BackgroundThe cell type(s) mediating the maternal influence on allergic disease in children remain unclear. We set out to define the relationship between maternal allergy and frequencies of cord blood (CB) basophils, and plasmacytoid dendritic cells (pDCs); to characterize surface-bound IgE and FcεRI expressions on these cells; and to investigate the association between maternal and CB serum IgE levels with surface-bound IgE and FcεRI expressions.MethodsOne hundred and three mother/infant dyads were recruited prenatally, and maternal allergic history was recorded. Maternal blood was collected prior to delivery, and CB was collected after birth. Flow cytometry was used to identify CB basophils and pDCs and to determine surface-bound IgE and FcεRI expressions.ResultsFrequencies of CB basophils and pDCs were low and not related to maternal history of allergy. Percentages of CB basophils with surface-bound IgE were significantly higher in infants of allergic mothers compared with infants of non-allergic mothers . IgE on CB basophils correlated with CB IgE levels , but not with maternal IgE levels . IgE on CB pDCs was low and not significantly associated with maternal or CB IgE levels. Similarly, FcεRI expression by CB basophils and pDCs was not significantly associated with maternal or CB IgE levels.ConclusionsFrequencies of CB basophils and pDCs are not influenced by maternal allergy. CB basophils and pDCs have surface-bound IgE and express FcεRI; however, only IgE on CB basophils appears influenced by maternal allergy. Recruiting strategy and eligibility criteria are described in the electronic repository (ER).Pregnant women were divided into two groups based on the absence or presence of allergic disease defined by a physician’s diagnosis of asthma, allergic rhinitis, atopic dermatitis, or food allergy, and associated symptoms  within the past 12 months. The study was approved by the Institutional Review Board at Hartford Hospital, and informed written consent was obtained from all prospective mothers and on behalf of their infants.Upon admission to Labor and Delivery, maternal blood was obtained by venipuncture. Following delivery, CB samples were obtained from the umbilical cord-placenta unit by means of needle puncture of the umbilical cord vein cleansed with alcohol. Total serum IgE concentrations were determined by Phadia  using the ImmunoCap method. The detection limit for IgE in maternal and CB specimens was 2.00 KU/l and 0.10 KU/l, respectively. As a surrogate marker for maternal blood contamination, sera from CB samples were analyzed for total IgA levels using a commercial ELISA kit . The detection limit for IgA was 0.50 μg/ml (based on a serum dilution of 1:100), and CB samples containing ≥10 μg/ml IgA were excluded from the study Cord blood was obtained for isolation and cryopreservation of CB peripheral blood mononuclear cells (CBMCs) using a procedure adapted from the Immune Tolerance Network (PBMC CPT v.009) online protocol and a published protocol by Basford et al. +high BDCA-2- and CD123+high BDCA-2+ cells, respectively Cord blood basophils and pDCs were identified as the CD123In some experiments, CBMCs were pre-incubated with 50 μg monoclonal human IgE (HE1)  or 400 IU WHO IgE preparation  for 1 h at 18–25°C prior to staining. This was carried out to investigate the ability of CB basophils and pDCs to bind exogenous IgE and whether or not bound IgE blocked binding of the FcεRI detection antibody.To determine the primary outcome of the relationship between maternal allergy and levels of CB basophils and pDCs, CB samples containing a minimum of 200 basophils and 200 pDCs were analyzed. Frequencies of CB basophils and pDCs were expressed as percentages of total CBMCs. To determine secondary outcomes, CB basophils and pDCs were analyzed for surface-bound IgE and FcεRI expressions. Percentages of CB basophils and pDCs identified by flow cytometry with bound IgE or that expressed FcεRI were compared between infants of allergic and non-allergic mothers. In addition, the association between maternal and CB serum IgE levels with surface-bound IgE and FcεRI expressions was investigated.Statistical analyses were performed using Prism 4 . For measured variables, statistical comparisons were performed by the nonparametric Mann–Whitney test. Chi-square was used to compare the distribution of weight gain between allergic and non-allergic mothers, and the Fisher’s exact test was applied to compare the remaining categorical variables between groups. Correlation analyses were performed using Pearson’s correlation. Statistical significance was defined as a p-value ≤ 0.05.The number of mother/infant dyads recruited, enrolled, and excluded is described in Pregnant women with a history of allergic disease demonstrated higher levels of serum total IgE than pregnant women without a history of allergic disease  . CB IgE Frequencies of CB basophils and pDCs for the different groups of infants are shown in We first determined the ability of CB basophils and pDCs to bind exogenous IgE. CBMC preparations were pre-incubated in the absence or presence of exogenous IgE. Following pre-incubation, cells were stained to identify CB basophils and pDCs, and to define each cell type’s capacity to bind IgE or level of FcεRI expressions. CB basophils and pDCs from a patient with low levels of endogenous surface-bound IgE were capable of binding monoclonal human IgE (HE1) or polyclonal WHO IgE . FurtherWe next determined the percentages of CB basophils and pDCs with bound IgE or FcεRI expression from study subjects. The percentages of CB basophils with bound IgE were significantly greater in infants of allergic mothers compared with infants of non-allergic mothers  . There wTo examine the association between maternal and CB serum IgE levels with bound IgE and FcεRI expressions by CB basophils or pDCs, data from the allergic and non-allergic mother/infant dyads were grouped together. There were no significant correlations between maternal serum IgE levels and percentages of CB basophils or pDCs with bound IgE  (data not shown). In contrast, there was a significant correlation between CB IgE levels and percentages of CB basophils with bound IgE  of total IgE in the fetal compartment. Screening for cell-bound IgE, in addition to other parameters such as CB IgE


Callithrix jacchus) monkey as a “super-model” of neuroscientific research. Here, we established organotypic tissue culture of the adult marmoset monkey retina with particle-mediated gene transfer of GFP to survey the morphological diversity of RGCs. We successfully incubated adult marmoset monkey retinas for 2 to 4 days ex vivo for transient expression of GFP. We morphologically examined 121 RGCs out of more than 3240 GFP-transfected cells in 5 retinas. Among them, we identified monostratified or broadly stratified ganglion cells , and bistratified ganglion cells . By this survey, we also found a candidate for bistratified DSGC whose dendrites were well cofasciculated with ChAT-positive starburst dendrites, costratified with ON and OFF ChAT bands, and had honeycomb-shaped dendritic arbors morphologically similar to those in rabbits. Our genetic engineering method provides a new approach to future investigation for morphological and functional diversity of RGCs in the monkey retina.The mammalian retina has more diversity of neurons than scientists had once believed in order to establish complicated vision processing. In the monkey retina, morphological diversity of retinal ganglion cells (RGCs) besides dominant midget and parasol cells has been suggested. However, characteristic subtypes of RGCs in other species such as bistratified direction-selective ganglion cells (DSGC) have not yet been identified. Increasing interest has been shown in the common marmoset ( Callithrix jacchus) monkey has become a “super-model” of neuroscientific research ex vivo for the marmoset monkey retina have been established, the methods should be powerful genetic engineering tools ex vivo for investigating monkey retinas. The aim of this study was to establish an organotypic tissue culture of the adult marmoset monkey retina with a genetic engineering technique of transient gene expression for surveying morphological diversity of RGCs.The mammalian retina has more diversity of retinal neurons than scientists had once believed in order to establish complicated vision processing Callithrix jacchus), ranging in weight from 250 to 300 g, obtained from CLEA Japan Inc. and maintained in the Marmoset Research Facility, National Institute for Basic Biology (NIBB) Bioresouce Center. Animals were housed indoors at Marmoset Research Facility of NIBB, in stainless lattice cages (69×65×72 cm or 104×65×154 cm) that permitted auditory and olfactory contact between animals in different groups. The cages contained fabric hammocks, wooden perches and nest boxes. The animals were fed CMS-1M  supplemented with vitamin D, vitamin C, Lactobacillus preparation and honey at 12∶30–13∶30 h; in addition, food was usually available in the cages at all times. Marmoset jelly , boiled quail eggs, Calorie Mate block  and apples were provided as daily special additional diet. Water was available ad libitum. Lights were on from 6∶30 to 18∶30 h, and room temperature and humidity were maintained at approximately 24°C and 30–70%, respectively.Experiments were carried out on four  adult common marmosets  code of practice for the care and use of animals and were approved by the institutional ethics committee of NIPS (No. 11A172) and by Animal Experimental Committee of National Institutes of Natural Sciences .2/5% CO2) Ames' medium (Sigma-Aldrich) prior to hemisection. The vitreous was removed with fine forceps. When it was difficult to remove the vitreous, opened eyecups were soaked for 10 min in hyaluronidase  -containing carboxygenated Ames' medium. Retinas were teased off the sclera and the pigment epithelium with fine forceps.Animals were pretreated with medetomidine  and ketamine  and were then overdosed with sodium pentobarbital intrapenetorially or intracardially . The eyes were removed and placed in carboxygenated  over the ganglion cell side. During culture in the CO2 incubator, the medium was agitated constantly at 55 rpm using an orbital shaker  and was exchanged daily.Adult marmoset retinal culture was perfomed according to the published protocol for the interphase culture system of adult rodent retinas Gene gunning was carried out as previously described To evaluate retinal cell viability after culture, we labeled cells undergoing apoptosis (and dead cells) with YO-PRO-1 (Invitrogen) after 3-day culture of marmoset retinas as previously described Forty-two-hour-cultured retinas were fixed in phosphate buffered saline (PBS), pH 7.4, containing 4% paraformaldehyde for 1 hour at room temperature. For vertical sections shown in Images were captured using a confocal laser scanning microscope . Nikon PlanApo VC20x and 40x lenses were used. Stacks of confocal digital images at 0.2- or 0.5-μm z-axis spacing were acquired with appropriate filter sets. The brightness and contrast of the images were adjusted by using ImageJ  or Photoshop CS (Adobe). Confocal z-stacks were imported into Imaris software (Bitplane) and maximum intensity projection was performed using the section view.Tracings of the dendritic tree and the soma of each ganglion cell were made using Neurolucida software (MicroBrightField) as previously described 2, 1 mM MgCl2, and 11 mM EGTA (pH adjusted to 7.2 with KOH). A piece of retina was placed in a recording chamber, ganglion cell layer up, and continuously perfused at a rate of 1 to 2 mL/min with oxygenated extracellular solution . The extracellular solution was continuously oxygenated with 5% CO2/95% O2 and kept between 32 and 35°C. The recording pipette was connected to the input stage of a patch-clamp amplifier, Axopatch 200B (Axon Instruments), and signals were sampled at 10 kHz with DigiData 1322A interface-type and pCLAMP8 software (Axon Instruments). The liquid junction potential was measured as 11 mV (Vm  =  Vp−11 mV) and corrected after recordings. Subsequent analysis was done by custom-made procedures in Igor Pro (WaveMetrics).Conventional recording techniques were used for patch-clamp