximal signaling but no downstream signaling through the MAPKs or Akt. We determined that this signaling blockade was caused by rapid degradation of the SFK LynA, which resulted in a loss of function that could not be compensated for by the other SFKs. We were able to rescue downstream signaling by priming the macrophages, which led to the upregulation of LynA. Receptor clustering enabled the participation of the other SFKs in the activation of downstream MAPK, Akt, and calcium signaling independently of LynA. From the data presented in this article, we propose a model to explain how macrophages are prevented from responding to weak stimuli, how inflammation increases macrophage sensitivity to weak stimuli, and how receptor clustering rewires SFK signaling to enable macrophage activation.We derived macrophages from the bone marrow of CskAS mice and verified that they express normal levels of myeloid and macrophage surface markers  from  markers . Within rylation , 3-IB-PPdep . To test the effects of inflammatory priming on SFK-mediated signaling, we exposed BMDMs to IFN-γ for 12–16 hr prior to treatment with zymosanough Erk , arrow aough Erk , arrows.Priming BMDMs with IFN-γ generally increased membrane-proximal signaling in response to 3-IB-PP1. We observed more robust phosphorylation of DAP12, Syk, LAT, PLCγ1, PLCγ2, Cbl, PI3K, HS1, Vav, and Fak in primed cells than in unprimed cells . Pulse-sdep and was therefore kinetically uncoupled from downstream signaling through Erk and Akt. Additionally, even the transient, low-level proximal signaling in response to 3-IB-PP1 in unprimed cells was quantitatively equivalent to or higher in magnitude than the signaling in response to zymosandep. Finally, as mentioned above, cotreatment with 3-IB-PP1 suppressed signaling through the CSF-1R pathway  was more sustained in primed cells . This ob pathway . TogetheWe first considered the possibility that signaling proteins involved in negative feedback, themselves activated by the SFKs, are somehow bypassed in primed cells treated with 3-IB-PP1. Surprisingly, we found that priming actually enhanced the phosphorylation of proteins that negatively regulate signaling, including the ITIM-containing protein Sirpα, the inositol phosphatase SHIP1, and the tyrosine phosphatase SHP-1 , which lAS BMDMs by means of direct cross-talk between IFN-γ and Erk signaling pathways or by a priming-mediated transcriptional change. We therefore treated BMDMs with IFN-γ in combination with cycloheximide to block translation, but we found that cycloheximide exposure was toxic and blocked all signaling through Erk. As an alternative, we tested different IFN-γ incubation times to look at the kinetics of the signaling enhancement. We found that 3-IB-PP1 failed to induce robust Erk phosphorylation in cells treated with IFN-γ for 4 hr or less  and a phosphorylation site (Y32)  Stanley. We therHaving established that LynA is selectively targeted for degradation, we looked more closely at the activation kinetics of the SFKs during the course of 3-IB-PP1 treatment . We idenAlthough both Lyn and Hck lost inhibitory-tail phosphorylation during 3-IB-PP1 treatment, the inhibitory tail of Lyn was dephosphorylated preferentially (5% Lyn vs 24% Hck tail phosphorylation remaining after 1 min) . SimilarThe active form of LynA was short-lived during 3-IB-PP1 treatment , mirroridep or 3-IB-PP1 treatment. In unprimed BMDMs treated with 3-IB-PP1, LynA was almost completely degraded within this time frame. LynA protein was also subject to degradation in IFN-γ-primed cells during 3-IB-PP1 treatment, but due to its increased basal expression LynA protein persisted at significant levels through the 3- to 5-min time frame of Erk activation  and compared this to signaling induced by receptor clustering (mediated by the Dectin-1 ligand zymosandep). Treatment with 3-IB-PP1 induced robust membrane-proximal signaling, but signal propagation was blocked downstream of PLCγ and PI3K, with no apparent activation of Erk, JNK, Akt, or NFAT. We determined that propagation of receptor-independent signaling induced by 3-IB-PP1 requires the SFK LynA, but we observed that LynA protein was almost completely degraded within the first three minutes after 3-IB-PP1 addition. This blockade was overcome by priming macrophages with IFN-γ or GM-CSF before 3-IB-PP1 treatment, which led to increased expression of LynA, thus buffering it against total degradation and leading to a more sustained presence of LynA during the first minutes of 3-IB-PP1 treatment. The other