recordings as described previously GFP-transfected ganglion cells, each piece of the cultured marmoset retinas was stained with ChAT antibody, and density of ON SAC in each part was analyzed.To determine retinal eccentricities and dendritic stratification in the inner plexiform layer (IPL) of As in previous reports 2 (up to 1.5 mm from the fovea by our estimation), “mid-peripheral retina” refers to density of ON SAC of 650–900 cells/mm2 (>1.5 mm to 4 mm by our estimation), and “far peripheral retina” refers to density of ON SAC below 650 cells/mm2 (>4 mm by our estimation).Since not all of the small pieces of cultured retina included the fovea, retinal eccentricity of the observed cells was estimated retrospectively by determining the density of ChAT-immunopositive SAC in comparison with the density data for ChAT-immunopositive SAC as shown in In the vertical section of the retina , ON and To quantify dendritic field size of each ganglion cell, a convex polygon was drawn by linking the outermost tips of dendrites, and the area was measured by ImageJ (NIH). Then the area was converted to diameter by assuming a circular dendritic field , 4 and 5GFP-F-transfected cells and ChAT-positive starburst amacrine cells, shown in To check the cofasciculation of dendrites between All analyses were performed by ImageJ (NIH).2 incubator at 37°C. Particle-mediated acute gene transfer (“gene gun”) was conducted from the retinal ganglion cell side. In the interphase chamber, we successfully incubated adult marmoset monkey retina tissue for 2 to 4 days ex vivo. Although light responses were not observed after incubation for several days because of loss of photosensitivity under room light, retinal ganglion cells in incubated retina tissue on which patch clamping was successfully performed were all electrophysiologically active (n = 7) and showed action potentials responding to current injection as well as transient Na+ currents under voltage clamp recordings after 72-hour incubation  with YO-PRO-1 after 3-day (72 hours) culture of marmoset retina pieces GFP variant (GFP-F) We transfected a As shown in GFP-transfected cells in 5 retinas (8.13 cell/mm2) as shown in GFP-transfected cells did not imply actual density of the populations, we counted the cells of each subtype out of the 121 identified cells  To find a candidate for bistratified DSGC, we searched for cells with the following criteria GFP transfection revealed that diverse subtypes of retinal ganglion cells exist in addition to the dominant midget and parasol cells in the marmoset monkey retina. By this surveying method, we found a candidate for bistratified DSGC in the marmoset monkey retina. The advantage of the incubation and GFP-F gene transfection method is that hundreds of retinal ganglion cells per a retina piece can be labeled simultaneously. The method could be easily combined with immunohistochemistry. Thus, it enabled us to find a rare subtype of ganglion cells in the marmoset monkey retina as a candidate for directional selective bistratified ganglion cells whose dendrites were costratified and cofasciculated with ChAT-immunopositive bands. Since we used a CMV promoter for expressing GFP-F in this study, the gene expression itself was not selectively targeted for certain cell types.Our organotypic tissue culture of the adult marmoset monkey retina with genetic engineering methods opens a new era of retinal neuroscience of the adult monkey retina. By combining with transient gene transfection using particle-mediated gene transfer, durable use of such a valuable material could be maximized. In the present study, an extensive survey of more than three thousands of retinal ganglion cells by Candidates for directional selective ganglion cells in primates have been previously suggested by their characteristic morphologies, such as “G11 cells” in the human retina by Kolb and coworkers Electrophysiological recordings must be done to confirm this cell's functional property in the marmoset monkey retina. To do so, we need to overcome problems such as how to maintain light-responsiveness during the organotypic culture. Light-responsiveness tended to be diminished during the organotypic culture because the pigment epithelium was removed. One possible attempt could be to add retinal to the incubation medium to keep photocycles of photoreceptors active. If light-responsiveness can be successfully maintained, our genetic engineering method would provide a new approach to future investigation for morphological and also functional diversity of RGCs in the monkey retina.


To prospectively explore the therapeutic use of these cells, CD8+ cells were isolated from patients that had been treated with cellular immunotherapy and IMRT, expanded in vitro, and injected into recurrent metastatic sites in 13 individuals who underwent the same immunoradiotherapeutic regimens but failed to respond. CRs were achieved in 34 of 58 of such recurrent lesions while PRs in 17 of 58. These data support the expanded use of immunoradiotherapy in advanced cancer patients exhibiting progressive disease.Patients afflicted with advanced cancers were treated with the intratumoral injection of autologous immature dendritic cells (iDCs) followed by activated T-cell infusion and intensity-modulated radiation therapy (IMRT). A second round of iDCs and activated T cells was then administered to patients after the last radiation cycle. This complete regimen was repeated for new and recurring lesions after 6 weeks of follow-up. One year post therapy, outcome analyses were performed to evaluate treatment efficacy. Patients were grouped according to both the number and size of tumors and clinical parameters at treatment initiation, including recurrent disease after standard cancer therapy, Stage IV disease, and no prior therapy. Irrespective of prior treatment status, 23/37 patients with ≤ 5 neoplastic lesions that were ≤ 3 cm in diameter achieved complete responses (CRs), and 5/37 exhibited partial responses (PRs). Among 130 individuals harboring larger and more numerous lesions, CRs were observed in 7/74 patients that had received prior SCT and in 2/56 previously untreated patients. Some patients manifested immune responses including an increase in CD8 One approach has been to conduct clinical trials to develop new drugs targeting a particular type of cancer. These drugs are often, if not routinely, pushed to a dosage nearing toxic levels in order to achieve an antitumor response. In many cancer centers, an alternative approach with considerably less toxicity (and thus morbidity) is being explored, an approach designed to stimulate antitumor immune responses in the host. These studies are progressing along with advances in our understanding of the complexities of the immune response and the potential to manipulate these responses to constrain malignant disease. One of the important recent advances in this area is the recognition that dendritic cells (DCs) are obligate contributors to the elicitation of efficient immune responses.-It is well established that DC-based vaccines, comprising DCs expanded and loaded with tumor-associated antigens (TAAs) ex vivo, can effectively induce cellular immunity in vitro as well as in vivo. However, so far limited clinical benefits have been achieved even upon the elicitation of a demonstrable immune response to TAAs. There are numerous reasons that may account for such a lack of clinical efficacy, including the well-documented immunosuppressive nature of the tumor microenvironment.--A variation on this approach that may offer improved efficacy is to combine immunotherapy with conventional treatments designed to decrease tumor burden and control or diminish the activity of tumor-infiltrating immunosuppressive cells.The clinical protocol described herein was developed to explore the efficacy of combining an immunotherapeutic approach with radiation to treat patients with advanced malignancies. The immunotherapeutic components of this regimen comprised autologous immature dendritic cells (iDCs) generated from the monocytes isolated from peripheral blood mononuclear cells (PBMC), as well as patient-derived, activated T cells. These were prepared from the residual T cells present among monocyte-depleted PBMCs and expanded in the presence of high-dose interleukin (IL)-2 in flasks coated with anti-CD3 antibodies.We previously reported the safety and feasibility of this protocol in a small scale clinical trial involving 26 patients affected by a variety of advanced cancers.+CD56+ cells among circulating lymphocytes. Cells with this phenotype have been reported by others to have dual cytotoxic activities as mediated by natural killer (NK) cells as well as by CD8+ cytotoxic T cells.,+ T cells were isolated from the peripheral blood of 13 patients who manifested recurrent disease in spite of the immunoradiotherapeutic protocol described above. After IL-2-mediated expansion in vitro, autologous CD8+ T cells were injected into 58 metastatic lesions, eliciting a CR in 59% and a PR in 29% of the cases.In the course of treatment, various parameters were monitored to detect changes that might reflect the elicitation of an immune response. We observed an increase in CD8Taken together, our data demonstrate the potential of combining immunotherapy with a conventional treatment modality to effectively treat cancer patients with advanced disease.The therapeutic regimen employed in this study, including the intratumoral administration of autologous iDCs, the infusion of patient-derived, IL-2 activated T cells and IMRT, is described in the Materials and Methods section and is illustrated in ,A more detailed description of patients bearing a relatively low number of small tumors is presented in + cells, including a cell subset that also expressed the T-cell marker CD8. These CD3+CD8+CD56+ T cells were present among circulating PBMCs. An example of the expression profile of these markers, as evinced by flow cytometry, on the PBMCs of a gastric cancer patient is shown in +CD56+ immune cells are capable of killing autologous and allogeneic cancer cells by mechanisms that are known to mediate the cytotoxicity of natural killer (NK) cells as well as through T-cell receptor (TRC) engagement (manuscript in preparation). In light of these observations, we naturally hypothesized that patient-derived immune cells displaying this cytotoxic T lymphocyte (CTL) phenotype and function could be used therapeutically. This concept was experimentally tested using the protocol illustrated in While monitoring patients subjected to the immunotherapeutic regimen described above for changes in immunological parameters, we observed an increase in circulating CD56+CD8+CD56+ surface markers (5x108 cells) were injected into tumors with a diameter < 10 mm. Autologous CTLs from 13 patients with a variety of cancer types were injected into a total of 58 neoplastic lesions (involving various organs) over an 8-mo period. Anatomical sites and local clinical responses after 1 or 2 CTL injections are reported in CTLs were prepared from the PBMCs of patients who manifested incomplete responses to previous cycles of iDCs, activated T cells, and IMRT, as evinced by new lesions identified by PET-CT. IL-2-expanded lymphocytes expressing combinations CD3A representative example of the therapeutic response observed after CTL injection is shown in We developed a protocol combining immunotherapy and radiation to treat patients with a myriad of advanced cancers involving various organs and anatomic sites. Our objective was to establish a therapeutic framework that could be applied to recurrent and/or metastatic cancers appearing at multiple sites. This study was presaged by preclinical studies conducted by us and others that suggested the potential therapeutic efficacy of this combinatorial treatment modality and providing rationale for clinical applications. PET-CT was primarily used to evaluate clinical responses in the course of treatment. Other objective criteria have also been used to evaluate the effectiveness of this anticancer therapy. These data have been reported in a prior publication and include the levels of circulating tumor markers over the course of treatment.,+CD56+ cells accumulating upon treatment are able to kill autologous  cancer cells (manuscript in preparation). Previously, we examined serum samples from some of our patients for the presence of antibodies against TAAs. Antibodies targeting mesothelin, a TAA expressed by some malignancies, were identified in post-treatment serum samples.As previously reported, we observed little to no toxicity in response to this immunoradiotherapeutic regimen.The ability of intratumorally injected DCs to solicit an antitumor immune response has been reported by others. Chi and colleagues observed signs of an immune response in hepatocellular carcinoma patients who received an intratumoral injection of iDCs combined with radiation, including antibodies against α-fetoprotein and increased NK-cell activity.-It has been well established that cytokines and chemokines play a fundamental role in the ability of iDCs to both engulf antigens from the microenvironment and migrate to lymphoid organs, the crucial immunological tissues where adaptive immune responses are elicit.-,Radiation has been reported to stimulate the antitumor immune responses elicited by immunotherapy in pre-clinical studies as well as in several clinical trials.In addition to the well-documented immunosuppressive environment of most solid tumors, another major impediment to successful cancer immunotherapy is the imbalance between tumor burden and the capacity of the host to develop an immune response that suffices to eliminate the bulk of malignant cells. The importance of tumor burden in the clinical response to immunotherapy is well illustrated in our study, as the most favorable responses developed in patients with limited numbers of lesions that were 3 cm or less in size. This appeared to be an important factor contributing to clinical outcome regardless of tumor origin and irrespective of whether or not the patient had received and failed prior treatment or presented with advanced disease and had not received conventional therapy.+CD56+ lymphocytes upon treatment. Other investigators have previously reported that immune cells with this phenotype have cytolytic activity against cancer cells and that CD8+ cells co-expressing CD56 may be generated in vitro by culturing PBMCs in media containing the powerful cytokine IL-2.-+ T cells and render them capable of killing cancer cells through both receptor-ligand interactions that generally mediate the cytotoxic activity of NK cells and TCR-dependent pathways resulting in direct target cell lysis. A comparison of the cytolytic capacity of cytokine-induced killer cells, lymphokine-activated killer cells and tumor-infiltrating lymphocytes has been provided by Jakel and colleagues.+CD56+ cells may exert therapeutic activity. To test this hypothesis, we isolated CD8+ T cells from the peripheral blood of patients treated with the immunoradiotherapeutic regimen described above and expanded them in the presence of anti-CD3 monoclonal antibodies and high concentrations of IL-2 in vitro. These cells were then directly injected into metastatic lesions that had reoccurred after the initial round of treatment. A substantial proportion of these lesions manifested CRs 3 mo after treatment and an additional CTL injection further increased the number of CRs and PRs. Our data demonstrate the antitumor efficacy of this subset of immune cells and support further investigation of their potential application for the treatment of metastatic cancers.In order to gain insights into that nature of the immune response generated by our immunoradiotherapeutic regimen, we monitored the distribution of subsets of lymphocytes in the peripheral blood with a particular focus on the different cell populations that could be mediate cytolytic activity. In many patients, we observed an increased number of circulating CD8In summary, we report here the treatment of patients with advanced cancers with a combinatorial approach integrating immunotherapy and local irradiation. The rationale for our immunoradiotherapeutic approach came from prior observations by us and others, including results from both in vitro and in vivo studies. The most favorable outcome was observed in patients with advanced recurrent and metastatic cancers harboring low a tumor burden. Immune cell populations that developed in the course of treatment course were shown to have exert antitumor activity expanded in vitro and re-injected into new or recurrent lesions. The intratumoral injection of these CTL into new metastases of limited size proved to be quite effective in patients that had recurrent disease following the administration of iDCs coupled to IMRT. It is important to emphasize that little to no toxicity was observed in patients undergoing this treatment. We shall continue to monitor the clinical and immunological responses of the advanced cancer patients treated with our immunoradiotherapeutic regimen with the objective of increasing our knowledge in order to develop more efficacious therapies for such patients in the future.All patients with either recurrent disease after standard treatment or stage IV disease and no prior treatment were self-referred to either the Shukokai Clinic or the Tokyo Clinic and Research Institute. Patients gave written informed consent for the procedures and protocols after detailed explanation and discussion, according to the Declaration of Helsinki. The consent forms and procedures were approved by the Institutional Review Boards of the respective institutions.An overview of the therapeutic protocol is provided in 8 cells/mL and cryopreserved (−80°C) in AIM-V® medium  containing 10% dimethylsulfoxide (DMSO). Prior to use, iDCs, activated T cells and CTLs were thawed in a 37°C water bath, washed twice in AIM-V® medium and re-suspended at the concentrations required for injection.Leukapheresis were performed on a COBE Spectra blood separator (Gambro KK), using the program for collection of mononuclear cell population (version 7.1). Anticoagulant citrate dextrose solution  was used for anticoagulation. The inlet rates were 40–60 mL/min with a collection rate of 1 mL/min and a separation factor of 700. PBMCs were isolated by gradient separation, suspended at 1.5x108 cells) were suspended in 20 mL AIM-V® medium and distributed in 5 mL aliquots to four T75 cm2 polystyrene flasks containing 10 mL of AIM-V® medium. After 2 h of incubation at 37°C, non-adherent (monocyte-depleted) cells were removed and destined to activated T-cell preparation (see below). Fifteen mL of DC growth medium, that is, AIM-V® medium supplemented with 800 IU/mL granulocyte macrophage colony-stimulating factor  and 500 U/mL IL-4 (BD PharMingen), was added to each flask containing adherent cells. Flasks were incubated at 37°C with 5% CO2. On day 3, growth media was refreshed. On day 7, iDCs were harvested and either prepared for injection, or re-suspended in AIM-V® freezing medium containing 20% autologous serum and 10% DMSO, then cryopreserved in BICELL containers (Nihon Freezer Co.).Thawed PBMCs  in 35 mL AIM-V® medium supplemented with 1,000 IU/mL IL-2 and 10% autologous serum. Flasks were incubated for 7 d at 37°C with 5% CO2. Three hours prior to harvesting, 1 µg/mL ionomycin (Sigma) was added to the medium. Harvested cells were extensively washed and prepared for infusion or cryopreserved, as described above.Approximately 6–9x102 for 2 d. The supernatants were harvested by centrifugation at 300 g for 7 min and stored at 4°C for later use. LCM was injected to patients together with iDCs.Detailed protocols to prepare products of activated lymphocytes for use as an adjuvant is described elsewhere.® medium supplemented with 10% autologous serum at a concentration of 5x106 cells/mL and incubated at 37°C in 5% CO2 for 24 h. After washing, cells were re-suspended in isolation buffer and CD8+ cells were isolated using Dynabeads®UntouchedTM Human CD8 T Cells kit , according to the manufacturer’s instruction. After isolation, CD8+ cells were washed twice and re-suspended in AIM-V® medium supplemented with 2000 IU/mL IL-2 and 5% autologous serum. CD8+ cells were adjusted to a concentration of 1x106 cells/mL and cultured for 5 d in flasks coated with OKT3 antibodies , harvested and cultured for an additional 25 d in uncoated flasks. Finally, cells were harvested and either prepared for injection or cryopreserved at −80°C for subsequent use.Cryopreserved PBMCs were suspended in 20 mL AIM-VThe presence of microbial contaminants was tested by incubating cultured cells on agar at 37°C with subsequent inspection for bacterial growth. Endotoxin levels (< 0.5 EU/mL) were determined using a commercially available chromogenic endotoxin assay kit , according to the manufacturer’s instructions. Only sterile and endotoxin-free preparations were used clinically.A standard fluorescence cytometry labeling protocol was used to determine the surface phenotype of iDCs using fluorochrome-conjugated monoclonal antibodies against CD11c, CD14, CD40, CD80, CD83, CD86, and HLA-DR (BD PharMingen). Activated T cells were evaluated for CD3, CD4, CD8, CD25, CD56, and CD154 (CD40L) expression after culture. CTLs were evaluated for CD3, CD4, CD8, and CD56 expression following culture. Data were analyzed using the Cell Quest software package (BD Biosciences). The immunophenotype of iDCs, activated T cells and CTLs is summarized in ® medium was added to each thawed vial. Vials were then incubated for 2 min at 22 ± 3°C (room temperature), transferred to 50 mL of medium and centrifuged at 300 g for 7 min to remove DMSO. iDCs were suspended in 10% LCM, then prepared in one microtube and placed on ice for transport. Activated T cells were suspended in 100 mL of normal saline containing 1.25% albumin and 10% LCM and infused i.v.. CTLs for intratumoral injection were prepared as described above.iDCs and activated T cells were thawed in a 37°C water bath approximately 1 h prior to injection. One mL of AIM-VIMRT was delivered to each iDC-injected neoplastic lesions. The total dose, delivered in fractions, was determined to be such that the biologically effective dose was 72 Gy, according to the linear quadratic model. Each fraction was optimized such that the radiation-induced late toxicity to surrounding normal tissue would be equal to or less than Grade 2 based on the National Cancer Institute Common Terminology Criteria (NCI-CTC) version 2.0 system.Patients were monitored for signs and symptoms of adverse reactions to therapy, including vital signs , diaphoresis, arthralgia, and swelling at injection sites, as well as blood chemistry and hematological analyses. Other signs and symptoms that might be associated with radiation or the injection of cells by insertion of a needle through various organs in order to reach metastatic lesions were also monitored. Blood tests showed no changes in blood cell counts or biochemistry, as determined by hemogram and biochemical data. Basically all treatments were well tolerated with no adverse reactions with the exception of 9 patients that developed transient temperature elevation (> 38°C) after IMRT.PET-CT imaging was used to assess clinical response to treatment based on the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines. The first exam was conducted 6 weeks after the end of the last treatment cycle with periodic follow-up PET-CT exams. Responses to treatment were defined as follows: complete response (CR), complete disappearance of tumor at the treated site and no new lesions at distant sites; partial response (PR), 30% reduction in the size of targeted lesions; progressive disease (PD), increase in size of targeted lesion and/or appearance of new lesions; and stable disease (SD), little to no changes in size of targeted lesion and no new lesions.Additional material