macrophage SFKs, Hck and Fgr, were also activated in response to 3-IB-PP1 treatment but were not capable of compensating for the lack of LynA activity after LynA degradation or in Lyn-deficient cells. In contrast, receptor clustering by zymosandep induced signaling through the MAPKs and Akt independently of LynA function and inflammatory priming.In this study, we examined signaling in BMDMs induced by activating the SFKs in the absence of receptor clustering , FITC-conjugated anti-F4/80 , and PE-conjugated CD11b . BD and eBioscience antibodies to CD45 were used as single-stained controls. Data were collected on a BD LSRFortessa flow cytometer running FACSDiva software and analyzed in FlowJo . Figures were made in Adobe Creative Suite .dep particles per ml were stored at −20°C and thawed, briefly sonicated, pelleted, and resuspended before use.Zymosan (Sigma) was depleted of TLR2 agonist as described previously . Brieflydep, all stimuli were applied by pulse spinning at 37°C. BMDMs were stimulated with 10 zymosandep particles per cell 1640 medium were added to adherent BMDMs at 37°C. In experiments with zymosanper cell , 10 μM 3nhibitor , 2–50 ngnhibitor , 20 μM Pnhibitor  or the a4, 0.01 mg/ml Aprotinin, 0.01 M NaF, 0.01 mg/ml Leupeptin, 0.01 mg/ml Pepstatin A, 2 mM phenylmethanesulfonyl fluoride (PMSF), and 0.4 mM EDTA. After scraping the plates, cells and detergent were incubated 30 min on ice. The lysate was then cleared by ultracentrifugation for 15 min at 50,000 rpm at 4°C in a Beckman  TLA120.2 rotor. The lysates were precleared for 30 min at 4°C with Protein G Sepharose beads  and normal rabbit or goat serum as appropriate . Protein G-Sepharose beads were covalently conjugated to Lyn or Hck antibodies (see table below) using dimethyl pimelimidate (Sigma). Antibody-bound beads were added to the lysate and mixed 1.5 hr at 4°C to immunoprecipitate Hck or Lyn. Finally, the samples were applied to micro bio-spin chromatography columns , washed, and eluted with SDS Sample Buffer.Stimulated BMDMs were lysed in 1% Lauryl Maltoside Lysis Buffer containing 150 mM NaCl, 0.01% sodium azide, and 10 mM Tris, pH 7.6 with 2 mM NaVO3. Antibodies were applied in solutions containing 20 mM Tris, pH 8.0; 125 mM NaCl; and 0.05% Tween-20 (plus 2% BSA for primary or 0.5% powered milk for secondary antibody solution). High-stringency washes after antibody binding were in similar Tris-buffered saline and Tween-20 (TBST) buffer with 200 mM NaCl. Antibodies were obtained from Cell Signaling Technology , Santa Cruz , ProMab Biotechnologies , Promega , Life Technologies, and Sigma-Aldrich). Primary staining was performed with the following antibodies:After transfer from SDS-PAGE to Immobilon P membrane (EMD Millipore), blots were blocked with 3% bovine serum albumin (BSA) in 25 mM Tris, pH 8.0; 125 mM NaCl; and 0.02% NaNHorseradish peroxidase- (HRP) conjugated secondary antibodies were from Southern Biotech , and blots were developed using Thermo Scientific SuperSignal Femto reagent. Blots were visualized on a Kodak Imagestation. Some blots were reprobed after HRP inactivation by azide treatment and freezing.Where applicable, immunoblots were analyzed quantitatively by performing densitometry with ImageJ software . Briefly, images were background-subtracted, and strips of the blot corresponding to each band were demarcated and analyzed for each time point/gel lane. Error bars reflect independent analysis of blots from at least three independent experiments. Figures were prepared in Graphpad Prism . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled “LynA regulates an inflammation-sensitive signaling checkpoint in macrophages” for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.This manuscript reports an interesting phenomenon associated with macrophage priming. Using a technique that allows them to pharmacologically inhibit the CSK kinase, they report rapid activation of SFK activity and robust phosphorylation of immediate downstream effectors but inability to activate ERK, suggesting some type of block. This is in contrast to activation by zymosan, which achieves similar levels of phosphorylated downstream intermediates but also allows for strong ERK activation. The ability of CSK inhibition to activate ERK could be overcome by macrophage priming suggesting that priming allows for some rewiring. Analysis of the mechanism revealed that CSK inhibition activates SFK and results in preferential inhibition of the LynA isoform. Presumably, the loss of LynA prevents a sustained enough of a signal