Lifetime cumulative MDMA use was significantly associated with poorer performance on visuospatial memory and perceptual attention. Heavy MDMA users (>100 tablets lifetime use) interacted with candidate gene polymorphisms in explaining individual differences in cognitive performance between MDMA users and controls. MDMA users carrying COMT val/val and SERT s/s had poorer performance than paired controls on visuospatial attention and memory, and MDMA users with CYP2D6 ultra-rapid metabolizers performed worse than controls on semantic fluency. Both MDMA lifetime use and gene-related individual differences influence cognitive dysfunction in ecstasy users.This study is aimed to clarify the association between MDMA cumulative use and cognitive dysfunction, and the potential role of candidate genetic polymorphisms in explaining individual differences in the cognitive effects of MDMA. Gene polymorphisms related to reduced serotonin function, poor competency of executive control and memory consolidation systems, and high enzymatic activity linked to bioactivation of MDMA to neurotoxic metabolites may contribute to explain variations in the cognitive impact of MDMA across regular users of this drug. Sixty ecstasy polydrug users, 110 cannabis users and 93 non-drug users were assessed using cognitive measures of Verbal Memory , Visual Memory , Semantic Fluency, and Perceptual Attention . Participants were also genotyped for polymorphisms within the  There is compelling evidence that MDMA produces selective long-lasting serotonergic neuroadaptations, including regulatory changes in the expression of the serotonin transporter Despite these findings about serotonin neuroadaptations, there is still debate on the question if MDMA use is reliably associated with neuropsychological impairment, regardless of the effects of concomitant use of other substances . Literature on this topic is characterized by considerable heterogeneity of results, which is attributable to the large amount of confounders inherent to research on the deleterious effects of MDMA SERT and 5HT2A receptor gene polymorphisms, have demonstrated significant associations with cognition and may therefore importantly impact MDMA use related neuropsychological effects COMTCYP2D6 polymorphisms BDNF and glutamate genes) SERT s/s and 5HT2a Tyr genotypes), high enzymatic activity linked to the bioactivation of MDMA to neurotoxic metabolites , and poor competency of executive control and memory consolidation systems  can contribute to explain variations in the cognitive impact of MDMA across regular users of this drug.A number of genes affecting serotonin function, including This study seeks to clarify the association between MDMA cumulative use and cognitive dysfunction, and the potential role of a number of relevant genetic polymorphisms in explaining individual differences in the cognitive effects of MDMA. To reach this aim, we examined cognitive performance in a sample of MDMA users recruited from a homogeneous socio-demographic context and thoroughly assessed to rule out psychiatric comorbidities. The sample also includes considerable variability in MDMA use patterns, which allowed us to characterize dose-related effects of cumulative MDMA use on cognitive performance. Neuropsychological testing was focused on those cognitive domains that have been consistently linked to MDMA use across studies: verbal and visual memory, attention/processing speed and executive functions. We hypothesize: (i) that heavier MDMA use would correlate with poorer neuropsychological performance in a dose-dependent fashion; (ii) that heavy MDMA users would perform poorer than cannabis and healthy comparison individuals on neuropsychological tests of processing speed, memory and fluency (indicating robust effects of MDMA on cognition regardless of co-abuse of cannabis); and (iii) that MDMA use would exacerbate cognitive performance decrements in individuals carrying genotypes associated with lower functionality of the serotonin, glutamate and dopamine systems.Two hundred sixty-three Caucasian participants were recruited, of whom 60 were MDMA polydrug users, 110 were cannabis users and 93 were non-users. MDMA users were further classified into two subgroups according to their lifetime MDMA use applying a cut-off of 100 tablets (more than 100 tablets defined as heavy users) This study was approved by and conducted in accordance with the local ethics committee (CEIC-IMAS). Upon arrival to the research centre , participants were informed of the ensuing protocol and provided informed consent before participating in the study.All participants were subjected to an initial exploration that included a detailed medical history, biochemical analyses, physical examination, urine and hair toxicology screens, and a brief neurological examination. They were also assessed with a structured psychiatric interview specifically designed to diagnose lifetime use of different drugs and psychiatric comorbidity among substance users PRISM  administWe administered the Vocabulary test from the WAIS-III (estimated IQ) Genomic DNA was extracted from the peripheral blood leukocytes of all the participants using Flexi Gene DNA kit  according to the manufacturer's instructions.5HTTLPR genotyping was performed using polymerase chain reaction (PCR) as described in 5HTTLPR (A/G) polymorphism (rs25531) was detected by MspI restriction enzyme digestion 5HTTLPR PCR was digested in a 10 µl reaction assay containing 1× NEBuffer 2 and 3 U MspI at 37°C for 3 h and a final inactivation step of 20 minutes at 65°C. The resulting fragments were detected on an automatic ABI 3730XL capillary sequencer and analyzed by GeneMapper Software v3.5 (Applied Biosystems). Product sizes for the digest were: long A (LA) = 337 bp, short A (SA) = 292 bp, long G (LG) = 162 bp, and short G (Sg) = 162 bp.Escherichia coli Exonuclease I (New England Biolabs) and 0.64× shrimp alkaline phosphatase buffer. The mixture was incubated at 37°C for 15 min, followed by deactivation for 15 min at 80°C. Sequencing reactions were performed with BigDye v3.1 (Applied Biosystems) in 10 µl total volume containing 1 µl template (approximately 25 ng), 3.2 pmol primer, 1 µl Applied Biosystems 5× DNA sequencing buffer, 2 µl BigDye v3.1, and water. The reactions were cycled at 94°C for 3 min, followed by 30 cycles at 96°C for 10 sec, 50°C for 5 sec, and 60°C for 4 min. Reactions were then purified with PureLink Quick Gel extraction kit (Invitrogen) according to manufacturer's instructions. Samples were analyzed on a Prism 3730xl DNA Analyzer (Applied Biosystems).In some cases, were MspI digestion gave unclear results the samples were sequenced to assign the correct genotype. Sequencing was performed in both, the sense and antisense orientations. The excess primers and deoxynucleotides in the polymerase chain reaction (PCR) products were then degraded by adding a 2 µl of a solution of 0.8 units of shrimp alkaline phosphatase , 4 units of COMT val108/158met (rs4680) and BDNF val66met (rs6265) single nucleotide polymorphism (SNP) allelic variants were determined using the 5′ exonuclease TaqMan assay with ABI 7900HT Sequence Detection System  supplied by Applied Biosystems. Primers and fluorescent probes were obtained from Applied Biosystems . Reaction conditions were those described in the ABI PRISM 7900HT user's guide. Endpoint fluorescent signals were detected on the ABI 7900, and the data were analyzed using SDS software, version 2.3 (Applied Biosystems).The CYP2D6, GRIN2B C2664T (rs1806201), the 5HT2A His452Tyr (rs6314) and T102C (rs6313) genotypes were performed using the PHARMAchip™ DNA array  The COMT val158met genotype, on one hand, and the 5HTTLPR genotype, on the other. Since several significant correlations with the neuropsychological variables were found (as described in the following section), in the sequel, a distinction was made between regular and heavy ecstasy users taking the consumption of 100 ecstasy pills during lifetime as cutoff. Since the principal interest was to study the association between cognitive performance and both drug consumption and each of the genotypes of interest, ANCOVA models were fitted for all neuropsychological variables and each genotype separately. These models included drug consumption group and the respective genotype as predictive variables of interest as well as gender and the WAIS-III Vocabulary index score; the Vocabulary index was used as a proxy of cultural level and verbal IQ. These two variables, sex and WAIS-III Vocabulary index, were included in all ANCOVA models in order to rule out the possible confusion due to differences observed among the drug consumption groups with respect to sex and education/IQ. Initially, all models did also include the two-way interaction between genotype and drug consumption. Whenever the interaction could be discarded, both factors were studied separately using the ANCOVA model excluding interaction. If a significant effect was observed of either factor, post-hoc multiple comparisons were carried out in the framework of the corresponding model using the Tukey test. If, by contrast, the interaction was significant, the effect of drug consumption was studied separately for each genotype expression and, vice versa. Again, the Tukey test for multiple comparisons was applied for these analyses in the framework of the ANCOVA models including interaction.Sample characteristics, including drug consumption, are described by means of either mean and standard deviation  or absolute and relative frequencies . To quantify differences among the three groups, a generalization of Cohen's d, the standard mean difference for more than two groups, was computed. Following the suggestions of Cohen, values of 0.25 and 0.4 indicate medium and large effect sizes, respectively Statistical significance was set at 0.05 for the two-way genotype-drug consumption interaction within all ANCOVA models in order to reduce the probability of a possible Type II error. For those variables showing a significant interaction effect, results from follow-up pairwise comparisons were thresholded at p< = 0.01 to protect against Type I error was applied. The statistical software package R , version 2.11.1, was used for all analyses. In particular, R Package multcomp Socio-demographic variables, drug use characteristics and genotype distributions are presented in COMT val158met polymorphism in the cannabis group (p<0.01).Genotype distributions by group are presented in 5HTTLPR and the COMT val158met polymorphisms. Significant differences were observed regarding the genotype distributions for the COMT val158met polymorphism (p<0.05) when the distinction between heavy and light MDMA users was taken into account. There were a higher number of individuals with the val/val genotype in the heavy MDMA users group (42.9%) compared to the control group (29.0%). The val/met genotype was also overrepresented in the cannabis group (63.6%) compared to the control group (48.4%) (p<0.05).No significant differences were observed in the genotype distributions among the different groups, except for the MDMA lifetime use showed a significant negative association with ROCFT Copy Accuracy , Immediate Recall , and Delayed Recall . MDMA lifetime use was also significantly correlated with SDMT . No other correlations reached statistical significance.Results are presented in We found no effects of group, genotype or the group x genotype interaction on performance indices from this test.p<0.001), cannabis users , and non-users .We found a main group effect; paired contrasts indicated that heavy MDMA users had lower scores than light MDMA users . When the val/met genotype was examined, heavy MDMA users with this genotype had lower scores than light MDMA users , cannabis users  or non-users  with the same genotype.We observed a significant group x genotype interaction for the COMT val158met and 5HTTLPR genotypes, we also found an effect of the group x genotype interaction . MDMA users carrying the S/S+val/val genotype scored poorer than non-users carrying the same genotype . In addition, concerning the L+met genotype, MDMA users scored poorer than Cannabis users .When considering the combination of the p<0.01), cannabis users , and non-users .We found a main effect of group, with heavy MDMA users having significantly lower scores than light MDMA users ; MDMA individuals with the S/S+val/val genotype had significantly lower scores than non-users carrying the same genotype .When examining the combination of the p<0.01), with heavy MDMA users having significantly lower scores than cannabis users , and non-users . We also found a main effect of 5HT2A genotype, indicating that individuals carrying the His/Tyr variant had significantly poorer performance than those with the His/His genotype .We found a main group effect . Pairwise comparisons showed that MDMA users carrying the S/S+val/val combination had significantly lower scores than non-users .The study of the interaction between the We found no significant main effect of group on performance in this test.5HTTLPR genotype interaction , with heavy MDMA users carrying the S/S genotype performing poorer than S/S non-users .We found a significant group x 5HTTLPR and the COMT val158met genotypes . Pairwise comparisons showed that MDMA users carrying the S/S+val/val combination performed significantly more poorly than MDMA L+val/val carriers .In addition, we observed a significant group x genotype interaction for the combination of CYP2D6 phenotype interaction . In non-users, individuals who were Intermediate/Poor for the CYP2D6 performed worse than those who were Ultra-rapid/Extensive . In contrast, MDMA users with the Ultra-rapid/Extensive phenotype had significantly lower scores than those with the same phenotype in the non-user group .The semantic word fluency was unaffected by group or genotypes alone. However, there was a significant effect of the group x COMT and SERT genes showed that MDMA users carrying the COMT val/val and SERT S/S genotypes have poorer performance on tests of visuospatial memory and perceptual attention (ROCFT and SDMT). Delayed recall was also modulated by a gene polymorphism in the 5HT2a receptor, such that Tyr carriers had poorer performance irrespective of drug use. Finally, we found an interaction between MDMA use and CYP2D6 extra-high metabolic activity phenotype and lower performance on verbal fluency.Our findings show detrimental effects of both MDMA lifetime use and variations in candidate genes on a number of neuropsychological measures, with particular relevance of visuospatial attention and memory. With respect to dose-related effects, we found that greater lifetime use of MDMA is negatively correlated with performance on visuospatial memory (ROCFT) and attention and perceptual speed (SDMT) tests. These results were further supported by group comparisons, which showed that heavy MDMA users (lifetime use>100 tablets) have significantly poorer visuospatial memory than light MDMA users, cannabis users and non-users. Importantly, we found a number of gene x MDMA interaction effects. Results for per se; this is consistent with the finding that MDMA use is also negatively associated with SDMT-indexed perceptual attention, and with decreased gray matter volumes and SERT availability in posterior brain cortices The main MDMA dose-related findings and MDMA x gene interactions were found in the ROCFT. This is a complex task involving visuospatial attention and planning/organization skills during the copy, and planning and episodic memory skills during immediate and delayed recall COMT and SERT genotypes. Our results consistently showed that MDMA users carrying the COMT val/val and SERT S/S genotypes perform significantly more poorly on ROCFT Copy, Immediate and Delayed Recall, and on SDMT, such that there was consistency between gene x drug effects on different probes involving perceptual attention and visuospatial planning/memory skills. These results fit with evidence showing that the COMT gene is significantly associated with visuospatial planning ability –gene-carriers with high enzymatic activity display poorer performance COMT is one of the genes in the deleted region– displaying robust deficits in the ROCFT SERT genotype on visuospatial attention/planning performance indexed by a Mental Rotation task after tryptophan depletion –which mimics MDMA-induced serotonin reductions SERT availability in occipital, hippocampal and parietal regions COMT val/val and SERT S/S genotypes interact to produce greater detrimental effects on perceptual attention and planning/organization, ultimately affecting visuospatial memory skills. However, these results would need follow-up in larger samples, and they need to be interpreted with caution considering that the role of the COMT gene on cognition and brain functioning is still controversial 5HT2a gene polymorphism, such that Tyr carriers have poorer performance irrespective of drug use; this is in agreement with previous observations on the association between this genotype and the consolidation process of episodic memory The association between MDMA use and perceptual attention and visual memory/organization skills seems to be modulated by CYP2D6 polymorphisms may modulate MDMA-induced neurotoxic effects BDNF val/met, the 5HT2a T102C, and the GRIN2B C2664T– were explored but failed to show significant results in relation to cognitive performance. Nonetheless, the GRIN2B genotype showed a trend to significance in relation to verbal memory , raising the possibility that effects of these genes on cognition may emerge in larger samples sizes.In agreement with our initial assumptions, MDMA users with CYP2D6 high metabolic activity phenotypes performed more poorly on the semantic fluency test. These results are in agreement with recent findings about the link between higher CYP2D6 activity and impaired executive performance, including semantic fluency, in methamphetamine users COMT and SERT genes) and verbal fluency (CYP2D6). Strengths from the study include the successful recruitment of a large number of non-treatment seeking MDMA users who self-report an adequate academic/ professional and social functioning, thus avoiding socio-demographic confounders and approximating research to the reality of MDMA use beyond clinical settings; the carefully conducted medical and psychiatric explorations to discard potential confounders related to physical illness or psychopathology; and the use of well-validated neuropsychological measures taxing key cognitive domains related to MDMA use. Limitations include the relatively small sample size for a genetic study –especially when combining some of the rare genotypes, and the elevated number of statistical comparisons, which may have inflated the risk of Type I error. Nonetheless, we should note that our drugs x gene interaction findings are in agreement with initial assumptions, biologically plausible and overly consistent with previous literature, and consistent across independent but conceptually related neuropsychological probes and indices. Therefore, although these results would need to be further explored and replicated, we arguably reckon that the probability that they stem from false positive effects is low.In conclusion, this study reliably demonstrates dose-related effects of MDMA use on visual attention, organization and memory. In addition, we show an interaction between MDMA use and different gene polymorphisms in determining poorer performance of MDMA users in tests of visual attention and memory (


Stethojulis bandanensis, which maintains tuna-like optimum cruising speeds (up to 1 metre s−1) while using 40% less energy than expected for their body size. Displaying an exceptional aerobic scope (22-fold above resting), streamlined rigid-body posture, and wing-like fins that generate lift-based thrust, S. bandanensis literally flies underwater to efficiently maintain high optimum swimming speeds. Extreme energetic performance may be key to the colonization of highly variable environments, such as the wave-swept habitats where S. bandanensis and other wing-finned species tend to occur. Challenging preconceived notions of how best to power aquatic locomotion, biomimicry of such lift-based fin movements could yield dramatic reductions in the power needed to propel underwater vehicles at high speed.Underwater locomotion is challenging due to the high friction and resistance imposed on a body moving through water and energy lost in the wake during undulatory propulsion. While aquatic organisms have evolved streamlined shapes to overcome such resistance, underwater locomotion has long been considered a costly exercise. Recent evidence for a range of swimming vertebrates, however, has suggested that flapping paired appendages around a rigid body may be an extremely efficient means of aquatic locomotion. Using intermittent flow-through respirometry, we found exceptional energetic performance in the Bluelined wrasse  Underwater locomotion is challenging due to the high friction and resistance imposed on a body moving through water and energy lost in the wake during undulatory propulsion Reef fish predominantly swim via the labriform gait, which involves moving solely the pectoral fins for propulsion Stethojulis bandanensis and Cheilinus fasciatus. Chosen for their pectoral fin morphologies  to wing-shaped (2.01 in S. bandanensis) fin shape extremes that have previously been linked to the distinct forms of resistance-based and lift-based labriform swimming , respectively Uopt) at which GCOT is minimized for each species Integrating new data into a meta-analysis of comparative swimming energetics in bony fishes, we aimed to test the energetic efficiency of resistance- and lift-based labriform swimming for high-speed underwater locomotion. Firstly, we used intermittent flow-through respirometry to measure aerobic energy use during prolonged swimming activity in two species of coral reef fishes from the Great Barrier Reef, Australia: Stethojulis bandanensis  and Redbreasted wrasse Cheilinus fasciatus  were collected from the wild and tested for their swimming performance at the Lizard Island Research Station. Individuals were hand-collected during September 2008 from reefs around Lizard Island, Great Barrier Reef by divers on SCUBA (Self-Contained Underwater Breathing Apparatus) using ultra-fine barrier nets. Transported to the Lizard Island Research Station within 2 hours of capture, fish were maintained in flow-through seawater aquaria at ambient temperatures (27–28°C) and fasted for 36 hours prior to their swimming trial to maximize energy available for swimming and minimize potential respirometer fouling. All individuals were swum within 3 days of capture.This study was carried out in strict accordance with the protocols approved by The Australian National University Animal Experimentation Ethics Committee (F.BTZ.03.06) for this specific project. All efforts were made to minimize animal suffering through careful collection, handling, and swimming trials based upon the natural rheotaxic behaviour and self-motivation of individuals. Bluelined wrasse 2MO) by each fish was measured with a computerized, intermittent-flow respirometry system S. bandanensis and 4.6±0.01 cm for C. fasciatus) fitted well within the dimensions of the flow tank working section , which was calibrated by vane-wheel flow probe  against the voltage output of the propeller motor controller. Honeycomb collimators and curved baffles were used to produce a smooth laminar flow in the working section of the flow tank. Fish were monitored continuously throughout each trial to ensure they swam in this calibrated flow without any use of the walls or corners. Temperature inside the flow tank was maintained at 27.7±0.1°C via a computer-actuated cooling coil housed downstream of the working section. Oxygen partial pressure (2Po) in the flow tank was measured with an optic-fibre sensor and oxygen meter  linked to a computer to continuously record 2Po measurements in real time. The respirometer was set to periodically flush the flow tank with aerated water from the bath for 6 minutes, followed by a 2-minute closed mixing period then 7 minutes of closed respirometry when oxygen measurements were taken at a rate of 1 s−1 to yield an 2MO at each set flow speed.Rate of oxygen consumption (−1 (4.7–7.9 cm s−1). This initial speed provided adequate water mixing whilst allowing the fish to rest on the flow tank bottom without swimming. Oxygen measurements began immediately at the set flushing cycle described above, with these 2MO measurements at 0.5 total body lengths s−1 used to calculate standard metabolic rate . Flow speed was then incrementally increased by 0.5 total body lengths s−1 every 15 minutes and 2MO recorded for each set speed. Incremental speed increases were continued until the fish could no longer hold position and became impinged on the downstream grid. To ensure trials measured aerobic locomotion, at the conclusion of each trial, flow speed was reduced to 1 total body length s−1 and 2MO was recorded through an additional two flushing cycles. Any post-trial repayments of excessive post-exercise oxygen consumption  were measured by comparison of post-trial 2MO against 2MO at 1 total body length s−1 at the beginning of the trial. After the fish was removed, the flow tank was resealed and oxygen consumption in the empty respirometer measured to determine background levels, which were subtracted from the 2MO values for the fish swum previously .Each fish was measured for body depth, width, mass and total length before placement in the respirometer flow tank to calculate solid blocking effects, where they were allowed to acclimate for a minimum of 5 hours at a flow speed of 0.5 total body lengths s2MO) was determined from the slope of the linear regression of the 2Po decline over time for each measurement cycle, using the formula 2MO =  sVrespα where s is the slope, Vresp the volume of the respirometer minus the volume of the fish , and α is the solubility of oxygen in seawater. Only measurements with a regression coefficient of determination (r2) greater than 0.94 were used in the analyses. SMR was calculated from a frequency histogram of the raw 2MO data within the acclimation period , by fitting two normal curves to separate the SMR peak, when the fish was at rest, from higher 2MO observations that corresponded with spontaneous activity 2MO – SMR)  =  a + b U c, where a, b and c are constants and U is corrected swimming speed in total body lengths per second. This power equation was then transformed following Uopt): Uopt  =  [(a+SMR)/(c-1)b]1/c, where Uopt is the speed that minimizes energy expenditure per unit of travel distance. Finally, the gross cost of transport  was calculated for each individual by first multiplying total MO2 consumption at Uopt by the conversion factor 14.1 J mg−1 O2 and then using the equation 2O(opt) (optg U)−1, where M2O(opt) is the gross rate of oxygen consumption at optU (converted to units of J kg−1 hr−1), g the acceleration due to gravity (9.8 m s−2), and optU as calculated above (but converted to units of m hr−1).Oxygen consumption rate (S. bandanensis) and denoting this as 1.00, then diving this by the exponent value for each other species. Secondly, Uopt for our study species was placed within a log-log plot of Uopt versus body mass for a range of six scombrid (tuna-like) and seventeen other non-scombrid fishes  following −1 m−1) and body mass was used to examine the overall energetic cost incurred by each species swimming at Uopt following Comparative performance of our study species was examined in the context of a range of bony fishes using published energetic estimates, either taken directly from reported figures in each paper (which often required conversion to the same units stated above) or calculated from fitted equations. Firstly, the exponent in the hydrodynamics-based power equation was used to provide a size-independent measure locomotion efficiency (rate of increase in energy consumption with increasing swimming speed) among a range of fishes. Relative efficiency was calculated by taking the lowest exponent value for a species  while they forage across the reef Challenging long-held notions about the costs of underwater locomotion S. bandanensis seems to arise from mechanical efficiency; the comparative performance of S. bandanensis and C. fasciatus matched predictions based on the relative thrust-producing efficiency of their different pectoral fin shapes and movements S. bandanensis achieves high-speed efficiency by sweeping their wing-like (high AR) fins in a figure-eight pattern to produce lift-based thrust on all fin strokes. Rowing paddle-shaped pectoral fins (low AR) in the power-recovery stroke cycle means that C. fasciatus produces thrust only half the time Extreme performance in S. bandanensis is brought into extreme relief by comparison with a range of vertebrates spanning multiple families and forms of locomotion. For instance, S. bandanensis displayed the widest factorial scope of active metabolism ever seen in a bony fish  at a speed four times faster than non-scombrid fishes of similar size, without incurring the increased energy use typically seen in tuna-like fishes swimming at this speed. It is interesting to note that other coral reef fishes using labriform swimming also display faster than expected optimal swimming speeds for their size, with similarly low costs of transport, suggesting this may be a generalized feature of pectoral-based propulsion. Collectively, these coral reef fishes adopt oscillatory fin strokes around a rigid-body posture, which is thought to bring hydrodynamic drag down to minimum S. bandanensis currently stands out as the highest performing swimmers for their size with respect to measures of scope, optimum swimming speed and energy consumption.Energetic performance by C. fasciatus, we tend to find these fish occupying calm water habitats sheltered from incident wave energy and storms. In contrast, S. bandanensis and other wing-finned fish species tend to be in great abundance in habitats subject to highly variable and extreme water flows generated by direct exposure to wave energy S. bandanensis appears to be equipped with the physiological traits needed to maintain their metabolic delivery of energy across a wide range of activities and hydrodynamic conditions. While S. bandanensis may be able to ride through such challenges, species such as C. fasciatus and other coral reef fishes of low aerobic scope Extremes in energetic performance appear to be linked with ecological extremes in these coral reef fishes. For species with low speed efficiency, such as Madeleine robot turtle Such functional innovation in swimming prompts a rethink of the possibilities and limits to the cost of high speed performance in underwater locomotion. In fishes, we see that relative swimming performance is not necessarily related to their perceived lifestyle, with reef fishes capable of exceptional swimming performance despite their supposed sedentary, reef-associated existence Table S1Meta-data for comparative analysis of the energetic swimming performance of reef fishes, scombrid, and non-scombrid fishes.(DOC)Click here for additional data file.


Coexpression has been frequently used to explore modules of functionally related genes in eukaryotic genomes. However, we found that genetically interacting mammalian genes identified through radiation hybrid (RH) genotypes tend not to be coexpressed across tissues. This pattern remained unchanged after controlling for potential confounding factors, including chromosomal linkage, chromosomal distance, and gene duplication. Because >99.9% of the genetically interacting genes were identified according to the higher co-retention frequencies, our observation implies that coexpression is not necessarily an indication of the need for the co-presence of two genes in the genome, which is a prerequisite for cofunctionality of their coding proteins in the cell. Therefore, coexpression information must be applied cautiously to the exploration of the functional relatedness of genes in a genome. Coexpression refers to the coherent transcription of genes in spatial, temporal, or environmental dimensions Proteins that are produced together undoubtedly require the co-presence of their coding genes in the genome. However, regulation of protein abundance does not necessarily occur at the transcriptional level D, see 6 “genetic interactions” among >18,000 genes To understand the biological implications of gene coexpression, we examined whether elevated coexpression predicts the need for the co-presence of genes in the genome, which is a prerequisite for the cofunctionality of their protein products. Exploiting genotypes of human, mouse, rat, and dog radiation hybrid (RH) panels, researchers recently calculated the co-retention frequencies of all mammalian gene pairs with an intergenic distance D, see  of ≥10 mln[(1+CoExp)/(1−CoExp)] (see ln[(1+CoExp)/(1−CoExp)] indicate higher coexpression. If coexpression predicts preference for the co-presence for two genes in the mammalian genome, then higher coexpression /(1−CoExp)]) is expected to be found in pairs of “genetically interacting genes,” as defined in Lin et al. (2010) (hereafter referred to as “co-retained gene pairs” [CRGPs]), than in other “non-co-retained” gene pairs (nCRGPs) ] see . Larger (nCRGPs) .ln[(1+CoExp)/(1−CoExp)] values between CRGPs were significantly lower than those between nCRGPs  was only approximately half of that for nCRGPs  . Compared to linked nCRGPs, the linked CRGPs had significantly larger D values  poses intrinsic biases in chromosomal linkage. Regardless of the cause, the bias in chromosomal linkage is an important factor that needs to be controlled in our analyses.We can potentially explain the tendencies of D of linked genes sufficiently explains the lower coexpression of CRGPs /(1−CoExp)] values than did the nCRGPs /(1−CoExp)] values than nCRGPs in nearly all groups . When linkage was defined by mouse genome coordinates and coexpression was measured based on expression levels across 58 mouse tissues, the result remained consistent with To determine whether our observations are specific to human, a parallel analysis was conducted on mouse data see . We usedCoExp to define high coexpression  CRGPs consistently showed a lower proportion of highly coexpressed gene pairs than nCRGPs. Parallel analysis with mouse genome coordinates and gene expression data generated a result consistent with In several previous studies e.g. transcriptional interference In summary, our analysis showed no indication that coexpression between genes indicates a need for co-presence in the genome. In contrast, we consistently observed patterns indicating that co-retained genes tend to avoid coexpression in both human and mouse genomes. The result presented in this study implies that factors unrelated to functionality . The intergenic distance D was calculated as the distance in nucleotides between the transcriptional start sites of two genes.analysis . ChromosCoExp was defined by the Spearman's correlation coefficient (ρ) or Pearson's correlation coefficient (r) of expression levels across human tissues. Because CoExp measured by Spearman's ρ yielded statistically more significant results, those results are presented in the main text. Consistent results derived from using Pearson's r to calculate CoExp are shown as Expression levels in 63 normal human tissues or 58 normal mouse tissues were obtained from Gene Atlas v2 CoExp, the expression profile “dissimilarity” between genes was calculated from the Euclidean distance n is the number of tissues, and A or gene B, respectively, in tissue i. The relative transcriptional abundance was calculated from the expression level of a gene in the tissue examined divided by the summation of expression levels of that gene in all of the tissues in the dataset d indicates a higher level of coexpression. Use of d yielded a result Click here for additional data file.Figure S2Regenerated CoExp is calculated by Pearson's r of expression levels between genes. See legend of (PDF)Click here for additional data file.Figure S3Regenerated (A) (PDF)Click here for additional data file.Figure S4Regenerated (A) Fig. S2C and (B)Fig. S2D by excluding gene pairs that are paralogous from the analysis.(PDF)Click here for additional data file.Figure S5Regenerated CoExp is measured using mouse gene expression data and linkage and D are defined using mouse genome coordinates. See legend of (PDF)Click here for additional data file.Figure S6Regenerated CoExp is calculated by Pearson's r of expression levels between genes. See legend of (PDF)Click here for additional data file.Figure S7Regenerated CoExp is measured using mouse microarray data and linkage and D are defined using mouse genome coordinates. See legend of Fig. 2 for detailed description.(PDF)Click here for additional data file.Figure S8CRGPs and nCRGPs. See legend of Regenerated (PDF)Click here for additional data file.Figure S9d, the Euclidean distance of the relative transcriptional abundance between genes. A lower d indicates a higher level of coexpression. See legend of Regenerated (PDF)Click here for additional data file.


Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions.In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes.By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides. Endolysosomes are cellular organelles that play a key role in protein turnover and homeostasis of the cell. They are made up of a complex set of enzymes that break down proteins, lipids and sugars that are all essential for normal functioning of the cell . A genetSeveral methods have been used to study their functions in the cell. The approaches taken to study endolysosome contribution to protein degradation include using recombinant cathepsins in peptide degradation assays , studyinEach of the above mentioned approaches have some caveats. Endolysosomes comprise a complex set of proteases that are still not completely characterized. Studying the role of a single protease and their contribution to antigen presentation using recombinant proteins is not representative of the complex milieu inside the organelle. Gene knock-out or siRNA studies are helpful in identifying the precise role of a particular protein but do not help in addressing the interactions between the different proteins in the organelle. Though using purified endolysosomes will address the above-mentioned concerns, it involves a laborious, multistep process and results in a much lesser yield. In studies involving limited amount of material such as primary cells from patients, it may not be feasible to purify endolysosomes.In this study, we describe a simple approach to test the contribution of endolysosomal proteases to protein degradation. Since endolysosomal proteases are active in acidic pH , we testWe purified endolysosomes from PBMC isolated from the blood of healthy donors. Following differential centrifugation enrichment of lysosomes was confirmed by Western blot by probing for lysosomal associated membrane protein 1 (LAMP1) and cathepsin S. Quantification of the western blot bands showed a six-fold enrichment of cathepsin S in the endolysosomal fraction. This fraction was also devoid of cytosolic protein such as proteasome subunit α7 and actin, and displayed a five-fold reduced expression of endoplasmic reticulum protein, calnexin , a 19 amino acid long fragment from p24 protein in HIV-1 clade B encompassing epitope B57KF11 Figure (ISPRTLNAWVKV), a 17 amino acid long peptide from the HIV p24 protein encompassing epitope B57ISW9  mostly enter dendritic cells through endolysosomes for degradation followed by presentation of peptides by MHC-I and MHC-II leading to the priming of CD8 T cell responses and CD4 T helper cell responses . KnowledIn summary, we have shown that using crude PBMC cellular extracts at low pH, endolysosomal protease activity can be measured in a simple and efficient manner, results of which can be used in several applications. We demonstrated that by varying the pH in crude cellular extracts, we could specifically activate certain cathepsins at each pH and thereby monitor protease activity over the course of endolysosomal maturation. We demonstrated the usefulness of this assay in the context of antigen processing and showed comparable results to using purified endolysosomes.Buffy coats from blood donated by anonymous healthy donors were purchased from Massachusetts General Hospital. Partners Human Research Committee  approved the use of buffy coats under protocol no. 2005P001218.Antibodies for Lysosome Associated Membrane Protein 1 (LAMP1) (sc-20011) and cathepsin S (sc-271619) were purchased from Santa Cruz, beta actin (ab8227) and calnexin (ab22595) from Abcam and proteasome α 7 subunit (BML-PW8110-0100) from Enzo Lifesciences. All chemicals were purchased from Sigma and fluorogenic substrates were purchased from Enzo lifesciences. Highly purified HIV peptides were purchased from Massachusetts General Hospital peptide core facility or from Biosynthesis.8 cells/ml [Endolysosomes were purified from crude PBMC lysate by differential centrifugation. PBMCs were isolated from human buffy coats using Ficoll centrifugation . Cells rcells/ml  were lyscells/ml . The celFollowing endolysosomal purification, 10μl of each fraction and the post nuclear supernatant were loaded on a 4-12% SDS PAGE gradient gel (Invitrogen) run at a constant 185V. The proteins were transferred to a PVDF membrane (Fisher Scientific) using a semi-dry blotting system (Bio-Rad) for 35 min at 20V. The membrane was blocked using a blocking buffer  for 1 hoActivities of proteases were tested using a fluorogenic enzyme substrate specific for each endolysosomal cathepsin . For end405(sample)-A405(blank)) × 0.05 × 0.3 × DF/A405(standard)x time x Venz, where DF is dilution factor and Venz is volume of the pure enzyme.The presence of acid phosphatase in endolysosomal fractions was confirmed using a kit from Sigma (CS0740). 10 μl of each fraction was incubated with the substrate in the provided substrate buffer for 10 min at 37°C. 200 μl of the stop solution (2M sodium hydroxide) was added and the absorbance at 405 nM was measured. A blank without any lysate serving as a negative control and pure enzyme serving as a positive control was also included in the experiment. The amount of acid phosphatase present was calculated using the formula, Units/ml = /min is plotted.Click here for fileISPRTLNAWVKV) was subjected to degradation using PBMC or purified endolysosomes at pH 4.0 or pH 5.5. The fragments produced were identified by mass spectrometry and the number of similar (light grey) and unique (dark grey) peptides between the two conditions was determined. Peptides that were not detected are indicated in grey letters. The surface of the peak of each peptide is indicated for each experimental condition. (B) The sum of all peptide peak intensities was determined and the distribution of peak intensities in similar (light grey) and unique (dark grey) fragments was calculated (upper). Of the total number of peptide fragments detected in each condition, the percentage of fragments with (dark grey) and without (light grey) the B57ISW9 epitope was calculated (middle panel). Similarly, the percentage of fragments that measured 13–19 amino acids in length (dark grey), 8–12 amino acids (white) and lesser than 7 amino acids (black) was determined. In all three cases, the average of the results from two MS runs of the same experiment was plotted.Degradation of peptides with crude lysate at pH 4.0 is equivalent to using purified endolysosomes for the oligopeptide B57-5ISW9-3. A) A long HIV peptide, 5-ISW9-3 (MVHQAClick here for file


The urokinase-type plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol (GPI) anchored membrane protein, regulates urokinase (uPA) protease activity, chemotaxis, cell-cell interactions, and phagocytosis of apoptotic cells. uPAR expression is increased in cytokine or bacteria activated cell populations, including macrophages and monocytes. However, it is unclear if uPAR has direct involvement in the response of inflammatory cells, such as neutrophils and macrophages, to Toll like receptor (TLR) stimulation. In this study, we found that uPAR is required for optimal neutrophil activation after TLR2, but not TLR4 stimulation. We found that the expression of TNF-α and IL-6 induced by TLR2 engagement in uPAR-/- neutrophils was less than that in uPAR+/+ (WT) neutrophils. Pretreatment of neutrophils with PI-PLC, which cleaves GPI moieties, significantly decreased TLR2 induced expression of TNF-α in WT neutrophils, but demonstrated only marginal effects on TNF-α expression in PAM treated uPAR-/- neutrophils. IκB-α degradation and NF-κB activation were not different in uPAR-/- or WT neutrophils after TLR2 stimulation. However, uPAR is required for optimal p38 MAPK activation after TLR2 engagement. Consistent with the in vitro findings that uPAR modulates TLR2 engagement induced neutrophil activation, we found that pulmonary and systemic inflammation induced by TLR2, but not TLR4 stimulation is reduced in uPAR-/- mice compared to WT counterparts. Therefore, our data suggest that neutrophil associated uPAR could be a potential target for treating acute inflammation, sepsis, and organ injury related to severe bacterial and other microbial infections in which TLR2 engagement plays a major role. Host immune cells, including macrophages and neutrophils, recognize and respond to microbial pathogen invasion using pattern recognition receptors (PRRs), including Toll like receptors (TLRs), Nod-like receptors (NLRs), and RIG-I-like receptors (RLRs) The urokinase-type plasminogen activator receptor (uPAR) was initially identified through its interaction with urokinase-type plasminogen activator (uPA), and consists of three globule like domains , but lacks trans-membrane and cytoplasmic structures A primary function of uPAR is binding to uPA and thereby concentrating the protease activity of uPA on the leading edge of migrating cells uPAR expression is increased in cytokine or bacteria activated cell populations including macrophages and monocytes, and contributes to the infiltration of inflammatory cells into infected tissues or organs In our previous studies, we found that uPA and plasminogen activator inhibitor 1 (PAI-1) enhance the inflammatory response of neutrophils to TLR4 stimulation Bone marrow neutrophils isolated from wild type (WT) and uPAR knockout (uPAR-/-) mice were stimulated with the specific synthetic TLR2 ligand, PAM3CSK4 (PAM). As shown in -α expression. However, PI-PLC pretreatment had only minimal effects on PAM induced TNF-α expression in uPAR-/- neutrophils. These data suggest that uPAR, but not other GPI anchored proteins, has a major role in neutrophil activation upon TLR2 stimulation. Of note, PI-PLC treatment decreased uPAR levels on WT neutrophils (uPAR is a GPI anchored protein and can be released from the plasma membrane by the GPI-specific phospholipases C (PI-PLC) and D trophils .To determine if direct interactions between uPAR and its ligands are required for the involvement of uPAR in TLR2 induced neutrophil activation, WT neutrophils were pre-treated with anti-uPAR or control antibodies and then stimulated with PAM. As shown in We have shown above that the secreted levels of TNF-α by PAM stimulated uPAR-/- neutrophils are significantly less than those generated by PAM stimulated WT neutrophils. We next asked if the diminished expression of TNF-α by uPAR-/- neutrophils is due to decreased transcriptional activation. To address this issue, WT and uPAR-/- neutrophils were treated with PAM for 0, 2, or 4 hours and the levels of mRNA for TNF-α and IL-1β were determined by realtime PCR. As shown in Upon TLR2 stimulation, the TLR2 intracellular domain recruits the adaptor molecule, MyD88, as well as IRAK1 and IRAK4, leading to the activation of kinases, including IKK, p38, and Erk. IKK kinases phosphorylate IκB-α, causing its ubiquitination and degradation by the 26S proteasome and resulting in NF-κB nuclear translocation TLR2 stimulation results in activation of p38 and ERK MAPKs in vitro conditions. As neutrophils play a major role in mediating acute inflammatory lung injury In the