to allow for ERK activation.There are many important findings in this manuscript. First, the data clearly show how dynamic the tyrosine phosphorylation of the SFK is as changes in phosphorylation are seen after 3 seconds. Second, the data show fundamental difference in LynA vs LynB, suggesting that LynA is responsible for positive signaling while LynB is responsible for negative regulator signaling. Third, it is shown that LynA is preferentially phosphorylated by CSK compared to other SFK and that LynA is also preferentially degraded. Fourth, the manuscript shows how rapidly SFK are lost after their activation. This is a little studied but important area. Lastly, the manuscript shows that an important effect of priming is up regulation of Lyn expression.The manuscript could be improved by addressing several issues that are raised by the data:1) The LynA degradation mechanism controls macrophage activation in response to pharmacological Csk inhibition, but does it also apply in response to a physiological stimulus?2) It is stated that the increased expression of LynA after priming results in delayed degradation. Is it really delayed degradation or that the larger pool is able to sustained signaling longer because it takes longer to degrade to the threshold level.3) It is proposed that LynA has an insert domain that contains a potential ubiquitination site and that this may account for the increased degradation of LynA compared to LynB. Is there any evidence to support this? Moreover, the authors point to a potential site of ubiquitination in this insert domain – is this site functionally relevant? Would a K40 mutant prevent LynA ubiquitination and degradation, and rescue activation of Erk and Akt.4) Are both LynA and B unregulated after priming? If so, why doesn't the increased expression of LynB attenuate or block the increased signaling mediated by LynA?5) If the inability of CSK inhibition to activate ERK is via increased degradation of LynA, it might be predicted that the kinetics of phosphorylation of immediate downstream substrates might be more transient. Is it?6) The authors need to discuss the possibility that the effect of interferon-γ is simply quantitative. Is it possible that the ability of the cytokine to enable some activation of Erk and Akt is simply due to increased tyrosine phosphorylation? The manuscript could be improved by addressing several issues that are raised by the data:1) The LynA degradation mechanism controls macrophage activation in response to pharmacological Csk inhibition, but does it also apply in response to a physiological stimulus?dep. Representative data are shown in the To mimic a soluble, physiological stimulus like cell debris, we treated BMDMs with laminarin, a soluble form of β-glucan that interacts with Dectin-1 but forms sub-threshold-sized receptor clusters . These ndep  It is stated that the increased expression of LynA after priming results in delayed degradation. Is it really delayed degradation or that the larger pool is able to sustained signaling longer because it takes longer to degrade to the threshold level.Thank you for pointing out that our argument was not clear in the initial manuscript. We were actually arguing that the larger pool of LynA is able to sustain signaling longer because it takes longer to degrade to the threshold level. We have updated the manuscript to resolve this confusion. Please refer to the last paragraphs of the Results and first paragraph of the Discussion.3) It is proposed that LynA has an insert domain that contains a potential ubiquitination site and that this may account for the increased degradation of LynA compared to LynB. Is there any evidence to support this? Moreover, the authors point to a potential site of ubiquitination in this insert domain – is this site functionally relevant? Would a K40 mutant prevent LynA ubiquitination and degradation, and rescue activation of Erk and Akt.ASBMDMs treated with 3-IB-PP1. These data have been added as a new figure supplement (We have strengthened our evidence for direct modification of LynA by ubiquitination (in contrast to Hck and the inactive forms of the SFKs) by using immunoblotting to detect laddering of LynA and activation-loop-phosphorylated SFKs in whole-cell lysates from Cskpplement . Please We do not yet have direct evidence that ubiquitination at the unique residue K40 targets LynA specifically for degradation, although we mention that this is the only lysine residue present in LynA and absent in LynB.-/- cells was toxic, whereas cells transduced instead with LacZ or Hck survived blasticidin selection. Although we were disappointed that we encountered this technical limitation, this observation of differential survival does support our model, since enhancing Lyn activity appears to affect the basal state of macrophages but enhancing Hck activity does not.We attempted to obtain experimental support for our model by infecting mouse bone marrow with lentivirus containing LynA, LynAK40R, Hck, or LacZ control DNA. We expected to observe enhanced signaling in response to 3-IB-PP1 in cells overexpressing LynA or expressing any LynAK40R, which lacks the uniquely predicted ubiquitination site. In fact, we found that the introduction of LynA or LynAK40R into WT or even LynWe are planning future experiments involving mass spectrometry, mutation of the other ubiquitination sites of Lyn, and using CRISPR-Cas methodology to knock the Lyn mutation into macrophages while maintaining physiological expression levels. These lines of inquiry, however, lie beyond the scope of this study and the appropriate timeframe for resubmission.4) Are both LynA and B unregulated after priming? If so, why doesn't the increased expression of LynB attenuate or block the increased signaling mediated by LynA?LynA and LynB are indeed both upregulated equally during priming, as we show in 5) If the inability of CSK inhibition to activate ERK is via increased degradation of LynA, it might be predicted that the kinetics of phosphorylation of immediate downstream substrates might be more transient. Is it?dep, so quantity of signaling cannot be the complete story. Please see the subsection “Priming macrophages with inflammatory cytokines overcomes their dependence on receptor clustering for downstream signaling”.We appreciate the reviewers' observation and have amended the manuscript to point this out. Indeed (as shown in 6) The authors need to discuss the possibility that the effect of interferon-is simply quantitative. Is it possible that the ability of the cytokine to enable some activation of Erk and Akt is simply due to increased tyrosine phosphorylation?This comment is closely related to our discussion above of major point 5. The reviewers are correct that there are quantitative differences in signaling in primed vs. unprimed cells, depending on the level of LynA persisting during the first 5 min of 3-IB-PP1 treatment . The qua


HTLV I is a retrovirus that can result in decreased effectiveness of immune response and an increased incidence of coinfections. HTLV I CNS coinfections with rare clinical manifestations that may relate to impaired cellular immunity are presented.Case1: a 15-year-old patient presented with difficulty in standing up and walking on her own , with visual disturbances, and refractory epileptic seizures; travel history included trips to the Caribbean (Isla Margarita).Case2: a 24-year-old patient presented with difficulty in standing up and walking without support, with sudden hearing loss, peripheral paresthesia, and generalized weakness, after having experienced grand mal seizures several days before; travel history included trips to the Caribbean (her fiancé was captain of a cruise ship), the Amazon rainforest and Antarctica.Case1: brain MRI imaging revealed localized, non-Gadolinium enhancing lesions in the right thalamus and occipital lobe and cortex edema. EBV DNA and HTLV I/II RNA were detected in the CSF and EBV VCA IgG antibodies in the serum. Following administration of supportive treatment and gancyclovir IV for EBV encephalitis, the patient was discharged with residual mobility impairment.Case2: brain MRI imaging revealed several medium sized, non-Gadolinium enhancing lesions in the white matter of both hemispheres . India ink examination and latex agglutination for Cryptococcus spp. were positive in the CSF. Cryptococcus gattii was consequently isolated from CSF cultures. HTLV I/II antibodies and HTLV I/II RNA were both detected in the serum only. Following a 4-month hospitalization, the patient was discharged with clinical improvement, but with relative speech and mobility impairment.Neurologic manifestations can be encountered in HTLV I/II monoinfected and coinfected patients, regardless of HAM/TSP. In CNS coinfections, the definite diagnosis for HAM/TSP cannot be established by current diagnostic criteria, reflecting the need for further criteria to also include coinfections. Quantitation of HTLV I/II proviral load and investigation of factors related to cellular immunity might augment diagnosis and prognosis.