above experiments, we demonstrated that uPAR participates in modulating TLR2 induced neutrophil activation under To define the role of uPAR in the regulation of TLR2 induced inflammatory responses, WT and uPAR-/- mice were given intraperitoneal PAM and serum levels of TNF-α measured 24 hours later. As shown in Given the participation of uPAR in TLR2 induced activation of neutrophils, we next asked if uPAR is also required for neutrophil activation upon TLR4 engagement. In these experiments, WT and uPAR-/- neutrophils were cultured with the TLR4 ligand, LPS, and the secreted levels of TNF-α measured. As shown in in vitro experiments did not demonstrate a role for uPAR in modulating TLR4 induced neutrophil activation, we wished to confirm this lack of interaction between TLR4 and uPAR under in vivo conditions. For this purpose, WT and uPAR-/- mice were given LPS intratracheally and inflammatory responses in the lungs determined 24 hours later. As shown in Although our The above experiments demonstrated that uPAR participates in TLR2, but not TLR4 mediated neutrophil activation. To determine if uPAR has similar effects on macrophage activation, peritoneal macrophages from WT and uPAR-/- mice were treated with PAM or LPS. As shown in To confirm the lack of participation of uPAR in TLR2 or TLR4 induced macrophage activation, we knocked down uPAR in WT macrophages using specific siRNA targeting uPAR. As shown in To explore mechanisms that might explain the apparently different contribution of uPAR to TLR2 activation in neutrophils and macrophages, we examined the relative expression of uPAR in neutrophils and macrophages. Surprisingly, we found that uPAR from macrophages demonstrated a smaller molecular weight than does uPAR from neutrophils .uPAR is a GPI anchored protein that lacks transmembrane and intracellular domains. The primary function of uPAR is to serve as the receptor for uPA, thus concentrating the protease activity of uPA in the proximity of cell surface. uPAR also has protease-independent activities in that it participates in the regulation of cell migration, chemotaxis, cell-cell interaction, and phagocytosis of apoptotic cells In these experiments, we found that the presence of uPAR results in enhanced production of the pro-inflammatory cytokines by neutrophils stimulated with PAM, a specific TLR2 ligand. The differential responses to PAM stimulation of WT and uPAR-/- neutrophils were significant in each independent experiment, although statistical analyses were not performed on results combined from multiple independent experiments due to different cell number used each time. However, there was no apparent difference in IκB-α degradation and subsequent NF-κB activation in PAM treated WT and uPAR-/- neutrophils. Consistent with such findings, there was no difference in the transcription of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6,, as determined by evaluation of mRNA levels, in PAM treated WT and uPAR-/- neutrophils. These data suggest that uPAR is not involved in signaling events downstream of IKK kinase activation in TLR2 stimulated neutrophils, but rather participates in modulating posttranscriptional regulation of the expression of cytokines.We found no involvement of uPAR in modulating IκB-α degradation in TLR2 activated neutrophils. However, there was markedly less activation of p38 in PAM treated uPAR-/- neutrophils than in PAM treated WT cells. Although TLR stimulation leads to activation of both IKK kinases and MAPKs, previous studies showed that such activation can be divergent in response to some extracellular stimuli We found no apparent involvement of uPAR in TLR4 induced neutrophil activation or in acute lung injury produced by LPS exposure. The mechanisms responsible for the different involvement of uPAR with TLR2 and TLR4 signaling may relate to the distinct requirement for co-factors in TLR4 associated cellular activation Unlike the potentiating role of uPAR for TLR2 induced neutrophil activation, there was no evidence that uPAR is involved in macrophage activation after TLR2 engagement. The differing participation of uPAR during TLR2 induced activation of neutrophils and macrophages is surprising as, to our knowledge, there is no cellular molecule that differentially regulates the responses of macrophage and neutrophils to TLR stimulation. However, we found that uPAR on the neutrophil surface demonstrated slower migration than did macrophage associated uPAR during PAGE electrophoresis, consistent with a higher molecular weight for neutrophil uPAR. We sequenced uPAR cDNA from neutrophils and macrophages and found that the cDNAs from the two cell populations were identical (data not shown). These data suggest that uPAR on the neutrophil surface differs from that on the macrophage because of posttranslational modifications. These findings are consistent with previous studies in various cell populations demonstrating the presence of different forms of uPAR in vitro, it is likely that diminished activation of neutrophils in vivo contributes to the attenuated inflammatory response and lung injury in PAM treated mice. Previous studies suggested that uPAR plays a detrimental role in hyperoxia-induced lung injury and that uPAR deficiency is associated with diminished neutrophil influx into both lung tissues and bronchoalveolar spaces, accompanied by decreased pulmonary injury We found that uPAR-/- mice are protected from TLR2 mediated inflammation and lung injury. Neutrophils play a central role in mediating inflammation in response to infection and tissue injury Excessive and uncontrolled inflammation after bacterial infection is a leading factor contributing to sepsis, and associated multiple organ dysfunction and death Escherichia coli 0111:B4 and rabbit anti-actin antibodies were from Sigma-Aldrich. PAM3CSK4 (PAM) was from Invivogene. Antibodies specific for phosphorylated ERK, phosphorylated-p38, total p38, and IκB-α were from Cell Signaling. Goat anti-uPAR was from R&D. Rabbit anti-GAPDH antibodies were from Santa Cruz. Control siRNA and uPAR siRNA were purchased from Dharmacon.Custom cocktail antibodies and negative selection columns for neutrophil isolation were purchased from StemCell Technologies. Brewer thioglycollate was from Sigma-Aldrich. LPS from uPAR knockout mice that have been backcrossed with C57/BL6 mice for at least nine generations were obtained from Dr. Margaret Gyetko (University of Michigan). Age-and sex-matched control C57/BL6 mice were purchased from NCI-Fredrick. 8-10 week old animals were used for experiments. Animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.Isolation of bone marrow neutrophils was performed as previously described The murine ALI model was used as previously described BAL samples were obtained from PAM or LPS-treated mice by securing a polyethylene catheter in the trachea and then lavaging the lungs three times with 1 ml of iced PBS. Levels of TNF-α in the BAL were determined by ELISA. Total cell counts were measured in the BAL fluid with a hemocytometer and protein concentrations were determined with a Bio-Rad protein assay kit.Solutions of PAM (200 µg PAM/mouse in 200 µl PBS) were injected intraperitoneally. At 6 hours after the injections, mice were sacrificed and blood was collected from the retro-orbital venous plexus.Western blotting assays were performed as previously described Immunoreactive TNF-α was quantified using DuoSet ELISA Development kits (R&D Systems) according to the manufacturer's instructions.32P] dATP (Perkin-Elmer) using T4 polynucleotide kinase as follows: κB sequence, 5′-GCCATGGGGGGATCCCCGAAGTCC-3′ (promega).Nuclear extracts were prepared and assayed by EMSA as previously described WT macrophages were plated in 24 or 48 well plates. One day after cell plating, the cells were transfected with 40 nM control siRNA or uPAR siRNA using HiperFect transfection reagents (Qiagen) according to the manufacturer's instructions. Three days after transfection, the cells were collected for Western blotting or treated with PAM or LPS.n = 3–4). One representative experiment is shown for most conditions. Each experiments were performed 2–3 times independently, using cells from different groups of mice on different days and showed similar results. One way analysis of variance (ANOVA) for multiple groups followed by the Tukey-Kramer test, or Student's t test for comparisons between two groups was used. p<0.05 was considered to be statistically significant.For each experiment, macrophages or neutrophils were isolated and pooled from groups of mice (


HBB: (c*+96T>C 3’UTR+1570 T>C) on the mildβ-thalassemia intermedia pheonotype, in the recent issueof the Journal . I congratulate theauthors for exploring at the molecular level at least oneof the thalassemia minima that fits well with the presentclinical thalassemia nomenclature. Based on their clinicaldescription, I would not consider the proband as thalassemiaintermedia, as until 20 years of age he did notrequire blood transfusions [570 T>C oI would like to suggest the following arbitrarily modifiednomenclature for clinical thalassemia syndromes:Thalassemia major (according to transfusion requirement):a) Severe: transfusion interval less than 1 month;b) Moderate: transfusion interval more than 1 month.Thalassemia intermedia (according to transfusionrequirement):a) Moderate: transfusion interval less than 1 year;b) Mild: transfusion interval more than 1 year.Thalassemia trait:a) Severe: with mild hepatosplenomegaly;b) Mild: without hepatosplenomegaly;Thalassemia minima: absence of clinical and hematologicalfindings.SincerelyŞinasi Özsoylu, MDRetired Professor of Pediatrics, Hematology, and HepatologyHonorary Fellow of the American Academy of Pediatrics since1995Honorary Member of the American Pediatric Society since 1993 The authors of this paper have no conflicts of interest,including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materialsincluded.First of all we thank Professor Dr. Özsoylu for his contributionand interest in our report. Intermedia is a betathalassemia group characterized by severity varying fromthalassemia major to asymptomatic carriers. It seems thataccording to clinical classification it is possible to describeour patient as severe thalassemia trait rather than mildthalassemia intermedia. On the other hand, the primaryreason we consider our patient as thalassemia intermediais that he is compound heterozygous for 3’UTR+1570 T>Cand Cod 8(-AA) beta-globin gene mutations. We thinkmild thalassemia intermedia is the appropriate descriptionof our patient, considering that he has two differentmutations on his 2 different copies of the beta globingene, as well as mild phenotypic findings. We think thatthe description of severe thalassemia trait is more suitablefor patients with only 1 beta globin gene mutation withclinical findings in addition to the thalassemia minor phenotype.Again, we appreciate your letter, as it afforded us theopportunity to explain in greater detail our rationale forthe use of genetic data in addition to clinical criteria in theclassification of thalassemias.